Common lizards break Dollo’s law of irreversibility: genome-wide phylogenomics support a single origin of viviparity and re-evolution of oviparity

Dollo’s law of irreversibility states that once a complex trait has been lost in evolution, it cannot be regained. It is thought that complex epistatic interactions and developmental constraints impede the re-emergence of such a trait. Oviparous reproduction (egg-laying) requires the formation of an eggshell and represents an example of such a complex trait. In reptiles, viviparity (live-bearing) has evolved repeatedly but it is highly disputed if oviparity has re-evolved. Here, using up to 194,358 SNP loci and 1,334,760 bp of sequence, we reconstruct the phylogeny of viviparous and oviparous lineages of common lizards and infer the evolutionary history of parity modes. Our phylogeny supports six main common lizard lineages that have been previously identified. We find strong statistical support for a topological arrangement that suggests a reversal to oviparity from viviparity. Our topology is consistent with highly differentiated chromosomal configurations between lineages, but disagrees with previous phylogenetic studies in some nodes. While we find high support for a reversal to oviparity, more genomic and developmental data are needed to robustly test this and assess the mechanism by which a reversal might have occurred

Differential reproductive investment in co-occurring oviparous and viviparous common lizards (Zootoca vivipara) and implications for life-history trade-offs with viviparity

Live-bearing reproduction (viviparity) has evolved from egg-laying (oviparity) independently many times and most abundantly in squamate reptiles. Studying life-history trade-offs between the two reproductive modes is an inherently difficult task, as most transitions to viviparity are evolutionarily old and/or are confounded by environmental effects. The common lizard (Zootoca vivipara) is one of very few known reproductively bimodal species, in which some populations are oviparous and others viviparous. Oviparous and viviparous populations can occur in sympatry in the same environment, making this a unique system for investigating alternative life-history trade-offs between oviparous and viviparous reproduction. We find that viviparous females exhibit larger body size, smaller clutch sizes, a larger reproductive investment, and a higher hatching success rate than oviparous females. We find that offspring size and weight from viviparous females was lower compared to offspring from oviparous females, which may reflect space constraints during pregnancy. We suggest that viviparity in common lizards is associated with increased reproductive burden for viviparous females and speculate that this promoted the evolution of larger body size to create more physical space for developing embryos. In the context of life-history trade-offs in the evolution of viviparity, we suggest that the extent of correlation between reproductive traits, or differences between reproductive modes, may also depend on the time since the transition occurred

Ecosystem size predicts eco-morphological variability in a postglacial diversification

Identifying the processes by which new phenotypes and species emerge has been a long-standing effort in evolutionary biology. Young adaptive radiations provide a model to study patterns of morphological and ecological diversification in environmental context. Here, we use the recent radiation (ca. 12k years old) of the freshwater fish Arctic charr (Salvelinus alpinus) to identify abiotic and biotic environmental factors associated with adaptive morphological variation. Arctic charr are exceptionally diverse, and in postglacial lakes there is strong evidence of repeated parallel evolution of similar morphologies associated with foraging. We measured head depth (a trait reflecting general eco-morphology and foraging ecology) of 1,091 individuals across 30 lake populations to test whether fish morphological variation was associated with lake bathymetry and/or ecological parameters. Across populations, we found a significant relationship between the variation in head depth of the charr and abiotic environmental characteristics: positively with ecosystem size (i.e., lake volume, surface area, depth) and negatively with the amount of littoral zone. In addition, extremely robust-headed phenotypes tended to be associated with larger and deeper lakes. We identified no influence of co-existing biotic community on Arctic charr trophic morphology. This study evidences the role of the extrinsic environment as a facilitator of rapid eco-morphological diversification

The genetic architecture underlying the evolution of a rare piscivorous life history form in brown trout after secondary contact and strong introgression

Identifying the genetic basis underlying phenotypic divergence and reproductive isolation is a longstanding problem in evolutionary biology. Genetic signals of adaptation and reproductive isolation are often confounded by a wide range of factors, such as variation in demographic history or genomic features. Brown trout ( ) in the Loch Maree catchment, Scotland, exhibit reproductively isolated divergent life history morphs, including a rare piscivorous (ferox) life history form displaying larger body size, greater longevity and delayed maturation compared to sympatric benthivorous brown trout. Using a dataset of 16,066 SNPs, we analyzed the evolutionary history and genetic architecture underlying this divergence. We found that ferox trout and benthivorous brown trout most likely evolved after recent secondary contact of two distinct glacial lineages, and identified 33 genomic outlier windows across the genome, of which several have most likely formed through selection. We further identified twelve candidate genes and biological pathways related to growth, development and immune response potentially underpinning the observed phenotypic differences. The identification of clear genomic signals divergent between life history phenotypes and potentially linked to reproductive isolation, through size assortative mating, as well as the identification of the underlying demographic history, highlights the power of genomic studies of young species pairs for understanding the factors shaping genetic differentiation

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

Cryptic diversity and deep divergence in an upper Amazonian leaflitter frog, Eleutherodactylus ockendeni

Supplementary files available: Additional file 1: Specimen information. Museum catalogue numbers (QCAZ) for E. ockendeni and two outgroup species, GenBank accession numbers for cyt b and/or 16S fragment, and locality of origin for each individual used in the study.- Additional file 2: TN-corrected p-distance among cyt b haplotypes. TN-corrected p-distances among cyt b haplotypes of E. ockendeni, grouped by clade.[Background] The forests of the upper Amazon basin harbour some of the world's highest anuran species richness, but to date we have only the sparsest understanding of the distribution of genetic diversity within and among species in this region. To quantify region-wide genealogical patterns and to test for the presence of deep intraspecific divergences that have been documented in some other neotropical anurans, we developed a molecular phylogeny of the wide-spread terrestrial leaflitter frog Eleutherodactylus ockendeni (Leptodactylidae) from 13 localities throughout its range in Ecuador using data from two mitochondrial genes (16S and cyt b; 1246 base pairs). We examined the relation between divergence of mtDNA and the nuclear genome, as sampled by five speciesspecific microsatellite loci, to evaluate indirectly whether lineages are reproductively isolated where they co-occur. Our extensive phylogeographic survey thus assesses the spatial distribution of E. ockendeni genetic diversity across eastern Ecuador.[Results] We identified three distinct and well-supported clades within the Ecuadorean range of E. ockendeni: an uplands clade spanning north to south, a northeastern and central lowlands clade, and a central and southeastern clade, which is basal. Clades are separated by 12% to 15% net corrected p-distance for cytochrome b, with comparatively low sequence divergence within clades. Clades marginally overlap in some geographic areas (e.g., Napo River basin) but are reproductively isolated, evidenced by diagnostic differences in microsatellite PCR amplification profiles or DNA repeat number and coalescent analyses (in MDIV) best modelled without migration. Using Bayesian (BEAST) and net phylogenetic estimates, the Southeastern Clade diverged from the Upland/ Lowland clades in the mid-Miocene or late Oligocene. Lowland and Upland clades speciated more recently, in the early or late Miocene.[Conclusion] Our findings uncover previously unsuspected cryptic species diversity within the common leaflitter frog E. ockendeni, with at least three different species in Ecuador. While these clades are clearly geographically circumscribed, they do not coincide with any existing landscape barriers. Divergences are ancient, from the Miocene, before the most dramatic mountain building in the Ecuadorean Andes. Therefore, this diversity is not a product of Pleistocene refuges. Our research coupled with other studies suggests that species richness in the upper Amazon is drastically underestimated by current inventories based on morphospecies.Peer reviewe

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‐seq 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 non‐differentially 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

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

Geographic separation and genetic differentiation of populations are not coupled with niche differentiation in threatened Kaiser's spotted newt (Neurergus kaiseri)

The combination of niche modelling and landscape genetics (genomics) helps to disentangle processes that have shaped population structure in the evolutionary past and presence of species. Herein, we integrate a comprehensive genomic dataset with ecological parameters and niche modelling for the threatened Kaiser's newt, a newt species adapted to mountain spring-ponds in Iran. Genomic analysis suggests the existence of two highly differentiated clades North and South of the Dez River. Genetic variation between the two clades (76.62%) was much greater than within clades (16.25%), suggesting that the Dez River prevented gene flow. River disconnectivity, followed by geographic distance, contributed mostly to genetic differentiation between populations. Environmental niche and landscape resistance had no significant influence. Though a significant difference between climatic niches occupied by each clade at the landscape-scale, habitat niches at the local-scale were equivalent. 'Niche similarity analysis' supported niche conservatism between the two clades despite the southward shift in the climatic niche of the Southern clade. Accordingly, populations of different clades may occupy different climatic niches within their ancestral niche. Our results indicate that the change of climatic conditions of geographically and genetically separated populations does not necessarily result in the shift of an ecological niche

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