mitochondrial and nuclear phylogenies of cervidae (mammalia, ruminantia): systematics, morphology,...

Molecular Phylogenetics and Evolution 40 (2006) 101–117www.elsevier.com/locate/ympev

Mitochondrial and nuclear phylogenies of Cervidae (Mammalia, Ruminantia): Systematics, morphology, and biogeography

Clément Gilbert a,b,c, Anne Ropiquet a,b, Alexandre Hassanin a,b,¤

a UMR 5202—Origine, Structure et Evolution de la Biodiversité, Département Systématique et Evolution, Muséum National d’Histoire Naturelle, Case postale N° 51, 55, rue BuVon, 75005 Paris, France

b Service de Systématique Moléculaire, Département Systématique et Evolution, Muséum National d’Histoire Naturelle, 43, rue Cuvier, 75005 Paris, Francec Evolutionary Genomics Group, Department of Botany and Zoology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa

Received 14 November 2005; revised 2 February 2006; accepted 19 February 2006Available online 3 April 2006

Abstract

The family Cervidae includes 40 species of deer distributed throughout the northern hemisphere, as well as in South America and South-east Asia. Here, we examine the phylogeny of this family by analyzing two mitochondrial protein-coding genes and two nuclear introns for 25species of deer representing most of the taxonomic diversity of the family. Our results provide strong support for intergeneric relationships.To reconcile taxonomy and phylogeny, we propose a new classiWcation where the family Cervidae is divided in two subfamilies and Wve tribes.The subfamily Cervinae is composed of two tribes: the tribe Cervini groups the genera Cervus, Axis, Dama, and Rucervus, with the PèreDavid’s deer (Elaphurus davidianus) included in the genus Cervus, and the swamp deer (Cervus duvauceli) placed in the genus Rucervus; thetribe Muntiacini contains Muntiacus and Elaphodus. The subfamily Capreolinae consists of the tribes Capreolini (Capreolus and Hydropotes),Alceini (Alces), and Odocoileini (Rangifer + American genera). Deer endemic to the New World fall in two biogeographic lineages: the Wrstone groups Odocoileus and Mazama americana and is distributed in North, Central, and South America, whereas the second one is composedof South American species only and includes Mazama gouazoubira. This implies that the genus Mazama is not a valid taxon. Molecular dat-ing suggests that the family originated and radiated in central Asia during the Late Miocene, and that Odocoileini dispersed to North Amer-ica during the Miocene/Pliocene boundary, and underwent an adaptive radiation in South America after their Pliocene dispersal across theIsthmus of Panama. Our phylogenetic inferences show that the evolution of secondary sexual characters (antlers, tusk-like upper canines, andbody size) has been strongly inXuenced by changes in habitat and behaviour.© 2006 Elsevier Inc. All rights reserved.

Keywords: Cervidae; Ruminantia; Phylogeny; Taxonomy; Evolution; Biogeography; Sexual dimorphism; Mitochondrial DNA; Nuclear DNA

1. Introduction

With 40 species of deer, the family Cervidae constitutesthe second most speciose family of artiodactyls after theBovidae (Grubb, 1993). Widely divergent in size, habitat,and behaviour, Cervidae are united by a series of synapo-morphies, including the possession of antlers in males(Janis and Scott, 1987). In the classiWcation of Grubb(1993), the 16 genera of Cervidae are arranged in four sub-

* Corresponding author. Fax: +33 1 40 79 30 63.E-mail address: [email protected] (A. Hassanin).

1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2006.02.017

families: Cervinae, Muntiacinae, Hydropotinae, and Odo-coileinae. The subfamily Cervinae includes four genera: (1)Dama in Eurasia with two species of fallow deer, (2) Axiswith four Asian species (e.g., hog deer and chital), (3) Ela-phurus davidianus (Père David’s deer) in China, and (4) thebroadly distributed genus Cervus, with nine species in Asiaand Cervus elaphus, which is widespread throughout thewhole northern hemisphere. The subfamily Muntiacinaecontains two Asian genera: Muntiacus, with nine species ofmuntjacs, and the monotypic Elaphodus cephalophus(tufted deer). The subfamily Hydropotinae is only repre-sented by the antlerless Hydropotes inermis (Chinese waterdeer). Finally, the subfamily Odocoileinae is the most

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mailto: [email protected]

102 C. Gilbert et al. / Molecular Phylogenetics and Evolution 40 (2006) 101–117

heterogeneous group, with nine genera currently deWned:(1) Alces (moose) and (2) Rangifer (reindeer), which areboth largely dispersed in North America and Eurasia, (3)the Eurasian genus Capreolus with two species of roe deer,(4) Odocoileus (mule deer and white-tailed deer) and (5)Mazama (six species of brocket deer), which are both pres-ent in North and South America, and the four South Amer-ican genera: (6) Blastocerus (marsh deer), (7) Hippocamelus(two species of huemuls), (8) Ozotoceros (pampas deer), and(9) Pudu (two species of pudus).

Other morphological classiWcations and phylogeniesagree with the monophyly of the 16 genera of Cervidae, butmany inconsistencies remain concerning the intergenericrelationships. In the Wrst classiWcation of Cervidae, Brooke(1878) recognized the four subfamilies retained by Grubb(1993), but two higher taxa were also proposed on the basisof diVerences in the metacarpals: (1) the Plesiometacarpa-lia, which unites Cervinae and Muntiacinae, as both possessonly the proximal part of the lateral metacarpals; and (2)the Telemetacarpalia, which includes Odocoileinae andHydropotinae, as both possess only the distal part of thelateral metacarpals. In contrast, Groves and Grubb (1987)did not consider Telemetacarpalia to be a valid taxon, asthey suggested that Hydropotes was the sister group to allother Cervidae because it does not possess antlers.

The subfamily Odocoileinae is the most problematictaxon, and intergeneric relationships are particularly con-troversial within this group. Brooke (1878) grouped Rang-ifer with the six genera endemic to America (Blastocerus,Hippocamelus, Mazama, Odocoileus, Ozotoceros, andPudu), because all of them possess a vomerine septum thatcompletely separates the choana. Simpson (1945) did notfollow this hypothesis and split the Odocoileinae intothe Wve tribes Odocoileini (Blastocerus, Hippocamelus,Mazama, Odocoileus, Ozotoceros, and Pudu), Alcini (Alces),Capreolini (Capreolus), Hydropotini (Hydropotes), andRangiferini (Rangifer). Following Simpson (1945), McK-enna and Bell (1997) placed Alces and Capreolus in theirown tribe Alceini (diVerent spelling from “Alcini”; Simp-son, 1945) and Capreolini, but they included Rangifer in thetribe Odocoileini and separated Hydropotes in the subfam-ily Hydropotinae. In an analysis of morphological charac-ters, Webb (2000) excluded Alces from the Odocoileinaeand recognized only two tribes in this subfamily: Rangife-rini, which contains Rangifer, Hippocamelus, and Pudu; andOdocoileini, which includes Odocoileus, Blastocerus,Mazama, and Ozotoceros. Additional subfamilies have beenproposed on the basis of antler characteristics: Pocock(1923) proposed the inclusion of Rangifer in its own sub-family Rangiferinae, because the reindeer is the only specieswith antlered females; and Kraglievich (1932) includedMazama and Pudu in the subfamily Mazaminae, as theyshare simple, one tined antlers.

The discrepancies between morphological phylogeniesof Cervidae can be explained by the fact that all these stud-ies have used diVerent matrices of characters (Groves andGrubb, 1987; Meijaard and Groves, 2004). It is also very

diYcult to evaluate the reliability of these hypotheses,because none of them has used a clearly deWned methodo-logical approach. Moreover, the usefulness of morphologi-cal characters in deciphering the phylogeny of Cervidae,and to a larger extent of ruminants, has been repeatedlyquestioned because of their high level of homoplasy (Gen-try, 1994; Groves and Grubb, 1987; Hassanin and Douzery,2003; Janis and Scott, 1987; Scott and Janis, 1987).

Several molecular studies have also been published onCervidae. The cytochrome b (Cyb) analysis of Randi et al.(1998) supported the monophyly of Plesiometacarpalia butnot that of Telemetacarpalia. The multigene analysis ofHassanin and Douzery (2003) favored the major Plesio-/Telemetacarpalia dichotomy, but only six genera of Cervi-dae were included in this study. Within the subfamilyMuntiacinae, the analyses of several mitochondrial markers(D-loop, Cyb, 12S and 16S rRNA, ND4 and ND4L) haveprovided a strong support for the monophyly of Muntiacusand interspeciWc relationships (Amato et al., 2000; Wangand Lang, 2000). The monophyly of the subfamily Cervinaehas been supported by analyzing a large species samplewith mitochondrial sequences of the control region (D-loop) and Cyb gene (Bonnet, 2001; Liu et al., 2003; Ludtet al., 2004; Pitra et al., 2004; Randi et al., 1998; Randi et al.,2001). Within Cervinae, all Cyb studies have shownthe polyphyly of Cervus and Axis, with C. eldi groupedwith Elaphurus, A. axis allied with C. duvauceli andC. schomburgki, and A. porcinus linked to C. timorensis(Liu et al., 2003; Ludt et al., 2004; Pitra et al., 2004). Thepolyphyly of Cervus was also reported in two analyses ofthe D-loop (Bonnet, 2001; Randi et al., 2001), but Axis wasfound monophyletic in Bonnet (2001).

The monophyly of Odocoileinae sensu Grubb (1993) hasbeen questioned based on molecular studies. Indeed, Capre-olus should be excluded from this group, as it was foundgrouped with Hydropotes in Cyb studies (Pitra et al., 2004;Randi et al., 1998), and in the multigene analysis of Hassa-nin and Douzery (2003). This molecular hypothesis sug-gests that antlers were completely lost in the lineage leadingto the extant Hydropotes (Randi et al., 1998). In addition,Cyb sequences have suggested that the American generaMazama and Odocoileus are closely related, and that theyshare aYnities with Rangifer (Randi et al., 1998). The posi-tion of the moose (Alces) is unresolved: it is supported aseither the sister group to the clade uniting American deerand Rangifer, or allied with Capreolus and Hydropotes(Randi et al., 1998).

Molecular investigations have greatly helped in delimit-ing clades within Cervidae, and in understanding the evolu-tion of morphological characters. Nevertheless, because ofinsuYcient gene and taxon sampling, in particular for deerof America, many nodes remain unresolved, impeding theinterpretation of many aspects of the biogeographic historyof the family. In particular, questions regarding the originof American deer and their colonization of South Americahave never been addressed. Here, we propose to Wll thesegaps by combining the strength of a large taxonomic sam-

C. Gilbert et al. / Molecular Phylogenetics and Evolution 40 (2006) 101–117 103

ple including 25 species of Cervidae (representing 15 gen-era) with the phylogenetic signal from both mitochondrialand nuclear markers. To provide a consistent signal span-ning the whole evolutionary history of the family, two rap-idly evolving mitochondrial genes, i.e., cytochrome b (Cyb)and the subunit II of the cytochrome oxidase (CO2), arecombined with two slower nuclear markers, i.e., intron 2 ofthe �-lactalbumin (�LAlb) and intron 1 of the proteinkinase C iota gene (PRKCI). This enables us to address thefollowing four phylogenetic questions: (1) Are the supra-generic taxa previously described in the literature (Plesio-/Telemetacarpalia; Cervinae; Muntiacinae; Odocoileinae)monophyletic? (2) Within Cervinae, are the genera Cervusand Axis monophyletic? (3) What is the phylogenetic posi-tion of the South American genera? (4) Among Americandeer, are the genera Mazama and Odocoileus monophy-letic? In addition, divergence times are estimated with therelaxed Bayesian molecular clock approach by using sev-eral calibration points. The results are compared with avail-able data on fossils, paleoclimatic changes, and theevolution of landscapes to provide a comprehensive sce-nario for the biogeography and morphological evolution ofCervidae during the Neogene.

2. Materials and methods

2.1. Taxonomic sample

The ingroup includes 25 species of Cervidae representing15 genera. As the monophyly of Muntiacus was conWrmedwith mitochondrial sequences (Amato et al., 2000; Wangand Lang, 2000), the monophyly of the subfamily Muntiac-inae is here tested by including one species of Muntiacusand the monospeciWc genus Elaphodus. The subfamily Cer-vinae was densely sampled (12 species) to clarify the statusof the genera Axis and Cervus. All genera of Odocoileinaeexcept Ozotoceros were incorporated in the analyses to bet-ter understand the evolution of American deer. The out-group contains four species belonging to four diVerent taxaof the suborder Ruminantia: Antilocapra americana(Antilocapridae), Moschus moschiferus (Moschidae), Gaz-ella granti (Bovidae, Antilopinae), and Tragelaphus imber-bis (Bovidae, Bovinae) (Table 1).

2.2. DNA extraction, ampliWcation, and sequencing

Most of the tissues used in this study come from the col-lections of the National Museum of Natural History ofParis (Muséum National d’Histoire Naturelle, MNHN)(Table 1). Cells and blood samples were digested in CTAB(hexade Cyl Trimethyl Ammonium Bromide) using the pro-tocol detailed in Winnepenninckx et al. (1993); DNA waspuriWed in chloroform isoamyl alcohol, and was then pre-cipitated with isopropanol. For bone samples, DNA wasextracted following Hassanin et al. (1998). First, bones wereground in liquid nitrogen and digested in a lysis solution(Tris–HCl 10 mM; EDTA 0.5 M, pH 8.5; SDS 0.5%; pro-

teinase K 200 �g/ml). Second, the supernatant was dialysed,and DNA was puriWed several times by using phenol andchloroform. Finally, DNA was precipitated with isopropa-nol.

Four genes were sequenced: two mitochondrial protein-coding genes, i.e., Cyb (1140 bp) and CO2 (582 bp), and twonuclear fragments, i.e., intron 2 of �-lactalbumin (�LAlb),which is 462 bp long in Cervus elaphus (Accession No.AY122017), and intron 1 (and two small exonic regions) ofthe gene encoding the protein kinase C iota (PRKCI),which is 514 bp long in C. elaphus (Accession No.AY846793). Most sequences were obtained using severaloverlapping PCR ampliWcations. The exact matchingbetween the overlapping portions of two diVerent PCRfragments has been checked as an evidence of authenticityof sequences. Most primers come from previous studies:Hassanin et al. (1998) and Hassanin and Douzery (1999)for Cyb; Hassanin and Douzery (2003) for �LAlb; Hassaninand Ropiquet (2004) for CO2, and Ropiquet and Hassanin(2005a) for PRKCI. AmpliWcations were done in 50 �l usingthe following PCR standard conditions: buVer 10£ withMgCl2 (1.5 mM), 5 �l; dNTPs, 5 �l (6.6 mM); Taq Appligen,0.3 �l (2.5 U); and primers, 2.5 �l at 10 �M. The standardPCR program used was: 94 °C for 4 min; 94 °C for 1 min,50–60 °C for 1 min, 72 °C for 1 min (30 cycles). AmpliWca-tion products were puriWed using the Montage PCR Cen-trifugal Filter Devices (Millipore). All sequences wereobtained by double-strand DNA cycle sequencing with aCEQ2000 Dye terminator cycle Sequencing Quick Start kitin a CEQ2000 Beckman (v4.3.9) sequencer. The resultingoutput was edited using Sequencher 4.1 (Gene Codes, AnnArbor, Michigan).

2.3. Phylogenetic analyses

Alignments were done by eye on Bioedit v5.0.6 (Hall,2001). The mitochondrial protein-coding genes, i.e., Cyband CO2, did not pose any problem of primary homologybecause no gaps were included in the alignment. In con-trast, four nuclear regions which were found ambiguous forthe position of gaps were excluded from analyses: 3 nucleo-tides (nt) and 9 nt, respectively, at position 70 and 218 of the�LAlb sequence of C. elaphus (Accession No. AY122017),and 14 nt and 17 nt at position 148 and 244 of the PRKCIsequence of C. elaphus (Accession No. AY846793). We alsoexcluded Wve autapomorphic single nucleotides (pos. 113,119, 179, and 246 of �LAlb and pos. 304 of PRKCI). Thealignments are available in the supplementary material S1on the MPE’s web page. Bayesian and maximum likelihood(ML) analyses were performed on each of the four genesseparately and on a matrix combining these four genes. Themodels of sequence evolution were selected by usingMrMODELTEST (v2.2) (Nylander, 2004). These modelsare GTR + I + � for CO2 and Cyb, HKY + � for �LAlb andPRKCI, and GTR +I + � for the concatenated dataset.

Bayesian analyses were performed using MrBayes(v3.0b4) (Huelsenbeck and Ronquist, 2001). To ensure a

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. Gilbert et al. / M

olecular Phylogenetics and E

volution 40 (2006) 101–117

Table 1Origin of the

a This stud lantes of the MNHN; Zoothèque: osteological collection of theMNHN; ISE . (1994); (3) Ludt et al. (2004); (4) Randi et al. (1998); (5) Hassaninand Douzer Davis (2001).

Cyb PRKCI �LAlb

Cervinae DQ379302a DQ379329a DQ379348a

DQ379301a DQ379367a DQ379349a

AF423202 (3) DQ379330a DQ379350a

DQ379303a DQ379331a DQ379351a

AY244490 (3) AY846793 (8) AY122017 (5)AY157735 (Unp) Negative PCR DQ379353a

AY035876 (3) DQ379332a DQ379352a

AF423200 (3) DQ379333a DQ379354a

AF423201 (3) DQ379334a DQ379355a

AJ000022 (4) DQ379335a DQ379356a

DQ379304a DQ379336a DQ379357a

AF423194 (3) DQ379337a DQ379358a

Muntiacinae DQ379305a DQ379339a DQ379359a

NC_004069 (Unp) AF165677 (9) AY122018 (5)

Alcinae AJ000026 (4) DQ379338a DQ379360a

Capreolinae AJ000024 (4) DQ365692a AY122021 (5)AJ000028 (4) DQ379340a AY122020 (5)

Odocoileina DQ379306a DQ379341a DQ379361a

DQ379307a DQ379342a DQ379362a

AJ000027 (4) DQ379343a DQ379363a

DQ379308a DQ379344a DQ379364a

AF091630 (4) DQ379345a AY122022 (5)DQ379370a DQ379346a DQ379365a

DQ379309a DQ379347a DQ379366a

AJ000029 (4) AF165693 (9) AY122019 (5)

Moschidae AY121995 (5) DQ365693a AY122033 (5)

Bovidae AF036279 (6) AF165733 (9) AY122025 (5)AF034723 (7) AF165749 (9) AY122029 (5)

Antilocaprid AF091629 (6) AF165669 (9) AY122014 (5)

sequences

y; Cyto: cytogenetic collection of the MNHN; Spot: tissue collection of the MNHN; MJP: Ménagerie du jardin des pM: Institut des Sciences de l’Evolution de Montpellier; Unp: Unpublished; (1) Honeycutt et al. (1995); (2) Miyamoto et al

y (2003); (6) Hassanin and Douzery (1999); (7) Hassanin et al. (1998); (8) Ropiquet and Hassanin (2005a); (9) Matthee and

Taxa Common name Collection reference CO2

Axis axis Chital Cyto 02.090 DQ379310a

porcinus Hog deer Cyto 02.054 DQ379311a

Cervus albirostris Thorold’s deer Cyto 02.052 DQ379312a

duvauceli Swamp deer Cyto 02.057 DQ379313a

elaphus Red deer Spot 01.176 DQ365689a

eldi Eld’s deer Cyto 02.060 DQ379314a

nippon Sika MJP 12887 C2 DQ379315a

timorensis Timor’s deer Cyto 02.65 DQ379316a

unicolor Sambar W. Robichaud T0009A DQ379317a

Dama dama Fallow deer Cyto 02-104 DQ379318a

mesopotamica Persian fallow deer Cyto 02.077 DQ379319a

Elaphurus davidianus Père David’s deer Cyto 02.070 DQ379320a

Elaphodus cephalophus Tufted deer Zoothèque 1896–689 DQ379321a

Muntiacus reevesi Reeves’ muntjac — NC_004069 (Unp)

Alces alces Moose Vincennes’s Zoo DQ379322a

Capreolus capreolus Roe deer Cyto 2000.256 DQ365690a

Hydropotes inermis Chinese water deer ISEM T4307 DQ379323a

e Blastocerus dichotomus Marsh deer Zoothèque Trophy 175 DQ379324a

Hippocamelus antisensis Huemul Zoothèque 1957–1302 DQ379325a

Mazama americana Red brocket Cyto 94.072 DQ379326a

gouazoubira Gray brocket ISEM T1627 DQ379368a

Odocoileus hemionus Mule deer ISEM T176 DQ379369a

virginianus White-tailed deer Cyto 02.133 U18816 (1)Pudu puda Pudu Spot 330 DQ379327a

Rangifer tarandus Reindeer ISEM T45353 DQ379328a

Moschus moschiferus Musk deer Spot 1258 DQ365691a

Tragelaphus imberbis Lesser kudu — U18815 (1)Gazella granti Grant’s gazelle — U18824 (1)

ae Antilocapra americana Pronghorn — U62571 (2)


good exploration of the posterior probabilities (PP) surfaceand to reach the stationary distribution of Markov chains,Wve chains were run for 2,000,000 generations and sampledevery 100 generations. Ten thousand sampled trees werediscarded as “burn in.” Each analysis was repeated twice tocheck that the chains always converged towards the samelikelihood score. Unambiguous indels (insertions or dele-tions) were coded as binary characters. Two diVerent char-acter partitions were therefore used for each nucleardataset: (1) DNA sequences were analyzed with the modelselected by MrMODELTEST, and (2) indels were analyzedusing the parsimony options. Five character partitions wereused for the combined analysis: the four diVerent geneswere analyzed by applying four diVerent GTR + I + � mod-els, and indels were analysed with the parsimony options.For the Bayesian bootstrap analysis, 100 pseudoreplicatesof the combined matrix were Wrst created using SEQBOOT3.5c (Felsenstein, 2004). Bootstrap Bayesian values (BPB)were obtained by constructing the consensus of the 100Bayesian trees with CONSENSE 3.5c (Felsenstein, 2004).The bootstrap ML analyses (BPML) were performed usingPHYML 2.1b1 (Guindon and Gascuel, 2003) with 1000replicates. We used the same models as for the Bayesiananalyses.

2.4. Molecular dating

Divergence times were calculated according to therelaxed Bayesian molecular clock approach for multigenedatasets described in Thorne et al. (1998) and Thorne andKishino (2002) and implemented in MULTIDIVTIME.

Divergence times were estimated for each four genes sep-arately and for the combined matrix. The mean of the dis-tribution of the root’s age (rttm) was set at rttmD 25Million Years Ago (MYA) with a standard deviation (rtt-msd) set at rttmsdD 10 MYA. To approximate the mean ofthe prior distribution of the rate of evolution at the root ofthe tree (rtrate), we followed the procedure recommendedin the documentation of the software. We therefore usedthe following values: rtrateD 0.07 for the Cyb, 0.06 forCO2, 0.013 for �LAlb, 0.008 for PRKCI, and 0.05 for thecombined analysis. The Markov chains were sampled10,000 times every 100 generations and the “burn in”period was set at 100,000 generations.

Three calibration points were used for the analyses: theWrst refers to the oldest fossil of Cervidae (20§ 2 MYA;Ginsburg, 1988), the second refers to the oldest fossil attrib-uted to the subfamily Muntiacinae or tribe Muntiacini(8§1 MYA; Dong et al., 2004) and the third refers to theoldest fossil of the clade Rangifer + American genera ortribe Odocoileini (5§ 1 MYA; Vislobokova, 1980) (seeparagraphs 4.1 and 4.2). The use of such constraintsinvolves the assumption that the age of the oldest fossilattributed to a node is a good approximation of the mini-mum age of this node. The divergence times were estimatedusing the three possible pairs of calibration points: (1) Cer-vidae and Muntiacinae, (2) Cervidae and (Rangifer +

American genera), and (3) Muntiacinae and (Rangifer +American genera).

2.5. Reconstruction of the ancestral morphotype of Cervidae

The tree resulting from the combined analysis (Fig. 4)was used for studying whether sexual characters (type ofantlers, upper canines, and body size) have evolved inrelation to changes in behaviour and habitat . The fourdiscrete states used for antlers were those previouslydeWned by Pocock (1933) as follows: (1) one tine, (2) twotines, (3) three tines, and (4) four or more tines. The pres-ence/absence and shape of upper canines were observedon a series of skulls available in the MNHN collections ofParis (Catalog numbers of the specimens are available inthe supplementary material S2 on the MPE’s web page).Two character states were considered for the type of habi-tat: (1) open habitat, which includes grasslands, marsh-lands, and open forests; and (2) closed habitat, whichincludes dense forests and marshes with reeds. We codedtwo categories of body size, i.e., minimal shoulder sizehigher or smaller than 650 mm. The presence/absence ofsexual dimorphism in body size was coded by taking intoaccount the body mass for each sex (Geist and Bayer,1988; Loison et al., 1999; Merino et al., 2005; Mooringet al., 2004). Habitat types and shoulder heights weretaken from Nowak (1999). The matrix of character statesis available in Fig. 4. The evolution of these characterswas optimized using the two parsimony options ACCTRAN and DELTRAN of the software PAUP 4.0b10(SwoVord, 2003). Only one ancestral character state wasfound ambiguous because of contradictions betweenACCTRAN and DELTRAN inferences.

3. Results

3.1. Phylogenetic analyses

The four genes have Wrst been analyzed separately(Fig. 1) and then concatenated to constitute a matrixincluding 2718 nucleotides and 6 unambiguous indels (fourin PRKCI and two in �LAlb; see in Fig. 2).

3.1.1. Monophyly of the family CervidaeEach of the four genes strongly supports the monophyly

of the Cervidae whatever the type of analysis (0.99 < PP < 1,and 70 < BPML < 100). The family is characterized by 11exclusive synapomorphies (the positions of all synapomor-phies are deWned on the sequences of C. elaphus, see acces-sion numbers in Table 1): the most striking is a 16nucleotides deletion (CATAAAAGGCAACAGG) at posi-tion (pos.) 355 of the �LAlb; seven are transversions(�LAlb: T!G and A!C in pos. 164 and 330; PRKCI:A!T and G!C in pos. 167 and 286; CO2: A!T, pos. 6;Cyb: C!A and G!T in pos. 114 and 712), and three aretransitions (�LAlb: C!T, pos. 86; PRKCI: A!G in pos. 7and 212).


3.1.2. The Plesio-/Telemetacarpalia dichotomyThe analysis combining the four markers reveals a main

dichotomy within Cervidae separating the Plesiometacarpa-lia, which includes the subfamilies Cervinae and Muntiacinae(PPD1; BPB/MLD100), and the Telemetacarpalia, which

groups all other Cervidae (members of the subfamilies Odo-coileinae and Hydropotinae) (PPD1; BPBD83; BPMLD70).The monophyly of Plesiometacarpalia was recovered in theindependent analyses of Cyb (PPD0.92; BPMLD75), �LAlb(PPD0.99; BPMLD52), and PRKCI (PPD0.58; BPMLD32).

Fig. 1. Phylogenetic analyses of the four markers: CO2, Cyb, �LAlb and PRKCI. The trees were obtained with the Bayesian approach using the evolution-ary model selected under MrModeltest 2.2, i.e., GTR+I+� for mitochondrial markers, and HKY+� for nuclear markers. The indels found in the nucleargenes were coded as binary characters, and analyzed in a diVerent partition from the nucleotides characters (see Section 2). The values on each node corre-spond to the Bayesian posterior probabilities (PP > 0.5) and bootstrap proportions calculated with the maximum likelihood method (BPML).

Alces alces

Capreolus capreolusHydropotes inermis

1/96

Mazama americana

Mazama gouazoubiraPudu puda

Rangifer tarandus

Odocoileus hemionusOdocoileus virginianus

0.68/50

Blastocerus dichotomusHippocamelus antisensis

0.65/530.76/61

1/80

Muntiacus reevesi

Cervus albirostris

Cervus duvauceli

Elaphodus cephalophus

Elaphurus davidianusCervus eldi

Cervus timorensisCervus unicolor

Cervus elaphusCervus nippon

1/97

Dama damaDama mesopotamica1/100

Axis axisAxis porcinus

1/93

0.67

0.99/52

1/93

Tragelaphus imberbis0.81/84

1/87

0.99/80

0.98/69

1/85

1/85

0.97/61

0.52

0.58/32

Antilocapra americanaMoschus moschiferus

Gazella granti

1/84

0.84/43

0.88/81

1/90

0.64/47

αLAlb PRKCI

0.82/39

0.98/91

0.99/90

0.8/39

1/100

1/86

1/100

0.98/84

1/1000.97/74

1/911/99

0.92/75

0.50/50

0.8/60

0.85/76

1/91

1/96

0.9/90

1/990.8/60

0.75/69

1/100

0.67/57

1/100

Antilocapra americanaMoschus moschiferus

Axis axisCervus duvauceli

Axis porcinus

Dama damaDama mesopotamica0.98/87

Elaphurus davidianusCervus eldi

Cervus timorensisCervus unicolor

0.84/71

Cervus elaphus

Cervus albirostrisCervus nippon

1/97

0.57/550.86/96

0.64/74

0.56/53

Elaphodus cephalophusMuntiacus reevesi

Rangifer tarandus

Alces alcesMazama americana

Odocoileus hemionusOdocoileus virginianus1/83

1/99

Blastocerus dichotomusHippocamelus antisensis

Mazama gouazoubiraPudu puda

0.89/71

1/881/71

Capreolus capreolusHydropotes inermis0.63/50

0.77/26

0.52

0.99/70

Gazella grantiTragelaphus imberbis

0.77/36

0.86/47

CO2 Cyb

PP/BPML


The monophyly of Telemetacarpalia is also recovered by theanalysis of PRKCI (PPD0.64; BPMLD47). Moreover, allmembers of this clade are diagnosed by a deletion of 12nucleotides (AATACCCCGTA) at pos. 238 of �LAlb, andby a transition A!G in pos. 251 of PRKCI.

3.1.3. Within PlesiometacarpaliaAccording to the combined analysis, the Plesiometacarpa-

lia are divided in two strongly supported clades which corre-spond to the subfamilies Cervinae (PPD1; BPB/MLD100)and Muntiacinae (PPD1; BPBD77; BPMLD72). The Cybdata alone strongly supports the two subfamilies (PPD1;BPMLD99 and 96). They are also recovered with PRKCI,and although this marker provides a weak quantitativesignal, it contains a molecular signature for each subfamily,i.e, an insertion of two nucleotides (AT) in pos. 81 for Cervi-nae, and a transition A!G in pos. 433 for Muntiacinae.

Within Cervinae, the monophyly of the genus Dama isfound in the independent analyses of the four genes

(0.75 < PP < 1; 69 < BPML < 100), and in the combinedanalysis (PPD 1; BPB/ML D 100). In addition, Dama isdiagnosed by a transversion T!G in pos. 375 of PRKCI.The genus Axis was found monophyletic with the com-bined analysis (PPD 1; BPB/ML D 100), as well as withindependent analyses of Cyb, �LAlb, and PRKCI (PPD 1;85 < BPML < 100). Moreover, both species of Axis sharesix exclusive synapomorphies, including four transitions(Cyb: T!C, pos. 884; �LAlb: A!G and G!A in pos.125 and 362; PRKCI: T!C, pos. 461, and two G!Ttransversions Cyb: pos. 462; PRKCI: pos. 142). By con-trast, the genus Cervus was found polyphyletic in the com-bined analysis, and in the independent analyses ofmitochondrial markers: C. duvauceli is allied with Axis(PPD 0.96; BPB/ML D 90/91); C. eldi is linked to Elaphurus(PPD 0.87; BPB/ML D 91/97); and the latter clade plus allother species of Cervus (C. timorensis, C. unicolor, C. albi-rostris, C. elaphus, and C. nippon) form the sister group toDama (PPD 0.96; BPB/ML D 93/88). Nuclear genes do not

Fig. 2. Bayesian tree resulting from the analysis combining the four markers CO2, Cyb, �LAlb and PRKCI. The selected model is GTR + I + � for bothBayesian and maximum likelihood (ML) analyses. The values shown for each node correspond to posterior probabilities (PP), Bayesian and ML boot-strap proportions (BPB and BPML, respectively) in the order indicated in the rectangle. The diVerent symbols indicate the four diagnostic indels which canbe found in the two nuclear genes: D deletion of ATT in PRKCI, D deletion of AATACCCTGTA in �LAlb, D insertion of AT in PRKCI and D deletion of ACATAAAAGGCAACAG in �LAlb.

PLESIOMETACARPALIA

Antilocapra americana

Dama dama

Dama mesopotamica1/100/100

Cervus timorensis

Cervus unicolor1/100/100

Cervus albirostris

Cervus elaphus

Cervus nippon1/52/56

1/93/96

1/100/100

Elaphurus davidianus

Cervus eldi

0.99

0.96/93/88

Cervus duvauceli

Axis axis

Axis porcinus1/100/100

0.96/90/91

1/100/100


Muntiacus reevesi1/77/72

1/97/99

Capreolus capreolus

Hydropotes inermis1/100/100

Alces alces

Rangifer tarandus

Mazama americana

Odocoileus hemionus

Odocoileus virginianus1/100/100

1/100/100

Blastocerus dichotomus

Mazama gouazoubira

Hippocamelus antisensis

Pudu puda0.51/52/46

1/99/98

1/98/97

1/100/99

0.73

1/83/70

1/100/100

Moschus moschiferus

Gazella granti

Tragelaphus imberbis0.99/50/50

1/80/63

0.87/91/97

0.75/52/51

CERV

BOVIDAE

IDAE

/86/89

PP/BPB

/BPML

TELEMETACARPALIA


produce a strong signal for the position of the variousspecies of Cervus: only two nodes are supported by PPvalues superior to 0.50, and they are incongruent with themitochondrial analyses: C. nippon is allied with C. elaphusin the �LAlb tree (PPD 1; BPML D 97; diagnostic transi-tion T!C in pos. 220), whereas it is grouped with C. albi-rostris in the mtDNA trees (CO2/Cyb: PPD 1/0.5;BPML D 97/50; diagnostic transition A!G in pos. 111 ofCO2); C. timorensis is allied with Elaphurus in the PRKCItree (PPD 0.97; BPML D 61; diagnostic transversion A!Tin pos. 362), whereas it is allied with C. unicolor in themtDNA trees (CO2/Cyb: PPD 0.84/1, and BPML D 71/100;diagnostic transition A!G in pos. 36 of Cyb).

3.1.4. Within TelemetacarpaliaThe subfamily Odocoileinae sensu Grubb (1993) is

found to be paraphyletic because Hydropotes (Hydropoti-nae) is robustly allied with Capreolus. This clade is found inthe combined analysis (PPD 1; BPB/MLD 100), and in all theseparate analyses of the four genes (0.63 < PP < 1;50 < BPML < 99). Moreover, this grouping is supported bythree transitions (�LAlb: A!G and C!T in pos. 130 andpos. 43; PRKCI: A!G, pos. 109) and two transversions(Cyb: A!T, pos. 318; �LAlb: C!A, pos. 357). The Ameri-can genera, i.e., Blastocerus, Hippocamelus, Mazama,Odocoileus, and Pudu, form a monophyletic assemblage inthe combined analysis (PPD1; BPB/MLD 98/97), and inthe mitochondrial analyses (CO2/Cyb: PPD 1/0.98;BPMLD 71/91). Their grouping with Rangifer receivesstrong support in the Cyb, �LAlb, and PRKCI analyses(PPD 1; 80 < BPML < 86) and in the combined analysis(PPD 1; BPB/MLD 100/99). The position of Alces is ambigu-ous: it is allied with Capreolus and Hydropotes in the Cybtree (PPD0.8; BPMLD 60), and in the bootstrap analyses ofthe combined matrix (BPB/MLD 61/74), whereas it isgrouped with Rangifer and American genera in the PRKCItree (PPD0.84; BPMLD 43), and in the Bayesian analysis ofthe combined analysis (PPD0.73).

The monophyly of Odocoileus is strongly supported bythe mitochondrial genes (CO2/Cyb: PPD 1; BPMLD 83/100), and in the combined analysis (PPD 1; BPB/MLD100),and it is also weakly favored by the nuclear �LAlb(PPD 0.68; BPMLD50). In contrast, the genus Mazama isfound polyphyletic in the combined analysis and in theindependent analyses of mtDNA markers: Mazama ameri-cana is grouped with the genus Odocoileus (combined anal-ysis/CO2/Cyb: PPD1, BPD99–100), resulting in a cladepresent in both North and South America, whereasMazama gouazoubira clusters with the genera Blastocerus,Hippocamelus, and Pudu (combined analysis/CO2/Cyb:PPD 0.99–1, BPD88–99), forming a strictly South Ameri-can clade.

3.2. Divergence times estimates

Three pairs of calibration points were used for estimat-ing divergence times (Fig. 3): in the Wrst one, the node Cer-

vidae at 20§ 2 MYA was combined with the nodeMuntiacinae (DMuntiacini) at 8§1 MYA; in the secondone, the node Cervidae at 20§2 MYA was combined withthe node (Rangifer + American genera) (DOdocoileini) at5§ 1 MYA; in the third one, the node Muntiacinae at 8§ 1MYA was combined with the node (Rangifer + Americangenera) at 5§ 1 MYA. The results show that the datesobtained with this third combination of calibration pointsare much younger than in the two Wrst analyses (Fig. 3).For instance, the origin of the family Cervidae was esti-mated between 7.7 and 9.6 MYA instead of 16.5–18 MYA,and the origin of American deer at around 4.2–5.7 MYA,instead of 9.1–12.9 MYA.

The dates obtained with each marker analyzed sepa-rately were very close to those obtained in the combinedanalysis (data not shown). However, we observed greatvariations in the intervals of credibility: they were verylarge for nuclear genes, shorter for mitochondrial genes,and very short for the combined analysis. These results con-Wrm that the intervals of credibility decrease when moreinformative sites are included in the analyses. This observa-tion is in favor of the combination of as many markers aspossible, as emphasized by Yang and Yoder (2003).

In our phylogenetic analyses, the genus Alces was foundas either the sister group to the clade composed of Rangiferand American genera, or as the sister group to Hydropotes

Fig. 3. Divergence times resulting from the three diVerent molecular dat-ing analyses. This graph illustrates the 95% credibility intervals (in ordi-nates) for the dates obtained for the nodes of the combined tree presentedin Fig. 2 (in abscissa) by using three diVerent combinations of calibrationpoints: D Cervidae (20 § 2 MYA) + Muntiacini ( D Muntiacinae)(8 § 1 MYA); D Cervidae (20 § 2 MYA) + Odocoileini ( D(Rangifer + American genera) (5§ 1 MYA); DOdocoileini (5§ 1MYA) + Muntiacini (8§ 1 MYA). This shows that only the combinationof the calibration points Odocoileini (5 § 1 MYA) + Muntiacini (8§ 1MYA) provides date estimates that are consistent with the fossil record ofMuntiacini and American deer (see text for more details). The linesbetween the date estimates are indicated only to facilitate the comparisonsbetween the diVerent analyses and palaeontological/geological dates.

1

3

5

7

9

11

13

15

17

Oldest Muntiacini (7-9)

Isthmus of Panam

a (3-3.5)

Oldest American

fossils (5)

DatesMYA

Nodes

Muntiacini

SouthAm

ericandeer

American

deer

Cervidae

Dama

Cervus

Mazam

a +Odocoileus

Axis +Rucervus

CerviniCapreoliniO

docoileiniCapreolini +

Alceini

CapreolinaeCervinae


and Capreolus. The molecular dating analyses were there-fore performed on these two topologies. The dates obtainedwith Alces sister to the clade (Rangifer + American genera)are all slightly older than those obtained with Alces sister to(Hydropotes + Capreolus), but they diVer only by 0.15 mil-lion years in average (data not shown). In particular, thesetwo sets of analyses provided very close dates for the com-mon ancestor of Alces + (Capreolus + Hydropotes) (7.4MYA) and the one of Alces + (Rangifer + American genera)

(7.5 MYA). These results suggest therefore that these threelineages diverged from each other during a very short timeframe.

3.3. Ancestral morphotype of Cervidae

Fig. 4 shows the distributions of diVerent characterstates corresponding to antlers, upper canines, body size,sexual weight dimorphism, and habitat. These inferences

Fig. 4. Synthetic tree of the family Cervidae. The tree is a consensus derived from the Bayesian and maximum likelihood analyses of the matrix combiningall four markers (CO2, Cyb, �LAlb and PRKCI). The nodes supported by a Bootstrap value below 70 in the combined analysis are not represented. Thedate estimates were calculated by using the tree shown in Fig. 2 and are detailed in Table 2. The time scale comes from the Geological Society of America(1999, available at http://www.geosociety.org/science/timescale/timescl.pdf). The symbols indicate the distributions of diVerent character states corre-sponding to antlers ( D one tine; D two tines; D three tines; D four tines or more), D tusk-like upper canines, body size (S D Small, orminimum shoulder size <650 mm; T D Tall or minimum shoulder size >650 mm), sexual dimorphism in weight ( D monomorphism; D dimor-phism), and habitat type (O D open; C D closed). As detailed in Section 2, the evolution of these characters was inferred using PAUP 4.0b10(SwoVord, 2003).

10 (MYA) 5.3 1.8 0.01

MIOCENE PLIOCENE PLEISTOCENE

Muntiacus reevesi


Dama dama

Cervus unicolor

Cervus elaphus

Cervus nippon

Cervus albirostris

Axis porcinus

Axis axis

Rangifer tarandus

Mazama americana

Odocoileus hemionus

Odocoileus virginianus

Blastocerus dichotomus

Mazama gouazoubira

Pudu puda

Hippocamelus antisensis

Hydropotes inermis

Capreolus capreolus

MUNTIACINI

CERV

ALCEINI

CAPREOLINI

CAPREOLINAE

CERVINI

COILEINI

ODO

INAE

CERVIDAE

Cervus timorensis

Cervus eldi

Cervus davidianus

TO

TO

TO

TO

TO

TO

TO

TO

TO

TOTO

TO

TO

TO

TO

TO

S C

CS

TO

O

S C

TO

TOC

S OC

TO

TO

TO

TO

TO

TO

TO

TO

TO

TO

TO

TO

TO

TO

TO

TO

S C

S C

S C

S C

S C

Rucervus duvauceli

Alces alces

Dama mesopotamica

http://www.geosociety.org/science/timescale/timescl.pdf

http://www.geosociety.org/science/timescale/timescl.pdf


suggest that in the ancestor of Cervidae, the males werelarge (shoulder height > 650 mm), bigger than females, withthree-tined antlers, without upper canines (although onlythe tusk-like upper canines are illustrated on Fig. 4, thepresence/absence of upper canines was also tested), andlived in open habitats. The same combination of characterstates is also found for most nodes, but for the ancestors of(Muntiacus + Elaphodus) and (Capreolus + Hydropotes), ouranalyses suggest a completely diVerent pattern: they lived inclosed habitats, and the males were small (shoulderheight < 650 mm), similar in body mass to females, withtwo-tined antlers. In addition, tusk-like upper canines wereacquired independently in Hydropotes and in the commonancestor of (Muntiacus + Elaphodus). More generally, allother species found in dense habitats (Mazama americana,M. gouzoubira, Pudu) are characterized by a reduction ofantler size, a small body size, and sexual weight monomor-phism.

4. Discussion

4.1. Phylogeny and taxonomy of the family Cervidae

In this study, we combine both nuclear and mitochon-drial markers and the largest sample of genera so far pub-lished to provide a good phylogenetic resolution spanningthe whole evolutionary history of the family Cervidae.The results show that the family is divided into two mainclades corresponding to the Plesiometacarpalia and Tele-metacarpalia proposed by Brooke (1878). Plesiometacar-palia possess only the proximal part of the lateralmetacarpals II and V, whereas Telemetacarpalia possessonly the distal part of these metacarpals. This basaldichotomy was previously found in the molecular study ofHassanin and Douzery (2003) based on a much smallertaxonomic sample. As the telemetacarpal condition is alsoobserved in musk deer (Moschidae) (Scott and Janis,1987) and in the fossil ruminants Merycodontidae (Frick,1937), it is likely that this character state is plesiomorphicwithin Cervidae. If it is true, only the plesiometacarpalcondition should be considered as a synapomorphy,which would actually be exclusive of the Plesiometacarpa-lia. However, this hypothesis is not supported by Bouv-rain et al. (1989), who argue that the plesiometacarpalcondition cannot derive from the telemetacarpal condi-tion. They considered these two character states to haveevolved independently from an ancestral morphotypepossessing complete metacarpals (holometacarpal condi-tion). As the telemetacarpal condition is observed inMoschidae, which is the sister family of Bovidae (Hassa-nin and Douzery, 2003), the hypothesis of Bouvrain et al.(1989) implies that the evolution of this character ishomoplasic within ruminants. Bouvrain et al. (1989) alsoobserved that the temporal canal of all Telemetacarpaliais broadly opened in its medial region, whereas it is closedin other ruminants. Our results conWrm that this characterconstitutes a synapomorphy of Telemetacarpalia.

Our analyses clearly divide the Plesiometacarpalia intotwo main clades corresponding to the subfamilies Cervinaeand Muntiacinae in the classiWcation of Grubb (1993), or tothe tribes Cervini and Muntiacini in the classiWcation ofMcKenna and Bell (1997). This dichotomy was previouslyproposed on the basis of molecular data (e.g., Cronin et al.,1996; Randi et al., 1998), but our study is the Wrst one incor-porating all genera of Plesiometacarpalia. The grouping ofMuntiacus and Elaphodus in the subfamily Muntiacinae (orin the tribe Muntiacini) has never been seriously questionedbased on morphological data, even if Groves and Grubb(1987) noted that the general skull shape of Muntiacusresembles that of Axis porcinus more than that of Elapho-dus. Our study is the Wrst one conWrming the monophyly ofthis taxon on a molecular basis: it is recovered in the com-bined analysis (PPD1; BPB/MLD77/72), and independentlyby two markers (Cyb and PRKCI). The grouping of Mun-tiacus and Elaphodus is morphologically coherent, as it issupported by one osteological synapomorphy, i.e., thefusion of the large cuneiform and cubonavicular in the tar-sus (Garrod, 1877). However, this character is not exclusiveto the muntjacs, as these tarsal bones are also fused in theSouth American Pudu (Pocock, 1910). Since both muntjacsand Pudu share similar habitats in dense forests, the fusionof tarsal bones may be interpreted as being a convergentadaptation for locomotion resulting from the colonizationof forested habitats.

The monophyly of the subfamily Cervinae sensu Grubb(1993) (or tribe Cervini sensu McKenna and Bell, 1997)is strongly supported in the combined analysis (PPD1;BPB/MLD 100), and is recovered by two markers (Cyb andPRKCI). This result is in agreement with previous studiesbased on Cyb or D-loop sequences (Bonnet, 2001; Douzeryand Randi, 1997; Pitra et al., 2004; Randi et al., 1998, 2001).From the morphological point of view, Pocock (1910) pro-posed that the hairy tuft on the upper half of the metapo-dials could be used to diagnose this clade.

The genus Axis contains four species, and two of themare included in our sample: the spotted deer (A. axis) andthe hog deer (A. porcinus). Both species are found in Nepal,Sri Lanka and India, where their distributions sometimesoverlap (see for example Biswas, 1999); in addition, thedistribution of A. axis includes Bangladesh, while that ofA. porcinus covers Indochina, Pakistan, and China (Grubband Gardner, 1998). These two species are quite diVerent insize and coat color (Nowak, 1999). Geist (1998) is evendoubtful of their close aYnity, because they exhibit diver-gent behaviours during sexual arousal. The analyses of Cybsequences seem to conWrm this hypothesis, as Axis wasfound paraphyletic in the studies of Liu et al. (2003) andPitra et al. (2004), with A. axis related to C. duvauceli, andA. porcinus allied with C. timorensis. By contrast, the analy-ses of D-loop support the monophyly of the genus Axis, andits grouping with Cervus duvauceli (Bonnet, 2001). Ouranalyses agree with this study: the monophyly of Axis isstrongly supported by three independent markers, includ-ing the Cyb gene, and two nuclear introns (�LAlb and


PRKCI), and Axis is grouped with C. duvauceli. All previ-ous Cyb studies were actually performed by using thesequence of A. porcinus produced by Ludt et al. (2004)(Accession No. AY035874). Our results suggest thereforethat this Cyb sequence was obtained either from carry-overcontamination, or by using tissue from a misidentiWed spec-imen. This hypothesis is also corroborated by our interpre-tation of cytogenetic data. All species of Cervinae sensuGrubb (1993) possess a fundamental number of 70, (Fon-tana and Rubini, 1990). According to Bonnet (2001), A.porcinus (2nD 68) has retained the ancestral karyotype ofCervinae, and A. axis (2nD66) diVers from this ancestralkaryotype by only one autapomorphic centric fusion. Inaddition, C. timorensis (2nD 60) and C. unicolor (2nD58 or56) share four centric fusions. These data therefore favorthe grouping of C. timorensis with C. unicolor rather thanwith A. porcinus, as proposed by Ludt et al. (2004).

Previous molecular studies based on D-loop and Cybsequences have concluded that the genus Cervus is polyphy-letic, because C. eldi was allied with Elaphurus (Liu et al.,2003; Pitra et al., 2004 and Randi et al., 2001), whereas C.duvauceli and C. schomburgki were grouped with Axis(Pitra et al., 2004). Our analyses conWrm the polyphyly ofCervus. Highlighting the crucial need for a taxonomic revi-sion of the subfamily Cervinae (or tribe Cervini), Randiet al. (2001) proposed the recognition of only the three gen-era Cervus, Axis, and Dama, with Elaphurus synonymizedwith the genus Cervus. Here, we show that Cervus duvauceliis grouped with the genus Axis, as previously found byPitra et al. (2004). We suggest therefore that C. duvaucelishould be placed in the genus Rucervus, as deWned byHodgson (1838).

The clade Telemetacarpalia is well supported in the com-bined analysis (PPD1; BPB/ML D83/70), but it is onlyretrieved with one marker independently (PRKCI). It ishowever deWned by a strong molecular synapomorphy, i.e.,a deletion of 12 nucleotides in �LAlb, conWrming the Wnd-ings of Hassanin and Douzery (2003) based on only fourTelemetacarpalia rather than 11 in the present study.

All members of Telemetacarpalia belong to the sub-family Odocoileinae except the Chinese water deer(Hydropotes inermis), which is the only species of the sub-family Hydropotinae. Indeed, the subfamily Odocoileinaesensu Grubb (1993) is found to be paraphyletic becauseHydropotes is robustly grouped with the roe deer (Capreo-lus) in the combined analysis (PPD 1; BPB/ML D 100) andin all independent analyses of the four genes (Fig. 1). Incontrast to Groves and Grubb (1987), who argue thatHydropotes is divergent from all other deer, this groupingconWrms previous molecular investigations based on vari-ous mitochondrial and nuclear markers (Douzery andRandi, 1997; Hassanin and Douzery, 2003; Randi et al.,1998). In addition to the clade uniting Hydropotes andCapreolus, two other lineages emerge from our analyses:the Wrst one includes Rangifer and all American genera(Blastocerus, Hippocamelus, Mazama, Odocoileus, andPudu), and the second one is composed only of the genus

Alces. The relationships between these three lineagesremain unclear. This lack of resolution may be explainedby the fact that they diverged from each other in a veryshort amount of time.

To reconcile our phylogenetic results with taxonomy, wepropose a subdivision of the family Cervidae into the twosubfamilies Cervinae and Capreolinae, as recognized byPocock (1910). This arrangement is equivalent to the Ple-sio-/Telemetacarpalia dichotomy proposed by Brooke(1878). The subfamily Cervinae is composed of the twotribes Cervini and Muntiacini. Within the subfamily Capre-olinae, we propose to retain three tribes: the tribe Capreo-lini includes Capreolus and Hydropotes, the tribe Alceini iscomposed of Alces alone, and the tribe Odocoileini con-tains Rangifer and all American genera, as previously pro-posed by McKenna and Bell (1997). The tribe Odocoileiniis recovered in analyses of three independent markers (Cyb,�LAlb, and PRKCI), and is characterized by a deletion of atriplet ATT in PRKCI. Brooke (1878) previously noted thatall Odocoileini share a vomerine septum dividing the cho-ana into two chambers.

Within the tribe Odocoileini, the South American generaconstitute a monophyletic group which is divided into twoclades by the mitochondrial genes. Surprisingly, each ofthese clades includes one species of Mazama. The Wrst cladelinks Mazama americana to the genus Odocoileus (PPD 1;BPB/MLD100 in the combined analysis), i.e., two taxa foundin South and North America. The second clade includes alltaxa restricted to South America, i.e., Mazama gouazoubira,Blastocerus, Hippocamelus, and Pudu (PPD1; BPB/MLD99/98).

This topology is in disagreement with the morphologi-cal phylogeny of Webb (2000), which proposes the exis-tence of two tribes: the tribe Rangiferini, which includesRangifer, Hippocamelus, and Pudu; and the tribe Odoco-ileini, which groups together all other American genera,i.e., Mazama, Ozotoceros, Blastocerus, and Odocoileus.However, the conclusions of Webb (2000) are critical forthree reasons: (1) only Capreolus was used as outgroup topolarize the transformations of character states, (2) mostof the characters supporting the two tribes are quantita-tive (antlers laterally compressed, stylohyoid cupenlarged, reduced P2, enlarged bullae), and (3), the loss ofupper canines is considered to be a synapomorphy of thetribe Odocoileini sensu Webb (2000), whereas our analy-ses show that this character state is plesiomorphic in thefamily Cervidae (see paragraph 3.3).

As discussed in Eisenberg (2000) and Medellin et al.(1998), the taxonomy of the genus Mazama is very con-fusing and no comprehensive study has been publishedon inter-speciWc relationships. Strikingly, the mitochon-drial genes analyzed in this study strongly suggest that M.americana and M. gouazoubira are not closely related,thereby implying the polyphyly of the genus Mazama(Figs. 1 and 2). The monophyly of Mazama has neverbeen questioned on the basis of morphological charac-ters. However, M. americana and M. gouazoubira possess


several diVerences concerning coat color (reddish brownversus grayish brown), antler characteristics (rugose ver-sus ridged) (Medellin et al., 1998), and body size, as M.gouazoubira is smaller than M. americana (shoulderheight: 55–60 versus 60–70 cm; weight: 22 (average) ver-sus 29–35 kg) (Eisenberg, 2000). In addition, their karyo-types show large diVerences in diploid numbers: 2nD 42–53 in M. americana, and 2nD 70 in M. gouazoubira(Eisenberg, 2000). However, the two species share a verysimilar morphotype characterized by small body size(minimum shoulder height < 65 cm) and the possession ofsimple one-tined antlers, among other similarities.Assuming that Mazama is polyphyletic, our morphologi-cal analyses show that this morphotype must haveevolved convergently in the two taxa (Fig. 4). If it is true,the polyphyly of Mazama would constitute a striking caseof morphological convergence within mammals. None-theless, before drawing deWnitive conclusions, thishypothesis needs to be corroborated by a more compre-hensive analysis including all the six species of the genusMazama (Grubb, 1993), and new nuclear markers provid-ing more information on the relationships between Amer-ican species.

4.2. New classiWcation (Fig. 4)

Family Cervidae Goldfuss, 1820:374. DeerSubfamily Cervinae Goldfuss, 1820:374 [D Plesiometa-carpalia Brooke, 1878].Tribe Cervini Goldfuss, 1820:374.-Cervus Linnaeus, 1758:66[including Elaphurus Milne-Edwards, 1866:1090–1091].-Axis C.H. Smith in GriYth, Smith and Pidgeon,1827:312–313.- Rucervus Hodgson, 1838:154.-Dama Frisch, 1775:table.Tribe Muntiacini Pocock, 1923:207.- Muntiacus RaWnesque, 1815:56.- Elaphodus Milne-Edwards, 1871:93.

Subfamily Capreolinae Brookes, 1828:62 [D Telemeta-carpalia Brooke, 1878].Tribe Alceini Brookes, 1828:61.- Alces Gray, 1821:307.Tribe Capreolini Brookes, 1828:62.- Capreolus Gray, 1821:307.- Hydropotes Swinhoe, 1870:264.Tribe Odocoileini Pocock, 1923:206.- Blastocerus Gray, 1850: 68- Hippocamelus Leuckart, 1816:24.- Mazama RaWnesque, 1817:363.1

- Odocoileus RaWnesque, 1832:109.

1 More data are needed to draw deWnitive conclusion about the genusMazama. Pending a comprehensive revision of Mazama, this classiWcationprovisionally regards the genus as it is currently deWned, i.e., including sixspecies (Grubb, 1993).

- Ozotoceros Ameghino, 1891:243.2

- Pudu Gray, 1852:242.- Rangifer, C. H. Smith, in GriYth, Smith and Pidgeon,1827: 304.

4.3. Calibration points and date estimates

In a Wrst approach, divergence times were estimated byusing two calibration points corresponding to the Wrstappearance of Muntiacini and Cervidae in the fossil record,at 8§1 MYA and 20§2 MYA, respectively. The calibrationpoint Cervidae (20§2 MYA) was previously used in severalstudies (Douzery and Randi, 1997; Hassanin and Douzery,2003; Ludt et al., 2004; Randi et al., 1998, 2001), and refers tothe oldest antlers found in the lower Miocene of Eurasia,with the genera ÐLagomeryx and ÐProcervulus (Ginsburg,1988). The dates obtained are inconsistent with the paleobi-ogeographic data (Fig. 3): 11.5–15.5 MYA for the tribeOdocoileini, 9.1–12.8 MYA for the American clade, and 7.3–10.8 MYA for the South American clade, whereas the oldestfossils of Cervidae in the New World were found in NorthAmerica at the Miocene/Pliocene boundary, at around 5MYA, with ÐBretzia and ÐEocoileus (Fry and Gustafson,1974; Webb, 2000). Thus, these dates would imply a huge gapin the fossil record of North America. Such a gap seemsunlikely given that the fossil mammalian fauna of NorthAmerica is one of the best documented in the world (Stehliand Webb, 1985). Similarly, the divergence times obtainedfor the tribe Cervini are older than the ages suggested by thefossil record (Di Stefano and Petronio, 2002): 8–11.9 MYAfor Cervini, whereas they Wrst appear at the Mio-Plioceneboundary; 4.3–6.8 MYA for Cervus and 5.5–8.7 MYA forAxis, whereas these two genera appear in the Early Pleisto-cene. In the same way, coupling the calibration points Cervi-dae (20§2 MYA) and Odocoileini (5§1 MYA) yields adate between 10.6 and 15.2 MYA for the origin of Muntia-cini, which is older than the Wrst fossils of Muntiacini (7–9MYA) (Dong et al., 2004) (Fig. 3). The dates obtained by cal-ibrating the age of the family Cervidae at 20 MYA wouldthus imply a large gap in the fossil record or numerous misin-terpretations of older fossils. These results suggest thereforethat the fossils used as calibration point for the node Cervi-dae, i.e., ÐLagomeryx and ÐProcervulus, are not closelyrelated to extant Cervidae. Interestingly, their taxonomic sta-tus is controversial, Wrst because the shape of their antlers issingular among fossil deer, and second because it is not surewhether they were deciduous or not (Azanza, 1993). Thesetwo issues have caused controversy regarding their system-

2 Although the genus Ozotoceros was not included in the present study,three arguments suggest that it belongs to the tribe Odocoileini: Wrst, itsforefoot is typically telemetacarpalian; second, its skull shows the synapo-morphy of Odocoileini, i.e., the choana is divided by an extension of the vo-mer; and third, its geographic distribution limited to Argentina, Bolivia,Brazil, Paraguay and Uruguay (Grubb and Gardner, 1998) suggests that itbelongs to the South American clade, as proposed in previous classiWcations(Brooke, 1878; Grubb, 1993; McKenna and Bell, 1997; Simpson, 1945).


atic position within ruminants (Vislobokova et al., 1989):they have been placed in the family Cervidae (Chow andShih, 1978; McKenna and Bell, 1997), in their own familyÐLagomerycidae (Pilgrim, 1941), and they have been relatedto the family GiraYdae (e.g., Simpson, 1945; Stirton, 1944).

In contrast to ÐLagomeryx and ÐProcervulus, our twoother calibration points refer to fossils whose taxonomicpositions are based on unambiguous morphological charac-ters: the synapomorphy of the tribe Odocoileini, i.e., thepresence of a vomerine septum dividing the choana, appearsin the Early Pliocene with ÐPavlodaria (Vislobokova, 1980)and the oldest antlers of muntjacs appear in the Late Mio-cene (between 7 and 9 MYA) (Dong et al., 2004). The use ofthese two calibration points produced date estimates thatagree with the palaeontological data available for Cervidae(Fig. 3). For instance, we found 4.2–5.7 MYA for the Ameri-can clade; 3.4–4.9 MYA for the South American clade; 4.2–6.0 MYA for the tribe Cervini; and 1.9–3.4 MYA and 2.3–3.7 MYA for the genera Axis and Cervus (as deWned in theclassiWcation proposed in paragraph 4.2) (Table 2). Wetherefore base the rest of the discussion on the date estimatesobtained with this combination of calibration points.

4.4. Origin and diversiWcation of deer in the Late Miocene of Asia

Both the fossil record and geographic distribution ofCervinae suggest that this group arose in the Miocene ofAsia. The oldest remains of Cervinae were found in CentralAsia, at the Mio/Pliocene boundary for the tribe Cerviniwith ÐCervocerus novorossiae (Di Stefano and Petronio,2002), and during the Late Miocene for the tribe Muntia-cini with ÐMuntiacus leilaoensis (Dong et al., 2004). In addi-tion, all extant species of Cervinae are found only in Asia,with the exception of Cervus elaphus and Dama dama.However, three arguments support the hypothesis that Cer-vus and Dama recently dispersed to Europe (and, in the caseof Cervus elaphus, North America): (1) these two species arealso distributed in Asia; (2) they appear within a clade ofexclusively Asian species; (3) the phylogeographic study of

Table 2Divergence times estimated within the family Cervidae

Age MYA (SD) 95% Cred. Int.

M. americana + Odocoileus 2.2 (§0.3) 1.6–2.8Cervus 2.9 (§0.3) 2.3–3.7Dama 3 (§0.4) 2.2–4Axis 2.6 (§0.4) 1.9–3.4South American clade 4.1 (§0.4) 3.4–4.9American clade 4.9 (§0.4) 4.2–5.7Cervini 5 (§0.5) 4.2–6Capreolini 5.6 (§0.6) 4.5–6.9Odocoileini 5.8 (§0.2) 5.3–6Muntiacini 7.3 (§0.3) 7–8Alceini+Capreolini 7.4 (§0.5) 6.4–8.4Odocoileinae 7.8 (§0.4) 6.9–8.7Cervinae 7.9 (§0.4) 7.2–8.9Cervidae 8.5 (§0.5) 7.7–9.6

Ludt et al. (2004) concluded that Cervus originated in Cen-tral Asia.

Although the subfamily Capreolinae is now found in Eur-asia and America, the fossil record suggests that it diversiWedin Central Asia, and dispersed thereafter to America. Indeed,the three tribes of Capreolinae emerged in Central Asia withÐProcapreolus during the Miocene/Pliocene boundary forCapreolini (Di Stefano and Petronio, 2002), ÐPavlodaria dur-ing the Early Pliocene of North Eastern Kazakhstan forOdocoileini (Vislobokova, 1980), and ÐCervalces and Alcesduring the Pliocene for Alceini (Breda and Marchetti, 2005;Heintz and Poplin, 1981; Kahlke, 1990). As both the subfam-ilies Cervinae and Capreolinae have an Asian origin, ourdata support the hypothesis that the family Cervidae origi-nated in Asia. Our dating estimates favor a Late Mioceneorigin for deer (7.7–9.6 MYA), which reduces the age usuallyassumed for the family Cervidae by more than half (Douzeryand Randi, 1997; Hassanin and Douzery, 2003; Ludt et al.,2004; Randi et al., 1998, 2001).

The origin of the Cervidae and their tribal diversiWcationoccurred during the Late Miocene of Asia. This period wascharacterized by dramatic changes in the environment andlandscapes of Asia. The pulse in the uplift of the Tibetan pla-teau began around 11 MYA, had peak values at 9 MYA, andlasted until 7.5 MYA (Amano and Taira, 1992). It coincideswith a global increase in seasonality and aridity (Flower andKennett, 1994), which resulted in the spread of grasslands inAsia and East Africa (Cerling et al., 1997; Morgan et al.,1994; Quade and Cerling, 1995). It is striking to note thatmany other groups of ruminants diversiWed in Asia duringthe Late Miocene, including Caprini sensu lato (goats, sheep,and allies), Boselaphini (species related to the nilgai and four-horned antelope), and Bubalina (buValoes) (Hassanin andRopiquet, 2004; Ropiquet and Hassanin, 2005a,b). As allthese groups include browser/grazer species, the competitionresulting from their overlapping diversiWcations in Asia musthave played a key role in the evolution of Cervidae.

4.5. Colonization of America

Given that the two subfamilies of Cervidae originated inAsia, their presence in America must be interpreted by oneor more dispersal events from Asia across Beringia. Thefossil record indicates that deer did not enter North Amer-ica until the latest Miocene (Webb, 2000). Two distinctodocoileine genera appeared at around 5 MYA: ÐEocoileusin Florida, and ÐBretzia in northeastern Nebraska. Theirclose aYnities with the Early Pliocene ÐPavlodaria fromNorth Eastern Kazakhstan (Vislobokova, 1980) suggestthat they were recent immigrants to the New World (Webb,2000). This scenario is supported by our molecular dating,as the common ancestor of the American Odocoileini isdated between 4.2 and 5.7 MYA. This implies that it waspossible to cross Beringia during the latest Miocene. Thepaleontological data support this hypothesis, as Camelidaedispersed from North America to Asia between 6.3 and 5.8MYA (Van Der Made et al., 2002). These dispersals could


have been favored by the spread of increasingly open anddrier habitats during the Late Miocene (Cerdeño, 1998).

Since Odocoileini occurred early in America, they mayhave had time to diversify in diVerent habitats, and somespecies may have developed tolerance to warm and dampenvironments, allowing their dispersal into the Neotropics.The deer found in South America exhibit a wide range ofmorphological variation and live in diVerent ecologicalhabitats, which is characteristic of an adaptive radiation:Mazama occurs in tropical forests, Blastocerus lives inmarshy areas, Hippocamelus and Pudu dwell in the ChileanAndes and Ozotoceros inhabits the Pampas (Nowak, 1999).Moreover, a great diversity of fossils suddenly appears atthe Plio-/Pleistocene boundary of South America with atleast four diVerent genera, i.e., ÐAntifer, ÐEpieuryceros,ÐMorenelaphus, and ÐParaceros (Castellanos, 1945; Krag-lievich, 1932). This radiation is traditionally explained bythe arrival of their common ancestor in South America at3–3.5 MYA, i.e., after the formation of the Isthmus of Pan-ama (Eisenberg, 1987; Marshall et al., 1979; Stehli andWebb, 1985). Our molecular analyses suggest, however,that the evolution of deer in America during the Pliocenewas more complex. Indeed, the genus Mazama is found tobe polyphyletic: M. americana is allied with Odocoileus,whereas M. gouazoubira is grouped with the generaendemic to South America, i.e., Blastocerus, Hippocamelus,and Pudu. Our interpretation is that South America wascolonized at least twice: a Wrst time by the ancestor of theSouth American clade in the Early Pliocene, and a secondtime, by both M. americana and O. virginianus at the Plio-Pleistocene boundary. In agreement with this scenario, thecommon ancestor of the species endemic to South Americais dated between 3.4–4.9 MYA, which is compatible withthe completion of the Pliocene land bridge. The fossilrecord indicates that the dwarf forms related to the generaPudu and Mazama evolved as small-bodied Neotropicforms during the Plio-Pleistocene, and descended fromlarger Nearctic ancestors possessing more developed ant-lers (Eisenberg, 2000; Webb, 2000). Our morphologicalinferences support this hypothesis, but they suggest thatsimilar adaptations were acquired independently in M.americana, and in the brockets and pudus of the SouthAmerican clade (Fig. 4).

Although Alces and Rangifer have a wide Holarctic dis-tribution today, their presence in America probablyresulted from a recent dispersal event during the EarlyPleistocene. The diversiWcation of Alceini in Asia and thesubsequent Pleistocene dispersal of Alces to North Americaare well documented by the fossil record (Breda andMarchetti, 2005; Heintz and Poplin, 1981; Kahlke, 1990).Such evidence, however, is lacking for Rangifer, as no fos-sils are known that predate the Pleistocene. But as Rangiferis also a specialist of the Arctic tundra, we suppose that itdispersed to America during the Pleistocene, i.e., at thesame epoch than Alces and the three bovid genera Oream-nos, Ovibos, and Ovis. According to our analyses, thelineages leading to Alces and Rangifer diverged from other

Capreolinae at around 6.4–8.4 and 5.3–6.0 MYA, respec-tively. Because fossils of these two genera are not foundbefore the Pliocene for Alces, and the Pleistocene for Rang-ifer, an important gap is inferred for the fossil record inAsia.

4.6. Evolution of sexual dimorphism

In the various species of deer, males can be generally dis-tinguished from females by the presence of antlers and/ortusk-like upper canines, larger body size and mass, and/ordiVerences in coat coloration. Antlers are made of a decidu-ous bony core covered by velvet skin, which fully regener-ates each year from the permanent pedicles (Lie and Suttie,2001). Structurally, they diVer from all other cranialappendages found in ruminants (Scott and Janis, 1987) andcan thus be considered as an autapomorphy of the familyCervidae. Three main reasons have been proposed forexplaining the origin of antlers in male deer: defense againstpredators, display structures to be appreciated by females,or weapons that serve during intermale Wghts for territoryand/or access to mating with more than one female (Jar-man, 2000). Our analyses suggest that the common ancestorof Cervidae lived in open habitats, that females were antler-less, and that males were large (shoulder height > 650 mm),bigger than females, with three-tined antlers, but withoutenlarged upper canines. Interestingly, closed habitats havebeen independently colonized by Capreolini, Muntiacini,Mazama americana, Mazama gouazoubira, and Pudu, andall these taxa have developed similar morphological adap-tations, including the reduction and simpliWcation of ant-lers in males (absent in Hydropotes, or with one or two tinesrather than three or four in other species), and the acquisi-tion of a small body size, accompanied by sexual weightmonomorphism. We can infer that these reductions wereselected positively because (1) large males with long andramiWed antlers are expected to move much more slowly inclosed habitats, and (2) the display function of antlers isexpected to be much less eVective in habitats where visibil-ity is considerably reduced. In parallel with these reduc-tions, males of Hydropotes and Muntiacini have acquiredtusk-like upper canines, which are used as display orna-ments and weapons for Wghting with their congeners duringthe rut (Cooke and Farrell, 1998; Hutchins et al., 2004).Thus, canines clearly replace antlers in the sexual behaviourof these species. This supports the hypothesis that the pri-mary role of antlers is for use in sexual competition duringthe breeding season, when males Wght each other to gainaccess to females in estrus. This important function mayexplain why antlers have been maintained in all cervidsexcept Hydropotes. InterspeciWc diVerences in the morphol-ogy of antlers may have occurred because of the evolutionof divergent Wghting behaviors (Caro et al., 2003; Geist,1966; Lundrigan, 1996). As the reproductive success ofmales is supposed to be directly correlated with strengthand weapon size (Clutton-Brock, 1989), it is likely thatlarge males with important armament have been positively


selected during the evolution of Cervidae. This wouldexplain why antlers are particularly developed and ramiWedin males of species dwelling in open habitats (Fig. 4).

The only species in which females have acquired antlersis Rangifer tarandus. As antlers may serve as weaponagainst predators (Geist, 1968), and because a high percent-age of mortality in reindeer is due to wolves and bears(Crête et al., 2001), predation may have constituted a suY-cient force for the selection of antlers in females. However,whereas this predation pressure is even more pronouncedfor the genus Alces (Wayne Kuzyk, 2002) and also exists inthe genus Odocoileus, females of these genera do not pos-sess antlers.

As an alternative hypothesis, we suggest that antleredfemales may have been positively selected because of gregari-ousness. In fact, intraspeciWc competition for food is expectedto be important in the large mixed-sex herds of reindeer,especially during harsh winters when the snow level is highfor extended periods of time. Schaefer and Mahoney (2001)actually found that through a 1000 km range, the percentageof antlered females was correlated positively with averageannual snowfall and mean snow depth at the end of March.These results support the hypothesis that antlers on femalesprovide functional advantages in interference competitionfor winter food (Schaefer and Mahoney, 2001). Antlers infemale reindeer could thus have evolved as an adaptationlinked to intraspeciWc competition for food during winter,but not to anti-predator defense.

Acknowledgments

We thank Jean-Luc Berthier, Céline Canler, FrançoisCatzeXis, Philippe Chardonnet, Raphaël Cornette, JacquesCuisin, Emmanuel Douzery, Mathieu Fritz, Françoise Her-gueta-Claro, Jacques Rigoulet, William Robichaud, MichelTranier, and Vitaly Volobouev for providing tissues orDNA samples. We also acknowledge Evelyne Bremond-Hoslet, Jean-Marc Bremond, Pedro Cordeiro and WoodyCotterill for their help with the bibliography. We acknowl-edge the two anonymous reviewers for comments on themanuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.ympev.2006.02.017.

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