ABSTRACT
A full understanding of how ecological factors drive the fixation of genetic changes during speciation is obscured by the lack of appropriate models with clear natural history and powerful genetic toolkits. In a recent study, we described an early stage of ecological speciation in a population of the generalist species Drosophila yakuba (melanogaster subgroup) on the island of Mayotte (Indian Ocean). On this island, flies are strongly associated with the toxic fruits of noni (Morinda citrifolia) and show a partial degree of pre-zygotic reproductive isolation. Here, I mine the nuclear and mitochondrial genomes and provide a full morphological description of this population. Only 29 nuclear sites (< 4 × 10−7 of the genome) are fixed in this population and absent from 3 mainland populations and the closest relative D. santomea, but no mitochondrial or morphological character distinguish Mayotte flies from the mainland. This result indicates that physiological and behavioral traits may evolve faster than morphology at the early stages of speciation. Based on these differences, the Mayotte population is designated as a new subspecies, Drosophila yakuba mayottensis subsp. nov., and its strong potential in understanding the genetics of speciation and plant-insect interactions is discussed.
Introduction
Speciation is a continuing process that culminates in the evolution of complete reproductive isolation and fixed genetic differences between nascent species. Understanding the evolutionary forces driving populations along this continuum (e.g., selection vs. drift) is a major biological problem.Citation1 Modern studies revealed that different forces differently act on various parts of the genome causing a heterogeneous rate of genetic divergence and differentiation.Citation2 This leads to questions about the nature of the earliest genetic differences to become fixed during speciation, the forces driving their fixation, and how they affect reproductive isolation.Citation3
A great deal of our understanding of the genetic basis of speciation and speciation genes draws from studies on Drosophila flies. Most studies have, however, focused on one aspect of the speciation process, i.e. the evolution of reproductive isolation, leading to breakthroughs in the understanding of the genetics of hybrid inviability.Citation4,5 However, the study of speciation should not be equated with reproductive isolation, and hybrid inviability is usually a late step in the speciation process.Citation3,4 In spite of significant progress,Citation6 our knowledge of the natural history of most laboratory model Drosophila species that have been used in speciation genetics research are often quite poor. Indeed, Drosophila research has been criticized for its neglect of the ecological context driving the early stages of speciation, and other models having clear natural history, such as Heliconius butterflies for example, have been proposed to fill this gap.Citation4 However, these models still lack the advanced Drosophila genetic toolkits that can elegantly identify the function of several diverging genes.
A particular case that seemed to be most promising in understanding the genetics of the ecological speciation in Drosophila is the specialization of Drosophila sechellia on the toxic fruits of noni (Morinda citrifolia) on the Seychelles islands in the Indian Ocean.Citation7 D. sechellia is closely-related to Drosophila melanogaster, has its genome fully sequenced and annotated, and can be reared in the laboratory. It has diverged nearly 250,000 y ago from its closest relative, the generalist fly Drosophila simulans, and from which it is partially pre- and post-zygotically isolated.Citation8 In spite of evidence for ancient and recent hybridization between the 2 species in Seychelles,Citation8,9 the 2 species are quite distinct. Orgogozo and SternCitation10 counted from the literature 5 morphological, 3 physiological and 6 behavioral differences between the 2 species, in addition to the higher attraction, oviposition preference and tolerance to noni toxins in D. sechellia. The capability of crossing D. sechellia with D. simulans and producing fertile hybrid females has led to several quantitative trait loci (QTLs) studies aiming at mapping the genes underlying these differences,Citation11-15 and Drosophila genetics tool elegantly illustrated the function of some genes controlling their phenotypic differences.Citation16-18 However, the old divergence between the 2 speciesCitation8 and the low level of genetic variation within D. sechelliaCitation19 make difficult to finely map these differences or infer the order by which they arose and potentially affected reproductive isolation.
In a recent study, my colleagues and I published the discovery of a parallel case of insular specialization on toxic noni that was also accompanied by partial reproductive isolation.Citation20 It concerns a population of the generalist species, Drosophila yakuba, whose last common ancestor with D. sechellia, D. simulans and D. melanogaster, lived more than 10 million years ago.Citation21 This population lives on Mayotte, an island of the Comoros archipelago, which is also in the Western Indian Ocean like the Seychelles but closer to the African mainland. However, unlike D. sechellia, we inferred the colonization of Mayotte to be relatively recent, ∼29,000 y ago. On Mayotte, the flies were exclusively bred from fallen noni fruits, an unusual habitat for D. yakuba flies that are usually easily collected by fermenting banana traps on the mainland. We confirmed this difference in the laboratory showing that Mayotte flies have higher preference for noni and also higher tolerance to its toxins than mainland flies. Also, genome-wide scan identified high differentiation peaks between Mayotte and the mainland with many peaks falling within identified noni tolerance QTLs of D. sechellia, suggesting some common genetic pathways driving parallel ecological specialization. The differences between D. sechellia and Mayotte D. yakuba on the degrees of specialization and reproductive isolation therefore offer a rare opportunity where early and late steps in speciation may be distinguished. Unlike other non-model organisms for which ecological speciation continuums have also been demonstrated (e.g., sticklebacks, Heliconius butterflies, Rhagoletis flies, etc.),Citation2 most of the advanced genetic and laboratory tools available for D. melanogaster could be applied to both D. sechellia and Mayotte D. yakuba.
Here, I mine the complete genome sequence of mainland and Mayotte D. yakuba and compare their mitochondria and morphology in search of the earliest fixed differences between them. Neither the morphology nor the mitochondrion provides diagnostic characters, whereas among 78,020,004 nuclear genomic sites, only 29 SNPs at 17 genes were specific to Mayotte D. yakuba. I discuss the relevance of these differences and use them, along with the geographical, physiological and behavioral characteristics of this population to define it as a subspecies that will likely become a promising model for ecological speciation genetics.
Materials and methods
Genomic analysis
For the nuclear genomes, I used the alignment to the D. yakuba reference genome r.1.3. (www.flybase.org)Citation22 for a line from Côte d'Ivoire that we generated for D. yakuba from Mayotte (SRA project no. SRP063516),Citation20 20 inbred lines from Cameroon and Kenya (SRP029453)Citation23 and a line of D. santomea, the closest relative of D. yakuba (SRP052875).Citation24 For the mitogenomes, I aligned reads from the pooled 22 lines of D. yakuba to the reference D. yakuba mitogenome (GenBank accession no. NC_001322)Citation25 using the same alignment protocol.Citation20 The consensus sequence was then aligned to complete mitogenomes for 29 and 18 lines of D. yakuba and its closest relative D. santomea, respectively, that were recently publishedCitation26 using MuscleCitation27 as implemented in MEGA6.Citation28 The D. yakuba lines represent 7 populations from mainland Africa (Côte d'Ivoire, Cameroon, Gabon, Kenya, Tanzania and Zimbabwe) and the Atlantic island of Sao Tomé. To observe polymorphism in the pooled sample of Mayotte, Popoolation2Citation29 was used to generate a synchronized mpileup file, to which a column describing sequence variation in the 29 African lines was added using a customized Perl script.
MEGA was used to generate a neighbor-joining (NJ) phylogenetic tree for the mitogenomes under the Kimura 2-parameter model and after 1000 bootstrap iterations. For this analysis, the mitochondrion of D. erecta (GenBank accession number BK006335)Citation22 was used as an outgroup. Character-based approachCitation30 was applied for both mitochondrial and nuclear genome analyses using a customized Perl script which parses the synchronized mpileup files to search sites with nucleotides that are unique to the Mayotte population, i.e., fixed in this population and totally absent in other African genomes including the reference genome. Only sites with at least 44 sequencing depth were evaluated. These sites were then used as molecular diagnostics for Mayotte D. yakuba.
Morphological analysis
Males were obtained from mass cultures of 3 populations of D. yakuba from Mayotte, Kunden (Cameroon) and Andasibe (Madagascar) as well as a culture of D. santomea from Sao Tomé. For each population, the genitalia of 10 males were dissected, mounted on microscopic slides in DMHF mounting medium (Entomopraxis A9001) and observed under a Zeiss light microscope as in Yassin and Orgogozo.Citation31 Male genital structures were also compared to descriptions of D. yakuba from Côte d'Ivoire,Citation32 KenyaCitation33 and LiberiaCitation34 covering the whole range of the species.
Results
Nuclear genome
Among 78,020,004 sites, only 36 SNPs belonging to 20 genes contained nucleotides that are fixed in Mayotte D. yakuba and absent from Kenya, Cameroon and the reference genome from Côte d'Ivoire (). Of these, 21 SNPs (10 genes) were on chromosome X (14,652,942 sites) indicating enrichment for this chromosome at early stages of speciation (Fisher exact test P < 3.1 × 10−7). With the exception of a single SNP, all X-linked fixed SNPs fell within a window of only 0.6 Mb, which constituted the largest peak of differentiation based on our previous FST analysis1 and overlapped with a major QTL conferring larval tolerance to noni toxin in D. sechellia.Citation15
Table 1. Fixed nucleotidic differences between Mayotte D. yakuba (D. y. mayottensis) and 3 populations of mainland D. yakuba (D. y. yakuba) with their ancestral states from D. santomea genome shown. The three populations of D. y. yakuba represent 21 lines from Côte d'Ivoire, Cameroon and Kenya. Gene names are given according to D. melanogaster ortholog. Sites shared between D. y. mayottensis and D. santomea are underlined.
For the autosomes, no fixed SNPs distinguishing the island population was found on chromosome 3. The six SNPs on chromosome arm 2L fell within a 20 kb region constituting another peak of differentiation. The SNPs belonged to 2 enzymatic genes, orthologous to the lipase CG4267 and the serine protease CG17243 in D. melanogaster. On chromosome arm 2R, 4 fixed SNPs fell within a 1 kb region within the egalitarian (egl) gene. Although all SNPs consisted of fixed differences between Mayotte and the mainland, one trinucleotide SNP at the Egfr gene consisted of an A/C polymorphism that was specific to Mayotte with both nucleotides absent from the mainland.
Interestingly, 29 out of the 36 sites (17 genes) were also unique to the Mayotte population if compared to D. santomea, suggesting these sites to be likely derived in the Mayotte lineage ().
Mitochondrial genome
Cytoplasmic introgression is common between D. yakuba and its 2 sibling species, D. santomea that is endemic to the Atlantic island of Sao Tomé and D. teissieri which is confined to the humid rainforests on the African mainland.Citation26 Consequently, mitogenomes should have little taxonomic utility in this species complex. Consistently, the Mayotte D. yakuba mitochondrion branched within the cluster of D. yakuba/D. Santomea mitochondria showing no distinct feature (). Interestingly, the closest mitochondrion to Mayotte D. yakuba belonged to an East African population (Zimbabwe) with a bootstrap value of 84.6%. There is no nuclear genome available at the moment for Zimbabwe D. yakuba. Unfortunately, no mitogenome is available neither from Mozambique, Comoros or Madagascar islands, which may be given their geographical proximity closer to Mayotte D. yakuba and more informative to infer its history.
Figure 1. Neighbor-joining tree of 29 and 18 mitogenomes of Drosophila yakuba yakuba and D. santomea, respectively, from Llopart et al.Citation26 and the consensus mitogenome of D. y. mayottensis. Bootstrap values are shown above nodes. The tree is rooted using D. erecta mitogenome but the branch leading to the root is not shown because of its long length.
Morphology
No distinct morphological character can distinguish Mayotte D. yakuba from their conspecifics. Considering body pigmentation, which represents the most differentiating trait between D. yakuba and its closest relative D. santomea,Citation35 Mayotte males have the last 2 abdominal tergites darkly pigmented like all D. yakuba flies. For male genitalia, D. yakuba differs from D. santomea in the shape of a 2 accessory articulating structures known as posterior parameresCitation35 (also known as inner paraphyses)Citation36 and the presence of 2 basal protrusions called phallic spines,Citation33 phallic spursCitation31 or ventral branches.Citation34 Both structures are of the D. yakuba type in the Mayotte population indicating that morphology is not informative at this stage of divergence ().
Figure 2. Male phallic apparatus in Drosophila yakuba mayottensis showing the placement of the 2 structures distinguishing D. y. yakuba and D. santomea: posterior parameres (dotted outline, above) and phallic spurs (dashed outline, below). Arrowheads indicate the distinguishing protrusions being longer in both subspecies of D. yakuba. ae = aedeagus; hyp = hypandrium; phap = phallic apodeme.
Taxonomy
In addition to the quantitative physiological differences between Mayotte and mainland D. yakuba regarding the higher preference for and tolerance to noni of the former population that we have previously described,Citation20 I report here some qualitative fixed differences at the nuclear genome level but not at the mitochondrial or morphological levels. Together with the particular geographical locality of Mayotte D. yakuba, I use these differences to split D. yakuba into 2 subspecies, as follows.
Drosophila (Sophophora) yakuba yakuba Burla, 1954:161
Type locality – Côte d'Ivoire.
Diagnosis – Nucleotide differences shown at .
Description – As in BurlaCitation32 and Bock and Wheeler.Citation37
Distribution – Africa: Côte d'Ivoire, Cameroon, Kenya and Sao Tomé, and presumably Benin, Congo, Gabon, Ghana, Madagascar, Malawi, Mozambique, Niger, Nigeria, Sierra Leone, South Africa, Tanzania, Uganda and Zimbabwe.
Drosophila (Sophophora) yakuba mayottensis David, Debat & Yassin, new subspecies
Type locality – Mayotte island (Comoros archipelago). A male holotype and 5 male and 5 female paratypes all drawn from F1 laboratory flies of isofemale lines collected by Jean R. David, Vincent Debat and Nelly Gidazweski in the Bay of Soulou (Grande Terre island, Mayotte) in January 2013 are deposited in the Muséum National d'Histoire Naturelle in Paris.
Diagnosis – Similar to D. y. yakuba, with male and female last abdominal tergites dark, but differs from it with the genomic differences shown in , its geographical location and its higher ecological specialization on noni fruits reported in Yassin et al.Citation20
Description – (male holotype) Similar to D. y. yakuba. Arista with 5 branches above and 2 below in addition to terminal fork. Pedicel yellow, flagellomere I grayish. Frons narrow and yellow, ocellar triangle brownish and shiny. Face yellow, first and second oral bristles nearly of equal lengths, carina narrow. Cheeks yellow and very narrow. Eyes bright red.
Mesonotum and scutellum yellow. Pleura and legs pale yellow. Eight rows of acrostichal hairs. Anterior scutellars convergent. Anterior to posterior katepisternal bristles (sterno index) about 0.6, with a third, middle bristle, subequal to the anterior one. Sex combs with 7 black, curved bristles on the first tarsomere of the front legs. Wing hyaline to light brown, wing veins light brown.
Abdomen yellow, with dark stripes on the posterior margin of the second to fourth tergite. Fifth and sixth tergite shiny, black.
Epandrium black, with 5 bristles above and a tuff of long bristles on the ventral epandrial lobe, and a vestigial, triangular posterior lobe. Surstylus with 13 stout spines and one long bristle. Cercus boomerang, constricted on the middle, hirsute on its entire length with the bristles on the dorsal portion being shorter and stouter. Hypandrium yellow, rounded, with fine hairs and 2 long bristles on the gonopod, notched on the middle. Outer paraphyses (anterior parameres) with 3 short bristles on the tip. Inner paraphyses (posterior parameres) long, swan-shaped at the tip. Aedeagus membranous, pointed at tip and papillate on its entire length, with 2 sharp protrusions at its base (ventral branches or phallic spurs) ().
Distribution – Africa: Mayotte island (Comoros archipelago).
Discussion
In the genus Drosophila, where the genetics of speciation has been intensively studied in multiple pairs of closely-related species, few taxa were identified as subspecies. This was attributed to the low level of morphological distinction between allopatric populations within Drosophila species compared to other subspecies-rich holometabolous insects such as lepidopterans or coleopterans.Citation38 Of the nearly 1,000 Drosophila species, only 24 have been subdivided into allopatric subspecies showing a relative degree of reproductive isolation (TAXODROS as of 19 November 2015). These subspecies were defined on the basis of morphological differences such as male pigmentationCitation39 or genitaliaCitation40, chromosomalCitation41-43 or allozyme traitsCitation44,45, since reproductive isolation alone is not enough to designate a subspecies. However, unlike morphological and molecular data, behavioral, karyological and enzymological variation cannot be traced on preserved name-holding type specimens making them of lesser identification utility.Citation46
Drosophila yakuba mayottensis is the first subspecies to be described in the melanogaster subgroup, which consists of 9 African or of African origin species closely related to D. melanogaster.Citation7 This designation is justified in terms of geographical locality, ecological specialization, partial reproductive isolation and the molecular diagnostics given in . Indeed, the genotypic species concept defines a species by ‘a stable multilocus genetic differentiation in the face of potential gene flow’.Citation47 However, subspecies are reversible since an increase of gene flow may lead to their disappearance and the abortion of the speciation process that has been initiated in their lineage.Citation48,49 Two major endeavors therefore should be undergone in the future. First, we should sequence more nuclear genomes from the African mainland in order to test for the absence of D. y. mayottensis diagnostic nucleotides there. And second, we should functionally test the relevance of these nucleotides to noni adaptation and/or premating reproductive isolation, therefore ecological speciation.
Ecological speciation should not be confounded with sympatric speciation, and indeed natural selection can lead to the rapid divergence of allopatric populations, such as between islands and mainland, due to their different habitats at the early stages of speciation. The fundamental question then becomes, when the 2 populations come in secondary contact, would they continue to be ecologically and reproductively isolated? Our recent population genomics analysis suggested a demographic scenario in which the ancestor of D. y. mayottensis colonized Mayotte long before human introduction of other fruit resources.Citation20 At this time, noni might have been the only predictable source for the flies and natural selection drove the specialization. Further studies on the demographic history of noni on Mayotte and neighboring regions should be conducted to test this hypothesis, i.e. when did noni arrive on the island? Moreover, human agriculture and forest exploitation have drastically changed the flora of Mayotte that only 5% of the original vegetation presently exists.Citation50 Consequently, the drosophilid fauna of Mayotte and neighboring islands is currently dominated by human-commensal species.Citation51,52 This would increase the chances of the introduction of mainland D. y. yakuba to the island, which may outcompete or abort the speciation process. On the other hand, noni itself is currently cultivated and exploited for its pharmaceutical benefits on the African mainland,Citation53 which may ultimately lead to other cases of specialization reminiscent to the ‘parallel speciation’ cases of sticklebacksCitation54 or Timema stick insects.Citation55 Further ecological studies on noni and D. yakuba on Mayotte, neighboring islands and the mainland are thus strongly needed.
The ecological parallelism between D. sechellia and D. yakuba mayottensis is a unique opportunity to distinguish species differences due to natural selection for a similar host from other differences arising during the speciation process. A great endeavor would be to determine the link between noni preference or toxin tolerance loci and the loci underlying mate choice in D. y. mayottensis females. This link should indicate how natural selection could affect reproductive isolation via pleiotropy or linkage, and also inform whether pre-copulatory isolation shares common basis between the 2 cases. Our analysis identified several common noni toxin tolerance genes or genetic pathways between D. sechellia and D. y. mayottensis, with some being implied in specialization on other host plants in some other drosophilids (such as leaf-mining Scaptomyza)Citation56,57 but also in several herbivorous insects, which constitute a majority of animal diversity. This makes this new subspecies a vey promising model to understand the genetics and evolution of insect-plant interactions in Drosophila and beyond.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
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