Distribution pattern and phylogeography of tree rats Chiromyscus (Rodentia, Muridae) in eastern Indochina

Abstract

The study combines available data on species distribution in eastern Indochina to investigate the phylogeographical genetic and morphological 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 , COI ) and nuclear ( IRBP , RAG1 and GHR ) genes. The study presents the most complete and up-to-date data on the distribution and phylogeography of the genus in eastern Indochina. As revealed by mitochondrial genes, C. langbianis 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 morphological 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.

Full text

Distribution pattern and phylogeography of tree rats Chiromyscus
(Rodentia, Muridae) in eastern Indochina
Alexander E. Balakirev1,2, Alexei V. Abramov1,3, Viatcheslav V. Rozhnov1,2
1 Joint Russian-Vietnamese Tropical Research and Technological Centre, 63 Nguyen Van Huyen, Nghia Do, Cau Giay, Hanoi, Vietnam
2 A.N. 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/DD5BE6F7-403E-4E16-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, COI) and nuclear (IRBP, RAG1 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. langbianis 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
signicant 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 insuciency 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
Genus Chiromyscus Thomas, 1925 is currently assigned
to the Dacnomys division of the tribe Rattini (Musser and
Carleton 1993, 2005). It was rst 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
(Thomas, 1891), C. langbianis and C. thomasi Balakirev,
Abramov & Rozhnov, 2014.
To date, Thomas’ tree rat, C. thomasi, is 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
Zoosyst. Evol. 97 (1) 2021, 83–95 | DOI 10.3897/zse.97.57490
Copyright Alexander E. Balakirev et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Alexander E. Balakirev et al.: Phylogeography of tree rats of Chiromyscus in eastern Indochina
84
(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 is known from Myanmar and
easternmost India (Musser 1973) and southern Vietnam
(Balakirev et al. 2014).
The evolution of the genus Chiromyscus was aected
by the natural history of this region. Continuous forest
cover in Indochina existed during a considerable pro-
portion of the Pliocene and Pleistocene (Meijaard 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 eects on the dis-
tribution patterns of the fauna and ora are more complex
(van den Bergh et al. 2001; Meijaard 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 is 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 eld 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 eld 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
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
eld 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 puried either by a DNA Purication
Kit (Fermentas, Thermo Fisher Scientic Inc., Pittsburgh,
PA, USA) or by direct ethanol precipitation. We targeted
ve 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
(COI), which is generally used for species diagnoses and
for DNA barcoding for a number of mammals (Hebert et
al. 2003); a portion of the rst exon of the Interphotore-
ceptor Retinoid Binding Protein gene (IRBP, also known
as Rbp3, up to 1233 bp); the rst exon of the Recombina-
tion Activation Factor gene (RAG1, 1244 bp); and a por-
tion of exon 10 of the Growth Hormone Receptor gene
(GHR, 815 bp).
PCR amplication and sequencing
Cyt b was amplied using H15915R, CytbRglu (Kocher
et al. 1989; Irvin et al. 1991) and CytbRCb9H primers
(Robins et al. 2007). The COI gene was amplied 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 µl volume
that contained 2.5–3 µl 10 x standard PCR buer, 50 mM
of each dNTP, 2 mM MgCl2, 10 pmol of each primer,
1 unit of Taq DNA polymerase (Fermentas, Thermo

Zoosyst. Evol. 97 (1) 2021, 83–95
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Fisher Scientic Inc., Pittsburgh, PA, USA) and 0.5 µl
(25–50 ng) of total DNA template per tube. The reaction
was performed using a Tercik (DNK-Tehnologia, Novo-
sibirsk, Russia) thermocycler. IRBP (1000–1610 bp in
length) was amplied 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 amplied using the primers GHRF1 and
GHRendAlt. This PCR product was re-amplied using
the nested GHRF1 primer paired with GHR750R and the
GHRF50 primer paired with GHRendAlt. A 1244 bp por-
tion of the RAG1 gene was obtained using primers S70
and S71, as described by Steppan et al. (2004).
The PCR products were puried using a DNA Purica-
tion Kit (Fermentas, Thermo Fisher Scientic 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–
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.

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Alexander E. Balakirev et al.: Phylogeography of tree rats of Chiromyscus in eastern Indochina
86
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
1C. langbianis N lineage China, Yunnan, Xishuangbanna 22.0°N, 100.8°E
5C. langbianis N lineage Vietnam, Tuyen Quang, Khong May 102 22.383°N, 105.339°E
6C. langbianis N lineage Vietnam, Vinh Phuc, Tam Dao 850 21.452°N, 105.636°E
7C. langbianis N lineage Vietnam, Lang Son, Huu Lien 230 21.661°N, 106.362°E
8C. langbianis N lineage Vietnam, Nghe An, Pu Hoat 840 19.756°N, 104.796°E
13 C. langbianis N lineage Laos, Khammouane 17.5°N, 105.33°E
13 C. langbianis N lineage Laos, Khammouane, Pha Deng 17.57°N, 105.23°E
14 C. langbianis N lineage Vietnam, Quang Binh, Le Thuy, Sa Khia 156 17.068°N, 106.601°E
15 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
3C. thomasi N lineage Vietnam, Lao Cai, Bat Xat, Y Ty 1830 22.624°N, 103.629°E
4C. thomasi N lineage Vietnam, Son La, Muong Coi 547 21.343°N, 104.749°E
5C. thomasi N lineage Vietnam, Tuyen Quang, Khong May 102 22.383°N, 105.339°E
2C. thomasi S lineage Laos, Houay Sai, Houay Khot Station 20.267°N, 100.4°E
9C. 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, Khammouane, Pha Deng 17.57°N, 105.23°E
15 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 11.583°N, 105.933°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-tting 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 6×106
generations for the Cyt b dataset, 2×106 for the COI, 4×106
for the RAG1 datasets and 5×106 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 at 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
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 ×104, 15 ×104, 50 ×103, 10
×103 and 12.5 ×103 trees for Cyt b, COI, RAG1, GHR and
IRBP, respectively) during the MCMC procedure by Mr-
Bayes v.3.2. The ve 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 5×106 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 dierent
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

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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 dierences amongst sites
(ve categories (+G, parameter = 0.9185)). The rate vari-
ation model allowed for some sites to be evolutionarily
invariable ([+I], 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 dierences were not espe-
cially tested; the possible sexual bias was compensated for
by equalisation of representatives of dierent 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 rst 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 (CLM1-3), rst
upper molar breadth (BM1), rst 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
dierences 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 b sequences. The trees were well supported (PP =
1) (Fig. 2). Dierent geographical populations of
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.

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Alexander E. Balakirev et al.: Phylogeography of tree rats of Chiromyscus in eastern Indochina
88
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 rst
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 intraspecic 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 COI
gene of 43 samples revealed another additional specic
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
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. langbianis 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 identied.
Thus, only species-level clusters were reliably traced
by the RAG1 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 identied
by the GHR gene (Suppl. material 1: Fig. S4), of which
we included 52 samples. Within C. langbianis, 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 signicantly
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 IRBP
gene tree, of which we had 49 samples (Suppl. material
1: Fig. S5). Within the C. langbianis 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.langbianis
(Hainan)
C.langbianis
(Northern)
C.langbianis
(Southern)
C.chiropus C.thomasi
(Northern)
C.thomasi
(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 COI
gene sequence (680 bp). Standard error (S.E.) estimates are shown above the diagonal.
C.langbianis
(Northern)
C.langbianis
(Southern)
C.langbianis
(Cambodia)
C.chiropus
(Lam_Dong)
C.chiropus
(others)
C.thomasi
(Northern)
C.thomasi
(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

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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. langbi-
anis from the Dalat Plateau recovered longer branches. In
general, it can be concluded that C. langbianis and C. chi-
ropus showed signicant 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 conict-
ing phylogenetic signals, especially for segments within
C. langbianis. In general, the low posterior probability
values for internal branches and the conicting phylo-
genetic signals in many lineages can be explained by a
signicantly 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
Figure 3. A. The phylogenetic time tree (Cyt b/COI/RAG1/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.

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Alexander E. Balakirev et al.: Phylogeography of tree rats of Chiromyscus in eastern Indochina
90
inside C. langbianis. 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. langbianis (two
of four phylogroups discovered were available) and
C. thomasi that were identied 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 signicantly dierent (p < 0.05 or lower) from each
other in 18 and 7 cranial characters for C. langbianis and
C. thomasi, respectively.
In a principal components analysis (PCA) drawing on
20 craniodental measurements, the rst two axes captured
60.9% (mainly reecting 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. langbianis 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 rst 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).
Discussion
Taxonomic implications
The concordance of morphological and genetic traits and
a good separation of samples in 3D factor space indicate
the morphological specicity 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. langbianis must be
undoubtedly assigned to subspecies C. l. 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 langbianis 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. langbianis. 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 subspecic level
(Baker 2006). However, despite their considerable ages,
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.

Zoosyst. Evol. 97 (1) 2021, 83–95
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91
these groups have no visually remarkable morphological
dierences (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 conned 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 condent 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 diversication 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 dierent population, not the one that inhabits
the Dalat highlands. Instead, it probably originated from
an additional northern refugium. The colonisation of
Hainan by C. langbianis 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 1 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 nding
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 signicantly 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. langbianis phylogroups and apparently is
associated with antecedent global natural factor uctu-
ations. However, the recent distribution pattern of these
species indicates that their natural history diers signi-
cantly amongst the populations that originate from dier-
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. langbianis. At the
same time, C. thomasi (both Northern and Southern phy-
logroups) appears to be distributed sympatrically with the
Northern phylogroup of C. langbianis in most of eastern
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 dierences among
sites (5 categories (+G, parameter = 0.9185); The rate variation model allowed for some sites to be evolutionarily in-
variable ([+I], 54.21% sites). The time tree was computed using 1 calibration constraints.
Calibration Clade A Clade B Clade C Clade D Clade E Clade F Clade G
Mus/Rattus
divergence
point
Chiromyscus/
Niviventer common
ancestor
C. thomasi
divergence
point
C. chiropus
divergence
point
Northern/Southern
lineages of C. thomasi
divergence point
Cambodian lineage
of C. langbianis
divergence point
Southern lineage
of C. langbianis
divergence point
Hainan lineage
of C. langbianis
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

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Alexander E. Balakirev et al.: Phylogeography of tree rats of Chiromyscus in eastern Indochina
92
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 reect 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 signicant 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. langbianis, may in-
dicate that these recent populations diverged signicantly
earlier. This nding 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 oshore 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
dierences from the southern Vietnamese populations and
these are treated as conspecic. 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-
cic level at least. However, these phylogenetic groups do
not demonstrate obvious univocal diagnostic dierences
in cranial features suitable for species diagnoses without
special statistical analysis. Our study shows that the recent
phylogenetic structure of C. langbianis 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. langbianis. 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 eld 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 eort
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 (Estación Biológica de Doñana, 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 eld 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.

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Supplementary material 1
Tables S1–S6, Figures S1–S5
Authors: Alexander E. Balakirev
Data type: phylogenetic, morphological
Explanation note: Tables, samples and other reerence
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://doi.org/10.3897/zse.97.57490.suppl1