evolutionary systematics of the indian mouse mus famulus bonhote, 1898: molecular (dna/dna...

Zoological Journal of the Linnean Society

, 2003,

137

, 385–401. With 5 figures

© 2003 The Linnean Society of London,

Zoological Journal of the Linnean Society,

2003,

137

, 385–401

385

Blackwell Science, Ltd

Oxford, UK

ZOJZoological Journal of the Linnean Society

0024-4082The Lin-nean Society of London, 2003

137

Original Article

Pascale Chevret et al.Evolutionary systematics of

Mus famulus

Corresponding author. Pascale Chevret. Tel.

+

33 4 67 14 32 54; fax:

+

33 4 67 14 36 10; e-mail: [email protected]

Evolutionary systematics of the Indian mouse

Mus famulus

Bonhote, 1898: molecular (DNA/DNA hybridization and 12S rRNA sequences) and morphological evidence

PASCALE CHEVRET

1

, PAULINA JENKINS

2

and FRANÇOIS CATZEFLIS

1

1

Laboratoire de Paléontologie, Paléobiologie, et Phylogénie, Institut des Sciences de l’Evolution (UMR 5554, CNRS), CC 064, Université Montpellier 2, F-34095 Montpellier cedex 5, France

2

The Natural History Museum, Mammal Group, Cromwell Road, London SW7 5BD, UK

Received January 2001; accepted for publication October 2002

The genus

Mus

encompasses 38 species of mice divided into four subgenera:

Mus

,

Pyromys

,

Nannomys

and

Coelomys

.Each of these four taxa is characterized by discrete morphological as well as biochemical traits. We used two differentmolecular approaches to determine the relationships between these subgenera: DNA/DNA hybridization and 12SrRNA mitochondrial sequences. We compared the resulting phylogenies from each method and with phylogeniesderived from morphological data. The degree of resolution of each molecular approach is discussed. The two molec-ular studies indicate that

Mus

,

Pyromys

,

Nannomys

and

Coelomys

are clearly distinct monophyletic groups, as pre-viously indicated by morphological data and other biochemical and molecular approaches. There is one divergencebetween previous morphological and the molecular and morphological studies presented here: the position of theIndian species

Mus famulus

. This taxon, which was formerly included in the subgenus

Coelomys

, is demonstratedhere to belong to the subgenus

Mus.

We also propose the following relationships within

Mus sensu lato

:

Mus

and

Pyromys

are the closest relatives, followed by

Nannomys

and

Coelomys

, whose relationships are still unclear. Thisarrangement is more robustly supported by DNA/DNA hybridization than by 12S rRNA data. A molecular time scalefor the evolution within

Mus sensu lato

is proposed, using as a reference the

Mus/Rattus

divergence estimated bythe fossil record at around 12 mya. © 2003 The Linnean Society of London,

Zoological Journal of the Linnean Soci-ety

, 2003,

137

, 385–401.

ADDITIONAL KEYWORDS: Murinae –

Mus

– scnDNA hybridization – 12S rRNA – systematics – India.

INTRODUCTION

The genus

Mus

Linnaeus, 1758, the common mouse(subfamily Murinae: Rodentia), comprises four sub-genera, well characterized morphologically (Marshall,1977, 1988; Nowak, 1991; Musser & Carleton, 1993):•

Mus

sensu stricto, house, field and commensalmice, nine species, mainly from Eurasia; feral anddomestic, world-wide

-

populations of

M

.

muscu-lus/domesticus

belong to this taxon•

Coelomys

Thomas, 1915a; shrew mice, five asiaticspecies

•

Pyromys

Thomas, 1911; spiny mice, five asiaticspecies;

•

Nannomys

Peters, 1876; pygmy mice, 19 Africanspecies.The taxonomy and systematics of mice (genus

Mussensu lato

) have long been studied using differentapproaches such as morphology (Misonne, 1969;Marshall, 1977, 1981), biochemistry (Bonhomme

et al

., 1984; Bonhomme, 1986; She

et al

., 1990) andmolecular biology (Catzeflis & Denys, 1992; Lundrigan& Tucker, 1994; Sourrouille

et al

., 1995). Some bio-chemical studies emphasized very large divergencesbetween representatives of the subgenera and led theauthors to consider them to be distinct genera(Bonhomme

et al

., 1984; Bonhomme, 1986, 1992).Among molecular methods, DNA/DNA hybridiza-

tion (Rice & Strauss, 1973; Benveniste

et al

., 1977;She

et al

., 1990; Catzeflis & Denys, 1992), as wellas study of the mitochondrial 12S ribosomal gene

386

P. CHEVRET

ET AL

.

© 2003 The Linnean Society of London,

Zoological Journal of the Linnean Society,

2003,

137

, 385–401

(Sourrouille

et al

., 1995), have been used to determinethe relationships between different species of

Mussensu lato

. The aim of this paper is to enhance theresults already available for this genus by addingsome new data and to focus particularly on the sys-tematic position of one of the shrew mice,

Mus famu-lus

Bonhote, 1898.

Mus famulus

is a poorly known asiatic species, onlyrecorded from the Nilgiri Mountains in South India.Based on skull characters, the few available specimens(the holotype and one individual housed in the NaturalHistory Museum, London) were attributed to the sub-genus

Coelomys

by Marshall (1977). Other taxaincluded in

Coelomys

are

M

.

vulcani

(Robinson & Kloss,1919),

M

.

crociduroides

(Robinson & Kloss, 1916),

M

.

pahari

Thomas (1916) and

M

.

mayori

(Thomas, 1915a)

.

Recent field work in Tamil Nadu, South India, provided10 specimens of a beautiful, long-nosed, small-eyedmouse that we referred to

Mus famulus

. These sampleswere included in a general study of

Mus

as a represen-tative of

Coelomys

, in order to determine the relation-ships within

Mus sensu lato

. In this paper, the taxonname

Mus

will be used to designate the subgenus

Mus

,whereas

Mus sensu lato

will be used to encompass the

whole genus. This study includes six species currentlyassigned to

Mus

, two to

Pyromys

, two to

Nannomys

andthree to

Coelomys

, and will investigate sequence aswell as DNA/DNA hybridization data, together with amorphological survey of dental and cranial characters.Two additional species, one assigned to

Nannomys

andone to

Coelomys

, were included in the morphologicalanalysis.

MATERIALS AND METHODS

DNA samples were extracted from 95% ethanol-preserved tissues housed in the collection of PreservedMammalian Tissues of the Institut des Sciences del’Evolution, Montpellier (Catzeflis, 1991). Table 1 listsall the taxa involved in the molecular study, their geo-graphical origins, collectors’ names and sequence orreference number. Voucher specimens of

Mus famulus

are lodged in the following institutions: MuséumNational d’Histoire Naturelle, Paris [MNHN],Museum of Vertebrate Zoology, Berkeley [MVZ] andThe Natural History Museum, London [BM(NH)].DNA was extracted from tissue samples of the follow-ing specimens: MNHN-1995-1315 and 1316, MVZ

Table 1.

List of species included in the molecular studies, with the geographical origin, collectors’ names and sequence ref-erences or numbers. The laboratory strains (described in Potter, 1986) are housed in the Laboratoire Génome, Populations,Interactions at the University Montpellier II, France (Bonhomme

et al

., 1984).

Taxon Geographic origin CollectorAccession number(12S rRNA sequence)

Subgenus

MusMus caroli

Thailand Laboratory strain KAR AJ279437

Mus cervicolor

Thailand Laboratory strain CRP AJ279441

Mus cookii

Thailand Laboratory strain COK Sourrouille

et al

., 1995

Mus musculus

Austria Laboratory strain MAI Sourrouille

et al.

, 1995

Mus spicilegus

Yugoslavia Laboratory strain ZYD AJ279439

u

Mus spretus

Spain Laboratory strain SEI AJ279438Subgenus

PyromysPyromys saxicola

India F. Catzeflis Sourrouille

et al

., 1995

Pyromys platythrix

India Laboratory strain PTX Sourrouille

et al

., 1995Subgenus

NannomysNannomys

cf

setulosus

Gabon V. Nancé Sourrouille

et al

., 1995*

Nannomys mattheyi

Burkina Faso T. Madalena Sourrouille

et al

., 1995Subgenus

CoelomysCoelomys pahari

Thailand Laboratory strain PAH Sourrouille

et al

., 1995*

Coelomys crociduroides

Sumatra M. Ruedi Sourrouille

et al

., 1995

Mus

?

Coelomys

?

famulus

India F. Catzeflis AJ279442Outgroups

Malacomys longipes

Gabon V. Nancé AJ279440

u

Rattus norvegicus

Kobyashi

et al.

, 1981

Rattus rattus

Pakistan W. Din Not done

uUnlabelled species.*Taxa that were not available for the DNA/DNA hybridization experiments.

EVOLUTIONARY SYSTEMATICS OF MUS FAMULUS 387

© 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137, 385–401

182985 and 182986 and BM (NH) 2000.78. For all thespecies included in this study we completed DNA/DNA hybridization data (She et al., 1990; Catzeflis &Denys, 1992) and 12S rRNA datasets (Sourrouilleet al., 1995) in order to present trees built with themaximum number of species in common. Neverthe-less, two species, Nannomys mattheyi Petter, 1969 andCoelomys crociduroides, were not available for theDNA hybridization experiments and appear only in

the 12S rRNA dataset. In both studies, we used twogenera of Murinae, Malacomys and Rattus, as out-groups. Table 2 records the specimens studied in themorphological analysis.

The Mus famulus specimens were caught in October1990 in two localities in Tamil Nadu state, India. Aval-lanchi (11∞23¢ N 76∞36¢ E) (three specimens) and Kota-giri (11∞26¢ N 76∞53¢ E) (seven specimens) both lie inthe Nilgiri Mountains, at 2100 and 1500 m above sea

Table 2. List of specimens included in the morphological analysis

Taxon Accession number Type status

Subgenus MusMus caroli BM (NH) 1902.10.7.17 Holotype

BM (NH) 1971.3166–3167Mus cervicolor BM (NH) 1845.1.8.383 Lectotype

BM (NH) 1976. 1857–1859Mus cookii BM (NH) 1913.11.18.2 Holotype

BM (NH) 1918.7.1.2BM (NH) 1918.7.4–5BM (NH) 1923.1.8.5 Holotype of Leggada palnica Thomas, 1923

Mus musculus BM (NH) 1962. 1748– 1749, 1751Mus spicilegus BM (NH) 1894.7.26.1–2 SyntypesMus spretus BM (NH) 1919.7.7. 1862 Holotype

Subgenus PyromysMus platythrix BM (NH) 1855.12.26.282 Holotype

BM (NH) 1928.9.1.84–87Mus saxicola BM (NH)32d Lectotype

BM (NH) 1975.1687–1692Subgenus Nannomys

Mus mattheyi MNHN 1972–581Mus minutoides BM (NH) 1965.1087–1089Mus setulosus BM (NH) 1901.11.21.29

BM (NH) 1914.1.24.28Subgenus Coelomys

Mus crociduroides BM (NH) 1919.11.5.62 HolotypeBM (NH) 1919.11.5.60BM (NH) 1919.11.5.63

Mus famulus BM (NH) 1897.11.12.1 HolotypeBM (NH) 1919.6.2.38BM (NH) 2000.78–80MNHN 2000–252MNHN 2000-265-270

Mus mayori BM (NH) 1914.12.1.7 HolotypeBM (NH) 1915.3.1.243–245BM (NH) 1914.12.1.8 Holotype of Coelomys bicolor Thomas, 1915b

Mus pahari BM (NH) 1915.9.1.199 HolotypeBM (NH) 1916.3.25.113–116

OutgroupsMalacomys longipes BM (NH) 1976.1516–1517Rattus norvegicus BM (NH) 1970. 1781

BM (NH) 1970. 1787Rattus rattus BM (NH) 1919.11.7.73

BM (NH) 1919.11.7.75

388 P. CHEVRET ET AL.


level, respectively. All shrew-mice were trapped inremnants of evergreen tropical primary forest knownas ‘long-wood shola forests’. Other small terrestrialmammals caught in the same trap-lines were the sori-cids Suncus murinus, Suncus montanus and Suncusdayi (Ruedi et al., 1996; Jenkins et al., 1998) and themurines Mus musculus Linnaeus, 1758, Rattus rattusand Millardia meltada. Vouchers of wild-caught orlaboratory-born specimens of M. famulus are depos-ited at the Museum of Vertebrate Zoology, Berkeley(MVZ-182985-182992), at the Museum Nationald’Histoire Naturelle, Paris (MNHN 1995-1315-1320,2000-265-270) and at The Natural History Museum,London [formerly the British Museum (Natural His-tory)] (BMNH 2000.78-80). The Avallanchi–Kotagirispecimens agree in pelage colour and morphology withthe holotype of Mus famulus, although on averagethey are larger and more robust than the few availablespecimens in the reference collection, thus extendingthe known size range of the species. Size variation isshown in Table 3.

With regard to subgeneric affiliation, we observedthat neither the two reference Mus famulus speci-mens, nor our 1990-caught animals exhibit some ofthe cranial characters (broad interpterygoid space;narrow, reclined zygomatic plate), which Marshall(1977) used for diagnosing the subgenus Coelomys,although other characters listed by him and Corbet &Hill (1992) are present (broad interorbital region).

DNA/DNA HYBRIDIZATION

DNA of each species, one to three samples per species,was purified and sheared into fragments of c. 500 basepairs (bp) length (range 200–1000 bp). The non-repeated nuclear DNA fractions were isolated byremoving onto hydroxyapatite columns (BIO-GELHTP, Biorad Laboratories) the highly repeatedsequences which reassociated at Cot 1000 (Cot:product of the DNA concentration by the time of reas-sociation) in 0.48 M phosphate buffer at 55∞C. Thesenon-repeated DNA fractions were chemically labelledwith 125I.

DNA/DNA hybrids, formed by one part of labelledDNA (tracer) and 400–1000 parts of non-labelled totalDNA (driver), were allowed to reassociate after heatdenaturation to a Cot of 16 000 at 60∞C in 0.48 M phos-phate buffer. The thermal elutions were begun at55∞C, with 2.5∞C increments up to 95∞C, and the rawdata are the radioactive counts eluted at each of the 17temperatures in the 55–95∞C range. The proceduresare the same as those published in our previous stud-ies (Catzeflis, 1990; Catzeflis & Denys, 1992; Chevretet al., 1993, 1994).

Several statistics can be calculated to estimate thedifferences between the thermal elution curves of

homoduplex (tracer and driver of the same species)and heteroduplex (tracer and driver of different spe-cies) hybrids. In this paper we used Tm and Mode,which are less variable than the other statistics for themuroid rodents (Catzeflis, 1990). Tm is the tempera-ture at which 50% of the hybrid DNA has been disso-ciated between 62.5 and 95∞C, and Mode is the highestpoint of the melting curve of radioactive counts vs.temperature. Delta values are the difference betweenthe Tm or Mode of the homologue minus the value ofthe one of an heterologue.

PHYLOGENY RECONSTRUCTION

We used two different approaches to validate the treesderived from our DNA/DNA hybridization distance

Table 3. Selected external and cranial measurements inmm and weight in g of Mus famulus. Presented as mean,standard deviation and range with number of specimens inparentheses. Measurements of the holotype, where avail-able, are provided in square brackets

Occipito-nasal length 24.76 ± 0.823.3–26.0 (9)[23.3]

Condylo-incisive length 23.6 ± 0.4922.7–24.3 (8)

Palatal formina length 4.58 ± 0.274.1–4.9 (9)[4.9]

Diastema length 6.43 ± 0.246.1–6.9 (9)[6.1]

Length of upper toothrow 3.93 ± 0.113.8–4.1 (9)[4.1]

Interorbital breadth 4.28 ± 0.164.1–4.5 (9)[4.1]

Braincase breadth 10.62 ± 0.2510.2–11.0 (9)[10.4]

Head and body length 90.68 ± 5.9690.4–100 (8)[100]

Tail length 87.42 ± 7.8871–94 (6)[71]

Hindfoot length 19.81 ± 0.9718.5–21 (8)[20]

Ear length 15.69 ± 1.5412–17 (8)[12]

Weight 22.88 ± 4.423–30.4 (5)



matrices: bootstrap (Krajewski & Dickerman, 1990)and jacknife (Lanyon, 1985; Lapointe et al., 1994).

BootstrapBootstrap procedures were applied to 12*12 distancematrices, that included only tracers. The bootstrap,as described by Krajewski & Dickerman (1990) andSheldon et al. (1992), is performed on uncorrected dis-tance values. It samples with replacement the repli-cate measures in each cell of a matrix. For each step,a pseudo-replicate matrix is constructed by recalculat-ing the average distance for each cell. As our matricesare incomplete (28% missing cells), we had to symme-trize and complete each pseudoreplicate matrix, usingthe additive procedure of Landry et al. (1996). Eachsymmetrized and completed pseudo-replicate matrixwas treated by the FITCH program (PHYLIP 3.5c pack-age, Felsenstein, 1993), which estimates the best-fittree. This cycle was repeated 100 times for each of thetwo matrices (delta-Tm and delta-Mode), and theresulting topologies were recorded. Next, a majority-rule consensus tree was derived from the replicatebootstrap topologies by the CONSENSE program inPHYLIP (Felsenstein, 1993). Each node of the resultingconsensus tree was characterized by the frequency atwhich the dichotomy of interest was found among the100 pseudoreplicate trees.

JacknifeWe used also 13*13 matrices, which included one non-labelled taxon: Mus spretus Lataste, 1883. They werecompleted with the additive method described byLandry et al. (1996). The FITCH algorithm was usedto reconstruct phylogeny. The trees were validated bysimple jacknife (Lanyon, 1985), one deletion at a time,and weighted jacknife (Lapointe et al., 1994) with theMAJACK package, provided by F. J. Lapointe. Thismethod realized all possible deletions (from 1 to 9),which represents in our case 7813 different sub-matrices, and yields three complementary trees: anaverage consensus tree, a minimum and a maximumtree.

Divergence date estimationsThe delta-Tm values of the completed 13*13 matrixwere transformed into percentage base pair mismatch(bpm) estimated by the relation of 1∞C delta-Tm = 1.18% pbm (Springer et al., 1992). Theseestimates were finally transformed into percent nucle-otide substitutions (% nucl. subst.) with the Jukes &Cantor formula (1969), which corrects for multiplesubstitutions. The % nucl. subst. values were cali-brated against the geological time provided by the fos-

sil record, in our case the Mus–Rattus dichotomyestimated at c. 12 mya (Jacobs & Pilbeam, 1980;Jaeger et al., 1986; Jacobs & Downs, 1994). A tree wasthen constructed with the KITSCH program (PHYLIP

3.5, Felsenstein, 1993), which implies a molecular-clock hypothesis.

DNA SEQUENCING

Sequences and alignmentThe newly acquired sequences complete the taxonomicsampling of Mus 12S rRNA sequences already pub-lished in Sourrouille et al. (1995) in order as far as pos-sible to obtain similar representatives as in the DNA/DNA hybridization datasets (Table 1). We used thesame primers as Sourrouille et al. (1995) to amplify andsequence the complete 12S rRNA gene. PCR productswere purified with the Wizard™ ‘PCR preps DNA puri-fication system’ (Promega) and directly sequenced with[33P]ddNTP and ‘Thermosequenase radiolabelled ter-minator cycle sequencing kit’ from Amersham.

The sequences were manually aligned using the EDprogram (MUST package, Philippe, 1993). The align-ment was refined in order to minimize the number ofindels (insertions-deletions) in stems (Douzery &Catzeflis, 1995; Springer & Douzery, 1996) using themodel of Gutell for Mus (Sullivan et al., 1995).

We checked for saturation by comparing observedand inferred (derived from maximum parsimony)nucleotide differences (Hassanin et al., 1998), whichoccurred in four partitions: transitions and transver-sions in stems and loops.

Phylogeny reconstructionWe performed distance (NJ = Neighbour-joining,Saitou & Nei, 1987), maximum parsimony (PAUP

4.0b4a, Swofford, 1999) and maximum likelihood(TREEPUZZLE, Version 5.0, Strimmer & von Haeseler,1996) analyses. The robustness of the inferred treeswas assessed by bootstrap percentage (BP) for dis-tance and parsimony, Bremer Support Index (BSI)(Bremer, 1988) for parsimony and Reliability Percent-age support values for maximum likelihood. The dif-ferent analyses were performed (i) using all thesubstitutions (TV + TI, indels were excluded), (ii) aweighted analysis undertaken with the parametersderived from the saturation analysis, and (iii) with theinclusion of indels.

MORPHOLOGICAL ANALYSIS

Cranial, dental and external characters were scoredfrom the specimens belonging to the taxa included inthe molecular analyses, with the addition of represen-tatives of Coelomys mayori and Nannomys minutoides



Smith, 1834, see Table 2. As for the molecular analy-ses, taxa used for the outgroup were Malacomyslongipes, Rattus norvegicus and R. rattus. Dentalnomenclature follows Rosevear (1969: 21). Characterpolarity for most of the cranial characters was deter-mined following Carleton (1980). Characters used inthe analysis were scored as follows (see Table 4 forcharacter matrix).1. First upper molar (M1): (0) smaller than combined

second (M2) and third (M3) upper molars; (1)longer than combined M2 and M3.

2. M3: (0) moderate in size; (1) reduced but largerthan T8 and T9 of M2; (2) very reduced, smallerthan T8 and T9 of M2. A simple linear progressionis deduced for this character.

3. Anterior loph on T2 of M1: (0) absent or poorlymarked; (1) present, well developed; (2) forms dis-tinct cusp. Development of an additional cusp isconsidered to represent the derived condition.

4. T1 of M1: (0) in line with, or slightly posterior to T2and T3; (1) posterior to T2 and T3; (2) markedlyposterior to T2 and T3.

5. M2: (0) moderate in size; (1) reduced.6. Upper incisor (I1): (0) opisthodont; (1) orthodont;

(2) pro-odont.7. I1: (0) subapical notch poorly developed or absent;

(1) well developed.8. Subsquamosal foramen: (0) absent; (1) present but

small; hamular process short; (2) large; hamularprocess long and downcurved.

9. Posterolateral palatal pits: (0) absent or 1 or 2small foramina present; (1) multiple foraminarecessed in pit.

10. Length of incisive foramina: (0) penetratingbetween anterior root of M1; (1) penetratingbetween root 2 of M1; (2) not reaching anteriorroot.

11. Suture between maxillary and palatine: (0) levelwith anterior of M2 or junction between M1 andM2; (1) level with posterior of M1; (2) level withmid-region of M1.

12. Palatine foramina: (0) rounded, at junction ofmaxillary and palatine; (1) oblong, penetratingboth maxillary and palatine; (2) large and smallforamina present; (3) long and slit-like.

13. Anterior border of mesopterygoid fossa: (0)medium, rounded; (1) narrow, tapered; (2) broad.

14. Lateral pterygoid fossa: (0) level with or slightlyrecessed above level of bony palate; (1) obviouslyrecessed above level of bony palate.

15. Supraorbital ridges: (0) absent; (1) present overposterior region of orbit, but temporal ridgesabsent; (2) distinct, extending from frontals toparietals.

16. Zygomatic notch: (0) absent or barely evident; (1)moderate notch present; (2) forms a deep recess.

17. Pelage: (0) soft, guard hairs normal; (1) harsh intexture, spinous guard hairs present. The pres-ence of spines is believed to be a derived featureoccurring in many rodent groups.

Table 4. Matrix for 17 morphological characters in 18 murine taxa. Charactersand their states are described in the text.

Taxon

Character

1 2 3 4 5 6 7 8 9 10

11

12

13

14

15

16

17

Mus caroli 1 1 1 2 0 1 1 2 0 0 0 0 0 1 1 2 0Mus cervicolor 1 1 1 2 0 0 1 2 1 1 0 2 0 1 1 2 0Mus cooki 1 1 1 2 0 1 1 2 1 0 0 2 2 1 1 2 0Mus musculus 1 1 0 2 0 1 1 2 1 0 0 1 1 0 1 2 0Mus spicilegus 1 1 0 2 0 ? ? ? 1 0 0 1 ? 0 1 2 0Mus spretus 1 1 1 2 0 1 1 2 1 0 0 1 1 0 1 2 0Mus saxicola 1 0 2 1 0 1 0 1 ? 1 0 3 0 0 2 2 1Mus platythrix 1 0 1 1 0 1 0 1 1 0 0 2 0 0 2 2 1Mus setulosus 1 2 1 2 0 1 1 2 0 0 1 3 2 1 1 1 0Mus minutoides 1 2 1 2 0 0 1 2 ? 0 1 3 0 1 1 2 0Mus mattheyi 1 2 1 2 0 1 1 2 0 0 1 3 0 1 1 2 0Mus pahari 1 2 0 1 0 1 0 2 1 2 1 1 2 0 0 1 1Mus mayori 1 2 0 1 0 0 0 1 1 2 2 3 2 1 2 2 1Mus crociduroides 1 2 0 1 1 0 1 2 ? 2 2 1 2 0 0 0 0Mus famulus 1 1 1 2 0 1 1 2 1 0 0 2 2 1 1 2 0Malacomys longipes 0 0 0 0 0 0 0 0 1 2 0 2 2 0 1 2 0Rattus norvegicus 0 0 0 0 0 0 0 0 1 0 0 2 2 1 2 2 0Rattus rattus 0 0 0 0 0 0 0 0 ? 0 0 2 2 1 2 2 0



Branch and bound analyses were carried out usingmaximum parsimony (PAUP 4.0b3a, Swofford, 1999),with a maximum trees setting of 100, all characterstreated as unordered. The resulting trees were com-pared with those obtained from the molecular analy-ses and conclusions drawn about possible specific andsubgeneric relationships.

RESULTS

DNA/DNA HYBRIDIZATION

We present the results of 12 tracers (Tables 5 and 6):the Indian specimens named Mus famulus, five spe-cies of Mus: musculus, spicilegus Petényi, 1882, cervi-color Hodgson, 1845, caroli Bonhote, 1902 and cookiiRyley, 1914; two of Pyromys: platythrix Bennett, 1832and saxicola Elliot, 1839; Coelomys pahari, one spe-cies of Nannomys and two other Murinae, Malacomysand Rattus. Mus spretus, which was not labelled, wasadded to the analysis, as it was included in the 12SrRNA dataset; this species was used as a driver in sev-eral experiments (Tables 5 and 6). Some of the resultshave already been published, namely those based onthe tracers Mus musculus, M. spicilegus and M. caroli(She et al., 1990), M. spicilegus, Nannomys setulosusPeters (1876) and Pyromys platythrix (Catzeflis &Denys, 1992). The tracers M. cervicolor, M. cookii, M.famulus, C. pahari, P. saxicola, Malacomys longipesand Rattus rattus have been produced for this paper.The results are somewhat incomplete because not allspecies involved in the most recent experiments, forexample M. famulus, P. saxicola and Malacomys lon-gipes, were available for the previous ones (Tables 5and 6).

All analyses provide strong support for the Musgroup, which is retrieved in all trees. This subgenusappears with 100% bootstrap values in the 12*12matrices (Fig. 1A and B), in the average jacknife of the13 taxa matrices (Fig. 1C and D), and in all jacknifetrees with single deletions. Figure 1(A and B) showsthat the subgenus Mus is grouped with Pyromys (74%for Tm and 78% for mode), and that Nannomys clus-ters with Coelomys (64% for delta-tm, 93% for delta-mode). The two species of Pyromys are very closelyrelated; the bootstrap support for Pyromys as well asfor Mus is 100%. The jacknife trees (Fig. 1C and D)indicate also that Pyromys and Mus are more closelyrelated but propose conflicting hypotheses for the posi-tion of Nannomys and Coelomys. We thus cannotclearly define the branching order of Coelomys andNannomys. The different approaches used show eitherNannomys and Coelomys pahari together (Fig. 1A andB), Nannomys basal (Fig. 1C) or C. pahari basal(Fig. 1D). According to these data, the basal diver-gence within Mus sensu lato should be depicted as

a multitomy: Mus and Pyromys – Nannomys –Coelomys.

Interestingly, the two species traditionally allocatedto Coelomys (M. pahari, M. famulus) involved in thisstudy are not closely related. Coelomys pahari is anearly offshoot of the Mus sensu lato radiation and M.famulus is nested within Mus, either basal to this sub-genus (Fig. 1B), or closely related to M. musculus andM. spicilegus (Fig. 1A, C and D).

At the specific level within Mus, there are two maingroups. An Asian group comprises cervicolor, cookiiand caroli, with cervicolor and cookii more closelyrelated (Fig. 1), whereas the Palearctic group com-prises musculus, spretus and spicilegus. Mus famulusis either associated with this Palearctic group(Fig. 1A, C and D) or is basal to the Mus group(Fig. 1B). The hybridization data do not allow us tospecify the relationships between musculus, spretusand spicilegus (Fig. 1C and D).

The tree reconstructed with a molecular clockhypothesis (Fig. 2) proposes the same general topol-ogy, with Mus more closely related to Pyromys, thenNannomys and finally Coelomys pahari. However, theancestral segment that defines Coelomys pahari isvery short and not robustly supported as previouslyindicated. The different divergence times are indi-cated in Table 7. Coelomys and Nannomys diverged ataround 6.5 mya, then Pyromys at 5 mya and the sub-genus Mus split into two groups at around 3 mya. Thedivergence between spicilegus, spretus and musculustook place around 1–1.5 mya and famulus divergedfrom its nearest relatives around 2.8 mya.

12S rRNA SEQUENCES

Sequence evolutionThe alignment of the complete 12S rRNA sequencescomprises 989 nucleotides. After excluding positionswith indels, the final alignment includes 909 sites.Based on the secondary structure, this matrix wasdivided in stems (447 sites, with 17 parsimony-informative ones) and loops (462 sites, with 84parsimony-informative ones). The graphical saturationanalyses (comparing observed to inferred changes)indicate that there is no saturation in stems, with slopevalues of 1.0 (transversions) and 0.95 (transitions) andfew saturation in loops with slopes = 0.62 (transver-sions) and 0.61 (transitions). However, we checked theeffect of this moderate saturation by using step matri-ces in the maximum parsimony analysis. We alsochecked the effect of including the indels in our dataset.This was done on the complete alignment, except twohighly variable segments of 8 and 34 sites, which couldnot be unambiguously aligned. We performed the sameanalyses with distance, parsimony and maximum like-lihood on this larger dataset of 942 sites.



Tab

le 5

.R

esu

lts

of D

NA

/DN

A h

ybri

diza

tion

exp

erim

ents

: Raw

Tm

val

ues

(hom

olog

ue

com

pari

son

s) a

nd

delt

a-T

m v

alu

es (h

eter

olog

ue

com

pari

son

s), w

ith

sta

nda

rdde

viat

ion

s an

d n

um

ber

of h

ybri

ds

Tra

cers

/D

rive

rsM

us

fam

ulu

sM

us

caro

liM

us

cerv

icol

orM

us

cook

iiM

us

mu

scu

lus

Mu

ssp

icil

egu

sP

yrom

yspl

atyt

hri

xP

yrom

yssa

xico

laC

oelo

mys

pah

ari

Nan

nom

ysM

alac

omys

Rat

tus

ratt

us

Mu

s fam

ulu

s85

.23

NA

NA

2.99

NA

NA

NA

5.96

6.54

7.07

9.90

12.7

40.

34/n

= 9

0.26

/n =

20.

13/n

= 3

0.18

/n =

30.

09/n

= 3

0.57

/n =

20.

54/n

= 2

Mu

s ca

roli

4.46

81.5

33.

923.

094.

174.

51N

A6.

827.

057.

8410

.44

NA

0.36

/n =

30.

29/n

= 7

0.06

/n =

30.

33/n

= 3

0.32

/n =

20.

11/n

= 2

0.18

/n =

20.

35/n

= 2

0.36

/n =

30.

46/n

= 2

Mu

s cerv

icol

or3.

693.

0885

.18

1.84

3.82

4.28

NA

5.96

NA

6.92

10.2

5N

A0.

04/n

= 3

0.39

/n =

30.

08/n

= 8

0.27

/n =

30.

24/n

= 2

0.10

/n =

20.

19/n

= 2

0.06

/n =

30.

19/n

= 2

Mu

s co

okii

4.11

3.41

2.70

85.0

33.

934.

096.

506.

547.

217.

2310

.16

12.4

70.

23/n

= 3

0.37

/n =

40.

08/n

= 3

0.25

/n =

50.

29/n

= 2

0.08

/n =

20.

24/n

= 3

0.36

/n =

30.

26/n

= 2

0.01

/n =

20.

62/n

= 2

0.41

/n =

2M

us mu

scu

lus

4.11

4.02

4.52

3.98

83.8

12.

186.

936.

807.

317.

3210

.56

12.3

30.

10/n

= 3

0.11

/n =

30.

24/n

= 3

0.61

/n =

30.

30/n

= 4

0.12

/n =

10

0.28

/n =

40.

37/n

= 4

0.30

/n =

20.

06/n

= 2

0.62

/n =

20.

30/n

= 2

Mu

s spic

ileg

us

3.37

3.72

NA

NA

1.55

85.5

3N

A6.

41N

A7.

16N

AN

A0.

22/n

= 2

0.17

/n =

20.

15/n

= 5

0.22

/n =

50.

18/n

= 2

0.06

/n =

2P

yrom

yspl

atyt

hri

x6.

406.

02N

A4.

996.

206.

9283

.94

0.52

NA

7.10

9.45

12.1

10.

22/n

= 4

0.27

/n =

30.

28/n

= 2

0.01

/n =

20.

08/n

= 3

0.54

/n =

50.

23/n

= 4

0.11

/n =

20.

69/n

= 2

0.11

/n =

2P

yrom

yssa

xico

la6.

40N

A6.

45N

AN

AN

AN

A84

.74

6.67

6.95

9.81

12.2

60.

11/n

= 3

0.17

/n =

30.

15/n

= 6

0.11

/n =

20.

10/n

= 3

0.11

/n =

20.

01/n

= 2

Coe

lom

yspa

har

i8.

317.

167.

845.

988.

639.

307.

757.

5484

.87

8.31

11.1

812

.74

0.25

/n =

40.

24/n

= 3

0.12

/n =

30.

91/n

= 2

0.49

/n =

20.

11/n

= 4

0.30

/n =

20.

41/n

= 4

0.31

/n =

40.

26/n

= 3

0.07

/n =

30.

48/n

= 3

Nan

nom

ys7.

48N

A7.

236.

91N

AN

A10

.09

6.77

7.19

85.1

110

.22

NA

0.15

/n =

40.

12/n

= 3

0.34

/n =

20.

73/n

= 3

0.15

/n =

30.

01/n

= 2

0.11

/n =

50.

25/n

= 3

Mal

acom

ysN

A9.

8710

.96

NA

NA

NA

NA

10.8

3N

AN

A82

.49

11.7

30.

20/n

= 2

0.18

/n =

30.

86/n

= 4

0.37

/n =

60.

28/n

= 2

Rat

tus

ratt

us

13.9

5N

A13

.62

NA

NA

NA

NA

NA

12.5

713

.75

NA

81.2

40.

17/n

= 3

n =

10.

08/n

= 2

0.18

/n =

20.

71/n

= 8

uM

us

spre

tus

3.71

3.96

NA

NA

1.81

1.83

6.03

6.30

NA

7.29

NA

12.3

40.

15/n

= 2

0.36

/n =

20.

04/n

= 2

0.13

/n =

30.

38/n

= 2

0.01

/n =

20.

00/n

= 2

0.23

/n =

2

NA

: Non

-ava

ilab

le d

ata.

uU

nla

bell

ed t

axon

.



Tab

le 6

. R

esu

lts

of D

NA

/DN

A h

ybri

diza

tion

exp

erim

ents

: R

aw m

ode

valu

es (

hom

olog

ue

com

pari

son

s) a

nd

delt

a-m

ode

valu

es (

het

erol

ogu

e co

mpa

riso

ns)

, w

ith

stan

dard

dev

iati

ons

and

nu

mbe

r of

hyb

rids

Tra

cers

/D

rive

rsM

us

fam

ulu

sM

us

caro

liM

us

cerv

icol

orM

us

cook

iiM

us

mu

scu

lus

Mu

ssp

icil

egu

sP

yrom

yspl

atyt

hri

xP

yrom

yssa

xico

laC

oelo

mys

pah

ari

Nan

nom

ysM

alac

omys

Rat

tus

ratt

us

Mu

s fam

ulu

s88

.16

NA

NA

3.07

NA

NA

NA

5.48

6.43

6.57

11.3

221

.13

0.23

/n =

90.

16/n

= 2

0.31

/n =

30.

37/n

= 3

0.03

/n =

30.

03/n

= 2

0.50

/n =

2M

us

caro

li4.

0384

.99

3.74

2.80

4.19

4.40

NA

5.92

6.75

7.42

11.2

2N

A0.

25/n

= 3

0.31

/n =

70.

08/n

= 3

0.40

/n =

30.

08/n

= 2

0.31

/n =

20.

30/n

= 2

0.09

/n =

20.

24/n

= 3

1M

us cerv

icol

or3.

802.

7687

.47

1.84

3.94

4.35

NA

5.59

NA

6.90

11.2

9N

A0.

24/n

= 3

0.18

/n =

30.

11/n

= 8

0.18

/n =

30.

14/n

= 2

0.42

/n =

20.

48/n

= 2

0.10

/n =

30.

37/n

= 2

Mu

s co

okii

3.77

3.50

2.41

87.5

24.

184.

244.

855.

546.

296.

5111

.09

21.6

40.

41/n

= 3

0.40

/n =

40.

15/n

= 3

0.24

/n =

50.

04/n

= 2

0.40

/n =

20.

44/n

= 3

0.14

/n =

30.

31/n

= 2

0.08

/n =

20.

63/n

= 2

0.64

/n =

2M

us mu

scu

lus

4.14

4.44

4.12

3.70

86.7

71.

916.

586.

147.

077.

4012

.11

20.2

00.

03/n

= 3

0.13

/n =

30.

29/n

= 3

0.37

/n =

30.

25/n

= 4

0.32

/n =

10

0.48

/n =

40.

41/n

= 4

0.08

/n =

20.

02/n

= 2

0.37

/n =

21

Mu

s spic

ileg

us

3.78

3.90

NA

NA

1.89

88.2

0N

A5.

70N

A7.

24N

AN

A0.

09/n

= 2

0.28

/n =

20.

16/n

= 5

0.45

/n =

50.

02/n

= 2

0.04

/n =

2P

yrom

yspl

atyt

hri

x6.

266.

48N

A4.

816.

246.

5787

.21

0.38

NA

6.64

11.2

921

.36

0.17

/n =

40.

41/n

= 3

0.53

/n =

20.

08/n

= 2

0.40

/n =

30.

36/n

= 5

0.13

/n =

40.

17/n

= 2

0.44

/n =

20.

23/n

= 2

Pyr

omys

saxi

cola

6.33

NA

6.01

NA

NA

NA

NA

87.4

56.

546.

4911

.03

21.3

10.

26/n

= 3

0.17

/n =

30.

15/n

= 6

0.06

/n =

20.

26/n

= 3

0.08

/n =

20.

11/n

= 2

Coe

lom

yspa

har

i7.

577.

717.

425.

958.

538.

527.

216.

4787

.48

7.61

11.3

021

.41

0.25

/n =

40.

09/n

= 3

0.13

/n =

31.

38/n

= 2

0.06

/n =

20.

39/n

= 4

0.16

/n =

20.

37/n

= 4

0.34

/n =

40.

16/n

= 3

0.31

/n =

30.

58/n

= 3

Nan

nom

ys7.

45N

A6.

946.

52N

AN

A10

.37

6.29

7.36

88.2

111

.71

NA

0.27

/n =

40.

35/n

= 3

0.75

/n =

20.

22/n

= 3

0.22

/n =

30.

16/n

= 2

0.07

/n =

50.

67/n

= 3

Mal

acom

ysN

A12

.22

11.3

9N

AN

AN

AN

A10

.57

NA

NA

85.3

719

.39

0.28

/n =

20.

42/n

= 3

0.30

/n =

40.

44/n

= 6

0.74

/n =

3R

attu

sra

ttu

s16

.00

NA

16.0

0N

AN

AN

AN

AN

A16

.11

16.2

5N

A85

.27

0.80

/n =

2n

= 1

0.35

/n =

20.

35/n

= 2

0.36

/n =

8uM

us

spre

tus

3.64

54.

093

NA

NA

2.12

1.91

35.

875

5.65

8N

A7.

308

NA

21.4

40.

08/n

= 2

0.1/

n =

20.

3/n

= 2

0.51

/n =

30.

18/n

= 2

0.05

/n =

20.

07/n

= 2

0.31

/n =

2

NA

: Non

-ava

ilab

le d

ata.

uU

nla

bell

ed t

axon

.



Phylogenetic resultsFor the 12S rRNA datasets, 15 species are included,with the addition of Nannomys mattheyi and Coelomyscrociduroides which were not available for the DNA/DNA hybridization experiments.

Figure 3 presents a synthetic tree with the differentrobustness indices, obtained when using all substitu-tions. Whatever the approach (distance, parsimony,weighted parsimony, likelihood), the subgenera

Coelomys, represented by pahari and crociduroides,and Pyromys, represented by saxicola and platythrix,are supported with the highest values (BP from 98 to100%, and BSI = + 13 and + 17). The African subgenusNannomys has lower support (70–87, BSI = + 2) andfinally Mus sensu stricto is defined by very low supportvalues in parsimony and distance but is not supportedby the maximum likelihood analysis, hence a trichot-omy. When indels are included in the analysis, the

Figure 1. Phylogenetic trees derived from the DNA/DNA hybridization analysis. A and B: Consensus trees resulting fromthe bootstrap analysis of delta-Tm (A) and delta-mode (B) 12*12 matrices. BP values are indicated when different from100%. The lengths of the branches correspond to one tree arbitrarily selected among those of the consensus. C and D: Aver-age consensus trees resulting from the weighted jacknife procedure for delta-Tm (C) and delta-mode (D) 13*13 matrices.The thin lines represent nodes that were not present in maximum and minimum consensus trees or that are not supportedfor all combinations of single deletion analysis. uUnlabelled taxa. The names in bold indicate the differences that can beobserved between the two distance estimators (Tm, Mode).



results were almost the same (data not shown), exceptthat the monophyly of Mus has stronger support(distance = 62, MP = 31). The relationships betweenthe different genera are different from the ones indi-cated by DNA/DNA hybridization experiments, withone group that comprises Nannomys, Pyromys andCoelomys (Fig. 3) as opposed to the two Mus groups.

Relationships within the subgenus Mus again indi-cate the existence of two main groups, an Asian group(caroli, cookii and cervicolor), and a Palearctic group(spretus, spicilegus, musculus), with which Mus fam-ulus is associated. As already indicated by the DNA/DNA hybridization experiments, Mus famulus is notrelated to the subgenus Coelomys, represented here bythe species pahari and crociduroides. Instead, the ani-mal from Kotagiri–Avallanchi is nested within Mus,and related to the Palearctic group (spretus, spicilegusand musculus). It should be noted that the ancestralsegment defining the Asian group is not supported bythe parsimony analysis (Fig. 3, BP = 15), only by thedistance and maximum likelihood analysis (Fig. 3,BP = 56, RP = 72). Within this group, Mus cookii andM. caroli cluster tightly. Within the Palearctic group,

Figure 2. Tree reconstructed with a molecular clockhypothesis calibrated with the Mus/Rattus divergenceestimated at around 12 mya. Dating values for the differ-ent dichotomies are indicated on Table 7.

Table 7. Divergence times for the different dichotomiesindicated on Fig. 2. The estimations are calibrated byassuming 12 My for the Mus/Rattus divergence

Divergence(%)

Time(My)

Mus/Rattus 12Mus cookii/M. cervicolor 3.38 1.9Mus caroli 5.28 2.9Mus spicilegus/M. spretus 2.22 1.2Mus musculus 2.58 1.4Mus famulus 5.12 2.8Mus musculus group/

Mus cervicolor group5.80 3.2

Mus/Pyromys 9.18 5.1Pyromys saxicola/ P. platythrix

0.74 0.4

Nannomys 10.34 5.7Coelomys pahari 11.82 6.5Malacomys 16.74 9.3Rate of change (%/My) 1.81

Figure 3. Synthetic tree derived from the 12S rRNAdatasets with the inclusion of all substitutions (TV + TI).The thin lines indicate nodes that are not robustly sup-ported by all kinds of analysis. The robustness of the dif-ferent nodes are indicated as follows: [BP(BPweightedanalysis)/BSI (Parsimony)]/[BP(NJ)/Reliability Percentage(ML)].



Figure 4. Fifty per cent majority rule consensus of 52trees derived from the morphological analysis. Each most-parsimonious tree is 54 steps long, and has a ConsistencyIndex of 0.52, a Retention Index of 0.72, and a RescaledConsistency Index of 0.37. Values given below the branchesrepresent the percentage of trees containing the specifiedclades.

M. famulus appears basal, and the taxa spicilegus,spretus and musculus are defined by a well supportedancestral segment (Fig. 3). As conflicting topologies forthese three latter taxa are observed according to thekind of substitutions or criterion used (data notshown), we prefer to depict the relationships betweenspicilegus, spretus and musculus as unresolved tri-chotomy, as previously seen in the DNA/DNA hybrid-ization experiments.

The 12S rRNA dataset provides a branchingpattern of the four subgenera that seems ratherdifferent from the one derived from the DNA/DNAhybridization. Moreover, this mitochondrial gene doesnot provide support for the monophyly of the subgenusMus.

MorphologyIn the branch and bound analysis, 52 trees wereretained and the length of the most parsimonious treewas 54. The Consistency Index for these trees is 0.52,the Retention Index 0.72, and the Rescaled Consis-tency Index 0.37.

Monophyletic groupings are evident for the taxaM. setulosus, M. mattheyi and M. minutoides, all cur-rently assigned to the subgenus Nannomys, for M.pahari, M. mayori and M. crociduroides, included inthe subgenus Coelomys, and for M. musculus, M. spi-cilegus and M. spretus, all belonging to the Palaearcticgroup of the subgenus Mus. The 50% majority ruleconsensus tree is shown in Fig. 4, and reveals variousgroupings. The clade M. setulosus, M. mattheyi andM. minutoides (subgenus Nannomys) occurs in allretained trees; M. musculus, M. spicilegus and M.spretus occur together with a frequency of 77%; 38most-parsimonious trees (73%) support a clade of M.cookii and M. famulus. Overall there is good support(71%) for a monophyletic group of the subgenusCoelomys, although only including M. pahari, M.mayori and M. crociduroides. Surprisingly, there is nosupport for the subgenus Pyromys, as represented byM. saxicola and M. platythrix. Likewise, althoughthere is good support for a clade of M. setulosus, M.mattheyi and M. minutoides (subgenus Nannomys),this is subsumed within the larger group of taxacurrently considered to represent the subgenus Mus.Furthermore M. caroli, supposedly a member of thesubgenus Mus, is most frequently associated withmembers of the subgenus Nannomys. Most interest-ingly, all evidence points to the inclusion of M. famu-lus within the subgenus Mus, rather than as amember of the subgenus Coelomys. The bootstrap con-sensus tree performed with a heuristic search (seeFig. 5) shows weak to moderate support for clades ofNannomys, Pyromys and for the Palaearctic group ofthe subgenus Mus.

COMPARISON OF MOLECULAR AND MORPHOLOGICAL RESULTS

There is limited congruence between the molecularand morphological analyses, although all agree inshowing strong support for the subgenus Coelomys asrepresented by M. pahari and M. crociduroides (andM. mayori) and moderately good support for the sub-genus Pyromys. A Palaearctic clade of M. musculus,M. spicilegus and M. spretus is evident in all analyses;however, support for an Asiatic clade is lacking inthe morphological analysis. Although M. famulusis clearly grouped with the subgenus Mus in allanalyses, it is basal to the Palaearctic clade in the



molecular analyses, yet aligned with the Asiaticspecies, particularly M. cookii, in the morphologicalanalyses.

DISCUSSION

DNA/DNA HYBRIDIZATION AND 12S rRNA DATASETS

Neither method (this paper; She et al., 1990; Catzeflis& Denys, 1992; Sourrouille et al., 1995) can clearlydecipher the relationships between the four subgeneracurrently recognized in Mus. Besides considering animproved taxonomic sampling, we should now envis-age the use of other molecular markers, such as slow-

evolving nuclear genes. The relationship between Musand Pyromys proposed by the different DNA/DNAhybridization datasets (this paper; She et al., 1990;Catzeflis & Denys, 1992) is apparently rejected by the12S rRNA datasets (this paper; Sourrouille et al.,1995). These two studies proposed another possiblegrouping within Mus sensu lato; however, with twogroups, Mus on one side and Pyromys, Nannomys andCoelomys on the other (Fig. 3, this study; Fig. 2 inSourrouille et al., 1995)

The monophyly of Nannomys has more support herethan was previously obtained by Sourrouille et al.(1995). This might be because of the exclusion of allsites containing indels from our analysis; also, unlikethe analysis of Sourrouille et al. (1995), we includehere a larger taxonomic sampling of Mus sensu lato(15 species instead of eight). African pygmy mice of thesubgenus Nannomys comprise 19 species (Musser &Carleton, 1993) and our sampling is clearly inade-quate to address this point. On a chromosomal basis,the few species so far analysed are very similar to eachother, leading Jotterand-Bellomo (1986) to infer themonophyly of African pygmy mice. Genetically, somespecies of Nannomys are closely related, such as N.minutoides and N. setulosus (Catzeflis & Denys, 1992),which differ (DNA/DNA hybridization) by about 2.5∞Cdelta-Mode, a value comparable to that measuredbetween Asian members of the subgenus Mus, such asM. cookii and M. caroli (Table 6).

An interesting difference between the results of ourtwo molecular approaches concerns the relationshipswithin the Asian group of Mus. DNA/DNA hybridiza-tion strongly indicates a robust (100%, Fig. 1A and98%, Fig. 1B) cervicolor/cookii association, whereasthe 12S rRNA sequences suggest, although with mod-erate support (from 56% in parsimony to 94% in dis-tance), a caroli/cookii association (Fig. 3).

Both approaches are congruent for the relationshipsof Mus famulus, which is included in the subgenusMus, and for its closer relationship to the Palearcticrather than the Asian species. Thus, the animals fromthe South Indian localities of Kotagiri and Avallanchiare not related to the subgenus Coelomys, representedhere by the species pahari and crociduroides (Figs 1and 3). The morphological analysis likewise supportsM. famulus as a member of the subgenus Mus,although more closely related to the Asian group.Moreover, the wild-caught mice that we call Mus fam-ulus have a karyotype of 40 acrocentric chromosomes(unpubl. data, courtesy of J. Britton-Davidian, Mont-pellier), that has the same diploid and fundamentalnumbers as all species of the subgenus Mus consid-ered in this study. For these reasons, in contradictionto the opinion of Marshall (1977), we consider that theSouth Indian species M. famulus is a member of thesubgenus Mus.

Figure 5. Bootstrap 50% majority rule consensus treefrom analysis of the morphological data. Bootstrap supportvalues (100 heuristic bootstrap replicates, 10 addition rep-licates, random addition sequence) are shown above thebranches.



SUBGENERIC RELATIONSHIPS

Few studies have simultaneously compared the foursubgenera of mice, and usually included one speciesper subgenus (except for Mus sensu stricto). She et al.(1990) presented electrophoretic data that wereunable to decipher the relationships within Mus sensulato, whereas their DNA/DNA hybridization data indi-cated (Nannomys was not represented) that Mus weremore closely related to Pyromys than to Coelomys (asour more complete dataset does). Catzeflis & Denys(1992) also found that Mus and Pyromys were moreclosely related than either to Nannomys or Coelomys.The results of the 12S rRNA datasets propose anotherhypothesis with a Nannomys–Pyromys and Coelomysclade opposed to Mus, this association was neverfound with other approaches. A better taxonomicsampling of these three subgenera may be necessaryin order to understand their relationships, as well asthe use of other molecular markers such as nucleargenes.

For both approaches used here, the amount ofmolecular divergence is substantial between the foursubgenera when compared with values in othermurines, such as between the different genera of thePraomys complex (Chevret et al., 1994). As alreadysuggested by Bonhomme et al. (1984) and Bonhomme(1986, 1992), based on isozyme data, the four subgen-era of mice might be considered to be distinct genera.Our data would agree with this suggestion, were it notfor the insufficient taxonomic sampling available todate.

Table 8 provides key characters that may be used todistinguish between the subgenera Mus, Pyromys andCoelomys as represented by the species included inthis study. No attempt is made to provide such char-acters to differentiate the subgenus Nannomys, as

only three of the 19 species are represented in thisstudy.

RELATIONSHIPS WITHIN THE SUBGENUS MUS

Because of the importance of mice in biomedical andgenetic research, numerous studies have addressedthe relationships of species closely related to thedomestic mouse, that is to taxa within the subgenusMus. Following Bonhomme et al. (1984), differentstudies have concurred in recognizing an Asian(caroli, cervicolor, cookii) and a Palearctic group(musculus, spretus, spicilegus). Our analyses confirmthis dichotomy, although the molecular and mor-phological results indicate different affinities for M.famulus.

In the Asian group, DNA/DNA hybridization and12S rRNA inferences provide conflicting resultsregarding the relationships between the species car-oli, cookii and cervicolor. Bonhomme (1986), usingisozyme electrophoresis, suggested that cookii and cer-vicolor were sister taxa. Later, She et al. (1990), with alarger taxonomic sample and different approaches(isozymes, DNA/DNA hybridization, mitochondrialDNA RFLP), provided conflicting results suggestingan unresolved trichotomy. Lundrigan & Tucker (1994),with a study of the Sry nuclear genes and a combina-tion of their data and some from She et al. (1990), indi-cated that cookii and cervicolor might be more closelyrelated than either is to caroli, a result that we alsoobserve here through DNA/DNA hybridization. Thus,the nuclear data (DNA/DNA hybridization and Srygene) and the biochemical data are in conflict with theresults of a mitochondrial gene, here 12S rRNA.Clearly, additional markers from both genomes areneeded in order to clarify these relationships.

Table 8. Key characters to distinguish between the Eurasian subgenera of Mus

Character Pyromys Coelomys Mus

Supraorbitalridges

Present, extending fromfrontals to parietals

Absent or present, extendingfrom frontals to parietals

Present but restricted to posteriorportion of orbit

Subsquamosalforamen

Small, hamular processshort

Large, hamular process long orsmall, hamular process short

Large, hamular process long

Incisive foramina Medium to long, penetratebetween M1 – M1

Short, terminate before anteriorroot of M1

Medium to long, penetratebetween M1 – M1

Suture betweenmaxillary andpalatine

Level with junctionbetween M1 and M2 toanterior region of M2

Level with posterior or midregion of M1

Level with junction between M1

and M2 to anterior region of M2

M3 Moderate in size Very reduced in size, smallerthan T7 and T8 of M2

Reduced in size, larger than T7and T8 of M2

T3 of M1 Posterior to T1 and T2 Posterior to T1 and T2 Displaced markedly posterior toT1 and T2

Dorsal pelage Guard hairs spinous Guard hairs spinous or pelagesoft

Pelage soft, lacks spinous guardhairs



The same uncertainty concerns the three species(musculus, spicilegus and spretus) of the Palearcticgroup, even if there is a weak support for a spretus-spicilegus clade. The data of She et al. (1990) do notclarify this matter. The Sry gene alone (Lundrigan& Tucker, 1994) indicates a possible relationshipbetween spicilegus and spretus, but when combinedwith the data of She et al. (1990), these three speciesappear as a polytomy, as was previously suggested byBonhomme (1992).

CONCLUSION

The 12S rRNA, DNA/DNA hybridization and morpho-logical analyses indicate that four subgenera, Mus,Pyromys, Nannomys and Coelomys, may be character-ized effectively. They could eventually be considered tobe different genera if this theory is confirmed by theinclusion of additional species in subsequent analyses.All analyses indicate that Mus famulus is not relatedto the subgenus Coelomys and should be thereforeincluded in Mus subgenus. The monophyly of Nanno-mys, the African pygmy mouse, of Mus, and of theEurasian group of Mus (caroli, cookii and cervicolor)has not been demonstrated with strong support withthe 12S rRNA datasets. Moreover, DNA/DNA hybrid-ization and 12S rRNA provide different relationshipsbetween the four subgenera. In order to improve thesupport of the weakly supported nodes and to clarifythe subgeneric relationships, several more species andsequencing of more nucleotides are needed before anyconclusion may be reached.

This study, as well as previous ones, indicatesthat to decipher the relationships within Mus sensulato, not only additional taxa but also more appro-priate molecular markers (slow-evolving nucleargenes) containing sufficient phylogenetic signal aredesirable.

ACKNOWLEDGEMENTS

We thank all the people who collected or donated thetissue samples used in this study: François Bonho-mme, Waheedud Din, Tizziano Maddalena, ValérieNancé and Manuel Ruedi. We are grateful to JacquesCuisin, Muséum National d’Histoire Naturelle, for theloan of specimens. The collecting trip in South Indiawas funded through NIH-NIAID grant AI29834attributed to F. Bonhomme, Montpellier, and labora-tory consumables were funded by a grant from GREG(Groupe de Recherche pour l’Etude des Génomes) to F.Catzeflis. We thank the federal, state and regionalagencies in India (New Delhi, Madras and Ootaca-mund) for providing collecting permits in differentareas of the Nilgiris Mountains, Tamil Nadu State.This is contribution ISEM-2001-124 of Institut des

Sciences de l’Evolution de Montpellier (UMR 5554CNRS).

NOTE ADDED IN PROOF

We have recently learned of a paper in press by Auf-fray et al., Phylogenetic position and description of anew species of mouse of subgenus Mus (Rodentia,Mammalia). Zoologica Scripta (in press), which shouldbe consulted for completeness.

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