The evolution of reproductive isolation in the Drosophila yakuba complex of species.

Received Date : 08-Aug-2014 Accepted Date : 15-Jan-2015 Article type : Research Papers

The evolution of reproductive isolation in the Drosophila yakuba complex of species David A. Turissini1, Geoffrey Liu1, Jean R. David2, 3 and Daniel R. Matute1* 1

Biology Department, University of North Carolina, Chapel Hill

2

Laboratoire Evolution, Genomes, Speciation (LEGS), CNRS, 91198 Gif sur Yvette cedex,

France; Université Paris-Sud 11, 91405 Orsay cedex, France. 3

Département Systématique et Evolution, Museum National d'Histoire Naturelle (MNHN), UMR

7205 (OSEB), 45 rue Buffon, 75005 Paris, France.

*Corresponding author: Daniel R. Matute Biology Department, University of North Carolina 250 Bell Tower Road, Chapel Hill, NC, 27599. Tel: 919-962-2077 Fax: 919-962-1625 E-mail: [email protected]

Running title: Fertile hybrids in the yakuba species complex

Keywords: hybridization, reproductive isolation, reinforcement, mitochondrial introgression, melanogaster subgroup

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jeb.12588 This article is protected by copyright. All rights reserved.

ABSTRACT In the Drosophila melanogaster subgroup, the yakuba species complex, D. yakuba, D. santomea and D. teissieri have identical mitochondrial genomes in spite of nuclear differentiation. The first two species can be readily hybridized in the laboratory, and produce fertile females and sterile males. They also form hybrids in natural conditions. Nonetheless, the third species, D. teissieri, was thought to be unable to produce hybrids with either D. yakuba or D. santomea. This in turn posed the conundrum of why the three species shared a single mitochondrial genome. In this report we show that D. teissieri can indeed hybridize with both D. yakuba and D. santomea. The resulting female hybrids from both crosses are fertile, while the hybrid males are sterile. We also characterize six isolating mechanisms that might be involved in keeping the three species apart. Our results open the possibility of studying the history of introgression in the yakuba species complex and dissecting the genetic basis of interspecific differences between these three species by genetic mapping.

INTRODUCTION Reproductive isolation demarcates when the speciation process is complete and separate species have arisen and is an important factor in the persistence of new species (Mayr 1942, Coyne and Orr 2004). Drosophila is one of the premier models for the study of the genetic basis of reproductive isolation (Nosil and Schluter 2011, Maheshwari and Barbash 2012 and references therein). Nonetheless, the extent to which hybridization occurs in nature remains unknown in many clades of Drosophila (Barbash 2010). The study of reproductive isolation in Drosophila focuses on either species pairs that have recently diverged or on pairs that diverged a long time ago. Each type of study aims to understand different biological phenomena. The study of reproductive isolation in recently diverged species reveals what biological features are important in the origin of new species or what features contribute to the persistence of species subject to introgression (Wu and Ting 2004, Orr et al. 2004). Investigating reproductive isolation between deeply diverged species reveals how genome evolution leads to sterility and inviability when long-separated alleles come together in hybrids.

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Two Drosophila complexes from the melanogaster subgroup have been pivotal to our understanding of the genetic basis of reproductive isolation in recently diverged species. The simulans subcomplex is composed of three sister species (D. simulans, D. sechellia and D. mauritiana) all of which diverged approximately 0.2 million years ago, (Kliman et al. 2000, McDermott and Kliman 2009, Garrigan et al. 2012). D. simulans is a semi-domestic species which originated in South West Africa (Lachaise and Silvain 2004). D. sechellia is endemic to the Seychelles archipelago (Tsacas and Bächli 1981, R’Kha et al. 1991), and D. mauritiana is endemic to the Mauritius and Rodrigues islands, also in the Indian Ocean (Tsacas and David 1974, David et al. 1989). Crosses between any pair of the three species from the clade produce sterile hybrid males and fertile hybrid females that can breed with males from either parental species (Coyne 1989, Hollocher and Wu 1996, Araripe et al. 2010). These species still show the potential for gene exchange as at least two of the species (Garrigan et al. 2012, Brand et al. 2013), D. simulans and D. sechellia, hybridize in their natural habitat (Matute and Ayroles, 2014). The second clade of recently diverged Drosophila, the species pair D. yakuba/D. santomea, also produces sterile hybrid males and fertile female hybrids. Drosophila yakuba is a human-commensal species that is widespread throughout sub-Saharan Africa (Burla and Genéve 1954, Lachaise et al 1988). Drosophila santomea, the closest relative of D. yakuba, is endemic to the highlands of São Tomé, a volcanic island off coast of Cameroon (Lachaise et al. 2000). On the extinct volcano of Pico de São Tomé, D. yakuba occurs at low elevations (below 1,450m) and is found in open and semi-dry habitats (Llopart et al. 2005). In contrast, D. santomea occupies the mist forests of the island at elevations between 1,153m and 1,800m (Llopart et al. 2005a, Llopart et al. 2005b). The two species recently entered secondary contact and occasionally hybridize in the midlands of Pico de São Tomé (Llopart et al. 2005a, Llopart et al. 2005b).

In more highly diverged species, the study of mechanisms that cause intrinsic postzygotic isolation, such as sterility and inviability, reveal how genomes evolve functional differences. Such cases provide very little insight into the origin of new species but shed light on the extent of genome divergence that can accumulate within clades of organisms that maintain the same body plan. The most studied case of hybrid breakdown is the inviability observed in D. melanogaster/D. simulans hybrids. In crosses between these two species, the hybrid progeny has


the sex of the D. melanogaster parent. The cross ♀D. melanogaster × ♂ simulans clade produces only sterile females while the hybrid males die as larvae that are unable to pupate (Sanchez and Dübendorfer 1983, Orr et al. 1997, reviewed in Sawamura and Tomaru 2002). The reciprocal cross produces hybrid males, while females die as embryos (Sawamura and Yamamoto 1993). Studies of these reciprocal crosses have led to the identification of alleles involved in hybrid breakdown (reviewed in Gérard and Presgraves 2009). The cross between the most diverged species in the melanogaster subgroup that can produce hybrids is between D. melanogaster females and D. santomea males (Matute et al. 2009, Gavin-Smyth et al. 2013). These hybrids have revealed the existence of extensive genetic divergence and the prevalence of epistasis between alleles from the divergent genomes with negative effects on fitness (Matute et al. 2010, Matute and Gavin-Smyth 2014).

Intermediate cases where hybridization between highly diverged species still results in gene flow are of particular interest. Population genetic studies suggest that hybridization and introgression might be common in the yakuba species complex (Bachtrog et al. 2009). Drosophila yakuba and D. santomea have identical mitochondrial genomes despite nuclear differentiation (Lachaise et al. 2000, Llopart et al. 2005, Bachtrog et al. 2009, Llopart et al. 2014). Mitochondrial homogeneity across species seems to result from recent mitochondrial gene flow (reviewed in Chan and Levin 2005, Toews and Brelsford 2012; Llopart et al. 2005a, Llopart et al. 2005b). Nonetheless, a third species, D. teissieri also has the same mitochondrial genome as D. santomea and D. yakuba. Levels of nuclear and mitochondrial polymorphism indicate that reduced mitochondrial divergence results from gene flow and strong selective sweeps in the three species (Monnerot et al. 1990, Bachtrog et al. 2009). Since mitochondrial introgression can only occur through the generation of hybrid females and repeated backcrossing, the common mitochondrial genome is strong evidence that the three species interbreed (or have recently interbred). To date, D. teissieri has been successfully hybridized with neither D. yakuba nor D. santomea.

Drosophila teissieri is distributed throughout tropical Africa, although it is thought to have occupied a much larger range in the past prior to humans expansion into the forests of SubSaharan Africa (Lachaise et al. 1981, Lachaise et al. 1988, Lachaise and Silvain 2004). The


species was first described in 1971 (Tsacas 1971) and even though it is usually found at high elevations, it is commonly found in the same locations (i.e., baited traps) where D. yakuba is collected (Devaux and Lachaise 1987). Coalescent and phylogenetic analyses date the divergence between D. teissieri and the yakuba/santomea species pair to around 1 MYA (Monnerot et al. 1990, Long and Langley 1993, Bachtrog et al. 2006). The nuclear genomes of D. yakuba and D. teissieri differ by numerous fixed inversions, and it has long been considered that these inversions might preclude hybridization (Lemeunier and Ashburner 1976, Lemeunier et al. 1986, Krimbas and Powell 1992).

Despite evidence of introgression across the species boundary, efforts to produce hybrids between D. teissieri and D. yakuba (or D. santomea) have thus far been unsuccessful. If the mitochondrial replacement results from recent admixture, at least one of the inter-species crosses should produce fertile hybrids in spite of the molecular and chromososomal divergences. In this report, we show that crosses from both directions can produce D. yakuba/D. teissieri and D. santomea/D. teissieri hybrids. Hybrid males are all sterile, but the females are notably fertile. We also report six reproductive isolating mechanisms that might be involved in the reduction of gene flow between these potentially-interbreeding species. We discuss our findings in the context of the role of fixed inversions in hybrid sterility and the potential role of natural selection on the evolution of reproductive isolation in Drosophila. Our results confirm the possibility of interspecific gene flow across the whole yakuba species complex and pave the way for further genetic investigations between these species.

METHODS Stocks We generated genetically heterogeneous strains of each species (i.e., synthetic lines) by combining virgin males and females from several isofemale lines. Drosophila santomea SYN 2005 was generated by J.A. Coyne by combining six isofemale lines collected in 2005 at the field station Bom Successo (elevation 1150 m). Drosophila yakuba SYN2005 was generated by combining five isofemale lines collected by J.A. Coyne in 2005 on the Pico de São Tomé (elevation 880m). D. teissieri SYN was generated by combining six isofemale lines (CT01,


CT02, CT03, collected in Cameroon; and Bata 1, Bata 2, Bata 3 collected in continental Equatorial Guinea). All stocks were kept in large numbers (over 50 flies per generation) after they were created. All rearing was done on standard cornmeal/Karo/agar medium at 24ºC under a 12 h light/dark cycle.

To confirm that the production of hybrids with D. yakuba and D. santomea was not specific to the D. teissieri SYN line, we used two additional lines: BRZ08 (isofemale line; Brazzaville, Republic of Congo) and Mount Selinda (mass culture, Zimbabwe). We confirmed the results obtained with the D. yakuba SYN 2005 line using a mass strain of D. yakuba from Kunden (Cameroon) established in 1967.

Finally, we used an attached-X stock from both D. yakuba and D. santomea (henceforth refered to as C(1)RM). In attached-X stocks, the two copies of the X-chromosome are physically fused and do not segregate at meiosis and are passed together into a single egg. This egg can be fertilized by a sperm carrying another X-chromosome, leading to a triploid metafemale that does not survive, or by a sperm carrying a Y-chromosome, leading to a female that is homozygous for the X-chromosome but heterozygous for the autosomes. These females also carry a Ychromosome and hybrids produce male progeny carrying the X-chromosome from the father. (Embryos with two Y-chromosomes fail to hatch.) These stocks were generated by X-ray irradiation and their precise genotype was D. santomea ♀C(1)RM gn; ♂ wild-type, and D. yakuba ♀C(1)RM y, wor; ♂ wild-type (Coyne et al. 2004).

Genome sequence and divergence We collected a D. santomea female in 2009 in the highlands of São Tomé outside of the hybrid zone with D. yakuba. Aditionally, we collected a D. teissieri female in Bata, Equatorial Guinea in 2009. We established an isofemale line (i.e., a laboratory stock derived from the progeny of a single inseminated field-collected female) from each of the two collected females. After 5 generations of establishing the isofemale lines, a virgin female was collected, aged to 4 days, and frozen. DNA was extracted from each female using previously described methods. Briefly, DNA was extracted with a QIAGEN DNA tissue extraction kit avoiding vortexing and using cut pipette tips. In both cases, the DNA concentration was over 20ng/uL. DNA was This article is protected by copyright. All rights reserved.

fragmented with a Nextera library preparation kit and DNA was sequenced with short read sequencing (Illumina HISeq 2,500). The resulting reads were 100bp. Reads were mapped to the D. yakuba reference genome using bwa mem 0.7.10 (Li and Durbin 2009).

Making hybrids Pure species males and females were collected as virgins under CO2 anesthesia and kept for three days in single-sex groups of 20 flies in 30mL, food-containing vials. On the morning of day four, we placed forty males and twenty females together at room temperature (21°–23°C) to mate en masse on corn meal media. We set up 100 crosses per species combination for a total of 900 crosses (six interspecific crosses and three interspecific crosses). Vials were inspected every five days to assess the presence of larvae. We transferred all the pure species adults to a new vial (without anesthesia) every ten days. This procedure was repeated until the cross did not produce any more progeny. For vials that had progeny, we added a 0.5% propionic acid solution to the food along with a pupation substrate to the vial (Kimberly Clark, Kimwipes Delicate Task). All hybrids were collected using CO2 anesthesia. The presence/absence of hybrid progeny was scored as a binomial outcome for each individual cross. The frequencies at which crosses produced progeny were compared by fitting a logistic binomial regression using R (R Development Core Team 2005). The response was whether a vial produced progeny or not, while the identity of the cross was the only fixed effect. The significance of the heterogeneity was assessed with a Wald test with one degree of freedom (df) using the function wald.test in the ‘aod’ R package (Lesnoff and Lancelot 2012).

Temperature preferences Thermal preferences were estimated by allowing flies to distribute themselves along a thermal gradient in a thermocline. The design of the apparatus was based on that of Sayeed and Benzer (1996) and is the same experimental setting as in Matute et al. (2008). The experimental setting consists of a rectangular plexiglass chamber (12 cm x 45 cm x 1 cm) with an aluminum floor. The inside of the apparatus can be divided into seven chambers, each 10.5 x 6 x 1 cm, by pushing a rod connected to six plexiglass partitions. The removable plexiglass lid on the apparatus has a small hole above the center of each segment for inserting a thermocouple to measure the temperature of the aluminum floor in each partition. To keep flies off the ceiling and


Sayeed, O., and S. Benzer. 1996. Behavioral genetics of thermosensation and hygrosensation in Drosophila. Proc. Natl. Acad. Sci. USA 93: 6079-6084. Serbus, L. R., C. Casper-Lindley, F. Landmann, and W. Sullivan. 2008. The genetics and cell biology of Wolbachia-host interactions. Annu. Rev. Genet. 42: 683-707. Sturtevant, A. H. 1920. Genetic studies on Drosophila simulans. I. Introduction. Hybrids with Drosophila melanogaster. Genetics 5: 488. Takahata, N., and M. Slatkin. 1984. Mitochondrial gene flow. Proc. Natl. Acad. Sci. USA, 81:1764-1767. Tamura, K., S. Subramanian, and S. Kumar. 2004. Temporal patterns of fruit fly (Drosophila) evolution revealed by mutation clocks. Mol. Biol. Evol. 21: 36-44. Toews, D. P., and A. Brelsford. 2012. The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol. 21: 3907-3930. Tsacas, L., 1971. Drosophila teissieri, nouvelle espèce africaine du groupe melanogaster et note sur deux autres espèces nouvelles pour l'Afrique (Dipt: Drosophilidae). Bull. Soc. Entomol. Fr. 76: 35–45. Tsacas, L., and G. Bächli. 1981. Drosophila sechellia, N. Sp., huitième espèce du sous-groupe melanogaster des iles Seychelles (Diptera, Drosophilidae). Rev. Fr. Entomol. 3: 146–150. Tsacas, L., and J. David. 1974. Drosophila mauritiana n. sp. du groupe melanogaster de l'île Maurice. Bull. Soc. Entomol. Fr. 79: 42–46. Turelli, M., and A.A. Hoffmann. 1995. Cytoplasmic incompatibility in Drosophila simulans: dynamics and parameter estimates from natural populations. Genetics 140: 1319–1338. Wasserman, M., and Wasserman, F. 1992. Inversion polymorphism in island species of Drosophila. In Evolutionary biology (pp. 351-381). Springer US. Werren, J. H. 1997. Biology of Wolbachia. Annu. Rev. Entomol. 42: 587-609. Wu, C. I., and C. T. Ting. 2004. Genes and speciation. Nature Reviews Genetics 5: 114-122. Yang, Z. 2007. PAML 4: a program package for phylogenetic analysis by maximum likelihood. Molecular Biology and Evolution 24: 1586-1591. Yukilevich, R. 2012. Asymmetrical patterns of speciation uniquely support reinforcement in Drosophila. Evolution 66: 1430-1446. Zabalou, S., S. Charlat, A. Nirgianaki, D. Lachaise, H. Merçot, and K. Bourtzis. 2004. Natural Wolbachia infections in the Drosophila yakuba species complex do not induce


each composed of 100 heterospecific and 100 conspecific matings, per cross. The heterospecific mating frequency per trial (block) was transformed to Is, an index of premating isolation which was calculated as:

(Coyne and Orr 1989)

We assessed whether there were differences in Is with a linear model where the identity of the cross was the only fixed effect. The residuals of the model were not normally distributed (Shapiro-Wilk test; W = 0.769, P < 1 × 10-10) and the data were resilient to transformation. We thus compared the values of Is in each of the heterospecific crosses between D. yakuba, D. santomea, and D. teissieri with a Kruskal-Wallis rank sum test with multiple post-hoc comparisons (function kruskalmc, R package ‘pgirmess’, Giraudoux and Giraudoux 2013).

Gametic isolation We watched single heterospecific pairs for 8 hours and kept the females that mated successfully for each of the six possible hybrid crosses. We repeated this approach until we collected 30 females from each of the heterospecific and conspecific crosses. We kept all females who mated (either to con- or heterospecific males) to measure gametic isolation. To prevent females from remating, males were removed from the vial by aspiration after mating. Each mated female was allowed to oviposit for 24 h in a vial, the female was transferred to a fresh vial, and the number of laid eggs were counted. Counting was repeated daily for 10 days. The production of eggs after each type of heterospecific mating was transformed to Ig, an index of premating isolation which was calculated as:

where produced in each vial, and

is the number of eggs is the average number of

eggs produced by conspecific matings of the maternal species involved in the hybrid cross. Ig values were compared across crosses using a Kruskal-Wallis test followed by multiple


comparisons (function kruskalmc, R package 'pgirmess', Giraudoux and Giraudoux 2013). This approach counts the total number of eggs regardless of whether they are viable or not.

Postzygotic isolation: Hybrid inviability We quantified the levels of hybrid inviability for all the F1 interspecific crosses involving D. teissieri and for the three pure species. (Hybrid inviability beween D. yakuba and D. santomea was reported in Matute and Coyne 2010.) After 2–3 days of crossing, both male and female flies were transferred to a collection cup with a yeasted apple juice plate to allow oviposition. In all crosses, embryonic lethality was quantified by counting the number of hatched and unhatched fertilized eggs (brown eggs) of an overnight deposition following 24 h incubation at 25°C. Briefly, viability was scored as the number of empty egg cases, while lethality was scored as unhatched eggs with discernible larval structures. We followed the same procedure to quantify lethality rates for intraspecific crosses. We quantified lethality for at least three replicates per cross and compared levels of inviability using a Kruskal-Wallis rank sum test (function kruskal.test, R package ‘stats’,R Development Core Team, 2005). Postzygotic isolation: male and female hybrid sterility Male sterility was measured by dissecting hybrid males and assessing whether they had motile sperm. All males were collected upon eclosion and were aged to 4 days. We removed the testis of each male and mounted them on chilled Ringer’s solution (4ºC). We mounted 20 pairs of testis per slide and the presence/absence of sperm was scored immediately. All scoring was done blindly (i.e., the scorer did not know the genotype of the cross when scoring). We applied the same approach to measure the proportion of sterile males in F1 and backcrossed hybrids. We compared the proportion of fertile males for each backcrossed genotype by fitting a logistic binomial regression in which whether the male was fertile or sterile was the response, and the genotype was the only fixed effect. The significance of the heterogeneity was assessed with a Wald test (df =1, R package ‘aod’, Lesnoff and Lancelot 2012). Female sterility was also measured as a binary trait, namely, the ability to produce progeny when virgin hybrid females were mated with males from both parental species. To do so, we collected hybrid females within the first eight hours after eclosion and kept them in groups of 20 during the first three days. On day four, each female was transferred to a 30mL corn


meal vial and was housed with 5 males of each parental species. We tended each vial following standard fly husbandry practices. If a female produced progeny, we did not identify the male that each female chose to mate (or whether the female mated with both), but only the ability of each female to produce progeny. We compared the proportion of fertile females for each backcrossed genotype by fitting a logistic binomial regression in which whether the female was fertile or sterile was the response, and the genotype was the only fixed effect. The significance of the heterogeneity was assessed with a Wald test (R package‘aod’, Lesnoff and Lancelot 2012, df =1). When wild type stocks are crossed, hybrid females from heterospecific crosses carry X chromosomes from the two parental species. This means that hybrid females are heterozygous for all X-linked loci, and all recessive alleles that might cause hybrid sterility when interacting with dominant autosomal alleles from either species will be masked. We further explored the role of the X-chromosome as a cause of hybrid sterility in hybrid females. We used D. yakuba and D. santomea stocks that carried fused X-chromosomes (attached-X). We used these mutants stocks to assess whether hybrid females homozygous for the X-chromosome for both D. yakuba, and D. santomea were fertile or sterile using the fertility measurements described above (Figure S1). Composite index of reproductive isolation We used an index to assess the sequenctial effect of the multiple intrinsic reproductive isolating mechanisms between species pairs. The potential levels of gene flow between D. yakuba and D. teissieri and between D. santomea and D. teissieri were calculated as the product of the reduction in gene flow resulting from each isolating mechanism:

Gene flow = (1-Is) × (1-Ig) × (1- lethalityegg) × (1- lethalitylarval) × (1-larval lethalitypupal) × (1sterility)

where Is is the index of behavioural isolation, Ig is the index of gametic isolation, and sterility is the average value between male and female sterility in F1s weighted by the frequency of each sex.


Ks measurements Ks was measured separately for the D. simulans and D. yakuba clades, and alignments were created using the D. simulans and D. yakuba reference genomes and one line each for the other species. The D. yakuba sequence and gene annotation information were downloaded from Flybase (ftp://ftp.flybase.net/genomes/Drosophila_yakuba/dyak_r1.3_FB2014_03/ ; Clark et al. 2007). The D. santomea and D. teissieri lines were Qiuja630.39 and Bata2 respectively, collected in the South side of São Tomé and the Bata airport in Equatorial Guinea. The D. simulans genomic sequence and gene annotation were obtained from(http://genomics.princeton.edu/AndolfattoLab/w501_genome.html (Hu et al. 2012), DenisMCL was the D. sechellia line (collected in the island of Denis in the Seychelles archipelago), and mau12w was the D. mauritiana line used (sequence downloaded from NCBI: SRR1555246, SRR483621) (Garrigan et al. 2014). Illumina reads from each species were mapped to the reference genome for their respective clade with BWA 0.7.10 (Li and Durbin 2009). A pileup file was generated using Samtools 0.1.19 (Li et al. 2009), and a consensus sequence was called at each site using a custom perl script. The most common allele was chosen for each site with sites with tied most common allele counts being set to ‘N’, and only sites with a coverage >= 10x were included. Genomic and genic alignments were created from the reference genomes and consensus sequences using a custom perl script. Ks was measured using codeml from the PAML 4.8 package (Yang 2007).

RESULTS D. yakuba and D. santomea have been known to produce fertile hybrid females since the discovery of D. santomea (Lachaise et al. 2000). D. teissieri is the sister to the D. yakuba/D. santomea species pair and was thought to not produce hybrids with either species. We explored whether we could produce F1 hybrids between D. teissieri and D. yakuba, and between D. teissieri and D. santomea.

In order to estimate the divergence between D. yakuba, D. santomea, and D. teissieri, we used genome-wide data and calculated Ks (rate of synonymous substitutions) using PAML. The distribution of Ks for the three pairs of species is shown in Figures 1 and S2. We found that the divergence between D. yakuba and D. santomea was on the order of the divergence among the


three D. simulans species subcomplex (Ksyak-san = 0.0479, Kssim-sech = 0.0501, Kssim-mau = 0.0471). Second, we found that as expected the neutral divergence between D. yakuba and D. teissieri is roughly equivalent to that between D. santomea and D. teissieri (Ksyak-tei = 0.1116; Kssan-tei = 0.1079). The divergence between these two species pairs is twice as much as the divergence between the more closely related pair, D. santomea and D. yakuba. We also compared the divergence between D. yakuba and D. teissieri, and D. santomea and D. teissieri with the divergence between D. melanogaster and D. simulans (Matute et al. 2010) and found comparable levels of neutral divergence between the two species pairs (Ksmel-sim = 0.101). We next estimated the potential for hybridization between the species of the yakuba species subcomplex. We set up 100 matings for each type of cross between the three species (three conspecific and six heterospecific). We found that both directions of crosses between D. teissieri and D. santomea produced hybrids. The same is true for crosses between D. teissieri and D. yakuba. Table 1 shows the frequency at which all the possible con- and heterospecific crosses produced progeny; these frequencies were significantly different among crosses (Binomial logistic regression followed by a Wald test: χ2 = 186.3 df = 5, P < 1 × 10-6; Table S1). Crosses involving D. teissieri produce progeny less frequently than heterospecific crosses between D. yakuba and D. santomea (nine out of ten pairwise comparisons: P < 0.001; Table S1). The abdominal pigmentation patterns of all the produced hybrids are shown in Figures 2 and 3.

We confirmed that the production of hybrids was not due to specific mutations in the D. teissieri SYN background. We used two alternative D. teissieri strains, BRZ8, collected in Brazzaville (Republic of Congo) and Mount Selinda (Zimbabwe, Tsacas 1970). This last one was the type strain used by for the description of the species. We also used an additional D. yakuba line, collected in Kunden (Cameroon). As expected, these lines also produced hybrids when mated with both D. yakuba and D. santomea. Nonetheless, the frequency of hybridization was much lower than in hybridizations with D. teissieri SYN, probably resulting from less vigorous courting of females due to inbreeding effects in isofemale lines (Table S2).


We counted the amount of progeny produced by each of the conspecific and heterospecific crosses (Figure 4) and detected heterogeneity in the amount of progeny produced between crosses (Kruskal-Wallis χ2 = 156.960, df = 8, P = 1 × 10-10; pairwise comparisons are shown in Table S3). Naturally, all heterospecific crosses produced far fewer offspring than conspecific crosses (Figure 4, Table S3). Heterospecific crosses involving D. teissieri (♀ D. teissieri × ♂ D. yakuba, ♀ D. teissieri × ♂ D. santomea, ♀ D. yakuba × ♂ D. teissieri, ♀ D. santomea × ♂ D. teissieri) produced fewer offspring than heterospecific crosses involving D. yakuba and D. santomea (♀ D. santomea × ♂ D. yakuba, ♀ D. yakuba × ♂ D. santomea). All pairwise comparisons are shown in Table S3.

We also counted the number of females and males produced by each cross. The three types of conspecific crosses produced the same number of females and males (Table 2, genotypes 1-3, 10-11), as did heterospecific crosses between D. yakuba and D. santomea (Table 2, genotypes 4-5). However, all heterospecific D. teissieri crosses produced more hybrid females than hybrid males (Table 2, genotypes 6-9). Heterospecific crosses involving attached-X females produced significantly more males than females (Table 2, genotypes 12-15). Most backcrosses produced progeny that followed a 1:1 sex ratio (Table 2, genotypes 16-27). The deviations in the expected sex ratio in heterospecific crosses between D. teissieri and D. santomea (or D. yakuba) indicate either the existence of hybrid inviability or of sex-ratio segregation distortion specific to hybrid crosses.

These results indicate that the heterospecific crosses between the members of the yakuba species complex produced fewer offspring, especially fewer males, than conspecific crosses. Counting progeny, however, in heterospecific crosses conflates all possible mechanisms of reproductive isolation. Given the potential for these species to interbreed, we studied what reproductive isolating mechanisms exist between D. teissieri and the other two species of the yakuba subcomplex.


(i) Temperature preferences There is evidence that species-specific thermal preference is one reproductive isolating mechanism at work in the yakuba species complex (Matute et al. 2009). D. yakuba and D. santomea come into secondary contact in the midlands of the volcanic island of São Tomé and hybridize at a low frequency. The two species segregate in different habitats of the island partially because they show different temperature preferences: D. santomea prefers cooler temperatures, while D. yakuba prefers higher temperatures (Matute et al. 2009). We assessed whether D. teissieri had different temperature preferences from the other two species. We used previously described methods and measured the temperature preference of the three species (see Methods, experimental runs were done for each sex, and included 200 flies from each species). The results are shown in Figure 5. We fitted a linear mixed model in which the temperature preference was the response, the sex and species of each fly were the fixed effects, and the experimental replicate was considered a random effect. The sex factor was significant (F1,18 = 78.722 , P < 1 × 10-4) indicating slight differences in temperature preferences between males and females. More importantly, we detected much bigger differences between species (F2,18 = 6548.262, P < 1 × 10-4). We also detected a significant interaction between sex and species (F2,18 = 130.963, P < 1 × 10-4). Post-hoc tests showed that of the three species, D. yakuba has the highest temperature preference (mean = 25.47ºC, SD= 2.85; Table S4), while D. santomea (mean = 22.56ºC, SD = 2.46) and D. teissieri (mean = 22.20ºC, SD = 2.58) have lower temperature preferences (Table S4). These results are consistent with the known geographic distribution of D. teissieri; even though it has a large range in Africa, it tends to be located at cooler mid and high elevations (> 500 m) than D. yakuba flies found in the same area. Even though temperature preferences are not a complete mechanism of reproductive isolation, they reveal that the behavioral ecology of these three species differ resulting in different habitat preferences.

(ii) Behavioral isolation The second tier of reproductive isolation that might occur after habitat isolation is female discrimination against heterospecific males. We measured how female choice could lead to behavioral isolation in two different ways. First, we assessed female behavior in no-choice experiments. We measured copulation frequency in heterospecific crosses when virgin females


were exposed to a male from another species. All heterospecific mating assays were run in parallel with two conspecific mating assays, one for each of the two species involved in the heterospecific cross, to make sure that females were receptive at the time of mating (N = 50 assays per cross). From the copulation frequency data, we calculated Is according to Coyne and Orr (1989). This index ranges from 0 to 1 and the higher values indicate strong discrimination against heterospecifics and low values indicate equal prevalence of hetero- and conspecific matings.

The three pairs of species show strong premating isolation in no-choice experiments (i.e., none of the Is values were equal to zero, Wilcoxon rank sum test with continuity correction, P < 1 × 10-10). We also detected a significant level of heterogeneity among heterospecific crosses in their magnitude of behavioral isolation (Kruskal-Wallis χ2 = 117.215, df = 5, P < 1 × 10-10). Of all heterospecific matings, the cross between ♀ D. yakuba and ♂ D. santomea was the most often successful, and the cross between ♀ D. yakuba and ♂ D. teissieri was the least successful (Table 3, Table S5). We used these pairwise comparisons to study the evolutionary history of behavioral isolation in the yakuba subcomplex.

First, we determined whether behavioral isolation was, as expected, stronger between more distantly related species. Pairwise comparisons between reciprocal crosses indicated that ♀D. yakuba ×♂ D. santomea crosses are much more likely than ♀D. santomea × ♂ D. yakuba crosses thus confirming previous results that indicate the existence of asymmetrical behavioral isolation (Table 3, Table S5). These two hybridizations were much more likely than any hybridization involving D. teissieri (Table 3, Table S5) suggesting that, as expected, crosses between the more divergent species are less common.

In order to determine whether any of the interspecific crosses with D. teissieri was more likely to occur (and thus shed some light on the origin of the widespread mitochondrial haplotype), we used the same pairwise comparisons. We detected no asymmetry in the interspecific reciprocal crosses between D. teissieri and D. yakuba (Table 3, Table S5), or between D. teissieri and D. santomea (Table 3, Table S5). It is worth noting that since these last


two sets of crosses are rare, we have little power to detect differences, and the potential lack of asymmetry should be taken with reservations.

We next explored whether reinforcing selection (i.e., the evolution of enhanced prezygotic isolating mechanisms as a byproduct of selection against maladaptive hybridization) has played a role in the evolution of behavioral reproductive isolation. The hypothesis is that if reinforcement has acted, then the pair that shares a geographical location, D. yakuba and D. teissieri should show stronger behavioral isolation than the pair with equal divergence but without overlapping geographic ranges, D. teissieri and D. santomea. ♀ D. santomea × ♂ D. teissieri crosses are much more successful than ♀ D. yakuba × ♂ D. teissieri crosses (Is-♀san × ♂tei

= 0.986, Is-♀yak × ♂tei = 0.999; Pairwise comparison test after Kruskal-Wallis, P < 0.05, Table

S5), indicating that D. yakuba females are more likely than D. santomea females to discriminate against D. teissieri males. This difference is asymmetric as crosses between ♀ D. teissieri and ♂ D. santomea are as likely to succeed as crosses between ♀ D. teissieri and ♂ D. yakuba (Is♀tei × ♂san

= 0.987, Is-♀tei × ♂yak = 0.992; Pairwise comparison test after Kruskal-Wallis, P > 0.05,

Table S5), indicate that D. teissieri females show no difference in their levels of discrimination towards D. santomea and D. yakuba males (Table S5).

We measured the magnitude of behavioral isolation in choice experiments as well as when females were exposed to a heterospecific and a conspecific male at the same time. Invariably, females choose conspecific males when they had the chance to discriminate (Table S6). The combination of these results indicates that even though behavioral isolation in the form of female choice is strong, it is not an absolute mechanism of reproductive isolation.

(iii) Gametic isolation Since the ejaculate and the female tract are under the constant influence of sexual selection, ejaculate × female interactions might go awry in heterospecific crosses and reduce the production of hybrid progeny (Manier et al. 2013). We studied post-mating phenomena after matings with a single heterospecific male (non-competitive gametic isolation) as measured by the number of eggs produced after heterospecific matings. We counted eggs and not adult


progeny to minimize the effects of hybrid inviability at different developmental stages. The mean number of offspring produced by each female over ten days following a heterospecific mating (and conspecific controls; N = 15 for each type of cross) is shown in Figure S3. In all cases, heterospecific matings produced fewer offspring than conspecific matings (Table 3, Table S7). Crosses between D. yakuba and D. santomea produced more progeny than heterospecific crosses involving D. teissieri but fewer than all conspecific crosses (Table 3, Table S7). All heterospecific crosses produced progeny for a shorter period of time than did conspecific crosses (Figure S3), indicating that sperm use and/or survival is less efficient in all heterospecific than in conspecific crosses.

We applied a series of comparisons similar to those described for behavioral isolation. First, we studied whether gametic isolation was, as expected, stronger in the more distant pairs. Drosophila yakuba showed higher levels of non-competitive gametic isolation from D. teissieri males than from D. santomea males (Kruskal-Wallis test with multiple comparison correction; P < 0.05). Similarly, D. santomea females were more gametically isolated from D. teissieri males than from D. yakuba males (Table S7, P < 0.05). Drosophila teissieri females, on the other hand, showed similar levels of gametic isolation from D. yakuba and D. santomea males (Table S7; P > 0.05). These results suggest that, indeed, gametic isolation is stronger in the pairs that are more divergent.

Second, we aimed to detect wheter there was asymmetric non-competitive gametic isolation between the three species pairs. As previously reported, the magnitude of gametic isolation is higher when ♀ D. santomea are mated to ♂ D. yakuba than in the reciprocal cross (Table S7, P < 0.05). We found no evidence of assymetric reproductive isolation between reciprocal crosses between D. santomea and D. teissieri (Table S7, P > 0.05) or between D. yakuba and D. teissieri (Table S7, P > 0.05).

Next we tested whether reinforcement has acted on gametic isolation. We compared the magnitude of the index of gametic isolation (Ig) in D. santomea × D. teissieri crosses with the magnitude of Ig in D. yakuba × D. teissieri crosses. We also compared the magnitude of Ig between D. teissieri × D. santomea and D. teissieri × D. yakuba crosses. We found that there


were no significant differences between any of the two pairs of crosses (Table S7, P > 0.05) indicating no effect of natural selection in gametic isolation.

The results from the studies of gametic isolation indicate that there is divergence in the interactions between the female tract and male ejaculate in the yakuba species complex. Such divergence is more pronounced in heterospecific crosses involving males from the most distantly related species, D. teissieri.

(iv) Postzygotic isolation: hybrid inviability We studied whether hybrid individuals were less likely to survive to adulthood than pure species individuals. We studied hybrid viability in three broadly defined developmental stages: embryo, larvae, and pupae. The relative viability at each of the developmental stages for each of the conspecific crosses and the four hybrid crosses involving D. teissieri is shown in Table 3 and Figure 6. Heterospecific crosses suffered more inviability than conspecific crosses at all developmental stages (embryonic viability: Kruskal-Wallis χ2 = 18.841, df = 6, P = 4.44 × 10-3; larval viability: Kruskal-Wallis χ2= 16.865, df = 6, P = 9.794 × 10-3; pupal viability: KruskalWallis χ2= 18.772, df = 6, P = 4.566 × 10-3). All pairwise comparisons are shown in Table S8. The existence of inviability at these three stages indicates that there has been molecular divergence between D. teissieri and D. santomea (and D. yakuba) in key developmental factors affecting proper development.

(v) Postzygotic isolation: hybrid male sterility To study hybrid male sterility in the D. yakuba/D. santomea/D.teissieri species complex, we determined what proportion of males from each of the six possible F1 and the 12 possible backcrosses were sterile. We dissected hybrid males and scored whether they had sperm in their seminal vesicles. The results of these dissections are shown in 4. As opposed to pure species progeny (Table 4, genotypes 1-3, genotypes 10-11), where the overwhelming majority of males are fertile, all hybrid males from the four possible crosses were sterile (Table 4, genotypes 4-9, genotypes 12-15). The seminal vesicles of the F1 males did not harbor motile sperm, indicating that there are defects during spermatogenesis in all F1 hybrid males from all crosses (Figure S4).


We also produced males through backcrossing by taking advantage of the fact that F1 females are fertile in all six interspecific hybridizations (see below). We compared the proportion of sterile males from the 12 possible backcrosses using a logistic binomial regression. The frequency of fertile males did not exceed 10% in any of the backcrosses that involved D. teissieri (Table 4, genotypes 16-27). The response of the linear model was whether males were fertile or not, and the genotype was the only fixed effect. This test revealed differences in the levels of male fertility in the backcrosses (χ2 = 206.1, df = 11, P = 1 × 10-10). We compared the levels of fertility of males from each type of backcross with a post-hoc analysis (Tukey HSD, Table S9). Because the crosses of each species pair in both directions produce fertile F1 females that we backcrossed to both parental species, we could determine whether the cytoplasmatic elements of any of the three species affected sterility (Table 4: pairwise comparisons 16 vs.18, 17 vs. 19, 20 vs. 22, 21 vs. 23, 24 vs. 27, and 25 vs. 26). Since none of these comparisons was significant, it is unlikely that the epistatic interactions involving either the mitochondrial genome or maternally deposited genes are responsible for hybrid sterility in these males. This is not surprising since this study was motivated by the shared mitochondrial genome of the three species. Instead, hybrid male sterility is likely caused by epistasis between autosomal factors, or between X-linked and autosomal alleles.

(vi) Postzygotic isolation: female sterility En masse backcrosses of F1 hybrid females revealed that at least some of them are fertile. We measured the proportion of these F1 hybrid females that were able to produce progeny. To do so, we housed a single virgin hybrid female with five males from each of the parental species. We tested 100 females from each of the 25 female genotypes shown in Table 5 (both hybrid and pure species). In these experiments, we did not assess the identity of the father but scored only whether or not the female was able to produce progeny. Not surprisingly, pure species females (Table 5, genotypes 1-3) produced progeny in all the attempted crosses. The vast majority of F1 hybrid females (Table 5, genotypes 4-9) were fertile (unlike the hybrid males) and produced progeny when they were mated to males of the parental species. The same was true for females produced by backcrossed F1 hybrid females (Table 5, genotypes 16-27). A binomial logistic model shows that there are no significant differences in terms of female fertility between


backcrossed female genotypes (Binomial logistic regression followed by a Wald test, χ2= 9.4, df = 11, P = 0.58).

Finally, we explored the effect of the X-chromosome on hybrid female fertility by using a genetic tool called an attached-X-chromosome (see methods for a description of the stocks). We crossed attached-X females from D. santomea and D. yakuba to D. teissieri males (Figure S1). The results of these crosses are listed in Table 5 (genotypes 10-15). We found a large proportion of D. santomea/D. teissieri F1 hybrid females carrying the attached-X chromosome from D. santomea are sterile. We also observed a high frequency of sterility in D. yakuba/D. teissieri hybrid females carrying the attached-X chromosome from D. yakuba. These results indicate the presence of recessive alleles on the D. santomea and D. yakuba X-chromosomes that interact with the D. teissieri genome and contribute to hybrid female sterility. These alleles are only expressed and cause female sterility when they are in their homozygous state and seem to be masked by the D. teissieri X-chromosome in hybrid crosses between wild-type lines.

Composite index of reproductive isolation We calculated the effect of three sequential intrinsic mechanisms of reproductive isolation in each of the four possible crosses involving D. teissieri (Table 6). The levels of gene flow allowed by the sum of the intrinsic mechanisms in all interspecific crosses is close to zero indicating low potential for gene exchange between D. teissieri and the D.yakuba/D. santomea species pair.

DISCUSSION Drosophila hybrids have been pivotal to the study of the biological mechanisms that allow potentially hybridizing species to persist in the face of gene flow. Drosophila teissieri produces hybrids with the other species of the yakuba species subcomplex (D. yakuba and D. santomea). The most recent common ancestor of these three species is hypothesized to have diverged about 1 million years ago (Bachtrog et al. 2006 but see Tamura et al. 2004). Our genomewide calculations reveal that the divergence between D. teissieri and D. yakuba (and thus D. santomea) is comparable with that between D. melanogaster and D. simulans. Interspecific crosses between the latter pair produces only hybrids of the sex of the melanogaster parent and hybrids are sterile (for an exception see Davis et al. 1996). The species of the yakuba


subcomplex are the most distantly related natural populations in the melanogaster subgroup of species to produce fertile female hybrids

Here we report six different mechanisms of reproductive isolation between these species pairs. Given the high levels of reproductive isolation between D. teissieri and the yakuba/santomea species pair, it is not surprising that previous attempts to produce these hybrids were unsuccessful. Lee and Watanabe (1987) succeeded in obtaining interspecific matings between D. teissieri females and D. yakuba males, but obtained no hybrid progeny. To our knowledge, there has been no other successful interspecific mating or hybrids produced between D. teissieri and D. yakuba (or D. santomea).

Our results have five implications for the general study of evolutionary biology. First, they confirm suspicions that the three species of the yakuba species complex can hybridize. The fact that these species produce fertile hybrid females capable of backcrossing raises the question of whether there has been gene flow between them. D. yakuba and D. santomea hybridize in nature, and show signatures of introgression in a few nuclear loci (Llopart et al. 2005). Comparative multilocus scans of the nuclear and mitochondrial genomes indicate that the mitochondrial genomes of the three species are almost identical in spite of nuclear differentiation (Monnerot et al. 1990, Bachtrog et al. 2006), suggesting recent introgression across the species boundaries. This report confirms that hybridization (and introgression) is possible not only between D. santomea and D. yakuba, but also with the more divergent D. teissieri.

Mitochondrial replacements are common across all taxa even at deep divergences (e.g., Bastos-Silveira et al. 2012). What were the evolutionary forces that led to a mitochondrial replacement in these three species? After admixture, the time required for an introgressed mitochondrial genotype to become common is inversely proportional to the hybridization rate (Takahata and Slatkin 1984). If the hybridization rate is low (as is likely the case between D. teissieri and the D. yakuba/D. santomea species pair), then even weak selection against the introgressed mitochrondria will be enough to prevent the fixation of the mitochondrial genotype (Takahata and Slatkin 1984).


Since a mitochondrial replacement driven by pure drift is unlikely, there are other explanations. Two possibilities have been proposed. The first involves introgression of the mitochondria from one species into the other(s) with positive selection on one or several mitochondrial alleles favoring allele frequency increases resulting in hitchhiking of the rest of the mitochondrial genome. At least two independent events of mtDNA introgression have occurred from D. yakuba into D. santomea, including an early invasion of the D. yakuba mitochondrial genome that fully replaced the D. santomea mtDNA native haplotypes and a more recent, ongoing invasion centered in the São Tomé hybrid zone. This last event has the signatures of positive selection and seems to be adaptive (Llopart et al. 2014).

The second possibility relates to cytoplasmatic incompatibility caused by Wolbachia or other intracellular bacteria (Serbus et al.2008). These bacteria are maternally inherited and are able to manipulate the reproduction of their hosts to enhance their own transmission (Werren 1997). The endosymbiotic bacteria are known to have an influence on mitochondrial evolution, but have a weaker influence on autosomal loci (Rousset et al. 1992, Charlat et al 2003). A new invasion of Wolbachia in one of the species, followed by hybridization, and then colonization of the intracellular bacteria across the species boundary could lead to a mitochondrial sweep without the need to invoke natural selection on mitochondrial loci. In D. simulans, several Wolbachia strains have spread in parallel over vast portions of the species’ geographic range in just a few generations (Hoffmann et al. 1986, Baba-Aissa et al. 1988, Binnington and Hoffmann 1989, Rousset et al. 1992, Turelli and Hoffman 1995). This process has lead to many different mtDNA haplotypes but no differentiation at nuclear loci (Dean et al. 2003, Lachaise et al. 2004). Nonetheless, none of the species of the yakuba complex are known to be fixed for a particular Wolbachia strain (Lachaise et al. 2000, Zabalou et al. 2004, Charlat et al. 2004). Also, of the Wolbachia strains isolated from D. yakuba and D. santomea, none seem to cause cytoplasmatic incompatibility (Charlat et al. 2004, Zabalou et al. 2004). We cannot currently disentangle between a mitochondrial replacement driven by selection or by cytoplasmatic incompatibility, or the concurrent action of both. Regardless of the actual cause, all possible explanations for the shared mitochondrial genome require the existence of hybridization among the three species, a prerequisite that had until now remained unproven


A second implication of our results is that the species from the yakuba complex can produce hybrids even though they carry multiple fixed inversions (Lemenuier and Ashburner 1976). Inversions can cause hybrid sterility in chromosomal heterozygotes through the production of unbalanced gametes in a wide variety of organisms (Noor et al. 2001, Rieseberg 2001, Anton et al. 2005). Sterility by structural misalignining of inversions is also a common condition in Drosophila (Naveira et al 1984, Wasserman and Wasserman 1992). Our results dispel the idea that the fixed inversions between D. yakuba and D. teissieri (and consequently between D. santomea and D. teissieri) preclude the possibility of admixture. Numerous chromosomal rearrangements are present on all but the fourth chromosome (Lemenuier and Ashburner 1976). The role of these fixed chromosomal inversions is a topic to be researched in the future, and we cannot rule out heterozygosity as a potential cause of male hybrid sterility. Nonetheless, our data show that hybrid females are fertile, even though they are heterozygous for all fixed inversions, indicating that inversion heterozygosity has no detectable effect on female fertility.

A third implication is the high number of reproductive isolating mechanisms between these three species. We consulted an exhaustive compilation of the magnitude of reproductive isolation among Drosophila species (Yukilevich 2012) and determined that out of 103 hybridizations in the Drosophila genus for which females are fertile and males are sterile, the cross between D. teissieri × D. santomea (and D. teissieri × D. yakuba) ranks as the 36th most divergent (when divergence is measured with Nei’s divergence, Table S10). We found six reproductive isolating mechanisms between D. teissieri and the D. yakuba/D. santomea species pair. A composite index of reproductive isolation revealed that the identified reproductive isolating mechanisms cause a reduction in gene flow close to 100% between D. teissieri and D. yakuba/D. santomea. One notable reproductive isolating mechanism is the stronger behavioral isolation between D. teissieri and D. yakuba when compared to D. teissieri and D. santomea. D. yakuba and D. teissieri share their geographical distributions (but not the same habitat) while the ranges of D. santomea and D. teissieri do not overlap at all (Lachaise et al. 1981, Lachaise et al. 1988). Two possibilities can explain the differences in the magnitude of reproductive isolation between the sympatric pair (D. teissieri/D. yakuba) and the allopatric pair (D. teissieri/D. santomea). The first one is differential extinction and fusion, a scenario in which two species


come into secondary contact, and the magnitude of reproductive isolation is polymorphic across demes. Upon secondary contact, only those demes that initially showed the strongest reproductive isolation will persist as differentiated species. The second possibility is that natural selection has made reproductive isolation stronger as a byproduct to minimize maladaptive hybridization, an evolutionary process known as reinforcement. The results shown here are incompatible with differential fusion because in this phenomenon, not only prezygotic, but also postmating-prezygotic, and postzygotic are expected to be stronger as well. Our results are consistent with the action of reinforcement as a driver of the evolution of mating choice that lead to stronger behavioral isolation between D. yakuba and D. teissieri than between D. santomea and D. teissieri (Noor 1997, Noor 1999). This comparative approach between species is even more powerful than the use of reproductive character displacement (i.e., the comparison of levels of reproductive isolation in sympatry vs. in allopatry) as a proxy for reinforcing selection because it cannot be obscured as in cases where increased isolation evolved in areas of secondary contact, and then spread throughout the whole range of a species. In such cases, the comparison of allopatric and sympatric lines reveal no signature of reinforcement.

From a technical standpoint, we have discovered that fertile female hybrids can be produced in the lab by species that are known to encounter each other in nature. The ability of these species to produce fertile hybrids and to backcross makes it possible to generate introgression lines and study interspecific genetic differences among these three species. Such introgressions could provide help elucidate the genetic architecture of hybrid sterility and other mechanisms involved in keeping these three species apart, especially in areas of secondary contact where hybridization and genetic exchange are possible. Habitat choice is a notable example of the interspecific differences between the three species of the yakuba subcomplex. Drosophila yakuba and D. teissieri show a stark habitat differentiation, as the first one tends to be associated with open habitats, while the latter one is found only in montane forests (Lachaise et al. 2004). Despite niche preference, the two species are known to coexist at mid-elevation transitional forests, such as the okoumé forest of Middle Ogougue in Central Gabón (Lachaise et al. 2004). So stronger prezygotic isolation could be selected for in areas of secondary contact. Future expeditions will determine whether these species hybridize in nature and to what extent gene flow has occurred between the different species of the yakuba complex.


ACKNOWLEDGEMENTS We would like to thank K. L. Gordon, V. Orgogozo, M. F. Przeworski, J.A. Coyne, for scientific discussions and comments at every stage of the manuscript. A. Llopart and P. Andolfatto shared stocks with us and we are thankful to them.

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FIGURE LEGENDS FIGURE 1. Phylogeny of seven of the nine species of the melanogaster supercomplex of species. The divergence value between species (Ks) is shown above each pair.

FIGURE 2. Pure species and F1 hybrid individuals in D. santomea/ D. teissieri crosses. A. D. santomea females. B. D. santomea males. C. D. teissieri females. D. D. teissieri males. E. F1 D. santomea/D. teissieri females. F. F1 D. santomea/D. teissieri males. G. F1 D. teissieri/D. santomea females. H. F1 D. teissieri/D. santomea males. I. F1 D. santomea C(1)RM/D. teissieri females. J. F1 D. santomea C(1)RM /D. teissieri males. FIGURE 3. Pure species and F1 hybrid individuals in D. yakuba/ D. teissieri crosses. A. D. yakuba females. B. D. yakuba males. C. D. teissieri females. D. D. teissieri males. E. F1 D. yakuba /D. teissieri females. F. F1 D. yakuba /D. teissieri males. G. F1 D. teissieri/D. yakuba females. H. F1 D. teissieri/D. yakuba males. I. F1 D. yakuba C(1)RM/D. teissieri females. Note that these hybrid females carry a C(1)RM chromosome from D. yakuba labeled with a yellowmarker. J. F1 D. yakuba C(1)RM /D. teissieri males. FIGURE 4. Production of progeny in pure species and interspecific crosses. All crosses involving D. teissieri are shown in light grey. Conspecific crosses are shown in dark grey. Crosses were ordinated from lowest to highest median.

FIGURE 5. Temperature preference of D. santomea, D. yakuba, and D. teissieri. The shown frequencies correspond to the number o flies at each temperature when the three replicates for each genotype are summed. A. Females. B. Males.

FIGURE 6. Relative viability of hybrid individuals at three different developmental stages. A. Embryonic viability. B. Larval viability. C. Pupal viability. The genotype of the female is


always shown first. y: D. yakuba; s: D. santomea; t: D. teissieri. White boxes show the viability of conspecific crosses. Grey boxes show the viability of heterospecific crosses.

FIGURE S1. Mating scheme for testing the effect of the D. santomea and D. yakuba Xchromosomes in sterility in hybrids with D. teissieri. A. Wild-type D. santomea females and D. teissieri males produce fertile F1 females and sterile males. B. Attached-X D. santomea females and D. teissieri males produce sterile F1 females and sterile males. FIGURE S2. Divergence between six species pairs in the melanogaster species supercomplex. Histrograms show the distribution of Ks values across the whole genome when Ks is calculated for each gene individually. Divergence for each species pair was calculated as the mean Ks value across all genes (red line). A. D. simulans vs. D. sechellia. B. D. simulans vs. D. mauritiana. C. D. sechellia vs. D. mauritiana. D. D. yakuba vs. D. santomea. E. D. yakuba vs. D. teissieri. F. D. santomea vs. D. teissieri. FIGURE S3. Egg production in conspecific and heterospecific matings during the next ten days after mating. Interspecific crosses involving D. teissieri produce progeny only for a few days after mating. Conspecific crosses: Squares, dotted lines; Interspecific crosses between D. yakuba and D. santomea: Triangles, dashed lines; interspecific crosses involving D. teissieri: circles, solid lines. FIGURE S4. Dissected testes from each of the three pure-species types of males and six types of F1 hybrid males.


TABLES TABLE 1. Number of crosses that produced progeny. We attempted nine different combinations of species, three conspecific and six heterospecific. Each hybridization attempt entailed housing 20 females and 20 males of the described genotypes. We tried 100 assays for each combination. Cross

N

Successful hybridizations

♀ D. yakuba SYN2005 × ♂ D. yakuba SYN2005

100

100

♀ D. santomea SYN2005 × ♂ D. santomea SYN2005 100

100

♀ D. teissieri SYN × ♂ D. teissieri SYN

100

100

♀ D. yakuba SYN2005 × ♂ D. santomea SYN2005

100

95

♀ D. santomea SYN2005 × ♂ D. yakuba SYN2005

100

83

♀ D. yakuba SYN2005 × ♂ D. teissieri SYN

100

9

♀ D. teissieri SYN × ♂ D. yakuba SYN2005

100

8

♀ D. santomea SYN2005 × ♂ D. teissieri SYN

100

40

♀ D. teissieri SYN × ♂ D. santomea SYN2005

100

19

TABLE 2. Sex ratios for pure species, F1 hybrids and backcrosses. The significance of the expected 1:1 female-male sex ratio was assessed using a χ2 test with one degree of freedom. Significant comparisons are marked with a star.

Fraction Genotype

Females

Males

of males

χ2

P-value

1

D. yakuba SYN2005

322

315

49.451

0.077

0.782

2

D. santomea SYN2005

431

422

49.472

0.095

0.758

3

D. teissieri SYN

533

515

49.141

0.309

0.578

4

F1 (♀ yak SYN2005 × ♂san SYN2005)

354

311

46.767

2.780

0.095

5

F1 (♀ san SYN2005 × ♂ yak SYN2005)

514

455

46.956

3.592

0.058

6 7 8 9

14.51 F1 (♀ yak SYN2005 × ♂ tei SYN)

402

301

42.817

1

0.000

*

F1 (♀ tei SYN × ♂yak SYN2005)

521

431

45.273

8.508

0.004

*

11.57 F1 (♀ san SYN2005 × ♂ tei SYN)

260

188

41.964

1

0.001

*

F1 (♀ tei SYN × ♂ san SYN2005)

271

198

42.200

11.36

A Major Locus Controls a Genital Shape Difference Involved in Reproductive Isolation Between Drosophila yakuba and Drosophila santomea.

Analysis of incipient reproductive isolation within a species of Drosophila.

Correlated evolution of male and female reproductive traits drive a cascading effect of reinforcement in Drosophila yakuba.

Genetics of sexual isolation in females of the Drosophila simulans species complex.

Evolution of the transposable element mariner in Drosophila species.

Reproductive isolation among allopatric Drosophila montana populations.

Evolution of the expression of the Gld gene in the reproductive tract of Drosophila.

Rapid evolution of reproductive isolation between incipient outcrossing and selfing Clarkia species.

Postmating Reproductive isolation between strains of Drosophila willistoni.

Landscape of standing variation for tandem duplications in Drosophila yakuba and Drosophila simulans.

Insertional mutagenesis in Drosophila. II. P element mediated transformation of Drosophila yakuba.

Genome Evolution in Three Species of Cactophilic Drosophila.

Parallel evolution of local adaptation and reproductive isolation in the face of gene flow.

Fine scale mapping of genomic introgressions within the Drosophila yakuba clade.

Distinguishing migration from isolation using genes with intragenic recombination: detecting introgression in the Drosophila simulans species complex.

Mating patterns and post-mating isolation in three cryptic species of the Engystomops petersi species complex.

Highly variable reproductive isolation among pairs of Catostomus species.

Asymmetrical sexual isolation but no postmating isolation between the closely related species Drosophila suboccidentalis and Drosophila occidentalis.

Evolution of the transposable element mariner in the Drosophila melanogaster species group.

Aggregation pheromone ofDrosophila mauritiana, Drosophila yakuba, andDrosophila rajasekari.

Sex pheromones and reproductive isolation in five mirid species.

Isolation of a multifunctional complex containing the first three enzymes of pyrimidine biosynthesis in drosophila melanogaster.

Micro-plasticity of genomes as illustrated by the evolution of glutathione transferases in 12 Drosophila species.

The rate test of speciation: estimating the likelihood of non-allopatric speciation from reproductive isolation rates in Drosophila.