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q 2004 The Society for the Study of Evolution. All rights reserved.
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AND
METHODS
N1
Pure species
bog ER
bog Sutatausa #5 strain
bog Susa #6 strain
per MSH 1993 strain
ps Flagstaff 1993 strain
ps James Reserve 032 strain
ps Mather 17 strain
101
100
100
137
164
131
108
N2
% Fertile
101
100
100
137
164
131
108
100
99
100
100
98
90
97
Fly Stocks
All initial crosses were done to the D. persimilis line from
Mount St. Helena, California, collected in 1993 (see Noor
1995). The D. pseudoobscura lines used were Flagstaff 1993
(collected in 1993 from Flagstaff, Arizona), Mather 17 (collected in 1997 from Mather, California), and James Reserve
032 (collected from the James Reserve in southern California,
provided by Wyatt Anderson). The D. p. bogotana lines used
were Sutatausa #5, Susa #6 (both collected in 1997 by Diana
Alvarez), and a white-eye mutant subculture of the el Recreo
line (collected in 1978, provided by H. Allen Orr).
Crosses and fertility assays
Two classes of progeny were scored in this study: BCps
and BCbog, shortened from backcross to D. pseudoobscura and backcross to D. p. bogotana. BCps progeny were
generated by crossing females from a D. pseudoobscura line
to D. persimilis males, and backcrossing the F1 females to
the same D. pseudoobscura line. BCbog progeny were generated by crossing females from a D. p. bogotana line to D.
persimilis males, and backcrossing the F1 females to the same
D. p. bogotana line. All crosses were carried out at 20 6 18C,
85% relative humidity, on standard sugar/yeast/agar medium.
The male backcross progeny were aged for eight days after
Genotyping assays
Previously, we demonstrated that alleles at different loci
within the inverted regions do not recombine in F1 hybrid
females (Noor and Smith 2000). Therefore, in this study, we
only genotyped one microsatellite from within each inversion
for all backcross hybrid males. DNA was extracted using the
protocol of Gloor and Engels (1992). The following microsatellite markers were genotyped for the three chromosome
arms- XL: DPSX002 or runt; XR: DPSX010; and 2: bicoid.
Primer sequences and amplification conditions are published
(Noor et al. 2000; Noor et al. 2001b). PCRs were visualized
either on 2% TBE agarose gels or on acrylamide gels on a
LiCor 4200 DNA sequencer/analyzer. Genotypes were entered into StatView for data analysis.
RESULTS
Males from the inbred lines of the pure-species bore motile
sperm (hereafter, fertile; see Table 1). In contrast, at least
100 F1 males from every interspecies cross were scored, and
all of them without exception did not bear motile sperm (hereafter, sterile). Among backcross hybrid males (BCps and
BCbog), about 35% of males were fertile.
We limited our analyses to those backcross hybrid males
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FIG. 2. Predictions of genetic test presented here, where only backcross progeny homozygous or hemizygous for all three inversions
from the D. pseudoobscura subspecies were scored for fertility. The figure denotes the four major chromosomes of D. pseudoobscura
and D. persimilis. Inversions separating these species are indicated by ovals. Chromosomal regions derived from D. persimilis are presented
in grey, and D. pseudoobscura-derived chromosomal regions are presented in white. Regions that may have experienced recent natural
interspecies introgression are hatched. These introgressing areas include all the collinear autosomal regions derived from D. pseudoobscura
pseudoobscura and D. persimilis. Arrows denote example interactions between pairs of loci that might result in hybrid male sterility. (a)
Backcross to D. p. pseudoobscura (BCps). (b) Backcross to D. p. bogotana (BCbog).
gions of the genome introgressing between D. p. pseudoobscura and D. persimilis, and hybrid-sterility-conferring alleles
being then eliminated from these regions. Introgression in
these collinear regions has been confirmed with DNA sequence data (Wang et al. 1997; Machado et al. 2002; Machado and Hey 2003; Hey and Nielsen 2004), and the present
study suggests a consequence of this introgression: loss of
factors conferring sterility. The inverted regions, however,
retain alleles conferring hybrid sterility and allow these species to persist.
The chromosomal rearrangement model test presented here
is robust to several possible evolutionary histories of these
taxa. Drosophila p. pseudoobscura and D. persimilis may have
been isolated through most of their divergence but came into
secondary contact after the split of D. p. pseudoobscura and
D. p. bogotana (see Fig. 1). Both the D. pseudoobscura subspecies would have borne alleles conferring sterility in hybridizations with D. persimilis, but such alleles in collinear
regions would have been eliminated by hybridization, recombination, and selection from D. p. pseudoobscura. An
alternative scenario could involve hybridization between D.
pseudoobscura and D. persimilis that predates the split of D.
p. pseudoobscura and D. p. bogotana. In that case, D. p.
pseudoobscura would have only accumulated alleles conferring sterility in its regions inverted relative to D. persimilis,
but D. p. bogotana accumulated such alleles in collinear regions only in its 150,000 years of isolation. These scenarios
can be distinguished through detailed examinations of the
sequences of sterility alleles separating D. p. bogotana and
D. persimilis that are currently present in collinear regions
of the genome.
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Three related models suggest how chromosomal rearrangements can facilitate species persistence through their effects
on recombination. Noor et al. (2001a) suggested that disadvantageous alleles (either through conferring differential
adaptation or reproductive isolation) would be eliminated after hybridization from collinear regions because of asymmetric effects: one allele might be disadvantageous in one
species while the alternate allele would be advantageous or
bear no cost in both species. As such, the former allele would
be eliminated while the latter would spread. In contrast, inverted regions, based on the large number of genes encompassed and linked, are more likely to bear multiple alleles
such that the whole region possesses a symmetric effect: one
allele disadvantageous in one species while the alternate allele is disadvantageous in the other species. Thus, neither
inversion can spread in a heterospecific genetic background.
Rieseberg (2001) independently proposed a very similar
model based on the summation of minor effects: inverted
regions are less likely to introgress between species than
collinear regions because the former may bear large number
of individual genes that each individually confer slight reproductive isolation or differential adaptation (Ortz-Barrientos et al. 2002). Finally, Navarro and Barton (2003a)
illustrated how advantageous alleles associated with incompatibilities between hybridizing populations would accumulate via a snowball process if associated with a nonrecombining barrier to introgression such as a chromosomal
rearrangement. These models make generally similar predictions, although Navarro and Bartons (2003a) model suggests
that incompatibilities are driven by positive selection and
predicts a molecular signature of natural selection in rearranged chromosomal regions (see Navarro and Barton
2003b).
Based on these models and our results, we hypothesize
mutations that confer hybrid male sterility between D. p.
pseudoobscura and D. persimilis arose in both inverted and
collinear regions of the genome. However, following hybridization, when such alleles introgressed between these
species, they were eliminated from collinear regions by recombination and natural selection. In contrast, sterility-conferring alleles were allowed to accumulate in collinear regions between D. p. bogotana and D. persimilis because these
species never hybridize. This contrast provides an elegant
negative control to the earlier observation of hybrid sterility being associated with inversions in D. p. pseudoobscura
and D. persimilis and supports a prediction unique to the three
new chromosomal speciation models.
Researchers have long known chromosomal rearrangements are associated with speciation or species persistence,
but the present results are more supportive of this association
resulting from the recombinational effects of rearrangements
rather than direct meiotic difficulties associated with rearrangements. Drosophila p. bogotana and D. p. pseudoobscura
are identical in chromosomal arrangement, so a meiotic model would not predict an excess of sterility in one of these
species over the other in hybridizations with D. persimilis.
Given the widespread pattern of species diversity with chromosomal rearrangements and the many observations of the
semipermeability of species boundaries (e.g., Harrison 1993;
Clarke et al. 1996; Butlin 1998; Jiang et al. 2000; Martinsen
et al. 2001; Besansky et al. 2003; Stre et al. 2003; Emelianov et al. 2004; Panithanarak et al. 2004), we postulate
that recombinational suppression may be a potent force in
preserving hybridizing sympatric species, and suggest the
model be tested across a broad taxonomic spectrum.
ACKNOWLEDGMENTS
This research was supported by a Howard Hughes Medical
Institute summer undergraduate research fellowship to KMB,
National Science Foundation grants 9980797, 0211007, and
0314552, and Louisiana Board of Regents Governors Biotechnology Initiative grant 005 to MAFN, and a Sigma Xi
grant-in-aid of research to LMH. We thank W. Anderson for
providing the strain from the James Reserve; H. A. Orr for
providing the mutant ER strain; S. D. Schully, R. Harrison,
C. Henzler, D. Ortz-Barrientos, L. Rieseberg, and an anonymous referee for constructive comments on this manuscript;
and L. Lohmiller and R. Beauvais for technical assistance.
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