Common lizard primary oviduct cell culture: A model system for the genetic and cellular basis of oviparity and viviparity.

Reproduction by egg-laying (oviparity) or live-bearing (viviparity) is a genetically determined trait fundamental to the biology of amniotes. Squamates are an emerging model for the genetics of reproductive mode yet lack cell culture models valuable for exploring molecular mechanisms. Here, we report a novel primary culture model for reproductive biology: cell cultures derived from the oviduct tissues (infundibulum, uterus and vagina) of oviparous and viviparous common lizards (Lacertidae: Zootoca vivipara). We maintained and expanded these cultures for over 100 days, including repeated subculturing and successful revival of cryopreserved cells. Immunocytochemical investigation suggested expression of both epithelial and fibroblast-like proteins, and RNA sequencing of cultured cells as compared to in vivo oviduct tissue showed changes in gene expression in response to the cell culture environment. Despite this, we confirmed the maintenance of distinct gene expression patterns in viviparous and oviparous cells after 60+ days of cell culture, finding 354 differentially expressed genes between viviparous and oviparous cells. Furthermore, we confirmed the expression of 15 viviparity-associated candidate genes in cells maintained for 60+ days in culture. Our study demonstrates the feasibility and utility of oviduct cell culture for molecular analysis of reproductive mode and provides a tool for future genetic experiments.

Genetic basis and expression of ventral colour in polymorphic common lizards.

Colour is an important visual cue that can correlate with sex, behaviour, life history or ecological strategies, and has evolved divergently and convergently across animal lineages. Its genetic basis in non-model organisms is rarely known, but such information is vital for determining the drivers and mechanisms of colour evolution. Leveraging genetic admixture in a rare contact zone between oviparous and viviparous common lizards (Zootoca vivipara), we show that females (N = 558) of the two otherwise morphologically indistinguishable reproductive modes differ in their ventral colouration (from pale to vibrant yellow) and intensity of melanic patterning. We find no association between female colouration and reproductive investment, and no evidence for selection on colour. Using a combination of genetic mapping and transcriptomic evidence, we identified two candidate genes associated with ventral colour differentiation, DGAT2 and PMEL. These are genes known to be involved in carotenoid metabolism and melanin synthesis respectively. Ventral melanic spots were associated with two genomic regions, including a SNP close to protein tyrosine phosphatase (PTP) genes. Using genome re-sequencing data, our results show that fixed coding mutations in the candidate genes cannot account for differences in colouration. Taken together, our findings show that the evolution of ventral colouration and its associations across common lizard lineages is variable. A potential genetic mechanism explaining the flexibility of ventral colouration may be that colouration in common lizards, but also across squamates, is predominantly driven by regulatory genetic variation.

Genomic underpinnings of head and body shape in Arctic charr ecomorph pairs.

Across its Holarctic range, Arctic charr (Salvelinus alpinus) populations have diverged into distinct trophic specialists across independent replicate lakes. The major aspect of divergence between ecomorphs is in head shape and body shape, which are ecomorphological traits reflecting niche use. However, whether the genomic underpinnings of these parallel divergences are consistent across replicates was unknown but key for resolving the substrate of parallel evolution. We investigated the genomic basis of head shape and body shape morphology across four benthivore-planktivore ecomorph pairs of Arctic charr in Scotland. Through genome-wide association analyses, we found genomic regions associated with head shape (89 SNPs) or body shape (180 SNPs) separately and 50 of these SNPs were strongly associated with both body and head shape morphology. For each trait separately, only a small number of SNPs were shared across all ecomorph pairs (3 SNPs for head shape and 10 SNPs for body shape). Signs of selection on the associated genomic regions varied across pairs, consistent with evolutionary demography differing considerably across lakes. Using a comprehensive database of salmonid QTLs newly augmented and mapped to a charr genome, we found several of the head- and body-shape-associated SNPs were within or near morphology QTLs from other salmonid species, reflecting a shared genetic basis for these phenotypes across species. Overall, our results demonstrate how parallel ecotype divergences can have both population-specific and deeply shared genomic underpinnings across replicates, influenced by differences in their environments and demographic histories.

Viviparity is associated with larger female size and higher sexual size dimorphism in a reproductively bimodal lizard.

Squamate reptiles are central for studying phenotypic correlates of evolutionary transitions from oviparity to viviparity because these transitions are numerous, with many of them being recent. Several models of life-history theory predict that viviparity is associated with increased female size, and thus more female-biased sexual size dimorphism (SSD). Yet, the corresponding empirical evidence is overall weak and inconsistent. The lizard Zootoca vivipara, which occupies a major part of Northern Eurasia and includes four viviparous and two non-sister oviparous lineages, represents an excellent model for testing these predictions. We analysed how sex-specific body size and SSD is associated with parity mode, using body length data for nearly 14,000 adult individuals from 97 geographically distinct populations, which cover almost the entire species' range and represent all six lineages. Our analyses controlled for lineage identity, climatic seasonality (the strongest predictor of geographic body size variation in previous studies of this species) and several aspects of data heterogeneity. Parity mode, lineage and seasonality are significantly associated with female size and SSD; the first two predictors accounted for 14%-26% of the total variation each, while seasonality explained 5%-7%. Viviparous populations exhibited a larger female size than oviparous populations, with no concomitant differences in male size. The variation of male size was overall low and poorly explained by our predictors. Albeit fully expected from theory, the strong female bias of the body size differences between oviparous and viviparous populations found in Z. vivipara is not evident from available data on three other lizard systems of closely related lineages differing in parity mode. We confront this pattern with the data on female reproductive traits in the considered systems and the frequencies of evolutionary changes of parity mode in the corresponding lizard families and speculate why the life-history correlates of live-bearing in Z. vivipara are distinct. Comparing conspecific populations, our study provides the most direct evidence for the predicted effect of parity mode on adult body size but also demonstrates that the revealed pattern may not be general. This might explain why across squamates, viviparity is only weakly associated with larger size.

Genomic basis of fishing-associated selection varies with population density.

Fisheries induce one of the strongest anthropogenic selective pressures on natural populations, but the genetic effects of fishing remain unclear. Crucially, we lack knowledge of how capture-associated selection and its interaction with reductions in population density caused by fishing can potentially shift which genes are under selection. Using experimental fish reared at two densities and repeatedly harvested by simulated trawling, we show consistent phenotypic selection on growth, metabolism, and social behavior regardless of density. However, the specific genes under selection-mainly related to brain function and neurogenesis-varied with the population density. This interaction between direct fishing selection and density could fundamentally alter the genomic responses to harvest. The evolutionary consequences of fishing are therefore likely context dependent, possibly varying as exploited populations decline. These results highlight the need to consider environmental factors when predicting effects of human-induced selection and evolution.

Environmental correlates of adaptive diversification in postglacial freshwater fishes.

Determining how environmental conditions contribute to divergence among populations and drive speciation is fundamental to resolving mechanisms and understanding outcomes in evolutionary biology. Postglacial freshwater fish species in the Northern Hemisphere are ideal biological systems to explore the effects of environment on diversification in morphology, ecology, and genetics (ecomorph divergences) within lakes. To date, various environmental factors have been implicated in the presence of multiple ecomorphs within particular lakes or regions. However, concerted evidence for generalizable patterns in environmental variables associated with speciation across geographical regions and across species and genera has been lacking. Here, we aimed to identify key biotic and abiotic factors associated with ecological divergence of postglacial freshwater fish species into multiple sympatric ecomorphs, focusing on species in the well-studied, widespread, and co-distributed genera Gasterosteus, Salvelinus, and Coregonus (stickleback, charr, and whitefish, respectively). We found that the presence of multiple sympatric ecomorphs tended to be associated with increasing lake surface area, maximum depth, and nutrient availability. In addition, predation, competition, and prey availability were suggested to play a role in divergence into multiple ecomorphs, but the effects of biotic factors require further study. Although we identified several environmental factors correlated with the presence of multiple ecomorphs, there were substantial data gaps across species and regions. An improved understanding of these systems may provide insight into both generalizable environmental factors involved in speciation in other systems, and potential ecological and evolutionary responses of species complexes when these variables are altered by environmental change.

Investigating the genetic basis of vertebrate dispersal combining RNA-seq, RAD-seq and quantitative genetics.

Although animal dispersal is known to play key roles in ecological and evolutionary processes such as colonization, population extinction and local adaptation, little is known about its genetic basis, particularly in vertebrates. Untapping the genetic basis of dispersal should deepen our understanding of how dispersal behaviour evolves, the molecular mechanisms that regulate it and link it to other phenotypic aspects in order to form the so-called dispersal syndromes. Here, we comprehensively combined quantitative genetics, genome-wide sequencing and transcriptome sequencing to investigate the genetic basis of natal dispersal in a known ecological and evolutionary model of vertebrate dispersal: the common lizard, Zootoca vivipara. Our study supports the heritability of dispersal in semi-natural populations, with less variation attributable to maternal and natal environment effects. In addition, we found an association between natal dispersal and both variation in the carbonic anhydrase (CA10) gene, and in the expression of several genes (TGFB2, SLC6A4, NOS1) involved in central nervous system functioning. These findings suggest that neurotransmitters (serotonin and nitric oxide) are involved in the regulation of dispersal and shaping dispersal syndromes. Several genes from the circadian clock (CRY2, KCTD21) were also differentially expressed between disperser and resident lizards, supporting that the circadian rhythm, known to be involved in long-distance migration in other taxa, might affect dispersal as well. Since neuronal and circadian pathways are relatively well conserved across vertebrates, our results are likely to be generalisable, and we therefore encourage future studies to further investigate the role of these pathways in shaping dispersal in vertebrates.

Deep genome-wide divergences among species in White Cloud Mountain minnow Tanichthys albonubes (Cypriniformes: Tanichthyidae) complex: Conservation and species management implications.

Identifying cryptic species is important for the assessments of biodiversity. Further, untangling mechanisms underlying the origins of cryptic species can facilitate our understanding of evolutionary processes. Advancements in genomic approaches for non-model systems have offered unprecedented opportunities to investigate these areas. The White Cloud Mountain minnow (Tanichthys albonubes) is a popular freshwater pet fish worldwide but its wild populations in China are critically endangered. Recent research based on a few molecular markers suggested that this species in fact comprised seven cryptic species, of which six were previously unknown. Here, we tested six of these cryptic species and quantified genomic interspecific divergences between species in the T. albonubes complex by analyzing genome-wide restriction site-associated DNA sequencing (RADseq) data generated from 189 individuals sampled from seven populations (including an outgroup congeneric species, T. micagemmae). We found that six cryptic species previously suggested were well supported by RADseq data. The genetic diversity of each species in the T. albonubes complex was low compared with T. micagemmae and the contemporary effective population sizes (Ne) of each cryptic species were small. Phylogenetic analysis showed seven clades with high support values confirmed with Neighbor-Net trees. The pairwise divergences between species in T. albonubes complex were deep and the highly differentiated loci were evenly distributed across the genome. We proposed that the divergence level of T. albonubes complex is at a late stage of cryptic speciation and lacking gene flow. Our findings provide new insights into cryptic speciation and have important implications for conservation and species management of T. albonubes complex.

Parallelism in eco-morphology and gene expression despite variable evolutionary and genomic backgrounds in a Holarctic fish.

Understanding the extent to which ecological divergence is repeatable is essential for predicting responses of biodiversity to environmental change. Here we test the predictability of evolution, from genotype to phenotype, by studying parallel evolution in a salmonid fish, Arctic charr (Salvelinus alpinus), across eleven replicate sympatric ecotype pairs (benthivorous-planktivorous and planktivorous-piscivorous) and two evolutionary lineages. We found considerable variability in eco-morphological divergence, with several traits related to foraging (eye diameter, pectoral fin length) being highly parallel even across lineages. This suggests repeated and predictable adaptation to environment. Consistent with ancestral genetic variation, hundreds of loci were associated with ecotype divergence within lineages of which eight were shared across lineages. This shared genetic variation was maintained despite variation in evolutionary histories, ranging from postglacial divergence in sympatry (ca. 10-15kya) to pre-glacial divergence (ca. 20-40kya) with postglacial secondary contact. Transcriptome-wide gene expression (44,102 genes) was highly parallel across replicates, involved biological processes characteristic of ecotype morphology and physiology, and revealed parallelism at the level of regulatory networks. This expression divergence was not only plastic but in part genetically controlled by parallel cis-eQTL. Lastly, we found that the magnitude of phenotypic divergence was largely correlated with the genetic differentiation and gene expression divergence. In contrast, the direction of phenotypic change was mostly determined by the interplay of adaptive genetic variation, gene expression, and ecosystem size. Ecosystem size further explained variation in putatively adaptive, ecotype-associated genomic patterns within and across lineages, highlighting the role of environmental variation and stochasticity in parallel evolution. Together, our findings demonstrate the parallel evolution of eco-morphology and gene expression within and across evolutionary lineages, which is controlled by the interplay of environmental stochasticity and evolutionary contingencies, largely overcoming variable evolutionary histories and genomic backgrounds.

Alternative splicing and gene expression play contrasting roles in the parallel phenotypic evolution of a salmonid fish.

Understanding the contribution of different molecular processes to evolution and development is crucial for identifying the mechanisms of adaptation. Here, we used RNA-sequencing data to test the importance of alternative splicing and differential gene expression in a case of parallel adaptive evolution, the replicated postglacial divergence of the salmonid fish Arctic charr (Salvelinus alpinus) into sympatric benthic and pelagic ecotypes across multiple independent lakes. We found that genes differentially spliced between ecotypes were mostly not differentially expressed (<6% overlap) and were involved in different biological processes. Differentially spliced genes were primarily enriched for muscle development and functioning, while differentially expressed genes were involved in metabolism, immunity and growth. Furthermore, alternative splicing and gene expression were mostly controlled by independent cis-regulatory quantitative trait loci (<3.4% overlap). Cis-regulatory regions were associated with the parallel divergence in splicing (16.5% of intron clusters) and expression (6.7%-10.1% of differentially expressed genes), indicating shared regulatory variation across ecotype pairs. Contrary to theoretical expectation, we found that differentially spliced genes tended to be highly central in regulatory networks ("hub genes") and were annotated to significantly more gene ontology terms compared to nondifferentially spliced genes, consistent with a higher level of pleiotropy. Together, our results suggest that the concerted regulation of alternative splicing and differential gene expression through different regulatory regions leads to the divergence of complementary processes important for local adaptation. This provides novel insights into the importance of contrasting but putatively complementary molecular processes in rapid parallel adaptive evolution.

Phylogenomic inference of species and subspecies diversity in the Palearctic salamander genus Salamandra.

The salamander genus Salamandra is widespread across Europe, North Africa, and the Near East and is renowned for its conspicuous and polymorphic colouration and diversity of reproductive modes. The phylogenetic relationships within the genus, and especially in the highly polymorphic species S. salamandra, have been very challenging to elucidate, leaving its real evolutionary history and classification at species and subspecies levels a topic of debate and contention. However, the distribution of diversity and species delimitation within the genus are critically important for identifying evolutionarily significant units for conservation and management, especially in light of threats posed by the pathogenic chytrid fungus Batrachochytrium salamandrivorans that is causing massive declines of S. salamandra populations in central Europe. Here, we conducted a phylogenomic analysis from across the taxonomic and geographic breadth of the genus Salamandra in its entire range. Bayesian, maximum likelihood and network-based phylogenetic analyses of up to 4905 ddRADseq-loci (294,300 nucleotides of sequence) supported the distinctiveness of all currently recognised species (Salamandra algira, S. atra, S. corsica, S. infraimmaculata, S. lanzai, and S. salamandra), and all five species for which we have multiple exemplars were confirmed as monophyletic. Within S. salamandra, two main clades can be distinguished: one clade with the Apenninic subspecies S. s. gigliolii nested within the Iberian S. s. bernardezi/fastuosa; and a second clade comprising all other Iberian, Central and East European subspecies. Our analyses revealed that some of the currently recognized subspecies of S. salamandra are paraphyletic and may require taxonomic revision, with the Central- and Eastern-European subspecies all being poorly differentiated at the analysed genomic markers. Salamandra s. longirostris - sometimes considered a separate species - was nested within S. salamandra, consistent with its subspecies status. The relationships identified within and between Salamandra species provide valuable context for future systematic and biogeographic studies, and help elucidate critical evolutionary units for conservation and taxonomy.

Evolutionary origins of viviparity consistent with palaeoclimate and lineage diversification.

It is of fundamental importance for the field of evolutionary biology to understand when and why major evolutionary transitions occur. Live-bearing young (viviparity) is a major evolutionary change and has evolved from egg-laying (oviparity) independently in many vertebrate lineages and most abundantly in lizards and snakes. Although contemporary viviparous squamate species generally occupy cold climatic regions across the globe, it is not known whether viviparity evolved as a response to cold climate in the first place. Here, we used available published time-calibrated squamate phylogenies and parity data on 3,498 taxa. We compared the accumulation of transitions from oviparity to viviparity relative to background diversification and a simulated binary trait. Extracting the date of each transition in the phylogenies and informed by 65 my of global palaeoclimatic data, we tested the nonexclusive hypotheses that viviparity evolved under the following: (a) cold, (b) long-term stable climatic conditions and (c) with background diversification rate. We show that stable and long-lasting cold climatic conditions are correlated with transitions to viviparity across squamates. This correlation of parity mode and palaeoclimate is mirrored by background diversification in squamates, and simulations of a binary trait also showed a similar association with palaeoclimate, meaning that trait evolution cannot be separated from squamate lineage diversification. We suggest that parity mode transitions depend on environmental and intrinsic effects and that background diversification rate may be a factor in trait diversification more generally.

The pitfalls and virtues of population genetic summary statistics: Detecting selective sweeps in recent divergences.

During evolution, genomes are shaped by a plethora of forces that can leave characteristic signatures. A common goal when studying diverging populations is to detect the signatures of selective sweeps, which can be rather difficult in complex demographic scenarios, such as under secondary contact. Moreover, the detection of selective sweeps, especially in whole-genome data, often relies heavily on a narrow set of summary statistics that are affected by a multitude of factors, frequently leading to false positives and false negatives. Simulating genomic regions makes it possible to control these demographic and population genetic factors. We used simulations of large genomic regions to determine how different secondary contact and sympatric speciation scenarios affect the footprint of hard and soft selective sweeps in the presence of varying degrees of gene flow and recombination. We explored the ability of an array of population genetic summary statistics to detect the footprints of these selective sweeps under specific demographies. We focussed on metrics that do not require phased data or ancestral sequences and therefore have wide applicability. We found that a newly developed beta diversity measure, B¯GD utperformed all other metrics in detecting selective sweeps and that FST also performed well. High accuracy was also found in Δπ and genotype distance-derived metrics. The performance of most metrics strongly depended on factors such as the presence of an allopatric phase, migration rates, recombination, population growth, and whether the sweep was hard or soft. We provide suggestions for locating and analysing the response to selective sweeps in whole-genome data.

Complex and divergent histories gave rise to genome-wide divergence patterns amongst European whitefish (Coregonus lavaretus).

Pleistocene glaciations dramatically affected species distribution in regions that were impacted by ice cover and subsequent postglacial range expansion impacted contemporary biodiversity in complex ways. The European whitefish, Coregonus lavaretus, is a widely distributed salmonid fish species on mainland Europe, but in Britain it has only seven native populations, all of which are found on the western extremes of the island. The origins and colonization routes of the species into Britain are unknown but likely contributed to contemporary genetic patterns and regional uniqueness. Here, we used up to 25,751 genome-wide polymorphic loci to reconstruct the history and to discern the demographic and evolutionary forces underpinning divergence between British populations. Overall, we found lower genetic diversity in Scottish populations but high differentiation (FST  = 0.433-0.712) from the English/Welsh and other European populations. Differentiation was elevated genome-wide rather than in particular genomic regions. Demographic modelling supported a postglacial colonization into western Scotland from northern refugia and a separate colonization route for the English/Welsh populations from southern refugia, with these two groups having been separated for more than ca. 50 Ky. We found cyto-nuclear discordance at a European scale, with the Scottish populations clustering closely with Baltic population in the mtDNA analysis but not in the nuclear data, and with the Norwegian and Alpine populations displaying the same mtDNA haplotype but being distantly related in the nuclear tree. These findings suggest that neutral processes, primarily drift and regionally distinct pre-glacial evolutionary histories, are important drivers of genomic divergence in British populations of European whitefish. This sheds new light on the establishment of the native British freshwater fauna after the last glacial maximum.

Functional colour genes and signals of selection in colour-polymorphic salamanders.

Coloration has been associated with multiple biologically relevant traits that drive adaptation and diversification in many taxa. However, despite the great diversity of colour patterns present in amphibians the underlying molecular basis is largely unknown. Here, we use insight from a highly colour-variable lineage of the European fire salamander (Salamandra salamandra bernardezi) to identify functional associations with striking variation in colour morph and pattern. The three focal colour morphs-ancestral black-yellow striped, fully yellow and fully brown-differed in pattern, visible coloration and cellular composition. From population genomic analyses of up to 4,702 loci, we found no correlations of neutral population genetic structure with colour morph. However, we identified 21 loci with genotype-phenotype associations, several of which relate to known colour genes. Furthermore, we inferred response to selection at up to 142 loci between the colour morphs, again including several that relate to coloration genes. By transcriptomic analysis across all different combinations, we found 196 differentially expressed genes between yellow, brown and black skin, 63 of which are candidate genes involved in animal coloration. The concordance across different statistical approaches and 'omic data sets provide several lines of evidence for loci linked to functional differences between colour morphs, including TYR, CAMK1 and PMEL. We found little association between colour morph and the metabolomic profile of its toxic compounds from the skin secretions. Our research suggests that current ecological and evolutionary hypotheses for the origins and maintenance of these striking colour morphs may need to be revisited.

Cryptic species in White Cloud Mountain minnow, Tanichthys albonubes: Taxonomic and conservation implications.

Cryptic species describe two or more species that had mistakenly been considered to be a single species, a phenomenon that has been found throughout the tree of life. Recognizing cryptic species is key to estimating the real biodiversity of the world and understanding evolutionary processes. Molecular methods present an unprecedented opportunity for biologists to question whether morphologically similar populations are actually cryptic species. The minnow Tanichthys albonubes is a critically endangered freshwater fish and was classified as a second-class state-protected animal in China. Previous studies have revealed highly divergent lineages with similar morphological characters in this species. Herein, we tested for cryptic species across the ranges of all known wild populations of this minnow. Using multilocus molecular (one mitochondrial gene, two nuclear genes and 13 microsatellite loci) and morphological data for 230 individuals from eight populations, we found deep genetic divergence among these populations with subtle morphological disparity. Morphological examination found variance among these populations in the number of branched anal-fin rays. Based on genetic data, we inferred eight monophyletic groups that were well supported by haplotype network and population clustering analyses. Species delimitation methods suggested eight putative species in the T. albonubes complex. Molecular dating suggested that these cryptic species diverged in the period from the Pliocene to the Pleistocene. Based on these findings, we propose the existence of seven cryptic species in the T. albonubes complex. Our results highlight the need for a taxonomic revision of Tanichthys. What is more, the conservation status of and conservation strategies for the T. albonubes complex should be reassessed as soon as possible.

Native bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix populations in the Pearl River are threatened by Yangtze River introductions as revealed by mitochondrial DNA.

Bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix have been two economically important aquaculture species in China for centuries. In the past decades, bighead and silver carp have been introduced from the Yangtze River to many river systems in China, including the Pearl River, in annual, large-scale, stocking activities to enhance wild fisheries. Nonetheless, few studies have assessed the ecological or genetic impacts of such introductions on native conspecific fish populations. An mtDNA D-loop segment of 978 bp from 213 bighead carp samples from 9 populations and a 975 bp segment from 204 silver carp samples from 10 populations were obtained to evaluate genetic diversity and population integrity. Results from a haplotype network analysis revealed that most haplotypes of the Pearl River clustered with those of Yangtze River origin and only a small proportion were distinct, suggesting that both the native Pearl River bighead and silver carp populations are currently dominated by genetic material from the Yangtze River. The genetic diversity of Pearl River populations is high in both species because of this inter-population gene flow, but the diversity of native Pearl River populations is low. To preserve the native genetic diversity, stocking of non-native fingerlings should cease immediately and native Pearl River bighead and silver carp fish farms should be established. This research demonstrates the danger to native biodiversity across China because of the substantial, ongoing stock-enhancement activities without prior genetic assessment.

Parallel selection on ecologically relevant gene functions in the transcriptomes of highly diversifying salmonids.

BACKGROUND: Salmonid fishes are characterised by a very high level of variation in trophic, ecological, physiological, and life history adaptations. Some salmonid taxa show exceptional potential for fast, within-lake diversification into morphologically and ecologically distinct variants, often in parallel; these are the lake-resident charr and whitefish (several species in the genera Salvelinus and Coregonus). To identify selection on genes and gene categories associated with such predictable diversifications, we analysed 2702 orthogroups (4.82 Mbp total; average 4.77 genes/orthogroup; average 1783 bp/orthogroup). We did so in two charr and two whitefish species and compared to five other salmonid lineages, which do not evolve in such ecologically predictable ways, and one non-salmonid outgroup. RESULTS: All selection analyses are based on Coregonus and Salvelinus compared to non-diversifying taxa. We found more orthogroups were affected by relaxed selection than intensified selection. Of those, 122 were under significant relaxed selection, with trends of an overrepresentation of serine family amino acid metabolism and transcriptional regulation, and significant enrichment of behaviour-associated gene functions. Seventy-eight orthogroups were under significant intensified selection and were enriched for signalling process and transcriptional regulation gene ontology terms and actin filament and lipid metabolism gene sets. Ninety-two orthogroups were under diversifying/positive selection. These were enriched for signal transduction, transmembrane transport, and pyruvate metabolism gene ontology terms and often contained genes involved in transcriptional regulation and development. Several orthogroups showed signs of multiple types of selection. For example, orthogroups under relaxed and diversifying selection contained genes such as ap1m2, involved in immunity and development, and slc6a8, playing an important role in muscle and brain creatine uptake. Orthogroups under intensified and diversifying selection were also found, such as genes syn3, with a role in neural processes, and ctsk, involved in bone remodelling. CONCLUSIONS: Our approach pinpointed relevant genomic targets by distinguishing among different kinds of selection. We found that relaxed, intensified, and diversifying selection affect orthogroups and gene functions of ecological relevance in salmonids. Because they were found consistently and robustly across charr and whitefish and not other salmonid lineages, we propose these genes have a potential role in the replicated ecological diversifications.

Phylogeny and species delimitation of near Eastern Neurergus newts (Salamandridae) based on genome-wide RADseq data analysis.

We reconstruct the molecular phylogeny of Near Eastern mountain brook newts of the genus Neurergus (family Salamandridae) based on newly determined RADseq data, and compare the outcomes of concatenation-based phylogenetic reconstruction with species-tree inference. Furthermore, we test the current taxonomy of Neurergus (with four species: Neurergus strauchii, N. crocatus, N. kaiseri, and N. derjugini) against coalescent-based species-delimitation approaches of our genome-wide genetic data set. While the position of N. strauchii as sister species to all other Neurergus species was consistent in all of our analyses, the phylogenetic relationships between the three remaining species changed depending on the applied method. The concatenation approach, as well as quartet-based species-tree inference, supported a topology with N. kaiseri as the closest relative to N. derjugini, while full-coalescent species-tree inference approaches supported N. crocatus as sister species of N. derjugini. Investigating the individual signal of gene trees highlighted an extensive variation among gene histories, most likely resulting from incomplete lineage sorting. Coalescent-based species-delimitation models suggest that the current taxonomy might underestimate the species richness within Neurergus and supports seven species. Based on the current sampling, our analysis suggests that N. strauchii, N. derjugini and N. kaiseri might each be subdivided into further species. However, as amphibian species are known to be composed of deep conspecific lineages that do not always warrant species status, these results need to be cautiously interpreted in an integrative taxonomic framework. We hypothesize that the rather shallow divergences detected within N. kaiseri and N. derjugini likely reflect an ongoing speciation process and thus require further investigation. On the contrary, the much deeper genetic divergence found between the two morphologically and geographically differentiated subspecies of N. strauchii leads us to propose that N. s. barani should be considered a distinct species, Neurergus barani Öz, 1994.

Allopatric diversification and evolutionary melting pot in a North African Palearctic relict: The biogeographic history of Salamandra algira.

North Africa is a climatically and topographically complex region with unique biotic assemblages resulting from the combination of multiple biogeographic realms. Here, we assess the role of climate in promoting intra-specific diversification in a Palearctic relict, the North African fire salamander, Salamandra algira, using a combination of phylogenetic and population genetic analyses, paleoclimatic modelling and niche overlap tests. We used mitochondrial DNA (Cyt-b), 9838 ddRADseq loci, and 14 microsatellite loci to characterize patterns of genetic diversity and population structure. Phylogenetic analyses recover two major clades, each including several lineages with mito-nuclear discordances suggesting introgressive patterns between lineages in the Middle Atlas, associated with a melting pot of genetic diversity. Paleoclimatic modelling identified putative climatic refugia, largely matching areas of high genetic diversity, and supports the role of aridity in promoting allopatric diversification associated with ecological niche conservatism. Overall, our results highlight the role of climatic microrefugia as drivers of populations' persistence and diversification in the face of climatic oscillations in North Africa, and stress the importance of accounting for different genomic regions when reconstructing biogeographic processes from molecular markers.