A Chromosome-Level Genome Assembly and Annotation for the Clouded Apollo Butterfly (Parnassius mnemosyne): A Species of Global Conservation Concern.

The clouded apollo (Parnassius mnemosyne) is a palearctic butterfly distributed over a large part of western Eurasia, but population declines and fragmentation have been observed in many parts of the range. The development of genomic tools can help to shed light on the genetic consequences of the decline and to make informed decisions about direct conservation actions. Here, we present a high-contiguity, chromosome-level genome assembly of a female clouded apollo butterfly and provide detailed annotations of genes and transposable elements. We find that the large genome (1.5 Gb) of the clouded apollo is extraordinarily repeat rich (73%). Despite that, the combination of sequencing techniques allowed us to assemble all chromosomes (nc = 29) to a high degree of completeness. The annotation resulted in a relatively high number of protein-coding genes (22,854) compared with other Lepidoptera, of which a large proportion (21,635) could be assigned functions based on homology with other species. A comparative analysis indicates that overall genome structure has been largely conserved, both within the genus and compared with the ancestral lepidopteran karyotype. The high-quality genome assembly and detailed annotation presented here will constitute an important tool for forthcoming efforts aimed at understanding the genetic consequences of fragmentation and decline, as well as for assessments of genetic diversity, population structure, inbreeding, and genetic load in the clouded apollo butterfly.

Batrachochytrium dendrobatidis strain affects transcriptomic response in liver but not skin in latitudinal populations of the common toad (Bufo bufo).

Batrachochytrium dendrobatidis (Bd) is a fungal pathogen that has decimated amphibian populations worldwide for several decades. We examined the changes in gene expression in response to Bd infection in two populations of the common toad, Bufo bufo, in a laboratory experiment. We collected B. bufo eggs in southern and northern Sweden, and infected the laboratory-raised metamorphs with two strains of the global panzoonotic lineage Bd-GPL. Differential expression analysis showed significant differences between infected and control individuals in both liver and skin. The skin samples showed no discernible differences in gene expression between the two strains used, while liver samples were differentiated by strain, with one of the strains eliciting no immune response from infected toads. Immune system genes were overexpressed in skin samples from surviving infected individuals, while in liver samples the pattern was more diffuse. Splitting samples by population revealed a stronger immune response in northern individuals. Differences in transcriptional regulation between populations are particularly relevant to study in Swedish amphibians, which may have experienced varying exposure to Bd. Earlier exposure to this pathogen and subsequent adaptation or selection pressure may contribute to the survival of some populations over others, while standing genetic diversity in different populations may also affect the infection outcome.

Body size mediates latitudinal population differences in the response to chytrid fungus infection in two amphibians.

Factors behind intraspecific variation in sensitivity to pathogens remain poorly understood. We investigated how geographical origin in two North European amphibians affects tolerance to infection by the chytrid fungus Batrachochytrium dendrobatidis (Bd), a generalist pathogen which has caused amphibian population declines worldwide. We exposed newly metamorphosed individuals of moor frog Rana arvalis and common toad Bufo bufo from two latitudinal regions to two different BdGPL strains. We measured survival and growth as infections may cause sub-lethal effects in fitness components even in the absence of mortality. Infection loads were higher in B. bufo than in R. arvalis, and smaller individuals had generally higher infection loads. B. bufo had high mortality in response to Bd infection, whereas there was little mortality in R. arvalis. Bd-mediated mortality was size-dependent and high-latitude individuals were smaller leading to high mortality in the northern B. bufo. Bd exposure led to sub-lethal effects in terms of reduced growth suggesting that individuals surviving the infection may have reduced fitness mediated by smaller body size. In both host species, the Swedish Bd strain caused stronger sublethal effects than the British strain. We suggest that high-latitude populations can be more vulnerable to chytrids than those from lower latitudes and discuss the possible mechanisms how body size and host geographical origin contribute to the present results.

Avian malaria parasite infections do not affect personality in the chestnut thrush (Turdus rubrocanus) on the Qinghai-Tibet Plateau.

Personality traits, the consistent individual behavioral differences, are currently gaining much attention in studies of natural bird populations. However, associations between personality traits and parasite infections are not often investigated. Even less attention has been given to studies of birds in the high-elevation region such as the Tibetan plateau. This research aims to examine the relationship between avian malaria parasites and two personality traits in a population of the Chestnut Thrush (Turdus rubrocanus) breed in the Tibetan plateau. Our results revealed no evidence of sex bias in malaria parasite prevalence. Furthermore, we found no effect of infection status on two personality scores: activity and boldness. Additionally, no effects on the activity level or boldness were observed for different parasite lineages of Haemoproteus, Leucocytozoon, the sex of the birds, or their interactions. Similarly, we did not find any relationship between activity level and boldness with nestling numbers, sex, or their interactions. Notably, individuals with a larger number of offspring tended to display greater boldness. Our findings indicate that blood parasite infections are common in this population but do not significantly impact the personality of the birds.

Understanding the evolution of immune genes in jawed vertebrates.

Driven by co-evolution with pathogens, host immunity continuously adapts to optimize defence against pathogens within a given environment. Recent advances in genetics, genomics and transcriptomics have enabled a more detailed investigation into how immunogenetic variation shapes the diversity of immune responses seen across domestic and wild animal species. However, a deeper understanding of the diverse molecular mechanisms that shape immunity within and among species is still needed to gain insight into-and generate evolutionary hypotheses on-the ultimate drivers of immunological differences. Here, we discuss current advances in our understanding of molecular evolution underpinning jawed vertebrate immunity. First, we introduce the immunome concept, a framework for characterizing genes involved in immune defence from a comparative perspective, then we outline how immune genes of interest can be identified. Second, we focus on how different selection modes are observed acting across groups of immune genes and propose hypotheses to explain these differences. We then provide an overview of the approaches used so far to study the evolutionary heterogeneity of immune genes on macro and microevolutionary scales. Finally, we discuss some of the current evidence as to how specific pathogens affect the evolution of different groups of immune genes. This review results from the collective discussion on the current key challenges in evolutionary immunology conducted at the ESEB 2021 Online Satellite Symposium: Molecular evolution of the vertebrate immune system, from the lab to natural populations.

A chromosome-level genome assembly for the Rock Ptarmigan (Lagopus muta).

The Rock Ptarmigan (Lagopus muta) is a cold-adapted, largely sedentary, game bird with a Holarctic distribution. The species represents an important example of an organism likely to be affected by ongoing climatic shifts across a disparate range. We provide here a high-quality reference genome and mitogenome for the Rock Ptarmigan assembled from PacBio HiFi and Hi-C sequencing of a female bird from Iceland. The total size of the genome is 1.03 Gb with a scaffold N50 of 71.23 Mb and a contig N50 of 17.91 Mb. The final scaffolds represent all 40 predicted chromosomes, and the mitochondria with a BUSCO score of 98.6%. Gene annotation resulted in 16,078 protein-coding genes out of a total 19,831 predicted (81.08% excluding pseudogenes). The genome included 21.07% repeat sequences, and the average length of genes, exons, and introns were 33605, 394, and 4265 bp, respectively. The availability of a new reference-quality genome will contribute to understanding the Rock Ptarmigan's unique evolutionary history, vulnerability to climate change, and demographic trajectories around the globe while serving as a benchmark for species in the family Phasianidae (order Galliformes).

How genomics can help biodiversity conservation.

The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics.

The era of reference genomes in conservation genomics.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics.

Demographic history and divergence of sibling grouse species inferred from whole genome sequencing reveal past effects of climate change.

BACKGROUND: The boreal forest is one of the largest biomes on earth, supporting thousands of species. The global climate fluctuations in the Quaternary, especially the ice ages, had a significant influence on the distribution of boreal forest, as well as the divergence and evolution of species inhabiting this biome. To understand the possible effects of on-going and future climate change it would be useful to reconstruct past population size changes and relate such to climatic events in the past. We sequenced the genomes of 32 individuals from two forest inhabiting bird species, Hazel Grouse (Tetrastes bonasia) and Chinese Grouse (T. sewerzowi) and three representatives of two outgroup species from Europe and China. RESULTS: We estimated the divergence time of Chinese Grouse and Hazel Grouse to 1.76 (0.46-3.37) MYA. The demographic history of different populations in these two sibling species was reconstructed, and showed that peaks and bottlenecks of effective population size occurred at different times for the two species. The northern Qilian population of Chinese Grouse became separated from the rest of the species residing in the south approximately 250,000 years ago and have since then showed consistently lower effective population size than the southern population. The Chinese Hazel Grouse population had a higher effective population size at the peak of the Last Glacial Period (approx. 300,000 years ago) than the European population. Both species have decreased recently and now have low effective population sizes. CONCLUSIONS: Combined with the uplift history and reconstructed climate change during the Quaternary, our results support that cold-adapted grouse species diverged in response to changes in the distribution of palaeo-boreal forest and the formation of the Loess Plateau. The combined effects of climate change and an increased human pressure impose major threats to the survival and conservation of both species.

King or royal family? Testing for species boundaries in the King Cobra, Ophiophagus hannah (Cantor, 1836), using morphology and multilocus DNA analyses.

In widespread species, the diverse ecological conditions in which the populations occur, and the presence of many potential geographical barriers through their range are expected to have created ample opportunities for the evolution of distinct, often cryptic lineages. In this work, we tested for species boundaries in one such widespread species, the king cobra, Ophiophagus hannah (Cantor, 1836), a largely tropical elapid snake distributed across the Oriental realm. Based on extensive geographical sampling across most of the range of the species, we initially tested for candidate species (CS) using Maximum-Likelihood analysis of mitochondrial genes. We then tested the resulting CS using both morphological data and sequences of three single-copy nuclear genes. We used snapclust to determine the optimal number of clusters in the nuclear dataset, and Bayesian Phylogenetics and Phylogeography (BPP) to test for likely species status. We used non-metric multidimensional scaling (nMDS) analysis for discerning morphological separation. We recovered four independently evolving, geographically separated lineages that we consider Confirmed Candidate Species: (1) Western Ghats lineage; (2) Indo-Chinese lineage (3) Indo-Malayan lineage; (4) Luzon Island lineage, in the Philippine Archipelago. We discuss patterns of lineage divergence, particularly in the context of low morphological divergence, and the conservation implications of recognizing several endemic king cobra lineages.

Whole genome sequencing reveals high differentiation, low levels of genetic diversity and short runs of homozygosity among Swedish wels catfish.

The use of genetic markers in the context of conservation is largely being outcompeted by whole-genome data. Comparative studies between the two are sparse, and the knowledge about potential effects of this methodology shift is limited. Here, we used whole-genome sequencing data to assess the genetic status of peripheral populations of the wels catfish (Silurus glanis), and discuss the results in light of a recent microsatellite study of the same populations. The Swedish populations of the wels catfish have suffered from severe declines during the last centuries and persists in only a few isolated water systems. Fragmented populations generally are at greater risk of extinction, for example due to loss of genetic diversity, and may thus require conservation actions. We sequenced individuals from the three remaining native populations (Båven, Emån, and Möckeln) and one reintroduced population of admixed origin (Helge å), and found that genetic diversity was highest in Emån but low overall, with strong differentiation among the populations. No signature of recent inbreeding was found, but a considerable number of short runs of homozygosity were present in all populations, likely linked to historically small population sizes and bottleneck events. Genetic substructure within any of the native populations was at best weak. Individuals from the admixed population Helge å shared most genetic ancestry with the Båven population (72%). Our results are largely in agreement with the microsatellite study, and stresses the need to protect these isolated populations at the northern edge of the distribution of the species.

The effects of drift and selection on latitudinal genetic variation in Scandinavian common toads (Bufo bufo) following postglacial recolonisation.

Clinal variation is paramount for understanding the factors shaping genetic diversity in space and time. During the last glacial maximum, northern Europe was covered by glacial ice that rendered the region uninhabitable for most taxa. Different evolutionary processes during and after the recolonisation of this area from different glacial refugia have affected the genetic landscape of the present day European flora and fauna. In this study, we focus on the common toad (Bufo bufo) in Sweden and present evidence suggesting that these processes have resulted in two separate lineages of common toad, which colonised Sweden from two directions. Using ddRAD sequencing data for demographic modelling, structure analyses, and analysis of molecular variance (AMOVA), we provide evidence of a contact zone located between Uppland and Västerbotten in central Sweden. Genetic diversity was significantly higher in southern Sweden compared to the north, in accordance with a pattern of decreased genetic diversity with increasing distance from glacial refugia. Candidate genes under putative selection are identified through outlier detection and gene-environment association methods. We provide evidence of divergent selection related to stress response and developmental processes in these candidate genes. The colonisation of Sweden by two separate lineages may have implications for how future conservation efforts should be directed by identifying management units and putative local adaptations.

Small-scale population divergence is driven by local larval environment in a temperate amphibian.

Genomic variation within and among populations is shaped by the interplay between natural selection and the effects of genetic drift and gene flow. Adaptive divergence can be found in small-scale natural systems even when population sizes are small, and the potential for gene flow is high, suggesting that local environments exert selection pressures strong enough to counteract the opposing effects of drift and gene flow. Here, we investigated genomic differentiation in nine moor frog (Rana arvalis) populations in a small-scale network of local wetlands using 16,707 ddRAD-seq SNPs, relating levels of differentiation with local environments, as well as with properties of the surrounding landscape. We characterized population structure and differentiation, and partitioned the effects of geographic distance, local larval environment, and landscape features on total genomic variation. We also conducted gene-environment association studies using univariate and multivariate approaches. We found small-scale population structure corresponding to 6-8 clusters. Local larval environment was the most influential component explaining 2.3% of the total genetic variation followed by landscape features (1.8%) and geographic distance (0.8%), indicative of isolation-by-environment, -by-landscape, and -by-distance, respectively. We identified 1000 potential candidate SNPs putatively under divergent selection mediated by the local larval environment. The candidate SNPs were involved in, among other biological functions, immune system function and development. Our results suggest that small-scale environmental differences can exert selection pressures strong enough to counteract homogenizing effects of gene flow and drift in this small-scale system, leading to observable population differentiation.

Occurrence of Batrachochytrium dendrobatidis in Sweden: higher infection prevalence in southern species.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused worldwide declines in amphibian populations. While Bd is widespread in southern and central Europe, its occurrence and distribution in northernmost Europe is mostly unknown. We surveyed for Bd in breeding anurans in Sweden by sampling 1917 amphibians from 101 localities and 3 regions in Sweden (southern, northern and central). We found that Bd was widespread in southern and central Sweden, occurring in all 9 investigated species and in 45.5% of the 101 localities with an overall prevalence of 13.8%. No infected individuals were found in the 4 northern sites sampled. The records from central Sweden represent the northernmost records of Bd in Europe. While the proportion of sites positive for Bd was similar between the southern and central regions, prevalence was much higher in the southern region. This was because southern species with a distribution mainly restricted to southernmost Sweden had a higher prevalence than widespread generalist species. The nationally red-listed green toad Bufotes variabilis and the fire-bellied toad Bombina bombina had the highest prevalence (61.4 and 48.9%, respectively). Across species, Bd prevalence was strongly positively, correlated with water temperature at the start of egg laying. However, no individuals showing visual signs of chytridiomycosis were found in the field. These results indicate that Bd is widespread and common in southern and central Sweden with southern species, breeding in higher temperatures and with longer breeding periods, having higher prevalence. However, the impact of Bd on amphibian populations in northernmost Europe remains unknown.

Genomic analysis of demographic history and ecological niche modeling in the endangered Chinese Grouse Tetrastes sewerzowi.

BACKGROUND: The Quaternary had worldwide consequences in forming the contemporary diversity of many populations, species and communities, which is characterized by marked climatic oscillations between glacial and interglacial periods. The origin and evolution of biodiversity in mountainous areas are highly dependent on historical orogenesis and associated climatic changes. The Chinese grouse Tetrastes sewerzowi is a forest-dwelling species endemic to the mountains to the east of the Qinghai-Tibet Plateau, which has been listed as Near Threatened with a decreasing trend by the IUCN because of ongoing deforestation and fragmentation of coniferous forests. It is important to place current population status into a broader ecological and evolutionary context to understand their demographic history. RESULTS: Analyses of the Chinese Grouse genome revealed fluctuations throughout the Pleistocene in effective population size. Populations decreased during early to middle Pleistocene but showed an expansion during late Pleistocene which was then followed by a sharp decline during the last glacial maximum (LGM). Ecological niche modeling indicated that a suitable habitat shift between high altitude regions to low altitude regions was due to a changing climate. This result parallels patterns of population size change in Chinese Grouse estimated from PSMC modelling, which suggested an expansion in population size from the last interglacial period (LIG) and then a peak and a bottleneck occurring at the last glacial maximum (LGM). Furthermore, the present-day distribution of Chinese Grouse is greatly reduced and fragmented. It will likely become even more fragmented in the future since coniferous forest cover is threatened in the region of their distribution and the availability of such habitat restricts their ecological niche. CONCLUSIONS: The Chinese Grouse have experienced substantial population size changes from the beginning to the LIG and reached a peak before the LGM. A sharp decrease and bottleneck occurred during the LGM, when the coniferous forests were subjected to extensive loss. The results inferred from the whole genome sequencing and species distribution models both support historical population fluctuations. The distribution of the Chinese Grouse is strongly dependent on the coniferous forest cover. To protect the fragmented coniferous forests is an essential action to protect the Chinese Grouse.

Antimicrobial peptide and sequence variation along a latitudinal gradient in two anurans.

BACKGROUND: While there is evidence of both purifying and balancing selection in immune defense genes, large-scale genetic diversity in antimicrobial peptides (AMPs), an important part of the innate immune system released from dermal glands in the skin, has remained uninvestigated. Here we describe genetic diversity at three AMP loci (Temporin, Brevinin and Palustrin) in two ranid frogs (Rana arvalis and R. temporaria) along a 2000 km latitudinal gradient. We amplified and sequenced part of the Acidic Propiece domain and the hypervariable Mature Peptide domain (~ 150-200 bp) in the three genes using Illumina Miseq and expected to find decreased AMP genetic variation towards the northern distribution limit of the species similarly to studies on MHC genetic patterns. RESULTS: We found multiple loci for each AMP and relatively high gene diversity, but no clear pattern of geographic genetic structure along the latitudinal gradient. We found evidence of trans-specific polymorphism in the two species, indicating a common evolutionary origin of the alleles. Temporin and Brevinin did not form monophyletic clades suggesting that they belong to the same gene family. By implementing codon evolution models we found evidence of strong positive selection acting on the Mature Peptide. We also found evidence of diversifying selection as indicated by divergent allele frequencies among populations and high Theta k values. CONCLUSION: Our results suggest that AMPs are an important source of adaptive diversity, minimizing the chance of microorganisms developing resistance to individual peptides.

Genome Assembly of the Common Pheasant Phasianus colchicus: A Model for Speciation and Ecological Genomics.

The common pheasant (Phasianus colchicus) in the order Galliformes and the family Phasianidae, has 30 subspecies distributed across its native range in the Palearctic realm and has been introduced to Europe, North America, and Australia. It is an important game bird often subjected to wildlife management as well as a model species to study speciation, biogeography, and local adaptation. However, the genomic resources for the common pheasant are generally lacking. We sequenced a male individual of the subspecies torquatus of the common pheasant with the Illumina HiSeq platform. We obtained 94.88 Gb of usable sequences by filtering out low-quality reads of the raw data generated. This resulted in a 1.02 Gb final assembly, which equals the estimated genome size. BUSCO analysis using chicken as a model showed that 93.3% of genes were complete. The contig N50 and scaffold N50 sizes were 178 kb and 10.2 Mb, respectively. All these indicate that we obtained a high-quality genome assembly. We annotated 16,485 protein-coding genes and 123.3 Mb (12.05% of the genome) of repetitive sequences by ab initio and homology-based prediction. Furthermore, we applied a RAD-sequencing approach for another 45 individuals of seven representative subspecies in China and identified 4,376,351 novel single nucleotide polymorphism (SNPs) markers. Using this unprecedented data set, we uncovered the geographic population structure and genetic introgression among common pheasants in China. Our results provide the first high-quality reference genome for the common pheasant and a valuable genome-wide SNP database for studying population genomics and demographic history.

Endless forms of sexual selection.

In recent years, the field of sexual selection has exploded, with advances in theoretical and empirical research complementing each other in exciting ways. This perspective piece is the product of a "stock-taking" workshop on sexual selection and sexual conflict. Our aim is to identify and deliberate on outstanding questions and to stimulate discussion rather than provide a comprehensive overview of the entire field. These questions are organized into four thematic sections we deem essential to the field. First we focus on the evolution of mate choice and mating systems. Variation in mate quality can generate both competition and choice in the opposite sex, with implications for the evolution of mating systems. Limitations on mate choice may dictate the importance of direct vs. indirect benefits in mating decisions and consequently, mating systems, especially with regard to polyandry. Second, we focus on how sender and receiver mechanisms shape signal design. Mediation of honest signal content likely depends on integration of temporally variable social and physiological costs that are challenging to measure. We view the neuroethology of sensory and cognitive receiver biases as the main key to signal form and the 'aesthetic sense' proposed by Darwin. Since a receiver bias is sufficient to both initiate and drive ornament or armament exaggeration, without a genetically correlated or even coevolving receiver, this may be the appropriate 'null model' of sexual selection. Thirdly, we focus on the genetic architecture of sexually selected traits. Despite advances in modern molecular techniques, the number and identity of genes underlying performance, display and secondary sexual traits remains largely unknown. In-depth investigations into the genetic basis of sexual dimorphism in the context of long-term field studies will reveal constraints and trajectories of sexually selected trait evolution. Finally, we focus on sexual selection and conflict as drivers of speciation. Population divergence and speciation are often influenced by an interplay between sexual and natural selection. The extent to which sexual selection promotes or counteracts population divergence may vary depending on the genetic architecture of traits as well as the covariance between mating competition and local adaptation. Additionally, post-copulatory processes, such as selection against heterospecific sperm, may influence the importance of sexual selection in speciation. We propose that efforts to resolve these four themes can catalyze conceptual progress in the field of sexual selection, and we offer potential avenues of research to advance this progress.

Genetic basis of amphibian larval development along a latitudinal gradient: Gene diversity, selection and links with phenotypic variation in transcription factor C/EBP-1.

Ectotherm development rates often show adaptive divergence along climatic gradients, but the genetic basis for this variation is rarely studied. Here, we investigated the genetic basis for phenotypic variation in larval development in the moor frog Rana arvalis from five regions along a latitudinal gradient from Germany to northern Sweden. We focused on the C/EBP-1 gene, a transcription factor associated with larval development time. Allele frequencies at C/EBP-1 varied strongly among geographical regions. Overall, the distribution of alleles along the gradient was in concordance with the dual post-glacial colonization routes into Scandinavia, with a large number of alleles exclusively present along the southern colonization route. Only three of 38 alleles were shared between the routes. Analysis of contemporary selection on C/EBP-1 showed divergent selection among the regions, probably reflecting adaptation to the local environmental conditions, although this was especially strong between southern and northern regions coinciding also with lineages from different colonization routes. Overall, the C/EBP-1 gene has historically been under purifying selection, but two specific amino acid positions showed significant signals of positive selection. These positions showed divergence between southern and northern regions, and we suggest that they are functionally involved in the climatic adaptation of larval development. Using phenotypic data from a common garden experiment, we found evidence for specific C/EBP-1 alleles being correlated with larval development time, suggesting a functional role in adaptation of larval development to large-scale climatic variation.