The de-extinction of species using genome-editing approaches depends on acquiring high-quality genomic information from the extinct target. However, the degraded nature of the ancient DNA (aDNA) that is typical for most extinct species, poses significant challenges to achieving comprehensive genome reconstruction. A systematic evaluation of the minimum sequencing effort that is required to reliably map the genome under varying DNA quality conditions to different reference genome remains lacking across different extinct species. Here, we systematically assess the impact of sequencing depth on genome coverage, heterozygosity estimation, and variant calling accuracy, when mapping both true aDNA data generated from the extinct Christmas Island rat (Rattus macleari), as well as in silico simulated modern- and ancient-like data generated from a modern relation (the brown rat, Rattus norvegicus), to the black rat (Rattus rattus) reference genomes. Our results demonstrate that even sequencing depths of 100× fail to yield stable heterozygosity estimates, and leave approximately 3.38% to 4.03% of its genome uncovered. These uncovered regions contained functionally relevant SNPs and indels, highlighting the limitations of reconstructing extinct genomes using reference sequences from extant relatives. Furthermore, simulations using computationally generated "degraded haploid and diploid" data based on the high-quality brown rat genome, revealed that false-positive SNPs primarily arise from insufficient coverage and low data quality, rather than aDNA damage (e.g. miscoding lesions, size of fragments, etc.) per se. These findings underscore the need to tailor sequencing depth standards by considering sample type, degradation level, and sequencing error profiles. This study provides a theoretical framework and methodological support for optimizing data strategies in aDNA research, and ultimately informing de-extinction efforts.

Global change is reshaping the distribution of biodiversity and the functioning of ecosystems. Predicting the long-term consequences of such changes remains a challenge due to a need for a clear understanding of the mechanisms underpinning ecosystem-level responses, as well as the role of geographical and environmental contingencies. We propose that these gaps can be addressed for freshwater ecosystems using a globally distributed experiment with standardized observations and disturbances. Specifically, this paper outlines the structure of PondNet - a globally distributed network of pond mesocosm experiments - designed to investigate how aquatic food webs respond to environmental change across broad geographical gradients. Pond mesocosms are affordable, low maintenance and easily replicated model ecosystems, with broadly predictable trophic architectures and community size-structure. PondNet would implement state-of-the-art environmental DNA biodiversity assessments for standardized taxonomic identification across biogeographical regions. A major operational bottleneck for developing a global understanding of environmental change effects on ecosystem functioning is the current lack of standardized experiments across coordinated infrastructures. We propose that by building on existing distributed experiments, we can assemble a modular participation scheme that ensures broad biogeographical coverage whilst accounting for varying levels of resource commitments from local hosts. PondNet aims to answer two overarching questions: 1) how general are community, food web and ecosystem-level responses to climate change across scales (i.e. local environmental gradients to biogeographical regions)?, and 2) to what extent are such responses contingent on local climate, environment, and regional species pools? PondNet will contribute to developing predictive models that can be adaptively improved from testing with data from globally replicated experiments and monitoring programmes.

In a world with an increase in human population, food consumption, and the generation of organic waste, insects are emerging as a promising tool to convert organic waste material into human food or animal feed. The insect microbiome is known to play a key role in the degradation of organic substrates, but little is known about the metabolic potential of the microbiome of industrially reared fly larvae. We investigated the microbial composition and metabolic potential of the house fly (Musca domestica) larva gut microbiome from larvae grown on three different waste and by-product-based substrates. We found that bacteria associated with the larval gut were enriched for functions related to microbial stress mechanisms, indicating strong selection of the gut microbiome by house fly larvae. In addition, the gut microbiome of larvae reared on sludge-based substrate had higher diversity when weighting for rare species and a higher coverage of "carbohydrate transport and metabolism" genes compared to brewery by-product-based substrate. A positive correlation between coverage of "pyridoxal-P synthesis" and larval survival and substrate conversion efficiency suggests that microbial synthesis of vitamin B6 could enhance larval performance. Additionally, a negative correlation between coverage of the "Entner-Doudoroff pathway" and "homoprotocatechuate degradation" and substrate conversion indicates microbial competition for sugars and aromatic amino acids. Together, these results reveal how the host selects on gut microbiomes with metabolic potential that is optimized toward the conversion of substrates that may be ultimately valuable for commercial insect production. IMPORTANCE:Fly larvae are expected to play an important role in future food and feed production through the conversion of low-value biomass into high-quality protein. The gut microorganisms of fly larvae are expected to play an important role in bioconversion and could potentially be manipulated to improve biomass conversion. In this study, the importance of the gut bacteria of house fly larvae for bioconversion was investigated by metagenomic sequencing, which provided information on the bacterial abundance and potential functional roles in the larval gut. The results reveal that the functional potential of gut bacteria is affected by larval feed and correlates with larval performance, highlighting the importance of the gut microbiome for efficient biomass conversion.

Hummingbirds (Trochilidae) represent the second largest avian family, with ~356 species occupying diverse habitats across the Neotropical and Nearctic regions. Their extensive diversification includes notable adaptations to extreme environments, with nearly one-third of extant taxa belonging to the predominantly high-altitude Coquettes and Brilliants. Although recent work proposed a monophyletic Andean clade uniting these groups, consistent with rapid radiation during Andean orogeny, we find no support for this relationship. Using 2,949 nuclear loci sampled from 47 species spanning all nine major hummingbird clades, we recover a different evolutionary pattern: a stepwise sequence of diversification in which Brilliants are sister to a broader assemblage comprising Coquettes, the genus Patagona, Emeralds, Mountain Gems, and the recently diverged Bees. By assembling complete mitochondrial genomes, we additionally detect significant discordance between nuclear topologies and those derived from the mitogenome and Z chromosome. Analyses of gene-tree heterogeneity show that incomplete lineage sorting is pervasive across the phylogeny, with particularly strong impacts on branches associated with Andean diversification. Divergence-time estimation further indicates that the major Andean radiation (including Brilliants, Coquettes, Emeralds, Bees, and Mountain Gems) originated around ~14 Ma, with the three younger clades diversifying ~12 Ma, coinciding with both the mid Miocene Andean uplift and the mid-Miocene Climate Transition that increased habitat heterogeneity and likely promoted rapid speciation. To support future phylogenomic efforts, we identify a reduced set of highly informative, independent protein-coding loci and present a near-complete species-level phylogeny constrained by our autosomal backbone. Our findings highlight the importance of integrating loci with distinct inheritance modes to detect and interpret phylogenetic incongruence in rapid radiations. ### Competing Interest Statement The authors have declared no competing interest. Villum Fonden, https://ror.org/05nqkay65, 25925, VKR023446 Carlsberg Foundation, 2011\_01\_0578 Lundbeck Foundation, https://ror.org/03hz8wd80, R52-5062 Danish National Research Foundation, DNRF96 Fundação para a Ciência e Tecnologia, https://ror.org/00snfqn58, SFRH/ BPD/70654/2010 Alexander Wetmore fund Smoketree Trust Natural Sciences and Engineering Research Council of Canada, RGPIN-2018-06747 Independent Research Fund Denmark, 10.46540/5244-00031B U.S. National Science Foundation, 1513629

Conservation can prevent species extinction via demographic recovery, yet it remains debated whether this translates into genomic recovery and restored fitness. The Mauritius kestrel ( Falco punctatus ) declined to four known wild birds in 1974 before intensive management recovered the population. Using 130 genomes spanning nearly 200 years, lifetime reproductive success data, and simulations, we reconstructed genomic change across the species' collapse and recovery. Long-term small population size had already removed some harmful variation before the crash, a process expected to buffer populations from severe inbreeding depression. Yet the recent bottleneck sharply increased inbreeding, exposed additional harmful variants, and left a signature of genomic erosion associated with reduced reproductive success. The long conservation history of the Mauritius kestrel shows how population collapse and recovery can leave a compounding genetic threat, in which partial genetic purging, continuing genomic erosion, and conservation dependence unfold together in rescued species.

The second plague pandemic (early 14th-early 19th centuries), which was caused by Yersinia pestis, had a profound demographic, socio-economic and cultural impact across Eurasia and North Africa. Many regions in Europe and the Middle East are estimated to have lost 40-60% of their human populations, with some areas suffering even higher mortality. Whether exposure to Y. pestis drove strong positive selection on protective genetic variants in the human genome, and how it shaped migration patterns, remains debated, despite several recent studies based on ancient DNA. Here, we analyse a markedly larger, higher coverage, and geographically diverse dataset based on shotgun sequencing of genomes from 529 ancient individuals to a mean depth 8.8x dating to either before or after the arrival of the pandemic at three sites in northern Europe: Trondheim (Norway), Lund (Sweden) and Vilnius (Lithuania). Genome-wide scans for signatures of selection provide no evidence for strong positive selection acting on specific genetic variants driven by Y. pestis exposure: we neither replicate selection signatures reported by previous studies nor identify new genome-wide significant candidates. However, for all three sites, we observe evidence for a reduction in long-range immigration, indicated by a drop in the diversity of ancestry that followed the arrival of Y. pestis and broadly coincided with the end of the Viking Age, Christianisation and the onset of the Little Ice Age. Our results shed important light on the demographic impact of major sociohistorical changes that occurred during the late Medieval period in Scandinavia and the Baltic region and link Christianisation to increased diversity in ancestry before the pandemic.

Dire wolves (Aenocyon dirus) are extinct predators of Pleistocene North America. Although phenotypically similar to living wolves (Canis lupus), dire wolves have yet to be placed confidently in the canid family tree. We generated 3.4× and 12.8× paleogenomes from two well-preserved dire wolves dating to >13,000 and >72,000 years ago and estimated consensus species trees for these and 10 canid species. Our results revealed that ∼2/3 of dire wolf ancestry is most likely derived from a lineage sister to the clade comprising the gray wolf, coyote, and dhole and the remaining ∼1/3 from a lineage near the base of Canini diversity. Our results underscore the power of paleogenomes to resolve long-standing taxonomic questions and contribute to growing evidence of the role of post-speciation gene flow as an evolutionary force.

Sticklebacks (Gasterosteidae) encompass model organisms which are of particular interest for evolutionary and ecological genomics. Within Gasterosteidae, chromosome number is variable (2n=40-46) and independent fusions of homologous chromosomes have been proposed. The sea stickleback (or fifteen-spined stickleback, Spinachia spinachia ) has the lowest known number of chromosomes (2n=40) and hence is crucial in understanding chromosome evolution among sticklebacks, but is so far missing in genomic datasets. Here, we present a high-quality diploid genome assembly of S. spinachia . PacBio HiFi and Hi-C reads were assembled into a genome of 407.5 Mb in size, consisting of 20 chromosomes, with an N50 of 6.6 Mb and 98.96% complete single-copy BUSCO genes. A phylogenetic tree inferred across five stickleback species and four outgroup genomes from 19,156 genes, alongside synteny analyses and ancestral chromosome reconstructions, confirms S. spinachia as the sister species to the four-spined stickleback ( Apeltes quadracus ) and not as the sister group to all other sticklebacks as once thought. It has one species-specific chromosome fusion and shares two fusions with the three-spined stickleback ( Gasterosteus aculeatus ), none of which are present in its sister species. One of these fusions is also present in Pungitius , leading to reinterpretion of this fusion as ancestral to Gasterosteidae, with subsequent fission in Apeltes . This implies a lower ancestral chromosome number in Gasterosteidae (2n=44) than previously thought. The other fusion shared with G. aculeatus presents a case of convergence. Our results suggest that karyotype evolution in Gasterosteidae has been shaped by ancestral chromosome fusion, convergent fusion, and secondary fission.

Abstract The extinct giant deer ( Megaloceros giganteus ) was one of the most striking megafaunal species of the Late Quaternary, distinguished by its enormous palmated antlers reaching up to 3.5 m across, the largest known among both living and extinct cervids. Despite its iconic status, little is known about its genomic history prior to extinction ∼8 thousand years ago (kya). We generated the first nuclear palaeogenomes for Megaloceros , represented by nine individuals from Germany (∼40 kya) and Ireland (∼11 kya), mapped to a new chromosome-level reference genome of the fallow deer ( Dama dama ). Phylogenomic analyses placed Megaloceros as sister to Dama (divergence ∼3.5 Ma) and revealed evidence of gene flow with ancestral Cervus lineages. Population analyses identified clear differentiation between German and Irish lineages, with higher genetic diversity in the German individuals. Two genes under strong positive selection, BNIPL and SLC10A7, are associated with apoptosis regulation and skeletal development/bone mineralisation, respectively, and may relate to the species’ large body and antler size. Demographic reconstructions indicate a long-term decline in effective population size, extremely low heterozygosity, little evidence of extensive runs of homozygosity, and an elevated burden of predicted deleterious alleles. Together, these results suggest that Megaloceros entered the terminal Pleistocene in a genomically fragile state, offering new insight into the biology and evolutionary legacy of one of the largest and most distinctive cervids that ever lived.

Growing evidence of the microbiome’s importance for plant ecology highlights the need for conservation strategies that consider both plants and their microbial partners, the holobiont. Population genomics are valuable tools for designing conservation strategies but rarely accounts for host- or site-specific microbial interactions. Orchids, with their dependence on specific mycorrhizal fungi, exemplify the need to include the microbiome in conservation planning. We here combine population genomics with exploration of root mycobiomes for three closely related orchid taxa (Platanthera chlorantha, P. bifolia var. bifolia and P. bifolia var. latissima) across 22 populations in Denmark to identify ecologically and evolutionary significant units. Our data revealed that Platanthera chlorantha and P. bifolia s.l. hosted different mycobiomes, suggesting a strong host effect on the fungal symbionts. Population genomic analyses identified three different clusters, one matching P. bifolia var. bifolia and two clusters dividing P. chlorantha into two geographically separate units. The genomic profile of P. bifolia var. latissima was similar to that of P. bifolia var. bifolia but indicated some introgression from P. chlorantha. By combining the mycobiome and population genomic data we reveal that the three detected clusters were associated with different mycobiomes, resulting in a significant correlation between host genomics and mycobiome. Root mycobiomes were correlated with variation in soil nutrients, suggesting a role of the orchids’ fungal partners in adaptation to local edaphic conditions. We propose to identify evolutionary significant units in Platanthera in Denmark based on the novel combination of host genomic and mycobiome profiles, in a conservation hologenomics approach.

Abstract Scalesia (Asteraceae) is the largest endemic plant genus of the Galápagos archipelago and an example of adaptive radiation. While Scalesia species are highly varied in habit and morphology, most remarkable is their variety of leaf shapes, especially in the differential presence of leaf lobing/serration, a derived trait that evolved multiple times as a likely adaptation to the islands’ hot and dry equatorial climate. Using population-level genomic data from 396 individuals representing all 15 recognized Scalesia species, we characterize this young radiation (around 1 million years ago), and reveal that their substantial morphological divergence and ecological specialization are primarily based on shared genetic variation. To further elucidate the repeated adaptive evolution of leaf lobing in Scalesia, we integrate genomic and leaf morphometric data, with transcriptomes from different developmental stages, and conclude that leaf lobing evolved through diversifying selection. Natural selection occurs independently on different regulators in the pathway controlling development of adaxial-abaxial leaf polarity, highlighting the importance of the founder populations’ high genetic diversity maintained via allopolyploidy. Finally, our findings have implications for the conservation of Scalesia’s threatened biodiversity, as unexpectedly high intra-specific genetic structure and long-term isolation among populations indicate widespread nascent speciation.

Abstract Neurological, neurodegenerative, and psychiatric disorders impose substantial morbidity and disability worldwide, yet their molecular basis remains incompletely understood, in part due to limited access to human brain tissue. The Danish Brain Collection, comprising brains from individuals who lived in Danish psychiatric institutions from the 1940s to the 1980s, represents a unique but largely untapped resource for retrospective molecular investigation. Here, we assess the feasibility of extracting and sequencing DNA and RNA from decades-old FFPE brain tissue. We systematically evaluate how extraction and library preparation strategies influence nucleic acid yield and quality, and show that RNA end-repair prior to library preparation substantially enhances transcript diversity, improving data quality from highly degraded samples. Despite extensive fragmentation, we recover biologically informative transcriptomic profiles, including protein-coding and microRNA expression profiles that retain clear tissue specificity. These results establish the Danish Brain Collection as a viable resource for genomic and transcriptomic analyses and demonstrate the broader potential of archival FFPE tissues for large-scale molecular studies.

Domestication represents one of the largest biological shifts of life on Earth, and for many animal species, behavioral selection is thought to facilitate early stages of the process. The gut microbiome of animals can respond to environmental changes and have diverse and powerful effects on host behavior. As such, we hypothesize that selection for tame behavior during early domestication, may have indirectly selected on certain gut microbiota that contribute to the behavioral plasticity necessary to adapt to the new social environment. Here, we explore the gut microbiome of foxes from the tame and aggressive strains of the "Russian-Farm-Fox-Experiment". Microbiota profiles reveal a significant depletion of bacteria in the tame fox population that have been associated with aggressive and fear-related behaviors in other mammals. Our metagenomic survey allows for the reconstruction of microbial pathways enriched in the gut of tame foxes, such as glutamate degradation, which converge with host genetic and physiological signals, revealing a potential role of functional host-microbiota interactions that could influence behaviors associated with domestication. Overall, by characterizing how compositional and functional potential of the gut microbiota and host behaviors co-vary during early animal domestication, we provide further insight into our mechanistic understanding of this adaptive, eco-evolutionary process.

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.

The Eurasian cave lion was abundant across the Northern Hemisphere before the Late Pleistocene megafaunal extinctions. However, the extent of the distinction between cave and modern lions and their adaptive differences have remained unclear. Using 12 cave lion genomes spanning more than 100,000 years, we show that modern and cave lions were distinct evolutionary lineages with separate demographic histories and unique non-synonymous variants. We also identify evidence of ancient gene flow between them, with the best modern lion proxy for this ancestry being an extinct Southwest Asian population. This admixture correlates with global ice extent, with 3.2%-4.4% modern lion ancestry detected in a ∼20,000-year-old cave lion from Central East Asia. These findings provide insight into the evolutionary history of the cave lion, once one of the Northern Hemisphere's most ecologically impactful megafaunal species.

The early-life development of the gut microbiome in broiler chickens is a dynamic ecological process with significant implications for host physiology and productivity. Using 388 genome-resolved metagenomic and 61 metatranscriptomic samples across two replicated trials, we analysed the compositional and functional succession of the caecal microbiome in chickens from hatching to slaughter age. We reconstructed 822 bacterial genomes and distilled gene annotations into comprehensive metabolic traits that captured the functional capacities of each genome. We observed that the increase in microbial diversity with chicken age was accompanied by a decline in community-level average metabolic capacity, driven by a shift from metabolically versatile generalists (Lachnospiraceae) to hitherto uncultured, genome-reduced specialists (RF39, RF32, and UBA1242). However, the specific identity of the dominant genome-reduced specialists varied among individuals, resulting in contrasting associations with host body weight. At slaughter age, only 10 UBA660 (RF39) bacteria were positively associated with body weight, while other genome-reduced lineages, such as UBA1242 (Christensenellales), were among 190 negatively associated bacteria. Gene expression analyses revealed that despite their reduced functional repertoire, UBA660 exhibited greater metabolic activity than UBA1242, particularly in the production of two key metabolites for host nutrition and intestinal homeostasis: the essential amino acid lysine and the signaling molecule indole-3-acetate. These findings provide new insights into the functional ecology of the chicken gut microbiome and highlight the relevance of cultivation approaches to retrieve underexplored and uncultured bacterial taxa, which could open new avenues for microbiome-based strategies aimed at improving poultry growth and health in intensive production systems.

The sable (Martes zibellina) and pine marten (Martes martes) are two Palearctic mustelids with long-recognized hybrids (kidases), whose fertility was controversial for years. Early genetic studies confirmed the existence of hybrids beyond F1, but limited marker resolution prevented detailed characterization of hybrid ancestry. Both species were hunted for centuries, but anthropogenic pressures during the 20th-century caused severe bottlenecks in the sable. Hunting bans and large-scale reintroduction programs restored sable populations across much of its range, including the sympatric zone, potentially affecting hybridization. We resequenced 30 individuals from most of the sables' range and the Eastern part of pine marten's. Among samples, we found a broad spectrum of hybrid types with mosaic recombinant chromosomes that confirm hybrid fertility and indicate crossover is not suppressed in kidases. This necessitates re-evaluation of previous research, as we detected notable discrepancies between short tandem repeat-based ancestry and whole-genome analysis. We revealed mitochondrial DNA introgression from sables into most pine martens, indicating displacement of native pine marten mitochondrial sequences. Pine marten heterozygosity is relatively low (∼0.5 to 0.6 hetSNPs/kbp), while sable's diversity (∼1.5 to 1.8 hetSNPs/kbp) is unexpectedly high given its demographic history, likely reflecting successful reintroduction programs. We dated species divergence at 1.52 [confidence interval (CI): 1.05 to 2.06] Mya, and identified candidate genes potentially associated with hybrid fertility issues. This study is the first to elucidate marten hybridization at the whole-genome level, opening new research directions for understanding hybridization among Holarctic martens, the genetic consequences of reintroduction programs, and comparative adaptomics.

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.

Lions and tigers, as dominant apex predators, likely became competitors when lions expanded from Africa into Eurasia approximately one million years ago (Ma), forming a lion-tiger transition belt from the Middle East through Central Asia to the Russian Far East. At the easternmost edge of this zone, the Japanese Archipelago has long been considered a Late Pleistocene tiger refugium, supported by large felid subfossils traditionally attributed to tigers (Panthera tigris), though their taxonomic identity remained unresolved. To clarify the origin, evolutionary history, and biogeography of Japan's Pleistocene felids, we analyzed 26 ancient specimens previously assumed to be tigers. Using mitochondrial and nuclear genome hybridization capture and sequencing, paleoproteomics, Bayesian molecular dating, and radiocarbon dating, we found that all ancient Japanese "tiger" remains yielding molecular data were, unexpectedly, cave lions (Panthera spelaea). One specimen from Yamaguchi Prefecture, western Japan, was radiocarbon dated to 36,000-34,891 cal. BP. These cave lions likely dispersed to the Japanese Archipelago between ~72.7 and 37.5 thousand years ago (ka), when a land bridge connected northern Japan to the mainland during the Last Glacial Period. Our findings challenge the long-held view that tigers once took refuge in Japan, showing instead that cave lions were widespread in northeast Asia during this period and were the Panthera lineage that colonized Japan, reaching even its southwestern regions despite habitats previously thought to favor tigers.