Phylogenetic Relationships among European Red Deer, Wapiti, and Sika Deer Inferred from Mitochondrial DNA Sequences

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Molecular Phylogenetics and EvolutionVol. 15, No. 1, April, pp. 115–123, 2000doi:10.1006/mpev.1999.0731, available online at http://www.idealibrary.com on

Phylogenetic Relationships among European Red Deer, Wapiti,and Sika Deer Inferred from Mitochondrial DNA Sequences

Ryu Kuwayama and Tomowo Ozawa

Department of Earth and Planetary Sciences, Graduate School of Science, Nagoya University,Furo cho, Chikusa-ku, Nagoya 464-8602, Japan

Received March 31, 1999; revised August 23, 1999

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We determined the mitochondrial cytochrome b geneequences (1140 bp) of one subspecies of the Europeaned deer (Cervus elaphus in Europe), three subspeciesf the wapiti (C. elaphus in Asia and North America),nd six subspecies of the sika deer (C. nippon inapan). Our phylogenetic analysis revealed the mono-hyly of the European red deer, that of the wapiti, andhat of the sika deer. The wapiti, however, was showno be more closely related to the sika deer than to theuropean red deer. This is in conflict with traditionalorphological results, which suggest a close sister

roup relationship between the wapiti and the Euro-ean red deer. The divergence time between the Euro-ean red deer and the wapiti plus the sika deer wasstimated to be approximately 0.80 Ma, and that be-ween the wapiti and the sika deer was estimated to be.57 Ma. The sika deer was subdivided into two subspe-ies groups, and the wapiti was also found to consist ofn Asian group and a North American group. r 2000

cademic Press

Key Words: European red deer; wapiti; sika deer;ervus; mtDNA phylogeny; Pleistocene

INTRODUCTION

The genus Cervus is the major lineage in the subfam-ly Cervinae (Artiodactyla, Cervidae), exceeding otherervids in range of distribution and number of speciesFlerov, 1952; Whitehead, 1972, 1993). Based on mor-hological analyses among living Cervus, the Europeaned deer (C. elaphus in Europe), the wapiti (C. elaphusn Asia and North America), and the sika deer (C.ippon in eastern Asia and Japan) are monophyleticnd thought to represent a derived morphology (Cor-et, 1978; Putman, 1988). In overall morphology, theyre closely similar to one another with the exception ofody size and antler morphology (Geist, 1971; Zima etl., 1990).The European red deer is considered to be more

losely related to the wapiti than to the sika deer based
n morphological characters, such as the degree of m
115

ranching of antlers and body size (Geist, 1971; Grovesnd Grubb, 1987). On the other hand, Lydekker (1898)uggested that the wapiti had a closer relationship withhe sika deer than with the European red deer based onther morphological characters, such as pelage colora-ion and uncupped antlers. Although recent molecularhylogenetic studies indicate that the European redeer, the wapiti, and the sika deer are monophyletic,he phylogenetic relationship among these three taxaemains unresolved (Emerson and Tate, 1993; Cronint al., 1996; Polziehn and Strobeck, 1998). The phyloge-etic relationship among the sika deer is also controver-ial because each subspecies of this deer shows variableorphologic characters in antler form and body size

Imaizumi, 1970; Groves and Grubb, 1987).In this study, we sequenced the entire cytochrome b

ene from a total of 10 individuals. We performed ahylogenetic analysis to clarify the phylogenetic rela-ionships of this clade. These data were then used tonalyze the process of divergence and dispersal of thesepecies of Cervus with reference to their Pleistoceneossil records.

MATERIALS AND METHODS

Tissue samples for preparation of DNA were ob-ained from the European red deer in England (C.laphus scoticus), two subspecies of the wapiti in AsiaC. e. kansuensis and C. e. xanthopygus) and one inorth America (C. e. canadensis), and six subspecies of

he sika deer in Japan (C. nippon yesoensis, C. n.entralis, C. n. nippon, C. n. mageshimae, C. n. kera-ae, and C. n. pulchellus) (Table 1). Total genomic DNA

amples were extracted from these tissue samplessing standard procedures. The cytochrome b geneegion (1140 bp) was amplified by the polymerase chaineaction (PCR) (Saiki et al., 1988) using the sets ofligonucleotide primers shown in Table 2.Twenty to 30 ng of template DNA was subjected to 40

ycles of PCR amplification in 200 µl of reaction mix-ure containing 10 mM Tris (pH 8.3), 1.5 mM MgCl2, 50
M KCl, 0.01% gelatine, each deoxynucleotide at 200
1055-7903/00 $35.00Copyright r 2000 by Academic PressAll rights of reproduction in any form reserved.

µ(d7

Npmq

uatd

gcfdatt‘bp((pbtKlcicc

m(h(rfdEA

CCCCCCCCCC

CN

L

L

L

L

LL

L

HH

HHHH

H

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116 KUWAYAMA AND OZAWA

M, each primer at 0.5 µM, and 1 U Taq polymeraseTakara). Each cycle consisted of 40 s at 94°C forenaturation, 1 min at 48°C for annealing, and 1 min at2°C for extension.The amplified DNA fragments were purified on 2%usieve agarose gels and then subcloned into plasmidUC118 after terminal blunt-ending with Klenow frag-ent (Frohman, 1994). Each plasmid clone was se-

uenced with appropriate sequencing primers (Table 2)

TAB

Taxa Examine

Scientific name Common name Tissue

ervus elaphus scoticus European red deer Hairervus elaphus kansuensis Wapiti Hairervus elaphus xanthopygus Wapiti Hairervus elaphus canadensis Wapiti Hairervus nippon yesoensis Sika deer Muscleervus nippon centralis Sika deer Muscleervus nippon nippon Sika deer Muscleervus nippon mageshimae Sika deer Muscleervus nippon keramae Sika deer Hairervus nippon pulchellus Sika deer Hair

TABLE 2

Primers for Amplification and Sequencing of theytochrome b Gene Region Showing Each Primerame and Sequence

Primername Sequence

14724 58-CGAAGCTTGATATGAAAAACCATCGTTG-38 (Irwin etal., 1991)

14841 58-AAAAAGCTTCCATCCAACATCTCAGCAT-GATGAAA-38 (Irwin et al., 1991)

15144 58-ATAGCCACAGC(C/A)TTCATAGG(A/C)TA(C/T)GTCCT-38

15306 58-CGATTCTTCGC(C/T)TTCCACTT(C/T)ATCCT(A/T/C)CCATT-38

15408 58-A(C/T)AGA(C/T)AAAAT(C/T)CC(A/C)TT(C/T)CA-3815606 58-TTTGCATACGCAATCCTACGATCA(A/G)T(C/T)CC(A/

C/T)AA(C/T)AA-3815702 58-ACATCCAAACAACGAAGCATAATATT(C/T)CG(A/

C)CC-3814927 58-GTGACAGAGGAGAATGCTGT-38 (Irwin et al., 1991)15149 58-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-38

(Kocher et al., 1989)15347 58-GGGTT(A/G)TT(G/T)GATCCTGTTTCGTG-3815494 58-TAGTTGTC(A/T)GGGTCTCC(G/T)A(A/G)-3815603 58-GCTAG(G/T)AC(G/T)CCTCCTAGTTT-3815752 58-TCTACTGG(C/T)TG(G/T)CC(T/G/C)CC(A/

G)ATTCATGT-3815915 58-GGAATTCATCTCTCCGGTTTACAAGAC-38

Note. In the primer names, L and H refer to the sequence of lightnd heavy strands, respectively, and the numbers correspond to the8-end positions of the primers in the numbering system for humanitochondrial DNA (Anderson et al., 1981). In this numbering system

he 1140-bp sequence corresponds to the sequence from 14,747 to
s5,886.
sing the dideoxy-chain termination method (Sanger etl., 1977). Multiple independent clones were sequencedo exclude any mutations that might have arisenuring PCR.DNA sequence data were aligned along with ortholo-

ous sequences for the sika deer from Honshu (C. n.entralis) (Chikuni et al., 1994), the European red deerrom Switzerland (C. e. hippelaphus), and the falloweer (Dama dama in the subfamily Cervinae) (Randi etl., 1998). The latter species was used as outgroup ofhe European red deer, the wapiti, and the sika deer. Inhis report, we use the names ‘‘C. n. centralis 1’’ and‘C. n. centralis 2’’ to refer to the C. n. centralis examinedy Chikuni et al. (1994) and that examined by us in theresent study, respectively. Neighbor-joining (NJ)Saitou and Nei, 1987) and maximum-parsimony (MP)Fitch, 1971) analyses were performed using the com-uter program PAUP*4.01b (Swofford, 1996). The num-er of nucleotide substitutions between each pair ofaxa was estimated by the two-parameter method ofimura (Kimura, 1980). Confidence values for internal

ineages were assessed by 1000 bootstrap pseudorepli-ates (Felsenstein, 1985). We used a data set includingnformation on the third codon position of protein-oding genes, as these sites show little change inompositional bias among taxa.

RESULTS

Sequence similarity. Table 3 summarizes the align-ent of the mitochondrial cytochrome b sequences

1140 bp) of the 10 Cervus taxa examined along withomologous sequences for the sika deer from Honshu

C. n. centralis 1) (Chikuni et al., 1994), the Europeaned deer from Switzerland (C. e. hippelaphus), and theallow deer (D. dama) (Randi et al., 1998). Newlyetermined sequences have been deposited in DDBJ,MBL, and GenBank under accession numbersB021090–AB021099. The matrix in Table 4 shows

1

in This Study

Natural distribution Source

at Britain Kushiro Zoo, Hokkaidosu, China, etc. Kanazawa Zoo, Yokohama

nchuria and Mongolia Yagiyama Zoo, Sendaith America Kushiro Zoo, Hokkaidokaido, Japan Wild deershu, Japan Wild deershu and Shikoku, Japan Wild deeregashima and Mageshima Is., Japan Wild deerama Is., Japan Wild deershima I., Japan Fukuoka Zoo, Fukuoka

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equence similarities and genetic distances among the

cwtSE

2Tfs

C ATGAAATTTCGCCCCCCCCCCCD

C TCCTCTGTCACCCCCCCCCCCCD

C ATGTAGGACGACCCCCCCCCCCD

C ATATGTCCTACCCCCCCCCCCCD

117PHYLOGENY OF EUROPEAN RED DEER, WAPITI, AND SIKA DEER

ytochrome b gene sequences for these 13 taxa. Pair-ise sequence differences ranged from 0 to 6.0% among

he European red deer, the wapiti, and the sika deer.equence differences were relatively small among the

TAB

Sequences of the Mitochondrial C

. e. canadensis ATGATCAATACCCGAAAAACCCACCCATTAATAAAA

. e. kansuensis .............................................................................................

. e. xanthopygus .............................................................................................

. n. mageshimae ....C...C.T..........................................C...........C........A........

. n. pulchellus ....C...C.T..........................................C...........C........A........

. n. keramae ....C...C.T..........................................C...........C........A........

. n. nippon ....C...C.T..........................................C...........C........A........

. n. centralis 1 ....C...C.T..........................................C...........C........A........

. n. centralis 2 ....C...C.T..........................................C...........C........A........

. n. yesoensis ....C...C.T..........................................C...........C........A........

. e. scoticus ....C.....T................C..............................................A............

. e. heppelaphus ....C.....T................C.G............................................A..........ama dama ..........T.........T........G........C....................T..............A.........

. e. canadensis GCTCCTTACTAGGAATTTGTCTAATCCTACAAATTC

. e. kansuensis ......................................................................A.....................

. e. xanthopygus .....C....................T...............................................................

. n. mageshimae ..................................C......................................C..............C

. n. pulchellus ..........................T.......C......................................C..............

. n. keramae ..................................C......................................C..............C

. n. nippon ..................................C......................................C..............C

. n. centralis 1 .....C............................C..T................................T..C...........

. n. centralis 2 .....C............................C..T................................T..C...........

. n. yesoensis .....C............................C..T................................T..C...........

. e. scoticus ....A.........G.C.................C....................G..............T............

. e. heppelaphus ....AC........G.C.................C....................G..............T..........ama dama .....C.............CT.............C.............................C.....T............

. e. canadensis CCATATCTGTCGAGATGTCAATTATGGTTGAATTAT

. e. kansuensis ...................T................................................................T.....

. e. xanthopygus ........................................................................T...........T.....

. mageshimae ..........................................................................................C

. n. pulchellus ..........................................................................................C

. n. keramae ..........................................................................................C

. n. nippon .......................................................A..................................C

. n. centralis 1 ......................C..............C....................................................

. n. centralis 2 ......................C..............C....................................................

. n. yesoensis ......................C..............C....................................................

. e. scoticus .............................C..............T.................................T..........

. e. heppelaphus .............................C................................................T..G..T..ama dama .........C.....C........C........C...........G...........A...........C..T......

. e. canadensis GGCCTATACTACGGGTCATACACTTTTCTAGAGAC

. e. kansuensis ....................T......A..............................................................

. e. xanthopygus ....................T.......................T.............................................

. n. mageshimae ..T..G........A.....T................................T..............C...............

. n. pulchellus ..T..G........A......................................T..............C................

. n. keramae ..T..G........A......................................T..............C................

. n. nippon ..T..G........A......................................T..............C................

. n. centralis 1 .....G........A.....T................................T.................................

. n. centralis 2 .....G........A.....T................................T.................................

. n. yesoensis .....G........A.....T................................T.................................

. e. scoticus .....G........A.....T..............G...............G.T..T........................

. e. heppelaphus .....G........A.....T..............G.....T.........G.T..T.......................ama dama ..T...........A.......................G..............T....................T...........

uropean red deer (2.1%), among the wapiti (1.4 to d

.1%), and among the sika deer (0 to 3.0%) subspecies.he European red deer sequence, however, differed

rom that of the wapiti by 5.2 to 5.9%. This largeequence difference suggests that the European red

3

ochrome b Gene Region (1140 bp)

100TGTAAACAACGCATTTATTGACCTCCCAGCCCCATCGAATATTTCATCCTG.......................................................................................................T.

200CAGGCCTATTCCTAGCAATACACTATACATCCGATACAATAACAGCATTT

..........................................................

............C.....................

300ATACATACACGCAAACGGGGCATCAATATTTTTCATCTGCCTATTCATAC

...

...

..

..

...................G...........C.....T......

400AAACATCGGAGTAATCCTCCATTTACAGTTATAGCCACAGCATTCGTAGG

......C..............................................................G...........C.......

LE

yt

AT........................................................................

TCA.................

C..............

......

......

...........C.............

TCG.............................................................................

ATG......................................................................C.

eer and the wapiti are taxonomically different species,

atadvCa

ysgIdt

C GGTCTGAGGAGGCCCCCCCCCCCD

C TACTCTTCCTTCCCCCCCCCCCD

C TTAGGTATCTTACCCCCCCCCCCCD

C CACCCCCTCACCCCCCCCCCCD


lthough there are some conflicting views among scien-ists on this subject (Corbet, 1978; Putman, 1988; Lowend Gardiner, 1989; Zima et al., 1990). Within the sikaeer subspecies, pairwise sequence differences wereery small among C. n. mageshimae, C. n. pulchellus,. n. keramae, and C. n. nippon (0.1 to 0.6%), and

TABLE 3—

. e. canadensis CATGAGGACAAATATCATTCTGAGGAGCAACAGTC

. e. kansuensis .........................................................................................A..

. e. xanthopygus .................................G.......................................................A

. n. mageshimae ...............................................................T.........................A

. n. pulchellus .........................................................................................A..

. n. keramae .........................................................................................A..

. n. nippon ...........................................C.............................................A

. n. centralis 1 ...........................................C...................T.........................A

. n. centralis 2 ...........................................C...................T.........................A

. n. yesoensis ...........................................C...................T.........................A

. e. scoticus ...................................C...........................T.....G..................

. e. heppelaphus ...............................................................T.....T...................Aama dama .................................T........T..............C...........T............T...

. e. canadensis CTTTTCAGTAGATAAAGCAACCCTAACCCGATTCTT

. e. kansuensis ...................................T........................................................

. e. xanthopygus ...................................T........................................................

. n. mageshimae ...................................T.....C..............T............A.................

. n. pulchellus ...................................T.....C..............T............A.................

. n. keramae ...................................T.....C..............T............A.................

. n. nippon ...................................T.....C..............T............A.................

. n. centralis 1 ...C...............................T....................T...................T..........

. n. centralis 2 ...C...............................T....................T...................T..........

. n. yesoensis ...C...............................T....................T...................T..........

. e. scoticus ............C......................T......................................................

. e. heppelaphus ............C......................T......................................................ama dama ............C.........T....T............................A.....C.....T..G....T....

. e. canadensis CACGAGACAGGATCTAATAACCCAACAGGAATCCC

. e. kansuensis .....A........C...............................................T...........C............

. e. xanthopygus ..............C.................T.............................T...........C............

. n. mageshimae ..............C..C.....................G......................T...........C.........

. n. pulchellus ..............C..C.....................G......................T...........C.........

. n. keramae ..............C..C.....................G......................T...........C.........

. n. nippon ..............C..C.....................G......................T...........C.........

. n. centralis 1 ..............C..C.....................G......................T...........C C.....

. n. centralis 2 ..............C..C.....................G......................T...........C.C......

. n. yesoensis ..............C..C.....................G......................T...........C.C......

. e. scoticus .....A...........................T.........................T..T.....T.....C.........

. e. heppelaphus .....A...........................T.........................T..T.....T..C..C........ama dama ..............C...........................T.T...T....T.....T..T..C.....C..C...

. e. canadensis TTCTAATACTCTTCCTAATATTACTAGTATTATTCGC

. e. kansuensis .....G........................................C........................................T

. e. xanthopygus .....G........................................C..........................................

. n. mageshimae .....G..........T....C......................C...........T...........C............G

. n. pulchellus .....G..........G...........................C...........T...........C............G

. n. keramae .....G..........G...........................C...........T...........C............G

. n. nippon .....G..........G...........................C...........T...........C............G

. n. centralis 1 .....G......................................C...........T...............................

. n. centralis 2 .....G......................................C...........T...............................

. n. yesoensis .....G......................................C...........T...............................

. e. scoticus ....TG........T.............................C..A........T.....T.....C............

. e. heppelaphus .....G........T.............................C..A..............T.....C............Cama dama .C...T.T......T.....AC.......C....T.........CT...................A..C..T.

mong C. n. centralis 1, C. n. centralis 2, and C. n. d

esoensis (0 to 0.2%). There were, however, 2.9 to 3.0%equence differences between the former subspeciesroup and the latter subspecies group of the sika deer.n this report, we use the names ‘‘the southwestern sikaeer’’ and ‘‘the Hokkaido–Honshu sika deer’’ to refer tohe former and latter subspecies groups of the sika

ontinued

500ACCAACCTTCTCTCAGCAATTCCATACATTGGCACAAACCTAGTCGAATG....................G....G....G...........G............

600CTTTCCACTTTATTCTCCCATTTATCATCGCAGCACTCGCTATAGTACACT..........................................................T........T...

700CAGACGCAGACAAAATCCCCTTCCACCCTTACTATACGATTAAAGATATC

.......

..........C..........C..........C..........C...........C..........C..........C........T...........C.........T.....C...C..T

800CAGATCTGCTTGGAGACCCAGACAACTATACCCCAGCAATCCACTCAACA...........................................................................................................T.....

C

ATT...................................................

.A..........AA

CG...........................................................................

AT.....C.....C...C....C....C....C.............................T.......

AC......................................................C...............

eer, respectively.

w1mp

pwCw

C TCTATCCTAATCCCCCCCCCCCCD

C AGACCTACTAACCCCCCCCCCCD

C GTCCTTATACCCCCCCCCCCCCD

CCCCCCCCCCCCD

na


Molecular phylogeny. Molecular phylogenetic treesere constructed using the maximum-parsimony (Fitch,971) and the neighbor-joining (Saitou and Nei, 1987)ethods based on the DNA sequence data of all codon

TABLE 3—

. e. canadensis CATTAAACCTGAATGATATTTCCTATTTGCATACGC

. e. kansuensis ..............................................G...................................G......

. e. xanthopygus ..............................................G......................A............G.....

. n. mageshimae ...C.................................................................A............G.....

. n. pulchellus ...C.................................................................A............G.....

. n. keramae ...C.................................................................A............G.....

. n. nippon ...C.................................................................A............G.....

. n. centralis 1 ...C..............C..................................................A............G...

. n. centralis 2 ...C..............C..................................................A............G...

. n. yesoensis ...C..............C..................................................A............G...

. e. scoticus T....................................................................A............G.....

. e. heppelaphus T.................C..................................................A............G...ama dama T........C........C......................................T...T..........G.........G

. e. canadensis TTGATTCTCATGCCTCTTCTTCACACATCCAAACAA

. e. kansuensis ...........................G...............................C.............................

. e. xanthopygus ...........................G...............................C....................T.......

. n. mageshimae C....C.....................G...............................C............G...........

. n. pulchellus C....C.....................G...............................C............G...........

. n. keramae C....C.....................G...............................C............G...........

. n. nippon C....C.....................G...............................C............G...........

. n. centralis 1 ...........................G...............................C.............................

. n. centralis 2 ...........................G...............................C.............................

. n. yesoensis ...........................G...............................C.............................

. e. scoticus ..A........................................................C..T.........................

. e. heppelaphus ..A........A...............................................C............................ama dama C.A.....T..A..CT.C..C.........................................T.....T........

. e. canadensis CACTCACATGAATTGGAGGACAGCCAGTTGAATAC

. e. kansuensis ......................................C..........................G..C...................

. e. xanthopygus .................................................................G.........................

. n. mageshimae ......................A..........................................G..C.......C.........

. n. pulchellus ......................A..........................................G..C.......C.........

. n. keramae ......................A..........................................G..C.......C.........

. n. nippon ......................A..........................................G..C.......C.........

. n. centralis 1 ......................A...........T..............................G.TC................

. n. centralis 2 ......................A...........T................................TC..................

. n. yesoensis ......................A...........T..............................G.TC................

. e. scoticus ....T........C........A.....C..............C.....................G.......T..C...

. e. heppelaphus .............C........A...........T........C.....................G.......T..C....ama dama ....T........C........A.....C.....T..T.......CC..........................T...C

1140. e. canadensis AATTACCAGCACAATCGAAAATAACCTCCTAAAATG. e. kansuensis ......................................... e. xanthopygus .....................C................... n. mageshimae G..C.................C................... n. pulchellus G..C.................C................... n. keramae G..C.................C................... n. nippon G..C.................C................... n. centralis 1 G........................................ n. centralis 2 G........................................ n. yesoensis G........................................ e. scoticus ...C.................C................... e. heppelaphus ...C.................C..................ama dama .GCC..............G........T............

Note. The DNA sequences of the 10 Cervus taxa determined in thisippon centralis 1) (Chikuni et al., 1994), the European red deer in Swl., 1998). A dot (.) indicates that the nucleotide at that position is iden

ositions and just the third codon positions (Fig. 1). All o

hylogenetic trees showed the same topology, exceptithin the clade of C. n. pulchellus, C. n. keramae, and. n. nippon. The monophyly of the European red deeras confirmed with 100% bootstrap support in all of

ontinued

900CCTACGATCAATTCCCAACAAACTAGGAGGGGTCTTAGCCCTAATCTCA

....................................................................G.............C.....G...

1000CAGCATGATATTCCGACCATTTAGCCAATGCCTATTCTGAATCTTAGTAGC.......................................................................T......................

1100CTTTATTATTATTGGACAACTAGCATCTATCTTATACTTTTTCATTATCCTA..........................................................................C...........C......T..............

GA

dy are shown along with those from the sika deer in Honshu (Cervusrland (C. e. hippelaphus), and the fallow deer (Dama dama) (Randi etal to that of C. elaphus canadensis.

C

AAT..............C.......................C....C....C..C......C.....

CG..............................................................T......G..C

CC.........................................................................

AA

stuitzetic

ur phylogenetic analyses. The monophyly of the wapiti

afiswhmtaHbwcr

ta(adto1doMEe

aafig1scg0

fibnsmectfcJbxccAc

cts(epttcdttwb

p(

1111


nd the monophyly of the sika deer were also con-rmed, with 94 to 98% and 90 to 96% bootstrapupport, respectively. A surprising finding was that theapiti and the sika deer were grouped together, withigh bootstrap support ranging from 91% in the maxi-um-parsimony trees to 96% in the neighbor-joining

ree based on all codon positions. Among the sika deer,southwestern sika deer clade and a Hokkaido–

onshu sika deer clade were identified with 100%ootstrap probabilities. The monophyly of the Asianapiti (C. e. kansuensis and C. e. xanthopygus) was also

onfirmed, with relatively low bootstrap probabilities,anging from 52 to 71%.Divergence times. Paleontological studies suggest

hat the ancestor of the living red deer (C. elaphus) firstppeared during the early Villafranchian StagePliocene Epoch) in Europe (Lydekker, 1898; Theniusnd Hofer, 1960) along with the ancestor of the falloweer (D. dama) (Kurten, 1968). These workers suggesthat the ancestors of these taxa closely resembled eachther morphologically at that time (Thenius and Hofer,960; Kurten, 1968). It is therefore considered that theivergence between the red deer and the fallow deerccurred around the Plio-Pleistocene boundary of 1.60a. In this study, the divergence times between theuropean red deer, the wapiti, and the sika deer werestimated with reference to this date.The divergence time between the European red deer

nd the wapiti plus the sika deer was estimated to bepproximately 0.80 Ma, which is concordant with therst appearance of the wapiti in Asia during the Mindellacial period (0.60–0.40 Ma) (Vangengeim and Sher,970). The divergence time between the wapiti and theika deer was estimated to be 0.57 Ma. This is notontradictory to the first appearance of the sika deer (C.rayi), believed to be the direct ancestor of C. nippon, at

TAB

Percentage Similarity of Nucleotide Sequences (abover Site between Each Pair of Sequences Estimated bbelow the Diagonal)

1 2 3 4 5

1 C. e. canadensis — 98.1 97.9 95.4 95.62 C. e. kansuensis 0.020 — 98.6 95.9 96.13 C. e. xanthopygus 0.021 0.014 — 95.9 96.34 C. n. mageshimae 0.049 0.043 0.043 — 99.55 C. n. pulchellus 0.046 0.040 0.038 0.005 —6 C. n. keramae 0.045 0.039 0.039 0.004 0.0017 C. n. nippon 0.047 0.041 0.041 0.006 0.0038 C. n. yesoensis 0.044 0.037 0.038 0.030 0.0319 C. n. centralis1 0.044 0.037 0.038 0.030 0.0310 C. n. centralis2 0.042 0.037 0.038 0.030 0.0311 C. e. scoticus 0.059 0.056 0.055 0.056 0.0572 C. e. hippelaphus 0.062 0.057 0.055 0.061 0.0623 Dama dama 0.108 0.115 0.114 0.120 0.124

.46–0.23 Ma (Young, 1932; Qi, 1989). Although the g

rst appearance of the sika deer in Japan is uncertainecause of its relatively poor fossil record and taxo-omic ambiguity, Otsuka (1988) suggested that theika deer (C. grayi) first appeared in Japan during theiddle Pleistocene. The split between the southwest-

rn sika deer and the Hokkaido–Honshu sika deerould be dated at 0.41 Ma. Although the divergenceime between the two groups of the sika deer is unclearrom the fossil records, it seems that the sika deer hasomprised these two groups since the Pleistocene inapan (Shikama, 1941, 1949). The divergence timeetween the Asian wapiti (C. e. kansuensis and C. e.anthopygus) and the North American wapiti (C. e.anadensis) was estimated to be 0.28 Ma, which isonsistent with the first appearance age of the wapiti inlaska (Illinoian glacial period: 0.26–0.15 Ma) indi-ated by the fossil record (Guthrie, 1966).

DISCUSSION

Taxonomy and phylogeny. The European red deer isonsidered to be more closely related to the wapiti thano the sika deer, based on morphological charactersuch as the degree of branching of antlers and body sizeGeist, 1971; Groves and Grubb, 1987). Some research-rs, however, emphasize that the wapiti has a closerhylogenetic relationship with the sika deer than withhe European red deer, based on morphological charac-ers such as the adult pelage coloration and the un-upped antler (Lydekker, 1898) and on immunologicalata (Harrington, 1985). This study demonstrates thathe sika deer (C. nippon), which has ancestral charac-ers in antler morphology, is most closely related to theapiti, which possesses the most derived type of antlerranching pattern among the red deer (C. elaphus).Considering that the sika deer comes into contact

4

he Diagonal) and Number of Nucleotide Substitutionshe Two-Parameter Method of Kimura (Kimura, 1980)

6 7 8 9 10 11 12 13

.7 95.6 95.8 95.8 96.0 94.4 94.1 90.2

.2 96.1 96.4 96.4 96.4 94.7 94.6 89.6

.2 96.1 96.3 96.3 96.3 94.7 94.8 89.7

.6 99.4 97.1 97.1 97.1 94.6 94.2 89.2

.9 99.7 97.0 97.0 97.1 94.6 94.1 88.999.8 97.1 97.1 97.1 94.6 94.2 89.0

.002 — 97.1 97.1 97.1 94.5 94.0 89.0

.030 0.030 — 100.0 99.8 94.4 94.8 89.6

.030 0.030 0.000 — 99.8 94.4 94.8 89.6

.030 0.030 0.002 0.002 — 94.4 94.8 89.8

.056 0.058 0.059 0.059 0.059 — 97.9 89.3

.061 0.063 0.055 0.055 0.055 0.021 — 89.9

.123 0.123 0.115 0.115 0.113 0.119 0.112 —

LE

e ty t

9596969999

—0000000

eographically with the wapiti in northeastern Asia

aap


FIG. 1. Phylogenetic trees constructed by maximum-parsimony (a and b) and the neighbor-joining (c and d) methods for the 12 Cervus taxand one outgroup. They were constructed based on data from all codon positions (a and c) and third codon positions only (b and d). The numbersbove or below the nodes in the trees are bootstrap proportions from 1000 pseudoreplicates. Scale bars represent branch length (substitutions
er site) for the NJ trees (c and d).

(ipthcnMls

ts(NikHatomGgt(atalcpcIao

lmsparctctp

sasrP

eh

wdSssfppfas1dmim

i1attlssokYs(

vt(hWtaiwgi1Joiw

SPfftF


Whitehead, 1972, 1993) and that these deer can hybrid-ze (MiroI’ubov, 1949; Geptner et al., 1961), it would beossible that they form a mosaic in mitochondrial generees as a result of their hybridization. There is,owever, little possibility of mitochondrial genome ex-hange between the wapiti and the sika deer, becauseo mosaic is found in our molecular phylogenetic trees.oreover, their hybridization is considered to be very

imited because of the large difference in body sizehown by the two taxa (Groves and Grubb, 1987).We found two distinctive subspecies groups within

he sika deer in our phylogenetic trees, as was alsouggested by previous molecular phylogenetic studiesTamate et al., 1995, 1998; Tamate and Tsuchiya, 1995;agata et al., 1995). One is the southwestern sika deer,

ncluding C. n. mageshimae, C. n. pulchellus, C. n.eramae, and C. n. nippon, and the other is theokkaido–Honshu sika deer, including C. n. centralisnd C. n. yesoensis. C. n. yesoensis has been consideredo have morphological characters different from anyther sika deer in Japan and to be grouped with theainland sika deer (C. n. hortulorum) (Imaizumi, 1970;roves and Grubb, 1987). However, the cytochrome bene sequence of C. n. yesoensis is completely identicalo that of one individual of C. n. centralis. Imaizumi1970) described the sika deer from Tsushima Island asfull species (C. pulchellus) on the basis of the charac-

eristic antler morphology. This deer, however, wasligned with the southwestern sika deer in our molecu-ar phylogenetic trees. Moreover, C. n. keramae isonsiderably smaller than any other sika deer andossesses simple two-forked antlers, but this deer isonsidered to have been introduced into the Keramaslands from mainland Kyushu just in medieval timesnd was included among the southwestern sika deer inur phylogenetic trees.Traditionally, cervid taxonomic classification and phy-

ogenetic analysis have been based mainly on antlerorphology, which is diagnostic for each species and

ubspecies (Lydekker, 1898, 1915, etc.). However, mor-hological characteristics, especially in the antler formnd the body size of deer, are very plastic in response toegional environmental conditions (Lister, 1989). Re-ently, the opinion has been expressed that the tradi-ional morphology-based taxonomy is inconsistent withervid molecular phylogeny (Miyamoto et al., 1990) andhat a taxonomic classification based on molecularhylogeny should be accepted.Divergence and dispersal events. Based on the pre-

ent molecular phylogenetic study, the fossil record,nd relevant geologic information, we evaluated pos-ible divergence and dispersal events for the Europeaned deer, the wapiti, and the sika deer during theleistocene to be as follows.The red deer (C. elaphus) first appeared during the

arly Gunz glacial period in Europe (Kurten, 1968). It
as been thought that only the wapiti migrated east- S
ard from Europe and diverged from the European redeer in the Mindel glacial period (Vangengeim andher, 1970; Geist, 1971). The present study, however,hows that the common ancestor of the wapiti and theika deer migrated eastward from Europe and divergedrom the European red deer in the early Mindel glacialeriod. The geographical isolation between the Euro-ean red deer and the wapiti plus the sika deer resultedrom the geographic barrier of the mountainous arearound the Himalayas, which is reflected by the pre-ent distribution of these two groups (Whitehead, 1972,993). The common ancestor of the wapiti and the sikaeer is considered to have adapted to the colder,ountainous environment in Asia and to have evolved

ts characteristic body size and more complicated antlerorphology probably according to Bergman’s law.The ancestor of the sika deer (C. grayi) first appeared

n eastern Asia in the late Mindel glacial period (Young,932; Qi, 1989). The present study suggests that thisncestor would have diverged from the wapiti at thatime and would have adapted to its ecological habitat inemperate forests by dwarfing and simplifying its ant-er form. The sika deer would have split into theouthwestern sika deer and the Hokkaido–Honshuika deer around 0.41 Ma. The former type of sika deerccupied their current distribution in Kyushu, Shi-oku, Tsushima, Tanegashima, Mageshima, andakushima Islands, Japan, whereas the latter type ofika deer dispersed to Hokkaido and Honshu, JapanShikama, 1941; Whitehead, 1972, 1993).

A population of the wapiti migrated to North Americaia the Bering land bridge from Asia and diverged fromhe Asian wapiti in the early Illinoian glacial periodGuthrie, 1966). Although the wapiti is also thought toave migrated to North America from Asia during theisconsin glacial period (Guthrie, 1966; Geist, 1971),

he divergence time (0.28 Ma) between the Asian wapitind the North American wapiti inferred from this studyndicates that there would not have been extensiveapiti migration to North America after the Illinoianlacial period. A population of the wapiti also appearedn the late Pleistocene of Japan (Kuwayama et al.,998), but they were not able to establish a niche in theapanese Islands because the sika deer had alreadyccupied the most suitable niche in the forests of theslands by that time (Shikama, 1941); consequently, theapiti disappeared from Japan.

ACKNOWLEDGMENTS

We are deeply indebted to Dr. Christopher C. Austin of the Institute oftatistical Mathematics, Dr. Thomas A. Demere of the Department ofaleontology, San Diego Natural History Museum, and Dr. E. Nakajima

or their critical reading of an earlier draft of this paper. We also thank theollowing institutions and persons for providing tissue samples of deer forhe present study: Kushiro Zoo, Yokohama Kanazawa Zoo, Yagiyama Zoo,ukuoka Zoo, Japan, Mr. K. Matsushita of Tanegashima, and Mr. K.
hiroma of the University of the Ryukyus.

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Documents

Phylogenetic Relationships among European Red Deer, Wapiti, and Sika Deer Inferred from Mitochondrial DNA Sequences