Mammalia 2015; 79(2): 201–213

Vuong Tan Tu, Raphaël Cornette, José Utge and Alexandre Hassanin* First records of lorelieae (Chiroptera: ) from Vietnam

Abstract: In 2011, three specimens from an unknown DOI 10.1515/mammalia-2013-0101 species of tube-nosed (genus Murina) were collected Received July 2, 2013; accepted April 30, 2014; previously published in montane moist forest at altitudes between 1117 and online June 7, 2014 1682 m in the Ngoc Linh Nature Reserve of Vietnam. We sequenced the mitochondrial COI gene from Ngoc Linh and performed comparisons with Murina sequences Introduction available in the nucleotide databases. The results sug- gested that the three unidentified specimens belong to The subfamily includes the tube-nosed bats Murina lorelieae, a species recently described from a and hairy-winged bats of the three genera Murina, Har- single specimen collected in southern China. Nucleotide piocephalus, and (Kuo et al. 2006). The system- distances between specimens from Ngoc Linh and south- atics of the genus Murina is regularly renewed since half ern China are exceptionally low for M. lorelieae (1.25%) in of the species have been described in the past 10 years: comparison with three other Murina species, i.e., Murina Murina harrisoni (Csorba and Bates 2005), Murina tiensa cyclotis, Murina feae, and Murina huttoni (3.9–5.5%). (Csorba et al. 2007), Murina harpioloides (Kruskop and We suggest that M. lorelieae is adapted to montane for- Eger 2008), Murina eleryi (Furey et al. 2009), Murina ests, which may have facilitated long-distance dispersal bicolor, Murina gracilis, Murina recondita (Kuo et al. events between southern China and Vietnam during gla- 2009), Murina beelzebub, Murina cineracea, Murina wal- cial periods of the Pleistocene. Morphological compari- stoni (Csorba et al. 2011), Murina jaintiana, Murina plu- sons based on body-size measurements and geometric viallis (Ruedi et al. 2012), Murina chrysochaetes, Murina morphometric analyses of the skulls showed differences lorelieae, Murina shuipuensis (Eger and Lim 2011), between Vietnamese specimens and the Chinese holo- Murina annamitica, and Murina fionae (Francis and Eger type of M. lorelieae. We proposed that the Vietnamese 2012). However, the taxonomic status and geographical specimens belong to a distinct subspecies, M. lorelieae distribution of some species are highly controversial. ngoclinhensis. For instance, Csorba et al. (2011) considered that South- east Asian bats previously identified as Murina tubi- Keywords: DNA barcode; morphology; Murininae; South- naris should be split into two distinct species, Murina east Asia; subspecies. belzeebub or Murina cineracea, whereas Francis and Eger (2012) treated M. cineracea as a junior synonym of Murina feae. In the end of the year 2011, a joint bat expedition

*Corresponding author: Alexandre Hassanin, Muséum national between the Institute of Ecology and Biological Resources d’Histoire naturelle (MNHN), Institut de Systématique, Evolution, (IEBR) and Muséum national d’Histoire naturelle, (MNHN, Biodiversité (ISYEB), UMR 7205, CP51, 55, rue Buffon, 75005 Paris, Paris) took place in the Ngoc Linh Nature reserve in the France; Sorbonne Universités, UPMC Univ Paris 06, UFR927, Kon Tum province of Central Vietnam (Figure 1). During 75005 Paris, France; and MNHN, UMS 2700, CP26, 43, rue Cuvier, the expedition, three adult tube-nosed bats, one male 75005 Paris, France, e-mail: [email protected] and two females, were collected and identified as the Vuong Tan Tu: MNHN, ISYEB UMR 7205, CP51, 55 rue Buffon, 75005 Paris, France; MNHN, UMS 2700, CP26, 43, rue Cuvier, same morphospecies. At first sight, their general appear- 75005 Paris, France; and Institute of Ecology and Biological ance was similar to that of Murina cyclotis. However, Resources, Vietnam Academy of Science and Technology, 18, external measurements and the detailed examination Hoang Quoc Viet road, Cau Giay district, Hanoi, Vietnam of both dorsal and ventral pelages suggested that these Raphaël Cornette: MNHN, ISYEB UMR 7205, CP51, 55 rue Buffon, specimens belong to a species of Murina not recorded 75005 Paris, France; and MNHN, UMS 2700, CP26, 43, rue Cuvier, 75005 Paris, France from Vietnam. José Utge: MNHN, UMS 2700, CP26, 43, rue Cuvier, 75005 Paris, Playing the important part of recent studies on bats, France DNA barcoding was recognized as a useful taxonomic 202 V.T. Tu et al.: First records of Murina lorelieae from Vietnam

Figure 1 Geographic location of Ngoc Linh Nature Reserve (Kon Tum province, Vietnam) and photograph of the live specimen of Murina lorelieae ngoclinhensis ssp. nov. (holotype MNHN 2013-1078, ). The map was created using Online Map Creation (http://www.planiglobe.com).

method to accelerate the discovery of new species (Francis vegetation types including lowland tropical rainforest et al. 2010). This approach is based on the comparisons at elevations < 1000 m and montane tropical rainforest of DNA sequences of the 5′ fragment of the mitochondrial above an altitude of 1000 m. Despite the conversion of cytochrome c oxidase subunit I (COI) gene. Fortunately, forest to agriculture in parts of the low altitude range the COI sequences of almost all Indochinese species of of the nature reserve, Ngoc Linh retains a large area of Murina are available in the international nucleotide data- undisturbed primary forest, mostly found at high ele- bases (EMBL/GenBank/DDBJ and BOLD). These data vations (Trai et al. 1999). These habitats contain high allow the rapid species identification of unknown speci- levels of biodiversity and endemism, many of those were mens collected in this region. recently described or are to be discovered (Bain and Therefore, we sequenced the COI barcode fragment Nguyen 2004, Abramov et al. 2006, Kruskop et al. 2006, from tube-nosed bats collected in the Ngoc Linh Nature Jenkins et al. 2007, Orlov 2009). Reserve in order to make rapid comparisons with all COI Bats were captured in the field using four-bank harp sequences previously generated for Murina species. Then, traps in combination with several Ecotone mist nets. Bats we performed morphological comparisons among the were then measured, photographed, and initially identi- four Murina species collected during our expedition in fied following the field guides (Borissenko and Kruskop Ngoc Linh. 2003, Francis 2008). All captured bats were adults as confirmed by the presence of fully ossified metacarpal- phalangeal joints. Materials and methods

Field survey DNA barcoding analyses

The Ngoc Linh (Kon Tum) Nature Reserve (geographic Total genomic DNA was extracted from muscle samples coordinates: 14°45′–15°15′N and 107°21′–108°20′E) using QIAGEN DNeasy Tissue Kit (Qiagen, Hilden, covers an area of 41.42 km2 in the Kon Tum Plateau of Germany) following the manufacturer’s protocol with the the Central Highlands of Vietnam (Figure 1). Topologi- final volume of 200 μl eluted DNA in AE buffer. cally, the region is composed mostly of mountainous For this study, the 5′ fragment of the mitochondrial and hilly terrain, including the second highest mountain COI gene was amplified and sequenced with two primers: in Vietnam, Mount Ngoc Linh (2598 m). The complex UTyr and C1L705 (Hassanin et al. 2012). The polymerase topology and climate of the region support a variety of chain reactions (PCR) were carried out in a volume of 20 V.T. Tu et al.: First records of Murina lorelieae from Vietnam 203

μl containing 3 μl of PCR buffer 10 × with MgCl2, 2 μl of specimens to the nearest 0.1 mm. Mass – the weight dNTPs (6.6 mm), 1 μl of each of two primers (10 μm), and of bat in gram; all the following measurements were 0.1 μl of Taq polymerase (2.5 U, Qiagen, Hilden, Germany). based primarily on Eger and Lim (2011): TL: total length The PCR was run using the C1000 Touch thermal cycler – from the tip of the face/chin to the tip of the tail; FA: (BIO-RAD) as follows: 4 min at 94°C; the denaturation/ forearm length – from the elbow to the wrist with both annealing/elongation process was set with 5 cycles of joints folded; Tib: length of tibia – from the knee to the 30 s at 94°C, 60 s at 60°C, and 60 s at 72°C, followed by 30 ankle; Ear: ear length – from the base of the ear, where cycles of 30 s at 94°C, 60 s at 50°C, and 60 s at 72°C. Final it attaches to the head, to the tip of the pinna; Tragus: elongation followed for 5 min at 72°C. PCR products were length of tragus – from the point where the proximal purified using ExoSAP Kit (GE Healthcare, Buckingham- edge of the tragus joins the bottom of the ear to the tip of shire, UK) and then sequenced in both directions using the tragus; HF: hind foot length – from the heel to the tip an automated DNA Sequencer (Applied Biosystems 3100). of the longest toe, including the claw; 3DM, 4DM, 5DM: These two last steps were performed at the Centre National length of third, fourth, and fifth metacarpals taken from de Séquençage (Genoscope) in Evry (France). Sequences the wrist to the end of the respective metacarpals; 3D1P, were edited and assembled using Codoncode Alignment 3D2P, 4D1P, 4D2P, 5D1P, 5D2P: length of the first and Version 3.7.1 (CodonCode Corporation). second phalanges of the respective third, fourth, and The COI sequences newly generated (accession fifth digits. numbers KF772775–KF772784) were compared to those Cranial measurements (Table 1, Supplementary available in the EMBL/GenBank/DDBJ nucleotide data- material 2) include GLS: greatest length of skull – from bases. A phylogenetic tree of the genus Murina was the posterior edge of the skull to the front of the inci- reconstructed using the Bayesian method. The outgroup sors; CIL: condylo-incisive length – from the occipi- species were chosen on the basis of previous molecular tal condyles to the front of the incisors; PAL: palatal studies on the subfamily Murininae (e.g., Ruedi et al. length – from the anterior palatal emargination to the 2012). Accordingly, Myotis muricola and Kerivoula hard- midpoint of the posterior palatal emargination (with wicki were used as the most distant outgroup species to palatal spike, if present); ZB: zygomatic breadth – great- root the tree because they belong to two different sub- est width across the zygoma; MB: mastoid breadth – families, Myotinae and Kerivoulinae, respectively. Two greatest breadth across the mastoids; BBC: braincase other species of different genera of the subfamily Murini- breadth – greatest breadth across the braincase; POC: nae were also included in the analyses: Harpiocephalus postorbital constriction – the least breadth of the con- harpia and Harpiola isodon (Simmons 2005, Kuo et al. striction posterior to the orbits; IC: least interorbital 2006). DNA sequences were aligned manually on Se-Al breadth – shortest distance between the orbits meas- v2.0a11 (A. Rambaut. Sequence Alignment Editor Version ured at the rostrum; CM3: length of maxillary toothrow 2.0 alpha 11. 2002; http://evolve.zoo.ox.ac.uk/). The COI – from the front of the canine to the posterior edge of dataset represents a total alignment of 657 nucleotides the 3rd upper molar; M2M2: breadth across upper molars and 108 taxa. The best-fitting model of sequence evolution – greatest breadth measured across the outer edges of was selected under jModelTest (Posada 2008) using the the second upper molars; CC: greatest breadth across Akaike information criterion. Bayesian analyses were then the upper canines; ML: greatest length of mandible – conducted using the selected GTR+G model on MrBayes greatest length measured from the posterior edge of the v3.2 (Ronquist et al. 2012). The posterior probabilities (PP) mandibular condyles to the front of the lower incisors; were calculated using four independent Markov chains CM3: length of mandibular toothrow – from the front of run for 10,000,000 Metropolis-coupled MCMC genera- the canine to the posterior edge of the 3rd lower molar; tions, with tree sampling every 1000 generations, and a HCP: height of coronoid process – measured from the burn-in of 25%. Mean pairwise distances were calculated inferior surface of the angular process of the ramus to with PAUP version 4b10 (Swofford 2002) using Kimura’s the tip of the coronoid process. two-parameter (K2P) model. Landmark-based geometric morphometric analyses (Zelditch et al. 2012) were used to describe and quantify more accurately shape differences in skulls of Murina Morphological comparisons collected in Ngoc Linh. The holotype of Murina lorelieae was also included in the analyses. Two-dimensional External measurements (Table 1, Supplementary coordinates were recorded using tpsDig2 (Rohlf 2010) on material 2) were taken from 10 living bats or museum digital images of four datasets, representing lateral and 204 V.T. Tu et al.: First records of Murina lorelieae from Vietnam ventral views of crania, and lateral and occlusal views of (PP = 1), a species recently described from a single male mandibles. The number and locations of landmarks are specimen collected in Diding Headwater Forest Nature indicated in Supplementary materials 3–7. Morphometric Preserve in southern China (Eger and Lim 2011; hereafter analyses were computed using “Rmorph” (Baylac 2012) referred to as Diding). The three Ngoc Linh specimens have library in “R” (R Development Core Team 2011). Shape identical COI sequences, and they differ from the Chinese variations were estimated using General Procruste Analy- holotype by only 1.25%, which is smaller than minimal ses (GPA) (Rohlf and Slice 1990). The differences in shape intraspecific distances calculated between allopatric pop- were displayed using principal component analyses (PCA) ulations of Ngoc Linh and southern China for three other and multivariate regressions along axes (Monteiro 1999). species of Murina, i.e., 4.84% for Murina cyclotis, 3.96% The first PCA axes obtained from each of the four datasets for Murina feae, and 3.38% for Murina huttoni (Table 2). were extracted and then combined in a new PCA in order The molecular data indicate therefore that Ngoc Linh to synthesize our different results. specimens belong to Murina lorelieae. In addition, the low COI distances calculated between Vietnamese specimens and the Chinese holotype of M. lorelieae suggest that these allopatric populations were isolated from each other more Results and discussion recently than allopatric populations of the three other species of Murina (see below for a possible explanation). Phylogenetic analyses based on COI DNA sequences Morphological comparisons The Bayesian tree reconstructed from the nucleotide align- ment of COI sequences is presented in Figure 2. The genus In the genus Murina, dentition differences are currently Murina appears as a paraphyletic group, due to the inclu- used as key characters for distinguishing two groups with sive position of Harpiola, a genus sometimes considered no phylogenetic value (Corbet and Hill 1992, Koopman as a subgenus of Murina (e.g., Simmons 2005). However, 1994): the “cyclotis group,” which contains 10 species, these deep relationships are not robust (PP < 0.5), suggest- i.e., Murina cyclotis, Murina annamitica, Murina fionae, ing that additional molecular markers, including nuclear Murina harrisoni (including Murina tiensa), Murina markers, should be sequenced for testing the monophyly huttoni, Murina lorelieae, Murina peninsularis, Murina of the genus Murina. pluvialis, Murina puta, and Murina rozendaali; and the All recognized species for which at least two speci- “suilla group,” which includes 21 species, i.e., Murina mens were sequenced were found to be monophyletic suilla, Murina aurata, Murina beelzebub, Murina bicolor, with maximum support values (PP = 1): Murina aenea, Murina chrysochaetes, Murina eleryi, Murina feae (includ- Murina annamitica, Murina cyclotis, Murina eleryi, Murina ing Murina cineracea), Murina fusca, Murina gracilis, feae, Murina fionae, Murina harrisoni, Murina hilgendorfi, Murina harpioloides, Murina hilgendorfi, Murina jaintiana, Murina huttoni, Murina peninsularis, Murina suilla, Murina Murina leucogaster, Murina recondita, Murina ryukyuana, ussuriensis, and Murina walstoni. Maximal intraspecific Murina shuipuensis, Murina silvatica, Murina tenebrosa, distances in the species of Murina range from 0.4% in M. Murina tubinaris, Murina ussuriensis, and Murina walstoni hilgendorfi to 6.9% in M. cyclotis. The smallest interspe- (Csorba et al. 2011, Eger and Lim 2011, Francis and Eger cific distance was found between Murina leucogaster and 2012, Ruedi et al. 2012). The three putative Vietnamese Murina shuipuensis, (2.6%), the species recently described specimens of M. lorelieae exhibit the dental character- in southern China (Eger and Lim 2011). However, all other istics of the “cyclotis group”: the crown area of the first distances between species of Murina range from 9% to upper premolar (P2) represents about two thirds that of the 19.8% (see Supplementary material 1 for details). These second (P4); and both upper incisors (I2 and I3) are visible distances are higher than those calculated in Kerivoulinae in lateral view (Figures 3 and 4). These features collectively and Myotinae, two other subfamilies of Vespertilionidae serve to distinguish the new specimens from members of (Francis et al. 2010). These comparisons suggest therefore the “suilla group,” in which I2 is situated more anteriorly that the mtDNA genome of Murininae is characterized by to I3, such that it is visible in lateral view, and the crown higher rates of nucleotide substitution. area of P2 is half or less that of P4 (see Figures 3A, 4A of Our mtDNA analyses indicate that the three unidenti- M. feae for an example). fied specimens from Ngoc Linh are grouped into the same Within the “cyclotis group,” the three new speci- clade (PP = 1), which is the sister group of Murina lorelieae mens mostly resemble the holotype of Murina lorelieae V.T. Tu et al.: First records of Murina lorelieae from Vietnam 205

Myotis muricola HM914942 Kerivoula hardwickii HM540687 Harpiocephalus harpia HM540283 Harpiola isodon HM540286 Vietnam: Ngoclinh (Kon Tum) 0.6 HM540980 Vietnam: Yokdon (Dak Lak) 1 HM540982 Vietnam: Yokdon (Dak Lak) Murina harrisoni 1 HM540981 Vietnam: Yokdon (Dak Lak) 0.4 1 HM540983 Laos: Nam Et (Louangphrabang) HM540984 China: Shiwandashan (Guangxi) 1 HM540976 Laos: NakaiNam Theun (Khammouan) 0.5 HM540979 China: Shuhuangshan (Hunan) JQ601542 Vietnam: Ngoclinh (Quang Nam) 1 1 MNHN 2013-1082 Vietnam: Ngoclinh (Kon Tum) 1 MNHN 2013-1083 Vietnam: Ngoclinh (Kon Tum) Murina huttoni 1 0.3 IEBR-Tu021211.1 Vietnam: Ngoclinh (Kon Tum) HM540978 China: Daweishan (Hunan) 0.3 0.3 JQ601452 China: Daweishan (Hunan) 1 0.3 0.9 JQ601454 China: Daweishan (Hunan) JQ601455 China: Daweishan (Hunan) 1 JF442842 Russia: Chernyshevka (Anuchinskii dist.) 0.9 JF442848 Russia: Lazovskyy (Lazo dist.) Murina ussuriensis 1 JF442843 Russia: Terney dist. JF442844 Russia:Nevelsk dist. 0.6 JF442850 Russia: Dolinsk dist. 1 Murina chrysochaetes HM540986 China: Diding (Guangxi) Murina harpioloides HM540975 Vietnam: Bidup (LamDong) Murina aenea 1 ADQ50994 Malaysia: Endau Rompin (Johor) HM540929 Malaysia: Sepilok (Sabah) 0.6 0.6 0.6 HM540989 Malaysia: Tabin (Sabah) Murina suilla 1 HM540990 Malaysia: Madai (Sabah) HM540991 Malaysia: Sepilok (Sabah) 1 HM540958 Laos: Phou Phanang (Vientiane) 0.8 HM540959 Laos: DongKanthung (Champasak) Murina walstoni 1 0.8 Q601537 Vietnam: ThacMai (Dong Nai) HM540962 Laos: Dong Amphan (Attapeu) 0.7 HM540970 Laos: NakaiNam Theun (Khammouan) 1 HM540967 Laos: NakaiNam Theun (Khammouan) Murina annamitica 1 0.9 HM540968 Laos: Nakai (Khammouan) HM540971 Laos: NamHa (LouangNamtha) 1 HM540993 Laos: Nam Et (Houaphan) 1 HM540995 Laos: NakaiNam Theun (Khammouan) 0.5 JQ601526 Laos: NakaiNam Theun (Khammouan) 0.7 1 HM540999 Laos: Dong Amphan (Attapeu) HM541000 Vietnam: Ngoc Linh (QuangNam) 1 0.6 JQ601539 Vietnam: Ngoc Linh (QuangNam) Murina feae 0.2 (sensu Francis and Eger, 2012) JQ601541 Vietnam: Ngoc Linh (QuangNam) 1 0.2 JQ601540 Vietnam: Ngoc Linh (QuangNam) 0.7 HM540994 Laos: NakaiNam Theun (Khammouan) 1 HM540997 Laos: NakaiNam Theun (Khammouan) 1 JQ601463 China: Diding (Guangxi) 0.5 JQ601519 China: Shiwandashan (Guangxi) 1 MNHN 2013-1080 Vietnam: Ngoc Linh (Kon Tum) IEBR-Tu071211.1 Vietnam: Ngoc Linh (Kon Tum)

0.2 JN082179 Diding (China) Murina lorelieae 1 0.3 IEBR-Tu281111.1 Ngoclinh (Kon Tum) MNHN 2013-1078 Ngoclinh (Kon Tum) 1 MNHN 2013-1079 Ngoclinh (Kon Tum) 1 Murina cf. cyclotis HM540939 India: Tamil Nadu 0.5 0.4 Murina cf cyclotis HM540955 Thailand: Krabi Murina fionae 1 HM540965 Laos: NakaiNam Theun (Khammouan) HM540966 Vietnam: Ngoclinh (Quang Nam) 0.3 HM540940 Laos: NakaiNam Theun (Khammouan) 0.8 JQ601530 Laos: Phou Hi Poun (Khammouan) 0.6 JQ601528 Laos: Phou Hi Poun (Khammouan) 0.9 HM540947 Laos: Phou Khao Khouay (Vientiane) 1 HM540944 Laos: Nakai (Khammouan) JQ601531 Laos: Phou Hi Poun (Khammouan) 0.7 JQ601532 Laos: Phou Khao Khouay (Vientiane) 1 HM540941 Laos: Nam Et (Houaphan) 1 JQ601533 Laos: Hin Nam No (Khammouan) JQ601534 Laos: Hin Nam No (Khammouan) 1 HM540948 China: Shiwandashan (Guangxi) HM540949 China: Diding (Guangxi) 0.2 Murina cyclotis 1 HM540951 China: Diding (Guangxi) 1 1 0.1 JQ601478 China: Diding (Guangxi) 0.9 0.1JQ601518 China: Shiwandashan (Guangxi) JQ601468 China: Diding (Guangxi) 0.4 0.2 0.3 JQ601524 China: Shiwandashan (Guangxi) JQ601464 China: Diding (Guangxi) 1 JQ601525 China: Shiwandashan (Guangxi) 0.8 1 HM540950 China: Diding (Guangxi) 1 JQ601469 China: Diding (Guangxi) 0.6 0.8 JQ601461 China: Diding (Guangxi) 0.8 HM540953 Vietnam: Huulien (Langson) 0.6 1 JQ601535 Vietnam: Tamdao (Vinh Phuc) 1 JQ601496 China: Diding (Guangxi) JQ601536 Vietnam: Nahang (Tuyen Quang) 0.5 HM540945 Laos: Dong Amphan (Attapeu) 1 HM540952 Laos: Dong Amphan (Attapeu) MNHN 2013-1081 Vietnam Ngoclinh (Kon Tum) 0.9 0.7 IEBR-Tu031211.1 Vietnam Ngoclinh (Kon Tum) 1 JQ601538 Vietnam Ngoclinh (Quang Nam) 0.2 0.7 JQ601544 Vietnam Ngoclinh (Quang Nam) HM540946 Laos: Xe pian (Champasak) Murina peninsularis 1 HM540972 Malaysia: Kuala Lompat (Pahang) HM540973 Malaysia: Kuala Lompat (Pahang) 1 HM540931 Laos: NakaiNam Theun (Khammouan) Murina eleryi 1 HM540933 Vietnam: Ngoclinh (Quang Nam) HM540935 China: Diding (Guangxi) 0.5 HM540937 China: Diding (Guangxi) 0.1 JF442833 China: Heilongjiang (Pingshan) 0.03 JF442836 Russia: Anuchino JF442834 Russia:Shira 0.2 0.1 JF442840 Russia: UstKansk Murina hilgendorfi 1 JF442835 Russia: Anuchino 0.1 JF442839 Russia: UstKansk 0.6 JF442841 Russia: Maslyanino 1 Murina leucogaster HM540987 China: Diding (Guangxi) Murina shuipuensis JN082180 China: Libo (Guizhou)

0.04

Figure 2 Bayesian tree of the subfamily Murininae reconstructed from the analysis of 108 DNA sequences of the mitochondrial cytochrome c oxidase subunit I gene. The values on the nodes indicate posterior probabilities. by sharing similarity in both external and dental features. surfaces, copper reddish-brown and dirty white, respec- They have long shiny hairs (8 mm ventrally and 13–15 mm tively (Figure 5). Dorsal hairs have a tri-colored pattern: dorsally) with distinct colorations on dorsal and ventral dark gray at the base, pale in the middle, and reddish 206 V.T. Tu et al.: First records of Murina lorelieae from Vietnam

A

B

C

D

10 mm

Figure 3 Lateral views (left) and occlusal views (right) of skulls of the four Murina species collected in Ngoc Linh Nature Reserve (Vietnam). (A) Murina feae (MNHN 2013-1080, ); (B) Murina cyclotis (IEBR-031211.1, ); (C) Murina lorelieae ngoclinhensis ssp. nov. (holotype MNHN 2013-1078, ); and (D) Murina huttoni (MNHN 2013-1082, ).

brown at the tip. The ventral underfur is bicolored: dark equal in height, and I2 significantly exceeds I3 in height gray in about two thirds of the length and whitish at the (Francis and Eger 2012). tip (Figure 5). Dentally, I2 and I3 are slightly equal in height The morphological comparisons of the three Vietnam- in frontal view. The mesostyles of M1 and M2 are well devel- ese specimens of Murina lorelieae suggest the existence of oped. The canines (upper and lower) are longer than the a sexual size dimorphism in favor of females, as it is gen- second premolars (Figures 3 and 4). These features also erally observed in other species of Murina (data from Kuo distinguish the three specimens from other members of et al. 2009, Francis and Eger 2012). The male collected in the “cyclotis group.” For instance, the pelage of Murina Ngoc Linh is larger than the holotype of M. lorelieae, with annamitica is very similar to that of the new putative a bigger body mass (5 vs. 4 g), and longer forearms (33 vs. materials of M. lorelieae, but in this species, P2 and P4 are 31 mm), tibia (19 vs. 14.6 mm), and skull (GLS: 16.23 vs. V.T. Tu et al.: First records of Murina lorelieae from Vietnam 207

A

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D

Figure 4 Front view of upper incisors (left) and occlusal views of upper left toothrows (right) of the four Murina species collected in Ngoc Linh Nature Reserve (Vietnam). (A) Murina feae (MNHN 2013-1080, ); (B) Murina cyclotis (IEBR-031211.1, ); (C) Murina lorelieae ngoclin- hensis ssp. nov. (holotype MNHN 2013-1078, ); and (D) Murina huttoni (MNHN 2013-1082, ).

15.5 mm) (Table 1). However, such variations in body size the differences in skull shape, we performed landmark- measurements ( ± 10–20%) were previously mentioned in based geometric morphometric analyses. Figure 6 shows a few other species of the “cyclotis group,” such as Murina the PCA constructed using the first PCA axes obtained cyclotis and Murina harrisoni (Francis and Eger 2012). from each of the four datasets corresponding to lateral and In addition, the skull’s shape of Ngoc Linh specimens occlusal views of mandible, and lateral and ventral views of M. lorelieae (Figure 3) differs from that of the Chinese of cranium (Supplementary materials 3–7). The first PCA holotype. In lateral view, it is elongate and gradually rises axis tends to separate Murina huttoni from Murina feae. from the rostrum to the forehead. By contrast, the skull The differences in shape variation are mainly explained by of the Chinese holotype is characterized by an abruptly the lateral view of the cranium (correlation: 0.89) and the rising profile (Eger and Lim 2011). To better characterize two views of the mandible (correlations of 0.92 and 0.90 208 V.T. Tu et al.: First records of Murina lorelieae from Vietnam

AB CD

Figure 5 Pelage and detailed pattern of hairs (scale = 5 mm) on ventral (top) and dorsal (bottom) surfaces of the four Murina species col- lected in Ngoc Linh Nature Reserve (Vietnam). From left to right: (A) Murina feae (MNHN 2013-1080, ); (B) Murina cyclotis (IEBR-Tu031211.1, ); (C) Murina lorelieae ngoclinhensis ssp. nov. (holotype MNHN 2013-1078, ); and (D) Murina huttoni (MNHN 2013-1082, ).

for lateral and occlusal views, respectively). The second similar shape, which differs from that of the Chinese holo- PCA axis tends to separate M. lorelieae, and especially the type of M. lorelieae. holotype, from other species of Murina. The differences in Our morphological comparisons have therefore shape variation are mainly explained by the ventral view revealed important differences in body size and skull of cranium (correlation: 0.97). The results show therefore shape between the Vietnamese specimens and the Chinese that the four species collected in Ngoc Linh can be distin- holotype of M. lorelieae. Hence, we propose that the Ngoc guished on the basis of their skull shape. In addition, the Linh specimens of M. lorelieae belong to a different sub- three Vietnamese specimens of M. lorelieae have a very species, hereafter named “ngoclinhensis.” V.T. Tu et al.: First records of Murina lorelieae from Vietnam 209

M. feae − IEBR MNHN 0.02 Tu071211.1 2013-1080 MNHN 2013-1082 IEBR Tu031211.1 0.01 M. huttoni M. cylotis MNHN IEBR 2013-1081 Tu021211.1 0 MNHN 2013-1083

MNHN PC2 -0.01 2013-1079 MNHN 2013-1078 IEBR Tu281111.1 -0.02 M. l. ngoclinhensis

M. l. lorelieae -0.03 Holotype

-0.06 -0.04 -0.02 0 0.02 0.04 PC1

Figure 6 Principal component analysis based on geometric morphometric analyses of the skulls of the four Murina species collected in Ngoc Linh. The Chinese holotype of Murina lorelieae was also included in the analyses.

Murina lorelieae ngoclinhensis Tu and Vietnam, 15°04.766 N; 107°49.833 E, elevation 1117 m Hassanin, ssp. nov. a.s.l., adult female, in alcohol skull removed, collected 28 November 2011 by Alexandre Hassanin and Vuong Holotype Tan Tu, accession number of COI sequence: KF772779. –– MNHN 2013-1079 (Field number VN1566, tissue code MNHN 2013-1078 (Field number VN1563, tissue code VN11- VN11-1223), Ngoc Linh Nature Reserve, Kon Tum, 1220), adult male, in alcohol skull removed, collected 3 Vietnam, 15°03.884 N, 107°49.888 E, 1682 m a.s.l., December 2011 by Alexandre Hassanin and Vuong Tan adult female, in alcohol skull removed, collected Tu. Mass: 5 g. Measurements (in mm) for the holotype are 3 December 2011 by Alexandre Hassanin and as follows: FA: 33.00; Tib: 19.00; Ear: 14.00; Tragus: 8.08; Vuong Tan Tu, accession number of COI sequence: 3DM: 30.19; 4DM: 29.03; 5DM: 29.07; 3D1P: 14.17; 3D2P: KF772781. 12.81; 4D1P: 10.48; 4D2P: 9.04; 5D1P: 11.07; 5D2P: 10.80; GLS: 16.23; CIL: 14.57; ZB: 8.83; MB: 7.65; BBC: 7.40; POC: 4.30; IC: 5.20; CM3: 5.34; M2M2: 5.50; CC:3.87; ML: 10.84; Etymology

CM3: 5.64; HCP: 4.23. The sequence of the mitochondrial gene COI has been deposited in the EMBL/GenBank/DDBJ The name “ngoclinhensis” is derived from the Mount Ngoc nucleotide databases with accession number KF772780. Linh of the Ngoc Linh Nature Reserve, Kon Tum province, Vietnam, from which all specimens of this subspecies were collected. Type locality

Ngoc Linh Nature Reserve, Kon Tum province, Vietnam. Diagnosis 15°03.884 N, 107°49.888 E, elevation 1682 m a.s.l. Murina lorelieae ngoclinhensis is externally similar to the typical form, M. l. lorelieae, but significantly larger in body Paratypes size (Table 1). In addition, the skull shape of the new sub- species is elongate and gradually rises from the rostrum to –– IEBR-Tu281111.1 (Field number VN1504 – tissue code the forehead (Figure 3), while that of the holotype is char- VN11-1161), Ngoc Linh Nature Reserve, Kon Tum, acterized by an abruptly rising profile. 210 V.T. Tu et al.: First records of Murina lorelieae from Vietnam

Table 1 External, cranial, and dental measurements (in mm, except mass in g) of the four specimens of Murina lorelieae: Murina lorelieae lorelieae from South China, Murina lorelieae ngoclinhensis from Vietnam (see Materials and methods for acronyms).

Characters M. l. lorelieae M. l. ngoclinhensis ssp nov. Holotype Holotype Paratype Paratype MNHN 2013-1078 IEBR-Tu281111.1 MNHN 2013-1079 Field No: VN1563 Field No: VN1504 Field No: VN1566 (Tis. VN11-1220) (Tis. VN11-1161) (Tis. VN11-1223)

Mass 4.0 5.0 5.5 6.5 TL 74.0 71.0a 84.0 85.0 FA 30.8 33.0 35.6 34.9 Tib 14.6 19.0 19.3 19.1 Ear 15.0 14.0 15.6 16.0 Tragus 9.0 8.1 8.0 8.1 3DM 30.03 30.19 32.94 34.87 3D1P 12.79 14.17 15.26 14.94 3D2P 11.3 12.8 14.1 13.5 4DM 29.16 29.03 31.62 30.77 4D1P 9.4 10.5 11.4 10.9 4D2P 7.6 9.0 9.9 9.0 5DM 29.09 29.07 32.56 31.84 5D1P 9.88 11.07 12.27 11.90 5D2P 7.36 10.80 11.65 11.31 GLS 15.52 16.23 16.65 16.57 CIL 14.11 14.57 15.76 15.21 PAL 6.81 6.65 7.27 7.03 ZB 8.75 8.83 9.47 9.09 MB 7.8 7.6 8.1 7.8 BBC 7.58 7.40 8.06 7.45 POC 4.25 4.30 4.41 4.21 IC 5.18 5.20 5.40 5.11 CM3 4.91 5.34 5.74 5.46 M2M2 5.40 5.50 5.54 5.45 CC 3.87 3.87 4.24 3.98 ML 9.69 10.84 11.38 11.03

CM3 4.91 5.64 6.01 5.98 HCP 3.62 4.23 4.67 4.60 aThe tail of this specimen is unusually shorter due to either congenital or accidental reasons.

Description maxillary toothrows are convergent anteriorly (Figure 3). The dentition is quite robust. The second upper incisor (I3) This is a small bat species, generally similar to Murina is situated posterior to the first (I2), and I2 is visible lat- lorelieae lorelieae (Table 1). The pelage is characterized erally. I2 and I3 are subequal in height (Figure 4) and are by long shiny hairs (8 mm ventrally and 13–15 mm dor- much less than half the height of the upper canine (C1). 1 sally), with distinct colorations on dorsal and ventral Upper (C ) and lower (C1) canines are well developed, surfaces, copper reddish-brown and dirty white, respec- exceed the height and subequal the basal areas of the cor- 4 tively. Dorsal hairs are tricolored: dark gray basally, pale responding second premolar (P and P4). The crown area in the middle and reddish brown at the tip. Ventral hairs and the height of the first upper premolar (P2) are two are bicolored: dark gray in about two thirds of the length thirds more than those of P4 (Figures 3 and 4). The first and whitish at the tip (Figure 5). The skull is domed. The (M1) and second (M2) upper molars have well-developed lateral profile of the anterior part of the skull gradually mesostyles and curved labial (outer) faces (Figure 4). The rises from the rostrum to the forehead (Figure 3). The sag- paracone, metacone, and protocone of the first (M1) and ittal crest is lacking; the lambdoid crests are visible. The second (M2) upper molars are distinctly defined. V.T. Tu et al.: First records of Murina lorelieae from Vietnam 211

Table 2 Nucleotide distances based on the Kimura 2-parameter (K2P) model between Murina species found in both Ngoc Linh Nature Reserve (Vietnam) and southern China.

Taxon 1 2 3 4 5 6 7

1 M. lorelieae Ngoc Linh 2 M. lorelieae S. China 0.013 3 M. feae Ngoc Linh 0.166 0.169 4 M. feae S. China 0.165 0.166 0.041 5 M. huttoni Ngoc Linh 0.172 0.172 0.187 0.179 6 M. huttoni S. China 0.170 0.169 0.180 0.181 0.039 7 M. cyclotis Ngoc Linh 0.162 0.162 0.151 0.154 0.164 0.167 8 M. cyclotis S. China 0.164 0.162 0.161 0.164 0.169 0.172 0.055

Distribution, ecological notes, and Forest Nature Preserve in southern China, with 1660 mm ­conservation status of mean annual precipitation, and 23°C of annual mean temperature (Robbins et al. 2006). All three specimens of Murina lorelieae ngoclinhensis were In Diding, Murina lorelieae was collected with six collected in harp traps that were set across the paths or other species of Murininae: Murina chrysochaetes, Murina trails in the wet montane evergreen forest at altitudes cyclotis, Murina eleryi, Murina feae, Murina leucogaster, between 1117 and 1682 m. At this elevation, the climate and Harpiocephalus harpia (Eger and Lim 2011). In Ngoc is different from that found in elevations lower than 500 Linh, Murina lorelieae occurred with four other species m: it is cool and humid in daytime and much colder and of Murininae: M. cyclotis, M. feae, Murina huttoni, and foggy at nighttime. At the studied site, the forest was fairly ­Harpiola isodon. As indicated previously, the smaller undisturbed with good vegetation coverage and abundant nucleotide distance calculated between Ngoc Linh banana trees in some slopes nearby small streams. and Diding specimens of M. lorelieae shows that these The comparisons of hairs between the four Murina allopatric populations were isolated from each other species found in Ngoc Linh showed that Murina lorelieae more recently than in other species Murina (M. cyclotis has a much longer fur, especially on the dorsum (Figure 5). and M. feae). Such result suggests that M. lorelieae had Possessing long hairs might be regarded as an adaptation a stronger ability to disperse over long distances during to mountain climate, as it has been observed in other bat the Pleistocene. During the Last Glacial Maximum, from species of high elevations, such as the long-haired rou- 26,500 to 19,000 years ago (Clark et al. 2009), climatic con- sette (Stenonycteris lanosus) in Africa or Blanford’s fruit ditions became cooler and drier in Southeast Asia. As a bat (Sphaerias blanfordi) in South Asia (Bates et al. 2008, consequence, montane forests descended to lower eleva- Mickleburgh et al. 2008). Since all specimens of M. lore- tions and expanded, whereas the distribution of lowland lieae were collected at high elevations, 976 m in Diding rainforests contracted into a few glacial refugia (Cannon (southern China) and over 1100 m in Ngoc Linh, we there- et al. 2009, Woodruff 2010, Turner and Cernusak 2011). fore suggest that M. lorelieae is a montane forest dweller. Each of the glacial periods probably showed a similar The finding of Murina lorelieae in Ngoc Linh, at pattern and caused the expansion of montane forests approximately 1000 km south from the type locality, sug- and the contraction of lowland forests. During glacial gests that M. lorelieae might be a widespread species. episodes, populations of the widespread bat species pre- Therefore, the species is likely distributed in relatively ferring lowland rainforests were affected by forest frag- intact montane forests of the Annamite Range, the moun- mentation and restricted to a few refugia. By contrast, tain range of eastern Indochina that extends approxi- populations of bat species adapted to montane forests, mately 1200 km from around 20° N, along the border such as M. lorelieae, are thought to have expanded. This between Vietnam and Lao PDR, and until the Da Lat biogeographic scenario may explain a higher capacity Plateau in south-central Vietnam (Sterling and Hurley for long-distance dispersal in M. lorelieae than in other 2005). In this mountain range, rainfall varies annually species of the genus Murina. between 1500 and 3850 mm, and the mean annual tem- This species is the 13th recently recognized Vietnam- perature is about 20°C (http://wwf.panda.org/). These ese Murininae (Hendrichsen et al. 2001, Csorba et al. 2007 conditions are similar to those found in Diding Headwater Csorba et al. 2011, Kruskop and Eger 2008, Furey et al. 212 V.T. Tu et al.: First records of Murina lorelieae from Vietnam

2009, Francis and Eger 2012). This record continually sup- Museum of Moscow, M.V. Lomonosov State University, Moscow, ports that the area of Mount Ngoc Linh of the Annamite Russia. pp. 203. Cannon, C.H., R.J. Morley and A.B.G. Bush. 2009. The current Range is a regional hotspot for biodiversity and endemism refugial rainforests of Sundaland are unrepresentative of their (Sterling and Hurley 2005), and Indochina hosts the biogeographic past and highly vulnerable to disturbance. Proc. highest diversity of the subfamily Murniniae (Francis and Natl. Acad. Sci. USA 106: 11188–11193. Eger 2012). As a resident of montane forests, Murina lore- Clark, P.U., A.S. Dyke, J.D. Shakun, A.E. Carlson, J. Clark, B. lieae might temporarily avoid the threats affecting bats Wohlfarth, J.X. Mitrovica, S.W. Hostetler and A.M. McCabe. found in lowland habitats, even though the high rate of 2009. The last glacial maximum. Science 325: 710–714. Corbet, G.B. and J.E. Hill. 1992. 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