Genome assemblies are in the process of becoming an increasingly important tool for understanding genetic diversity in threatened species. Unfortunately, due to limited budgets typical for the area of conservation biology, genome assemblies of threatened species, when available, tend to be highly fragmented, represented by tens of thousands of scaffolds not assigned to chromosomal locations. The recent advent of high-throughput chromosome conformation capture (Hi-C) enables more contiguous assemblies containing scaffolds spanning the length of entire chromosomes for little additional cost. These inexpensive contiguous assemblies can be generated using Hi-C scaffolding of existing short-read draft assemblies, where N50 of the draft contigs is larger than 0.1% of the estimated genome size and can greatly improve analyses and facilitate visualization of genome-wide features including distribution of genetic diversity in markers along chromosomes or chromosome-length scaffolds. We compared distribution of genetic diversity along chromosomes of eight mammalian species, including six listed as threatened by IUCN, where both draft genome assemblies and newer chromosome-level assemblies were available. The chromosome-level assemblies showed marked improvement in localization and visualization of genetic diversity, especially where the distribution of low heterozygosity across the genomes of threatened species was not uniform.

Pusa sibirica, the Baikal seal, is the only extant, exclusively freshwater, pinniped species. The pending issue is, how and when they reached their current habitat—the rift lake Baikal, more than three thousand kilometers away from the Arctic Ocean. To explore the demographic history and genetic diversity of this species, we generated a de novo chromosome-length assembly, and compared it with three closely related marine pinniped species. Multiple whole genome alignment of the four species compared with their karyotypes showed high conservation of chromosomal features, except for three large inversions on chromosome VI. We found the mean heterozygosity of the studied Baikal seal individuals was relatively low (0.61 SNPs/kbp), but comparable to other analyzed pinniped samples. Demographic reconstruction of seals revealed differing trajectories, yet remarkable variations in Ne occurred during approximately the same time periods. The Baikal seal showed a significantly more severe decline relative to other species. This could be due to the difference in environmental conditions encountered by the earlier populations of Baikal seals, as ice sheets changed during glacial–interglacial cycles. We connect this period to the time of migration to Lake Baikal, which occurred ~3–0.3 Mya, after which the population stabilized, indicating balanced habitat conditions.

The black-footed ferret (Mustela nigripes) narrowly avoided extinction to become an oft-cited example of the benefits of intensive management, research, and collaboration to save a species through ex situ conservation breeding and reintroduction into its former range. However, the species remains at risk due to possible inbreeding, disease susceptibility, and multiple fertility challenges. Here, we report the de novo genome assembly of a male black-footed ferret generated through a combination of linked-read sequencing, optical mapping, and Hi-C proximity ligation. In addition, we report the karyotype for this species, which was used to anchor and assign chromosome numbers to the chromosome-length scaffolds. The draft assembly was ~2.5 Gb in length, with 95.6% of it anchored to 19 chromosome-length scaffolds, corresponding to the 2n = 38 chromosomes revealed by the karyotype. The assembly has contig and scaffold N50 values of 148.8 kbp and 145.4 Mbp, respectively, and is up to 96% complete based on BUSCO analyses. Annotation of the assembly, including evidence from RNA-seq data, identified 21,406 protein-coding genes and a repeat content of 37.35%. Phylogenomic analyses indicated that the black-footed ferret diverged from the European polecat/domestic ferret lineage 1.6 million yr ago. This assembly will enable research on the conservation genomics of black-footed ferrets and thereby aid in the further restoration of this endangered species.

The stone marten (Martes foina) is an important species for cytogenetic studies in the order Carnivora. ZooFISH probes created from its chromosomes provided a strong and clean signal in chromosome painting experiments and were valuable for studying the evolution of carnivoran genome architecture. The research revealed that the stone marten chromosome set is similar to the presumed ancestral karyotype of the Carnivora, which added an additional value for the species. Using linked-read and Hi-C sequencing, we generated a chromosome-length genome assembly of a male stone marten (Gansu province, China) from a primary cell line. The stone marten assembly had a length of 2.42 Gbp, scaffold N50 of 144 Mbp, and a 96.2% BUSCO completeness score. We identified 19 chromosomal scaffolds (2n = 38) and assigned them chromosome ids based on chromosome painting data. Annotation identified 20,087 protein-coding gene models, of which 18,283 were assigned common names. Comparison of the stone marten assembly with the cat, dog, and human genomes revealed several small syntenic blocks absent on the published painting maps. Finally, we assessed the heterozygosity and its distribution over the chromosomes. The detected low heterozygosity level (0.4 hetSNPs/kbp) and the presence of long runs of homozygosity require further research and a new evaluation of the conservation status of the stone marten in China. Combined with available carnivoran genomes in large-scale synteny analysis, the stone marten genome will highlight new features and events in carnivoran evolution, hidden from cytogenetic approaches.

Mustelinae are among the most diverse and taxonomically complex subfamilies within the Mustelidae, yet their evolutionary history and genetic diversity remain largely unexplored at the whole-genome level. Here, we present the first comprehensive comparative and phylogenomic study of this lineage, integrating nuclear and mitochondrial genomes from 10 species across the Holarctic and Indomalayan realms. Our dataset includes two novel genome assemblies (Mustela strigidorsa, M. sibirica) and an improved genome for M. nivalis, enabling robust cross-species analyses of genome size, chromosomal evolution, genetic diversity, and demographic history. We uncover striking inter- and intraspecific variation in genome-wide heterozygosity and genome size, with evidence of marked homozygosity in some Asian lineages (M. eversmanii, M. sibirica, M. strigidorsa) and remarkable genetic diversity in widespread species such as M. nivalis and M. erminea. Phylogenomic results support the previously suggested split of M. richardsonii from M. erminea, but we found no evidence for speciation within M. nivalis. Ancestral reconstruction of chromosomal rearrangements revealed key chromosomal fissions that shaped the Mustelinae radiation, including early events predating the divergence of modern Mustela species. The results confirmed the suggested ancestral karyotypes of Mustela (2n = 44) and Mustelinae (2n = 42). Finally, demographic reconstructions exposed species-specific responses to Quaternary climatic cycles, ranging from long-term resilience in M. nivalis to repeated population bottlenecks in M. putorius and M. sibirica. Collectively, our findings establish a genomic foundation for future evolutionary and conservation genomic research on this emblematic Mustelidae lineage.

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.