Romain Savary

HOMOLOGOUS SEX CHROMOSOMES IN THREE DEEPLY DIVERGENT ANURAN SPECIES

Comparative genomic studies are revealing that, in sharp contrast with the strong stability found in birds and mammals, sex determination mechanisms are surprisingly labile in cold‐blooded vertebrates, with frequent transitions between different pairs of sex chromosomes. It was recently suggested that, in context of this high turnover, some chromosome pairs might be more likely than others to be co‐opted as sex chromosomes. Empirical support, however, is still very limited. Here we show that sex‐linked markers from three highly divergent groups of anurans map to Xenopus tropicalis scaffold 1, a large part of which is homologous to the avian sex chromosome. Accordingly, the bird sex determination gene DMRT1, known to play a key role in sex differentiation across many animal lineages, is sex linked in all three groups. Our data provide strong support for the idea that some chromosome pairs are more likely than others to be co‐opted as sex chromosomes because they harbor key genes from the sex determination pathway.

Low rates of X‐Y recombination, not turnovers, account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup)

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X‐Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex‐linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X‐Y recombination in males. Phylogenetic analyses of sex‐linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X‐Y homomorphy and fine‐scale sequence similarity in these species do not stem from recent sex‐chromosome turnovers, but from occasional X‐Y recombination.

Sex-chromosome differentiation parallels postglacial range expansion in European tree frogs (Hyla arborea)

Occasional XY recombination is a proposed explanation for the sex‐chromosome homomorphy in European tree frogs. Numerous laboratory crosses, however, failed to detect any event of male recombination, and a detailed survey of NW‐European Hyla arborea populations identified male‐specific alleles at sex‐linked loci, pointing to the absence of XY recombination in their recent history. Here, we address this paradox in a phylogeographic framework by genotyping sex‐linked microsatellite markers in populations and sibships from the entire species range. Contrasting with postglacial populations of NW Europe, which display complete absence of XY recombination and strong sex‐chromosome differentiation, refugial populations of the southern Balkans and Adriatic coast show limited XY recombination and large overlaps in allele frequencies. Geographically and historically intermediate populations of the Pannonian Basin show intermediate patterns of XY differentiation. Even in populations where X and Y occasionally recombine, the genetic diversity of Y haplotypes is reduced below the levels expected from the fourfold drop in copy numbers. This study is the first in which X and Y haplotypes could be phased over the distribution range in a species with homomorphic sex chromosomes; it shows that XY‐recombination patterns may differ strikingly between conspecific populations, and that recombination arrest may evolve rapidly (<5000 generations).

Maintenance of Ancestral Sex Chromosomes in Palearctic Tree Frogs: Direct Evidence from Hyla orientalis

Contrasting with the situation found in birds and mammals, sex chromosomes are generally homomorphic in poikilothermic vertebrates. This homomorphy was recently shown to result from occasional X-Y recombinations (not from turnovers) in several European species of tree frogs (Hyla arborea, H. intermedia and H. molleri). Because of recombination, however, alleles at sex-linked loci were rarely diagnostic at the population level; support for sex linkage had to rely on multilocus associations, combined with occasional sex differences in allelic frequencies. Here, we use direct evidence, obtained from anatomical and histological analyses of offspring with known pedigrees, to show that the Eastern tree frog (H. orientalis) shares the same pair of sex chromosomes, with identical patterns of male heterogamety and complete absence of X-Y recombination in males. Conservation of an ancestral pair of sex chromosomes, regularly rejuvenated via occasional X-Y recombination, seems thus a widespread pattern among Hyla species. Sibship analyses also identified discrepancies between genotypic and phenotypic sex among offspring, associated with abnormal gonadal development, suggesting a role for sexually antagonistic genes on the sex chromosomes.

Empirical evidence for large X-effects in animals with undifferentiated sex chromosomes.

Reproductive isolation is crucial for the process of speciation to progress. Sex chromosomes have been assigned a key role in driving reproductive isolation but empirical evidence from natural population processes has been restricted to organisms with degenerated sex chromosomes such as mammals and birds. Here we report restricted introgression at sex-linked compared to autosomal markers in a hybrid zone between two incipient species of European tree frog, Hyla arborea and H. orientalis, whose homologous X and Y sex chromosomes are undifferentiated. This large X-effect cannot result from the dominance or faster-X aspects of Haldane’s rule, which are specific to degenerated sex chromosomes, but rather supports a role for faster-heterogametic-sex or faster-male evolutionary processes. Our data suggest a prominent contribution of undifferentiated sex chromosomes to speciation.

A population genomics approach shows widespread geographical distribution of cryptic genomic forms of the symbiotic fungus Rhizophagus irregularis.

Arbuscular mycorrhizal fungi (AMF; phylum Gomeromycota) associate with plants forming one of the most successful microbe–plant associations. The fungi promote plant diversity and have a potentially important role in global agriculture. Plant growth depends on both inter- and intra-specific variation in AMF. It was recently reported that an unusually large number of AMF taxa have an intercontinental distribution, suggesting long-distance gene flow for many AMF species, facilitated by either long-distance natural dispersal mechanisms or human-assisted dispersal. However, the intercontinental distribution of AMF species has been questioned because the use of very low-resolution markers may be unsuitable to detect genetic differences among geographically separated AMF, as seen with some other fungi. This has been untestable because of the lack of population genomic data, with high resolution, for any AMF taxa. Here we use phylogenetics and population genomics to test for intra-specific variation in Rhizophagus irregularis, an AMF species for which genome sequence information already exists. We used ddRAD sequencing to obtain thousands of markers distributed across the genomes of 81 R. irregularis isolates and related species. Based on 6 888 variable positions, we observed significant genetic divergence into four main genetic groups within R. irregularis, highlighting that previous studies have not captured underlying genetic variation. Despite considerable genetic divergence, surprisingly, the variation could not be explained by geographical origin, thus also supporting the hypothesis for at least one AMF species of widely dispersed AMF genotypes at an intercontinental scale. Such information is crucial for understanding AMF ecology, and how these fungi can be used in an environmentally safe way in distant locations.

Stocking activities for the Arctic charr in Lake Geneva: Genetic effects in space and time

Abstract Artificial stocking practices are widely used by resource managers worldwide, in order to sustain fish populations exploited by both recreational and commercial activities, but their benefits are controversial. Former practices involved exotic strains, although current programs rather consider artificial breeding of local fishes (supportive breeding). Understanding the complex genetic effects of these management strategies is an important challenge with economic and conservation implications, especially in the context of population declines. In this study, we focus on the declining Arctic charr (Salvelinus alpinus) population from Lake Geneva (Switzerland and France), which has initially been restocked with allochtonous fishes in the early eighties, followed by supportive breeding. In this context, we conducted a genetic survey to document the evolution of the genetic diversity and structure throughout the last 50 years, before and after the initiation of hatchery supplementation, using contemporary and historical samples. We show that the introduction of exotic fishes was associated with a genetic bottleneck in the 1980–1990s, a break of Hardy–Weinberg Equilibrium (HWE), a reduction in genetic diversity, an increase in genetic structure among spawning sites, and a change in their genetic composition. Together with better environmental conditions, three decades of subsequent supportive breeding using local fishes allowed to re‐establish HWE and the initial levels of genetic variation. However, current spawning sites have not fully recovered their original genetic composition and were extensively homogenized across the lake. Our study demonstrates the drastic genetic consequences of different restocking tactics in a comprehensive spatiotemporal framework and suggests that genetic alteration by nonlocal stocking may be partly reversible through supportive breeding. We recommend that conservation‐based programs consider local diversity and implement adequate protocols to limit the genetic homogenization of this Arctic charr population.

Dual RNA-seq reveals large-scale non-conserved genotype x genotype specific genetic reprograming and molecular crosstalk in the mycorrhizal symbiosis

Arbuscular mycorrhizal fungi (AMF) impact plant growth and are a major driver of plant diversity and productivity. We quantified the contribution of intra-specific genetic variability in cassava (Manihot esculenta) and Rhizophagus irregularis to gene reprogramming in symbioses using dual RNA-sequencing. A large number of cassava genes exhibited altered transcriptional responses to the fungus but transcription of most of these plant genes (72%) responded in a different direction or magnitude depending on the plant genotype. Two AMF isolates displayed large differences in their transcription, but the direction and magnitude of the transcriptional responses for a large number of these genes was also strongly influenced by the genotype of the plant host. This indicates that unlike the highly conserved plant genes necessary for the symbiosis establishment, plant and fungal gene transcriptional responses are not conserved and are greatly influenced by plant and fungal genetic differences, even at the within-species level. The transcriptional variability detected allowed us to identify an extensive gene network showing the interplay in plant-fungal reprogramming in the symbiosis. Key genes illustrated that the two organisms jointly program their cytoskeleton organisation during growth of the fungus inside roots. Our study reveals that plant and fungal genetic variation plays a strong role in shaping the genetic reprograming in response to symbiosis, indicating considerable genotype x genotype interactions in the mycorrhizal symbiosis. Such variation needs to be considered in order to understand the molecular mechanisms between AMF and their plant hosts in natural communities.

Within-species phylogenetic relatedness of a common mycorrhizal fungus affects evenness in plant communities through effects on dominant species

Arbuscular mycorrhizal fungi (AMF) have been shown to influence plant community structure and diversity. Studies based on single plant - single AMF isolate experiments show that within AMF species variation leads to large differential growth responses of different plant species. Because of these differential effects, genetic differences among isolates of an AMF species could potentially have strong effects on the structure of plant communities. We tested the hypothesis that within species variation in the AMF R. irregularis significantly affects plant community structure and plant co-existence. We took advantage of a recent genetic characterization of several isolates using double-digest restriction-site associated DNA sequencing (ddRADseq). This allowed us to test not only for the impact of within AMF species variation on plant community structure but also for the role of the R. irregularis phylogeny on plant community metrics. Nine isolates of R. irregularis, belonging to three different genetic groups (Gp1, Gp3 and Gp4), were used as either single inoculum or as mixed diversity inoculum. Plants in a mesocosm representing common species that naturally co-exist in European grasslands were inoculated with the different AMF treatments. We found that within-species differences in R. irregularis did not strongly influence the performance of individual plants or the structure of the overall plant community. However, the evenness of the plant community was affected by the phylogeny of the fungal isolates, where more closely-related AMF isolates were more likely to affect plant community evenness in a similar way compared to more genetically distant isolates. This study underlines the effect of within AMF species variability on plant community structure. While differential effects of the AMF isolates were not strong, a single AMF species had enough functional variability to change the equilibrium of a plant community in a way that is associated with the evolutionary history of the fungus.

Genomic Evidence for Cryptic Speciation in Tree Frogs From the Apennine Peninsula, With Description of Hyla perrini sp. nov

Despite increasing appreciation of the “speciation continuum”, delimiting and describing new species is a major yet necessary challenge of modern phylogeography to help optimize conservation efforts. In amphibians, the lack of phenotypic differences between closely-related taxa, their complex, sometimes unresolved phylogenetic relationships, and their potential to hybridize all act to blur taxonomic boundaries. Here we implement a multi-disciplinary approach to evaluate the nature of two deeply-diverged mitochondrial lineages previously documented in Italian tree frogs (Hyla intermedia s. l.), distributed north and south of the Northern Apennine Mountains. Based on evidence from mitochondrial phylogenetics, nuclear phylogenomics, hybrid zone population genomics, niche modelling analyses, and biometric assessments, we propose that these lineages be considered distinct, cryptic species. Both mitochondrial and nuclear data affirm that they belong to two monophyletic clades of Pliocene divergence (~3.5 My), only admixing over a relatively narrow contact zone restricted to the southeast of the Po Plain (50-100km). These characteristics are comparable to similarly-studied parapatric amphibians bearing a specific status. Inferred from their current geographic distribution, the two Italian tree frogs feature distinct ecological niches (<15% of niche overlap), raising questions regarding potential adaptive components contributing to their incipient speciation. However, we found no diagnostic morphological and bioacoustic differences between them. This system illustrates the speciation continuum of Western-Palearctic tree frogs and identifies additional cryptic lineages of similar divergence to be treated as separate species (H. cf. meridionalis). We recommend combined approaches using genomic data as applied here for the future taxonomic assessment of cryptic diversity in alloparapatric radiations of terrestrial vertebrates, especially in controversial taxa. Finally, we formally described the northern Italian tree frogs as a new species, Hyla perrini, sp. nov.