We present here a high-quality genome assembly of a male mountain hare ( Lepus timidus Linnaeus), from Ilomantsi, Eastern Finland, utilizing an isolated fibroblast cell line as the source for high quality DNA and RNA. Following the previously published brown hare reference genome assembly, the mountain hare is the second Finnish pilot species for the European Reference Genome Atlas (ERGA) initiative, a collaborative effort to generate reference genomes for European biodiversity. The genome was assembled using 21x PacBio HiFi sequencing data and scaffolded using the Hi-C chromosome structure capture approach. After manual curation, the primary assembly length was 2,695,305,354 bp with N50 125,755,317 bp. The largest scaffold was 181 Mbp and the scaffold N50 127 Mbp, contributing to a primary assembly consisting of 85 scaffolds and an alternate assembly with 109 scaffolds. The scaffolds include 23 autosomes, numbered according to their size, as well as X and Y chromosomes, matching the known karyotype. Telomeric regions were present on at least one end of 19 of the chromosomes. The genome has a high degree of completeness based on the BUSCO score (mammalia_odb10 database), Complete: 95.1 % [Single copy: 92.3 %, Duplicated: 2.7 %], Fragmented 0.8 %, and Missing 4.1 %. The mitochondrial genome of the cell line was sequenced and assembled separately. The assembly meets the Earth BioGenome Project criteria for a reference-standard genome assembly. Compared to the previous pseudo-reference genome assembly of L. timidus ssp. hibernicus Bell, assembled using the rabbit genome, this new reference genome represents the nominate subspecies and the species-specific chromosomal conformation. The published genome assembly will provide a solid foundation for future genomic research on Lagomorpha, including the insights into the genomic basis of adaptations to snowy and cold environments. Furthermore, it opens opportunities for experimental analysis of mountain hare gene functions.

Mesozooplankton is a key component of the ocean, regulating global processes such as the carbon pump, and ensuring energy transfer from lower to higher trophic levels. Yet, knowledge on mesozooplankton diversity, distribution and connectivity at global scale is still fragmented. To fill this gap, we applied DNA metabarcoding to mesozooplankton samples collected during the Malaspina-2010 circumnavigation expedition across the Atlantic, Indian and Pacific oceans from the surface to bathypelagic depths. We highlight the still scarce knowledge on global mesozooplankton diversity and identify the Indian Ocean and the deep sea as the oceanic regions with the highest proportion of hidden diversity. We report no consistent alpha-diversity patterns for mesozooplankton at a global scale, neither across vertical nor horizontal gradients. However, beta-diversity analysis suggests horizontal and vertical structuring of mesozooplankton communities mostly attributed to turnover and reveals an increase in mesozooplankton beta-diversity with depth, indicating reduced connectivity at deeper layers. Additionally, we identify a water mass type-mediated structuring of mesozooplankton bathypelagic communities instead of an oceanic basin-mediated as observed at upper layers. This suggests limited dispersal at deep ocean layers, most likely due to weaker currents and lower mixing of water mass types, thus reinforcing the importance of oceanic currents and barriers to dispersal in shaping global plankton communities.

The temperate climate-adapted brown hare (Lepus europaeus) and the cold-adapted mountain hare (Lepus timidus) are closely related and interfertile species. However, their skin fibroblasts display distinct gene expression profiles related to fundamental cellular processes. This indicates important metabolic divergence between the two species. Through targeted metabolomics and metabolite tracing, we identified species-specific variations in glycerol 3-phosphate (G3P) metabolism. G3P is a key metabolite of the G3P shuttle, which transfers reducing equivalents from cytosolic NADH to the mitochondrial electron transport chain (ETC), consequently regulating glycolysis, lipid metabolism, and mitochondrial bioenergetics. Alterations in G3P metabolism have been implicated in multiple human pathologies including cancer and diabetes. We observed that mountain hare mitochondria exhibit elevated G3P shuttle activity, alongside increased membrane potential and decreased mitochondrial temperature. Silencing mitochondrial G3P dehydrogenase (GPD2), which couples the conversion of G3P to the ETC, uncovered its species-specific role in controlling mitochondrial membrane potential and highlighted its involvement in skin fibroblast thermogenesis. Unexpectedly, GPD2 silencing enhanced wound healing and cell proliferation rates in a species-specific manner. Our study underscores the pivotal role of the G3P shuttle in mediating physiological, bioenergetic, and metabolic divergence between these hare species.

The temperate climate adapted brown hare ( Lepus europaeus ) and the cold-adapted mountain hare ( Lepus timidus ) are evolutionarily closely related and interfertile. Still, their cultured skin fibroblasts show clear differences in the expression of genes related to basic cellular metabolism. To study this further, we utilized targeted metabolomics analysis, metabolite tracing, and high-resolution respirometry, and identified significant differences in metabolic pathways associated with adaptive thermogenesis, including a higher rate of glycerol 3-phosphate (G3P) production in the mountain hare. We therefore investigated mitochondrial heat production and its dependence on G3P in the two hare species. The mountain hare maintained lower mitochondrial temperature and had weaker thermal change following OXPHOS inhibition. Manipulating mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) levels demonstrated its role in mitochondrial thermogenesis and revealed species-specific function in maintaining mitochondrial membrane potential. This study unveils previously undocumented and unexpected species differences in the mitochondrial properties of fibroblasts that could indicate differences in metabolic adaptability. These findings also demonstrate the utility of cell culture models in assessing trait differences between species and their evolutionary significance, contributing to a deeper understanding of metabolic adaptation in animals and underscoring the potential of in vitro approaches for eco-physiological studies.### Competing Interest StatementThe authors have declared no competing interest.

We present here a high-quality genome assembly of the brown hare (Lepus europaeus Pallas), based on a fibroblast cell line of a male specimen from Liperi, Eastern Finland. This brown hare genome represents the first Finnish contribution to the European Reference Genome Atlas pilot effort to generate reference genomes for European biodiversity. The genome was assembled using 25X PacBio HiFi sequencing data and scaffolded utilizing a Hi-C chromosome structure capture approach. After manual curation, the assembled genome length was 2,930,972,003 bp with N50 scaffold of 125.8 Mb. 93.16% of the assembly could be assigned to 25 identified chromosomes (23 autosomes plus X and Y), matching the published karyotype. The chromosomes were numbered according to size. The genome has a high degree of completeness based on the BUSCO score (mammalia_odb10 database), Complete: 96.1% [Single copy: 93.1%, Duplicated: 3.0%], Fragmented 0.8%, and Missing 2.9%. The mitochondrial genome of the cell line was sequenced and assembled separately. The final annotated genome has 30,833 genes of which 21,467 code for a polypeptide. The brown hare genome is particularly interesting as this species readily hybridizes with the mountain hare (Lepus timidus L.) at the species contact zone in northern Eurasia, producing fertile offspring and resulting in gene flow between the two species. In addition to providing a useful comparison for population studies, the genome can offer insight into the chromosomal evolution among Glires in general and Lagomorpha in particular. The chromosomal assembly of the genome also demonstrates that the cell line has not acquired karyotypic changes during culture.

Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

Resource specialization and ecological speciation arising through host-associated genetic differentiation (HAD) are frequently invoked as an explanation for the high diversity of plant-feeding insects and other organisms with a parasitic lifestyle. While genetic studies have demonstrated numerous examples of HAD in insect herbivores, the rarity of comparative studies means that we still lack an understanding of how deterministic HAD is, and whether patterns of host shifts can be predicted over evolutionary timescales. We applied genome-wide single nucleotide polymorphism and mitochondrial DNA sequence data obtained through genome resequencing to define species limits and to compare host-plant use in population samples of leaf- and bud-galling sawflies (Hymenoptera: Tenthredinidae: Nematinae) collected from seven shared willow (Salicaceae: Salix) host species. To infer the repeatability of long-term cophylogenetic patterns, we also contrasted the phylogenies of the two galler groups with each other as well as with the phylogeny of their Salix hosts estimated based on RADseq data. We found clear evidence for host specialization and HAD in both of the focal galler groups, but also that leaf gallers are more specialized to single host species compared with most bud gallers. In contrast to bud gallers, leaf gallers also exhibited statistically significant cophylogenetic signal with their Salix hosts. The observed discordant patterns of resource specialization and host shifts in two related galler groups that have radiated in parallel across a shared resource base indicate a lack of evolutionary repeatability in the focal system, and suggest that short- and long-term host use and ecological diversification in plant-feeding insects are dominated by stochasticity and/or lineage-specific effects.

Abstract Studies on host–parasite systems that have experienced distributional shifts, range fragmentation, and population declines in the past can provide information regarding how parasite community richness and genetic diversity will change as a result of anthropogenic environmental changes in the future. Here, we studied how sequential postglacial colonization, shifts in habitat, and reduced host population sizes have influenced species richness and genetic diversity of Corynosoma (Acanthocephala: Polymorphidae) parasites in northern European marine, brackish, and freshwater seal populations. We collected Corynosoma population samples from Arctic, Baltic, Ladoga, and Saimaa ringed seal subspecies and Baltic gray seals, and then applied COI barcoding and triple‐enzyme restriction‐site associated DNA (3RAD) sequencing to delimit species, clarify their distributions and community structures, and elucidate patterns of intraspecific gene flow and genetic diversity. Our results showed that Corynosoma species diversity reflected host colonization histories and population sizes, with four species being present in the Arctic, three in the Baltic Sea, two in Lake Ladoga, and only one in Lake Saimaa. We found statistically significant population‐genetic differentiation within all three Corynosoma species that occur in more than one seal (sub)species. Genetic diversity tended to be high in Corynosoma populations originating from Arctic ringed seals and low in the landlocked populations. Our results indicate that acanthocephalan communities in landlocked seal populations are impoverished with respect to both species and intraspecific genetic diversity. Interestingly, the loss of genetic diversity within Corynosoma species seems to have been less drastic than in their seal hosts, possibly due to their large local effective population sizes resulting from high infection intensities and effective intra‐host population mixing. Our study highlights the utility of genomic methods in investigations of community composition and genetic diversity of understudied parasites.

Speciation is a fundamental evolutionary process, which results in genetic differentiation of populations and manifests as discrete morphological, physiological and behavioural differences. Each species has travelled its own evolutionary trajectory, influenced by random drift and driven by various types of natural selection, making the association of genetic differences between the species with the phenotypic differences extremely complex to dissect. In the present study, we have used an in vitro model to analyse in depth the genetic and gene regulation differences between fibroblasts of two closely related mammals, the arctic/subarctic mountain hare (Lepus timidus Linnaeus) and the temperate steppe-climate adapted brown hare (Lepus europaeus Pallas). We discovered the existence of a species-specific expression pattern of 1623 genes, manifesting in differences in cell growth, cell cycle control, respiration, and metabolism. Interspecific differences in the housekeeping functions of fibroblast cells suggest that speciation acts on fundamental cellular processes, even in these two interfertile species. Our results help to understand the molecular constituents of a species difference on a cellular level, which could contribute to the maintenance of the species boundary.

Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood. Here, we investigate the role of Topoisomerase 3α (Top3α) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3α knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin OH and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3α knockdown and overexpression affected mitochondrial RNA transcription, leading to a decrease in steady-state levels of mitochondrial transcripts. Together, our results indicate that the mitochondrial isoform of Top3α is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3α is also influencing the progression of transcription, with its absence affecting downstream transcript levels.

We provide the first whole genome sequences from three specimens of the mountain hare subspecies the heath hare (Lepus timidus sylvaticus), along with samples from two mountain hares (Lepus timidus timidus) and two brown hares (Lepus europaeus) from Sweden. The heath hare has a unique grey winter pelage as compared to other mountain hares (white) and brown hares (mostly brown), and face regional extinction, likely due to competitive exclusion from the non-native brown hare. Whole genome resequencing from the seven hare specimens were mapped to the Lepus timidus pseudoreference genome and used for detection of 11,363,883 polymorphic nucleotide positions. The data presented here could be useful for addressing local adaptations and conservation status of mountain hares and brown hares in Sweden, including unique subspecies.

Increasing anthropogenic pressures on the coastal marine environments impact these ecosystems via a variety of mechanisms including nutrient loading, leading to eutrophication and increases in algal blooms. Here, we use a metagenomics approach to assess the taxonomic and functional changes of the microbial community throughout a nutrient enriched mesocosm phytoplankton bloom. We tested four different nutrient treatments consisting of either nitrate and phosphate or nitrate, phosphate and silicate, administered on the first day or continuously for the first two weeks of the experiment. Our results show a shift in the taxonomic composition of the community over time that is dependent on the nutrient addition regime. Significant differences in the functional potential of the communities were detected, with an interaction between bloom period (pre-bloom, bloom and post-bloom) and nutrient treatment (p = 0.004). A sharp drop in functional similarity was observed in the first week in all treatments and after 20 days had not returned to pre-bloom levels. Changes within energy metabolism pathways showed a remarkable enrichment of the dissimilatory nitrate reduction pathway in the post-bloom period. Eukaryotic oxidative phosphorylation and photosynthetic antenna proteins were more abundant during the bloom, especially in the continuous treatment with silicate. Our results suggest that continuous (i.e. chronic) nutrient enrichment has a larger effect on the functioning of marine systems compared to a single (i.e acute) addition. A deep understanding of the functional and taxonomic shifts in the community during blooms is essential to reverse or mitigate human impacts on coastal environments.

Brown hares ( Lepus europaeus Pallas) are able to hybridize with mountain hares ( L. timidus Linnaeus) and produce fertile offspring, which results in cross-species gene flow. However, not much is known about the functional significance of this genetic introgression. Using targeted sequencing of candidate loci combined with mtDNA genotyping, we found the ancestral genetic diversity in the Finnish brown hare to be small, likely due to founder effect and range expansion, while gene flow from mountain hares constitutes an important source of functional genetic variability. Some of this variability, such as the alleles of the mountain hare thermogenin (uncoupling protein 1, UCP1 ), might have adaptive advantage for brown hares, whereas immunity-related MHC alleles are reciprocally exchanged and maintained via balancing selection. Our study offers a rare example where an expanding species can increase its allelic variability through hybridization with a congeneric native species, offering a route to shortcut evolutionary adaptation to the local environmental conditions.

Three subspecies of the ringed seal (Pusa hispida) are found in northeastern Europe: P. h. botnica in the Baltic Sea, P. h saimensis in Lake Saimaa in Finland, and P. h. ladogensis in Lake Ladoga in Russia. We investigated the poorly-known cestode helminth communities of these closely related but ecologically divergent subspecies using COI barcode data. Our results show that, while cestodes from the Baltic Sea represent Schistocephalus solidus, all worms from the two lakes are identified as Ligula intestinalis, a species that has previously not been reported from seals. The observed shift in cestode communities appears to be driven by differential availability of intermediate fish host species in marine vs. freshwater environments. Both observed cestode species normally infect fish-eating birds, so further work is required to elucidate the health and conservation implications of cestode infections in European ringed seals, whether L. intestinalis occurs also in marine ringed seals, and whether the species is able to reproduce in seal hosts. In addition, a deep barcode divergence found within S. solidus suggests the presence of cryptic diversity under this species name.

While free-living herbivorous insects are thought to harbor microbial communities composed of transient bacteria derived from their diet, recent studies indicate that insects that induce galls on plants may be involved in more intimate host-microbe relationships. We used 16S rDNA metabarcoding to survey larval microbiomes of 20 nematine sawfly species that induce bud or leaf galls on 13 Salix species. The 391 amplicon sequence variants (ASVs) detected represented 69 bacterial genera in six phyla. Multi-variate statistical analyses showed that the structure of larval microbiomes is influenced by willow host species as well as by gall type. Nevertheless, a "core" microbiome composed of 58 ASVs is shared widely across the focal galler species. Within the core community, the presence of many abundant, related ASVs representing multiple distantly related bacterial taxa is reflected as a statistically significant effect of bacterial phylogeny on galler-microbe associations. Members of the core community have a variety of inferred functions, including degradation of phenolic compounds, nutrient supplementation, and production of plant hormones. Hence, our results support suggestions of intimate and diverse interactions between galling insects and microbes and add to a growing body of evidence that microbes may play a role in the induction of insect galls on plants.

Brown hares ( Lepus europaeus Pallas) are able to hybridize with mountain hares ( L. timidus Linnaeus) and produce fertile offspring, which results in cross-species gene flow. However, not much is known about the functional significance of this genetic introgression. Using targeted sequencing of candidate loci combined with mtDNA genotyping, we found the ancestral genetic diversity in the brown hare to be small, likely due to founder effect and range expansion, while gene flow from mountain hares constitutes an important source of functional genetic variability. Some of this variability, such as the alleles of the mountain hare thermogenin (uncoupling protein 1, UCP1 ), is likely of adaptive advantage for brown hares, whereas immunity-related MHC alleles are reciprocally exchanged and maintained via balancing selection. Our study offers a rare example where an expanding species can ease its expansion load through hybridization and obtain beneficial alleles to shortcut evolutionary adaptation to the novel environmental conditions.

Oxidative stress can be modeled using various different experimental approaches, such as exposing the cells or organisms to oxidative chemicals. However, the actual effects of these chemicals, outside of the immediate measured effect, have attracted relatively little attention. We show here that three commonly used oxidants, menadione, potassium bromate, and hydrogen peroxide, while known to function differently, also elicit different types of responses in HEK293T cells. Menadione and bromate exposure mainly trigger an integrated stress response, whereas hydrogen peroxide affects cellular processes more diversely. Interestingly, acute oxidative stress does not universally cause notable induction of DNA repair or antioxidant defense mechanisms. We also provide evidence that cells with previous experience of oxidative stress show adaptive changes in their responses when the stress is renewed. Our results urge caution when comparing studies where different sources of oxidative stress have been used or when generalizing the findings of these studies to other oxidant types or tissues.

Hymenoptera is a hyperdiverse insect order represented by over 153,000 different species. As many hymenopteran species perform various crucial roles for our environments, such as pollination, herbivory, and parasitism, they are of high economic and ecological importance. There are 99 hymenopteran genomes in the NCBI database, yet only five are representative of the paraphyletic suborder Symphyta (sawflies, woodwasps, and horntails), while the rest represent the suborder Apocrita (bees, wasps, and ants). Here, using a combination of 10X Genomics linked-read sequencing, Oxford Nanopore long-read technology, and Illumina short-read data, we assembled the genomes of two willow-galling sawflies (Hymenoptera: Tenthredinidae: Nematinae: Euurina): the bud-galling species Euura lappo and the leaf-galling species Eupontania aestiva. The final assembly for E. lappo is 259.85 Mbp in size, with a contig N50 of 209.0 kbp and a BUSCO score of 93.5%. The E. aestiva genome is 222.23 Mbp in size, with a contig N50 of 49.7 kbp and a 90.2% complete BUSCO score. De novo annotation of repetitive elements showed that 27.45% of the genome was composed of repetitive elements in E. lappo and 16.89% in E. aestiva, which is a marked increase compared to previously published hymenopteran genomes. The genomes presented here provide a resource for inferring phylogenetic relationships among basal hymenopterans, comparative studies on host-related genomic adaptation in plant-feeding insects, and research on the mechanisms of plant manipulation by gall-inducing insects.

The perception that the inheritance of phenotypic traits operates solely through genetic means is slowly being eroded: epigenetic mechanisms have been shown to induce heritable changes in gene activity in plants1,2 and metazoans1,3. Inheritance of DNA methylation patterns provides a potential pathway for environmentally induced phenotypes to contribute to evolution of species and populations1–5. However, in basal metazoans, it is unknown whether inheritance of CpG methylation patterns occurs across the genome (as in plants) or as rare exceptions (as in mammals)4. Here, we show that DNA methylation patterns in a reef-building coral are determined by genotype and developmental stage, as well as by parental environment. Transmission of CpG methylation from adults to their sperm and larvae demonstrates genome-wide inheritance. Variation in the hypermethylation of genes in adults and their sperm from distinct environments suggests intergenerational acclimatization to local temperature and salinity. Furthermore, genotype-independent adjustments of methylation levels in stress-related genes were strongly correlated with offspring survival rates under heat stress. These findings support a role of DNA methylation in the intergenerational inheritance of traits in corals, which could extend to enhancing their capacity to adapt to climate change. Intergenerational inheritance of traits in corals can help species survive environmental change. Examination of intergenerational DNA methylation profiles in a reef-building coral shows there to be genome-wide inheritance, with the potential for adaptive capacity to environmental stressors.