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Home , Hamster, Lesser bandicoot rat

J. Genet. Vol. 71, Nos 1 & 2, August 1992, pp. 43-56. Printed in .

Molecular phylogeny of Rodenfia in the descent of genus

S. BARNABAS 1'2'*, S. KRISHNAN 1 and L BARNABAS 1 1Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India 2post-Graduate School of Biological Studies, Ahmednagar College, Ahmednagar 414001, India MS received 17 March 1992; revised 2 July 1992 Abstract. The interrelationships of murids and other families as well as the evolutionary descent of multiple/l-globins of murines are deduced from parsimony trees of relevant gtobin sequences. Our results show that Caviidae arises first, followed by Sciuridae which joins . In the murid line of descent Spalacinae arises first followed by two branches, one representing Cricentinae and and the other . Althongh the rates of evolution of globin genes in the different rodent families are different, the mnrid branches show more or less a uniform rate of evolution of// globins. We have used this information to show that - divergence occurred around 20 million years ago. The evolutionary rationale for the presence and the expression of different//-globin genes in murid populations is also discussed. Based on mitochondrial DNA restriction fragment analysis, the between- relationships of rattus rufescens. Bandicota indica and Bandicota bengalensis have been assessed and the time of divergence of the two estimated. Keywords. Globin phylogeny; rodent evolution; murid descent; mt DNA analysis, Bandicota interrelationships.

1. Introduction

A number ofeutherian lineages survived the mass extinction at the Cretaceous/Tertiary boundary around 65 million years (m.y.) ago and underwent a burst of cladogenesis at the end of the event. These eutherians had a bush-like pattern of origin and radiation. Among these is included the Order Rodentia which tended to have a faster rate of evolution as compared to other . The fossil record gives us a glimpse of the origin and evolution of murids. There was an Early Oligocene radiation of primitive muroids in North America, as indicated by the fossil muroids of Chadronian deposits (Flynn et al. 1985). They appear to be absent from the Early Oligocene of Asia, but it seems probable that they were present and perhaps originated in Asia earlier than the Middle Oligocene at which time a diverse variety of muroids are found in Mongolia (Kowalski 1974). The 16 to 1.8 m.y. old muroids from the Siwalik group of rocks of northern Pakistan are part of a Miocene radiation from which most of the extant muroids are descended. The ancestry of most of the surviving muroids lies in the family , which evolved during tile Late Oligocene and excludes the Oligocene muroids from North America which are considered to be an endemic group. Antemus from the Miocene Chinji Formation (13 m.y. ago) in Pakistan, is regarded as the most primitive murid genus (Jacobs 1979). In the Siwaliks fossil record it is succeeded by another primitive murid, Progonomys and preceded by muroids which have been called 'primitive dendromurids'. Perhaps

*For correspondence 43 44 S. Barnabas, S. Krishnan and ,J. Barnabas

the modern murids and also cricetomyids and dendromurids are all derived from widely distributed 'primitive dendromurids'. In the absence of reasonably collaborated relationships among rodent groups, the internal systematics of muroids and other rodent families are not well settled. The phylogenetic relationships within murines are also not clearly established. In the present paper, we have first established the interrelationships and molecular evolution of muroids and other rodent groups by constructing a phylogenetic tree based on combined alpha and beta globin sequences. The evolutionary descent of multiple beta globin genes of rats and mice were also discerned by constructing a phylogenetic tree of the different beta globin sequences of murines and other . Based on the phylogenetic trees we have estimated the evolutionary rates of globins in different rodent lineages and observed that the rate of globin evolution in muroid lineage is more or less uniform. Based on this observation we have established the time of mouse-rat divergence from the rodent stem. Since the sequence data on globins of bandicoot rats are not available, we have used mitochondrial DNA restriction analysis as a means to get insight into the evolutionary relationship of these murine with other murids in the rodent phylogeny. In a preliminary report on mitochondrial DNA restriction analysis of Rattus rattus rt!fescens (the common commensal rat of western India), Bandicota indica indica (th e larger bandicoot rat) and Bandicota bengalensis Kok (the lesser bandicoot rat), it h~{s been shown that shared mitochondrial DNA fragments justify the inclusion of Rattus and Bandicota in the same but Rattus appears to be quite divergent from the genus Bandicota in which the sequence divergence among the two bandicoot species is 3.6% (Barnabas 1989). We have analysed in detail the mitochondrial DNA restriction fragment profile of B. indica and B. bengalensis along with that of R. r. rufescens using thirteen type II restriction endonucleases with hexanucleotide recognition sites. Based on restriction h'agment profile we havediscussed the between-species relationships of the three species of murine rats. We have also established the time of evolutionary divergence of the two species of bandicoot rats using equation (21) of the mathematical model of Nei and Li (1979) for studying the genetic variation in terms of restriction endonucleases.

2. Materials and methods

2.1 Construction of phylogenetic trees

Our treeing algorithm called ZIPSEARCHwhich is based on the zip parsimony approach of Barnabas et al. (1978) has two operations called zipping and unzipping, and is adapted from a mathematically proven maximum parsimony method (Moore et al. 1973). Our algorithm incorporates an exhaustive swapping procedure to carry out an iterative search to arrive at the parsimony solutions (Krishnan et al. 1990). We start from an aligned set of currently available globin sequences and an initial network derived by the neighbour-joining method of Saitou and Nei (1987) and apply null and synonymy elimination to this network as outlined earlier (Barnabas et al. 1972). To this network we apply ZIPSEAP,CH which involves the following operations. For each aligned amino acid position, zipping operation assigns values (corresponding nucleotides) at the interior points which have the potential of minimising the network Molecular phylogeny of Rodentia in the descent of Bandicota 45 length. It also determines the minimum possible network length from the assigned values and sums it over the entire sequence to get the total network length. By using our swapping procedure we carry out extensive search in the topological space using a combination of breadth first and depth first searches to arrive at the most parsimonious network. To this network we apply unzipping which identifies specific nucleotides, derived from amino acid data, at each interior point and determines the linklengths of the network for all the aligned positions of the sequence. The network is converted into a phylogenetic tree by assuming that the ancestor of Caviidae-Sciuridae-Muridae is the most ancestral branching in the rodent lineage (figure la, b).

GOLDEN

MOUSE

22

RAT

MOLE RAT ( EHRENBERG'S

ALPINE MARMOT

HUMAN 32~~~2 EUROPE AN 21 SUSLIK 12

LANGUR 5~,13 GROUND SQUIRREL ( TOWN SEND'S )

16 GUINEA-

L LAMA Figure l. (a) Phylogenetic tree of tandemly placed ~- and fl-globin sequences of rodents with those of primates and ungulates as outgroups. The tree length = 367. The sequences were taken from the Protein Identification Resource data base. Numbers represent nucleotide replacements between ancestor and descendant sequences. The sequences of the following species have been used: auratus (), Ondatra zibethicus (muskrat), Mus museulus (mouse), Marmota marmota marmota (Alpine, marmot), Spermophilns citellus (European sustik), Spermophilus townsendii (Townsend's ground squirrel), Spalax leucodon ehrenbergi (Ehrenberg's mole-rat), Rattus norve~jicus (rat), Cavia porcellus (), Equus caballus (horse), Lama guanicoe glama (llama), Homo sapiens (human), Presbytis entellus (Hanuman langur). 46 S. Barnabas, S. Krishnan and J. Barnabas

MOUSE MOUSE l~s i3 d major RAT r~ ]1: \ / ~/ RAT B mojor MOUSE 15d minor. 1~ /2 ' /RAT 1 [5 minor o ro MOUSE [3 p mmor~66 ] 4 -..( .J...... / RAT 2 ~B re,nor

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GROUND SQUIRREL HUMAN ( TOWNSEND'S ) PINE MARMOT LANGUR

. 30 EUROPEAN SUSLIK

HORSE 14 8 GUINEA- PIG

LLAMA Figure 1. (h) Phylogenetic tree of the fl-globins of murids and other rodents. The variant forms of fl-globins of rats and mice are taken from: rat flII (Satoh et al. 1987); rat [I major (Radosavljevic and Crkvenjakov 1989); rat 1 fl minor (Stevanovic and Crkvenjakov 1989); rat 2 fl minor (Wong et al. 1988);mouse ,8 dmajor (Konkel et al. 1979); mouse fls (Erhart et al. 1985); mouse fl dminor (Konkel et al. 1979); mouse fl pminor (Gilman 1976).

2.2 Mitochondrial DNA analysis

Rattus rattus rufescens and Bandicota indica were trapped in Ahmednaga r and Pune while Bandicota bengalensis was trapped in Bombay. The animals were sacrificed and the livers were removed and stored at -20~ for extraction of mitochondrial DNA. Mitochondrial DNA (mt DNA) was prepared fi'om the livers Of the murids according to the methods described by Brown and Vinograd (1974) and Spolsky and Uzzell (1984) with some modifications. The liver tissue was crushed in a glass homogenizer with a teflon pestle in 10 mM NaC1, 10 mM Tris (pH 7.8), l mM EDTA and sucrose was added Molecular phylogeny of Rodentia in the descent of Bandicota 47

to a concentration of 0.25M. Crude mitochondria were isolated by differential centrifugation; suspended in 0.25 M sucrose, 5 mM MgCI2, 5 mM NaC1, 10raM Tris (pH 7.2) and treated with 40/zg/ml DNase I and 250/~lg/ml RNase A for one hour. The reactions were terminated by adding EDTA to a concentration of 0.05 M and by chilling. The mitochondria were pelleted at 10,000r.p.m. for 15min, suspended in 100 mM NaC1, 10 mM EDTA, 50 mM Tris (pH 8.5) and lysed by incubation with 1~ sodium dodecyl sulphate for ten minutes at room temperature. Lysed mitochondria were extracted with a mixture of equal volumes of equilibrated phenol and chloro- form +isoamyl alcohol (24:1) and then with only chloroform + isoamyl alcohol. The mt DNA was now purified by sedimentation equilibrium centrifugation in ethidium bromide/cesium chloride gradient. The density of the aqueous layer was adjusted to 1.57 g/ml with solid CsC1 and ethidium bromide was added to a final concentration of 370/.zg/ml. The samples were centrifuged at 38,000 r.p.m, in a Beckman SW 60 or Ti 50 rotor for 36 h. The lower band consisting of supercoiled mt DNA was collected and extracted with salt-saturated isopropanol to remove ethidium bromide and dialyzed against 0.01 M EDTA, 0.01 M Tris (pH8.0). Sodium acetate was added to a concentration of 0.25 M and mt DNA was precipitated by addition of two volumes of ethanol and keeping overnight at - 20~ The mt DNA was then pelleted, dried and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). In later operations, a shorter method was followed and the lysed mitochondria were extracted with phenol and chloroform. Sodium acetate was added to the aqueous layer and mt DNA precipitated by addition of two volumes of ethanol. The mt DNAs were digested with thirteen type II restriction endonucleases with hexanucleotide recognition sequence, following the conditions indicated by the suppliers (Amersham, UK, and Centre for Biochemicals, Delhi). The enzymes are avaI, BamHI, BglII, ClaI, EcoRI, EcoRV, HaeII, HindIII, KpnI, PvuII, ScaI, XbaI and XhoI. About ten-fold excess enzyme was added to bring about complete digestion. The mixture was heated at 65~ for ten minutes prior to loading on a gel. Digested DNAs were resolved on varying percentages of agarose gels to separate all the fragments. HindIII digest of 2 DNA and HaelII digest of 4)-XI74RF DNA were used as molecular weight markers. The gels were stained With ethidium bromide, visualized under UV and photographed. The sizes of the fr'agments were measured with the help of the molecular weight markers run in each gel and the common fragments between the three murid species were scored.

3. Results

111 our phylogenetic analysis, we have used ungulates and primates as outgroup mammals to rodents, on the assumption that these three lineages diverged from each other at the time of radiation of eutherian mammals near the end of the Mesozoic period around 85 m.y. ago (Romer 1966; McKenna 1975; Goodman et aI. 1987). The branching order of muroid lineage and other rodent groups based on a phylogenetic tree of tandemly arranged r and fl-globin sequences is shown in figure la. The evolution of fl-globins in the descent of murines from the rodent ancestor is given in figure lb. Both these phylogenetic trees show essentially the same branching order of rodents. Since parsimony trees are unrooted the figures do not reflect ancestor- 48 S. Barnabas, S. Krishnan and J. Barnabas

Table 1. Nucleotide replacements of globin sequences from rodent ancestor.

NR c~+fl NR[3

For 287 sites For 100 sites For 146 sites For 100 sites Rodent lineage in 65m.y. in 100m.y. in 65m.y. in 100m.y.

Caviinae Guinea-pig 57 30.78 30 31'50 Sciurinae European suslik 41 22.14 21 22.05 Alpine marmot 36 19.44 20 21'00 Ground squirrel 37 19.98 19 19"95 Spalacinae Ehrenberg's mole rat 36 19.44 23 24'15 Cricentinae. Golden hamster 70 37.80 38 39'90 Arvicolinae Musk rat 66 35.64 39 40.95 Murinae Rat 78 42.12 36 (/3 major) 37.80 36 (/3 II) 37.80 41 (//minor 1) 43'05 41 (/3 minor 1) 43.05 Mouse 46 19.5 35 (/3 dmajor) 36.75 34 (/3s) 35.70 35 (/3 dminor) 36.75 38 (/3 pminor) 39'90

descendant relationship of rodents. However, based on paleontological evidences, we presume that the most common ancestor of primates, ungulates and rodents in figure 1 represents the eutherian ancestor and that of Caviidae, Sciuridae and Muridae represents the rodent ancestor. Evolutionary rates of globin sequences presented in table 1 were calculated from our phylogenetic trees (figure la, b). The tandemly combined c~ and//sequences consisted of 287 sites, 141 for c~ and 146 for//. The evolutionary rates are represented as nucleotide replacements (NR) for 100 sites for 100 million years, on the assumption that Rodentia arose around 65 m.y. ago (Shoshani et al. 1985). Restriction enzyme cleavage patterns of mt DNAs of the three murine animals with the thirteen restriction enzymes are given in figure 2. It can be seen from the figure that the mt DNAs from all the three species were linearized by BamHI and PvuII, but otherwise too few fragments are shared between Rattus and Bandicota species though they belong to the same family (Muridae) and sub-family (Murinae). Also out of the thirteen endonucleases used, AvaI, EcoRI, HindIII, ScaI, and XhoI gave a total of thirty-six fragments, out of which ten fragments are shared between the two Bandicota species (figure 2). No common fragments were observed in digestions with BgllI, ClaI, EcoRV, HaeII, KpnI, XbaI. Molecular phylogeny qf Rodentia in the desce~tt o[" Bandicota 49

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4. Discussion

4.1 Globin phylogenj~ of rodents in the descent of murids

Several suprafamilial classifications of rodents are known (Hartenberger 1985). Traditionally rodents have been divided into three major groups: sciuromorphs, myomorphs and hystricomorphs. The most parsimonious phylogeny for vertebrates based on amino acid sequences requiring 5024 nucleotide replacements by Goodman et al. (1987) places guinea-pig (hystricomorph) in rodent lineage which in turn shares a common ancestor with lagomorphs and primates; and shows ungulates as the most ancestral eutherian branch. In a recent phylogenetic analysis of 15 protein sequences involving 1998 aligned amino acid sites, Graur et al. (1991) have shown that guinea-pig diverged before the separation of primates and artiodactyles from the myomorph rodents. In figure 1, guinea-pig diverges from the rodent stem followed by two branches, one representing sciuromorphs and the other myomorphs. If we include chicken globin sequences in figure la, the resultant tree shows guinea-pig as the nearest neighbour of chicken. But a larger data set of tandemly arranged ~ and fl globin sequences ofeutherian mammals with either kangaroo or chicken as outgroup species (unpublished results) confirm the results of Goodman et al. (1987). Clearly a larger body of data of molecular sequences will be needed to clarify the position of guinea-pig. As seen in figure 1, sciurids represented by ground squirrel, marmot and suslik emerge as a monophyletic branch from the rodent stem after the separation of Caviidae and show a relatively small number of nucleotide replacements among them. Sciurid branch in turn joins Muroidae. An earlier analysis of globin sequences using maximum parsimony method (Shoshani et al. 1985) has shown that Sciuridae and Muroidae group together and the resultant branch joins Caviidae. It can be seen in figure 1 that in the murid line of descent, Spalacinae (Ehrenberg's mole rat) arises first followed by two branches, one representing Cricentinae (golden hamster) and Arvicolinae (musk rat) and the other Murinae (mouse and rat). Based on the cladistic analysis of electromorphs of 24 protein loci in 76 species of rodents, Bonhomme et al. (1985), have shown that , Glirimorpha and Caviomorpha separate into three distinct groups. Although inter-generic separation was not clear cut, their analysis nevertheless showed that arvicolids and cricetids are sister groups.

4.2 Evolutionary rationale Jbr the expression of different beta globin genes in murid populations

It is evident from figure 1b that murine animals show a variety of/'/globin types. Earlier, we had studied the globin gene profile of murids common to western India. Our results had shown that Rattus rattus rt!fescens, like Rattus norvegicus (Garrick et al. 1978), has six hemoglobin phenotypes made up of two c~ chains and three fl chains whereas Bandicota indica shows two hemoglobin phenotypes with a single c~ chain and two fl chains (Pratap et al. 1980). The two e chains of rat appear to differ from each other in at least seven amino acid replacements (Garrick et al. 1975). Similarly the amino acid sequences of different fl-globins ofRattus norvegicus, namely fl major (Satoh et al. 1987; Radosavljevic and Crkvenjakov 1989) and fl minor (Ohshita and Hozurni 1987; Wong et al. 1988; Stevanovic and Crkvenjakov 1989) have also been established. The three 52 S. Barnabas, S. Krishnan and J. Bru'nabas

different/~ chains seen in rat populations are probably due to gene triplication (Garrick et aI. 1978; Pratap et al. 1978, 1980). The B. indica populations examined by us earlier (Pratap et al. 1980) showed two categories of phenotypes: one in which the two component hemoglobins occurred in the ratio 50:50 and the other in which the two components had the ratio 75:25. In each of these, one c~ and two // chains were present indicating duplication at the fl locus. Analysis at the protein level has shown that in Lepore hemoglobins (Baglioni 1962; Barnabas and Muller 1962), a hybrid hemoglobin is present as a result of unequal crossover between linked/3 and 6 chain genes. Similarly, variation in gene number in within-species ct-globin in horse (Clegg 1974); monkeys, apes and man (Nute 1974), Macaca radiata (Mathew et al. 1984) and Bubalus bubalis (Balani and Banabas 1965; Hanbarhatty et al. 1983), has been attributed to the presence of duplicate globin genes and unequal crossover between them. Zimmer et al. (1980) have presented evidence for concerted evolution and evolutionary change in the gene number at the c~ chain loCus of adult hemoglobins of chimpanzees. Their studies on five chimpanzees showed that out of the ten chromosomes, eight had three ct chain genes, one had two c~ genes and one had a single c~ gene. Evolution in concert via gene conversion has also been reported in the two human c~ globin genes (Lauer et aI. 1981), the two human -~, globin genes (Slightom et al. 1980; Shen et aI. 1980) and the two human ~ globin genes (Proudfoot et al. 1982). In the general model of concerted evolution proposed by Zimmer et al. (1980), the identical copies of the gene present in ancestral populations could accumulate mutations to give rise to a non-identical doublet. Unequal crossover between duplicate genes followed by mutations could give rise to progeny bearing one gene and three genes per chromosome. Thus it is possible that one-gene, two-gene and three-gene states could exist in a given population either singly or in combination. A second crossover between any two of the states could generate a variety of polymorphic genes and any one or more of these could also get fixed in the population. This model can be applied to understand the presence and expression of multiple copies of/~ globin genes in rats and mice. Thus the presence of three different beta globins in rat populations would imply that the three-gene state has been selected for. Similarly, we can also explain the presence of two types of hemoglobin phenotypes in B. indica populations. Thus we envisage that the ancestral population orB. indica had a non-identical doublet (1-2 type) of beta globin genes which as a result of crossover followed by fixation generated the phenotypes present in the Bandicota populations. Thus the homozygote for the 1-2 type of beta genes in conjunction with a single alpha gene could produce the 50:50 hemoglobin phenotype, whereas the heterozygote for 1-2 type and 1-1-2 type could produce the 75:25 hemoglobin phenotype. The underlying assumption here is that the globin genes present in one of the chromosomes are responsible for the production of 503/o of the total globin. The inbred strains of mice (Mus musculus) produce three alleles at the fl chain locus: d (diffuse), s (single) and p (Hutton et al. 1962). The d and p alleles are closely linked doublets which express a major and a minor beta chain while the s allele produces a single chain (Gilman 1976). In C57BL/10 mouse, the s allele complex has two non- allelic closely similar genes, fls and [J" (Erhart et al. 1985). To account for the presence of d and s allele complexes in mice, Erhart et al. (1985) have proposed an evolutionary scheme in which, from the duplication Of a single ancestral fl-globin gene followed by mutations, two non-allelic genes arose and two distinct ancestral haplotypes (fl* and fl*', fl,i and [jd,) were established; then further evolution followed by gene conversions Molecular phylogeny of R~ in the descent of Bandicota 53

among the evolutionary forerunners (fl~, fl~' and rid) eventually gave rise to fl~ -- fl' and fld,,~j __ fla,,i,, allele complexes in mouse populations. In general, figure lb is compatible with the proposed model. It can be seen that the fl single chain is phylogenetically closer to fl dmajor chain than to flminor chains.

4.3 Evolutionary rates of globin genes in rodents

In our analysis, rates of evolution in the three groups of rodents, namely Caviidae, Sciuridae and Muridae are different. While sciurids show the slowest rate, the murids, with the exception of Spalax (Spalacinae) show the highest. Both the ct + fl sequences and the fl sequences show around 31 NR's for 100 sites for 100m.y. in guinea-pig as compared to around 21 in sciurids. In the murid branch, fl globins evolve at a faster rate than the ~ globins except in rat where the latter shows a higher rate. In rat and mouse, fl minor globins have relatively a higher rate of evolution than fl major globins. Since the average rates of fl globins in golden hamster, musk rat, rat and mouse, from the rodent ancestor are more or less uniform (39.16) we have used this information to arrive at a tentative time of divergence of rat and mouse as well as the time of the emergence of musk rat and golden hamster. Using the average rate of evolution of fl globins in rat and mouse from their common ancestor (7.53 for 100 sites) we estimated the time of divergence of these two species to be around 19.23 m.y. This is compatible with the generally accepted time of mouse-rat divergence of 20 m.y. (Shoshani et al. 1985). Similarly we calculated the emergence of the branch leading to the musk rat and golden hamster from the rodent stem to be around 35-85 m.y. before present. Flynn et al. (1985) have pointed out that Spalacinae occur in the fossil record in the early Miocene: and that Oligocene Tachyoryctoidines may indicate a much earlier origin of Spalacidae.

4.4 Divergence estimate of the two species of bandicoot rats

The genus Bandicota has only the above mentioned two species which were regarded as different genera earlier; B. indica was put under Bandicota and B. bengalensis was called Gunomys. They were later included in the same genus, Bandicota (Ellerman 1961). are found in India, Ceylon, Indo-China, Malaya and other neighbouring islands and are the largest rats of the Asiatic mainland with head and body varying from 160 to 360mm; B. bengalensis being smaller than the B. indica. They are good diggers and construct elaborate with storage chambers, each being inhabited by a single . They usually leave their burrows at night. They are aggressive by nature, emitting a harsh nasal bark when disturbed. Rattus rattus is world-wide in distribution and is the common Rattus species in the Indo-Malayan region. It has two forms, the white-bellied wild form and the dull-bellied commensal form (Rattus rattus rt!fescens). The,mammalian mt DNA is a circular molecule of about 16,000 base pairs in these rodents; itis maternally inherited, does not undergo recombination and its mutation rate is higher than that of nuclear DNA. These properties make it a very useful tool for studying the relationships among closely related organisms. The mt DNAs of the three murids were digested with thirteen restriction endonucleases in order to locate shared fragments between the three species. The proportion of DNA fragments shared by populations is expected to be correlated with the degree of genetic divergence between 54 S. Barnabas, S. Krishnan and J. Barnabas

them. Since the two genera Rattus and Bandicota share very few common fragments in the restriction profile of eleven restriction enzymes and are only linearised by two restriction enzymes (figure 2), we believe that Rattus and Bandicota are quite divergent from each other. An electrophoretic profile of twenty-four protein loci, for a large number ofmurids and non-murid muroids found Rattus to be the most divergent genus (Bonhomme et al. 1985). Mus and Rattus both included in Muridae show a divergence estimate of about thirty percent (Jakovcic et al. 1975). Our results also suggest that Muridae is a very diverse group and Rattus is an outgroup genus to Bandicota. Based on figure 2, the proportion of shared fragments (F) was estimated according to the equation, F = 2nxy/(nx + ny) (Nei and Li 1979), where nx and ny are the number of fragments in populations x and y, respectively, while nxy is the number of fragments shared by the two populations, and the value of F was converted to estimates of nucleotide divergence. Thus the percentage sequence divergence between B. indica and B. bengalensis is about 3.6~ (when nxy = 10 and about 2.63/o when nxy = 12, if the single fragments obtained from BamHI, PvuII digestions are included among the shared fragments). If the mean rate of divergence in the mt DNA molecule is assumed to be 2~ per million years (Wilson et al. 1985) then the divergence estimate for B. indica and B. bengalensis is 1.8 m.y.

Acknowledgements

We would like to thank Prof. A. S. Kolaskar for giving us access to the PIR data base. One of us (S.B.) thanks the University Grants Commission for the award of UGC - Research Scientist's position and Dr. R. A. Mashelkar, National Chemical Laboratory, for the opportunity to work at NCL. She also thanks Mr. M. P. Karunakaran for procuring the tissues.

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