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A TEST OF THE CHROMOSOMAL REARRANGEMENT MODEL OF

SPECIATION IN DROSOPHILA PSEUDOOBSCURA

Author(s): Kirsten M. Brown, Lisa M. Burk, Loren M. Henagan, and Mohamed A. F. Noor

Source: Evolution, 58(8):1856-1860.

Published By: The Society for the Study of Evolution

DOI: http://dx.doi.org/10.1554/04-174

URL: http://www.bioone.org/doi/full/10.1554/04-174

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1856

  2004 The Society for the Study of Evolution. All rights reserved.

 Evolution,  58(8), 2004, pp. 1856–1860

A TEST OF THE CHROMOSOMAL REARRANGEMENT MODEL OF SPECIATION IN

 DROSOPHILA PSEUDOOBSCURA

KIRSTEN   M. BROWN, LISA   M. BURK , LOREN   M. HENAGAN,   AND   MOHAMED   A. F. NOOR 1

 Department of Biologi cal Sciences, Louisiana State University , Life Science s Bldg., Baton Rouge, Louisiana 708031 E-mail: mnoor@lsu.ed u

 Abstract.   Recent studies suggest that chromosomal rearrangements play a significant role in speciation by preventingrecombination and maintaining species persistence despite interspecies gene flow. Factors conferring adaptation orreproductive isolation are maintained in rearranged regions in the face of hybridization, while such factors are elim-inated from collinear regions. As a direct test of this rearrangement model, we evaluated the genetic basis of hybridmale sterility in a sympatric species pair,  Drosophila pseudoobscura pseudoobscura an d D. persimilis, and an allopatricspecies pair, D. pseudoobscura bogotana and  D. persimilis. Our results are consistent with the proposed model: virtuallyall of the sterility factors in the former pair are associated with three inverted regions, whereas sterility factors arepresent in the collinear regions in the latter pair. These findings indicate recombination and selection may haveeliminated sterility factors outside the inverted regions between   D. p. pseudoobscura   and   D. persimilis, suggestingchromosomal rearrangements may facilitate species persistence despite hybridization.

Key words.   Chromosomal inversions,  Drosophila persimilis,  Drosophila pseudoobscura, hybrid sterility, speciation.

Received March 12, 2004. Accepted May 5, 2004.

Because the process of speciation is rarely observed, we

strive to understand this process by surveying many pairs or

groups of taxa and identifying consistencies among them.

For example, cytogenetic studies in the last century proved

that closely related species were often fixed for different chro-

mosomal rearrangements such as inversions or translocations

(Stebbins 1958; White 1969; King 1993). Researchers be-

lieved such rearrangements lead to meiotic disruptions in

heterozygotes (hybrids), and therefore contribute directly to

hybrid sterility. Although there is some support for this hy-

pothesis (e.g., Gropp et al. 1982; Rieseberg et al. 1995;

Hauffe and Searle 1998; Delneri et al. 2003), two difficulties

with this proposal became apparent in the next several years.First, new arrangements must have arisen in the heterozygous

state. If these new arrangements were strongly underdomi-

nant, they would have been immediately eliminated. Strong

genetic drift or meiotic drive (Walsh 1982) would be required

to allow a strongly underdominant new arrangement to

spread. If the new arrangements were only weakly under-

dominant, then they would not be significant contributors to

hybrid sterility. Second, several empirical studies of the fit-

ness of rearrangement heterozygotes suggested that they are

often as fit, or nearly as fit, as homozygotes for an arrange-

ment (Nachman and Myers 1989; Coyne et al. 1993; Reed

et al. 1995). As such, ‘‘chromosomal speciation’’ was con-

sidered largely untenable (Butlin 1993; Coyne 1994).A few years ago, a new formulation of chromosomal spe-

ciation was introduced by Rieseberg (2001), Noor et al.

(2001a), and Navarro and Barton (2003a). These authors sug-

gested that chromosomal rearrangements facilitate the per-

sistence of hybridizing species because they lock together

sets of genes conferring adaptation or reproductive isolation,

and prevent them from breaking apart through recombination

in hybrids. Recombination had long been recognized as a

force that would facilitate the fusion of hybridizing taxa (e.g.,

Felsenstein 1981; Barton and Bengtsson 1986; Trickett and

Butlin 1994), but chromosomal rearrangements could inhibit

recombination and avert fusion (see Ortı́ z-Barrientos et al.2002).

In support of this idea, Rieseberg et al (1999) showed thatrates of gene flow are higher between collinear than betweenrearranged chromosomes in   Helianthus   sunflower hybridzones. Sixteen of the twenty-six rearranged segments werealso significantly associated with pollen sterility. Similarly,in a literature survey of   Drosophila  species separated by hy-brid sterility, Noor et al. (2001a) found that virtually all taxanot separated by inversions were allopatric, whereas mosttaxa differing by one or more inversions were sympatric,suggesting inversions make the persistence of closely related

species more likely.The North American species pair   Drosophila pseudoob-

scura   and   D. persimilis   has been studied extensively withregard to the role of inversions in reproductive isolation and

species persistence. These species are separated by fixed ornearly fixed paracentric inversions on three of their six chro-mosome arms spanning various lengths: of approximately100 cytological bands across the genome, six cytologicalbands are inverted on the left arm of the  X -chromosome ( XL),11 cytological bands are inverted on the right arm of the  X -chromosome ( XR), and five cytological bands are inverted in

the center of the second chromosome (Tan 1935). However,recombination still occurs across the uninverted regions of 

these chromosomes (Dobzhansky and Tan 1936; Sturtevant

and Dobzhansky 1936; Noor and Smith 2000; Machado etal. 2002). Noor et al. (2001a,b) found that all forms of re-

productive isolation, and indeed other characters separating

these species (e.g., Noor and Coyne 1996; Williams et al.2001), map almost exclusively to the three regions inverted

between them. Similarly, Machado et al (2002; Machado and

Hey 2003) found fixed DNA sequence differences in rear-

ranged regions but extensive haplotype sharing in collinear

regions. Noor et al. (2001a) speculated that gene flow be-tween   D. pseudoobscura  and   D. persimilis  eliminated or pre-

vented the evolution of alleles conferring hybrid sterility be-

tween these species outside the rearranged regions.

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FIG. 1. Estimated phylogeny of the   Drosophila   species studied.The question mark indicates that hybridization between   D. pseu-doobscura  and   D. persimilis   may have predated the split of   D. p.bogotana  and   D. p. pseudoobscura.

TABLE  1. Fertility in pure-species and backcross hybrid males o Drosophila pseudoobscur a pseudoobscu ra   (ps),   D. p. bogotana(bog), and  D. persimilis   (per).  N 1 denotes the total number of fliedissected and genotyped.  N 2 indicates the number used for calculating the fraction fertile. Among the backcross hybrids, only thosehomozygous/hemizygous for all three inversions from one speciewere used for the calculation of fertility.

Males   N 1   N 2 % Fertile

Pure species

bog ERbog Sutatausa #5 strainbog Susa #6 strainper MSH 1993 strain

101100100137

101100100137

10099

100100

ps Flagstaff 1993 strainps James Reserve 032 strainps Mather 17 strain

164131108

164131108

989097

Backcross hybrids homo/hemizygous for all arrangements from onespecies

Backcross to bog (ER)Backcross to bog (Sutatausa #5)Backcross to bog (Susa #6)Backcross to ps (Flagstaff 1993)

Backcross to ps (Mather 17)Backcross to ps (JR032)

483451494

1147

498532

886446

166

6628

68647487

8993

Here, we provide a direct test of the new chromosomalspeciation model. Drosophila pseudoobscura bogotana (here-after, bog) occurs in Colombia and diverged from   D. pseu-doobscura pseudoobscura   (hereafter, ps) approximately

150,000 years ago (see Fig. 1 and Schaeffer and Miller 1991).These two subspecies bear the same sequence arrangements,

and thus also differ from  D. persimilis  (hereafter, per) by thesame three inversions. However, in contrast to ps, bog isallopatric to per and has thus not hybridized with it at leastin the past 150,000 years. Therefore, we predict that bog andper will be separated by alleles conferring hybrid sterilityboth within and outside the inversions, whereas ps and perwill have all their sterility associated with alleles within in-versions. Thus, the hybridization of bog and per effectivelyacts as a ‘‘negative control’’ for the effect of sympatry andhybridization in ps and per.

MATERIALS AND   METHODS

Fly Stocks

All initial crosses were done to the   D. persimilis   line fromMount St. Helena, California, collected in 1993 (see Noor1995). The   D. pseudoobscura   lines used were Flagstaff 1993(collected in 1993 from Flagstaff, Arizona), Mather 17 (col-lected in 1997 from Mather, California), and James Reserve032 (collected from the James Reserve in southern California,provided by Wyatt Anderson). The  D. p. bogotana lines usedwere Sutatausa #5, Susa #6 (both collected in 1997 by DianaAlvarez), and a white-eye mutant subculture of the el Recreoline (collected in 1978, provided by H. Allen Orr).

Crosses and fertility assays

Two classes of progeny were scored in this study: BCpsand BCbog, shortened from ‘‘backcross to  D. pseudoobscu-ra’’ and ‘‘backcross to  D. p. bogotana.’’ BCps progeny weregenerated by crossing females from a   D. pseudoobscura  lineto   D. persimilis   males, and backcrossing the F1   females to

the same  D. pseudoobscura   line. BCbog progeny were gen-erated by crossing females from a  D. p. bogotana   line to   D.

 persim ilis  males, and backcrossing the F1 females to the same D. p. bogotana  line. All crosses were carried out at 20 1C,85% relative humidity, on standard sugar/yeast/agar medium.The male backcross progeny were aged for eight days after

eclosion in groups of 5–30 individuals before fertility assaysApproximately 500 males were scored from each backcross

Fertility of pure strain males, F 1  males, BCbog males, andBCps males was assayed by dissection of testes in insecRinger’s solution using the method of Coyne (1984). A malewas scored as fertile if he had any motile sperm and sterileif no motile sperm were observed. Although other means exisfor quantifying fertility (White-Cooper 2004), Coyne’s(1984) method has been shown to be conservative (Campbelland Noor 2001). Dissected males were frozen in labeled 0.6

ml microcentrifuge tubes.

Genotyping assays

Previously, we demonstrated that alleles at different locwithin the inverted regions do not recombine in F1   hybridfemales (Noor and Smith 2000). Therefore, in this study, weonly genotyped one microsatellite from within each inversionfor all backcross hybrid males. DNA was extracted using theprotocol of Gloor and Engels (1992). The following microsatellite markers were genotyped for the three chromosomearms- 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 visualizedeither on 2% TBE agarose gels or on acrylamide gels on aLiCor 4200 DNA sequencer/analyzer. Genotypes were en-tered 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 least100 F1  males from every interspecies cross were scored, andall of them without exception did not bear motile sperm (hereafter, sterile). Among backcross hybrid males (BCps andBCbog), 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 inversionsfrom the   D. pseudoobscura   subspecies were scored for fertility. The figure denotes the four major chromosomes of   D. pseudoobscuraand  D. persimilis. Inversions separating these species are indicated by ovals. Chromosomal regions derived from D. persimilis are presentedin grey, and  D. pseudoobscura-derived chromosomal regions are presented in white. Regions that may have experienced recent naturalinterspecies introgression are hatched. These introgressing areas include all the collinear autosomal regions derived from D. pseu doobscura pseudoobscu ra  a nd  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).

that were homozygous or hemizygous for all three inversions

from ps or bog. If all hybrid sterility results from interactionsamong alleles between inverted regions, then the resultantmales would be all or virtually all fertile. In contrast, if thereare alleles conferring sterility outside the inverted regions,then several backcross hybrids will be sterile (Fig. 2).

Among BCps hybrid males, 87–93% were fertile, sug-gesting little or no effect of genes in collinear regions onhybrid male sterility. In fact, in the ps line from James Re-serve, (nonsignificantly) more backcross hybrid males werefertile than pure-species males. In contrast, among BCboghybrid males, 64–74% were fertile, suggesting a substantialeffect of genes in collinear regions on hybrid male sterility.The fertility difference between the BCps and BCbog linesis statistically significant (Mann-Whitney  U -test,  P  0.05).Further, comparison between any individual BCps and anyindividual BCbog line also showed significantly more ste-

rility in the BCbog line (chi-square tests,   P     0.05).

DISCUSSION

Virtually all hybrid male sterility was associated with thethree inverted regions separating the hybridizing species pair

 D. persimilis   and   D. pseudoobscura pseudoobscura. Back-cross hybrids that were homozygous or hemizygous for allthree inverted regions from D. pseudoobscura were nearly all

fertile. In contrast, we found evidence of alleles conferringhybrid male sterility in collinear regions of the allopatricspecies pair   D. persimilis   and   D. pseudoobscura bogotana.Approximately one-third of hybrids homozygous or hemi-zygous for the three inverted regions from   D. p. bogotana

were sterile. This difference is consistent with collinear re-

gions of the genome introgressing between   D. p. pseudoob-

scura and  D. persimilis, and hybrid-sterility-conferring alleles

being then eliminated from these regions. Introgression in

these collinear regions has been confirmed with DNA se-

quence data (Wang et al. 1997; Machado et al. 2002; Ma-chado 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 spe-

cies 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  sub-

species would have borne alleles conferring sterility in hy-

bridizations with  D. persimilis, but such alleles in collinear

regions would have been eliminated by hybridization, re-

combination, and selection from   D. p. pseudoobscura. Analternative 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 confer-

ring sterility in its regions inverted relative to   D. persimilis,

but   D. p. bogotana  accumulated such alleles in collinear re-

gions 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. persim ilis   that are currently present in collinear regions

of the genome.

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Three related models suggest how chromosomal rearrange-ments can facilitate species persistence through their effectson recombination. Noor et al. (2001a) suggested that dis-advantageous alleles (either through conferring differentialadaptation or reproductive isolation) would be eliminated af-ter hybridization from collinear regions because of asym-

metric effects: one allele might be disadvantageous in onespecies while the alternate allele would be advantageous orbear no cost in both species. As such, the former allele wouldbe eliminated while the latter would spread. In contrast, in-verted regions, based on the large number of genes encom-passed and linked, are more likely to bear multiple allelessuch that the whole region possesses a symmetric effect: oneallele disadvantageous in one species while the alternate al-lele is disadvantageous in the other species. Thus, neitherinversion can spread in a heterospecific genetic background.Rieseberg (2001) independently proposed a very similarmodel based on the summation of minor effects: invertedregions are less likely to introgress between species thancollinear regions because the former may bear large number

of individual genes that each individually confer slight re-productive isolation or differential adaptation (Ortı́z-Bar-

rientos et al. 2002). Finally, Navarro and Barton (2003a)illustrated how advantageous alleles associated with incom-patibilities between hybridizing populations would accu-mulate via a ‘‘snowball process’’ if associated with a non-recombining barrier to introgression such as a chromosomalrearrangement. These models make generally similar predic-tions, although Navarro and Barton’s (2003a) model suggeststhat incompatibilities are driven by positive selection andpredicts a molecular signature of natural selection in rear-ranged chromosomal regions (see Navarro and Barton2003b).

Based on these models and our results, we hypothesize

mutations that confer hybrid male sterility between   D. p. pseudoobscu ra   and  D. persimilis   arose in both inverted andcollinear regions of the genome. However, following hy-bridization, when such alleles introgressed between thesespecies, they were eliminated from collinear regions by re-

combination and natural selection. In contrast, sterility-con-ferring alleles were allowed to accumulate in collinear re-gions between D. p. bogotana and  D. persimilis because thesespecies never hybridize. This contrast provides an elegant‘‘negative control’’ to the earlier observation of hybrid ste-rility being associated with inversions in  D. p. pseudoobscura

and D. persimilis and supports a prediction unique to the threenew chromosomal speciation models.

Researchers have long known chromosomal rearrange-

ments are associated with speciation or species persistence,but the present results are more supportive of this associationresulting from the recombinational effects of rearrangementsrather than direct meiotic difficulties associated with rear-rangements. Drosophila p. bogotana  and  D. p. pseudoobscuraare identical in chromosomal arrangement, so a meiotic mod-el would not predict an excess of sterility in one of thesespecies over the other in hybridizations with   D. persimilis.

Given the widespread pattern of species diversity with chro-mosomal rearrangements and the many observations of thesemipermeability 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; Sætre et al. 2003; Eme-

lianov 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 Medica

Institute summer undergraduate research fellowship to KMB

National Science Foundation grants 9980797, 0211007, and

0314552, and Louisiana Board of Regents Governor’s Bio-

technology 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. Ortı́ z-Barrientos, L. Rieseberg, and an anon

ymous referee for constructive comments on this manuscript

and L. Lohmiller and R. Beauvais for technical assistance.

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Corresponding Editor: R. Harrison