Inferring phylogenies and distributions of upper-Andean and Amazonian frogs from the genus Pristimantis using new and

previously published sequence data.

Pristimantis acuminatus

Name: Robyn Cuthbertson Matriculation number: 0906976c Level 4 Honours Project Supervisor: Dr Kathryn Elmer Submission date: 22 March 2013

Contents
Abstract ................................................................................................................................ 2 Introduction .......................................................................................................................... 3 Methods ................................................................................................................................ 6 Data collection .................................................................................................................. 6 Phylogenetic analyses........................................................................................................ 6

Results .................................................................................................................................. 8 Convergence assessment ................................................................................................... 8

Discussion........................................................................................................................... 13 Phylogenetic relationships ............................................................................................... 13 Model and analyses ......................................................................................................... 17 Convergence on stationarity ............................................................................................ 18 Wider implications .......................................................................................................... 18

Acknowledgements ............................................................................................................. 20 References .......................................................................................................................... 21 Appendix I .......................................................................................................................... 28 Appendix II ........................................................................................................................ 34 Appendix III ....................................................................................................................... 36

Abstract
The genus Pristimantis is the largest of the amphibian genuses with 454 species currently described and this number is likely to increase with more recently developed techniques for recognition of cryptic species and the introduction of new molecular techniques. The deep evolutionary history has been examined and the evolutionary relationship with Eleutherodactylus has been discussed by Heinicke, Duellman and Hedges (2007), inferring that Pristimantis split from the Eleutherodactylines 37 million years ago, with rapid speciation 24 million years ago and also with the uplift of the Andes, where mountain building first appeared around 23 Ma, and the most pronounced peaks followed between 12 and 4.5 Ma (Hoorn et al., 2010). My study involved the alignment and phylogenetic analysis of 112 sequences, followed by the investigation of the distributions of species in a selected clade. This revealed some relationships between species which support those found by Heinicke, Duellman and Hedges (2007) and some which differ new evolutionary relationships may be the result of the introduction of 30 new sequences into the analyses. Wide distribution of some of the species possibly suggest cryptic species complexes and indicate further studies need to be done in order to fully delimitate species such as Pristimantis ockendeni and Pristimantis chalceus. The implications for my study and those similar, for example Heinicke, Duellman and Hedges (2007), on the knowledge of evolutionary history and relationships can have an impact on the knowledge of the methods of diversification (for example due to topographical complexities) which in turn can give an indication of the history of the landscape. It can also affect conservation as subdivision of distributions due to delimitation of species can change the species conservation requirements.

Introduction

Frogs of the genus Pristimantis (Jimnez de la Espada 1870), the South American rain frogs, are distributed mainly in Central and South America . They are nocturnal and primarily arboreal (Kok and Kalamandeen, 2008), and are small in length like most direct developing frogs (range from 20-50mm) (Heinicke, Duellman and Hedges, 2007). They are also polychromatic which can cause problems in identification (Kok and Kalamandeen, 2008). Pristimantis is widely accepted to be the most speciose genus of the Neotropical amphibia, with an estimated species number of 454 (AmphibiaWeb 2013). This can be validated with comparison of other genuses in the family Strabomantidae: for example the genus Oreobates with 21 species listed, and the genus Bryophryne with just eight (AmphibiaWeb, 2013). With the increasing availability of molecular information it has become of great interest to researchers to utilise this information in order to infer phylogenies, the overall aim of which is to discover the topology of the tree and infer evolutionary relationships . However, the correct phylogenies can be affected by the model chosen to investigate the evolutionary history (Bollback, 2002). There are a variety of different methods, each with strengths and weaknesses (Yang and Rannala, 2012). The process of molecular phylogenetics that is, using DNA sequences to investigate the evolutionary histories and relationships between species (Hay, Ruvinsky, Hedges, and Maxson, 1995; Darst and Cannatella, 2003; Fouquet et al., 2007) allows for more precise phylogenetic inference than morphological classifications can allow. Species concepts have been discussed recently in terms of reclassifying species, with changes from the typical biological species concept to phylogenetic and evolutionary 3

species concepts (Hanken, 1999). South America has many areas of high species diversity (Myers 2000), with important protected areas such as Manu in Peru and Madidi in Bolivia to preserve niche biodiversity hotspots (Padial and de la Riva, 2009). There are numerous hypotheses as to why these areas of diversity exist, such as riverine barriers (Gascon, Lougheed and Bogart, 1996) and paleogeographic characteristics (Rsnen et al., 1996; Hughes and Eastwood, 2006). Wang et al., (2009) discuss two causes of speciation: firstly, where geographic and physical landscape barriers restrict the movement of individuals and so constrain gene flow between populations, in turn causing genetic drift and/or selection to result in divergence of phenotype and secondly, where gene flow against immigrant phenotypes is the result of natural or sexual selection, which causes divergence, and demonstrates that geographic isolation, although thought to be the primary reason for diversification and speciation, may not be as fundamental a theory as previously thought. Generally, it is established that geography has had a major impact on species diversity in South America, especially in the Andes and Amazonia (Elmer, Dvila and Lougheed, 2007). Recently Heinicke, Duellman and Hedges (2007) refined the Eleutherodactylines into three clades Middle American, the Caribbean, and finally the South American clade. It is the later in which Pristimantis is included, and it has been inferred that this genus split from the rest of the Eleutherodactylines 37 million years ago (Heinicke, Duellman and Hedges, 2007). The range of Pristimantis is central to the Andes, caused by changes in plate tectonics in the Palaeocene 65-34 million years ago (Hoorn et al., 2010). In terms of geological history this is very recent and it is apparent that Pristimantis became speciose in a short space of evolutionary time, diversifying rapidly from 24 million years ago (Heinicke, Duellman and Hedges, 2007). This idea is supported by changes in climate resulting in genetic isolation resulting in further speciation of the species and the pronounced uplift of the Andes in the last 4

12 4.5 million years (Hoorn et al., 2010). The genus invaded Central America on at least 11 separate occasions, and some of these occurred before the suggested closing of the Isthmus of Panama (Pinto-Sanchez et al., 2012) around 3 million years ago (Heinicke, Duellman and Hedges, 2007) for example, Pristimantis ridens was also established in Central America before the presence of the land bridge (Wang, Crawford and Bermingham, 2008), again consistent with the hypothesis of rapid radiation from South America . Arguments for high diversity of poison frogs in the Amazon basin due to rapid dispersal from neighbouring areas have also been made (Santos et al., 2009) and so this may also have occurred in Pristimantis. It is clear that even the current estimations of Pristimantis species numbers may be lower than the actual number of species present (Fouquet et al, 2007; Lehr, Moravec and Gagliardi Irrutia, 2010) due to unexplored regions, polymorphic species, cryptic species and species that have extremely similar morphologies. Recent analyses of species in the Pristimantis genus include those by Elmer and Cannatella (2008), Siqueira et al., (2008), Padial and de la Riva (2009), and Arteaga-Navarro and Guayasamin (2011), and with further work done in the area of phylogenetics and more scientific exploration in the region it is likely that there will be a continuation of these redefinitions of relationships in the genus. This study will attempt to determine the phylogenies of the South American clade of Pristimantis using recently acquired and previously-published sequence data, and comparisons with other papers will be made in order to infer the likelihood of the accuracy of results. Using Bayesian analyses, I will closer inspect a single clade in terms of altitude - in doing so I aim to understand better the patterns of diversity and species richness of the genus and investigate possible multi-complexes of species.

Methods Data collection

This study used data from 121 sequences of 12S ribosomal RNA (12S) and 16S ribosomal RNA (16S) mitochondrial genes. Of these, 30 were new sequences were provided by Dr Kathryn Elmer (see Appendix II) and 91 were published sequences were downloaded from NCBI database (see Appendix I). The South American clade group formed by Heinicke et al., 2007 (species numbers 162-248) was the source for which accession numbers were chosen and the outgroup was selected from the same study (see Appendix I).

Phylogenetic analyses
The removal of one outgroup species and the joining of 12S and 16S sequences from the same species resulted in a total of 112 sequences analysed with a total of 1017 sites. The sequences were aligned using the default settings of the ClustalW option in Geneious 6.0.6 (Biomatters, 2012), where the cost matrix used was IUB, the gap open cost was 15, and the gap extend cost was 6.66. MEGA 5.1 was used to extend the flanking regions of the sequences in order to make them the same length as 12S and 16S sequences have hyper variable regions which cannot be aligned. Ambiguous regions in the alignment were then removed using Gblocks 0.91b (Castresana, 2000), in which the default block parameters were: minimum number of sequences for a conserved position: 9; minimum number of sequences for a flank position: 14; maximum number of contiguous non-conserved positions: 8; minimum length for a block: 10; allowed gap positions: 0. The resulting alignment was comprised of 1017 sites. The MrBayes plugin (Hulsenbeck and Ronquist, 2001) in Geneious 6

6.0.6 was used to perform Bayesian analyses to reconstruct phylogenies with the GTR + I + model of evolution (adhering to the methods used by Heinicke, Duellman and Hedges, 2007). Two parallel runs of the Monte Carlo Markov chain algorithm were conducted with 1,100,000 generations (chain lengths) each. The burn-in length was 160,000 and branch lengths were unconstrained. The subsampling frequency was 200. The posterior output was examined to assess the convergence of likelihoods onto the stationary distribution of the two separate runs . A 50% consensus tree was formed using the resulting raw tree data (11002 trees) in Geneious 6.0.6 (Biomatters, 2012). The posterior probability (or clade credibility) values for the branch nodes were examined . Inferred relationships were examined. A clade encompassing 32 sequences was chosen for further analysis this clade was chosen based on the high proportion of new sequence data and species of interest included . For this clade, the altitudinal ranges were obtained from AmphibiaWeb (2013), IUCN (2012) and from the individually collected specimen information. A bar graph was created to compare these altitudinal ranges to further investigate their biodiversity and possible relationships.

Results Convergence assessment

The parameter estimates included the log likelihood score (LnL), total tree length (TL), the 4 stationary state frequencies (piT, piA, piG, and piC) and the 6 reversible substitution rates (rA-T, rA-G, rA-C, rC-G, rC-T, and rG-T), and the shape of the gamma distribution of rate variation across sites (alpha). The comparison of these parameters from each of the 2 runs shows that the results of each run were similar asides from the autocorrelation time which increased fairly substantially. The correlation graph shows a random distribution, or white noise of points. The density and relative density graphs for each run show similar patterns. The trace and sample trace was stochastic around the mean. These indicate convergence on stationarity in the Bayesian analyses and tree space by MCMC (see Appendix III).

Phylogeny assessment

Clade selected for further analysis

Fig. 1: 50% consensus tree from 11002 raw trees created using the MrBayes plugin in Geneious 6.0.6. (Biomatters 2012) showing the inferred phylogenies of 111 Pristimantis species sequences and 1outgroup sequence. Bayesian posterior probability values are shown.

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Altitude (meters above sea level) 500 1000 1500 2000 3000 3500

Altitudinal assessment

2500

Fig 2: Comparison of altitudinal distributions of species in the clade encompassing species from P. orestes to P. diadematus QCAZ18015. For published data, the altitudinal range was taken from IUCN (2012) and AmphibiaWeb (2013), and for new sequence data the altitude where the individual was located was taken from the active specimen information sheet provided by K. Elmer.
P. orestes P. simonbolivari P. trachyblepharis (QCAZ18235) P. trachyblepharis (QCAZ18236) P. chalceus P. luteolateralis P. librarius (QCAZ25589) P. unistrigatus P. sp. (QCAZ25768) P. ockendeni P. sp (QCAZ25582) P. sp (QCAZ25588) P. ceuthospilus P. cajamarcensis P. martiae (QCAZ25403) P. sp (QCAZ18233) P. achuar holotype (QCAZ25463) P.achuar (QCAZ25457) P. altamnis (QCAZ25808) P. sp.(QCAZ25576) P. kichwarum (QCAZ25579) P. croceoinguinis (QCAZ18231) P. croceoinguinis P. imitatrix P. malkini (QCAZ25444) P. lirellus P. altamazonicus (QCAZ25407) P. altamazonicus P. altamazonicus (QCAZ25535) P. diadematus (QCAZ18017) P. diadematus (QCAZ18014) P. altamazonicus (QCAZ25382) P. diadematus (QCAZ18015)

Distribution assessment
P. ockendeni: P. simonbolivari:

P. cajamarcensis:

P. orestes:

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P. ceuthospilus:

P. luteolateralis:

P. chalceus:

Fig 3. Distributions of 7 species of Pristimantis (P. ockendeni, P. ceuthospilus, P. simonbolivari, P. cajamarcensis, P. chalceus, P. luteolateralis, and P. orestes), from the clade encompassing species from P. orestes to P. diadematus, with altitudinal distributions above 1500m.a.s.l. All images from IUCN (2012). Species with altitudinal distributions above 1500m.a.s.l were chosen as occurrences at these altitudes are generally rarer and investigation into their distribution could possibly show multicomplexes of species.

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Discussion Phylogenetic relationships

74 of the 99 posterior probability values (75%) in the 50% consensus tree were over 80%, indicating the high likelihood of relationships in the majority of the nodes . However, there are several incidences where the posterior probability values were less than 70%, including several of the basal relationships. Unfortunately, because of low support, the deep relationships of these species cannot be confidently confirmed with this analysis . These deep relationships are likely to be ancient, and confidence in these phylogenies could mean a better understanding of the evolutionary histories of the genus overall. There is one occurance of phylogenetic polytomy these occur where there is a lack of information, the inability to determine the branching order, or possibly speciation events that occured at once (Garland and Diaz-Uriarte, 1999). In this case there is phylogenetic polytomy of P. nigrovittattus (QCAZ25789). I believe this was due to errors in the analysis rather than speciation from the rest of the species involved at the same time as the outgroup Phrynopys bracki. The new sequence Pristimantis altamazonicus QCAZ25382 was grouped separately from the other 2 new P. altamazonicus and the 1 published sequence of P. almatazonicus instead it was grouped with P. diadematus. This may have been due to discrepancies in sequence analysis, but as the distributions of the species are similar it is also possible that the inconsistent sequence of P. altamazonicus was simply mis-identified and should be named P. diadematus. This sequence was also found at a higher altitude than the rest of the species in the P. diadematus clade this could indicate that it is in fact a different species altogether, but further analyses would be needed to investigate this. 13

P. sp. QCAZ 25588 and P. sp. QCAZ 25582 are likely to be the same species as there was high support for a close relationship and they were also found in the same area . The most closely related species according to my analyses is Pristimantis ockendeni. Heinicke, Duellman and Hedges (2007) analysis found that the closest relative to P. ockendeni was P. unistrigatus this species was found in the similar position in my analyses but was not more closely related to P. sp. QCAZ 25588 and P. sp. QCAZ 25582 compared to P. ockendeni (see Fig. 4). With the wide distribution and the recognition that P. ockendeni is yet to be fully delimited is likely that P. sp (QCAZ25582 and QCAZ25588) are that of one of the species that has been or is yet to be delimited from Pristimantis ockendeni.

Fig. 4. Simple representation of the comparison of relationships between Pristimantis ockendeni, Pristimantis unistrigatus and two unidentified Pristimantis species in my own analysis and the analysis of Heinicke, Duellman and Hedges (2007).

It is likely that the sequence P. sp. QCAZ25392 is P. conspicillatus due to the high posterior probabiliy values for their relationship.The inferred relationships of P. condor, P. conspicillatus (QCAZ18007, QCAZ25392) and P. croceoinguinis (QCAZ25544) are similar to the inferred relationships proposed by Pinto-Snchez (2012) (see Fig. 5). They also find that P. malkini is the sister species to P. citrogaster with relatively high support, but my Bayesian analysis showed P. malkini to be more closely related to P. lirellus and P. imitatrix,

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with higher support. It is less similar to the relationships proposed by Heinicke, Duellman and Hedges (2007); they show P. croceoinguinis to be a sister species to P. citrogaster, and P. condor to be a derived species, whereas my analyses show P. citrogaster and P. croceoinguinis to be sister species (see Fig. 6). With a follow-up study I would consider removing the P. croceoinguinis sequence from the analysis in order to see if the relationships between P. condor, P. conspicillatus and P. citrogaster remained the same if so we could conclude that these relationships are more likely to be correct than those proposed by Heinicke, Duellman and Hedges (2007).

Fig. 5. Simple representation of the comparison of relationships between 3 species of Pristimantis in my own analysis and the analysis of Pinto- Snchez (2012) P conspicillatus here includes the sequences P. sp. QCAZ18007, QCAZ25392 and the published sequence of P. conspicillatus.

Fig. 6. Simple representation of the comparison of relationships between 4 species of Pristimantis in my own analysis and the analysis of Heinicke, Duellman and Hedges (2007)

With further investigation of the species listed in Heinicke, Duellman and Hedges (2007) as Pristimantis sp. AY326002, I found that this sequence is listed as an Eleutherodactylus species on NCBI. However, this was defined by Darst and Canatella 15

(2004) before the analyses done by Heinicke, Duelmann and Hedges (2007), and so I currently accept the laters definition of the sequence. P. acuminatus and P. lacrimosus were not included in the study by Heinicke, Duellman and Hedges (2007) nor by Pinto-Sanchez et al., (2007) it is possible that this study reveals newly discovered relationships, but it would be useful for further investigations of the relationships between these species and those surrounding them in the inferred phylogenies as the posterior probability value for splitting of P. lacrimosus was extremely low (52.3) and so this relationship cannot be established with confidence.

Fig. 7. Simple representation of the comparison of relationships between 5 species of Pristimantis in my own analysis and the analysis of Heinicke, Duellman and Hedges (2007).

The 32 species wide clade that was used for altitudinal analyses was chosen as it encompasses some of the species that have been most recently studied, namely P. achuar, P. altamnis, and P. kichwarum (and the previously mentioned P. sp.) and the majority of the information included in this clade was from the new sequences . The distribution map for P. ockendeni shows a vast range, but this species is undergoing scrutinisation. The small distributions of the recently delimited P. kichwarum, P. altamnis and P. achuar by Elmer and Canatella (2008) indicate that any occurence of P. ockendeni which has a large distribution should be studied further as the presence of cryptic

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species, each with a smaller seperate distribution, is likely. This may also be true of other species with wide distributions. The distribution of P. chalceus appears to be vast, but of the 25 localities in Ecuador, 20 of them occur below 1000m.a.s.l. (AmphibiaWeb, 2013) and so those at higher altitudes are probably uncommon. Even so, this wide distribution could indicate that the species name is an umbrella term for species that have yet to be delimited. All the sampling was performed in Ecuador, but for species which appear to have distributions which need to be further defined sampling further afield would be useful as individuals in those areas may give an indication of further delimited species.

Model and analyses

Each model available for phylogenetic analyses has its weaknesses. The GTR + I + model was chosen as it is more parameter rich than models such as the Jukes Cantor model (Hulsenbeck, Ronquist, Neilson and Bollback, 2001) and has been used in a number of studies involving phylogenetic relationships (Heinicke, Duellman and Hedges 2007; Santos et al., 2009; Pinto-Sanchez et al., 2012) and as the comparisons would involve two of these studies it was beneficial for the same model to be used. It has a relaxed assumption of equal base frequencies. The I symbol indicates that the base frequencies are stationary, and the symbol indicates a gamma distribution of rates for sites (Neilson, Lohman and Sullivan, 2001). The MCMC (Markov chain Monte Carlo) algorithm is used by MrBayes to estimate the posterior probabilities of trees (Hulsenbeck and Ronquist, 2001) and is useful as it takes phylogenetic uncertainty into account. However, it has been shown that even the widely used Bayesian methods may not be fully accurate but may be too liberal, especially if the model used is not complex enough (Suzuki, Glazko and Nei, 2002). With more time, analyses such 17

as maximum likelihood and bootstrap probability would be performed to deduce any discrepancies that may have occurred due to the use of the model and Bayesian methods chosen.

Convergence on stationarity
The output results show convergence on stationarity when addressing the correlation, density and trace plots of the runs. The shape of the correlation graph showed no affinity for increasing or decreasing over time, with a white noise pattern of points, another indication of convergence. Similarly, initially the shape of the trace and sample trace graphs are stochastic around the mean, showing that variation of the likelihood around the mean is high, but then nearing the end of the analysis it varies less so indicating the approach to stationarity. With analyses run for a longer period of time it is likely that the variation around the mean would decrease further and give greater confidence in the output. The standard deviation of the mean was 0.915 and 1.083 for run 1 and run 2 respectively it is expected that during analysis this value should approach 0. Overall, the likelihood of reaching stationarity in this project was high, but with a repetition of the analysis the program should be run for longer in order for better confidence in this respect .

Wider implications
As well as the inferrred deep evolutionary history of the genus, assessing the distribution of Pristimantis is useful in determining which diversification hypotheses is most likely to be correct - for example, it has been stated that diversification was ancient and due to tectonic events (with the formation of the Andes) rather than climatic change (Elmer, Dvila and Lougheed, 2007; Heinicke, Duellman and Hedges, 2007). 18

The importance of conservation is amplified by studies similar to my own as it is difficult to know what real damage anthropogenic practices may have on undiscovered cryptic species as rapid declines continue (Stuart et al., 2004, Bickford et al., 2007). For example, the subdivision of wide distribution ranges into multiple smaller regions due to the occurrence of newly delimited species can intensify the pressure on each new species, or mean that different conservation practices are needed (Kohler et al., 2005) this may be true for the example in this study of Pristimantis ockendeni. Globally, 1933 species of amphibians are listed on the IUCN Red List of Threatened Species as Vulnerable, Endangered or Critically Endangered (IUCN, 2012) due to known practices such as habitat destruction and fragmentation, infectious disease, and many enigmatic declines (Stuart et al ., 2004). The continued efforts to revise the taxonomy of the Pristimantis genus will enhance the knowledge of species requirements and the knowledge of the genus overall.

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Acknowledgements
I would like to thank Dr Kathryn Elmer for her continued support throughout my time working on this project. Her knowledge of the subject was indeed an inspiration and gave me drive to learn more about the subject. I would also like to thank Roderic Page for his knowledge of the program Geneious and Roman Biek for the access to the licence for the same program, and Hans Recknagel for his insight into the program Gblocks and for his assistance when things were not going smoothly. I would also like to thank Isabel Coombs for the use of the laptop used for the majority of the preliminary analyses. Thanks also to my friends and family for putting up with my stress-filled messages and phone calls. Title page photo credit: Santiago R. Ron, Museo de Zoologa, Universidad Catlica del Ecuador.

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IUCN (International Union for Conservation of Nature), Conservation International & NatureServe, 2004. Pristimantis luteolateralis. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2 IUCN (International Union for Conservation of Nature), Conservation International & NatureServe, 2004. Pristimantis ockendeni. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2 IUCN (International Union for Conservation of Nature), Conservation International & NatureServe, 2004. Pristimantis orestes. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2 IUCN (International Union for Conservation of Nature), Conservation International & NatureServe, 2004. Pristimantis simonbolivari. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2 Kass, R. W., Carlin, B. P., Gelman, A. and Neal, R. M., 1998. Markov Chain Monte Carlo in Practice: A Roundtable Discussion. The American Statistician, 52(2), 93-100 Khler, J., Vietes, D. R., Bonett, R. M., Garca, F. H., Glaw, F., Steinke, D., and Vences, M., 2005. New Amphibians and Global Conservation: A Boost in Species Discoveries in a Highly Endangered Vertebrate Group. Bioscience, 55(8), 693-696 Kok, P. J. R., and Kalamandeen, M., 2008. Introduction to the taxonomy of the amphibians of Kaieteur National Park, Guyana. ABC Taxa, 5, 238-243

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Lehr, E., Moravec, J., and Gagliardi Urrutia, L. A. G., 2010. A new species of Pristimantis (Anura: Strabomantidae) from the Amazonian lowlands of northern Peru . Salamandra, 46(4), 197-203 Myers, N., Mittermeier, R. A., Mittermeier, C. G., da Fonseca, G. A. B., and Kent, J., 2000. Biodiversity hotspots for conservation priorities . Nature, 403, 853-858. Neilson, M., Lohman, K., and Sulliovan, J., 2001. Phylogeography of the tailed frog (Ascaphus truei): Implications for the biogeography of the Pacific Northwest. Evolution, 55(1), 147-160 Padial, J. M ., and de la Riva, I., 2009. Integrative taxonomy reveals cryptic Amazonian species of Pristimantis (Anrua: Strabomantidae). Zoological Journal of the Linnean Society, 155(1), 97-122 Pinto-Snchez, N. R., Ibez, R., Madrin, S., Sanjur, O. I., Bermingham, E., and Crawford, A.J., 2012. The Great American Biotic Interchange in frogs: Multiple and early colonization of Central America by the South American genus Pristimantis (Anura: Craugastoridae). Molecular Phylogenetics and Evolution. 62, 954-972 Pyron, R. A., and Wiens, J. J., 2011. A large-scale phylogeny of Amphibia including over 2800 species, and revised classification of extant frogs, salamanders, and caecilians . Molecular Phylogenetics and Evolution, 61(2), 543-583 Rsnen, M. E., Linna, A.M., Santos, J.C.R., and Negri, F.R., 1995. Late Miocene tidal deposits in the Amazonian foreland basin. Science, 269, 386-390

25

Ron, S. R., Guayasamin, J. M., and Yanez-Munoz, M. H., 2013. AmphibiaWeb Ecuador. Version 2013.0. Museum of Zoology, Catholic University of Ecuador. Available at: <http://zoologia.puce.edu.ec/Vertebrados/anfibios/AnfibiosEcuador> Accessed 13 February 2013 Ronquist, F., Huelsenbeck, J. P., and van der Mark, P., 2005. MrBayes 3.1 Manual, Draft 5/26/2005 [online] Available at <http://www.cse.unsw.edu.au/~binftools/birch/birchhomedir/dat/GDE/GDEHELPsolaris-amd64/dat/dGDE/GDEHELP-osx-x86_64/doc/mrbayes/mb3.1_manual.pdf> Accessed 9 March 2012 Sayers, E. W., Barrett, T., Benson, D. A., Bryant, S. H,, Canese, K., Chetvernin, V., Church, D. M., DiCuccio, M., Edgar, R., Federhen, S., Feolo, M., Geer, L, Y., Helmberg, W., Kapustin, Y., Landsman, D., Lipman, D. J., Madden, T. L., Maglott, D. R., Miller, V., Mizrachi, I., Ostell, J., Pruitt, K. D., Schuler, G. D., Sequeira, E., Sherry, S.,T, Shumway, M ., Sirotkin, K., Souvorov, A., Starchenko, G., Tatusova, T. A., Wagner, L., Yaschenko, E., and Ye, J., (2009). Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2009 Jan;37(Database issue):D5-15. Epub 2008 Oct 21. Siqueira, S., Aguiar Jr, O., Strssmann, C., Del-Grande, M. L., Recco-Piemtel, S. M., 2008. Chromosomal analysis of three Brazilian eleutherodactyline frogs (Anura: Terrarana), with suggestion of a new species. Zootaxa, 1860, 51-59

26

Stuart, N. S., Chanson, J. S., Cox, N. A., Young, B. E., Rodrigues, A. S. L., Fischman, D. L., and Waller, R. W., 2004. Status and Trends of Amphibian Declines and Extinctions Worldwide. Science, 306, 1783-1786 Suzuki, Y., Glazko, G. V., and Nei, M., 2002. Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics. Proceedings of the National Academy of Sciences of the United States of America. 99(25), 16138-16143 Wang, I. J., and Summers, K., 2009. Genetic structure is correlated with phenotypic divergence rather than geographic isolation in the highly polymorphic strawberry poison-dart frog. Molecular Ecology, 19(3), 447-458 Wang, I. J., Crawford,A. J., and Bermingham, E., 2008. Phylogeography of the Pygmy Rain Frog (Pristimantis ridens) across the lowland wet forests of isthmian Central America. Molecular Phylogenetics and Evolution, 47(3), 992-1004 Yang, Z., and Rannala, B., 2012. Molecular phylogenetics: principles and practice. Nature Reviews Genetics, 13, 303-314

27

Appendix I: Table of species names and accession numbers used (Heinicke, Duellman and Hedges, 2007).
No. Species Museum voucher Lab tissue number Genbank accession no. 12S 16S

Pristimantis 162 walkeri Pristimantis 163 luteolateralis Pristimantis 164 parvillus Pristimantis 165 chalceus Pristimantis 166 ockendeni Pristimantis 167 unistrigatus Pristimantis 168 cajamarcensis Pristimantis 169 ceuthospilus Pristimantis 170 lirellus Pristimantis 171 imitatrix Pristimantis 172 croceoinguinis Pristimantis 173 altamazonicus Pristimantis 174 orestes Pristimantis 175 simonbolivari Pristimantis 176 riveti

Origin Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007)

KU218116

267231

EF493518

KU177807

267863

EF493517

KU177821

267864

EF493351

KU177638

267865

EF493675

KU222023

267253

EF493519

KU218057

267227

EF493387

KU217845

267211

EF493823

EF493663

KU212216

267198

EF493520

KU212226

267200

EF493521

KU215476

267205

EF493824

EF493667

KU217862

267213

EF493669

EF493665

KU215460

267204

EF493670

KU218257

267249

EF493388

KU218254

267248

EF493671

KU218035

267224

EF493348

28

Pristimantis 177 versicolor Pristimantis 178 phoxocephalus Pristimantis 179 spinosus Pristimantis 180 cryophilius Pristimantis 181 rhodoplichus Pristimantis 182 wiensi Pristimantis 183 petrobardus Pristimantis 184 melanogaster Pristimantis 185 simonsii Pristimantis 186 appendiculatus Pristimantis 187 pycnodermis Pristimantis 188 dissimulatus Pristimantis 189 calcarulatus Pristimantis 190 orcesi Pristimantis 191 glandulosus Pristimantis 192 inusitatus Pristimantis 193 acerus

Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007)

KU218096

267228

EF493389

KU218025

267222

EF493349

KU218052

267225

EF493673

KU217863

267214

EF493672

KU219788

267250

EF493674

KU219796

267251

EF493377

EF493668

KU212293 MHNSMWE D56846

267202

EF493825

EF493367

267438

EF493826

E F493664

n/a

n/a

AM039709

AM039641

KU177637

267866

EF493524

KU218028

267223

EF493680

KU179090

267867

EF493522

KU177658

267868

EF493523

KU218021

267221

EF493679

KU218002

267217

EF493676

KU218015

267218

EF493677

KU217786

267207

EF493678

29

Pristimantis 194 schultei Pristimantis 195 bromeliaceus Pristimantis 196 subsigillatus Pristimantis 197 nyctophylax Pristimantis 198 shrevei Pristimantis 199 euphronides Pristimantis 200 rozei Pristimantis 201 gentryi Pristimantis 202 truebae Pristimantis 203 curtipes Pristimantis 204 vertebralis Pristimantis 205 buckleyi Pristimantis 206 devillei Pristimantis 207 surdus Pristimantis 208 quinquagesimus Pristimantis 209 duellmani Pristimantis 210 thymalopsoides

Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007)

KU212220

267199

EF493681

KU291702

171051

EF493351

KU218147

267246

EF493525

KU177812

267869

EF493526

No voucher

266036

EF493692

BWMC6918

266624

EF493527

No voucher

102308

EF493691

KU218109

267230

EF493511

KU218013

267229

EF493512

KU217871

267215

EF493513

KU177972

267870

EF493689

KU217836

267210

EF493350

KU217991

267216

EF493688

KU177847

267871

EF493687

KU179374 n/a KU217998

267872 n/a KU21799 8

EF493690

AY326003

KU177861

267873

EF493514

30

Pristimantis 211 ocreatus Pristimantis 212 pyrrhomerus Pristimantis 213 festae Pristimantis 214 leoni Pristimantis 215 verecundus Pristimantis 216 celator Pristimantis 217 chloronotus Pristimantis 218 thymelensis Pristimantis 219 supernatis Pristimantis 220 sp. Pristimantis 221 urichi Pristimantis 222 latidiscus Pristimantis 223 colomai Pristimantis 224 cruentus Pristimantis 225 ridens Pristimantis 226 cremnobates Pristimantis 227 w-nigrum

Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007)

KU208508

267439

EF493682

KU218030

267441

EF493683

KU218234

267247

EF493515

KU218227

267437

EF493684

QCAZ12410

267646

EF493686

KU177684

267874

EF493685

n/a

n/a

AY326007

QCAZ16428

267644

EF493516

n/a

n/a

AY326005

n/a

n/a

AY326002

USNM33609

101646

EF493699

KU218016

267219

EF493698

QCAZ17101 AMNHA124 44 AMNHA124 551

267635

EF493354

267876

EF493697

267877

EF493355

KU177252

267878

EF493528

n/a

n/a

AY326004

31

Pristimantis 228 actites Pristimantis 229 lanthanites Pristimantis 230 crenunguis Pristimantis 231 labiosus Pristimantis 232 sp. Pristimantis 233 conspicillatus Pristimantis 234 condor Pristimantis 235 citriogaster Pristimantis 236 achatinus Pristimantis 237 lymani Pristimantis 238 fenestratus Pristimantis 239 bipunctatus Pristimantis 240 skydmainos Pristimantis 241 toftae Pristimantis 242 rhabdolaemus Pristimantis 243 pluvicanorus Pristimantis 244 sagittulus

Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007)

KU217830

267209

EF493696

KU222001

267252

EF493695

KU177730

267879

EF493693

EF493666

QCAZ19771 MHNSMLR 4341

267640

EF493694

266049

EF493356

QCAZ28448

267636

EF493529

KU217857

267212

EF493701

KU212278

267201

EF493700

KU217809

267208

EF493827

EF493660

KU218019 MHNSM929 8

267220

EF493392

266046

EF493703

KU291638 MHNSM100 71

171021

EF493702

266052

EF493393

KU215493

267206

EF493353

KU173492

267875

EF493706

n/a

n/a

AY843586

KU291635

171098

EF493705

32

Pristimantis 245 stictogaster Pristimantis 246 aniptopalmatus Pristimantis 247 peruvianus Pristimantis 248
..... Phrynopus

caprifer

Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007) Heinicke, Duellman and Hedges (2007)

KU291659

171080

EF493704

KU291627 MHNSM926 7

171070

EF493390

266050

EF493707

KU177680

267880

EF493391

260 bracki

USNM28691 9

171045

EF493709

33

Appendix II: New sequence information provided by Dr Kathryn Elmer

QCAZ number 25457 25463 16748 19664 25382 25407 25535 25808 Elevation (m.a.s.l) 239 239 230 230 973 245 239 610

Species name

Locality Kapawi lodge Kapawi lodge EC Yasuni EC Yasuni FMT La Selva Kapawi lodge Comunidad Asociacin Chonta Yacu Estacion Biologica Jatun Sacha, Napo Shaime, ZamoraChinchipe Kapawi lodge Estacion Biologica Jatun Sacha, Napo Estacion Biologica Jatun Sacha, Napo Estacion Biologica Jatun Sacha, Napo Estacion Biologica Jatun Sacha, Napo Azuela

Latitude S 0232'32" S 0232'32" S 040' 36'' S 040' 36'' S 0121'26" S 0029'89" S 0232'32" N 0006.69

Longitude W 7651'50" W 7651'50" W 7623'43'' W 7623'43'' W 7751'73" W 7622'43" W 7551'50" W 7722.46

Pristimantis achuar Pristimantis achuarholotype Pristimantis acuminatus Pristimantis acuminatus Pristimantis altamazonicus Pristimantis altamazonicus Pristimantis altamazonicus Pristimantis altamnis Pristimantis conspicillatus Pristimantis croceoinguinis Pristimantis croceoinguinis Pristimantis diadematus Pristimantis diadematus Pristimantis diadematus Pristimantis kichwarum Pristimantis lacrimosus

18007

S 0104'840"

W 7736502

419

18231

S 0418'92"

W 7839'84"

907

25544

S 0232'32"

W 7551'50" W 7736502

239

18014

S 0104'840"

419

18015

S 0104'841"

W 7736503

420

18017

S 0104'842"

W 7736504

421

25579

S 0103.740

W 7736879

390

15982

S 010'00"

W 7739'00"

unknown

34

Pristimantis lanthanites Pristimantis librarius Pristimantis malkini Pristimantis martiae Pristimantis nigrovitattus Pristimantis ockendeni Pristimantis ockendeni Pristimantis sp. Pristimantis sp. Pristimantis sp. Pristimantis sp. Pristimantis sp. Pristimantis sp. Pristimantis variabilis

25389 25589

La Selva Auca 14 Rd Estacion Biologica Jatun Sacha, Napo La Selva Lumbaqui Cuyabeno Estacion Biologica Jatun Sacha, Napo Shaime, ZamoraChinchipe Shaime, ZamoraChinchipe Shaime, ZamoraChinchipe La Selva Auca 14 Rd Auca 14 Rd Cuyabeno

S 0029'89" S 0041'85"

W 7622'43" W 7643'79"

245 255

25444

S 0104'840"

W 7736'502"

419

25403 25789 25576

S 0029'89" N 0006'69" S 0000'8"

W 7622'43" W 7722'46" W 7610'4"

245 610 230

25768

S 0104'840"

W 7736'502"

419

18233

S 0418'92"

W 7839'84"

907

18235

S 0418'93"

W 7839'85"

908

18236 25392 25582 25588 28430

S 0418'94" S 0029'89" S 0041'85" S 0041'86" S 0000'8"

W 7839'86" W 7622'43" W 7643'79" W 7643'80" W 7610'4"

909 245 255 256 230

35

Appendix III: Output tables and graphs from MrBayes (Hulsenbeck and Ronquist, 2001)
Table 1. Summary statistics produced from the MrBayes analysis.

Summary statistic Mean Standard deviation of mean Median 95% HPD lower 95% HPD upper Auto-correlation time (ACT) Effective sample size (ESS)

LnL_run1 -17914.636 0.915 -17914.23 -17938.889 -17892.597 5380.765 173.804

LnL_run2 -17911.164 1.083 -17911.767 -17938.827 -17882.131 5153.555 181.467

Fig. S1. Correlation of LnL_run1 and LnL_run2.

36

Fig. S2. Comparison of density between LnL_run1 and LnL_run2.

Fig. S3. Comparison of relative density between LnL_run1 and LnL_run2.

37

Fig. S4. Trace plot

Fig. S5. Trace plot (sample only)

38