Detection of novel coronaviruses from dusky fruit bat (Penthetor lucasi) in Sarawak, Malaysian Borneo.

BACKGROUND: Sarawak has one of the highest diversity of fruit bats species (family Pteropodidae) in Malaysia, with 19 species described. Most coronavirus (CoV) studies have mainly focused on insectivorous bats, resulting in a lack of information on CoVs present in frugivorous bats. In addition, bat CoV surveillance activities are lacking in Malaysia. OBJECTIVES: Our study focuses on determining the presence of bat CoVs in dusky fruit bat (Penthetor lucasi). METHODS: Guano samples belonging to P. lucasi were collected from Wind Cave Nature Reserve. The samples were screened for the presence of CoVs using validated hemi-nested consensus RNA-dependent RNA polymerase consensus primers. RESULTS: The bat CoV positivity rate was 38.5% (n = 15/39), with the viruses belonging to two subgenera: Alphacoronavirus (α-CoV) and Betacoronavirus (ß-CoV). Phylogenetic analysis revealed that CoVs from 14 samples of P. lucasi belong to the genus α-CoV and may represent previously described genetic lineages in insectivorous bats in Wind Cave. However, only one sample of P. lucasi was detected with ß-CoV which is closely related to subgenus Nobecovirus, which is commonly seen in frugivorous bats. CONCLUSIONS: This study provides the first available data on CoVs circulating in P. lucasi.

Solving the taxonomic identity of Hipposideros cineraceus sensu lato (Chiroptera: Hipposideridae) in the Thai-Malay Peninsula, with the description of a new species.

A new species of small Hipposideros in the bicolor group is described based on specimens from Thailand and Malaysia. It can be distinguished from other small Hipposideros in Southeast Asia by a combination of external, craniodental, and bacular morphology, as well as echolocation call frequency. The new species has a distinct rounded swelling on the internarial septum of the noseleaf, with a forearm length of 35.3-42.6 mm, greatest skull length of 15.94-17.90 mm, and a call frequency of maximum energy of 132.3-144.0 kHz. Although clearly different in morphology, the new species forms a sister clade with H. kunzi and H. bicolor in the phylogenetic trees based on mitochondrial DNA. In addition, this study reports echolocation and genetic data, with a confirmed record of H. einnaythu from Thailand for the first time. The new species most closely resembles H. einnaythu. However, it differs in the details of the noseleaf and craniodental morphology, and it has a genetic distance of 9.6% and 10.4% based on mitochondrial COI and ND2, respectively. It is currently documented from five localities: two in peninsular Thailand, at Hala Forest in Yala Province, and Phru To Daeng Swamp Forest in Narathiwat Province, one from peninsular Malaysia at Krau Wildlife Reserve in Pahang, and another two in Sabah, Malaysian Borneo at Gunung Kinabalu, and near Madai Caves. However, it is likely that many previous records of "H. cineraceus" from Borneo refer to this species. Most records of the species are from lowland evergreen rainforest, though one record from Sabah was at 1800m. The roosting sites for this new species are currently unknown. Future research with a combination of data such as genetics, echolocation and morphology would be necessary to further determine the species geographic distribution in Southeast Asia.

ChiroVox: a public library of bat calls.

Recordings of bat echolocation and social calls are used for many research purposes from ecological studies to taxonomy. Effective use of these relies on identification of species from the recordings, but comparative recordings or detailed call descriptions to support identification are often lacking for areas with high biodiversity. The ChiroVox website (https://www.chirovox.org) was created to facilitate the sharing of bat sound recordings together with their metadata, including biodiversity data and recording circumstances. To date, more than 30 researchers have contributed over 3,900 recordings of nearly 200 species, making ChiroVox the largest open-access bat call library currently available. Each recording has a unique identifier that can be cited in publications; hence the acoustic analyses are repeatable. Most of the recordings available through the website are from bats whose species identities are confirmed, so they can be used to determine species in recordings where the bats were not captured or could not be identified. We hope that with the help of the bat researcher community, the website will grow rapidly and will serve as a solid source for bat acoustic research and monitoring.

Molecular data from the holotype of the enigmatic Bornean Black Shrew, Suncusater Medway, 1965 (Soricidae, Crocidurinae), place it in the genus Palawanosorex.

Although Borneo has received more attention from biologists than most other islands in the Malay Archipelago, many questions regarding the systematic relationships of Bornean mammals remain. Using next-generation sequencing technology, we obtained mitochondrial DNA sequences from the holotype of Suncusater, the only known specimen of this shrew. Several shrews collected recently in Sarawak are closely aligned, both morphologically and mitochondrially, with the holotype of S.ater. Phylogenetic analyses of mitochondrial sequences indicate that the S.ater holotype and new Sarawak specimens do not belong to the genus Suncus, but instead are most closely related to Palawanosorexmuscorum. Until now Palawanosorex has been known only from the neighboring Philippine island of Palawan. Additional sequences from nuclear ultra-conserved elements from the new Sarawak specimens strongly support a sister relationship to P.muscorum. We therefore transfer ater to Palawanosorex. The new specimens demonstrate that P.ater is more widespread in northern Borneo than previously recorded. Continued sampling of Bornean mammal diversity and reexamination of type material are critical in understanding the evolutionary history of the biologically rich Malay Archipelago.

Fungal communities in bat guano, speleothem surfaces, and cavern water in Madai cave, Northern Borneo (Malaysia).

The island of Borneo is a global biodiversity hotspot. However, its limestone caves are one of its least-studied ecosystems. We report for the first time the fungal species richness, diversity and abundance from Madai cave, situated in north-eastern Borneo. Environmental samples from inside the cave environment were collected (guano, speleothem, and cavern water) via opportunistic sampling. The dilution method was performed for isolation of fungi. Morphological characterisation and molecular analysis of the ITS region were utilised for the identification of isolates. Fifty-five pure cultures of fungi were attained, comprising 32 species from 15 genera, eight orders, and two divisions, Ascomycota and Basidiomycota. Ascomycetes dominated the fungal composition, accounting for 53 (96%) out of 55 total isolates. Penicillium spp. accounted for more than 47.1% of fungal abundance in all sample types. However, Aspergillus spp. had the highest occurrence rate, being isolated from all environmental samples except one. Purpureocillium lilacinum was isolated most frequently, appearing in five separate samples across all three substrates. Annulohypoxylon nitens, Ganoderma australe, Pyrrhoderma noxium, and Xylaria feejeensis were discovered and reported for the first time from the cave environment. This study provides additional data for further research on the mycoflora of Sabah's various ecosystems, especially limestone caves.

Evolutionary pattern of Macaca fascicularis in Southeast Asia inferred using Y-chromosomal gene.

BACKGROUND: We analyzed a combined segment (2032-bp) of the sex-determining region and the testis-specific protein of the Y-chromosome (Y-DNA) gene to clarify the gene flow and phylogenetic relationships of the long-tailed macaques (Macaca fascicularis) in Southeast Asia. Phylogenetic relationships were constructed using the maximum likelihood, Bayesian inference, and the median-joining network from a total of 164 adult male M. fascicularis from 62 localities in Malaysia, including sequences from the other regions from previous studies. RESULTS: Based on Y-DNA, we confirm the presence of two lineages of M. fascicularis: the Indochinese and Sundaic lineages. The Indochinese lineage is represented by M. fascicularis located northwards of the Surat Thani-Krabi depression region and is introgressed by the Macaca mulatta Y-DNA. The Sundaic lineage is free from such hybridization event, thus defined as the original carrier of the M. fascicularis Y-DNA. We further revealed that the Sundaic lineage differentiated into two forms: the insular and the continental forms. The insular form, which represents the ancestral form of M. fascicularis, consists of two haplotypes: a single homogenous haplotype occupying the island of Borneo, Philippines, and southern Sumatra; and the Javan haplotype. The more diverse continental form consists of 17 haplotypes in which a dominant haplotype was shared by individuals from southern Thai Peninsular (south of Surat Thani-Krabi depression), Peninsular Malaysia, and Sumatra. Uniquely, Sumatra contains both the continental and insular Y-DNA which can be explained by a secondary contact hypothesis. CONCLUSIONS: Overall, the findings in this study are important: (1) to help authority particularly in Malaysia on the population management activities including translocation and culling of conflict M. fascicularis, (2) to identify the unknown origin of captive M. fascicularis used in biomedical research, and; (3) the separation between the continental and insular forms warrants for the treatment as separate management units.

Preserve a Voucher Specimen! The Critical Need for Integrating Natural History Collections in Infectious Disease Studies.

Despite being nearly 10 months into the COVID-19 (coronavirus disease 2019) pandemic, the definitive animal host for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causal agent of COVID-19, remains unknown. Unfortunately, similar problems exist for other betacoronaviruses, and no vouchered specimens exist to corroborate host species identification for most of these pathogens. This most basic information is critical to the full understanding and mitigation of emerging zoonotic diseases. To overcome this hurdle, we recommend that host-pathogen researchers adopt vouchering practices and collaborate with natural history collections to permanently archive microbiological samples and host specimens. Vouchered specimens and associated samples provide both repeatability and extension to host-pathogen studies, and using them mobilizes a large workforce (i.e., biodiversity scientists) to assist in pandemic preparedness. We review several well-known examples that successfully integrate host-pathogen research with natural history collections (e.g., yellow fever, hantaviruses, helminths). However, vouchering remains an underutilized practice in such studies. Using an online survey, we assessed vouchering practices used by microbiologists (e.g., bacteriologists, parasitologists, virologists) in host-pathogen research. A much greater number of respondents permanently archive microbiological samples than archive host specimens, and less than half of respondents voucher host specimens from which microbiological samples were lethally collected. To foster collaborations between microbiologists and natural history collections, we provide recommendations for integrating vouchering techniques and archiving of microbiological samples into host-pathogen studies. This integrative approach exemplifies the premise underlying One Health initiatives, providing critical infrastructure for addressing related issues ranging from public health to global climate change and the biodiversity crisis.

Description, molecular characteristics and Wolbachia endosymbionts of Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. (Nematoda: Filarioidea) from the Bornean bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae) of Sarawak, Malaysia.

BACKGROUND: The genus Onchocerca Diesing, 1841 includes species of medical importance, such as O. volvulus (Leuckart, 1893), which causes river blindness in the tropics. Recently, zoonotic onchocercosis has been reported in humans worldwide. In Japan, O. dewittei japonica Uni, Bain & Takaoka, 2001 from wild boars is a causative agent for this zoonosis. Many filarioid nematodes are infected with Wolbachia endosymbionts which exhibit various evolutionary relationships with their hosts. While investigating the filarial fauna of Borneo, we discovered an undescribed Onchocerca species in the bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae). METHODS: We isolated Onchocerca specimens from bearded pigs and examined their morphology. For comparative material, we collected fresh specimens of O. d. dewittei Bain, Ramachandran, Petter & Mak, 1977 from banded pigs (S. scrofa vittatus Boie) in Peninsular Malaysia. Partial sequences of three different genes (two mitochondrial genes, cox1 and 12S rRNA, and one nuclear ITS region) of these filarioids were analysed. By multi-locus sequence analyses based on six genes (16S rDNA, ftsZ, dnaA, coxA, fbpA and gatB) of Wolbachia, we determined the supergroups in the specimens from bearded pigs and those of O. d. dewittei. RESULTS: Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. is described on the basis of morphological characteristics and its genetic divergence from congeners. Molecular characteristics of the new species revealed its close evolutionary relationship with O. d. dewittei. Calculated p-distance for the cox1 gene sequences between O. borneensis n. sp. and O. d. dewittei was 5.9%, while that between O. d. dewittei and O. d. japonica was 7.6%. No intraspecific genetic variation was found for the new species. Wolbachia strains identified in the new species and O. d. dewittei belonged to supergroup C and are closely related. CONCLUSIONS: Our molecular analyses of filarioids from Asian suids indicate that the new species is sister to O. d. dewittei. On the basis of its morphological and molecular characteristics, we propose to elevate O. d. japonica to species level as O. japonica Uni, Bain & Takaoka, 2001. Coevolutionary relationships exist between the Wolbachia strains and their filarial hosts in Borneo and Peninsular Malaysia.

Molecular Identification of a Novel Hantavirus in Malaysian Bronze Tube-Nosed Bats (Murina aenea).

In the past ten years, several novel hantaviruses were discovered in shrews, moles, and bats, suggesting the dispersal of hantaviruses in many animal taxa other than rodents during their evolution. Interestingly, the coevolutionary analyses of most recent studies have raised the possibility that nonrodents may have served as the primordial mammalian host and harboured the ancestors of rodent-borne hantaviruses as well. The aim of our study was to investigate the presence of hantaviruses in bat lung tissue homogenates originally collected for taxonomic purposes in Malaysia in 2015. Hantavirus-specific nested RT-PCR screening of 116 samples targeting the L segment of the virus has revealed the positivity of two lung tissue homogenates originating from two individuals, a female and a male of the Murina aenea bat species collected at the same site and sampling occasion. Nanopore sequencing of hantavirus positive samples resulted in partial genomic data from S, M, and L genome segments. The obtained results indicate molecular evidence for hantaviruses in the M. aenea bat species. Sequence analysis of the PCR amplicon and partial genome segments suggests that the identified virus may represent a novel species in the Mobatvirus genus within the Hantaviridae family. Our results provide additional genomic data to help extend our knowledge about the evolution of these viruses.

Population Genetics Analyses of the Endangered Proboscis Monkey from Malaysian Borneo.

The proboscis monkey, Nasalis larvatus, is an endemic species to the island of Borneo. It is listed in the IUCN Red List as Endangered with a decreasing population trend. Nevertheless, biological information, especially on the genetic diversity of the species, is still incomplete. Its fragmented distribution poses difficulties in gathering genetic samples along with its widespread distribution across Borneo. This study aims to determine the genetic variation and structure of N. larvatus with an emphasis on Malaysian Borneo populations to elucidate its gene flow. The genetic variation and structure of N. larvatus were examined using 50 sequences of the 1,434-bp cytochrome oxidase subunit I (COI) gene region of mitochondrial DNA. The COI sequences revealed low genetic variation among N. larvatus populations in Malaysian Borneo. This low genetic variability could be the result of inbreeding pressure that may have occurred due to the absence of population expansion in this species over the last 30,000 years. This is supported in our analysis of molecular variance, which showed that groups of N. larvatus are significantly differentiated possibly due to natural geographic barriers. This study provides baseline information on the genetic diversity among proboscis monkey populations in Borneo for the future genetic assessment of the species.

Microclimate and Physiological Effects in the Roosts of Cave Dwelling Bats: Implications in Roost Selection and Conservation in Sarawak, Malaysian Borneo.

Bats play crucial ecological and economic roles. However, this group of mammals is largely threatened due to anthropogenic activities inside or around their caves. In the present study, we investigate the effects of cave microclimate and physiological parameters on bat roost preference in Fairy Cave Nature Reserve (Fairy Cave NR). The microclimate and physiological parameters including temperature (°C), relative humidity (RH), light intensity (lux), air flow, passage dimension (m), roost height (m) and distance of roost from nearest entrance (m) were measured. Results showed that Emballonura monticola, Hipposideros diadema, Hipposideros larvatus and Penthetor lucasi roost in Fairy Cave NR. These bats can be distinguished by their colony size, roost posture, spatial arrangement and position of their roost. Penthetor lucasi makes up the largest colony in the bright zone, whereas E. monticola has the smallest colony, occupying the twilight zone throughout the cave. Members from the family Hipposideridae roost in the dark portion of the cave with zero light intensity. Emballonura monticola utilizes the hottest roost in the cave compared to the other microbats, whereas H. diadema inhabits the coolest roost. Physiological parameters such as light intensity, passage dimension, and distance from nearest entrance are significant parameters in roosting preferences. These parameters should be monitored to ensure the sustainability of not only the aforementioned species, but also other species that roost in nearby caves of Bau limestone (Wind Cave Nature Reserve: with 13 recorded species).

Rodent Species Distribution and Hantavirus Seroprevalence in Residential and Forested areas of Sarawak, Malaysia.

Rodents belong to the order Rodentia, which consists of three families in Borneo (i.e., Muridae, Sciuridae and Hystricidae). These include rats, mice, squirrels, and porcupines. They are widespread throughout the world and considered pests that harm humans and livestock. Some rodent species are natural reservoirs of hantaviruses (Family: Bunyaviridae) that can cause zoonotic diseases in humans. Although hantavirus seropositive human sera were reported in Peninsular Malaysia in the early 1980s, information on their infection in rodent species in Malaysia is still lacking. The rodent populations in residential and forested areas in Sarawak were sampled. A total of 108 individuals from 15 species of rodents were collected in residential (n = 44) and forested ( n = 64) areas. The species diversity of rodents in forested areas was significantly higher (H = 2.2342) compared to rodents in residential areas (H = 0.64715) (p < 0.001 of Zar-t test based on the Shannon index). Rattus rattus and Sundamys muelleri were present at high frequencies in both localities. An enzyme-linked immunosorbent assay (ELISA) showed that hantavirus-targeting antibodies were absent from 53 tested serum samples. This is the first report of hantavirus seroprevalence surveillance in rodent populations in Sarawak, East Malaysia. The results suggested that hantavirus was not circulating in the studied rodent populations in Sarawak, or it was otherwise at a low prevalence that is below the detection threshold. It is important to remain vigilant because of the zoonotic potential of this virus and its severe disease outcome. Further studies, such as molecular detection of viral genetic materials, are needed to fully assess the risk of hantavirus infection in rodents and humans in this region of Malaysia.

The systematic position of Hypsugo macrotis (Chiroptera: Vespertilionidae) and a new record from Peninsular Malaysia.

The Southeast Asian species of Hypsugo are rare bats, except for H. cadornae and H. pulveratus, which are distributed throughout the Indomalayan region. Hypsugo macrotis is restricted to Peninsular Malaysia, Sumatra, Java and adjacent islands, and is known only from a handful of specimens. Here we report a new locality record of the species from Seremban, Peninsular Malaysia, which also represents the first known building-dweller colony of any Hypsugo from the region. We discuss the taxonomic status of two morphologically similar species, H. macrotis and H. vordermanni, and provide the first COI and cyt b gene sequences for H. macrotis and reconstruct the species' phylogenetic relationships.

Taxonomic implications of geographical variation in Rhinolophus affinis (Chiroptera: Rhinolophidae) in mainland Southeast Asia.

BACKGROUND: Rhinolophusaffinis sensu lato isdistributed throughout Southeast Asia. The taxonomic status of forms attributed to the species is unclear due to the limited sample size with incomplete datasets and the taxa have high variation in morphology and echolocation call frequency. The aim of the study was to evaluate the distribution and taxonomic status of the subspecific forms of R. affinis in mainland Southeast Asia using large sample size with multiple datasets, including morphological, acoustic, and genetic data, both to elucidate taxonomic relationships and to test for congruence between these datasets. RESULTS: Three morphological forms were confirmed within the region; two concur with previously recognized taxa, namely R.affinis macrurus andR.affinis superans,and are strongly supported by morphological and genetic data. The third form is morphologically distinct, but its taxonomic status remains unclear. It is probable that this third form represents a distinct taxonomic entity; however, more data are required to confirm this. R. a. macrurus is known from the north of peninsular Thailand, Cambodia, Myanmar, Laos, and Vietnam (Indochinese subregion); R. a. superans is found throughout the Thai-Malay Peninsula (Sundaic subregion); whilst the third form is presently known from east central Myanmar (Shan state) and lower northern Vietnam (Nghe An Province). CONCLUSIONS: Our results suggest that at least three morphological forms occur in mainland Southeast Asia including one form which appears to be new to science. Echolocation call data for R. affinis are not a robust taxonomic tool as it shows a significant degree of variation which is not explained or supported by genetic and morphological findings. This study highlights significant levels of morphological variation in mainland Southeast Asia and provides an essential basis for further studies aiming to understand the population genetics, phylogeography, and taxonomy of the species.

Timeframes of speciation, reticulation, and hybridization in the bulldog bat explained through phylogenetic analyses of all genetic transmission elements.

Phylogenetic comparisons of the different mammalian genetic transmission elements (mtDNA, X-, Y-, and autosomal DNA) is a powerful approach for understanding the process of speciation in nature. Through such comparisons the unique inheritance pathways of each genetic element and gender-biased processes can link genomic structure to the evolutionary process, especially among lineages which have recently diversified, in which genetic isolation may be incomplete. Bulldog bats of the genus Noctilio are an exemplar lineage, being a young clade, widely distributed, and exhibiting unique feeding ecologies. In addition, currently recognized species are paraphyletic with respect to the mtDNA gene tree and contain morphologically identifiable clades that exhibit mtDNA divergences as great as among many species. To test taxonomic hypotheses and understand the contribution of hybridization to the extant distribution of genetic diversity in Noctilio, we used phylogenetic, coalescent stochastic modeling, and divergence time estimates using sequence data from cytochrome-b, cytochrome c oxidase-I, zinc finger Y, and zinc finger X, as well as evolutionary reconstructions based on amplified fragment length polymorphisms (AFLPs) data. No evidence of ongoing hybridization between the two currently recognized species was identified. However, signatures of an ancient mtDNA capture were recovered in which an mtDNA lineage of one species was captured early in the noctilionid radiation. Among subspecific mtDNA clades, which were generally coincident with morphology and statistically definable as species, signatures of ongoing hybridization were observed in sex chromosome sequences and AFLP. Divergence dating of genetic elements corroborates the diversification of extant Noctilio beginning about 3 Ma, with ongoing hybridization between mitochondrial lineages separated by 2.5 myr. The timeframe of species' divergence within Noctilio supports the hypothesis that shifts in the dietary strategies of gleaning insects (N. albiventris) or fish (N. leporinus) are among the most rapid instances of dietary evolution observed in mammals. This study illustrates the complex evolutionary dynamics shaping gene pools in nature, how comparisons of genetic elements can serve for understanding species boundaries, and the complex considerations for accurate taxonomic assignment.

Genetic diversity of neotropical Myotis (chiroptera: vespertilionidae) with an emphasis on South American species.

BACKGROUND: Cryptic morphological variation in the Chiropteran genus Myotis limits the understanding of species boundaries and species richness within the genus. Several authors have suggested that it is likely there are unrecognized species-level lineages of Myotis in the Neotropics. This study provides an assessment of the diversity in New World Myotis by analyzing cytochrome-b gene variation from an expansive sample ranging throughout North, Central, and South America. We provide baseline genetic data for researchers investigating phylogeographic and phylogenetic patterns of Myotis in these regions, with an emphasis on South America. METHODOLOGY AND PRINCIPAL FINDINGS: Cytochrome-b sequences were generated and phylogenetically analyzed from 215 specimens, providing DNA sequence data for the most species of New World Myotis to date. Based on genetic data in our sample, and on comparisons with available DNA sequence data from GenBank, we estimate the number of species-level genetic lineages in South America alone to be at least 18, rather than the 15 species currently recognized. CONCLUSIONS: Our findings provide evidence that the perception of lower species richness in South American Myotis is largely due to a combination of cryptic morphological variation and insufficient sampling coverage in genetic-based systematic studies. A more accurate assessment of the level of diversity and species richness in New World Myotis is not only helpful for delimiting species boundaries, but also for understanding evolutionary processes within this globally distributed bat genus.