Absence of phenotypic differentiation can challenge the study of diversification by concealing evolutionary divergence through morphological similarity. Such cryptic species diversity is found particularly often in extreme environments, including arid mountain ranges. In the Hajar Mountains of southeastern Arabia, the Pristurus rupestris species complex comprises divergent lineages with notable phenotypic variation. Here, we investigate the evolutionary dynamics of this radiation by integrating geometric morphometric analyses with whole-genome sequencing and phylogenomic reconstructions. We analyzed five species-P. ali, P. assareen, P. feulneri, P. omanensis, and P. rupestris-including all previously identified genetic lineages within those species, and generated a new reference genome for an Old World sphaerodactylid gecko (P. rupestris sensu stricto). Morphometric analyses of comprehensive two- and three-dimensional morphological datasets indicate strong overlap in both body and head shape. Despite considerable phenotypic similarities, our results reveal deep interspecific divergences exceeding 16 million years and contrasting demographic histories. While we detected introgression restricted to specific lineage pairs, phenotypic crypticity persists across the entire complex, suggesting stabilizing selection in harsh environments. Our study highlights the P. rupestris species complex as an ideal model for investigating the role of environmental constraints in limiting morphological divergence and shaping diversification dynamics in arid mountain ecosystems.

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.

BACKGROUND:With over 10,000 recognized species, birds constitute one of the most diverse and widely distributed vertebrate groups. Although avian genomics has advanced rapidly over the past decade, substantial gaps remain across the global avifauna. Filling these gaps is essential for understanding macroevolutionary patterns, population structure, and the molecular basis of ecological and behavioral diversity. Worldwide museum collections represent invaluable resources for filling these gaps, yet the typically degraded DNA and limited quantities from historical specimens have posed significant challenges for generating high-quality genome assemblies. RESULTS:Here, the Bird Genome 10 K Project adopted low-input sequencing strategies that reduce costs while improving assembly quality compared with earlier order- and family-level genomes. Using mainly stLFR, complemented by 10X Genomics and standard next-generation sequencing, we assembled 177 avian genomes from museum specimens and tissue collections representing 161 genera, including 102 newly sequenced at the genomic level. The assemblies average ∼1.2 Gb in size, with scaffold N50 = 8.03 Mb, contig N50 = 120 kb, 93% BUSCO completeness, and Merqury Quality Value score of 56. CONCLUSIONS:These genomes greatly expand avian taxonomic coverage and demonstrate the efficiency of low-input sequencing for generating high-quality assemblies from limited and often degraded material sourced from museum specimens. This resource provides a foundation for comparative genomics, conservation genetics, and evolutionary studies across the avian tree of life.

We present a chromosome-level genome assembly of a female Norwegian white-throated dipper ( Cinclus cinclus cinclus ) generated using Oxford Nanopore Technologies (ONT) long reads and Hi-C scaffolding. The assembly comprises two pseudo-haplotypes, hap1 (1186 Mb) and hap2 (1115 Mb), with 96.7% and 94.4% of sequences assigned to chromosome-scale scaffolds, respectively. Both pseudo-haplotypes contain 40 autosomes, with the Z and W sex chromosomes assigned to hap1. Compared with the PacBio HiFi-based C. c. gularis reference assembly bCinCin1.1.pri, which contains 38 autosomes, sequence represented as a single dot-chromosome (chr 36) is resolved into three distinct dot-chromosomes (chr 36, 39, and 40), a configuration supported by Hi-C contact patterns. BUSCO completeness was high for hap1 (99.2%) and hap2 (95.0%), with 19,003 and 17,746 predicted protein-coding genes, respectively. Compared with the HiFi-based C. c. gularis reference and HiFi-based assemblies generated from the same individual, the ONT-derived assemblies were substantially less fragmented and recovered more sequence from the smallest chromosomes. Synteny was otherwise largely conserved between subspecies. HiFi depletion increased strongly from macrochromosomes to micro- and dot-chromosomes, and HiFi-depleted regions were enriched for repeats and predicted non-B-DNA-associated features, particularly G-quadruplexes and direct repeats, whereas ONT coverage remained comparatively stable. These results show that conventional genome-wide assembly metrics can obscure substantial differences in the recovery of repeat-rich avian dot-chromosomes and highlight the value of chromosome-aware evaluation and ONT sequencing for recovering these regions.

Reference genome assemblies are essential infrastructure for investigating phylogeny and population/conservation genetics of wild organisms. Birds serve as model vertebrates in ecology and evolutionary biology due to their well-documented natural histories and extensive community science data. We release a set of 350 newly assembled avian genomes, which, when combined with 97 previously published genomes, represent 447 of the bird species recorded in Denmark, the Faroe Islands, and Greenland-the largest regional dataset of a vertebrate group to date. These genomes are published for various research activities. This data release advances the global effort to build comprehensive and accessible biodiversity genomic resources for the research community.

Abstract We describe a chromosome-level, pseudo-haplotype-resolved genome assembly from a female bluethroat (Luscinia s. svecica). The assembly comprises two pseudo-haplotypes of 1,461 and 1,171 Mb, with 77.4% and 88.4% scaffolded into 40 autosomal chromosomes and the W and Z sex chromosomes in pseudo-haplotype one. Assembly completeness is high (BUSCO 99.2% and 94.9%), with 22,462 and 18,769 annotated protein-coding genes for pseudo-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. We find that MHC loci include both the canonical organization of tandemly duplicated MHCIIβ genes with a single MHCIIα, and a distinct arrangement in which MHCI and MHCIIβ loci are interspersed. Substantial structural differences between the two assembled chromosome 35 homologs are resolved across the major histocompatibility complex region.

Genetic research is unevenly distributed across the globe, with most research done in temperate zones. To better understand the birdlife in an under-represented, arid subtropical country, Qatar, we blood sampled birds and salvaged tissue from dead birds, then sequenced a mitochondrial marker (cytochrome c oxidase subunit I, COI, the "DNA barcoding" gene). We chose the DNA barcoding gene because it has previously proven useful for preliminary explorations of evolutionary history. We obtained DNA barcodes for 115 birds (34 species, 12 orders). Our data suggest that the existing DNA barcode reference library, built largely from sampling in the Americas, Europe, and east Asia, is generally sufficient for species identification in Qatar. Based on DNA barcode similarity, Qatar provides overwintering habitat to some species with apparent strong migratory connectivity and others with weaker migratory connectivity. Among locally breeding species, we found no evidence of hybridization between House Sparrows Passer domesticus (n = 16 males) and Spanish Sparrows P. hispaniolensis (n = 14 males), breeding simultaneously and in the same habitats, although in other locations of range overlap, habitat segregation and timing of breeding are hypothesized to be the primary reproductive barrier between them. Our results highlight the benefits of expanding the geographic range of genetic and ecological research.

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.

Identifying inconspicuous hybrids in the wild can be challenging and might cause a low rate of detectability for phenotypically similar species. Here, we report on a record of a hybrid between Marsh Tit (Poecile palustris) and Willow Tit (P. montanus) from Southern Norway detected using photographs, sound recordings and molecular analyses. This integrative approach allowed the sex determination of the hybrid individual and the identification of both parental species. Our record represents the first genetically confirmed evidence of hybridization between these two species in the wild.

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.

The health and fitness of vertebrates are constantly challenged by environmental stresses, which include exposure to diseases that pose significant evolutionary pressures on immune genes. The bluethroat ( Luscinia svecica ), has been extensively studied for its haemosporidian parasite diversity across Europe showing a high diversity and prevalence rate. Given possible evolutionary pressure exerted by these pathogens, the present study used high-throughput amplicon sequencing to explore the genetic diversity and selection mechanisms of three immunity genes (MHC I exon 3, TLR3 and TLR4) in three European bluethroat populations. Specifically, we seek to (1) characterize the genetic diversity of these immunity genes, (2) detect selection signatures shaping their diversity, and (3) investigate the relationship between these immunity genes and haemosporidian parasites ( Plasmodium and Leucocytozoon ). Selection analysis was conducted using FUBAR and SLAC methods, whereas GLM regression was employed to explore the correlation between MHC genes and haemosporidian parasites. Despite geographical and ecological differences, nucleotide and haplotype diversities were similar across all populations. Most frequent haplotypes were found to be shared by both red-spotted ( L. s. svecica ) and white-spotted ( L. s. cyanecula ) bluethroat subspecies. The “insular” Krkonoše population, despite being biogeographically peripheral and experiencing minor inbreeding, did not show significantly reduced immunogenetic diversity. Selection analysis revealed a higher presence of purifying selection in TLR genes and a combination of purifying and diversifying selection in the MHC gene, reflecting their evolutionary constraints and functional importance, while haemosporidian parasite pressure was not a major driver of genetic diversity. ### Competing Interest Statement The authors have declared no competing interest.

There is a need for baseline information about how much plastics are ingested by wildlife and potential negative consequences thereof. We analysed the frequency of occurrence (FO) of plastics >1 mm in the stomachs of five pursuit-diving seabird species collected opportunistically. Atlantic puffins (Fratercula arctica) found emaciated on beaches in SW Norway had the highest FO of plastics (58.8 %), followed by emaciated common guillemots (Uria aalge; 9.1 %) also found beached in either SW or SE Norway. No plastics were detected in razorbills (Alca torda), great cormorants (Phalacrocorax carbo), and European shags (Gulosus aristotelis) taken as bycatch in northern Norway. This is the first study to report on plastic ingestion of these five species in northern Europe, and it highlights both the usefulness and limitations of opportunistic sampling. Small sample sizes, as well as an unbalanced sample design, complicated the interpretation of the results.

Mitochondrial DNA copy number varies across species, individuals and cell types. Two avian cell types carrying a relatively low number of mitochondria are the red blood cells and spermatozoa. While previous studies investigating variation of mitochondrial abundance in animal sperm have generally used quantitative PCR (qPCR), this method shows potential limitations when quantifying low abundant targets. 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. Using synthetic DNA, we found that both dPCR and ddPCR displayed lower Limit of Detection and Limit of Quantification than qPCR. Using DNA extracted from blood and sperm cells of Eurasian Siskin, we found that qPCR, dPCR and ddPCR reliably quantified mitochondrial DNA in sperm samples, but showed significant differences when analyzing typically lower levels of mtDNA in blood. We found that ddPCR consistently showed lower variation among replicates. These analyses provide critical insights and recommendations for future studies aiming to quantify target mtDNA. Our study indicates that dPCR and ddPCR are the preferred methods when working with samples with low abundance of mtDNA. ### Competing Interest Statement The authors have declared no competing interest.

Monitoring haemosporidian parasites in birds is essential to comprehend the dynamics of avian malaria, a disease that significantly affects bird populations worldwide. This study concentrated on the prevalence and diversity of haemosporidian parasites in 198 specimens from two subspecies of the Eurasian bluethroat (Luscinia svecica), aiming to explore the genetic diversity and species richness of haemosporidian fauna across the host populations. By utilizing next-generation amplicon high-throughput sequencing (NGS), we observed a marked increase in the detection of haemosporidian diversity, revealing cryptic variants and species previously unidentified by Sanger sequencing. A high prevalence of Plasmodium was seen in all studied sites, accompanied by a less frequent Leucocytozoon infection in the red-spotted subspecies and minimal occurrence of Haemoproteus. Both previously known and new, low prevalence cryptic variants were detected, underscoring the complexity of haemosporidian infections in avian hosts. The use of species delimitation tools provided a detailed understanding of haemosporidian species diversity, their coexistence within hosts, and their phylogenetic relationships. Despite the varying ecological characteristics of the study sites, no significant difference in haemosporidian alpha diversity among populations was found. However, significant differences in beta diversity were identified, suggesting that habitat characteristics and geographic distance influence parasite distribution. These findings highlight the importance of advanced molecular techniques in revealing the hidden diversity of parasites, offering valuable insights into the ecology and evolution of haemosporidian infections. Given the threatened status of one of the host’s populations, knowledge on local diversity of haemosporidian parasites also has implications for possible conservation strategies.

Small and isolated populations are at risk of local extinction, either due to the reduction of genetic diversity or due to stochastic events. We assessed genetic diversity in populations of the red-spotted (Luscinia svecica svecica) and white-spotted bluethroat (L. s. cyanecula) subspecies from six European sites, focusing on a peripheral and declining, red-spotted population from Central Europe (Krkono & scaron;e Mountains, Czech Republic). Analysis of population structure using mitochondrial sequences (cytochrome b) and ddRAD genomic data revealed that the two subspecies form clearly defined clusters, but traces of possible admixture were found in three populations of the white-spotted bluethroat. Demographic history reconstruction indicates past population range expansion in both subspecies, while the occurrence of short (0.4 Mbp) runs of homozygosity (ROH) segments suggests possible inbreeding 50 to 200 generations ago. Interestingly, although established by a small number of individuals, the Krkono & scaron;e population show reduced genetic diversity in only one measure of summary statistics, possibly due to sustained gene flow. Correspondingly, nine highly homozygous genes were recovered within a 2.6-Mbp long ROH region on the Chr 5. Thus, we found only a small reduction of genetic diversity in a population facing extinction, contrary to the expectations for a geographically distant and small population.

Cover photo: Adult Great Cormorant of the subspecies Phalacrocorax carbo sinensis in breeding plumage. Photo: Frode Falkenberg. Great Cormorants Phalacrocorax carbo are now regularly seen in inland watercourses in southeast Norway, after the P. c. sinensis subspecies first established breeding colonies in coastal south Norway in 1996. Although both the sinensis and carbo subspecies occur, the ratio between them is unknown. We tested the accuracy of subspecific identification based on biometrics and genetic analyses in 75 Great Cormorants that drowned in fishing nets in a lake in southeast Norway during the period 2009-2020. Primarily based on the gular pouch angle (GPA), we classified 40 individuals to the carbo subspecies and 35 individuals to the sinensis subspecies. Eight of the carbo individuals and 14 of the sinensis individuals were within the overlapping range of GPA and were therefore classified to subspecies by supplementary measurements (bill depth minimum and bill length). Genetic analyses were based on seven polymorphic microsatellite markers. Assuming one panmictic population, we performed Structure analyses to separate the genotypes into two assumed genetic clusters. The two clusters, carbo and sinensis, could only be separated when adding information about morphological subspecies identities for the carbo, sinensis and carbo/sinensis groups. The sinensis and the carbo/sinensis groups belonged almost entirely to one genetic cluster, whereas the carbo group consisted of individuals with varying proportions of mixed ancestry. Furthermore, we found significant genetic differentiation between the carbo and sinensis groups, and between the carbo and the carbo/sinensis groups, but no significant differentiation between the sinensis and the carbo/sinensis groups. Our results suggest that gene flow is more common from sinensis into carbo than vice-versa, and that the use of GPA < 73° has limitations for the identification of Great Cormorant subspecies in areas where both forms occur. We conclude that the numbers of carbo vs. sinensis individuals in our sample were 32 and 43, respectively.

Individual heterozygosity may influence the expression of fitness‐related traits, via genome‐wide or local genetic effects. Earlier studies have shown negative relationships between heterozygosity and sperm variation, predominantly in captive, highly inbred populations. Little is known about the possible influence of variation in heterozygosity on sperm traits in wild, outbred populations. We studied two populations of the bluethroat, one from the widely distributed northern subspecies Luscinia. s. svecica and the other from the smaller, more patchily distributed subspecies breeding along the French coast of Brittany L. s. namnetum. The two subspecies differed significantly in body size, plumage colour, sperm traits and the degree of genetic diversity. However, there was no evidence that sperm traits (total length and motility) were influenced by the degree of heterozygosity at the individual level. In contrast, we found that male body size was positively related to heterozygosity across both populations, indicating a possible relationship between overall genetic diversity and general vigour or ability to obtain food. We conclude that sperm traits are unrelated to levels of heterozygosity in the studied outbred and weakly genetically depauperate bluethroat populations.

Genes of the major histocompatibility complex (MHC) are crucial for adaptive immunity in jawed vertebrates, and theory predicts that there should be mate choice for optimizing MHC constitution in the offspring. In a previous study, we demonstrated a non-random female choice of extra-pair males in the bluethroat ( Luscinia svecica ), yielding offspring that was closer to an intermediate MHC class II (MHCII) allele count than their within-pair halfsiblings. The present study tests whether social pairs with only within-pair young (WPY) in their brood, in the same study population, had a combined MHC-constitution closer to a presumed intermediate optimum, than social pairs with extra-pair young (EPY), with a corresponding pattern in their offspring. As expected, we found that WPY from pure WPY-broods were more MHC-optimal than WPY from mixed broods, but only in broods of young (second year) males. Correspondingly, there was a tendency for social pairs with only WPY in their brood to be more MHC-compatible than social pairs with EPY in their brood, when the male was young. Older bluethroat males have considerably larger testes than young males, and their higher sperm competitiveness could help them secure paternity in their own brood, also when they are not MHC-compatible. In other words, in the sexual conflict over paternity, females may be more likely to realise their preference for a MHC-compatible mate when paired to a young male. As a possible fitness indicator, immune responsiveness to an injected antigen (PHA) was elevated for offspring closer to “the golden mean” in MHCII allele count. Significance statement This study contributes to our understanding of MHC-based mate choice in extra-pair mating systems, by showing that female bluethroats ( Luscinia svecica ) with an MHCII-compatible social mate tend to have no extra-pair young in their brood, but only when the social male is young. This elucidates a possible sexual conflict, in which older social males are able to override female preferences and prevent other males from gaining paternity in their brood through higher sperm production. Studying systems in which extra-pair paternity occurs offers an insight into the genetic benefits of mate choice, as extra-pair males, in contrast to social males, generally contribute only sperm. Further, the strict and thorough genotyping scheme applied in this study enabled us to demonstrate a preference for “the golden mean” in MHC-diversity in a species with one of the highest MHC class II-diversity known to date.

In the NHMO zoological collections, specimens from the Arctic include about 9,000 mammals and 7,100 birds, whereas the Insect Collection holds about 105,000 specimens plus more than hundred jars with unsorted material. The Fish Collection contains approximately 1,400 specimens, while the Herptile Collection (amphibians & reptiles) holds only thirty-one specimens of three taxa. Many of these specimens originate from expeditions to E Greenland, N Canada, Svalbard, Novaya Zemlya, Finnmark, and NE Siberia in the period 1898 to 1966. Furthermore, the DNA Bank has about 5,600 tissue and extracted DNA samples, mostly sampled from wild animals during the last decades but also from specimens in the voucher collections. Most of the Arctic specimens have been digitized and are available in online data portals like GBIF, except for the Insect Collection, where only the type material and about 30 percent of the total specimens are digitized.

Sperm cells are variable both within and among species. To be able to accurately measure sperm cells and understand their function, it is important that sperm cells are preserved in a manner that maintains their structural integrity. Formalin is a widely used fixative and storage medium for sperm cells, but few studies have examined the effect of fixation and long-term storage on their morphological integrity. Ethanol is also a common fixation and storage agent for tissue samples, and here we examine if fixation and storage in formalin or ethanol alters sperm cell size and structural integrity. We found no significant effects of the fixation process on fresh sperm cells fixed in formalin or ethanol. Further, there were no consistent length changes in sperm cells stored in formalin or ethanol over a period of 227 days, or in sperm cells stored in formalin for three years. A comparison across 13–14 years of storage time showed a small but significant reduction in sperm cell length of 0.93%. Furthermore, sperm cells initially fixed in formalin remained quite stable in dry storage on glass slides for a minimum of six months (we found a mean reduction in sperm cell length of 0.18% after 6 months). The proportion of sperm cells with head damage was, however, much higher for samples stored in ethanol than for those stored in formalin. Overall, 70% of sperm cells had acrosome damage in ethanol versus only 3% in formalin. Finding intact sperm cells for measuring length, therefore, required greater effort in ethanol samples than in formalin samples. Our findings indicate that use of sperm cells from long-term storage for the study of sperm morphometrics is justified for either fixative, although formalin clearly preserves the sperm cells better.