TD LauraMirallesLopez

Universidad de Oviedo
Departamento de Biología Funcional

Programa de Doctorado: Biología Funcional y Molecular
Papel de las barreras biogeográficas e historia

evolutiva en la estructura poblacional actual de
especies marinas cosmopolitas.
Tesis doctoral
Laura Miralles López
Oviedo 2014
©Laura Miralles López, 2014.
Diseño de portada
Laura Miralles
Fotografía de portada
Ángel Medina
Maquetación
Laura Miralles
Edición
Servicio de Publicaciones de la Universidad de Oviedo

RESUMEN DEL CONTENIDO DE TESIS DOCTORAL
1.- Título de la Tesis

Español/Otro Idioma: Inglés:
PAPEL DE LAS BARRERAS THE ROLE OF BIOGEOGRAPHIC
BIOGEOGRÁFICAS E HISTORIA EVOLUTIVA BARRIERS AND EVOLUTIONARY HISTORY
EN LA ESTRUCTURA POBLACIONAL IN THE PRESENT POPULATION
ACTUAL DE ESPECIES MARINAS STRUCTURE OF MARINE COSMOPOLITAN
COSMOPOLITAS SPECIES
2.- Autor
Nombre: DNI/Pasaporte/NIE:
LAURA MIRALLES LÓPEZ -
Programa de Doctorado: BIOLOGÍA FUNCIONAL Y MOLECULAR
Órgano responsable: BIOLOGÍA FUNCIONAL
RESUMEN (en español)
Las especies son grupos de poblaciones naturales e intercruzables que están

FOR-MAT-VOA-010-BIS
aislados reproductivamente de otros grupos análogos. En el medio marino, las barreras

al flujo génico entre especies no son tan evidentes como en el terrestre. Es
imprescindible identificarlas porque tanto la variabilidad genética de las especies como
su estructura genética poblacional y por supuesto, la especiación, están modeladas por
barreras presentes y pasadas en la historia evolutiva de cualquier grupo taxonómico.
La hibridación y la introgresión son procesos que suceden regularmente en la
naturaleza, íntimamente relacionados con los procesos de especiación. La hibridación
ocurre cuando las barreras de aislamiento reproductor entre dos especies son débiles y
estas se aparean entre sí. Cuando los descendientes híbridos son viables y fértiles se
produce la introgresión genética. Actualmente muchos casos de hibridación en la
naturaleza son atribuidos a la degradación del medio o a la acción humana. Los cambios
causados por las actividades humanas afectan a todos y cada uno de los ecosistemas del
planeta. Los océanos se encuentran entre los más vulnerables. La contaminación, la
sobrepesca, la acuicultura, la introducción de especies, el tráfico marítimo y el
calentamiento global son algunos de los muchos factores de impacto negativo que
afectan al medio marino. Entre otros efectos promueven o eliminan barreras al flujo
génico, cambiando de este modo las relaciones entre especies y alterando las
probabilidades de ocurrencia de hibridación e introgresión.
Para la realización de esta tesis doctoral se seleccionaron tres modelos
biológicos con gran capacidad de dispersión e historias evolutivas diferenciales, que
permiten abordar los mecanismos que promueven procesos de especiación en el océano
Atlántico: la anjova Pomatomus saltatrix, única especie en su género y familia, como
ejemplo de especiación lenta o no especiación; las cinco especies de merluzas Euro-
Africanas del género Merluccius como ejemplo de especiación múltiple en gradientes
ambientales; y las dos especies de calderones del género Globicephala, como modelo de
especies con especiación reciente. Se analizaron secuencias de ADN mitocondriales: el
gen de la subunidad I de la citocromo oxidasa c (COI), el gen del citocromo b, y la
región control. También se utilizaron loci nucleares: el gen de la región determinante
del sexo en el cromosoma Y (SRY), el gen del ARN ribosomal 5S, y un total de 25
microsatélites. Mediante estas herramientas genéticas se realizaron reconstrucciones
filogenéticas, inferencias demográficas históricas mediante coalescencia, identificación
de barreras al flujo génico entre unidades genéticas poblacionales y específicas, y
cuantificación de introgresión genética. Se determinaron los factores ecológicos
implicados en las barreras al flujo génico mediante análisis multivariante, de
similitudes, de componentes principales, no métricos y representaciones
multidimensionales.
Los resultados se reflejan en siete artículos científicos. Para Pomatomus saltatrix
se infiere por primera vez la existencia de migración transoceánica en el océano
Atlántico como factor preventivo de especiación vicariante, contrarrestando el efecto de
barreras reproductivas pasadas que aparecieron durante las glaciaciones. También se
describen sesgos asociados al sexo en la migración y la dispersión de esta especie, así
como el papel de la actividad humana, en este caso la acuicultura, para suprimir la
diferenciación entre unidades poblacionales distantes en el mar Mediterráneo.
En los dos clados que componen las merluzas Euro-Africanas se ha podido
identificar un mecanismo de especiación ecológica basado en un gradiente batimétrico,
donde la profundidad de puesta parece ser el factor principal que explica la especiación
de este grupo. La hibridación introgresiva entre especies pertenecientes a clados
distintos (Merluccius capensis y M. paradoxus) se ha asociado a alteraciones climáticas,
como el fenómeno de El Niño de Benguela, sin excluir el posible papel de otras
presiones antropogénicas como las pesquerías.
Finalmente, la hibridación introgresiva principalmente unidireccional encontrada
en los calderones hacia el calderón común, Globicephala melas, en el noreste del
Atlántico, ha podido ser interpretada en relación con el efecto de la acción humana,
probablemente una combinación de las cacerías tradicionales al norte de la distribución,
el tráfico marítimo y el calentamiento global, que están contribuyendo a suprimir las
barreras interespecíficas naturales y ponen en peligro su integridad como especie en esta
zona.
El conjunto de resultados de esta Tesis Doctoral han permitido identificar
factores ecológicos como la profundidad como causas principales de especiación. Si
bien en el pasado los cambios climáticos parecen haber promovido barreras al flujo
génico en especies con gran capacidad dispersiva, los resultados encontrados en los tres
modelos analizados sugieren que en la actualidad tanto las alteraciones climáticas como
la actividad antropogénica están contribuyendo a la ruptura o fragilidad de las barreras
entre unidades genéticas. Las consecuencias esperables son la homogeneización
genética espacial a gran escala, implicando una pérdida de biodiversidad y un aumento
de la diversidad intraespecífica. Se acompañan recomendaciones para la conservación
de especies de importancia económica y social, basadas en la moderación de las
actividades antropogénicas más que en el énfasis en santuarios marinos, probablemente
de menor importancia relativa en especies de gran capacidad de dispersión.
RESUMEN (en Inglés)
Species are groups of natural interbreeding populations that are reproductively

isolated from other analogous groups. In the marine realm, barriers to gene flow are not
as evident as in land. It is essential to identify them because species genetic variability
as well as genetic population structure, and obviously speciation, are shaped by present
and historical barriers in the evolutionary history of any taxonomic group.
Hybridization and introgression are processes that occur with regularity in
nature and they are closely related to speciation processes. Hybridization happens when
barriers to reproductive isolation between two species are weak and they can interbreed.
Introgression occurs when the hybrid offspring is viable and fertile. Nowadays, most of
hybridization processes in nature are assigned to ecosystem degradation or due to
human action. Changes made by human activities affect every single ecosystem of the
planet. Oceans are one of the most vulnerable ecosystems. Contamination, overfishing,
aquaculture, species introduction, marine traffic and global warming are some of many
negative antropogenic factors that affect the marine realm. Between other effects, those
factors promote or erase barriers to gene flow. Then, it changes the relationships
between species and alters the likelihood of possible hybridization and introgression.
Three different study models were selected for the fulfillment of this PhD thesis.
The selected models have a wide dispersal capacity and different evolutionary histories
that would deal with mechanisms that promote speciation processes in the Atlantic
ocean: the Bluefish Pomatomus saltatrix, the only species in its genus and family as an
example of slow speciation or no-speciation; the five Euro-African true-hakes from the
genus Merluccius as an example of diversified speciation across gradients, and the two
cetacean species of the genus Globicephala with a recent speciation. Different
mitochondrial DNA sequences were analysed: cytochrome oxidase c subunit I gene,
cytochrome b gene and d-loop gene. Nuclear loci were also employed: sex determining
region in Y chromosome gene (SRY) and ribosomal ARN 5s gene, and a total of 25
microsatellite loci. Phylogenetic reconstructions, coalescent historic effective
population size inferences with, identification of barriers to gene flow between
population and specific genetic units, and hybridization and introgression
quantifications were done with the previously described genetic markers. Moreover,
ecological factors that are concerned in the gene flow barriers were determined by
multivariate analysis, analyses of similarity, principal components, non-metric analyses
and multidimensional representations.
Results were reflected on seven scientific articles. For Pomatomus saltatrix, the
existence of trans-oceanic migration across both sides of the Atlantic Ocean has been
suggested as a preventing factor of vicariant speciation by counteracting the effect of
historical reproductive barriers during the glaciations. Moreover, sex-biased migration
and dispersal has been described for this species. Also, it has been studied that human
action, through aquaculture, may alter the regional differentiation of this species in the
Mediterranean Sea.
A model of ecological speciation, based on a bathymetric gradient, was
identified in the two clades of Euro-African hakes, with spawning-depth set as the main
factor behind the speciation of this group. Introgressive hybridization between species
from different clades (Merluccius capensis and M. paradoxus) were related to climatic
alterations, like the Benguela Niño, without excluding the role of other antropogenic
pressures like fisheries.
Finally, the unidirectional introgressive hybridization found into the long-finned
pilot whale, Globicephala melas, off northeastern Atlantic Ocean could be probably due
to a combination of effects of human activity such as cultural grinds, marine traffic and
global warming, that erase the natural interspecific barriers and set under risk its
integrity as a species in this area.
The results of this PhD thesis let the identification of ecological factors such as
depth as the main causes of speciation. While past climate changes seem to have
promoted barriers to gene flow in species with high dispersive capacity, the results
found in the three analyzed models suggested that current climate alterations and
anthropogenic activities are contributing to the fragility or breakdown of barriers
between genetic units. The expected consequences are spatial genetic homogenization at
a wide scale, that implying a loss of biodiversity and an increase of intraspecific
diversity. Recommendations for the conservation of economically and culturally
important species were included, based on a moderation of anthropogenic activities
rather than the emphasis on marine sanctuaries, probably with relatively minor
importance for species with high dispersal capacity.
SR. DIRECTOR DE DEPARTAMENTO DE BIOLOGÍA FUNCIONAL

Agradecimientos
Como todo gran viaje que se preste, cuando estás a punto de llegar al final
empiezas a recordar qué es lo que te ha llevado hasta ahí, por qué decidiste tomar
ese rumbo, quienes se han cruzado en tu camino y quiénes han hecho de ese viaje
una aventura inolvidable.
A día de hoy, puedo decir alto y claro que este viaje a Asturias ha sido mi aventura
más importante hasta el momento y estoy enormemente agradecida de haber
conocido a un montón de personas que me han ayudado a ser quien soy, al igual
que también lo estoy por haber tenido a mi lado a otro montón de personas que ya
conocía de antes, de otros viajes, de otros lugares y de otras aventuras. Es difícil
resumir en unas pocas palabras todo ese agradecimiento, pero voy a intentarlo:
En primer lugar y no se me ocurre un lugar mejor, quiero dar las gracias a mi jefa y
directora: Eva García Vázquez. No sólo por la gran oportunidad que me diste al
poder realizar esta Tesis Doctoral, sino por todas las demás cosas que he podido
hacer en este tiempo que he estado contigo, desde invenciones de juegos frikis,
hasta coordinar decenas de Erasmus por los ríos más perdidos e inaccesibles de
Asturias. Gracias por tus conocimientos, por tu comprensión, por tu ideas, por tus
correcciones, por tu fuerza y por tu actitud positiva siempre. Gracias por confiar en
mi. Gracias por dejarme incluir las ballenas en la Tesis y por hacer posible que fuera
al otro lado del planeta, al paraíso, a por ellas. Gracias por formarme como
científica. Un millón de gracias por guiarme y enseñarme tantas cosas, no sólo de
ciencia, sino de la vida.
Gracias a toda la gente del Área de Genética, compañeros de Laboratorio, de la

hora de la comida, profesores, amigos. Todos los que están y los que se fueron
yendo poco a poco. De todos aprendí un poquito y su paso por mi Tesis fue
enriquecedor tanto a nivel académico como personal. ¡¡Gracias chicos!!
También quiero agradecer a Santiago Lens, del Instituto Español de Oceanografía,

su ayuda en la parte de mamíferos marinos. Gracias por acogerme en Vigo y por
aportar tu granito de arena en esta Tesis. Estoy muy contenta de haberte conocido
y haber podido trabajar contigo.
Con mi francés manchego: Merci à tous mes amis polynésiennes et toutes les
personnes avec qui j'ai travaillé à le CRIOBE en Moorea pour qu’ils ont fait mon
séjour international, la meilleure expérience de ma vie. Surtout je veux remercier
Serge Planes, pour rendre possible cette aventure et pour fait réaliser cette rêve.
J'espère qu'un jour je peux revenir à Moorea. Je serai éternellement reconnaissant,
Serge. Aussi, je voulais remercier mon amie Agnès. Merci de m'aider avec les
baleines, d'être mon amie, d'être à Tahiti avec moi, et de joindre à moi dans cette
aventure. A très bientôt.
A mis amigas bio-locas de toda la vida: Ana, Irene, María José y Sandra. La verdad
es que ha sido un placer compartir esta aventura con vosotras, iba a decir que
desde la distancia, pero eso nunca fue un problema. Siempre estuvisteis y estáis
ahí. Habéis sido un gran apoyo y ayuda. ¡¡Gracias chicas!! Además, tengo que
admitir que ha sido muy alentador y está siendo muy emocionante que nos
vayamos a doctorar a la vez, ¡¡mucho ánimo Ana y Sandra, que no os queda nada
para ser doctoras vosotras también!!
A Ester, mi capitana preferida y mi gran apoyo en multitud de ocasiones a lo largo

de esta singladura y se que a lo largo de toda una travesía mucho más larga, la vida.
Al patrón Ramón, por ser un verdadero piratilla y alegrarme cada vez que hablamos
o nos vemos. Al gran Enrique, por todas esas veces que has venido a Asturias y
hemos compartido auténticos manjares de la tierra recordando nuestras hazañas
marineras.
A Catarina y Mareike, my flatmates, porque no sólo fuisteis mis compañeras de

piso, sino mis confidentes, mis amigas, mis compañeras de piscina, de paseos, de
fiestas, de compras... I miss you a lot, my girls.
Gracias a todos mis amigos, que me han dado múltiples (y variados) apoyos
durante esta Tesis. No os imagináis lo agradecida que estoy por todas vuestras
palabras a lo largo de estos 4 años. Vosotros también formáis parte de esta tesis y
aunque ya os he dicho personalmente, quería dejarlo escrito para la posteridad.
Gracias.
A las familias Aquanau y Medina, porque hicisteis de Alicante mi casa y me hiciste

un miembro más de vuestra familia: la hija pródiga, vuestra bióloga. Porque fue un
verdadero placer poder haceros partícipes de mi trabajo y haber trabajado juntos.
Ha sido un orgullo haber compartido todo tipo de experiencias con vosotros y
sobre todo me siento privilegiada de haber podido aprender y crecer con vosotros.
No os imagináis cuanto os echo de menos… ¡¡Tenemos que ir a bucear ya!!
Hablando de familia y viajando entre Alicante y Asturias, voy a hacer una parada en
Almansa porque también quiero agradecer a mi familia, la de sangre que dirían
ellos o de genes que diría yo (tíos, primos, abuelos) por vuestro cariño y apoyo,
porque se que estáis orgullosos de mi y eso me da mucha fuerza para seguir
siempre adelante. Finalizando el viaje, ya en la “tierrina”, también quería dedicarle
unas palabras a mi nueva familia de Salinas, porque me hicieron sentir como en
casa desde el primer momento y me han dado muchísimo cariño.
Por supuesto, no podían faltar las personas a las que más agradecida estoy y lo
estaré siempre, por todo su apoyo incondicional: mis padres y mis hermanos.
Porque siempre han estado ahí, a pesar de que con todo su dolor tuvieron que
dejarme ir y poner kilómetros por medio para que yo pudiera cumplir este sueño.
Por todos esos minutos de teléfono y videoconferencias, por todos esos “te echo
de menos” que no nos hemos dicho (pero si los hemos sentido), por todos esos “tu
puedes” que salían cuando más los necesitaba, por recordarme que hay tiempo
para todo y un tiempo para cada cosa… y por tantísimas cosas más: MIL MILLONES
DE GRACIAS, OS QUIERO TANTO… Sin vosotros, ni vuestro apoyo, esto nunca habría
sucedido.
Por último, quería agradecerle enormemente a la persona que ha estado a mi lado

día a día en este último tramo, por haber vivido conmigo cada etapa, cada
problema y cada alegría de esta Tesis. Gracias por tu paciencia, por tu
comprensión, por tu energía, por aguantarme (que no es poco… jajajaja...), por
hacerme reír, por quererme, por cuidarme, por un millón de cosas más y sobre
todo, por enseñarme que mi paso por Asturias es mucho más que una Tesis
Doctoral. Porque tú me has dado la fuerza que necesitaba para poder culminar esta
importante etapa de mi vida y has hecho del tramo más difícil, el más bonito de
todos. Gracias Javi.
Gracias también a los Yoyis, a mi Escubi y a mis chuchis, que tanta compañía, sustos
y alegrías me han dado.
Muchas gracias a todos, sin vosotros esto habría sido mucho más difícil,
probablemente imposible... Os quiero.
Laura
A ti, porque te quiero.
Mamá, papá, Chencho, Marisa y Javi

“Nothing in Biology makes sense
except in the light of Evolution”
T. Dobzhansky, 1972.
!
Índice
1
2
Índice
!
!
Resumen!!!______________________________________________!!!!9!
Summary!!!______________________________________________!!!!13!
Introducción!!!___________________________________________!!!!17!
Especies!y!especiación!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!21!
Barreras,!aislamiento!y!especiación!en!el!medio!marino!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!24!
Hibridación!e!introgresión!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!26!
El!ser!humano!en!los!procesos!de!especiación!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!28!
Modelos!para!el!estudio!de!la!especiación!en!el!medio!marino!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!31!
Hipótesis!de!partida!!!!_____________________________________!!!39!
Objetivos!!!______________________________________________!!!43!
Material!y!métodos!!______________________________________!!!!47!
Regiones!marinas!en!estudio!y!obtención!de!muestras!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!49!
Extracción!de!ADN!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!53!
Marcadores!genéticos!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!54!
Datos!morfológicos!y!ecológicos!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!62!
Análisis!de!datos!genéticos!y!software!utilizado!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!62!
3
!
Resultados!!!______________________________________________!65!
Capitulo!1!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!69!
Capitulo!2!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!81!
Capitulo!3!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!91!
Capitulo!4!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!99!
Capitulo!5!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!111!
Capitulo!6!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!121!
Capitulo!7!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!129!
Discusión!!!______________________________________________!!141!
Barreras!al!flujo!génico!en!la!especiación!marina!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!143!
El!concepto!de!especie!en!los!modelos!estudiados!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!149!
La!acción!humana!en!los!procesos!de!especiación!marina!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!150!
Recomendaciones!para!la!conservación!!!_____________________!!155!
Tesis:!Reconsideración!de!las!hipótesis!de!partida!!!_____________!!159!
Conclusiones!!!!__________________________________________!!165!
Conclusions!!!!___________________________________________!!167!
References!!!____________________________________________!!169!
Glosario!!_______________________________________________!!185!
Anexos!!!!_______________________________________________!!!!!!I!
1.ZPermisos!CITES!de!las!muestras!de!calderones!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!III!
2.Z!Ficha!de!morfometría!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!IX!
3.Z!Portada!revista!Fisheries!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!XI!
4.Z!Divulgación!en!los!medios!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!XII!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
4
!
5
6
Resumen / Summary
7
Resumen
8
Resumen
Resumen
Las especies son grupos de poblaciones naturales e intercruzables que están

aislados reproductivamente de otros grupos análogos. En el medio marino, las
barreras al flujo génico entre especies no son tan evidentes como en el terrestre. Es
imprescindible identificarlas porque tanto la variabilidad genética de las especies
como su estructura genética poblacional y por supuesto, la especiación están
modeladas por barreras presentes y pasadas en la historia evolutiva de cualquier
grupo taxonómico.
La hibridación y la introgresión son procesos que suceden regularmente en

la naturaleza y están íntimamente relacionados con los procesos de especiación. La
hibridación ocurre cuando las barreras de aislamiento reproductor entre dos
especies son débiles y estas se aparean entre sí. Cuando los descendientes híbridos
son viables y fértiles se produce la introgresión genética. Actualmente muchos
casos de hibridación en la naturaleza son atribuidos a la degradación del medio o a
la acción humana. Los cambios causados por las actividades humanas afectan a
todos y cada uno de los ecosistemas del planeta. Los océanos se encuentran entre
los más vulnerables. La contaminación, la sobrepesca, la acuicultura, la
introducción de especies, el tráfico marino y el calentamiento global son algunos de
los muchos factores de impacto antropogénico negativo que afectan al medio
marino. Entre otros efectos promueven o eliminan barreras al flujo génico,
cambiando de este modo las relaciones entre especies y alterando las
probabilidades de ocurrencia de hibridación e introgresión.
9
Resumen
Para la realización de esta Tesis Doctoral se seleccionaron tres modelos

biológicos con gran capacidad de dispersión e historias evolutivas diferenciales, que
permiten abordar los mecanismos que promueven procesos de especiación en el
océano Atlántico: la anjova Pomatomus saltatrix, única especie en su género y
familia, como ejemplo de especiación lenta o no especiación; las cinco especies de
merluzas Euro-Africanas del género Merluccius como ejemplo de especiación
múltiple en gradientes ambientales; y las dos especies de calderones del género
Globicephala, como modelo de especies con especiación reciente. Se analizaron
secuencias de ADN mitocondriales: el gen de la subunidad I de la citocromo oxidasa
c (COI), el gen del citocromo b, y la región control. También se utilizaron loci
nucleares: el gen de la región determinante del sexo en el cromosoma Y (SRY), el
gen del ARN ribosomal 5S, y un total de 25 microsatélites. Mediante estas
herramientas genéticas se realizaron reconstrucciones filogenéticas, inferencias
demográficas históricas mediante coalescencia, identificación de barreras al flujo
génico entre unidades genéticas poblacionales y específicas, y cuantificación de
introgresión genética entre dichas unidades. Se determinaron los factores
ecológicos implicados en barreras al flujo génico mediante análisis multivariante,
de similitudes, de componentes principales, no métricos y representaciones
multidimensionales.
Los resultados se reflejan en siete artículos científicos. Para Pomatomus

saltatrix se infiere por primera vez la existencia de migración transoceánica en el
océano Atlántico como factor preventivo de especiación vicariante,
contrarrestando el efecto de barreras reproductivas pasadas que aparecieron
durante las glaciaciones. También se describen sesgos asociados al sexo en la
migración y la dispersión de esta especie, así como el papel de la actividad humana,
en este caso la acuicultura, para suprimir la diferenciación entre unidades
poblacionales distantes en el mar Mediterráneo.
En los dos clados que componen las merluzas Euro-Africanas se ha podido

identificar un mecanismo de especiación ecológica basado en un gradiente
batimétrico, donde la profundidad de puesta parece ser el factor principal que
10
Resumen
explica la especiación de este grupo. La hibridación introgresiva entre especies

pertenecientes a clados distintos (Merluccius capensis y M. paradoxus) se ha
asociado a alteraciones climáticas, como el fenómeno de El Niño de Benguela, sin
excluir el posible papel de otras presiones antropogénicas como las pesquerías.
Finalmente, la hibridación introgresiva principalmente unidireccional

encontrada en los calderones hacia el calderón común, Globicephala melas, en el
noreste del Atlántico, ha podido ser interpretada en relación con el efecto de la
acción humana, probablemente una combinación de las cacerías tradicionales al
norte de la distribución, el calentamiento global y el tráfico marino, que están
contribuyendo a suprimir las barreras interespecíficas naturales y ponen en peligro
su integridad como especie en esta zona.
El conjunto de resultados de esta Tesis Doctoral han permitido identificar

factores ecológicos, como la profundidad, como causas principales de especiación.
Si bien en el pasado los cambios climáticos parecen haber promovido barreras al
flujo génico en especies con gran capacidad dispersiva, los resultados encontrados
en los tres modelos analizados sugieren que en la actualidad tanto las alteraciones
climáticas como la actividad antropogénica están contribuyendo a la ruptura o
fragilidad de las barreras entre unidades genéticas. Las consecuencias esperables
son la homogeneización genética espacial a gran escala, implicando una pérdida de
biodiversidad y un aumento de la diversidad intraespecífica. Se acompañan
recomendaciones para la conservación de especies de importancia económica y
social, basadas en la moderación de las actividades antropogénicas más que en el
énfasis en santuarios marinos, probablemente de menor importancia relativa en
especies de gran capacidad de dispersión.
11
12
Summary
Summary
Species are groups of natural interbreeding populations that are

reproductively isolated from other analogous groups. In the marine realm, barriers
to gene flow are not as evident as in land. It is essential to identify them because
species genetic variability as well as genetic population structure, and obviously
speciation, are shaped by present and historical barriers in the evolutionary history
of any taxonomic group.
Hybridization and introgression are processes that occur with regularity in

nature and they are closely related to speciation processes. Hybridization happens
when barriers to reproductive isolation between two species are weak and they
can interbreed. Introgression occurs when the hybrid offspring is viable and fertile.
Nowadays, most of hybridization processes in nature are assigned to ecosystem
degradation or due to human action. Changes made by human activities affect
every single ecosystem of the planet. Oceans are one of the most vulnerable
ecosystems. Contamination, overfishing, aquaculture, species introduction, marine
traffic and global warming are some of many negative antropogenic factors that
affect the marine realm. Between other effects, those factors promote or erase
barriers to gene flow, which changes the relationships between species and alters
the likelihood of possible hybridization and introgression.
Three different study models were selected for the fulfillment of this PhD
Thesis. The selected models have a wide dispersal capacity and different
evolutionary histories that would deal with mechanisms that promote speciation
processes in the Atlantic ocean: the Bluefish Pomatomus saltatrix, the only species
13
Summary
in its genus and family as an example of slow speciation or no-speciation; the five
Euro-African true-hakes from the genus Merluccius as an example of diversified
speciation across gradients, and the two cetacean species of the genus
Globicephala with a recent speciation. Different mitochondrial DNA sequences
were analysed: cytochrome oxidase c subunit I gene, cytochrome b gene and d-
loop gene. Nuclear loci were also employed: sex determining region in Y
chromosome gene (SRY) and ribosomal 5s gene, and a total of 25 microsatellite
loci. Phylogenetic reconstructions, coalescent historic effective population size
inferences with, identification of barriers to gene flow between population and
specific genetic units, and hybridization and introgression quantification between
those units were done with the previously described genetic markers. Moreover,
ecological factors that are concerned in the gene flow barriers were determined by
multivariate analysis, analyses of similarity, principal components, non-metric
analyses and multidimensional representations.
Results were reflected on seven scientific articles. For Pomatomus saltatrix,

the existence of trans-oceanic migration across both sides of the Atlantic Ocean has
been suggested as a preventing factor of vicariant speciation by counteracting the
effect of historical reproductive barriers during the glaciations. Moreover, sex-
biased migration and dispersal has been described for this species. Also, it has been
studied that human action, through aquaculture, may alter the regional
differentiation of this species in the Mediterranean Sea.
A model of ecological speciation, based on a bathymetric gradient, was

identified in the two clades of Euro-African hakes, with spawning-depth set as the
main factor behind the speciation of this group. Introgressive hybridization
between species from different clades (Merluccius capensis and M. paradoxus)
were related to climatic alterations, like the Benguela Niño, without excluding the
role of other antropogenic pressures like fisheries.
Finally, the unidirectional introgressive hybridization found into the long-

finned pilot whale, Globicephala melas, off northeastern Atlantic Ocean could be
14
Summary
probably due to a combination of effects of human activity such as cultural grinds,

global warming and marine traffic, that erase the natural interspecific barriers and
set under risk its integrity as a species in this area.
The results of this PhD Thesis let the identification of ecological factors such
as depth as the main causes of speciation. While past climate changes seem to
have promoted barriers to gene flow in species with high dispersive capacity, the
results found in the three analyzed models suggested that current climate
alterations and anthropogenic activities are contributing to the fragility or
breakdown of barriers between genetic units. The expected consequences are
spatial genetic homogenization at a wide scale, that implying a loss of biodiversity
and an increase of intraspecific diversity. Recommendations for the conservation of
economically and culturally important species based on a moderation of
anthropogenic activities rather than the emphasis on marine sanctuaries, probably
with relatively minor importance for species with high dispersal capacity, were
included.
15
16
Introducción
17
Introducción
18
Introducción
Introducción
La especiación o formación de las especies ha sido un tema de gran interés

para el ser humano desde la antigüedad hasta hoy en día, incluyendo antiguas
religiones (Génesis, Capítulo 1, La Biblia; ayatolá Makarem Shirazi, El Corán) y
pasando por perspectivas científicas (Darwin 1859). Ha sido y será fuente de
debate tanto por especialistas en el tema, así como por gente profana en la
materia de todas las edades. Como todo tema de gran interés, los procesos de
formación de las especies son también una importante fuente de controversia, no
sólo entre las grandes y extremas corrientes de pensamiento, sino incluso entre las
diferentes teorías existentes dentro de cada una de ellas. Así pues, dentro de la
corriente científica y evolutiva, el concepto de especiación fue cambiando a lo largo
de la historia.
En 1809, Jean Baptiste Lamarck ofreció una interpretación filogenética de una

de las más antiguas representaciones de la naturaleza: la “Scala Naturae”,
propuesta por Platón y Aristóteles, que tomó mayor importancia en el
Renacimiento. Se trataba de una escalera de la vida que podía ser escalada por la
evolución e iba desde las formas más inferiores hasta las superiores. Pero su teoría
no fue aceptada en su época, tanto por razones científicas como políticas y
sociales. No fue hasta 1859 cuando Charles Darwin escribió “El origen de las
especies”, cuyo título completo, aunque menos conocido, es “El origen de las
especies por medio de la selección natural, o la preservación de las razas
favorecidas en la lucha por la vida”. En esta obra se proponía un mecanismo de
especiación basado en fuertes evidencias sobre la evolución de los organismos que,
19
Introducción
entre otros factores,

consideraba el papel de la
selección natural. Unos años
más tarde, en 1866, Ernst
Haeckel (seguidor de Darwin)
produjo “El árbol de la vida”
(Figura 1) en el que se percibía
la evolución de los organismos
a través del tiempo como un
proceso de ramificación,
literalmente. A pesar de que la
teoría de la evolución de
Darwin despertó mucho
interés, la falta de
conocimientos sobre los
Figura 1: Árbol de la vida de Ernst Haeckel
mecanismos de herencia
genética hizo que dicha teoría no fuera realmente evidente en su época. En 1900,
Mendel descubrió la herencia de los caracteres discretos. Unos años más tarde, los
trabajos de Fisher (1930), Wright (1931) y Haldane (1932) contribuyeron al
desarrollo de la “Síntesis Moderna” (Dobzhansky 1937; Huxley 1942; Mayr 1942;
Simpson 1944), que no sólo unificaba los conceptos de la herencia genética con los
mecanismos de la evolución, sino que representaba también la conexión entre
multitud de ramas de la biología como genética, paleontología, botánica,
sistemática, citología, etc. Actualmente, los avances técnicos y la aparición de
nuevas herramientas en biología molecular han hecho posibles grandes progresos
en el campo de la evolución. Sin embargo, a día de hoy, la especiación sigue siendo
un tema de gran interés (Dieckmann y Doebeli 1999; Gavrilets 2004; Mallet 2008;
entre otros) sobre el que aún no está todo dicho y quedan muchas preguntas por
resolver. Así, esta Tesis Doctoral se centrará en los procesos de evolución y, más
concretamente, de especiación en el medio marino.
20
Introducción
Especies y especiación
La especiación es la formación de nuevas especies; por tanto, para poder

hablar de especiación es necesario definir previamente qué es una especie.
El concepto de especie es un fundamento básico para cualquier rama de la

biología (Barberá 1994). Sin embargo, a pesar de ser fundamental, el concepto per
se ha estado rodeado de controversia a lo largo de su historia, desde su primera
definición por Aristóteles, pasando por Aquilino, Linneo, Buffon, Darwin y otros
autores; llegando en ocasiones a definiciones incompatibles entre ellas. No hay en
biología un concepto tan fundamental y rodeado de tanta controversia como el
concepto de especie. La aparición del concepto biológico de especie (Mayr 1942)
parecía solucionar un problema con más de dos milenios de antigüedad. No
obstante, el concepto biológico de especie ha tenido muchas críticas y fue revisado
numerosas veces, incluso por el propio autor, principalmente debido a que, según
Mayr, una especie no se puede definir (Mayr 2000), sí puede ser descrita, pero no
definida ni delimitada con rotundidad. Para intentar subsanar este problema,
diferentes ramas de la biología han aportado diferentes conceptos de especie
basados cada uno en diferentes propiedades biológicas. El concepto biológico de
especie (Mayr 1942) está basado en el aislamiento reproductivo, el ecológico (Van
Valen 1976) en la separación de nichos, el genético (revisado en Baker y Bradley
2006) en el aislamiento genético, el evolutivo (Wiley 1978) en linajes ancestro-
descendientes, el filogenético (Cracraft 1989) en caracteres derivados, y así hasta
más de una veintena de conceptos de especie (Mayden 1997). Sin embargo, la
utilización de uno u otro de los diferentes conceptos hace variar el número de
especies reconocidas (Baker y Bradley 2006) y, a día de hoy, siguen siendo
numerosas las revisiones a cerca del tema (e.g. Häusen 1987; Kimbel y Martin
1993; Mayr 2000; Hausdorf 2001; De Queiroz 2007). El concepto biológico de
especie, que es uno de los más aceptados y usados en biología (Mayr 2000), se
define como:
21
Introducción
“Las especies son grupos de poblaciones naturales, real o

potencialmente intercruzables, aislados reproductivamente de otros
grupos análogos” (Mayr 1942)
Utilizando el concepto biológico de especie como referencia, la especiación

ocurre cuando una población inicial evoluciona a dos subgrupos que están aislados
reproductivamente (Bolnick 2004). Así, basándose en el grado de aislamiento entre
las poblaciones se llegó a la enunciación de los modelos de especiación (Figura 2):
Figura 2: Esquema de los modelos y mecanismos de evolución en la especiación alopátrica, peripátrica,

parapátrica y simpátrica. Las especies se representan en diferentes tonalidades de azul.
- Especiación alopátrica: Aislamiento por barreras geográficas en el que existe

una separación espacial de las poblaciones. La nueva especie surge por
adaptación divergente, en la cual la selección natural y la deriva génica hacen
que se acumulen mutaciones diferentes en cada subpoblación.
22
Introducción
- Especiación simpátrica: No hay aislamiento geográfico, las poblaciones

comparten el mismo espacio. La nueva especie surge a partir de
polimorfismos (ver Glosario), entre los cuales aparecen mecanismos de
aislamiento genético. También podría ocurrir por especiación ecológica
diferencial, es decir, aunque compartan la misma zona geográfica los
individuos de una y otra subpoblación se especializan en el aprovechamiento
de nichos ecológicos diferentes (Barluenga y col. 2006).
- Especiación parapátrica y peripátrica: La nueva especie surge en hábitats

marginales adyacentes o cercanos, habitualmente en los límites de
distribución de una población central de mayor tamaño.
En los últimos años, ha tomado fuerza un nuevo concepto de especiación: la

especiación ecológica. Un número de estudios cada vez mayor sugiere que la
selección basada en condiciones ecológicas ambientales es uno de los mecanismos
clave para la especiación en los ecosistemas marinos (Keller y Seehausen 2012;
Nosil 2012; Jennings y col. 2013; Prada y col. 2013). En la especiación ecológica se
forma un continuo que va desde la adaptación genética a diferentes ambientes
dentro de una misma especie, hasta la diversidad genética entre especies que
suelen estar cercanamente emparentadas (Skulason y Smith 1995; Schluter 2001;
Coyne y Orr 2004). A lo largo de este continuo, las barreras reproductivas son
débiles y puede haber flujo génico entre las unidades reproductoras. Sobre este
flujo se superpondría la selección de los genes beneficiosos para una de las
variantes ambientales, lo que en un ambiente heterogéneo llevará a la adaptación
divergente de las especies (Seehausen y col. 2008; Wu 2001). El aislamiento
reproductivo aparecerá como un subproducto de la divergencia ecológica. En otras
palabras, los gradientes ambientales pueden causar diferenciación poblacional,
selección y pueden promover la adaptación a condiciones ambientales específicas
lo que conllevará con el tiempo a la especiación.
23
Introducción
Barreras, aislamiento y especiación en el medio marino
Un cambio evolutivo rápido, así como la adaptación a un medio diferente, no

son suficientes para que ocurra especiación. Tiene que haber una sincronización
entre la divergencia poblacional y la evolución del aislamiento reproductivo (Orr y
Smith 1998). Indudablemente, las barreras geográficas producen especiación por
aislamiento a gran escala evolutiva (Rocha y Bowen 2008). Uno de los primeros
científicos en reconocer la importancia de las barreras geográficas para la
especiación en el medio marino fue el ictiólogo David Starr Jordan que utilizó
especies separadas por el istmo de Panamá como ejemplo de divergencia por
aislamiento geográfico (Jordan 1905, 1908). Más tarde, Dobzhansky (1937) y Mayr
(1942) describieron la teoría más aceptada sobre especiación: la especiación
geográfica o alopátrica. El caso más estudiado de especiación alopátrica es el de la
formación del istmo de Panamá, pero hay muchos otros casos como por ejemplo el
cierre del mar de Thetis, que separó al mar Rojo y el Mediterráneo (Bellwood y
Wainwright 2002).
Por el contrario, la especiación simpátrica se define como especiación sin

aislamiento geográfico (Mayr 1942) o especiación entre poblaciones con migración
libre (Coyne y Orr 2004). Entender cómo ocurre la especiación en ausencia de
barreras geográficas ha sido una fuente tanto de interés, como de debate (Orr y
Smith 1998). A nivel general, la especiación con flujo génico es difícil, ya que el
intercambio de genes entre poblaciones frena la diferenciación, previene el
aislamiento reproductivo y por tanto la especiación (Mayr 1963; Coyne y Orr 2004).
Pero a día de hoy, la especiación simpátrica se considera una ruta posible de
especiación y se acepta la evolución de barreras reproductivas en poblaciones que
habiten zonas sin barreras geográficas (Servedio y Noor 2003; Bolnick y Fitzpatrick
2007).
Teniendo en cuenta la posible existencia de diferentes barreras, dependiendo

del mecanismo de especiación, la estructura poblacional de las especies marinas
puede ser modelada tanto por obstáculos físicos que impidan el flujo génico
24
Introducción
(Partanello y col. 2007; Perez-Losada y col. 2007), como por barreras reproductoras
mediadas por estrategias vitales (Zardoya y col. 2004) o rasgos de comportamiento
intrínsecos a las especies (Perrin y Mazalov 2000). Hasta hace pocas décadas, se
pensaba que en el medio marino, las corrientes y la aparente falta de barreras
geográficas facilitaban el intercambio genético entre poblaciones de peces
(Palumbi 1994), contrarrestando los procesos de diferenciación. Los océanos eran
vistos como hábitats abiertos, en los que el aislamiento por distancia es el
mecanismo principal que promueve la especiación (Palumbi 1994). Sin embargo,
numerosos estudios han demostrado la existencia de barreras que producen
fragmentación genética intraespecífica (ver Glosario). Algunos mecanismos físicos
que pueden promover la especiación en el medio marino son las características
hidrográficas (Palumbi 1994), la convergencia de diferentes masas de agua
(Borrero-Perez y col. 2011) y las corrientes (Machado-Schiaffino y col. 2010), entre
otros. También hay factores físico-químicos que pueden actuar como barreras,
como por ejemplo la temperatura (e.g. Crow y col. 2007), la luz (Vermeij y Bak
2002) y/o la salinidad (e.g. Nielsen y col. 2004; Milano y col. 2014). Por otro lado,
conductas de comportamiento, como la filopatria (ver Glosario) o el instinto de
regresar a casa (homing) para reproducirse, pueden causar diferenciación
poblacional para algunas especies de peces (e.g. Castillo y col. 2005).
Hay que tener en cuenta también la perspectiva temporal. La variabilidad

genética de las especies, así como su estructura genética poblacional, están
modeladas por las barreras presentes, por las pasadas y por su historia paleo-
ecológica (Partanello y col. 2007; Perez-Losada y col. 2007). Hewitt (1996, 2000)
demostró que la última glaciación tuvo un efecto muy profundo en la diversidad
genética del planeta. Por tanto, para un mejor entendimiento de los mecanismos
de especiación en el medio marino es importante no solamente caracterizar la
estructura y dinámica poblacionales, sino también las estrategias de la historia vital
de las especies, los factores ambientales del pasado y del presente (Zardoya y col.
2004), así como las barreras físicas y de comportamiento que impidan la dispersión
y el flujo génico entre poblaciones actuales.
25
Introducción
Hibridación e introgresión
La hibridación entre especies se conoce desde tiempos de Linneo (Mallet

2005) y ha sido discutida frecuentemente por los evolucionistas (Mayr 1963; Arnold
1997; Coyne y Orr 2004; Mallet 2005, 2007, 2008, etc.). La hibridación ocurre
cuando las barreras de aislamiento reproductor entre dos poblaciones diferentes
no son infranqueables y las dos poblaciones se aparean entre sí. Por lo general en
la hibridación interespecífica, la descendencia suele ser estéril y poco viable,
aunque en algunos casos los descendientes híbridos son viables y fértiles,
produciéndose la introgresión genética y con el tiempo, pudiendo llegar a formar
una nueva especie. (Jones y col. 1995; Mavárez y col. 2006; Chapman y Burke
2007). En general, se asume que la hibridación es rara, pues de lo contrario sería
imposible la diferenciación entre especies (Mallet 2005). Es más, según el concepto
biológico de especie, las especies están aisladas reproductivamente unas de otras.
Habitualmente se presupone que su importancia evolutiva es escasa, debido a que
la hibridación suele producir descendientes infértiles, terminándose el intercambio
genético entre especies en la primera generación (F1) (Mallet 2008). Sin embargo,
gracias a los marcadores moleculares de alta resolución empleados hoy en día, la
hibridación introgresiva está bien establecida y reconocida (Arnold 1997; Coyne y
Orr 2004; Mallet 2005, 2007, 2008; Hegarty y col. 2008; Jiggins y col. 2008).
La hibridación, tanto interespecífica como intraespecífica (ver Glosario), es

un mecanismo de evolución potencialmente importante en los seres vivos,
incluyendo plantas y animales (DeMarais y col. 1992; Mallet 2005, 2007, 2008). Los
botánicos reconocieron su importancia en la producción de nuevas especies y
variedades, así como en la incorporación de variación genética a partir de taxones
heterólogos (Anderson 1949; Grant 1981). En cambio, hasta hace poco, no se
pensaba que la hibridación jugase un papel importante en la evolución animal
(DeMarais y col. 1992; Mallet 2007, 2008). Hoy en día, con nuevas evidencias
genéticas, se ha visto que la hibridación y la especiación híbrida son más comunes
de lo que se creía, tanto en animales como en plantas (Mallet 2007, 2008). En
26
Introducción
general, la hibridación en animales es más rara que en plantas (Mallet 2008). Sin
embargo, Palumbi (1992, 1994) sugiere que en el medio marino podría ser mayor,
ya que la mayoría de los peces tiene un modo de dispersión planctónico y por
corrientes, que se asimila mucho al método de dispersión de las plantas terrestres.
Por otro lado, aunque se asume que los mamíferos hibridan muy raramente (Grant
y Grant 1992), los cetáceos ofrecen muchos casos de hibridación no sólo entre
especies (Willis y col. 2004; Glover y col. 2013), sino incluso entre diferentes
géneros (Berubé y Aguilar 1998).
La hibridación natural y la introgresión entre especies ocurre con regularidad

y especialmente en grupos de rápida diversificación y radiación (Price y Bouvier
2002; Seehausen 2004; Grant y col. 2005; Mallet 2005, 2007, 2008; Gourbière y
Mallet 2010). La hibridación puede contribuir a la especiación a través de la
formación de nuevos taxones híbridos, mientras que la introgresión diferencial
puede promover divergencia adaptativa y, por tanto, facilitar la especiación
(Abbott y col. 2013). La hibridación puede dar lugar a una población nueva
recombinante, que con el tiempo puede convertirse en suficientemente divergente
como para formar una nueva especie (Abbott y col. 2013). Así, la especiación
híbrida supone que la hibridación tiene un papel principal en el proceso de
especiación. Un creciente número de evidencias empíricas sugieren que la
especiación híbrida es un potente generador de biodiversidad en animales (Grant y
Grant 1992; Dowling y Secor 1997; Arnold 2006, entre otros). Ésta, por definición,
ocurre en simpatría ya que la hibridación requiere flujo génico (Mallet 2007; Barton
2013). Por tanto, la especiación híbrida sucede cuando los genotipos híbridos
pueden establecerse como poblaciones distintas, que están aisladas
reproductivamente de los parentales (Barton 2013).
La diferenciación genética entre las especies se debe, en parte, a la evolución

adaptativa en ecosistemas heterogéneos (Seehausen y col. 2008); sin embargo, la
inestabilidad ambiental produciría el efecto contrario. El ejemplo más accesible a
día de hoy sobre condiciones cambiantes y adaptación es el clima. El continuo
27
Introducción
cambio climático, incluyendo el calentamiento global, el deshielo y la subida del

nivel del mar están cambiando los ecosistemas de todo el planeta, con mayor
énfasis en los ecosistemas templados y las zonas polares, con consecuencias
devastadoras para muchas de las especies que viven allí (e.g. Kelly y col. 2010;
Edwards y col. 2011). Multitud de especies se encuentran bajo condiciones
extremadamente adversas y alteran su elección de pareja promoviendo la
hibridación (Seehausen y col. 2008; Badyaev 2005), como por ejemplo el caso de
los osos polares (Kelly y col. 2010).
Como contrapunto a su apariencia espontánea en la naturaleza, la mayoría de

los casos de hibridación son atribuidos actualmente a la degradación del medio,
por ejemplo, como resultado de transferencia de especies entre regiones mediadas
por el ser humano (Mallet 2005). La idea no es nueva, Mayr ya reconocía este
hecho en 1942 (Mayr 1942, 1963). Algunos autores (Seehausen y col. 2008)
mantienen que la hibridación introgresiva es causa de extinción de especies, sobre
todo en los casos de contactos secundarios. La invasión del genoma por genes
procedentes de otras especies puede ser vista como un problema de identidad
para la especie receptora, pero no se debería de olvidar el hecho de que esa
invasión genética en muchos casos es natural, y aunque a veces sea inducida por
circunstancias extremas, en última instancia contribuye a la adaptación y la
diversificación (Mallet 2005).
El ser humano en los procesos de especiación
En los últimos siglos, la mayoría de los ecosistemas del planeta están siendo
alterados debido a la acción humana, cambiando su composición, estructura y
funcionamiento (Vitousek y col. 1997; Sala y col. 2000; Mooney y Cleland, 2001).
Los impactos de las actividades derivadas de la acción humana hacen que los
ecosistemas naturales sean cada vez más homogéneos, reduciendo la diversidad de
hábitats y simplificando su estructura (Candolin y col. 2006; Hendry y col. 2006;
28
Introducción
Seehausen 2006). Todos estos procesos de reducción de ecosistemas podrían

conducir a una reducción de la biodiversidad (Seehausen y col. 2008), ya que se
reduce la adaptación divergente entre especies y, por tanto, los límites entre ellas.
Hay muchas otras actividades humanas que afectan a la biodiversidad del planeta y
a la especiación, que van desde las más obvias y fáciles de percibir, como las
introducciones de especies, las extinciones por sobre-explotación o eliminación de
hábitats, las invasiones biológicas, etc., hasta otras actividades menos obvias de
percibir que afectan también a los procesos de evolución (Myers y Knoll 2001;
Rosenzweig 2001; Seehausen y col. 2008). Por ejemplo, la hibridación
interespecífica es uno de los procesos evolutivos altamente susceptible a la acción
humana porque, en muchos casos, especies no simpátridas no entrarían en
contacto secundario (ver Glosario) si no fuera por el ser humano (Mallet 2005;
Seehausen y col. 2008). Es frecuente encontrar en la literatura ejemplos sobre
hibridación que han sido atribuídos a la modificación del medio como resultado de
la acción del hombre (Mayr 1963; Coyne y Orr 2004; Crego-Prieto y col. 2013). Más
aún, la hibridación es más frecuente en ecosistemas dañados y condiciones
cambiantes, porque en situaciones adversas las especies suelen alterar su elección
de pareja (Seehausen y col. 2008). Distinguir entre la hibridación natural y la
promovida por la acción del ser humano es crucial para la conservación, pero no
siempre es posible conseguirlo (Allendorf y col. 2001).
Los cambios a escala evolutiva causados por las actividades humanas afectan
a todos y cada uno de los ecosistemas del planeta, y los océanos son quizás de los
más alterados. Diversos estudios (e.g. Sibert y col. 2006; Mutsert y col. 2008;
Polacheck 2008; Game y col. 2009; entre otros) resaltan el impacto negativo de las
actividades humanas en el medio marino. Según Halpern y col. (2008) no existe
ninguna zona en todo el océano que esté libre del efecto e impacto antropogénico.
La contaminación, la sobrepesca, la acuicultura, la introducción de especies, el
tráfico marino y el calentamiento global son algunos de los muchos factores
negativos causados por el ser humano que afectan a los océanos. Un claro ejemplo
es la influencia humana sobre las trayectorias evolutivas de las especies de
29
Introducción
pesquerías, donde se ha visto (tanto a escala temporal reciente, como evolutiva) la

selección por tamaño en los peces sujetos a una mayor explotación pesquera
(Conover y Munch 2002; Pauly y col. 2005; Figura 3).
Figura 3: Diferencias en la longitud de los peces e invertebrados sujetos a explotación pesquera entre
1950 y 1990. Los colores rojo, naranja y amarillo muestran un descenso en centímetros de más de
100cm, mayor de 50cm o mayor de 5cm respectivamente, las tonalidades verdes indican que no ha
habido cambio, las blancas que no se dispone de información y las azules que ha habido un aumento
(Pauly y col. 2005)
A pesar del amplio abanico de ejemplos de los efectos negativos derivados de

la acción humana sobre los ecosistemas marinos, a día de hoy los océanos siguen
siendo grandes desconocidos y, por tanto, las consecuencias evolutivas de estos
efectos a larga escala se desconocen también. Se recomienda, así, mucha más
investigación en profundidad para conocer estos fenómenos (Smith y Bernatchez
2008), entender su alcance y poder predecir sus consecuencias. En este contexto se
inscribe la presente Tesis Doctoral.
30
Introducción
Modelos para el estudio de la especiación en el medio marino
Debido a la enorme complejidad de factores naturales y antropogénicos que

inciden sobre la especiación en el medio marino, la elección de modelos para
abordar este tema no es fácil. Parece adecuado contrastar grupos taxonómicos que
tengan una historia evolutiva muy diferente, para poder inferir causas y
mecanismos globales por comparación. En esta Tesis se seleccionaron tres modelos
diferentes, agrupados taxonómicamente por géneros. Cada modelo de estudio fue
elegido por poseer distinto grado de especiación, así como distintas características
biológicas que permiten tener una visión amplia de los mecanismos que están
actuando a diferentes niveles en los procesos de especiación en el medio marino.
a. Género Pomatomus:
Reino: Animal. Filo: Cordados. Clase: Actinopterigios. Orden: Perciformes.

Suborden: Percoideos. Superfamilia: Percoidea. Familia Pomatomidae. Género:
Pomatomus.
Pomatomus saltatrix es un pez (Figura 4) de interés económico en pesquerías

y también en pesca deportiva a lo largo de gran parte de su distribución (Ceyhan y
col. 2007; Robillard y col. 2008; entre otros). Pomatomus saltatrix es un gran
depredador pelágico que habita aguas de los océanos Atlántico, Índico y Pacífico,
incluyendo sus mares adyacentes como el Mediterráneo, Egeo y Mar Negro (Briggs
1960; Juanes y col. 1996; Pottern y col. 1989; Tortonese 1986). Realiza migraciones
estacionales relacionadas con la temperatura y el fotoperiodo en algunas regiones,
como las aguas frente a la costa este de los Estados Unidos (Juanes y col. 1996).
31
Introducción
Figura 4: Dibujo de Pomatomus saltatrix (Whitehead 1978) también conocido en castellano

como anjova, anchova o chova, dependiendo de la localidad en la que nos encontremos.
Pomatomus saltatrix es el único miembro en su género y familia, lo que

podría ser una clara señal de que el intercambio y flujo génico entre poblaciones
estén evitando su especiación a nivel regional. Sería un ejemplo paradigmático del
modelo clásico de especiación que se espera en el medio marino, es decir, su
ausencia por falta de barreras a la dispersión y el flujo génico (Palumbi 1994), que
se traduciría en una gran única población (o metapoblación, si el ambiente no es
homogéneo). Existen estudios regionales sobre la diferenciación genética regional
en esta especie, de los cuales destacan el estudio de Goodbread y Gaves (1996)
sobre la diferenciación entre muestras del Atlántico e Indo-Pacífico y el estudio de
Pardiñas y col. (2010) acerca de la diferenciación entre el Atlántico y Mediterráneo.
En el momento de comenzar esta Tesis Doctoral, se desconocía tanto su estructura
poblacional a gran escala, como la intensidad de migración entre poblaciones y, por
tanto, la existencia o no de posibles barreras a lo largo de su distribución.
Pomatomus saltatrix parece ser un buen modelo de estudio, cuyos resultados
aportarán información sobre los mecanismos que estén impidiendo la especiación
en este género y el papel de las posibles barreras biogeográficas en la estructura
poblacional actual de esta especie cosmopolita.
32
Introducción
b. Género Merluccius:
Reino: Animal. Filo: Cordados. Clase: Actinopterigios. Orden: Gadiformes.

Familia: Merlucciidae. Género: Merluccius.
En el otro extremo del rango de modelos de especiación marina en

vertebrados, se encontrarían probablemente las merluzas, con similar capacidad de
dispersión que Pomatomus, es decir, con gran capacidad migratoria (Alheit y
Pitcher 1995). Sin embargo, las merluzas han de tener ciertas características que
las han hecho proclives a la diferenciación, ya que el género Merluccius engloba a
12 especies de merluzas (Alheit y Pitcher 1995) que habitan aguas de los océanos
Atlántico y Pacífico, en ambos hemisferios. El origen y dispersión de las merluzas
alrededor del mundo es aún controvertido y teorías basadas sólo en la deriva
larvaria parecen no ser convincentes para explicar la gran variedad de especies
(Inada 1981; Alheit y Pitcher 1995; Campo 2009). Las merluzas emergieron en el
norte del Atlántico hacia la mitad del Oligoceno (Kabata y Ho 1981; Inada 1981;
Fedotov y Bannikov 1989; Campo y col. 2007), dividiéndose este linaje ancestral en
dos linajes coincidentes con las dos regiones geográficas del Atlántico, es decir, el
linaje del este en Europa y África y el del oeste en América (Roldán y col. 1999;
Quinteiro y col 2000; Grant y Leslie 2001; Campo y col. 2009). Por otro lado,
probablemente, las merluzas llegaron al Pacífico a través del Istmo de Panamá
cuando estaba abierto (Inada 1981; Ho 1990; Campo y col. 2007).
Las merluzas son peces demersales de gran importancia económica a lo largo de

toda su distribución, siendo el recurso pesquero más importante para muchos
países (Alheit y Pitcher 1995). En esta Tesis se analizaron las cinco especies de
merluzas que conforman el complejo de merluzas euroafricanas y habitan aguas
del este del Atlántico en Europa y África: Merluccius merluccius, M. senegalensis,
M. polli, M. capensis y M. paradoxus en orden respectivamente desde el norte al
sur (Figura 5). M. merluccius es la merluza europea, la especie que se distribuye
más al norte de las merluzas euroafricanas. Importante recurso pesquero
desde tiempos históricos, la merluza europea es muy apreciada comercialmente
33
Introducción
Figura 5: Merluzas euroafricanas: a. Merluccius merluccius, b. M. polli, c. M. capensis, d. M. paradoxus y

e. M. senegalensis. Imágenes tomadas de Cohen y col. 1990
34
Introducción
(Cohen y col. 1990; Alheit y Pitcher 1995). Las especies que se encuentran más
al sur son M. capensis y M. paradoxus, también conocidas como merluzas del
Cabo, dos especies del sur de África. Las merluzas del Cabo son el recurso pesquero
más importante para Namibia y Sudáfrica. En esta pesquería no se diferencia entre
especies y ambas son pescadas, comercializadas y gestionadas conjuntamente
(Alheit y Pitcher 1995). Finalmente, en el centro de África se encuentran otras dos
especies: M. polli, con una distribución amplia desde Mauritania hasta Angola, y M.
senegalensis, con una distribución más restringida pero solapante tanto con M.
polli al sur, como con M. merluccius al norte. En aguas de Mauritania y Senegal
estas tres últimas especies (M. merluccius, M. polli y M. senegalensis) son
capturadas conjuntamente en pesca de arrastre (Cohen y col. 1990).
Por todo esto, el género Merluccius parece especialmente interesante como

modelo de especiación con una fuerte radiación de especies de gran similitud
morfológica y distribuciones solapantes, cada una con características y
comportamientos propios. Su estudio podría aclarar los procesos de radiación
específica y, por tanto, los mecanismos de especiación rápida. El solapamiento
geográfico en las distribuciones de las especies podría aportar información sobre
los posibles mecanismos evolutivos de especiación en simpatría, o bien sobre los
procesos que pueden darse ante un contacto secundario entre especies, si fuera
este el caso.
c. Género Globicephala:
Reino: Animal. Filo: Cordados. Clase: Mamíferos. Orden: Cetacea. Suborden:

Odontoceti. Familia: Delphinidae. Género: Globicephala.
El género Globicephala engloba solamente dos especies de cetáceos

conocidos como calderones o ballenas piloto: Globicephala melas, llamada en
castellano calderón común y G. macrorhynchus, conocida como calderón tropical
(Figura 6).
35
Introducción
Figura 6: Globicephala melas (arriba) y G. macrorhynchus (abajo), donde se aprecian sus
principales diferencias morfológicas, como la longitud de la aleta pectoral .
El calderón tropical (G. macrorhynchus), como su nombre indica, habita aguas

tropicales y subtropicales, mientras que el calderón común (G. melas) se encuentra
en aguas templadas y sub-antárticas. La distribución anti-tropical del calderón
común es todavía un tema de especulación (Oremus y col. 2009), ya que
representa un caso muy especial en la naturaleza, al ser una de las dos únicas
especies reconocidas, junto a los zifios Mesoplodon mirus, que tienen una
distribución anti-tropical con poblaciones aisladas en los dos hemisferios y que son
consideradas la misma especie (Dalebout y col. 2007; Oremus y col. 2009). Davies
(1960) situó el último contacto entre las poblaciones del norte y del sur del
calderón común hace 10.000-15.000 años, durante las últimas glaciaciones del
Pleistoceno, cuando las aguas ecuatoriales estaban más frías. Por lo que la
temperatura parecía ser un factor clave para la distribución de esta especie, como
fue confirmado posteriormente por Fullard y col. (2000). Aunque las dos especies
de calderones tienen distribuciones separadas a lo largo de los océanos del planeta,
36
Introducción
existen algunas zonas de contacto, como la zona templada del norte del Atlántico,
donde las dos especies conviven y pueden ser avistadas indistintamente. Los
calderones tienen una fuerte estructura, que hasta hace poco se consideraba
matriarcal (Amos y col. 1993) y a día de hoy, gracias a estudios genéticos más
precisos, parece ser simplemente social, donde hembras no emparentadas pueden
cuidar de las crías (Oremus y col. 2013).
La elección de este modelo de estudio reside en que el género Globicephala
podría situarse en el medio de los dos modelos de estudio antes expuestos, al
tratarse de un género con gran capacidad migratoria pero pocas especies. Es decir,
sería un ejemplo intermedio entre el género Merluccius con numerosas especies y
el género Pomatomus con una única especie en su género. Sin embargo, la
dispersión de los calderones no tiene lugar de forma separada en la fase juvenil y la
fase adulta, como es paradigma en la especiación marina (Seehausen 2004), ya que
se mueven en grupos sociales donde el cuidado parental tiene lugar hasta una edad
relativamente avanzada de las crías y una vez alcanzan la madurez sexual no suelen
abandonar el grupo en el que nacen (Oremus y col. 2013). Además, los calderones,
como mamíferos, poseen reproducción con fecundación interna, frente a los
modelos anteriores con fecundación externa.
Por otro lado, se sabe que la especiación de la familia Delphinidae es muy
reciente (LeDuc y col. 1999; McGowen y col. 2009) y bastante controvertida en
cuanto al número de especies (Caballero y col. 2008; Slater y col. 2010). Por tanto,
este modelo de estudio representaría un caso de especiación muy reciente, frente
al género Pomatomus con aparentemente una especiación lenta y entre ambos se
encontrarían las merluzas, con una especiación también reciente, aunque más
antigua que los calderones.
37
38
Hipótesis de partida
39
40
Teniendo en cuenta las características biológicas, distribuciones geográficas e

historia evolutiva de los tres modelos propuestos en esta Tesis, las hipótesis de
partida que se plantean sobre sus mecanismos de especiación son las siguientes:
1: En el género Pomatomus, que se ha elegido como ejemplo de especiación

lenta al no haber evidencias de especiación detectada hasta el momento, se espera
encontrar indicios de diferenciación genética solamente entre poblaciones muy
distantes, aunque con flujo génico entre ellas suficiente como para impedir el
aislamiento completo. Esto puede deberse a dos factores: migración intensa, o
poblaciones muy grandes, en las que la deriva génica no tenga efecto detectable a
escala temporal evolutiva media. El análisis de diferenciación genética a diferentes
escalas geográficas permitirá averiguar si hay indicios de aislamiento por distancia y
por tanto especiación vicariante. Mediante inferencias coalescentes combinadas
con un análisis de estrategias de historia vital se espera determinar si la falta de
diferenciación se debe a una migración relativamente intensa o a un tamaño
efectivo poblacional lo bastante grande para impedir dicha diferenciación.
2: En las especies africanas del género Merluccius, en cuyas especies

americanas se ha descrito una posible especiación híbrida y hay evidencias
genéticas de hibridación introgresiva, se espera encontrar igualmente signos de
hibridación entre especies solapantes. Dado que las especies africanas pertenecen,
al menos, a dos linajes claramente diferenciados genéticamente, se espera que las
barreras interespecíficas sean de tipo ecológico o etológico y no vicariantes; el
41
estudio detallado de las zonas de solapamiento permitiría inferir el tipo de barrera

más probable entre especies.
3: En el género Globicephala, se espera encontrar evidencias de cierto flujo

génico entre especies, al tratarse de un proceso de especiación muy reciente. En
otros géneros de cetáceos hay numerosas evidencias de hibridación interespecífica
e incluso entre diferentes géneros, pero hasta el momento de comenzar esta tesis
no hay conocimiento de hibridación en este género. Dado que las distribuciones de
los calderones están íntimamente relacionadas con la temperatura, se propone un
modelo de especiación probablemente ecológico muy reciente, donde es esperable
la aparición de un cierto grado de hibridación en las zonas geográficas solapantes
entre las dos especies.
42
Objetivos
43
Objetivos
44
Objetivos
Objetivos
El objetivo general de esta tesis es el estudio y caracterización de procesos de

especiación en el medio marino, así como de las posibles barreras actuales e
históricas que afectan al flujo génico y promueven la especiación en el océano. Las
barreras consideradas principalmente son de tipo vicariante, ecológico y/o
etológico.
Los objetivos específicos de la Tesis, que serán abordados en los diferentes

capítulos del apartado de Resultados, son:
1.- Caracterizar la estructura genética poblacional de Pomatomus saltatrix e

identificar las posibles barreras actuales e históricas al flujo génico, a lo largo de su
distribución en el hemisferio norte y en escalas geográficas diferentes: a través del
océano Atlántico (gran escala), entre los extremos de la cuenca del Mediterráneo
(escala intermedia), y entre localidades dentro de cada región (pequeña escala).
2.- Mediante inferencias coalescentes, revisión de características biológicas y

etológicas y uso de marcadores genéticos con diferente tipo de herencia, estimar
los tamaños poblacionales y las tasas de migración a diferentes escalas espaciales,
identificando posibles factores principales que ralenticen la especiación en
Pomatomus.
3.- Combinando marcadores genéticos de herencia diferente, mitocondriales

y nucleares, y el análisis de características ecológicas y etológicas de las distintas
45
Objetivos
especies, inferir los principales mecanismos de especiación implicados en la

radiación euroafricana del género Merluccius.
4.- Mediante marcadores genéticos, analizar las zonas simpátricas de especies

de Merluccius solapantes para investigar la posible existencia de zonas híbridas
similares a las descritas para las especies americanas y, en su caso, identificar
posibles factores ecológicos o etológicos que promuevan la ruptura de barreras
interespecíficas.
5.- Mediante marcadores nucleares y mitocondriales, analizar la

diferenciación genética entre las dos especies del género Globicephala a lo largo de
su distribución atlántica en el hemisferio norte, con especial énfasis en las zonas de
contacto de las dos especies, para identificar las barreras interespecíficas
existentes, su posible tipo (por distancia, ecológicas o etológicas) y, en su caso,
inferir las posibles causas de su debilitamiento o ruptura.
46
Material y métodos
47
Material y métodos
48
Material y métodos
Material y métodos
En este apartado se describen de forma general los tipos de muestras

analizadas, su procedencia, las distintas técnicas empleadas para su análisis y un
breve resumen de los análisis estadísticos de los datos. Posteriormente, en cada
capítulo del apartado Resultados se especificará de manera más detallada toda la
metodología utilizada, así como los análisis estadísticos requeridos en cada artículo.
Regiones marinas en estudio y obtención de muestras
Cada modelo de estudio cuenta con diferentes zonas de muestreo y protocolos

para la obtención de muestras que serán resumidos a continuación.
a. Género Pomatomus:
Se obtuvieron 225 muestras de tejido de Pomatomus saltatrix de 9

localidades a lo largo de su distribución en el hemisferio norte. Los puntos de
muestreo para esta especie, al igual que el número de individuos utilizados en el
estudio se pueden ver en la Figura 7. Estas muestras se obtuvieron gracias al
proyecto Nacional “ESPEZ: Especiación en peces marinos. Barreras reproductivas
ecológicas, cromosómicas y geográficas en los géneros Merluccius y Pomatomus,
con el género Salmo como referencia” MICINN CGL 2009-08279 y al proyecto
“Bluefish/Striped Bass Research Program” de la Administración Nacional Oceánica y
Atmosférica (NOAA) de los Estados Unidos de América.
49
Material y métodos
Figura 7: Puntos de muestreo y número de muestras empleadas en el estudio de Pomatomus saltatrix.
Para la obtención de las muestras de tejido, una vez capturados los

ejemplares adultos se les cortó un trozo de aleta o branquia de unos 3 cm2
aproximadamente que fueron guardados en tubos con etanol al 70% hasta su
análisis.
b. Género Merluccius:
Se obtuvieron muestras de tejido de 467 ejemplares adultos de merluzas,

procedentes de 5 especies diferentes (Figura 8). Las muestras de merluzas se
obtuvieron gracias al proyecto nacional MICINN CGL 2009-08279 y al proyecto
europeo MARINEGGS QLK5-CT1999- 01157. Las muestras de tejido proceden de
aleta y/o músculo de ejemplares adultos y su conservación hasta el análisis
genético fue idéntica a la descrita para Pomatomus saltatrix anteriormente.
50
Material y métodos
Figura 8: Numero de ejemplares y áreas de distribución de las merluzas euroafricanas. En la imagen no

se señalan los puntos de muestreo.
c. Género Globicephala:
Dada la naturaleza de este género, las muestras de calderones fueron

especialmente difíciles de conseguir, por su escasez y tramitaciones legales
(permisos CITES correspondientes; ANEXO 1). Las muestras provienen de animales
varados y/o biopsiados, además de museos, bancos de tejidos, centros de
investigación y asociaciones de la red nacional de varamientos; ningún animal fue
sacrificado para la realización de este estudio. Se consiguieron un total de 151
muestras de calderones (de las cuales sólo 120 pudieron ser utilizadas)
procedentes de 5 zonas principales de muestreo (Figura 9), algunas de ellas
subdivididas, a su vez, en varios puntos. Las muestras de las islas Faroe proceden
51
Material y métodos
del Faroese Museum of Natural History. Las muestras de la Península Ibérica

proceden del banco de tejidos de mamíferos marinos de la Sede en Vigo del
Instituto Español de Oceanografía (IEO), de la Agencia de Medio Ambiente y Agua
de la Junta de Andalucía y de un varamiento registrado por la asociación
GREMMAR. Las muestras de las islas Azores fueron conseguidas gracias al proyecto
MAPCET-M2.1.2/F/012/2011 (FEDER), the Competitiveness Factors Operational
(COMPETE), QREN European Social Fund, y Proconvergencia Açores/EU Program.
Las muestras de las islas Canarias se consiguieron a través de dos asociaciones de
cetáceos: Canarias Conservación, en Tenerife, y SECAC, en Lanzarote. Por último,
las muestras de Polinesia Francesa fueron obtenidas en una estancia internacional
en el centro de investigación CRIOBE (Centre de Recherche Insulaire et Observatoire
de l'Environnement) en la isla de Moorea, a través de una colaboración con el
Departamento de Ciencias Biológicas de la Universidad de Auckland, Australia.
Figura 9: Zonas de muestreo a lo largo de la distribución de las ballenas piloto y número de ejemplares
analizados. En color verde se encuentra la zona de distribución de Globicephala melas; en color azul claro
la distribución de G. macrorhynchus; y en color azul oscuro las zonas de solapamiento de las dos especies.
La toma de muestras para la extracción de ADN se realizó de diferentes tipos

de tejidos (incluyendo piel, músculo, grasa) y de dientes (Figura 10). El protocolo de
52
Material y métodos
obtención de muestras en el caso de animales varados consistía en la escisión de

un trozo de piel, grasa o músculo (preferentemente músculo), dependiendo del
estado de degradación del animal, y se conservaban en etanol absoluto hasta su
extracción. En el caso de animales vivos se realizaba una pequeña biopsia, que
también se conservaba en etanol hasta la extracción de ADN. Las muestras
procedentes del Museo Nacional de las Islas Faroe se encontraban previamente
almacenadas en etanol absoluto; en cambio, las muestras del banco de tejidos del
IEO de Vigo estaban depositadas a -20°C.
Figura 10: Diente de Globicephala melas (Izquierda: macho de una longitud de 5.7 m;
Derecha: hembra de 3.5m)
Extracción de ADN
La extracción de ADN se realizó utilizando la resina Chelex® (Bio-Rad

Laboratories) y proteinasa K, siguiendo el protocolo descrito por Estoup y col.
(1996). Para ello, se coloca una pequeña cantidad del tejido disponible (unos 3mm
aproximadamente) en un tubo de 1.5ml al que se le añaden 500 μl de una solución
de Chelex 100® al 10% previamente calentada a 60°C junto con 7 μl de proteinasa K
(20mg/ml). Después se incuba durante una hora y media a 55°C con agitación. Por
último, se deja durante 20 minutos a 100°C para inactivar la proteinasa K. Con este
53
Material y métodos
método, el ADN extraído queda en el sobrenadante y la resina junto con los restos
celulares, en el fondo. Este proceso de extracción, rápido y económico, permite la
extracción del ADN en un par de horas, ya que evita los periodos de incubación
comunes en otros métodos más complejos. Una vez extraído el ADN se guarda a
4°C para conservarlo hasta su utilización a corto plazo, mientras que para su
almacenamiento a largo plazo se depositan a -20°C.
La extracción de ADN en calderones se realizó en su mayoría con el método
previamente descrito de la resina Chelex® (Bio-Rad Laboratories) y proteinasa K,
siguiendo el protocolo descrito por Estoup y col. (1996). En los casos en los que la
muestra de tejido provenía de animales varados con un alto grado de
descomposición y el método anterior fallaba, se extrajo el ADN mediante columnas
de gel de sílice (QIAmp DNA Mini Kit, Quiagen), manualmente y siguiendo las
instrucciones descritas por el fabricante. El método se divide en dos procesos:
digestión, que favorece la disociación del tejido, y extracción del ADN. Este
procedimiento es más eficaz, aunque más caro y conlleva más tiempo, al tener
periodos de incubación. Finalmente, la extracción de ADN en el caso de los dientes
se realizó mediante una técnica de extracción no invasiva descrita por Rohland y
col. (2004) para muestras de ADN antiguo, donde el diente no sufría ningún daño
en su estructura, lo que es imprescindible cuando se trataba de ejemplares de
museo. El protocolo consiste en una serie de incubaciones y lavados para extraer el
ADN, sin dañar la estructura del diente.
Marcadores genéticos
Los marcadores genéticos son caracteres simples heredables con múltiples

estados para cada carácter (Sunnucks 2000). En otras palabras, son un fragmento
de ADN conocido, heredable y polimórfico (ver Glosario). Estos segmentos de ADN,
con una localización identificable, necesitan ser amplificados mediante la reacción
en cadena de la polimerasa (PCR) para su análisis. La elección de los marcadores
genéticos más adecuados para cada estudio se llevó a cabo teniendo en cuenta no
54
Material y métodos
sólo la variabilidad genética de las muestras, sino también las características

genéticas y evolutivas de cada marcador, como sugiere Carvalho (1998). El uso de
varios marcadores independientes aumenta la sensibilidad de los análisis genéticos
(Sunnucks 2000). Además, la utilización conjunta de marcadores nucleares y
mitocondriales multiplica la efectividad de las herramientas genéticas en los
estudios poblacionales (Brunner y col. 1998), ya que ambos tipos de marcadores
pueden llegar a las mismas conclusiones o puede ocurrir que se identifique cierta
discrepancia entre marcadores, dados sus diferentes patrones de herencia (Bowen
y col. 1992). Estos diferentes patrones de transmisión y evolución hacen que tanto
el ADN mitocondrial como el nuclear puedan mostrar diferentes aspectos
biológicos e históricos de las poblaciones (Sunnucks 2000) y su uso conjunto
permite detectar eventos, como por ejemplo la dispersión o la supervivencia
diferencial entre sexos. Por todo ello, en esta Tesis se utilizaron conjuntamente
marcadores nucleares y mitocondriales.
a. Marcadores mitocondriales
Los marcadores mitocondriales son adecuados para estudios poblacionales a

gran escala geográfica, ya que debido a su herencia materna (con alguna
excepción), haploide y no recombinante, el tamaño poblacional es la cuarta parte
de los genes nucleares y por tanto la acción de la deriva génica puede causar una
diferenciación poblacional más marcada (Birky y col. 1989). Además, es una
molécula con regiones de evolución rápida y otras regiones de evolución lenta, lo
que permite poder abordar cuestiones filogenéticas a diferentes niveles (Zhang y
Hewitt 1996). Se secuenciaron tres marcadores mitocondriales:
i. Gen de la subunidad I de la citocromo oxidasa c (COI)

Gen encargado de codificar la subunidad 1 de la última enzima en la
cadena respiratoria mitocondrial, que cataliza la conversión de una
molécula de oxígeno en dos de agua. Es el gen elegido por la iniciativa
55
Material y métodos
internacional del “Código de barras de la vida” (Barcoding of Life,

www.boldsystems.org) que tiene como propósito la identificación genética
de todas las especies y la creación de una base de datos global a nivel
mundial (Hebert y col. 2003).
En esta Tesis Doctoral se empleó este marcador para la identificación
de especies así como también para estudios filogenéticos y poblacionales.
Para la amplificación del COI se utilizaron dos tipos de cebadores. Por un
lado, los cebadores universales para peces descritos por Ward y col. (2005)
y, por otro lado, una pareja de cebadores diseñados específicamente para
Pomatomus saltatrix a partir de secuencias de las bases de datos, ya que
mediante el uso de los cebadores universales para peces no se obtenía una
buena amplificación del fragmento de ADN correspondiente. Los
cebadores diseñados específicamente para Pomatomus saltatrix son:
COI-F Pom.: 5´- TTGGTGCATGAGCTGGTATG-3´
COI-R Pom.: 5´-AAGAATGGGGTCTCCTCCAC-3´
ii. Gen del citocromo b

El gen del citocromo b ha sido ampliamente empleado en animales,
desde estudios poblacionales hasta filogenéticos (ejemplos en peces:
Lovejoy y de Araújo 2000; Lydeard y Roe 1997; Meyer y Wilson 1990;
Normark y col. 1991; Rocha-Olivares y col. 1999; entre otros muchos). Es
uno de los genes mitocondriales mejor conocidos en cuanto a su
estructura y función de su proteína (Esposti y col. 1993).
En esta Tesis se utilizó este gen para completar los análisis de
Pomatomus saltatrix empleando los cebadores H151 y L148, el protocolo
y las condiciones descritas en Kocher y col. (1989) para su amplificación.
iii. Región control - “D-loop”

El “D-loop” es un fragmento no codificante localizado en la región
control mitocondrial, que es la región más hipervariable y de más rápida
56
Material y métodos
tasa de evolución dentro del ADN mitocondrial (e.g. Hoezel y col. 1991).
La región control en vertebrados se subdivide comúnmente en tres
dominios que difieren entre sí por su composición y por su tasa de
evolución (Lee y col. 1995; Baker y Marshall 1997). Numerosos estudios,
tanto poblacionales como evolutivos, se han centrado en el uso de este
marcador genético, así como en la región control completa (Lee y col.
1995).
En esta tesis se utilizó esta región hipervariable del ADN mitocondrial
para el estudio genético del género Globicephala utilizando los cebadores
M13-Dlp-1.5 (Baker y col. 1996) y Dlp-8G (Dalebout y col. 2005) y
siguiendo el protocolo y condiciones descritas en Oremus y col. (2009).
b. Marcadores nucleares
Mientras que los marcadores mitocondriales son utilizados para resolver

incertidumbres taxonómicas y evolutivas, los marcadores nucleares son, en
general, herramientas más potentes para analizar eventos recientes o
contemporáneos (Wan y col. 2004). A diferencia de los marcadores
mitocondriales, los marcadores nucleares son diploides y tienen herencia
biparental, por lo que permiten reconocer individuos híbridos y estudiar
introgresión genética.
i. Microsatélites
Los microsatélites son fragmentos de ADN compuestos por repeticiones
en tándem de pares de bases. Se encuentran repartidos por el genoma de
forma aleatoria (Toth y col. 2000). Son ampliamente empleados en
estudios de genética de poblaciones porque son muy polimórficos,
codominantes, de herencia mendeliana simple y en teoría, neutrales
(Jarne y Lagoda 1996). Estos marcadores elevadamente polimórficos son
57
Material y métodos
capaces de detectar diferenciaciones poblacionales pequeñas, incluso en

especies con gran flujo génico (Waples, 1998), lo que los convierte en
marcadores ideales para estudiar la estructura poblacional y dinámica de
poblaciones, para estudios de parentesco y pedigrees, para evaluar la
diversidad genética y para estudiar la historia reciente de poblaciones y
especies (Zhang y Hewitt 2003).
En esta Tesis se utilizaron un total de 25 microsatélites que fueron
puestos a punto para 8 especies diferentes. Las condiciones finales
empleadas para el género Pomatomus se resumen en la Tabla 1; en la
Tabla 2, para el género Merluccius; y en la Tabla 3, para el género
Globicephala.
Tabla 1: Microsatélites para Pomatomus saltatrix con las condiciones finales que se pusieron a punto
para desarrollar esta Tesis. Modificadas de Dos Santos y col. (2003).
Locus Temperatura [MgCl2] (mM) Fluorocromo Referencia
Elf 17 60.2°C 2 VIC Dos Santos y col. 2008
Elf 19 52°C 1.5 NED Dos Santos y col. 2008
Elf 37 54.5°C 1.5 6FAM Dos Santos y col. 2008
Elf 39 56°C 1.5 NED Dos Santos y col. 2008
Elf 44 63.5°C 1.5 VIC Dos Santos y col. 2008
Elf 46 54°C 1.5 6FAM Dos Santos y col. 2008
Elf 49 60°C 2 6FAM Dos Santos y col. 2008
Elf 50 51°C 1 NED Dos Santos y col. 2008
58
Material y métodos
Tabla 2: Microsatélites para el género Merluccius, separados por especies y con las condiciones para el
desarrollo de esta Tesis. Temperatura en grados centígrados (°C) en la primera línea y concentración de
magnesio ([ ]) en mM en la segunda línea de cada recuadro de la tabla.
Locus M.mer. M.cap. M.pax. M.polli M.sen. Referencia
52°C 54°C 54°C 52°C 52°C Morán y col. 1999

HK3
[1] [1.5] [1.5] [2] [2]
53°C 54°C 54°C 58°C 58°C Morán y col. 1999

HK9
[1] [1.5] [1.5] [2.5] [2.5]
50°C 52°C 52°C 52°C 52°C Morán y col. 1999

HK20
[1] [1.5] [1.5] [1.5] [2]
52°C 52°C 52°C 54°C 54°C Morán y col. 1999

HK29
[1.5] [2] [2] [2.5] [2]
52°C 52°C 52°C 54°C 54°C Morán y col. 1999

HK34
[1.5] [2.5] [2] [2.5] [2]
60°C 60°C 60°C 58°C 58°C Rico y col. 1997

WO1
[1] [1.5] [1.5] [2] [2]
56°C 61°C 61°C Machado-Schiaffino y

Maus7 - - Garcia-Vazquez, 2009
[1] [2.5] [2.5]
60°C 60°C 63°C 60°C 60°C Machado-Schiaffino y

Maus32 Garcia-Vazquez, 2009
[1.5] [2.5] [2] [2.5] [2.5]
60°C 54.5 60°C Machado-Schiaffino y

Maus30 - - Garcia-Vazquez, 2009
[1.5] [2] [2.5]
59
Material y métodos
Tabla 3: Microsatélites utilizados para el estudio del género Globicephala con las condiciones finales
empleadas para el desarrollo de esta Tesis.
Locus Temperatura [MgCl2] (mM) Fluorocromo Referencia
EV37MN 47°C 1 PET Valsecchi y Amos 1996
EV94MN 55°C 1 6FAM Valsecchi y Amos 1996
199/200 48°C 1 VIC Amos y col. 1993
415/416 47°C 1 6FAM Amos y col. 1993
417/418 47°C 1 NED Amos y col. 1993
409/470 52°C 1 VIC Amos y col. 1993
468/469 50°C 1 NED Amos y col. 1993
464/465 50°C 1 PET Amos y col. 1993
ii. Gen del ARN ribosomal 5S

La identificación visual de híbridos de merluza no es fácil debido a su
gran parecido morfológico. Para su identificación genética se llevó a
cabo la amplificación del gen de ARN ribosomal de la unidad 5S con los
cebadores descritos por Pendás y col. (1994) y siguiendo las condiciones
descritas en Pérez y Garcia-Vazquez (2004). En dicho trabajo de Pérez y
Garcia-Vazquez (2004), se describe un protocolo rápido y simple para la
identificación de especies de merluza, basado en la longitud del
espaciador o espaciadores no transcritos del gen ribosomal del ARN 5S.
Cada especie de merluzas tiene uno o varios fragmentos de diferentes
tamaños (Tabla 4), con lo que los híbridos interespecíficos de primera
generación deben tener los fragmentos característicos de ambos
parentales.
60
Material y métodos
Tabla 4: Fragmentos 5s para merluzas euroafricanas (Campo y col. 2009)
Especie Tamaño fragmentos
Merluccius merluccius 371
M. senegalensis 365
M. capensis 371
M. paradoxus 371 + 492
M. polli 371 + 501
iii. Gen de la región determinante del sexo en el cromosoma Y

(SRY)
El cromosoma Y ha emergido en los últimos años como un nuevo y
eficiente marcador genético (Hurles y Jobling 2001). En mamíferos, el
cromosoma Y juega un papel crucial en la determinación del sexo a
través de la acción de un único gen, el gen SRY (Berta y col. 1990;
Gubbay y col. 1990). El gen SRY ha sido utilizado para trazar linajes
paternos e inferir filogenias en mamíferos, desde ballenas (Nishida y col.
2003, 2007) hasta macacos (Tosi y col. 2000, 2003), entre otros.
En esta Tesis se utilizó el gen SRY para el sexado de ballenas,
utilizando los cebadores y siguiendo el protocolo descrito en Nishida y
col. (2003) mediante PCR. La presencia de amplificación positiva implica
que se trataba de un macho, y la ausencia (pero con amplificación
positiva para el resto de marcadores genéticos empleados) indica que es
una hembra.
61
Material y métodos
Datos morfológicos y ecológicos
Para esclarecer los límites entre especies y mecanismos de especiación, se

recogieron variables ecológicas del comportamiento reproductivo de las merluzas
Euro-Africanas, a partir de fuentes bibliográficas (profundidades de puesta,
distribuciones máximas y mínimas de especies, picos de reproducción, tiempo de
reproducción, tamaño al alcanzar la madurez sexual tanto de hembras como de
machos, temperatura y otras).
Para los calderones se recopilaron datos de morfometrías (ver Glosario y
ANEXO 2), que se obtuvieron de los animales varados donde era posible tomar las
medidas de las aletas y resto del cuerpo, así como contar el número de dientes
(carácter distintivo entre las especies de calderones). Pero dada la dificultad de la
recopilación de estos datos y su escasez, el estudio de la morfometría de las
ballenas piloto no se pudo incluir finalmente en los artículos científicos publicados.
Análisis de datos genéticos y software utilizado
Para los cálculos de diversidad genética se utilizaron los programas: DNAsp

v.4.50.3 (Rozas y col. 2003) para secuencias; Microsatellite Analyser MSA versión
4.05 (Dieringer y Schlstterer 2003), GENEPOP Versión Online (Raymond y Rousset
1995), GENETIX Versión 4.03 (Belkhir et al. 2004) y FSTAT Versión 2.9.3.2 (Goudet
2001) para microsatélites; y Arlequin versión 3.0 (Excoffier y col 2005) para ambos
tipos de marcadores genéticos.
Los análisis de la estructura poblacional, en el caso de las secuencias de ADN,
se llevaron a cabo mediante reconstrucciones filogenéticas en forma de árboles
con los programas PHYLIP versión 3.69 (Felsenstein 2005) y BEAST version 1.6.1
(Drummond y Rambaut 2007); y en forma de redes con el software Network
versión 4.5.1.6 (http://fluxus-engineering.com). En el caso de los microsatélites se
empleó el software STRUCTURE versión 2.3.1 (Pritchard y col. 2000). Los análisis de
diferenciación poblacional: FSTs, AMOVAs, test de asociación de Mantel, test de
62
Material y métodos
neutralidad y tests de expansión espacial y/o demográfica fueron calculados con el

software Arlequin versión 3.0 (Excoffier y col 2005). Para descartar posibles cuellos
de botella en las poblaciones se empleó el software BOTTLENECK versión 1.2.02
(Cornuet y Luikart 1997).
Para la identificación de especies mediante secuencias de ADN se emplearon
los programas online NCBI-BLAST (Altschul y col. 1990) y DNA Surveillance (Ross y
col. 2003), este último únicamente para cetáceos.
La identificación de híbridos se llevó a cabo mediante el software NewHybrids
versión 1.0 (Anderson and Thompson 2002), empleando datos de microsatélites.
El flujo génico entre diferentes poblaciones o diferentes especies fue
calculado utilizando varios tipos de marcadores (mitocondriales y nucleares) y
varias aproximaciones estadísticas. Se empleó el cálculo bayesiano basado en
teoría de coalescencia del software MIGRATE 3.0 (Beerli 2004), así como el cálculo
de máximas probabilidades del mismo programa. El flujo génico también fue
calculado con el método de alelos únicos utilizando el software GENEPOP Versión
Online (Raymond y Rousset 1995).
Para determinar posibles barreras geográficas para la dispersión genética se
utilizó el software BARRIER versión 2.2 (Manni y col 2004).
Las asignaciones genéticas de individuos a su posible población de origen se
realizó utilizando tres metodologías diferentes: dos mediante cálculos de
estadística bayesiana siguiendo las referencias de Rannala y Mountain (1997) y
Baudouin y Lebrun (2001) utilizando el software GeneClass2 (Piry y col. 2004), y un
tercero utilizando el método de máximas probabilidades con el software ONCOR
(Kalinoski y col 2007).
Las reconstrucciones de tamaños poblacionales históricos, así como
estimaciones de tiempos de divergencia entre especies o linajes dentro de una
misma especie, se llevaron a cabo bajo el marco de la estadística bayesiana y
basados teoría de coalescencia con el software BEAST versión 1.6.1 (Drummond y
Rambaut 2007). Los datos a priori necesarios para el uso de este programa fueron
calculados con jModeltest versión 0.11 (Posada 2009).
63
Material y métodos
Mayor detalle de los métodos estadísticos utilizados en cada análisis, así como
una mayor información sobre material y métodos empleados, se encuentran
especificados en la metodología de cada artículo científico que compone la
sección de Resultados de esta Tesis Doctoral.
64
Resultados
65
Resultados
66
Resultados
Resultados
Capítulo 1: Miralles L, Juanes F, Pardinas AF and Garcia-Vazquez E. (2014)

Palaeoclimate shaped Bluefish (Pomatomus saltatrix, L.) structure in the Northern
hemisphere. Fisheries. Aceptado. En prensa.
Capítulo 2: Miralles L., Juanes F. and Garcia-Vazquez E. (2014) Trans-oceanic sex-

biased migration in Bluefish. Transactions of the American Fish Society, 143 (5):
1308-1315, DOI: 10.1080/00028487.2014.935480
Capítulo 3: Miralles L., Mrugala A., Sanchez-Jerez P., Juanes F. and Garcia-Vazquez
E. (2014) Potential evolutionary impact of Mediterranean aquaculture on the wild
predator Pomatomus saltatrix L. Journal of Sea Research. En revisión.
Capítulo 4: Miralles L. and Garcia-Vazquez E. (2014) The deep history of Euro-

African hakes. Evolutionary Ecology. En revisión.
Capítulo 5: Miralles L, Machado-Schiaffino G and Garcia-Vazquez E. (2013) Genetic

markers reveal a gradient of hybridization between Cape hakes (Merluccius
capensis and Merluccius paradoxus) in their sympatric geographic distribution.
Journal of Sea Research. http://dx.doi.org/10.1016/ j.seares.2013.11.009
Capítulo 6: Miralles L, Lens S, Rodriguez-Folgar A, Carrillo M, Martin V, Mikkelsen B,

Garcia-Vazquez E. 2013. Interspecific Introgression in Cetaceans: DNA Markers
Reveal Post-F1 Status of a Pilot Whale. PLoS ONE 8(8): e69511.
DOI:10.1371/journal.pone.0069511
Capítulo 7: Miralles L., Oremus M., Silva M.A., Planes S. and Garcia-Vazquez E.
(2014) Merging Species in Pilot Whales: Globicephala melas under introgressive
hybridization process. Molecular Ecology. En revisión.
67
Resultados
68
Resultados
Capítulo 1:
Palaeoclimate shaped Bluefish
(Pomatomus saltatrix, L.) structure in the Northern
hemisphere.
Miralles, L. , Juanes, F., Pardiñas, A. F., Garcia-Vazquez, E.
Fisheries
69
70
M iralles et al. 2014 / Nor t hern hem isph er e popu la t ion st r uct u re of Bluefish / Fish eries
Palaeoclimate shaped Bluefish (Pomatomus saltatrix, L.) structure in the Northern

hemisphere.
L. Miralles 1*, F. Juanes2, A.F. Pardiñas 1, E. Garcia-Vazquez1.
1. Department of Functional Biology. University of Oviedo, 33006 Oviedo, Spain
2. Department of Biology, University of Victoria, Victoria V8W3N5, BC, Canada
ARTICLE INFO ABSTRACT
Bluefish (Pomatomus saltatrix L.), a highly migratory cosmopolitan predator, is the only extant representative of the family
Keywords: Pomatomidae. It has been the subject of many studies due to its commercial and recreational value, but much less research has been
conducted on its global population structure. Here we investigate the population structure of this species and the effects of present
Pomatomus saltatrix and past oceanographic barriers to dispersal in its North Atlantic, Mediterranean, Marmara, and Black seas populations. We
Bluefish employed mitochondrial (cytochrome b and cytochrome oxidase subunit I genes) and nuclear (eight microsatellite loci) DNA as
Phylogeography molecular markers. Three main genetic units of Bluefish were identified: American (West Atlantic waters), Spanish (East Atlantic-
Ocean barriers Western Mediterranean regions), and Turkish (Eastern Mediterranean, Marmara and Black seas). Our results suggested that
Glaciations Bluefish is panmictic in the northwest Atlantic Ocean but not in the Mediterranean Sea. The common ancestor of the studied
Population structure populations was traced back to the interglacial cycle Aftonian II, while the separation between clades was estimated to have
occurred during glacial periods, likely due to migrations to refuges and the closure of the Mediterranean Sea. In conclusion,
paleoclimate seems to have been fundamental for shaping the present genetic lineages of Pomatomus saltatrix.
Introduction
As a consequence of its broad distribution and the existence of
Ocean currents and the apparent lack of physical barriers in the potential oceanographic barriers, the species may be composed of
marine realm seem to facilitate extensive gene flow among marine multiple different populations but there is limited information
fish populations (Palumbi 1994). Pelagic and demersal fishes are available (Goodbred and Graves 1996; Turan et al. 2006; Pardiñas et
expected to exhibit little intraspecific genetic structuring even over al. 2010). Bluefish is one of the most important recreational and
large geographic distances (Ward et al. 1994) and marine commercial species along the east coast of the United States
environments are often seen as open habitats in which isolation by (Robillard et al. 2008) and is the target species of an important
distance is the main mechanism that may promote speciation (Palumbi artisanal fishery in all Turkish seas: Marmara, Aegean, and Black
1994). However, several studies have demonstrated the existence of (Ceyhan et al. 2007). Failure to detect and recognise population units
marine physical barriers that produce intraspecific genetic can lead to local overfishing and ultimately to severe declines of
fragmentation in marine systems. Population structuring of highly fisheries (Hutchings 2000; Knutsen et al. 2009), thus an accurate
migratory marine fish can be promoted by currents (Machado- definition of Bluefish population structure is particularly important
Schiaffino et al. 2010), salinity gradients (Nielsen et al. 2004), and necessary for fisheries management (Utter 1991; Wilson 2003).
temperature boundaries (Crow et al. 2007), convergence of distinct The contemporary distribution of Bluefish is coincident with sea
water masses (Borrero-Perez et al. 2011), behaviour (Campos-Telles surface temperatures of 18-27ºC (Juanes et al. 1996) and it has been
2011), or historical past events (Shen et al. 2011). Some barriers to suggested that shifts in its ranges and contacts between populations
gene flow are well defined by coastal shapes and features. Examples have resulted from historical changes in water temperature (Goodbred
are the Gibraltar Strait for some Sparidae species (Bargelloni et al. and Graves 1996). The sensitive behavioural response of Bluefish to
2003), the Siculo-Tunisian strait for Sea Bass (Bahri-Sfar et al. 2000), temperature variations may provide new insights into the evolutionary
the Florida Keys for gobies (Avise 1992), and the hydrographic consequences of the glacier-interglacier cycles and migrations into
isolation of the Aegean and Ionian and Adriatic Seas for numerous refuges for marine migratory species.
species (Partanello et al. 2007; Perez-Losada et al. 2007). On the other The objective of this study was to document the present
hand, behavioral traits such as homing can also account for population population structure of Bluefish in its northern distribution across the
differentiation in some species (e.g., Castillo et al. 2005). Finally, North Atlantic ocean and Mediterranean, Marmara, and Black seas,
while the genetic variability and population genetic structure of a identify possible ocean barriers to dispersal, and reconstruct the
species are shaped by both present and historical marine barriers, they phylogeography of Bluefish to understand the role of climate for
are also affected by paleoecological history (Partanello et al. 2007; determining historical and present barriers to gene flow along the
Perez-Losada et al. 2007). Hence, to better understand speciation Atlantic and Mediterranean basins.
mechanisms in the marine environment, it is important to characterize
not only population dynamics and structure, but also life-history
strategies, environmental factors from past and present, and physical Materials and Methods
barriers to dispersal (Zardoya et al. 2004).
Bluefish (Pomatomus saltatrix) is a cosmopolitan, migratory, Sampling
pelagic predator distributed over continental shelves and in estuaries A total of 120 samples of Pomatomus saltatrix collected from 8
of temperate waters of the Atlantic, Indian, and Pacific Oceans and different locations (Figure 1) between 2004 and 2009 were analyzed.
adjacent seas, including the Mediterranean, Aegean, and Black seas These samples represent at most two consecutive generations since
(Briggs 1960; Tortonese 1986; Pottern et al. 1989; Juanes et al. 1996). the maturity age is 2.4 in males and 1.9 in females (Dhieb et al.,
2006). Four locations were in the northwest (NW) Atlantic Ocean, on
*
Corresponding author: Laura Miralles Tel: +34-985102726; Fax: +34- the American coast: New Jersey, Maryland, North Carolina, and
985103534; E-mail: lml.miralles@gmail.com Florida and four locations were in the East Atlantic and
71
Figure 1: Sampling locations: Geographical distribution of sampling points in the Atlantic Ocean and the Mediterranean, Marmara, and Black Seas. NJ: New Jersey (USA); MD:
Maryland (USA); NC: North Carolina (USA); F: Florida (USA); C: Cadiz (Spain); B: Barcelona (Spain); TC: Canakkale (Turkey); TI: Istanbul (Turkey)
and in the Mediterranean basin: Cadiz (Atlantic Spanish coast) and Genetic diversity:
Barcelona (West Mediterranean, Spanish coast), and Canakkale and Sequences of each mitochondrial gene (cytb and COI) were
Istanbul (Marmara and Black seas, Turkish coast). aligned using ClustalW (Thompson et al. 1994) from the BioEdit
Sequence Alignment Editor (Hall 1999) and were visually inspected
DNA extraction, amplification and sequencing: to avoid base-calling errors. The incongruence length difference (ILD)
DNA was extracted with Chelex (Bio-Rad®) following Estoup tests (Farris et al. 1995) were performed in PAUP* 4.0 (Swofford
et al. (1996). Two mitochondrial genes and eight microsatellite loci 1999) with 1, 000 replicates and p-value of 0.05. The two mtDNA
were amplified. The mitochondrial cytochrome b (Cyt b) gene gene sequences were concatenated and haplotypes defined with
sequences were obtained following the protocol described by Kocher DNAsp v.4.50.3 (Rozas et al. 2003). MtDNA haplotype (H) and
et al. (1989) with the primers H151 and L148 described therein. The nucleotide diversity (pi) were calculated for each location using the
cytochrome oxidase subunit I (COI) gene was amplified with primers software Arlequin version 3.0 (Excoffier et al. 2005).
designed for Pomatomus saltatrix: COI-R Pom.: 5´- Microsatellite allele sizes were estimated using the
AAGAATGGGGTCTCCTCCAC-3´ and COI-F Pom.: 5´- GeneMapper® Software Version 4.0 (Applied Biosystems). Loci with
TTGGTGCATGAGCTGGTATG-3´; with the software PRIMER3 scoring errors, large allele dropout, and null alleles were discarded
(Rozen and Skaletsky 2000). Several potential primers set were employing the program MICROCHECKER (Van Oosterhout et al.
generated; we selected one set of primers that covered the maximum 2004). Conformity to Hardy-Weinberg equilibrium was calculated
number of base pairs. The polymerase chain reactions (PCR) to obtain using the exact probability test with GENEPOP software (Raymond
the COI sequences were performed using the GeneAmp PCR system and Rousset 1995). Microsatellite variation (number of alleles per
2700 (Applied Biosystems). Total reaction volume was 40 μl and the locus, allelic richness, and observed and expected heterozygosity) was
reaction mix contained approximately 50 ng of DNA, 20 pmol of each calculated with the programs GENETIX Version 4.03 (Belkhir et al.
primers, 10 mM Tris-HCL pH 8.8, 250 μM of each dNTP, 5U of 2004) and FSTAT Version 2.9.3.2 (Goudet 2001).
DNA Taq polymerase (Promega, Madison, Wisconsin) and 2.5 mM
MgCl2. The PCR conditions were: initial denaturing at 95ºC for 5 Population differentiation and structure:
min, then 35 cycles of denaturing at 95ºC for 30 s, annealing at 58ºC A median-joining (Bandelt et al. 1999) haplotype network was
for 30 s and an extension of 72ºC for 30 s, and a final extension at constructed using the concatenated mtDNA genes to visualize the
72ºC for 20 min. PCR products were visualized in 2% agarose gels intra-specific relationships of the different haplotypes and their
with 10 mg/ml of ethidium bromide. Stained bands were excised from relative frequencies in the sampled populations with the program
the gel and DNA fragments were purified with an Eppendorf Network 4.5.1.6 (http://fluxus-engineering.com) with default settings.
PerfectPrep Gel CleanUp® kit. Then, purified DNA was precipitated Network software reconstructed all possible, shortest, least complex,
using standard 2-propanol precipitation and re-suspended in phylogenetic trees (maximum parsimony) from a data set under
formamide prior to sequencing. Sequencing was performed in an ABI different algorithms.
PRISM 3100 Genetic Analyzer (Applied Biosystems), with BigDye Genetic divergence between populations was estimated using
3.1 Terminator system, in the Unit of Genetic Analysis of the population pairwise FST values calculated using nuclear and mtDNA
Scientific-Technical Services of the University of Oviedo (Spain). data with the program Arlequin version 3.0 (Excoffier et al. 2005).
Eight tetranucleotide microsatellites were assayed: elf 17, elf 19, The statistical significance of FST values between samples was
elf 37, elf 39, elf 44, elf 46, elf 49 and elf 50 (Dos Santos et al. 2008). calculated with 1,000 permutations and 10,000 steps in Markov chain.
Reaction and conditions were modified from those described by the This software was also employed for molecular analysis of variance
authors. PCRs consisted of: 95ºC for 5 min, 35 cycles of 95ºC for 30s, (locus by locus and standard AMOVAs) using 1,000 permutations.
the annealing temperature (Table 1) for 30s, 72ºC for 30s, followed by Fu’s FS test (Fu 1997) for selective neutrality was calculated in
72ºC of 20 min. Concentration of MgCl2 for each loci and fluorescent Arlequin v.3.0. For neutral markers, this test can be employed to
label were also described in Supplementary Information Table S1. detect changes in population size. Significant and negative FS values
PCRs were performed in a total volume of 20 μl in individual PCR can be interpreted as signatures of population expansion (Dodson et
reactions for each locus with the GeneAmp PCR system 2700 and al. 2007). Mismatch analysis was also used to explore the spatial and
2720 Thermal Cycler (Applied Biosystems). Products were visualized demographic evolution of the studied Bluefish populations with the
in 50 ml 2% agarose gels with 2.5 μl of ethidium bromide (10 mg/ml) Raggedness index (Harpending 1994) and the Sum of Squared
for verification and genotyped using an ABI PRISM 3100 Genetic Deviation (SSD; Schneider and Excoffier 1999). Both demographic
Analyzer (Applied Biosystems), with GS500 LIZ 3130 size standard, and spatial mismatch analysis were calculated with Arlequin v.3.0 and
in the Unit of Genetic Analysis of the Scientific-Technical Services of were based on the null hypothesis of expansion, thus non-significant
the University of Oviedo (Spain). values reveal population expansion.
72
Population structure across the study area was assessed using the priors (kappa, gamma-shape, proportion of invariant sites, etc.) were
program STRUCTURE 2.3.1 (Pritchard et al. 2000) by using inferred by using jModeltest software version 0.11 (Posada 2008) and
microsatellite loci data. This software estimates the minimum number its implementation of the Akaike information criterion (AIC; Akaike
of population units with genetic identity in the dataset under a 1974). The mutation rates employed were 2% per million years (MY)
Bayesian framework. This dataset was analyzed under the “Admixture for Cyt b (Brown et al. 1979) and 1.2% per MY for COI
model” which assumes that individuals may have mixed ancestry. The (Bermingham et al. 1997). Tracer version 1.5 (Rambaut and
parameter set consisted of a burn-in period of 30,000 steps followed Drummond 2007) was used to check that chains had converged to a
by 300,000 Markov Chain Monte Carlo (MCMC) iterations and 7 stationary distribution. The analysis was repeated with longer runs (50
runs for each K (number of genetic units estimated); the number of K million MCMC steps) when datasets did not accomplish this
was estimated following Evanno et al. (2005). condition.
Mantel tests of association between genetic differentiation
values (pairwise FST values) and geographical Euclidean distances
(linear distances between sampling locations) was calculated with
Arlequin version 3.0 (Excoffier et al. 2005). Mantel tests were done to
Results
determine if the considered populations follow an isolation-by- Genetic diversity:
A total of 46 haplotypes for the concatenated COI-Cyt b genes
distance model. To determine possible geographical barriers to
were detected among the studied samples. Most (68.75%) were
dispersal, we employed the software BARRIER v.2.2 (Manni et al.
observed solely among American samples (Northwest Atlantic Ocean)
2004) that can identify geographically continuous and discontinuous
while the rest were from the eastern area (Table 1; GenBank ID:
assemblages of samples from a spatial landscape. Geographical
JQ039400-JQ039435for COI and JQ039436-JQ039465 for Cyt b
coordinates of each sampling area were mapped into a matrix
haplotypes). High haplotype diversity and low nucleotide diversity
connected by Delauney triangulation (Brassel and Reif 1979). Barriers
was found in general for all the regions, but differences between
in the triangulation were identified using mitochondrial and nuclear
localities revealed a gradient from west to east across the studied area.
pairwise FST distances. The analysis employed was based on
American samples exhibited higher diversities than European ones,
Monmonier’s maximum distance algorithm (Manni et al. 2004) to
and western Mediterranean samples were more diverse than those
identify regions with sharp genetic change or discontinuity.
from the eastern Mediterranean (Table 1). The highest number of
haplotypes and haplotype and nucleotide diversities corresponded to
Phylogeny and evolutionary history of Pomatomus saltatrix
northwest Atlantic samples.
Population divergence time estimations were done under a
The eight microsatellite loci considered were amplified for the
Bayesian Markov Chain Monte Carlo (MCMC) framework using the
same 120 individuals across the sampling area. MICROCHECKER
two mtDNA genes with the softwareBEAST version 1.6.1
did not detect dropouts or scoring errors, but null alleles were found
(Drummond and Rambaut 2007). Following a burn-in of 3 million
for one locus (Elf44) in different populations. Therefore, Elf44 was
cycles, rates were sampled once every 1,000 cycles from 30 million
excluded from the data set. High genetic variability was found at the
MCMC steps for an Extended Bayesian Skyline tree prior with a
seven loci. The number of alleles per locus ranged from 18 (Elf17) to
stepwise model for mitochondrial DNA and strict clock model.
29 (Elf37– See Supplementary Information Table S1). All sampling
Bayesian intraspecific phylogenies are based on coalescent theory
areas were in Hardy-Weinberg equilibrium and there were no
(Kingman 1982) and allow the inference of past population dynamics
significant differences between expected and observed
and parameters from contemporary gene sequences. The best
heterozygosities in any location (Table 1)..
evolutionary model of both sequences (COI and Cyt b) and their
Table 1: Diversity indices for mitochondrial DNA and microsatellite loci of Pomatomus saltatrix samples. Diversity indices of the concatenated Cytb-COI genes as H,
Haplotype diversity;; (π), Nucleotide diversity;; Nh, Number of haplotypes (private/singletons as exclusive of a locality / one single copy respectively) and for microsatellite loci
asNA, average number of alleles per locus. AR, allelic richness. He and Ho, heterozygosity observed and expected respectively and FIS values. N, sample size.
73
Table 2: Populations pairwise Fst differences: Pairwise FST estimates between Bluefish samples based on mtDNA (below diagonal) and nuclear DNA based on microsatellite
loci (above diagonal). Significant P values are in bold. Locality acronyms as described in Table 1.
Population differentiation and structure: structure results (STRUCTURE, mitochondrial and nuclear FST and
Differentiation between the two sides of the Atlantic Ocean was AMOVAs) suggested some permeability across the three genetic units
observed in the haplotype network (Figure 2), where the main The Gibraltar Strait was not a barrier to gene flow for Pomatomus
separation between mitochondrial lineages corresponded clearly to the saltatrix since the Spanish samples on the two sides of the Strait
geographical differentiation between the two sides of the Atlantic (Cadiz and Barcelona) were not significantly different.
Ocean, giving separate American and European clades, with further
internal division. The presence of unique haplotypes may be caused Phylogeny and evolutionary history of Pomatomus saltatrix
by recent differentiation and factors such as geological or The time to the most recent common ancestor (TMRCA) of the
demographic factors and mutations would explain the haplotype sampled Bluefish was estimated to be 480,000 years ago (95%
structure. Highest Posterior Density (HPD) = 0.28–0.72 MY), while the
Population structure of Bluefish based on both mitochondrial TMRCA for the NW Atlantic Ocean was dated as 252,000 years
and nuclear DNA was consistent with three different genetic units in (95%HPD=0.13–0.39 MY) and 148,300 years (95%HPD=0.05–0.27
the sampling area: northwest Atlantic, and west and east MY) for the Mediterranean samples. The population growth rate
Mediterranean. FST population pairwise comparisons revealed estimated with the Extended Bayesian Skyline model for the North
significant differences between northwest Atlantic and other locations American samples reached its maximum approximately 40,100 years
and also between east and west Mediterranean samples (Table 2) for ago, while for the European clade the maximum occurred 23,420
both types of markers. The northwest Atlantic group consisted of all years ago (Figure 5). Both estimates suggest that the northwest
the North American localities: Florida, Maryland, North Carolina, and Atlantic clade is more ancient than the Mediterranean. The Bayesian
New Jersey. The west Mediterranean cluster included Cadiz (from the tree obtained for the northwest Atlantic Ocean suggested that there are
Atlantic Ocean) and Barcelona (west Mediterranean), and the east two separated American lineages which are composed of a mixture of
Mediterranean group was composed by the two Turkish localities, all localities. The TMRCA of the two American lineages was
Istanbul and Canakkale. estimated to be approximately 175,000 and 131,000 years. The
Bayesian analysis confirmed that Bluefish from this study substructure detected with the Bayesian methods and not with FST is
belonged to three different genetic units (K=3), corresponding to the likely due to the more sophisticated approaches of Bayesian inference.
same three clusters detected with the genetic distances but with a FST – based approaches are well understood, widely used, and easily
moderate level of admixture between them (Figure 3). There are also a applied but Bayesian-based analyses allow more precise estimates
secondary maximum at K=8, supported by much lower likelihood (See Pearse and Crandall 2004 for a review)
than K=3, suggesting a hierarchical island model (Evanno et al. 2005)
of 8 different populations clustered in 3 main sets. The analysis of
Figure 2: Haplotypes network: Median-Joining network with the relationships among
molecular variance confirmed these three genetic units. Both the 48 haplotypes defined by concatenated Cyt b and COI mitochondrial genes. Circles
AMOVAs, standard (for mtDNA) and locus by locus (for sizes are proportional to the frequency of each haplotype. Different locations are
microsatellite loci; Table 3), showed significant differences among the represented in different colors. Branches are proportional to mutations between
three groups and within populations, but not among populations haplotypes.
within the three previously defined groups. In the standard AMOVA,
the highest percentage of variation was observed between groups
(71%; P<0.001), while the highest percentage of variation in the locus
by locus AMOVA was within populations (93.72%; P < 0.001). These
results were consistent with STRUCTURE and mitochondrial and
nuclear FST results.
Demographic analyses indicated that all the populations except
Canakkale were in expansion (Table 4). In the Mantel test, a high and
significant correlation was detected between genetic and Euclidean
distances between samples (r = 0.734; p<0.001), suggesting a pattern
of isolation by distance for Pomatomus saltatrix. The software
BARRIER (Manni et al. 2004) identified two main boundaries
coincident with the two longer geographical distances between
samples. The two detected barriers were supported with both types of
molecular markers (Figure 4). However, all the previous population
74
Figure 3: Population structure across the sampling area:

(A.) Likelihood and number of cluster detected (K) based on nuclear DNA with STRUCTURE 2.3.1 (Pritchard et al. 2000) following Evanno et al. (2005).
(B.) Percentage of individual membership detected with STRUCTURE 2.3.1 (Pritchard et al. 2000). Each genetic unit is represented by one color (American samples in blue, Spanish
in green, and Turkish in red). Each vertical bar represents one individual. Acronyms of sampling localities are below the plot and described in Figure 1 and Table 1
Figure 4: Barriers to dispersal: Detected spatial genetic discontinuities along the distribution of the samples based on the Monmonier’s maximum difference algorithm are marked with
red arrows and green letters: A is the first detected barrier and B the second barrier.
75
Table 3: Molecular analyses of variance: Based on microsatellite loci (locus by Discussion

locus AMOVA) and mitochondrial sequence variation (standard AMOVA).
Significant P values in bold. This study provides a genetic analysis of North Atlantic and
Mediterranean Bluefish populations. Three clear genetic units were
identified, and may follow a hierarchical island model. The North
American samples were clustered together, without any significant
difference between sampling sites, suggesting that Bluefish is
panmictic in the northwest Atlantic Ocean. Many studies based on
distribution and morphological characteristics had investigated the
number of stocks in the East Coast of the United States. In previous
studies, the number of stocks identified ranged from six to two
(Lund 1961; Lassiter 1962; Lund and Maltezos 1970), but other
investigations concluded that either two distinct spawning groups
or one stock with two distinct spawning periods occurred in the
region (Norcross et al. 1974; Kendall and Walford 1979; Hare and
Cowen 1993; Smith et al. 1994). Later, Graves et al. (1993)
Table 4: Population expansion test: Mismatch analysis of demographic and spatial concluded that there was a single population based on
expansion and neutrality tests: SSD, Schneider and Excoffier’s test of sudden
expansion (Schneider and Excoffier 1999); Raggedness index (Harpending 1994); FS: mitochondrial DNA, and a morphometric study (Austin et al. 1999)
Fu’s test of neutrality (Fu 1997), respectively. Values indicating population expansion corroborated the one-stock hypothesis despite the evidence of
are marked in bold. Locality acronyms as described in Table 1. phenotypic plasticity. Our study confirms the one-stock hypothesis
based on both nuclear and mitochondrial DNA, but the Bayesian
mitochondrial DNA results suggested that there are two different
lineages within the American panmictic population that may have
evolved at different times.
Our results support two genetic discontinuities for Bluefish:
one in the middle of the Atlantic Ocean and the other in the
Mediterranean Sea. However, regional permeability and migration
can be inferred to occur across both of them. The Mediterranean
barrier could be at the level of the Siculo-Tunisian Strait, and may
be due to the hydrographic isolation of the Aegean and Ionian and
Adriatic Seas, or either of them, but not in the Gibraltar Strait or
Alboran Sea. The Siculo-Tunisian strait is a barrier to gene flow for
other species (Stefanni and Thorley 2003; Zardoya et al. 2004), and
in most cases the major genetic break or limitation to larval
dispersal between the Eastern and Western Mediterranean occurs in
the straits separating the Adriatic, Aegean, and ⁄∕ or Black Seas
Figure 5: Maximum population growth rates for the studied Bluefish during the
last 50,000 years: Population growth rates (in percentage) estimated with the (Nikula and Vainola 2003; Costagliola et al. 2004; Domingues et
Extended Bayesian Skyline of BEAST software for the European and American al. 2005; Peijnenburg et al. 2006; Perez-Losada et al. 2007;
Bluefish clades. Zulliger et al. 2009). The accurate location/s of the barrier/s for this
species cannot be deduced from the present results.
The most probable factor influencing Bluefish population
genetic structure seems to be geographical, such as the distance to
continents and shallow waters. Small fish are commonly found in
shallow coastal waters (e.g., May and Maxwell 1986) and apparent
population genetic isolation associated with different continents
has been reported before (Goodbred and Graves 1996). Bluefish is
a highly migratory species (Juanes et al. 1996). Water temperature
and photoperiod are major factors described to influence movement
patterns in Bluefish (Juanes et al. 1996; Olla and Studholme 1972;
Wilk 1977) but other factors, like water salinity, could contribute to
shape their population structure. We have compared annual sea
surface temperature as well as annual temperature at 200 m depth
(maximum depth for Pomatomus saltatrix) with the Bluefish
genetic structure detected in this study. In this case, changes in
temperature might not explain the barriers detected here, nor the
genetic structure of different Bluefish populations (See maps of
temperature in Supplementary Information Image S1). Similarly,
salinity gradients at the surface and at 200 m depth did not explain
the genetic structure and barriers detected (See Supplementary
Information Image S1). Neither factors showed differences along
the genetic units detected, suggesting that those factors are not a
barrier in our sampling area. In contrast, both annual salinity and
water temperature along the North Atlantic Ocean and
Mediterranean Sea could explain the permeability across the two
boundaries detected and could allow Bluefish to migrate. This
connection across barriers could prevent vicariant speciation, as
Pomatomus saltatrix is the sole member of its genus and family.
76
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In conclusion, the present study suggests that the Dhieb, K., Ghorbel, M., Jarboui O., and Bouain, A. 2006. Interactions between
reproduction and fisheries in Bluefish, Pomatomus saltatrix
paleoecological history (e.g., glaciations and interglacial periods in (Pomatomidae), from Gulf of Gabes (Tunisia). Cybium 4:355–364.
the late Pleistocene) has been crucial for shaping the present Dodson, J.J., S. Tremblay, F. Colombani, and J.E. Carscadden. 2007. Trans-
genetic variability and population structure of Pomatomus saltatrix Arctic dispersals and the evolution of circumpolar marine fish species
in the North Atlantic Ocean and in the Mediterranean basin. complex, the Capelin (Mallotus villosus). Molecular Ecology 16:5030-
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Domingues, V.S., G. Bucciarelli, V.C. Almada, and G. Bernardi. 2005.
Historical colonization and demography of the Mediterranean damselfish,
Acknowledgments Chromis chromis. Molecular Ecology 14: 4051–4063.
Dos Santos, S.M.R., A.W. Klopper, C.J. Oosthuizen, and P. Bloomer. 2008.
Isolation and characterization of polymorphic tetranucleotide
We are grateful to Maria del Carmen Sarasquete (CSIC, microsatellite loci in the pelagic perciform fish Pomatomus saltatrix
Spain) for Cadiz samples, to Tevfik Ceyhan for Turkish samples, to (Linnaeus, 1766) from South Africa. Molecular Ecology Resources
8:1065–1067.
Peter Clarke and John Murt for Florida samples, to Keith Dunton Drummond, A.J., and A. Rambaut. 2007. "BEAST: Bayesian evolutionary
for New York samples, to Ryan Woodland for Maryland samples, analysis by sampling trees." BMC Evolutionary Biology 7:214.
to Jim Morley for North Carolina samples, and to Ivan G. Pola and Estoup, A., C.R. Largiader, E. Perrot, and D. Chourrout. 1996. Rapid one-tube
DNA extraction protocol for reliable PCR detection of fish polymorphic
Greg Puncher for helping in laboratory tasks. This work was markers and transgenes. Molecular Marine Biology and Biotechnology
supported by the Spanish National Grant CGL2009-08279. 5(4):295-298.
American sampling was funded through the Bluefish/Striped Bass Evanno, G., S. Regnaut, and J. Goudet. 2005. Detecting the number of clusters of
Research Program (NOAA). Laura Miralles holds a PCTI Grant individuals using the software STRUCTURE: a simulation study.
Molecular Ecology 14:2611-2620.
from the Asturias Regional Government, referenced BP 10-004. Excoffier, L., G. Laval, and S. Schneider. 2005. Arlequin ver. 3.0: an integrated
software package for population genetics data analysis. Evolutionary
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Farris, J.S., M. Kallersjo, A.G. Kluge, and C. Bult. 1995. Constructing a
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M iralles et al. 2014 / Nor t her n hem isph er e popu la t ion st r uct u re of Bluefish / Fisheries
Suplementary information:
Figure S1: Temperature and salinity annual gradients
Maps were taken from World Ocean Atlas 2009. Temperature is represented in Celsius degrees (Locamini et al. 2010). Salinity is represented in
Practical Salinity Scale (Antonov et al. 2010)
79
M iralles et al. 2014 / Nor t her n hem isph er e popu la t ion st r uct u re of Bluefish / Fisheries
Table S1: Microsatellite loci PCR condition and number of alleles per locus. TA (ºC): Annealing temperature in Celsius degrees; [MgCl2](mM):
Concentration of Magnesium in mili Molar; Dye: fluorochrome used for genotyping; NA: number of alleles per locus.
80
Resultados
Capítulo 2:
Interoceanic Sex-Biased Migration in Bluefish.
Miralles, L. , Juanes, F., Garcia-Vazquez, E.
Transactions of the American Fisheries Society
81
82
Transactions of the American Fisheries Society 143:1308–1315, 2014
! American Fisheries Society 2014
ISSN: 0002-8487 print / 1548-8659 online
DOI: 10.1080/00028487.2014.935480
NOTE
Interoceanic Sex-Biased Migration in Bluefish
L. Miralles*
Department of Functional Biology, University of Oviedo, C/Julian Claveria s/n 33006 Oviedo, Asturias, Spain
F. Juanes
Department of Biology, University of Victoria, 3800 Finnerty Road, Victoria, British Columbia V8W 3N5, Canada
E. Garcia-Vazquez
Department of Functional Biology, University of Oviedo, C/Julian Claveria s/n 33006 Oviedo, Asturias, Spain
Downloaded by [85.152.212.49] at 16:04 03 October 2014
influenced by life history strategies (Zardoya et al. 2004) and

behavioral traits, such as sex-biased dispersal. Sex-biased dis-
Abstract
The Bluefish Pomatomus saltatrix is a highly migratory species persal occurs when individuals of one sex tend to be philopat-
that is composed of different stocks and populations along its ric (i.e., return to the natal area to reproduce; e.g., Campos-
nearly cosmopolitan distribution. The Bluefish is the only mem- Tellez et al. 2011) while those of the other sex tend to disperse
ber of its genus and family, and high migration rates could pre- (e.g., Cano et al. 2008). Although the term “migration” is
vent vicariant speciation across its wide geographical often used to mean “movement,” it has several other meanings
distribution. However, the extent of gene flow between distant
populations is unknown. We employed two mitochondrial genes (e.g., see Dingle 1996). Here, we use the term migration to
(cytochrome-c oxidase subunit I and cytochrome b) and eight refer to dispersal (emigration) from and recruitment (immigra-
nuclear microsatellite loci to study population structure and infer tion) to a population.
dispersal of this important commercial and recreational fish Sex-biased dispersal is common in animals (Perrin and
across its Northern Hemisphere distribution. Higher gene flow Mazalov 2000), including fishes. Groups such as sharks (e.g.,
estimates for nuclear loci (of biparental inheritance) than for
mitochondrial loci (of maternal inheritance) suggested sex-biased White Shark Carcharodon carcharias: Pardini et al. 2001;
dispersal, which could be explained by greater female homing or Bonfil et al. 2005), most salmonids (e.g., Campos-Tellez et al.
fidelity to spawning sites and greater dispersal of males. Males 2011), Weakfish Cynoscion regalis (Thorrold et al. 2001), and
could contribute more to transoceanic connectivity of Bluefish many others have been shown to exhibit differences in migra-
populations in the North Atlantic Ocean, thus shaping the tory and homing behaviors between sexes. Differences
observed pattern of spatial genetic structure of the Bluefish in its
Northern Hemisphere distribution. between sexes in life history traits also promote differences in
dispersal. For example, if males mature later, they might
migrate longer distances than females (e.g., Palo et al. 2004),
as was suggested for Bluefish Pomatomus saltatrix (Morley
et al. 2013); the opposite is probably also true. Inferences
Gene flow and connectivity among populations are key about sex-biased dispersal from observed population structur-
drivers of population dynamics and patterns of genetic struc- ing depend on whether the examined genes are of maternal,
ture in marine metapopulations (Cowen et al. 2000). Migra- paternal, or biparental inheritance (e.g., Prugnolle and de
tion, including dispersal (emigration) and recruitment Meeus 2002). For example, if pairwise genetic differentiation
(immigration), modulates the persistence and demography of index (FST) values (measures of genetic distance between pop-
species (Hanski 1999), buffers populations against environ- ulations) are higher for maternally inherited mitochondrial
mental fluctuations (Friedenberg 2003) or stochastic events genes than for biparentally inherited nuclear DNA, this is often
(Cadet et al. 2003), and reduces the likelihood of local extinc- interpreted as an indication of higher female fidelity to particu-
tions (Poethke et al. 2002). Migration patterns of marine fish lar groupings or reproductive locations (Hueter et al. 2005;
can also shape patterns of population structure and may be Karl et al. 2011).
*Corresponding author: lml.miralles@gmail.com

Received February 1, 2014; accepted June 9, 2014
1308
83
NOTE 1309
Bluefish are fast-growing predators that undergo seasonal locations were in the Mediterranean Basin: (1) the Mediterra-
migrations related to temperature and photoperiod and also nean Sea off the coast of Barcelona, Spain (BCN); (2) the Mar-
exhibit long displacements for spawning in some regions (e.g., mara Sea off the coast of Çanakkale, Turkey (TC); and (3) the
U.S. Atlantic coast), undertaking spring or summer move- Black Sea off the coast of Istanbul, Turkey (TI). Small pieces
ments to higher latitudes, where they are often the target of of muscle or fin (»1 cm3) were dissected from each individual
recreational fisheries (Juanes et al. 1996). Bluefish reproduc- and were preserved in 100% ethanol prior to laboratory
tive behavior consists of mass spawning with external fertili- analyses.
zation. Bluefish populations inhabit continental shelves and Extraction, amplification, and sequencing of DNA.—Blue-
estuaries in temperate waters of the Atlantic, Indian, and fish DNA was extracted with Chelex (Bio-Rad) following the
Pacific oceans and adjacent seas (Briggs 1960; Tortonese method of Estoup et al. (1996). Two mitochondrial genes and
1986; Pottern et al. 1989; Juanes et al. 1996; Shepherd et al. eight microsatellite loci were amplified. The mitochondrial
2006). In the Northern Hemisphere, Bluefish are distributed cytochrome-b (cyt-b) gene sequences were obtained with the
across the Atlantic Ocean and the Mediterranean Basin, primers H151 and L148 following the protocol and PCR con-
including the adjacent Marmara, Aegean, and Black seas. The ditions described by Kocher et al. (1989). The cytochrome-c
Bluefish is the sole member of the genus Pomatomus and the oxidase subunit I (COI) gene was amplified with primers
family Pomatomidae, and its cosmopolitan distribution designed for Bluefish (COI-R-Pom: 50 -AAGAATGGGGTC-
(Juanes et al. 1996) indicates a high dispersal potential. How- TCCTCCAC-30 ; COI-F-Pom: 50 - TTGGTGCATGAGCTGG-
ever, the species seems to be spatially structured at continental TATG-30 ) with PRIMER3 software (Rozen and Skaletsky
and regional scales (Turan et al. 2006; Pardi~nas et al. 2010). 2000) and manually adjusted. The PCRs to obtain the COI
On the other hand, subtle differences in life history traits sequences were performed using the GeneAmp PCR Sys-
between sexes have been documented (Table 1). In general, tem2700 (Applied Biosystems, Inc. [ABI]). Total reaction vol-
Bluefish males mature slightly later than females; because the ume was 40 mL, and the reaction mix contained
sex that matures later tends to migrate longer distances (Palo approximately 50 ng of DNA, 20 pmol of each primer,
et al. 2004; Morley et al. 2013), we expect that male Bluefish 10 mM of Tris-HCl (pH 8.8), 250 mM of each deoxynucleo-
disperse slightly farther than females. To test the hypothesis tide triphosphate, 5 units of Taq DNA polymerase (Promega,
that males have a higher dispersal potential than females, we Madison, Wisconsin), and 2.5 mM of MgCl2. The PCR condi-
used population genetic analyses of mitochondrial DNA tions were initial denaturing at 95! C for 5 min; 35 cycles of
(mtDNA) sequences and hypervariable nuclear microsatellite denaturing at 95! C for 30 s, annealing at 58! C for 30 s, and an
loci to examine Bluefish samples spanning the Atlantic Ocean extension at 72! C for 30 s; and a final extension at 72! C for
and Mediterranean Sea. 20 min. The PCR products were visualized in 50-mL 2% aga-
rose gels with ethidium bromide at 10 mg/mL. Stained bands
were excised from the gel, and DNA fragments were purified
METHODS with an Eppendorf PerfectPrep Gel CleanUp kit. Purified
Study area and sampling.—In total, 123 Bluefish samples DNA was precipitated using standard 2-propanol precipitation
were collected from eight locations across the species’ northern and was re-suspended in formamide prior to sequencing. Frag-
distribution (Figure 1). Four of the sampling locations were in ments were forward sequenced at the Genetic Analysis Unit,
the northwest Atlantic Ocean along the U.S. coast: New Jersey University of Oviedo, Spain, by using an ABI Prism 3100
(NJ), Maryland (MD), North Carolina (NC), and Florida (FL). Genetic Analyzer with the BigDye Terminator 3.1 system.
One sampling location was in the northeast Atlantic Ocean: in Eight tetranucleotide microsatellites—elf17, elf19, elf37,
the Bay of Cadiz (CZ) off the coast of Spain. Three sampling elf39, elf44, elf46, elf49, and elf50 (Dos Santos et al. 2008)—
TABLE 1. Reported differences in Bluefish life history traits between sexes, as determined in three regions (Tunisia, Turkey, and the U.S. Atlantic).
Marine region Trait Males Females Reference
Tunisia Age at maturity (years) 2.4 1.9 Dhieb et al. 2006

Sex ratio Monthly variation Monthly variation
Size at maturity (cm) 18.1 17.1
Turkey Von Bertalanffy growth (cm) 48.0 51.0 Ceyhan et al. 2007
Sex ratio Females dominate all age-groups
U.S. Atlantic Age at maturity (years) 1.2 1.1 Salerno et al. 2001
Size at maturity (cm) 33.9 33.4
Winter sex percentage (%) 40.4 59.6 Morley et al. 2013
Summer sex percentage (%) 25.6 74.4
84
1310 MIRALLES ET AL.
FIGURE 1. Geographical distribution of Bluefish sampling points in the Atlantic Ocean and the Mediterranean, Marmara, and Black seas (NJ D New Jersey;
MD D Maryland; NC D North Carolina; F D Florida; C D Cadiz, Spain; B D Barcelona, Spain; TC D Çanakkale, Turkey; TI D Istanbul, Turkey).
were assayed with the conditions described by Dos Santos followed an isolation-by-distance model—that is, to infer how
et al. (2008). Products were visualized in 2% agarose gels they are structured in the study area. Minimum evolution and
with 2.5 mL of ethidium bromide (10 mg/mL) to confirm neighbor-joining trees based on genetic distance were per-
amplifications and were genotyped using an ABI Prism 3100 formed with PHYLIP version 3.69 (Felsenstein 2005) for
Genetic Analyzer with a GS500 LIZ 3130 size standard at the microsatellites and mtDNA.
Genetic Analysis Unit, University of Oviedo. Gene flow and migration rates—Migration rates between
Genetic diversity, population differentiation, and struc- populations were calculated for mtDNA and nuclear DNA with
ture.—Mitochondrial sequences were aligned using ClustalW MIGRATE version 3.0 (Beerli 2004). The program is based on
(Thompson et al. 1994) from the BioEdit Sequence Alignment coalescent theory (Beerli and Felsenstein 2001), relaxing
Editor (Hall 1999) for each gene. After sequence congruence Wright’s (1951) assumptions that the population does not grow
was checked with the incongruence length difference test (Far- or shrink, that every individual has the same chance to repro-
ris et al. 1995) implemented in PAUP* version 4.0 (Swofford duce, and that every generation of adults is replaced by their off-
1999), the two genes were concatenated and haplotypes were spring. MIGRATE estimates Q D xNem and M D m/m, where Q
defined with DNAsp version 4.50.3 (Rozas et al. 2003). Mito- is the mutation-scaled population size; x is the inheritance
chondrial DNA haplotype diversity (H) and nucleotide diver- parameter (x D 4 for nuclear DNA [here microsatellite loci] and
sity (p) were calculated for each location by using Arlequin 1 for mtDNA); Ne is the effective population size; M is the
version 3.11 (Excoffier et al. 2005). mutation-scaled effective immigration rate; m is the immigra-
Microsatellites were genotyped by employing GeneMapper tion rate; and m is the mutation rate. The migration estimate is
version 4.0 (ABI). Scoring errors, large-allele dropout, and often expressed as xNm, which is Q and M multiplied together.
null alleles were checked with Micro-Checker (Van Oosterh- We used this formula, employing the x for each type of data, to
out et al. 2004). Genepop (Raymond and Rousset 1995) was calculate the effective number of immigrants per generation
employed to test departures from Hardy–Weinberg equilib- from nuclear DNA and mtDNA. To ensure that the results
rium, with a adjusted using a Bonferroni correction. Microsat- would not reflect spurious local likelihood peaks, three runs
ellite variation (number of alleles per locus, allelic richness, were performed with the maximum likelihood method using 10
observed heterozygosity, and expected heterozygosity) was long chains (50,000 recorded steps with increments of 100) and
calculated with Genetix version 4.03 (Belkhir et al. 2001) and five replicates. The MIGRATE software with the previously
Fstat version 2.9.3.2 (Goudet 2001). defined settings was run three independent times to ensure that
Genetic divergence between populations was estimated final chains were estimating the same value of Q for each data
from the population pairwise values of FST obtained with Arle- set (nuclear DNA and mtDNA data sets).
quin version 3.11 (Excoffier et al. 2005) using 1,023 permuta-
tions. Arlequin was also employed for Mantel tests of
association, which were performed between pairwise FST val- RESULTS
ues based on nuclear DNA and mtDNA to determine differen-
ces between the two genetic markers. Mantel tests were also Genetic Diversity
conducted between FST values and geographical Euclidean Overall, 46 haplotypes for the concatenated COI–cyt-b
distances to determine whether the considered populations sequences were defined in our Bluefish samples. Notably, sites
85
NOTE 1311
TABLE 2. Genetic variation in Bluefish at each sampling location (N D sample size; NA D average number of microsatellite alleles per locus; AR D allelic rich-
ness; He D expected heterozygosity; Ho D observed heterozygosity; Nh D number of mitochondrial haplotypes; H D haplotype diversity; p D nucleotide
diversity).
Locality Acronym N NA AR He Ho Nh H p
New Jersey NJ 25 14.1 13.19 0.8853 0.7021 14 0.9264 § 0.0388 0.0045 § 0.0026
Maryland MD 20 13.1 12.52 0.8714 0.7576 16 0.9692 § 0.0209 0.0051 § 0.0029
North Carolina NC 11 16.4 7.96 0.8399 0.7549 8 0.9273 § 0.0665 0.0051 § 0.0030
Florida FL 11 10.1 9.09 0.8593 0.6598 9 0.9636 § 0.0510 0.0056 § 0.0033
Cadiz, Spain CZ 35 14.6 13.68 0.8014 0.7170 11 0.8414 § 0.0441 0.0025 § 0.0016
Barcelona, Spain BCN 6 6.7 6.02 0.7143 0.7619 6 1.0000 § 0.0962 0.0031 § 0.0021
Çanakkale, Turkey TC 7 6.7 4.09 0.7194 0.8299 1 0.0000 § 0.0000 0.0000 § 0.0000
Istanbul, Turkey TI 7 6.0 4.08 0.7211 0.6922 3 0.5238 § 0.2086 0.0006 § 0.0006
Total 122 23.28 20.75 0.839 0.741 46 0.9344 § 0.0140 0.0086 § 0.0023
in the western Atlantic exhibited more haplotypes and higher was excluded from analyses. Samples were in Hardy–Weinberg
H and p values than eastern Atlantic sites. The number of hap- equilibrium and exhibited similar levels of variation as mea-
lotypes per site varied from 1 in the TC samples to 16 in the sured by allelic richness and heterozygosity (Table 2), despite
MD samples. The concatenated fragments were 881 bp long the fact that BCN, TC, and TI sample sizes were smaller than
and were composed of 570 bp from the COI gene plus 311 bp the rest; this suggests that the study is adequate and that our
from the cyt-b gene (GenBank accession numbers JQ039400– results are robust.
JQ039435 for COI; JQ039436–JQ039465 for cyt-b). Frag-
ments had 10 variable sites that differentiated the two sides of
the Atlantic Ocean and another 39 variable positions that Population Differentiation and Population Structure
defined all described haplotypes. We generally found high H Pairwise FST comparisons revealed three different genetic
and low p in all regions (Table 2). clusters—American (NJ, NC, MD, and FL), Spanish (BCN and
Micro-Checker did not detect dropouts or scoring errors in CZ), and Turkish (TI and TC)—for both mitochondrial and
the eight microsatellite loci considered, but null alleles were nuclear markers (Table 3). However, we did detect differences
found for elf44 in different sampling locations, so this locus between the two markers, as mitochondrial FST values were
TABLE 3. Pairwise estimates of the genetic differentiation index (FST; below the diagonal) between Bluefish samples based on mitochondrial DNA or micro-
satellite loci. Associated P-values are given above the diagonal; significant P-values are in bold italics. Sampling locality acronyms are defined in Table 2.
Locality NJ MD NC FL CZ BCN TC TI
Mitochondrial DNA
NJ 0.18919 0.15315 0.07207 0.00000 0.00000 0.00000 0.00000
MD 0.02436 0.71171 0.62162 0.00000 0.00000 0.00000 0.00000
NC 0.04170 ¡0.02772 0.90090 0.00000 0.00000 0.00000 0.00000
FL 0.05955 ¡0.02595 ¡0.05941 0.00000 0.00000 0.00000 0.00000
CZ 0.75687 0.74427 0.75280 0.74692 0.91892 0.01892 0.00000
BCN 0.70537 0.68326 0.67787 0.65902 ¡0.06545 0.00000 0.00000
TC 0.74745 0.73286 0.74596 0.73079 0.19134 0.18157 0.99099
TI 0.75434 0.74041 0.75938 0.74326 0.23288 0.17656 0.00001
Nuclear microsatellite loci
NJ 0.15137 0.57324 0.50684 0.00000 0.00977 0.00000 0.00000
MD 0.01008 0.25391 0.67285 0.00195 0.03809 0.00000 0.00000
NC 0.00367 0.01153 0.20312 0.01855 0.09473 0.00098 0.00391
FL 0.00498 0.00240 0.01777 0.05469 0.00781 0.00391 0.02734
CZ 0.02203 0.01930 0.01969 0.01357 0.41699 0.00098 0.00195
BCN 0.03900 0.03542 0.02785 0.04297 0.00572 0.00000 0.00293
TC 0.06407 0.08319 0.07205 0.06358 0.07385 0.05565 0.12891
TI 0.06734 0.07287 0.06623 0.04482 0.07825 0.04119 0.01869
86
main geographical units defined above. As expected from the

previously described results, estimates of gene flow (mean
effective number of immigrants per generation and for each
genetic unit) were significantly different for mtDNA and
microsatellites (Figure 3) in all cases (paired t-test: P ! 0.05),
using the corresponding inheritance parameters (x) in the cal-
culations to make the different markers comparable. Effective
number of immigrants, mean Q values, and their 95% confi-
dence intervals are shown in Figure 3. We observed asymmet-
rical gene flow among regions for the two marker types.
Values ranged from 0.02 to 1.62 immigrants/generation for
mtDNA and from 0.98 to 3.49 immigrants/generation for
microsatellite loci. For the Mediterranean Sea samples, the
asymmetry was in opposite directions for the two markers:
more intense westward migration was indicated by microsatel-
lite loci, and eastward migration was indicated by mtDNA.
For samples from the Atlantic Ocean, asymmetry between
markers was not detected. In both cases, the estimated gene
flow was more intense from the USA to Spain. The main dif-
ference between markers in the Atlantic Ocean samples was
that the total number of immigrants (detected with microsatel-
lite loci) from Spain to the USA was 91 times stronger than
the number of female immigrants (detected with mtDNA).
Unexpectedly, for both markers, estimates of gene flow across
the Atlantic Ocean were similar in magnitude to estimates of
gene flow across the Mediterranean Sea, despite the large dif-
FIGURE 2. Neighbor-joining trees based on genetic differentiation index ference in geographical scale.
(FST) values calculated from Bluefish (A) mitochondrial DNA and (B) nuclear
microsatellite loci. Sampling locality acronyms are defined in Figure 1.
DISCUSSION
always higher than nuclear FST values. Two combinations of Based on indirect evidence from genetic analyses, the
samples from Spain and the USA were not significantly differ- results of this study suggest the occurrence of transoceanic
ent: CZ versus FL (P D 0.05469) and BCN versus NC (P D effective migration for Bluefish across the Atlantic Ocean.
0.09473). The differences between genetic markers were also Transoceanic passage would be possible for this species given
reflected in the neighbor-joining trees that were constructed its migratory lifestyle, as has been shown for other species
based on genetic distances (Figure 2). The position of the such as sharks (Bonfil et al. 2005) and tunas (Block et al.
branch containing the Spanish samples (CZ [eastern Atlantic] 2001). Larval transport in the Gulf Stream (as suggested by
and BCN [western Mediterranean Sea]) varied in the trees built Hare and Cowen 1993) does not seem sufficient to account for
from the different markers, clustering with the eastern Mediter- the transoceanic differences we have documented here, given
ranean clade for mtDNA and with the American clade for that (1) Bluefish larvae complete their development near the
microsatellites (Figure 2). The minimum evolution trees (data surface and (2) juveniles are generally found on continental
not shown) were identical to the neighbor-joining trees. shelves, bays, estuaries, and shallow waters (Kendal and Wal-
Mantel tests revealed a low correlation between mitochon- ford 1979; Juanes et al. 1996). Instead, active migration by
drial and nuclear genetic markers (r D 0.496, P D 0.02) sug- adult Bluefish is a more likely explanation. Bluefish migration
gesting a low but correlated pattern in genetic distances from may be temperature dependent (see Goodbred and Graves
maternally inherited (mtDNA) and biparentally inherited 1996), and glacial periods have been identified as isolation
(nuclear) markers. Furthermore, a high and significant correla- stages for this species (Pardi~nas et al. 2010); therefore, current
tion was detected between genetic distances and Euclidean interglacial conditions may be promoting Bluefish dispersal.
distances between samples (r D 0.734, P < 0.001), implying a Pairwise FST values (measures of genetic distance between
pattern of isolation by distance for Bluefish. populations) based on mtDNA were all higher than those
based on nuclear DNA. Higher pairwise FST values for mater-
Gene Flow and Migration nally inherited mitochondrial genes than for biparentally
To estimate gene flow and improve the analysis by avoiding inherited nuclear DNA are often interpreted as indicating
small sample sizes, the samples were clustered into the three higher female fidelity to particular groupings or reproductive
87
NOTE 1313
FIGURE 3. Gene flow across the detected Bluefish genetic units. Gray circles represent population units and are proportional to theta (Q; the mutation-scaled
population size) values. Gene flow as number of migrants per generation (Nm) is represented with arrows (95% confidence intervals are shown in parentheses
below the mean values): (A) number of female migrants per generation (Nfm) based on mitochondrial DNA; and (B) number of migrants per generation (Nim)
based on nuclear microsatellite loci.
locations (Hueter et al. 2005; Karl et al. 2011). Moreover, (Salerno et al. 2001; Dhieb et al. 2006), thus affording greater
much lower estimates of gene flow were obtained for mtDNA swimming ability. The results of this study not only support
than for microsatellites, suggesting some homing fidelity in our expectation but also suggest that slight differences in life
females (lower migration rates) but not males (Hueter et al. history traits can have important consequences for population
2005). Both results suggest that Bluefish females have genetic structuring. Behavior, together with life history differences,
barriers to dispersal. Further support for sex-specific migration can explain the differing migration estimates for maternally
comes from a recent study using the scales of adult Bluefish and biparentally inherited markers in this species. Such differ-
collected in NC, where males disproportionately (relative to ences could also lead to sex-specific selective constraints, such
females) migrated out of the South Atlantic Bight in the sum- as higher dispersal costs or lower postmigratory breeding suc-
mer (Morley et al. 2013). Other highly migratory fish species, cess for females (Cano et al. 2008). Unfortunately, little work
including salmonids, seem to exhibit sex-biased dispersal has focused on adult Bluefish behavior; our results suggest
(Campos-Telles et al. 2011), but this is the first time that hom- that additional work on this subject is greatly needed.
ing fidelity and sex-biased dispersal have been suggested for In addition to trans-Atlantic migration, trans-Mediterranean
Bluefish. migration apparently also occurs in Bluefish. Highly asymmet-
Understanding the dynamics of sex-specific movements has ric gene flow, which was more intense from west to east when
both ecological and evolutionary implications. Although Blue- estimated from microsatellites and vice versa when estimated
fish do not exhibit sexual dimorphism (Pottern et al. 1989), from mtDNA (Figure 3), again suggests differences in dis-
small differences in life history traits between males and persal behavior between males and females. Differences in the
females have been reported (Table 1). From these differences asymmetric pattern found in the Mediterranean Sea relative to
and based on the hypothesis that maturity influences migration the Atlantic Ocean could be due to the Ne (proportional to Q
strategy (Palo et al. 2004; Morley et al. 2013), we expected values, which are represented in Figure 3 with different circle
that male Bluefish would be able to disperse slightly farther sizes). In other words, the genetic units with higher Ne may
than females because males mature later and at larger sizes provide more migrants to the smaller populations. For
88
example, based on our mtDNA results, more migrants go to populations. [GENETIX, software for Windows for population
Spain (with the lowest Q value) from both the USA and Tur- genetics.] Universit!e de Montpellier II, Montpellier, France.
key because these latter populations have larger values of Ne. Block, B. A., H. Dewar, S. B. Blackwell, T. D. Williams, E. D.
The general trend was similar in the case of nuclear DNA. Dif- Prince, C. J. Farwell, and A. Boustany. 2001. Migratory move-
ferences in the relative level of asymmetry between the two ments, depth preferences and thermal biology of Atlantic Bluefin
Tuna. Science 293:1310–1314.
markers as seen across the Mediterranean Sea, where the
Bonfil, R., M. Meyer, M. C. Scholl, R. Johnson, S. O’Brien, H. Oos-
asymmetry is more marked, could also be due to differences in
thuizen, and S. Swanson. 2005. Transoceanic migration, spatial
Ne. Physical tag–recapture experiments (of the sort summa- dynamics and population linkages of White Sharks. Science
rized by Shepherd et al. 2006, but including sex-specific infor- 310:100–103.
mation) would confirm these results based on population Briggs, J. C. 1960. Fishes of worldwide (circumtropical) distribution.
genetic methodology. Copeia 1960:171–180.
In conclusion, our results depict patterns of genetic popula- Cadet, C., R. Ferriere, J. A. J. Metz, and M. van Baalen. 2003. The
tion structure of Bluefish in the species’ northern distribution, evolution of dispersal under demographic stochasticity. American
consistent with sex-biased dispersal and transoceanic migra- Naturalist 162:427–441.
tion. Different patterns between the two genetic markers, Campos-Telles, M. P., R. G. Collevatti, M. C. da Costa, R. B. Bar-
including lower migration rates and stronger genetic barriers them, N. J. da Silva, A. C. Souza Neto, and J. A. F. Diniz-Filho.
2011. A geographical genetics framework for inferring homing
inferred from mtDNA (maternally inherited) relative to those
reproductive behavior in fishes. Genetica 139:243–253.
inferred from microsatellite loci (biparentally inherited), sug-
Cano, J. M., H. S. M€akinen, and J. Meril€a. 2008. Genetic evidence for
gest male-mediated gene flow among regions and greater phil- male-biased dispersal in the Three-Spined Stickleback (Gasteros-
opatry in females. Trans-Atlantic migratory movements and teus aculeatus). Molecular Ecology 17:3234–3242.
subsequent gene flow could explain the lack of divergence in Ceyhan, T., O. Akyol, A. Ayaz, and F. Juanes. 2007. Age, growth,
Bluefish populations, thereby maintaining a single species and reproductive season of Bluefish (Pomatomus saltatrix) in the
across the Atlantic Ocean and in the family Pomatomidae. Marmara region, Turkey. ICES Journal of Marine Science
64:531–536.
Cowen, R. K., K. M. M. Lwiza, S. Sponaugle, C. B. Paris, and D. B.
Olson. 2000. Connectivity of marine populations: open or closed?
ACKNOWLEDGMENTS Science 287:857–859.
We are grateful to Maria del Carmen Sarasquete (Spanish Dhieb, K., M. Ghorbel, O. Jarboui, and A. Bouain. 2006. Interactions
National Research Council) for the CZ samples; Tevfik Cey- between reproduction and fisheries in Bluefish, Pomatomus salta-
han for the Turkish samples; Peter Clarke and John Murt for trix (Pomatomidae), from Gulf of Gabes (Tunisia). Cybium
the FL samples; Keith Dunton for the NY samples; Mark 4:355–364.
Wuenschell for the NJ samples; Ryan Woodland for the MD Dingle, D. H. 1996. Migration: the biology of life on the move.
samples; Jim Morley for the NC samples; and Ivan G. Pola Oxford University Press, Oxford, UK.
and Greg Puncher for help in laboratory tasks. This work was Dos Santos, S. M. R., A. W. Klopper, C. J. Oosthuizen, and P.
Bloomer. 2008. Isolation and characterization of polymorphic tet-
supported by the Spanish National Grant Number CGL2009-
ranucleotide microsatellite loci in the pelagic perciform fish
08279. Sampling in U.S. waters was funded through the Blue-
Pomatomus saltatrix (Linnaeus, 1766) from South Africa. Molec-
fish/Striped Bass Research Program (National Oceanic and ular Ecology Resources 8:1065–1067.
Atmospheric Administration). Laura Miralles holds a PCTI Estoup, A., C. R. Largiader, E. Perrot, and D. Chourrout. 1996. Rapid
(Plan de Ciencia Tecnolog!ıa e Innovaci!on del Principado de one-tube DNA extraction protocol for reliable PCR detection of
Asturias) and FICYT (Fundaci!on para el Fomento de la Inves- fish polymorphic markers and transgenes. Molecular Marine
tigaci!
on Cient!ıfica Aplicada y la Tecnolog!ıa en Asturias) Biology and Biotechnology 5:295–298.
Grant (BP 10-004) from the Asturias Regional Government. Excoffier, L., G. Laval, and S. Schneider. 2005. Arlequin version 3.0:
an integrated software package for population genetics data anal-
ysis. Evolutionary Bioinformatics Online 1:47–50.
Farris, J. S., M. Kallersjo, A. G. Kluge, and C. Bult. 1995. Construct-
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90
Resultados
Capítulo 3:
Potential evolutionary impact of Mediterranean
aquaculture on the wild predator
Pomatomus saltatrix L.
Miralles, L. , Mrugala, A., Sanchez-Jerez, P., Juanes, F., Garcia-Vazquez, E.
Journal of Sea Research
91
92
M iralles et al.
Potential evolutionary impact of Mediterranean aquaculture on the wild predator

Pomatomus saltatrix L.
L. Miralles 1*, A. Mrugala1, P. Sanchez-Jerez2, F. Juanes3, E. Garcia-Vazquez1.
2. Department of Marine Science and Applied Biology, University of Alicante, 03080 Alicante, Spain
3. Department of Biology, University of Victoria, Victoria V8W3N5, BC, Canada
Aquaculture impacts on wild populations have been considered principally at the conspecific level, since farm escapes can
alter the genetic pattern of surrounding conspecific populations. However, aquaculture may cause wider effects on interspecific
Keywords: populations. To explore this question we used Bluefish Pomatomus saltatrix, which exhibits two distinct genetic units in the
Mediterranean: East and West. This fast-growing and voracious fish is attracted to aquaculture sites (particularly for Seabream and
Pomatomus saltatrix Sea Bass) to prey on farmed fish. We compared the genetic structure and diversity of adult Bluefish caught inside an aquaculture
Bluefish farm on the western Mediterranean coast with individuals caught in the open sea from areas much more distant from aquaculture
Fish farm facilities. Bluefish were genetically assigned to their origin populations using seven microsatellite loci and mitochondrial
Heterospecific impact cytochrome oxidase subunit I as molecular markers. As expected, most of individuals caught from inside the farm were assigned to
Genetic Diversity the local genetic unit. However, ten percent of individuals were assigned to the distant and different genetic unit inhabiting Turkish
Mediterranean Sea waters, the Eastern Mediterranean populations. Farm-collected Bluefish were also more genetically diverse. These results suggest
that aquaculture acts as an attractor for Bluefish and can affect genetic diversity and the phylogeography of this and other
accompanying species that aggregate around marine farms.
Introduction Wild fishes tend to concentrate around aquaculture facilities

(Dempster et al., 2002). Hatcheries and aquaculture cages also act as
Impacts of aquaculture on natural ecosystems are wide-ranging, magnets for predators like bottlenose dolphins (Diaz-Lopez et al.,
from aesthetic aspects to undesirable effects on wild surrounding 2005; Diaz-Lopez & Bernal-Shirai, 2007), monk seals (Güçlüsoy &
populations (Black, 2000; Fernandes et al., 2002; Diana, 2009). . One Savas, 2003), cormorants (Diaz-Lopez et al., 2008; Liordos &
of the most studied effects on wild populations is the genetic Goutner, 2008; Akyol & Ertosluk, 2010) and many wild fish species
interaction between farm escapees and wild conspecifics (e.g. Hindar (Dempster et al., 2002; Valle et al., 2007). Changes in the predatory
et al., 1991; Youngson et al., 2001; Read & Fernandes, 2003; Naylor community alter the equilibrium of their ecosystem and may
et al., 2005). Variants from domestic fish can be introduced into potentially result in their collapse (e.g. McCann, 2007; Baum &
native genetic pools thus reducing local adaptation, as reported for Worm, 2009), but the consequences for the phylogeography of
different species following farm escapes (e.g. Youngson et al., 2001; predator species have not been explored yet.
Utter & Epifanio, 2002). Farm escapes and deliberate releases of In this study, we use Bluefish Pomatomus saltatrix L. as a model
hatchery stocks also have the potential to alter population structuring to investigate the effects of aquaculture farms on the population
and phylogeographic patterns of aquatic species. Examples of such structuring of attracted predators. Bluefish is a fast-growing piscivore
alterations are abundant (salmonids (e.g. Machordom et al., 2000, (e.g. Juanes & Conover, 1994) that has been reported to break into sea
Bernatchez, 2001, Naylor et al., 2005), flatfish (e.g. Danancher & cages and feed on farmed fish (Seabream Sparus aurata) in the west
Garcia-Vazquez, 2011), shellfish (e.g. Gaffney, 2006), algae (e.g. Mediterranean Sea (Alicante, Spain; Sanchez-Jerez et al. 2008).
Voisin et al., 2005). Bluefish exhibit spatial population differentiation across the
Interspecific impacts of aquaculture have also been reported; Mediterranean Sea (Miralles et al., 2014; Pardiñas et al., 2010), and
these include reduction of local native populations by competition are thus a good candidate for testing the effects of attraction at long-
with exotic farmed species (Naylor et al., 2000; McGinnity et al., distance scales since individuals from different areas may be
2003), as well as increases in interspecific hybridization (Verspoor, identified by their genetic pattern. We captured Bluefish that had
1988; Youngson et al., 1993), sometimes due to alterations of broken into farm sea cages and were feeding on farmed Seabream;
behaviour in domestic variants (Hutchings & Fraser, 2009; Castillo et determined their geographical origin employing genetic markers;
al., 2010). In general, however, the study of aquaculture genetic calculated their genetic diversity and compared it with Bluefish from
impacts has focused on wild populations of the same and closely populations not associated with fish farms. Our hypothesis was that
related species that can hybridize following escapes. However, other Bluefish populations from inside the farm are a mixture of genetic
types of heterospecific impacts could also be derived from lineages coming from different geographical areas, thus altering the
aquaculture. For example, hitchhiker species transported with normal phylogeographic pattern of the species.
domestic stocks move with them around the world with their
phylogeography modified as a consequence (e.g. Voisin et al., 2005).
For accompanying species, an aspect that has rarely been considered
Materials and Methods
is the potential of aquaculture attractiveness for changing the
phylogeographic pattern of attracted species. If such attractiveness is
Sampling
strong enough to attract individuals from a long distance or change
We used spearfishing to collect 42 Bluefish inside a Spanish
migratory routes and occurs repeatedly, we migh expect that the
Seabream (Sparus aurata) and Sea Bass (Dicentrarchus labrax) marine
population structure and, at a longer term, the phylogeographic pattern
farm located in Guardamar (Alicante province, Spanish Mediterranean
of the attracted species change.
coast), 3.7 km from shore (Sanchez-Jerez et al., 2008), during 2004
*
Corresponding author: Laura Miralles Tel: +34-985102726; Fax: +34- and 2005. Reference population samples were taken from a total of
985103534; E-mail: lml.miralles@gmail.com
93
M iralles et al.
of 159 Bluefish collected from four distant areas far from aquaculture 3.0 (Excoffier et al., 2005) (10 000 permutations, 100 000 steps in
farms and from the two recently described distinct genetic units in the Markov chain).
Mediterranean Basin (Miralles et al., 2014). From east to west,
reference locations were Istanbul (Marmara Sea) and Canakkale Population assignment based on genotypes:
(Aegean Sea), both from the east Mediterranean Bluefish population, Bluefish assignation to a population of origin was carried out
at approximately 2900 and 2700 km respectively from Guardamar. employing three different methodologies to get more robust
From the west Mediterranean population, the reference sampling conclusions based on consistent results. We used GeneClass2 (Piry et
locations were Barcelona (West Mediterranean Sea) and Cadiz (Gulf al., 2004) for Bayesian assignment with a 0.05 score threshold. Two
of Cadiz, Atlantic Ocean) at 500 and 900 km respectively from methods were assayed: Rannala & Mountain (1997) and Baudouin &
Guardamar. We dissected small pieces of muscle (1 cm3) from each Lebrun (2001). Assignation results were checked by Monte-Carlo re-
individual and preserved them in absolute ethanol prior to laboratory sampling through the simulation algorithm of Paetkau et al. (2004)
analyses. with 10 000 individual simulations and type I error of 0.01. We used
DNA extraction, amplification and sequencing: ONCOR (Kalinowski et al., 2007) for maximum-likelihood
DNA was extracted following a Chelex-based protocol (Estoup assignment tests. Individuals were assigned to a population based on
et al., 1996). Seven hypervariable microsatellite loci were PCR- the probability of that population containing the individual’s
amplified following the conditions described by Dos Santos et al. genotype.
(2008). Allele sizes were determined from PCR products using an
ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) in the Unit
of Genetic Analysis of the University of Oviedo (Spain).
Microsatellite alleles were scored with the program GeneMapper®
Results
Version 4.0 (Applied Biosystems).
Bluefish collected inside the Guardamar fish farm exhibited very
The mitochondrial cytochrome oxidase subunit I (COI) gene was
high genetic diversity at both microsatellite and mitochondrial DNA,
amplified with the primers, conditions and protocol described in
higher than all the other sampling locations clustered together (Fig 1)
Miralles et al. (2014). Sequencing was performed in the genetic
and significant in a Wilcoxon Signed-Rank Test (W-value = 0 and
analyzer reported above, with a BigDye 3.1 Terminator system.
significant at p≤ 0.05). We found a total of 12 COI sequence
haplotypes (Accession numbers JQ039400-JQ039406; JQ039425-
Population genetic diversity and differentiation estimates:
JQ039429 in the GenBank; www.ncbi.nlm.nih.gov/genbank/); half of
We used MICROCHECKER 2.2.3 (Van Oosterhout et al., 2004)
them were found in specimens caught within the farm. Inside-farm
to check for null alleles, scoring errors and allele drop-out.
Bluefish had higher COI diversity (Fig 1 left) than the rest of the
Microsatellite variation (number of alleles per locus, allelic richness
Bluefish analysed in this study together and obtained from four
and observed and expected heterozygosities) was calculated with
different distant locations. Inside-farm Bluefish were also more
GENETIX Version 4.03 (Belkhir et al., 2001) and FSTAT Version
diverse at microsatellite loci than outside-farm samples (Fig 1 right),
2.9.3.2 (Goudet, 2001). Exact tests for the departure from Hardy-
exhibiting higher allelic richness, more loci with Wahlund effects
Weinberg equilibrium were performed with GENEPOP version 1.2
(significant excess of homozygotes over Hardy-Weinberg equilibrium
(Raymond & Rousset, 1995) using Bonferroni corrections.
expectations), and less private alleles. All these results suggest that the
COI sequences were edited with BioEdit Sequence Alignment
high diversity of Bluefish caught inside the farm was due to
Editor (Hall, 1999) and aligned with ClustalW (Thompson et al.,
population mixture.
1994). DNAsp v5 (Librado & Rozas, 2009) was employed to calculate
Concerning Bluefish population structure, genetic distances
the number of haplotypes (Nh), haplotype diversity (Hd) and
(pair-wise Φst values) confirmed the east and west Mediterranean
nucleotide diversity (π). Pair-wise Φst values, indicators of genetic
genetic clusters as expected (Miralles et al., 2014; Pardiñas et al.,
distance between populations, were calculated with Arlequin version
2010)..
Fig 1. Genetic diversity parameters

for Bluefish from inside Spanish
seabream farm compared with all
other Mediterranean samples:
Variation at mitochondrial DNA
(left) and microsatellite loci (right).
Nh: number of haplotypes; n: number
of individuals; Hd: Haplotype
diversity;; π: Nucleotide diversity;;
AR: Allelic Richness; Wahlund: %
loci with significant Wahlund effect
(excess of homozygotes, indicator of
population mixture). Values were
transformed mathematically for
easier visualization in the graph as:
Nh/n (x2);; π (x700);; AR and Private
alleles (/20).
94
M iralles et al.
Fig 2. Assignation scores of Bluefish from inside the aquaculture farm. Each bar represents one individual from A1 to A42. Assignation to local population (West
Mediterranean) is in blue and assignation to East Mediterranean is in orange. A: Rannala & Mountain (1997) method. B: Baudouin & Lebrun (2001) method. C: ONCOR
(Kalinowski et al., 2007) method.
Figure 1 (black and white).
The three population assignation methods showed that the expected from other observations on predators (Dempster et al., 2002;
majority of Bluefish caught inside the farm belonged to the local Valle et al., 2007).
reference population, the west Mediterranean genetic unit (Fig 2). A comparison of the three different methodologies can be done
Surprisingly, a high percentage of individuals was assigned to Turkish with the present results. ONCOR assignations are stricter than the two
populations (Fig 2 and Table 1), varying from 7.14 to 11.9% methods from GeneClass. The Rannala & Mountain (1997) method,
depending on the methodology used. The three methods identified frequently employed to assign fish to farms by other authors (Glover
assigned the same three individuals to the Turkish population (A09, et al., 2008, 2009), yielded the highest assignation values to Turkish
A21, A35), and two more were identified by at least one method with populations (Table 1 and Fig 2A). The Baudouin & Lebrun (2001)
Geneclass 2: A36 with Rannala & Mountain and A42 with Baudouin method is intermediate to the two others. The most robust analysis is
& Lebrun (2001). to combine all the methodologies to get the best resolution. From this
perspective, a minimum of 7% farm-collected individuals would be
Discussion immigrants.
Given the continuous global increase in marine aquaculture, the The results found in this study suggest that the observed
study of its effects on the wider ecosystem should be of concern. In differentiation of Bluefish between the two sides of the Mediterranean
this study, we tackle possible evolutionary changes of surrounding Sea (Miralles et al., 2014; Pardiñas et al. 2010) might be in a
accompanying species due to aquaculture, using a predator as a model regressive process if the two genetic units fuse together. Our findings
species. Although we used different assignation methodologies with highlight an unexpectedly strong impact of commercial aquaculture
different approaches, we obtained the same results: Bluefish from on the phylogeography of this wild predator species, at a considerable
inside the aquaculture farm belong to different genetic units than those geographic scale across the Mediterranean Sea.
from the two sides of the Mediterranean Sea. They likely came from The ultimate causes of farm attraction for bluefish are not well
all regions of the Mediterranean Sea, including distant Turkish known. Components from fish meal (pellets) and noise produced by
localities and the closer North-East Atlantic Ocean (Cadiz, Spain). farmed fish may attract predators (Bjørn et al., 2009; Arechavala-
Then Bluefish from inside the farm would be a mixture of immigrants. Lopez et al., 2010), but these factors have not been studied in Bluefish
Accordingly, our results suggest that the Bluefish found inside the yet. However, increasing density of farms in the western
farm have higher genetic diversity and form a mixed population Mediterranean Sea (FAO, 2012) could attract more Bluefish from
containing lineages from both founder populations. Our genetic other regions and accelerate the fusion of the two current genetic
assignation tests confirmed that these farms attract Bluefish, as units. Bluefish populations seem to be expanding northwards in the
95
M iralles et al.
Table 1: Immigrant frequencies for different assignation scores for each methodology: A summary comparing the three assignation methodologies based on four assignment
thresholds (90%, 80%, 70% or 60% probability of belonging to a putative population). GC2 – RM: Rannala & Mountain (1997) method; GC2 – BL: Baudouin & Lebrun (2001) method;
ONCOR: ONCOR (Kalinowski et al., 2007) method; GC2 + ONCOR: A combination of the three methodologies
90% 80% 70% 60%
GC2 - RM 0.07 0.10 0.10 0.10
GC2 - BL 0.05 0.05 0.07 0.10
ONCOR 0.02 0.05 0.05 0.07
GC2 + ONCOR 0.02 0.10 0.10 0.12
Bernatchez, L. (2001). The evolutionary history of brown trout (Salmo trutta L.)
inferred from phylogeographic, nested clade, and mismatch analyses of
Mediterranean Sea, which has been principally attributed to ocean mitochondrial DNA variation. Evolution 55: 351–379.
warming (Bombace, 2001, Dulcic et al., 2005, Sabates et al., 2012). Bjørn P.A., Uglem, I., Kerwath, S., Sæther, S.B., Nilsen, R. (2009). Spatiotemporal
Bluefish range expansion is coincident with a general change in distribution of Atlantic cod (Gadus morhua L.) with intact and blocked
Mediterranean fish biodiversity (Azzurro et al., 2011). Our results olfactory sense during the spawning season in a Norwegian fjord with
intensive salmon farming. Aquaculture 286, 36–44.
suggest that the increase in aquaculture production across the Black, K.D. (2000). Environmental impacts of aquaculture. Sheffield biological
Mediterranean Sea could be an additional factor explaining the sciences. Sheffield. 214pp.
general expansion of this predator. And because bluefish, when Bombace, G. (2001). Influence of climatic changes on stocks, fish species and marine
ecosystems in the Mediterranean Sea. Archivio de Oceanografia e Limnologia
attracted to fish farms attack the farmed fish, reducing aquaculture 22, 67–72.
productivity, early detection of their presence as well as monitoring Castillo, A.G.F., Beall, E., Moran, P., Martinez, J.L., Garcia-Vazquez, E. (2010).
of their movements will be useful for anticipating their potential Indirect benefits for female Salmon from mating with Brown trout. Journal of
impact. Heredity 101 (4), 461-468.
Danancher, D., Garcia-Vazquez, E. (2011). Genetic population structure in flatfishes
Finally, our study provides a new perspective on aquaculture and potential impact of aquaculture and stock enhancement on wild
effects on the surrounding ecosystem. Aquaculture facilities can populations in Europe. Reviews in Fish Biology and Fisheries 21(3), 441-462.
impact the the phylogeography of wild predators, such as Dempster, T., Sanchez-Jerez, P., Bayle-Sempere, J.T., Giménez-Casalduero, F., Valle,
C. (2002). Attraction of wild fish to sea-cage fish farms in the south-western
Pomatomus saltatrix, that are not farmed but are attracted to farms. Mediterranean Sea: spatial and short-term temporal variability. Marine
In conclusion, based on genetic assignations of Bluefish caught Ecology Progress Series 242, 237–252.
inside and outside a Spanish Seabream farm we have revealed that Diana, J.S. (2009). Aquaculture production and biodiversity conservation. BioScience
there is a mixture of East and West Mediterranean lineages inside a 59, 27-38.
Díaz-López, B., Bernal-Shirai, J.A. (2007). Bottlenose dolphin (Tursiops truncatus)
sea-farm located in Guardamar (West Mediterranean Sea). This presence and incidental capture in a marine fish farm on the north-eastern coast
mixed population exhibits a much higher genetic diversity than of Sardinia (Italy). Journal of Marine Biological Association of the United
populations not associated with farms. We suggest that marine Kingdom 87, 113–117.
Diaz-Lopez, B., Bunke, M., Bernal, J.A. (2008). Marine aquaculture off Sardinia
farms can be a cause of modification of genetic variation of Island (Italy) ecosystem effects evaluated through a trophic mass-balance
accompanying species at different levels, from local population model. Ecological Modeling 212, 292-303.
diversity and structuring to phylogeography, especially when Díaz-López, B., Marini, L., Polo, F. (2005). The impact of a fish farm on a bottlenose
attraction occurs at a long distance as it seems to happen in the dolphin population in the Mediterranean Sea. Thalassas 21, 53–58.
Dos Santos, S.M.R., Klopper, A.W., Oosthuizen, C.J., Bloomer, P. (2008). Isolation
Mediterranean. and characterization of polymorphic tetranucleotide microsatellite loci in the
pelagic perciform fish Pomatomus saltatrix (Linnaeus, 1766) from South
Africa. Molecular Ecology Resources 8, 1065–1067.
Dulčić, J., Kraljević, M., Pallaoro, A., Glamuzina, B. (2005). Unusual catch of
Acknowledgments bluefish Pomatomus saltatrix (Pomatomidae) in Tarska cove (northern
Adriatic). Cybium 29(2), 207-208.
We are very grateful to T. Ceyhan for providing the Turkish Estoup, A., Largiader, C.R., Perrot, E., Chourrout, D. (1996). Rapid one-tube DNA
extraction for reliable PCR detection of fish polymorphic markers and
samples. The study was supported by the MICINN CGL-2009- transgenes. Molecular Marine Biology and Biotechnology 5, 295–298.
08279 Grant (Spain). Laura Miralles holds a PCTI Grant from the Excoffier L., Laval, G., Schneider, S. (2005). Arlequin ver. 3.0: An integrated
Asturias Regional Government, referenced BP 10-004. software package for population genetics data analysis. Evolutionary
Bioinformatics Online 1, 47–50
FAO (2012) http://www.fao.org/fishery/culturedspecies/Sparus_aurata/es Last access:
January 2014.
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Resultados
Capítulo 4:
The deep history of Euro-African hakes.
Miralles, L. , Garcia-Vazquez, E.
Evolutionary Ecology
99
100
M iralles an d Garcia-Vazqu ez / E ast Atlan tic h ak e speciation
THE DEEP HISTORY OF EUROAFRICAN HAKES.

L. Miralles 1* , E. Garcia-Vazquez1.
* Corresponding author: Laura Miralles Tel: +34-985102726; Fax: +34 985103534; E-mail: lml.miralles@gmail.com
The genus Merluccius is a biologically and economically important group of 12 hake species that exhibit a controversial
Keywords: reticulated speciation. M. merluccius, M. senegalensis, M. polli, M. capensis and M. paradoxus comprise the ancient Euro-African
hake clade. The objective of this work was to identify the main mechanisms of speciation in this group of hakes by employing
True hakes hypervariable microsatellite loci and using multidimensional environmental analyses. Our results were consistent with previous
Merluccius studies and showed two lineages within the Euro-African clade: M. merluccius, M. capensis and M. senegalensis in one cluster
Ecological speciation (being M. merluccius and M. senegalensis sister species), and M. polli and M. paradoxus in the other. Ecological and genetic studies
Phylogeny yielded to the same cluster of species, based on a bathymetric separation. Depth has been identified as the main factor of ecological
Depth speciation. A possible parallel spatial expansion of the two lineages at different depths could be an explanation of the actual
distribution of Euro-African hakes. Partially overlapping spawning niches, plus altered contact during drastic climate events may
explain hybridization reported between these species. Overlapping spawning zones should be priority areas for monitoring in order
to preserve current biodiversity and sustainable fisheries of these valuable species.
Introduction 2014), and between silver and offshore hake in North America
How species arise is one of the most fundamental questions in (Machado-Schiaffino et al 2010). Furthermore, an ancestral hybrid
evolutionary biology (Jennings et al 2013). Marine pelagic and origin has been proposed for M. australis (Campo et al 2009),
demersal fishes, such as true-hakes Merluccius spp., were originally involving one ancestor from the Pacific and other from the Atlantic
thought to disperse widely and have little population structuring, even Ocean. The oldest true hake ancestor likely emerged in the North
over large geographic distances (Scheltema 1986; Ward et al 1994) Atlantic during the Oligocene (Kabata and Ho 1981; Inada 1981;
because marine environments are often seen as open habitats. Fedotov and Bannikov 1989) and probably split into two different
However, many studies have shown that dispersal is more constrained lineages: American in the west and Euro-African in the east (Roldan
than originally inferred (e.g. Warner and Cowen 2002; Palumbi 2004; et al 1999; Quinteiro et al 2000; Grant and Leslie 2001).
Milano et al. 2014). Some mechanisms that might limit dispersal and This study is focused on the eastern Atlantic hakes, also known
consequently promote speciation in the marine realm include as the Euro-African hakes, in order to identify the key factors that
hydrografic features (Palumbi 1994; Helberg 2009), distance promote speciation in this group of the genus Merluccius. Individuals
(isolation by distance – Bradbury and Bentzen 2007) and from the five species that inhabit European and African waters
environmental factors such as temperature (e.g. Crow et al 2007) and (Merluccius merluccius, M. polli, M. senegalensis, M. capensis and
salinity (e.g. Nielsen et al 2004; Milano et al. 2014). Environmental M. paradoxus - Figure 1) were genetically and ecologically analyzed
gradients can cause population divergence, selection and may promote to determine possible barriers between species and speciation
adaptation to those conditions. An increasing number of studies mechanisms.
suggest that ecologically-based selection among environments is one
of the key mechanisms of speciation in marine ecosystems (Keller and
Seehausen 2012; Nosil 2012; Jennings et al 2013; Prada et al 2013); it Materials and Methods
known as ecological speciation.
The genus Merluccius (true hakes) is a good model to study Genetic analyses:
marine speciation. They have a rapid radiation with a highly A total of 312 hakes were collected from 2001-2003 from three
conserved and similar external morphology (Quinteiro et al 2000). areas along Europe and Africa coasts: Merluccius merluccius (n=50)
True hakes are 12 Merluccius species distributed across the Atlantic from South Spain; M. senegalensis (n=50) and M. polli (n=62) from
and Pacific oceans. Five species inhabit European and African waters Mauritania; M. capensis (n=75) and M. paradoxus (n=75) from the
(from North to South: M. merluccius, M. senegalensis, M. polli, M. Agulhas bank in South Africa.
capensis, M. paradoxus), three along Atlantic American coasts (M. DNA was extracted with Chelex (Bio-Rad®) following Estoup
albidus, M. bilinearis, M. hubbsi), and four are distributed in the et al. (1996). Nine microsatellite loci were assayed: Mmer-Hk3,
Pacific Ocean (M. productus, M. angustimanus, M. gayi, M. australis) Mmer-Hk9, Mmer-Hk20, Mmer-Hk29, Mmer-Hk34 (Morán et al
(Alheit and Pitcher 1995). Commonly, two species overlap for a 1999), Mmer-UEAW01 (Rico et al 1997), Maus07, Maus30 and
considerable part of their distribution range (Quinteiro et al 2000). For Maus32 (Machado-Schiaffino and Garcia-Vazquez 2009). PCR
example, the two Cape hakes (the shallow-water Cape hake M. conditions were summarized in Supplementary information (Table
capensis and the deep-water Cape hake M. paradoxus) overlap off S1). Genotypes were separated using an ABI PRISM 3100 Genetic
southern Africa; the silver hake (M. bilinearis) and the offshore hake Analyzer (Applied Biosystems), in the Unit of Genetic Analysis of the
(M. albidus) off the North America; the Senegalese hake (M. University of Oviedo (Spain). Microsatellites were genotyped
senegalensis) overlap in its northern distribution with the European employing the GeneMapper® Software Version 4.0 (Applied
hake (M. Merluccius) and in its southern distribution with the Biosystems). Scoring errors, large allele dropout and null alleles were
Benguela hake (M. polli) off west Africa (Alheit and Pitcher 1995). checked with the program MICROCHECKER (Van Oosterhout et al
Introgressive hybridization was reported in some of those overlapping
regions, like between Cape hakes along African coasts (Miralles et al
101
Figure 1: Euroafrican hake distribution and morphology. Merluccius merluccius is represented in red, M. senegalensis in yellow, M. polli in green, M. capensis in dark blue and
M. paradoxus in light blue. Hake morphology draws were taken from Cohen et al. 1990
2004). Microsatellite variation (number of alleles per locus, allele - Spawning time, as spawning season duration in months:
ranges, allelic richness and observed and expected heterozygosity) data for all species were taken from Alheit and Pitcher (1995).
was calculated with GENETIX Version 4.03 (Belkhir et al 2001) and - Number of spawning peaks: data for M. merluccius, M. polli
FSTAT Version 2.9.3.2 (Goudet 2001). Allele ranges for each species and M. senegalensis were taken from Alheit and Pitcher (1995); for
at each locus were represented in plots to visualize the distribution of M. capensis and M. paradoxus from Lloris et al (2003).
alleles. Genetic divergence between populations was estimated using - Male size at maturity: data for M. merluccius, M. polli, M.
population pairwise FST obtained with Arlequin version 3.0 senegalensis and M. paradoxus were taken from Alheit and Pitcher
(Excoffier et al 2005). (1995); for M. capensis from Lloris et al (2003).
The suitability of the microsatellite panel for species - Female size at maturity: data for M. merluccius, M. polli, M.
differentiation, thus their value for use in phylogenetic analysis, was senegalensis and M. paradoxus were taken from Alheit and Pitcher
assessed using the program STRUCTURE 2.3.1 (Pritchard et al 2000). (1995); for M. capensis from Lloris et al (2003).
This software estimates, under a Bayesian framework, the minimum - Temperature: data for M. paradoxus and M. capensis were
number of population units with genetic identity in the dataset. This taken from Alheit and Pitcher (1995); for M. merluccius from Alvarez
dataset was analyzed with the “Admixture model” which assumes that et al (2004); for M. polli from Bianchi (1992); for M. senegalensis
individuals may have mixed ancestry. The parameter set consisted of from Cohen et al (1990)
a burn-in period of 50,000 steps followed by 500,000 Markov chain More variables were previously selected, but only those that
Monte Carlo (MCMC) iterations and ten runs for K = 5. Five clearly were not significantly auto-correlated (P>0.01, |r|≥0.8;; Berry and
differentiated genetic units (K) are expected, one corresponding to Feldman 1995;; Bowerman and O’Connell 1990) were considered for
each species (Miralles et al 2014). Molecular phylogeny represented the analyses. All data were normalized and transformed to Euclidean
as a Neighbour-Joining (NJ) tree was inferred from microsatellite loci distances prior to environmental multi-analyses.
genetic distances FST and reconstructed with PHYLIP v. 3.69 Non-metric multidimensional scaling (NMDS) based on
(Felsenstein 2005). Euclidean-distance was employed to display the selected variables of
each hake species in a final 2D and 3D spatial representation of
Ecological analyses: species using NMDS test implemented in PAST version 3.0 (Hammer
Ecological variables were selected based on intrinsic et al 2001) for 2D plot and PRIMER v6 (Clarke et al 2006) for 3D
reproduction characteristics for each species, that might explain plot. An analysis of similarities (One-way ANOSIM) was done with
speciation. A multivariate analysis was implemented for the following PRIMER6 (Clarke et al 2006) to test for significant differences
variables: between groups. The test statistic called R ranges from 0 to 1; large
- Depth for the maximum abundance of individuals: data for positive R (up to 1) signifies dissimilarity between previously defined
M. merluccius, M. capensis and M. paradoxus were taken from Alheit groups. Data were analyzed with 9999 permutations. A principal
and Pitcher (1995); for M. polli and M. senegalensis from Lloris et al components analysis (PCA) was carried out using PAST v.3.0
(2003). software (Hammer et al 2001) and 9999 permutations in order to find
- Spawning depth: data for M. merluccius were taken from the main hypothetical variables (or components) which account for
Alheit and Pitcher (1995); for M. capensis and M. paradoxus from variance in the multivariate dataset.
Stenevick et al (2008); for M. polli and M. senegalensis from
Fernandez-Peralta et al (2011).
102
Table 1: Microsatellite variation: NA: Number of allele; AR: Allele richness; He: Expected heterozygosity
HK3 HK9 HK20 HK29 Hk34 W01 Maus 32 Multilocus
M.merluccius
NA 11 33 18 19 18 14 5 16.857
AR 9.681 27.804 16.233 17.920 15.828 13.678 4.532 15.056
He 0.816 0.968 0.895 0.921 0.910 0.908 0.262 0.802

M.senegalensis
NA 2 40 15 7 20 5 2 12.857
AR 2.000 35.730 13.909 6.486 19.566 4.666 1.058 11.964
He 0.505 0.974 0.894 0.686 0.946 0.442 0.016 0.629
M. polli
NA 9 37 2 10 7 13 2 11.429
AR 8.22 30.040 1.581 9.475 7.000 12.169 2.000 10.069
He 0.695 0.956 0.106 0.867 0.727 0.857 0.401 0.640
M.capensis
NA 6 41 21 19 26 11 3 18.143
AR 4.249 29.020 16.201 16.285 19.283 9.487 2.766 13.898
He 0.571 0.971 0.914 0.929 0.937 0.861 0.106 0.753
M.paradoxus
NA 6 51 23 15 16 14 12 19.571
AR 4.587 34.078 16.119 12.175 16.000 11.578 9.989 14.932

He 0.316 0.977 0.892 0.887 0.935 0.889 0.753 0.800
Results concordant with previous phylogenetic studies done by Quintero et al

Genetic variation and phylogeny: (2000) and Campo et al (2007; 2009) with mitochondrial (control
The panel of nine microsatellite loci did not exhibit positive region, 12S, 16S and cytochrome b) and nuclear (5S) gene sequences.
amplification for some species at two loci: Maus07 and Maus30, and In all genetic reconstructions, two main clades were identified: clade
were removed from further analysis. The final panel of seven A with M. merluccius, M. capensis and M. senegalensis and clade B
microsatellite loci were highly variable (Table 1) and did not show with M. polli and M. paradoxus.
significant differences between observed and expected
heterozygosities. Null alleles, dropouts and linkage disequilibrium Species separation with ecological traits:
were not found (p> 0.05 in all cases). The locus HK9 was the most Data values were summarized in table 2, where we can observe
variable across species and the locus Maus32 was the least. The that the considered traits more or less overlapped between species.
species with higher number of alleles and higher allele richness were Both 2D and 3D NMDS representations separated the five Euro-
M. paradoxus and M. merluccius, respectively (Table 1). The allele African hake species into two groups (Figure 5). These two groups
range of each species at each locus was represented in plots (Figure were the same two clades separated by genetic data: M. merluccius,
2). For the microsatellite HK3, M. polli and M. paradoxus (deep M. capensis and M. senegalensis in one cluster and M. polli and M.
hakes) have the longer alleles and M. capensis and M. senegalensis paradoxus in other. Furthermore, the analysis of similarities (One-way
the smallest ones. The opposite occurred in microsatellites W01 and ANOSIM) revealed that the two groups were clearly separated (R =
HK34, with bigger alleles for M. capensis and M. senegalensis 0.9167, P=0.0963) and the two genetic clusters were highly dissimilar
(Figure 2). The species with wider allele range across loci was M. based on ecological variables. The two main variables that explain the
Merluccius (4 loci: HK3, HK29, HK34 and W01) followed by M. variance of the ecological dataset were spawning depth which
paradoxus (3loci: HK9, HK20 and Maus32). accounted a 97.697% of variance and depth for the maximum
Good discrimination power (99.7%) for the microsatellite panel abundance of individuals, with a lower contribution of 2.282% of
considered here was reflected in STRUCTURE plot (Figure 3) where variance (Table 3). Both factors accounted the 99.979 % of total
all species were well separated. This analysis also revealed that M. variance and presented the importance of depth for hake species.
Merluccius shares some membership with M. capensis (mean =
6.48%; with SD = 1.07), but interespecific introgression between the
two species can be completely discarded due to their non-overlapping
distribution, with M. merluccius in the Northern and M. capensis in Discussion
the Southern Hemisphere. Highly significant FST-values between
This ecological and genetic merge study describes for the first
species represented in the NJ tree (Figure 4), also confirmed enough
time a bathymetric gradient as an ecological speciation mechanism in
resolution for species discrimination. The microsatellite NJ tree
Euro-African hakes, coincident with phylogenetic reconstructions.
obtained from pairwise genetic distances between species were
103
Figure 2: Allele ranges of each hake species at each locus. Species are represented in colors as Figure 1 (Merluccius merluccius is represented in red, M.
senegalensis in yellow, M. polli in green, M. capensis in dark blue and M. paradoxus in light blue). Allele ranges are represented in number of base pairs (bp).
104
Table 2: Reproductive variables for ecological analyses: M.A.: Maximum abundance; S.A.M.: Size at maturity. Depth is represented in meters, time in months,
size in centimeters, temperature in Celsius degrees and genetics in phylogenetic clusters (A and B)
M. merluccius M. senegalensis M. polli M. capensis M. paradoxus
Depth (M.A.) 220 350 400 250 400

Spawning depth 150 300 550 240 850
Spawning peaks 2 1 1 2 1
Spawning time 12 7 6 12 12
S.A.M. male 31.45 28.15 27 40 42.5
S.A.M. female 37.9 31.95 32 42.5 42.5
Temperature 12 12 8.95 12 8
Genetics A A B A B
The main novelty of our results is the identification of depth as

Table 3: Principal components (PC) analysis: var.: variance; M.A.: the main variable that explains the differences of Euro-African hakes.
Maximum abundance; S.A.M.: Size at maturity. Different spawning depth could be the key mechanism for speciation
in this group. Bathymetric gradient (depth, temperature, pressure, etc.)
has been recently proposed as a speciation factor in a few other
PC Eigenvalue % variance Cumulative var. marine organisms (Ingram 2011; Jennings et al 2013; Prada et al
2013), and Euro-African hakes would join this small but growing
1- Spawning depth 80419.7 97.697 97.697 group. The total concordance of the ecological-based and genetically-
based cluster reconstructions and the fact that spawning depth is the
2- Depth (M.A.) 1878.65 2.282 99.979 main factor that explains the ecological clustering strongly supports
this idea. All previous phylogenetic works and our study of Euro-
3- S.A.M. male 16.8082 0.020 99.999 African hake phylogeny (Figure 4) separate the two deep-water hakes
(M. paradoxus and M. polli – Alheit and Pitcher 1995) in one clade,
away from the rest of true hakes. A possible parallel spatial expansion
at different depths of the two lineages of hakes could be an
Figure 3: Phylogenetic DNA reconstruction. Neighbor-joining microsatellite explanation of the actual distribution and phylogeny of Euro-African
distance tree. Bootstrap values are on the branches. Principal hakes. A bathymetric gradient also implies temperature and pressure
changes with depth. Deep-water hakes inhabit cooler waters (Figure
6). Temperatures for M. polli (7.6 -10.3ºC – Bianchi 1992) and M.
paradoxus (4-8ºC – Alheit and Pitcher 1995) are lower than for the
rest of Euro-African hakes (around 12ºC - Alheit and Pitcher 1995;
Cohen et al. 1990). Moreover, bathymetric pressure could be another
important factor in hake’s development. A previous recent study
reveals that European small hakes (M. merluccius smaller than 10cm)
have preference for depths, reaching the deeper waters at that early
life-stage (Bartolino et al 2008). More research is needed on these
different specific factors for a finer knowledge of speciation in hakes.
.
.
Figure 4: Individual species membership estimated with STRUCTURE software. Each vertical bar represents one individual. Membership to each species is
represented in percentage and in different colors. Each species is represented in colors as in Figure 1 (Merluccius merluccius is represented in red, M. senegalensis
in yellow, M. polli in green, M. capensis in dark blue and M. paradoxus in light blue)
105
Figure 5: Non-metric 3D and 2D multidimensional scaling (MDS) plots. microsatellite loci were previously employed to elucidate
Environmental multivariate ordination using Euclidean distances to display the
quantitative reproduction variables of the two genetic clusters: Clade A (M.
American hake phylogeny by Campo et al (2009). Here, we
Merluccius, M. capensis and M. paradoxus) is shown in light blue circles and Clade B employed seven microsatellite loci to represent a phylogenetic
(M. polli and M. paradoxus) in dark blue squares. reconstruction of Euro-African hakes and it is concordant with
recent previous studies based on different types of genetic markers
(Quinteiro et al 2000, 2001; Campo et al 2007; 2009).
Our results are consistent with a North Atlantic origin of the
genus Merluccius (Kabata and Ho; Inada 1981; Fedotov y
Bannikov 1989; Campo y col 2007) and suggest that M.
Merluccius could be the most ancient species within this group.
Speciation can occur by mutation and random allele drift,
accompanied by the accumulation of reproductive isolation as a by-
product (Gavrilets 2003). M. merluccius allele ranges for most loci
(HK3, HK29, HK3 and WO1) were containing the allele ranges of
other species (Figure 2). This species had also higher allele
richness. Since the simplest approach to model allopatric speciation
is to assume that populations accumulate substitutions (Gavrilets
2003), the most ancient species would have more alleles than the
youngest. From this perspective, M. merluccius could be the most
ancient species whereas M. polli would be the most differentiated
and might be the youngest species of this group because its allele
ranges are more different and it exhibits lower genetic variability.
However this is highly speculative and could be also explained
from population bottlenecks affecting this particular species,
therefore it should be taken as an exploratory hypothesis. In any
case, the genetic variability of M. polli showed that it is the most
differentiated species in the Euro-African clade.
Spawning depth was considered as the main factor that
explains the ecological and genetic clustering. The differences in
spawning depth for the Euro-African species are drawn in Figure 6.
A total separation of spawning areas in the overlapping
distributions does not occur in any case. Consequently,
interespecific hybrids are expected to occur in such areas, as
already reported for the two Cape hakes (Miralles et al 2014) as a
result of incomplete reproductive isolation. Ancient (Campo et al
2009) and recent (Machado-Schiaffino et al 2009; Miralles et al
2014) hybridization seems to be another key mechanism of
speciation in hakes. Alterations due to phenomena like El Niño and
its changes in oxygen content of ocean water displace hake to
different depths to survive (Hamukuaya et al 1998) and would
change the proportion of hybrids and introgression in those
regions. Therefore, regions with overlapping distribution of hakes
Previous genetic studies (Stepien and Rosenblatt 1996; should be priority for monitoring surveys because hybrid zones are
Roldan et al 1999; Quinteiro et al 2000; Grant and Leslie 2001; reported to be fragile and unstable (Moore 1977; Buggs 2007).
Campo et al 2007, 2009) agree with our results in the separation of African hake fisheries are sustainable (Alheit and Pitcher 1995),
two Euro-African lineages of hakes, one formed by M. paradoxus and a rapid detection and evaluation of genetic changes in those
and M. polli and another comprising M. merluccius, M. vulnerable zones will help to a sustainable management for
senegalensis and M. capensis. However, those previous studies did preserving biodiversity.
not agree in the internal clustering of the group M. capensis, M.
senegalensis, M. Merluccius. From allozyme data (Roldan et al
1999; Grant and Leslie 2001), M. senegalensis and M. capensis are Acknowledgments
sister taxa, whereas from mitochondrial (Quinteiro et al 2000,
2001; Campo et al 2007) and other nuclear genes (Campo et al This work was supported by the Spanish project MICINN
2009), M. merluccius and M. senegalensis are sister species. We CGL2009-08279. Hake samples were kindly provided by Dr.
support the latter hypothesis by employing hipervariable Robin Tilney (Department of Environmental Affairs, Cape Town,
microsatellite loci for phylogenetic reconstruction. Microsatellite South Africa) and the EU Project MARINEGGS QLK5-CT1999-
loci are highly polymorphic genetic markers that are able to detect 01157. L.M. holds a FICYT-PCTI Severo Ochoa Grant from
weak population differentiation even in species with strong gene Regional Government of Asturias, referenced BP 10-004. This is a
flow (Waples 1998); therefore they are highly informative. Five contribution from the Asturias Marine Observatory.
106
Figure 6: Bathymetric distribution of Euroafrican hakes and its correlation with temperature. Depth distribution for each species is represented in colors as
in Figure 1 (Merluccius Merluccius is represented in red, M. senegalensis in yellow, M. polli in green, M. capensis in dark blue and M. paradoxus in light blue).
The total distribution range of each hake species is shown in color lines. The most abundant distribution is reproduced in full-color. The spawning depths of each
species are represented in black squares with grey color inside; overlapping spawning depths are in black. Below, a bathymetric temperature profile from ODW. In
both graphs, depth is presented in meters, North latitude is on the left side and South latitude on the right.
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108
Table S1: PCR microsatellite conditions for each Merluccius species.

For each loci the temperature is represented in Celsius degrees (°C) in the upper part of the row and below Magnesium
concentration [] in mM.
Locus M.mer. M.cap. M.pax. M.polli M.sen. Reference
52°C 54°C 54°C 52°C 52°C Morán y col. 1999

HK3
[1] [1.5] [1.5] [2] [2]
53°C 54°C 54°C 58°C 58°C Morán y col. 1999

HK9
[1] [1.5] [1.5] [2.5] [2.5]
50°C 52°C 52°C 52°C 52°C Morán y col. 1999

HK20
[1] [1.5] [1.5] [1.5] [2]
52°C 52°C 52°C 54°C 54°C Morán y col. 1999

HK29
[1.5] [2] [2] [2.5] [2]
52°C 52°C 52°C 54°C 54°C Morán y col. 1999

HK34
[1.5] [2.5] [2] [2.5] [2]
60°C 60°C 60°C 58°C 58°C Rico y col. 1997

WO1
[1] [1.5] [1.5] [2] [2]
56°C 61°C 61°C Machado-Schiaffino y Garcia-

Maus7 - - Vazquez, 2009
[1] [2.5] [2.5]
60°C 60°C 63°C 60°C 60°C Machado-Schiaffino y Garcia-

Maus32 Vazquez, 2009
[1.5] [2.5] [2] [2.5] [2.5]
60°C 54.5 60°C Machado-Schiaffino y Garcia-

Maus30 - - Vazquez, 2009
[1.5] [2] [2.5]
109
110
Resultados
Capítulo 5:
Genetic markers reveal a gradient of hybridization
between Cape hakes (Merluccius capensis and
Merluccius paradoxus) in their sympatric geographic
distribution.
Miralles, L. , Machado-Schiaffino, G., Garcia-Vazquez, E.
111
112
Author's personal copy
Journal of Sea Research 86 (2014) 69–75
Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/seares
Genetic markers reveal a gradient of hybridization between cape hakes

(Merluccius capensis and Merluccius paradoxus) in their sympatric
geographic distribution
Laura Miralles a, Gonzalo Machado-Schiaffino b, Eva Garcia-Vazquez a,⁎
a
Department of Functional Biology, University of Oviedo, C/Julian Claveria s/n., 33006 Oviedo, Spain
b
Department of Biology, University of Konstanz, Universitätsstrasse 10, 78457, Germany
a r t i c l e i n f o a b s t r a c t
Article history: The cape hakes Merluccius capensis and Merluccius paradoxus are important fishing resources for African
Received 9 January 2013 countries such as Namibia and South Africa. In this study we have genetically analyzed adult samples
Received in revised form 24 September 2013 from the overlapping distribution of these species. Eight microsatellite loci, the nuclear 5S rDNA locus
Accepted 14 November 2013
and the Cytochrome Oxidase subunit I (COI) gene were employed as molecular markers. A North–South
Available online 26 November 2013
gradient of interspecific hybridization was found, with discordant mitochondrial and nuclear genotypes
Keywords:
at the northernmost edge of M. paradoxus distribution. These results suggest intense introgression in
Cape hakes North Benguela off the Namibian coast. Independent hake stock assessment is recommended in this region
Hybrid zones for sustainable management of this valuable resource.
Benguela © 2013 Elsevier B.V. All rights reserved.
Merluccius capensis
Merluccius paradoxus
1. Introduction another possible outcome is hybrids becoming a new lineage (see

Seehausen, 2004).
Hybrid zones may occur when two formerly separated species Hybridization is not expected to occur with the same frequency in
meet again (Avise and Wollenberg, 1997; Hewitt, 2001). They often all the areas where two species are sympatric. Hybrids are more fre-
arise at biogeographic borders and may occur for different taxa in quent in marginal populations, where mate choice may be relaxed
what it is called a suture zone (Hewitt, 2000). In the marine realm (e.g. Ritchie, 2007), and in the colonization front when one of the
hybrids between different animal species are relatively frequent species is displacing or expanding its distribution (e.g. Carson and
(e.g. Gardner, 1997; Miralles et al., 2013; Palumbi, 1994; Srinivasa Templeton, 1984; Horreo et al., 2011). It also happens where the
Rao and Lakshmi, 1999) because, among other reasons, many species two sympatric species are unequally abundant (e.g. Arnold, 1997;
have mass spawning and/or interspecific reproductive barriers may Hobbs et al., 2009). In these cases asymmetric hybridization would
be weak. However, marine hybrid zones have been considered rare, be expected, the rarer species providing frequently the female in hy-
perhaps because they have not been sufficiently studied (Arnold, brid crosses (e.g. Wirtz, 1999).
1997; Gardner, 1997). They have been reported for a few species, such Identification of hybrid zones is especially important for species
as mussels of the genus Mytilus (e.g. Bierne et al., 2003, Riginos and subjected to exploitation because they may require a distinct man-
Cunningham, 2005), redfish of the genus Sebastes (e.g. Roques et al., agement. Allendorf et al. (2001) have classified hybrid zones in six
2001), hakes of the genus Merluccius (Machado-Schiaffino et al., different types based on their origin (natural versus anthropogenic)
2010) and some coral reef fishes (Hobbs et al., 2009). A variety of genet- and on the extent of introgression, with differential management
ic consequences can result from hybridization (Seehausen, 2004, 2006). and conservation priorities proposed for each of them. Cape hakes
In cases of hybridization but no introgression, no genetic consequences (Merluccius capensis and Merluccius paradoxus) are two of the most
are expected (this would be an evolutionary dead end). When there is economically and ecologically important African fishing resources
introgression through unidirectional gene flow, one species will lose (Alheit and Pitcher, 1995; Boyer and Hampton, 2001), and have
its genetic identity. Introgression through bi-directional gene flow will been subjected to sustainable management initiatives for the last de-
potentially result in reverse speciation (Seehausen, 2006). Finally, cades (e.g. Butterworth and Rademeyer, 2005; Hutchings et al.,
2009a). They overlap in the large part of their distributions, along
⁎ Corresponding author at: Department of Functional Biology, University of Oviedo,
the coastlines of Namibia and South Africa (Fig. 1), but they inhabit at
C/Julian Claveria s/n., 33006 Oviedo, Spain. Tel.: +34 985102726; fax: +34 985103534. different depths. M. capensis is known as the shallow cape hake while
E-mail address: egv@uniovi.es (E. Garcia-Vazquez). M. paradoxus is called the deep cape hake (Alheit and Pitcher, 1995).
1385-1101/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seares.2013.11.009
113
70 L. Miralles et al. / Journal of Sea Research 86 (2014) 69–75
locus and the Cytochrome Oxidase subunit I (COI) gene for genetic
estimation of their hybrid status.
2. Materials and methods
2.1. Sampling
A total of 296 cape hakes, M. capensis and M. paradoxus, were

collected during 2002–2003 from three different areas in the over-
lapping zone of both species in the south Atlantic Ocean (Fig. 1):
two within the Benguela current (North and South, 11–14°E 22–26°S
and 15–18°E 30–33°S, respectively) and one within the Agulhas current
(20–24°E 34–36°S). They were taxonomically identified de visu by local
experts. Tissue samples (muscle or fin biopsy of approx. 1 mm3) were
obtained from each individual and stored in absolute ethanol until
analysis.
2.2. Genetic analysis
Eight microsatellite loci were assayed: Mmer-Hk3, Mmer-Hk9,

Mmer-Hk20, Mmer-Hk29, Mmer-Hk34 (Morán et al., 1999), Mmer-
UEAW01 (Rico et al., 1997), Maus7 and Maus32 (Machado-Schiaffino
and Garcia-Vazquez, 2009). PCR conditions and protocols were slightly
modified from Machado-Schiaffino et al. (2010) for optimizing amplifi-
cation in M. capensis and M. paradoxus (Table 1). PCR products were
separated using an ABI PRISM 3100 Genetic Analyzer (Applied
Biosystems), with BigDye 3.1 Terminator system, in the Unit of
Genetic Analysis of the University of Oviedo (Spain). Genotypes were
determined employing the GeneMapper® Software Version 4.0.
The nuclear 5S rDNA coding gene was genotyped as described by
Perez and Garcia-Vazquez (2004). M. capensis yields only one fragment
of 371 nucleotides and M. paradoxus provides two fragments of 371 and
Fig. 1. Merluccius capensis and Merluccius paradoxus distribution range (above). Sampling
494 nucleotides. Fragment sizes were determined in 2% agarose gels by
areas in Agulhas, South Benguela and North Benguela are marked in dark in the enlarged
section (below). comparison with a DNA mass ladder.
The mitochondrial COI gene was amplified employing the primers
COIFish-F1 and COIFish-R1 (Ward et al., 2005). PCR reactions were
Cape hakes' population structure has been described by von der Heyden carried out accordingly with the protocols described by Ward et al.
et al. (2007b): there are no barriers to dispersal between Namibian and (2005). PCR products were visualized, purified and sequenced as de-
South African waters for M. capensis while for M. paradoxus there are scribed in Machado-Schiaffino et al. (2010). PCR products were visual-
significant spatial population genetic differences. Spawning of the two ized in 50 ml 2% agarose gels 3 μl of ethidium bromide (10 mg/ml).
cape hakes overlaps temporally. In South African waters, spawning oc- Stained bands were excised from the gel and DNA was purified with a
curs from August to March with two apparent peaks, the first at the end Wizard SV Gel and PCR Clean up system (Promega) prior to sequencing.
of the year for both species and the second in the austral autumn mainly Automated fluorescence sequencing was performed on an ABI PRISM
for M. paradoxus (Assorov and Berembeim, 1983; Botha, 1986). In 3100 Genetic Analyzer (Applied Biosystems) in the Unit of Genetic
Namibian waters, M. capensis spawns throughout the year, more in- Analysis of the University of Oviedo (Spain).
tensely between July and October, while by now there is no evidence
of M. paradoxus spawning there (Alheit and Pitcher, 1995; Assorov 2.3. Data analysis
and Berembeim, 1983; Kainge et al., 2007). Although little is known
about the spawning behavior of these two species, reproductive bar- Microsatellite scoring errors, large allele dropout and null alleles
riers between them seem to exist, at least partially, due to depth. Von were checked employing MICROCHECKER (van Oosterhout et al.,
der Heyden et al. (2007a) and Stenevik et al. (2008) found eggs of 2004). GENEPOP (Raymond and Rousset, 1995) was employed to test
M. paradoxus distributed in deeper waters than M. capensis eggs for linkage disequilibrium and departure from Hardy–Weinberg equi-
(with an average depth of 231 m and 348 m for M. capensis and librium. Microsatellite variation parameters such as expected and ob-
M. paradoxus respectively). However, displacement of cape hakes served heterozygosity were calculated with GENETIX Version 4.03
has been reported in response to change in the oxygen content of (Belkhir et al., 2004). FSTAT Version 2.9.3.2 (Goudet, 2001) was used
bottom waters, M. capensis entering in contact with M. paradoxus to calculate microsatellite allelic richness. To identify individuals from
(Hamukuaya et al., 1998). Since hybrid zones have been reported each pure species, hybrids of first generation and backcrosses we
for other overlapping species of this genus (the North American hakes employed NewHybrids (Anderson and Thompson, 2002), with settings
Merluccius albidus and Merluccius bilinearis; Machado-Schiaffino et al., of 300 000 Monte Carlo Markov Chain (MCMC) iterations after a burn-
2010), it is theoretically possible that the same phenomenon occurs in period of 30 000 iterations. The Bayesian software STRUCTURE v.2.3.1
also for cape hakes. (Pritchard et al., 2000) was used to estimate the membership of each in-
The objective of this study was to examine the extent and direction dividual to each species with the “Admixture model” and K = 2 (two
of possible introgressive hybridization and to identify potential hybrid expected genetic units, one corresponding to each species), which as-
zones in cape hakes. For this purpose, adults of both species were sumes that individuals may have mixed ancestry. Settings were a
sampled from different areas across the overlapping distribution burn-in period of 100 000 steps followed by 1 000 000 MCMC iterations.
and genotyped for eight microsatellite loci, the nuclear 5S rDNA Since there is no clear consensus about the proportion of membership
114
L. Miralles et al. / Journal of Sea Research 86 (2014) 69–75 71
Table 1
Description of the eight microsatellite loci assayed for Merluccius capensis (C) and Merluccius paradoxus (P) and PCR conditions. Ta: annealing temperature.
Primer name Reference Repeat motif GenBank accession no. Ta (°C) Mg 2+ (mM)
Mmer-HK3 (Morán et al., 1999) GT AF136627 54 1.5

Mmer-HK9 (Morán et al., 1999) GA AF136628 54 1.5
Mmer-HK20 (Morán et al., 1999) GT AF137595 52 1.5
Maus7 (Machado-Schiaffino and Garcia-Vazquez, 2009) (GT)5AA(GT)3AA EU703880 C: 61 2.5
(GT)7TA(GT)10 P: 60
Maus32 (Machado-Schiaffino and Garcia-Vazquez, 2009) (CA)9(TG)2 EU703883 C: 60 C: 2.5
P: 63 P: 2
UEAW01 (Rico et al., 1997) (CA)2 (CA)11 X87461 60 1.5
Mmer-HK29 (Morán et al., 1999) GT AF137597 52 2
Mmer-HK34 (Morán et al., 1999) GT AF137596 52 C: 2.5
P: 2
considered as a signal of introgression (Allendorf et al., 2001), for top), indicating some degree of introgression. In South Benguela
conservative interpretation we have considered N 25% the threshold (Fig. 3 bottom left), 13 M. capensis individuals (26.5% of analyzed
for significant membership of a species as in Machado-Schiaffino samples) exhibited mixed membership (introgression), whereas
et al. (2010). We have run STRUCTURE five times with K = 2. We M. paradoxus specimens had no introgression. Finally, numerous in-
have also followed the methodology described by Schwartz and dividuals of both M. capensis and M. paradoxus sampled from North
Beheregaray (2008) with two runs, using the species defined in the Benguela had mixed membership (Fig. 3 bottom right): 36% and
first run as a prior for the second run. This is a test for each individual 20% respectively. All the individuals identified as F1 and backcrosses
having an ancestor of the other species in the last two generations by NewHybrids exhibited mixed membership with STRUCTURE. All
(Pritchard et al., 2000). the samples yielded 5S rDNA amplification patterns concordant
COI sequences were edited using the BioEdit Sequence Alignment with their assignation to a species (this marker cannot distinguish
Editor software (Hall, 1999). The edited sequences were compared between M. paradoxus and hybrids).
with standard sequences of each species with the online software The 622 nucleotide-long COI gene sequence obtained in this study
NCBI-BLAST (Altschul et al., 1990). was polymorphic and differed between species, as expected. The differ-
Species divergence time was estimated from COI sequences under a ent haplotypes from the analyzed samples are available at GenBank
Bayesian MCMC framework using BEAST version 1.6.1 (Drummond and (http://www.ncbi.nlm.nih.gov/genbank/) with the accession numbers
Rambaut, 2007). Bayesian intraspecific phylogenies are based on coales- JF268612-JF268620. Comparing the sequences obtained with reference
cent theory (Kingman, 1982) and allow the inference of past population sequences from GenBank (JF493884.1 and JF493889.1 for M. capensis
dynamics and parameters from contemporary gene sequences. Fol- and M. paradoxus respectively), all the individuals were identified de
lowing a burn-in of 10 million cycles, rates were sampled every 1 visu and by microsatellite genotypes as M. capensis exhibited typical
000 cycles from 60 million MCMC steps for an Extended Bayesian M. capensis COI genes. It is an indicator that hybrids and introgressed
Skyline tree with a stepwise model for mitochondrial DNA and strict M. capensis had been produced from crosses between M. capensis
clock model. The substitution model of COI sequences and their females and M. paradoxus males. On the other hand, all the 30
priors (previously known information) were defined by jModeltest M. paradoxus individuals sampled from North Benguela and one from
software version 0.11 (Posada, 2008) using the Akaike information South Benguela exhibited typical M. capensis COI sequences. Therefore,
criterion (AIC; Akaike, 1974). The COI gene mutation rate employed these individuals were nuclear–mitochondrial discrepant. It can be ex-
was 1.2% per MY (Bermingham et al., 1997). Three runs were performed plained by recurrent backcrosses of descendants of M. capensis ×
to ensure that results do not reflect spurious probabilities. Tracer ver- M. paradoxus hybrid females with M. paradoxus (Fig. 4).
sion 1.5 (Rambaut and Drummond, 2007) was used to check that chains Concerning the level of mitochondrial variation in the studied
converged to a stationary distribution and to visualize the results areas, the Agulhas samples exhibited less haplotypes and lower di-
obtained. versities (Hd and π) than the Benguela samples (Table 2). According
to the results explained above, FST values between pairs of samples
3. Results were discordant for microsatellites and mitochondrial DNA mainly
due to the North Benguela sample of M. paradoxus (Table 3). North
Microsatellite loci were employed to assign individuals to a species Benguela M. paradoxus was not different from any M. capensis sample
and to identify first-generation hybrids and brackcrosses with the pro- for mitochondrial DNA (Table 3, below diagonal) but was significantly
grams NewHybrids and STRUCTURE. Two microsatellites (Mmer-Hk29 different from the other M. paradoxus. For microsatellites (Table 3,
and Mmer-Hk34; Morán et al., 1999) exhibited null alleles and were above diagonal), North Benguela M. paradoxus were significantly dif-
not used for the study of interspecific introgression. Null alleles and ferent from two M. capensis samples (not from South Benguela
dropouts were not found for other microsatellites. The six loci retained M. capensis) and also from Agulhas M. paradoxus samples, but not
for the study were highly variable (Table 2) and did not show significant from South Benguela M. paradoxus.
differences between observed and expected heterozygosity, neither From the COI sequences found in this study, the estimated time
linkage disequilibrium for any sample (p N 0.05 in all cases). With for the most recent common ancestor (tMRCA) of South African
NewHybrids software, hybrids of M. capensis and M. paradoxus cape hakes samples was 3.4 Ma ago (MYA) with a standard deviation
were not identified in the Agulhas Bank sampling area (Fig. 2 bottom). of 3.63 × 10− 3 MYA and 95% HPD of 2.437–4.471 MYA.
However, 4% hybrids were found in South Benguela (Fig. 2 middle).
Greater hybridization was found in North Benguela sample (Fig. 2 4. Discussion
top), with 5% hybrids issued from M. capensis females and 8.5% individ-
uals backcrossed to M. paradoxus. This North–South gradient of inter- The results of this study revealed hybridization and introgression
specific hybridization was confirmed with STRUCTURE software. In between the two cape hake species, with a clear North–South gradient.
the Agulhas Bank area, one M. paradoxus individual (1.3% of analyzed Higher proportion of introgressed individuals was found in the north
samples) exhibited 27% individual membership of M. capensis (Fig. 3 (North Benguela exhibited the highest). It is geographically associated
115
Table 2
Diversity indices for microsatellite loci and mitochondrial DNA of the Merluccius capensis and Merluccius paradoxus samples analyzed.
M. capensis M. paradoxus
Agulhas S.B. N.B. Agulhas S.B. N. B.

(n = 75) (n = 49) (n = 50) (n = 75) (n = 17) (n = 30)
Microsatellite loci
A. R. 11.653 11.097 10.592 11.682 13.537 11.407
N. A. 19.833 16.500 15.667 21.167 13.667 14.833
He 0.730 0.717 0.718 0.779 0.800 0.683
Ho 0.675 0.543 0.626 0.742 0.790 0.560
Mitochondrial DNA
Hd 0.378 0.800 0.667 0.464 0.378 0.833
(π) 0.00096 0.00268 0.00161 0.00161 0.00151 0.00214
Nh 3 4 5 3 3 6
S.B., South Benguela; N.B., North Benguela; n, number of individuals; A.R., Allelic Richness; N.A., Average Number of Alleles per locus and population; He, Expected heterozygosity; Ho,
Observed heterozygosity; Hd, Haplotype diversity; (π), Nucleotide diversity; Nh, Number of haplotypes.
with the border of M. paradoxus distribution (Fig. 1). Moreover, (Hutchings et al., 2009b). Also it is intensely affected by the Benguela
M. paradoxus seems to have captured the M. capensis mitochondrial ge- Niño and anoxic periods (Boyer and Hampton, 2001; Monteiro et al.,
nome in that region, as described in a few cases for other fish like Arctic 2008; Rouault et al., 2007). From the distribution of their eggs in the
char (Bernatchez et al., 1995) and also for North American hakes water column, it seems that M. paradoxus spawn in deeper waters
(Machado-Schiaffino et al., 2010). Although discordant mitochondrial than M. capensis (Stenevik et al., 2008; Von der Heyden et al., 2007a).
and microsatellite population patterns can be explained based on differ- It is possible that adverse environmental conditions (in the bottom
ent potential for natural selection, lack of mutation-drift equilibrium and/or in the surface) force repeatedly spawning overlaps of these spe-
and/or sex-biased dispersal (DiBattista et al., 2012), the present case cies in North Benguela, thus allowing interspecific mattings.
of species status discordance between markers could be due to repeated On the other hand, adverse environmental factors may not be neces-
generations of backcrosses of hybrids M. capensis × M. paradoxus to sary for explaining these results. Hybridization occurs naturally
M. paradoxus, leading to a molecular leakage classified as Type 2 hybrid- between other hake species (e.g. Machado-Schiaffino et al., 2010) and
ization or natural introgression by Allendorf et al. (2001). North Ben- it could be an evolutionary mechanism in the genus Merluccius
guela (Namibian waters) could therefore be considered a hybrid zone (Campo et al., 2009). Hybridization is most common and successful in
for these species. recently diverged species (Mallet, 2005), as it is the case of these
From the technical point of view, this study may encompass some hakes (e.g. Campo et al., 2009; Roldán et al., 1999). Grant and Leslie
ascertainment bias because the microsatellites employed were de- (2001) suggested that most hake species diverged around 2–3 MYA
veloped for other Merluccius species (Merluccius merluccius and and Quinteiro et al. (2000) estimated the divergence time of M. capensis
Merluccius australis). Ascertainment bias can complicate cross- and M. paradoxus between 3.8 and 4.5 MYA. Our estimation of species
species comparisons of genetic diversity (e.g. Annos et al., 2003) be- divergence sets the time for the most recent common ancestor in
cause the species from which DNA was used for microsatellite primer 3.4 MYA, very similar to previous estimates (Becker et al., 1988;
development often shows higher genetic diversity than other species Quinteiro et al., 2000). The gradient of introgression, more intense at
for which the same primers are used. However in this study genetic the edge of M. paradoxus distribution, could be explained as a conse-
diversity was similar in the two species (Table 2), therefore ascer- quence of relaxed sexual selection that could be expected in marginal
tainment bias, if occurring, affected likely similarly the two species populations (e.g. Ritchie, 2007), and/or as a strategy in the colonization
here compared. front (e.g. Carson and Templeton, 1984; Horreo et al., 2011). If
Different factors can be invoked for explaining the hybridization M. paradoxus expanded northwards from South Africa as it could be de-
found in North Benguela. On one hand, environmental alterations duced from the phylogeny of the genus (Campo et al., 2007, 2009;
(natural and/or anthropogenic) promote the breakdown of interspe- Roldán et al., 1999), the phenomenon of hybridization would be essen-
cific barriers (e.g. Gilman and Behm, 2011; Crego-Prieto et al., 2012; tially natural in this case (Type 2 from Allendorf et al., 2001). These in-
and references therein). The North Benguela region is subjected to teresting evolutionary hypotheses deserve further investigations.
stressful processes such as overfishing and the Benguela regime shift Comparing this pair of species with other sympatric Merluccius and
Fig. 2. Hybridization detected with NewHybrids software. Percentage of each species (Merluccius capensis in dark blue and Merluccius paradoxus in light blue), hybrids (intermediate blue)
and backcrosses to M. paradoxus (white) per location.
116
Fig. 3. Individual membership of Cape hake samples from the considered regions, estimated with STRUCTURE software. Membership to each species is represented as Merluccius capensis
in dark blue and Merluccius paradoxus in light blue. Each vertical bar represents one individual.
combining genetic data with life history trait patterns could enlighten Past M. capensis overfishing in Namibian waters (Isarev, 1983, 1988)
the mechanisms involved in speciation at sea, that are still largely un- combined with more general climate factors affecting this species (e.g.
known (Norris and Hull, 2012). Rikhter and Golubiatnikova, 1997), could have led to hybridization
An alternative explanation could be scarcity of one of the two species due to reduced abundance of this species. This hypothesis would also
(e.g. Arnold, 1997; Frisch and van Herwerden, 2006; Hobbs et al., 2009). explain the asymmetric hybridization found in our results, issued from
Fig. 4. Example of a possible scenario to explain the nuclear–mitochondrial discordance found in this study. First-generation hybrids (F1) have 50% nuclear genes of each parental species.
The nuclear genome of the non-recurrent parental species will be diluted in successive backcrosses. However, Merluccius capensis mitochondrial DNA (marked as ♀), of maternal origin,
will remain the same across generations.
117
Table 3
Genetic differentiation between populations. Pairwise FST estimates between hake samples based on microsatellite loci (above diagonal) and mtDNA (below diagonal). Significant values
are in bold. S.B., South Benguela; N.B., North Benguela.
M. capensis M. paradoxus
Agulhas S.B. N.B. Agulhas S.B. N.B.
M. capensis Agulhas – 0.0034 0.0030 0.0612 0.0422 0.0176

S.B. 0.0372 – 0.0016 0.0524 0.0329 −0.0002
N.B. −0.0256 −0.0505 – 0.0505 0.0295 0.0162
M. paradoxus Agulhas 0.9834 0.9728 0.9788 – 0.0131 0.0241
S.B. 0.8789 0.8432 0.87411 −0.0199 – 0.0000
N.B. 0.0045 0.0068 0.0017 0.9748 0.8652 –
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120
Resultados
Capítulo 6:
Interspecific Introgression in Cetaceans: DNA
Markers Reveal Post-F1 Status of a Pilot Whale.
Miralles, L. , Lens, S., Rodrı´guez-Folgar, A., Carrillo, M., Martıń, V., Mikkelsen, B.,
Garcia-Vazquez, E.
Plos One
121
122
Interspecific Introgression in Cetaceans: DNA Markers
Reveal Post-F1 Status of a Pilot Whale
Laura Miralles1*, Santiago Lens2, Antonio Rodrı́guez-Folgar3, Manuel Carrillo4, Vidal Martı́n5,
Bjarni Mikkelsen6, Eva Garcia-Vazquez1
1 Department of Functional Biology, University of Oviedo, Oviedo, Asturias, Spain, 2 Instituto Español de Oceanografı́a, Vigo, Galicia, Spain, 3 G.R.E.M.MAR Dolphin Rescue
and Research Group of Marine Mammals, Cámpelo Parroquia de San Juan de Poio, Galicia, Spain, 4 Canarias Conservación Cetacean Research Society, La Laguna, Canary
Islands, Spain, 5 Sociedad para el Estudio de los Cetáceos en el Archipiélago Canario (SECAC), Yaiza, Canary Islands, Spain, 6 Faroese Museum of Natural History, Tórshavn,
Faroe Islands
Abstract
Visual species identification of cetacean strandings is difficult, especially when dead specimens are degraded and/or species
are morphologically similar. The two recognised pilot whale species (Globicephala melas and Globicephala macrorhynchus)
are sympatric in the North Atlantic Ocean. These species are very similar in external appearance and their morphometric
characteristics partially overlap; thus visual identification is not always reliable. Genetic species identification ensures correct
identification of specimens. Here we have employed one mitochondrial (D-Loop region) and eight nuclear loci
(microsatellites) as genetic markers to identify six stranded pilot whales found in Galicia (Northwest Spain), one of them of
ambiguous phenotype. DNA analyses yielded positive amplification of all loci and enabled species identification. Nuclear
microsatellite DNA genotypes revealed mixed ancestry for one individual, identified as a post-F1 interspecific hybrid
employing two different Bayesian methods. From the mitochondrial sequence the maternal species was Globicephala melas.
This is the first hybrid documented between Globicephala melas and G. macrorhynchus, and the first post-F1 hybrid
genetically identified between cetaceans, revealing interspecific genetic introgression in marine mammals. We propose to
add nuclear loci to genetic databases for cetacean species identification in order to detect hybrid individuals.
Citation: Miralles L, Lens S, Rodrı́guez-Folgar A, Carrillo M, Martı́n V, et al. (2013) Interspecific Introgression in Cetaceans: DNA Markers Reveal Post-F1 Status of a
Pilot Whale. PLoS ONE 8(8): e69511. doi:10.1371/journal.pone.0069511
Editor: Tom Gilbert, Natural History Museum of Denmark, Denmark
Received April 19, 2013; Accepted June 11, 2013; Published August 19, 2013
Copyright: ! 2013 Miralles et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Laura Miralles holds a PCTI Grant from the Asturias Regional Government, referenced BP 10-004. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: lml.miralles@gmail.com
Introduction Genetic species identification in cetaceans is generally based on

maternally inherited mitochondrial DNA (mtDNA). A cetacean
In a progressively threatened oceanic environment where large sequence database, DNA Surveillance [8], has been created to
species are more and more endangered, cetacean monitoring is help in this purpose. It contains reference sequences for all known
increasingly important for estimating population censuses and cetacean species. In addition, other databases like for example
early detecting signals of species depletion [1]. However, visual GenBank [9] have also reference sequences for cetaceans –and
species identification is not always accurate. Some species are many other organisms. Although mtDNA is more frequently used
morphologically similar and their distribution ranges overlap. A for cetacean identification [7], [8] nuclear markers may be also
correct taxonomic identification is indeed important for practical needed for this purpose. Some species naturally hybridize e.g. [10],
issues of management and conservation of cetacean species. [11], [12]. Hybrids may exhibit morphologically ambiguous
The two recognised pilot whale species, Globicephala melas and G. phenotypes e.g. [13] and therefore nuclear (biparentally inherited)
macrorhynchus, are cetaceans of charismatic behaviour. They are genetic markers are needed for accurate identification of pure
highly social and exhibit post-reproductive female care. They are species and their hybrids [14]. Nuclear markers are also
sympatric across a North Atlantic latitudinal area from American recommended in cases of PCR contamination [14], e.g. bacterial
to European coasts [2], [3]. Based on their osteology, Van Bree [4] contamination of cetacean tissues. In this study, we have used
demonstrated that they are two clearly distinct species; however, mitochondrial and nuclear genetic markers to determine the
their external appearance is similar and the morphometric species of stranded individuals and to investigate the possible
characteristics employed to visual species discrimination partially existence of some genetic mixture between the two pilot whale
overlap [5]. Therefore species identification based on external species found in waters off northern Spain.
morphology may be difficult and in some cases impossible [6].
Moreover, dead stranded individuals are sometimes highly Materials and Methods
degraded and their distinctive traits may be lost. For these
reasons, as in other forensic zoological studies, DNA-based Six stranded pilot whales (Table 1), 19 reference Globicephala
identification is necessary [7], [8]. macrorhynchus from Canary Islands and 20 reference Globicephala
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1 August 2013 | Volume 8 | Issue 8 | e69511
First Adult Post-F1 Hybrid in Cetaceans
melas from Faroe Islands (donated by the Faroese Museum of

Natural History) were analysed (Figure 1). All animal samples were
obtained from dead specimens: dead strandings and museum
collection. We obtained the CITES permit (ESBI00001/12I) and
all the permissions from the Faroese Museum of Natural History
to analyse the Faroese samples. No one animal suffered nor was
injured or killed for this study. The protocol employed was
approved by the Committee on the Ethics of Animal Experiments
of the University of Oviedo.
DNA was extracted with a Chelex-based protocol [15]. The
mitochondrial control region (D-loop) was amplified following
Oremus et al. [16]. Sequences were edited with BioEdit Sequence
Alignment Editor [17]. NCBI-BLAST [18] and DNA Surveillance
[8] online software were employed for species identification. The
number of haplotypes, haplotypic and nucleotidic diversities were
calculated with DNAsp v5 [19]. A Neighbour-Joining tree with
1 000 bootstrap re-sampling was reconstructed from sequences
with PHYLIP v.3.69 [20].
Eight microsatellite loci (EV37MN; EV94MN; 199/200; 415/
416, 417/418, 409/470; 468/469 and 464/465) were amplified as
in Fullard et al. [21] and genotyped employing GeneMapperH
Software. A multi-tube method [22] was employed to validate the
allele scores. Each microsatellite locus was individually amplified
four times in three different thermal cycler machines (Applied
Biosystems 2720 Thermal Cycler). Scoring errors, large allele
dropout and null alleles were checked with MICROCHECKER Figure 1. Pilot whale NE Atlantic distribution and sampling
[23]. Linkage disequilibrium tests were performed with GENE- areas. Faroe Islands (n = 20), Galicia region (n = 6) and Canary Islands
POP version 4.2 [24]. Variation parameters (number of alleles; (n = 19).
doi:10.1371/journal.pone.0069511.g001
allele richness; minimum, maximum and mean allele length;
expected and observed heterozygosities; FIS) and distances 100 000 MCMC iterations) that identifies first and second
between genotypes for populations (Nei distances) and individuals generations hybrids and backcrosses.
(Fuzzy set similarity distances) were calculated with Microsatellite
Analyser MSA 4.05 [25]. FST distances were calculated with Results and Discussion
Arlequin v.3.5.1.3 [26] with 1 023 permutations and 0.05
significance level. Neighbour-Joining trees based on genetic The markers employed exhibited sufficient variation for
distances and bootstrap (1 000 bootstrapping) were reconstructed discriminating between the two pilot whale species in reference
with PHYLIP v. 3.69 [20]. Species assignment was done using samples. The mitochondrial D-loop haplotypes were clearly
three different methods widely employed for microsatellites e.g. species-specific, as expected [12]. Intraspecific polymorphism
[27]. The likelihood-based Bayesian method of Rannala and was found for the two species, with seven and two haplotypes
Mountain [28] was performed with GeneClass2 [29] with 0.05 for G. macrorhynchus and G. melas respectively (Table 2). Eight
score threshold. Two fully Bayesian methods were also employed: microsatellite loci were assayed, from which two (EV94MN and
one with the program STRUCTURE 2.3.1 [30] (under the 468/469) exhibited possible null alleles in our dataset (detected
‘‘Admixture model’’ which assumes that individuals may have with MICROCHECKER) and were discarded from further
mixed ancestry; burn-in period of 100 000 steps followed by analyses. For the six remaining microsatellite loci, null alleles
1 000 000 Markov Chain Monte Carlo (MCMC) iterations and and linkage disequilibrium were found to be non-significant,
five runs for k = 2 -two species), and other with NewHybrids 1.0 allowing their use for genetic assignment. Allelic frequencies of the
[31] software (with 500 000 sweeps after a burn-in period of six selected microsatellite loci were deposited in LabArchives, LLC
Table 1. Stranded pilot whale samples analyzed.
Reference Visu P Sex Size Date Location
Galicia01 69/85 G. melas 2 F 450 22/05/1996 42u349420N 09u059070W

Galicia02 72/77 G. melas 3 M 496 14/12/1995 42u169390N 08u299500W
Galicia03 74/84 G. melas 2 F 450 17/05/1996 42u539330N 09u159510W
Galicia04 75/103 G. macrorhynchus 3 F 391 9/09/1998 43u449000N 07u409190W
Galicia05 77/N Not possible 4 M 553 20/03/2005 -
Galicia06 78/G G. melas 2 F 360 8/12/2011 42u23.429N 08u49.894W
Visual identification done by experts in cetaceans. P: State of preservation proposed by the European Cetacean Society (ECS) ranged from 2 (freshly dead) to 4 (highly
degraded); M: male; F: Female; -: unknown location (accidental capture by-catch).
doi:10.1371/journal.pone.0069511.t001

124
Table 2. Mitochondrial and nuclear variability of reference samples.
DNA marker G. melas G. macrorhynchus All
D-loop AN KC542368 KC542369 KC542370 to KC542376 KC542368 to KC542376

Nh 2 7 9
Hd 0.105 0.692 0.681
P 0.00018 0.00209 0.01243
EV37NM A 6 7 8
AR 5.916 6.640 7.168
Ho 0.750 0.722 0.713
He 0.700 0.722 0.740
FIS 20.046 0.029
199/200 A 2 9 10
AR 2.000 8.473 7.483
Ho 0.150 0.333 0.365
He 0.219 0.417 0.517
FIS 0.337 0.286
415/416 A 4 6 9
AR 3.987 5.810 7.916
Ho 0.800 0.667 0.767
He 0.648 0.792 0.776
FIS 20.211 20.039
417/418 A 2 4 4
AR 2.000 3.989 3.795
Ho 0.350 0.400 0.466
He 0.499 0.480 0.568
FIS 0.321 20.011
409/470 A 7 10 11
AR 6.678 10.000 8.819
Ho 0.750 0.800 0.850
He 0.789 0.560 0.761
FIS 0.075 20.173
464/465 A 7 8 11
AR 6.742 8.000 8.595
Ho 0.789 0.667 0.752
He 0.792 0.639 0.792
FIS 0.031 0.067
All loci A 28 44 53
Ho 0.598 0.769 0.652
He 0.608 0.768 0.692
FIS 0.041 0.109 0.029
For the mitochondrial control region: AN, accession numbers in the GenBank; Nh, number of haplotypes; Hd, Haplotype diversity; P, Nucleotide diversity. For the
microsatellite loci: A, number of alleles; AR, mean allelic richness; Ho and He, observed and expected heterozygosity respectively per locus and population; FIS, FIS-value
per locus and population. P-values were not significant in any case.
(DOI: 10.6070/H43F4MHJ) as well as all genotypes (DOI: loci considered (Table 3); except for two loci that failed to amplify
10.6070/H4765C78). The number of alleles per locus ranged in one specimen (Galicia01). Genetic assignment was coincident
from 2 to10 (Table 2). No significant differences between expected with visual species identification when available (Table 4), and
and observed heterozygosities and low FIS values were found consistent for nuclear and mitochondrial markers. The male of
(Table 2). Highly significant FST-values between species (0.2957, ambiguous phenotype Galicia05 exhibited private alleles of the
P,0.00001) confirmed enough resolution for species discrimina- two parental species for 4 loci (Table 3 and Figures S1, S2, S3, S4,
tion. S5, S6): one exclusive allele of G. melas for EV37MN and 199/200,
The six stranded pilot whale here analysed yielded positive one exclusive allele of G. macrorhynchus at 464/465 locus and two
amplification at the D-Loop sequence and the six microsatellite alleles of G. macrorhynchus at 415/416 locus. These are unambig-

125
Figure 2. Individual membership of pilot whale samples from the considered regions estimated with STRUCTURE software. Each
vertical bar represents one individual. Membership to G. macrorhynchus in dark blue and to G. melas in light blue. The numbers identifying stranded
individuals are indicated above the corresponding vertical bars; the Post-F1 hybrid is in red.
uous signals of post-F1 status. This individual was assigned with Galicia05 appears in the middle of the two species. Its clustering
NewHybrids to a cross between F2 and G. melas (Table 4). The with a reference G. melas individual was not supported by
STRUCTURE software also revealed mixed ancestry for bootstrapping, which was very low. In contrast the tree exhibited
Galicia05 (57% membership of G. melas, 43% G. macrorhynchus; high bootstrapping in the rest of the nodes. These results therefore
Figure 2). From the mitochondrial DNA its maternal species was identify the first known hybrid between the two pilot whale species.
G. melas. As in other studies [27], the two fully Bayesian methods These two species join the pairs blue whales and fin whales; Dall’s
(STRUCTURE and NewHybrids software) performed better than and harbour porpoises; narwhals and belugas, and Risso’s and
partially Bayesian assignment tests (GeneClass), which did not bottle-nosed dolphins in the short list of sympatric cetaceans that
assign Galicia05 significantly to any species. The hybrid status of hybridize [32]. A post-F1 hybrid foetus was described between
this individual is clearly visualized in the NJ tree reconstructed blue and fin whales [13], but this is the first post-F1 adult cetacean
from nuclear markers (Figure 3): in the microsatellite-based tree, documented until now and strongly suggests the possibility of
Table 3. DNA markers of the stranded pilot whales analyzed.
GenBank AN Mitochondrial DNA EV37MN 199/200 415/416 417/418 409/470 464/465
Galicia01 KC542377 G. melas 184 , 184 114 , 114 236 , 236 - - 150 , 152
Galicia02 KC542368 G. melas 186 , 186 114 , 114 234 , 236 187 , 187 180 , 188 150 , 152
Galicia04 KC542370 G. macrorhynchus 192 , 196 126 , 142 228 , 232 183 , 183 188 , 190 142 , 152
GenBank AN, accession number of the D-Loop sequence obtained for each whale, available at http://www.ncbi.nlm.nih.gov/genbank/ Exclusive alleles of G. melas and
G. macrorhynchus are marked in italics and bold respectively. Results were confirmed with a multi-tube method to validate the allele scores. The suspected hybrid
(Galicia05) has private alleles of both species.
Table 4. Species assignment of stranded pilot whales based on genetic markers.
Mitochondrial DNA Nuclear microsatellite loci
NCBI-BLAST DNA-Surveillance GC2 STRUCTURE NewHybrids
Galicia01 G. melas G. melas G. melas G. melas (0.99) G. melas

Galicia04 G. macrorhynchus G. macrorhynchus G. macrorhynchus G. macrorhynchus (0.98) G. macrorhynchus
Galicia05 G. melas G. melas Not significant G. melas (0.57) F26G. melas
G. macrorhynchus (0.43)
From mitochondrial D-Loop: online assignation with NCBI-BLAST [17] and DNA-Surveillance [8] software. From nuclear microsatellite loci: NewHybrids [28], GC2
GeneClass2 [26], STRUCTURE 2.3.1 [27] (membership to a species in parenthesis).

126
Figure 3. Mitochondrial and nuclear phylogenetic trees of the analyzed samples. Neighbour Joining trees reconstructed based on: A)
mitochondrial D-Loop haplotypes; B) microsatellite loci genotypes. G. macrorhynchus is represented in dark blue and G. melas in light blue. Galician
strandings are in black except the Post-F1 hybrid (Galicia05) that is in red. Bootstrapping is given for each node.
interspecific introgression in marine mammals, a good example of Figure S3 415/416 microsatellite chromatograms.
Darwinian continuum between varieties and species [32]. Graph order as in Figure S1.
Finally, the present results emphasize the need of including (TIF)
nuclear markers in reference databases aimed at identifying
Figure S4 417/418 microsatellite chromatograms.
cetacean species [8]. SNPs and nuclear sequence data, as well as
Graph order as in Figure S1.
hypervariable microsatellite loci, can be used for this purpose. As
proposed long time ago by Palumbi and Cipriano [14], nuclear (TIF)
markers will help to understand the extent of interspecific Figure S5 409/470 microsatellite chromatograms.
hybridization in these marine mammals and its implications for Graph order as in Figure S1.
conservation. (TIF)
Figure S6 464/465 microsatellite chromatograms.
Supporting Information
Graph order as in Figure S1.
Figure S1 EV37MN microsatellite chromatograms. First (TIF)
graph Globicephala melas, second Globicephala macrorhynchus, and third
sample Galicia 05. Author Contributions
(TIF)
Conceived and designed the experiments: EGV SL. Performed the
Figure S2 199/200 microsatellite chromatograms. experiments: LM. Analyzed the data: LM. Contributed reagents/
Graph order as in Figure S1. materials/analysis tools: SL ARF MC VM BM. Wrote the paper: LM
(TIF) EGV.

127
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2986.

128
Resultados
Capítulo 7:
Merging species in pilot whales: Globicephala melas
under introgressive hybridization process.
Miralles, L., Oremus, M., Silva, M.A., Planes, S., Garcia-Vazquez, E.
Molecular Ecology
129
130
M iralles et al. / In trogression in N orth ern Pilot Wh ales
Merging species in pilot whales:

Globicephala melas under a process of introgressive hybridization.
L. Miralles 1* , M. Oremus2, M. A. Silva3, 4, 5, S. Planes6, E. Garcia-Vazquez1.
2. 2 16 rue Henri Niautou, 98800 Noumea, New Caledonia.
3. 3 Center of the Institute of Marine Research (IMAR) and Department of Oceanography and Fisheries, University of the Azores, Portugal.
4. 4 Laboratory of Robotics and Systems in Engineering and Science (LARSyS), Portugal.
5. 5 Biology Department, Woods Hole Oceanographic Institution, United States of America.
6. 6 Laboratoire d’Excellence “CORAIL”, Centre de Recherche Insulaire et Observatoire de l'Environnement (CRIOBE). USR 3278 CNRS-EPHE-UPVD. BP 1013 Papetoai,
98729 Moorea, Polynésie Française.
* Corresponding author: Laura Miralles Tel: +34-985102726; Fax: +34 985103534; E-mail: lml.miralles@gmail.com
Pilot whales are two sympatric cetacean species (Globicephala melas and G. macrorhynchus) in which distributions partially
Keywords: overlap in some areas like the North Atlantic Ocean. Previous studies in this area have reported some overlap in the species distinctive
external traits which represent clines instead of clear distinctive features. Also the recent publication of the first pilot whale post-F1
Pilot whales hybrid led us to hypothesize the occurrence of a hybrid zone for the Northeast Atlantic pilot whales. In this work, strong evidences of
Globicephala recurrent hybridization between pilot whales were detected with genetic molecular markers (mitochondrial dloop and 8 microsatellite
Hybridization loci) and supported the hypothesis that the North-East Atlantic G. melas is a species under an incipient introgressive hybridization
Introgression process. Our study revealed genetic introgression only in G. melas, not in G. macrorhynchus. Four hybrids were found and one post-F1
North Atlantic hybrid even exhibited mitochondrial-nuclear discordance. The high introgressive hybridization detected for G. melas, together with
lower genetic variation and the current increase of environmental and human-mediated stressors suggest that this species could be at
risk in its northern distribution. The evolutionary consequences of this hybridization event are difficult to predict, but to preserve the
two species of pilot whales it seems urgent to monitor the genetic variability of G. melas and to develop a conservation program.
Introduction Hybridization is thought to be more frequent in damaged, novel

Pilot whales are two sympatric cetacean species commonly environment where species may be colonising, or fast-changing
known as long-finned (Globicephala melas) and short-finned ecosystems, because in adverse conditions species may alter their
(Globicephala macrorhynchus) pilot whales. They belong to the mate choice (Seehausen et al. 2008); an example could be the northern
family Delphinidae, a family with recent speciation (LeDuc et al. latitudes, particularly the Polar zones (Kelly et al. 2010). Ongoing
1999; McGowen et al. 2009) and a controversial number of species climatic changes over-expressed in the northern latitudes, including
(Caballero et al. 2008; Slater et al. 2010). Pilot whales can form elevated temperatures, glacial ice melting and rising sea level, are
aggregations of hundreds of individuals, exhibit parental care and a reshaping those ecosystems, with potential devastating consequences
strong social structure (Oremus et al. 2012, 2009). Their home ranges for the species adapted to such environment (Edwards et al. 2011). In
partially overlap in some areas (Fig.1) where both species co-exist. particular, the distribution of pilot whales appears correlated with sea
Van Bree (1971) demonstrated that pilot whales are two clearly surface temperature (Fullard et al. 2000), thus climate change will
distinct species based in osteological differences. Molecular affect their northern populations.
phylogenetic studies also support their species status (Oremus et al. With the present study, we want to investigate hybridization in
2009; May-Collado & Agnarsson 2006; LeDuc et al. 1999). However, Globicephala species and determine if there are any hybrid zones in
their external appearance is similar and the morphological the most affected areas by climate change, or if the hybridization
characteristics used to discriminate between species, mainly the previously reported between the two pilot whales (Miralles et al.
pectoral fin length, partially overlap (Bloch et al. 1993). In the North 2009) was just a sporadic event. We aim to localize possible
Atlantic Ocean, Bloch et al. (1993) reported some ambiguity between hybridization hotspots, to describe the direction and intensity of
the distinctive external traits of Faroese long-finned pilot whales and hybridization and to analyze the extent of possible introgression.
short-finned pilot whales, which describe clines instead of clear
distinctive features. Furthermore, several studies revealed that pilot
whales from Faroe Islands were different from their conspecifics of Materials and Methods
the Atlantic coast of North America (Ottensmeyer & Whitehead 2003;
Bloch & Lastein 1993). These differential characteristics of the A total of 151 pilot whales samples were collected in 1997-2012,
northernmost population of G. melas together with the recent from five different locations and clustered into two different
publication of the first post-F1 hybrid in pilot whales (Miralles et al. geographical settings: vicariant areas where only one species occurs
2013) led us to hypothesize the occurrence of a hybrid zone for the (Faroe Islands for G. melas and French Polynesia for G.
Northeast Atlantic pilot whales. macrorhynchus) and sympatric areas inhabited by both species
Natural hybridization between sympatric species occurs rarely in (Iberian Peninsula, Azores Islands and Canary Islands). Tissue
nature (Mallet 2008) and is particularly infrequent in mammals samples were taken from stranded animals, biopsies and museum
(Larsen et al. 2010). However, many cases of hybridization have been collections. No animal was injured or killed for this study. We
reported in cetaceans to date (Glover et al. 2013; Willis et al 2004; obtained the CITES permit (ESBI00001/12I) and all the permissions
and many others), not only between species within a genus but also from the Faroese Museum of Natural History to bring the Faroese
across different genera (Berube & Aguilar 1998). Moreover, note that samples, as well as French Polynesian samples (CITES permit
cetacean hybridization might be underestimated due to the difficulties 13NZ000012; original permit number FR-02-987-0083-E) to Spain.
of sampling (Mallet 2008; Berube & Aguilar 1998).
131
Figure 1: Pilot whales distribution and sampling areas:

The map show the known distributions for Pilot whales: Globicephala melas in green, Globicephala macrorhynchus in blue and overlapping areas in purple. Sampling locations and
number of samples with positive DNA amplifications that were used in this study are marked in dark.
DNA was extracted with a Chelex-based protocol (Estoup et al. were performed with Bayesian approximation, burn-in of 500 000,
1996). The mitochondrial control region (D-loop) was amplified eight long chains (50 000 recorded steps with increment of 100) and
following Oremus et al. (2009). Sequences were edited and aligned five replicates. For each dataset, the software was run three times to
using ClustalW (Thompson et al. 1994) from the BioEdit Sequence assure that final chains were estimating the same value of Theta (Ө).
Alignment Editor (Hall 1999). NCBI-BLAST (Altschul et al. 1990) Eight microsatellite loci (EV37MN; EV94MN; 199/200;
online software was employed for species identification. The number 415/416, 417/418, 409/470; 468/469 and 464/465) were amplified as
of haplotypes, haplotypic and nucleotidic diversities were calculated in Fullard et al. (2000) and genotyped employing GeneMapper®
with DNAsp v5 (Librado & Rozas 2009). A median-joining (Bandelt Software. A multi-tube method (Allentoft et al. 2011) was employed
et al. 1999) haplotype network was constructed to visualize the intra- to validate the allele scores. Each microsatellite locus was individually
specific relationships of the different mitochondrial haplotypes and amplified 3 times in 3 different thermal cycler machines (Applied
their relative frequencies in the sampled populations with the program Biosystems 2720 Thermal Cycler). Microsatellites were genotyped
Network 4.5.1.6 (http://fluxus-engineering.com) with default settings. employing the GeneMapper® Software Version 4.0 (Applied
Network software reconstructed all possible shortest least complex Biosystems). Scoring errors, large allele dropout and null alleles were
phylogenetic trees (maximum parsimony) from a data set under tested for employing the program MICROCHECKER (Van
different algorithms. Oosterhout et al. 2004). Conformity with Hardy-Weinberg
Population divergence time estimations were done under a equilibrium was calculated using the exact probability test with
Bayesian Markov Chain Monte Carlo (MCMC) framework using GENEPOP software (Raymond & Rousset 1995). Microsatellite
BEAST version 1.6.1 (Drummond & Rambaut 2007) and following a variation (number of alleles per locus, allelic richness and observed
burn-in of 5 million cycles, rates were sampled once every 1 000 and expected heterozygosity) was calculated with the programs
cycles from 50 million MCMC steps for an Extended Bayesian GENETIX Version 4.03 (Belkhir et al. 2001) and FSTAT Version
Skyline tree with a stepwise model for mitochondrial DNA and strict 2.9.3.2 (Goudet 2001).
clock model. Bayesian intraspecific phylogenies are based on The Bayesian software STRUCTURE v.2.3.1 (Pritchard et al.
coalescent theory (Kingman 1982) and allow the inference of past 2000) was employed for estimating the membership of each
population dynamics and parameters from contemporary gene individual to each species with the “Admixture model” and K=2 (two
sequences. The best evolution model and their priors (kappa, gamma- expected genetic units one corresponding to each species), which
shape, proportion of invariant sites, etc.) were defined by jModeltest assumes that individuals may have mixed ancestry. The parameter set
software version 0.11 (Posada 2009) using the Akaike information consisted of a burn-in period of 70,000 steps followed by 700,000
criterion (Akaike 1974). The mutation rates employed were 1.5 % per Markov chain Monte Carlo (MCMC) iterations and seven runs for
million years (Hoelzel et al. 2007; 1991; Baker et al. 1993). Tracer each K. Since there is no clear consensus about the proportion of
version 1.5 (Drummond & Rambaut 2007) was used to check that membership considered as a signal of introgression (Allendorf et al.
chains had converged to a stationary distribution. 2001), for conservative interpretation we have considered >25% the
Gene flow across the two species was calculated for threshold for significant membership of a species following Miralles
mitochondrial DNA with MIGRATE 3.0 (Beerli 2004) which et al. (2013). The software NewHybrids (Anderson & Thompson
estimates Ө = xNeμ and M=m/μ (Ne, effective size;; m, immigration 2002) was employed for identifying individuals from each pure
rate;; μ, mutation rate) in each species based on coalescent theory species, hybrids of first and second generation and backcrosses. The
(Beerli & Felsenstein 2001) and relaxing the original assumption of MCMC (Monte Carlo Markov Chain) was run for 300 000 iterations
Wright (1951). If Ө and M are multiplied together, the number of after a burn-in period of 30 000 iterations. Gene flow across species
immigrants per generation can be calculated as gene flow. To ensure was also calculated with microsatellite data using the private allele
that results do not reflect spurious local likelihood peaks, three runs methods implemented in GENEPOP (Raymond & Rousset 1995). To
132
detect recent bottleneck events we employed the software Table 1: Microsatellite variation for each species:
BOTTLENECK version 1.2.02 (Cornuet & Luikart 1997) with default NA: Number of Alleles; AR: Allele Range; EA: Exclusive Alleles; He:
settings. This software detects possible bottleneck events based in Expected heterozygosity; Ho: Observed heterozygosity; FIS: Inbreeding
coefficient.
reductions of effective population sizes from the allele data
frequencies. G. melas G. macrorhynchus
Samples with positive amplification for all mitochondrial and
microsatellite loci were sexed by amplifying the SRY gene included EV37NM NA 7 10
in the Y chromosome (Nishida et al. 2007). PCR products were AR 184-198 164-200
visualized in 2% agarose gel.
Annual sea surface temperature map for 2013 were generated EA - 3
with Aqua MODIS Ocean Color from NASA He 0.776 0.797
(http://oceancolor.gsfc.nasa.gov/). GISS surface temperature analysis
(GISTEMP) taken from NASA were employed to calculate annual Ho 0.772 0.661
temperature change trends from 1951 to 2013 and annual temperature FIS 0.040 0.146
change trends from 1988 to 2013; both calibrated with a base period
199/200 NA 3 10
from 1951 to 1980 (Hansen et al. 2010)
AR 110-132 120-142
Results and Discussion EA 2 9
We have PCR-amplified one mitochondrial (D-Loop region; He 0.314 0.862

maternally inherited) and eight nuclear (microsatellites; biparentally Ho 0.304 0.797
inherited) loci in 120 DNA samples (with positive amplifications for
all molecular markers). Eight microsatellite loci were assayed, from FIS 0.110 0.098
which two (EV94MN and 468/469) may exhibited possible null 415/416 NA 7 11
alleles in our dataset (detected with MICROCHECKER) and were
AR 214-238 212-242
discarded from further analyses. For the six remaining microsatellite
loci, null alleles and linkage disequilibrium were non-significant, EA - 4
allowing their use for genetic studies. The number of alleles per locus He 0.663 0.854
ranged from 3 to13, all loci had exclusive alleles for each species
except EV37NM and 415/416 for G. melas, and allele ranges of all Ho 0.597 0.853
loci for the two species were different (Table 1). No significant FIS 0.088 0.030
differences between expected and observed heterozygosities and low
FIS values were found (Table 1). 417/418 NA 4 7
The results provided strong evidences of recurrent hybridizations AR 179-189 173-187
between long-finned and short-finned pilot whales. Nuclear genetic
EA 1 4
markers revealed unidirectional introgression of G. macrorhynchus
DNA into G. melas, and not in the opposite direction (Fig.2). The two He 0.524 0.704
pilot whales species are merging in the North Atlantic and G. melas Ho 0.529 0.698
are in a process of introgressive hybridization. Four post-F1 hybrids
were identified (Fig.2; Table 2) in G. melas from their genotypes at FIS 0.031 0.038
microsatellite loci with a high discrimination power (99.7%). The 409/470 NA 12 13
hybridization (6.89% hybrids in G. melas samples) and introgression
(average of 5.35% in G. melas; whereas only 0.21% in G. AR 176-200 174-204
macrorhynchus) rates in the area of concern is very high for mammals EA 1 4
in general (Mallet 2005; Gray 1971), but may not be for cetaceans
He 0.782 0.888
(Berube & Aguilar 1998). A surprisingly high genetic interchange
measure as effective immigrants between species of 1.1309 Ho 0.704 0.878
individuals per generation was estimated using the private allele
FIS 0.062 0.058
method. From a coalescence method the estimate was 0.4286
individuals per generation in only one direction: G. melas 464/465 NA 10 12
incorporating G. macrorhynchus genes. In our study, three of the four AR 138-158 118-152
detected hybrids (Table 2) were originated from crosses between a G.
melas female and G. macrorhynchus male (they have the EA 2 4
mitochondrial DNA type of G. melas). The forth hybrid was He 0.775 0.889
descendant of the reciprocal cross but was morphologically identified
and genetically assigned to G. melas from nuclear markers, so Ho 0.727 0.870
evidencing morphological and mitochondrial-nuclear discordance FIS 0.055 0.035
normally caused by repeated backcrosses with G. melas. This points
All loci NA 43 63
provide evidence that introgression is almost unidirectional and goes
into G. melas. AR 110-238 118-242
From our sequence data, the estimated divergence time between EA 6 28
the pilot whales species occurred about 648 500 years ago (standard
deviation of 4817.4 years and a 95% HDP between 363 600 and 961 He 0.644 0.828
900). This is a short time since the last common ancestor and would Ho 0.616 0.779
explain relatively weak interspecific reproductive barriers (Seehausen
et al. 2008; Mallet 2005; Palumbi 1994). However, unidirectional FIS 0.053 0.068
genetic introgression into G. melas still remains unexplained and the
133
Figure 2: Genetic introgression in Globicephala species:

Introgression detected with STRUCTURE 2.3.1 software and represented as membership of each Globicephala species. Each bar represents one individual and
each color means one species: green for G. melas and blue for G. macrorhynchus. Hybrids are indicated with a black asterisk and the nuclear-mitochondrial
discordant with a red asterisk. Sampling locations are indicated below the graph.
results obtained support that hybridization and introgression in reported in France for the first time (Alfonsi et al. 2013). Temperature
pilot whales are recent. Why is G. melas more affected than G. change was less intense in the North-West Atlantic Ocean (Fig.3),
macrorhynchus? What mechanisms could trigger it? Could be due to although hybrids might also occur in that area since a recent
environmental or human pressures? photographic study found some individuals that could not be
Climate change can be one of such environmental pressures morphologically classified as one of the two species (Rone & Pace
since pilot whales distribution seems to be correlated with sea surface 2011). Under this scenario it seems that climate change is widening
temperature (Fullard et al. 2000). Pilot whale mating occurs when two the area of co-occurrence of the two species, especially during the
or more pods meet or when adult males pay short visits to other pods reproductive peak in summer. But the question remains, why would
(Amos et al. 1993). In the North Atlantic Ocean, reproduction G. melas be more prone to introgressive hybridization than G.
happens during the warmer months and the observation of larger macrorhynchus? The answer must rely on demographic events
groups coincides with higher proportion of mixed pods (Alves et al. together with human and environmental pressures affecting this
2013; Sergeant 1962). During summer, when temperature increases, species. Hybridization is known to occur more frequently in areas
the temperature distribution limit for G. macrorhynchus could move where population density is low, and where species are near the edge
northwards. It would create a northern or maybe a wider seasonal of their ecological range (Arnold 1997). When isolated populations
contact area for the two species coincident with mating period. The and species enter into contact and hybridize the rarer species uses to
global climate change has increased the ocean temperature around provide the female (e.g. Wirtz 1999). It is what happened to the North
2°C degrees since 1951 in the area where introgression was detected Atlantic G. melas and the hybrid calves stay in their maternal pods
(Fig.3B). This change was also high in the last 25 years (Fig.3C). due to the social matrilineal structure of these species. If hybrids
Accordingly, the northern distribution limit for G. macrorhynchus in exhibit reduced fitness it could go extinct (Kelly et al. 2010); if not,
1988 was described in the western edge of the Cantabrian Sea (Nores introgression could happen. With our results, we can state that
& Perez 1988), but in 2013 strandings of this species have been introgression happened in G. melas.
.
Table 2: Hybridization in pilot whales: Hybridization scores in assignment proportions per species and for each descendent of hybrid calculated with two
different methods (NewHybrids 1.0 and STRUCTURE 2.3.1 software). GME: Globicephala melas; GMA: Globicephala macrorhynchus; Memb.: Membership;
F: Faroe Islands; IP: Iberian Peninsula; F2: second generation; Bx: Backcrossing.
New Hybrids 1.0 STRUCTURE 2.3.1
Hybrid Pure GME Pure GMA Memb.GME Memb.GMA
G. macrorhynchus 0 0.000 0.985 0.002 0.998
G. melas 4 0.966 0.000 0.913 0.087
Hybrid F 07 F2 GME 0.524 0.000 0.465 0.535
Hybrid F 12 Bx GME 0.711 0.000 0.650 0.350
Hybrid F 33 Bx GME 0.671 0.000 0.742 0.258
Hybrid IP 53 F2 GME 0.752 0.000 0.674 0.326
134
Table 3: Diversity indices for each species and location

N: sample size; M: Male; F: Female; Hd, Haplotype diversity;; (π), Nucleotide diversity;; Nh, Number of haplotypes. NA, average number of alleles per locus. AR,
allelic richness. He and Ho, heterozygosity observed and expected respectively. *The only individual G. macrorhynchus from the Iberian Peninsula was not taken
into account in these calculations.
D-LOOP Microsatellite loci

Species Sex
(M : F)
Localities N Hd (π) Nh NA AR He Ho
G. macrorhynchus 62 32 : 30 0.667 0.002 16 64 62.690 0.828 0.779

Azores Islands 16 14 : 2 0.517 0.001 4 45 28.200 0.822 0.783
Canary Islands 26 14 : 12
0.697 0.002 11 50 27.299 0.806 0.804
French Polynesia 20 4 : 16
0.582 0.001 3 44 26.003 0.776 0.782
G. melas 57 50 : 7 0.474 0.001 12 45 44.268 0.644 0.616

Faroe Islands 50 48 : 2 0.263 0.001 9 41 20.389 0.644 0.617
Iberian Peninsula 7* 2:5 0.928 0.003 6 21 17.680 0.603 0.525
With our results, the level of population variation could indicate exponentially in the last decades and has become very intense
a past or present reduced population size of G. melas in comparison (Kaluza et al. 2010; Vieites et al. 2004; Johnson & Wheatley 1972).
with G. macrorhynchus. Historical population size estimations More and more vessels cross the main foraging, resting and mating
revealed that G. melas from the North Atlantic suffered drastic decline areas of Northeast Atlantic pilot whales and likely represent an
events in the last centuries (Fig. 4). In the last decline we can observe important stressor for them. Marine traffic could be a metabolic
that it became negative after a stationary event. This high decrease of stressor for pilot whales as it is already reported in the literature to
historical population sizes was coincident in time with the first records affect negatively to many other cetacean species [e.g. Rolland et al.
of Faroese grinds in the literature (Brakes et al. 2004) around 700 2013; Lusseau et al. 2009; Hastie et al. 2003; between others].
years ago (Fig.4B). Furthermore, although sample sizes were very Although it is highly speculative, it might also be a behavioral stressor
similar for the two species, G. melas exhibited lower haplotype and promoting hybridization as populations under high stress tend to
nucleotide diversity than G. macrorhynchus, also less variation for the hybridize (Badyaev 2005). In addition to traffic, the European
nuclear markers analyzed (Table 3). These differences between Atlantic Ocean has been considered the hottest oil spill spot
species could be explained by a recent reduction of G. melas worldwide (Peterson et al. 2003). The negative effects of crude oil on
populations, selection or other possible factors (Alexander 2006; marine organisms have been largely reported [e.g. Peterson et al.
Whitehead 2005). In addition, the G. melas haplotype network (Fig.5) 2003; Engelhardt 1983], even promoting hybridization in fish (Crego-
revealed a star-like phylogeny indicator of recent population Prieto et al. 2012).
expansion (Lavery et al. 1996). However, bottlenecks and genetic or With the data obtained in the present study we cannot
cultural hitchhiking also leave similar footprints in DNA (Oremus et discriminate between natural or human-mediated hybridization.
al. 2009; Maynard-Smith & Haigh 1974). Although we did not detect Reductions of populations, climate change, marine traffic and other
any recent bottleneck event in our study, for G. melas it could be a possible factors could be affecting negatively to G. melas and might
false negative caused by the incorporation of new alleles through promote recurrent hybridization. Whatever the reason, this process
hybridization. Moreover, missing haplotypes in the haplotype network could be a signal of introgression. The evolutionary consequences of
(Fig.3) may indicate lineage extinction and support the hypothesis of a this reticulated evolution event are difficult to predict. If the detected
possible bottleneck or reduction event. Matrilineal social structure interspecific gene flow and introgression continue for a long time, and
together with mass mortality can reduce mitochondrial DNA diversity mating encounters between species become more frequent due to
(Alexander 2006); the genetic signature found in our study could be at continued climate warming, the species G. melas could disappear
least in part a consequence of the Faroese traditional hunts and events from the North-East Atlantic Ocean, as it already disappeared from
of mass strandings, where large groups of several generations of the North Pacific about 800-1200 years ago in Japan (Kasuya 1975)
maternally-related individuals die. and 3500-2500 years ago in Alaska (Frey et al. 2005). To preserve this
Another factor that might affect G. melas hybridization could be species it seems urgent to develop a monitoring program to survey the
the intense maritime traffic in the Northeast Atlantic Ocean (Fig. S1), genetic variability of G. melas and to create a conservation program
crossed by many cargo ships, ferries, fishing and motor and sailing for considering possible marine protected areas, refuges or sanctuaries
boats, whale-watching tours, etc. Maritime traffic has been increasing with no disturbances for this species.
135
.
Figure 3: Sea surface temperature and global temperature changes. Figure4: Pilot whales historical effective sizes.
A. Annual sea surface temperature for 2013 in Celsius degrees. Reconstruction of the historical effective size of both pilot whales species.
Data taken from Aqua MODIS Ocean Color from NASA. The graph at the bottom shows the last 10000 years. G. macrorhynchus is
B. GISS surface temperature analysis (GISTEMP) taken from represented in dark grey and G. melas is in light grey.
NASA by using annual temperature change trends from 1951 to
2013 and calibrated with a base period from 1951 to 1980
(Hansen et al. 2010)
C. GISS surface temperature analysis (GISTEMP) taken from
NASA by using annual temperature change trends from 1988 to
2013 and calibrated with a base period from 1951 to 1980
(Hansen et al. 2010)
Scale bar under each plot. Gray color means data missing.
Figure 5: Mitochondrial haplotypes network.

Median-Joining network which represent the relationships among D-loop
mitochondrial haplotypes. Circle sizes are proportional to the frequency of
each haplotype. Different locations are represented in different colors. Each
species is clustered in a square. Mutations are represented as perpendicular
bars in the branches. Inferred haplotypes are represented by a small red
circle.
136
Acknowledgments Arnold M.L. (1997) Natural Hybridization and Evolution. Oxford: Oxford
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M iralles et al. / Introgression in N orth ern Pilot Whales
Figure S1: Annual marine traffic across the world:

Annual global cargo shipping routes (only cargo ships bigger than 10 000 Tm were represented). It is easy to see how dense
marine cargo traffic is in the North Atlantic Ocean. Image edited from Kaluza et al. (2010).
139
140
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141
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142
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Discusión
Barreras al flujo génico en la especiación marina
A lo largo de esta Tesis Doctoral se ha visto que en el medio marino, no

todas las barreras al flujo génico son tan obvias como la descrita por el ictiólogo
David Starr Jordan (1905, 1908), el istmo de Panamá, ni tampoco el aislamiento por
distancia es el único mecanismo que explica la especiación en el mar (Palumbi
1994). También se ha visto que la distancia puede ser una barrera o no,
dependiendo de multitud de factores, no tan fáciles de percibir como las barreras
geográficas.
El caso de Pomatomus saltatrix es especialmente enriquecedor porque

ilustra cómo una pequeña migración, promovida sólo por los machos, puede servir
para eliminar el efecto de barreras históricas al flujo génico, así como el
aislamiento por distancia. Las barreras en esta especie se remontan a las
glaciaciones y afectaron a su estructura poblacional actual dibujando los linajes
existentes hoy en día. Sin embargo, el hecho de que Pomatomus saltatrix sea una
especie muy migradora, donde los machos se dispersan incluso a través del océano
Atlántico, promueve el suficiente flujo génico como para que las barreras
regionales sean permeables y no se produzca la especiación. Es decir, el
intercambio de genes entre poblaciones frena la diferenciación, impide el
aislamiento y por tanto la especiación en Pomatomus saltatrix. La migración
efectiva es un factor muy importante en los procesos de especiación (Coyne y Orr
2004) y en los organismos marinos, migración y conectividad son procesos clave
para la dinámica y la estructura de sus poblaciones (Cowen y col. 2000). No sólo
modulan la persistencia y demografía de las especies (Hanski 1999) o reducen la
143
Discusión
probabilidad de extinciones (Poethke y col. 2002) debidas a eventos estocásticos

(Cadet y col. 2003) o fluctuaciones ambientales (Friedenberg 2003), sino que
además actúan evitando la diferenciación regional, el aislamiento y finalmente, la
especiación a largo plazo. Las diferencias en el comportamiento reproductor de
machos y hembras, con migración y dispersión diferencial de ambos sexos, no
parecen impedir la homogenización de la especie Pomatomus saltatrix. El hecho de
que las hembras tengan mayor fidelidad por particulares grupos o zonas de puesta
produciría cierta diferenciación regional, pero no un aislamiento total, que sería
evitado por la mayor dispersión de los machos. La dispersión diferencial entre
sexos es común en animales (Perrin y Mazalov 2000) incluidos los peces, donde hay
un elevado número de ejemplos, como los tiburones (e.g. Pardini y col. 2001) o la
mayoría de los salmónidos (Campos-Tellez y col. 2011), entre otros muchos.
En el caso de las merluzas euroafricanas se ha inferido que una de las

principales barreras entre especies podrían ser los gradientes batimétricos. Según
los resultados de esta Tesis, estas especies proporcionarían un claro ejemplo de
especiación ecológica por profundidad. La radiación de las merluzas euroafricanas
podría explicarse basándose en parámetros ecológicos, entre los cuales destacaría
la batimetría (ver Glosario) y más concretamente, la profundidad de puesta.
Especies que comparten la misma área geográfica, pero desovan a diferentes
profundidades, pertenecen a linajes distintos a lo largo de toda la extensa
distribución que ocupa el género en ambos hemisferios de la costa oriental del
Atlántico. Las parejas simpátricas pertenecientes a distintos linajes serían
Merluccius capensis y M. paradoxus en aguas de Namibia y Sudáfrica; M. capensis y
M. polli en las aguas frente a Angola; M. senegalensis y M. polli en aguas de
Mauritania y Senegal, siendo en todos los casos la primera especie de un linaje y la
segunda de otro, y desovando respectivamente en zonas menos y más profundas
(Stenevick y col. 2008; Fernandez-Peralta y col. 2011; Figura 11). Así pues, podría
explicarse la actual distribución de las merluzas euroafricanas como una expansión
espacial paralela de dos linajes a dos profundidades diferentes. Las merluzas
euroafricanas se sumarían, por tanto, a la reciente, pero aún escasa lista de
144
Discusión
especies cuya especiación está basada en condiciones ecológicas (Ingram 2011;

Keller y Seehausen 2012; Nosil 2012; Jennings y col. 2013; Prada y col. 2013; Milano
y col. 2014) y entre ellas, al pequeño grupo basado en gradientes batimétricos
(Ingram 2011; Jennings y col. 2013; Prada y col. 2013).
Figura 11: distribución geográfica (A.) y en profundidad (B.) de las merluzas euroafricanas. En el
diagrama B. se muestra en color sólido la distribución en profundidad de la máxima abundancia de cada
especie, en color rayado la distribución general. La profundidad de puesta está marcada con un cuadro
negro punteado y las zonas de solapamiento de puestas de dos especies está marcado en negro sólido.
145
Discusión
Dado que es probable que las merluzas euroafricanas y los calderones hayan
tenido una especiación ecológica, cabe mencionar en este momento que durante la
especiación ecológica, el aislamiento reproductivo aparece secundariamente como
un subproducto de la divergencia ecológica (Ingram 2011). Así, en esta Tesis se ha
observado que las barreras interespecíficas tanto entre merluzas, como entre
calderones tienen una cierta permeabilidad, y en los dos casos mencionados, las
barreras fueron sobrepasadas permitiendo la hibridación y la introgresión, a pesar
de tratarse de dos modelos de estudio muy distintos. También cabe destacar que
las barreras, en ambos casos, son muy diferentes: en el caso de las merluzas, la
profundidad es una barrera vertical, compartiendo la misma área geográfica pero
diferentes profundidades. En cambio, en el caso de los calderones la barrera
biogeográfica que les separa es de distancia geográfica y, probablemente, de
temperatura. Lo más distintivo de estos dos tipos de barreras es que para especies
migradoras, en general, es mayor el aislamiento por profundidad que por distancia
(e.g. Bucklin y col. 1987; France y Kocher 1996; Etter y col. 2005; Zardus y col. 2006;
Raupach y col. 2007; Miller y col. 2011). Sin embargo, comparando las tasas de
hibridación introgresiva de ambos modelos de estudio y teniendo en cuenta sus
características e historias evolutivas, vemos que es mayor la permeabilidad de las
barreras en merluzas (profundidad), que en calderones (distancia). Seguramente,
explicable debido a las características biológicas de cada especie: las merluzas son
peces con reproducción externa y los calderones son mamíferos con reproducción
interna y acoplamiento de aparatos reproductores. Además, tanto circunstancias
naturales como antrópicas pudieron contribuir a este debilitamiento en sinergia.
También se ha visto en numerosos estudios que la distancia filogenética entre
taxones y la hibridación están negativamente correlacionadas (Arnold 1997; Coyne
y Orr 1997; Edmands 2002; Price y Bouvier 2002). Sin embargo, la hibridación se
encontró en merluzas, donde M. capensis y M. paradoxus pertenecen a linajes
distintos y diferenciados, así como en calderones donde las especies están más
cercanamente emparentadas, tanto taxonómicamente como en el tiempo (Figura
12). A pesar de la distinta naturaleza de las barreras, así como de los modelos de
146
Discusión
estudio, el resultado en ambos casos fue que las barreras entre especies se
debilitaron permitiendo la hibridación interespecífica.
Figura 12: Reconstrucciones filogenéticas de las especies donde se encontró hibridación

interespecífica y estimas del tiempo de divergencia calculado como el tiempo al ancestro común
más cercano (tMRCA) entre las especies que hibridan. M.A.: Millones de Años. A. Especies del
género Merluccius, con los linajes dibujados en diferentes colores. B. Especies pertenecientes al
género Globicephala.
La hibridación es consecuencia de la ruptura de barreras entre taxones y en

todos los modelos de estudio empleados en esta Tesis se han encontrado procesos
de hibridación. En Pomatomus saltatrix se encontró hibridación intraespecífica
entre las poblaciones regionales de la especie, e hibridación interespecífica entre
las merluzas del Cabo (entre Merluccius paradoxus y M. capensis) y entre
calderones (Globicephala melas y G. macrorhynchus).
En el caso de Pomatomus saltatrix es esperable que suceda al ponerse en

contacto machos y hembras de poblaciones separadas, ya que pertenecen a la
misma especie y se sabe que la hibridación ocurre inevitablemente durante la
especiación (Abbott y col. 2013; Barton 2013). La hibridación entre poblaciones
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Discusión
genéticamente similares dentro de una misma especie puede tener un papel

importante en la adaptación divergente entre poblaciones (Allendorf y col. 2001).
Se ha discutido bastante sobre la hibridación intraespecífica (Allendorf y col. 2001),
ya que las poblaciones de una misma especie comparten alelos, así que las nuevas
variantes genéticas importadas evitarán problemas de reducción de la variabilidad
genética en las poblaciones (Cuenco 1993; Keenan 2000). También se ha sugerido
que la introducción de variación genética nueva en una población puede resultar
beneficiosa y, por tanto, la selección natural hará que sea posible aumentar el
fitness de la población (Cuenco 1999). Sin embargo, en ciertas circunstancias el
intercambio genético entre poblaciones intraespecíficas podría causar una
depresión reproductiva extrínseca (Allendorf y col. 2001) derivada de la pérdida de
adaptaciones locales imprescindibles para la viabilidad de las poblaciones locales
(Storfer 1999).
Aunque los híbridos son por lo general raros, unos pocos híbridos pueden
proporcionar un puente para el paso de ciertos alelos entre especies (Mallet 2005).
En cambio, cuando esos híbridos pasan de ser unos pocos y son muchos, se puede
dar la especiación híbrida, es decir, la aparición de una nueva especie mediante
hibridación. En la especiación híbrida, además de la hibridación per se también
deben darse el aislamiento reproductor y la divergencia ecológica respecto a las
especies parentales para que suceda con éxito (Mayr 1963). De lo contrario, los
genotipos recombinantes se diluirán con las formas parentales a través del flujo
génico. Por lo general, los híbridos tienen cierta divergencia de los parentales y
están mejor adaptados a los ambientes intermedios (Anderson 1949). De hecho, es
la presencia de ese hábitat intermedio favorable lo que ha sido bastante discutido
como el primer factor limitante para la especiación híbrida (Templeton 1981). En el
caso de los otros dos géneros estudiados, Merluccius y Globicephala, no se puede
predecir si los eventos de hibridación encontrados contribuirán o no a
desencadenar un proceso de especiación híbrida.
La introgresión genética ha sido un tema bastante debatido, ya que hasta la

última década se creía que era sumamente infrecuente en la naturaleza (Mallet
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Discusión
2005). Actualmente, con la aparición de nuevas técnicas moleculares y genéticas

está bien reconocida (Coyne y Orr 2004; Mallet 2005, 2007, 2008; Hegarty y col.
2008; Jiggins y col. 2008) y en esta Tesis ha sido descrita en los capítulos 5, 6 y 7 de
Resultados. Parece que la primera generación híbrida (F1) es la más rara y difícil de
producir (Mallet 2005). Una vez que ocurre, si el primer híbrido (F1) es viable y
fértil es muy probable que se reproduzca con una de las especies parentales por
retrocruzamiento, y que el híbrido retrocruzado sea casi imposible de distinguir
morfológicamente de los parentales (Nasbit y col. 2003; Mallet 2005). De hecho, en
esta Tesis los híbridos encontrados son de generaciones post-F1, demostrando que
existe introgresión genética tanto entre las merluzas del Cabo como en los
calderones.
El concepto de especie en los modelos estudiados
A pesar de que el concepto biológico de especie es ampliamente utilizado y

reconocido en biología (Mayr 2000), las especies no son unidades discretas aisladas
unas de otras, por lo que hoy en día se debería ver a las especies como estados
poco diferenciados y estacionales dentro de un continuo jerárquico que es la
biodiversidad (Mallet 2005) que se encuentra en continua evolución y cambio. Así
pues, el concepto genético de especie propuesto por Baker y Bradley (2006),
basado en sus estudios en mamíferos, es mucho más adecuado para abordar los
trabajos de especiación en general, de especies con hibridación en particular, y aún
más concretamente, en los modelos de estudio de esta Tesis Doctoral. Hay
multitudes de definiciones y revisiones de lo que puede ser interpretado como el
concepto genético de especie (Bateson 1909; Taverner 1920; Muller 1939; Simpson
1943; Dobzhansky 1950; Mayr 1969; Nei 1976; Baker and Bickham 1986; Masters
and Spencer 1989; Avise and Ball 1990; Mayden 1997; Schilthuizen 2000; Butlin
2005). El concepto propuesto por Baker y Bradley (2006) define a las especies como
un grupo de poblaciones naturales que pueden entrecruzarse pero que están
aisladas genéticamente, frente al concepto biológico de especie según el cual las
149
Discusión
especies están aisladas reproductivamente. Por tanto, los dos conceptos difieren
en el tipo de aislamiento. El concepto genético es especialmente útil en especies
con hibridación, por ejemplo en el caso de las merluzas estudiado en esta Tesis.
Bajo el concepto biológico de especie, las merluzas del Cabo no se considerarían
dos especies distintas al no existir aislamiento reproductivo y tener una gran
hibridación e introgresión a lo largo de su distribución y serían consideradas una
misma especie. En cambio, bajo el concepto genético, se seguirían considerando
dos especies porque aunque no haya aislamiento reproductivo total, las especies sí
están relativamente aisladas y cada una tiene su propia identidad genética. Esa
distinción entre “genéticamente aislado y reproductivamente aislado” y
“genéticamente aislado pero no reproductivamente” es muy significativa para el
entendimiento de muchos procesos evolutivos, incluidos la especiación (Baker y
Bradley 2006). Por ello, para el estudio de especiación en el medio marino
considero mucho más apropiado el concepto genético de especie.
La especiación, ha sido uno de los centrales enigmas de la biología (Darwin

1859; Dobzhansky 1937; Mayr 1942; White 1978; Coyne y Orr 2004; Dieckmann y
col. 2004; Gavrilets 2004). En todo este tiempo, el papel del aislamiento y del flujo
genético ha sido uno de los temas más polémicos en los procesos de especiación
(Rocha y Bowen 2008). A día de hoy, se reconoce que la especiación puede ocurrir
sin aislamiento y con flujo génico entre poblaciones, por tanto, no siempre ha de
seguir el patrón de una especie que se divide en dos (cladogénesis) como sugieren
multitud de autores (e.g. Bolnick 2004; Rocha y Bowen 2008).
La acción humana en los procesos de especiación marina
El medio marino está afectado por múltiples factores como la pesca, la

producción pesquera, los contaminantes, el tráfico marítimo (Halpen y col. 2008),
etc. En general, los impactos humanos hacen que los ecosistemas naturales sean
cada vez más homogéneos (Candolin y col. 2006; Hendry y col. 2006; Seehausen
2006). Esto no sólo lleva a una reducción de biodiversidad por adaptación y
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Discusión
evolución divergente (Seehausen y col. 2008), sino que además, como se ha visto
en esta Tesis Doctoral, puede ir seguida de homogenización genética de las
especies si se promueve la hibridación.
En esta Tesis se han visto tres casos diferentes de hibridación en los que la
actividad humana podría haber mediado. i) La hibridación entre poblaciones
promovida por las granjas de acuicultura, en el caso de Pomatomus saltatrix. ii) En
la hibridación interespecífica entre las merluzas del Cabo, además de un conjunto
de variables naturales (el fenómeno del Niño de Benguela y periodos anóxicos del
agua), algunas actividades humanas, como la sobrexplotación pesquera de las
costas de Namibia en los años 70, podría también haber ejercido algún efecto. iii)
Finalmente, la hibridación interespecífica de los calderones podría explicarse por
multitud de impactos humanos: caza, calentamiento global, tráfico marítimo… A
pesar de que la hibridación natural es un proceso que ocurre con regularidad,
especialmente en grupos de rápida diversificación y radiación (Price y Bouvier
2002), es importante identificar los casos en los que la acción o intervención del ser
humano puede estar influyendo para en su caso establecer medidas conducentes a
reducir este impacto.
El estudio de los efectos de las granjas marinas de dorada y lubina del

Mediterráneo sobre el predador Pomatomus saltatrix (Figura 13) proporciona una
nueva visión sobre los efectos de la acción humana en los ecosistemas de
explotación pesquera mediante acuicultura. Los impactos de la acuicultura sobre
los ecosistemas naturales han sido revisados por multitud de autores (e.g. Black
2000; Fernandes y col. 2002; Diana 2009). Uno de los efectos más estudiados es la
interacción entre los escapes de las granjas y sus conespecíficos salvajes (e.g.
Hindar y col. 1991; Youngson y col. 2001; Read y Fernandes 2003; Naylor y col.
2005). Por otro lado, los impactos interespecíficos descritos van desde el
incremento de hibridación con especies cercanas (Verspoor 1988; Youngson y col.
1993; Horreo y col. 2014), hasta reducciones de las poblaciones nativas locales
debido a la competición con especies exóticas escapadas en las granjas (Naylor y
col. 2000; McGinnity y col. 2003), e incluso ejemplos de alteraciones del
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Discusión
Figura 13: Pomatomus saltatrix capturado dentro de las granjas de acuicultura junto
a doradas cultivadas en Alicante, España. Foto tomada por P.Sanchez durante el
muestreo del estudio de Pomatomus saltatrix del Capítulo 3 de Resultados.
comportamiento inducidos por la domesticación (Hutchings y Fraser 2009; Castillo

y col. 2010). El estudio de Pomatomus saltatrix de esta Tesis detecta un posible
efecto a nivel evolutivo sobre las poblaciones de los depredadores salvajes que
hasta el momento nadie había abordado y que supone una novedad en el campo
de la ecología evolutiva marina. Creo que es muy importante para la ciencia, para
la conservación e incluso para la sociedad, concebir que las instalaciones de cría de
peces en el mar pueden influenciar la filogeografía de depredadores salvajes, como
Pomatomus saltatrix, que no son cultivados pero si son atraídos por los peces
criados en las granjas. Este modelo de estudio puede ser extrapolable a otros casos
aún no estudiados, especialmente teniendo en cuenta el gran número de
depredadores atraídos a las granjas marinas: delfines (Diaz-Lopez y col. 2005; Diaz-
Lopez y Bernal-Shirai 2007), focas monje (Güçlüsoy y Savas 2003), cormoranes
(Diaz-Lopez y col. 2008; Liordos y Goutner 2008; Akyol y Ertosluk 2010) y muchos
peces salvajes (Dempster y col. 2002; Valle y col. 2007). En el escenario actual con
152
Discusión
un enorme incremento global de la acuicultura, los estudios basados en los efectos

sobre el ecosistema deberían ser considerados como una prioridad, ya que, por
ejemplo, los cambios en la comunidad de depredadores puede alterar y colapsar el
ecosistema completo (e.g. McCann 2007; Baum y Worm 2009). Más aún, cambios
evolutivos como el aquí descrito pueden pasar inadvertidos y sus consecuencias a
largo plazo son impredecibles a día de hoy.
Además de la explotación pesquera y del incremento de la acuicultura, las

especies marinas deben enfrentarse a otras amenazas causadas por la actividad
humana que pueden afectar a su historia evolutiva a través de los procesos de
especiación. El ejemplo de los calderones desarrollado en esta Tesis Doctoral
podría apuntar en el sentido de un impacto simultáneo de varios factores distintos.
La explotación, la reducción de poblaciones, el cambio climático, el tráfico
marítimo… todo ello (y seguramente más factores) podían estar poniendo en
peligro la integridad de la especie Globicephala melas en el norte de su
distribución, al noreste del Atlántico. No puede excluirse que la principal razón del
contacto secundario pueda ser el clima. La distribución de los calderones tienen
una fuerte relación con la temperatura del agua (Fullard y col. 2000) y el
calentamiento global les está afectando, cambiando su distribución más al norte
(Nores y Perez 1988 frente a Alfonsi y col. 2013). Precisamente, es la especie
norteña en la que se encontró hibridación e introgresión. Esta, además, está sujeta
a explotación en cacerías en las islas Faroe desde hace unos 700 años (Brakes y col.
2004), reduciendo sus poblaciones constantemente y por tanto reduciendo su
variabilidad genética. Además, los calderones del Atlántico norte comparten su
espacio con un intenso tráfico marítimo, que ha crecido exponencialmente en los
últimos años (Johnson y Wheatley 1972; Vieites y col. 2004; Kaluza y col. 2010),
siendo la zona más concurrida del planeta (Capítulo 7, Resultados). El tráfico
marítimo puede ser un estresante metabólico para estos animales, al igual que lo
es para otras especies de cetáceos (algunos ejemplos son Hastie y col. 2003;
Lusseau y col. 2009; Rolland y col. 2013, entre otros), y también puede afectar a su
conducta reproductiva. Otro posible factor de estrés podrían ser los vertidos de
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Discusión
petróleo. La parte europea del océano Atlántico ha sido considerada el mayor

punto del mundo de vertidos de petróleo (Peterson y col. 2003). Sus efectos
negativos han sido ampliamente informados en numerosos estudios (e.g.
Engelhardt 1983; Peterson y col. 2003) e incluyen el aumento de hibridación en
peces (Crego-Prieto y col. 2012, 2013). Se espera de las especies bajo un fuerte
estrés o en ecosistemas cambiantes y/o dañados que alteren su conducta
reproductiva e hibriden (Badyaev 2005; Seehausen y col. 2008). La hibridación
repetida, inferida de la introgresión y los retrocruzamientos encontrados en esta
Tesis, llevan a pensar que el incremento de los factores de estrés sobre las
poblaciones más vulnerables (en este caso Globicephala melas) podían llevar a la
despeciación y fusión de las dos especies en el noreste del Atlántico, o en el caso
más desfavorable, a su extinción como ocurrió anteriormente en el norte del
Pacífico hace unos 3.500-2.500 en Alaska (Frey y col. 2005) y en Japón hace unos
800-1.200 años (Kasuya 1975).
154
Recomendaciones para la
conservación
155
Recomendaciones para la conservación
156
Los resultados obtenidos en esta Tesis Doctoral señalan que tanto las
actividades humanas directamente, como sus impactos en el medio marino pueden
afectar a la especiación natural de peces y de cetáceos, a diferentes niveles, tanto
evitando como favoreciendo la especiación. El ser humano ha interferido en los
procesos naturales, causado efectos evolutivos en las especies y sus consecuencias,
a día de hoy, son impredecibles. Por ello, es imprescindible una mayor
concienciación ciudadana, así como un mayor estudio científico en aras de la
conservación de las especies, especialmente en las zonas frágiles de su distribución,
como por ejemplo las áreas donde hay hibridación.
En esta Tesis Doctoral se han identificado varias zonas híbridas para el

importante recurso pesquero de las merluzas del Cabo y para los carismáticos
cetáceos conocidos como calderones o ballenas piloto. Una vez identificadas,
deberían manejarse y gestionarse como zonas vulnerables para la conservación.
Estas zonas son las aguas frente a Namibia y las aguas frente a la costa noroeste de
Sudáfrica para las merluzas del Cabo y las aguas del noreste del Atlántico (desde
Galicia, en España, hasta las Islas Faroe incluidas) para los calderones, en especial
para el calderón común. Dado que el ser humano depende de los ecosistemas
marinos para la obtención de importante bienes y servicios (Halpern y col. 2008)
junto con la gran capacidad de dispersión de las especies estudiadas, sería poco
realista intentar catalogar estas zonas como zonas de exclusión para la pesca,
santuarios para las especies o áreas marinas protegidas. Por ello, se recomienda un
uso moderado del ecosistema para actividades humanas, más reducido del actual,
157
en estas zonas vulnerables. Además, se sugiere un control más exhaustivo de las

capturas de merluzas del Cabo, identificando genéticamente las especies para un
manejo mucho más adecuado del stock, así como su monitorización continua para
ver el estado actual y poder regular las cuotas de pesca en base a esos resultados.
Por otro lado, en el caso de los calderones, se recomienda una moderación del
tráfico marítimo que afecta a esa zona, una reducción de las cacerías culturales de
las Islas Faroe y una monitorización genética de las poblaciones norteñas para
estudiar la intensidad de la introgresión y poder así predecir las posibles tendencias
evolutivas de la especie.
Para promover el uso de estas medidas preventivas y recomendaciones para

la conservación, se van a enviar los resultados de esta Tesis Doctoral a los órganos
e instituciones correspondientes. En el caso de las merluzas del Cabo se enviarán al
Directorado de Pesquerías Sudafricano (Department of Agriculture, Forestry and
Fisheries, DAFF), al Consejo Internacional para la Exploración del Mar (International
Council for the Exploration of the Sea, ICES), a la Asociación Americana de
Pesquerías (American Fisheries Society, AFS), a las Naciones Unidas (Food and
Agriculture Organization of the United Nations, FAO) y, dado que son un
importante recurso pesquero en España, los resultados obtenidos ya han sido
enviados y discutidos con los científicos especializados en la gestión de este recurso
pesquero pertenecientes al Instituto Español de Oceanografía (IEO). En el caso de
los calderones, los resultados se enviarán a la Comisión Ballenera Internacional
(International Whale Commission, IWC), a la Sociedad para la Conservación de
Ballenas y Delfines (Whale and Dolphin Conservation Society, WDC), al Consejo
Internacional para la Exploración del Mar (ICES) y a la Unión Europea (Animal
Health and Welfare, European Commission).
158
TESIS
159
Tesis: Reconsideración de las hipótesis de partida
160
Tesis: Reconsideración de las hipótesis de partida
Reconsideración
de las hipótesis de partida
1: En el género Pomatomus, se encontró, como se esperaba en la hipótesis de

partida, una diferenciación genética entre las áreas muestreadas más alejadas
conformando tres unidades genéticas: americana (muestras de la costa atlántica de
Estados Unidos), ibérica (muestras del Atlántico y del Mediterráneo de las costas
españolas) y turca (muestras de Turquía). El flujo génico entre las tres unidades
genéticas detectadas, debido a su gran capacidad de dispersión que parece ser
mayor para los machos, parece impedir la especiación vicariante en este género.
2: En las especies africanas del género Merluccius, tal y como se esperaba en

la hipótesis de partida, se encontró hibridación introgresiva entre las dos merluzas
del Cabo, a pesar de pertenecer a linajes relativamente distantes. La profundidad
de puesta sugerida como barrera ecológica interespecífica confirmaría la
especiación ecológica como mecanismo significativo en este género.
3: En el género Globicephala la hipótesis de partida también se confirmó al

encontrarse hibridación e introgresión entre ambas especies, siendo más intensa
en la especie boreal G. melas. Es posible que, como en otros géneros de cetáceos,
las barreras interespecíficas sean incompletas y exista un cierto flujo génico entre
especies dada su reciente especiación.
161
162
Conclusiones / Conclusions
163
Conclusiones
164
Conclusiones
Conclusiones
1. Se ha encontrado, mediante marcadores genéticos, evidencia de diferenciación

poblacional entre regiones en la especie Pomatomus saltatrix, que parece ser
promovida no sólo por el actual aislamiento por distancia, sino también por
barreras antiguas que se han datado mediante coalescencia y coinciden con las
últimas glaciaciones. El flujo génico entre poblaciones, detectado incluso a nivel
interoceánico, parece contrarrestar el proceso de especiación vicariante en este
género.
2. Comparando los resultados de marcadores genéticos mitocondriales y

nucleares, se ha inferido un patrón de dispersión diferencial entre sexos para
Pomatomus saltatrix, siendo los machos los que promoverían el intercambio
genético entre las diferentes poblaciones.
3. Mediante asignación genética, se ha detectado la presencia de una elevada

proporción de Pomatomus saltatrix procedentes de regiones alejadas dentro de
jaulas flotantes de lubina y dorada. La gran variación genética de los individuos
asociados a las jaulas flotantes, superior a la de las poblaciones circundantes,
sugiere que la acuicultura ejerce un efecto atractivo sobre esta especie y está
erosionando la diferenciación poblacional, induciendo mayor flujo génico
interregional.
4. En las especies euroafricanas del género Merluccius se ha encontrado, a partir

de análisis multivariantes, que el gradiente batimétrico parece ser la principal
variante ambiental que explique su distribución. La asociación de los distintos
165
Conclusiones
linajes genéticos con diferentes rangos de profundidad para las puestas sugiere
que el aislamiento reproductivo entre especies se debería a esta barrera
ecológica.
5. Se ha detectado un gradiente de hibridación introgresiva bidireccional entre las

dos especies de merluzas del Cabo (Merluccius capensis y M. paradoxus), que
pertenecen a dos linajes diferentes dentro del grupo de merluzas euroafricanas.
La hibridación detectada se distribuye en un gradiente progresivamente
creciente desde Sudáfrica a Namibia, siendo la zona afectada por el fenómeno
climático El Niño de Benguela la de mayor introgresión entre especies.
6. Mediante marcadores genéticos, se ha evidenciado hibridación introgresiva

entre las dos especies de calderones (género Globicephala) del océano Atlántico
en el hemisferio norte. La introgresión genética ha sido detectada en una única
dirección, siendo la especie afectada Globicephala melas. Unida a una menor
variabilidad genética, apuntan a una mayor vulnerabilidad de esta especie, cuya
integridad se vería amenazada por varias actividades antrópicas como cacerías
tradicionales y tráfico marítimo.
166
Conclusions
Conclusions
1. Evidences of regional population differentiation were found in Pomatomus

saltatrix by employing genetic markers, that appears to be promoted not only by
present isolation by distance but also by historical barriers that have been dated
with coalescence and are coincident with the last glaciations. Detected gene
flow between populations, even at inter-oceanic level, seems to counteract the
process of vicariant speciation in this genus.
2. Comparing the results of mitochondrial and nuclear genetic markers, a pattern

of sex-biased dispersal has been inferred in Pomatomus saltatrix, in which males
promote the genetic interchange between different populations.
3. The presence of a high proportion of Pomatomus saltatrix from remote regions

inside floating aquaculture cages of Sea bass and Sea bream has been detected
through genetic assignation. The high genetic variation of individuals from inside
floating cages, higher than the surrounding populations, suggests that
aquaculture exerts an effect of attraction on this species that is erasing
population differentiation and inducing greater interregional gene flow.
4. Based on multivariate analyses, a bathymetric gradient seems to be the main

environmental variable that explains the distribution of the Euro-African species
of the genus Merluccius. The association of different genetic lineages with
different spawning depth ranges suggests that reproductive isolation between
species might be due to this ecological barrier.
167
Conclusions
5. A bidirectional introgressive hybridization gradient was detected between the

two Cape hakes (Merluccius capensis and M. paradoxus), that belong to two
different lineages of Euro-African hakes. The detected hybridization is spread in
a progressive gradient from South Africa to Namibia, being the area affected by
the climatic event of the Benguela Niño the higher introgression zone between
species.
6. Introgressive hybridization between the two Pilot whales species (genus

Globicephala) off the North Atlantic Ocean was evidenced with genetic markers.
Genetic introgression was detected in only one direction, into the species
Globicephala melas. Coupled with a low genetic variability, it sets this species as
vulnerable, whose integrity could be threatened due to several antropic
activities such as grinds and maritime traffic.
168
References
169
References
170
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184
Glosario
185
Glosario
186
Glosario
Glosario
Batimetría: estudio de las profundidades marinas. Es el semejante a la altimetría

en el medio terrestre, pero localizada en el medio marino.
Codominancia: o dominancia compartida, sucede cuando dos alelos diferentes

están presentes en un genotipo y se expresa tanto la información del padre como
de la madre, es decir, ningún alelo es dominante o recesivo.
Conespecífico: Se considera que dos o más individuos, poblaciones o taxones son

conespecíficos si pertenecen a la misma especie.
Contacto secundario: ocurre cuando dos poblaciones o especies recientemente

separadas vuelven a entrar en contacto.
Filogenia: estudio histórico del desarrollo evolutivo de un grupo de organismos
Filogeografía: estudio de los procesos históricos y evolutivos que pueden ser

responsables de la distribución geográfica contemporánea de los organismos.
Filopatria: tendencia que presentan muchas especies de animales a permanecer en

el mismo territorio en que nacieron, o a volver al mismo para reproducirse.
Flujo génico: transferencia de información genética de una población a otra.

También expresado con frecuencia como migración efectiva.
Homing: tendencia que presentan muchos animales de volver a casa (territorio

donde nacieron). Ver también: Filopatria.
187
Glosario
Hibridación: Cruzamiento reproductivo entre individuos de poblaciones

genéticamente diferentes, sin importar el estado taxonómico de las poblaciones de
origen (especies, subespecies, etc.)
Interespecífico/a: Entre especies
Intraespecífico/a: Dentro de la misma especie
Introgresión: Flujo génico entre poblaciones cuyos individuos hibridan.
Morfometría: estudio basado en la forma y las medidas de cualquier objeto o ser

vivo, de cetáceos en este caso particular, para su clasificación e identificación.
Polimórfico/a: con gran variación genética (ver Polimorfismo)
Polimorfismo: variación genética entre individuos. Puede ir desde una simple

variación en una base nitrogenada entre individuos o ser más complejo.
188
189
190
Anexos
I
II
ANEXO 1.-
Permisos CITES de importación y exportación de muestras de calderones desde

países no europeos a España
a.- Permiso de importación de las muestras de las Islas Faroe.
b.- Permiso de exportación de las muestras de la Polinesia Francesa,

exportadas desde la Universidad de Auckland, Nueva Zelanda.
III
IV
a.-
V
VI
b.-
VII
VIII
ANEXO 2.-
Ficha utilizada para la toma de datos morfométricos de la asociación GREMMAR

con datos del varamiento de un calderón (Muestra: Galicia 06).
IX
X
ANEXO 3.-
Imagen aceptada para la portada de la revista Fisheries
XI
XII
ANEXO 4.-
Repercusión en los medios de comunicación de los resultados obtenidos en esta

Tesis Doctoral:
- Agencia Europea de Noticias Científicas “CORDIS: Community Research and

Development Information Service”: http://cordis.europa.eu/news/rcn/36215_en.html
- Portada de la pagina web de la Universidad de Oviedo durante Octubre 2013 y

nota de prensa de la Universidad: http://www.uniovi.es/-/investigadores-de-la-universidad-
de-oviedo-y-el-ieo-describen-por-primera-vez-a-nivel-mundial-el-hallazgo-de-un-hibrido-fertil-de-
cetaceo
- Portada de la página web del Instituto Español de Oceanografía (IEO) y nota de

prensa: http://www.ieo.es/prensa/NP_161013_hibridocetaceo.pdf
- Repercusión mediática de las notas de prensa de la Universidad de Oviedo e

Instituto Español de Oceanografía (IEO): Selección de los siete medios
nacionales más relevantes de más de una centena de noticias generadas en
octubre de 2013:
. Agencia EFE (http://www.efeverde.com/noticias/describen-por-primera-vez-el-hallazgo-

de-un-hibrido-viable-de-cetaceo/ ),
. RTVE (http://www.rtve.es/noticias/20131016/hallan-calderon-hibrido-primero-conocido-
entre-especies-etaceos/766101.shtml&ei=nI1eUu67M4LetAbt4oE4&usg=AFQjCNG5nMm
fTPSGJBMzKA8vyaG9ftFCHA),
. Europa Press (http://www.europapress.es/sociedad/noticia-descubierto-hibrido-calderon-
comun-tropical-20131016113531.html ),
. Diario ABC (http://www.abc.es/natural-biodiversidad/20131017/abci-calderon-hibrido-
201310171051.html),
. Diario La Razón (http://www.larazon.es/detalle_normal/noticias/3993110/verde/
descubierto-un-hibrido-de-calderon-comun-y-tropical#.Ttt19ge0JV8L4KA ),
. Informativos Telecinco (http://www.telecinco.es/informativos/sociedad/Descubierto-
hibrido-calderon-comun-tropical_0_1685250162.html ),
. Yahoo España Noticias (https://es.yahoo.com/?err=404&err_url=http%3a%2f%2fes.
noticias.yahoo.com%2fdescriben-hallazgo-h%25C3%25ADbrido-viable-cet%25C3%25A1ceo-
175107720.html),
- Radio y prensa regionales:
. Radio parpayuela, entrevista: http://www.parpayuela.com/

. La Nueva España, entrevista: http://www.lne.es/asturias/2013/07/17/miralles-
oceano-mayor-caja-sorpresas/1443033.html
. Asturias Mundial: http://www.asturiasmundial.com/noticia/50846/relevancia-mundial-
del-hibrido-fertil-de-cetaceo-investigado-en-asturias/
XIII
XIV

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Universidad de Oviedo

Departamento de Biología Funcional

Papel de las barreras biogeográficas e historia

Laura Miralles López

Servicio de Publicaciones de la Universidad de Oviedo

1.- Título de la Tesis

RESUMEN (en español)

Las especies son grupos de poblaciones naturales e intercruzables que están

aislados reproductivamente de otros grupos análogos. En el medio marino, las barreras

RESUMEN (en Inglés)

Species are groups of natural interbreeding populations that are reproductively

SR. DIRECTOR DE DEPARTAMENTO DE BIOLOGÍA FUNCIONAL

Gracias a toda la gente del Área de Genética, compañeros de Laboratorio, de la

También quiero agradecer a Santiago Lens, del Instituto Español de Oceanografía,

A Ester, mi capitana preferida y mi gran apoyo en multitud de ocasiones a lo largo

A Catarina y Mareike, my flatmates, porque no sólo fuisteis mis compañeras de

A las familias Aquanau y Medina, porque hicisteis de Alicante mi casa y me hiciste

Por último, quería agradecerle enormemente a la persona que ha estado a mi lado

Mamá, papá, Chencho, Marisa y Javi

except in the light of Evolution”

Las especies son grupos de poblaciones naturales e intercruzables que están

La hibridación y la introgresión son procesos que suceden regularmente en

Para la realización de esta Tesis Doctoral se seleccionaron tres modelos

Los resultados se reflejan en siete artículos científicos. Para Pomatomus

En los dos clados que componen las merluzas Euro-Africanas se ha podido

explica la especiación de este grupo. La hibridación introgresiva entre especies

Finalmente, la hibridación introgresiva principalmente unidireccional

El conjunto de resultados de esta Tesis Doctoral han permitido identificar

Species are groups of natural interbreeding populations that are

Hybridization and introgression are processes that occur with regularity in

Results were reflected on seven scientific articles. For Pomatomus saltatrix,

A model of ecological speciation, based on a bathymetric gradient, was

Finally, the unidirectional introgressive hybridization found into the long-

probably due to a combination of effects of human activity such as cultural grinds,

La especiación o formación de las especies ha sido un tema de gran interés

En 1809, Jean Baptiste Lamarck ofreció una interpretación filogenética de una

entre otros factores,

La especiación es la formación de nuevas especies; por tanto, para poder

El concepto de especie es un fundamento básico para cualquier rama de la

“Las especies son grupos de poblaciones naturales, real o

Utilizando el concepto biológico de especie como referencia, la especiación

Figura 2: Esquema de los modelos y mecanismos de evolución en la especiación alopátrica, peripátrica,

- Especiación alopátrica: Aislamiento por barreras geográficas en el que existe

- Especiación simpátrica: No hay aislamiento geográfico, las poblaciones

- Especiación parapátrica y peripátrica: La nueva especie surge en hábitats

En los últimos años, ha tomado fuerza un nuevo concepto de especiación: la

Barreras, aislamiento y especiación en el medio marino

Un cambio evolutivo rápido, así como la adaptación a un medio diferente, no

Por el contrario, la especiación simpátrica se define como especiación sin

Teniendo en cuenta la posible existencia de diferentes barreras, dependiendo

Hay que tener en cuenta también la perspectiva temporal. La variabilidad

La hibridación entre especies se conoce desde tiempos de Linneo (Mallet

La hibridación, tanto interespecífica como intraespecífica (ver Glosario), es

La hibridación natural y la introgresión entre especies ocurre con regularidad

La diferenciación genética entre las especies se debe, en parte, a la evolución

cambio climático, incluyendo el calentamiento global, el deshielo y la subida del

Como contrapunto a su apariencia espontánea en la naturaleza, la mayoría de

El ser humano en los procesos de especiación

Seehausen 2006). Todos estos procesos de reducción de ecosistemas podrían

pesquerías, donde se ha visto (tanto a escala temporal reciente, como evolutiva) la

A pesar del amplio abanico de ejemplos de los efectos negativos derivados de

Modelos para el estudio de la especiación en el medio marino

Debido a la enorme complejidad de factores naturales y antropogénicos que