Chromosome-length genome assemblies and cytogenomic analyses of pangolins reveal remarkable chromosome counts and plasticity.

We report the first chromosome-length genome assemblies for three species in the mammalian order Pholidota: the white-bellied, Chinese, and Sunda pangolins. Surprisingly, we observe extraordinary karyotypic plasticity within this order and, in female white-bellied pangolins, the largest number of chromosomes reported in a Laurasiatherian mammal: 2n = 114. We perform the first karyotype analysis of an African pangolin and report a Y-autosome fusion in white-bellied pangolins, resulting in 2n = 113 for males. We employ a novel strategy to confirm the fusion and identify the autosome involved by finding the pseudoautosomal region (PAR) in the female genome assembly and analyzing the 3D contact frequency between PAR sequences and the rest of the genome in male and female white-bellied pangolins. Analyses of genetic variability show that white-bellied pangolins have intermediate levels of genome-wide heterozygosity relative to Chinese and Sunda pangolins, consistent with two moderate declines of historical effective population size. Our results reveal a remarkable feature of pangolin genome biology and highlight the need for further studies of these unique and endangered mammals.

Waking the undead: Implications of a soft explosive model for the timing of placental mammal diversification.

The explosive, long fuse, and short fuse models represent competing hypotheses for the timing of placental mammal diversification. Support for the explosive model, which posits both interordinal and intraordinal diversification after the KPg mass extinction, derives from morphological cladistic studies that place Cretaceous eutherians outside of crown Placentalia. By contrast, most molecular studies favor the long fuse model wherein interordinal cladogenesis occurred in the Cretaceous followed by intraordinal cladogenesis after the KPg boundary. Phillips (2016) proposed a soft explosive model that allows for the emergence of a few lineages (Xenarthra, Afrotheria, Euarchontoglires, Laurasiatheria) in the Cretaceous, but otherwise agrees with the explosive model in positing the majority of interordinal diversification after the KPg mass extinction. Phillips (2016) argues that rate transference errors associated with large body size and long lifespan have inflated previous estimates of interordinal divergence times, and further suggests that most interordinal divergences are positioned after the KPg boundary when rate transference errors are avoided through the elimination of calibrations in large-bodied and/or long lifespan clades. Here, we show that rate transference errors can also occur in the opposite direction and drag forward estimated divergence dates when calibrations in large-bodied/long lifespan clades are omitted. This dragging forward effect results in the occurrence of more than half a billion years of 'zombie lineages' on Phillips' preferred timetree. By contrast with ghost lineages, which are a logical byproduct of an incomplete fossil record, zombie lineages occur when estimated divergence dates are younger than the minimum age of the oldest crown fossils. We also present the results of new timetree analyses that address the rate transference problem highlighted by Phillips (2016) by deleting taxa that exceed thresholds for body size and lifespan. These analyses recover all interordinal divergence times in the Cretaceous and are consistent with the long fuse model of placental diversification. Finally, we outline potential problems with morphological cladistic analyses of higher-level relationships among placental mammals that may account for the perceived discrepancies between molecular and paleontological estimates of placental divergence times.

Resolution of a concatenation/coalescence kerfuffle: partitioned coalescence support and a robust family-level tree for Mammalia.

Recent phylogenetic analyses of a large dataset for mammalian families (169 taxa, 26 loci) portray contrasting results. Supermatrix (concatenation) methods support a generally robust tree with only a few inconsistently resolved polytomies, whereas MP-EST coalescence analysis of the same dataset yields a weakly supported tree that conflicts with many traditionally recognized clades. Here, we evaluate this discrepancy via improved coalescence analyses with reference to the rich history of phylogenetic studies on mammals. This integration clearly demonstrates that both supermatrix and coalescence analyses of just 26 loci yield a congruent, well-supported phylogenetic hypothesis for Mammalia. Discrepancies between published studies are explained by implementation of overly simple DNA substitution models, inadequate tree-search routines and limitations of the MP-EST method. We develop a simple measure, partitioned coalescence support (PCS), which summarizes the distribution of support and conflict among gene trees for a given clade. Extremely high PCS scores for outlier gene trees at two nodes in the mammalian tree indicate a troubling bias in the MP-EST method. We conclude that in this age of phylogenomics, a solid understanding of systematics fundamentals, choice of valid methodology and a broad knowledge of a clade's taxonomic history are still required to yield coherent phylogenetic inferences.

Range-Wide Snow Leopard Phylogeography Supports Three Subspecies.

The snow leopard, Panthera uncia, is an elusive high-altitude specialist that inhabits vast, inaccessible habitat across Asia. We conducted the first range-wide genetic assessment of snow leopards based on noninvasive scat surveys. Thirty-three microsatellites were genotyped and a total of 683 bp of mitochondrial DNA sequenced in 70 individuals. Snow leopards exhibited low genetic diversity at microsatellites (AN = 5.8, HO = 0.433, HE = 0.568), virtually no mtDNA variation, and underwent a bottleneck in the Holocene (∼8000 years ago) coinciding with increased temperatures, precipitation, and upward treeline shift in the Tibetan Plateau. Multiple analyses supported 3 primary genetic clusters: (1) Northern (the Altai region), (2) Central (core Himalaya and Tibetan Plateau), and (3) Western (Tian Shan, Pamir, trans-Himalaya regions). Accordingly, we recognize 3 subspecies, Panthera uncia irbis (Northern group), Panthera uncia uncia (Western group), and Panthera uncia uncioides (Central group) based upon genetic distinctness, low levels of admixture, unambiguous population assignment, and geographic separation. The patterns of variation were consistent with desert-basin "barrier effects" of the Gobi isolating the northern subspecies (Mongolia), and the trans-Himalaya dividing the central (Qinghai, Tibet, Bhutan, and Nepal) and western subspecies (India, Pakistan, Tajikistan, and Kyrgyzstan). Hierarchical Bayesian clustering analysis revealed additional subdivision into a minimum of 6 proposed management units: western Mongolia, southern Mongolia, Tian Shan, Pamir-Himalaya, Tibet-Himalaya, and Qinghai, with spatial autocorrelation suggesting potential connectivity by dispersing individuals up to ∼400 km. We provide a foundation for global conservation of snow leopard subspecies, and set the stage for in-depth landscape genetics and genomic studies.

Genetically based low oxygen affinities of felid hemoglobins: lack of biochemical adaptation to high-altitude hypoxia in the snow leopard.

Genetically based modifications of hemoglobin (Hb) function that increase blood-O2 affinity are hallmarks of hypoxia adaptation in vertebrates. Among mammals, felid Hbs are unusual in that they have low intrinsic O2 affinities and reduced sensitivities to the allosteric cofactor 2,3-diphosphoglycerate (DPG). This combination of features compromises the acclimatization capacity of blood-O2 affinity and has led to the hypothesis that felids have a restricted physiological niche breadth relative to other mammals. In seeming defiance of this conjecture, the snow leopard (Panthera uncia) has an extraordinarily broad elevational distribution and occurs at elevations above 6000 m in the Himalayas. Here, we characterized structural and functional variation of big cat Hbs and investigated molecular mechanisms of Hb adaptation and allosteric regulation that may contribute to the extreme hypoxia tolerance of the snow leopard. Experiments revealed that purified Hbs from snow leopard and African lion exhibited equally low O2 affinities and DPG sensitivities. Both properties are primarily attributable to a single amino acid substitution, ß2His→Phe, which occurred in the common ancestor of Felidae. Given the low O2 affinity and reduced regulatory capacity of feline Hbs, the extreme hypoxia tolerance of snow leopards must be attributable to compensatory modifications of other steps in the O2-transport pathway.

Accelerated evolution of CES7, a gene encoding a novel major urinary protein in the cat family.

Cauxin is a novel urinary protein recently identified in the domestic cat that regulates the excretion of felinine, a pheromone precursor involved in sociochemical communication and territorial marking of domestic and wild felids. Understanding the evolutionary history of cauxin may therefore illuminate molecular adaptations involved in the evolution of pheromone-based communication, recognition, and mate selection in wild animals. We sequenced the gene encoding cauxin, CES7, in 22 species representing all major felid lineages, and multiple outgroups and showed that it has undergone rapid evolutionary change preceding and during the diversification of the cat family. A comparison between feline cauxin and orthologous carboxylesterases from other mammalian lineages revealed evidence of strong positive Darwinian selection within and between several cat lineages, enriched at functionally important sites of the protein. The higher rate of radical amino acid replacements in small felids, coupled with the lack of felinine and extremely low levels of cauxin in the urine of the great cats (Panthera), correlates with functional divergence of this gene in Panthera, and its putative loss in the snow leopard. Expression studies found evidence for several alternatively spliced transcripts in testis and brain, suggesting additional roles in male reproductive fitness and behavior. Our work presents the first report of strong positive natural selection acting on a major urinary protein of nonrodent mammals, providing evidence for parallel selection pressure on the regulation of pheromones in different mammalian lineages, despite the use of different metabolic pathways. Our results imply that natural selection may drive rapid changes in the regulation of pheromones in urine among the different cat species, which in turn may influence social behavior, such as territorial marking and conspecific recognition, therefore serving as an important mechanism for the radiation of this group of mammals.

Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis.

BACKGROUND: The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis. RESULTS: Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks. CONCLUSIONS: Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis.

Status and distribution of jaguarundi in Texas and Northeastern México: Making the case for extirpation and initiation of recovery in the United States.

The jaguarundi (Puma yagouaroundi) is a small felid with a historical range from central Argentina through southern Texas. Information on the current distribution of this reclusive species is needed to inform recovery strategies in the United States where its last record was in 1986 in Texas. From 2003 to 2021, we conducted camera-trap surveys across southern Texas and northern Tamaulipas, México to survey for medium-sized wild cats (i.e., ocelots [Leopardus pardalis], bobcats [Lynx rufus], and jaguarundi). After 350,366 trap nights at 685 camera sites, we did not detect jaguarundis at 16 properties or along 2 highways (1050 km2) in Texas. However, we recorded 126 jaguarundi photographic detections in 15,784 trap nights on 2 properties (125.3 km2) in the northern Sierra of Tamaulipas, Tamaulipas, México. On these properties, latency to detection was 72 trap nights, with a 0.05 probability of detection per day and 0.73 photographic event rate every 100 trap nights. Due to a lack of confirmed class I sightings (e.g., specimen, photograph) in the 18 years of this study, and no other class I observations since 1986 in the United States, we conclude that the jaguarundi is likely extirpated from the United States. Based on survey effort and results from México, we would have expected to detect jaguarundis over the course of the study if still extant in Texas. We recommend that state and federal agencies consider jaguarundis as extirpated from the United States and initiate recovery actions as mandated in the federal jaguarundi recovery plan. These recovery actions include identification of suitable habitat in Texas, identification of robust populations in México, and re-introduction of the jaguarundi to Texas.

Methane, arsenic, selenium and the origins of the DMSO reductase family.

Mononuclear molybdoenzymes of the dimethyl sulfoxide reductase (DMSOR) family catalyze a number of reactions essential to the carbon, nitrogen, sulfur, arsenic, and selenium biogeochemical cycles. These enzymes are also ancient, with many lineages likely predating the divergence of the last universal common ancestor into the Bacteria and Archaea domains. We have constructed rooted phylogenies for over 1,550 representatives of the DMSOR family using maximum likelihood methods to investigate the evolution of the arsenic biogeochemical cycle. The phylogenetic analysis provides compelling evidence that formylmethanofuran dehydrogenase B subunits, which catalyze the reduction of CO2 to formate during hydrogenotrophic methanogenesis, constitutes the most ancient lineage. Our analysis also provides robust support for selenocysteine as the ancestral ligand for the Mo/W atom. Finally, we demonstrate that anaerobic arsenite oxidase and respiratory arsenate reductase catalytic subunits represent a more ancient lineage of DMSORs compared to aerobic arsenite oxidase catalytic subunits, which evolved from the assimilatory nitrate reductase lineage. This provides substantial support for an active arsenic biogeochemical cycle on the anoxic Archean Earth. Our work emphasizes that the use of chalcophilic elements as substrates as well as the Mo/W ligand in DMSORs has indelibly shaped the diversification of these enzymes through deep time.

The horse Y chromosome as an informative marker for tracing sire lines.

Analysis of the Y chromosome is the best-established way to reconstruct paternal family history in humans. Here, we applied fine-scaled Y-chromosomal haplotyping in horses with biallelic markers and demonstrate the potential of our approach to address the ancestry of sire lines. We de novo assembled a draft reference of the male-specific region of the Y chromosome from Illumina short reads and then screened 5.8 million basepairs for variants in 130 specimens from intensively selected and rural breeds and nine Przewalski's horses. Among domestic horses we confirmed the predominance of a young'crown haplogroup' in Central European and North American breeds. Within the crown, we distinguished 58 haplotypes based on 211 variants, forming three major haplogroups. In addition to two previously characterised haplogroups, one observed in Arabian/Coldblooded and the other in Turkoman/Thoroughbred horses, we uncovered a third haplogroup containing Iberian lines and a North African Barb Horse. In a genealogical showcase, we distinguished the patrilines of the three English Thoroughbred founder stallions and resolved a historic controversy over the parentage of the horse 'Galopin', born in 1872. We observed two nearly instantaneous radiations in the history of Central and Northern European Y-chromosomal lineages that both occurred after domestication 5,500 years ago.

Horse Y chromosome assembly displays unique evolutionary features and putative stallion fertility genes.

Dynamic evolutionary processes and complex structure make the Y chromosome among the most diverse and least understood regions in mammalian genomes. Here, we present an annotated assembly of the male specific region of the horse Y chromosome (eMSY), representing the first comprehensive Y assembly in odd-toed ungulates. The eMSY comprises single-copy, equine specific multi-copy, PAR transposed, and novel ampliconic sequence classes. The eMSY gene density approaches that of autosomes with the highest number of retained X-Y gametologs recorded in eutherians, in addition to novel Y-born and transposed genes. Horse, donkey and mule testis RNAseq reveals several candidate genes for stallion fertility. A novel testis-expressed XY ampliconic sequence class, ETSTY7, is shared with the parasite Parascaris genome, providing evidence for eukaryotic horizontal transfer and inter-chromosomal mobility. Our study highlights the dynamic nature of the Y and provides a reference sequence for improved understanding of equine male development and fertility.

Loss of genetic diversity among ocelots in the United States during the 20th century linked to human induced population reductions.

Ocelots (Leopardus pardalis) in the United States currently exhibit low levels of genetic diversity. One hypothesis for this observation is that habitat fragmentation, resulting from human induced changes in the landscape during the 20(th) century, created island populations with highly reduced gene flow and increased genetic drift and inbreeding. In an effort to investigate this, we used a portion of the mitochondrial control region and 11 autosomal microsatellite loci to examine historical levels of genetic diversity and infer temporal changes in ocelot populations between 1853 and 2005. Levels of genetic diversity were higher in historical ocelot populations than in extant populations from Texas. The earliest documented loss of mitochondrial haplotype diversity occurred at Laguna Atascosa National Wildlife Refuge. The second extant population inhabiting private lands in Willacy County retained higher levels of genetic diversity through the 1990s, but subsequently lost diversity over the next decade. A similar pattern was observed for autosomal microsatellite loci. This supports the argument that low levels of genetic diversity in Texas are related to human induced population reductions and fragmentation, both of which threaten the remaining ocelots in the United States. At this time, the best means of mitigating the continued erosion of genetic variation are translocation of individuals either from larger populations in Mexico to Texas, or between the Texas populations.

Macroevolutionary dynamics and historical biogeography of primate diversification inferred from a species supermatrix.

Phylogenetic relationships, divergence times, and patterns of biogeographic descent among primate species are both complex and contentious. Here, we generate a robust molecular phylogeny for 70 primate genera and 367 primate species based on a concatenation of 69 nuclear gene segments and ten mitochondrial gene sequences, most of which were extracted from GenBank. Relaxed clock analyses of divergence times with 14 fossil-calibrated nodes suggest that living Primates last shared a common ancestor 71-63 Ma, and that divergences within both Strepsirrhini and Haplorhini are entirely post-Cretaceous. These results are consistent with the hypothesis that the Cretaceous-Paleogene mass extinction of non-avian dinosaurs played an important role in the diversification of placental mammals. Previous queries into primate historical biogeography have suggested Africa, Asia, Europe, or North America as the ancestral area of crown primates, but were based on methods that were coopted from phylogeny reconstruction. By contrast, we analyzed our molecular phylogeny with two methods that were developed explicitly for ancestral area reconstruction, and find support for the hypothesis that the most recent common ancestor of living Primates resided in Asia. Analyses of primate macroevolutionary dynamics provide support for a diversification rate increase in the late Miocene, possibly in response to elevated global mean temperatures, and are consistent with the fossil record. By contrast, diversification analyses failed to detect evidence for rate-shift changes near the Eocene-Oligocene boundary even though the fossil record provides clear evidence for a major turnover event ("Grande Coupure") at this time. Our results highlight the power and limitations of inferring diversification dynamics from molecular phylogenies, as well as the sensitivity of diversification analyses to different species concepts.

Noninvasive genetic population survey of snow leopards (Panthera uncia) in Kangchenjunga conservation area, Shey Phoksundo National Park and surrounding buffer zones of Nepal.

BACKGROUND: The endangered snow leopard is found throughout major mountain ranges of Central Asia, including the remote Himalayas. However, because of their elusive behavior, sparse distribution, and poor access to their habitat, there is a lack of reliable information on their population status and demography, particularly in Nepal. Therefore, we utilized noninvasive genetic techniques to conduct a preliminary snow leopard survey in two protected areas of Nepal. RESULTS: A total of 71 putative snow leopard scats were collected and analyzed from two different areas; Shey Phoksundo National Park (SPNP) in the west and Kangchanjunga Conservation Area (KCA) in the east. Nineteen (27%) scats were genetically identified as snow leopards, and 10 (53%) of these were successfully genotyped at 6 microsatellite loci. Two samples showed identical genotype profiles indicating a total of 9 individual snow leopards. Four individual snow leopards were identified in SPNP (1 male and 3 females) and five (2 males and 3 females) in KCA. CONCLUSIONS: We were able to confirm the occurrence of snow leopards in both study areas and determine the minimum number present. This information can be used to design more in-depth population surveys that will enable estimation of snow leopard population abundance at these sites.

The historical biogeography of Mammalia.

Palaeobiogeographic reconstructions are underpinned by phylogenies, divergence times and ancestral area reconstructions, which together yield ancestral area chronograms that provide a basis for proposing and testing hypotheses of dispersal and vicariance. Methods for area coding include multi-state coding with a single character, binary coding with multiple characters and string coding. Ancestral reconstruction methods are divided into parsimony versus Bayesian/likelihood approaches. We compared nine methods for reconstructing ancestral areas for placental mammals. Ambiguous reconstructions were a problem for all methods. Important differences resulted from coding areas based on the geographical ranges of extant species versus the geographical provenance of the oldest fossil for each lineage. Africa and South America were reconstructed as the ancestral areas for Afrotheria and Xenarthra, respectively. Most methods reconstructed Eurasia as the ancestral area for Boreoeutheria, Euarchontoglires and Laurasiatheria. The coincidence of molecular dates for the separation of Afrotheria and Xenarthra at approximately 100 Ma with the plate tectonic sundering of Africa and South America hints at the importance of vicariance in the early history of Placentalia. Dispersal has also been important including the origins of Madagascar's endemic mammal fauna. Further studies will benefit from increased taxon sampling and the application of new ancestral area reconstruction methods.

Impacts of the Cretaceous Terrestrial Revolution and KPg extinction on mammal diversification.

Previous analyses of relations, divergence times, and diversification patterns among extant mammalian families have relied on supertree methods and local molecular clocks. We constructed a molecular supermatrix for mammalian families and analyzed these data with likelihood-based methods and relaxed molecular clocks. Phylogenetic analyses resulted in a robust phylogeny with better resolution than phylogenies from supertree methods. Relaxed clock analyses support the long-fuse model of diversification and highlight the importance of including multiple fossil calibrations that are spread across the tree. Molecular time trees and diversification analyses suggest important roles for the Cretaceous Terrestrial Revolution and Cretaceous-Paleogene (KPg) mass extinction in opening up ecospace that promoted interordinal and intraordinal diversification, respectively. By contrast, diversification analyses provide no support for the hypothesis concerning the delayed rise of present-day mammals during the Eocene Period.

The mitochondrial genome sequence of the Tasmanian tiger (Thylacinus cynocephalus).

We report the first two complete mitochondrial genome sequences of the thylacine (Thylacinus cynocephalus), or so-called Tasmanian tiger, extinct since 1936. The thylacine's phylogenetic position within australidelphian marsupials has long been debated, and here we provide strong support for the thylacine's basal position in Dasyuromorphia, aided by mitochondrial genome sequence that we generated from the extant numbat (Myrmecobius fasciatus). Surprisingly, both of our thylacine sequences differ by 11%-15% from putative thylacine mitochondrial genes in GenBank, with one of our samples originating from a direct offspring of the previously sequenced individual. Our data sample each mitochondrial nucleotide an average of 50 times, thereby providing the first high-fidelity reference sequence for thylacine population genetics. Our two sequences differ in only five nucleotides out of 15,452, hinting at a very low genetic diversity shortly before extinction. Despite the samples' heavy contamination with bacterial and human DNA and their temperate storage history, we estimate that as much as one-third of the total DNA in each sample is from the thylacine. The microbial content of the two thylacine samples was subjected to metagenomic analysis, and showed striking differences between a wild-captured individual and a born-in-captivity one. This study therefore adds to the growing evidence that extensive sequencing of museum collections is both feasible and desirable, and can yield complete genomes.

Molecular and genomic data identify the closest living relative of primates.

A full understanding of primate morphological and genomic evolution requires the identification of their closest living relative. In order to resolve the ancestral relationships among primates and their closest relatives, we searched multispecies genome alignments for phylogenetically informative rare genomic changes within the superordinal group Euarchonta, which includes the orders Primates, Dermoptera (colugos), and Scandentia (treeshrews). We also constructed phylogenetic trees from 14 kilobases of nuclear genes for representatives from most major primate lineages, both extant colugos, and multiple treeshrews, including the pentail treeshrew, Ptilocercus lowii, the only living member of the family Ptilocercidae. A relaxed molecular clock analysis including Ptilocercus suggests that treeshrews arose approximately 63 million years ago. Our data show that colugos are the closest living relatives of primates and indicate that their divergence occurred in the Cretaceous.