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Genes Genet. Syst. (2015) 90, p. 21–30 Molecular phylogenetic characterization of common murine from Manipur, Northeast

Dhananjoy S. Chingangbam1, Joykumar M. Laishram1 and Hitoshi Suzuki2* 1Plant Breeding and Genetics Department, Central Agricultural University, Iroishemba, Imphal, Manipur 795004, India 2Graduate School of Environmental Earth Science, Hokkaido University, North 10, West 5, Kita-ku, Sapporo, Hokkaido 060-0810,

(Received 11 July 2014, accepted 8 February 2015)

The Indian subcontinent and Southeast are hotspots of murine biodiver- sity, but no from the Arakan Mountain system that demarcates the border between the two areas has been subjected to molecular phylogenetic analyses. We examined the mitochondrial cytochrome b gene sequences in six murine species (the Rattus rattus species complex, R. norvegicus, R. nitidus, Berylmys manipulus, sp. and Mus musculus) from Manipur, which is located at the western foot of the mountain range. The sequences of B. manipulus and Niviventer sp. examined here were distinct from available congeneric sequences in the databases, with sequence divergences of 10–15%. Substantial degrees of intrapopulation divergence were detected in R. nitidus and the R. rattus species complex from Manipur, implying ancient habitation of the species in this region, while the recent introduction by modern and prehistoric human activities was suggested for R. norvegicus and M. musculus, respectively. In the nuclear gene Mc1r, also analyzed here, the R. rattus species complex from Manipur was shown to possess allelic sequences related to those from the Indian subcontinent in addition to those from East Asia. These results not only fill gaps in the phylo- genetic knowledge of each taxon examined but also provide valuable insight to bet- ter understand the biogeographic importance of the Arakan Mountain system in generating the species and genetic diversity of murine rodents.

Key words: Berylmys, cytochrome b, India, Mc1r, Rattus

species, as are the R. rattus SC and M. musculus, but INTRODUCTION despite their importance the population genetic structure Murine rodents, members of the subfamily , and evolutionary episodes have not yet been assessed. comprise ~500 species and their evolutionary radiation in The zoogeographic Indomalayan Region has a high the major homeland of the Indomalayan Region is an level of terrestrial biodiversity (Srinivasulu and important biogeographic topic. Molecular phylogenetic Srinivasulu, 2012; Barley et al., 2014) and its continental relationships and population genetic structuring have part can be divided into two regions, the Indian subcon- been assessed in certain groups of murine genera and spe- tinent and mainland , with the Arakan cies, respectively, such as the genus Mus (Shimada et al., Mountain system on the border of India and 2010), the house M. musculus (Suzuki et al., 2013), acting as a geographic barrier. Widespread taxonomic and the Rattus rattus species complex (e.g., Pagès et al., groups are ideal systems for testing biogeographic 2010; Aplin et al., 2011; Conroy et al., 2013; Yasuda et al., hypotheses, because they offer the opportunity of examin- 2014), but a large portion of murine taxa have not yet ing how biogeographic barriers, as well as dispersal and been examined. Even in extensively examined taxa, colonization, affect diversification and determine biogeo- some geographic areas, such as Northeast India, remain graphic distribution patterns (e.g., Irschick et al., 1997; to be investigated. In addition, two species, R. Barley et al., 2014). Among the taxa occurring in norvegicus and the Himalayan rat R. nitidus, are known Northeast India, a most atractive taxon is the R. rattus as commensal rodents and recognized as invasive alien SC, because the geographic area is a possible suture zone of the two major forms, “R. rattus” and “R. tanezumi”, Edited by Toshihiko Shiroishi whose native ranges are the Indian subcontinent (mainly * Corresponding author. E-mail: [email protected] in central and southern parts) and the remaining 22 D. S. CHINGANGBAM et al.

Indomalayan Region (e.g., , , , ence code and the skull and skin were stored at the Japan, Korea, , , , and Central Agricultural University, Imphal (CAUI), under ), respectively (Yosida, 1980; Musser and Carleton, the code name CAUI (Table 1). Approval was obtained 2005). In addition, generic taxa of purely non-commensal from the Institutional Ethics Committee (IEC) for use of forest-dwelling rodents, such as Berylmys and Niviventer, , and IEC protocols were followed throughout the should be examined to assess the role of the mountain study. Morphological and taxonomic studies were car- range in speciation processes. ried out according to Corbet and Hill (1992), Agrawal We conducted a molecular phylogenetic study on six (2000), Aplin et al. (2003) and Alfred (2005). The skulls species collected from Manipur, in the easternmost were prepared according to Herbreteau et al. (2011) to part of the union of India. We determined the nucleotide ascertain the true identities of the specimens. All mea- sequences of mitochondrial cytochrome b (Cytb; 1140 bp) surements were taken using Fisher’s digital calipers. in B. manipulus, M. musculus, Niviventer sp., R. nitidus, Tissue samples were preserved in > 95% ethanol for DNA R. norvegicus and the R. rattus SC collected from extraction. Manipur. We also examined the variation in chromo- In the present study, we collected rat specimens of the some number in representative individuals of the six genus Rattus from habitats in Manipur (Table 1), using species. A nuclear gene, the melanocortin-1-receptor live traps in 2012 and 2013. In Manipur, belonging (Mc1r) gene, which is one of the most important genes in to the R. rattus SC are abundant in the hills and valley the evolution of pigmentation in vertebrates, including regions; in particular, they congregate at Loktak Lake. rodent species (Nachman et al., 2003; Hoekstra et al., Rattus nitidus is indigenous to mainland Southeast Asia 2005), has been used to assess the phylogenetic groups of (Aplin et al., 2003; Musser and Carleton, 2005) and is the R. rattus SC (Kambe et al., 2011, 2012; Suzuki, 2013; found exclusively in hilly regions. We collected a single Yasuda et al., 2014), and we examined the variation of individual, with the tip of its tail cut off, belonging to Mc1r sequences (954 bp) in this taxon. The present Niviventer sp. (head and body, 135 mm; tail, > 115 mm; study was performed to fill some of the gaps in our under- hind foot, 27 mm; ear, 18 mm). Even accounting for the standing of the inter- and intraspecies phylogenetic rela- missing tail tip, this individual had a relatively short tail tionships of the six species of rats and mice mentioned (possible tail ratio < 110%), which differed from other spe- above. This study also contributed to a better under- cies occurring in the Himalayas (Corbet and Hill, 1992)— standing of the biogeographic nature of Northeast India. N. eha (> 140%), N. fulvescens (> 115%) and N. langbianis (140%)—and was likely to be consistent with that of N. niviventer (100%–115%). Two species of Berylmys are MATERIALS AND METHODS known from Manipur, B. mackenziei and B. manipulus Samples All of the specimens used in this study were (Corbet and Hill, 1992; Agrawal, 2000). Our young spec- collected from 11 localities in Manipur (Fig. 1). Before imens from Singda (Locality 3 in Fig. 1), with incomplete initiating any work, each specimen was assigned a refer- tails, CAUI 2024 (head and body, 115 mm; ear, 23 mm;

AB 1 2 Ukhrul The Indian 3 4 5 6 Imphal Subcontinent 7 Thoubal 8 Lineage II 10 9 Manipur Mainland Loktak Lake 11 SE Asia Lineage I

50 km

Fig. 1. The geographic position of Manipur lying at the western foot of the Arakan Mountain range, which demarcates the two faunal groups of the Indian subcontinent and mainland Southeast Asia (A). The known natural ranges of the two mitochondrial phylogroups of Lineages I and II of the R. rattus species complex (see Aplin et al., 2011 for details; see also Yasuda et al., 2014 for ) are shown with dotted lines. The collection localities of the samples used in this study are shown with the serial numbers defined in Table 1; filled and open circles represent the district headquarters and actual collection spots, respectively (B). Phylogeny of Manipur rats and mice 23

Table 1. Samples used in this study

Specimen Date of Diploid Mc1r Species Collection locality** Habitat code* collection number sequence*** Rattus rattus CAUI 2008 Langol (4) Valley 2012/7/26 42 AB973118 (I/I) species complex CAUI 2047 Langol (4) Valley 2013/11/5 42 – CAUI 2048 Langol (4) Valley 2013/11/5 42 – CAUI 2030 Iroisemba (5) Valley 2013/2/1 42 – CAUI 2037 Iroisemba (5) Valley 2013/1/30 42 – CAUI 2041 Iroisemba (5) Valley 2013/4/2 42 – CAUI 2046 Iroisemba (5) Valley 2013/4/17 42 – CAUI 2038 Thoubal (8) Valley 2013/6/3 42 – CAUI 2056 Heirok (9) Valley 2013/5/8 42 – CAUI 345 Loktak (10) Valley 2011/8/13 42 AB973117 (I/II) CAUI 2013 Loktak (10) Valley 2011/8/13 42 AB973119 (II/II) CAUI 2014 Loktak (10) Valley 2011/8/13 42 AB973120 (I/II) CAUI 2035 Khudei Khunou (11) Hill 2012/9/28 42 – Rattus norvegicus CAUI 2010 Keishampat (7) Valley 2012/2/1 42 AB973121 CAUI 2058 Keishampat (7) Valley 2013/8/28 42 – Rattus nitidus CAUI 344 Mao (1) Hill 2011/7/25 42 AB973122 CAUI 2012 Ukhrul (2) Hill 2012/7/25 42 AB973123 CAUI 2015 Ukhrul (2) Hill 2012/7/25 42 AB973124 Berylmys manipulus CAUI 2024 Singda (3) Hill 2012/9/28 40 – CAUI 2025 Singda (3) Hill 2012/9/28 40 – Niviventer sp. CAUI 2020 Ukhrul (2) Hill 2012/9/8 46 – Mus musculus CAUI 2007 Imphal (6) Valley 2012/6/16 40 AB973125 CAUI 2057 Keishampat (7) Valley 2013/8/16 40 – CAUI 2011 Thoubal (8) Valley 2012/6/17 40 AB973126 * DNA code of CAUI (Central Agricultural University, Imphal) number. ** Specimens were collected from Manipur, India. The serial numbers are shown in parentheses (see Fig. 1B for details). *** DDBJ/EMBL/GenBank Accession number. Diploid pairs of haploid groups are shown in parentheses. hind foot, 28 mm) and CAUI 2025 (head and body, karyotyping. Chromosome number and morphology 165 mm; ear, 24 mm; hind foot, 31 mm), were identified were recorded from 50 Giemsa-stained metaphase plates as B. manipulus, based on a comparison with other from each specimen directly under an Olympus BX-41 Berylmys specimens (n = 20) from Singda, preserved in microscope at ×100 as well as from 10 photographs of formalin in our laboratory in the Central Agricultural selected cells. The types of chromosome were deter- University, Imphal. They have hind foot lengths of 28– mined according to Levan et al. (1964) in which the chro- 38 mm, with other morphological characteristics of the mosomes were arranged in descending order of tail equal to or longer than the head and body length, the chromosome length, the seven small metacentric pairs body having a dense, crisp, iron-gray pelage above and were separated from the acrocentrics and subtelocentrics white below, the distal one third of the tail being white were inserted among the acrocentrics according to total and the rest brown, and having five pairs of mammae, chromosome length. and they have been assigned as B. manipulus (Agrawal, 2000). Gene sequence analysis DNA samples were extracted by the standard phenol/chloroform method (Sambrook et Karyotype analysis Chromosomes were harvested al., 1989) and stored at the Central Agricultural Univer- from the bone marrow cells of the femur using a standard sity, Imphal. PCR was performed using Taq DNA poly- colchicine-hypotonic air-drying technique. The chromo- merase (GeNei Merck, Mumbai, India) and a model 2720 somes were stained with 4% Giemsa for conventional Thermal Cycler (Applied Biosystems). The complete 24 D. S. CHINGANGBAM et al. mitochondrial Cytb coding sequence (1140 bp) was ampli- showed some simple karyotypic features, such as 13 pairs fied using the universal primers L14115 and H15300 of acrocentrics, 7 pairs of meta- or submetacentrics, and (Irwin et al., 1991). The amplified fragments were used acrocentric X and Y chromosomes with a fundamental as templates for sequence determination with the above number of 56, with slight deviations. With regard to universal primers and two internal primers. The chromosome variation in the longest chromosome, which sequences of the entire coding region of the Mc1r gene is known to be either acrocentric or subtelocentric (954 bp) were determined with primers reported previ- (Yosida, 1973), our samples from Manipur were of the for- ously (Shimada et al., 2009). Both DNA strands of the mer type. The “ordinary” chromosome numbers were PCR products were sequenced using an automated observed in the other species, B. manipulus (2n = 40), M. method with a BigDye terminator Cycle Sequence Kit musculus (2n = 40), Niviventer sp. (2n = 46), R. nitidus (ver. 3.1; Applied Biosystems) and an automated (2n = 42) and R. norvegicus (2n = 42) (Table 1). sequencer (model 3130; Applied Biosystems). Sequence fragments obtained with different primers were assem- Mitochondrial gene sequences We determined Cytb bled using MEGA5 software (Tamura et al., 2011), and gene sequences (1140 bp) in 24 rats and mice from the sequences were aligned by eye. Novel sequences Manipur (Table 1). We constructed ML and NJ trees were deposited in the DDBJ/EMBL/GenBank DNA data- using the Cytb sequences, together with database bases under accession numbers AB973093–AB973126. sequences, to assess the phylogenetic relationships within and between species (Fig. 2). The divergence times were Data analysis and tree building We constructed max- assessed by BEAST analysis (Rambaut and Drummond, imum-likelihood (ML) and neighbor-joining (NJ) trees 2009) and TMRCAs for internal nodes of interest are using MEGA5. In ML analyses, the substitution model shown in Fig. 2. that better described the substitution patterns of the Cytb The 13 individuals of the R. rattus SC from Manipur (HKY+G; Hasegawa et al., 1985) data was determined by yielded eight distinct haplotypes, with sequence diver- MEGA5. In ML analysis, the tree topology search was gence of 0.6%–0.9% (Fig. 2). The eight Manipur haplo- evaluated using simultaneous nearest-neighbor inter- types were integrated into the clade of R. rattus SC change (NNI). In NJ analysis, the Kimura two-parameter Lineage II (Fig. 2) defined in a previous study (Aplin et model (K2) was used. The reliability of nodes was al., 2011), and they differed from haplotypes of the other assessed using 1000 bootstrap replicates. Lineage II sequences compared, with sequence divergence BEAUti and BEAST v1.7.5 (Drummond et al., 2012) of 0.6%–1.5%. The Tajima’s D value for the 18 individ- was used for Bayesian Markov-chain Monte-Carlo ual rats of Lineage II was significantly negative (–1.4; P < (MCMC) analyses (10000000 chain length) of the Cytb 0.05), suggesting rapid population growth. The expan- sequence data to estimate the time to the most recent sion parameter τ was calculated to be 6.9. common ancestor (TMRCA) and 95% highest posterior In phylogenetic analysis with the Cytb sequence, the density (HPD). An expected divergence time of 1.7 mil- sequence of R. norvegicus from Manipur was shown to be lion years ago (MYA) was used for the ancestral node virtually identical to those reported for rats from other comprising M. spretus and M. musculus (Suzuki et al., countries. The Cytb sequences of the three R. nitidus 2004). After a 1000-tree burn-in, 10000 trees were used rats differed substantially, yielding two distinct haplo- for the analyses. The Bayesian tree was generated types with 1.5%–1.7% sequence divergence, one of which under the relaxed clock model (HKY substitution). The was similar to those from Southeast Asia, with 0.2%–0.4% convergence of the MCMC chains and the effective sample sequence divergence. The single specimen of Niviventer size (ESS) values exceeding 200 for all parameters were sp. (CAUI 2020) was placed in the clade of the N. assessed using Tracer v1.5 (Rambaut and Drummond, confucianus species group, with substantial genetic dis- 2009). tances of 10%–12% from any other sequences of this The population expansion event was assessed by group that have been deposited in the databases with a Tajima’s D value using Arlequin version 3.5 (Excoffier variety of taxon names (i.e., N. confucianus, N. coxingi, N. and Lischer, 2010). The same software was used for esti- bukit, N. tenaster, and N. niviventer) and were apparently mation of the expansion parameter τ. distinct from N. fulvescens, with a genetic distance of 17%. Comparison of Berylmys Cytb sequences revealed that B. manipulus, endemic to Northeast India, was RESULTS equally distinct from B. berdmorei and B. bowersi, with Karyotype We performed karyotype analyses using 10%–12% genetic divergence. bone marrow cells and conventional methods (Table The three Cytb sequences of M. musculus from Manipur 1). The four individuals belonging to the R. rattus SC were integrated into the major subclade of M. m. castaneus, were shown to have 2n = 42, indicative not of R. rattus, CAS-1. In addition, the Manipur sequences were more but of R. tanezumi. The R. rattus SC with 2n = 42 closely related to the sequence of mouse from Phylogeny of Manipur rats and mice 25

99/99 RrC II India CAUI 345 RrC II India CAUI 2008 RrC II India CAUI 2038 RrC II India CAUI 2035 RrC II India CAUI 2041 RrC II India CAUI 2056 RrC II India CAUI 2037 93/94 RrC II Inida CAUI 2014 RrC II India CAUI 2046 Lineage II RrC II India CAUI 2013 RrC II India CAUI 2047 “R. tanezumi” RrC II India CAUI 2048 RrC II India CAUI 2030 species complex RrC II China AB096841 85/83 RrC II VietNam AB355900

100/99 RrC II Japan: Kagoshima AB211040 RrC II USA JQ823487

97/96 Rattus rattus RrC II VietNam AB355901 RrC I Japan HS2343 AB211039 96/89 Lineage I 99/99 RrC I Japan HS132 AB033702 “R. rattus” RrC III JN675599 1.5-1.9 mya 99/77 100/99 RrC III JN675600 73/67 RrC III JN675601 55/- RrC VI JN675624

99/85 99/82 RrC IV HM217443 Rattus argentiventer Indonesia AB033701 Rattus pyctoris JN675511 (HS1471) R. norvegicus India CAUI 2010 62/- 100/94 R. norvegicus India CAUI 2058 1.0 mya 100/99 R. norvegicus Strain SD AB033713 Rattus norvegicus R. norvegicus R9 Thailand HM217429

99/92 R. norvegicus R9 Thailand HM217370 100/99 R. nitidus India CAUI 344

88/84 R. nitidus India CAUI 2012 71/50 100/86 R. nitidus India CAUI 2015 R. nitidus Rattus nitidus 100/81 Laos HM217479 Clade A R. nitidus Laos HM217478 R. nitidus Laos HM217474 2.1-2.4 mya 100/100 Berylmys bowersi HM217413 Berylmys bowersi KC878024

100/100 Berylmys manipulus India CAUI 2024 94/84 Berylmys manipulus Berylmys manipulus India CAUI 2025 58/- Berylmys berdmorei HM217432 100/98 Berylmys berdmorei HM217431 Niviventer fulvescens VietNam GU457005 Niviventer coxingi Taiwan EF053024 73/94 Niviventer bukit VietNam GU456986 84/54 99/97 Niviventer confucianus China HQ225803 Niviventer confucianus 51/51 Niviventer cf. niviventer India CAUI 2020 Species Group Niviventer niviventer VietNam GU456998 Niviventer tenaster VietNam GU457016 Mus spretus 1.3-1.9 mya AB033700 97/- Mus spicilegus AB125775 100/100 Mus macedonicus AB125770

100/72 Mus musculus Germany AB649455 94/65 1.7 mya Mus musculus Peru AB649478

100/65 Mus musculus Taiwan AB125773 CAS-2 Mus musculus AB649486 CAS-4 99/99 Mus musculus Japan AB205291 (CAS-1a) 99/65 Mus musculus Mus musculus India CAUI 2057 95/93 CAS Mus musculus India CAUI 2007 CAS-1 70/93 Mus musculus India CAUI 2011 53/- Mus musculus Indonesia AB205276 (CAS-1b)

0.02 Fig. 2. A NJ tree for six taxa of the murine rodents, including the R. rattus species complex (RrC), collected from Northeast India. Numbers at nodes indicate bootstrap values by the NJ and ML methods (1000 replicates, > 50%). The taxon names corre- spond to those in Table 1. CAUI DNA codes are those of the authors’ personal collections. Shaded boxes indicate taxa collected from Northeast India. Resultant TMRCA values from BEAST analysis are marked on the nodes focused upon in this study. We used a calibration point for dating (the node marked with a star), 1.7 MYA for the basal divergence of M. musculus species group, including M. spretus (Suzuki et al., 2004). The scale bar indicates the genetic distance. 26 D. S. CHINGANGBAM et al.

Group I: “R. rattus” Group II: “R. tanezumi”

2014A

Hap 11 2008A 2014B, 345B

Hap 1 2013A 2013B 345A Hap 8

2008B Hap 9 Hap 2

Hap 10 Hap 4 Hap 12 Hap 6 Hap 3

0.0010 Hap 7 Hap 5

Fig. 3. A neighbor-net network with the nuclear melanocortin 1 receptor gene (Mc1r) sequences for four individuals of the R. rattus SC collected from Northeast India (CAUI 345, 2008, 2013 and 2014; see Table 1 for details). Representative haplotype sequences for the two species members of the taxon, R. rattus (Haps 4–11) and R. tanezumi (Haps 1–3 and Hap 12) were obtained from DNA data- bases (Kambe et al., 2011). The scale bar indicates the genetic distance. than to that of the Japanese mouse, representing the two groups, consistent with our previous studies (Kambe et locally restricted phyletic groups CAS-1b and CAS-1a, al., 2011, 2012). The Manipur sequences were located at respectively (Fig. 2) (Suzuki et al., 2013). The Tajima’s six different positions in the network, showing their D value calculated for the three mice together with 17 apparent demarcation into two clusters designated mice belonging to CAS-1b (Suzuki et al., 2013) was signif- “rattus” (Group I) and “tanezumi” (Group II). icantly negative (–2.1; P < 0.01), suggesting rapid popu- lation growth. The τ value was calculated as 1.9. DISCUSSION Mc1r sequences We determined the Mc1r sequences Geographically, Northeast India is potentially a key (954 bp) in four individuals from the R. rattus SC, location to better understand phylogenetic relationships together with R. nitidus, R. norvegicus and M. musculus both between and within species of murine rodents in (Table 1), which will be valuable in future phylogeo- South Asia. Here, we performed phylogenetic analyses graphic inferences as nuclear markers and for assessing on six rodent taxa from Manipur, located at the western functional effects of each amino acid change on coat color foot of the mountain range of the predicted geographic alteration during the course of evolution. In this study, border demarcating the Indian subcontinent and main- we addressed the phylogeographic relationships of the R. land Southeast Asia: two taxa occurring in natural habi- rattus SC from Manipur, using the Mc1r sequence as a tats (Berylmys and Niviventer) and the remaining four phylogeographic marker. The Mc1r sequences of the showing a trend of human influence in their secondary four rats from Manipur were prone to polymorphic sites habitation (M. musculus, R. nitidus, R. norvegicus and and haplotype sequences were created based on minimiz- the R. rattus SC). The present study provided not only ing the number of haplotypes. We ignored singletons in data regarding the phylogenetic positions of the rodents the separation of haplotypes and in the construction of a from Northeast India but also information valuable for network tree. The neighbor-net tree was generated from assessing the biogeographic importance of this area. the Mc1r sequences from Manipur and 12 sequences iden- The genus Berylmys comprises four species, B. berdmorei, tified previously (alleles 1–12; Kambe et al., 2011, B. bowersi, B. mackenziei and B. manipulus (Corbet and 2012). The resulting tree (Fig. 3) exemplified two dis- Hill, 1992; Agrawal, 2000). The northeastern part of tinct clusters representing the “rattus” and “tanezumi” India is rich in this genus, harboring B. manipulus with Phylogeny of Manipur rats and mice 27 a distribution confined to Northeast India, and B. indicative of R. tanezumi, and not R. rattus. The Cytb berdmorei and B. bowersi with broad distribution ranges phylogenetic analysis reported here supported these con- in the Indomalayan Region. The latter two species have clusions (Fig. 2), indicating that all haplotypes recovered been examined in terms of mitochondrial Cytb sequences fell within the clade of Lineage II with a 100% support (Pagès et al., 2010; Aplin et al., 2011); however, no molec- value. Notably, the substantial level of nucleotide diver- ular data are available for the two local species. To our sity in the mitochondrial DNA (mtDNA) from Manipur knowledge, this is the first reported molecular analysis of implies ancient habitation of this species in this geo- the Northeast Indian species B. manipulus (Fig. 2). graphic area, providing robust evidence that the geo- Comparison of the Cytb sequences revealed that B. graphic area of the western foot of the Arakan Mountain manipulus was equally distinct from B. berdmorei range functioned as part of the homeland of this species. (TMRCA: 2.4 MYA, 95%HPD 1.9–3.0) and B. bowersi More interestingly, together with previous results (Chinen (TMRCA: 2.1 MYA, 95%HPD 1.6–2.6), with 13% and 16% et al., 2005; Aplin et al., 2011; Kambe et al., 2012), it is genetic divergence, respectively. These findings suggest now evident that mtDNA Lineage II dispersed across the that Northeast India has fostered its own lineages of for- Arakan Mountain range, linking the western and eastern est dwellers over a long period. sides and covering the far eastern area, the Japanese The phylogenetic relationships of the genus Niviventer Archipelago. are difficult to unravel, partly because species misidenti- From the shallow branching pattern of the phylogenetic fications are abundant in database sequences and also tree (Fig. 2) and the result of Tajima’s D test (significantly because of the existence of several cryptic species negative), it is reasonable to assume that the Lineage II (Balakirev and Rozhnov, 2010; Pagès et al., 2010; He and phylogroup experienced an ancient range expansion Jiang, 2013). According to Corbet and Hill (1992), in the event, as has been noted in our previous study (Aplin et Himalayan region and its neighboring region of Northeast al., 2011), which also stated that such ancient coloniza- India, the two species N. fulvescens and N. niviventer are tion events resulted in the deep and geographically highly both present, in addition to N. eha and N. langbianis. In structured mitochondrial diversity of the R. rattus species molecular phylogenetic analyses of Niviventer species complex in the Indian subcontinent and Southeast Asia. diversity using more than 200 mitochondrial Cytb Contrary to the initial prediction, the eight Mc1r sequences, He and Jiang (2013) discussed the presence of sequences examined from four individuals from Manipur four or five major clades, including the two clades of the were split into two clusters representing “R. rattus” (four N. confucianus species group and N. fulvescens species sequences) and “R. tanezumi” (four sequences), corre- group. To date, no Cytb sequence has been reported for sponding to the mtDNA Lineages I and II, respectively individuals from the Himalayan region and Northeast (Fig. 3). One individual (CAUI 2013) from Loktak was India. In this study, we have a single sequence deter- found to have the R. tanezumi type in both the mitochon- mined for Niviventer sp. from Manipur, which provides drial and nuclear sequences, while the other three from several valuable insights regarding the zoogeography of Loktak (CAUI 345, 2014) and Langol (CAUI 2008) had this genus. The Cytb sequence is the first determined for the R. tanezumi type of Cytb and the R. rattus type of a member of this genus in the northeastern peripheral Mc1r in their genomes. These results suggest that region and suggests the existence of a distinct lineage genetic hybridization is occurring between the two geo- belonging to the N. confucianus species group with an graphic groups of “R. rattus” (2n = 38) and “R. tanezumi” estimated divergence time of 1.3–1.9 MYA (Fig. 2), indi- (2n = 42) in the northeastern part of India. This again cating that Northeast India was the site of this ancient highlights the specific features of the geographic area divergence of the species group of forest-dwelling rats. that have assisted the dispersal of murine rodents across Issues related to the evolution of the , the R. different geographic domains, perhaps through connectiv- rattus SC, for which many molecular studies are avail- ity via mountain ranges of South Asia, including the able, are of interest. The R. rattus SC is predicted to Arakan Mountain range. have homelands in Asia, with six distinct mitochondrial One of our great concerns related to the murine rodents Cytb lineages (I–VI) with different geographic affinity is the prehistoric human influence on their secondary (Chinen et al., 2005; Pagès et al., 2010; Aplin et al., distributions. Generally, the northeastern tip of India is 2011). The area comprising eastern and southern India, thought to have been a key corridor for human migrations representing Lineage I, is the likely geographic focus of between these two subcontinental areas (e.g., Nei and the karyotype group of 2n = 38, while the geographic area Roychoudhury, 1993; Cavalli-Sforza et al., 1994; Reddy et covering the easternmost part of India to southern China al., 2007). In contrast, Cordaux et al. (2004) suggested represents the natural range of Cytb Lineage II, repre- that the Northeast Indian passageway acted as a geo- senting the chromosome 2n = 42 form, the so-called R. graphic barrier between the Indian subcontinent and tanezumi (Fig. 1A). We confirmed these conclusions, as East/Southeast Asia, at least since the arrival of Tibeto- our specimens from Manipur had the karyotype 2n = 42, Burman speakers in Northeast India, on the basis of 28 D. S. CHINGANGBAM et al. human mtDNA and Y chromosome studies. The two the karyotype of R. nitidus of the male individuals was commensal taxa R. rattus SC and M. musculus have been shown to have eight small metacentric pairs, two subte- subjected to intensive phylogeographic analyses of the locentric pairs, 10 acrocentrics pairs, and acrocentric X prehistoric and historical dispersals in association with and Y chromosomes, exhibiting similar cytogenetic fea- humans (e.g., Aplin et al., 2011; Suzuki et al., 2013) but tures to the conspecific Chinese population (Li et al., few molecular studies had included samples from 2008). Substantial levels of sequence divergence (~1.4%) Northeast India. The house mouse, M. musculus, is were found between the two Cytb haplotypes recovered known to have its homeland in India and the western from three Manipur R. nitidus individuals. This sug- neighboring countries of Iran, , and Pakistan, gests a long-standing presence of this species in this geo- and then experienced geographic expansion from three graphic area, which may thus be part of the homeland of different source areas, including somewhere on the this species. On the other hand, one of the lineages Indian subcontinent, that shaped the current broad dis- (CAUI 2012, 2015; Fig. 2) was closely related to R. nitidus tributions of the M. m. castaneus (CAS) subspecies (e.g., from Southeast Asia (Pagès et al., 2011), with which it Suzuki et al., 2013). Mus musculus castaneus harbors formed clade A. This may be explained by range expan- four distinct mtDNA lineages, one of which (CAS-1) had sion through natural population dynamics, associated experienced human-associated geographic expansion with prehistoric human movements, or stowaway intro- from the predicted source area on the Indian subcontinent duction in the modern age. The numbers of differences to its eastern neighboring geographic areas via different are two to four sites among clade A and the degree of routes, resulting in two phyletic groups, CAS-1a from sequence divergence (0.2%–0.4%) is comparable to that in Yunnan and Japan and CAS-1b from a broad area of the CAS-1 clade of M. musculus (0.1%–0.2%), the diver- Southeast Asia and South China (Suzuki et al., 2013). sity of which is thought to have been created by prehis- The two Cytb sequences of M. musculus from Manipur toric human movement (Suzuki et al., 2013). In R. were integrated into the major subclade of CAS-1, further norvegicus, in contrast, our sequence of Cytb from indicating a close relationship with the haplotype from Manipur was identical to that of the laboratory strain SD Indonesia (CAS-1b) rather than that from Japan (CAS-1a; (Fig. 2). This suggests a modern-age stowaway introduc- Fig. 2). The expansion parameter τ for CAS-1b with the tion of this species to Manipur. A worldwide survey of R. three Manipur sequences and 17 from the databases nitidus and R. norvegicus is needed to address these ques- (Suzuki et al., 2013) was estimated to be 1.9 (see Results). tions more fully. Notably, the value is substantially lower than the τ value In summary, the mtDNA lineages of the murine species of 6.9 for the R. rattus SC from Manipur and other areas examined are suggested to have emerged in Northeast of Lineage II (n = 18, Fig. 2), indicating that the two com- India on different time scales, from the ancient (B. mensal murine rodents—the house mouse and black rat— manipulus, Niviventer sp.) to comparatively recent, from Manipur have different population expansion sce- including the modern age (M. musculus, R. nitidus, R. narios. Further studies of M. musculus will enhance our norvegicus, R. rattus SC). Certain groups of commensal understanding of the geographic importance of Northeast murine rodents appear to have been affected by human India with respect to prehistoric human movements. activities, as seen in the cases of R. norvegicus and M. Rattus norvegicus is currently found worldwide, while R. musculus, while others, such as R. nitidus and the R. nitidus and R. pyctoris have broad and enclosed geographic rattus SC, displayed bimodal patterns, with the presence distributions in Southeast Asia and the Himalayan range, of both indigenous lineages with long histories and newly respectively, with limited overlapping areas in Northeast introduced lineages, perhaps through human activities. India (Agrawal, 2000). Specifically, R. nitidus is indige- The non-commensal species of Berylmys and Niviventer nous to mainland Southeast Asia and occurs in South showed apparent differences from those of their geo- China (including Hainan Island), Vietnam, Laos, north- graphic neighbors in Southeast Asia. The present study ern Thailand, Burma, Bangladesh, Nepal, and provides preliminary information for assessing the bio- northern India; the species is also found on the islands of geographic significance of the region, in terms of whether Central Sulawesi, Luzon Island in the , Pulau it acted as a passage corridor or as a barrier for different Seram in the Molluccas, the Vogelkop Peninsula of the species. Regardless, it remains true that Northeast Province of Papua, and the islands, probably due to India is important geographically as well as for assess- human-mediated introductions (Aplin et al., 2003; Musser ment of prehistoric human activities. and Carleton, 2005). Despite its widespread distribu- tion, molecular information about R. nitidus is limited We thank Vivekananda S. Yaikhom, Ibemhal D. Asem, and (Pagès et al., 2010; Balakirev and Rozhnov, 2012). The Surchandra S. Thangjam of the Plant Breeding and Genetics Department, Central Agricultural University, Imphal, for their exact locations of the homeland and source area(s) for the assistance with PCR amplification and for valuable suggestions. extensive dispersal events have not yet been addressed by We are also grateful for extensive comments from Ian Smith and molecular phylogenetic studies. In the present study, anonymous reviewers. This study was supported by the Depart- Phylogeny of Manipur rats and mice 29 ment of Biotechnology (DBT) under DBT Research Associateship Y., Cosson, J. F., and Morand, S. (2011) Protocols for Field 2010, and in part by a grant-in-aid for Scientific Research (B) to and Laboratory Rodent Studies. Retrieved from CERoPath H. S. 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