Species-specific SNP arrays for non-invasive genetic monitoring of a vulnerable bat.

Genetic tagging from scats is one of the minimally invasive sampling (MIS) monitoring approaches commonly used to guide management decisions and evaluate conservation efforts. Microsatellite markers have traditionally been used but are prone to genotyping errors. Here, we present a novel method for individual identification in the Threatened ghost bat Macroderma gigas using custom-designed Single Nucleotide Polymorphism (SNP) arrays on the MassARRAY system. We identified 611 informative SNPs from DArTseq data from which three SNP panels (44-50 SNPs per panel) were designed. We applied SNP genotyping and molecular sexing to 209 M. gigas scats collected from seven caves in the Pilbara, Western Australia, employing a two-step genotyping protocol and identifying unique genotypes using a custom-made R package, ScatMatch. Following data cleaning, the average amplification rate was 0.90 ± 0.01 and SNP genotyping errors were low (allelic dropout 0.003 ± 0.000) allowing clustering of scats based on one or fewer allelic mismatches. We identified 19 unique bats (9 confirmed/likely males and 10 confirmed/likely females) from a maternity and multiple transitory roosts, with two male bats detected using roosts, 9 km and 47 m apart. The accuracy of our SNP panels enabled a high level of confidence in the identification of individual bats. Targeted SNP genotyping is a valuable tool for monitoring and tracking of non-model species through a minimally invasive sampling approach.

Close-kin mark-recapture informs critically endangered terrestrial mammal status.

Reliable information on population size is fundamental to the management of threatened species. For wild species, mark-recapture methods are a cornerstone of abundance estimation. Here, we show the first application of the close-kin mark-recapture (CKMR) method to a terrestrial species of high conservation value; the Christmas Island flying-fox (CIFF). The CIFF is the island's last remaining native terrestrial mammal and was recently listed as critically endangered. CKMR is a powerful tool for estimating the demographic parameters central to CIFF management and circumvents the complications arising from the species' cryptic nature, mobility, and difficult-to-survey habitat. To this end, we used genetic data from 450 CIFFs captured between 2015 and 2019 to detect kin pairs. We implemented a novel CKMR model that estimates sex-specific abundance, trend, and mortality and accommodates observations from the kin-pair distribution of male reproductive skew and mate persistence. CKMR estimated CIFF total adult female abundance to be approximately 2050 individuals (95% CI (950, 4300)). We showed that on average only 23% of the adult male population contributed to annual reproduction and strong evidence for between-year mate fidelity, an observation not previously quantified for a Pteropus species in the wild. Critically, our population estimates provide the most robust understanding of the status of this critically endangered population, informing immediate and future conservation initiatives.

Molecular systematic analysis demonstrates that the threatened southern bell frog, Litoria raniformis (Anura: Pelodryadidae) of eastern Australia, comprises two sub-species.

In south-eastern Australia, the pelodryadid Litoria aurea Group (sensu Tyler & Davies 1978) comprises three species: Litoria aurea (Lesson, 1829), Litoria raniformis (Keferstein, 1867), and Litoria castanea (Steindachner, 1867). All three species have been subject to declines over recent decades and taxonomic uncertainty persists among populations on the tablelands in New South Wales. We address the systematics of the Group by analysing mitochondrial and nuclear DNA sequences to assess divergence in the Litoria raniformis from across its current range in New South Wales (NSW), Victoria, South Australia (SA) and Tasmania. We also included samples of Litoria castanea from a recently rediscovered population in the southern tablelands of NSW. Our phylogenetic and population genetic analyses show that Litoria raniformis comprises northern and southern lineages, showing deep mitochondrial DNA sequence divergence (7% net average sequence divergence) and can be diagnosed by fixed allelic differences at more than 4,000 SNP loci. Samples of the northern lineage were collected from the Murray-Darling Basin while those of the southern lineage were collected from south-eastern South Australia, southern and south-eastern Victoria and Tasmania. Analysis of the morphology and bioacoustics did not unequivocally delineate the two lineages. The presence of a hybrid backcross individual in western Victoria at the northern margin of the southern lineage, leads us to assign sub-species status to the two lineages, L. r. raniformis for the northern lineage and L. r. major for the southern lineage. Our data do not unequivocally resolve the taxonomic status of L. castanea which will require molecular genetic analyses of museum vouchers from those parts of the range where L. castanea and L. raniformis are no longer extant. Our data also suggest that human mediated movement of frogs may have occurred over the past 50 years. Our genotyping of vouchers collected in the 1970s from the Mount Lofty Ranges in South Australia detected mitochondrial haplotypes of both sub-species and SNP analysis showed that a single Tasmanian specimen was a backcross with L. r. raniformis ancestry. Movement of L. r. raniformis into Tasmania and both sub-species into the Mount Lofty Ranges are not likely due to passive movements of animals through agricultural commerce, but due to the attractiveness of the species as pets and subsequent escapes or releases, potentially of the larval life stage.

Sheath-tailed bats (Chiroptera: Emballonuridae) from the early Pleistocene Rackham's Roost Site, Riversleigh World Heritage Area, and the distribution of northern Australian emballonurid species.

Sheath-tailed bats (Family Emballonuridae) from the early Pleistocene Rackham's Roost Site cave deposit in the Riversleigh World Heritage Area, north-western Queensland are the oldest recorded occurrence for the family in Australia. The fossil remains consist of maxillary and dentary fragments, as well as isolated teeth, but until now their precise identity has not been assessed. Our study indicates that at least three taxa are represented, and these are distinguished from other Australian emballonurids based on morphometric analysis of craniodental features. Most of the Rackham's Roost Site emballonurid remains are referrable to the modern species Taphozous georgianus Thomas, 1915, but the extant species T. troughtoni Tate, 1952 also appears to be present, as well as a very large, as-yet undetermined species of Saccolaimus Temminck, 1838. We identify craniodental features that clearly distinguish T. georgianus from the externally very similar T. troughtoni. Results suggest that the distributions of T. georgianus and T. troughtoni may have overlapped in north-western Queensland since at least the early Pleistocene.

Development and optimisation of molecular assays for microsatellite genotyping and molecular sexing of non-invasive samples of the ghost bat, Macroderma gigas.

The ghost bat (Macroderma gigas) is endemic to Australia but is under threat, with scarce information available on the genetic health of remaining populations. Here, we develop molecular assays for microsatellite genotyping and molecular sexing of non-invasive samples as a genetic monitoring tool to identify individuals, measure genetic diversity and investigate spatial and temporal patterns of habitat use by ghost bats. We identified novel microsatellites through high-throughput sequencing on the Illumina MiSeq platform. Of 48 loci tested, six markers were added to five previously developed microsatellite loci. We developed three Y-linked (DDX3Y, Zfy and SRY) and one X-linked markers (Zfx) to enable molecular identification of sex. To assess performance, all 11 microsatellite and four sex-linked markers were amplified in three multiplex reactions in 160 M. gigas faecal samples from the Pilbara region, Western Australia. The combined markers offered a high level of individual discrimination (PIDsibs = 0.00002) and we detected 19 bats in total (11 males, 4 females and 4 sex undetermined). The number of alleles per locus ranged from 5 to 14 and the average observed and expected heterozygosity across loci were Ho = 0.735 (0.58-0.91) and uHe = 0.785 (0.59-0.89) respectively. Our molecular assays allowed identification of individuals from faecal samples at multiple time points and spatial locations and enabled us to elucidate patterns of habitat usage at the study site. This study highlights the value of our molecular assays as a potential capture-mark-recapture technique for population monitoring for this species.

Post K-Pg diversification of the mammalian order Eulipotyphla as suggested by phylogenomic analyses of ultra-conserved elements.

The origin of the mammalian order Eulipotyphla has been debated intensively with arguments around whether they began diversifying before or after the Cretaceous-Palaeogene (K-Pg) boundary at 66 Ma. Here, we used an in-solution nucleotide capture method and next generation DNA sequencing to determine the sequence of hundreds of ultra-conserved elements (UCEs), and conducted phylogenomic and molecular dating analyses for the four extant eulipotyphlan lineages-Erinaceidae, Solenodontidae, Soricidae, and Talpidae. Concatenated maximum-likelihood analyses with single or partitioned models and a coalescent species-tree analysis showed that divergences among the four major eulipotyphlan lineages occurred within a short period of evolutionary time, but did not resolve the interrelationships among them. Alternative suboptimal phylogenetic hypotheses received consistently the same amount of support from different UCE loci, and were not significantly different from the maximum likelihood tree topology, suggesting the prevalence of stochastic lineage sorting. Molecular dating analyses that incorporated among-lineage evolutionary rate differences supported a scenario where the four eulipotyphlan families diversified between 57.8 and 63.2 Ma. Given short branch lengths with low support values, traces of rampant genome-wide stochastic lineage sorting, and post K-Pg diversification, we concluded that the crown eulipotyphlan lineages arose through a rapid diversification after the K-Pg boundary when novel niches were created by the mass extinction of species.

A common name for the bat family Rhinonycteridae-the Trident Bats.

Recent elevation in the rank of J.E. Gray's (1866) 'Leaf-nosed Bats' the Rhinonycterina to family level recognised the phylogenetic uniqueness of bats in the extant genera Cloeotis, Paratriaenops, Rhinonicteris and Triaenops, and the fossil genera Brachipposideros and Brevipalatus (Foley et al. 2015). In the systematic summary of that paper, attention was drawn to the issue of correct nomenclature because of past ambiguity around the appropriate spelling of the type genus Rhinonicteris (see also Simmons 2005; Armstrong 2006). However, no suggestion was made for the common name of the Rhinonycteridae, and that used for the Hipposideridae was simply duplicated-'Old World Leaf-nosed Bats'. It would be helpful for this newly distinguished family to have its own appellation-to avoid unnecessary confusion in the wider literature, and to recognise its distinctiveness and evolutionary history.

How and why overcome the impediments to resolution: lessons from rhinolophid and hipposiderid bats.

The phylogenetic and taxonomic relationships among the Old World leaf-nosed bats (Hipposideridae) and the closely related horseshoe bats (Rhinolophidae) remain unresolved. In this study, we generated a novel approximately 10-kb molecular data set of 19 nuclear exon and intron gene fragments for 40 bat species to elucidate the phylogenetic relationships within the families Rhinolophidae and Hipposideridae. We estimated divergence times and explored potential reasons for any incongruent phylogenetic signal. We demonstrated the effects of outlier taxa and genes on phylogenetic reconstructions and compared the relative performance of intron and exon data to resolve phylogenetic relationships. Phylogenetic analyses produced a well-resolved phylogeny, supporting the familial status of Hipposideridae and demonstrated the paraphyly of the largest genus, Hipposideros. A fossil-calibrated timetree and biogeographical analyses estimated that Rhinolophidae and Hipposideridae diverged in Africa during the Eocene approximately 42 Ma. The phylogram, the timetree, and a unique retrotransposon insertion supported the elevation of the subtribe Rhinonycterina to family level and which is diagnosed herein. Comparative analysis of diversification rates showed that the speciose genera Rhinolophus and Hipposideros underwent diversification during the Mid-Miocene Climatic Optimum. The intron versus exon analyses demonstrated the improved nodal support provided by introns for our optimal tree, an important finding for large-scale phylogenomic studies, which typically rely on exon data alone. With the recent outbreak of Middle East respiratory syndrome, caused by a novel coronavirus, the study of these species is urgent as they are considered the natural reservoir for emergent severe acute respiratory syndrome (SARS)-like coronaviruses. It has been shown that host phylogeny is the primary factor that determines a virus's persistence, replicative ability, and can act as a predictor of new emerging disease. Therefore, this newly resolved phylogeny can be used to direct future assessments of viral diversity and to elucidate the origin and development of SARS-like coronaviruses in mammals.

Late pleistocene Australian marsupial DNA clarifies the affinities of extinct megafaunal kangaroos and wallabies.

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.

Molecular phylogeny, biogeography, and habitat preference evolution of marsupials.

Marsupials exhibit great diversity in ecology and morphology. However, compared with their sister group, the placental mammals, our understanding of many aspects of marsupial evolution remains limited. We use 101 mitochondrial genomes and data from 26 nuclear loci to reconstruct a dated phylogeny including 97% of extant genera and 58% of modern marsupial species. This tree allows us to analyze the evolution of habitat preference and geographic distributions of marsupial species through time. We found a pattern of mesic-adapted lineages evolving to use more arid and open habitats, which is broadly consistent with regional climate and environmental change. However, contrary to the general trend, several lineages subsequently appear to have reverted from drier to more mesic habitats. Biogeographic reconstructions suggest that current views on the connectivity between Australia and New Guinea/Wallacea during the Miocene and Pliocene need to be revised. The antiquity of several endemic New Guinean clades strongly suggests a substantially older period of connection stretching back to the Middle Miocene and implies that New Guinea was colonized by multiple clades almost immediately after its principal formation.

Dynamic cerebral autoregulation and tissue oxygenation in amnestic mild cognitive impairment.

BACKGROUND: Vascular disease and dysfunction are associated with the higher risk of Alzheimer's disease hypothetically due to cerebral hypoperfusion. Brain perfusion is protected by cerebral autoregulation, which, under normal conditions, maintains a constant cerebral blood flow and brain tissue oxygenation. OBJECTIVE: To determine whether dynamic regulation of cerebral blood flow and tissue oxygenation is impaired in patients with amnestic mild cognitive impairment (aMCI). METHODS: Twenty-seven patients with aMCI and 15 control subjects with normal cognitive function underwent the measurements of cerebral hemodynamics, brain MR imaging, and neurocognitive assessment. Dynamic regulation of cerebral blood flow and tissue oxygenation were assessed by transfer function analysis of changes in mean blood pressure (MBP), normalized cerebral blood flow velocity (CBFV%), and cerebral tissue oxygenation index (TOI) at baseline and during a sit-stand maneuver. RESULTS: Patients with aMCI demonstrated lower cognitive performance in memory and executive function, accompanied by smaller entorhinal cortex volumes. At baseline, cerebral TOI was lower in patients with aMCI than in control subjects. Lower cerebral TOI was also correlated with lower cognitive performance in memory and executive function in all subjects. Transfer function gain and phase between MBP and CBFV% and between CBFV% and cerebral TOI were not different between the groups. Within aMCI patients, greater oscillations of cerebral TOI and higher transfer function gain between cerebral TOI and CBFV% were associated with the lower scores on delayed recall. CONCLUSION: Dynamic regulation of cerebral tissue oxygenation is associated with neurocognitive dysfunction in aMCI patients.

Cerebral autoregulation of blood velocity and volumetric flow during steady-state changes in arterial pressure.

The validity of using transcranial Doppler measurement of cerebral blood flow velocity (CBFV) to assess cerebral autoregulation (CA) still is a concern. This study measured CBFV in the middle cerebral artery using transcranial Doppler and volumetric cerebral blood flow (CBF) in the internal carotid artery (ICA) using color-coded duplex ultrasonography to assess CA during steady-state changes in mean arterial pressure (MAP). Twenty-one healthy adults participated. MAP was changed stepwise by intravenous infusion of sodium nitroprusside and phenylephrine. Changes in CBFV, CBF, cerebrovascular resistance (CVR=MAP/CBF), or cerebrovascular resistance index (CVRi=MAP/CBFV) were measured to assess CA by linear regression analysis. The relationship between changes in ICA diameter and MAP was assessed. All values were normalized as percentage changes from baseline. Drug-induced changes in MAP were from -26% to 31%. Changes in CBFV and CVRi in response to MAP were linear, and the regression slopes were similar between middle cerebral artery and ICA. However, CBF in ICA remained unchanged despite large changes in MAP. Consistently, a steeper slope of changes in CVR relative to CVRi was observed (0.991 versus 0.804; P<0.05). The ICA diameter changed inversely in response to MAP (r=-0.418; P<0.05). These findings indicate that CA can be assessed with transcranial Doppler measurements of CBFV and CVRi in middle cerebral artery. However, it is likely to be underestimated when compared with the measurements of CBF and CVR in ICA. The inverse relationship between changes in ICA diameter and MAP suggests that large cerebral arteries are involved in CA.

Modelling the prey detection performance of Rhinonicteris aurantia (Chiroptera: Hipposideridae) in different atmospheric conditions discounts the notional role of relative humidity in adaptive evolution.

We examined a recent notion that differences in echolocation call frequency amongst geographic groups of constant frequency (CF)-emitting bats is the result of a trade-off between maximising prey detection range at lower frequencies and enhancing small-prey resolution at higher frequencies in different atmospheric (relative humidity; RH) environments. Isolated populations of the endemic Australian orange leaf-nosed bat Rhinonicteris aurantia were used as an example since geographic isolation in different environments has been a precursor to differences in their characteristic echolocation call frequencies (mean difference c. 6 kHz; means of 114.64 and 120.99 kHz). The influence of both atmospheric temperature and RH on maximum prey detection range was explored through mathematical modelling. This revealed that temperature was of similar importance to relative humidity and that under certain circumstances, each could reduce the effect of the other on ultrasound attenuation rates. The newly developed models contain significant conceptual improvements in method compared to other recent approaches, and can be applied to the situation of any other species of bat. For a given set of atmospheric conditions, the prey detection range of R. aurantia was reduced slightly when call frequency increased by 6 kHz, but an increase in RH, temperature or both reduced detection range significantly. A similar trend was also evident in prey detection volume ratios calculated for the same conditions. Spatial volume ratios were applied to assess the impact of changed atmospheric conditions and prey size on foraging ecology. Reductions in detection range associated with increases in RH and/or temperature also varied in relation to the size (cross sectional area) of insect prey. Modelling demonstrated that small (6 kHz) movements in call frequency could not compensate for the changes in prey detection range and spatial detection volumes that result from significant changes in atmospheric temperature or RH. The notion that differences in RH are the primary cause leading to adaptive evolution and speciation in CF-emitting bats by precipitating intraspecific differences in the mean call frequency of geographically isolated bat populations was not supported by the results of this case study.

DNA content and distribution in ancient feathers and potential to reconstruct the plumage of extinct avian taxa.

Feathers are known to contain amplifiable DNA at their base (calamus) and have provided an important genetic source from museum specimens. However, feathers in subfossil deposits generally only preserve the upper shaft and feather 'vane' which are thought to be unsuitable for DNA analysis. We analyse subfossil moa feathers from Holocene New Zealand rockshelter sites and demonstrate that both ancient DNA and plumage information can be recovered from their upper portion, allowing species identification and a means to reconstruct the appearance of extinct taxa. These ancient DNA sequences indicate that the distal portions of feathers are an untapped resource for studies of museum, palaeontological and modern specimens. We investigate the potential to reconstruct the plumage of pre-historically extinct avian taxa using subfossil remains, rather than assuming morphological uniformity with closely related extant taxa. To test the notion of colour persistence in subfossil feathers, we perform digital comparisons of feathers of the red-crowned parakeet (Cyanoramphus novaezelandiae novaezelandiae) excavated from the same horizons as the moa feathers, with modern samples. The results suggest that the coloration of the moa feathers is authentic, and computer software is used to perform plumage reconstructions of moa based on subfossil remains.

Genetic and acoustic population structuring in the Okinawa least horseshoe bat: are intercolony acoustic differences maintained by vertical maternal transmission?

The origin and meaning of echolocation call frequency variation within rhinolophid bats is not well understood despite an increasing number of allopatric and sympatric examples being documented. A bimodal distribution of mean regional call frequency within the Okinawa-jima Island population of Rhinolophus cornutus pumilus (Rhinolophidae) provided a unique opportunity to investigate geographic call frequency variation early in its development. Individual resting echolocation frequencies, partial mitochondrial DNA D-loop sequences and genotypes from six microsatellite loci were obtained from 288 individuals in 11 colonies across the entire length of the island, and nearby Kume-jima Island. Acoustic differences (5-8 kHz) observed between the north and south regions have been maintained despite evidence of sufficient nuclear gene flow across the middle of the island. Significant subdivision of maternally inherited D-loop haplotypes suggested a limitation of movement of females between regions, but not within the regions, and was evidence of female philopatry. These results support a 'maternal transmission' hypothesis whereby the difference in the constant frequency (CF) component between the regions is maintained by mother-offspring transmission of CF, the restricted dispersal of females between regions and small effective population size. We suggest that the mean 5-8 kHz call frequency difference between the regions might develop through random cultural drift.

Evolution of craniofacial novelty in parrots through developmental modularity and heterochrony.

Parrots (order Psittaciformes) have developed novel cranial morphology. At the same time, they show considerable morphological diversity in the cranial musculoskeletal system, which includes two novel structures: the suborbital arch and the musculus (M.) pseudomasseter. To understand comprehensively the evolutionary pattern and process of novel cranial morphology in parrots, phylogenetic and developmental studies were conducted. Firstly, we undertook phylogenetic analyses based on mitochondrial ribosomal RNA gene sequences to obtain a robust phylogeny among parrots, and secondly we surveyed the cranial morphology of parrots extensively to add new information on the character states. Character mapping onto molecular phylogenies indicated strongly the repeated evolution of both the suborbital arch and the well-developed M. pseudomasseter within parrots. These results also suggested that the direction of evolutionary change is not always identical in the two characters, implying that these characters are relatively independent or decoupled structures behaving as separate modules. Finally, we compared the developmental pattern of jaw muscles among bird species and found a difference in the timing of M. pseudomasseter differentiation between the cockatiel Nymphicus hollandicus (representative of a well-developed condition) and the peach-faced lovebird Agapornis roseicollis (representative of an underdeveloped condition). On the basis of this study, we suggest that in the development of novel traits, modularity and heterochrony facilitate the diversification of parrot cranial morphology.