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The evolutionary history of Southern Hemisphere graylings (Retropinnidae) in Aotearoa New Zealand—including the number of colonisation events, the directionality and timing of dispersal, and their relationship to the Australian grayling—is poorly understood. The New Zealand grayling (Prototroctes oxyrhynchus) is the only known freshwater fish species to have gone extinct since human arrival in New Zealand. Despite its historical abundance, only 23 formalin-fixed specimens (both wet and dried) exist in museum collections globally, which were previously non-amenable to palaeogenetic analysis.Here, we used high-throughput DNA sequencing techniques, specifically designed for formalin-fixed specimens, to generate mitochondrial genomes of P. oxyrhynchus, and analysed these within a temporal phylogenetic framework of retropinnid and osmerid taxa.We recovered strong evidence for a sister relationship between the New Zealand and Australian grayling (P. mareana), with the two having a common ancestor around 13.8 Mya (95% HPD: 6.1–23.2 Mya), after the height of Oligocene marine inundation in New Zealand.Our temporal phylogenetic analysis suggests a single marine dispersal event between New Zealand and Australia, though the direction of dispersal is equivocal, followed by divergence into genetically and morphologically distinguishable species through isolation by distance.This study provides further insights into the possible drivers of the extinction of the New Zealand grayling, and highlights how advancements in palaeogenetic techniques can be used to test evolutionary hypotheses in extinct (and living) fish, which have been comparatively neglected in the field of ancient DNA.

Natural history collections worldwide contain a plethora of mollusc shells. Recent studies have detailed the sequencing of DNA extracted from shells up to thousands of years old and from various taphonomic and preservational contexts. However, previous approaches have largely addressed methodological rather than evolutionary research questions. Here, we report the generation of DNA sequence data from mollusc shells using such techniques, applied to Haliotis virginea Gmelin, 1791, a New Zealand abalone, in which morphological variation has led to the recognition of several forms and subspecies. We successfully recovered near-complete mitogenomes from 22 specimens including 12 dry-preserved shells up to 60 years old. We used a combination of palaeogenetic techniques that have not previously been applied to shell, including DNA extraction optimized for ultra-short fragments and hybridization-capture of single-stranded DNA libraries. Phylogenetic analyses revealed three major, well-supported clades comprising samples from: (1) The Three Kings Islands; (2) the Auckland, Chatham and Antipodes Islands; and (3) mainland New Zealand and Campbell Island. This phylogeographic structure does not correspond to the currently recognized forms. Critically, our nonreliance on freshly collected or ethanol-preserved samples enabled inclusion of topotypes of all recognized subspecies as well as additional difficult-to-sample populations. Broader application of these comparatively cost-effective and reliable methods to modern, historical, archaeological and palaeontological shell samples has the potential to revolutionize invertebrate genetic research.

Understanding how natural populations respond to climatic shifts is a fundamental goal of biological research in a fast-changing world. The Southern Ocean represents a fascinating system for assessing large-scale climate-driven biological change, as it contains extremely isolated island groups within a predominantly westerly, circumpolar wind and current system. Blue-eyed shags represent a paradoxical seabird radiation—a circumpolar distribution implies strong dispersal capacity yet their species-rich nature suggests local adaptation and isolation. Here we attempt to resolve this paradox in light of the history of repeated cycles of climate change in the Southern Ocean.

Natural Trap Cave (Bighorn Mountains, Wyoming) preserves an abundance of fossil remains from extinct Late Pleistocene fauna and is situated near a past migration route that likely connected populations in Eastern Beringia and the contiguous US—the ice-free corridor between the Cordilleran and Laurentide icesheets. Some palaeontological evidence supports a correspondingly high affinity between fauna recorded in Natural Trap Cave and Eastern Beringia versus elsewhere in the contiguous US, but this hypothesis has not yet been extensively tested using genetic data. In the present study, we analysed 16 horse specimens and one camel specimen from Natural Trap Cave. Of the horse specimens we analysed, we obtained 10 unique and previously unreported mitochondrial haplotypes belonging to two distinct (extinct) genetic clades—two haplotypes corresponded to a caballine horse (Equus sp.) and eight corresponded to the stilt-legged horse (Haringtonhippus francisci). With only one exception, it appears these newly sequenced individuals all shared a common ancestor more recently with Eastern Beringian individuals than with others from the contiguous US. In addition, mitochondrial data from a specimen assigned to Camelops sp. revealed that it shares a closer affinity with specimens from the Yukon Territory than those from Idaho or Nevada, though all appear to belong to a single species (“yesterday''s camel”; Camelops cf. hesternus). Together, these results are consistent with a high level of genetic connectivity between horse and camel populations in the Bighorn Mountains and Eastern Beringia during the Pleistocene. © 2021 Elsevier Ltd and INQUA

The Bering Land Bridge connecting North America and Eurasia was periodically exposed and inundated by oscillating sea levels during the Pleistocene glacial cycles. This land connection allowed the intermittent dispersal of animals, including humans, between Western Beringia (far north-east Asia) and Eastern Beringia (north-west North America), changing the faunal community composition of both continents. The Pleistocene glacial cycles also had profound impacts on temperature, precipitation, and vegetation, impacting faunal community structure and demography. While these palaeoenvironmental impacts have been studied in many large herbivores from Beringia (e.g., bison, mammoths, horses), the Pleistocene population dynamics of the diverse guild of carnivorans present in the region are less well understood, due to their lower abundances. In this study, we analyse mitochondrial genome data from ancient brown bears (Ursus arctos; n = 103) and lions (Panthera spp.; n = 39), two megafaunal carnivorans that dispersed into North America during the Pleistocene. Our results reveal striking synchronicity in the population dynamics of Beringian lions and brown bears, with multiple waves of dispersal across the Bering Land Bridge coinciding with glacial periods of low sea levels, as well as synchronous local extinctions in Eastern Beringia during Marine Isotope Stage 3. The evolutionary histories of these two taxa underscore the crucial biogeographic role of the Bering Land Bridge in the distribution, turnover, and maintenance of megafaunal populations in North America.

Subtropical and temperate rainforests of Central Eastern Australia are some of the largest remaining fragments of their kind globally. The biota of these rainforests appears to comprise two broad biogeographical elements: a more ancient (Miocene or older) and typically upland temperate (‘Gondwanan’) element and a younger (Plio-Pleistocene) lowland tropical element. We present the first phylogenetic synthesis of the spatiotemporal origins for the eight bird taxa endemic to Central Eastern Australian Rainforests. At least five of these eight focal taxa show Plio-Pleistocene divergences from their respective northern sister taxa, consistent with origins driven by recent expansion and contraction of lowland rainforest. In contrast, two more strictly upland species, the rufous scrub-bird (Atrichornis rufescens) and the logrunner (Orthonyx temminckii), diverged from their nearest living relatives during the Miocene, suggesting potentially longer histories of persistence and more temperate origins. Finally, we did not recover reciprocal monophyly in mitogenomes from the two extant lyrebirds, Albert’s lyrebird (Menura alberti) and the superb lyrebird (Menura novaehollandiae). The disparate divergence ages recovered among all eight taxa are consistent with the biota of the Central Eastern Australian Rainforests comprising isolates either of younger age and tropical lowland origins or of older age and temperate upland origins.

Conservation genomics research often relies on accurate sex information to make inferences about species demography, dispersal, and population structure. However, field determined sex data are not always available and can be subject to human error, while laboratory sex assignment methods such as PCR assays can often be costly and challenging for non-model species. Conservation genomics programs increasingly use reduced-representation genome sequencing to assess neutral and functional genetic diversity, population structure, gene flow and pedigrees in threatened species. Here we demonstrate that sex can be determined from reduced-representation sequencing data produced by the increasingly popular Diversity Arrays Technology sequencing workflow (DArT-seq) using a program originally designed for application to shotgun data. This program—sexassign—compares the “dosage” of sequencing reads mapping to autosomes versus the X chromosome. In the present study, sexassign was used to identify the sex of 60 field-collected Greater Stick-Nest Rat (Leporillus conditor) samples, despite the absence of an annotated reference genome for the species. This “read-dosage” approach is not only more accurate and affordable than traditional sex assignment methods, but can be applied to any diploid organism with a heterogametic sex determination system—including non-model and understudied species of conservation importance—by using FASTQs generated by DArT.

The many high-resolution tools that are uniquely applicable to specimens from the Quaternary period (the past ~2.5 Ma) provide an opportunity to cross-validate data and test hypotheses based on the morphology and distribution of fossils. Among these tools is palaeogenomics - the genome-scale sequencing of genetic material from ancient specimens - that can provide direct insight into ecology and evolution, potentially improving the accuracy of inferences about past ecological communities over longer timescales. Palaeogenomics has revealed instances of over- and underestimation of extinct diversity, detected cryptic faunal migration and turnover, allowed quantification of widespread sex biases and sexual dimorphism in the fossil record, revealed past hybridisation events and hybrid individuals, and has highlighted previously unrecognised routes of zoonotic disease transfer.

The origin of pygmy right whales (Caperea marginata), the smallest and most enigmatic of the living baleen whales, remains contentious. Morphological analyses largely continue to ally Caperea with right whales (balaenids) based on shared cranial features like a tall braincase and a narrow, highly arched rostrum. By contrast, molecular data and some anatomical evidence suggest a closer relationship with rorquals (balaenopterids), but fail to explain “the substantial issue of convergence posed by the many balaenid features of Caperea” (Berta & Demere, 2017). To resolve this question, we sequenced the nuclear genome of C. marginata (812,269,251 paired reads; 47X average depth of coverage, with 89.33% of the genome covered at ≥10X) and subjected it to a multispecies coalescent analysis including representatives of all baleen whale families. Our results confirm Caperea as sister to rorquals and, thus, the convergent origin of its right whale-like anatomy. Considering this overwhelming molecular evidence, we propose that the traditional taxonomic grouping of Caperea with right whales be abandoned.

We describe a new species of Polynesian sandpiper from Henderson Island, Prosobonia sauli sp. nov., based on multiple Holocene fossil bones collected during the Sir Peter Scott Commemorative Expedition to the Pitcairn Islands (1991–92). Prosobonia sauli is the only species of Prosobonia to be described from bone accumulations and extends the record of known extinct Polynesian sandpipers to four. It is readily differentiated from the extant Tuamotu Sandpiper P. parvirostris in several features of the legs and bill, implying ecological adaptations to different environments. The geographically nearest Prosobonia populations to Henderson Island were found on Mangareva, where it is now extinct. A previous record of a species of Prosobonia from Tubuai, Austral Islands, is here shown to belong to the Sanderling Calidris alba. Our analyses of newly sequenced genetic data, which include the mitochondrial genomes of P. parvirostris and the extinct Tahiti Sandpiper P. leucoptera, confidently resolve the position of Prosobonia as sister-taxon to turnstones and calidrine sandpipers. We present a hypothesis for the timing of divergence between species of Prosobonia and other scolopacid lineages. Our results further provide a framework to interpret the evolution of sedentary lineages within the normally highly migratory Scolopacidae.

The largest anthropogenic extinction events during the Holocene occurred on Pacific islands, where thousands of bird populations were lost. Although ancient DNA approaches have become widely used to monitor the genetic variability of species through time, few studies have been conducted to identify the potential cryptic loss of genetic and species diversity within Pacific seabird species. Here we used heterochronous sampling of mitochondrial DNA (Cytochrome b) in the genus Pterodroma from Norfolk Island to quantify potential loss of genetic and species diversity. We particularly focused on the providence petrel P. solandri whose main breeding colony (~ 1,000,000 breeding pairs) became extirpated from Norfolk Island following European settlement circa 1800. We sampled subfossil bones consistent with Pterodroma spp. from Norfolk Island, and performed genetic comparisons with other populations of P. solandri and congeneric species. The majority of subfossil Norfolk Island individuals exhibited the most common mitochondrial haplotype from Lord Howe Island P. solandri, suggesting no appreciable loss of genetic variation as a consequence of the Norfolk Island extirpation. Our findings provide an example where a large seabird population was rapidly extirpated by humans without loss of species-level genetic diversity, probably as a consequence of high connectivity with other populations. However, past connectivity was insufficient to prevent the extirpation itself, which has conservation implications for predicting the resilience of threatened seabirds. In contrast, ancient DNA analyses of smaller Pterodroma bones from Norfolk Island indicate the loss of a second species, potentially P. pycrofti, P. brevipes or another closely related, possibly undescribed taxon, from the Tasman Sea.

Cycles of glacial expansion and contraction throughout the Pleistocene drove increases and decreases, respectively, in the geographical range and population size of many animal species. Genetic data have revealed that during glacial maxima the distribution of many Eurasian animals was restricted to small refugial areas, from which species expanded to reoccupy parts of their former range as the climate warmed. It has been suggested that the extinct eastern moa ( Emeus crassus )—a large, flightless bird from New Zealand—behaved analogously during glacial maxima, possibly surviving only in a restricted area of lowland habitat in the southern South Island of New Zealand during the Last Glacial Maximum (LGM). However, previous studies have lacked the power and geographical sampling to explicitly test this hypothesis using genetic data. Here we analyse 46 ancient mitochondrial genomes from Late Pleistocene and Holocene bones of the eastern moa from across their post-LGM distribution. Our results are consistent with a post-LGM increase in the population size and genetic diversity of eastern moa. We also demonstrate that genetic diversity was higher in eastern moa from the southern extent of their range, supporting the hypothesis that they expanded from a single glacial refugium following the LGM.

A recent study of mammoth subfossil remains has demonstrated the potential of using relatively low-coverage high-throughput DNA sequencing to genetically sex specimens, revealing a strong male-biased sex ratio [P. Pečnerová et al., Curr. Biol. 27, 3505–3510.e3 (2017)]. Similar patterns were predicted for steppe bison, based on their analogous female herd-based structure. We genetically sexed subfossil remains of 186 Holarctic bison (Bison spp.), and also 91 brown bears (Ursus arctos), which are not female herd-based, and found that ∼75% of both groups were male, very close to the ratio observed in mammoths (72%). This large deviation from a 1:1 ratio was unexpected, but we found no evidence for sex differences with respect to DNA preservation, sample age, material type, or overall spatial distribution. We further examined ratios of male and female specimens from 4 large museum mammal collections and found a strong male bias, observable in almost all mammalian orders. We suggest that, in mammals at least, 1) wider male geographic ranges can lead to considerably increased chances of detection in fossil studies, and 2) sexual dimorphic behavior or appearance can facilitate a considerable sex bias in fossil and modern collections, on a previously unacknowledged scale. This finding has major implications for a wide range of studies of fossil and museum material.

Almost a half-century ago excavations at Natural Trap Cave (NTC) began to yield evidence of the steppe paleoecology along the western slope of the Bighorn Mountains in north central Wyoming. The first decade of fieldwork led to the discovery of a diverse fauna that existed at the end of the Last Glacial Maximum. Stratigraphic deposits below the entrance of the cave were studied soon after excavations began, but never formally published. Although stratigraphy, taphonomy, and depositional circumstances were briefly discussed over the following years, little has been done to correlate the numerous stratigraphic schemes used by various authors. In this study, four stratigraphic sections were measured and analysed to establish an easily modifiable lithostratigraphic system of nomenclature. We provide the first correlations of all stratigraphic nomenclature used throughout excavations at NTC to facilitate comparisons with current and previous collections and publications. By leveraging more than 100 radioisotopic dates we developed an age-depth model and chronostratigraphic framework to further interrogate spatiotemporal relationships between strata, paleoenvironmental proxies, and fossil assemblages. Deposition is shown to be discontinuous; sediment accumulation in the study area is restricted to the buildup through peak penultimate and Last Glacial maxima. More recent (

In the Southern Hemisphere, there are two exceptionally large noncontinental landmasses with very long histories of geographical isolation—New Zealand and Madagascar. Both landmasses have unique and distinctive fauna and flora that nonetheless present a number of intriguing parallels, including the long-term preservation of distant sister lineages to otherwise globally widespread groups of animals. Further, both landmasses were also home to >100-kg megafauna that became extinct only in the last thousand years—for example, moa in New Zealand, and elephant birds and giant lemurs in Madagascar—leaving many unanswered questions about the evolution, ecology, and biology of these vanished giants. The generally temperate climate of New Zealand, which is conducive to DNA preservation, has allowed nuclear genome sequencing from extinct species—“paleogenomics”—to be brought to bear in answering these questions (1); however, due to the comparatively warmer and more humid climate of Madagascar—poorer for DNA preservation (2)—it has remained uncertain whether similarly powerful paleogenomic datasets might be obtained for extinct species from Madagascar. Marciniak et al.’s (3) analysis of genomic data from a 1.5-thousand-year-old bone belonging to the giant extinct “koala lemur” ( Megaladapis edwardsi ) now puts Madagascar firmly on the paleogenomic map (4). The gorilla-sized Archaeoindris fontoynontii , which could grow to >150 kg (5), was one of as many … [↵][1]1Email: kieren.mitchell{at}adelaide.edu.au. [1]: #xref-corresp-1-1

The tropical archipelago of Wallacea contains thousands of individual islands interspersed between mainland Asia and Near Oceania, and marks the location of a series of ancient oceanic voyages leading to the peopling of Sahul—i.e., the former continent that joined Australia and New Guinea at a time of lowered sea level—by 50,000 years ago. Despite the apparent deep antiquity of human presence in Wallacea, prior population history research in this region has been hampered by patchy archaeological and genetic records and is largely concentrated upon more recent history that follows the arrival of Austronesian seafarers ~3000–4000 years ago (3–4 ka). To shed light on the deeper history of Wallacea and its connections with New Guinea and Australia, we performed phylogeographic analyses on 656 whole mitogenomes from these three regions, including 186 new samples from eight Wallacean islands and three West Papuan populations. Our results point to a surprisingly dynamic population history in Wallacea, marked by two periods of extensive demographic change concentrated around the Last Glacial Maximum ~15 ka and post-Austronesian contact ~3 ka. These changes appear to have greatly diminished genetic signals informative about the original peopling of Sahul, and have important implications for our current understanding of the population history of the region. 1. Introduction 2. Materials and Methods 2.1. Sample Collection and Ethics 2.2. Mitochondrial Sequence Generation 2.3. Combined Wallacea–Sahul Dataset 2.4. Phylogenetic Parameter Estimation 2.5. Using Ancestral Node Dates from Geographically Exclusive Clades to Infer Demographic History 2.6. Migration Model Inference and Testing 2.7. Simulating and Estimating the Timing of Migration Events 3. Results 3.1. Summary of New Mitochondrial Haplogroups from Wallacea and West Papua 3.2. Phylogeographic Analyses 4. Discussion 4.1. Post-LGM Population Expansions and Movements 4.2. Redistribution of Papuan mtDNA Lineages Following Austronesian Contact 4.3. Comparison with Wallacean Archaeological and Linguistic Records 5. Conclusions

Viperinae is a subfamily of viperid snakes whose fossil record in the Mediterranean islands is, until now, restricted to 12 palaeontological deposits on seven islands. Revision of the material excavated 30 years ago from the Middle/Late Pleistocene–Holocene deposit of Es Pouàs [Eivissa (= Ibiza), Balearic Islands, western Mediterranean] revealed about 6000 bones of a small-sized viper across different stratigraphic levels. Its morphological characteristics are different enough to known species of Vipera to warrant the description of a new species, but the nearly complete mitochondrial genome obtained from this snake based on a sample dated to 16 130 ± 45 bp, suggested it belonged to a new insular population of Lataste’s viper (Vipera latastei), Vipera latastei ebusitana subsp. nov. Phylogenetic analysis indicates that the dispersal of the ancestors of V. l. ebusitana to Eivissa, most probably from a north-east Iberian population, occurred via overwater colonization

We show that the Wotjulum frog, Litoria watjulumensis (Copland, 1957), comprises two deeply divergent mitochondrial DNA lineages that are also reciprocally monophyletic for a nuclear gene locus and have discrete distributions. The taxa are differentiated in multivariate analysis of shape but show no appreciable differences in colour and pattern. The two taxa differ substantially in the degree of female biased sexual size dimorphism, with the western taxon showing considerably more pronounced dimorphism. We subsequently resurrect Litoria (Hyla) spaldingi (Hosmer, 1964) for populations from east of the Daly River system in the Northern Territory through to western Queensland and restrict L. watjulumensis to populations from the Kimberley region of north-western Australia and the Victoria River system of the western Northern Territory. The complex advertisement call of L. spaldingi is described for the first time.

SummaryTwo genera and multiple species of short-faced bear from the Americas went extinct during or toward the end of the Pleistocene, and all belonged to the endemic New World subfamily Tremarctinae [1-7]. Two of these species were giants, growing in excess of 1,000 kg [6, 8, 9], but it remains uncertain how these extinct bears were related to the sole surviving short-faced bear: the spectacled bear (Tremarctos ornatus). Ancient mitochondrial DNA has recently suggested phylogenetic relationships among these lineages that conflict with interpretations based on morphology [1, 10-12]. However, widespread hybridisation and incomplete lineage sorting among extant bears mean that the mitochondrial phylogeny frequently does not reflect the true species tree [13, 14]. Here we present ancient nuclear genome sequences from representatives of the two extinct short-faced bear genera, Arctotherium and Arctodus. Our new data support a third hypothesis for the relationships among short-faced bears, which conflicts with existing mitochondrial and morphological data. Based on genome-wide D-statistics, we suggest that the extant spectacled bear derives substantial ancestry from Pleistocene hybridisation with an extinct short-faced bear lineage, resulting in a discordant phylogenetic signal between the mitochondrion and portions of the nuclear genome.

Two genera and multiple species of short-faced bear from the Americas went extinct during or toward the end of the Pleistocene, and all belonged to the endemic New World subfamily Tremarctinae. Two of these species were giants, growing in excess of 1,000 kg, but it remains uncertain how these extinct bears were related to the sole surviving short-faced bear: the spectacled bear (Tremarctos ornatus). Ancient mitochondrial DNA has recently suggested phylogenetic relationships among these lineages that conflict with interpretations based on morphology. However, widespread hybridisation and incomplete lineage sorting among extant bears mean that the mitochondrial phylogeny frequently does not reflect the true species tree. Here we present ancient nuclear genome sequences from representatives of the two extinct short-faced bear genera, Arctotherium and Arctodus. Our new data support a third hypothesis for the relationships among short-faced bears, which conflicts with existing mitochondrial and morphological data. Based on genome-wide D-statistics, we suggest that the extant spectacled bear derives substantial ancestry from Pleistocene hybridisation with an extinct short-faced bear lineage, resulting in a discordant phylogenetic signal between the mitochondrion and portions of the nuclear genome.

Only five species of the once-diverse Rhinocerotidae remain, making the reconstruction of their evolutionary history a challenge to biologists since Darwin. We sequenced genomes from five rhinoceros species (three extinct and two living), which we compared to existing data from the remaining three living species and a range of outgroups. We identify an early divergence between extant African and Eurasian lineages, resolving a key debate regarding the phylogeny of extant rhinoceroses. This early Miocene (∼16 million years ago [mya]) split post-dates the land bridge formation between the Afro-Arabian and Eurasian landmasses. Our analyses also show that while rhinoceros genomes in general exhibit low levels of genome-wide diversity, heterozygosity is lowest and inbreeding is highest in the modern species. These results suggest that while low genetic diversity is a long-term feature of the family, it has been particularly exacerbated recently, likely reflecting recent anthropogenic-driven population declines., The authors acknowledge support from the Science for Life Laboratory, the Garvan Institute of Medical Research, the Knut and Alice Wallenberg Foundation, and the National Genomics Infrastructure funded by the Swedish Research Council and Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. We thank the Natural History Museum at the University of Oslo for providing the Javan rhinoceros sample. We thank the Museum of the Institute of Plant and Animal Ecology (UB RAS, Ekaterinburg) for providing the sample of Siberian unicorn. M.T.P.G. was supported by European Research Council (ERC) Consolidator grant 681396 (Extinction Genomics). E.D.L. was supported by Independent Research Fund Denmark grant 8021-00218B. A.C. was supported by an Australian Research Council Laureate Fellowship (FL140100260). T.M.B. is supported by funding from the ERC under the European Union’s Horizon 2020 research and innovation program (grant agreement 864203), grant BFU2017-86471-P (MINECO/FEDER, UE), “Unidad de Excelencia María de Maeztu” funded by the AEI (CEX2018-000792-M), Howard Hughes International Early Career, and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). L.D. was supported by the Swedish Research Council (2017-04647) and Formas (2018-01640)., With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2018-000792-M).

The red wolf (Canis rufus) of the eastern US was driven to near-extinction by colonial-era persecution and habitat conversion, which facilitated coyote (C. latrans) range expansion and widespread hybridization with red wolves. The observation of some grey wolf (C. lupus) ancestry within red wolves sparked controversy over whether it was historically a subspecies of grey wolf with its predominant "coyote-like" ancestry obtained from post-colonial coyote hybridization (2-species hypothesis) versus a distinct species closely related to the coyote that hybridized with grey wolf (3-species hypothesis). We analysed mitogenomes sourced from before the 20th century bottleneck and coyote invasion, along with hundreds of modern amplicons, which led us to reject the 2-species model and to investigate a broader phylogeographic 3-species model suggested by the fossil record. Our findings broadly support this model, in which red wolves ranged the width of the American continent prior to arrival of the grey wolf to the mid-continent 60-80 ka; red wolves subsequently disappeared from the mid-continent, relegated to California and the eastern forests, which ushered in emergence of the coyote in their place (50-30 ka); by the early Holocene (12-10 ka), coyotes had expanded into California, where they admixed with and phenotypically replaced western red wolves in a process analogous to the 20th century coyote invasion of the eastern forests. Findings indicate that the red wolf pre-dated not only European colonization but human, and possibly coyote, presence in North America. These findings highlight the urgency of expanding conservation efforts for the red wolf.

Conservation genomics research often relies on accurate sex information to make inferences about species demography, dispersal, and population structure. However, field determined sex data are not always available and can be subject to human error, while laboratory sex determination is costly and often challenging for non-model species. Conservation genomics programs increasingly use reduced-representation genome sequencing to assess neutral and functional genetic diversity, population structure, gene flow and pedigrees in threatened species. Here we demonstrate that sex can be determined from reduced-representation sequencing data produced by the increasingly popular Diversity Arrays Technology sequencing workflow (DArT-seq) using a program originally designed for application to shotgun data. This program – sexassign – compares the “dosage” of sequencing reads mapping to autosomes versus the X chromosome. In the present study, sexassign accurately determined the sex of 60 field-collected Greater Stick-Nest Rat (Leporillus conditor) samples, despite the absence of an annotated reference genome for the species. This “read-dosage” approach is not only more accurate and affordable than traditional sex determination methods, but can be applied to any diploid organism with a heterogametic sex determination system – including non-model and understudied species of conservation importance – by using FASTQs generated by DArT.

The Bering Land Bridge connecting North America and Eurasia was periodically exposed and inundated by oscillating sea levels during the Pleistocene glacial cycles. This land connection allowed the intermittent dispersal of animals, including humans, between Western Beringia (far north-east Asia) and Eastern Beringia (north-west North America), changing the faunal community composition of both continents. The Pleistocene glacial cycles also had profound impacts on temperature, precipitation, and vegetation, impacting faunal community structure and demography. While these paleoenvironmental impacts have been studied in many large herbivores from Beringia (e.g., bison, mammoths, horses), the Pleistocene population dynamics of the diverse guild of carnivorans present in the region are less well understood, due to their lower abundances. In this study, we analyze mitochondrial genome data from ancient brown bears (Ursus arctos; n = 103) and lions (Panthera spp.; n = 39), two megafaunal carnivorans that dispersed into North America during the Pleistocene. Our results reveal striking synchronicity in the population dynamics of Beringian lions and brown bears, with multiple waves of dispersal across the Bering Land Bridge coinciding with glacial periods of low sea levels, as well as synchronous local extinctions in Eastern Beringia during Marine Isotope Stage 3. The evolutionary histories of these two taxa underscore the crucial biogeographic role of the Bering Land Bridge in the distribution, turnover, and maintenance of megafaunal populations in North America.

Wallace's Line demarcates the transition between the differentiated regional faunas of Asia and Australia. However, while patterns of biotic differentiation across these two continental landmasses and the intervening island groups (Wallacea) have been extensively studied, patterns of long-term dispersal and diversification across this region are less well understood. Frogmouths (Aves: Podargidae) are a relictual family of large nocturnal birds represented by three extant genera occurring, respectively, in Asia, ‘Sahul’ (Australia and New Guinea) and the Solomon Islands, thus spanning Wallace's Line. We used new mitochondrial genomes from each of the extant frogmouth genera to estimate the timeline of frogmouth evolution and dispersal across Wallace's Line. Our results suggest that the three genera diverged and dispersed during the mid-Cenozoic between approximately 30 and 40 Mya. These divergences are among the oldest inferred for any trans-Wallacean vertebrate lineage. In addition, our results reveal that the monotypic Solomons frogmouth ( Rigidipenna inexpectata ) is one of the most phylogenetically divergent endemic bird lineages in the southwest Pacific. We suggest that the contemporary distribution of exceptionally deep divergences among extant frogmouth lineages may be explained by colonization of, and subsequent long-term persistence on, island arcs in the southwest Pacific during the Oligocene. These island arcs may have provided a pathway for biotic dispersal out of both Asia and Australia that preceded the formation of extensive emergent landmasses in Wallacea by at least 10 million years.

Dire wolves are considered to be one of the most common and widespread large carnivores in Pleistocene America1, yet relatively little is known about their evolution or extinction. Here, to reconstruct the evolutionary history of dire wolves, we sequenced five genomes from sub-fossil remains dating from 13,000 to more than 50,000 years ago. Our results indicate that although they were similar morphologically to the extant grey wolf, dire wolves were a highly divergent lineage that split from living canids around 5.7 million years ago. In contrast to numerous examples of hybridization across Canidae2,3, there is no evidence for gene flow between dire wolves and either North American grey wolves or coyotes. This suggests that dire wolves evolved in isolation from the Pleistocene ancestors of these species. Our results also support an early New World origin of dire wolves, while the ancestors of grey wolves, coyotes and dholes evolved in Eurasia and colonized North America only relatively recently. © 2021, The Author(s), under exclusive licence to Springer Nature Limited. Acknowledgements We thank the staff at the Carnegie Museum of Natural History, Cincinnati Museum Center, Danish Zoological Museum, Harrison Zoological Museum, Harvard Museum of Comparative Zoology, Idaho Museum of Natural History, Institute of Archaeology (Russian Academy of Sciences), Institute of Systematics and Animal Ecology (Russian Academy of Sciences), Institute of Zoology (Chinese Academy of Sciences), Instituto de Conservação da Natureza e das Florestas, Kansas Museum of Natural History, La Brea Tar Pits and Museum, Ludwig Maximilian University, McClung Museum, Museum of the Institute of Plant and Animal Ecology (Russian Academy of Sciences), Museum national d’Histoire naturelle, National Museums Scotland, Natural History Museum London, Naturalis Biodiversity Center, Naturhistorisches Museum Bern, Smithsonian National Museum of Natural History, Swedish Naturhistoriska Riksmuseet, SYLVATROP, US Bureau of Reclamation, University of California Museum of Paleontology, University of Texas at El Paso, University of Washington Burke Museum and the Zoological Institute (Russian Academy of Sciences; state assignment no. АААА-А19-119032590102-7) for access to specimens in their care; T. Barnosky, S. Bray, A. Farrell, R. Fischer, A. Harris, J. Harris, A. Henrici, P. Holroyd, R. MacPhee, T. Martin, A. Philpot, J. Saunders, J. Southon, G. Storrs, G. Takeuchi, X. Wang and C. Widga for assistance; and L. DeSantis for comments. A.M. used computational and storage services associated with the Hoffman2 Shared Cluster provided by UCLA Institute for Digital Research and Education’s Research Technology Group. DireGWC was sequenced using the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant. We acknowledge the assistance of the Danish National High-Throughput Sequencing Centre, BGI-Europe, the Garvan Institute of Medical Research and the Australian Cancer Research Foundation (ACRF) Cancer Genomics Facility for assistance in Illumina and BGIseq500 data generation. A.R.P. was supported by a Marie Curie COFUND Junior Research Fellowship (Durham University). A.M. was supported by an NSF grant (award number: 1457106) and the QCB Collaboratory Postdoctoral Fellowship (UCLA). L.A.F.F., J.H., A.H.-B. and G.L. were supported by either European Research Council grant (ERC-2013-StG-337574-UNDEAD and ERC-2019-StG-853272-PALAEOFARM) and/or Natural Environmental Research Council grants (NE/K005243/1 and NE/K003259/1). K.S. was supported by a grant from Barrett, the Honors College at Arizona State University. A.T.O. was supported by the Strategic Initiative Funds, Office of the President, Arizona State University to the Institute of Human Origins DNA and Human Origins at Arizona State University project. L.A.F.F. was supported by a Junior Research Fellowship (Wolfson College, University of Oxford) and L.A.F.F. and A. Carmagnini were supported by the Wellcome Trust (210119/Z/18/Z). S.G. was supported by Carlsbergfondet grant CF14–0995 and Marie Skłodowska-Curie Actions grant 655732-WhereWolf. M.T.P.G. was supported by ERC Consolidator grant 681396-Extinction Genomics. B.S. and J.K. were supported by IMLS MG-30-17-0045-17 and NSF DEB-1754451. A.H.-B. was supported by the Leverhulme Trust (ECF-2017-315). A. Cooper, K.J.M. and H.H. were supported by the Australian Research Council. A.T.S. and G.G. were supported by Australian Government Research Training Program Scholarships. A.T.L. was supported by the Peter Buck Postdoctoral Fellowship from the Smithsonian Institution’s National Museum of Natural History. Y.V.K. was supported by the by State Assignment of the Sobolev Institute of Geology and Mineralogy.

The Tasmanian tiger or thylacine (Thylacinus cynocephalus) was the largest carnivorous Australian marsupial to survive into the modern era. Despite last sharing a common ancestor with the eutherian canids ~160 million years ago, their phenotypic resemblance is considered the most striking example of convergent evolution in mammals. The last known thylacine died in captivity in 1936 and many aspects of the evolutionary history of this unique marsupial apex predator remain unknown. Here we have sequenced the genome of a preserved thylacine pouch young specimen to clarify the phylogenetic position of the thylacine within the carnivorous marsupials, reconstruct its historical demography and examine the genetic basis of its convergence with canids. Retroposon insertion patterns placed the thylacine as the basal lineage in Dasyuromorphia and suggest incomplete lineage sorting in early dasyuromorphs. Demographic analysis indicated a long-term decline in genetic diversity starting well before the arrival of humans in Australia. In spite of their extraordinary phenotypic convergence, comparative genomic analyses demonstrated that amino acid homoplasies between the thylacine and canids are largely consistent with neutral evolution. Furthermore, the genes and pathways targeted by positive selection differ markedly between these species. Together, these findings support models of adaptive convergence driven primarily by cis-regulatory evolution.

Aim The Tasmanian tiger, or thylacine, is an infamous example of a recent human-mediated extinction. Confined to the island of Tasmania in historical times, thylacines were hunted to extinction

The dormice (Gliridae) are a family of rodents represented by relatively few extant species, though the family was much more species-rich during the Early Miocene. Intergeneric phylogenetic relationships among glirids in some cases remain unresolved, despite extensive molecular and morphological analyses. Uncertainty is greatest with respect to the relationships among fossil taxa and how extinct lineages are related to modern species. The fossil genus Hypnomys from the Balearic Islands (western Mediterranean Sea) includes the Late Pleistocene–Holocene species Hypnomys morpheus, which has variously been considered a close relative or subgenus of the extant Eliomys. In the present study, we sequenced ancient mitochondrial DNA from H. morpheus, which suggests a sister relationship with the extant members of Eliomys. In addition, the pairwise sequence variation between Hypnomys and Eliomys is higher than that observed between congeneric glirid species (e.g., many Graphiurus spp.), which allows us to reject the hypothesis that Hypnomys is a subgenus of Eliomys. Our molecular dating analyses suggest that Hypnomys and Eliomys diverged 13.67 million years ago (95% highest posterior density [HPD] = 7.39–20.07). The relatively early split between these genera together with the molar morphology of early representatives of Hypnomys points to a Middle-Late Miocene origin from a continental glirid with a complex molar pattern, such as Vasseuromys or a closely related genus., This research was supported by a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (project MEDITADNA, PIOF‐GA‐2011‐300854, FP7‐PEOPLE), and it is included in the Research Project CGL2016‐79795, [“Cambios holocénicos en la biodiversidad animal de las islas de la Macaronesia y de Baleares. II,” CGL2016‐80000‐P (Dirección General de Investigación Científica y Técnica, Ministerio de Economía y Competitividad)] and SGR2017‐859 (AGAUR, Gencat).

Adopting the name Canis dingo for the Dingo to explicitly denote a species-level taxon separate from other canids was suggested by Crowther et al. (2014) as a means to eliminate taxonomic instability and contention. However, Jackson et al. (2017), using standard taxonomic and nomenclatural approaches and principles, called instead for continued use of the nomen C. familiaris for all domestic dogs and their derivatives, including the Dingo. (This name, C. familiaris, is applied to all dogs that derive from the domesticated version of the Gray Wolf, Canis lupus, based on nomenclatural convention.) The primary reasons for this call by Jackson et al. (2017) were: (1) a lack of evidence to show that recognizing multiple species amongst the dog, including the Dingo and New Guinea Singing Dog, was necessary taxonomically, and (2) the principle of nomenclatural priority (the name familiaris Linnaeus, 1758, antedates dingo Meyer, 1793). Overwhelming current evidence from archaeology and genomics indicates that the Dingo is of recent origin in Australia and shares immediate ancestry with other domestic dogs as evidenced by patterns of genetic and morphological variation. Accordingly, for Smith et al. (2019) to recognise Canis dingo as a distinct species, the onus was on them to overturn current interpretations of available archaeological, genomic, and morphological datasets and instead show that Dingoes have a deeply divergent evolutionary history that distinguishes them from other named forms of Canis (including C. lupus and its domesticated version, C. familiaris). A recent paper by Koepfli et al. (2015) demonstrates exactly how this can be done in a compelling way within the genus Canis—by demonstrating deep evolutionary divergence between taxa, on the order of hundreds of thousands of years, using data from multiple genetic systems. Smith et al. (2019) have not done this; instead they have misrepresented the content and conclusions of Jackson et al. (2017), and contributed extraneous arguments that are not relevant to taxonomic decisions. Here we dissect Smith et al. (2019), identifying misrepresentations, to show that ecological, behavioural and morphological evidence is insufficient to recognise Dingoes as a separate species from other domestic dogs. We reiterate: the correct binomial name for the taxon derived from Gray Wolves (C. lupus) by passive and active domestication, including Dingoes and other domestic dogs, is Canis familiaris. We are strongly sympathetic to arguments about the historical, ecological, cultural, or other significance of the Dingo, but these are issues that will have to be considered outside of the more narrow scope of taxonomy and nomenclature.

The relationships of the extinct New Zealand ravens (Corvus spp.) are poorly understood. We sequenced the mitogenomes of the two currently recognised species and found they were sister-taxa to a clade comprising the Australian raven, little raven, and forest raven (C.coronoides, C. mellori and C. tasmanicus respectively). The divergence between the New Zealand ravens and Australian raven clade occurred in the latest Pliocene, which coincides with the onset of glacial deforestation. We also found that the divergence between the two putative New Zealand species C. antipodum and C. moriorum probably occurred in the late Pleistocene making their separation as species untenable. Consequently, we consider Corax antipodum (Forbes, 1893) to be a subspecies of Corvus moriorum Forbes, 1892. We re-examine the osteological evidence that led 19th century researchers to assign the New Zealand taxa to a separate genus, and re-assess these features in light of our new phylogenetic hypotheses. Like previous researchers, we conclude that the morphology of the palate of C. moriorum is unique among the genus Corvus, and suggest this may be an adaptation for a specialist diet.

The New Zealand acanthisittid wrens are the sister-taxon to all other "perching birds" (Passeriformes) and - including recently extinct species - represent the most diverse endemic passerine family in New Zealand. Consequently, they are important for understanding both the early evolution of Passeriformes and the New Zealand biota. However, five of the seven species have become extinct since the arrival of humans in New Zealand, complicating evolutionary analyses. The results of morphological analyses have been largely equivocal, and no comprehensive genetic analysis of Acanthisittidae has been undertaken. We present novel mitochondrial genome sequences from four acanthisittid species (three extinct, one extant), allowing us to resolve the phylogeny and revise the taxonomy of acanthisittids. Reanalysis of morphological data in light of our genetic results confirms a close relationship between the extant rifleman (Acanthisitta chloris) and an extinct Miocene wren (Kuiornis indicator), making Kuiornis a useful calibration point for molecular dating of passerines. Our molecular dating analyses reveal that the stout-legged wrens (Pachyplichas) diverged relatively recently from a more gracile (Xenicus-like) ancestor. Further, our results suggest a possible Early Oligocene origin of the basal Lyall's wren (Traversia) lineage, which would imply that Acanthisittidae survived the Oligocene marine inundation of New Zealand and therefore that the inundation was not complete.

Background: Recombinase Polymerase Amplification (RPA) is a relatively new isothermal methodology for amplifying DNA. RPA is similar to traditional PCR in that it produces an amplicon that is defined by the annealing of two opposing oligonucleotide primers. However, while PCR relies on repeated heating and cooling cycles to denature and amplify DNA fragments, RPA is performed at a single moderate temperature and uses enzymatic activity to drive amplification. While RPA is commonly used in field-based monitoring of pathogens, it is unknown whether RPA is a viable alternative to PCR for the amplification of ancient DNA. Methods: In this study, PCR and RPA were used to amplify shotgun and mitochondrial DNA enriched libraries made from extracts from four ancient bison bone samples. Sequencing data from the amplified libraries were examined for biases in sequence content (read length and GC content), fraction of unique reads mapping to a reference sequence, and mitochondrial polymorphisms detection accuracy. Results: In comparison to PCR, RPA had a variable effect on sequence content, except in the mitochondrial DNA enriched libraries where RPA consistently reduced mean read length by approximately 30 bp. RPA increased the number of unique shotgun reads that mapped to a cattle nuclear reference by between 9% and 99% versus PCR. In contrast, RPA reduced the fraction of unique mitochondrial DNA enriched reads by > 26%, possibly due to the preferential amplification of small unmappable molecules.Both RPA and PCR data allowed the identification of similar variants in mitochondrial DNA enriched libraries, suggesting that the accuracy of the two amplification methods is comparable. Importantly, RPA was able to generate sequencing libraries at approximately a sixth of the cost of PCR. These results indicate that RPA is a viable alternative to PCR for amplification of shotgun libraries made from ancient DNA but may not be suitable for all ancient DNA applications.

Hybridization capture with in-solution oligonucleotide probes has quickly become the preferred method for enriching specific DNA loci from degraded or ancient samples prior to high-throughput sequencing (HTS). Several companies synthesize sets of probes for in-solution hybridization capture, but these commercial reagents are usually expensive. Methods for economical in-house probe synthesis have been described, but they do not directly address one of the major advantages of commercially synthesised probes: that probe sequences matching many species can be synthesised in parallel and pooled. The ability to make "phylogenetically diverse" probes increases the cost-effectiveness of commercial probe sets, as they can be used across multiple projects (or for projects involving multiple species). However, it is labour-intensive to replicate this with in-house methods, as template molecules must first be generated for each species of interest. While it has been observed that probes can be used to enrich for phylogenetically distant targets, the ability of this effect to compensate for the lack of phylogenetically diverse probes in in-house synthesised probe sets has not been tested. In this study, we present a refined protocol for in-house RNA probe synthesis and evaluated the ability of probes generated using this method from a single species to successfully enrich for the target locus in phylogenetically distant species. We demonstrated that probes synthesized using long-range PCR products from a placental mammal mitochondrion (Bison spp.) could be used to enrich for mitochondrial DNA in birds and marsupials (but not plants). Importantly, our results were obtained for approximately a third of the cost of similar commercially available reagents.

The recently extinct New Zealand adzebills (Aptornithidae, Aptornis spp.) were an enigmatic group of large flightless birds that have long eluded precise taxonomic assignment as they do not closely resemble any extant birds. Adzebills were nearly wingless, weighed approximately 16&ndash, 19 kg, and possessed massive adze-like reinforced bills whose function remains unknown. Using hybridisation enrichment and high-throughput sequencing of DNA extracted from subfossil bone and eggshell, near-complete mitochondrial genomes were successfully assembled from the two Quaternary adzebill species: the North Island Adzebill (Aptornis otidiformis) and South Island Adzebill (A. defossor). Molecular phylogenetic analyses confirm that adzebills are members of the Ralloidea (rails and allies) and are sister-taxon to the Sarothruridae, which our results suggest comprises the Madagascan wood rails (Mentocrex, two likely sp.) in addition to the tiny (&lt, 50 gram) rail-like Afro-Madagascan flufftails (Sarothrura, 9 spp.). Node age estimates indicate that the split between adzebills and Sarothruridae occurred ~39.6 Ma, suggesting that the ancestors of the adzebills arrived in New Zealand by long-distance dispersal rather than continental vicariance. This newly identified biogeographic link between physically distant New Zealand and Afro-Madagascar, echoed by the relationship between the New Zealand kiwi (Apterygiformes) and Madagascan elephant-birds (Aepyornithiformes), suggests that the adzebill&rsquo, s near relatives were formerly more widespread. In addition, our estimate for the divergence time between the two Quaternary adzebill species (0.2&ndash, 2.3 Ma) coincides with the emergence of a land-bridge between the North and South islands of New Zealand (ca. 1.5&ndash, 2 Ma). This relatively recent divergence suggests that North Island adzebills are the result of a relatively recent dispersal from the South Island, from which the earliest (Miocene) adzebill fossil has been described.

Myotragus balearicus was the last living representative of an extinct caprine lineage endemic to the Balearic Islands (Western Mediterranean), which became extinct following the arrival of humans around 4300 years ago. The tribal attribution of Myotragus based on morphological analyses has been complicated due to its unusual morphological characteristics, though most studies agree on its inclusion within Caprini (also including modern sheep and goats). Analyses of short fragments of ancient mitochondrial DNA have suggested Ovis (comprising extant sheep) as sister taxon to Myotragus, although other authors have suggested alternative placements within Caprini using the same data. In the present study, we present a complete high-depth mitochondrial genome of M. balearicus, which allowed us to test the previously proposed Ovis/Myotragus clade and revealed a closer relationship between Myotragus and the takin (Budorcas taxicolor). The results of our molecular clock analyses suggested that the split between Myotragus and Budorcas occurred around 7.1 Mya, which is compatible with the arrival of the ancestor of Myotragus to the Balearic Islands during the Messinian Salinity Crisis (5.97–5.33 Mya). We also conducted a preliminary phylogeographic analysis of M. balearicus in Mallorca, which revealed weak spatial and temporal structure within the population during the Holocene., This research was supported by a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (project MEDITADNA, PIOF-GA-2011-300854, FP7-PEOPLE) (Pere Bover), and Australian Research Council Laureate and Discovery Project funding (Alan Cooper). This work is also integrated within the research project CGL2016–79795-R (Josep A. Alcover). Pere Bover has a research contract from the Fundación Agencia Aragonesa para la Investigación y el Desarrollo (ARAID).

Understanding extinction events requires an unbiased record of the chronology and ecology of victims and survivors. The rhinoceros Elasmotherium sibiricum, known as the ‘Siberian unicorn’, was believed to have gone extinct around 200,000 years ago—well before the late Quaternary megafaunal extinction event. However, no absolute dating, genetic analysis or quantitative ecological assessment of this species has been undertaken. Here, we show, by accelerator mass spectrometry radiocarbon dating of 23 individuals, including cross-validation by compound-specific analysis, that E. sibiricum survived in Eastern Europe and Central Asia until at least 39,000 years ago, corroborating a wave of megafaunal turnover before the Last Glacial Maximum in Eurasia, in addition to the better-known late-glacial event. Stable isotope data indicate a dry steppe niche for E. sibiricum and, together with morphology, a highly specialized diet that probably contributed to its extinction. We further demonstrate, with DNA sequencing data, a very deep phylogenetic split between the subfamilies Elasmotheriinae and Rhinocerotinae that includes all the living rhinoceroses, settling a debate based on fossil evidence and confirming that the two lineages had diverged by the Eocene. As the last surviving member of the Elasmotheriinae, the demise of the ‘Siberian unicorn’ marked the extinction of this subfamily.

The musk ox (Ovibos moschatus) is the only surviving member of a group of Pleistocene North American musk ox genera (Praeovibos, Ovibos, Bootherium, Euceratherium, and Soergelia) whose taxonomy is uncertain. The helmeted musk ox (Bootherium bombifrons) and the woodland musk ox (Symbos cavifrons) have been synonymised as male and female forms of a single Nearctic species found from Alaska, in the north, to Texas, in the south. However, this reclassification has not been tested using molecular data, despite the potential to use ancient DNA to examine these late Pleistocene taxa. In the present study, we sequenced mitochondrial genomes from seven subfossil musk ox specimens (originally identified as Bootherium and/or Symbos), allowing us to evaluate the identity of these muskoxen, explore their phylogeography, and estimate the timeline for their evolution. We also used nuclear genomic data to determine the sex of six of our seven samples. Ultimately, our molecular data support the synonymisation of the North American muskoxen Bootherium and Symbos.

The red-toothed shrews (Soricinae) are the most widespread subfamily of shrews, distributed from northern South America to North America and Eurasia. Within this subfamily, the tribe Nectogalini includes the fossil species Nesiotites hidalgo recorded from the Late Pleistocene to Holocene of the Balearic Islands (Western Mediterranean). Although there is a consensus about the close relationship between the extinct red-toothed shrew genera Nesiotites and Asoriculus based on morphology, molecular data are necessary to further evaluate the phylogenetic relationships of the Balearic fossils. We obtained a near complete mitochondrial genome of N. hidalgo, allowing the first molecular phylogenetic analysis of this species. Analyses based on 15,167 bp of the mitochondrial genome placed N. hidalgo as close relative to the extant Himalayan shrew (Soriculus nigrescens), and a combined analysis using molecular and morphological data confirm that N. hidalgo and Asoriculus gibberodon are sister-taxa with S. nigrescens as the immediate outgroup. Molecular clock and divergence estimates suggest that the split between N. hidalgo and its closest living relative occurred around 6.44 Ma, which is in agreement with the previously proposed colonisation of the Balearic Islands from mainland Europe by nectogaline shrews during the Messinian Salinity Crisis (5.97–5.33 My ago). Our results highlight that it is possible to retrieve genetic data from extinct small mammals from marginal environments for DNA preservation. Additional finds from the fossil record of Soricinae from the Eurasian Late Miocene/Early Pliocene are needed to shed further light on the still confusing taxonomy and paleobiogeography of this clade.

Understanding extinction events requires an unbiased record of the chronology and ecology of victims and survivors. The rhinoceros Elasmotherium sibiricum, known as the 'Siberian unicorn', was believed to have gone extinct around 200,000 years ago-well before the late Quaternary megafaunal extinction event. However, no absolute dating, genetic analysis or quantitative ecological assessment of this species has been undertaken. Here, we show, by accelerator mass spectrometry radiocarbon dating of 23 individuals, including cross-validation by compound-specific analysis, that E. sibiricum survived in Eastern Europe and Central Asia until at least 39,000 years ago, corroborating a wave of megafaunal turnover before the Last Glacial Maximum in Eurasia, in addition to the better-known late-glacial event. Stable isotope data indicate a dry steppe niche for E. sibiricum and, together with morphology, a highly specialized diet that probably contributed to its extinction. We further demonstrate, with DNA sequencing data, a very deep phylogenetic split between the subfamilies Elasmotheriinae and Rhinocerotinae that includes all the living rhinoceroses, settling a debate based on fossil evidence and confirming that the two lineages had diverged by the Eocene. As the last surviving member of the Elasmotheriinae, the demise of the 'Siberian unicorn' marked the extinction of this subfamily.

The species-level systematics of the marsupial family Phalangeridae, particularly Phalanger, are poorly understood, due partly to the family’s wide distribution across Australia, New Guinea, eastern Indonesia, and surrounding islands. In order to refine the species-level systematics of Phalangeridae, and improve our understanding of their evolution, we generated 36 mitochondrial ND2 DNA sequences from multiple species and sample localities. We combined our new data with available sequences and produced the most comprehensive molecular phylogeny for Phalangeridae to date. Our analyses (1) strongly support the monophyly of the three phalangerid subfamilies (Trichosurinae, Ailuropinae, Phalangerinae); (2) reveal the need to re-examine all specimens currently identified as ‘Phalanger orientalis’; and (3) suggest the elevation of the Solomon Island P. orientalis subspecies to species level (P. breviceps Thomas, 1888). In addition, samples of P. orientalis from Timor formed a clade, consistent with an introduction by humans from a single source population. However, further research on east Indonesian P. orientalis populations will be required to test this hypothesis, resolve inconsistencies in divergence time estimates, and locate the source population and taxonomic status of the Timor P. orientalis.

Understanding the evolution of Australia's extinct marsupial megafauna has been hindered by a relatively incomplete fossil record and convergent or highly specialized morphology, which confound phylogenetic analyses. Further, the harsh Australian climate and early date of most megafaunal extinctions (39-52 ka) means that the vast majority of fossil remains are unsuitable for ancient DNA analyses. Here, we apply cross-species DNA capture to fossils from relatively high latitude, high altitude caves in Tasmania. Using low-stringency hybridization and high-throughput sequencing, we were able to retrieve mitochondrial sequences from two extinct megafaunal macropodid species. The two specimens, Simosthenurus occidentalis (giant short-faced kangaroo) and Protemnodon anak (giant wallaby), have been radiocarbon dated to 46-50 and 40-45 ka, respectively. This is significantly older than any Australian fossil that has previously yielded DNA sequence information. Processing the raw sequence data from these samples posed a bioinformatic challenge due to the poor preservation of DNA. We explored several approaches in order to maximize the signal-to-noise ratio in retained sequencing reads. Our findings demonstrate the critical importance of adopting stringent processing criteria when distant outgroups are used as references for mapping highly fragmented DNA. Based on the most stringent nucleotide data sets (879 bp for S. occidentalis and 2,383 bp for P. anak), total-evidence phylogenetic analyses confirm that macropodids consist of three primary lineages: Sthenurines such as Simosthenurus (extinct short-faced kangaroos), the macropodines (all other wallabies and kangaroos), and the enigmatic living banded hare-wallaby Lagostrophus fasciatus (Lagostrophinae). Protemnodon emerges as a close relative of Macropus (large living kangaroos), a position not supported by recent morphological phylogenetic analyses.