Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, Son La Province, north-western Vietnam, based on integrative insights from morphology, genetics and echolocation data

Biodiversity Data Journal 13: e165516 doi: 10.3897/BDJ.13.e165516 Research Article Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, Son La Province, northwestern Vietnam, based on integrative insights from morphology, genetics and echolocation data Yen H Vu , Hai T Bui , Toan T Giang , Luong K Vu , Luong T Nguyen , Masaharu Motokawa , Son T Nguyen ‡ University of Science, Vietnam National University (VNU), Hanoi, Vietnam § Institute of Biology, Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam | Vietnam Nation University of Forestry, Hanoi, Vietnam ¶ The Kyoto University Museum, Kyoto University, Kyoto, Japan # Graduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam Corresponding author: Son T Nguyen ([email protected]) Academic editor: Krizler Tanalgo Received: 17 Jul 2025 | Accepted: 24 Oct 2025 | Published: 04 Nov 2025 Citation: Vu Y, Bui H, Giang T, Vu L, Nguyen L, Motokawa M, Nguyen S (2025) Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, Son La Province, north-western Vietnam, based on integrative insights from morphology, genetics and echolocation data. Biodiversity Data Journal 13: e165516. https://doi.org/10.3897/BDJ.13.e165516 Abstract This study presents the results of bat diversity surveys in Xuan Nha Nature Reserve, north-western Vietnam. A total of 114 individuals, representing 19 species belonging to four families, were recorded. The Rhinolophidae family was the most species-rich, contributing eight species to the total diversity, followed by Hipposideridae, with four species. Eight species, including Rhinolophus episcopus,R. siamensis, R. cf. episcopus, R. perniger, Hipposideros griffini, Megaerops niphanae, Tylonycteris tonkinensis and Myotis muricola, were newly recorded for Xuan Nha NR. Species richness was high, but evenness was low, with Hipposideros poutensis and Rhinolophus pearsonii dominating captures. Eleven species were observed, represented by only one individual. The morphological examinations, with support from echolocation calls and preliminary genetic analysis, revealed the presence of morphologically conserved and potentially ‡,§ ‡ | ‡ § ¶ §,# © Vu Y et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. cryptic taxa. Reproductive data indicated early wet-season breeding for several species. Compared to previous surveys in the region, our study substantially expands the knowledge of chiropteran fauna in Xuan Nha. Given the high proportion of habitatspecialist and montane-associated species and the documented presence of a conservation-priority taxon, Hipposideros griffin, in Xuan Nha NR, continued biodiversity surveys incorporating molecular and acoustic methods are essential to refine species inventories and to provide information for conservation strategies for this biologically important landscape. Keywords biodiversity, Chiroptera, cytochrome b, species richness Introduction Vietnam, located in Indochina, is recognised as a hotspot country and amongst the world's richest regions for mammal diversity. Such biodiversity is attributed to the country's complex topography, diverse climatic conditions and distinct ecosystems (Sterling et al. 2006, Tordoff et al. 2012). Northern Vietnam, in particular, with numerous protected areas, serves as important refugia for both widespread and endemic bat species. Xuan Nha Nature Reserve (NR), in Xuan Nha Commune of Son La Province includes both primary and secondary evergreen forests, with limestone areas interspersed amongst them (The People’s Committee of Son La Province 2019). Regarding bat diversity, Nguyen et al. (2012) recorded a total of 19 species at this area; nevertheless, data on environmental conditions, as well as detailed morphological characteristics and genetic information of the recorded species, remain unavailable. In August 2024, within the framework of the Nagao NEF Project, we conducted an 11-day field survey in Xuan Nha NR. The results significantly expand current knowledge of bat diversity in the region by increasing the number of recorded species and providing understandings of undocumented taxa. This paper aims to update the chiropteran fauna of Xuan Nha NR by providing information related to morphological characteristics, echolocation calls and genetic information; and then we discuss species composition and diversity in north-western Vietnam to support future ecological and taxonomic research in the region. Material and methods Study area Xuan Nha NR (20°36'–20°48'N, 104°29'–104°50'E) is positioned in the south-western area of Son La Province, north-western Vietnam, bordering Laos to the west (Fig. 1) and is one of four nature reserves in Son La (The People’s Committee of Son La Province 2019). The Reserve is dominated by tropical forests and interspersed with substantial 2Vu Y et al areas of limestone forest ecosystems (Tordoff et al. 2004). Pha Luong Mountain (1970 m a.s.l.) is the highest peak and located on the mountainous ridge that delineates the Vietnam–Laos border, serving as an important ecological corridor for montane fauna (Center for Nature Conservation and Development (CEV) 2005). Along the mid-elevation transects (500–800 m a.s.l.), the surveyed routes crossed areas heavily altered by anthropogenic activity. Remnant patches of primary forest were fragmented and embedded in a matrix of cultivated lands and secondary vegetation (Fig. 2c). Dense thickets of low-trunk tracts predominate between 700 and 900 m a.s.l., while limestone outcrops was observed near a bat cave at approx. 910 m a.s.l. Above 1000 m a.s.l., undisturbed forest patches persist along the slopes (Fig. 2a). These forests have a canopy height of 20–30 m with a dense understory of saplings, climbing vines and dense shrubs. At elevations above 1500 m a.s.l., nearly pristine montane moss forests remain, characterised by high canopy trees (20–50 m), abundant epiphytes and closed canopy cover. The region has cooler temperatures and high humidity. Streams formed by precipitation patterns and seasonal runoff are typically 1.5–4.5 m wide and less than 0.7 m deep. Survey methods and sampling The main field survey was conducted from 4-15 August 2024, with additional data from a two-day survey on 25-26 March 2010. Bats were surveyed using mist nets and harp traps (Tidemann and Woodside 1978) set along four elevational bands (ca. 600, 700, 900 and 1100 m a.s.l.) (Fig. 1). Sixteen trapping sites were established, each operated for 1–2 nights depending on weather and accessibility. Harp traps (1.8 m high × 1.2 m wide) with Figure 1. Map of Vietnam showing the location of Xuan Nha NR within Son La Province. Red dots indicate bat sampling sites. The map was organised using QGIS 3.38.3 (https://www.qgis.org).  Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 3 four parallel rows of monofilament lines (2.5 cm spacing) were deployed at each site (Tidemann and Woodside 1978). Traps were strategically placed along bat flyways such as: forest trails, ridgelines, streams and cave entrances (Fig. 3a, b and d). At least one harp trap was installed per site and set before dusk, remaining open until midnight or later depending on bat activities. Traps were checked every 30–45 minutes, synchronised with mist net inspections, to minimise stress and injury to captured individuals. Mist nets were deployed at ground level across presumed flyways. Two net sizes were used: 9 × 3 m and 12 × 3 m, constructed from black nylon with four shelves (Fig. 3c). Nets were mounted on fiberglass or in combinations with locally sourced bamboo poles. Sampling was conducted from 18:00 h to 23:00 h and 04:00 h to 05:00 h, with 1–3 nets operated per site per night. Nets were closed during the day to avoid accidental captures of birds or livestock. Geographic coordinates and elevations were recorded using the Gaia GPS (WGS84 datum). Echolocation recordings and analyses Echolocation calls were recorded using an Echo Meter Touch 2 ultrasonic detector (Wildlife Acoustics, Inc., Maynard, Massachusetts, USA) connected to the smartphone device. Recordings were obtained under two conditions: (1) handheld recordings made immediately at the mist net site of captured individuals; (2) controlled recordings inside a temporary flight tent (4 × 4 × 2 m). All recordings were analysed visually using spectrograms to characterise call structure and frequency parameters. Echolocation calls were compared with references from previous studies in Vietnam (Nguyen et al. 2021, Győrössy et al. 2024) to assist species identification, particularly for cryptic taxa or species difficult to capture. Figure 2. Habitat natures in Xuan Nha NR. (a) Tropical evergreen forest at 1000 m a.s.l.; (b) Mixed habitat including forest edges and open areas at around 600 m a.s.l.; (c) Disturbed secondary forest at 900 m a.s.l.  4Vu Y et al Specimen examination Most specimens (109 individuals) were collected during the main survey in 2024, while only five individuals were captured on March 2010, with detailed information for each specimen presented in Suppl. material 1. Photographs of live specimens were taken using a Canon EOS Kiss X7 digital camera with an EF-S 18–55 mm f/3.5–5.6 kit lens. Standard morphometric measurements were followed Wilson and Mittermeier (2019). The reproductive condition of females was assessed following Racey (2009) and age class was determined by the degree of phalangeal epiphyseal fusion (Brunet-Rossinni and Wilkinson 2009). Pregnancy in females was checked by gentle abdominal palpation. Specimens were preserved in 95% ethanol during both the 11-days survey in 2024 and 2-days in 2010. Upon transfer to the laboratory, ethanol concentration was reduced to 70% for long-term storage. Skulls were extracted and cleaned and one to two individuals per species were selected for genetic analyses. Craniodental characters were measured under a stereoscopic microscope (SMZ 745, Nikon) using an electronic digital caliper (Mitutoyo NTD12-15PMX, 0.01 mm precision). A total of 20 metrics were measured following Vu et al. (2024) (Fig. 4; Suppl. material 2). Specimens were identified, based on external and cranial morphology, supplemented by DNA analysis and echolocation data, using comparative references (Borissenko and Kruskop 2003, Vu 2012,Kruskop 2013, Vuong et al. 2015, Vuong et al. 2017a, Vuong et al. 2017b, Vuong et al. 2018, Moratelli et al. 2019, Vu et al. 2024). Voucher specimens and associated tissue samples are currently deposited in the Department of Zoology, Institute of Biology (IB), VAST, Hanoi, Vietnam. Figure 3. Bat trapping deployment: (a, b, d) Double-harp traps set along forest trails and over streams; (c) Setup of mist nets.  Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 5 Molecular data and phylogenetic analyses DNA was isolated from tissue samples preserved in 99% ethanol using the DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany). The mitochondrial cytochrome b (Cyt b) gene was targeted for species-level identification. PCR amplification employed primer pair SoriF/SoriR (Bui et al. 2020a) with the following thermal cycling conditions: initial denaturation at 95°C for 5 min; 35 cycles of 95°C for 30 s, 55°C for 50 s and 72°C for 2 min; and a final extension at 72°C for 10 min. PCR products were purified and sequenced by 1st BASE (Selangor, Malaysia) using Sanger sequencing. Chromatograms were edited and assembled in Chromas Pro (Technelysium Pty Ltd., Australia) and MEGA 11 (Tamura et al. 2021). Sequences were aligned with MUSCLE in MEGA 11. The final alignment of the Cyt b fragment was 1140 bp. Phylogenetic analyses were performed using Maximum Likelihood (ML) under the GTR+G+I model, selected by the Bayesian Information Criterion (BIC) in ModelFinder implementation in IQ-TREE v. 1.6.12 (Nguyen et al. 2015). Node support was evaluated with 10,000 ultrafast bootstrap replicates (Hoang et al. 2018). The resulting phylogenetic trees were visualised using FigTree v.1.4.4 and edited using Adobe Photoshop 2023. Data resources Data package title: Occurrence dataset of bats (Mammalia, Chiroptera) from Xuan Nha Nature Reserve, Son La Province, north-western Vietnam. Resource link: https://doi.org/10.15468/7d54s5 Figure 4. (a) Dorsal, (b) ventral, (c) lateral views of the cranium; (d) lateral views of mandible displaying craniodental measurements. The diagram is constructed, based on the skull morphology of P. tenuis species.  6Vu Y et al Number of datasets: 1 Data set name: Occurrence dataset of bats (Mammalia, Chiroptera) from Xuan Nha Nature Reserve, Son La Province, north-western Vietnam. Data format: Darwin Core Event Description: This dataset presents species occurrence of bats collected from Xuan Nha NR, Son La Province, north-western Vietnam. Field surveys were conducted in August 2024 as part of the biodiversity research programme with the support of the Nagao Natural Environment Foundation (NEF). For every occurrence record, the dataset provides information on location (GPS coordinates), date, sex, reproductive condition and trap type. Results Species richness, diversity and distribution A total of 114 bat individuals were collected during two field surveys in Xuan Nha NR, representing 19 species from nine genera and four families: Hipposideridae, Pteropodidae, Rhinolophidae and Vespertilionidae. The Rhinolophidae was the most species-rich family, with eight species of one genus (R. pearsonii, R. perniger, R. affinis, R. pusillus, R. episcopus, R. siamensis, R. thomasi and R. cf. episcopus). This was followed by Hipposideridae, with four species of two genera (A. stoliczkanus, H. armiger, H. griffini and H. poutensis) and Vespertilionidae, with five species of four genera (K. cf. dongduongana, M. alticraniatus, M. muricola, P. tenuis and T. tonkinensis). The Pteropodidae family was represented by two species of two genera (C. sphinx and M. niphanae) (Table 1). H. poutensis was the most abundant species, with 39 individuals (34.2%). The second most common was R. pearsonii (32 individuals, 28.1%). Other frequently captured species included R. thomasi (7), C. sphinx (8) and M. niphanae (7). In contrast, eleven species were represented by a single individual, including R. pusillus, R. affinis, R. episcopus, R. siamensis, R. cf. episcopus, R. perniger, H. griffini, M. alticraniatus, M. muricola, T. tonkinensis and T. tenuis. Capture success varied by trap type: harp traps accounted for 88 individuals (77.2%), proving effective for Rhinolophus and Hipposideros species, while mist nets (26 individuals, 22.8%) exclusively captured frugivorous taxa, such as C. sphinx and M. niphanae. Species evenness was low, with captures dominated by a few abundant taxa. Signs of reproductive activity, including pregnancy, lactation or the presence of newborns, were observed in 15 females across five species, while 54 females showed no evidence of breeding. Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 7 No. Specific name n (M♂, F♀) Reproductive information Elevation (m) Habitat nature Species recorded in this study Previous Record (Nguyen et al. 2012) IUCN Status Hipposideridae Lydekker, 1891 1Hipposideros armiger (Hodgson, 1835) 3 (2♂, 1♀) 1♀ (Not reproductive) 2 (650 m), 1 (750 m) 2✓ ✓ LC 2Hipposideros griffini Vu, Puechmaille, Denzinger, Dietz, Csorba, Bates, Teeling & Schnitzler, 2012 1 (1♂, 0♀) — 1 (650 m) 2 ✓* — NT 3Hipposideros poutensis Allen, 1906 39 (21♂, 18♀) 3♀ (3 Lactating), 15♀ (Not reproductive) 1 (600 m), 4 (650 m), 8 (700 m), 15 (750 m), 7 (800 m), 2 (850 m), 2 (910 m) 1, 2, 3, 4 ✓ ✓ LC 4Hipposideros gentilis Andersen, 1918 —✓LC 5Aselliscus stoliczkanus (Dobson, 1871) 3 (0♂, 3♀) 3♀ (Not reproductive) 1 (750 m), 2 (800 m) 1, 2 ✓ ✓ LC Pteropodidae Brisson, 1762 6Cynopterus sphinx (Vahl, 1797) 8 (2♂, 6♀) 3♀ (2 Lactating, 1 Pregnant, 1 with newborn), 3♀ (Not reproductive) 1 (600 m), 5 (700 m), 2 (750 m) 2, 4 ✓ ✓ LC 7Megaerops niphanae Yenbutra & Felten, 1983 7 (3♂, 4♀) 2♀ (2 Lactating), 2♀ (Not reproductive) 3 (600 m), 2 (700 m), 2 (750 m) 2, 4 ✓* — LC 8Sphaerias blanfordi (Thomas, 1891) —✓LC Rhinolophidae Gray, 1825 9Rhinolophus affinis Horsfield, 1823 1 (0♂, 1♀) 1♀ (Not reproductive) 1 (750 m) 2 ✓ ✓ LC Table 1. List of bat species recorded from Xuan Nha NR. n (♂, ♀) = sample size (♂ = male, ♀ = female); Elevation: n (m) = number of individuals with elevation in metres. Recorded habitat: 1 = evergreen forest, 2 = disturbed secondary forest, 3 = cave areas, 4 = stream valley. Species records: ✓ = recorded, ✓* = newly recorded in the 2024, — = Not recorded. 8Vu Y et al No. Specific name n (M♂, F♀) Reproductive information Elevation (m) Habitat nature Species recorded in this study Previous Record (Nguyen et al. 2012) IUCN Status 10 Rhinolophus episcopus Allen, 1923 1 (1♂, 0♀) — 1 (600 m) 2 ✓* — LC 11 Rhinolophus siamensis Gyldenstolpe, 1917 1 (1♂, 0♀) — 1 (750 m) 2 ✓* — LC 12 Rhinolophus pearsonii Horsfield, 1851 32 (5♂, 27♀) 6♀ (5 Lactating, 1 Pregnant), 21♀ (Not reproductive) 1 (500 m), 4 (600 m), 1 (650 m), 1 (700 m), 20 (750 m), 5 (800-850 m) 2, 4 ✓ ✓ LC 13 Rhinolophus pusillus Temminck, 1834 1 (1♂, 0♀) — 1 (650 m) 2 ✓ ✓ LC 14 Rhinolophus cf. episcopus Allen, 1923 1 (1♂, 0♀) — 1 (750 m) 2 ✓* — LC 15 Rhinolophus thomasi K. Andersen, 1905 7 (5♂, 2♀) 1♀ (1 Lactating), 1♀ (Not reproductive) 2 (600 m), 5 (800 m) 2, 4 ✓ ✓ LC 16 Rhinolophus rouxii Temminck, 1835 —✓LC 17 Rhinolophus perniger Hodgson, 1843 1 (0♂, 1♀) 1♀ (Not reproductive) 1 (600 m) 2 ✓* — LC Vespertilionidae Gray, 1821 18 Kerivoula cf. dongduongana Vuong et al. 2018 4 (0♂, 4♀) 4♀ (Not reproductive) 4 (900 m) 1 ✓ ✓ LC 19 Murina cyclotis Dobson, 1872 —✓LC 20 Myotis alticraniatus Osgood, 1932 1 (0♂, 1♀) 1♀ (Not reproductive) 1 (800 m) 2 ✓ ✓ LC 21 Myotis muricola (Gray, 1864) 1 (0♂, 1♀) 1♀ (Not reproductive) 1 (950 m) 2 ✓* — LC 22 Pipistrellus tenuis (Temminck, 1840) 1 (1♂, 0♀) — 1 (800 m) 2 ✓ ✓ LC 23 Pipistrellus abramus (Temminck, 1840) —✓LC Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 9 Character Species (n) H. armiger (3) H. griffini (1) H. poutensis (39) A. stoliczkanus (3) C. sphinx (8) M. niphanae (7) M M W 7.14–7.34 7.25±0.07 11.18 5.06–5.2 5.14±0.06 4.55 5.79 5.02 5.38 ML 12.57– 12.79 12.68±0.08 22.98 9.13–9.49 9.31±0.16 8.68 10.43 8.54 8.81 CPH 2.61–2.73 2.66±0.05 5.72 2.83–3.05 2.95±0.1 2.06 3.11 2.24 2.42 cm L 7.33–7.69 7.54±0.13 13.48 5.42–5.51 5.47±0.04 4.66 5.65 4.45 4.23 cp L 2.32–2.79 2.55±0.17 5.38 2.33–2.39 2.36±0.03 1.87 2.07 1.43 1.21 p m L 5.67–6.07 5.86±0.16 10.03 — 3.27 4.02 3.41 3.12 m m L 4.93–5.18 5.05±0.1 8.16 — 2.95 3.37 2.85 2.81 Hipposideros poutensis Allen, 1906 In the study, 39 individuals of H. poutensis (21♂, 18♀) were recorded (Table 1, Suppl. material 1). Amongst females, only three showed active reproductive status as lactating, 3 3 3 4 4 3 1 3 Figure 6. (Left) Cranium of H. poutensis (A–C) and A. stoliczkanus (a–c) in dorsal (A, a), lateral (B, b) and ventral (C, c) views; mandible in dorsal (D, d) and lateral (E, e) views; (Right) Phylogeny based on Cyt b sequences of Hipposideridae bats. Bootstrap support values (BS) are shown at nodes.  16 Vu Y et al while the remaining 15 were non-reproductive. H. poutensis is a medium-sized leafnosed bat (Fig. 7a), characterised by a slender skull compared to H. armiger or H. griffini, with a developed sagittal crest, a narrow braincase and a slightly swollen rostrum. The specimen clustered within the H. poutensis lineage on the phylogenetic tree, with strong support and showed the closest genetic relationship with specimens collected from Bai Tu Long Island (OP142137), followed by population from Cat Ba Island (OP142143–OP142145) (Fig. 6, right). Pairwise genetic divergence ranged between 2% and 3.6%. These findings support the recent taxonomic distinction of H. poutensis from H. larvatus as reported by Yuzefovich et al. (2022), with both morphological and molecular analyses confirming its status as the distinct species. Based on recordings of a single male individual, this species emitted narrowband CF-FM calls (Fig. 5, right) with a peak frequency of 85.6 ± 0.7 kHz, starting at 88.7 kHz and ending at 70.7 kHz. Mean call duration was 6.2 ± 0.2 ms, with the longest call lasting 6.5 ms. Aselliscus stoliczkanus (Dobson, 1871) A. stoliczkanus is widely distributed across north-western and central Vietnam (Vuong et al. 2015). In this study, three specimens were identified as A. stoliczkanus, based on diagnostic external and cranial traits. These bats are small-sized, with a tricuspid posterior nose-leaf, short bodies and bicoloured dorsal fur (white bases and brown tips), while the ventral fur is paler (Fig. 7b). Cranial features include a slightly swollen rostrum and a relatively elongated snout compared to members of Hipposideridae (Fig. 6, left). The newly-obtained specimens clustered with reference A. stolickzanus sequences (BS = 89–98%) (Fig. 6, right). Genetic divergence between the study specimens and known references ranged from 0.5% to 1.5%. Figure 7. Selected Hipposideridae species recorded on Xuan Nha NR: (a) H. poutensis; (b) A. stolickzanus.  Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 17 Family Pteropodidae Two species of fruit bats were recorded during this survey (Table 1). C. sphinx, previously reported from the Reserve, was re-confirmed, while M. niphanae represents a new record for this locality. Both species exhibited signs of reproductive activity during August. Cynopterus sphinx (Vahl, 1797) C. sphinx is common, occurring across lowland and edge habitats. Adults show short orange-brown dorsal pelage, a greyish ventral side and a darker mantle region, more pronounced in males. Juveniles are paler with an overall greyish tone. The ears are brown with a distinctive whitish margin and the interfemoral membrane is narrow, but evident, with a short tail extending slightly beyond it. Compared with Megaerops, C. sphinx is larger-bodied, more robust, with a more developed interfemoral membrane and a diagnostic white ear margin (absent in Megaerops). Cranially, the skull is elongate, narrowing anteriorly (Fig. 8), with expanded zygomatic arches and a broad, elongated palate. The dentition consists of robust molars with slightly rounded cusps and elongated upper canines. Megaerops niphanae Yenbutra & Felten, 1983 This is a small pteropodid bat. The species is easily recognised by its soft, light brownishgrey pelage and absence of a tail. The flight membranes are pale grey with weak pigmentation, while the ears, muzzle and limbs are pale brownish, giving a subtly translucent appearance. Cranially, the skull is short (Fig. 8), with narrow zygomatic arches and a constricted postorbital region. The palate is smooth and lacks strong ridging and the molars are less robust than those of C. sphinx. Of four adult females captured in mid-elevation forests, two were lactating. Figure 8. Dorsal (A, a), lateral (B, b), ventral (C, c) views of the cranium and Dorsal (D, d), lateral (E, e) views of the mandible of C. sphinx and M. niphanae, respectively.  18 Vu Y et al Family Rhinolophidae Eight species of Rhinolophidae were documented. Four species (R. episcopus, R. siamensis, R. cf. episcopus and R. perniger) are newly recorded, while R. affinis, R. thomasi, R. pearsonii and R. pusillus had previously been reported by Nguyen et al. (2012). R. rouxii, listed in earlier surveys, was not detected. This species is now considered restricted to eastern Asia; therefore, its previous record from Xuan Nha NR is likely a misidentification. Reproductive evidence was observed in R. thomasi and R. pearsonii. Rhinolophus affinis Horsfield, 1823 Only a female R. affinis was recorded showing no signs of reproductive activity. This species is a medium-sized horseshoe bat. Externally, it has a broad horseshoe with a deep median emargination, rounded connecting process and a moderately convex sella lacking basal lappets; the lancet is subtriangular with an unreduced tip. The pelage is soft, dark greyish-brown. The skull is small, with well-developed lateral nasal compartments and narrow interorbital constriction. The dentition shows a slightly reduced P within the tooth row. In comparison with R. thomasi (Fig. 9, left), although both species share skull shape typical of the “R. megaphyllus” group (Ith et al. 2015), R. affinis has a slightly larger cranium, a proportionally longer mandible, broader rostrum and more laterally expanded arches. Their dental dimensions (C C W, M M W) are otherwise similar. Molecular data place the specimen within the R. affinis clade, with pairwise genetic distances of 2.5–2.75% (Fig. 9, right). 2 1 1 3 3 Figure 9. (Left) Dorsal (A, a, a’), lateral (B, b, b’), ventral (C, c, c’) views of the cranium and Dorsal (D, d, d’), lateral (E, e, e’) views of the mandible of R. affinis, R. thomasi and R. pusillus; (Right) ML tree based on Cyt b sequences of Rhinolophydae bats.  Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 19 Rhinolophus thomasi K. Andersen, 1905 One lactating female of R. thomasi was observed. Compared to R. affinis, R. thomasi has slightly smaller zygomatic, braincase breadths and its mandible is thinner and shorter. Externally, it shows a narrow, rectangular horseshoe and a broad, bluntly pointed lancet. The pelage is uniformly light grey with a metallic sheen, dense and velvety, with pale bases and darker tips (Fig. 10a). Echolocation calls of a female exhibited broadband FM–CF–FM structure dominated by CF components, with a peak frequency of 77.4 ± 0.2 kHz, starting at 78.6 kHz and ending at 66.7 kHz. Calls averaged 19.6 ± 0.3 ms in duration, with the longest call lasting 20.3 ms (Fig. 10d). Rhinolophus pearsonii Horsfield, 1851 A total of 32 individuals of R. pearsonii were captured and were the most common species of Rhinolophus in the survey. Six females were reproductively active (five lactating, one pregnant). This species has a broad rostrum and a well-developed braincase. Cranial dimensions include GTL 22.78–23.85 mm, CM L 8.97–9.78 mm and ML 16.27 ± 0.28 mm. The molars are marked by high, sharp cusps (Fig. 10c’). Echolocation calls of a male recorded in a tent exhibited broadband FM-CF-FM structure (Fig. 10d), with peak frequency 56.9 ± 0.7 kHz, ranging from 39.6 to 58.9 kHz. Calls averaged 21.6 ± 0.3 ms in duration, with the longest call lasting 22.3 ms. Rhinolophus perniger Hodgson, 1843 Previously, R. perniger was considered a subspecies within the R. luctus complex; however, studies of Volleth et al. (2017) demonstrated differences between R. perniger 3 Figure 10. Selected Rhinolophydae species recorded: (a) R. thomasi; (b) R. pusillus; (c) R. pearsonii; (c’) Five aspects of the cranium and mandible of R. pearsonii; (d) Ultrasonic echolocation calls of Rhinolophids.  20 Vu Y et al and R. luctus sensu stricto. In this study, a single female was captured and clustered with reference R. perniger sequences, showing 1.2% genetic divergence (Fig. 9, right). The individual was non-reproductive. R. perniger is a large horseshoe bat (FA 73.4 mm, Wt 48 g) with a massive skull (GTL 33.54 mm). The horseshoe-shaped nose-leaf is broad, covering the upper lip with a deep median notch. The fur is thick, dark grey dorsally and ventrally. Dentition is robust, with well-developed toothrows. Echolocation calls recorded from the captured female were broadband FM–CF–FM (Fig. 10d), with peak frequency 31.9 ± 0.7 kHz, starting at 32.4 kHz and ending at 22.7 kHz. Calls averaged 33.4 ± 0.7 ms in duration, with the longest call lasting 35.3 ms. Rhinolophus pusillus Temminck, 1834 This species is amongst the smallest horseshoe bats (Wt 4.2 g, FA 35.4 mm, CCL 11.54 mm), with proportionally small ears and nose-leaf and weakly developed supplementary leaflets (Fig. 10b). The lancet is elongated with a slight forward bend at the tip. The pelage is fine and silky, ranging from light brown to greyish-brown dorsally with a paler underside. The skull is small and delicate (Fig. 9, left), with a short, narrow rostrum, inflated braincase and slender zygomatic arches. The dentition is compact, with small, closely-spaced canines and premolars; the mandible is gently curved with diminutive lower teeth. In the phylogenetic analysis (Fig. 9, right), the specimen grouped within the R. pusillus clade; however, pairwise divergence from Vietnamese R. pusillus populations is high, ranging from 2.0% to 2.5%. This divergence shows a certain genetic differentiation of the studied individual relative to Vietnamese R. pusillus’s populations. Rhinolophus episcopus Allen, 1923 The male R. episcopus has morphometric measurements: HB 41.8 mm, TL 21.9 mm, HF 8.5 mm, EL 23.4 mm, FA 42.1 mm and Wt 6 g. The bat has light brown pelage, large ears and a well-developed nose-leaf. The horseshoe is broad, covering the muzzle, with small lateral leaflets and a visible median notch. The lancet is elongated with convex margins and a rounded tip (Fig. 11). The anterior median swellings are prominent and elongated, while the posterior swellings are short. The sagittal crest is weakly developed and the frontal depression is shallow. Supraorbital crests are well-defined with sharp ridges. In the R. macrotis complex, craniodental differences can be detected even amongst sympatric taxa. Comparative measurements between R. episcopus and R. cf. episcopus specimens collected from the same locality revealed that R. episcopus exhibits smaller CCL (13.79 mm vs. 14.16 mm) and BCH (6.73 mm vs. 6.95 mm) relative to R. cf. episcopus. Dental measurements such as CM L (6.38 mm vs. 6.71 mm) and CP L (2.85 mm vs. 3.05 mm) are smaller in R. episcopus. Conversely, R. episcopus displays a broader interorbital width (IOW). In this study, Cyt b sequencing was attempted for the specimen, but the obtained fragment (~ 500 bp) was insufficient in length and quality to be included in phylogenetic analyses. However, based on external morphology and craniodental traits, the identification of the specimen as R. episcopus is considered reliable. The echolocation call of this male was a broadband FM-CF-FM structure (Fig. 10 d). The maximum start frequency was 65.6 kHz and the minimum value was 58.2 kHz, 3 4 Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 21 with peak energy at 63.4 ± 0.5 kHz. The mean call duration was 25.2 ± 0.6 ms, with the longest call lasting 26.8 ms. Rhinolophus siamensis Gyldenstolpe, 1917 R. siamensis initially described as a subspecies of R. macrotis, has since been elevated to species level, with a wide distribution in Southeast Asia (Vuong et al. 2017b, Hutson et al. 2019). This individual represents a small-sized member of the macrotis complex. The FA is 37.3 mm, HB is 40.7 mm and the Wt is only 3.07 g, all lower than corresponding values for R. cf. episcopus. The skull of R. siamensis is not only shorter, but also more gracile, shows a narrower braincase, reduced rostrum width and a shorter mandible (Table 3). Morphologically, R. siamensis has relatively large ears, about half the forearm length and a broad horseshoe covering the muzzle with a distinct median notch. The sella projects forwards and the connecting process is broad and rounded, giving the nose-leaf a structure distinct from other small horseshoe bats. Its pelage is soft and woolly, brown dorsally and paler ventrally. Rhinolophus cf. episcopus Allen, 1923 In this study, R. cf. episcopus was represented by a single adult male. External measurements included FA of 43.2 mm, E of 23.1 mm and Wt of 4.51 g. The ears were large, approximately half the length of the forearm and the pelage was soft, woolly and brown dorsally with a slightly paler ventral surface. The cranial profile is broad, with MAW of 8.91 mm and ZYW of 8.17 mm. The mandible is well-developed, with a ML of 10.12 mm. The skull morphology shows a more heavily built cranial structure compared to other members of the macrotis group (Fig. 11). Molecular analysis placed the specimen within the R. macrotis complex clade. It clustered most closely with individuals provisionally identified as R. cf. episcopus from Vietnam in the study of Vuong et al. 2017b, with a Figure 11. Dorsal (A, a, a’), lateral (B, b, b’), ventral (C, c, c’) views of the cranium and Dorsal (D, d, d’) views of the mandible of R. episcopus, R. cf. episcopus and R. siamensis, respectively.  22 Vu Y et al genetic divergence of approximately 2.5–3%. This level of divergence offers a close relationship, while also showing the possibility of regional differentiation within R. cf. episcopus (Liu et al. 2019). Family Vespertilionidae Several species reported by Nguyen et al. (2012), including P. abramus, P. coromandra, P. javanicus, Ia io,T. fulvida (referred to as T. pachypus) and Murina cyclotis, were not detected in the present survey. Nevertheless, our study expands the species inventory of Xuan Nha NR by providing new records for T. tonkinensis and M. muricola. Kerivoula cf. dongduongana Vuong, Hassanin, Furey, Nguyen & Csorba, 2018 According to Vuong et al. (2018) and other studies, K. hardwickii sensu lato in Vietnam has been divided into four smaller, closely-related species that are morphologically difficult to distinguish. Based on external and craniodental measurements, our specimen is tentatively assigned to K. dongduongana; however, molecular analysis is necessary to confirm its precise species identity. Morphologically, it shows soft, long fur, with smokybrown dorsally and lighter greyish-brown ventrally. HB ranges 34.3–40.4 mm, FA 33.5– 35.4 mm, with brownish-black translucent wing membranes. K. cf. dongduongana presents a domed braincase with a concave frontal profile. Cranial measurements (GTL 14.03–14.14 mm, ML 6.65–6.75 mm) point out a small and compact cranial structure compared to larger Kerivoula species (Vuong et al. 2018, Liang et al. 2023). Myotis alticraniatus Osgood, 1932 M. alticraniatus was once considered a subspecies of M. siligorensis in Vietnam, but more recent taxonomic studies by Ruedi et al. (2021) have clarified its status as a distinct species. According to these studies, M. siligorensis is restricted to the Central and Eastern Himalayas, whereas bats occurring further east, including populations in China and Indochina, are now assigned to M. alticraniatus. A female specimen represents a well-preserved example of this small vespertilionid. It has a delicate skull with a low rostrum, domed braincase, steep frontal profile and slightly swollen occipital region. The zygomatic arches are thin and inwardly concave. Dorsal fur is buff to dark brown, with lighter greyish-brown ventrally. Ears are long and narrow, reaching or surpassing the muzzle tip. The upper premolars have a prominent size difference, particularly between P and P (Fig. 12). The bat has a delicate mandible with small lower premolars (p ) and its lower molars are semi-nyctalodont types. Myotis muricola (Gray, 1864) A female was collected at 950 m a.s.l. The pelage is soft and dense, pale brownish-grey dorsally and dirty white ventrally with dark hair roots (Fig. 13, left). Ears are long and narrow, the tragus slender and forward-bent and the feet small with short claws. The skull is small with an elongated rostrum, flattened braincase and developed sagittal crest (Fig. 13, right). The zygomatic arch is evenly curved and massive. C are wide and robust, exceeding the height of P . P are well-developed and aligned in the tooth row. P are 2 4 4 1 4 2 3 Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 23 small, displaced lingually and stand inwards on the inside of the upper tooth row. c are large, pointed and slightly higher than p . Lower molars are of the myotodont type. Genetic analysis of Cyt b confirmed the specimen within the M. muricola clade, with 1– 1.5% divergence from other conspecific sequences (Fig. 14, left). 1 4 Figure 12. Dorsal (A), lateral (B), ventral view (C) of cranium; Dorsal (D), lateral (E) of mandibles, Occlusal view of left upper (G) and right lower (H) toothrows of M. alticraniatus.  Figure 13. (Left) M. muricola recorded on Xuan Nha NR; (Right) Dorsal (A), lateral (B), ventral view (C) of cranium; Dorsal (D), lateral view (E) of mandibles, Occlusal view of left upper (G) and right lower (H) toothrows of M. muricola.  24 Vu Y et al Pipistrellus tenuis (Temminck, 1840) Externally, P. tenuis has dark brown dorsal pelage with a slightly paler ventral side and a short broad tragus (Fig. 15a). The skull is small and delicate, with a narrow rostrum, inflated braincase, weak crests and simple bicuspid upper canines (Fig. 15b). Sequencing of the Cyt b gene positioned the specimen firmly within the tenuis clade. Pairwise genetic distances ranged from 1.0% to 1.7% compared to P. tenuis individuals previously collected from the Tay Con Linh Mountain by Kruskop et al. (2024) (Fig. 14, left). Figure 14. (Left) ML phylogenetic tree, based on Cyt b sequences of the recorded Vespertilionidae bats; nodes with BS < 70% are not displayed; (Right) Ultrasonic echolocation call characteristics of T. tonkinensis.  Figure 15. (a) P. tenuis recorded on Xuan Nha NR; (b) Five aspects of the cranium and mandible of P. tenuis.  Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 25 • Ruedi M, Saikia U, Thabah A, Görföl T, Thapa S, Csorba G (2021) Molecular and morphological revision of small Myotinae from the Himalayas shed new light on the poorly known genus Submyotodon (Chiroptera: Vespertilionidae). Mammalian Biology 101: 465‑480. https://doi.org/10.1007/s42991-020-00081-3 • Srinivasulu B, Srinivasulu C (2018) In plain sight: Bacular and nose-leaf morphology supports distinct specific status of roundleaf bats Hipposideros pomona Andersen, 1918 and Hipposideros gentilis Andersen, 1918 (Chiroptera: Hipposideridae). Journal of Threatened Taxa 10 (8): 12018‑12026. https://doi.org/10.11609/jott.4111.10.8.12018-12026 • Sterling E, Hurley M, Le M (2006) Vietnam: A natural history. Yale University Press • Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution 38 (7): 3022‑3027. https://doi.org/ 10.1093/molbev/msab120 • The People’s Committee of Son La Province (2019) Geography of Son La Province. Truth National Political Publishing House, Hanoi, 931 pp. • Tidemann C, Woodside D (1978) A collapsible bat-trap and a comparison of results obtained with the trap and with mist-nets. Australian Wildlife Research 5 (3): 355‑362. https://doi.org/10.1071/WR9780355 • Tordoff A, Tran Q, Nguyen D, Le M (Eds) (2004) Sourcebook of Existing and Proposed Protected Areas in Vietnam. 2nd, Volume 1: Northern Vietnam. BirdLife International in Indochina and MARD, Hanoi. • Tordoff A, Baltzer M, Fellowes J, Pilgrim J, Langhammer P (2012) Key biodiversity areas in the Indo-Burma hotspot: Process, progress and future directions. Journal of Threatened Taxa 4 (8): 2779‑2787. https://doi.org/10.11609/jott.o3000.2779-87 • Volleth M, Nguyen S, Wu Y, Li Y, Yu W, Lin L, Arai S, Trifonov V, Liehr T, Harada M (2017) Comparative chromosomal studies in Rhinolophus formosae and R. luctus from China and Vietnam: Elevation of R. l. lanosus to species rank. Acta Chiropterologica 19 (1): 41‑50. https://doi.org/10.3161/15081109ACC2017.19.1.003 • Vuong T, Csorba G, Görföl T, Arai S, Nguyen S, Hoang T, Hasanin A (2015) Description of a new species of the genus Aselliscus (Chiroptera, Hipposideridae) from Vietnam. Acta Chiropterologica 17 (2): 233‑254. https://doi.org/10.3161/15081109ACC2015.17.2.002 • Vuong T, Csorba G, Ruedi M, Furey N, Nguyen S, Vu T, Bonillo C, Hassanin A (2017a) Comparative phylogeography of bamboo bats of the genus Tylonycteris (Chiroptera, Vespertilionidae) in Southeast Asia. European Journal of Taxonomy 274: 1‑38. https:// doi.org/10.5852/ejt.2017.274 • Vuong T, Hassanin A, Görföl T, Arai S, Fukui D, Hoang T, Nguyen S, Furey N, Csorba G (2017b) Integrative taxonomy of the Rhinolophus macrotis complex (Chiroptera, Rhinolophidae) in Vietnam and nearby regions. Journal of Zoological Systematics and Evolutionary Research 55 (3): 177‑198. https://doi.org/10.1111/jzs.12169 • Vuong T, Hassanin A, Furey M, Nguyen T (2018) Four species in one: multigene analyses reveal phylogenetic patterns within Hardwicke’s woolly bat, Kerivoula hardwickii-complex (Chiroptera, Vespertilionidae) in Asia. Hystrix, the Italian Journal of Mammalogy 29 (1): 111‑121. https://doi.org/10.4404/hystrix-00017-2017 • Vu T (2012) New records of Griffin’s Leaf-nosed bat (Hipposideros griffini Thong et al. 2012) from Vietnam. Vietnamese Journal of Biology 34 (3): 323‑327. https://doi.org/ 10.15625/0866-7160/v34n3.2463 32 Vu Y et al • Vu T, Puechmaille S, Denzinger A, Dietz C, Csorba G, Bates P, Teeling E, Schnitzler H (2012) A new species of Hipposideros (Chiroptera: Hipposideridae) from Vietnam. Journal of Mammalogy 93 (1): 1‑11. https://doi.org/10.1644/11-MAMM-A-073.1 • Vu T (2015) Bats of Cat Tien National Park: diversity, echolocation and taxonomic remarks. Vietnamese Journal of Biology 37 (3): 336‑343. https://doi.org/ 10.15625/0866-7160/v37n3.7418 • Vu T (2019) New records of Hipposideros griffini from lava caves and the threats to its conservation in Vietnam. Academia Journal of Biology 41 (4). https://doi.org/ 10.15625/0866-7160/v41n4.14487 • Vu Y, Bui H, Hoang T, Vu L, Nguyen S (2024) Multivariate analysis of craniodental morphology in mouse-eared bats (Chiroptera, Vespertilionidae, Myotis) from Vietnam. Biodiversity Data Journal 12: e122597. https://doi.org/10.3897/BDJ.12.e122597 • Wilson D, Mittermeier R (Eds) (2019) Handbook of the Mammals of the World. Volume 9: Bats. Lynx Edicions, Barcelona, 1008 pp. [ISBN 978-84-16728-19-0] • Yuzefovich A, Artyushin I, Scopin A, Nguyen S, Kruskop S (2022) Taxonomic diversity of the Hipposideros larvatus species complex (Chiroptera: Hipposideridae) in mainland Asia. Zootaxa 5200 (1): 73‑95. https://doi.org/10.11646/zootaxa.5200.1.6 Supplementary materials Suppl. material 1: Bat dataset table Authors: YHV, STN, HTB Data type: occurences Brief description: Bat dataset table from the field survey in Xuan Nha NR, Son La, Vietnam (March 2010 & August 2024). Dash (–): Not determined. Habitat nature: 1 = Evergreen forest, 2 = Disturbed secondary forest, 3 = Cave areas, 4 = Stream valley. Download file (44.13 kb) Suppl. material 2: List of craniodental measurements used in this study Authors: YHV, STN, HTB Data type: morphological Download file (19.07 kb) Study of bat diversity (Mammalia, Chiroptera) in Xuan Nha Nature Reserve, ... 33