Zavreliella inawaheia Sasa, Kitami & Suzuki,

The Phylogeography of Red Spiny Rats Maxomys surifer (Rodentia, Muridae) in Indochina with Comments on Taxonomy and Description of New Subspecies Alexander Evgenievich Balakirev1,2,*, Alexei Vladimirovich Abramov1,3, and Viatcheslav Vladimirovich Rozhnov1,2 1Joint Russian-Vietnamese Tropical Research and Technological Centre, Nguyen Van Huyen, Nghia Do, Cau Giay, Hanoi, Vietnam 2A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33, Moscow 119071, Russia. E-mail: rozhnov[email protected] 3Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, Saint Petersburg 199034, Russia. E-mail: [email protected] (Received 8 December 2016; Accepted 26 February 2017; Published 19 April 2017; Communicated by Jian-Nan Liu) Alexander Evgenievich Balakirev, Alexei Vladimirovich Abramov, and Viatcheslav Vladimirovich Rozhnov (2017) The phylogeographic pattern of Maxomys surifer across most of its geographic range was investigated based on existing sequencing from GenBank and new original data from Vietnam to evaluate its natural subdivision and taxonomic structure in Indochina and neighboring regions. Seven major phylogenetic clusters/groups are apparent on the cytochrome b (Cyt b) and cytochrome с oxydase subunit 1 gene (COI) trees, corresponding to geographical subpopulations of the species. Among them, distinct position of most divergent, clade Msur7 is also supported by analyses of nuclear (IRBP) gene. The taxonomic implication of these findings is tested by comparison of morphological features of this Northern (labeled by Msur7) and Southern Vietnamese populations widely distributed over the Indochina labeled by Msur3 mtDNA genetic marker. Direct comparisons of skulls measurements and multivariate analyses performed for these southern and northern populations showed that latter specimens are distinctive in being significantly larger in a number of cranial characters, with diagnostically smaller teeth relative to M. surifer from southern Vietnam, bearing also some traits in its external appearance, like relative tail length and coloration pattern. The pattern of genetic and cranial variation in M. surifer revealed in the present study suggests the existence of distinct genetic lineages and suspected longitudinal isolation, corresponding to morphologically distinctive forms. It is evident that at least some of these lineages merit subspecific status. We provide a taxonomical description elevating the northern Vietnamese populations to a new subspecies M. s. tonkinensis subsp. nov. We discuss the taxonomic implications, tentative range, and appropriate synonyms for all main genetic lineages over the range of M. surifer in the Sundaic region. Key words: Mammals, Rodents, Southeast Asia, Taxonomy, Biodiversity. *Correspondence: Tel: +84 0946059487. E-mail: [email protected] BACKGROUND Spiny rats of the genus Maxomys Sody, 1936 are widely distributed in evergreen and semievergreen forests of Southeast Asia (Corbet and Hill 1992; Nowak 1999). These rats are considered as the most abundant, morphologically and ecologically variable, and geographically widely distributed group of rats in Southeast Asia (Musser and Carleton 1993, 2005). In accordance with the currently accepted taxonomic understanding (Musser et al. 1979; Musser and Newcomb 1983; Musser and Holden 1991; Corbet and Hill 1992; Musser and Carleton 1993, 2005; Pavlinov 2005), Zoological Studies 56: 6 (2017) doi:10.6620/ZS.2017.56-06 1 the genus comprises 17 recent species. A new species was recently described from Borneo (Achmadi et al. 2012), and two more undescribed species are supposed to inhabit Sulawesi (Achmadi et al. 2013). The overwhelming majority of Maxomys species have rather narrow insular distributions, except for the red spiny rat Maxomys surifer (Miller, 1900). M. surifer is the only species of Maxomys whose natural range encompasses almost the entire continental Indochinese and Sundaic faunal regions, which may reflect its long evolutionary history and considerable ecological plasticity. The early presence of Maxomys surifer in the Indomalayan region is supported by late Pliocene to middle Pleistocene fossils, where Maxomys and related forms were discovered (Chaimanee 1998; van der Meulen and Musser 1999). The taxonomic composition and phylogenetic structure of M. surifer is far from being finally established. Previous studies (Musser and Newcomb 1983; Musser et al. 1979) demonstrated at least two groups of populations, descended from Sunda Islands and from peninsular Malaya, that differ considerably in morphological differences. The same was noticed by Kloss (1919) for Indochinese and Malayan specimens. Corbet and Hill (1992) detected a geographic pattern in pelage coloration. In spite of abundant materials, actual complexity of entire genus Maxomys composition and fragmentariness of data prevent from taxonomical assessment of these forms. It still also ambiguous whether distribution of morphological and molecular traits concordant with one another. Musser and Carleton (2005) recognize this group as a species complex in need of detailed taxonomical revision. Phylogenetic analyses of mtDNA cytochrome b (cyt b) and D-loop sequences by Gorog et al. (2004) identified six distinct lineages in what is now defined as M. surifer associated with 1) Java, 2) Sumatra, 3) Borneo, 4) the Malay Peninsula, 5) southern Vietnam, and 6) central Vietnam. Six mitochondrial DNA lineages, but of lower divergence level (both cyt b and COI gene associated) were also discovered for the species in Thailand by Latinne et al. (2013). Some of them proved to correspond to Malayan and South Vietnamese ones delimited by Gorog et al. (2004). A considerable amount of genetic data is currently available for this species complex, including new genetic data from Vietnam. We combine these data to assess the phylogeographic patterns across most of the geographic range of M. surifer and provide taxonomic changes resulting from genetic subdivisions. MATERIALS AND METHODS Field works were conducted by the Joint Russian-Vietnamese Tropical Research and Technological Centre in Southern and Central Vietnam from 2009-2015 in full agreement with current Vietnam regulations in field of Nature Protection and Biodiversity Conservation. We followed guidelines of the American Society of Mammalogists during the collection and handling of the animals used in this work (Gannon et al. 2011). DNA extraction, PCR amplification, and sequencing In total, 33 original specimens from 7 localities in Vietnam were collected in the present study and sampled for genetic analysis (Appendix 1, Fig. 1). Small fragments of liver and muscle tissue, fingertips, or earlaps were stored in 96% alcohol and used for DNA extraction. Total genomic DNA was extracted using a routine phenol/ chloroform/proteinase K protocol (Kocher et al. 1989; Sambrook et al. 1989). The DNA was further purified either by double ethanol precipitation or by using a DNA Purification Kit (Thermo Scientific). Targeted genes included a complete or substantial portion of the Cytochrome b gene (cyt b, 950-1140 bp), a portion of the first exon of Interphotoreceptor Retinoid Binding Protein gene (IRBP; up to 1600 bp) and 5’-proximal 680 bp portion of subunit I of the Cytochrome C Oxidase subunit 1 gene (COI), which is generally used for species diagnoses and for DNAbarcoding in Metazoa (Hebert et al. 2003). The cyt b was amplified using the primers H15915R, (Kocher et al. 1989; Irwin et al. 1991), CytbRglu and CytbRCb9H (Robins et al. 2007). The COI gene was amplified using the primers BatL5310 and R6036R (Robins et al. 2007) and universal conservative primers LCO1490 and HCO2198 (Hebert et al. 2003). The following PCR protocol was used to amplify both mtDNA fragments: initial denaturation for 1 min 30 sec at 95°C, followed by 40 cycles of denaturation for 30 sec at 95°C, annealing for 1 min at 52°C, and elongation for 30 sec at 72°C, followed by terminal elongation for 2 min at 72°C. The PCR reaction was performed in a 30-50 ml volume that contained 2.5-3 μml 10 x page 2 of 19Zoological Studies 56: 6 (2017) standard PCR buffer (Thermo Scientific), 50 mM of each dNTP, 2 mM MgCl2, 10-12 pmol of each primer, 1 unit of Taq DNA polymerase (Fermentas) and 0.5 μml (20-50 ng) of total DNA template per tube. The reaction was performed using a Tercik (DNK-Tehnologia) thermocycler. The IRBP gene (1000-1600 bp in length) was amplified using the IRBP125f, IRBP1435r, IRBP1125r and IRBP1801r primers, according to the protocol of Stanhope et al. (1992). PCR products were purified using a DNA Purification Kit (Thermo Scientific). The resulting double-stranded DNA products were directly sequenced in both directions using the Applied Biosystems 3130 Genetic Analyzer and the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with the same primers as have been used for routine PCR. We also brought into study all the samples of M. surifer from a number localities used by Achmadi et al. (2013), Pages et al. (2010), Latinne et al. (2013) and some others obtained by C.M. Francis and A.G. Servent (deposited in GenBank and BOLD databases, unpublished) taken from a number of sites in Vietnam, Thailand, Laos, Malay Peninsula, Borneo, Sumatra and Java. Sequenced specimens cover almost completely the geographic range of the species. As outgroups, we used some sequences from several other Maxomys spp. and Leopoldamys sabanus (see Appendix 1). All sequence data have been submitted to the GenBank databases (www.ncbi.nlm.nih.gov/ Genbank) under accession numbers KU057301KU057344. Sequence editing and phylogenetic analyses All the sequences in the dataset were aligned using BIOEDIT 3.0 (Hall 1999) and CLUSTAL W (incorporated into BIOEDIT and MEGA 5.05) software and were verified manually. Basic sequence parameter calculations (i.e., variable sites, parsimony-informative sites, base composition biases, nucleotide frequencies and nucleotide substitution tables), codon evolution model testing, and interand intra-population divergence (d, Tamura 3 parameter, T3P genetic divergence algorithm (Tamura et al. 2012)) evaluations were performed using MEGA 5.05 software (Tamura et al. 2011). Maximum parsimony (MP), maximum likelihood (ML), minimum evolution (ME), and neighbor-joining (NJ) were applied to phylogenetic reconstructions using MEGA 5.05 software. The best-fitting models of gene evolution out of 24 possible codon evolution models were determined using the Maximum Likelihood value (lnL), the Bayesian Information Criterion (BIC) and the corrected Akaike Information Criterion (AICc) as implemented in MEGA 5.05. The TN93+G+I substitution model was applied for the cyt b and COI genes, and the GTR+G substitution model was used for the IRBP gene (Nei and Kumar 2000). The calculated gamma shape parameters were 1.76, 1.53 and 1.11 for the cyt b, COI and IRBP genes respectively. The robustness of the tree was assessed using a bootstrap procedure with 1000 replications. All trees were constructed and visualized directly with MEGA 5.05 or with TREEVIEW 1.6.6 software (Page 1996). Divergence time approximation was performed by Maximum Likelihood method on the T3P model (Tamura 1992). Morphological analysis The morphological study was performed based on 138 skulls from 10 localities across Vietnam, including the genetically investigated vouchers (Appendix 2, Fig. 1). We used only adults for the analysis in order to minimize age variation. Age assessed by teeth wearing and cranial seams conditions. Specimens kept in the collections of the Zoological Museum of the Moscow State University (ZMMU, Moscow, Russia), the Zoological Institute of the Russian Academy of Sciences (ZIN, Saint Petersburg, Russia), and the Institute of Ecology and Biological Resources of the Vietnamese Academy of Science and Technology (IEBR, Hanoi, Vietnam). The skulls originated from ten localities in Vietnam: The northern most populations were represented by three localities: Ba Vi Nature Reserve, Ha Tay province (n = 24), Nghe An Province (n = 9) and Vu Quang Natural Park, Ha Tinh Province (n = 15) whereas seven more, namely Cat Tien Nature Park, Dong Nai Province (n = 11), Gia Lai Province (n = 18), Kon Tum Province (n = 6), Lo Go Xa Mat Nature Reserve, Tay Ninh Province (n = 6), Ma Da Forest, Dong Nai Province (n = 20), Phu Quoc Island, Kien Giang Province (n = 26) and Xuyen Moc, Ba Ria - Vung Tau Province (n = 3) are originated from southern regions. These localities cover the main part of species range in eastern Indochina (Appendix 2). Only intact skulls of adult specimens were measured irrespectively to the sex. Twenty measurements were taken on each skull using digital calipers to the nearest 0.1 mm, cranial measurements followed Musser and Newcomb page 3 of 19Zoological Studies 56: 6 (2017) Fig. 1. Localities of investigated specimens and geographic distribution of the mtDNA lineages of Maxomys surifer in Indochina and Sunda region. See the precise samples locations in Appendices 1 and 2. The following symbols indicate the locality source of specimens: filled squares, specimens collected for this study; open squares, specimens used in morphological analyses (thick-lined if genotyped, thin-lined when not); filled circles, specimens drawn from GenBank; open circles, Gorog et al. (2004) sampling sites for D-loop. Stars with names indicate the type localities for subspecies or synonyms of M. surifer. page 4 of 19Zoological Studies 56: 6 (2017) (1983) and Musser et al. (2006); occipitonasal length, or the greatest length of the skull (ONL), zygomatic breadth (ZB), interorbital breadth (IB), length of rostrum (LR), breadth of rostrum (BR), breadth of braincase (BBC), height of braincase (HBC), breadth of zygomatic plate (BZP), length of diastema (LD), length of incisive foramina (LIF), breadth of incisive foramina (BIF), palatal length (LBP) (palatal bridge), breadth across palate at first molars (BBP), postpalatal length (PPL), breadth of mesopterygoid fossa (BMF), length of bulla (LB), crown length of maxillary molar row (CLM1-3), crown breadth of M1 (BM1), crown length of mandibular row (CLm1-3), crown breadth of m1 (Bm1). This set of characters is mutually applied for investigation of cranil variation within Muridae. The principal components analysis (PCA) and the canonical discriminant analysis (CDA) have been used to evaluate a degree of cranial differentiation between geographical populations labeled by different genetic lineages. A one-way analysis of variance (ANOVA) was performed to test the differences among groups on all cranial variables. The Statistica 8.0 (StatSoft Inc., Tulsa, OK, USA) software has been used for all analytical procedures. RESULTS In total, datasets obtained comprise more then two hundred cyt b sequences with 78 unique haplotypes and 142 individuals with as many as 142 haplotypes for COI gene and 32 unique sequences for IRBP gene genes (See Appendix 1). No insertions, deletions or premature stop codons or any others signs of NUMT (pseudogenes) occurrence were observed for mtDNA. Phylogenetic analyses To examine the phylogenetic structure across the entire range M. surifer, we combined our genetic data from Vietnamese specimens to those of previous studies (Gorog et al. 2004; Achmadi et al. 2012, 2013; Latinne et al. 2013). Phylogenetic trees constructed based on cyt b and COI sequences presented generally the same topology with the only exclusion of SumatraJavanese branch in COI tree in lack of sampling (Figs. 2-3). Six major phylogenetic clusters/groups can be seen on the COI tree. One more, additional seventh branch also appears at geographically more representative cyt b tree. Most part of basal branches does not demonstrate reliable level of support due to higher level of genetic diversity and indicates ancient radiation of the group. While the M. surifer relationships are not supported at the deeper nodes of the tree, distinct geographically localized phylogroups are apparent and named Msur 1-7 (Figs. 1-3). The geographic distribution of these phylogenetic clades presented in figure 1. Phylogroup Msur1 includes samples from continental Malaysia and peninsular Thailand, cluster Msur2 includes the populations of western and northern Thailand and cluster Msur3 combines the populations from major part of Indochina (central and eastern Thailand, Cambodia, southern Laos and southern Vietnam). The lineage Msur4 is distributed over the most of Borneo, where one more distinct lineage Msur5 appears from a single locality on extreme east of the island. As it can be seen on the cyt b tree, an additional small subclade appeared in Borneo together with two another corresponding to Msur4 and Msur5 in COI tree. Another large clade Msur6 is evidently monophyletic but may be additionally subdivided into two subclades from Java and Sumatra islands respectively. An extremely poor sampling (only a few samples from two localities) hamper to outline its geographical distribution in considerable details. Finally, Msur7 is found in northern and central Vietnam and central Laos. This is the most divergent clade among M. surifer phylogroups. Its level of divergence is significantly higher than that demonstrated for other lineages. The genetic divergence for cyt b (d, T3P) of northern Vietnamese M. surifer from another six clades reaches to 0.09-0.11, whereas, for example, the distance of Malayan-Javanese populations from that of Borneo do not exceed 0.06-0.08 (and about 0.08 for corresponding populations of M. whiteheadi (Achmadi et al. 2013). The distinct position of Msur7 lineage is also supported by analyses of nuclear genes. The IRBP gene tree is shown on figure 4. In spite of scarcity of samples, the branch corresponding to Msur 7 in mitochondrial trees appeared as an independent one. This branch is reliably depicted by high support values whereas all the other samples do not demonstrates significant reciprocal monophyly. Morphological analysis A summary of the descriptive statistics of cranial variables for southern and northern groups is given in table 1. Multivariate analysis of cranial characters was done for populations of northern page 5 of 19Zoological Studies 56: 6 (2017) Fig. 2. The phylogenetic tree (cyt b, ML, 950 bp long) tree for the Maxomys surifer genetic lineages radiation. The bootstrap values (for different tree-constructing methods, NJ/ML/ME/MP, /*/ if 99-100, /-/ when below 50) are indicated above the nodes. The names for major phylogenetic lineages as indicated in figure 1. page 6 of 19Zoological Studies 56: 6 (2017) and central Vietnam representing the genetic clade Msur7 and for other ones from southern Vietnam belonging to distinct clade Msur3. Results of the PCA for mean values of ten geographic samples are shown in figure 5 and table 2. It can be seen the northern and southern populations diverge mainly along the first principal component PC1, reflecting considerable differences in overall cranial size. Direct comparisons of skulls from southern and northern lineages showed that northern specimens are distinctive in being significantly differ in many (six out of twenty) of cranial characters including appreciably larger general size of skull, with diagnostically smaller teeth relative to M. surifer from southern Vietnam. Thus, the specimens from northern populations show the largest average meanings of skull measurements, whereas those from southern and central Vietnam are appeared as the smallest one. Canonical discriminant analyses drawing on Fig. 3. The phylogenetic tree (COI, ML, 630 bp long) tree for the Maxomys surifer genetic lineages radiation. The bootstrap values (for different tree-constructing methods, NJ/ML/ME/MP, /*/ if 99-100, /-/ when below 50) are indicated above the nodes. The names for major phylogenetic lineages as indicated in figure 1. page 7 of 19Zoological Studies 56: 6 (2017) Fig. 4. The phylogenetic tree (IRBP, ML, 1080 bp long) tree for the Maxomys surifer genetic lineages radiation. The bootstrap values are indicated above the nodes. The names for major phylogenetic lineages as indicated in figure 1. page 8 of 19Zoological Studies 56: 6 (2017) Fig. 5. Ungrouped morphometric separation (principal components analysis) of ten Maxomys surifer samples, drawing from means of craniodental measurements. Northern populations marked as red, southern - by green, yellow and blue colors. Table 1. Descriptive statistics (mean, range, standard deviation) for skull measurements (in mm) for Vietnamese Maxomys surifer Characters Northern form (n = 48) Southern form (n = 90) ANOVA Mean Min Max Std.Dev. Mean Min Max Std.Dev. F p ONL 45.83 41.43 49.09 1.58 44.77 40.84 49.43 1.65 1.08 0.778 ZB 20.23 18.23 21.49 0.77 20.00 18.09 22.74 0.95 1.52 0.118 IB 7.33 6.79 7.90 0.26 7.03 6.24 8.17 0.41 2.34 0.002 LR 16.10 14.44 17.38 0.68 15.70 13.90 17.55 0.77 1.29 0.347 BR 8.10 7.42 9.32 0.38 8.01 7.05 9.84 0.52 1.82 0.026 BBC 17.10 16.46 17.99 0.38 16.73 15.68 17.76 0.47 1.56 0.098 HBC 12.46 11.75 13.44 0.36 11.86 10.98 12.90 0.38 1.12 0.685 BZP 4.10 3.43 4.58 0.28 4.25 3.67 4.89 0.24 1.35 0.222 LD 12.79 11.78 13.91 0.49 12.48 10.70 13.93 0.63 1.70 0.047 LIF 6.95 6.00 7.89 0.40 6.25 5.31 7.31 0.42 1.13 0.650 BIF 3.61 2.95 4.12 0.28 3.62 2.96 4.34 0.31 1.22 0.457 LBP 8.86 7.98 9.68 0.43 9.01 8.18 10.20 0.47 1.20 0.505 BBP 4.68 3.72 5.13 0.27 4.37 3.56 5.07 0.32 1.37 0.241 PPL 16.82 14.70 18.24 0.74 15.91 14.30 18.03 0.80 1.16 0.590 BMF 3.30 2.71 4.02 0.33 3.12 2.62 3.83 0.24 1.83 0.015 LB 5.00 4.47 5.53 0.24 5.01 4.44 5.52 0.18 1.87 0.012 CLM1-3 6.56 6.11 7.01 0.23 6.61 6.15 7.10 0.21 1.14 0.592 BM1 2.00 1.82 2.20 0.10 2.10 1.78 2.36 0.10 1.00 1.000 CLm1-3 6.20 5.74 6.70 0.23 6.37 5.90 6.81 0.20 1.27 0.330 Bm1 1.69 1.42 1.90 0.10 1.72 1.53 1.88 0.08 1.86 0.012 page 9 of 19Zoological Studies 56: 6 (2017) ethanol, collected 6.03.2014), ZMMU S-194711 (female, skull and skin, collected 7.03.2014), ZMMU S-194712 (male, skull and skin, collected 7.03.2014), ZMMU S-194713 (male, skull and skin, collected 7.03.2014), ZMMU S-194714 (male, skull, collected 7.03.2014), ZMMU S-194715 (male, body at ethanol, collected 8.03.2014), ZMMU S-194716 (male, body at ethanol, collected 8.03.2014), ZMMU S-194717 (male, skull, collected 8.03.2014), ZMMU S-194719 (male, skull, collected 9.03.2014, trapped near Ban Dom1 Village), ZMMU S-194720 (male, skull, collected 9.03.2014, trapped near Ban Dom1 Village), ZMMU S-194721 (female, body at ethanol, collected 9.03.2014), ZMMU S-194722 (male, skull, collected 10.03.2014), ZMMU S-194723 (male, skull, collected 10.03.2014), ZMMU S-194724 (female, skull, collected 10.03.2014), ZMMU S-194725 (male, skull, collected 11.03.2014), ZMMU S-194726 (female, body at ethanol, collected 12.03.2014). All collected by Alexander E. Balakirev and Tran Quang Tien from the same locality as the holotype or in closest vicinity. Etymology: The new subspecies is named after Tonkin, the former name of northern part of Eastern Indochina, with the Latin suffix -ensis (belonging to). Diagnosis: Medium-sized rat, larger on average in its external and cranial measurements than the nominotypical M. surifer with a longer tail (106-115% of body length for most individuals). Description and comparisons: Head and body length 170-222 mm, tail 193-227 mm, ear 24-29 mm, weight 127-210 g. figure 10 B. New subspecies differs from the M. s. finis which distributed over southern Indochina in the larger skull sizes and in the relatively short upper and lower toothrows. The most evident morphological features at skull construction are also be noticed the size and shape of incisive foramina. In contrast with M. s. finis, which usually has shorter, reniform openings with clearly pointed cranial edge of notch, M. s. tonkinensis has more elongated foramina with more or less rounded cranial and caudal edges of notch figures 7-9, its somewhat reassemble in shape to the foramina characteristic to representatives of genus Leopoldamys but much wider at caudal side. The most remarkable external feature is the pattern of tail coloration, namely the character of tail tip discoloration. This progressive discoloration, characteristic for all M. surifer starts appreciably earlier than it usually happens with representatives of southern populations, discolorations starts approximately from the middle of tail and terminal one third usually became completely white, whereas for M. s. finis discolorations launched from approximately terminal third with only very tip about 1/5 of tail length being completely discolored. Distribution: Northern Vietnam from Ha Tinh Province in its extreme south to Ba Vi (Hanoi area) in the north; central Laos (Khammouane and Bolikhamxai provinces). It may be probably found in southernmost Yunnan, China (Wang 2003) and northern Laos, but its presence there has to be approved by investigation of museum specimens or new field records. Acknowledgment: This work and the one new subspecies name have been registered with Zoo Bank under urn:lsid:zoobank.org:pub:017C3A9D9B40-4C8C-A621-7BF5883DD1D7. This study was realized with the support of the Joint RussianVietnamese Tropical Research and Technological Centre, Hanoi, Vietnam. We thank Dr. Victor V. Suntsov (A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia), Dr. Sergey V. Kruskop (Zoological Museum of Moscow State University, Moscow, Russia), Dr. Charles M. Francis (Canadian Wildlife Service, Environment Canada, Ottawa, Canada), Dr. Vu Dinh Thong (Zoological Museum of Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology) who allow us to access for samples and museum collections and datasets, Dr. Nguyen Dang Hoi, Dr. Bui Xuan Phuong, Mr. Tran Quang Tien and Ms. Pham Mai Phuong (all from the Joint RussianVietnamese Tropical Research and Technological Centre, Hanoi, Vietnam), who made considerable efforts in preparing for a number of expeditions and who supplied us with a significant number of specimens. We also want to express our warmest gratitude to Dr. Kristofer M. Helgen, Mr. Craig Ludwig and Mr. Renee Regan (all from the Smithsonian Institution; National Museum of Natural History, Division of Mammals, Washington, USA) for their kind assistance in the investigation and photography of holotype specimen. Finally, we thank the administrations of Ba Vi, Nam Cat Tien, Bi Dup-Nui Ba, Bu Gia Map, Lo Go Xa Mat, Dong Nai, Phu Quoc and Binh Chau National Parks and Nature Reserves for their aid in the management of our studies. This study was supported in part by the Research Program “Living nature: modern state and problems of development” of the Presidium of the Russian Academy of Sciences, by page 16 of 19Zoological Studies 56: 6 (2017) the Zoological Institute’ Program No.0125-2016004, the Russian Foundation for Basic Research (grant 16-04-00085) and the Russian Science Foundation (grant 17-14-01160). List of abbreviation (not explained in text) DNA - deoxyribonucleic acid ICZN - International Code of Zoological Nomenclature PCR - polymerase chain reaction REFERENCES Achmadi AS, Esselstyn JA, Rowe KC, Maryanto I, Abdullah MT. 2013. Phylogeny, diversity, and biogeography of Southeast Asian spiny rats (Maxomys). J Mammal 94(6):1412-1423. doi:10.1644/13-MAMM-A-092.1. 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List of specimens used for the genetic study: species name, geographic location, collection ID, GenBank accession No. (download) Appendix 2. List of Maxomys surifer and M. moi specimens used for morphological analysis. (download) page 19 of 19Zoological Studies 56: 6 (2017)