key: cord-0723186-crxsv3y7
authors: David, Jean R.; Ferreira, Erina A.; Jabaud, Laure; Ogereau, David; Bastide, Héloïse; Yassin, Amir
title: Evolution of assortative mating following selective introgression of pigmentation genes between two Drosophila species
date: 2022-04-13
journal: Ecol Evol
DOI: 10.1002/ece3.8821
sha: 318f5f0ac2d22cb521e8428636408de20118cffb
doc_id: 723186
cord_uid: crxsv3y7

Adaptive introgression is ubiquitous in animals, but experimental support for its role in driving speciation remains scarce. In the absence of conscious selection, admixed laboratory strains of Drosophila asymmetrically and progressively lose alleles from one parental species and reproductive isolation against the predominant parent ceases after 10 generations. Here, we selectively introgressed during 1 year light pigmentation genes of D. santomea into the genome of its dark sibling D. yakuba, and vice versa. We found that the pace of phenotypic change differed between the species and the sexes and identified through genome sequencing common as well as distinct introgressed loci in each species. Mating assays showed that assortative mating between introgressed flies and both parental species persisted even after 4 years (~60 generations) from the end of the selection. Those results indicate that selective introgression of as low as 0.5% of the genome can beget morphologically distinct and reproductively isolated strains, two prerequisites for the delimitation of new species. Our findings hence represent a significant step toward understanding the genome‐wide dynamics of speciation‐through‐introgression.

the diagnostic characters also contribute, either directly or through genetic linkage, to reproductive isolation. The search for such traits, which were dubbed "magic," has been a "holy grail" in speciation genetics Servedio et al., 2011; Smadja & Butlin, 2011; Thibert-Plante & Gavrilets, 2013) . However, how such traits form is enigmatic, and theory predicts that substantial degrees of geographical isolation and long times of divergence are necessary for the build-up of genetic barriers to reproduction . Therefore, it has been argued that adaptive introgression, that is, the exchange of beneficial alleles between species with intermediate levels of reproductive isolation (Hedrick, 2013) , could significantly shorten the time of speciation.

Introduced alleles could epistically interact with the host genome leading to the rapid formation of populations that are phenotypically distinct and reproductively isolated from the parental species (Abbott et al., 2013; Payseur & Rieseberg, 2016; Richards et al., 2019; Schumer et al., 2014) . In spite of the growing evidence for the ubiquity of interspecific gene flow unraveled by recent comparative genomic studies in plants and animals (Edelman et al., 2019; Lamichhaney et al., 2015; Leducq et al., 2016; Pease et al., 2016; Racimo et al., 2015; Schumer et al., 2018) , experimental tests for the role of adaptive introgression in the evolution of reproductive barriers are rare. Indeed, two recent reviews on experimental speciation had barely addressed the question of adaptive introgression (Fry, 2009; White et al., 2020) .

For nearly 100 years, Drosophila species have been a primary model for the experimental study of speciation (Castillo & Barbash, 2017; Mallet, 2006) . Introgression between species with incomplete reproductive isolation has long been used to identify the quantitative trait loci (QTL) responsible for phenotypic differences and reproductive barriers (e.g., Ding et al., 2016; Massey et al., 2021; Shahandeh & Turner, 2020; Tanaka et al., 2015) . In those experiments, two species are crossed and their fertile F 1 hybrid females are backcrossed to one parental species for one or a few generations. Introgressed genomic regions are then assessed using molecular markers and isogenic lines are produced via inbreeding to test for the statistical association with the phenotype of interest. Such short-term introgression does not inform us much on how introgression can lead to the origin of new species. Indeed, whereas F 1 hybrid males are sterile, a proportion of males issued from the first backcross are often fertile. When those males are left to mate with the backcross females, the proportion of sterile males progressively diminish each generation. In the absence of conscious selection on a particular introgressed phenotype, alleles from one parent, usually the one that was not used in the backcross, are gradually purged out in less than 20 generations (Amlou et al., 1997; David et al., 1976; Matute et al., 2020) . Contrary to those experimental observations, comparative genomics studies have unraveled strong evidence for genetic introgression between many Drosophila species pairs (Lohse et al., 2015; Mai et al., 2020; Schrider et al., 2018; Turissini & Matute, 2017) , with the traces of introgression sometimes persisting for millions of years (Suvorov et al., 2022) .

To test for the effect of adaptive introgression on speciation, one should identify an easily measurable phenotype distinguishing a pair of species, deliberately select it in backcross flies for several generations, and then quantify the degree of reproductive isolation of introgressed flies with both parental species. Unfortunately, most sister Drosophila species are usually recognizable only on the basis of subtle differences in their genitalia whose dissection and measuring are quite difficult and laborious (Yassin, 2021) . A striking exception is the case of the species pair of D. yakuba and D. santomea, which, in addition to genital differences, also shows a contrasting pigmentation pattern (Lachaise et al., 2000) . Both species lack the characteristic sexual dimorphism of pigmentation found in all other species of the melanogaster subgroup, where the last abdominal segments of the females are lighter than those of the males. Those segments are equally dark or equally light in both sexes of D. yakuba and D. santomea, respectively. Both species can mate readily in the laboratory, producing fertile hybrid females but sterile males, and there is strong evidence from field studies and population genomics that hybridization takes place also in the wild on the island of Sao Tomé where D. santomea is endemic (Cariou et al., 2001; Llopart et al., 2005 Llopart et al., , 2014 Turissini & Matute, 2017) .

Leveraging the crossability of the two species, short-term introgression experiments were used to identify the QTL underlying their morphological differences (Carbone et al., 2005; Liu et al., 2019; Nagy et al., 2018; Peluffo et al., 2015) and reproductive isolation (Cande et al., 2012; Moehring et al., 2006a Moehring et al., , 2006b . Introgressing dark pigmentation alleles of D. yakuba in the genome of the lightly pigmented D. santomea indicated that at least 5 loci were responsible for the striking pigmentation difference, namely the melanin-synthesis genes yellow (y), tan (t) and ebony (e) and the transcription factors Abdominal-B (Abd-B) and POU-domain motif 3 (pdm3) (Liu et al., 2019) . Remarkably, longterm introgression experiments between D. santomea and D. yakuba showed, that in the absence of conscious selection on any of their morphological differences, reproductive isolation with the parental species may persist for 10 generations (Comeault & Matute, 2018) , but at generation 20, introgressed flies completely resemble their D. yakuba parent with no trace of isolation (Matute et al., 2020) .

In 2015, our late colleague Jean R. David (1931 David ( -2021 started two long-term introgression experiments. In the first one, he deliberately introgressed light D. santomea alleles in the genome of dark D. yakuba, whereas in the second experiment he performed the opposite introgression, that is, introgressing dark D. yakuba alleles in the genome of light D. santomea. In this paper, we report the progress of his 5-year experiments and the results of sequencing two lines from the first experiment. We show through behavioral assays that introgression of as low as 0.5% of the genome has been sufficient to produce flies that were morphologically and behaviorally distinct from both parental species, even after 60 generations from the end of selection. We discuss the relevance of our work to the role of adaptive introgression in speciation.

Two experiments were conducted from reciprocal crosses between a strain of D. yakuba, which was collected by L. Tsacas from Kounden, Cameroon in 1966, and D. santomea from the type laboratory strain collected by D. Lachaise from Sao Tomé Island in 1998. Strains and experimental lines were reared at 21°C on a standard Drosophila medium kept in culture bottles at a density of ~1,000 flies. The timeline of each introgression experiment is presented in Figure 1 .

For the "light yakuba" experiment: virgin D. yakuba females were crossed to D. santomea males. Fertile F 1 females were mated to D.

yakuba Kounden males, and the progeny called backcross to yakuba (BCyak). Backcross flies contained a small proportion (not determined) of fertile males. Those flies were used as a mass population to produce a self-reproducing strain. After a second generation of mass culture, phenotypes were observed on anesthetized, 3-5 days old flies, and we assumed that most females had already copulated, many of them with fertile males. Selection was made on females only, who were far more variable than males. At each generation ~50 females with the lightest phenotype were transferred to lay eggs in new culture bottles. Precise phenotypic measurements were not done on regular basis and the progress of selection (if any) was not monitored. However, from our empirical observations, the selection was not efficient; each generation, the light females produced the same proportion of light and dark flies. This result persisted for more than a year (~15 generations). Then, some positive effects were observed: pigmentation of the females became lighter, and also some effects were found on the males, who also could be selected, leading to the establishment of an introgressed D. yakuba strain in 2016 (hereafter BCyak), quite lighter than the typical D. yakuba, especially for the females. However, after 2 years from the end of selection, female dark pigmentation slightly increased, attaining the levels of those found in F 1 hybrids. So a second round of selection on both males and females restarted in 2018, leading to two new derived introgression strains denoted BCyak CC and BCyak selD for flies selected for their light and dark abdomen, respectively.

For the "dark santomea" experiment: virgin D. santomea females were crossed to D. yakuba males. The fertile F 1 females were backcrossed to D. yakuba males, and the progeny was reared as a mass culture. Selection started by keeping females with a slightly dark abdomen, but the progress was very slow and took more than a year.

Interestingly, the dark pigmentation of the males increased more rapidly than that of the females, and after about half a year males were also included in selection. In 2016, an introgressed D. santomea strain, darker than the typical D. santomea, especially for males, was established and denoted BCsan.

Throughout the introgression experiments, no samples were archived frozen or in alcohol for genome sequencing and subsequent behavioral assays. Following the perturbations related to the COVID-19 pandemic lockdowns in early 2020, and the deterioration of Jean David's health later that year, only two strains, denoted BCyak and BCsan were present at the time of genome sequencing in December 2020 and behavioral assays. Those two strains along with those of the parental species were used for genome sequencing and subsequent mapping of introgressed loci. Sequencing revealed both strains to be predominated by the D. yakuba genome, sharing two introgressed F I G U R E 1 Timeline of the "light yakuba" and "dark santomea" introgression experiments showing the origin of the introgression strains for which pigmentation was scored D. santomea loci at genes known to affect pigmentation (see Results below). Because selection on dark D. yakuba alleles in a D. santomea background would not have only fixed light D. santomea alleles, we therefore hypothesized that the two strains were derived from the same "light yakuba" experiment. This was reconfirmed by checking their male genitalia, which were both of the "yakuba" type, in contrast to previous microscopic preparations of BCsan strain up to April 2020.

A contamination occurring after this date has likely replaced BCsan with one of the BCyak lines. Because the two strains, BCyak and BCsan, had two and three fixed D. santomea loci (see Results below), the two strains were then denoted BCyak-2 and BCyak-3, respectively.

Abdominal pigmentation was scored on parental species, reciprocal F 1 hybrids and the introgression lines following the scoring scheme of David et al., 1990) , that is, the width of black area at the posterior part of each tergite was visually scored by establishing 11 phenotypic classes from 0 (no black pigment) up to 10 (tergite completely black).

Abdominal tergites 2-7 as well as tergite 8 (the epigynium) were considered for females and tergites 2-6 as well as tergite 9 (the epandrium) were considered for males. For the introgression lines, scoring was made in 2016 at the end of selection and then once each 2 years (i.e., in 2018 and 2020). For each strain, ≥4 days old, 10 females and 10 males were used. Pigmentation scores are provided in Table S1 . All statistical analyses were conducted using R (R Core Team, 2016).

We also aimed to quantify subtle differences in pigmentation intensity between the two strains that were sequenced in 2020, that is, BCyak-2 and BCyak-3. For this, flies were killed in 70% ethanol and wings and legs removed using a pair of forceps. Each fly was then individually placed on its left side in 2 ml 70% ethanol solution in an excavated glass block and photographed under a binocular Leica stereoscope provided with a digital camera connected to a computer. Flies were photographed and grayscale intensity was measured using ImageJ (Abramoff et al., 2004) after manually defining the contour of each abdominal tergite.

The two parental species differ in their male genital traits, with the most easily traceable character being the loss of a pair of hypandrial (sternite 9) bristles in D. santomea (Nagy et al., 2018) . At the end of selection in 2016, we dissected the male genitalia of the introgression strains and found that the presence or absence of the hypandrial bristles followed the direction of the backcross, that is, present in BCyak and absent in BCsan. Male genitalia were then routinely dissected on a regular basis to guarantee the distinction between the lines of the two experiments.

For the two strains BCyak-2 and BCyak-3, genomic DNA was extracted from 30 flies using standard DNA extraction kit protocol Nucleobond AXG20 (Macherey Nagel 740544) with NucleoBond Buffer Set IV (Macherey Nagel 740604). DNA was then sequenced on Illumina Novaseq6000 platform (Novogene UK company limited). In order to update the current reference genome of D. yakuba v1.05 retrieved from Flybase (https://flyba se.org/, Thurmond et al., 2019) , we compared this version to a genome of the same D. yakuba strain that was sequenced and assembled using hybrid short-read (Illumina) and long-read (Oxford Nanopore) method (http://flyseq. org; Kim et al., 2021) . We used assembly-to-assembly command in Minimap2 (Li, 2018) to generate a PAF file, based on which we attributed each new ≥100 kb-long contig to the corresponding 1.05 chromosomal arm according to the longest homology tract. We also mapped each coding DNA sequence (CDS) to the new contigs using Blast (Altschul et al., 1997) in order to localize previously unmapped 1.05 contigs and genes. For each chromosome, assembled scaffolds were then ordered according to the cytological map of D. yakuba in (Lemeunier & Ashburner, 1976) . This resulted into a newly assembled reference genome of D. yakuba (cf. Table S2 ) that we used for mapping introgressed loci.

Minimap2-generated SAM files were converted to BAM format using samtools 1.9 software (Li et al., 2009) . The BAM files were then cleaned and sorted using Picard v.2.0.1 (http://broad insti tute.github.io/picar d/). We generated synchronized files for the 20 D. y. yakuba lines using Popoolation 2. We then used a customized Perl script to extrapolate allele frequencies to 2 diploid counts for each strain, after excluding sites with less than 10 reads and alleles with frequencies less than 25% for the total counts using a customized Perl script (cf. Ferreira et al., 2021) . We also excluded tri-allelic sites for each line. We then parsed the parental strains for divergent sites, that is, sites with distinct alleles fixed in each strain, and estimated the ancestry proportion at each site in the two introgressed strains in 50 kb-long windows. All sequences were deposited in NCBI's Sequence Read Archive (SRA) associated to the Bioproject (PRJNA820524).

We estimated precopulatory reproductive isolation between the two parental and the two introgressed strains, Bcyak-2 and BCyak-3, Copulations were observed also for 2 h, and once copulation started flies were anesthetized under slight CO 2 , and the identity of the mating and the un-mating flies identified. In some instances, for example, those involving a D. santomea male, no marking was needed. For most other cases, flies were individually left to feed in vials with artificial food blue or red colorants (Sainte Lucie co., France) 24 h before the start of the experiment as in Comeault and Matute (2018) . A chisquared test was then conducted for each strain pairing to test the deviation from parity between homo-and hetero-gamic successful matings.

For all behavioral analyses, flies were maintained in a temperature-regulated fly room with glass windows, that is, with natural cycles of night and day. Copulations were conducted on lab benches under light conditions. Previous experimentations showed no differences in mating choice between D.

yakuba and D. santomea under light and dark conditions.

The trajectories of pigmentation evolution during the two 5-year introgression experiments are given in Figure 2 yakuba (t test for the sum of segments 5 and 6 = 9.25, p < 4.3 × 10 −6 ) but still much darker than D. santomea (t = 10.85, p < 1.8 × 10 −6 ).

However, the last segment, that is, the epandrium or tergite 9, became almost completely light (t = 10.16, p < 1.7 × 10 −6 ), as in D.

santomea (t = 1.00, p = .34). For the "dark D. santomea" experiment, introgressed females (Figure 2g ) at the end of selection in 2016 were darker than the parental D. santomea (t test for the sum of segments 6 and 7 = 10.11, p < 3.3 × 10 −6 ), but not as dark as D. yakuba (t = 7.60, p < 1.8 × 10 −5 ). The males (Figure 2h ), on the other hand, had much darker posterior abdomen (t test for the sum of segments 5 and 6 = 21.34, p < 5.1 × 10 −9 ), yet still lighter than D. yakuba (t = 10.96, p < 4.1 × 10 −7 ). The last segments in both sexes were completely light as in D. santomea. Remarkably, introgressed females from both experiments significantly differed (t = 9.46, p < 3.5 × 10 −6 ), but not introgressed males (t = 1.99, p = .065).

After 2 years from the end of selection in 2016, both experiments tended toward pigmentation values of the ancestral backcross parent, but at a much slower rate. This was most pronounced in females of the "light yakuba" experiment (t = 2.79, p = .021), but not in males (t = 1.02, p = .321), and in males of the "dark santomea" experiment (t = 3.42, p < .004), but not in females (t = 1.63, p = 0.121). For the second round of selection in the "light yakuba" experiment, starting in 2018, the two strains BCyak CC and BCyak selD very slightly differed only for male pigmentation of segments 5 and 6 in 2020 (t = 2.19, p = .042). This indicated that selection has attained its limits very rapidly in 2016, but morphological differences between introgressed flies and their parental species persisted for more than 60 generations after selection.

As stated in the Materials and Methods, we sequenced in December 2020 the genome of the two remaining introgressed strains in the laboratory, which were named BCyak and BCsan. We then estimated the ancestry proportion of both parental species across the genome.

This showed that both strains belonged to the "light yakuba" experiments, bearing only 5%-6% alleles from D. santomea. The two strains showed almost the same profile of D. santomea introgression tracts, which were classified either as fixed or nearly fixed (D. santomea ancestry ≥75%) and intermediate (D. santomea ancestry ≥40%) (Table 1 ; The two strains were likely derived from the BCyak CC and BCyak selD strains, which corresponded to the second round of selection in the "light yakuba" experiment, and which by 2020 slightly differed in male pigmentation (see above). However, the two sequenced strains, BCyak-2 and BCyak-3, did not show significant difference in pigmentation, even when more numerical analyses were used to quantify melanization ( Figure 4) . Nonetheless, both strains showed significant differences with the two parental species for females' segment 7 and males' segment 5, and from a single parent for females' segment 6 and males' segment 6, resembling D. santomea for the former and D. yakuba for the later.

In no-choice experiments, homogamic mating occurred with almost the same frequency between pairs belonging to the same F I G U R E 2 (a-d) Photomicrographs of females and males of the parental species, light Drosophila santomea (a, c) and dark D. yakuba (b, d) . (e-h) Pigmentation introgression trajectories in the "light yakuba" (e, f) and the "dark santomea" (g, h) experiments. (e-h) Principal component analysis (PCA) of pigmentation scores on six successive abdominal segments per individual was conducted on combined males and females data but each sex per experiment was presented in a separate panel according to the coordinates of the two first principal components. In each panel, 95% confidence ellipses for the two parental species are shown in yellow (D. sanromea) and black (D. yakuba). Colors refer to F 1 hybrids issued from the cross between female yakuba × male santomea (brown), BCyak 2016 (turquoise), BCyak 2018 (dark green), BCyak selD_2020 (dark blue), BCyak CC_2020 (light blue), F 1 hybrids issued from the cross between female santomea x male yakuba (orange), BCsan 2016 (pink) and BCsan 2018 (red). Arrows indicate the trajectory of pigmentation changes in each panel (Table 3) . However, sex-dependent assortative mating was found for all crosses between D. yakuba and introgressed strains. In all those crosses, females always showed a higher preference for homogamic males, whereas no significant departure from parity was observed for males.

We reported here the results of 5-year experiments to reciprocally introgress genes causing morphological difference between a pair of sister species with a major difference in body pigmentation, and a strong, yet incomplete reproductive isolation. We showed that such introgression was possible and that the limits of selection were attained within only a single year (~15 generations), with the new phenotypes of the introgressed flies remaining distinct from the parental species. Remarkably and contrary to previous studies with no conscious selection on a morphological trait (Amlou et al., 1997; David et al., 1976; Matute et al., 2020) , assortative mating persisted in the introgressed flies even after 4 years from the end of selection (~60 generations). The success of selective introgression might strongly depend on the nature of the phenotype. Pigmentation can easily be scored and measured and its variation often has a simple, oligogenic architecture (Massey & Wittkopp, 2016) . By contrast, when Amlou et al. (1997) tried to introgress resistance to a fruit toxin from D. sechellia into D. simulans, their attempt failed, likely due to the difficulty of measuring toxicity and to the polygenic nature of survival as a phenotype. Indeed, many known cases of cross-species adaptive introgression involve color variation, for example, coat in wolves (Anderson et al., 2009) , skin and hair colors in humans (Dannemann & Kelso, 2017) , wing patterns in mimetic butterflies (Edelman et al., 2019) , winter-coats in hares (Giska et al., 2019) , plumage in pigeons (Vickrey et al., 2018) and wagtails (Semenov et al., 2021) , and beaks in Darwin's finches .

A parallel dynamics of introgressed trait trajectories was observed in both experiments, characterized by an initial phase of slow progress of introgression during selection. This progress was F I G U R E 3 Proportion of D. santomea ancestry averaged over 50-kb windows in two introgressed "light yakuba" lines (a) BCyak-2 and (b) BCyak-3. Vertical dotted lines refer to the location of the five pigmentation genes that were identified in Liu et al.'s (2019) "dark santomea" investigation (in black) as well as the location of the transcription factor Gug (in red)

most likely due to the nature of the trait, that is, pigmentation is a complex trait with major epistatic and dominance interactions, and the efficiency of selection being applied to a single sex, the female.

Male sterility tends to decrease across successive generations, as introgressed incompatibility genes are selected against. Because selection was conducted on females that were presumably mated, it is likely that fertile males bearing the ancestral phenotype have sired the progeny of those females. In agreement with this hypothesis, and with our knowledge of the major contribution of the X chromosome to pigmentation differences between the parental species ( Note: Twenty copulating pairs were tested per cross. For heterogamic crosses, significant deviation from the homogamic D. yakuba cross, that is, 17 successful crosses out of 20, was estimated using chi-squared test: *<.05, **<.01, and ***<.001. different sex-specific regulatory changes affecting similar sets of melanin-synthesis genes, introgression of those changes in the new backgrounds could epistatically resuscitate the lost dimorphism.

We were not able to sequence our introgressed "dark santomea" flies which were lost by mid-2020, but fortunately Liu et al. (2019) have conducted similar experiment and identified at least five genes whose D. yakuba alleles darken D. santomea male pigmentation. Our introgressed loci in the "light yakuba" flies overlapped with three out of these genes, namely the X-linked melanin-synthesis genes y and t and the autosomal transcription factor pdm3. By contrast, we did not detect signal of introgression on either the melanin-synthesis gene e or the homeotic transcription factor Abd-B, which were identified in "dark santomea" (Liu et al., 2019) . increases pigmentation, with the increase being more pronounced in females (Rogers et al., 2014) . Whereas pdm3 is a suppressor of y in D.

santomea (Liu et al., 2019) , Gug is an enhancer of t and a suppressor of e in D. melanogaster (Rogers et al., 2014) . Therefore, it is possible that the gain of female-specific pigmentation in D. yakuba was partly due to a down-regulation of pdm3 whereas the loss of male-specific pigmentation in D. santomea was partly due to a up-regulation of Gug.

The lack of significant difference in pigmentation between BCyak-2 and BCyak-3 argues against any role of the 3L locus, including Gug, on pigmentation. However, we note that pigmentation analysis of those two strains has been made in December 2021 after at least 18 months from the end of the second round of selection in the "light yakuba" experiment. Laboratory experiments and population analyses in Drosophila have suggested that balancing selection may act on pigmentation genes, hence restoring their alleles to intermediate frequencies when selection ends (Kalmus, 1945; L'Héritier & Teissier, 1937; Rendel, 1951) . For example, pigmentation polymorphism in D.

kikkawai, which is controlled by the pdm3 locus (Yassin, Delaney, et al., 2016) , is maintained by heterozygous advantage in experimental populations (Freire-Maia, 1964) . Similarly, ancient balancing selection on t was demonstrated in D. erecta . Further isolation from pdm3 and t of the introgressed locus on 3L and subsequent molecular dissection are therefore needed to understand its potential role in pigmentation evolution.

Color-based assortative mating could lead to the loss of sexual dimorphism and ultimately precopulatory reproductive isolation. Our results showed that fixation of as low as 0.8 Mb (~0.5% of the genome) during selection on pigmentation loci has altered mating propensities between pure and introgressed flies. The demonstration of color-based (dis)assortative mating in Drosophila has long been problematic (Kopp et al., 2000; . Our behavioral assays support the presence of color-based assortative mating between D. yakuba and D. santomea, but in a way that was asymmetric between the sexes and dependent on the degree of divergence. On the one hand, light male D. santomea had almost 5-fold success in mating with introgressed light D. yakuba females than with dark pure D. yakuba in no choice experiments. On the other hand, light females from both introgressed BCyak-2 and

BCyak-3 showed preference for their own light males over pure dark D. yakuba males. This suggests that the two X-linked y and t loci that were fixed in both strains probably play a role in colorbased assortative mating. However, female-limited assortative mating also existed between the introgressed strains BCyak-2 and

BCyak-3, in spite of their great coloration resemblance. The fixed autosomal locus in BCyak-3 may therefore also contain elements affecting behavior. In addition to its possible effect on pigmentation, the transcription factor Gug also interacts with another transcription factor, hairy (h), which is also located in the same fixed locus, in affecting the size of male genital organs that are used to grasp the females during mating, namely the surstyli (claspers) (Hagen et al., 2021) . The effect of pigmentation genes on mating behavior can be attained either directly through pleiotropy or indirectly genetic linkage to other mating phenotypes (Wellenreuther et al., 2014) . Pleiotropy should drive more pervasive associations between pigmentation and mating behavior than linkage. A possible source of genetic linkage could have been the physical proximity in the low recombining subtelomeric region of the X chromosome between y and the enhancer of scute (sc) which led to the loss of the hypandrial bristles and gain of extranumerary sex comb teeth in D. santomea males (Nagy et al., 2018) . Both characters may be involved in copulation and consequently contribute to mating success or choice. However, we found through regular dissections of the genitalia that this strong linkage was broken during the first

year of the selection experiment, dissociating pigmentation, and hyprandial bristles.

In conclusion, our result demonstrate that selective introgression on a morphological phenotype could rapidly lead to the evolution of pervasive behavioral isolation. They hence complement previous Drosophila experimental speciation studies, which showed that adaptation from standing variation to contrasting environments could lead the evolution of reproductive isolation (Fry, 2009 ).

Pigmentation also responds to diverse natural selection pressures (Bastide et al., 2014) including those that discriminate the ecological niches of D. santomea and D. yakuba such as temperature, desiccation, and UV intensity (Comeault & Matute, 2021; Matute & Harris, 2013; Matute et al., 2009) . Further experimental manipulations, for example, testing competition between pure and introgressed flies in different environments, coupled with the investigation of postcopulatory isolation barriers, will definitively shed more light on genome dynamics of homoploid speciation in animals, hence bridging experimental studies with empirical field observations in a primary model.

We thank two anonymous reviewers for their constructive criticisms.

We declare no conflicts of interest. 

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Genetic control and evolution of sexually dimorphic characters in Drosophila

Towards the completion of speciation: The evolution of reproductive isolation beyond the first barriers

Élimination des formes mutantes dans les populations de drosophile. Cas des drosophiles ebony

Evolutionary novelties in islands: Drosophila santomea, a new melanogaster sister species from São Tomé

Evolution of Darwin's finches and their beaks revealed by genome sequencing

Speciation driven by hybridization and chromosomal plasticity in a wild yeast

Relationships within the melanogaster species subgroup of the genus Drosophila (Sophophora) -II. Phylogenetic relationships between six species based upon polytene chromosome banding sequences

Minimap2: Pairwise alignment for nucleotide sequences

The sequence alignment/Map format and SAMtools

Changes throughout a genetic network mask the contribution of hox gene evolution

Pigmentation and mate choice in Drosophila

Genetics of a difference in pigmentation between Drosophila yakuba and Drosophila santomea

Sequential adaptive introgression of the mitochondrial genome in Drosophila yakuba and Drosophila santomea

An anomalous hybrid zone in drosophila

Genome-wide tests for introgression between cactophilic Drosophila implicate a role of inversions during speciation

Patterns of genomic differentiation in the Drosophila nasuta species complex

What does Drosophila genetics tell us about speciation?

The paradox behind the pattern of rapid adaptive radiation: How can the speciation process sustain itself through an early burst?

Distinct genetic architectures underlie divergent thorax, leg, and wing pigmentation between Drosophila elegans and D. gunungcola

Chapter two -The genetic basis of pigmentation differences within and between drosophila species

Current topics in developmental biology

Rapid and predictable evolution of admixed populations between two drosophila species pairs

The influence of abdominal pigmentation on desiccation and ultraviolet resistance in two species of Drosophila

Temperature-based extrinsic reproductive isolation in two species of drosophila

The genetic basis of postzygotic reproductive isolation between Drosophila santomea and D. yakuba due to hybrid male sterility

The genetic basis of Prezygotic reproductive isolation between Drosophila santomea and D. yakuba due to mating preference

Correlated evolution of two sensory organs via a single cisregulatory nucleotide change

A genomic perspective on hybridization and speciation

Phylogenomics reveals three sources of adaptive variation during a rapid radiation

A major locus controls a genital shape difference involved in reproductive isolation between Drosophila yakuba and Drosophila santomea

R: A language and environment for statistical computing. R Foundation for Statistical Computing

Evidence for archaic adaptive introgression in humans

Mating of ebony vestigial and wild type drosophila melanogaster in light and dark

Searching for sympatric speciation in the genomic era

A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation

Supervised machine learning reveals introgressed loci in the genomes of Drosophila simulans and D. sechellia

How common is homoploid hybrid speciation? Evolution

Natural selection interacts with recombination to shape the evolution of hybrid genomes

Asymmetric introgression reveals the genetic architecture of a plumage trait

Magic traits in speciation: 'Magic' but not rare?

The complex genetic architecture of male mate choice evolution between Drosophila species

A framework for comparing processes of speciation in the presence of gene flow

Drosophila genomes

Genetic architecture and functional characterization of genes underlying the rapid diversification of male external genitalia between Drosophila simulans and Drosophila mauritiana

What do we need to know about speciation?

Evolution of mate choice and the so-called magic traits in ecological speciation

FlyBase 2.0: The next generation

Fine scale mapping of genomic introgressions within the Drosophila yakuba clade

Introgression of regulatory alleles and a missense coding mutation drive plumage pattern diversity in the rock pigeon

Sexual selection and genetic colour polymorphisms in animals

The past and future of experimental speciation

Systematics in the (Post)genomic era: A look at the drosophila model

Ancient balancing selection at tan underlies female colour dimorphism in Drosophila erecta

The pdm3 locus is a hotspot for recurrent evolution of female-limited color dimorphism in drosophila

Additional supporting information may be found in the online version of the article at the publisher's website.How to cite this article: David, J. R., Ferreira, E. A., Jabaud, L., Ogereau, D., Bastide, H., & Yassin, A. (2022) . Evolution of assortative mating following selective introgression of pigmentation genes between two Drosophila species. Ecology and Evolution, 12, e8821. https://doi.org/10.1002/ece3.8821