THE KARYOTYPE EVOLUTION AND SPECIES DIFFERENTIATION IN THE GENUS RATTUS OF MANIPUR , INDIA *

Rattus is the mostly studied genus all over the world but species of the genus are not thoroughly reported from Manipur. The present paper deals with the morphometric, cytotaxonomic and phylogenetic studies of Manipur, India. The different species of Rattus namely Rattusrattus, Rattus brunneusculus, Rattus tanezumi, and Rattus nitidus were studied. Berylmys manipulus and B. mackenziei were taken from the genus Berylmys. The studies of the species reveal that the polymorphic chromosomes in the genus Rattus were pair numbers i.e. 1, 9, 10 and 13. The change of acrocentrics which are regarded as primitive/ancestral types of chromosomes into either subtelocentric or small metacentrics leads to speciation or simply new species particularly in Manipur. It is generally accepted cytologically that 2n=40 (Berylmys manipulus, B. mackenziei, Rattus kandianus etc) are derived from the 2n=42 through centric fusion/Robertsonian fusion, but the soft palate studies shows affinity of the species i.e. Berylmys manipulus and B. mackenziei from Manipur towards Niviventer. The present study is the first step towards the understanding of the relationship between these two genera-Rattus and Berylmys.


INTRODUCTION
The genus Rattus has been defined as a widely distributed and taxonomically mixed group including many species and subspecies throughout the World (Ellerman and Morrison-Scott, 1951;Vinogradov and Argyropulo, 1941;Corbet, 1978;Harrison and Bates, 1991).Out of a total of 4,629 species of mammals known in the world, 372 species occur in the Indian union (Corbet and Hill, 1992).Of these, 69 species are reported in Manipur (Mandal et al., 2005).Mandal et al. (2005) reported five species and seven subspecies: Rattus rattus brunneusculus (Hodgson), Rattus rattus bullocki Roonwal, Rattus rattus tistae Hinton, Rattut nitidus obsoletus Hinton, R. mackenziei (Thomas) and Rattus manipulus manipulus (Thomas).The latter two taxa were then included in the genus Berylmys as Berylmys mackenziei (Thomas) and Berylmys manipulus *Corresponding author.E-mail: dhananjoych@gmail.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (Thomas), respectively (Agrawal, 2000).Manipur is one of the passage to the South East Asia from Indian continent or vice-versa.Rattus rattus originated from South East Asia, whereas the India Continent is the old resident of the species (Suzuki et al., 2012).It is pertinent to study the different species of the genus Rattus and some species of the Berylmys.The present study is the first step towards understanding the chromosomal profiles of some species of the Rattus and to investigate some of the relationship with Berylmys particularly in the context of Manipur

MATERIALS AND METHODS
A total of 107 (55 males, 52 females) specimens of the genus Rattus were caught from 14 localities in five districts of Manipur using live traps during June-October, 2012 (Table 1).Before the work began, each specimen was given an accession code.The wet specimens, skulls and skins were deposited in the University Museum of Central Agricultural University, Imphal.The morphological studies like fur colours, Head-body Length, Tail length, length of hind foot etc. were carried out according to Agrawal (2000), Aplin et al. (2003) and Alfred (2005).The skulls were set (Herbreteau et al., 2011) to study the cranial features according to Agrawal (2000).Chromosomes were collected from the somatic cells from bone marrow cells of femur after treating the rats with colchicine for 2 h.The cells were then treated with KCl for 45 min, and fixed in acetic acid and methanol in the ratio 1:3 by volume.Finally the cell suspension was spread on slides pre-chilled in methanol and flame dried.
The chromosomes were stained using 4% Giemsa solution.Cbanding was done according to Sumner (1972) with some modification.Chromosome number and morphology were recorded from 50 metaphases per specimen directly under a 100X and photographs of selected cells were taken by camera attached in Olympus BX-41.The sorting types of chromosomes and karyotypes were done according to Yosida (1983).
Ethical approval was obtained from the Institutional Ethics Committee (IEC) of the Central Agricultural University whose protocols and guidelines about the using of living animals in science were strictly followed.

General characters
Medium-sized rat, with a completely uncoloured tail, generally longer than head and body length.Fur short and harsh, with many soft flattened spines which were from the plains and soft without spines in winter from mountain habitats.Dorsum are in various shades of brown or of two colours that is, (i) greyish, tipped with buff (indoor forms) and (ii) white or creamish, with a medial streak of grey or irregular grey patches (outdoor forms).Mammae is generally 5 pairs, if 6 th pair is present, then postaxial pairs will be close together (less than 10 mm apart).
Occipitonasal length of skull ranges from 36.9 to 46.5 mm; palate always more than one-half of occipitonasal length; extending posteriorly behind third upper molars; nasals relatively short, less than 38% of occipitonasal length and not extending behind ascending process of premaxillary tooth.the teeth row ranges from 5.5 to 7.5 mm and forms one of the main characters in the classification of species into subspecies through Shorttoothed forms (less than 6.6 mm in length) and longtoothed forms (more than 6.6 mm in length).
Morphological features of the rats from the Imphal West were agouti dorsal and venter yellowish white fur (Figure 1d).

Cytology
The 2n = 42 karyotype was found in three individuals (1 females and 2 males) collected from Imphal West district (Langol Locality).Their karyotype consists of eleven acrocentrics pairs (#1 to 9, 11 and 12); pair 10 was homomorphic subtelocentric pair, one heteromorphic pair to subtelocentric/acrocentrics (#13), seven pairs of small metacentric (#14 to 20) elements, and the two acrocentrics X and Y chromosomes.The rats from Keishampat (one male) showed polymorphic in pair #1 due to acrocentric and subtelocentric and rest of the karyotype was quite similar to Langol type.The X chromosome is comparable in size to the 12th autosomal pair while the Y is the shortest element of the male genome (Figure 3).

Rattus tanezumi Temminck, 1844 from Karang of
Loktak Lake (N 24° 54.770'' E 93° 83.300''), Bishnupur District The rats from the Karang (PBG 345) were identified as Rattus tanezumi with the help of mitochondrial DNA (COI).The Rattus tanezumi have a unique feature which is the presence of a smoky T-shaped around throat and as on upper parts of manus (Figure 1g to i)

Cytology
2n=42, without exception the karyotype consisted of thirteen acrocentrics pairs (#1 to 13), seven pairs of small metacentric pairs (#14 to 20) and acrocentrics XY, and sex chromosomes were acrocentrics.The X chromosome was 10th of autosomal chromosome in length and Y chromosome is the shortest in the genome (Figure 4).Chromosome numbers 15, 19 and 20 were heteromorphic due to metacentric and submetacentrics homologues.

Genaral characters
A bandicoot -like large rat, have an obscurely bicoloured tail, shorter than head and body length (80 to 95%) and relatively smaller ears (16 to 23).Fur coarse with spines, Dorsum dark brown and venter grey.Large skull, occipitonasal length 45 to 55 mm; supraorbital ridges powerful, extending backwards fairly straight up to occiput; palate long, more than one half of occipitonasal length (onl), extending posteriorly far behind third upper molars; maxillary toothrows less than 15% of onl and its width ranges from 2.0 to 2.3 mm; anterior palatal foramina, on average, 17% of onl and broad (3.0 to 4.6), ending far ahead of first upper molars; braincase narrow, cranial width less than 28% of onl, Mammae 5 or 6 (Agrawal, 2000).This species is robust and heavily built.The tail is always less than head and body length in adult specimens.The ear is short and when drawn forward, does not reach the eyes.External measurements are given in Table 2. Dorsal fur colour varies slightly from dark brownish to ochre (particularly in old specimens) and the dorsal hair bases are greyish.Tail is slightly is bicoloured and covered with short, sparse, dirty, whitish hairs.The soles of the fore and hind feet are completely naked.The upper surface of both the fore and hind feet are covered with tiny whitish hairs but the base of the hind upper surface has patches of grayish hairs.The hairs on ventral fur are dirty white but bases are greyish.The line of demarcation is quite distinct.The species has six pairs of mammae (2 pairs pectoral, 1 pair abdominal and 3 pairs inguinal).

Cytology:
The rats from Imphal West district with the parallel supra-orbital ridge are to be Rattus norvegicus (Berkenhout).The species has 2n=42 with exception to karyotype thirteen acrocentrics pairs (#1 to 13), seven small metacentric pairs (14 to 20) and acrocentrics sex chromosomes.The X chromosome was 11 th of autosomal chromosome in length and Y chromosome is the shortest in the male genome (Figure 6).

General character
In this species the fur is soft and quite thick, particularly in the typical race.The tail is nearly naked, with signs of growth of hairs throughout its length.Hind foot with five toes, all clawed; the hallux shorter than the fifth, which is shorter than the central three; apparently six plantar pads.Tail little longer than head and body as a rule (107% average for typical race, Hinton; 99% average for obsoletus ).Mammae 12, as a rule.

Colour:
The back with is usually dark brown, occasionally with a darker mid-dorsal patch or line.Feet usually yellowish or whitish, rarely dark.Tail normally dark as a whole (Ellerman, 1961).
The rats from the Mao (PBG 344) are identified as Rattus nitidus on the basis of mitochondrial DNA (COI) sequences.The sequence is submitted in the NCBI Gene Bank (NCBI accession number was JQ918374).
The species Berylmys berdmorei with which B. manipulus was originally confused, differs from it by the length of tail which is smaller than head and body and its distal half brown instead of white, and large inflated bulla (more than 7mm or more than 18% of onl).Total length of the tail is 14.5 and white part is 9 cm and % of the white is 62.06%.

Cytology:
The diploid count is 40 consisting of three subtelocentric pairs (# 1, 2, and 3), 8 acrocentric pairs (# 4-10, and 13) and one big metacentric pair ( #11) that is formed by the Robertsonian fusion of chromosome numbers 11 and 12 of the ancestral Asian black rat Rattus rattus, 7 small metacentric pairs (# 14-20), the X chromosome is acrocentric which is 9 th in length of the autosomes and Y chromosome is acrocentrics comparable to 13 th of the autosomes (Figure 8).1a).This is reflected in length of head and body,  hindfoot, occipitonasal and maxillary toothrows; bulla less than 15% of onl; diastema less than 31% of onl as in bowersii but shorter than manipulus.Body colour iron grey above, white below; distal one-third to one-half of tail white; hind foot brown, toes white.Mammae 5 pairs.

Cytology:
This rat is initially named as Rattus mackenziei (Thomas, 1916) but according Musser and Newcomb (1983), it had changed to Berylmys manipulus (Thomas, 1916).The diploid count is 40 consisting of three subtelocentric pairs (# 1, 2, and 3), eight acrocentric pairs (# 4 to 10, and 13) and one big metacentric pair (#11) that is formed by the Robertsonian fusion of chromosome numbers 11 and 12 of the ancestral Asian black Rattus rattus, 7 small metacentric pairs (# 14 to 20), the X chromosome is subtelocentric which is 6 th in length of the autosomes and Y chromosome is acrocentrics comparable to 9 th of the autosomes (Figure 9).The heterochromatic region as evidence from the Cbanded pattern points the difference between B. manipulus and B. mackenziei (Figure 10a and b).

Soft palate of the species in the study:
The soft palate photographs of different species were compared and it can be shown that the Rattus rattus, R. tanezumi, and R. nitidus have three chevron-shaped ridges while the B. manipulus has only four tapering ridges arising from the base of the molar teeth (Figure 1m

DISCUSSION
Ellerman and Morrison-scout (1951) classified R. rattus with black coat colour inhabiting the northern part of the world and R. rattus alexandrinus (Geof) with agouti coat colour inhabiting the southern part.Specimens of Ratus rattus species collected from hills have agouti as well as black colour while those from valley areas have agouti coat colour agreeing with the above two coat colours of this species.Tolga et al. (1998) reported four main colorations in this species.The first type, has a dark, slate blackish dorsal fur and greyish ventral fur, and is also referred to as R. rattus rattus.The second type, has dark brownish dorsal fur with yellowish white ventral fur, and is known as R. rattus elaxandrinus.The third type is dorsally the same as in the second type, but the ventral fur is light greyish and the upper sides of the feet are pale brownish, and it is referred to as R. r. alexandrinus.In these three types, the hair bases of dorsal and ventral furs are greyish.The fourth type, which is dorsally similar to the second and the third types, has ventral fur that is pure white, and is referred to as R. rattus frugivorus.In the first type, the tail and the ear are uniformly covered with short, sparse blackish hairs, the dorsal colour grows paler toward the flanks, but the line of demarcation along the flanks is not very distinct.If available published data are for sure, the present specimen from the Senapati District might be R. r. alexandrines while the rats from Thoubal and Imphal West might be R. rattus elaxandrinus.The morphological features frequently lead to misidentification in the cryptic species and so the specimen in the present study should be re-examined in future to confirm the true identity through sequencing the mitochondrial COI.
The chromosome compliment of the Rattus rattus in the present study shows the polymorphic chromosome numbers of 1 (heteromorphic in Imphal West, homomorphic in the Imphal East), 9 (homomorphic in Rattus brunneuculus) Another polymorphic chromosome pair is number 10 (Imphal West) which is found in Rattus bullocki.Hence all the polymorphic chromosomes in the present study are one of the polymorphic chromosomes of the Rattus rattus as reported by Yoshida (1983) except pair number #10.Depending upon the length of the acrocentric and subtelocentrics in the study, it can be referred that the subtelocentrics were derived from the acrocentric counterparts through the pericentric inversion as reported by Yosida (1983) and Pages et al. (2011).Gadi and Sharma (1983) reported 2n=42 for Rattus rattus, consisting of 7 small metacentric, 3 subtelocentric, 10 telocentric pairs and telocentric X and Y chromosomes which is agree with the present work except for the number of the subtelocentric.The number of the subtelocentrics may vary due to the pericentric inversion.Wilson and Reeder (2005) synonymised Rattus brunneusculus (Hogson, 1945) with Rattus tanezumi.In Indian context too Rattus brunneusculus was reported from Manipur instead of the Rattus tanezumi (Alfred, 2005) and Ellerman (1961) from The Loktak Lake.Alfred reports that the brunneusculus had longer tail, being 123-131% of head and body length but in our study it is 90%.Again Ellerman (1961) synonimised it with sikkimensis and argued that the typical brunneusculus has the underparts yellowish, not very typical for a wild race.Besides this the characteristic feature of both brunneusculus and sikkimensis is "the bullae are usually less than 17% of the occipitonasal length" but in our study it is 19.51%.Therefore, it is yet to decide the true taxonomic position of Rattus tanezumi particularly in Indian context.A further study in involving the three species through morphology, cytology and COI sequence are much to resolve the taxonomic conflicts of these three species in future.Here in the present study the Rattus brunneusculus is quite different from the tanezumi in both morphological and cytological point of view.In tanezumi the chromosomes are telocentric in all the autosomal as well as the sex chromosomes bit in the brunneusculus the chromosome numbers 1 and 9 were homomorphic subtelocentrics but sex chromosomes are similar.So much work should be done to discriminate these two taxa.Out of the five types of the karyotypes recognised by Yosida (1980), the Manipuri Rattus rattus should be regarded as Japanese types-(2n=42, with low C-banding; R. tanezumi).This shows that the heterochromatin diminution is an effective mechanism in the evolution of the karyotypes that led to obvious differentiation among the species and in the speciation process.
Rattus nitidus is indigenous to mainland Southeast Asia and occurs in south China (including Hainan Island), Vietnam, Laos, northern Thailand, Burma, Bangladesh, Nepal, Bhutan, and Northern India; it is also found on the islands of central Sulawesi, Luzon island of the Philippines, Pulau Seram in the Molluccas, the Vogelkop Peninsula of the Province of Papua, and the Palau Islands, probably due to human-mediated introductions (Aplin et al., 2003;Musser and Carleton, 2005).Li et al. (2008) and Gadi and Sharma (1983) reported the 2n=42 of Ratuus nitidus comprising of eight metacentric pairs, two subtelocentric pairs, and 10 acrocentric pairs as autosome compliments and acrocentrics XX from China.In the present study also the karyotype is consisting of ten acrocentric pairs (#2-8, 10-12), two subtelocentric pairs (#1, and 9), eight pairs of small metacentric pairs (# 13, 14-20) and acrocentric X and Y. Hence the Manipur Rattus nitidus is quite similar to the Chinese species in the autosomal compliments but the heterochromatic q-arms in the Xchromosomes are yet to be reported from the other part of the world.
According to Yosida (1983) the Asian black rat with 42 chromosomes is the ancestor of the Ceylonese black rat with 40 chromosomes and the Oceanian black rat with 38 chromosomes.He proposed that 40 chromosomes (Rattus rattus kandianus in the Sri Lanka, Beylmys manipulus and Berylmys mackenziei of present studies) and 38 chromosome karyotypes evolved from 42 chromosome karyotype through a first Robertsonian fusion of acrocentric pairs 11 and 12 and a second Robertsonian fusion between acrocentric pairs 4 and 7.It is considered that the progenitors of the members of the genus Rattus originally had acrocentric karyotype and the subtelocentric and metacentric members were derived by pericentric inversion of the acrocentric chromosomes (Yosida, 1983).Manipur is one of gateways of South Asian to the Indian continent, the ancestral Rattus rattus might have passed through, hybridized here and result into diversity of Black rats.The first Robertsonian fusion might had formed here in the Indo-Burma areas and travel through up to Western India and might have occurred there the second Robertsonian fusion and formed the rats with 2n=38.But the comparative studies of the softpalate shows other facts; these structures having three chevron-shaped ridges are present in the different species of the genus Rattus but the fingers like soft palate ridges of the Berylmys are not at all related to the genus Rattus.The features are quite common in the genus Niviventer and morphology too except for the pure white tail tips.The only karyotypic similarity between Rattus and Berylmys is the 14 metacentric chromosomes and differ in the number of the subtelocentris.Considering the differences in autosomal chromosomes and soft palate, there is not much identical in these two genera, but much works should take up involving the two genera in future.
Phylogeny of the present study begins as Rattus sabanus an ancestral karyotype and divergent karyotypes were obtained due to per-centric inversion of acrocentric chromosomes to subtelocentrics and much deviated karyotypes were seen among the different species of the genus Rattus in Manipur (Figure 11).Here the number of the meta-centric is the reference points to decide the primitiveness of the species.In Rattus tanezumi the number of metacentric chromosomes is 12 and rest of the Rattus species are 14 in number.The number of subtelocentrics not considered in the study.
The summary of the present study can be as follows: 1.The autosomal chromosome compliments of different species of the genus Rattus quite agree with the published data.2. The chromosomes compliments of different species of the genus Rattus are one of the polymorphic forms of the Rattus rattus.3. The studies of the species reveal that the polymorphic chromosomes in the genus Rattus were pair numbers 1, 9, 10 and 13. 4. The cytological studies of Berylmys species (2n=40) as reported by other authors need to be re-evaluated particularly in the Manipur rats. 5.The Manipur i rats are tanezumi type (2n= 42) not the Rattus type ( 2n= 38) in chromosomal basis.6. Change in single chromosome leads to differentiation of new species.7. The biometrics such as morphotaxonomy, cytotaxonomy and molecular are required for identification of taxon.

Figure 2 .
Figure 2. Metaphase a and karyotype b of Rattus rattus from Kharam Waiphei.

Figure 3 .
Figure 3. Metaphase a and karyotype b of Rattus rattus from Keishampat.

Figure 4 .
Figure.2.Metaphase a and karyotype b of Rattus rattus .

Figure 5 .
Figure 5. Metaphase a and karyotype b of Rattus brunneusculus.

Figure 6 .
Figure 6.Metaphase a and karyotype b of Rattus norvegicus.

Figure 7 .
Figure 7. Metaphase a and karyotype b of Rattus nitidus.Figure 6. Metaphase a and karyotype b of Rattus norvegicus.

Figure 6 .
Figure 7. Metaphase a and karyotype b of Rattus nitidus.Figure 6. Metaphase a and karyotype b of Rattus norvegicus.

Figure 7 .
Figure 7. Metaphase a and karyotype b of Rattus nitidus.
to q).

Figure 11 .
Figure 11.Phylogeny of the genus Rattus (without sex chromosomes), involving percentric inversion and Robertsonian fusion S=subtelocentric, M=large metacentric, m=small metacentric, A=acrocentric, numeral represents the chromosome numbers, black ones are of other authors.

Table 1 .
The different rat specimens collected from the different parts of Manipur.

Table 2 .
The morphometrics and crannial measurement of rat species of Manipur