The equilibrium of sex ratios in sexually reproducing species is often disrupted by various environmental and genetic factors, including endosymbionts like Wolbachia. In this study, we explore the highly female-biased sex ratio observed in the flea beetle, Altica lythri, and its underlying mechanisms. Ancient hybridization events between Altica species have led to mitochondrial DNA introgression, resulting in distinct mitochondrial haplotypes that go along with different Wolbachia infections (HT1-wLytA1, HT1*- uninfected, HT2-wLytA2, and HT3-wLytB). Notably, beetles with some haplotypes exclusively produce female offspring, suggesting potential Wolbachia-induced phenomena such as feminization of genetic males. However, the observed female bias could also be a consequence of the ancient hybridization resulting in nuclear-cytoplasmic conflicts between introgressed mtDNA and nuclear genes. Through transcriptomic analysis and the program SEX-DETector, we established markers for genotypic sex differentiation for A. lythri, enabling genetic sexing via qPCR. Our findings suggest that feminization of genetic males is contributing to the skewed sex ratios, highlighting the intricate dynamics of sex determination and reproductive strategies in this flea beetle. This study provides valuable insights into the dynamics of genetic conflicts, endosymbionts, and sex ratios, revealing the novel phenomenon of genetic male feminization in the flea beetle A. lythri.

Differences between sexes in growth, ecology and behavior strongly shape species biology. In some animal groups, such as spiders, it is difficult or impossible to identify the sex of juveniles based on external morphology. This information would be useful for field surveys, behavioral experiments, and ecological studies on e.g. sex ratios and dispersal. In species with sex chromosomes, sex can be determined based on the specific sex chromosome complement. Additionally, information on the sequence of sex chromosomes provides the basis for studying sex chromosome evolution. We combined cytogenetic and genomic data to identify the sex chromosomes in the sexually dimorphic spider Argiope bruennichi , and designed RT-qPCR sex markers. We found that genome size and GC content of this spider falls into the range reported for the majority of araneids. The male karyotype is formed by 24 acrocentric chromosomes with an X1X20 sex chromosome system, with little similarity between X chromosomes, suggesting origin of these chromosomes by X chromosome fission or early duplication of an X chromosome and subsequent independent differentiation of the copies. Our data suggest similarly sized X chromosomes in A. bruennichi . They are smaller chromosomes of the complement. Our findings open the door to new directions in spider evolutionary and ecological research.

Mutations are the ultimate source of heritable variation and therefore the fuel for evolution, but direct estimates of mutation rates exist only for few species. We estimated the spontaneous single nucleotide mutation rate among clonal generations in the waterflea Daphnia galeata with a short-term mutation accumulation approach. Individuals from eighteen mutation accumulation lines over five generations were deep sequenced to count de novo mutations that were not present in a pool of F1 individuals, representing the parental genotype. We identified 12 new nucleotide mutations in 90 clonal generational passages. This resulted in an estimated single nucleotide mutation rate of 0.745 x 10(-9) (95% c.f. 0.39 x 10(-9)-1.26 x 10(-9)), which is slightly lower than recent estimates for other Daphnia species. We discuss the implications for the population genetics of Cladocerans.

Resting stages of aquatic organisms that accumulate in the sediment over time are an exceptional resource that allows direct insights into past populations and addressing evolutionary questions. This is of particular interest in taxa that face relatively new environmental challenges, e.g., climate change and eutrophication, such as the Daphnia longispina species complex, a keystone zooplankton group in European freshwater ecosystems. However, genomic analysis might be challenging as DNA yield from many of these resting stages can be low and the material degraded. To reliably allow the resequencing of single Daphnia resting eggs from different sediment layers and characterize genomic changes through time, we performed whole-genome amplification to obtain DNA amounts suitable for genome resequencing and tested multiple protocols involving egg isolation, whole-genome amplification kits, and library preparation. A pre-sequencing contamination screening was developed, consisting of amplifying mitochondrial Daphnia and bacterial markers, to quickly assess and exclude possibly contaminated samples. In total, we successfully amplified and sequenced nine genomes from Daphnia resting eggs that could be identified as Daphnia longispina species. We analyzed the genome coverage and heterozygosity of these samples to optimize this method for future projects involving population genomic investigation of the resting egg bank.

We present four new complete mitochondrial genomes for Dasypoda hirtipes, Melitta schultzei, Capicola nanula and Samba griseonigra belonging to the basally branching bee family Melittidae covering four genera in three tribes (Melittini, Hesperaspini, Dasypodaini) and two subfamilies (Melittinae, Dasypodainae). The mitogenomes vary between 15,884 and 20,324 bp in length and consist of the typical set of 13 protein-coding genes, 22 tRNAs, two rRNAs and the control region. These new mitogenomes raise the number of available mitochondrial genomes for the family Melittidae to five and will help to shed light on the phylogenetic relationships within Melittidae and their position within the Anthophila.

Hybridization and introgression are recognized as an important source of variation that influence adaptive processes; both phenomena are frequent in the genus Daphnia, a keystone zooplankton taxon in freshwater ecosystems that comprises several species complexes. To investigate genome-wide consequences of introgression between species, we provide here the first high-quality genome assembly for a member of the Daphnia longispina species complex, Daphnia galeata. We further re-sequenced 49 whole genomes of three species of the complex and their interspecific hybrids both from genotypes sampled in the water column and from single resting eggs extracted from sediment cores. Populations from habitats with diverse ecological conditions offered an opportunity to study the dynamics of hybridization linked to ecological changes and revealed a high prevalence of hybrids. Using phylogenetic and population genomic approaches, we provide first insights into the intra- and interspecific genome-wide variability in this species complex and identify regions of high divergence. Finally, we assess the length of ancestry tracts in hybrids to characterize introgression patterns across the genome. Our analyses uncover a complex history of hybridization and introgression reflecting multiple generations of hybridization and backcrossing in the Daphnia longispina species complex. Overall, this study and the new resources presented here pave the way for a better understanding of ancient and contemporary gene flow in the species complex and facilitate future studies on resting egg banks accumulating in lake sediment.

Hatching resting stages of ecologically important organisms such as Daphnia from lake sediments, referred to as resurrection ecology, is a powerful approach to assess changes in alleles and traits over time. However, the utility of the approach is constrained by a few obstacles, including low and/or biased hatching among genotypes. Here, we eliminated such bottlenecks by investigating DNA sequences isolated directly (i.e. without hatching) from resting eggs found in the sediments of Lake Constance spanning pre-, peri-, and post-eutrophication. While we expected genome-wide changes, we specifically expected changes in alleles related to pathways involved in mitigating effects of cyanobacterial toxins. We used pairwise FST-analyses to identify transcripts that showed strongest divergence among the four different populations and a clustering analysis to identify correlations between allele frequency shifts and changes in abiotic and biotic lake parameters. In a cluster that correlated with the increased abundance of cyanobacteria in Lake Constance we find genes that have been reported earlier to be differentially expressed in response to the cyanobacterial toxin microcystin and to microcystin-free cyanobacteria. We further reveal the enrichment of gene ontology terms that have been shown to be involved in microcystin-related responses in other organisms but not yet in Daphnia and as such are candidate loci for adaptation of natural Daphnia populations to increased cyanobacterial abundances. In conclusion this approach of investigating DNA extracted from Daphnia resting stages allowed to determine frequency changes of loci in a natural population over time.

AbstractHybridization and introgression are recognized as an important source of variation that influence adaptive processes; both phenomena are frequent in the genusDaphnia,a keystone zooplankton taxon in freshwater ecosystems that comprises several species complexes. To investigate genome-wide consequences of introgression between species, we provide here the first high-quality genome assembly for a member of theDaphnia longispinaspecies complex,Daphnia galeata. We further re-sequenced 49 whole genomes of three species of the complex and their interspecific hybrids both from genotypes sampled in the water column and from single resting eggs extracted from sediment cores. Populations from habitats with diverse ecological conditions offered an opportunity to study the dynamics of hybridization linked to ecological changes and revealed a high prevalence of hybrids. Using phylogenetic and population genomic approaches, we provide first insights into the intra- and interspecific genome-wide variability in this species complex and identify regions of high divergence. Finally, we assess the length of ancestry tracts in hybrids to characterize introgression patterns across the genome. Our analyses uncover a complex history of hybridization and introgression reflecting multiple generations of hybridization and backcrossing in theDaphnia longispinaspecies complex. Overall, this study and the new resources presented here pave the way for a better understanding of ancient and contemporary gene flow in the species complex and facilitate future studies on resting egg banks accumulating in lake sediment.

Supplementary data for the Genome Biology and Evolution paper 10.1093/gbe/evab267

Bees (Anthophila) represent a critical taxon for the ecosystem service of pollination and hence have received great scientific attention. The more than 20,000 known species of bees are grouped in seven families. Yet, the relationships between bee families are not entirely clear, and specifically, the position of Melittidae and their monophyly has been discussed in the past. Here, we present the most comprehensive mitogenomic phylogeny of bees including five members of Melittidae from two subfamilies and three tribes. Our results suggest monophyly of the family Melittidae and further support their basal position. The data also support the two subfamilies, but are not in line with current assignment of tribes. Our results show, despite advancement with transcriptomic and whole genomic data, the value of mitochondrial data for the reconstruction of phylogenies.

Functional traits are measurable characteristics of an organism that have an impact on its fitness. Variation in functional traits between and among species has been suggested to represent the basis for competition and selection, thus allowing for evolution in natural populations. In freshwater ecosystems, the availability of essential polyunsaturated fatty acids (PUFAs), in particular omega 3- and omega 6-PUFAs, determines the food quality of phytoplankton for the herbivorous zooplanktonDaphnia, an unselective filter feeder. The content of such essential PUFAs in the phytoplankton is thus a functional phytoplankton trait affecting the trophic transfer efficiency and dynamics at the pelagic plant-herbivore interface. In turn, the susceptibility of consumers to become limited by the availability of essential PUFAs is a fitness-determining trait ofDaphniagenotypes, and variability of this herbivore trait may thus affect the daphnids' intrapopulation competition. To estimate the intrapopulation variation in susceptibility, we isolated clonal lines ofDaphnia longispinafrom a natural population and compared the strength of their limitation by dietary PUFA availability via standardised laboratory growth assays. We used a liposome supplementation technique to enrich a PUFA-poor green alga with essential omega 3- and omega 6-PUFAs and determined juvenile somatic growth rate of differentD. longispinagenotypes as a fitness proxy. As expected,D. longispinagenotypes that coexisted in a natural population differed markedly in their specific patterns of susceptibility to dietary PUFA availability. On average, theD. longispinapopulation was more strongly susceptible to limitations in the availability of the omega 6-PUFA arachidonic acid (20:4 omega 6) than to limitations in the availability of omega 3-PUFAs alpha-linolenic acid (18:3 omega 3) and eicosapentaenoic acid (20:5 omega 3). The ability to cope with PUFA limitation is thus a crucial trait that can probably affect intraspecific competition andDaphniapopulation structure. Therefore, we suggest that such intrapopulation variation in susceptibility to absence of dietary PUFAs might be one of the driving forces of natural selection and local adaptation among freshwater zooplankton.

Phenotypic variation plays an important role in how species cope with environmental challenges. Pinpointing which genes and genomic regions are underlying phenotypic variability thus helps to understand the processes of acclimation and adaptation. We used Daphnia as a system to identify candidates playing a role in phenotypic variation related to a predation risk environment with a genome‐wide association approach. Furthermore, a gene co‐expression network analysis allowed identifying clusters of co‐expressed genes which correlated to life history traits. To enhance the understanding of the functional roles of the transcripts, we identified orthologs and paralogs from related species and used ontologies to annotate the candidates of interest. Our study revealed that only one life history trait and two morphometric traits have a genetic association in the presence of predation risk (fish kairomones), whereas most genotype–phenotype associations were detected in a genotype–environment interaction analysis for reproduction‐related phenotypic traits. The gene co‐expression network analysis identified a total of 44 modules, of which one module correlated to another life history trait namely the ‘total number of broods’. The combined use of gene co‐expression network and transcriptome‐wide association analysis allowed the identification of 131 candidate transcripts associated with life history traits in Daphnia galeata . These results lay the ground for targeted studies to further understand phenotypic variability in this species.

AbstractPhenotypic plastic responses allow organisms to rapidly adjust when facing environmental challenges—these responses comprise morphological, behavioral but also life‐history changes. Alteration of life‐history traits when exposed to predation risk have been reported often in the ecological and genomic model organism Daphnia. However, the molecular basis of this response is not well understood, especially in the context of fish predation. Here, we characterized the transcriptional profiles of two Daphnia galeata clonal lines with opposed life histories when exposed to fish kairomones. First, we conducted a differential gene expression, identifying a total of 125 candidate transcripts involved in the predator‐induced response, uncovering substantial intraspecific variation. Second, we applied a gene coexpression network analysis to find clusters of tightly linked transcripts revealing the functional relations of transcripts underlying the predator‐induced response. Our results showed that transcripts involved in remodeling of the cuticle, growth, and digestion correlated with the response to environmental change in D. galeata. Furthermore, we used an orthology‐based approach to gain functional information for transcripts lacking gene ontology (GO) information, as well as insights into the evolutionary conservation of transcripts. We could show that our candidate transcripts have orthologs in other Daphnia species but almost none in other arthropods. The unique combination of methods allowed us to identify candidate transcripts, their putative functions, and evolutionary history associated with predator‐induced responses in Daphnia. Our study opens up to the question as to whether the same molecular signature is associated with fish kairomones‐mediated life‐history changes in other Daphnia species.

Sexual reproduction is pervasive in animals and has led to the evolution of sexual dimorphism. In most animals, males and females show marked differences in primary and secondary sexual traits. The formation of sex-specific organs and eventually sex-specific behaviors is defined during the development of an organism. Sex determination processes have been extensively studied in a few well-established model organisms. While some key molecular regulators are conserved across animals, the initiation of sex determination is highly diverse. To reveal the mechanisms underlying the development of sexual dimorphism and to identify the evolutionary forces driving the evolution of different sexes, sex determination mechanisms must thus be studied in detail in many different animal species beyond the typical model systems. In this perspective article, we argue that spiders represent an excellent group of animals in which to study sex determination mechanisms. We show that spiders are sexually dimorphic in various morphological, behavioral, and life history traits. The availability of an increasing number of genomic and transcriptomic resources and functional tools provides a great starting point to scrutinize the extensive sexual dimorphism present in spiders on a mechanistic level. We provide an overview of the current knowledge of sex determination in spiders and propose approaches to reveal the molecular and genetic underpinnings of sexual dimorphism in these exciting animals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Understanding the genetic basis of local adaptation has long been a focus of evolutionary biology. Recently there has been increased interest in deciphering the evolutionary role of Daphnia ’s plasticity and the molecular mechanisms of local adaptation. Using transcriptome data, we assessed the differences in gene expression profiles and sequences in four European Daphnia galeata populations. In total, ~33% of 32,903 transcripts were differentially expressed between populations. Among 10,280 differentially expressed transcripts, 5,209 transcripts deviated from neutral expectations and their population-specific expression pattern is likely the result of local adaptation processes. Furthermore, a SNP analysis allowed inferring population structure and distribution of genetic variation. The population divergence at the sequence-level was comparatively higher than the gene expression level by several orders of magnitude and consistent with strong founder effects and lack of gene flow between populations. Using sequence information, the candidate transcripts were annotated using a comparative genomics approach. Thus, we identified candidate transcriptomic regions for local adaptation in a key species of aquatic ecosystems in the absence of any laboratory induced stressor.

Phenotypic plastic responses allow organisms to rapidly adjust to environmental challenges. Although phenotypic plastic responses to predation risk have been reported for the ecological and genomic model organism Daphnia, their genetic basis is not well understood. Here, we characterized the transcriptional profile of Daphnia galeata when exposed to fish kairomones. First, we investigated the differential gene expression, identifying candidate transcripts being involved in shifts of life history traits. A total of 125 differentially expressed transcripts (40 up- and 85 downregulated) were identified. Second, we applied a gene co-expression network analysis to find clusters of tightly linked transcripts and reveal the genetic pathways underlying predator-induced responses. Our results showed that transcripts involved in remodeling of the cuticle, growth and digestion correlated with life history shifts in D. galeata. Furthermore, we compared our results with previous studies on other Daphnia species. This was achieved using an orthology approach on D. magna predator-induced response on the one hand, and reproduction associated genes in D. pulex on the other hand. The unique combination of methods including the comparative approach allowed for the identification of candidate transcripts, their functions and orthologs associated with predator-induced responses in Daphnia.