Historic black rat invasions into Sri Lanka lead to hybridization forming two sub-lineages in the Rattus rattus species complex

: In this work, we used two mitochondrial (16S rRNA and cytochrome- b ) and one nuclear (Rag 1) gene fragments to analyse phylogeographic history of black rats in Sri Lanka at subspecies level. Of the five subspecies recorded in Sri Lanka three ( Rattus rattus kelaarti , R.r. rufescens , R. r. kandianus ) are believed to have invaded Sri Lanka in prehistoric times and two ( R.r. alexandrinus , R. r. rattus ) in the recent past through trade ships from Europe and India. All subspecies, except R. r. kandianus , have restricted destribution with none occuring sympatrically. Rattus r. kandianus is widely destributed throughout the country. In the phylogeny, all black rats from Sri Lanka fell into the Rattus rattus complex lineage I. But, R.r. kelaarti , R.r. rufescens most of R.r. kandianus and one individual of R.r. alexandrinus formed a unique Sri Lankan sub-lineage within the lineage I. The results suggests hybridization with mitochondrial introgression of the endemic R.r. kandianus with all other subspecies separately. Introgression of R.r. alexandrinus mitochondrial DNA into a subpopulation of R.r. kandianus is responsible for placing few individuals of the latter in the widespead sub-lineage in the Lineage I with R.r. alexandrinus and R. rattus from other parts of the world. The fifth subspecies, R.r. rattus which was reported in small numbers from ports in 1930s, has most likely been replaced by R.r. alexandrinus.


INTRODUCTION
Genus Rattus, comprised of 66 species, is cosmopolitan in distribution and may probably have originated in mainland Asia (Watts & Baverstock 1994;Chaimanee & Jaeger 2001). The black rat, R. rattus (Linnaeus 1758), well known as a pest and a disease carrier, is native to the Indian Peninsula and has been introduced world-wide (Musser & Carleton 2005). There are more than 80 subspecies listed under R. rattus (Musser & Carleton 2005) however, the subspecies level diversification is largely ignored by taxonomists (Corbet & Hill 1991;Wilson & Reeder 2005).
In general, subspecies are phenotypically distinct, but not distinct enough to prevent hybridization among them. Subspecies has had a long history in taxonomy, dating back to the 19 th century. Initially called "varieties", taxonomists placed any distinct natural population that was not sufficiently different to be called a species in a subspecies (Mayr 1982). But, with the advent of molecular studies, the failure to identify subspecies as phylogenetically distinct entities started a long-running controversy of recognizing subspecies as a valid taxonomic unit (Mayr 1982;O'Brien & Mayr 1991;Ball & Avise 1992;Burbrink et al. 2000;Zink 2004). However, since by definition subspecies interbreed in the zone of contact, it may not always be possible to see phylogenetic distinctness (Patten 2010). Studies which included island subspecies have recovered high level of phylogenetic distinctness, explained by divergence in isolation and smaller population size (Hastings & Gavrilets 1999;Gavrilets 2004;Phillimore & Owens 2006). In addition, some studies have proved that subspecies are useful in estimating historic patterns of divergence among populations (O'Brien & Mayr 1991), populations which may become species with time. Many argue that geographic variation of species can be better understood with use of subspecies (Mayr 1982;Barrowclough 1982). Well-defined subspecies may also serve to track migration, dispersal and geographic origins of introduced populations (Zusi 1982).
With respect to R. rattus¸ numerous studies carried out throughout the world have tried to understand migratory patterns, hybridization or mitochondrial introgression and phylogeograghy of the group ( Chinen et al. 2005;Robins et al. 2007;Aplin et al. 2011;Lack et al. 2012;Conroy et al. 2013;Yasuda et al. 2014). However, only few studies even mention subspecies (Robins et al. 2007;Aplin et al. 2011;Robins et al. 2014).
In Sri Lanka there are three species of Rattus (R. rattus, R. norvegicus and R. montanus), of which R. rattus is subdivided into 5 subspecies: R. r. rattus, R. r. alexandrinus, R. r. rufescens, R. r. kandianus and R. r. kelaarti, with the latter two endemic to the country (Phillips 1980). Rattus r. rattus, commonly known as the black rat, is a slender, medium-sized rat with a slender tail longer than head and body and large naked ears. Colour is black or blackish grey on dorsal, which pales in to grey black in lower parts. It has been reported in small numbers around dock areas in sea-port towns: Colombo, Galle and Trincomalee. Rattus r. alexandrinus, commonly known as Egyptian houserat or ship rat, can be readily distinguished by its brown or brownish grey dorsal fur and dingy grey lower parts. Size and build is similar to R. r. rattus. It is recorded in moderate numbers in and around the docks and densely populated areas in Colombo and Trincomalee. Both these subspecies are known to have imported in ships from Europe and Indian ports (Phillips 1980). Rattus r. rufescens is a medium-sized long-tailed house rat found commonly in the dry zone. It can be easily distinguished from other subspecies by rusty tinge of grey fur of the belly. It has undoubtedly spread from South India to Sri Lanka (Phillips 1980). Rattus r. kandianus, is a subspecies endemic to Sri Lanka but found commonly almost everywhere on the island. Its upperparts are generally dark brown and underparts creamy white to light brown. But the colour is very variable among the individuals that occur in different altitudes. Fur texture, though very variable, is usually short, harsh and spiny in lowland populations and long and soft in highland populations. Rattus r. kelaarti, commonly known as Ceylon highland rat, is also endemic to Sri Lanka (Phillips 1980). Its dorsal colour is dark olive brown and underparts are greyish white. It can be distinguished from other subspecies by its longer, softer and darker fur and relatively short tail. It is found commonly but confined to jungles of higher hills of central mountains. Except R. r. kandianus, which occur sympatrically with all other subspecies, none of the others cross each other's paths.
Rattus rattus, being a species widely distributed in the world, has diversified into a complex, forming four phylogenetic lineages (Aplin et al. 2011). Lineage RrC LI (Rattus rattus Complex: Lineage I), mainly centred in western India has a broad distribution throughout Europe, Americas, Africa, Madagascar, Australia and Pacific islands. RrC LIII lineage is distributed in Himalayan foothills of Pakistan and Nepal. Lineages RrC LII and RrC LIV are found in Indo Malayan region. Aplin et al. (2011) has included a single sequence of R. r. kandianus from highlands of Sri Lanka in their phylogeny, which fell in the lineage RrC LIV. This lineage is typically found in lower Mekong river catchment in southern Laos, Thailand and southern Vietnam. Considering the unique faunal diversity in Sri Lanka, Aplin et al. (2011) deduced a prehistoric invasion of Sri Lanka by R. rattus. However, a later study based on cytochrome-b gene by Yasuda et al. (2014) revealed that specimens collected from Kandy district, Sri Lanka, fall in RrC LI not RrC LIV, forming two distinct sub-lineages. One sub-lineage (RrC LIa), which is widely distributed in the world, included few Sri Lankan specimens with R. rattus from other parts of the world. The other sublineage (RrC LIb), closely related to RrC LIa sub-lineage, included only specimens from Sri Lanka. They infered two migration events to Sri Lanka, one ancient and the other recent. They also suggested a possible hybridization between the two sub-lineages.
Considering the report of two migration events of R. rattus subspecies to Sri Lanka by Phillips (1980), we aimed to determine their phylogenetic relationships and phylogeographic history. Based on distribution of subspecies reported by Phillips (1980) and our own field experience, we hypothesised hybridization or mitochondrial introgression between the widespread R. r. kandianus with the other subspecies, including ones introduced through trade ships.

MATERIALS AND METHODS
Rattus rattus subspecies were collected from selected sites throughout the country during field surveys from 2003 to 2005, and 2015 to 2016 ( Fig.1; Table 1). One hundred mesh traps and Sherman traps baited with pieces of roasted coconut were used to collect specimens. They were identified using the descriptions given by Phillips (1980). All specimens are deposited in the Department of Zoology, Faculty of Science, University of Peradeniya.
A muscle sample was taken from each specimen and preserved in 90% ethanol for DNA extraction. Two mitochondrial (16S rRNA and cytochrome-b) and one nuclear (Rag 1) gene fragments were sequenced for molecular analysis. DNA was extracted from ethanolpreserved tissues using Promega wizard blood and tissue extraction kit following manufacturer's protocols. DNA was amplified by PCR using 25 µl reactions containing 2.0 µl of template, 1.0 µl of each primer (10 µM), and 12.5 µl GoTaq Green master mix and 8.5 µl nano water. Thermal cycling for the cytochrome-b fragment was as follows: 35 cycles of denaturation at 94° C for 30 s, annealing at 45° C for 30 s, and extension at 72° C for 1 min, with a final extension of 72° C for 5 min. The same conditions were used to amplify 16S gene, except for the annealing temperature, which was 48° C. Sequence length of cytochrome-b and 16S were 1140 and 547 bp, respectively. Thermal cycling for Rag 1 gene (819 bp) was as follows: 35 cycles of denaturation at 95° C for 45 s, annealing at 55° C for 45 s, and extension at 72° C for 1 min, with a final extension of 72° C for 5 min. Cytochrome-b and 16S were amplified using the primers MVZ 05/ MVZ 14 and 16S ar/ 16S br, respectively. Primer sequences are as follows: MVZ 05 5' CGA AGC TTG ATA TGA AAA ACC ATC GTTG 3'; MVZ 14 5' GGT CTT CAT CTY HGG YTT ACA AGAC 3'; 16S ar 5' CGC CTG TTT ATC AAA AAC AT 3'; 16S br 5' CCG GTC TGA ACT CAG ATC ACGT 3'. Primers used for Rag 1 PCR were: AmpRAG1 F 5' AGC TGCAGY CAR TAC CAY AAR ATG TA 3'; Amp RAG1R1 5'AAC TCA GCT GCA TTK CCA ATR TCACA 3'. Sequences were cleaned using ChromasPro 1.7.7, aligned using Clustal W in MEGA 6 software and phylogenies were generated using MEGA 6 software. Two species of Bandicota Gray, 1873 and two species of Mus Linnaeus, 1758 were also used in the analysis as they are closely related to Rattus species (Yasuda et al. 2014;Pages et al. 2010). Tatera indica (Hardwicke 1807) was used as the outgroup because of its suggested close relationship to murine rodents (Steppan et al. 2004;Jansa and Weksler 2004). Genetic distance values for cytochrome-b gene were calculated using MEGA 6 software. The GenBank accession numbers for the sequences generated anew for the study and sequences downloaded from the GenBank are given in the Table 1.  Mitochondrial gene trees and mitochondrial gene combined tree (cytochrome-b and 16S) resulted in the same topology except the placement of R. norvegicus in the 16S gene tree. Rag 1 gene was not included in the phylogeny because there were no sequence differences between any subspecies of R. rattus. In cytochrome-b gene tree, R. rattus subspecies fell into four distinct lineages. Rattus rattus from Sri Lanka fell into one lineage (RrC LI), which is reciprocally monophyletic and had two distinct sub-lineages. One sub-lineage (RrC LIb) included only Sri Lankan specimens with representatives from all four subspecies, whereas the other sub-lineage (RrC LIa) included two specimens of R. r. alexandrinus and three R. r. kandianus with R. rattus from other parts of the world (Fig.  2). All R. r. rufescens (n=6), all R. r. kelaarti (n=4), one out of three R. r. alexandrinus and 23 out of 27 R. r. kandianus in our collection fell into Sri Lankan unique RrC LIb sublineage. All relationships had high bootstrap support. Sri Lankan population of R. r. alexandrinus and R. r. kandianus which were in the sub-lineage RrC LIa fell together in a distinct clade with 97% bootstrap support.
There were 13 cytochrome-b haplotypes in RrC LIb sub-lineage. Of 34 individuals in RrC LIb sub-lineage, 18 were of a single haplotype which was common to all four subspecies. One haplotype was only found in R. r. rufescens and two others were shared by R. r. kandianus and R. r. rufescens, and R. r. kandianus and R. r. kelaarti. All other nine haplotypes were found from R. r. kandianus (Table 2). All Sri Lankan samples in RrC LIa were of a single unique haplotype.
Uncorrected percentage pairwise distances for cytochrome-b gene between the four main lineages of Rattus rattus (RrC LI, RrC LII, RrC LIII and RrC LIV) were 3.72-6.79%. That of between individuals of two sublineages of RrC LI was 1.79-2.61%. Individuals within RrC LIa and RrC Lib had genetic distances of 0.00-0.98% and 0.00-0.89%, respectively. Percentage pairwise distances among the subspecies present in Sri Lanka, regardless of the placement in the phylogeny, were between 0.00-2.52%, with some individuals of all four subspecies having 0.00 genetic difference for cytochrome-b. Sri Lankan subspecies in the RrC LIa formed a separate clade in the phylogeny with a genetic distance of 0.53-0.89% from others in the same sub-lineage.  39  63  165  174  297  303  318  348  393  477  573  694  702  703  768  771  807  816  924  1014  1039  1040   1  18 All four subspecies

DISCUSSION
Only four subspecies of R. rattus were captured (R. r. kandianus, R. r. alexandrinus, R. r. rufescens and R. r. kelaarti) during this study. We were not able to capture R. r. rattus during this study. Early surveys by Phillips (1980) reported the presence of R. r. rattus in small numbers around the dock areas in Colombo, Galle and Trincomalee. At present, it is probable that this subspecies is entirely replaced by R. r. alexandrinus that occur in docks.
Even though the four subspecies are morphologically distinguishable, genetic differences among them were not substantial (0.00-2.52%). This range does not exactly fall within the average genetic distance value of 0.09-2.34% reported for currently identified subspecies taxa (Bradley & Baker 2001). There were individuals of all four subspecies with zero genetic divergence for cytochrome-b. Accordingly, we can deduce that there is hybridization or mitochondrial introgression among all four subspecies. However, this is not the case for these four subspecies.
We have included 40 R. rattus individuals collected from around the country representing four subspecies in the phylogeny. They all fell in two sub-lineages of lineage RrC LI as reported earlier (Yasuda et al. 2014). The Sri Lankan unique sub-lineage, RrC LIb, contained only the four R. rattus subspecies from Sri Lanka, with all individuals of R. r. kelaarti and R. r. rufescens falling in it. All four subspecies having individuals with zero cytochrome-b divergence indicate that each subspecies interbreed with R. r. kandianus which is the widespread and common subspecies throughout the island. Rattus r. kandianus sharing two cytochrome-b haplotypes with R. r. rufescens and R. r. kelaarti also supports this. Rattus r. kandianus is the only subspecies occuring sympatrically with all the other subspecies, facilitating its hybridization with the others. All other subspecies have restricted destributions and none of them occurs sympatrically with each other.
Other sub-lineage, RrC LIa, included four R. r. kandianus and two R. r. alexandrinus individuals, with R. rattus from other parts of the world (Fig. 2). With respect to R. r. kandianus, both sub-lineages had individuals from different localities in the country, from lowland dry zone to highland wet zone. All three R. r. alexandrinus specimens were from Galle. Rattus r. rattus and R. r. alexandrinus have been introduced to the island through trade vessels from Europe and India and were reported from in and around the docks in Galle, Colombo and Trincomalee (Phillips, 1980). Other three subspecies had been in the island for a longer period of time and may have invaded Sri Lanka during the glacial sea-level lowstands when there were terrestrial connections between Sri Lanka and India (most recently until ca 10,000 ybp) or with the help of ancient trade boats. Hence, according to our phylogeny, most likely senario is hybridization or mitochondrial introgression between sub-populations of R.r. kandianus and R. r. alexandrinus. Introgression appears to have happened in both directions. Rattus r. kandianus being a subspecies widespread around the country, occur sympatrically with R. r. alexandrinus.
Hence, may have interbread with R. r. alexandrinus. Since we did not find R. r. rattus in any of the ports, it is not possible to evaluate its relationship with the other Sri Lankan subspecies. However, it is possible that they also may have hybridized with sub-populations of R. r. kandianus and R. r. alexandrinus.
Hybridization and mitochondrial introgression between different Rattus rattus lineages due to human intervened invasions has been reported in other parts of the world (Chinen et al. 2005;Lack et al. 2012;Conroy et al. 2013). Global picture of invations and hybridization will be useful in identifing disease transfer paths, pest control methods and even prehistoric human migrations (Matisoo-Smith & Robins 2004).
Rattus rattus rufescens was previously reported only from the dry zone of Sri Lanka (Phillips 1980). During our field collections we found two specimens from Gampola (Rathmalkaduwa) and Peradeniya, indicating the spread of this subspecies towards upland. Though R. r. rufescens is reported from Trinchomalee, they are not reported to occur sympatrically with R. r. alexandrinus and R. r. rattus in sea ports. During our field surveys also we did not find them living sympatrically.
Biological invasions result in novel interactions among taxa and can have significant evolutionary implications on both native and invading taxa (Chinen et al. 2005;Lack et al. 2012). The genetic distance between individuals of the Sri Lankan unique sub-lineage, RrC Lib is 0.00-0.89%. The genetic distance of cytochrome-b among the R. rattus living in Sri Lanka has increased up to 2.52% due to mitochondrial genes being introgressed from the later introduced R. r. alexandrinus into a sub-population of R. r. kandianus. A sub-population of R. r. alexandrinus has also changed due to mitochondrial introgression from R. r. kandianus.