Genome Biology and Evolution

In the genus Paramecium, the macronuclear genome is remarkably compact and optimized for gene expression. As a means to explore eukaryotic transcription in the context of a streamlined genome and shed light on the role of sequence architecture on gene expression and loss, we analyzed the distribution and diversity of candidate transcription initiation sites (TISs) in Paramecium sexaurelia, Paramecium tetraurelia and their outgroup, Paramecium caudatum. Our analysis suggests that for Paramecium, most genes have very short 5 prime UTRs (40 bp or less) and their transcription initiation regions (TIRs) have a median dispersion (akin to width) of 8-10 bp. The TIRs for the three species have high AT content. TIR dispersion is not to gene expression. However, mean TIS position relative to the translation start site per gene does in gene expression for the three species, and is often conserved between them. While mean TIS position and gene expression are linked, gene expression itself is the main driver of paralog retention in the aurelias. As compared to other eukaryotes, Paramecium has a uniquely well-defined and short main TIS region, and sequence motifs that likely diverge from the consensus in multicellular eukaryotes.

The extent of within-species variation in genome assembly size, and the relative contributions of neutral vs. adaptive processes to it, remains poorly understood. We assembled the pangenome for the nine-spined stickleback (Pungitius pungitius) from whole-genome resequencing data obtained from range-wide sampling of 1,478 individuals across 47 populations. Our analysis recovered approximately 234.15 Mb of novel sequence and identified 11,151 protein-coding genes annotated in non-reference-represented regions, which accounted for 28.8% of all annotated pangenomic genes, corresponding to a 40.6% expansion relative to the 27,450 protein-coding genes annotated in the updated v8 reference genome. The identified presence/absence variation (PAV) genes exhibited a higher incidence of deleterious and non-synonymous mutations than core genes. Freshwater populations with smaller effective population sizes had lost more genes and had smaller assembly sizes relative to larger marine populations. Larger genome sizes of marine populations were primarily associated with higher transposable element content. Across gene categories, patterns of molecular variation were consistent with weaker purifying selection in freshwater populations than in marine populations, while recurrent freshwater-biased loss of a subset of PAV genes suggested that gene loss is not entirely random. Together, these results indicate that demographic history explains the overall pattern of gene loss, whereas negative natural selection may contribute to repeated loss of a subset of genes.

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.

Gene duplication is a major driver of genomic innovation, but whether paralog accumulation scales uniformly with increasing genome size across the domain Archaea remains unclear. This study presents a domain-wide analysis of 1,829 archaeal genomes and their predicted proteomes to evaluate protein family expansions, their distribution, and lineage-specific patterns of gene duplication. Protein family expansions were classified as recent (90% sequence identity), intermediate (70%), or old (40%) duplications based on sequence-identity thresholds within each family. The findings show that gene duplication content is decoupled from overall proteome size at high sequence-identity levels, indicating that recent paralog retention does not correlate with proteome expansion. Archaeal lineages exhibit contrasting gene duplication profiles. All 1,829 genomes contained paralogs at the 40% sequence identity threshold, indicating the widespread presence of ancient or divergent duplication events. In contrast, paralogs detected at the 90% identity threshold, showed substantial variation across lineages. Their proportion reached up to 16.6% in groups such as Halobacteria, whereas only 74 genomes (4.04%) completely lacked paralogs at this threshold. Highly duplicated families are enriched for membrane transport systems (ABC/MFS), central metabolic enzymes (e.g., acetyl-CoA synthetase), a high proportion of uncharacterized functions, and mobile elements, predominantly transposases. Notably, we identified 314 transposase protein families in Archaea, distributed in 161 unique families in Archaea and 153 families shared with Bacteria, suggesting mechanisms of interdomain gene transfer. Overall, paralog accumulation in Archaea is a lineage-specific phenomenon shaped by targeted functional duplications and mobile element activity, rather than a simple consequence of proteome growth.

GC-biased gene conversion (gBGC) is a widespread recombination-associated process that favours the fixation of GC alleles independently of their fitness. In coding regions, it can oppose purifying selection and favour the accumulation of deleterious AT to GC mutations. In mammals where recombination hotspots are predominantly determined by PRDM9, the deleterious effect of gBGC is expected to be episodic due to the rapid turnover of hotspots. The deleterious impact of gBGC episodes on proteins has been well documented, particularly in rodents and primates. However, the evolutionary response of the affected proteins after the episodes end has been scarcely investigated. Theory predicts that a decline in gBGC intensity should trigger a compensatory response that enables previous damage to be repaired. Recently, we reported evidence for pervasive compensatory evolution following gBGC episodes in Hydromyini rodents. Here, we aimed to investigate whether this post- gBGC compensation is widespread among mammals in which gBGC is episodic. Using a phylogenetic approach, we analysed substitution patterns in coding exons from five mammalian taxa spanning a range of effective population size (Ne): Murini, Rattini, Cetacea, Cercopithecidae and Hominoidae. We detected thousands of gBGC episodes, characterised by an increased rate of non-synonymous AT to GC substitutions. We found evidence for compensatory evolution in non-primate taxa, and a lack of signal in Primates. Although the influence of Ne remains unclear, our study highlights both the evolutionary significance of post-gBGC compensation and the methodological challenges in detecting it.

Animal mitochondrial rRNAs are commonly viewed as structurally reduced, yet sponge mt SSU rRNAs range from compact to highly expanded structures. Using nine conserved structural anchors, we compared 216 taxonomically resolved records from four classes and 22 orders, including 16 freshwater Spongillida and 200 marine sponges. Twelve homologous hypervariable substructures were coded as structural types, and their ordered combinations as composite types. We identified 38 structural types and 62 composite types across molecules ranging from 853 to 2,019 nt. Hexactinellida and freshwater Spongillida were each uniform for a distinct composite type but differed markedly in overall structure: hexactinellid mt SSU rRNAs were compact, whereas those of Spongillida were long and contained four to five candidate insertion regions. These results show that a conserved scaffold can accommodate extensive lineage-associated structural variation and provide a practical framework for comparing highly divergent mitochondrial rRNAs.

Chromosome evolution varies widely across Aphidomorpha: several aphid lineages exhibit extensive interautosomal reshuffling, whereas comparisons involving grape phylloxera and Adelges suggest greater chromosome conservation outside Aphididae. However, all published adelgid genomes represent the genus Adelges, leaving conservation across deeper adelgid divergences unresolved. Here, we report a chromosome-scale genome for the pine bark adelgid, Pineus strobi, providing the first genome for the genus Pineus and extending genomic sampling to an early-diverging adelgid lineage. Synteny analyses revealed broad conservation of major linkage groups between Pineus and Adelges across approximately 90 million years. The five grape phylloxera chromosomes also showed broad correspondence to the ten adelgid chromosomes, consistent with a limited number of chromosomal fusions or fissions and relatively little exchange among major linkage groups. These findings strengthen evidence that such extensive interautosomal reshuffling is not characteristic of Aphidomorpha as a whole. We also recovered a complete 25.3 kb mitochondrial genome with expanded, repeat-rich noncoding regions and complete circular genomes for the obligate nutritional symbionts "Candidatus Annandia pinicola" and "Candidatus Hartigia pinicola." Comparisons with Pineus similis symbionts revealed conserved coding capacity and genome-wide synteny, supporting conservation of this dual nutritional symbiosis in pine-associated adelgids. Finally, we recovered the first complete Wolbachia genome reported from an adelgid. Because Wolbachia can induce parthenogenesis, its presence in P. strobi raises the possibility that it contributes to the maintenance or reinforcement of parthenogenesis in a species lacking a viable sexual generation. Together, these genomes provide an integrated resource for aphidomorph evolution and symbiosis. Significance statementSeveral aphid lineages exhibit extensive reshuffling among autosomes, whereas previous comparisons with grape phylloxera and Adelges cooleyi suggested greater chromosome conservation outside Aphididae; however, all published adelgid genomes have represented Adelges, leaving the long-term stability of chromosome organization across Adelgidae unresolved. The first chromosome-scale genome from Pineus reveals broad conservation of major linkage groups across approximately 90 million years of adelgid evolution and clear correspondence with the five chromosomes of the grape phylloxera, supporting chromosome conservation outside Aphididae. The accompanying mitochondrial and symbiont genomes provide an integrated resource for studying aphidomorph genome evolution and symbiosis.

A significant fraction of archaeal genomes have their genomic content distributed across two or more DNA molecules, yet the characteristics and evolution of these different replicons remain poorly understood. Here, we investigated the genomic and functional organization of secondary replicons in Halobacteriota using 211 complete genomes, comprising 766 replicons, including 211 primary chromosomes (chr1), and 555 secondary replicons. Comparative analyses of genomic features, including replicon size, GC content, and codon usage, revealed a continuous distribution of secondary replicons from plasmid-like to chromosome-like characteristics. Larger secondary replicons showed progressively more chromosome-like genomic and functional properties, including greater protein-family overlap with chr1 and a higher proportion of conserved gene families. At the phylum level, functional profiles substantially overlapped among replicon types, providing further support for a continuum rather than discrete functional classes. Larger replicons also showed increased protein-family duplication, while pangenome analysis revealed a greater proportion of conserved gene families, with accessory families most abundant at intermediate sizes and rare and singleton families declining with increasing replicon size. Together, these results support a model in which secondary replicons in Halobacteriota span a genomic and functional continuum from plasmid-like to chromosome-like states , with increasing size associated with greater conservation, gene duplication and progressively more chromosome-like gene content and functional profile.

Forms I&prime, I&Prime, and I RuBisCOs were recently identified in bacterial metagenome-assembled genomes (MAGs) and are significant for evolutionarily bridging form I, which is responsible for the major part of carbon fixation on Earth, and other forms. Pioneering studies focused mainly on the biochemical and structural characteristics of "form I-related" RuBisCOs and deduced the evolution of this protein family over phylogenetic relationships among form I and form I-related sequences. We here surveyed form I-related sequences in public databases and conducted phylogenetic analyses incorporating the updated diversity of form I-related RuBisCOs. This study yielded two major outcomes regarding RuBisCO evolution. First, the analyses presented here suggest uncertainties overlooked in the previously published studies. It appeared to be difficult to conclude (i) the phylogenetic relationship among forms I, I&prime, and I&Prime, (ii) the monophyly of form I&prime, or (iii) the monophyly of form I. Second, we identified a gene encoding form I RuBisCO in an archaeal MAG, and this protein is likely involved in the pentose bisphosphate pathway. The results presented here suggest that the evolutionary route leading to form I in RuBisCO evolution remains unsettled.

The classical model of chromosomal speciation is based on the observation that divergent species often differ in chromosome numbers and that heterokaryotypic hybrids show reduced fitness. Furthermore, positive correlations between the rates of karyotype evolution and speciation have been established in several groups of organisms. Yet, the mechanisms that make some lineages more prone to chromosomal fissions and fusions than others, and the causal role of such karyotype changes in speciation, are poorly understood. Here, we focus on the taxonomically and karyotypically diverse group of African monkeys, the guenons (tribe Cercopithecini), consisting of ~35 species with diploid chromosome numbers ranging from 48 to 72. We generated de novo genome assemblies for eight species with different karyotypes, and incorporated publicly available genomes from additionally three guenon species. Using the rhesus macaque as an outgroup, we identified 28 independent chromosomal fissions and 4 fusions in guenons. In line with previous cytogenetic studies, repeat composition around chromosomal breakpoints suggested that most fissions were non-centromeric, requiring the evolution of a new centromere in at least one of the fission products. In contrast to the expectation under strong underdominance, we found a paraphyletic distribution of several chromosomal fissions. Some of these phylogenetically discordant events could have resulted from interspecific hybridization, whereas others are more likely caused by incomplete lineage sorting, suggesting that fissioned and non-fissioned homologs segregated in ancestral guenon lineages for millions of years. Nevertheless, we find evidence for reduced introgression on rearranged chromosomes in an ancient cross-karyotypic gene flow event between Cercopithecus cephus and C. pogonias. Although this suggests that chromosomal fissions may have constituted barriers to gene flow in the guenon evolution, the signal was inconclusive or absent in two other investigated gene flow events. This study provides important first insights into the remarkable karyotypic diversification of guenons, and its role in generating reproductive barriers.

Whole-genome duplication (WGD; or polyploidy) has played a major role in the evolution of many lineages however, our understanding of the processes that shape genome evolution following WGD remains incomplete. While polyploidy duplicates the entire genome sequence, it is rediploidisation that establishes independent duplicated genes. Rediploidisation proceeds through suppression of meiotic recombination across polysomic loci thus restoring disomic inheritance, a process that is not synchronised across the genome. Despite its importance, the mechanisms underlying this process remain poorly understood. The slowly evolving genomes of paleopolyploid Acipenseriformes paddlefish and sturgeon provide an invaluable system for investigating this, as rediploidisation was highly asynchronous in these lineages. In both genomes ohnologs tend to segregate into blocks on the chromosomes according to rediploidisation timing, a pattern that suggests links between chromosomal structure and rediploidisation. Here, we analyse a newly-produced duplicate-resolved paddlefish genome assembly and show a strong concordance between genome rearrangement and rediploidisation timing. We also find that topologically associated domain (TAD) boundaries are associated with rediploidisation block boundaries. Together these results indicate that rediploidisation in acipenseriformes occurred through a process of genome rearrangements that was subject to functional constraints imposed by 3D genome architecture. We investigate the evolution of Hox clusters in these lineages, revealing a previously overlooked duplicate HoxC region in paddlefish, and both ancestral and lineage-specific Hox cluster rediploidisation with substantially different timings. These findings highlight a complex evolutionary history following WGD in Acipenseriformes with implications for understanding short-term adaptations to polyploidy as well as longer-term diversification of lineages.

Background: Chromosome-level genome assemblies are increasingly enabling tests of chromosome evolution, conserved synteny, and sex chromosome conservation across diverse animal lineages, including spiders. Results: Here, we present a chromosome-level genome assembly for the African hermit spider, Nephilingis cruentata, a species with extreme female-biased sexual size dimorphism and a cytogenetically inferred XX2 sex chromosome system. The final Hi-C-assisted assembly spans 1.72 Gbp, with 99.5% of bases assigned to 13 pseudochromosomes, a scaffold N50 of 131.6 Mbp, and a BUSCO completeness score of 98.8%. We annotated 20,021 protein-coding genes, and repetitive elements accounted for 42.7% of the genome. Sex-specific whole-genome resequencing identified Chr02 and Chr07 as candidate X chromosomes based on reduced male coverage, consistent with the expected XX2 system. Using comparative whole-genome alignments across existing chromosome-scale spider assemblies, we also show that sex-linked chromosomes retain broad homologous identity across sampled spider lineages but exhibit lower synteny conservation and greater chromosome-length divergence than autosomes. Conclusions: These results suggest that spider sex chromosomes are conserved in homologous identity but more labile in structure, providing a comparative framework for studying sex chromosome conservation and divergence across Araneae.

The eriophyoid mites (Acari: Eriophyoidea) represent an extreme case of genome streamlining, with genomes averaging just 32Mb, among the smallest of all animals. This clade of mites is highly diverse with more than 4000 species. Their morphologies are specialized for feeding on their host plants, including a simplified worm-like body plan with just two pairs of legs and modified mouth parts that can induce the formation of galls or plant deformities during feeding. Their generally strong host affinities make eriophyoid mites appealing for use as biological control agents, although their short generation times and small genomes could facilitate rapid evolution and impact their efficacy in management programs. Here, we sequenced the genome of the biological control mite Floracarus perrepae, producing a highly contiguous genome totalling just 23.1 Mb, among the smallest of all animals. We also assembled another non-eriophyid mite genome from accidental DNA bycatch (69.4Mb). We placed this new genomic resource in a phylogenetic context to reveal that mites have highly dynamic genome evolution, with a significant trend in genome downsizing in the eriophyoids. Our results suggest that this streamlining is associated with non-genic elements such as the suppression or excision of retrotransposons and purging of introns. As new sequencing techniques become available, novel genomic resources for tiny organisms such as F. perrepae will be more readily accessible, facilitating both fundamental genome evolutionary biology and applied sciences such as biological control programs which use eriophyoid mites for the management of invasive species.

Tandemly repeated sequences are noncoding, highly variable, yet integral components of eukaryotic genomes. Empirical research has so far largely focused on a few well-characterized, functional tandem repeats (TRs) or documented the broad diversity and rapid evolution of these repeats without explicitly testing the forces driving their evolution. By characterizing TR abundance and TR sequence composition within and between chromosomes and species in the stick insect genus Timema, we uncover that TR loads are enriched in genomic regions of high recombination but tend to be reduced in species with higher effective population sizes. These results suggest that there is a mutational effect of recombination that generates increased TR loads and that the bulk of TRs experience weak selection, leading to their rapid turnover over short evolutionary timescales. Finally, comparisons of centromere and non-centromere TRs reveal that the rapid divergence of centromeres is best explained by the combination of the fast-evolving nature of TRs per se and centromere-specific evolutionary forces. Overall, this study provides the first in-depth investigation of genome-wide TR evolution, while integrating the effects of recombination, genetic drift, and centromeric position along the genome.

The European leaf-toed gecko (Euleptes europaea) is a small, nocturnal gecko endemic to the western Mediterranean. As a phylogenetically distinctive member of the Gondwanan family Sphaerodactylidae, it represents an important species for studying Mediterranean island biogeography, adaptation, and reptile genome evolution. The species also occupies a key position for investigating the evolution of sex chromosomes, as geckos exhibit remarkable diversity and frequent transitions in sex-determination systems. We present a chromosome-level genome assembly of Euleptes europaea generated as part of the Vertebrate Genomes Project. The 1.8 Gb assembly has a scaffold N50 of 102.3 Mb (contig N50 27 Mb), with 21 chromosome-scale scaffolds corresponding to the known karyotype (2n = 42). The primary assembly has a BUSCO completeness of 97.80% (95.60% as single-copy), a k-mer completeness of 96.00%, and a k-mer quality value (QV) of 61.20. Repetitive elements account for 53.20% of the genome and genome annotation identified 18,633 protein-coding genes. This high-quality reference genome will facilitate studies of genome evolution, island adaptation, and sex chromosome evolution across geckos and other reptiles.

Clownfishes are a complex of 28 species that rapidly diversified after acquiring mutualism with host sea anemones around 15 million years ago. The genomic mechanisms behind this radiation are only beginning to emerge, and structural variants (SVs) have received little attention. Previous phylogenomic studies suggested two large inversions on chromosome 18, but their distribution across the clownfish phylogeny and their potential effects have not been formally explored. SVs can disrupt gene function and regulation or alter gene dosage, and they have been shown to play a central role in adaptive evolution and diversification across many taxa. Here, we characterized SVs across the clownfish radiation using long-read PacBio sequencing, generating high-quality genome assemblies for sixteen species spanning the main lineages of the genus. We identified and catalogued nearly 130,000 SVs spanning close to 570 Mb. Short insertions and deletions were the most numerous, but large inversions accounted for most of the affected sequence. No SV was consistently associated with host specialization. We nevertheless confirmed a single inversion of approximately 17 Mb on chromosome 18, clarifying earlier reports of two separate rearrangements at this locus, and identified an additional inversion on chromosome 9 whose distribution suggests a history of hybridization. This study provides the first genome-wide characterization of structural variation in clownfishes, revealing extensive SVs despite the group's rapid and recent radiation. It also delivers new, high-quality genomic resources that will support future research into this iconic group of coral reef fishes and their adaptation to sea anemones.

Rates of de novo mutations ({micro}) are highly variable among different taxa but less is known about their variability at the intraspecific level. Using a large data set (n=364 trios) from eight nine-spined stickleback (Pungitius pungitius) populations differing in their effective population sizes (Ne), we tested the prediction of the drift-barrier hypothesis (DBH) that {micro} scales negatively with increasing Ne. Indeed, {micro} was a negative function of Ne, also after correcting for phylogenetic non-independence of populations. While the range of variation in mutation rates across populations spanned more than a 4-fold range ({micro} = 1.60 - 6.99 x 10-9), we discovered one highly mutable family with a five-times higher mutation rate than the population average. Evidence was also found (i) for reduced efficiency of selection in small freshwater populations subject to strong genetic drift, (ii) that maternal age associates positively with {micro}, and shorter generation time elevates per-year mutation rates, (iii) that both replication errors and DNA repair efficiency contributed to {micro}, and that (iv) mutation rate variation has a polygenic basis. In general, the results provide support for the DBH identifying elevated mutation rates in populations subject to strong genetic drift.

We present draft genome assemblies for two poneroid ant species with distinctive life history traits: Platythyrea mocquerysi Emery, 1899 (Ponerinae) and Stigmatomma oregonense Wheeler, 1915 (Amblyoponinae). Platythyrea mocquerysi is a Malagasy ant in which larvae produce secretions from a ventral tubercle that are consumed by adults, representing an unusual form of larva-to-adult trophallaxis. Stigmatomma oregonense, a North American "dracula ant," feeds non-lethally on larval haemolymph. Using PacBio HiFi long-read sequencing, we assembled the P. mocquerysi genome (187 Mb, 307 scaffolds, N50 = 5.26 Mb, BUSCO 99.2% complete) and the S. oregonense genome (262 Mb, 1,189 scaffolds, N50 = 0.84 Mb, BUSCO 98.3% complete). These genomes encode 10,860 and 12,354 protein-coding genes, respectively. Repeat content is higher in S. oregonense than in P. mocquerysi, 54.3% against a lower bound of 15.7%. Most of the difference is in unclassified repeats, which we show to be diverged transposable elements. Comparative analysis with two additional poneroid genomes from closely related species reveals that genome size variation across poneroid ants is largely explained by transposable element dynamics, with notable expansions in several gene families including carboxylesterases, serine proteases, and lipid metabolism genes in Amblyoponinae. These genomes provide essential resources for investigating the molecular basis of unconventional brood management strategies in ants.

The allorecognition system of the hydroid Hydractinia symbiolongicarpus plays an essential role in mediating self vs. non-self recognition among cnidarian colonies, analogous to the role of the major histocompatibility complex (MHC) in bilaterian immune systems. Both complexes have contiguous stretches of similar, highly duplicated genes, suggesting a homologous relationship. Protein structural prediction methods such as AlphaFold2 also indicate structural similarity between the proteins encoded within the Hydractinia ARC and the MHC. We have determined the structure of the ARC in the related hydrozoan Podocoryna americana to determine the degree of conservation of the cnidarian allorecognition complex. Protein structural prediction methods have revealed a number of candidate allorecognition proteins that form a long contiguous complex. Phylogenies suggest homology between cnidarian allorecognition genes, as well as syntenic conservation of this gene complex across hydrozoans. Ongoing comparative genomic analyses across bilaterian species aim to clarify the relationship between the ARC and the MHC, thereby informing our understanding of the evolutionary origins of bilaterian adaptive immunity.

We present a high-quality chromosome-level reference genome for the Saimaa ringed seal (Pusa saimensis), an endangered freshwater pinniped endemic to Lake Saimaa, Finland. The assembly spans 2.353 Gb and comprises 15 autosomes together with the X and Y sex chromosomes. Using Oxford Nanopore Technologies (ONT) long-read sequencing and Hi-C scaffolding, we achieved a telomere-to-telomere assembly for all chromosomes, except the Y chromosome. Genome annotation identified approximately 21,800 protein-coding genes, consistent with other mammalian genomes. Assembly completeness was high, with BUSCO analysis recovering 99.6% of expected complete single-copy genes (98.2% single-copy and 1.3% duplicated). Comparative analyses revealed a highly conserved chromosomal architecture, with only minor syntenic differences relative to other pinniped chromosome-level assemblies. Previously described cytogenetic fusion events in Phocidae were confirmed (chromosomes 2 and 7). A translocation between chromosomes 6 and 7 distinguishes phocids from the otariids. In general, more distantly related taxa exhibit an increasing degree of intrachromosomal rearrangements. Notably, we identified a large intrachromosomal rearrangement on chromosome 2 that appears specific to the Saimaa ringed seal. Phylogenomic analysis based on 9,226 single-copy orthologues placed the Saimaa ringed seal as a sister lineage to the Baltic ringed seal (Pusa hispida botnica), while confirming also other established evolutionary relationships among pinnipeds. Comparative gene family analysis between the Saimaa ringed seal and the closely related grey seal (Halichoerus grypus) revealed lineage-specific differences driven by a limited number of gene families. In the Saimaa ringed seal, expansions were observed in ion transport, cytoskeleton, and regulatory genes, potentially reflecting adaptation to freshwater conditions. In contrast, the grey seal showed expansions in olfaction, immune-and spermatogenesis-associated gene families, including MAGE/MIA genes, consistent with differences in ecology and mating systems. This reference genome provides an important resource for studies of pinniped genome evolution, as well as conservation and population genomics of the Saimaa ringed seal, facilitating future work on genetic diversity, inbreeding, mutational load and adaptive potential in this highly endangered species.