SAG-RAD: A Method for Single-Cell Population Genomics of Unicellular Eukaryotes.

Sequencing of reduced representation libraries enables genotyping of many individuals for population genomic studies. However, high amounts of DNA are required, and the method cannot be applied directly on single cells, preventing its use on most microbes. We developed and implemented the analysis of single amplified genomes followed by restriction-site-associated DNA sequencing to bypass labor-intensive culturing and to avoid culturing bias in population genomic studies of unicellular eukaryotes. This method thus opens the way for addressing important questions about the genetic diversity, gene flow, adaptation, dispersal, and biogeography of hitherto unexplored species.

Genomic insights on conservation priorities for North Sea houting and European lake whitefish (Coregonus spp.).

Population genomics analysis holds great potential for informing conservation of endangered populations. We focused on a controversial case of European whitefish (Coregonus spp.) populations. The endangered North Sea houting is the only coregonid fish that tolerates oceanic salinities and was previously considered a species (C. oxyrhinchus) distinct from European lake whitefish (C. lavaretus). However, no firm evidence for genetic-based salinity adaptation has been available. Also, studies based on microsatellite and mitogenome data suggested surprisingly recent divergence (c. 2500 years bp) between houting and lake whitefish. These data types furthermore have provided no evidence for possible inbreeding. Finally, a controversial taxonomic revision recently classified all whitefish in the region as C. maraena, calling conservation priorities of houting into question. We used whole-genome and ddRAD sequencing to analyse six lake whitefish populations and the only extant indigenous houting population. Demographic inference indicated post-glacial expansion and divergence between lake whitefish and houting occurring not long after the Last Glaciation, implying deeper population histories than previous analyses. Runs of homozygosity analysis suggested not only high inbreeding (FROH up to 30.6%) in some freshwater populations but also FROH up to 10.6% in the houting prompting conservation concerns. Finally, outlier scans provided evidence for adaptation to high salinities in the houting. Applying a framework for defining conservation units based on current and historical reproductive isolation and adaptive divergence led us to recommend that the houting be treated as a separate conservation unit regardless of species status. In total, the results underscore the potential of genomics to inform conservation practices, in this case clarifying conservation units and highlighting populations of concern.

Underground speciation: Unraveling the systematics and evolution of the highly diverse tuco-tucos (genus Ctenomys) with genomic data.

Subterranean rodents of the genus Ctenomys (tuco-tucos) are endemic to South America and have experienced relatively recent radiation. There are about 67 recognized species that originated in approximately 1-2 MY. They stand out for their species richness, extraordinary chromosomal diversity, and wide range of habitat they occupy in the continent. Phylogenetic relationships among species of tuco-tucos have been challenging to resolve. Groups of closely-related species have been suggested, but their relationships must be resolved. This study estimates the phylogeny of the genus using massive sequencing, generating thousands of independent molecular markers obtained by RADseq, with a taxonomic sampling that includes 66% of the recognized species. The sequences obtained were mapped against the C. sociabilis genome, recovering up to 1,215 widely shared RAD loci with more than 19,000 polymorphic sites. Our new phylogenetic hypothesis corroborated the species groups previously proposed with cytochrome b gene sequences and provided a much greater resolution of the relationships among species groups. The frater group is sister to all other tuco-tucos, whereas some of the earlierliest proposals placed the sociabilis group as sister to all other tuco-tucos. Ctenomys leucodon, previously proposed as an independent lineage, is associated with the frater group with moderate statistical support. The magellanicus and mendocinus are sister groups in a major clade formed by the boliviensis, talarum, tucumanus, torquatus, and opimus groups. Ctenomys viperinus, included in the phylogeny for the first time, belongs to the tucumanus group. This multi-locus phylogenetic hypothesis provides insights into the historical biogeography of understanding this highly diverse genus.

Challenge accepted: Evolutionary lineages versus taxonomic classification of North American shrub willows (Salix).

PREMISE: The huge diversity of Salix subgenus Chamaetia/Vetrix clade in North America and the lack of phylogenetic resolution within this clade has presented a difficult but fascinating challenge for taxonomists to resolve. Here we tested the existing taxonomic classification with molecular tools. METHODS: In this study, 132 samples representing 46 species from 22 described sections of shrub willows from the United States and Canada were analyzed and combined with 67 samples from Eurasia. The ploidy levels of the samples were determined using flow cytometry and nQuire. Sequences were produced using a RAD sequencing approach and subsequently analyzed with ipyrad, then used for phylogenetic reconstructions (RAxML, SplitsTree), dating analyses (BEAST, SNAPPER), and character evolution analyses of 14 selected morphological traits (Mesquite). RESULTS: The RAD sequencing approach allowed the production of a well-resolved phylogeny of shrub willows. The resulting tree showed an exclusively North American (NA) clade in sister position to a Eurasian clade, which included some North American endemics. The NA clade began to diversify in the Miocene. Polyploid species appeared in each observed clade. Character evolution analyses revealed that adaptive traits such as habit and adaxial nectaries evolved multiple times independently. CONCLUSIONS: The diversity in shrub willows was shaped by an evolutionary radiation in North America. Most species were monophyletic, but the existing sectional classification could not be supported by molecular data. Nevertheless, monophyletic lineages share several morphological characters, which might be useful in the revision of the taxonomic classification of shrub willows.

Genomic resources for the Yellowfin tuna Thunnus albacares.

BACKGROUND: The Yellowfin tuna (Thunnus albacares) is a large tuna exploited by major fisheries in tropical and subtropical waters of all oceans except the Mediterranean Sea. Genomic studies of population structure, adaptive variation or of the genetic basis of phenotypic traits are needed to inform fisheries management but are currently limited by the lack of a reference genome for this species. Here we report a draft genome assembly and a linkage map for use in genomic studies of T. albacares. METHODS AND RESULTS: Illumina and PacBio SMRT sequencing were used in combination to generate a hybrid assembly that comprises 743,073,847 base pairs contained in 2,661 scaffolds. The assembly has a N50 of 351,587 and complete and partial BUSCO scores of 86.47% and 3.63%, respectively. Double-digest restriction associated DNA (ddRAD) was used to genotype the 2 parents and 164 of their F1 offspring resulting from a controlled breeding cross, retaining 19,469 biallelic single nucleotide polymorphism (SNP) loci. The SNP loci were used to construct a linkage map that features 24 linkage groups that represent the 24 chromosomes of yellowfin tuna. The male and female maps span 1,243.8 cM and 1,222.9 cM, respectively. The map was used to anchor the assembly in 24 super-scaffolds that contain 79% of the yellowfin tuna genome. Gene prediction identified 46,992 putative genes 20,203 of which could be annotated via gene ontology. CONCLUSIONS: The draft reference will be valuable to interpret studies of genome wide variation in T. albacares and other Scombroid species.

Population genomics of Corsican wildcats: Paving the way toward a new subspecies within the Felis silvestris spp. complex?

In the context of the current extinction crisis, identifying new conservation units is pivotal to the development of sound conservation measures, especially in highly threatened taxa such as felids. Corsican wildcats are known by Corsican people since a very long time but have been little studied. Meaningful information about their phylogenetic position is lacking. We used ddRADseq to genotype phenotypically homogenous Corsican wildcats at 3671 genome-wide SNPs and reported for the first time their genetic identity. We compared this genomic information to domestic cats Felis silvestris catus from Corsica and mainland France, European wildcats F. s. silvestris and Sardinian wildcats F. s. lybica. Our premise was that if the Corsican wildcat, as a phenotypic entity, also represents a genetic entity, it deserves conservation measures and to be recognized as a conservation unit. Corsican wildcats appeared highly genetically differentiated from European wildcats and genetically closer to Sardinian wildcats than to domestic cats. Domestic cats from Corsica and mainland France were closer to each other and Sardinian wildcats were intermediate between Corsican wildcats and domestic cats. This suggested that Corsican wildcats do not belong to the F. s. silvestris or catus lineages. The inclusion of more high-quality Sardinian samples and Near-Eastern mainland F. s. lybica would constitute the next step toward assessing the status of Corsican wildcat as a subspecies and/or evolutionarily significant unit and tracing back wildcat introduction history of in Corsica.

The worldwide invasion history of a pest ambrosia beetle inferred using population genomics.

Xylosandrus crassiusculus, a fungus-farming wood borer native to Southeastern Asia, is the most rapidly spreading invasive ambrosia species worldwide. Previous studies focusing on its genetic structure suggested the existence of cryptic genetic variation in this species. Yet, these studies used different genetic markers, focused on different geographical areas and did not include Europe. Our first goal was to determine the worldwide genetic structure of this species based on both mitochondrial and genomic markers. Our second goal was to study X. crassiusculus' invasion history on a global level and identify the origins of the invasion in Europe. We used a COI and RAD sequencing design to characterize 188 and 206 specimens worldwide, building the most comprehensive genetic data set for any ambrosia beetle to date. The results were largely consistent between markers. Two differentiated genetic clusters were invasive, albeit in different regions of the world. The markers were inconsistent only for a few specimens found exclusively in Japan. Mainland USA could have acted as a source for further expansion to Canada and Argentina through stepping stone expansion and bridgehead events. We showed that Europe was only colonized by Cluster II through a complex invasion history including several arrivals from multiple origins in the native area, and possibly including bridgehead from the United States. Our results also suggested that Spain was colonized directly from Italy through intracontinental dispersion. It is unclear whether the mutually exclusive allopatric distribution of the two clusters is due to neutral effects or due to different ecological requirements.

Meeting in Liguria: Hybridisation between Apennine endemic Euphorbia barrelieri and western Mediterranean E. nicaeensis led to the allopolyploid origin of E. ligustica.

The Mediterranean Basin is renowned for its extremely rich biota and is considered as one of the 25 Global Biodiversity Hotspots, but its diversity is not homogeneously distributed. Outstanding in the number of (endemic) species are the Ligurian Alps (Italy). At the foot of the Ligurian Alps, little above the Mediterranean Sea, a disjunct occurrence of Italian endemic Euphorbia barrelieri was reported. Using an array of integrative methods ranging from cytogenetic (chromosome number and relative genome size estimation), over phylogenetic approaches (plastid, ITS and RAD sequencing) to multivariate morphometrics we disentangled the origin of these populations that were shown to be tetraploid. We performed phylogenetic analyses of the nuclear ITS and plastid regions of a broad taxonomic sampling of Euphorbia sect. Pithyusa to identify possible species involved in the origin of the tetraploid populations and then applied various analyses of RADseq data to identify the putative parental species. Our results have shown that the Ligurian populations of E. barrelieri are of allotetraploid origin that involved E. barrelieri and western Mediterranean E. nicaeensis as parental species. We thus describe a new species, E. ligustica, and hypothesise that its adaptation to similar environments in which E. barrelieri occurs, triggered development of similar morphology, whereas its genetic composition appears to be closer to that of E. nicaeensis. Our study emphasises the importance of polyploidisation for plant diversification, highlights the value of the Ligurian Alps as a hotspot of biodiversity and endemism and underlines the importance of integrative taxonomic approaches in uncovering cryptic diversity.

Reproductive isolation between diploid and tetraploid individuals in mixed-cytotype populations of Lycium australe.

PREMISE: Whole-genome duplication is considered a major mechanism of sympatric speciation due to the creation of strong and instantaneous reproductive barriers. Although postzygotic reproductive isolation between diploids and polyploids is often expected, the extent of reproductive incompatibility must be empirically determined and compared to patterns of genetic isolation to fully characterize the reproductive dynamics between cytotypes. METHODS: We investigated reproductive compatibility between diploid and tetraploid Lycium australe in two mixed-cytotype populations using (1) controlled crossing experiments to evaluate fruit and seed production and (2) germination trials to test seed viability following homoploid and heteroploid crosses. We contrast these experiments with a single-nucleotide polymorphism (SNP) data set to measure genetic isolation between cytotypes and explore whether cytotype or population origin better explains patterns of genetic variation. Finally, we explore mating patterns using the observed germination rates of naturally produced seeds in each population. RESULTS: Although homoploid and heteroploid crosses resulted in similar fruit and seed production, reproductive isolation between co-occurring diploids and tetraploids was nearly complete, due to low seed viability following heteroploid crosses. Of 191,182 total SNPs, 21,679 were present in ≥90% of individuals and replicate runs using unlinked SNPs revealed strong clustering by cytotype and differentiation of tetraploids based on population origin. CONCLUSIONS: As often reported, diploid and tetraploid L. australe experience strong postzygotic isolation via hybrid seed inviability. Consistent with this result, cytotype explained a greater amount of variation in the SNP data set than population origin, despite some evidence of historical introgression.

Genomic evidence for three distinct species in the Erebia manto complex in Central Europe (Lepidoptera, Nymphalidae).

A problem to implement conservation strategies is that in many cases recognized taxa are in fact complexes of several cryptic species. Failure to properly delineate species may lead to misplaced priorities or to inadequate conservation measures. One such species complex is the yellow-spotted ringlet Erebia manto, which comprises several phenotypically distinct lineages, whose degree of genomic isolation has so far not been assessed. Some of these lineages are geographically restricted and thus possibly represent distinct units with conservation priorities. Using several thousand nuclear genomic markers, we evaluated to which degree the bubastis lineage from the Alps and the vogesiaca lineage from the Vosges, are genetically isolated from the widespread manto lineage. Our results suggest that both lineages are genetically as strongly differentiated from manto as other taxonomically well separated sibling species in this genus from each other, supporting a delineation of bubastis and vogesiaca as independent species. Given the restricted and isolated range of vogesiaca as well as the disjunct distribution of bubastis, our findings have significant implication for future conservation efforts on these formerly cryptic species and highlight the need to investigate the genomic identity within species complexes. Supplementary Information: The online version contains supplementary material available at 10.1007/s10592-023-01501-w.

A phylogeny of Antirrhinum reveals parallel evolution of alpine morphology.

Parallel evolution of similar morphologies in closely related lineages provides insight into the repeatability and predictability of evolution. In the genus Antirrhinum (snapdragons), as in other plants, a suite of morphological characters are associated with adaptation to alpine environments. We tested for parallel trait evolution in Antirrhinum by investigating phylogenetic relationships using restriction-site associated DNA (RAD) sequencing. We then associated phenotypic information to our phylogeny to reconstruct the patterns of morphological evolution and related this to evidence for hybridisation between emergent lineages. Phylogenetic analyses showed that the alpine character syndrome is present in multiple groups, suggesting that Antirrhinum has repeatedly colonised alpine habitats. Dispersal to novel environments happened in the presence of intraspecific and interspecific gene flow. We found support for a model of parallel evolution in Antirrhinum. Hybridisation in natural populations, and a complex genetic architecture underlying the alpine morphology syndrome, support an important role of natural selection in maintaining species divergence in the face of gene flow.

Population genomics of introduced Nile tilapia Oreochromis niloticus (Linnaeus, 1758) in the Democratic Republic of the Congo: Repeated introductions since colonial times with multiple sources.

During colonial times, Nile tilapia Oreochromis niloticus (Linnaeus, 1758) was introduced into non-native parts of the Congo Basin (Democratic Republic of the Congo, DRC) for the first time. Currently, it is the most farmed cichlid in the DRC, and is present throughout the Congo Basin. Although Nile tilapia has been reported as an invasive species, documentation of historical introductions into this basin and its consequences are scant. Here, we study the genetic consequences of these introductions by genotyping 213 Nile tilapia from native and introduced regions, focusing on the Congo Basin. Additionally, 48 specimens from 16 other tilapia species were included to test for hybridization. Using RAD sequencing (27,611 single nucleotide polymorphisms), we discovered genetic admixture with other tilapia species in several morphologically identified Nile tilapia from the Congo Basin, reflecting their ability to interbreed and the potential threat they pose to the genetic integrity of native tilapias. Nile tilapia populations from the Upper Congo and those from the Middle-Lower Congo are strongly differentiated. The former show genetic similarity to Nile tilapia from the White Nile, while specimens from the Benue Basin and Lake Kariba are similar to Nile tilapia from the Middle-Lower Congo, suggesting independent introductions using different sources. We conclude that the presence of Nile tilapia in the Congo Basin results from independent introductions, reflecting the dynamic aquaculture history, and that their introduction probably leads to genetic interactions with native tilapias, which could lower their fitness. We therefore urge avoiding further introductions of Nile tilapia in non-native regions and to use native tilapias in future aquaculture efforts.

Population genomic structure in Goodman's mouse lemur reveals long-standing separation of Madagascar's Central Highlands and eastern rainforests.

Madagascar's Central Highlands are largely composed of grasslands, interspersed with patches of forest. The historical perspective was that Madagascar's grasslands had anthropogenic origins, but emerging evidence suggests that grasslands were a component of the pre-human Central Highlands vegetation. Consequently, there is now vigorous debate regarding the extent to which these grasslands have expanded due to anthropogenic pressures. Here, we shed light on the temporal dynamics of Madagascar's vegetative composition by conducting a population genomic investigation of Goodman's mouse lemur (Microcebus lehilahytsara; Cheirogaleidae). These small-bodied primates occur both in Madagascar's eastern rainforests and in the Central Highlands, making them a valuable indicator species. Population divergences among forest-dwelling mammals will reflect changes to their habitat, including fragmentation, whereas patterns of post-divergence gene flow can reveal formerly wooded migration corridors. To explore these patterns, we used RADseq data to infer population genetic structure, demographic models of post-divergence gene flow, and population size change through time. The results offer evidence that open habitats are an ancient component of the Central Highlands, and that widespread forest fragmentation occurred naturally during a period of decreased precipitation near the last glacial maximum. Models of gene flow suggest that migration across the Central Highlands has been possible from the Pleistocene through the recent Holocene via riparian corridors. Though our findings support the hypothesis that Central Highland grasslands predate human arrival, we also find evidence for human-mediated population declines. This highlights the extent to which species imminently threatened by human-mediated deforestation may already be vulnerable from paleoclimatic conditions.

Genomic scale data shows that Parastacus nicoleti encompasses more than one species of burrowing continental crayfishes and that lineage divergence occurred with and without gene flow.

Delimiting species is a challenge, especially in scenarios of diversification with gene flow and when species are now allopatric where reproductive isolation cannot be directly tested. Continental burrowing crayfishes of the genus Parastacus present a disjoint distribution in southern South America. One of the species is P. nicoleti, which lives in underground waters in swampy and wooded areas of southern Chile. A previous assessment based on mitochondrial DNA sequences suggest that the taxon may represent a species complex. Here, using thousands of nuclear genomic single-nucleotide polymorphisms obtained via RADSeq from 81 specimens collected at 27 localities throughout the distributional range of the species, we apply an integrative species delimitation approach to test species boundaries and to investigate some aspects of the speciation process. Our analyses corroborate previous results; a scenario that we favor suggests that the P. nicoleti encompasses seven distinct species. Additionally, demographic analyses show that the distinct species have followed distinct trajectories in size change during the last 17.5 million years and that speciation in this group occurred both in strict isolation as well as in the presence of gene flow.

Species and population genomic differentiation in Pocillopora corals (Cnidaria, Hexacorallia).

Correctly delimiting species and populations is a prerequisite for studies of connectivity, adaptation and conservation. Genomic data are particularly useful to test species differentiation for organisms with few informative morphological characters or low discrimination of cytoplasmic markers, as in Scleractinians. Here we applied Restriction site Associated DNA sequencing (RAD-sequencing) to the study of species differentiation and genetic structure in populations of Pocillopora spp. from Oman and French Polynesia, with the objectives to test species hypotheses, and to study the genetic structure among sampling sites within species. We focused here on coral colonies morphologically similar to P. acuta (damicornis type ß). We tested the impact of different filtering strategies on the stability of the results. The main genetic differentiation was observed between samples from Oman and French Polynesia. These samples corresponded to different previously defined primary species hypotheses (PSH), i.e., PSHs 12 and 13 in Oman, and PSH 5 in French Polynesia. In Oman, we did not observe any clear differentiation between the two putative species PSH 12 and 13, nor between sampling sites. In French Polynesia, where a single species hypothesis was studied, there was no differentiation between sites. Our analyses allowed the identification of clonal lineages in Oman and French Polynesia. The impact of clonality on genetic diversity is discussed in light of individual-based simulations.

Landscape Connectivity and Genetic Structure in a Mainstem and a Tributary Stonefly (Plecoptera) Species Using a Novel Reference Genome.

Understanding how environmental variation influences population genetic structure can help predict how environmental change influences population connectivity, genetic diversity, and evolutionary potential. We used riverscape genomics modeling to investigate how climatic and habitat variables relate to patterns of genetic variation in 2 stonefly species, one from mainstem river habitats (Sweltsa coloradensis) and one from tributaries (Sweltsa fidelis) in 40 sites in northwest Montana, USA. We produced a draft genome assembly for S. coloradensis (N50 = 0.251 Mbp, BUSCO > 95% using "insecta_ob9" reference genes). We genotyped 1930 SNPs in 372 individuals for S. coloradensis and 520 SNPs in 153 individuals for S. fidelis. We found higher genetic diversity for S. coloradensis compared to S. fidelis, but nearly identical genetic differentiation among sites within each species (both had global loci median FST = 0.000), despite differences in stream network location. For landscape genomics and testing for selection, we produced a less stringently filtered data set (3454 and 1070 SNPs for S. coloradensis and S. fidelis, respectively). Environmental variables (mean summer precipitation, slope, aspect, mean June stream temperature, land cover type) were correlated with 19 putative adaptive loci for S. coloradensis, but there was only one putative adaptive locus for S. fidelis (correlated with aspect). Interestingly, we also detected potential hybridization between multiple Sweltsa species which has never been previously detected. Studies like ours, that test for adaptive variation in multiple related species are needed to help assess landscape connectivity and the vulnerability of populations and communities to environmental change.

Mapping QTLs for 1000-grain weight and genes controlling hull type using SNP marker in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum), an important pseudocereal crop, has high economic value due to its nutritional and medicinal properties. However, dehulling of Tartary buckwheat is difficult owing to its thick and tough hull, which has greatly limited the development of the Tartary buckwheat processing industry. The construction of high-resolution genetic maps serves as a basis for identifying quantitative trait loci (QTLs) and qualitative trait genes for agronomic traits. In this study, a recombinant inbred lines (XJ-RILs) population derived from a cross between the easily dehulled Rice-Tartary type and Tartary buckwheat type was genotyped using restriction site-associated DNA (RAD) sequencing to construct a high-density SNP genetic map. Furthermore, QTLs for 1000-grain weight (TGW) and genes controlling hull type were mapped in multiple environments. RESULTS: In total, 4151 bin markers comprising 122,185 SNPs were used to construct the genetic linkage map. The map consisted of 8 linkage groups and covered 1444.15 cM, with an average distance of 0.35 cM between adjacent bin markers. Nine QTLs for TGW were detected and distributed on four loci on chromosome 1 and 4. A major locus detected in all three trials was mapped in 38.2-39.8 cM region on chromosome 1, with an LOD score of 18.1-37.0, and explained for 23.6-47.5% of the phenotypic variation. The genes controlling hull type were mapped to chromosome 1 between marker Block330 and Block331, which was closely followed by the major locus for TGW. The expression levels of the seven candidate genes controlling hull type present in the region between Block330 and Block336 was low during grain development, and no significant difference was observed between the parental lines. Six non-synonymous coding SNPs were found between the two parents in the region. CONCLUSIONS: We constructed a high-density SNP genetic map for the first time in Tartary buckwheat. The mapped major loci controlling TGW and hull type will be valuable for gene cloning and revealing the mechanism underlying grain development and easy dehulling, and marker-assisted selection in Tartary buckwheat.

Evidence for multiple colonisations and Wolbachia infections shaping the genetic structure of the widespread butterfly Polyommatus icarus in the British Isles.

The paradigm of isolation in southern refugia during glacial periods followed by expansions during interglacials, producing limited genetic differentiation in northern areas, dominates European phylogeography. However, the existence of complex structured populations in formerly glaciated areas, and islands connected to mainland areas during glacial maxima, call for alternative explanations. We reconstructed the mtDNA phylogeography of the widespread Polyommatus Icarus butterfly with an emphasis on the formerly glaciated and connected British Isles. We found distinct geographical structuring of CO1 haplogroups, with an ancient lineage restricted to the marginal European areas, including Northern Scotland and Outer Hebrides. Population genomic analyses, using ddRADSeq genomic markers, also reveal substantial genetic structuring within Britain. However, there is negligble mito-nuclear concordance consistent with independent demographic histories of mitochondrial versus nuclear DNA. While mtDNA-Wolbachia associations in northern Britain could account for the geographic structuring of mtDNA across most of the British Isles, for nuclear DNA markers (derived from ddRADseq data) butterflies from France cluster between northern and southern British populations - an observation consistent with a scenario of multiple recolonisation. Taken together our results suggest that contemporary mtDNA structuring in the British Isles (and potentially elsewhere in Europe) largely results from Wolbachia infections, however, nuclear genomic structuring suggests a history of at least two distinct colonisations. This two-stage colonisation scenario has previously been put forth to explain genetic diversity and structuring in other British flora and fauna. Additionally, we also present preliminary evidence for potential Wolbachia-induced feminization in the Outer Hebrides.

New genomic resources for three exploited Mediterranean fishes.

Extensive fishing has led to fish stock declines throughout the last decades. While clear stock identification is required for designing management schemes, stock delineation is problematic due to generally low levels of genetic structure in marine species. The development of genomic resources can help to solve this issue. Here, we present the first mitochondrial and nuclear draft genome assemblies of three economically important Mediterranean fishes, the white seabream, the striped red mullet, and the comber. The assemblies are between 613 and 785 Mbp long and contain between 27,222 and 32,375 predicted genes. They were used as references to map Restriction-site Associated DNA markers, which were developed with a single-digest approach. This approach provided between 15,710 and 21,101 Single Nucleotide Polymorphism markers per species. These genomic resources will allow uncovering subtle genetic structure, identifying stocks, assigning catches to populations and assessing connectivity. Furthermore, the annotated genomes will help to characterize adaptive divergence.

Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs.

Subdivided Pleistocene glacial refugia, best known as "refugia within refugia", provided opportunities for diverging populations to evolve into incipient species and/or to hybridize and merge following range shifts tracking the climatic fluctuations, potentially promoting extensive cytonuclear discordances and "ghost" mtDNA lineages. Here, we tested which of these opposing evolutionary outcomes prevails in northern Iberian areas hosting multiple historical refugia of common frogs (Rana cf. temporaria), based on a genomic phylogeography approach (mtDNA barcoding and RAD-sequencing). We found evidence for both incipient speciation events and massive cytonuclear discordances. On the one hand, populations from northwestern Spain (Galicia and Asturias, assigned to the regional endemic R. parvipalmata), are deeply-diverged at mitochondrial and nuclear genomes (~4 My of independent evolution), and barely admix with northeastern populations (assigned to R. temporaria sensu stricto) across a narrow hybrid zone (~25 km) located in the Cantabrian Mountains, suggesting that they represent distinct species. On the other hand, the most divergent mtDNA clade, widespread in Cantabria and the Basque country, shares its nuclear genome with other R. temporaria s. s. lineages. Patterns of population expansions and isolation-by-distance among these populations are consistent with past mitochondrial capture and/or drift in generating and maintaining this ghost mitochondrial lineage. This remarkable case study emphasizes the complex evolutionary history that shaped the present genetic diversity of refugial populations, and stresses the need to revisit their phylogeography by genomic approaches, in order to make informed taxonomic inferences.