A method for rapid and simultaneous mapping of genetic Loci and introgression sizes in nematode species

Caenorhabditis briggsae is emerging as an attractive model organism not only in studying comparative biology against C. elegans, but also in developing novel experimentation avenues. In particular, recent identification of a new Caenorhabditis species, C. sp.9 with which it can mate and produce viable progeny provides an opportunity for studying the genetics of hybrid incompatibilities (HI) between the two. Mapping of a specific HI locus demands repeated backcrossing to get hold of the specific genomic region underlying an observed phenotype. To facilitate mapping of HI loci between C. briggsae and C. sp.9, an efficient mapping method and a genetic map ideally consisting of dominant markers are required for systematic introgression of genomic fragments between the two species. We developed a fast and cost-effective method for high throughput mapping of dominant loci with resolution up to 1 million bps in C. briggsae. The method takes advantage of the introgression between C. briggsae and C. sp.9 followed by PCR genotyping using C. briggsae specific primers. Importantly, the mapping results can not only serve as an effective way for estimating the chromosomal position of a genetic locus in C. briggsae, but also provides size information for the introgression fragment in an otherwise C. sp.9 background. In addition, it also helps generate introgression line as a side-product that is invaluable for the subsequent mapping of HI loci. The method will greatly facilitate the construction of a genetic map consisting of dominant markers and pave the way for systematic isolation of HI loci between C. briggsae and C. sp.9 which has so far not been attempted between nematode species. The method is designed for mapping of a dominant allele, but can be easily adapted for mapping of any other type of alleles in any other species if introgression between a sister species pair is feasible.     

Introgression and selection shaped the evolutionary history of sympatric sister‐species of coral reef fishes (genus: Haemulon)

Closely related marine species with large overlapping ranges provide opportunities to study mechanisms of speciation, particularly when there is evidence of gene flow between such lineages. Here, we focus on a case of hybridization between the sympatric sister‐species Haemulon maculicauda and H. flaviguttatum, using Sanger sequencing of mitochondrial and nuclear loci, as well as 2422 single nucleotide polymorphisms (SNPs) obtained via restriction site‐associated DNA sequencing (RADSeq). Mitochondrial markers revealed a shared haplotype for COI and low divergence for CytB and CR between the sister‐species. On the other hand, complete lineage sorting was observed at the nuclear loci and most of the SNPs. Under neutral expectations, the smaller effective population size of mtDNA should lead to fixation of mutations faster than nDNA. Thus, these results suggest that hybridization in the recent past (0.174–0.263 Ma) led to introgression of the mtDNA, with little effect on the nuclear genome. Analyses of the SNP data revealed 28 loci potentially under divergent selection between the two species. The combination of mtDNA introgression and limited nuclear DNA introgression provides a mechanism for the evolution of independent lineages despite recurrent hybridization events. This study adds to the growing body of research that exemplifies how genetic divergence can be maintained in the presence of gene flow between closely related species.     

Limited Introgression between Rock-Wallabies with Extensive Chromosomal Rearrangements

Chromosome rearrangements can result in the rapid evolution of hybrid incompatibilities. Robertsonian fusions, particularly those with monobrachial homology, can drive reproductive isolation amongst recently diverged taxa. The recent radiation of rock-wallabies (genus Petrogale) is an important model to explore the role of Robertsonian fusions in speciation. Here, we pursue that goal using an extensive sampling of populations and genomes of Petrogale from north-eastern Australia. In contrast to previous assessments using mitochondrial DNA or nuclear microsatellite loci, genomic data are able to separate the most closely related species and to resolve their divergence histories. Both phylogenetic and population genetic analyses indicate introgression between two species that differ by a single Robertsonian fusion. Based on the available data, there is also evidence for introgression between two species which share complex chromosomal rearrangements. However, the remaining results show no consistent signature of introgression amongst species pairs and where evident, indicate generally low introgression overall. X-linked loci have elevated divergence compared with autosomal loci indicating a potential role for genic evolution to produce reproductive isolation in concert with chromosome change. Our results highlight the value of genome scale data in evaluating the role of Robertsonian fusions and structural variation in divergence, speciation, and patterns of molecular evolution.     

Apparent 'sympatric' speciation in ecologically similar herbivorous beetles facilitated by multiple colonizations of an island

Coexistence of recently diverged and ecologically similar sister species in complete sympatry represents a particularly compelling case for sympatric speciation. This study investigates the possible sympatric origin of two coexisting bark beetle taxa that utilize the same host plant on the island of La Palma in the Canary Islands. Aphanarthrum subglabrum and Aphanarthrum glabrum ssp. nudum breed inside dead twigs of Euphorbia lamarckii plants and are closely related to the allopatric A. glabrum ssp. glabrum in Tenerife, El Hierro and La Gomera. We tested the various speciation hypotheses in a genealogical context, using mitochondrial gene fragments from Cytochrome Oxidase I and 16S, and nuclear gene fragments from Enolase, Elongation Factor 1alpha and Histone H3. Phylogenetic analyses of the combined nuclear DNA data strongly supported a sister relationship between two sympatric and reproductively isolated taxa in La Palma. However, network analyses of subdivided nonrecombinant segments of the Enolase locus indicated a closer relationship between the two allopatric A. glabrum subspecies, suggesting multiple colonizations of this island. A bimodal distribution of mtDNA haplotypes in La Palma further documented the independent colonization of this island, with asymmetric introgression of mtDNA between two lineages. Consequently, the sympatric origin of the La Palma species is concluded to have involved allopatric phases before the parallel colonization of this island and subsequent introgression at some loci. The clear genetic and morphological evidence for reproductive isolation between these species suggests that the sympatric completion of divergence was either due to initial genetic incompatibility, morphological character displacement in male genitalia, or a combination of these factors.     

Evolution of assortative mating following selective introgression of pigmentation genes between two Drosophila species

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.     

Pervasive genetic associations between traits causing reproductive isolation in Heliconius butterflies

Ecological speciation proceeds through the accumulation of divergent traits that contribute to reproductive isolation, but in the face of gene flow traits that characterize incipient species may become disassociated through recombination. Heliconius butterflies are well known for bright mimetic warning patterns that are also used in mate recognition and cause both pre- and post-mating isolation between divergent taxa. Sympatric sister taxa representing the final stages of speciation, such as Heliconius cydno and Heliconius melpomene, also differ in ecology and hybrid fertility. We examine mate preference and sterility among offspring of crosses between these species and demonstrate the clustering of Mendelian colour pattern loci and behavioural loci that contribute to reproductive isolation. In particular, male preference for red patterns is associated with the locus responsible for the red forewing band. Two further colour pattern loci are associated, respectively, with female mating outcome and hybrid sterility. This genetic architecture in which ‘speciation genes’ are clustered in the genome can facilitate two controversial models of speciation, namely divergence in the face of gene flow and hybrid speciation.     

Molecular population genetics and phenotypic sensitivity to ethanol for a globally diverse sample of the nematode Caenorhabditis briggsae

New genomic resources and genetic tools of the past few years have advanced the nematode genus Caenorhabditis as a model for comparative biology. However, understanding of natural genetic variation at molecular and phenotypic levels remains rudimentary for most species in this genus, and for C. briggsae in particular. Here we characterize phenotypic variation in C. briggsae’s sensitivity to the potentially important and variable environmental toxin, ethanol, for globally diverse strains. We also quantify nucleotide variation in a new sample of 32 strains from four continents, including small islands, and for the closest‐known relative of this species (C. sp. 9). We demonstrate that C. briggsae exhibits little heritable variation for the effects of ethanol on the norm of reaction for survival and reproduction. Moreover, C. briggsae does not differ significantly from C. elegans in our assays of its response to this substance that both species likely encounter regularly in habitats of rotting fruit and vegetation. However, we uncover drastically more molecular genetic variation than was known previously for this species, despite most strains, including all island strains, conforming to the broad biogeographic patterns described previously. Using patterns of sequence divergence between populations and between species, we estimate that the self‐fertilizing mode of reproduction by hermaphrodites in C. briggsae likely evolved sometime between 0.9 and 10 million generations ago. These insights into C. briggsae’s natural history and natural genetic variation greatly expand the potential of this organism as an emerging model for studies in molecular and quantitative genetics, the evolution of development, and ecological genetics.     

GENOME‐WIDE INVESTIGATION OF REPRODUCTIVE ISOLATION IN EXPERIMENTAL LINEAGES AND NATURAL SPECIES OF NEUROSPORA: IDENTIFYING CANDIDATE REGIONS BY MICROARRAY‐BASED GENOTYPING AND MAPPING

Inherent incompatibilities between genetic components from genomes of different species may cause intrinsic reproductive isolation. In evolution experiments designed to instigate speciation in laboratory populations of the filamentous fungus Neurospora, we previously discovered a pair of incompatibility loci (dfe and dma) that interact negatively to cause severe defects in sexual reproduction. Here we show that the dfe‐dma incompatibility also is a significant cause of genetic isolation between two naturally occurring species of Neurospora (N. crassa and N. intermedia). The strong incompatibility interaction has a simple genetic basis (two biallelic loci) and antagonistic epistasis occurs between heterospecific alleles only, consistent with the Dobzhansky–Muller model of genic incompatibility. We developed microarray‐based, restriction‐site associated DNA (RAD) markers that identified ～1500 polymorphisms between the genomes of the two species, and constructed the first interspecific physical map of Neurospora. With this new mapping resource, the approximate genomic locations of the incompatibility loci were determined using three different approaches: genome scanning, bulk‐segregant analyses, and introgression. These population, quantitative, and classical genetics methods concordantly identified two candidate regions, narrowing the search for each incompatibility locus to only ～2% of the nuclear genome. This study demonstrates how advances in high‐throughput, genome‐wide genotyping can be applied to mapping reproductive isolation genes and speciation research.     

Tracking the progression of speciation: variable patterns of introgression across the genome provide insights on the species delimitation between progenitor–derivative spruces (Picea mariana × P. rubens)

The genic species concept implies that while most of the genome can be exchanged somewhat freely between species through introgression, some genomic regions remain impermeable to interspecific gene flow. Hence, interspecific differences can be maintained despite ongoing gene exchange within contact zones. This study assessed the heterogeneous patterns of introgression at gene loci across the hybrid zone of an incipient progenitor–derivative species pair, Picea mariana (black spruce) and Picea rubens (red spruce). The spruce taxa likely diverged in geographic isolation during the Pleistocene and came into secondary contact during late Holocene. A total of 300 SNPs distributed across the 12 linkage groups (LG) of black spruce were genotyped for 385 individual trees from 33 populations distributed across the allopatric zone of each species and within the zone of sympatry. An integrative framework combining three population genomic approaches was used to scan the genomes, revealing heterogeneous patterns of introgression. A total of 23 SNPs scattered over 10 LG were considered impermeable to introgression and putatively under diverging selection. These loci revealed the existence of impermeable genomic regions forming the species boundary and are thus indicative of ongoing speciation between these two genetic lineages. Another 238 SNPs reflected selectively neutral diffusion across the porous species barrier. Finally, 39 highly permeable SNPs suggested ancestral polymorphism along with balancing selection. The heterogeneous patterns of introgression across the genome indicated that the speciation process between black spruce and red spruce is young and incomplete, albeit some interspecific differences are maintained, allowing ongoing species divergence even in sympatry. The approach developed in this study can be used to track the progression of ongoing speciation processes.     

Two genomic regions together cause dark abdominal pigmentation in Drosophila tenebrosa

Pigmentation is a rapidly evolving trait that is under both natural and sexual selection in many organisms. In the quinaria group of Drosophila, nearly all of the 30 species have an abdomen that is light in color with distinct markings; D. tenebrosa is the exception in that it has a completely melanic abdomen with no visible markings. In this study, we use a combination of quantitative genetic and candidate gene approaches to investigate the genetic basis of abdominal pigmentation in D. tenebrosa. We find that abdominal pigmentation is invariant across wild-caught lines of D. tenebrosa and is not sexually dimorphic. Quantitative genetic mapping utilizing crosses between D. tenebrosa and the light-colored D. suboccidentalis indicates that two genomic regions together underlie abdominal pigmentation, including the X-chromosome and an autosome (Muller Element C/E). Further support for their central importance in pigmentation is that experimental introgression of one phenotype into the other species, in either direction, results in introgression of these two genomic regions. Finally, the expression of the X-linked gene yellow in the pupae exactly foreshadows the adult melanization pattern in the abdomen of both species, suggesting that changes in the regulation of yellow are important for the phenotypic divergence of D. tenebrosa from the rest of the quinaria group. These results contribute to a body of work that demonstrates how changes in expression of highly conserved genes can cause substantial phenotypic differences even between closely related species.     

Incompatibility and competitive exclusion of genomic segments between sibling Drosophila species

The extent and nature of genetic incompatibilities between incipient races and sibling species is of fundamental importance to our view of speciation. However, with the exception of hybrid inviability and sterility factors, little is known about the extent of other, more subtle genetic incompatibilities between incipient species. Here we experimentally demonstrate the prevalence of such genetic incompatibilities between two young allopatric sibling species, Drosophila simulans and D. sechellia. Our experiments took advantage of 12 introgression lines that carried random introgressed D. sechellia segments in different parts of the D. simulans genome. First, we found that these introgression lines did not show any measurable sterility or inviability effects. To study if these sechellia introgressions in a simulans background contained other fitness consequences, we competed and genetically tracked the marked alleles within each introgression against the wild-type alleles for 20 generations. Strikingly, all marked D. sechellia introgression alleles rapidly decreased in frequency in only 6 to 7 generations. We then developed computer simulations to model our competition results. These simulations indicated that selection against D. sechellia introgression alleles was high (average s = 0.43) and that the marker alleles and the incompatible alleles did not separate in 78% of the introgressions. The latter result likely implies that most introgressions contain multiple genetic incompatibilities. Thus, this study reveals that, even at early stages of speciation, many parts of the genome diverge to a point where introducing foreign elements has detrimental fitness consequences, but which cannot be seen using standard sterility and inviability assays.     

Caenorhabditis briggsae and C. elegans: partial characterization of cuticle surface carbohydrates.

The presence of galactose, glucose, mannose, and N-acetylglucosamine on the exposed surface of the nematodes Caenorhabditis briggsae and C. elegans was indicated by specific binding of three iodinated plant lectins. Proteolysis experiments suggested the absence of digestible glycoproteins on the exposed surfaces of the two nematode species. High resolution micrographs of cuticle surface preparations labeled with cationized ferritin indicated that the negative charge-bearing molecules are more densely packed on the nematode surface than on animal plasma membranes.     

Differential introgression from a sister species explains high FST outlier loci within a mussel species

Scanning genomes for loci with high levels of population differentiation has become a standard of population genetics. F_ST outlier loci are most often interpreted as signatures of local selection, but outliers might arise for many other reasons too often left unexplored. Here, we tried to identify further the history and genetic basis underlying strong differentiation at F_ST outlier loci in a marine mussel. A genome scan of genetic differentiation has been conducted between Atlantic and Mediterranean populations of Mytilus galloprovincialis. The differentiation was low overall (F_ST = 0.03), but seven loci (2%) were strong F_ST outliers. We then analysed DNA sequence polymorphism at two outlier loci. The genetic structure proved to be the consequence of differential introgression of alleles from the sister‐hybridizing species Mytilus edulis. Surprisingly, the Mediterranean population was the most introgressed at these two loci, although the contact zone between the two species is nowadays localized along the Atlantic coasts of France and the British Isles. A historical contact between M. edulis and Mediterranean M. galloprovincialis should have happened during glacial periods. It proved difficult to disentangle two hypotheses: (i) introgression was adaptive, implying edulis alleles have been favoured in Mediterranean populations, or (ii) the genetic architecture of the barrier to edulis gene flow is different between the two M. galloprovincialis backgrounds. Five of the seven outliers between M. galloprovincialis populations were also outliers between M. edulis and Atlantic M. galloprovincialis, which would support the latter hypothesis. Differential introgression across semi‐permeable barriers to gene flow is a neglected scenario to interpret outlying loci that may prove more widespread than anticipated.     

Hybridisation among groupers (genus <Emphasis Type="Italic">Cephalopholis</Emphasis>) at the eastern Indian Ocean suture zone: taxonomic and evolutionary implications

Hybridisation is a significant evolutionary process that until recently was considered rare in the marine environment. A suture zone in the eastern Indian Ocean is home to numerous hybridising sister species, providing an ideal opportunity to determine how hybridisation affects speciation and biodiversity in coral reef fishes. At this location, hybridisation between two grouper (Epinephelidae) species: Cephalopholis urodeta (Pacific Ocean) and C. nigripinnis (Indian Ocean) was investigated to determine the genetic basis of hybridisation and to compare the ecology and life history of hybrids and their parent species. This approach aimed to provide insights into the taxonomic and evolutionary consequences of hybridisation. Despite clear phenotypic differences, multiple molecular markers revealed hybrids, and their parent species were genetically homogenous within and (thousands of kilometres) outside of the hybrid zone. Hybrids were at least as fit as their parent species (in terms of growth, reproduction, and abundance) and were observed in a broad range of intermediate phenotypes. The two species appear to be interbreeding at Christmas Island due to inherent biological and ecological compatibilities, and the lack of genetic structure may be explained by three potential scenarios: (1) hybridisation and introgression; (2) discordance between morphology and genetics; and (3) incomplete lineage sorting. Further molecular analyses are necessary to discriminate these scenarios. Regardless of which applies, C. urodeta and C. nigripinnis are unlikely to evolve in reproductive isolation as they cohabit where they are common (Christmas Island) and will source congeneric mates where they are rare (Cocos Keeling Islands). Our results add to the growing body of evidence that hybridisation among coral reef fishes is a dynamic evolutionary factor.     

Molecular data support the existence of two species of the Antarctic fish genus <Emphasis Type="Italic">Cryodraco</Emphasis> (Channichthyidae)

Antarctic notothenioids represent one of the few strongly supported examples of adaptive radiation in marine fishes. The extent of population connectivity and structure is unknown for many species, thereby limiting our understanding of the factors that underlie speciation dynamics in this radiation. Here, we assess the population structure of the widespread species Cryodraco antarcticus and its sister species Cryodraco atkinsoni, whose taxonomic status is currently debated. Combining both population genetic and phylogenetic approaches to species delimitation, we provide evidence that C. atkinsoni is a distinct species. Our analyses show that C. atkinsoni and C. antarcticus are recently diverged sister lineages, and the two species differ with regard to patterns of population structure. A systematic and accurate account of species diversity is a critical prerequisite for investigations into the complex processes that underlie the history of speciation in the notothenioid adaptive radiation.     

Genetic differentiation and adaptive evolution at reproductive loci in incipient Drosophila species

Accessory gland proteins (Acps) are part of the seminal fluid of male Drosophila flies. Some Acps have exceptionally high evolutionary rates and evolve under positive selection. Proper interactions between Acps and female reproductive molecules are essential for fertilization. These observations lead to suggestions that fast evolving Acps could be involved in speciation by promoting reproductive incompatibilities between emerging species. To test this hypothesis, we used population genetics data for three sibling species: D. mayaguana, D. parisiena and D. straubae. The latter two species are morphologically very similar and show only incipient reproductive isolation. This system allowed us to examine Acp evolution at different time frames with respect to speciation and reproductive isolation. Comparing data of 14 Acp loci with data obtained for other genomic regions, we found that some Acps show extraordinarily high levels of divergence between D. mayaguana and its two sister species D. parisiena and D. straubae. This divergence was likely driven by adaptive evolution at several loci. No fixed nucleotide differences were found between D. parisiena and D. straubae, however. Nevertheless, some Acp loci did show significant differentiation between these species associated with signs of positive selection; these loci may be involved in this early phase of the speciation process.     

Reproductive Mode and the Evolution of Genome Size and Structure in Caenorhabditis Nematodes

The self-fertile nematode worms Caenorhabditis elegans, C. briggsae, and C. tropicalis evolved independently from outcrossing male-female ancestors and have genomes 20-40% smaller than closely related outcrossing relatives. This pattern of smaller genomes for selfing species and larger genomes for closely related outcrossing species is also seen in plants. We use comparative genomics, including the first high quality genome assembly for an outcrossing member of the genus (C. remanei) to test several hypotheses for the evolution of genome reduction under a change in mating system. Unlike plants, it does not appear that reductions in the number of repetitive elements, such as transposable elements, are an important contributor to the change in genome size. Instead, all functional genomic categories are lost in approximately equal proportions. Theory predicts that self-fertilization should equalize the effective population size, as well as the resulting effects of genetic drift, between the X chromosome and autosomes. Contrary to this, we find that the self-fertile C. briggsae and C. elegans have larger intergenic spaces and larger protein-coding genes on the X chromosome when compared to autosomes, while C. remanei actually has smaller introns on the X chromosome than either self-reproducing species. Rather than being driven by mutational biases and/or genetic drift caused by a reduction in effective population size under self reproduction, changes in genome size in this group of nematodes appear to be caused by genome-wide patterns of gene loss, most likely generated by genomic adaptation to self reproduction per se.