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The Black Rat (Rattus rattus) spread out of Asia to become one of the world’s worst agricultural and urban pests, and a reservoir or vector of numerous zoonotic diseases, including the devastating plague. Despite the global scale and inestimable cost of their impacts on both human livelihoods and natural ecosystems, little is known of the global genetic diversity of Black Rats, the timing and directions of their historical dispersals, and the risks associated with contemporary movements. We surveyed mitochondrial DNA of Black Rats collected across their global range as a first step towards obtaining an historical genetic perspective on this socioeconomically important group of rodents. We found a strong phylogeographic pattern with well-differentiated lineages of Black Rats native to South Asia, the Himalayan region, southern Indochina, and northern Indochina to East Asia, and a diversification that probably commenced in the early Middle Pleistocene. We also identified two other currently recognised species of Rattus as potential derivatives of a paraphyletic R. rattus. Three of the four phylogenetic lineage units within R. rattus show clear genetic signatures of major population expansion in prehistoric times, and the distribution of particular haplogroups mirrors archaeologically and historically documented patterns of human dispersal and trade. Commensalism clearly arose multiple times in R. rattus and in widely separated geographic regions, and this may account for apparent regionalism in their associated pathogens. Our findings represent an important step towards deeper understanding the complex and influential relationship that has developed between Black Rats and humans, and invite a thorough re-examination of host-pathogen associations among Black Rats. © 2011 Aplin et al.
Sample collecting by KPA was carried out under CSIRO AEC Permit No: 00/01 – 28 titled “Taxonomic studies of agricultural pest rodents and their relatives in Southeast Asia”. All other samples were sourced from existing tissue collections associated with museum collections. Our sampling of Black Rats focussed on the Asian region which is widely regarded as the place of origin of both the genus Rattus and of Black Rats specifically [8]. However, we also attempted to include samples from all continents, and from major island groups (Table S2). In Asia, we sampled populations in rural contexts and in remnant forest habitats rather than in major towns to maximize our chances of sampling native lineages or those derived from prehistoric introductions. Populations in larger towns and ports are more likely to be of mixed origin due to introductions in modern times. Sampling coverage in Southeast Asia includes one significant gap spanning the Malay Peninsula and it is also sparse for the larger Sundaic Islands (Sumatra, Borneo). It is similarly sparse for the Indian subcontinent but adequate for southern India where chromosomal diversity is greatest [8]. Our sampling of Europe is also very incomplete. However, because of the strong historical evidence for dispersal of European ‘ship rats’ during the past 500 years or less [19], [38], we anticipated that our sampling in Africa, the Americas and Australia would be representative of diversity within the secondary ‘source area’ of Europe. We included samples of a variety of other species of Rattus including some that are considered to be close relatives of R. rattus [11]. For these potentially related species, we attempted to sample multiple individuals from across the species’ entire geographic range so as to establish a benchmark for typical levels of phylogeographic diversity within Rattus. The mitochondrial cytochrome b gene (cyt b) was selected for analysis because: 1) mitochondrial DNA has proven utility for revealing phylogeographic structure within mammalian taxa [70] as well as dispersal histories, including those associated with human translocation [71]; 2) a prior study of Japanese Black Rats [12] found an appropriate level of variation in cyt b between two karyomorphs; and 3) we have access to a large cyt b dataset for Rattus and related murines of the Tribe Rattini [25]. The majority of sequences were determined from liver samples obtained from modern specimens. A small number of sequences were obtained from skin and hair samples removed from museum vouchers (see below). The following primer combinations were used for particular DNA amplifications: A) mcytb1: CCA TCG TTG TAA TTC AAC TAT AG, mcytbL400: CAT GAG GAC AAA TAT CAT TCT GAG G, mcytbHb: GAA TGG GAG AAT GAA GTG GAA TGC G, mcytb4: CTT TGG CTT ACA AGA CCA AGG TAA; B) {“type”:”entrez-nucleotide”,”attrs”:{“text”:”L14724″,”term_id”:”402705″,”term_text”:”L14724″}}L14724: TGA YAT GAA AAA YCA TCG TTG, {“type”:”entrez-nucleotide”,”attrs”:{“text”:”H15915″,”term_id”:”880735″,”term_text”:”H15915″}}H15915: CAT TTC AGG TTT ACA AGA C; C) {“type”:”entrez-nucleotide”,”attrs”:{“text”:”L14724″,”term_id”:”402705″,”term_text”:”L14724″}}L14724: 5′ CGA AGC TTG ATA TGA AAA ACC ATC GTT G 3′, HRa1025: 5′ GGG TGT TCT ACT GGT TGG CCT CC 3′. The cyt b gene was sequenced for more than 200 individuals (Table S1), either in entirety or in part, depending on the quality of available tissues. A small number of sequences were obtained from GenBank. Most sequences included a common alignment of 945 bp and this was used for the primary analysis of 153 sequences providing unique combinations of haplotype and locality. A subsequent round of analyses used a shorter alignment of 596 bp to accommodate a further 14 sequences. Details of collecting localities are given in Table S3, References S1. We designed several sets of primers to amplify overlapping smaller fragments of the cyt b gene from museum vouchers of Rattus. Primers were designed from an alignment of six Rattus species and human cyt b – where feasible, primers included nucleotides that mismatched human sequences at the 3′ end of each primer to exclude amplification of human contamination. The six reference sequences are: Rattus praetor {“type”:”entrez-nucleotide”,”attrs”:{“text”:”DQ191487″,”term_id”:”77632550″}}DQ191487; Rattus tanezumi {“type”:”entrez-nucleotide”,”attrs”:{“text”:”DQ191488″,”term_id”:”77632552″}}DQ191488; Rattus exulans {“type”:”entrez-nucleotide”,”attrs”:{“text”:”DQ191486″,”term_id”:”77632548″}}DQ191486; Rattus everetti {“type”:”entrez-nucleotide”,”attrs”:{“text”:”DQ191485″,”term_id”:”77632546″}}DQ191485; Rattus rattus {“type”:”entrez-nucleotide”,”attrs”:{“text”:”AB033702″,”term_id”:”6139024″}}AB033702; and Rattus norvegicus {“type”:”entrez-nucleotide”,”attrs”:{“text”:”AB033713″,”term_id”:”6088103″}}AB033713. Predicted PCR product lengths were (including primers): 235 bp for RattuscytbF2 5′ TCA TCA GTT ACC CAC ATC TGC 3′ and RattuscytbR2 3′-ACC CTA GTC GAA TGA ATC TGA GG-5′; and 306 bp for RattuscytbF1 5′ATC ACA CCC TCT ACT CAA AA 3′ and RattuscytbR1 5′CTA ATY CGA TAC TTA CAT GCC 3′. DNA was extracted from small (1–2 mm2) pieces of dried skin using Qiagen DNeasy tissue kit as per the manufacturer’s instructions. Control extracts and PCR negative controls were included. We used Invitrogen’s Platinum Taq Hi Fidelity (MgSO4, buffer and enzyme) employing 50 cycles of amplification for PCR. Products of expected size were observed on agarose gels in the sample lanes, all control lanes were clean. PCR products were cleaned using magnetic cleanup system (Agencourt, Ampure) and sequenced using the same primers as for the PCR using ABI Big Dye 3.1 chemistry. The majority of sequences were determined from liver samples obtained from modern specimens. A small number of sequences were obtained from skin and hair samples removed from museum vouchers (see below). The following primer combinations were used for particular DNA amplifications: A) mcytb1: CCA TCG TTG TAA TTC AAC TAT AG, mcytbL400: CAT GAG GAC AAA TAT CAT TCT GAG G, mcytbHb: GAA TGG GAG AAT GAA GTG GAA TGC G, mcytb4: CTT TGG CTT ACA AGA CCA AGG TAA; B) {“type”:”entrez-nucleotide”,”attrs”:{“text”:”L14724″,”term_id”:”402705″,”term_text”:”L14724″}}L14724: TGA YAT GAA AAA YCA TCG TTG, {“type”:”entrez-nucleotide”,”attrs”:{“text”:”H15915″,”term_id”:”880735″,”term_text”:”H15915″}}H15915: CAT TTC AGG TTT ACA AGA C; C) {“type”:”entrez-nucleotide”,”attrs”:{“text”:”L14724″,”term_id”:”402705″,”term_text”:”L14724″}}L14724: 5′ CGA AGC TTG ATA TGA AAA ACC ATC GTT G 3′, HRa1025: 5′ GGG TGT TCT ACT GGT TGG CCT CC 3′. The cyt b gene was sequenced for more than 200 individuals (Table S1), either in entirety or in part, depending on the quality of available tissues. A small number of sequences were obtained from GenBank. Most sequences included a common alignment of 945 bp and this was used for the primary analysis of 153 sequences providing unique combinations of haplotype and locality. A subsequent round of analyses used a shorter alignment of 596 bp to accommodate a further 14 sequences. Details of collecting localities are given in Table S3, References S1. We designed several sets of primers to amplify overlapping smaller fragments of the cyt b gene from museum vouchers of Rattus. Primers were designed from an alignment of six Rattus species and human cyt b – where feasible, primers included nucleotides that mismatched human sequences at the 3′ end of each primer to exclude amplification of human contamination. The six reference sequences are: Rattus praetor {“type”:”entrez-nucleotide”,”attrs”:{“text”:”DQ191487″,”term_id”:”77632550″}}DQ191487; Rattus tanezumi {“type”:”entrez-nucleotide”,”attrs”:{“text”:”DQ191488″,”term_id”:”77632552″}}DQ191488; Rattus exulans {“type”:”entrez-nucleotide”,”attrs”:{“text”:”DQ191486″,”term_id”:”77632548″}}DQ191486; Rattus everetti {“type”:”entrez-nucleotide”,”attrs”:{“text”:”DQ191485″,”term_id”:”77632546″}}DQ191485; Rattus rattus {“type”:”entrez-nucleotide”,”attrs”:{“text”:”AB033702″,”term_id”:”6139024″}}AB033702; and Rattus norvegicus {“type”:”entrez-nucleotide”,”attrs”:{“text”:”AB033713″,”term_id”:”6088103″}}AB033713. Predicted PCR product lengths were (including primers): 235 bp for RattuscytbF2 5′ TCA TCA GTT ACC CAC ATC TGC 3′ and RattuscytbR2 3′-ACC CTA GTC GAA TGA ATC TGA GG-5′; and 306 bp for RattuscytbF1 5′ATC ACA CCC TCT ACT CAA AA 3′ and RattuscytbR1 5′CTA ATY CGA TAC TTA CAT GCC 3′. DNA was extracted from very small (1–2 mm2) pieces of dried skin using the Qiagen DNeasy tissue kit as per the manufacturer’s instructions. Control extracts and PCR negative controls were included. We used Invitrogen’s Platinum Taq Hi Fidelity (MgSO4, buffer and enzyme) employing 50 cycles of amplification for PCR. Products of expected size were observed on agarose gels in the sample lanes, all control lanes were clean. PCR products were cleaned using magnetic cleanup system (Agencourt, Ampure) and sequenced using the same primers as for the PCR using ABI Big Dye 3.1 chemistry. Authenticity of cyt b sequences obtained from museum vouchers was controlled and assessed using a number of standard ancient DNA criteria, including conducting all pre-PCR work (DNA extraction and PCR set-up) in a physically remote ancient DNA laboratory employing positive HEPA-filtered air pressure, bleach and UV decontamination and full PPE; including negative extraction controls and PCR negative controls alongside all sample extractions and PCR amplifications; intra-laboratory replication of all sequences from independent PCRs; and visual assessment of nucleotide substitutions to ensure they conform with molecular evolutionary expectations (ts>tv, 3rd codon>1st codon>2nd codon substitutions, synonymous>non-synonymous substitutions, absence of intra-sequence stop codons). Phylogenetic analyses were performed on manually aligned sequences under Bayesian Inference (BI) using MrBayes version 3.1.2 [72], and the Neighbour Joining (NJ) Method and Maximum Parsimony (MP) as implemented in MEGA version 3.1 [73]. Appropriate models of DNA evolution were determined using ModelTest [74]. For the BI analysis, MCMC were run for 5×106 generations with each codon position as a separate partition with the (GTR+I+Γ) model of nucleotide substitution. Convergence was assessed from multiple chains and by ensuring that effective sample sizes for parameters were >200. Visual inspection of a plot of log likelihood against generation was use to select ‘burnin’ trees to discard prior to summarising the search results with a majority rule consensus tree. For NJ and MP methods we performed 1000 pseudoreplicates to obtain estimates of nodal non-parametric bootstrap support. The tree generated under BI is shown in Fig. 2. For each of the analyses, we included representatives of various other species of Rattus (R. andamanensis, R. argentiventer, R. exulans, R. losea, R. nitidus, R. norvegicus, R. satarae) with the following objectives: 1) to test the monophyly or otherwise of R. rattus; 2) to identify the closest relatives of R. rattus within Rattus; and 3) to compare nucleotide diversity within R. rattus with the inter- and intra-specific nucleotide diversity in each species of Rattus and its close relative Bandicota. We also included representatives of other genera of the Rattini (Berylmys and Niviventer [14]) to serve as outgroups. The phylogenetic structure of better sampled sub-lineages was investigated using Median-Joining Network analyses [75] and the inferential methods developed in comparable studies of human dispersal history [76]. To quantify diversity and test for population expansions, we calculated a variety of population genetic parameters and statistics for different mtDNA lineages and geographic groupings (Table 3), using DnaSP version 5.10.1 [77]. Specifically, we calculated nucleotide diversity Pi, Tajima’s D [78], Fu and Li’s D* and F* [79], Fu’s Fs [80], and Ramos-Onsin and Rozas’s R2 [81]. We also produced graphs of pairwise mismatch distribution [82], using the coalescent simulation approach implemented in DnaSP version 5.10.1 [77] (Figs. 5, ,6,6, ,7).7). Estimates of tau ( = expansion time in mutational units) were converted into years before present (ybp) with the formula ybp = generation length multiplied by tau/2uk, where u = substitution rate per site per year; k = sequence length [83]. We used various prior estimates of u, drawn from studies of murine rodents in general or from analysis of the Rattus Division (see Table 4). For each of lineages I and II, separate analyses were carried out on populations within the inferred natural range vs populations resulting from range expansion into areas not originally occupied by any member of the Black Rat group. Where the karyotype of individual rats included in this study is known from prior studies, this information is included in Table S1. No karyotypes were determined specifically for our study. Cytochrome b sequences reported by Robins et al. [24] were added to the current dataset to enable further comparison of results. Six House Mouse (Mus musculus) cytochrome b sequences (GenBank accession numbers EF108340-5) were added to incorporate a calibration point from the murine fossil record. The widely used divergence of Mus and Rattus actually corresponds to the origin of tribal diversity within mainland Asian representatives of the subfamily Murinae [25]. The best substitution model was selected using ModelGenerator by comparison of Akaike Information Criterion 2 scores [84]. Bayesian phylogenetic analysis with timescale was performed with BEAST version 1.4 [85], using the divergence time estimation of 10.4–14 Mya [64] for the Mus/Rattus node. An uncorrelated lognormal relaxed-clock model [86] was used with HKY+I+G6 substitution model on the data partitioned into the three codon positions. MCMC analyses were run for 30,000,000 steps, with posterior samples drawn every 1000 steps after a burn-in of 3,000,000 steps. Due to the complex mixture of intra and inter-specific diversity in the dataset, the general constant size coalescent model was chosen as tree prior (see Fig. 3). However, applying a general model will not always conciliate such different levels of genetic diversity. In order to check for any bias in time estimations due to this issue, a similar BEAST analysis was conducted using only one representative per clade (these are identified in Table S4, References S1), and applying the Yule process speciation model as tree prior (see Fig. S1). Comparison of the two sets of results does not indicate significant differences in estimated dates according to 95% margins (Table S4, References S1). The BEAST input files are available from the authors upon request. Robins et al. [24] calculated the tMRCA for four clades that are present in our data (see Table S4, References S1), using whole mitochondrial sequences and a narrower calibration date range (Mus/Rattus, 11–12.3 Mya [87]). We defined a broader time interval for the fossil calibration point that better reflects uncertainties about the evolutionary history of this group [88]. Comparison of tMRCA estimations between the two studies shows slightly older dates in our analysis, but no significant differences considering the error margins. Two sources of uncertainty remain concerning the inferred timescale. Firstly, it is now well known that molecular rates are time-dependant [89]–[91], and a single, deep, calibration point at inter-specific level may not provide optimal accuracy for estimations at an intra-specific level. Only a combination of several calibration points at contrasting time scales would be likely to significantly increase dating accuracy [92]–[93]. Unfortunately, for this group we lack both a well-dated Quaternary fossil record or radiocarbon dated specimens from which ancient DNA has been derived. Secondly, our study and that of Robins et al. [24] are based only on the mitochondrial genome and thus is subject to intrinsic biases related to maternal inheritance and organelle location [94]–[95]. Information from multiple markers will be needed to derive more robust estimates of the timing of phylogenetic diversification in this recently evolved group.
