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Historical DNA obtained from voucher specimens housed in natural history museums worldwide have allowed the study of elusive, rare or even extinct species that in many cases are solely represented by museum holdings. This has resulted in the increase of taxonomic representation of many taxa, has led to the discovery of new species, and has yielded stunning novel insights into the evolutionary history of cryptic or even undescribed species. Peromyscus mekisturus, is a critically endangered cricetid rodent endemic to Mexico and is only known from two museum specimens collected in 1898 and 1947. Intensive field work efforts to attempt to determine if viable populations still exist have failed, suggesting that this species is extinct or is nearing extinction. In addition, a recent study using mitogenomes demonstrated that P. mekisturus forms a well-supported clade outside the genus Peromyscus and hypothesized that this taxon is the sister group of the genus Reithrodontomys. Here, we used target enrichment and high-throughput sequencing of several thousand nuclear ultraconserved elements and mitogenomes to reconstruct dated phylogenies to test the previous phylogenetic hypothesis. We analyzed the holotype and the only other known specimen of P. mekisturus and museum samples from other peromyscine rodents to test the phylogenetic position of the species. Our results confirm that the only two specimens known to science of P. mekisturus belong to the same species and support the hypothesis that this species belongs to an undescribed genus of cricetid rodents that is sister to the genus Reithrodontomys. We dated the origin of P. mekisturus together with other speciation events in peromyscines during the late Pliocene – early Pleistocene and related these events with the Pleistocene climatic cycles. In light of our results, we recommend a taxonomic re-evaluation of this enigmatic species to properly recognize its taxonomic status as a new genus. We also acknowledge the relevance of generating genomic data from type specimens and highlight the need and importance of continuing to build the scientific heritage of the collections to study and better understand past, present, and future biodiversity.

Animal specimens in natural history collections are invaluable resources in examining the historical context of pathogen dynamics in wildlife and spillovers to humans. For example, natural history specimens may reveal new associations between bat species and coronaviruses. However, RNA viruses are difficult to study in historical specimens because protocols for extracting RNA from these specimens have not been optimized. Advances have been made in our ability to recover nucleic acids from formalin-fixed paraffin-embedded samples (FFPE) commonly used in human clinical studies, yet other types of formalin preserved samples have received less attention. Here, we optimize the recovery of RNA from formalin-fixed ethanol-preserved museum specimens in order to improve the usability of these specimens in surveys for zoonotic diseases. We provide RNA quality and quantity measures for replicate tissues subsamples of 22 bat specimens from five bat genera (Rhinolophus, Hipposideros, Megareops, Cynopterus, and Nyctalus) collected in China and Myanmar from 1886 to 2003. As tissues from a single bat specimen were preserved in a variety of ways, including formalin-fixed (8 bats), ethanol-preserved and frozen (13 bats), and flash frozen (2 bats), we were able to compare RNA quality and yield across different preservation methods. RNA extracted from historical museum specimens is highly fragmented, but usable for short-read sequencing and targeted amplification. Incubation of formalin-fixed samples with Proteinase-K following thorough homogenization improves RNA yield. This optimized protocol extends the types of data that can be derived from existing museum specimens and facilitates future examinations of host and pathogen RNA from specimens.

Tree squirrels (Sciurinae, Sciurini) represent a diverse radiation that successfully colonized Europe, Asia and the Americas during the Miocene-Pliocene, but information on their evolutionary history remains unclear. In the Neotropics, they have been shown to exhibit the highest rate of diversification amongst all arboreal squirrels, with strikingly high species accumulation rates in the past 3 Mya. In this study, we investigated the tempo and mode of diversification of tree squirrels using a mitogenome dataset that includes 43 Sciurini species. Our results corroborate the date of origin of the tribe Sciurini around 14 Mya (13.4–15.5) but suggest that their ancestral area was most likely in North America. This is in contrast to previous findings that suggested that the ancestors of this tribe occupied Eurasia. We estimated that cladogenetic events leading to the Eurasian lineages occurred twice at 10.5 and 9.7 Mya. Current North American genera originated in a temporal window from 6.2–2.3 Mya, and the origin of the Neotropical radiation was estimated to have occurred around 6 Mya in northwestern South America, in the Pacific dominion. Remarkably, our results indicate that tree squirrels entered South America at an earlier date than previously estimated. This could have happened either through a land corridor connecting the Caribbean islands or through the Panamanian land bridge. Most cladogenetic events in Eurasia and North America appear to have occurred either late in the Miocene or in the Pleistocene, while the majority of Neotropical cladogenetic events occurred along the Pliocene—right after the South American invasion. We found a fairly constant speciation rate for tree squirrels (averaging 0.29), which contrasts with the peak of lineage accumulation observed in the Pliocene. The absence of fluctuations in the diversification rate may be the result of several extinction events that were responsible for equalizing the number of lineages maintained over time. Finally, we conclude that the South American invasion was not as recent as previously inferred, but the diversification there was indeed very fast.

Sedimentary ancient DNA has been proposed as a key methodology for reconstructing biodiversity over time. Yet, despite the concentration of Earth’s biodiversity in the tropics, this method has rarely been applied in this region. Moreover, the taphonomy of sedimentary DNA, especially in tropical environments, is poorly understood. This study elucidates challenges and opportunities of sedimentary ancient DNA approaches for reconstructing tropical biodiversity. We present shotgun-sequenced metagenomic profiles and DNA degradation patterns from multiple sediment cores from Mubwindi Swamp, located in Bwindi Impenetrable Forest (Uganda), one of the most diverse forests in Africa. We describe the taxonomic composition of the sediments covering the past 2200 years and compare the sedimentary DNA data with a comprehensive set of environmental and sedimentological parameters to unravel the conditions of DNA degradation. Consistent with the preservation of authentic ancient DNA in tropical swamp sediments, DNA concentration and mean fragment length declined exponentially with age and depth, while terminal deamination increased with age. DNA preservation patterns cannot be explained by any environmental parameter alone, but age seems to be the primary driver of DNA degradation in the swamp. Besides degradation, the presence of living microbial communities in the sediment also affects DNA quantity. Critically, 92.3% of our metagenomic data of a total 81.8 million unique, merged reads cannot be taxonomically identified due to the absence of genomic references in public databases. Of the remaining 7.7%, most of the data (93.0%) derive from Bacteria and Archaea, whereas only 0–5.8% are from Metazoa and 0–6.9% from Viridiplantae, in part due to unbalanced taxa representation in the reference data. The plant DNA record at ordinal level agrees well with local pollen data but resolves less diversity. Our animal DNA record reveals the presence of 41 native taxa (16 orders) including Afrotheria, Carnivora, and Ruminantia at Bwindi during the past 2200 years. Overall, we observe no decline in taxonomic richness with increasing age suggesting that several-thousand-year-old information on past biodiversity can be retrieved from tropical sediments. However, comprehensive genomic surveys of tropical biota need prioritization for sedimentary DNA to be a viable methodology for future tropical biodiversity studies.

A surprising amount of hidden phylogenetic diversity exists in the small to medium size, drab colored squirrels of the genus Sundasciurus. This genus is endemic to Sundaland and the Philippines, where it is widespread. An earlier revision of this genus found that the high elevation ‘populations’ of the widespread, lowland slender squirrel (S. tenuis) were different species. Previous phylogenies based on mitochondrial cytochrome b sequences also suggested that the widespread, lowland Low’s squirrel (S. lowii) and the narrow endemic Fraternal squirrel (S. fraterculus) are not reciprocally monophyletic. Additionally, deep divergences have been identified between lineages within Low’s squirrel that date to the early Pliocene. Here we focus on evaluating the relationships and differences within and between populations of these two nominal species using whole mitochondrial genome sequences, nuclear intron sequences, and morphology. We reassess the taxonomy of this group, revalidate the species status of Robinson’s squirrel (Sundasciurus robinsoniBonhote, 1903) support the species level recognition of the Natuna squirrel (Sundasciurus natunensisThomas, 1895) and identify three other lineages that require further study. We estimate times of divergence and integrate geologic history to find that most of the divergences are pre-Pleistocene, and thus predate the Pleistocene flooding of Sundaland. Biogeographic, and ecological factors may have played a more important role than climatic factors in generating these patterns. While divergence in allopatry seems to be the main process driving speciation in lowland Sundaland squirrels (Sundasciurus), ecomorphological and behavioral adaptations in this clade suggest an important role of niche divergence.

Key insights into the evolutionary history of recently extinct or critically endangered species can be obtained through analysis of genomic data collected using high-throughput sequencing and ancient DNA from museum specimens, particularly where specimens are rare. For instance, the evolutionary history of the critically endangered Puebla deer mouse, Peromyscus mekisturus, remains unclear due to discordance between morphological and molecular phylogenetic analyses. However, previous molecular analyses were based on PCR and Sanger sequencing of only a few mitochondrial genes. Here, we used ancient DNA from historical museum specimens followed by target enrichment and high-throughput sequencing of several thousand nuclear ultraconserved elements and whole mitochondrial genomes to test the validity of the previous phylogenetic placement of P. mekisturus. Based on UCEs and mitogenomes, our results revealed that P. mekisturus forms a well-supported distinct lineage outside the clade containing all other members of the Peromyscus melanophrys group. Additionally, the mitogenome phylogeny further supports the placement of P. mekisturus as the sister species of the genus Reithrodontomys. This conflicts with the previous mtDNA phylogenetic reconstruction, in which P. mekisturus was nested within the species P. melanophrys. Our study demonstrates that high-throughput sequencing of ancient DNA, appropriately controlling for contamination and degradation, can provide a robust resolution of the evolutionary history and taxonomic status of species for which few or no modern genetic samples exist. In light of our results and pending further analysis with denser taxon sampling and the addition of morphological data, a re-evaluation of the taxonomy and conservation management plans of P. mekisturus is needed to ensure that the evolutionary distinctiveness of this species is recognized in future conservation efforts.