Zoosyst. Evol. 97 (1) 2021, 83-95 | DOI 10.3897/zse.97.57490 Ate BERLIN Distribution pattern and phylogeography of tree rats Chiromyscus (Rodentia, Muridae) in eastern Indochina Alexander E. Balakirev!*, Alexei V. Abramov! *, Viatcheslav V. Rozhnov!? 1 Joint Russian-Vietnamese Tropical Research and Technological Centre, 63 Nguyen Van Huyen, Nghia Do, Cau Giay, Hanoi, Vietnam 2 AN. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33, Moscow 119071, Russia 3 Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, Saint Petersburg 199034, Russia http://zoobank.org/DD5BE6F 7-403 E-4E 16-BA21-D23F7C06CF7A Corresponding author: Alexander E. Balakirev (alexbalakirev@mail.ru) Academic editor: M.T.R. Hawkins # Received 11 August 2020 # Accepted 12 January 2021 Published 3 February 2021 Abstract The study combines available data on species distribution in eastern Indochina to investigate the phylogeographical genetic and mor- phological diversity of tree rats (Chiromyscus, Rodentia, Muridae) and to specify their natural ranges. We examined the diversity and distribution of tree rats over its range, based on recent molecular data for mitochondrial (Cyt b, CO/) and nuclear (JRBP, RAG/ and GHR) genes. The study presents the most complete and up-to-date data on the distribution and phylogeography of the genus in east- ern Indochina. As revealed by mitochondrial genes, C. /angbianis splits into at least four coherent geographically-distributed clades, whereas C. thomasi and C. chiropus form two distinctive mitochondrial clades each. Chiromyscus langbianis and C. chiropus show significant inconsistency in nuclear genes, whereas C. thomasi shows the same segregation pattern as can be traced by mitochondrial markers. The Northern and Southern phylogroups of C. thomasi appear to be distributed sympatrically with northern phylogroups of C. langbianis in most parts of eastern Indochina. The mitochondrial clades discovered are geographically subdivided and divergent enough to suspect independent subspecies within C. langbianis and C. thomasi. However, due to the insufficiency of obvious mor- phological traits, a formal description is not carried out here. The processes of recent fauna formation, species distribution patterns, dispersion routes and possible natural history in Indochina are discussed. Key Words biodiversity, Indochina, Southeast Asia, taxonomy, tree rats, Vietnam Introduction (Thomas, 1891), C. langbianis and C. thomasi Balakirev, Abramov & Rozhnov, 2014. Genus Chiromyscus Thomas, 1925 is currently assigned to the Dacnomys division of the tribe Rattini (Musser and Carleton 1993, 2005). It was first described, based on Mus chiropus (Thomas, 1891) from East Burma (= Myanmar) and for a long time was considered monotypic. Its close relationships with Niviventer langbianis (Robinson, Kloss, 1922) and N. cremoriventer (Miller, 1900) were initially suspected and discussed by Musser (Musser 1973, 1981; Musser and Carleton 1993, 2005). The genus was recently re-established by Balakirev et al. (2014) as comprising at least three recent species: C. chiropus To date, Thomas’ tree rat, C. thomasi, 1s known to be distributed from southwest China (Lan et al. 1994; Chen et al. 1995; Wang 2002) to eastern Myanmar and north- ern Thailand (Marshall 1977), Vietnam (Dang et al. 1994; Lunde and Nguyen 2001; Can et al. 2008, Balakirev et al. 2014) and Laos (Musser 1981; Corbet and Hill 1992; Aplin et al. 2003; Aplin and Lunde 2016), where it has long been known under the name C. chiropus. The Da- lat tree rat C. langbianis was described from the Dalat Plateau in southern Vietnam (Robinson and Kloss 1922) and is currently recorded throughout Vietnam and Laos Copyright Alexander E. Balakirev etal. 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. 84 Alexander E. Balakirev et al.: (Musser 1973; Dang et al. 1994; Lunde et al. 2003; Bal- akirev et al. 2011; Lu et al. 2015) and southern China (Lu et al. 2015), including Hainan Island (Ge et al. 2018). Fea’s tree rat C. chiropus 1s known from Myanmar and easternmost India (Musser 1973) and southern Vietnam (Balakirev et al. 2014). The evolution of the genus Chiromyscus was affected by the natural history of this region. Continuous forest cover in Indochina existed during a considerable pro- portion of the Pliocene and Pleistocene (Metjaard and Groves 2006), enabling the direct contact, dispersion and genetic exchange of western and eastern Indochinese populations, which are currently mostly interrupted by the extensive deforestation in areas of central Thailand and southern Cambodia. During Pleistocene glaciations, the forest edge was located at lower elevations (Cox and Moore 2000) and substantial forest contraction happened during the last glacial periods, as evidenced in Peninsular Malaysia and Palawan (Wurster et al. 2010). Cranbrook (2000) and Gathorne-Hardy et al. (2002) stressed that most areas in this region were covered with savannah vegetation unfavourable to arboreal species during long periods of the Quaternary epoch. However, the most re- cent surveys on the biogeography and paleoenvironments of the Sunda Shelf have suggested that the interactions between climate and sea level and their effects on the dis- tribution patterns of the fauna and flora are more complex (van den Bergh et al. 2001; Metjaard and Groves 2006) and environmental conditions changed repeatedly during the Pleistocene. These global processes of ecosystems change were likely to have had an impact on the recent genetic structure of Chiromyscus species. This group of rodents 1s still quite rare in museum collections, so little information is available about its natural diversity, dis- tribution and phylogenetic relationships and only a few specimens have been genetically characterised. In this pa- per, we combine available data, including novel data, on species distribution in eastern Indochina to investigate the phylogeography and diversity of these species, both ge- netic and morphological and specify their natural ranges. Material and methods Specimens and samples A great number of Chiromyscus specimens, obtained in Vietnam during a series of field expeditions of the Joint Russian-Vietnamese Tropical Research and Technologi- cal Centre between 2007 and 2018, were sampled for ge- netic analysis in full agreement with current Vietnamese regulations in the field of Nature Protection and Biodi- versity Conservation. We followed the guidelines of the American Society of Mammalogists during the collection and handling of the animals used in this survey (Gannon et al. 2011). The museum specimens were kept in the Zo- ological Museum, Moscow State University, Moscow, Russia (ZMMU) and the Zoological Institute, Russian zse.pensoft.net Phylogeography of tree rats of Chiromyscus in eastern Indochina Academy of Sciences, Saint-Petersburg, Russia (ZIN); genetic samples are part of collection of Joint Russian— Vietnamese Tropical Research and Technological Centre, Hanoi, Vietnam. New samples were combined with sequences available in GenBank, including our sequences previously submit- ted (Balakirev and Rozhnov 2010; Balakirev et al. 2011, 2012, 2014; Rowe et al. 2008; Pages et al. 2010; Zhang et al. 2016). In total, 79 specimens were investigated (47 C. langbianis, 15 C. thomasi and 17 C. chiropus). The geographic scope of the survey included 23 localities (Fig. 1, Table 1; see also Suppl. material 1: Table S1) scattered over China, Laos, Vietnam and Cambodia and constitute the known species distribution. Most of these sample specimens were collected personally by the au- thors (AVA, AEB). DNA extraction Small pieces of liver or muscle tissue were sampled in the field and stored in 96% ethanol. 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 a DNA Purification Kit (Fermentas, Thermo Fisher Scientific Inc., Pittsburgh, PA, USA) or by direct ethanol precipitation. We targeted five genes that were previously used for the phylogenetic analysis of Chiromyscus and were available for compar- ative analyses in GenBank. These genes included a com- plete Cytochrome b (Cyt b, 1140 bp); the 5’-proximal portion (680 bp) of subunit I of the Cytochrome Oxidase (COD), which 1s generally used for species diagnoses and for DNA barcoding for a number of mammals (Hebert et al. 2003); a portion of the first exon of the Interphotore- ceptor Retinoid Binding Protein gene (/RBP, also known as Rbp3, up to 1233 bp); the first exon of the Recombina- tion Activation Factor gene (RAG/, 1244 bp); and a por- tion of exon 10 of the Growth Hormone Receptor gene (GHAR, 815 bp). PCR amplification and sequencing Cyt b was amplified using H15915R, CytbRglu (Kocher et al. 1989; Irvin et al. 1991) and CytbRCb9H primers (Robins et al. 2007). The CO/ gene was amplified us- ing the universal conservative primers BatL 5310 and R6036R (Kocher et al. 1989; Irwin et al. 1991). The following universal PCR protocol was used to amplify mtDNA fragments: initial denaturation for 1 min 30 sec at 95 °C, 35 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 was performed in a 30-50 ul volume that contained 2.5—3 ul 10 x standard PCR buffer, 50 mM of each dNTP, 2 mM MgCl, 10 pmol of each primer, 1 unit of Taq DNA polymerase (Fermentas, Thermo Zoosyst. Evol. 97 (1) 2021, 83-95 ' Chiromyscus thomasi WW northern lineage southern lineage Chiromyscus langbianis northern lineage © southern lineage @ Hainan lineage @ Cambodian lineage Chiromyscus chiropus Figure 1. Scattering of Chiromyscus spp. genetic samples in eastern Indochina. Type localities of C. chiropus, C. langbianis, and C. thomasi are shown by black circles. Fisher Scientific Inc., Pittsburgh, PA, USA) and 0.5 ul (25-50 ng) of total DNA template per tube. The reaction was performed using a Tercik (DNK-Tehnologia, Novo- sibirsk, Russia) thermocycler. JZRBP (1000-1610 bp in length) was amplified using the IRBP125f, IRBP1435r, IRBP1125r and IRBP1801r primers according to Stan- hope et al. (1992). A nested PCR technique was applied to amplify GHR in accordance with Jansa et al. (2009). An approximately 1.0-kb portion of exon 10 from the GHR gene was amplified using the primers GHRF1 and GHRendAlt. This PCR product was re-amplified using the nested GHRF1 primer paired with GHR750R and the GHRF50 primer paired with GHRendAlt. A 1244 bp por- tion of the RAG/ gene was obtained using primers S70 and S71, as described by Steppan et al. (2004). The PCR products were purified using a DNA Purifica- tion Kit (Fermentas, Thermo Fisher Scientific Inc., Pitts- burgh, PA, USA). The resulting double-stranded DNA products were directly sequenced in both directions us- ing the Applied Biosystems 3130 Genetic Analyzer with the BigDye Terminator Cycle Sequencing Ready Reac- tion Kit (Applied Biosystems, Waltham, Massachusetts, USA). All obtained sequences were deposited in Gen- Bank (www.ncbi.nlm.nih.gov/Genbank; MK957014— zse.pensoft.net 86 Alexander E. Balakirev et al.: Phylogeography of tree rats of Chiromyscus in eastern Indochina Table 1. List of geographical localities of Chiromyscus specimens used for genetic and morphological analyses. No. (Fig. 1) Species DNA lineage Locality Elevation (m asl) Latitude / Longitude 1 C. langbianis N lineage China, Yunnan, Xishuangbanna 22.0°N, 100.8°E 5 C. langbianis N lineage Vietnam, Tuyen Quang, Khong May 102 221soaN; 105, 339°F 6 C. langbianis N lineage Vietnam, Vinh Phuc, Tam Dao 850 21.452°N, 105.636°E 7 C. langbianis N lineage Vietnam, Lang Son, Huu Lien 230 21.661°N, 106.362°E 8 C. langbianis N lineage Vietnam, Nghe An, Pu Hoat 840 19,.756°N, 104.796°E ile} C. langbianis N lineage Laos, Knhammouane TBSrN OS .30; 2 13 C. langbianis N lineage Laos, Khammouane, Pha Deng 1.7.57 N; LObG23*E 14 C. langbianis N lineage Vietnam, Quang Binh, Le Thuy, Sa Khia 156 17.068°N, 106.601°E ils: C. langbianis N lineage Vietnam, Kon Tum, Kon Plong 1030 14.722°N, 108.316°E 16 C. langbianis N lineage Vietnam, Kon Tum, Kon Chu Rang 1020 14.505°N, 108.541°E 17 C. langbianis N lineage Vietnam, Gia Lai, Kon Ka Kinh 900 14.203°N, 108.315°E 19 C. langbianis S lineage Vietnam, Lam Dong, Bi Doup-Nui Ba 1400-1800 12.179°N, 108.679°E 11 C. langbianis Hainan lineage China, Hainan, Jianfengling 18.74°N, 108.85°E 12 C. langbianis Hainan lineage China, Hainan, Baoting 18.641°N, 109.775°E 18 C. langbianis Cambodian lineage Cambodia, Kaoh Kong,Thmar Bang, Tatai Leu 11.961°N, 103.303°E 3 C. thomasi N lineage Vietnam, Lao Cai, Bat Xat, Y Ty 1830 22.624°N, 103.629°E 4 C. thomasi N lineage Vietnam, Son La, Muong Coi 547 21.343°N, 104.749°E 5 C. thomasi N lineage Vietnam, Tuyen Quang, Khong May 102 2233SS NOS 3397 2 C. thomasi S lineage Laos, Houay Sai, Houay Khot Station 20.267°N, 100.4°E 9 C. thomasi S lineage Vietnam, Nghe An, Xoong Con 141 19.252°N, 104.318°E 10 C. thomasi S lineage Vietnam, Nghe An, Pu Mat 200 18.957°N, 104.686°E 13 C. thomasi S lineage Laos, Knhammouane, Pha Deng 17.57°N, 105.23°E tS C. thomasi S lineage Vietnam, Kon Tum, Kon Plong 1030 14.722°N, 108.316°E 20 C. chiropus Vietnam, Lam Dong, Bao Loc 650 11.837°N, 107.64°E 21 C. chiropus Vietnam, Dong Nai, Ma Da 75 11.381°N, 107.062°E 22 C. chiropus Vietnam, Tay Ninh, Lo Go Xa Mat 1 5SS Ni 103,993°E 23 C. chiropus Vietnam, Ba Ria-Vung Tau, Binh Chau 68 10.55°N, 107.483°E MK957137). Niviventer spp., Rattus norvegicus and Mus musculus were used as outgroups. Molecular data analyses Individual sequences were edited manually using BioEd- it v. 7.1.11 (Hall 1999) and aligned by Clustal W soft- ware incorporated into BioEdit and MEGA 6. The basic sequence parameter calculations and the best-fitting evo- lution models and inter- and intrapopulation divergence evaluations were performed using MEGA 6 (Tamura et al. 2013). No pseudogenes were detected for the mitochon- drial genes. The optimal substitution models and their pa- rameters are summarised in Suppl. material 1: Table S2. Genetic distances (d) between groups under Tamuta-Nei gamma distributed invariant sites including (TN93+G+I) or Tamura 3-parameter (T3P) models (Tamura et al. 2004), (depending on the best model determined) were calculated, based on the Cytb and COI genes in MEGA 6. Bayesian phylogenetic trees were inferred using MrBayes v.3.2. (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003), two MCMCs for four chains with the default heating value and with a burn-in parameter equal to 25% of the initial number of runs. We applied 6x10° generations for the Cyt b dataset, 210° for the COJ, 4x10° for the RAG/ datasets and 5x10° for both GHR and IRBP datasets until the average standard deviation of split fre- quencies dropped below the level of 0.0025 after the runs for all datasets investigated. We used a flat Dirichlet prior for the relative nucleotide frequencies and for the relative rate parameters, a discrete uniform prior for the topolo- gies and an exponential distribution for the gamma shape zse.pensoft.net parameter and all branch lengths. The gamma shape pa- rameters for Bayesian Inference were evaluated directly from a general dataset by MrBayes v.3.2. A burn-in peri- od of one million generations was determined graphically using TRACER v.1.4 (Rambaut and Drummond 2007) to ensure convergence. Consensus trees were built from the last 25% of trees obtained (15 x10*, 15 x10*, 50 x10°, 10 x10° and 12.5 x10? trees for Cyt b, COL, RAGI, GHR and IRBP, respectively) during the MCMC procedure by Mr- Bayes v.3.2. The five individual genes were concatenated using the software SequenceMatrix v1.7.6 (Vaidya et al. 2011) to create a master alignment of 5,199 bp total (5,208 bp including three triplet insertions in Mus musculus GHR gene). A restricted dataset, including all species includ- ed in this study and consisting of samples with a com- plete data matrix, were used for concatenated sequences analyses. A total of 5x10° generations was applied during the MCMC procedure by MrBayes v.3.2. for concatenat- ed alignment until the average standard deviation value dropped to 0.0077. TREEROT v.3 (Sorenson and Franzo- sa 2007) was used to examine tree-bisection-reconnection branch-swapping (PBS) to assess the contribution of each data partition in the combined analysis (Baker and De- Salle 1997). This analysis was performed to test the sus- tainability of the primary internal nodes for the different gene analyses. The robustness of the trees was assessed by posterior probabilities (PP). Trees were visualised and prepared by FigTree v.1.4.3 (Rambaut 2012). Divergence time approximation was performed by Mega X (Kumar et al. 2018), a time tree inferred using the Reltime method (Tamura et al. 2012; 2018) and the General Time Reversible model and branch lengths eval- uated by MrBayes v.3.2. for the concatenated dataset. The Zoosyst. Evol. 97 (1) 2021, 83-95 0,02 0,02 0,03 0,09 0,1 K-63 FJ665441 Niviventer bukit 87 Hainan HM031673 Hainan KF 740122 Hainan. ; KI734674 Hainan 008 KT734675 Hainan P22627 KY068792 Hainan ang Son 154042 baa. Son 1501 KT 734678 Lang Son D4 KY068 782 Vinh Phuc 101502 KT734679 Lang Son Hainan 0,02 cluster IN ‘s can NAN ZSZ™ um gaged a fo) i) Northern cluster OQ QQRCAS BeeQs Fo, Dbhors fo} a4 Or al Tum ZIN96516 KT734680 Kon Tum 7433 Khammouane Laos —_ N=, 1 OP Sim 0,01 C.langbianis As IS: KO! O' Yunnan. -/ FJ665434 Bi Doup-Nui Ba BD-9 FJ665435 Bi Doup-Nui Ba Doup-Nui Ba | i Ba ive) O 57009 Bi Doup-Nui Ba Southern cluster (=O BS, 1S) Be OR Or = 0,02 BOA coe IO, ras) a ong Dong Nai Dong Nai , 4039 Dong Nai 34677 Lam Dong 54038 Ba Ria-Vung Tau F 154040 ort Ninh | ong Nai 13 Tu Ro Tuyen - Tuyen Quarig Q-117 Tuyen Quang 00 1 Lao ‘C; MGC-68 JQ755933 Son La MC-80 Toieeees SonLa _. ABTC 69097 EU349739 Houay Sai Laos 15-046 Kon Tum Northern 0,03 cluster al Southern cluster C.thomasi C.chiropus he An NA1 B16 Nghe An 18-076 Nghe -0,175 -0,15 -0,125 -0,1 -0,075 0,05 -0,025 0 Figure 2. The phylogenetic tree (Cyt b, Bayesian inference) for Chiromyscus genetic lineages radiation. The posterior probability values are presented at the nodes, and the branch lengths (scale bar at the bottom) are indicated above the nodes. The sample labels and locality numeration are indicated as in Fig. 1 and Suppl. material 1: Table S1. timetree was computed using one calibration constraint chosen as the divergence event between Mus and Rattus genera which are known to happen within 12.3—11.0 Mya (95% CI) (Benton and Donoghue 2007 with correction of Kimura et al. 2015). Discrete Gamma distribution was used to model evolutionary rate differences amongst sites (five categories (+G, parameter = 0.9185)). The rate vari- ation model allowed for some sites to be evolutionarily invariable ([+/], 54.21% sites). The analysis involved all 82 nucleotide sequences and 5208 nucleotide positions. Morphological data analyses In total, 63 intact skulls of adult Chiromyscus (15 C. chiro- pus, 35 C. langbianis and 13 C. thomasi) obtained from 19 genetically-investigated localities in Vietnam (see Table 1 and Suppl. material 1: Tables S1 for references) were mea- sured for morphometric comparison and analyses. Age was assessed by tooth wear and closure of cranial sutures. Due to the limited sampling, sexual differences were not espe- cially tested; the possible sexual bias was compensated for by equalisation of representatives of different sexes in the sample. The sex ratio did not exceed 15 percentage points. Twenty measurements were taken from each skull by means of digital calipers to the nearest 0.01 mm: greatest length of skull (ONL), braincase breadth (BBC), brain- case height (HBC), zygomatic breadth (ZB), interorbital breadth (IB), length of rostrum (LR), breadth of rostrum (BR), breadth of zygomatic plate (BZP), diastema length (LD), length of foramina incisive (LIF), breadth of foram- ina incisive (BIF), length of bony palate (LBP), breadth across the palatal bridge at the level of the first molar (BBP), distance from the anterior edge of the premaxillary to the posterior edge of the palatine (= postpalatal length, PPL), breadth of the mesopterygoid fossa (BMF), length of the bulla (LB), upper molar row length (CLM 1-3), first upper molar breadth (BM1), first lower molar breadth (Bm1) and lower molar row length (CLm1-3). The cranial measurements followed Musser et al. (2006) and Balaki- rev et al. (2011), Suppl. material 1: Fig. S1. The measure- ments dataset is available from AEB by request. Principal components analysis (PCA) and canonical discriminant function analysis (DFA) were used to eval- uate “distinctiveness” amongst the samples. A one-way analysis of variance (ANOVA) was performed to test the differences amongst groups on all cranial variables. The software programme Statistica 8.0 (StatSoft Inc., Tulsa, OK, USA) was used for all analytical procedures. Results Phylogenetic subdivision and relationships The most representative tree was constructed for 66 Cyt 6b sequences. The trees were well supported (PP = 1) (Fig. 2). Different geographical populations of zse.pensoft.net 88 Alexander E. Balakirev et al.: C. chiropus formed a single homogeneous cluster, while C. thomasi and C. langbianis populations were represent- ed by a few geographically-segregated clusters. C. thom- asi split into two clusters, which we conventionally called the Northern and Southern phylogroups. The haplotypes forming the Northern cluster were distributed locally over the north-western part of Vietnam, whereas South- ern haplotypes spread substantially more widely, stretch- ing to the south to the Tay Nguyen Plateau (also known as the Central Highlands) and westwards to the extreme northwest of Laos. Chiromyscus langbianis formed three coherent geographically-distributed clusters. The first from the Dalat Plateau, the terra typica of this species; named hereafter as the Southern lineage. Samples origi- nating from continental Indochina, southern Yunnan and north-eastern Vietnam in the north to the Tay Nguyen Pla- teau in the south formed a second cluster, the Northern lineage. Another cluster, which was sister to the Northern lineage, contained samples from Hainan Island (Figure 2). The genetic divergence within and between these groups is shown in Tables 2, 3. The estimated genetic dis- tances (d) for intraspecific C. langbianis and C. thomasi phylogroups were not very high and fell within the limits of 0.036 and 0.063. Another tree constructed using the mitochondrial CO/ gene of 43 samples revealed another additional specific phylogenetic lineage (Suppl. material 1: Figure S2), with divergence levels as high as those for the groups described above. (Table 3). It was formed by a single sample from the Cardamom Mountains, Cambodia. Unfortunately, this was the only sample of this group and, so far, there are no data about the distribution of this genetic lineage in southern Indochina. The clustering pattern of C. thomasi samples was similar to that obtained using Cyt b, with Phylogeography of tree rats of Chiromyscus in eastern Indochina C. chiropus demonstrating two reliable subclusters, com- bining the animals from the Dalat Plateau foothills (Lam Dong Province) and lowland populations (Tay Ninh, Dong Nai and Ba Ria-Vung Tau Provinces), respectively. Thus, mitochondrial genes supported C. /angbianis split into four geographically-distributed phylogroups, where- as C. thomasi and C. chiropus formed two distinctive phylogroups each. Phylogenetic reconstructions, based on nuclear genes, did not allow us to clarify the relationships and the tax- onomic rank of the distinctive phylogroups identified. Thus, only species-level clusters were reliably traced by the RAG/ gene tree constructed for the 26 available samples (Suppl. material 1: Fig. S3). The overall level of divergence was low and genetic distances did not ex- ceed 0.01. The same species-level groups were identified by the GHR gene (Suppl. material 1: Fig. S4), of which we included 52 samples. Within C. /angbianis, complex soft polytomy without notable geographic segregation was traced, whereas within C. thomasi, two clusters cor- responding to the mitochondrial phylogenetic lineages mentioned above were clearly demonstrated. At the same time, the considerable length of the branches was apparent for C. thomasi and for some specimens of C. langbianis from the Dalat Plateau. These branches were significantly longer than those characteristic of C. chiropus and most of the C. langbianis samples, a trait that may indicate a special pattern of its evolutionary history and, in partic- ular, the longer evolutionary age of these populations. Species-level clusters may also be traced in the /RBP gene tree, of which we had 49 samples (Suppl. material 1: Fig. $5). Within the C. /angbianis cluster, no geograph- ical segregation was traced, which may indicate incom- plete sorting of lineages; on the other hand, C. thomasi Table 2. Genetic distances (d, TN93+G+I, gamma = 1.48) for geographic populations and species of Chiromyscus as calculated based on the Cyt b gene sequence (1140 bp). Standard error (S.E.) estimates are shown above the diagonal. C.llangbianis C.langbianis —C_langbianis C.chiropus C.thomasi C.thomasi (Hainan) (Northern) (Southern) (Northern) (Southern) between group distances within groups distances d (TN93+G+I, Tamura-Nei) S.E. C.langbianis (Hainan) 0.005 0.008 0.012 0.015 0.016 0.0061 0.0013 C.langbianis (Northern) 0.036 0.007 0.011 0.015 0.015 0.0097 0.0015 C.langbianis (Southern) 0.063 0.052 0.013 0.016 0.016 0.0080 0.0017 C.chiropus 0.129 0.123 0.130 0.014 0.013 0.0074 0.0015 C.thomasi (Northern) 0.179 0.188 0.187 0.166 0.008 0.0039 0.0013 C.thomasi (Southern) 0.186 0.191 0.192 0.162 0.063 0.0071 0.0016 Table 3. Genetic distances (d; T3P, T92+I) for geographic populations and species of Chiromyscus as calculated based on the CO/ gene sequence (680 bp). Standard error (S.E.) estimates are shown above the diagonal. C.langbianis C.langbianis C.langbianis C.chiropus C.chiropus — C.thomasi C.thomasi (Northern) (Southern) (Cambodia) (Lam_Dong) (others) (Northern) (Southern) between group distances within groups distances d(T3P: GTR) S.E. C.langbianis (Northern) 0.0106 0.0100 0.0176 0.0243 0.0289 0.0305 0.0045 0.0020 C.langbianis (Southern) 0.035 0.0128 0.0183 0.0243 0.0296 0.0278 0.0022 0.0021 C.langbianis (Cambodia) 0.033 0.044 0.0190 0.0242 0.0264 0.0292 C.chiropus (Lam_Dong) 0.083 0.089 0.093 0.0121 0.0262 0.0277 0.0050 0.0024 C.chiropus (others) 0.124 0.130 0.127 0.040 0.0294 0.0307 0.0022 0.0021 C.thomasi (Northern) 0.156 0.161 0.138 0.155 0.172 0.0131 0.0110 0.0036 C.thomasi (Southern) 0.163 0.153 0.154 0.160 0.178 0.049 0.0037 0.0026 zse.pensoft.net Zoosyst. Evol. 97 (1) 2021, 83-95 Niviventer sp. Chiromuscus sums} 3.98 Myear 138-470 i 99/* : Leopoldamys sp. 99/4 eben, ee Redes 2.71 Myear | [1-69-3.67] “st eee xe | Dacnomys millardi we : Lenotrix canus ie o4/ an Margaretamys sp. 93/0.96| — wT pee Tonkinomys daovantieni | *x —— isha CW dine | 2 Rattus division cite Sag Melasmothrix naso INS ee Maxomys division t Saxatilomys paulinae oe 4 ak (1.248)| C |(2.70) 1 (0.54) B |(4.09) A |(4.85) (12.98) (11.65) 93 HIN127 Hainan (11) a “HIN128 Hainan (11) 066 HAN131 Hainan (11) : HNI12008 Hainan (11) 70) (0.95) 0.44 89 Hainan cluster OE gfIN11248 Hainan (12) *HIN12007 Hainan (11) YP22627 Hainan (11) oa TQ 51 Tuyen Quang (5) =" TQ 12 Tuyen Quang (5) 072 1 12 229 Lang Son (7) 12 237 Lang Son (7) 056 HN11249 Hainan (12) a8 TD40 Vinh Phuc (6) ZIN101501 Lang Son (7) ZIN101502 Lang Son (7) G 15 019 Kon Tum (15) 15 020 Kon Tum (15) 15 025 Kon Tum (15) 15 026 Kon Tum (15) 16 049 Kon Tum (15) 0S ho 15 027 Kon Tum (15) TDS50 Vinh Phuc (6) 15 033 Kon Tum (15) 16 035 Kon Tum (16) 15 038 Kon ‘Tum (15) Tx 16 010 Gia Lai (17) OTT 16 036 Kon Tum (16) 16 044 Kon Tum (15) CMF960411.5 Khammouane (13) CMF960411.4 Khammiouane (13) = QBI7 78 Quang Binh (14) ~~ R3795 Khammouane (13) R3796 Khammouane (13) QB17 3 Quang Binh (14) ZIN96516 Kon Tum (15) BN1409007 Yunnan (1) BN1409011 Yunnan (1) BD 48 Lam Dong (19) 7g, BD 26 Lam Dong (19) i BD 7 Lam Dong (19) BD 9 Lam Dong (19) BD 109 Lam Dong (19) BD 50 Lam Dong (19) BD 10 Lam Dong (19) BD10-6 Lam Dong (19) CCP-AO-0004 Kaoh Kong (18) | 12 042 Lam Dong (20) 1 , 12.062 Lam Dong (20) ; 12 083 Lam Dong (20) 077 12 063 Lam Dong (20) cbscaar_ 12.082 Lam Dong (20) : 12 087 Lam Dong (20) iL 12 086 Lam Dong (20) sar 12.068 Lam Dong (20) 06 bap 12 070 Lam Dong (20) a 12 037 Lam Dong (20) 88 = 12 031 Lam Dong (20) ZIN100961 Dong Nai (21) _ MD10-14 Dong Nai (21) MD10-15 Dong Nai (21) O01 1 BT10 2 Ba Ria-Vung Tau (23) aa «LO 7 Tay Ninh (22) 2 MD9-6 Dong Nai (21) Northern cluster 1 0.92 C.langbianis 0,96 Southern cluster O91 Cambodian lineage 0. C.chiropus AB-96 Dong Nai (21) 17 100 Lao Cai (3) O28 gerR TQ 110 Tuyen Quang (5) Op] LQ 50 Tuyen Quang (5) : TQ 13 Tuyen Quang (5) TQ 117 Tuyen Quang (5) <— MC 68 Son La (4) MC 80 Son La (4) D 9.01 15 046 Kon Tum (15) 1 16 045 Kon Tum (15) 18 029 Nghe An (10) 0.68 ome, 18 076 Nghe An (10) (0.43) = CMF960423.2 Khammouane (13) ae NAI8-16 Nghe An (10) “18 083 Nghe An (10) ABTC69097 Houay Sai (2) Northern Ee cluster Southern cluster C.thomasi Niviventer Rattus Mus Figure 3. A. The phylogenetic time tree (Cyt b/COY/RAGI/GHR/IRBP genes, concatenated analyses Bayesian inference) for Chiro- myscus genetic lineages radiation. The posterior probability values and average divergence time (Mya, in brackets) are presented at the nodes. Branches lengths are indicated above the branches. B. The position of Chiromyscus among most closely relative groups of rodents of SE Asia, marked by arrow (see Pages et al. 2016 for details). Footnote: The sample labels and locality numeration are indicated as in Fig. 1 and Suppl. material 1: Table S1. showed deep trichotomy. The samples corresponding to the Southern cluster of mitochondrial lineages were repre- sented here by two independent branches, which formed populations from the Tay Nguyen Plateau and populations distributed further to the north. Similar to the GHR gene tree (Suppl. material 1: Fig. S4), the inequality of the phy- logenetic branch lengths should garner attention. Howev- er, In contrast to the GHR gene, all samples of C. /angbi- anis from the Dalat Plateau recovered longer branches. In general, it can be concluded that C. /angbianis and C. chi- ropus showed significant homogeneity in nuclear genes, whereas C. thomasi had the same pattern of nuclear gene variation as traced by mitochondrial markers. In addition to low support levels for many nuclear gene clades, tree-bisection-reconnection branch-swap- ping (PBS) analysis indicates an existence of conflict- ing phylogenetic signals, especially for segments within C. langbianis. In general, the low posterior probability values for internal branches and the conflicting phylo- genetic signals in many lineages can be explained by a significantly slower evolution rate of nuclear genes (gen- erally weak phylogenetic signal) and incomplete lineage sorting that may be the result of symplesiomorphy. The tree which constructed the concatenated sequence (Fig. 3) is consistent with nuclear gene trees, but posterior proba- bilities values for some internal nodes are lower, mainly zse.pensoft.net 90 Alexander E. Balakirev et al C. chiropus C. langbianis (southern) C. langbianis (northern) C. thomasi (southern) C. thomasi (northern) = @ore¢e+ne .. Phylogeography of tree rats of Chiromyscus in eastern Indochina 4 ee @ 3 ee e 2 @ e + | 1 e =o e 0 e. eo? ¢ a 9 , _ 2 A - ¢ ) e o va | te it le -2 ooo e i x ey A pea “4 4 aa \ 2 z — Figure 4. Results of the multivariate analyses of Chiromyscus spp. from eastern Indochina. A. Ungrouped morphometric separation (PCA analysis); the data were drawn from 20 craniodental measurements. B. Grouped morphometric separation (DFA analysis) drawn from the same specimens and measurements. inside C. /angbianis. The Hainan cluster is not monophy- letic, as one of the samples was recovered in the Northern continental cluster. Morphological analyses The descriptive statistics of the craniodental measure- ments for phylogenetic lineages of C. /angbianis (two of four phylogroups discovered were available) and C. thomasi that were identified by the abovementioned analyses are summarised in Suppl. material 1: Tables S3, S4. Craniodental measurements for C. chiropus are given in Suppl. material 1: Table S5. As revealed by the inter- group F-test, the populations under study demonstrated notable peculiarities of cranial morphology. The samples were significantly different (p < 0.05 or lower) from each other in 18 and 7 cranial characters for C. Jangbianis and C. thomasi, respectively. In a principal components analysis (PCA) drawing on 20 craniodental measurements, the first two axes captured 60.9% (mainly reflecting general size) and 6.4% of the total variation, respectively. ONL, ZB, IB, LD, PPL and CLM1-3 were the six measurements that had the high- est correlations with PC 1 (Suppl. material 1: Table S6). In the PCA of skull measurements, all three species of Chiromyscus overlapped and C. /angbianis showed the largest range of variation amongst these species (Fig. 4A). Discriminant function analysis (DFA), which drew on the same variables, provided another means of illuminating the morphometric distinctions and the first two axes cap- tured 53.6% and 25.4% of the variation (Suppl. material 1: Table S3). The DFA yielded moderate to high discrimi- nation amongst all species and genetic lineages (Fig. 4B). zse.pensoft.net Discussion Taxonomic implications The concordance of morphological and genetic traits and a good separation of samples in 3D factor space indicate the morphological specificity of the studied populations. On the other hand, the concordant pattern of morphologi- cal, genetic and clear geographic subdivision of the mito- chondrial phylogroups allow us to question the taxonom- ic status of these populations; in particular, they allow us to attribute the observed genetic lineages to distinct taxa. The Northern genetic lineage of C. Jangbianis must be undoubtedly assigned to subspecies C. /. indosinicus Os- good, 1932. This taxon was described as Rattus indosini- cus by Osgood (1932) from Sapa in northern Vietnam, Lao Cai Province. The Northern genetic lineage of C. thom- asi has to be attributed to the nominotypical subspecies C. t. thomasi (this phylogroup includes the holotype of C. thomasi). The appropriate name for the Southern ge- netic lineage of C. thomasi is debatable. It may be associ- ated with Rattus indosinicus vientianensis Bourret, 1942, described from the surroundings of Vientiane, Laos. How- ever, Musser (1973) treated vientianensis as a younger synonym of /angbianis and, in our previous survey where the most recent genus revision has been made (Balakirev et al. 2014), we also supposed that nomen vientianensis should be associated with C. /angbianis. Unfortunately, we have no specimens from the neighbouring Vientiane and we cannot identify which of the species is distributed there. The holotype of vientianensis is unavailable. The genetic distances between the two phylogroups of C. thomasi correspond minimally to the subspecific level (Baker 2006). However, despite their considerable ages, Zoosyst. Evol. 97 (1) 2021, 83-95 o1 Table 4. Estimated time to most recent common ancestor (Mya) for Chiromyscus based on Reltime method and the General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.9185); The rate variation model allowed for some sites to be evolutionarily in- variable ({+/], 54.21% sites). The time tree was computed using | calibration constraints. Calibration Clade A Clade B Clade C Clade D Clade E Clade F Clade G Mus/Rattus Chiromyscus/ C. thomasi. C.chiropus _Northern/Southern Cambodian lineage Southern lineage Hainan lineage divergence Niviventercommon divergence’ divergence lineages of C. thomasi of C. langbianis of C. langbianis _ of C. langbianis point ancestor point point divergence point divergence point divergence point divergence point Mean 11.65 4.85 4.09 2.70 1.19 1.248 0.950 0.621 95% CI lower 11.0 2.89 1.78 0.73 0.545 0.041 0.032 0.020 95% CI upper 12.3 7.02 5.90 3.58 3.49 2.88 2.01 1.39 these groups have no visually remarkable morphological differences (see Suppl. material 1: Table S3), so we can- not give here formal description of a new subspecies and assert only that the southern populations of C. thomasi belong to a unique monophyletic genetic lineage. Phylogeography and recent fauna formation Tree rats are usually confined to forest environments and their dispersal is restricted by the forest edge. They usual- ly do not spread beyond these limits and never cross wide deforested areas as they do not feel confident on open ground surface (Musser 1981; Corbet and Hill 1992; our personal observations). The main kind of natural event that contributed to the dispersal, population segregation and speciation of mammalian fauna is, most probably, repeated disjunction-reconnection events of natural pop- ulations that were associated with areas covered by tropi- cal forest during the late Miocene-Holocene (Hall 1998). The distribution pattern and genetic diversification of the genetic lineages revealed in the genus Chiromyscus shed light on the natural history and range formation of these species. Based on genetic data, the population of C. langbianis inhabiting the Dalat Plateau is apparently older than other recent continental populations. It can be assumed that the Dalat Plateau served as its main refu- gium during the Holocene climate oscillations (see also Meschersky et al. 2016; Abramov et al. 2017 for another rodent species). Judging by observed genetic distances and the homogeneity of the Northern genetic lineage of C. langbianis, its expansion beyond the Plateau occurred fairly quickly. Moreover, there is a reason to suppose multiple refugia, as supported by the fact that the most northern Hainanese cluster occupies a basal position in relation to the continental lineages. Noteworthy, to date, the Hainanese haplogroup may occur amongst continental populations. This may indicate both incomplete lineage sorting in this pair of clusters and an ancient hybridisation event between insular and continental populations. Multi- ple reconnection events that occurred during the Pleisto- cene make the second scenario possible. This gives some reason to believe that colonisation of the Island initiat- ed from a different population, not the one that inhabits the Dalat highlands. Instead, it probably originated from an additional northern refugium. The colonisation of Hainan by C. /angbianis might have happened simulta- neously with those of other Muridae (Niviventer and Rat- tus), Which are currently represented by distinct insular populations (Pan et al. 2007; Li et al. 2008; Smith and Xie 2008). This event could be dated back to the Late Miocene. According to Voris et al. (2000), Hainan had been connected to the mainland when the sea level was 120-75 m below the current level, which has happened many times, with the longest connections occurring at approximately 0.25, 0.15 and 0.017 Mya. However, judg- ing by the estimated time of species level genetic lineage divergence (over | Mya, Fig. 3, Table 4), all of them were formed much earlier than these dates and cannot be asso- ciated with recent insularisation. It should be noticed that estimates, evaluated for divergence time for Muridae, are slightly higher than proposed earlier (Rowe et al. 2011; Pages et al. 2016); however, the genus Chiromyscus was represented there by only a single individual. Our finding provides evidence in support of more complex patterns of its evolutionary history. In any case, even if our estimates are closer to the higher limit of generic age determined earlier (Fabre et al. 2013; Pages et al. 2016), these timings for group split points are significantly older than the last events of the Hainan-Mainland reconnection. The latter supports the hypothesis of their formation in the con- tinental refugia during the Late Miocene. On the other hand, the occurrence of another original genetic lineage in southern Cambodia, which is an even more ancient separation than the Hainanese, indicates that there may have been several insularisation and resettlement events and that “distribution waves” originated from the Dalat and any other refugia during the Pleistocene. The split of C. thomasi into the Northern and South- ern phylogroups happened before the split of the corre- sponding C. /angbianis phylogroups and apparently is associated with antecedent global natural factor fluctu- ations. However, the recent distribution pattern of these Species indicates that their natural history differs signifi- cantly amongst the populations that originate from differ- ent dispersion centres/refugia. As far as can be traced by the data available, C. thomasi does not reach the Dalat Plateau and more southern regions inhabited by C. chi- ropus and the Southern lineage of C. /angbianis. At the same time, C. thomasi (both Northern and Southern phy- logroups) appears to be distributed sympatrically with the Northern phylogroup of C. /angbianis in most of eastern zse.pensoft.net 92 Alexander E. Balakirev et al.: Indochina. This indicates that their possible migration routes alongside the Annamite Range occurred in two op- posite directions, with C. thomasi moving northwards and C. langbianis moving southwards. The fact that C. thom- asi did not participate in mammal fauna formation on Hainan Island supports the recent natural area expansion of C. langbianis and are probably explained by ecological factors. Namely, these phenomena may reflect the eco- logical preferences of this species. C. thomasi is known to be more strictly associated with mountain forest forma- tions than C. langbianis, showing greater habitat versatil- ity, which apparently allowed the latter to spread much further eastwards along the plains of eastern Indochina. On the other hand, the significant genetic homogeneity of C. chiropus, which inhabits forest formations everywhere in the extreme south of Indochina and its basal position in relation to the genetic lineages of C. Jangbianis, may in- dicate that these recent populations diverged significantly earlier. This finding also indicates that forest refugia re- mained at the southern part of Indochina throughout the Holocene and even earlier. They could be associated not only with the Dalat Plateau, but also with the Cardamom Mountains, Bolaven Plateau and probably also with some of the offshore islands on the shelf of the Gulf of Siam. The distribution pattern of Chiromyscus species in the region also raises the problem of the initial intrusion and distribution of C. chiropus in eastern Indochina. The terra typica for this species is the Karen Mountains in eastern Myanmar, where the species inhabit mountainous forests. As we pointed out earlier (Balakirev et al. 2014), Burmese specimens do not demonstrate noticeable morphological differences from the southern Vietnamese populations and these are treated as conspecific. Unfortunately, there are still no genetic data from Burmese populations that would allow direct comparison of their genetic identity. Howev- er, the wide distribution of this species to the east in east- ern Indochina through the Yunnan and Annamite Ranges is hampered by the wide distribution of another species, namely, C. thomasi, which populates these regions. No cases of sympatry are currently documented, which may suggest a competitive exclusion in this pair of species. At the same time, the existence of a direct connection be- tween the Malacca and southern Indochina in the Holo- cene by a forest corridor cannot be excluded. Based on data of Meijaard et al. (2003) on tropical forest persistence and the distribution of forest-dependent species on islands of the South China Sea and a forest connection between southern Indochina and Malacca, a southern expansion route is probable. Nevertheless, there are no records on the current distribution of this species in the lowland areas in central Indochina to the west from 105°E. Conclusions We show that the genetic distances between phylogroups of C. langbianis and C. thomasi correspond to the subspe- cific level at least. However, these phylogenetic groups do zse.pensoft.net Phylogeography of tree rats of Chiromyscus in eastern Indochina not demonstrate obvious univocal diagnostic differences in cranial features suitable for species diagnoses without special statistical analysis. Our study shows that the recent phylogenetic structure of C. Jangbianis is the most recent within the genus and appears within several independent refugia that remained isolated throughout the Pleistocene. In turn, the phylogroups of C. thomasi are likely older than those of C. /angbianis. Environmental factors and species preferences followed recent natural ecological shifts which drove allopatry. However, C. chiropus demonstrates the greatest age; the ways of formation of the area of this spe- cies still remain obscure and are likely to be associated with changes in forest cover in Indochina and Malacca Penin- sula during the Pleistocene. The possibility of competitive interaction of these species in the process of formation of their recent natural areas also cannot be excluded. Acknowledgements This study was realised with the support of the Joint Rus- sian-Vietnamese Tropical Research and Technological Center, Hanoi, Vietnam. We thank Dr. Sergei V. Kruskop (Zoological Museum of Moscow State University, Mos- cow, Russia) and Olga V. Makarova (Zoological Insti- tute of Russian Academy of Sciences, Saint Petersburg, Russia) for giving access to the collections under their care. We are grateful to Dr. Viktor V. Suntsov and Dr. German V. Kuznetsov, whose field collections of skulls and skins we used to investigate morphology. We thank Dr. Nguyen Dang Hoi, Dr. Bui Xuan Phuong, Tran Quang Tien, Le Xuan Son and Tran Huu Coi (all from the Joint Russian- Vietnamese Tropical Research and Technologi- cal Center, Hanoi, Vietnam), who put considerable effort into the expedition’s preparations. We also thank the ad- ministrations of Huu Lien, Ke Go, Kon Chu Rang, Kon Plong, Vinh Cuu Ma Da, Pu Mat, Pu Hoat, Bi Doup-Nui Ba, Lo Go Xa Mat and Binh Chau National Parks and Nature Reserves for their help with managing our re- search. We are also very grateful to Dr. Miguel Camacho Sanchez (Estacion Biolégica de Dofiana, Sevilla, Spain) and Dr. Melissa T. R. Hawkins (Smithsonian Institu- tion, National Museum of Natural History, Washington, USA) for their helpful and constructive comments on an earlier version of the manuscript. The study was partly supported by the programme of the Ministry of Science and Higher Education of the Russian Federation (project AAAA-A19-119082990107-3). All authors participated in samples collection, AEB did the genetic analyses and wrote the main part of pa- per, AEB and AVA together performed the morphological analyses and prepared illustrations; VVR provided fund- ing and coordinated all our surveys in Vietnam. The study was performed in full agreement with cur- rent Vietnamese regulations in the field of Nature Pro- tection and Biodiversity Conservation. We followed the guidelines of the American Society of Mammalogists during the collection and handling of the animals. Zoosyst. Evol. 97 (1) 2021, 83-95 References Abramov AV, Balakirev AE, Rozhnov VV (2017) New insights into the taxonomy of the marmoset rats Hapalomys (Rodentia: Muridae). Raffles Bulletin of Zoology 65: 20-28. 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PNAS 107: 15508-15511. https://doi. org/10.1073/pnas. 1005507107 25 Zhang B, He K, Wan T, Chen P, Sun G, Liu S, Nguyen TS, Lin L, Jiang X (2016) Multi-locus phylogeny using topotype specimens sheds light on the systematics of Niviventer (Rodentia, Muridae) in China. BMC Evolutionary Biology 16(1): e261. https://doi.org/10.1186/ $12862-016-0832-8 Supplementary material | Tables S1—S6, Figures S1—S5 Authors: Alexander E. Balakirev Data type: phylogenetic, morphological Explanation note: Tables, samples and other refference materials. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.97.57490.suppl 1 zse.pensoft.net