Abstract We describe a chromosome-level, haplotype-resolved genome assembly from a female bluethroat ( Luscinia s. svecica ). The assembly comprises two pseudo-haplotypes of 1461 Mb and 1171 Mb, with 77.4% and 88.4% scaffolded into 40 autosomal chromosomes and the W and Z sex chromosomes (haplotype one). Assembly completeness is high (BUSCO 99.2% and 94.9%), with 22,462 and 18,769 annotated protein-coding genes for haplotypes one and two, respectively. The use of Oxford Nanopore Technologies sequencing enables resolution of genomic regions that are often fragmented in genome assemblies, including the hypervariable Major Histocompatibility Complex (MHC). We find that MHC loci include both the canonical organization of tandemly duplicated MHCIIβ genes with a single MHCIIA, and a distinct arrangement in which MHCI and MHCIIβ loci are interspersed in intermixed arrays, and that substantial structural differences between haplotypes are directly resolved in the assembly.

Tremendous variation in sperm morphology is observed across the animal kingdom. Within avian taxa, the songbirds (infraorder Passerides) have the largest variation in sperm morphology. Their spermatozoa move by using energy generated in the midpiece, which is formed by multiple mitochondria fusing together during spermatogenesis. However, very little is known regarding the number of mitochondria required to form the songbird midpiece. Based on previous research showing an association of midpiece length and mitochondrial DNA (mtDNA) copy number in the zebra finch Taeniopygia guttata, we hypothesize that songbird species with longer sperm midpieces have more copies of mtDNA. We estimated the sperm mtDNA copy number in 19 species from 10 families within Passerides, covering a broad range of midpiece sizes. Mitochondrial and nuclear DNA abundance were determined using droplet digital PCR (ddPCR) and the ratio between mitochondrial and single-copy nuclear genes was used to estimate mtDNA copy number per spermatozoon. We found that species differ in their average mtDNA copy number, but the variation was small and not significantly related to midpiece length. A possible explanation is that mitochondrial genomes are eliminated in the spermatids during spermatogenesis.

Mitochondrial DNA (mtDNA) copy number varies across species, individuals, and cell types. In birds, there are two types of cells with a relatively low number of mitochondria: red blood cells and spermatozoa. Previous studies investigating variation of mitochondrial abundance in animal sperm have generally used quantitative PCR (qPCR), but this method shows potential limitations when quantifying targets at low abundance. To mitigate such issues, we investigated and compared the reliability and accuracy of qPCR, digital PCR (dPCR) and droplet digital PCR (ddPCR) to quantify high and low concentration DNA. We used synthetic DNA targets, to calculate the limit of detection and the limit of quantification and found that with both dPCR and ddPCR, these limits were lower than with qPCR. Then, to compare quantification accuracy and repeatability, we used DNA extracted from blood and sperm cells of Eurasian siskin. We found that qPCR, dPCR and ddPCR all reliably quantified mitochondrial DNA in sperm samples but showed significant differences when analyzing the typically lower levels of mtDNA in blood, with ddPCR consistently showing lower variation among replicates. Our study provides critical insights and recommendations for future studies aiming to quantify target mtDNA and indicates that dPCR and ddPCR are the preferred methods when working with samples with low abundance of mtDNA.

Reproductive barriers limit gene flow and drive population divergence. Sperm morphology plays an important role in reproductive barriers, as successful fertilization depends on how well sperm perform in the female environment. Specifically, sperm length must be adapted to fit the female reproductive tract and storage organs. We investigated sperm length in species and ecotypes of Littorina, a genus of promiscuous marine snails with internal fertilization. Various Littorina species have ecotypes adapted to different shoreline environments where reproductive traits like sperm length divergence might play a role in reinforcing these barriers. Due to their promiscuity, sperm competition likely plays a role in Littorina, and sperm length variation can give insights regarding sperm competition intensities. This study examined eusperm length in four species (L. fabalis, L. littorea, L. obtusata, L. saxatilis) and two different ecotypes in both L. fabalis and L. saxatilis. The ecotypes of both L. fabalis and L. saxatilis differed in eusperm lengths, indicating the potential for this trait to be involved in prezygotic reproductive barriers between ecotypes of these species. Among-species differences in eusperm length variation were observed and may be a result of different sperm competition intensities.

Spermatozoa may provide insights into the evolutionary history, reproductive isolation, and mating systems of species. Here we combine sperm and genomic data to conduct the first comparative analysis of sperm differentiation among Darwin's finches, an iconic adaptive radiation with considerable gene flow across species borders. All eight study species had the typical form of songbird spermatozoa, but shorter than most other species in the Thraupidae family. There was no detectable differentiation in sperm length among four ground finch species (Geospiza) and two tree finch species (Camarhynchus). In both genera, autosomal single nucleotide polymorphisms (SNPs) revealed signatures of genetic admixture. The grey warbler-finch Certhidea fusca and the vegetarian finch Platyspiza crassirostris had significantly shorter sperm than the sister genera Geospiza and Camarhynchus from which they diverged about 0.90 and 0.43 Mya, respectively. The largest intergeneric divergences in sperm length were of the same magnitude as divergences observed within the speciation continuum in other songbirds over similar time spans. Relatively high among-male variation in sperm length indicates a moderate-to-low level of extrapair paternity and a divergence rate in sperm length that is lower than in more promiscuous songbirds. We conclude that sperm size evolution is too slow to drive prezygotic isolation in this radiation.

Sperm morphology varies considerably among species. Sperm traits may contribute to speciation if they diverge fast in allopatry and cause conspecific sperm precedence upon secondary contact. However, their role in driving prezygotic isolation has been poorly investigated. Here we test the hypothesis that, early in the speciation process, female promiscuity promotes a reduction in overlap in sperm length distributions among songbird populations. We assembled a data set of 20 pairs of populations with known sperm length distributions, a published estimate of divergence time, and an index of female promiscuity derived from extrapair paternity rates or relative testis size. We found that sperm length distributions diverged more rapidly in more promiscuous species. Faster divergence between sperm length distributions was caused by the lower variance in the trait in more promiscuous species, and not by faster divergence of the mean sperm lengths. The reduced variance is presumably due to stronger stabilizing selection on sperm length mediated by sperm competition. If divergent sperm length optima in allopatry causes conspecific sperm precedence in sympatry, which remains to be shown empirically, female promiscuity may promote prezygotic isolation, and rapid speciation in songbirds.

Key innovations are fundamental to biological diversification, but their genetic basis is poorly understood. A recent transition from egg-laying to live-bearing in marine snails ( Littorina spp.) provides the opportunity to study the genetic architecture of an innovation that has evolved repeatedly across animals. Individuals do not cluster by reproductive mode in a genome-wide phylogeny, but local genealogical analysis revealed numerous small genomic regions where all live-bearers carry the same core haplotype. Candidate regions show evidence for live-bearer–specific positive selection and are enriched for genes that are differentially expressed between egg-laying and live-bearing reproductive systems. Ages of selective sweeps suggest that live-bearer–specific alleles accumulated over more than 200,000 generations. Our results suggest that new functions evolve through the recruitment of many alleles rather than in a single evolutionary step.

The intertidal gastropod Littorina saxatilis is a model system to study speciation and local adaptation. The repeated occurrence of distinct ecotypes showing different levels of genetic divergence makes L. saxatilis particularly suited to study different stages of the speciation continuum in the same lineage. A major finding is the presence of several large chromosomal inversions associated with the divergence of ecotypes and, specifically, the species offers a system to study the role of inversions in this divergence. The genome of L. saxatilis is 1.35 Gb and composed of 17 chromosomes. The first reference genome of the species was assembled using Illumina data, was highly fragmented (N50 of 44 kb), and was quite incomplete, with a BUSCO completeness of 80.1% on the Metazoan dataset. A linkage map of one full-sibling family enabled the placement of 587 Mbp of the genome into 17 linkage groups corresponding to the haploid number of chromosomes, but the fragmented nature of this reference genome limited the understanding of the interplay between divergent selection and gene flow during ecotype formation. Here, we present a newly generated reference genome that is highly contiguous, with a N50 of 67 Mb and 90.4% of the total assembly length placed in 17 super-scaffolds. It is also highly complete with a BUSCO completeness of 94.1% of the Metazoa dataset. This new reference will allow for investigations into the genomic regions implicated in ecotype formation as well as better characterization of the inversions and their role in speciation.

In species with alternative reproductive tactics, there is much empirical support that parasitically spawning males have larger testes and greater sperm numbers as an evolved response to a higher degree of sperm competition, but support for higher sperm performance (motility, longevity and speed) by such males is inconsistent. We used the sand goby (Pomatoschistus minutus) to test whether sperm performance differed between breeding-coloured males (small testes, large mucus-filled sperm-duct glands; build nests lined with sperm-containing mucus, provide care) and parasitic sneaker-morph males (no breeding colouration, large testes, rudimentary sperm-duct glands; no nest, no care). We compared motility (per cent motile sperm), velocity, longevity of sperm, gene expression of testes and sperm morphometrics between the two morphs. We also tested if sperm-duct gland contents affected sperm performance. We found a clear difference in gene expression of testes between the male morphs with 109 transcripts differentially expressed between the morphs. Notably, several mucin genes were upregulated in breeding-coloured males and two ATP-related genes were upregulated in sneaker-morph males. There was a partial evidence of higher sperm velocity in sneaker-morph males, but no difference in sperm motility. Presence of sperm-duct gland contents significantly increased sperm velocity, and nonsignificantly tended to increase sperm motility, but equally so for the two morphs. The sand goby has remarkably long-lived sperm, with only small or no decline in motility and velocity over time (5 min vs. 22 h), but again, this was equally true for both morphs. Sperm length (head, flagella, total and flagella-to-head ratio) did not differ between morphs and did not correlate with sperm velocity for either morph. Thus, other than a clear difference in testes gene expression, we found only modest differences between the two male morphs, confirming previous findings that increased sperm performance as an adaptation to sperm competition is not a primary target of evolution.

AbstractThe Centre for Marine Evolutionary Biology (CeMEB) at the University of Gothenburg, Sweden, was established in 2008 through a 10‐year research grant of 8.7 m€ to a team of senior researchers. Today, CeMEB members have contributed >500 scientific publications, 30 PhD theses and have organised 75 meetings and courses, including 18 three‐day meetings and four conferences. What are the footprints of CeMEB, and how will the centre continue to play a national and international role as an important node of marine evolutionary research? In this perspective article, we first look back over the 10 years of CeMEB activities and briefly survey some of the many achievements of CeMEB. We furthermore compare the initial goals, as formulated in the grant application, with what has been achieved, and discuss challenges and milestones along the way. Finally, we bring forward some general lessons that can be learnt from a research funding of this type, and we also look ahead, discussing how CeMEB’s achievements and lessons can be used as a springboard to the future of marine evolutionary biology.

Chromosomal inversions have been identified in many natural populations and can be responsible for novel traits and fast adaptation. In zebra finch, an inversion in the Z chromosome has a pleiotropic effect on multiple traits but especially on sperm. Males heterokaryotypic for the inversion have sperm with a longer midpiece and higher velocity. They have higher fertility and siring success than homokaryotypic males. To understand the effect the Z inversion on these traits, we examined young males at different stages of testis development using two homokaryotypic lines and heterokaryotypes. We sampled both testis and liver tissue to identify gene expression differences. In testis, 520 differentially expressed genes were found and most of them were located on chromosome Z. They were functionally enriched for sperm-related traits. In liver, 420 differentially expressed genes were not enriched for any specific functions but showed also an overrepresentation on chromosome Z. In both tissues, this overrepresentation was located near the distal end of Z but also in the middle of the chromosome. For the heterokaryotype, we observed several genes with expression patterns more similar to one or the other karyotype, based on similarity to homokaryotype invidividuals. This was confirmed with SNPs for three genes, and interestingly one gene, dmgdh, had allele-specific expression originating from only one inversion-allele in the testis, yet both inversion alleles were expressed in the liver. This allele-specific difference in tissue-specific expression demonstrates a pleiotropic effect of the inversion and thus suggests a mechanism for divergent phenotypic effects resulting from an inversion.

Species invasions are a global problem of increasing concern, especially in highly connected aquatic environments. Despite this, salinity conditions can pose physiological barriers to their spread, and understanding them is important for management. In Scandinavia's largest cargo port, the invasive round goby (Neogobius melanostomus) is established across a steep salinity gradient. We used 12,937 SNPs to identify the genetic origin and diversity of three sites along the salinity gradient and round goby from western, central and northern Baltic Sea, as well as north European rivers. Fish from two sites from the extreme ends of the gradient were also acclimated to freshwater and seawater, and tested for respiratory and osmoregulatory physiology. Fish from the high-salinity environment in the outer port showed higher genetic diversity, and closer relatedness to the other regions, compared to fish from lower salinity upstream the river. Fish from the high-salinity site also had higher maximum metabolic rate, fewer blood cells and lower blood Ca2+. Despite these genotypic and phenotypic differences, salinity acclimation affected fish from both sites in the same way: seawater increased the blood osmolality and Na+ levels, and freshwater increased the levels of the stress hormone cortisol. Our results show genotypic and phenotypic differences over short spatial scales across this steep salinity gradient. These patterns of the physiologically robust round goby are likely driven by multiple introductions into the high-salinity site, and a process of sorting, likely based on behaviour or selection, along the gradient. This euryhaline fish risks spreading from this area, and seascape genomics and phenotypic characterization can inform management strategies even within an area as small as a coastal harbour inlet.

Conservative flowering behaviours, such as flowering during long days in summer or late flowering at a high leaf number, are often proposed to protect against variable winter and spring temperatures which lead to frost damage if premature flowering occurs. Yet, due the many factors in natural environments relative to the number of individuals compared, assessing which climate characteristics drive these flowering traits has been difficult. We applied a multidisciplinary approach to 10 winter-annual Arabidopsis thaliana populations from a wide climactic gradient in Norway. We used a variable reduction strategy to assess which of 100 climate descriptors from their home sites correlated most to their flowering behaviours when tested for responsiveness to photoperiod after saturation of vernalization; then, assessed sequence variation of 19 known environmental-response flowering genes. Photoperiod responsiveness inversely correlated with interannual variation in timing of growing season onset. Time to flowering appeared driven by growing season length, curtailed by cold fall temperatures. The distribution of FLM, TFL2 and HOS1 haplotypes, genes involved in ambient temperature response, correlated with growing-season climate. We show that long-day responsiveness and late flowering may be driven not by risk of spring frosts, but by growing season temperature and length, perhaps to opportunistically maximize growth.

Key innovations are fundamental to biological diversification, but their genetic architecture is poorly understood. A recent transition from egg-laying to live-bearing in Littorina snails provides the opportunity to study the architecture of an innovation that has evolved repeatedly in animals. Samples do not cluster by reproductive mode in a genome-wide phylogeny, but local genealogical analysis revealed numerous genomic regions where all live-bearers carry the same core haplotype. Associated regions show evidence for live-bearer-specific positive selection, and are enriched for genes that are differentially expressed between egg-laying and live-bearing reproductive systems. Ages of selective sweeps suggest live-bearing alleles accumulated gradually, involving selection at different times in the past. Our results suggest that innovation can have a polygenic basis, and that novel functions can evolve gradually, rather than in a single step.

When populations become geographically isolated, they begin to diverge in various traits and at variable rates. The dynamics of such trait divergences are relevant for understanding evolutionary processes such as local adaptation and speciation. Here we examine divergences in sperm and body structures in a polygynandrous songbird, the alpine accentor (Prunella collaris) between two allopatric high-altitude populations, in Morocco and Spain. The populations diverged around 82,000 years ago, as estimated with a coalescence-based phylogenetic analysis of genome-wide single-nucleotide polymorphisms. We found that birds in the two areas had nonoverlapping sperm lengths, which suggests adaptation to divergent female reproductive tract environments. Sperm length also showed an exceptionally low coefficient of among-male variation, a signal of strong stabilizing selection imposed by sperm competition. The evolutionary rate of sperm length was almost twice the rates for the most divergent morphological traits and more than three times higher than expected from literature data over a similar generational timescale. This rapid evolution of a key reproductive trait has implications for reproductive isolation and ultimately for speciation. Strong selection for different sperm length optima in allopatry predicts conspecific sperm precedence and disruptive selection in sympatry, hence a possible postcopulatory prezygotic barrier to gene flow.

Chromosomal inversions have been identified in many natural populations and can be responsible for novel traits and rapid adaptation. In zebra finch, a large region on the Z chromosome has been subject to multiple inversions, which have pleiotropic effects on multiple traits but especially on sperm phenotypes, such as midpiece and flagellum length. To understand the effect, the Z inversion has on these traits, we examined testis and liver transcriptomes of young males at different maturation times. We compared gene expression differences among three inversion karyotypes: AA, B*B* and AB*, where B* denotes the inverted regions on Z with respect to A. In testis, 794 differentially expressed genes were found and most of them were located on chromosome Z. They were functionally enriched for sperm-related traits. We also identified clusters of co-expressed genes that matched with the inversion-related sperm phenotypes. In liver, there were some enriched functions and some overrepresentation on chromosome Z with similar location as in testis. In both tissues, the overrepresented genes were located near the distal end of Z but also in the middle of the chromosome. For the heterokaryotype, we observed several genes with one allele being dominantly expressed, similar to expression patterns in one or the other homokaryotype. This was confirmed with SNPs for three genes, and interestingly one gene, DMGDH, had allele-specific expression originating mainly from one inversion haplotype in the testis, yet both inversion haplotypes were expressed equally in the liver. This karyotype-specific difference in tissue-specific expression suggests a pleiotropic effect of the inversion and thus suggests a mechanism for divergent phenotypic effects resulting from an inversion.

The objective of this study was to fine map the quantitative trait loci region of chromosome 27 which was previously reported as a major contributor of total genetic variation for resistance against cardiomyopathy syndrome in Atlantic salmon. The fine mapping using whole genome resequencing of individuals increased resolution of the targeted genomic region (Ssa27:10-12 Mbps), resulting in uncovering the two immune related genes (HA1F and MR1). Transcriptome analysis revealed only two differentially expressed genes, MR1 and HA1F across resistant vs susceptible individuals. The gene MR1 was highly expressed in the susceptible group with log2 fold change of 6.405 compared to resistant individuals. Contrary to MR1 expression, HA1F gene was lowly expressed (log2 fold change of 2.099) in susceptible group. Moreover, the proteomics results partially validated the results of transcriptomics with the product of MR1 being differentially produced across resistant vs susceptible individuals.

Understanding the effects of interspecific competition on genetic diversity will deepen our knowledge on species evolution. In the case of Calopteryx splendens and C. virgo, sympatric damselfly species, interspecific interference competition by C. virgo has remarkable effects on territoriality of C. splendens resulting in reproductive character displacement. Since territoriality is correlated with phenotype and mating success, we investigated the effects of interspecific interference competition on genetic diversity of C. splendens populations. Using amplified fragment length polymorphisms (AFLP), we determined the population genetic structure of 12 C. splendens populations and used the genetic diversity information to relate heterozygosity of C. splendens to abundance of C. virgo in sympatric populations. We found that heterozygosity of C. splendens males decreased with increasing abundance of C. virgo males. This result most likely reflects changes in effective population size due to interspecific interference competition and shows an effect on genetic structure in damselfly populations.

For externally fertilising organisms in the aquatic environment, the abiotic fertilisation medium can be a strong selecting force. Among bony fishes, sperm are adapted to function in a narrow salinity range. A notable exception is the family Gobiidae, where several species reproduce across a wide salinity range. The family also contains several wide-spread invasive species. To better understand how these fishes tolerate such varying conditions, we measured sperm performance in relation to salinity from a freshwater and a brackish population within their ancestral Ponto-Caspian region of the round goby, Neogobius melanostomus. These two ancestral populations were then compared to nine additional invaded sites across northern Europe, both in terms of their sperm traits and by using genomic SNP markers. Our results show clear patterns of ancestral adaptations to freshwater and brackish salinities in their sperm performance. Population genomic analyses show that the ancestral ecotypes have generally established themselves in environments that fit their sperm adaptations. Sites close to ports with intense shipping show that both outbreeding and admixture can affect the sperm performance of a population in a given salinity. Rapid adaptation to local conditions is also supported at some sites. Historical and contemporary evolution in the traits of the round goby sperm cells is tightly linked to the population and seascape genomics as well as biogeographic processes in these invasive fishes. Since the risk of a population establishing in an area is related to the genotype by environment match, port connectivity and the ancestry of the round goby population can likely be useful for predicting the species spread.

In externally fertilizing species the gametes of both males and females are exposed to the influences of the environment into which they are released. Sperm are sensitive to abiotic factors such as salinity, but they are also affected by biotic factors such as sperm competition. Here we compared the performance of sperm of three goby species, the painted goby, Pomatoschistus pictus, the two-spotted goby, P. flavescens and the sand goby, P. minutus. These species differ in their distributions, with painted goby having the narrowest salinity range and sand goby the widest. Moreover, data from paternity shows the two-spotted goby experiences the least sperm competition while in the sand goby sperm competition is ubiquitous. We took sperm samples from dissected males and exposed them to high salinity water (31 PSU) representing the North Sea and low salinity water (6 PSU) representing the brackish Baltic Sea Proper. We then used computer assisted sperm analysis (CASA) to measure the proportion of motile sperm and sperm swimming speed 10 min and 20 hrs after sperm activation. We found that sperm performance depended on salinity but there seemed to be no relationship to the species' geographical distribution in relation to salinity range. The species differed in the proportion of motile sperm but there was no significant decrease in sperm motility during 20 hrs. The sand goby was the only species with motile sperm after 72 hrs. This article is protected by copyright. All rights reserved.