Distinct genetic characteristics of Sri Lankan Rattus and Bandicota (Murinae, Rodentia) inferred from mitochondrial and nuclear markers

ABSTRACT

We examined genetic variation in black rats (the Rattus rattus complex) from Kandy District, Sri Lanka using mitochondrial cytochrome b (cytb, 1140 bp) and nuclear melanocortin 1 receptor (Mc1r, 954 bp) gene sequences together with database sequences. We confirmed the existence of two divergent mitochondrial lineages in Sri Lankan black rats, with genetic distance of 2.2% and estimated divergence time of 0.3 million years ago. Because one lineage is unique to the island and the other is closely related to R. rattus populations on the Indian subcontinent, two migration events of R. rattus from the subcontinent are inferred, one ancient and one recent. Mc1r analyses revealed 12 haplotypes among the Sri Lankan black rats. A median-joining network together with other available sequences separated the 12 haplotypes into two groups, one unique to the island and the other related to previously reported R. rattus sequences. Notably, most individuals possessed various combinations of both haplotype groups which had no association with the cytb clades. These results imply that old and new R. rattus lineages are now intermingled as a result of hybridization in Sri Lanka. Specimens of the lesser bandicoot rat (Bandicota bengalensis) collected from Sri Lanka (n = 24) were shown to have no genetic variability in the cytb sequence. Our results indicate that the two most abundant groups of commensal rats in Sri Lanka, black rats and lesser bandicoot rats, are the product of contrasting evolutionary histories on different timescales.

INTRODUCTION

The island nation of Sri Lanka harbors particularly high levels of species diversity and endemism among terrestrial mammals and, together with the Western Ghats of southwestern India with which it shares many faunistic elements, has been designated as one of the world’s biodiversity hotspots (Myers et al., 2000). Diversity is especially high among rats and mice of the subfamily Murinae, with 22 species having been recorded (Carleton and Musser, 2005; Bambaradeniya, 2006). However, this total includes a significant number of species that live as human commensals, and the status of these populations as either native or introduced is often ambiguous (Carleton and Musser, 2005; Bambaradeniya, 2006).

Two of the more ubiquitous and destructive of the Sri Lankan commensal rodents are black rats (members of the Rattus rattus Complex sensu Aplin et al., 2011) and bandicoot rats of the genus Bandicota. Both of these groups are thought to have undergone major range expansions in prehistoric to recent times, but both are also likely to have originated somewhere on the Indian subcontinent or surrounding region (Aplin et al., 2003; Carleton and Musser, 2005). Elucidation of their evolutionary history is a prerequisite for studies of mammalian biodiversity in Sri Lanka.

The phylogeny and phylogeography of black rats have attracted much recent attention (Pagès et al., 2010; Aplin et al., 2011) and these studies have provided a rich context for examining the affinities of Sri Lankan populations. In contrast, members of the genus Bandicota (usually called bandicoot rats) have been largely neglected in genetic studies other than as outgroups to the closely related Rattus (e.g., Pagès et al., 2010; Aplin et al., 2011). Members of both genera are known to be reservoirs for the pathogenic agents of major infectious diseases including hemorrhagic fever with renal syndrome (HFRS), hepatitis E, scrub typhus, rickettsial pox, leishmaniasis and leptospirosis (Meerburg et al., 2009; Aplin et al., 2011; Li et al., 2011), several of which have been reported in Sri Lanka (Vitarana et al., 1988; Gamage et al., 2011a, 2011b). Since these pathogens are often host species-specific (Bharti et al., 2003; Bi et al., 2008), a firm taxonomic and phylogenetic understanding of the rodent hosts is essential for understanding their evolutionary history, natural ecology and zoonotic behavior. To date, this foundation has been entirely lacking for infectious disease research in Sri Lanka.

In this study, we attempted to elucidate the phylogeographic histories and phylogenetic positions of R. rattus and Bandicota bengalensis from Sri Lanka using mitochondrial cytochrome b (cytb) gene sequences. In addition, for R. rattus, we used the nuclear gene marker melanocortin 1 receptor (Mc1r) to examine the hybridization history of genetically divergent populations within Sri Lanka. The results of our study provide new insights into the evolutionary history of Rattus and Bandicota in Sri Lanka.

MATERIALS AND METHODS

Sample collection

Samples were collected for epidemiological study of leptospirosis during 2009 and 2010 in rural areas of Yatinuwara and Udunuwara Divisional Secretariats (DS), Kandy District, Sri Lanka (Fig. 1) (Gamage et al., 2011a). Rats were collected in houses in Yatinuwara and Udunuwara DS, and bandicoot rats were collected from farms in Yatinuwara DS. We obtained lung tissue samples from a total of 21 black rats R. rattus (specimen code: R01-21) and 24 lesser bandicoot rats B. bengalensis (specimen code: B01-24). All of the R. rattus collected were agouti in dorsal coloration and no melanistic specimens were encountered. Samples were stored at –80℃ until laboratory analysis.

Fig. 1.

Sampling localities. Specimens of Rattus and Bandicota were collected from Yatinuwara and Udunuwara Divisional Secreatriats, Kandy District, Sri Lanka.

DNA isolation, amplification and sequencing

DNA was extracted from tissues using a DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. Two DNA fragments containing the N- and C-terminal halves of the mitochondrial cytb coding region (1140 bp) were amplified using the polymerase chain reaction (PCR) with the primer pairs L14115 (Yasuda et al., 2005) and H655A (5’-TGTGTAGTATGGGTGGAATGG-3’) and L497A (5’-CCTAGTAGAATGAATCTGAGG-3’) and H15300 (Yasuda et al., 2005), respectively. H655A and L497A were modified primers of H-15401 and L-15423 (Shinohara et al., 2004). Likewise, two DNA fragments containing the N- and C-terminal halves of the nuclear Mc1r coding region (954 bp) of R. rattus specimens were amplified with the primer pairs 5’Mc1r (–52) and 3’Mc1r (+504) and 5’Mc1r (+131) and 3’Mc1r (+1025), respectively (Shimada et al., 2009). Each PCR mix contained 0.5 U of Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with specific buffer, 0.5 mM dNTP mix, 0.4 μM of each primer (8 pmol/reaction), 2 mM MgSO₄ and 0.1–0.5 μg of template total genomic DNA in a final volume of 20 μl. The thermal cycling parameters for the PCR of cytb were as follows: 94℃ for 1 min and 35 cycles of 94℃ for 20 sec, 50℃ for 30 sec and 68℃ for 60 sec, followed by a final extension of 68℃ for 5 min. Those for the PCR of Mc1r were as described by Shimada et al. (2009). All PCR products were purified using MicroSpin^TM S-400 HR Columns (GE Healthcare) and were sequenced according to the manufacturer’s instructions using a Big Dye Terminator v3.1 Cycle Sequencing Kit (ABI) and an ABI 3100 automated sequencer with two PCR primers. The sequence data obtained in this study were deposited in DDBJ/EMBL/GenBank with accession nos. AB762700-AB762765.

Phylogenetic analysis

Phylogenetic analysis of the full cytb sequence dataset (Table 1) was conducted using four methods: neighbor joining (NJ) (Saitou and Nei, 1987), maximum parsimony (MP) (Swofford and Olsen, 1990), maximum likelihood (ML) (Felsenstein, 1981) and Bayesian inference (BI) (Huelsenbeck et al., 2001). For the ML method, the best-fit nucleotide-substitution model and parameters were determined using the AIC criterion (Posada and Buckley, 2004), as implemented in MEGA 5.1 (Tamura et al., 2011). The NJ and MP methods were implemented using MEGA 5.1, and the ML method was implemented using PHYML 3.0 (Guindon et al., 2005). Maximum composite likelihood distance and GTR + G distance were used for NJ and ML analyses, respectively. Bootstrap values (Felsenstein, 1985) were estimated for the NJ, MP and ML trees by resampling 10,000 iterations. For BI, the best-fit nucleotide-substitution model and parameters were determined using the AIC criterion as implemented in MrModeltest version 2.3 (Nylander, 2004), and the HKY + I + G model was selected. Trees were generated by the Metropolis-coupled Markov-chain Monte Carlo algorithm using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Each run consisted of four simultaneous chains, one cold and three incrementally heated, starting from a random tree. Chains were run for 10,000,000 generations and sampled once every 100 generations. The first 25,000 trees (25%) were discarded as burn-in. The reliabilities for inferred nodes were assessed with posterior probabilities. Genetic distance and diversity were calculated using MEGA 5.1. Divergence time for lineages ‘Ia’ and ‘Ib’ in R. rattus was estimated with a relaxed molecular-clock model (Drummond et al., 2006), using the software BEAST v1.7.5 (Drummond and Rambaut, 2007) with HKY + G + I as the model of nucleotide substitution and 50,000,000 MCMC iterations. Both Yule and coalescent priors were used as tree models because it was uncertain whether or not lineage “Ib” represents an independently evolving species. We used 2.78–3.53 million years ago and 440–550 kya (thousand years ago) as priors for the divergences of R. norvegicus-R. rattus and R. rattus-R. tanezumi, respectively (Robins et al., 2008; Aplin et al., 2011).

Table 1. Specimen list of cytochrome b gene sequences from DDBJ/EMBL/GenBank

species or lineage	country	accession	reference
RrC LI	Japan	AB211039	Chinen et al., 2005
	India	HM217367	Pagès et al., 2010
	India	GQ891573	Tollenaere et al., 2010
	India	GQ891572	Tollenaere et al., 2010
	India	GQ891569	Tollenaere et al., 2010
	India	GQ891570	Tollenaere et al., 2010
	India	GQ891571	Tollenaere et al., 2010
	Indonesia	AB033702	Suzuki et al., 2000
	Madagascar	HM217368	Pagès et al., 2010
	New Zealand	EU273707	Robins et al., 2008
	Oman	HM217366	Pagès et al., 2010
	Oman	GQ891576	Tollenaere et al., 2010
	Oman	GQ891575	Tollenaere et al., 2010
	Oman	GQ891574	Tollenaere et al., 2010
	Oman	GQ891577	Tollenaere et al., 2010
	Oman	GQ891578	Tollenaere et al., 2010
	South Africa	HQ157803	Bastos et al., 2011
	Tanzania	HM217365	Pagès et al., 2010
	Yemen	GQ891581	Tollenaere et al., 2010
	Yemen	GQ891582	Tollenaere et al., 2010
RrC LII		AB211040	Chinen et al., 2005
		HM217426	Pagès et al., 2010
RrC LIII		JN675599	Aplin et al., 2011
		JN675600	Aplin et al., 2011
		JN675601	Aplin et al., 2011
RrC LIV		HM217389	Pagès et al., 2010
		HM217372	Pagès et al., 2010
RrC LV		HM217421	Pagès et al., 2010
		HM217443	Pagès et al., 2010
		HM217454	Pagès et al., 2010
RrC LVI		HM217391	Pagès et al., 2010
		EF186492	Robins et al., 2007
		EF186514	Robins et al., 2007
R. argentiventer		AB033701	Suzuki et al., 2000
		HM217362	Pagès et al., 2010
		HM217364	Pagès et al., 2010
R. hoffmanni		EF186442	Robins et al., 2007
		EF186443	Robins et al., 2007
R. andamanensis		HM217396	Pagès et al., 2010
		HM217403	Pagès et al., 2010
R. exulans		HM217437	Pagès et al., 2010
		HM217470	Pagès et al., 2010
		HM217472	Pagès et al., 2010
R. norvegicus		AB033713	Suzuki et al., 2000
		HM217370	Pagès et al., 2010
		HM217429	Pagès et al., 2010
R. nitidus		HM217478	Pagès et al., 2010
		HM217474	Pagès et al., 2010
		HM217479	Pagès et al., 2010
B. indica		HM217390	Pagès et al., 2010
		HM217408	Pagès et al., 2010
		HM217425	Pagès et al., 2010
		HM217376	Pagès et al., 2010
		HM217378	Pagès et al., 2010
		HM217380	Pagès et al., 2010
		HM217386	Pagès et al., 2010
		HM217435	Pagès et al., 2010
		HM217447	Pagès et al., 2010
		HM217469	Pagès et al., 2010
		HM217476	Pagès et al., 2010
B. bengalensis		AM408336	Michaux et al., 2007
B. savilei		HM217427	Pagès et al., 2010
		HM217385	Pagès et al., 2010
		HM217387	Pagès et al., 2010
		HM217455	Pagès et al., 2010

Sequences of the Mc1r coding region from R. rattus were aligned and haplotypes were inferred using PHASE v 2.1 (Stephens et al., 2001; Stephens and Donnelly, 2003; Stephens and Scheet, 2005). The inferred haplotypes and 11 previously identified haplotypes from R. tanezumi (haplotypes 1–3) and R. rattus (haplotypes 4–11) (Kambe et al., 2011) were used to construct a median-joining (MJ) network (Bandelt et al., 1999) as implemented in Network^® version 4.6.0.0. (Fluxus Technology, 2010).

RESULTS

Sequence diversity and affinity of black rats

All phylogenetic analyses of the cytb gene sequences showed a consistent topology. An NJ tree with bootstrap supports calculated by NJ, MP and ML methods and posterior probabilities under BI is illustrated in Fig. 2. Lineage names within the R. rattus Complex (RrC) follow the proposed nomenclature of Aplin et al. (2011). All Sri Lankan black rats associate with RrC Lineage I. However, two sub-lineages are indicated, with strong support values for reciprocal monophyly; these are labeled as ‘RrC LIa’ and ‘RrC LIb’ (Fig. 2). Sub-lineage RrC LIa includes two rats (R09 and R15) from Sri Lanka and examples of R. rattus drawn from numerous localities worldwide. Sequences from R09 and R15 are particularly close to examples from Oman and India (Fig. 2). Sub-lineage RrC LIb consists exclusively of the remaining 19 Sri Lankan black rat sequences. Nucleotide diversity [π] within sub-lineages RrC LIa and RrC LIb is 0.66% (95% CI: 0.40–0.92) and 0.24% (95% CI: 0.10–0.38%), respectively. Average genetic distance between sub-lineages RrC LIa and RrC LIb is 2.21% (95% CI: 1.41–3.01%). As found in previous analyses (Pagès et al., 2010; Aplin et al., 2011), our phylogenetic analyses indicate that the sister lineage of the combined RrC LIa + RrC LIb is RrC LII (Fig. 2). We follow Chinen et al. (2005) in associating RrCII with the taxon R. tanezumi (Carleton and Musser, 2005). The divergence times between sub-lineages RrC LIa and RrC LIb were estimated to be 285 kya (95% CI: 152–415 kya) and 314 kya (95% CI: 220–404 kya) by Yule and coalescent priors, respectively.

Fig. 2.

A neighbor joining tree based on cytb gene sequences from various Rattus and Bandicota species. Numbers at nodes are support values for the respective clades determined by the methods of NJ/MP/ML/BI. The shaded regions in the phylogenetic tree represent individuals collected in Sri Lanka. Major clades within the Rattus rattus Complex are enumerated in accordance with the suggestion of Aplin et al. (2011).

Sequences of Mc1r from Sri Lankan R. rattus yielded 10 haplotypes (haplotypes a–j; Table 2). Genotypes of all individuals from Sri Lanka were homozygous for SNP 280G, which is consistent with their observed agouti fur color (Kambe et al., 2011). Nine of the 21 substitution sites were unique to rats from Sri Lanka, and no substitutions were shared by Sri Lankan rats and individuals of R. tanezumi as reported by Kambe et al. (2011). In contrast, three substitutions (C273T, A341G and C894T) were shared by Sri Lankan rats and R. rattus reported by Kambe et al. (2011) from Pakistan and Ogasawara Is., Japan.

Table 2. Summary of variable sites of 13 observed Mc1r gene sequences found in 21 black rats from Sri Lanka and 11 previously identified haplotypes (Kambe et al., 2011) together with 10 newly deduced haplotypes (a–j) in this study

sample ID	haplotype	site number																					genotype
		20	33	93	138	199	273	280	328	336	341	387	426	450	496	577	642	786	843	894	918	931
R12		C	G	A	C	C	C	G	G	G	A	A/G	A/C	G	G	G	G	A	C	C	C	G	(9, a)
R13		C	G	A/T	C	C	C	G	G	G	A	G	C	G	G	G	G	A	C	C/T	C	G	(9, i)
R14		C	G	A	C	C	C	G	G	G	A	G	C	G	G	G	G	A	C	C/T	C	G	(8, 9)
R19		C	G	A	C	C	C	G	G	G	A	G	C	G	G	G	G	A	C	C	C	G	(9, 9)
R16		C	G	A	C	C	C	G	G	G	A	A/G	C	G	G	G	A/G	A	C	C	C	G	(9, c)
R11/R21		C	G	A	C	C	C/T	G	G	G	A/G	A/G	C	G	G	G	G	A	C	C	C	G	(b, f)
R01/R03/R05/R17		C	G	A	C	C	C/T	G	G	G	A/G	G	C	G	G	G	G	A	C	C	C	G	(9, f)
R02/R18/R20		C	G	A	C	C	C/T	G	G	G	A/G	G	C	G	G	A/G	G	A	C	C	C	G	(e, f)
R04/R07/R08		C	G	A	C/T	C	C	G	G	G	A	A/G	C	G	G	G	G	A	C	C	C	G	(9, g)
R06		C	G	A	C/T	C/T	C	G	G	G	A	A/G	C	G	G	G	G	A	C	C	C	G	(9, h)
R10		C	G	A	C	C	C	G	G	G	A	A/G	C	G	A/G	G	G	A	C	C	C	G	(9, d)
R15		C	G	A	C	C	C	G	G	G	A	G	C	G	G	A/G	G	A	C	C	C	G	(9, e)
R09		C	A	A	C	C	C	G	G	G	A	A	C	G	G	G	G	A	C	C	C	G	(j, j)
	1	C	G	A	C	C	C	G	A	G	A	G	C	G	G	G	G	G	G	C	A	G
	2	T	G	A	C	C	C	G	G	G	A	G	C	G	G	G	G	G	G	C	A	G
	3	T	G	A	C	C	C	G	G	G	A	G	C	A	G	G	G	G	G	C	A	G
	4	C	G	A	C	C	C	G	G	A	A	G	C	G	G	G	G	G	C	C	C	G
	5	C	G	A	C	C	C	A	G	A	A	G	C	G	G	G	G	G	C	C	C	G
	6	C	G	A	C	C	C	G	G	A	A	G	C	G	G	G	G	A	C	C	C	G
	7	C	G	A	C	C	C	G	G	A	A	G	C	G	G	G	G	A	C	C	C	A
	8	C	G	A	C	C	C	G	G	G	A	G	C	G	G	G	G	A	C	T	C	G
	9	C	G	A	C	C	C	G	G	G	A	G	C	G	G	G	G	A	C	C	C	G
	10	C	G	A	C	C	C	G	G	G	A	G	C	G	G	G	G	A	C	C	C	A
	11	C	G	A	C	C	T	G	G	G	G	G	C	G	G	G	G	A	C	T	C	G
	a	C	G	A	C	C	C	G	G	G	A	A	A	G	G	G	G	A	C	C	C	G
	b	C	G	A	C	C	C	G	G	G	A	A	C	G	G	G	G	A	C	C	C	G
	c	C	G	A	C	C	C	G	G	G	A	A	C	G	G	G	A	A	C	C	C	G
	d	C	G	A	C	C	C	G	G	G	A	A	C	G	A	G	G	A	C	C	C	G
	e	C	G	A	C	C	C	G	G	G	A	G	C	G	G	A	G	A	C	C	C	G
	f	C	G	A	C	C	T	G	G	G	G	G	C	G	G	G	G	A	C	C	C	G
	g	C	G	A	T	C	C	G	G	G	A	A	C	G	G	G	G	A	C	C	C	G
	h	C	G	A	T	T	C	G	G	G	A	A	C	G	G	G	G	A	C	C	C	G
	i	C	G	T	C	C	C	G	G	G	A	G	C	G	G	G	G	A	C	T	C	G
	j	C	A	A	C	C	C	G	G	G	A	A	C	G	G	G	G	A	C	C	C	G

A total of 13 genotypes were identified (Table 2). Ten of the 13 genotypes contained haplotype 9, which is also common in Pakistan (Kambe et al., 2011). Haplotype 8, which was found in Pakistan and Ogasawara Is., Japan (Kambe et al., 2011), was also found in rat R14. Other haplotypes (haplotypes 1–7, 10 and 11) detected by Kambe et al. (2011) were not found in this study.

The MJ network for Mc1r gene sequences contains three clusters, labeled “A”, “B” and “C” (Fig. 3). Cluster “A” consists of haplotypes 1–3 from R. tanezumi. Cluster “B” consists of haplotypes 4–11 of Kambe et al. (2011) from R. rattus and three haplotypes inferred in this study. Cluster “C” consists of the other seven haplotypes inferred in this study. Haplotype 9 of Kambe et al. (2011) and haplotype b of this study differ by a single substitution and they occupy central positions of clusters “B” and “C”, respectively. Sri Lankan rats R09 and R15, both members of cytb sub-lineage RrC LIa, each possess unique genotypes. The genotype of R15 consists of haplotypes 9 and e, both of cluster “B”, while the genotype of R09 was homozygous for haplotype j of cluster “C” (Fig. 3 and Table 2). None of the haplotypes inferred for Sri Lankan rats were closely related to the haplotypes reported from R. tanezumi.

Fig. 3.

Median-joining network of Mc1r gene sequences. Open squares and triangles represent haplotypes reported by Kambe et al. (2011) from R. r. rattus (haplotypes 4–11) and R. tanezumi (haplotypes 1–3), respectively. Open circles represent haplotypes determined in this study (haplotypes a–j). Each branch between nodes represents a single nucleotide substitution. Solid circles represent unobserved intermediate haplotypes. Three clusters detected in the network are labeled A, B and C.

Sequence diversity and affinity of bandicoot rats

All 24 specimens of B. bengalensis collected in this study shared one cytb haplotype that falls outside clusters of sequences previously reported for each of B. indica and B. savilei (Table 1 and Fig. 2). One sequence previously reported as B. bengalensis by Michaux et al. (2007) falls within the B. indica cluster. Phylogenetic study based on representative sequences preserves the inferred paraphyly of B. bengalensis, with moderate or high support values (Fig. 2), and also indicates that B. savilei diverged first within the genus (Fig. 2). Whether or not B. bengalensis is genuinely paraphyletic for cytb with respect to B. indica depends on the veracity of the taxonomic identification of the specimen represented by sequence AM408336. We suspect that it may be a misidentified specimen of B. indica.

DISCUSSION

Phylogenetic positions and evolutionary histories of Sri Lankan black rats

Recent work on the genetic composition and affinity of black rats (Pagès et al., 2010; Aplin et al., 2011) has revealed a total of four deeply divergent mitochondrial lineages with inferred allopatric natural ranges centered on the Himalayan foothills (RrC LIII), southern India (RrC LI), upland Indochina and southern China (RrC LII), and lowland Indochina (possibly including the larger islands of the Malay Archipelago; RrC LIV). Two of these mtDNA lineages appear to correspond to karyotypically distinct populations that are sometimes treated as distinct species (Carleton and Musser, 2005): RrC LI with 2n = 38 R. rattus and RrC LII with 2n = 42 R. tanezumi. Populations of RrC LIII and RrC LIV are less well studied karyotypically but may be undifferentiated from RrC LII (Aplin et al., 2011). Of the four black rat mtDNA lineages, RrC LIV is the most deeply divergent, with >6% cytb sequence divergence from LI–III, which forms a monophyletic clade (Aplin et al., 2011). In contrast, the cytb sequence divergence between RrC LI R. rattus and RrC LII R. tanezumi is 3.8%, with an estimated divergence time of around 0.5 million years ago. Hybridization and introgression among the karyotypically distinct populations is inferred from intermediate karyotypes and patterns of genetic admixture for various localities in India, the western Pacific and Japan (Yosida, 1980; Chinen et al., 2005) and the USA (Lack et al., 2012; Conroy et al., 2013).

Black rats from Kandy District in the central highlands of Sri Lanka have been distinguished by some previous workers as an endemic subspecies, R. r. kandianus (Hinton, 1918; Ellerman, 1941). Yosida (1980) demonstrated that rats of this population possess a unique karyotype of 2n = 40, which he interpreted as a transitional state between the ancestral 2n = 42 of R. tanezumi and the 2n = 38 karyotype that occurs on the Indian mainland and has a worldwide distribution as a result of ship-borne dispersal out of Europe (Yosida, 1980). This idea was also supported by phylogenetic relationships inferred from isozyme profiles (Baverstock et al., 1983). Moreover, the uniqueness of Sri Lankan black rats was suggested by restriction enzyme analysis of mitochondrial DNA (mtDNA) (Brown and Simpson, 1981). The 2n = 38 karyotype was reported from black rat populations in the lowlands of Sri Lanka (Yosida et al., 1974, 1979).

Two Sri Lankan black rats with known 2n = 40 karyotypes were independently sequenced for cytb by Robins et al. (2007) and Aplin et al. (2011). Both yielded a single cytb haplotype that belongs unambiguously to RrC LIV (Aplin et al., 2011). This finding led Aplin et al. (2011) to postulate a prehistoric or early historic introduction to Sri Lanka of black rats from an Indochinese or Indonesian source. Despite this earlier result, we failed to detect any RrC LIV cytb haplotypes among our sample of black rats from Kandy District of Sri Lanka. Instead, our rats yielded cytb sequences of two different lineages. One of these lineages, found in a minority of rats, represents the globally distributed RrC LI of Aplin et al. (2011). The other, more frequently encountered, lineage is hitherto unreported and appears to be unique to Sri Lanka. This new lineage is phylogenetically closest to RrC LI, yet the two show reciprocal monophyly. The new Sri Lankan lineage is herein designated RrC LIb to distinguish it from the more widespread RrC LI, which we relabel as RrC LIa. Genetic distinction between two groups of Sri Lankan rats is also observed in the nuclear sequences of Mc1r, for which MJ networks show three discrete clusters of haplotypes having strong albeit imperfect association, with individual cytb affinities for RrC Lineages Ia, Ib and II. The Mc1r gene has not been investigated among members of RrC LIV.

Our results suggest that R. rattus is a part of the native mammalian fauna of Sri Lanka. The divergence time estimate of 285–314 kya between the uniquely Sri Lankan sub-lineage RrC LIb and the Indian mainland sub-lineage RrC LIa falls within the prolonged period of globally low sea levels associated with Marine Isotope Stage 8, spanning the period 245–300 kya (Ehlers and Gibbard, 2007). Black rats thus appear to have invaded Sri Lanka from southern India sometime during the upper Middle Pleistocene, probably taking advantage of one or more of the numerous land-bridge connections between Sri Lanka and the continental mainland (Bossuyt et al., 2004). Any natural dispersal of black rats between the two areas during subsequent glacial episodes must have been insufficient to counter the genetic consequences of geographic isolation on the two gene pools.

Our results also point to a second invasion of black rats during recent times, resulting in interbreeding and genetic introgression. This is indicated by the detection of mtDNA sub-lineage RrC LIa in rats from Kandy District, by the presence of Mc1r haplotypes associated with this sub-lineage, and possibly by the pattern of karyotypic diversity reported previously by Yosida et al. (1974, 1979). Documentation of this second phase of invasion is not unexpected given the near-global dispersal of black rats with sub-lineage RrC LIa that has occurred out of both European and Indian ports (Tollenaere et al., 2010; Aplin et al., 2011; Bastos et al., 2011). Our hypothesis of a dual invasion of Sri Lanka by black rats parallels a recent interpretation of elephant genetic diversity on the island (Vidya et al., 2009).

The significance of the 2n = 40 karyotype reported previously from Sri Lanka remains uncertain. Because we found the indigenous RrC sub-lineage Ib to be dominant in Kandy District, where the 2n = 40 karyotype occurs at high frequency (Yosida et al., 1974, 1979), we suspect that this karyotype is representative of the indigenous population of black rats. However, there are two grounds for caution. First, since we did not determine the karyotypes of rats for our study, we have no direct evidence to link sub-lineage RrC LIb with the 2n = 40 karyotype, and second, as noted above, the only individuals with confirmed 2n = 40 karyotypes have yielded mtDNA of RrC IV (Robins et al., 2007; Aplin et al., 2011). This contradictory evidence can be reconciled by postulating localized introgression into R. r. kandianus of mtDNA from a second recently invasive population of black rats, in this instance carrying mtDNA of RrC LIV (Aplin et al., 2011) – a hypothesis that can be tested by further genetic sampling of black rats in other localities within Sri Lanka and by determination of karyotypes as part of the research protocol.

The mis-match we observed between the cytb sub-lineages and the Mc1r haplotype group affinities of individual rats from Kandy District suggests that gene flow is occurring between the indigenous black rat population (‘R. r. kandianus’) and the more recent immigrants (R. r. rattus). This is consistent with the results of laboratory crosses performed by Yosida (1980), who showed that F1 and F2 hybrids were readily obtained between R. r. kandianus and R. r. rattus, albeit with reduced litter size at F2 (Yosida, 1980). An alternative interpretation is that the shared haplotypes are part of an ancestral gene pool, but this seems unlikely given the peripheral location of the shared haplotypes on the network and the otherwise highly structured nature of the MJ network. Taking into consideration both the preliminary genetic data and the results of previous crossbreeding experiments, we consider it very likely that gene flow is occurring between R. r. kandianus and R. r. rattus. Further studies using a wider suite of genetic markers will be needed to clarify the extent and evolutionary outcome of gene flow.

Phylogeny and diversity of the Sri Lankan Bandicota rats

Although B. bengalensis is clearly an invasive species in parts of Southeast Asia, Sri Lanka is thought to be within its natural distribution (Carleton and Musser, 2005). Our sample of 24 individuals of B. bengalensis all shared a single cytb haplotype, which is consistent with B. bengalensis being a recently introduced species in Sri Lanka. However, since B. bengalensis is a colonial species and all 24 individuals were collected at the same locality, the result may also be an artifact of sampling. Further sampling across more localities will be needed to understand the history of B. bengalensis populations in Sri Lanka.

Zoonoses of Sri Lanka

This study was initially sparked by a concern with the evolutionary history and ecology of zoonoses in Sri Lanka, where hantaviruses and leptospirosis have been reported as causative agents of disease (Vitarana et al., 1988; Gamage et al., 2011a, 2011b). Both of these pathogens, as well as many others, are likely transmitted by species of Rattus and Bandicota (Meerburg et al., 2009).

Our study has revealed that Sri Lankan black rats are of dual origin, with an indigenous population (R. r. kandianus) that probably entered Sri Lanka toward the end of the Middle Pleistocene and a more recent invasive population (R. r. rattus) that probably arrived within the historical period. Although interbreeding and genetic introgression are clearly occurring, our results suggest that a complete genetic admixture has yet to occur, at least in Kandy District where our study was conducted. For zoonotic disease studies, the insular populations thus provide good opportunities to assess several significant research topics, notably: 1) the extent of genetic divergence among the various major zoonoses harbored by indigenous vs. invasive black rats; and 2) how genetic differences between the two black rat populations affect the susceptibility to and transmission of any given disease.

B. indica is known as a host of Thailand virus (THAIV), a member of the hantaviruses, which are the causative agent of HFRS (Schmaljohn and Hjelle, 1997). In Sri Lanka, human cases of THAIV-related virus infections have been reported, but the animal host species has not been identified (Gamage et al., 2011b). Hantaviruses are known to be host-specific and to have coevolved with their host species (Plyusnin et al., 1996). The limited genetic studies carried out to date on the genus Bandicota indicate that all three recognized species are closely related (Fig. 2; also Pagès et al., 2010; Aplin et al., 2011). Either of the two species present in Sri Lanka, namely B. bengalensis and B. indica, and possibly both, may carry the pathogenic agent of THAIV-related virus infections. Further work is underway to explore these unanswered possibilities.

ACKNOWLEDGMENTS

We thank Drs. Angela Frost and Ken Aplin for their help with species identifications and their useful suggestions that improved the manuscript. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Heiwa Nakajima Foundation and the Global COE Program (Establishment of International Collaboration Center for Zoonosis Control).

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