High‐density linkage maps fail to detect any genetic component to sex determination in a Rana temporaria family

Sex chromosome differentiation in Rana temporaria varies strikingly among populations or families: whereas some males display well‐differentiated Y haplotypes at microsatellite markers on linkage group 2 (LG₂), others are genetically undistinguishable from females. We analysed with RADseq markers one family from a Swiss lowland population with no differentiated sex chromosomes, and where sibship analyses had failed to detect any association between the phenotypic sex of progeny and parental haplotypes. Offspring were reared in a common tank in outdoor conditions and sexed at the froglet stage. We could map a total of 2177 SNPs (1123 in the mother, 1054 in the father), recovering in both adults 13 linkage groups (= chromosome pairs) that were strongly syntenic to Xenopus tropicalis despite > 200 My divergence. Sexes differed strikingly in the localization of crossovers, which were uniformly distributed in the female but limited to chromosome ends in the male. None of the 2177 markers showed significant association with offspring sex. Considering the very high power of our analysis, we conclude that sex determination was not genetic in this family; which factors determined sex remain to be investigated.     

Molecular phenotyping of maternally mediated parallel adaptive divergence within Rana arvalis and Rana temporaria

When similar selection acts on the same traits in multiple species or populations, parallel evolution can result in similar phenotypic changes, yet the underlying molecular architecture of parallel phenotypic divergence can be variable. Maternal effects can influence evolution at ecological timescales and facilitate local adaptation, but their contribution to parallel adaptive divergence is unclear. In this study, we (i) tested for variation in embryonic acid tolerance in a common garden experiment and (ii) used molecular phenotyping of egg coats to investigate the molecular basis of maternally mediated parallel adaptive divergence in two amphibian species (Rana arvalis and Rana temporaria). Our results on three R. arvalis and two R. temporaria populations show that adaptive divergence in embryonic acid tolerance is mediated via maternally derived egg coats in both species. We find extensive polymorphism in egg jelly coat glycoproteins within both species and that acid‐tolerant clutches have more negatively charged egg jelly – indicating that the glycosylation status of the jelly coat proteins is under divergent selection in acidified environments, likely due to its impact on jelly water balance. Overall, these data provide evidence for parallel mechanisms of adaptive divergence in two species. Our study highlights the importance of studying intraspecific molecular variation in egg coats and, specifically, their glycoproteins, to increase understanding of underlying forces maintaining variation in jelly coats.     

Recent Y chromosome divergence despite ancient origin of dioecy in poplars (Populus)

All species of the genus Populus (poplar, aspen) are dioecious, suggesting an ancient origin of this trait. Despite some empirical counter examples, theory suggests that nonrecombining sex‐linked regions should quickly spread, eventually becoming heteromorphic chromosomes. In contrast, we show using whole‐genome scans that the sex‐associated region in Populus trichocarpa is small and much younger than the age of the genus. This indicates that sex determination is highly labile in poplar, consistent with recent evidence of ‘turnover’ of sex‐determination regions in animals. We performed whole‐genome resequencing of 52 P. trichocarpa (black cottonwood) and 34 Populus balsamifera (balsam poplar) individuals of known sex. Genomewide association studies in these unstructured populations identified 650 SNPs significantly associated with sex. We estimate the size of the sex‐linked region to be ~100 kbp. All SNPs significantly associated with sex were in strong linkage disequilibrium despite the fact that they were mapped to six different chromosomes (plus 3 unmapped scaffolds) in version 2.2 of the reference genome. We show that this is likely due to genome misassembly. The segregation pattern of sex‐associated SNPs revealed this to be an XY sex‐determining system. Estimated divergence times of X and Y haplotype sequences (6–7 Ma) are much more recent than the divergence of P. trichocarpa (poplar) and Populus tremuloides (aspen). Consistent with this, in P. tremuloides, we found no XY haplotype divergence within the P. trichocarpa sex‐determining region. These two species therefore have a different genomic architecture of sex, suggestive of at least one turnover event in the recent past.     

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.     

Genomic characterization of sex‐identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes

Fish have evolved a variety of sex‐determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex‐specific genotypic markers, double digest restriction site – associated DNA sequencing (ddRAD‐seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male‐specific loci met the criteria in at least one species and 17 in both. Conversely, no female‐specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex‐specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex‐specific and autosomal loci. A PCR‐RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex‐determining gene in other teleosts gonadal soma‐derived factor (gsdf) was present in the model fish chromosomes that spanned our sex‐specific markers.     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Freeze tolerance evolution among anurans: Frequency and timing of appearance

Despite numerous mechanistic studies on physiological responses supporting freeze tolerance in anurans, few have addressed the evolutionary significance of this trait. We thus investigated the phylogenetic relationships among anuran species whose freeze tolerance has been assessed and in combination with new data on freezing tolerance of two closely related species of the European brown frogs (Rana temporaria and Rana dalmatina). The species we studied exhibited short survival times in frozen state (around 8 h for both species). Phylogenetic analysis suggests that freeze tolerance evolved at least two times among Ranidae and one or two times among Hylidae and never in Bufonidae. Furthermore, in order to assess the timing of divergence of this character we used a relaxed molecular clock created, and found that the most recent separation between a freeze tolerant species and a freeze intolerant species dates from 15.9 ± 7.6 Myr (Rana arvalis and R. temporaria). The comparison between these two species thus represents the best current model to understand freeze tolerance evolution. Addressing the evolution of this trait with such large-scale approaches will not only improve our understanding of cold hardiness strategies, but might also create a framework guiding future comparative studies.     

Applicability of RAD‐tag genotyping for interfamilial comparisons: empirical data from two cetaceans

Restriction‐site‐associated DNA tag (RAD‐tag) sequencing has become a popular approach to generate thousands of SNPs used to address diverse questions in population genomics. Comparatively, the suitability of RAD‐tag genotyping to address evolutionary questions across divergent species has been the subject of only a few recent studies. Here, we evaluate the applicability of this approach to conduct genome‐wide scans for polymorphisms across two cetacean species belonging to distinct families: the short‐beaked common dolphin (Delphinus delphis; n = 5 individuals) and the harbour porpoise (Phocoena phocoena; n = 1 individual). Additionally, we explore the effects of varying two parameters in the Stacks analysis pipeline on the number of loci and level of divergence obtained. We observed a 34% drop in the total number of loci that were present in all individuals when analysing individuals from the distinct families compared with analyses restricted to intraspecific comparisons (i.e. within D. delphis). Despite relatively stringent quality filters, 3595 polymorphic loci were retrieved from our interfamilial comparison. Cetaceans have undergone rapid diversification, and the estimated divergence time between the two families is relatively recent (14–19 Ma). Thus, our results showed that, for this level of divergence, a large number of orthologous loci can still be genotyped using this approach, which is on par with two recent in silico studies. Our findings constitute one of the first empirical investigations using RAD‐tag sequencing at this level of divergence and highlights the great potential of this approach in comparative studies and to address evolutionary questions.     

Density dependent growth in adult brown frogs Rana arvalis and Rana temporaria – A field experiment

In species with complex life cycles, density regulation can operate on any of the stages. In frogs there are almost no studies of density effects on the performance of adult frogs in the terrestrial habitat. We therefore studied the effect of summer density on the growth rate of adult frogs during four years. Four 30 by 30 m plots in a moist meadow were used. In early summer, when settled after post-breeding migration, frogs (Rana arvalis and Rana temporaria that have a very similar ecology and potentially compete) were enclosed by erecting a fence around the plots. Frogs were captured, measured, marked and partly relocated to create two high density and two low density plots. In early autumn the frogs were again captured and their individual summer growth determined. Growth effects were evaluated in relation to two density measures: density by design (high/low manipulation), and actual (numerical) density. R. arvalis in plots with low density by design grew faster than those in high density plots. No such effect was found for R. temporaria. For none of the species was growth related to actual summer density, determined by the Lincoln index and including the density manipulation. The result suggests that R. arvalis initially settled according to an ideal free distribution and that density had a regulatory effect (mediated through growth). The fact that there were no density effects on R. temporaria (and a significant difference in its response to that of R. arvalis) suggests it is a superior competitor to R. arvalis during the terrestrial phase. There were no density effects on frog condition index, suggesting that the growth rate modifications may actually be an adaptive trait of R. arvalis. The study demonstrates that density regulation may be dependent on resources in frogs' summer habitat.     

Within‐population polymorphism of sex‐determination systems in the common frog (Rana temporaria)

In sharp contrast with birds and mammals, the sex chromosomes of ectothermic vertebrates are often undifferentiated, for reasons that remain debated. A linkage map was recently published for Rana temporaria (Linnaeus, 1758) from Fennoscandia (Eastern European lineage), with a proposed sex‐determining role for linkage group 2 (LG2). We analysed linkage patterns in lowland and highland populations from Switzerland (Western European lineage), with special focus on LG2. Sibship analyses showed large differences from the Fennoscandian map in terms of recombination rates and loci order, pointing to large‐scale inversions or translocations. All linkage groups displayed extreme heterochiasmy (total map length was 12.2 cM in males, versus 869.8 cM in females). Sex determination was polymorphic within populations: a majority of families (with equal sex ratios) showed a strong correlation between offspring phenotypic sex and LG2 paternal haplotypes, whereas other families (some of which with female‐biased sex ratios) did not show any correlation. The factors determining sex in the latter could not be identified. This coexistence of several sex‐determination systems should induce frequent recombination of X and Y haplotypes, even in the absence of male recombination. Accordingly, we found no sex differences in allelic frequencies on LG2 markers among wild‐caught male and female adults, except in one high‐altitude population, where nonrecombinant Y haplotypes suggest sex to be entirely determined by LG2. Multifactorial sex determination certainly contributes to the lack of sex‐chromosome differentiation in amphibians.     

SNP discovery using Paired‐End RAD‐tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae)

Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost‐effective approaches to uncover genome‐wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD‐PE (Restriction site Associated DNA Paired‐End sequencing) approach. RAD tags were generated from the PstI‐digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired‐end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N₅₀ = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD‐PE as an inexpensive genome‐wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.     

Identification of SNP markers for inferring phylogeny in temperate bamboos (Poaceae: Bambusoideae) using RAD sequencing

Phylogenetic relationships among temperate species of bamboo are difficult to resolve, owing to both the challenge of detecting sufficiently variable markers and their polyploid history. Here, we use restriction site–associated DNA sequencing to identify candidate loci with fixed allelic differences segregating between and within two temperate species of bamboos: Arundinaria faberi and Yushania brevipaniculata. Approximately 27 million paired‐end sequencing reads were generated across four samples. From pooled data, we assembled 67 685 and 70 668 de novo contigs from partial overlap among paired‐end reads, with an average length of 240 and 241 bp for the two species, respectively, which were used to investigate functional classification of RAD tags in a blastx search. Analysed separately by population, we recovered 29 443 putatively orthologous RAD tags shared across the four sampled populations, containing 28 023 sequence variants, of which c. 13 000 are segregating between species, and c. 3000 segregating between populations within each species. Analyses based on these RAD tags yielded robust phylogenetic inferences, even with data set constructed from surprisingly few loci. This study illustrates the potential for reduced‐representation genome data to resolve difficult phylogenetic relationships in temperate bamboos.     

Dmrt1 polymorphism covaries with sex‐determination patterns in Rana temporaria

Patterns of sex‐chromosome differentiation and gonadal development have been shown to vary among populations of Rana temporaria along a latitudinal transect in Sweden. Frogs from the northern‐boreal population of Ammarnäs displayed well‐differentiated X and Y haplotypes, early gonadal differentiation, and a perfect match between phenotypic and genotypic sex. In contrast, no differentiated Y haplotypes could be detected in the southern population of Tvedöra, where juveniles furthermore showed delayed gonadal differentiation. Here, we show that Dmrt1, a gene that plays a key role in sex determination and sexual development across all metazoans, displays significant sex differentiation in Tvedöra, with a Y‐specific haplotype distinct from Ammarnäs. The differential segment is not only much shorter in Tvedöra than in Ammarnäs, it is also less differentiated and associates with both delayed gonadal differentiation and imperfect match between phenotypic and genotypic sex. Whereas Tvedöra juveniles with a local Y haplotype tend to ultimately develop as males, those without it may nevertheless become functional XX males, but with strongly female‐biased progeny. Our findings suggest that the variance in patterns of sex determination documented in common frogs might result from a genetic polymorphism within a small genomic region that contains Dmrt1. They also substantiate the view that recurrent convergences of sex determination toward a limited set of chromosome pairs may result from the co‐option of small genomic regions that harbor key genes from the sex‐determination pathway.     

Characterization of a Y‐specific duplication/insertion of the anti‐Mullerian hormone type II receptor gene based on a chromosome‐scale genome assembly of yellow perch,Perca flavescens

Yellow perch, Perca flavescens, is an ecologically and economically important species native to a large portion of the northern United States and southern Canada and is also a promising candidate species for aquaculture. However, no yellow perch reference genome has been available to facilitate improvements in both fisheries and aquaculture management practices. By combining Oxford Nanopore Technologies long‐reads, 10X Genomics Illumina short linked reads and a chromosome contact map produced with Hi‐C, we generated a high‐continuity chromosome‐scale yellow perch genome assembly of 877.4 Mb. It contains, in agreement with the known diploid chromosome yellow perch count, 24 chromosome‐size scaffolds covering 98.8% of the complete assembly (N50 = 37.4 Mb, L50 = 11). We also provide a first characterization of the yellow perch sex determination locus that contains a male‐specific duplicate of the anti‐Mullerian hormone type II receptor gene (amhr2by) inserted at the proximal end of the Y chromosome (chromosome 9). Using this sex‐specific information, we developed a simple PCR genotyping assay which accurately differentiates XY genetic males (amhr2by⁺) from XX genetic females (amhr2by^?). Our high‐quality genome assembly is an important genomic resource for future studies on yellow perch ecology, toxicology, fisheries and aquaculture research. In addition, characterization of the amhr2by gene as a candidate sex‐determining gene in yellow perch provides a new example of the recurrent implication of the transforming growth factor beta pathway in fish sex determination, and highlights gene duplication as an important genomic mechanism for the emergence of new master sex determination genes.     

Sex reversal and primary sex ratios in the common frog (Rana temporaria)

Sex reversal has been suggested to have profound implications for the evolution of sex chromosomes and population dynamics in ectotherms. Occasional sex reversal of genetic males has been hypothesized to prevent the evolutionary decay of nonrecombining Y chromosomes caused by the accumulation of deleterious mutations. At the same time, sex reversals can have a negative effect on population growth rate. Here, we studied phenotypic and genotypic sex in the common frog (Rana temporaria) in a subarctic environment, where strongly female‐biased sex ratios have raised the possibility of frequent sex reversals. We developed two novel sex‐linked microsatellite markers for the species and used them with a third, existing marker and a Bayesian modelling approach to study the occurrence of sex reversal and to determine primary sex ratios in egg clutches. Our results show that a significant proportion (0.09, 95% credible interval: 0.04–0.18) of adults that were genetically female expressed the male phenotype, but there was no evidence of sex reversal of genetic males that is required for counteracting the degeneration of Y chromosome. The primary sex ratios were mostly equal, but three clutches consisted only of genetic females and three others had a significant female bias. Reproduction of the sex‐reversed genetic females appears to create all‐female clutches potentially skewing the population level adult sex‐ratio consistent with field observations. However, based on a simulation model, such a bias is expected to be small and transient and thus does not fully explain the observed female‐bias in the field.     

Maintaining their genetic distance: Little evidence for introgression between widely hybridizing species of Geum with contrasting mating systems

Within the plant kingdom, many genera contain sister lineages with contrasting outcrossing and inbreeding mating systems that are known to hybridize. The evolutionary fate of these sister lineages is likely to be influenced by the extent to which they exchange genes. We measured gene flow between outcrossing Geum rivale and selfing Geum urbanum, sister species that hybridize in contemporary populations. We generated and used a draft genome of G. urbanum to develop dd‐RAD data scorable in both species. Coalescent analysis of RAD data from allopatric populations indicated that the species diverged 2–3 Mya, and that historical gene flow between them was extremely low (1 migrant every 25 generations). Comparison of genetic divergence between species in sympatry and allopatry, together with an analysis of allele frequencies in potential parental and hybrid populations, provided no evidence of contemporary introgression in sympatric populations. Cluster‐ and species‐specific marker analyses revealed that, apart from four early‐generation hybrids, individuals in sympatric populations fell into two genetically distinct groups that corresponded exactly to their morphological species classification with maximum individual admixture estimates of only 1–3%. However, we did observe joint segregation of four putatively introgressed SNPs across two scaffolds in the G. urbanum population that was associated with significant morphological variation, interpreted as tentative evidence for rare, recent interspecific gene flow. Overall, our results indicate that despite the presence of hybrids in contemporary populations, genetic exchange between G. rivale and G. urbanum has been extremely limited throughout their evolutionary history.     

Chromosomal‐level genomes of three rice planthoppers provide new insights into sex chromosome evolution

The brown planthopper Nilaparvata lugens, white‐backed planthopper Sogatella furcifera, and small brown planthopper Laodelphax striatellus are three major insect pests of rice. They are genetically close; however, they differ in several ecological traits such as host range, migration capacity, and in their sex chromosomes. Though the draft genome of these three planthoppers have been previously released, the quality of genome assemblies need to be improved. The absence of chromosome‐level genome resources has hindered in‐depth research of these three species. Here, we performed a de novo genome assembly for N. lugens to increase its genome assembly quality with PacBio and Illumina platforms, increasing the contig N50 to 589.46 Kb. Then, with the new N. lugens genome and previously reported S. furcifera and L. striatellus genome assemblies, we generated chromosome‐level scaffold assemblies of these three planthopper species using HiC scaffolding technique. The scaffold N50s significantly increased to 77.63 Mb, 43.36 Mb and 29.24 Mb for N. lugens, S. furcifera and L. striatellus, respectively. To identify sex chromosomes of these three planthopper species, we carried out genome re‐sequencing of males and females and successfully determined the X and Y chromosomes for N. lugens, and X chromosome for S. furcifera and L. striatellus. The gene content of the sex chromosomes showed high diversity among these three planthoppers suggesting the rapid evolution of sex‐linked genes, and all chromosomes showed high synteny. The chromosome‐level genome assemblies of three planthoppers would provide a valuable resource for a broad range of future research in molecular ecology, and subsequently benefits development of modern pest control strategies.     

Identification of sex‐specific molecular markers using restriction site‐associated DNA sequencing

A major barrier to evolutionary studies of sex determination and sex chromosomes has been a lack of information on the types of sex‐determining mechanisms that occur among different species. This is particularly problematic in groups where most species lack visually heteromorphic sex chromosomes, such as fish, amphibians and reptiles, because cytogenetic analyses will fail to identify the sex chromosomes in these species. We describe the use of restriction site‐associated DNA (RAD) sequencing, or RAD‐seq, to identify sex‐specific molecular markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the lizard Anolis carolinensis. We performed RAD‐seq on seven male and ten female A. carolinensis and found one male‐specific molecular marker. Anolis carolinensis has previously been shown to possess male heterogamety and the recently published A. carolinensis genome facilitated the characterization of the sex‐specific RAD‐seq marker. We validated the male specificity of the new marker using PCR on additional individuals and also found that it is conserved in some other Anolis species. We discuss the utility of using RAD‐seq to identify sex‐determining mechanisms in other species with cryptic or homomorphic sex chromosomes and the implications for the evolution of male heterogamety in Anolis.     

Local adaptation with high gene flow: temperature parameters drive adaptation to altitude in the common frog (Rana temporaria)

Both environmental and genetic influences can result in phenotypic variation. Quantifying the relative contributions of local adaptation and phenotypic plasticity to phenotypes is key to understanding the effect of environmental variation on populations. Identifying the selective pressures that drive divergence is an important, but often lacking, next step. High gene flow between high‐ and low‐altitude common frog (Rana temporaria) breeding sites has previously been demonstrated in Scotland. The aim of this study was to assess whether local adaptation occurs in the face of high gene flow and to identify potential environmental selection pressures that drive adaptation. Phenotypic variation in larval traits was quantified in R. temporaria from paired high‐ and low‐altitude sites using three common temperature treatments. Local adaptation was assessed using Q_ST–F_ST analyses, and quantitative phenotypic divergence was related to environmental parameters using Mantel tests. Although evidence of local adaptation was found for all traits measured, only variation in larval period and growth rate was consistent with adaptation to altitude. Moreover, this was only evident in the three mountains with the highest high‐altitude sites. This variation was correlated with mean summer and winter temperatures, suggesting that temperature parameters are potentially strong selective pressures maintaining local adaptation, despite high gene flow.     

Post‐glacial colonization routes coincide with a life‐history breakpoint along a latitudinal gradient

Although adaptive divergence along environmental gradients has repeatedly been demonstrated, the role of post‐glacial colonization routes in determining phenotypic variation along gradients has received little attention. Here, we used a hierarchical Q_ST–F_ST approach to separate the roles of adaptive and neutral processes in shaping phenotypic variation in moor frog (Rana arvalis) larval life histories along a 1,700 km latitudinal gradient across northern Europe. This species has colonized Scandinavia via two routes with a contact zone in northern Sweden. By using neutral SNP and common garden phenotypic data from 13 populations at two temperatures, we showed that most of the variation along the gradient occurred between the two colonizing lineages. We found little phenotypic divergence within the lineages; however, all phenotypic traits were strongly diverged between the southern and northern colonization routes, with higher growth and development rates and larger body size in the north. The Q_ST estimates between the colonization routes were four times higher than F_ST, indicating a prominent role for natural selection. Q_ST within the colonization routes did not generally differ from F_ST, but we found temperature‐dependent adaptive divergence close to the contact zone. These results indicate that lineage‐specific variation can account for much of the adaptive divergence along a latitudinal gradient.