Bat diversity in the Cuc Phuong National Park, Vietnam - Results from VIETBIO field training and annotated species list.

Background: Biodiversity surveys are essential for both academic research and conservation. Integrative approaches that combine morphological, genetic and acoustic aspects for species identification can provide reliable information in taxonomy and evolution. This is especially relevant for those groups with a high degree of cryptic diversity such as bats. New information: Here, we present the results from a field survey carried out in the Cuc Phuong National Park (CPNP) during 2019 as part of the VIETBIO project and from the examination of specimen collections preserved at the museums of CPNP and the Institute of Ecology and Biological Resources (IEBR). In addition, we include an annotated species list, based on this survey and a literature review. We here confirm that CPNP is home to at least 47 bat species belonging to 23 genera and seven families. We recorded ten of these bat species during our field survey. Obtained data in genetics (sequencing a fragment of the mitochondrial gene COI) supported the morphological identification of the recorded species for which we were able to produce these data. In addition, we include echolocation recordings obtained during our field training with the hope that they may contribute valuable insights to future work concerning the surveyed species. Results from the field survey represent a relevant contribution to biodiversity assessment efforts and, thus, support conservation and management efforts to maintain bat diversity in Vietnam.

Genotype and Phenotype Characterization of Rhinolophus sp. Sarbecoviruses from Vietnam: Implications for Coronavirus Emergence.

Bats are a major reservoir of zoonotic viruses, including coronaviruses. Since the emergence of SARS-CoV in 2002/2003 in Asia, important efforts have been made to describe the diversity of Coronaviridae circulating in bats worldwide, leading to the discovery of the precursors of epidemic and pandemic sarbecoviruses in horseshoe bats. We investigated the viral communities infecting horseshoe bats living in Northern Vietnam, and report here the first identification of sarbecoviruses in Rhinolophus thomasi and Rhinolophus siamensis bats. Phylogenetic characterization of seven strains of Vietnamese sarbecoviruses identified at least three clusters of viruses. Recombination and cross-species transmission between bats seemed to constitute major drivers of virus evolution. Vietnamese sarbecoviruses were mainly enteric, therefore constituting a risk of spillover for guano collectors or people visiting caves. To evaluate the zoonotic potential of these viruses, we analyzed in silico and in vitro the ability of their RBDs to bind to mammalian ACE2s and concluded that these viruses are likely restricted to their bat hosts. The workflow applied here to characterize the spillover potential of novel sarbecoviruses is of major interest for each time a new virus is discovered, in order to concentrate surveillance efforts on high-risk interfaces.

New geographical and host records of bat fleas (Siphonaptera: Ischnopsyllidae) in Russia.

To better understand the distribution and host-parasite relationships, we explored 12 large regions of Russia and recorded new bat fleas (Insecta: Siphonaptera: Ischnopsyllidae) for Dagestan, Bashkiria, Mordovia, Khakassia and Buryatia Republics. Also we curate previously known data and registered new host-parasite associations for species belonging to genus Ischnopsyllus (Ischnopsyllus octactenus and I. variabilis ex Pipistrellus pygmaeus, I. intermedius ex Hypsugo savii, I. variabilis ex Myotis dasycneme, I. hexactenus ex Murina hilgendorfi) and species Myodopsylla trisellis. One of the associations we recorded in Altai republic is particularly interesting (Myodopsylla trisellis ­ Myotis blythii). Further investigation is required to study vector role of bat fleas and the effects of flea parasitism on their natural hosts.

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.

New findings of spinturnicid mites (Mesostigmata: Gamasina: Spinturnicidae) from the Caucasus.

The Caucasus is a large region in Eurasia consisting of four countries: Russia, Georgia, Azerbaijan, and Armenia. Although it is one of the biodiversity hotspots in the world, the bat ectoparasite fauna has been poorly studied. To promotionally fill in the gaps regarding bat ectoparasites, we conducted five field surveys on bats and their ectoparasites at nine localities within the region between April 2016 and March 2021. Eight species and subspecies of spinturnicid mites were recorded over the surveys: Eyndhovenia euryalis oudemansi, Spinturnix acuminata acuminata, S. emarginata, S. myoti, S. nobleti, S. plecotina, S. psi, and S. punctata. Among them, three species, Spinturnix emarginata, S. nobleti and S. punctate, are newly recorded from the Caucasus region, and one each of subspecies and species, Eyndhovenia euryalis oudemansi and S. plecotina, are newly recorded from Georgia. In addition, Myotis tschuliensis was recorded as a new host species of S. myoti.

Fireflies produce ultrasonic clicks during flight as a potential aposematic anti-bat signal.

Fireflies are known for emitting light signals for intraspecific communication. However, in doing so, they reveal themselves to many potential nocturnal predators from a large distance. Therefore, many fireflies evolved unpalatable compounds and probably use their light signals as anti-predator aposematic signals. Fireflies are occasionally attacked by predators despite their warning flashes. Bats are among the most substantial potential firefly predators. Using their echolocation, bats might detect a firefly from a short distance and attack it in between two flashes. We thus aimed to examine whether fireflies use additional measures of warning, specifically focusing on sound signals. We recorded four species from different genera of fireflies in Vietnam and Israel and found that all of them generated ultrasonic clicks centered around bats' hearing range. Clicks were synchronized with the wingbeat and are probably produced by the wings. We hypothesize that ultrasonic clicks can serve as part of a multimodal aposematic display.

First record of Eyndhovenia (Mesostigmata: Gamasina: Spinturnicidae) from Vietnam.

Eyndhovenia is one of the twelve genera of Spinturnicidae which are highly specialised parasites of bats. Previously known hosts of this genus comprised 17 species of Old World bats: Eptesicus serotinus, Hipposideros larvatus, Miniopterus schreibersi, Myotis blythi, M. emarginatus, Pipistrellus pipistrellus, P. gaisleri, Rhinolophus axillaris, R. blasii, R. clivosus, R. cornutus, R. euryale, R. ferrumequinum, R. hipposideros, R. megachyllus, R. mehelyi, R. rouxi. Within Asia, Eyndhovenia was only recorded from two countries, China and Thailand. Between 2018 and 2020, we conducted a series of bats surveys and recorded of this genus from intermediate horseshoe bat, Rhinolophus affinis, in Vietnam. The present study exhibits the new record in both parasitological and geographical aspects.

Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

Resolving evolutionary relationships among six closely related taxa of the horseshoe bats (Rhinolophus) with targeted resequencing data.

Recently diverged taxa are often characterised by high rates of introgressive hybridization and incomplete lineage sorting, both of which can complicate phylogenetic reconstructions of species histories. Here we use a sequence capture approach to obtain genome-wide data to resolve the evolutionary relationships, and infer the extent and timescale of hybridization and introgression events, among six recently diverged taxa of the horseshoe bat species complexes Rhinolophus sinicus and R. thomasi. We show that two different methods of species tree reconstruction applied to a set of ~1500 nuclear loci all recover species trees with similar topologies, differing from the previous phylogeny based on two nuclear loci. By comparing the tree topology obtained from the nuclear loci with that inferred from the mitochondrial genome, we observed at least three cases of conflict, each of which likely results from past introgression. Of these, the occurrence of a highly similar mitogenome sequence shared by individuals of two taxa in a sympatric region points to very recent mtDNA introgression. The other cases are characterised by greater divergence and strong phylogeographic structure among putative introgressed individuals and their source populations, and thus likely reflect more ancient hybridization events. These results also suggest that two of the subspecies (R. s. septentrionalis and the undescribed taxon R. s. ssp) are likely to represent full species, warranting full taxonomic descriptions. This work adds a growing number of studies showing the potential problems of relying solely on mitochondrial sequences, or a limited number of loci, to infer phylogenetic relationships among recently diverged taxa.

Species delimitation and evolutionary reconstruction within an integrative taxonomic framework: A case study on Rhinolophus macrotis complex (Chiroptera: Rhinolophidae).

Species delimitation and evolutionary reconstruction remain challenging for non-model species that have experienced reticulate evolution and exhibit conflicting patterns of differentiation among multiple lines of evidence, such as mitochondrial and nuclear data and phenotypes. Here, we applied an integrative taxonomic approach to a case study of Rhinolophus macrotis complex, whose taxonomic status remains controversial, to provide insight into the systematics and evolutionary history of these species. By integrating traditional genetic markers with different modes of inheritance, genome-wide SNPs as well as phenotypic characteristics, we clarified the presence of three closely related species, R. episcopus, R. siamensis, and R. osgoodi, within this complex, and proposed a new taxonomic treatment for R. osgoodi. Our results suggested that hybridization and introgression are the main causes of low mtDNA divergence in these species. Combined with the demographic inference, we deduced that glacial-interglacial cycles drove geographic isolation and secondary contacts of these species, then promoted hybridization and lineage fusion among them, finally resulting in a reticulate evolutionary pattern. Overall, our study highlights the importance of combining multiple types of data to delimit species, especially those with conserved morphology, and to reveal the sophisticated processes of speciation.

The first record of Ia io Thomas, 1902 (Mammalia: Chiroptera: Vespertilionidae) from the Sundaic Subregion, with a description of a new subspecies from peninsular Thailand.

The Great evening bat Ia io Thomas, 1902, previously considered as an endemic to the Indochinese subregion, is reported from the Sundaic subregion for the first time based on specimens collected from three localities in Surat Thani Province and Phang Nga Province, peninsular Thailand. It is described herein as a new subspecies based on its substantially larger body and skull size. The mitochondrial COI and cytochrome b genes reveal that the new subspecies has a genetic distance of 1.89% and 1.65%, respectively, from the nominate subspecies. Echolocation calls comprise four harmonics, with the maximum energy in the first harmonic (fmaxe) of 23.6-27.4 kHz. Notes on the population size as well as roosting and foraging behaviour are also provided.

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.

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.

Morphometric variation in the pusillus group of the genus Rhinolophus (Mammalia: Chiroptera: Rhinolophidae) in East Asia.

Based on 203 specimens belonging to the Rhinolophus "pusillus group" (Mammalia: Chiroptera: Rhinolophidae), univariate and multivariate morphometric analyses using 19 characters were performed to assess the confused species taxonomy. The results indicated that R. pusillus (including calidus, parcus, and szechuanus) in the continental region and Hainan Island of China and "R. cornutus" in Japan are morphologically divergent species. Rhinolophus cornutus should be further split into R. cornutus (including orii, pumilus, and miyakonis) in the main islands of Japan, the Amami and Okinawa Group of the central Ryukyu Archipelago, and Miyako Group of the southern Ryukyus; and R. perditus and R. imaizumii from the Yaeyama Group in the southern Ryukyus. Rhinolophus monoceros from Taiwan is morphologically more similar to species in Japan than to R. pusillus. In addition to R. pusillus, another form that is morphologically similar to species in Japan was recognized from Langzhong in Sichuan Province; this may represent an undescribed species, and further examination is necessary to determine its taxonomic status. Specimens from Guang'an in Sichuan Province, China, are also different from the others, and are characterized by the smallest skull size. Although further studies are required, these specimens were tentatively identified as R. subbadius.

A new species of Rhinolophus (Chiroptera: Rhinolophidae) from China.

A new species of the genus Rhinolophus is described from Yunnan Province, southwestern China. The new taxon belongs to the Rhinolophus "philippinensis-group" and is distinguished by differences in the nose-leaf structures, craniodental characteristics, and bacular features.