Long-term balancing selection for pathogen resistance maintains trans-species polymorphisms in a planktonic crustacean.

Balancing selection is an evolutionary process that maintains genetic polymorphisms at selected loci and strongly reduces the likelihood of allele fixation. When allelic polymorphisms that predate speciation events are maintained independently in the resulting lineages, a pattern of trans-species polymorphisms may occur. Trans-species polymorphisms have been identified for loci related to mating systems and the MHC, but they are generally rare. Trans-species polymorphisms in disease loci are believed to be a consequence of long-term host-parasite coevolution by balancing selection, the so-called Red Queen dynamics. Here we scan the genomes of three crustaceans with a divergence of over 15 million years and identify 11 genes containing identical-by-descent trans-species polymorphisms with the same polymorphisms in all three species. Four of these genes display molecular footprints of balancing selection and have a function related to immunity. Three of them are located in or close to loci involved in resistance to a virulent bacterial pathogen, Pasteuria, with which the Daphnia host is known to coevolve. This provides rare evidence of trans-species polymorphisms for loci known to be functionally relevant in interactions with a widespread and highly specific parasite. These findings support the theory that specific antagonistic coevolution is able to maintain genetic diversity over millions of years.

Tick range expansion to higher elevations: does Borrelia burgdorferi sensu lato facilitate the colonisation of marginal habitats?

BACKGROUND: Parasites can alter host and vector phenotype and thereby affect ecological processes in natural populations. Laboratory studies have suggested that Borrelia burgdorferi sensu lato, the causative agent of human Lyme borreliosis, may induce physiological and behavioural alterations in its main tick vector in Europe, Ixodes ricinus, which increase the tick's mobility and survival under challenging conditions. These phenotypic alterations may allow I. ricinus to colonise marginal habitats ('facilitation hypothesis'), thereby fuelling the ongoing range expansion of I. ricinus towards higher elevations and latitudes induced by climate change. To explore the potential for such an effect under natural conditions, we studied the prevalence of B. burgdorferi s.l. in questing I. ricinus and its variation with elevation in the Swiss Alps. RESULTS: We screened for B. burgdorferi s.l. infection in questing nymphs of I. ricinus (N = 411) from 15 sites between 528 and 1774 m.a.s.l to test if B. burgdorferi s.l. prevalence is higher at high elevations (i.e. in marginal habitats). Opposite of what is predicted under the facilitation hypothesis, we found that B. burgdorferi s.l. prevalence in I. ricinus nymphs decreased with increasing elevation and that Borrelia prevalence was 12.6% lower in I. ricinus nymphs collected at the range margin compared to nymphs in the core range. But there was no association between Borrelia prevalence and elevation within the core range of I. ricinus. Therefore the observed pattern was more consistent with a sudden decrease in Borrelia prevalence above a certain elevation, rather than a gradual decline with increasing elevation across the entire tick range. CONCLUSIONS: In conclusion, we found no evidence that B. burgdorferi s.l.-induced alterations of I. ricinus phenotype observed in laboratory studies facilitate the colonisation of marginal habitats in the wild. Rather, ticks in marginal habitats are substantially less likely to harbour the pathogen. These findings have implications for a better understanding of eco-evolutionary processes in natural host-parasite systems, as well as the assessment of Lyme borreliosis risk in regions where I. ricinus is newly emerging.

Whole-Genome Phylogenetic Reconstruction as a Powerful Tool to Reveal Homoplasy and Ancient Rapid Radiation in Waterflea Evolution.

Although phylogeny estimation is notoriously difficult in radiations that occurred several hundred million years ago, phylogenomic approaches offer new ways to examine relationships among ancient lineages and evaluate hypotheses that are key to evolutionary biology. Here, we reconstruct the deep-rooted relationships of one of the oldest living arthropod clades, the branchiopod crustaceans, using a kaleidoscopic approach. We use concatenation and coalescent tree-building methods to analyze a large multigene data set at the nucleotide and amino acid level and examine gene tree versus species tree discordance. We unequivocally resolve long-debated relationships among extant orders of the Cladocera, the waterfleas, an ecologically relevant zooplankton group in global aquatic and marine ecosystems that is famous for its model systems in ecology and evolution. To build the data set, we assembled eight de novo genomes of key taxa including representatives of all extant cladoceran orders and suborders. Our phylogenetic analysis focused on a BUSCO-based set of 823 conserved single-copy orthologs shared among 23 representative taxa spanning all living branchiopod orders, including 11 cladoceran families. Our analysis supports the monophyly of the Cladocera and reveals remarkable homoplasy in their body plans. We found large phylogenetic distances between lineages with similar ecological specializations, indicating independent evolution in major body plans, such as in the pelagic predatory orders Haplopoda and Onychopoda (the "Gymnomera"). In addition, we assessed rapid cladogenesis by estimating relative timings of divergence in major lineages using reliable fossil-calibrated priors on eight nodes in the branchiopod tree, suggesting a Paleozoic origin around 325 Ma for the cladoceran ancestor and an ancient rapid radiation around 252 Ma at the Perm/Triassic boundary. These findings raise new questions about the roles of homoplasy and rapid radiation in the diversification of the cladocerans and help examine trait evolution from a genomic perspective in a functionally well understood, ancient arthropod group. [Cladocera; Daphnia; evolution; homoplasy; molecular clock; phylogenomics; systematics; waterfleas.].

No evidence for genetic sex determination in Daphnia magna.

Mechanisms of sex determination (SD) differ widely across the tree of life. In genotypic sex determination (GSD), genetic elements determine whether individuals are male or female, while in environmental sex determination (ESD), external cues control the sex of the offspring. In cyclical parthenogens, females produce mostly asexual daughters, but environmental stimuli such as crowding, temperature or photoperiod may cause them to produce sons. In aphids, sons are induced by ESD, even though GSD is present, with females carrying two X chromosomes and males only one (X0 SD system). By contrast, although ESD exists in Daphnia, the two sexes were suggested to be genetically identical, based on a 1972 study on Daphnia magna (2n=20) that used three allozyme markers. This study cannot, however, rule out an X0 system, as all three markers may be located on autosomes. Motivated by the life cycle similarities of Daphnia and aphids, and the absence of karyotype information for Daphnia males, we tested for GSD (homomorphic sex chromosomes and X0) systems in D. magna using a whole-genome approach by comparing males and females of three genotypes. Our results confirm the absence of haploid chromosomes or haploid genomic regions in D. magna males as well as the absence of sex-linked genomic regions and sex-specific single-nucleotide polymorphisms. Within the limitations of the three studied populations here and the methods used, we suggest that our results make the possibility of genetic differences among sexes in the widely used Daphnia model system very unlikely.

Genomic characterization of selfing in the cyclic parthenogen Daphnia magna.

Inbreeding refers to the fusion of related individuals' gametes, with self-fertilization (selfing) being an extreme form of inbreeding-involving gametes produced by the same individual. Selfing is expected to reduce heterozygosity by an average of 50% in one generation; however, little is known about the empirical variation on a genome level surrounding this figure and the factors that affect variation. We selfed genotypes of the cyclic parthenogen Daphnia magna and analysed whole genomes of mothers and selfed offspring, observing the predicted 50% heterozygosity reduction on average. We also saw substantial variation around this value and significant differences among mother-offspring pairs. Crossover analysis confirmed the known trend of recombination occurring more often towards the telomeres. This effect was shown, through simulations, to increase the variance of heterozygosity reduction compared to when a uniform distribution of crossovers was used. Similarly, we simulated inbred line production after several generations of selfing and we observed higher variance in achieved homozygosity when we consider a higher recombination rate towards the telomeres. Our empirical and simulation study highlights that the expected mean values of heterozygosity reduction show remarkable variation, which can help understand, for example, differences among inbred individuals.

Combining genome-wide association study and FST -based approaches to identify targets of Borrelia-mediated selection in natural rodent hosts.

Recent advances in high-throughput sequencing technologies provide opportunities to gain novel insights into the genetic basis of phenotypic trait variation. Yet to date, progress in our understanding of genotype-phenotype associations in nonmodel organisms in general and natural vertebrate populations in particular has been hampered by small sample sizes typically available for wildlife populations and a resulting lack of statistical power, as well as a limited ability to control for false-positive signals. Here we propose to combine a genome-wide association study (GWAS) and FST -based approach with population-level replication to partly overcome these limitations. We present a case study in which we used this approach in combination with genotyping-by-sequencing (GBS) single nucleotide polymorphism (SNP) data to identify genomic regions associated with Borrelia afzelii resistance or susceptibility in the natural rodent host of this Lyme disease-causing spirochete, the bank vole (Myodes glareolus). Using this combined approach we identified four consensus SNPs located in exonic regions of the genes Slc26a4, Tns3, Wscd1 and Espnl, which were significantly associated with the voles' Borrelia infectious status within and across populations. Functional links between host responses to bacterial infections and most of these genes have previously been demonstrated in other rodent systems, making them promising new candidates for the study of evolutionary host responses to Borrelia emergence. Our approach is applicable to other systems and may facilitate the identification of genetic variants underlying disease resistance or susceptibility, as well as other ecologically relevant traits, in wildlife populations.

A fossil-calibrated phylogenomic analysis of Daphnia and the Daphniidae.

In the post-genomic era, much of phylogenetic analyses still relies on mitochondrial DNA, either alone or in combination with few nuclear genes. Although this approach often makes it possible to construct well-supported trees, it is limited because mtDNA describes the history of a single locus, and nuclear phylogenies based on a few loci may be biased, leading to inaccurate tree topologies and biased estimations of species divergence time. In this study, we perform a phylogenomic analysis of the Daphniidae family (Crustacea: Branchiopoda: Anomopoda) including some of the most frequently studied model organisms (Daphnia magna and D. pulex) whose phylogenetic relationships have been based primarily on an assessment of a few mtDNA genes. Using high-throughput sequencing, we were able to assemble 38 whole mitochondrial genomes and draft nuclear genomes for 18 species, including at least one species for each known genus of the family Daphniidae. Here we present phylogenies based on 636 nuclear single-copy genes shared among all sampled taxa and based on whole mtDNA genomes. The phylogenies we obtained were highly supported and showed some discrepancies between nuclear and mtDNA based trees at deeper nodes. We also identified a new candidate sister lineage of Daphnia magna. Our time-calibrated genomic trees, which we constructed using both fossil records and substitution rates, yielded very different estimates of branching event times compared to those based on mtDNA. By providing multi-locus, fossil-calibrated trees of the Daphniidae, our study contributes to an improved phylogenetic framework for ecological and evolutionary studies that use water fleas as a model system.

Small-scale spatial variation in infection risk shapes the evolution of a Borrelia resistance gene in wild rodents.

Spatial variation in pathogen-mediated selection is predicted to influence the evolutionary trajectory of host populations and lead to spatial variation in their immunogenetic composition. However, to date few studies have been able to directly link small-scale spatial variation in infection risk to host immune gene evolution in natural, nonhuman populations. Here, we use a natural rodent-Borrelia system to test for associations between landscape-level spatial variation in Borrelia infection risk along replicated elevational gradients in the Swiss Alps and Toll-like receptor 2 (TLR2) evolution, a candidate gene for Borrelia resistance, across bank vole (Myodes glareolus) populations. We found that Borrelia infection risk (i.e., the product of Borrelia prevalence in questing ticks and the average tick load of voles at a sampling site) was spatially variable and significantly negatively associated with elevation. Across sampling sites, Borrelia prevalence in bank voles was significantly positively associated with Borrelia infection risk along the elevational clines. We observed a significant association between naturally occurring TLR2 polymorphisms in hosts and their Borrelia infection status. The TLR2 variant associated with a reduced likelihood of Borrelia infection was most common in rodent populations at lower elevations that face a high Borrelia infection risk, and its frequency changed in accordance with the change in Borrelia infection risk along the elevational clines. These results suggest that small-scale spatial variation in parasite-mediated selection affects the immunogenetic composition of natural host populations, providing a striking example that the microbial environment shapes the evolution of the host's immune system in the wild.

Survival and divergence in a small group: The extraordinary genomic history of the endangered Apennine brown bear stragglers.

About 100 km east of Rome, in the central Apennine Mountains, a critically endangered population of ∼50 brown bears live in complete isolation. Mating outside this population is prevented by several 100 km of bear-free territories. We exploited this natural experiment to better understand the gene and genomic consequences of surviving at extremely small population size. We found that brown bear populations in Europe lost connectivity since Neolithic times, when farming communities expanded and forest burning was used for land clearance. In central Italy, this resulted in a 40-fold population decline. The overall genomic impact of this decline included the complete loss of variation in the mitochondrial genome and along long stretches of the nuclear genome. Several private and deleterious amino acid changes were fixed by random drift; predicted effects include energy deficit, muscle weakness, anomalies in cranial and skeletal development, and reduced aggressiveness. Despite this extreme loss of diversity, Apennine bear genomes show nonrandom peaks of high variation, possibly maintained by balancing selection, at genomic regions significantly enriched for genes associated with immune and olfactory systems. Challenging the paradigm of increased extinction risk in small populations, we suggest that random fixation of deleterious alleles (i) can be an important driver of divergence in isolation, (ii) can be tolerated when balancing selection prevents random loss of variation at important genes, and (iii) is followed by or results directly in favorable behavioral changes.

Genome complexity of harmful microalgae.

During the past decade, next generation sequencing (NGS) technologies have provided new insights into the diversity, dynamics, and metabolic pathways of natural microbial communities. But, these new techniques face challenges related to the genome size and level of genome complexity of the species under investigation. Moreover, the coverage depth and the short-read length achieved by NGS based approaches also represent a major challenge for assembly. These factors could limit the use of these high-throughput sequencing methods for species lacking a reference genome and characterized by a high level of complexity. In the present work, the evolutionary history, mainly consisting of gene transfer events from bacteria and unicellular eukaryotes to microalgae, including harmful species, is discussed and reviewed as it relates to NGS application in microbial communities, with a particular focus on harmful algal bloom species and dinoflagellates. In the context of genetic population studies, genotyping-by-sequencing (GBS), an NGS based approach, could be used for the discovery and analysis of single nucleotide polymorphisms (SNPs). The NGS technologies are still relatively new and require further improvement. Specifically, there is a need to develop and standardize tools and approaches to handle large data sets, which have to be used for the majority of HAB species characterized by evolutionary highly dynamic genomes.

The Genome of the "Great Speciator" Provides Insights into Bird Diversification.

Among birds, white-eyes (genus Zosterops) have diversified so extensively that Jared Diamond and Ernst Mayr referred to them as the "great speciator." The Zosterops lineage exhibits some of the fastest rates of species diversification among vertebrates, and its members are the most prolific passerine island colonizers. We present a high-quality genome assembly for the silvereye (Zosterops lateralis), a white-eye species consisting of several subspecies distributed across multiple islands. We investigate the genetic basis of rapid diversification in white-eyes by conducting genomic analyses at varying taxonomic levels. First, we compare the silvereye genome with those of birds from different families and searched for genomic features that may be unique to Zosterops. Second, we compare the genomes of different species of white-eyes from Lifou island (South Pacific), using whole genome resequencing and restriction site associated DNA. Third, we contrast the genomes of two subspecies of silvereye that differ in plumage color. In accordance with theory, we show that white-eyes have high rates of substitutions, gene duplication, and positive selection relative to other birds. Below genus level, we find that genomic differentiation accumulates rapidly and reveals contrasting demographic histories between sympatric species on Lifou, indicative of past interspecific interactions. Finally, we highlight genes possibly involved in color polymorphism between the subspecies of silvereye. By providing the first whole-genome sequence resources for white-eyes and by conducting analyses at different taxonomic levels, we provide genomic evidence underpinning this extraordinary bird radiation.

Defining conservation units in a stocking-induced genetic melting pot: unraveling native and multiple exotic genetic imprints of recent and historical secondary contact in Adriatic grayling.

The definition of conservation units is crucial for the sustainable management of endangered species, though particularly challenging when recent and past anthropogenic and natural gene flow might have played a role. The conservation of the European grayling, Thymallus thymallus, is particularly complex in its southern distribution area, where the Adriatic evolutionary lineage is endangered by a long history of anthropogenic disturbance, intensive stocking and potentially widespread genetic introgression. We provide mtDNA sequence and microsatellite data of 683 grayling from 30 sites of Adriatic as well as Danubian and Atlantic origin. We apply Bayesian clustering and Approximate Bayesian Computation (ABC) to detect microgeographic population structure and to infer the demographic history of the Adriatic populations, to define appropriate conservation units. Varying frequencies of indigenous genetic signatures of the Adriatic grayling were revealed, spanning from marginal genetic introgression to the collapse of native gene pools. Genetic introgression involved multiple exotic source populations of Danubian and Atlantic origin, thus evidencing the negative impact of few decades of stocking. Within the Adige River system, a contact zone of western Adriatic and eastern Danubian populations was detected, with ABC analyses suggesting a historical anthropogenic origin of eastern Adige populations, most likely founded by medieval translocations. Substantial river-specific population substructure within the Adriatic grayling Evolutionary Significant Unit points to the definition of different conservation units. We finally propose a catalog of management measures, including the legal prohibition of stocking exotic grayling and the use of molecular markers in supportive- and captive-breeding programs.

Mitochondrial and nuclear DNA survey of Zootoca vivipara across the eastern Italian Alps: evolutionary relationships, historical demography and conservation implications.

The European common lizard Zootoca vivipara exhibits reproductive bimodality, with populations being either viviparous or oviparous. In the central-eastern Italian Alps oviparous populations (Z. v. carniolica) and viviparous populations (Z. v. vivipara) partly overlap geographically. Studying the evolutionary relationship between these taxa presents an interesting opportunity to gain insight into the evolution of this trait. We aim to: i) test whether Z. v. carniolica, which is endangered, constitutes an ESU (Evolutionary Significant Unity); ii) infer mtDNA divergence time between the Z. v. carniolica clade and all the other Z. vivipara subspecies with the aid of an external calibration point; and iii) describe the phylogeographical and demographic scenarios in the area. To do so we sequenced about 200 individuals for mitochondrial variation; 64 of them were also analysed for three nuclear genes. Furthermore, we analysed the same nuclear markers in 17 individuals from the other oviparous subspecies Z. v. louislantzi and 11 individuals of Z. v. vivipara from widespread geographical origins. The mtDNA and nDNA loci that we examined supported the monophyly of Z. v. carniolica. The mtDNA-based estimate of divergence time between Z. v. carniolica and all the other subspecies indicated a separation at 4.5 Mya (95% CI 6.1-2.6), with about 5% of sequence divergence. Considering that Z. v. carniolica harbours higher genetic diversity, while Z. v. vivipara from central-eastern Alps shows a signature of recent population and spatial expansion, we argue that Z. v. carniolica represents a distinct evolutionary unit, with a presumably long-term evolutionary history of separation. Z. v. carniolica populations, occurring at higher latitudes and altitudes than insofar supposed, live in peat bogs, a seriously threatened habitat: taking into account also its evolutionary distinctness, specific conservation measures should be considered.