Proc. Nati. Acad. Sci. USA Vol. 87, pp. 6127-6131, August 1990 Evolution Phylogeny and evolution of antlered determined from mitochondrial DNA sequences (DNA systematics/Cervidae/phylogenetic robustness/fossils/biogeography) MICHAEL M. MIYAMOTO*t, FRED KRAUS*, AND OLIVER A. RYDERt *Department of Zoology, University of Florida, Gainesville, FL 32611; and *Research Department, Zoological Society of San Diego, San Diego, CA 92112 Communicated by Charles G. Sibley, May 21, 1990

ABSTRACT Mitochondrial DNA sequences of both ribo- outgroup H. inermis (Chinese , subfamily somal RNA genes and three adjacent transfer RNA genes were Hydropotinae)§. These newly obtained orthologues spanning obtained for the three extant subfamilies of antlered deer nearly 2.7 kilobase pairs of contiguous coding mtDNA pro- (Cervinae, Muntiacinae, and Odocoileinae) as well as for their vide the large number of phylogenetically informative char- antlerless sister group Hydropotinae (family Cervidae). Phy- acters needed to resolve subfamilial relationships among logenetic analysis of these sequences (each nearly 2.7 kilobase antlered deer. With these results, we have reevaluated the pairs in length) supports a cervine/muntiacine lade to the affinities of dicrocerines and the New World origins of exclusion of odocoileines. These results are statistically signif- odocoileines. icant, stable, and congruent with some independent data. Our mitochondrial DNA sequences, when coupled with other in- formation, indicate that the earliest fossil antlered deer are not MATERIALS AND METHODS closely related to living muntiacines or any other contemporary mtDNA from the four cervids was purified from brain or liver subfamily. From this information, we hypothesize an Old (12), cleaved into restriction fragments by BamHI or EcoRI, World, Late Miocene origin of Odocoileinae. and then cloned into the corresponding sites of plasmid vector pUC19 or pBR322 (13). Amplified plasmid DNA The subfamilies Cervinae (Old World deer), Muntiacinae carrying the 2.7-kilobase-pair region of interest was isolated ( and ), and Odocoileinae (New World from transformants of DHa5 or JM109 by the alkaline ex- deer) of the family Cervidae (order Artiodactyla, suborder traction method (14) and then directly sequenced either by Ruminantia) (table 1.V. in ref. 1) are unique in that males (as the chemical cleavage procedure [ (15)] or by the well as females of Rangifer) have antlers (2, 3). In the dideoxy double-strand method [, Hydropotes, and subfamilies Cervinae and Odocoileinae, antlers are usually Muntiacus (16, 17)]. The Cervus, Hydropotes, and Muntia- large and complex structures, consisting of a long, multi- cus orthologues were sequenced in both directions by a series branched distal element attached to a short nondeciduous of 14 primers (7 per complement), which were more or less pedicle or base. In contrast, the antlers of Muntiacinae are evenly spaced along the two strands. Similarly, the Odo- relatively small with a short, simple distal region connected coileus orthologue was sequenced in both directions. The to a long pedicle. This latter condition is considered primitive Needleman and Wunsch algorithm (18) was used to align the because it is found in the earliest fossil antlered deer (tribe cervid sequences to each other and to the more distantly Dicrocerini) from the Early and Middle Miocene of Eurasia related outgroup taurus [family , suborder Ru- (2-5). Because of their similar small, long-pedicled antlers, minantia (19)]. Parsimony analysis of the aligned sequences living muntiacines and dicrocerines have been assigned to the was conducted with PAUP (20) as described (21, 22); bootstrap same subfamily Muntiacinae (= Cervulinae) (4, 6-8). How- resampling used the program in PHYLIP (23). ever, this and other conclusions about antlered deer phylog- Branch lengths for the most-parsimonious solution were eny are rarely supported by synapomorphies (shared derived calculated two different ways: (i) from unambiguous changes characters) obtained from explicit cladistic analysis (9). The only (e.g., those with one most-parsimonious placement on morphological cladogram of Groves and Grubb (10) is an the phylogeny) and (ii) from all mutations, including those important exception. with multiple assignments (24). Such ambiguous mutations Previous phylogenetic studies of morphological and pale- were assigned to the most-parsimonious phylogeny accord- ontological information for the suborder Ruminantia have ing to the optimization procedure ofFitch (24). This approach demonstrated that the three extant subfamilies of antlered uses all parsimonious placements of an ambiguous mutation deer form a monophyletic group (3, 10). The sister group to to calculate a probability of change for each branch. These these three subfamilies is the antlerless Hydropotes inermis probabilities are then summed across all mutations to obtain (Chinese water deer), the sole living representative of Hy- the branch lengths. In this manner, each alternative place- dropotinae (1, 3, 5, 10, 11). Evolutionary relationships among ment contributes proportionally to the final assignments. antlered deer subfamilies remain unresolved (10). We sequenced the 12S and 16S ribosomal RNA (rRNA) genes and three flanking transfer RNA (tRNAPhe, tRNAval, RESULTS AND DISCUSSION and the 5' end of tRNAIeu) genes of mitochondrial DNA Pairwise comparisons of the four cervid sequences indicate (mtDNA) from Cervus unicolor (, subfamily that Cervus and Muntiacus are most similar (Fig. 1 and Table Cervinae), Muntiacus reevesi (Reeves' , subfamily 1). Cervus and Muntiacus differ by 5.6%, whereas Odo- Muntiacinae), and Odocoileus virginianus (white-tailed deer, coileus differs from them by an average of7.6%. On average, subfamily Odocoileinae) as well as from the closely related Abbreviation: MA, million years ago. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. M35874-M35877). 6127 Downloaded by guest on October 2, 2021 6128 Evolution: Miyamoto et al. Proc. Natl. Acad. Sci. USA 87 (1990)

I tRNAPHE 112S rRNA GTTGATGTAGCTTAACC--CAAAGCAAGGCACTGAAAATGCCTAGATGAGTCTCCCA-ACTCCATAAACACATAGGTTTGGTCCCAGCCTTCCTGTTAAC BTA A ACAAT A AATT-A C G A A T OVI G ACAAC A ATTA-A T C A A C CUN A AAAAT A ATTTAA C A A G C HIN A GCAAT G ACTA-G T T A A C MRE -______+ .______+ .______+ -______+ .______+ .______+ 4______. ------+ 100

TCTTAATAAACTTACACATGCAAGCATCTACACCCCAGTGAGAATGCCCTCTAGGTTATTAAAACTAAGAGGAGCTGGCATCAAGCACACACCCTGTAGC BTA CCC GC G T CT T A T C A C AA T T C - OVI CCT AT G C CG C AC T A T A G T A T - CUN CTT AT G C CG T A T C A T A G T A C - HIN CCT AT G C CA T A T TA T A G T A T - MRE ___+_____---_--4_------+------4---+ 200

TCACGACGCCTTGCTTAACCACACCCC-ACGGGAAACAGCAGTGACAAAAATTAAGCCATAAACGAAAG1rTTGACTAAGTTATATTAATTAGGGTTGGTA BTA T G TC G CG T T A TC GC T OVI C A AC G C G T C A CC AT C CUN T A TT G C G T C G CC AT T HIN C G TC G C G C CA CC AT T MRE --_-_-_-_-___-+-______+ -__ __+ -______+ .______+_-______+___- _4 ------+ 300

AATCTCGTGCCAGCCACCGCGGTCATACGATTAACCCAAGCTAACAGGAGTACGGCGTAAAACGTGTTAAkAGCACCATA-CCAAATAGGGTTAAATTCTA BTA T T T CAC G T CT G-CC GA T C CC OVI T T T CAC G A CT T-TC AA T C CC CUN T T T CAT G A AC ATAA GA T C TT HIN T T T TAT G A CT TTCC AA C T TC MRE -___ _+--_-_-+-4_--_-- ______+-______-+------+ 400

ACTAAGCTGTAAAAAGCCATGATTA-AAATAAAAATAAATGACGAAAGTGACCCTACAATAGCCGACGCAkCTATAGCTAAGACCCAAACTGGGATTAGAT BTA T A CA-AG CG TA A TT ACC T AA CG A T ovI T A TG-CA CA CA A TT ACC T AA CG G C CUN T A TATTA CG TA A TT ATC T AA TG A C HIN T A TG-CA CA TA A TT GTT T AA CG G C MRE --_-_-_-_-_-_-_-+-______+-______+__-____+__-+--++-+..-_-_-____4 _____+_--4---___+--+------+ 500

ACCCCACTATGCTTAGCCCTAAACACAGATAATTAC-ATAAACAAAATTATTCGCCAGAGTACTACTAGCGAACAGCTTAAAACTCAAAGGACTTGGCGGT BTA T C T A G AT-ATA C A CG T OVI C T C A G GT-GTA C A CG T CUN T C C A G AT-AAT C G CG T HIN C C C A G TCCACA C A CG T MRE --_____+_-______+- __+ I------+------.---+------4 ------4------600

GCTTTATATCCTTCTAGAGGAGCCTGTTCTATAATCGATAAACCCCGATAAACCTCACCAATTCTTGCTF kATACAGTCTATATACCGCCATCTTCAGCAA BTA T C G T CCC T A OVI C C A C TTC T A CUN T T T C CCC T G HIN T C G C TTC C A MRE -_++A.------_-__-_---_-_--_-__-__-_ -- 4------____----____-+_----4----+ 700

ACCCTAAAAAGGAAAAAAAGTAAGCGTAATTATGATACATAAAAACGTTAGGTCAAGGTGTAACCTATGAFAATGGGAAGAAATGGGCTACATTCTCTACA BTA AAC AC C T A G;AGT A T AT OVI TAC AC CAA G3AAC A T AT CUl AGC AT CAA GGAGT G T AT HIN AAT GC C A G G3GAT G T AC MRE --_-_-_-_-_-__-_-+-__-__-_____-+-4__ __--+- + - + - 4f------+------800

CCAAGAGAA--TCAAGCACGAAAGTTATTATGAAACCAATAACCAAAGGAGGATTTAGCAGTAAACTAACGAATAGAGTGCTTAGTTGAATTAGGCCATGA BTA CT AA --CTCTTT C TT G T C OVI CT AA --TCCAAC T TT A T C CUN TT AA --CTTAAT T TT A C A HIN TT AT ATTCATAT T TT A T C MRE _+ ____+ -_ _+ _-______-+-______+.___ -____ .______+______- +_ ___--_+_4______------900

AGCACGCACACACCGCCCGTCACCCTCCTCAAATAGATTCAGTGCATCTAACCCTATTTAAACGCACTAC GCTACATGAGAGGAGACAAGTCGTAACAAGG BTA T A GGCA AA ACACTT ATT A TAT CGT TT AATCAT A T OVI C G GGCA AA ACACTC ATT T TAC CGT TT AATCAT G C CUN C A AGCA AA ACGCTT ATT T AGT TGT TA IACTGT G C HIN C G AGTA GA ATGCTC ACT T TAC TAT TT AACCAT G T MRE .-_-_-_-_-_-_-_-+-_-_-_-_-____-+.______+.______+ -______+ - + . .______+.______-. ------+ 1000 I tRNAVAL TAAGCATACTGGAAAGTGTGCTTGGATAAATCAAGATATAGCTTAAA-CAAAGCATCCAGTTTACACCTAAGAAGACTTCAT-TCATTATGAATATCTTGA BTA CT C T T T C-AT TT T OVI A -T C T T T C-AT TC C CUN A -T C C T T C-AC CC C HIN A -T TT T T CTAT CC C MRE ---__-___-,-__- +--,______+__ ,___-+- +-+______------___ .------+------+1100 116S rRNA ACTAGACCTAGCCCAAAGATACCCTCTCGACTAAACAACCAAGA-TAGAATAAAACAAAACATTTAATCCC-CAATTTAAAGTATAGGAGATAGAAATCTA BTA T ACC AC AAT-CCA TTAT TT TC AGA- CT C- TA TCCIA-CTGTTA T T OVI C ATT GC AGC-CCA TCAC TT TC ATG- AC TA T A TTCIAACAACAA TT CUN T ATT AT AAT-TTA TTAC CT TT TTAA TT T- C G TCTIA-TAATT- T T HIN C ATT AT AGC-CGA TCAC TT CC ATT- TT T- T A CCTIh-CCATTA T T MRE --_-_-_-_-_-_-_-_-+._-_-_-_-_-_-_-_-+-__-_-_-_____-+-______+ + - + .------.------.+ 1200

-AGTACGGCGCTATAGAGAAAGTACCGCAAGGGAACGATGAAAGAAAAAAACTAAAAGTATAAAAAAGCAhAAGATTACCCCTTGTACCTTTTGCATAATG BTA AGA T G A T C A -TT - C A A T OVI -AA C G A T C A ATC - C A A T CUN TAA T A A TT G ATT - C A G C HIN -AA T G T T C A ATC - C G A T MRE --_-_-_-_-_-___-+___--4---- + .______+-_+___------_44___. +______--4-___-+------+ 1300

AATTAACTAGTATAAGACTTAACAAAATGAATTTTAGCTAAGCAGCCCGAAACCAGACGAGCTACTCACAAAACAGTTTACCAAGAACTAACTCATCTATG BTA G T - A T C T C T TCG A T G TC OVI G T - A T C T C T T(G A T G CC CUN G T - A C T T C T TlA G T A CC HIN G C - A T T T C T T(G A T G CC MRE --_-_-_-_-_-_-_-_-+-____+__--++-+--__ __+ -__ ------4+- .______+______-+------+ 1400

TGGCAAAATAGTGAGAAGATTTGTAAGTAGAGGTGACATGCCTAACGAGCCTGGTGATAGCTGGTTGTCC :AGAAAATGAATCTAAGTTCAGCTTTAAAGA BTA G CA A C T G A T A OVI A TG A C C A A T A CUN G TA A C T A A T A HIN A TG A C T A A T A MRE -____+____+____--+- _+______+______+______--4-4 - + - 4--4-----___------1500 FIG. 1. (Figure continues on the opposi te page.) Downloaded by guest on October 2, 2021 Evolution: Miyamoto et al. Proc. Natl. Acad. Sci. USA 87 (1990) 6129

-TACCAAAAATTCAAATAAACCCCACTGTAGCTTTAAAAGTTAGTCTAAAAAGGTACAGCCTTTTAGAAACGGATACAACCTTGACTAGAGAGTAAAATT BTA A T ATA C -TAT C TT G A C A C AACC OVI - A ATG C TTAT A TT A T T A C GTTT CUN _ A ATA C TCAC C TT A T T G C AATT HIN - A ATT C TTAT A TT A T C AT AACT MRE

TAACACT--ACCATAGTAGGCCTAAAAGCAGCCATCAATTAAGAAAGCGTTAAAGCTCAACAACAAAAATTAAATAGATTCCAACAACAAATGATTAACT BTA G T AT-- C CC C T A-ACTATC TA CC T CA ATAA C OVI A C TTAA C CT C T A-ATAGTA TA TT T TA ATAG C CUN A T AC-- C CT T T G-TTTACA TA TT T TT TAAA C HIN A T -CAA T TT C T A-ACAACC TA TC C CA GTAA C MRE -_-_-_-_-_-_-_-_+--___-_-_-_-__+-______+______+______----_--_-_--_-+ - +-+ 1700

CCTAGCCCCAATACTGGACTAATCTATTATAGAATAGAAGCAATAATGTTAATATGAGTACAAGAAAATTTCCTTrcrU''GCATAAGTCT-AAGTCuAGTGC BTA GCCTT - - A GT TC CC CG T AC OVI A-TCT T - G GT AC CC TA T AT CUN G-CTT - - A GT TA CT TA T AT HIN A-CCC T - A GT T- CT TG T AT MRE 1800

CTGATAATACTCTGACCACTAACAGTCAA-TAAAAATAATCCAACAATAAACAATTTATTGATTATACTGTTAACCCAACACAGGAGTGCATCTAAGGAA BTA TT C T CA AA G CCT ATT TA C C T T A G TA G CT OVI CT T T CA AA G CCT ACT TA C T A C A A T G A CT CUN TC T T CA AA G CTC ACT TG C T A T C A T A A CT HIN TT T T CA GA G CCT CCC TA T T AT A A T A A CT MRE - 4.___+______-4.-4.---___-- ______-_-+______--4_-+_-_-_-_-__+ _-___--__+- - +-4+ 1900

AGATTAAAAGAAGTAAAAGGAACTCGGCAAACAC-AAACCCCGCCTGTTTACCAAAAACATCACCTCCAGCATTCCCAGTATTGGAGGCATTGCCTGCCC BTA C C AA T G G T OVI T C AA T G A C CUN T C AA T G A C HIN T T TA T A A C MRE -_ __-+--+---+____--+- + - ---+__--___--__+--___-__------+-- + - + 2000

BTA C A T A T AA OVI C A G C G T AC CUN C A G C AA AA HIN C A G C A T AC MRE +4._-4.- _____+__-4.- 4. - 4. - 4 - 4 - 4 -_ _- 2100

TTACTGTCTCTTACTTCCAATCAGTGAAATTGACCTTCCCGTGAAGAGGCGGGAATGCACAAATAAGACGAGAAGACCCTATGGAGCTTTAACTAACCAA BTA ACTAT CTT G OVI ATACT CTT G CUN ATAAT CTT A HIN ATATT CTT G MRE -4._--_---_--__-_--__-___+ _-__-__-__-__-__+- 4.- -_ - ___+ -__ __-. - .--__+ 2200

CCCAAAGAGAATAGATTTAACCATTAAGGA-ATAACAACAATCTCCATGAGTTGGTAGTTTCGGTTGGGGTGACCTCGGAGAATAAAAAATCCTCCGAGC BTA T A- T A T CACTAACC AA C T T TC GAC AAC C T C GA T OVI C A- C A T TCATTACC AA C C C TT GGC AAC C T C AG A CUN C A- C A C TAACCACT TG C T T CT GGT AAC C T C AA T HIN C A- C A A TTATTTCT AA C C T CT GGC AAC C T C AA T MRE . 4.---4..--_--+_-__--__--_+--__--__-__+-__-_-__-_-_-_-_-+._4_.__._-__-_-______+ -_ -_ ._ -_ 2300

GATTTTAAAGACTAGACCCACAAGTCAAATCACTCTATCGCTCATTGATCCAAAAACTTGATCAACGGAACAAGTTACCCTAGGGATAACAGCGCAATCC BTA TC AA A ACA C T A A A OVI CT GA A ACA T T A AA CUN CT GC G ACA T T - A A HIN CC AA A ATA CC A T A MRE . 4. . 4. 4.___. 4. . 4. . ____-4.-4.--__--__-__-_-4.-4.4.-_+-_-- . __-__ 2400

TATTCAAGAGTCCATATCGACAATAGGGTTTACGACCTCGATGTTGGATCAGGACATCCTGATGGTGCAACCGCTATCAAAGGTTCGTTTGTTCAACGAT BTA C C CA A OVI C T C A A CUN T T T G G HIN C T C A A MRE - 4.--_-_-+_4. --_._ --_-_+____-__-__-_-_--_--_-_-__+_--4.._4__.. .4__..+4._-_._-_-__+-_--_-_-+-+ 2500

TAAAGTCCTACGTGATCTGAGTTCAGACCGGAGTAATCCAGGiTCGGTTTCTATCTATTACGTATTTCTCCCAGTACGAAAGGACAAGAGAAATAAGGCCA BTA AC T C G OVI AT C C A CUN GT C C A HIN AT C C A MRE - 4.__ __-4.. 4. . 4. - 4. . 4. - 4.- - 4.-4. ------__. 2600 tRNALEU ACTTTAAATCAAGCGCCTTAAGACAACCAATGATAACATCTCAACTGACAACACA---AAACCCTGCCCTAGAACAGGGCTTAGTTAAGGTGGCAGAGCC BTA TAG -C ATT ACT TTAC T T AGATAC AAC A TCTCG AA CT C OVI TCA -T ATT GTT ATCA T T AACCTC AAC A CTTTG GA CT C CUN AAA -A CCC ATT CTCA C T AAAATC AAC A CTCCG AA C T C HIN TCA -C ATT ATT ATTA T T AACTAT AAC G CCCTA AA T T - MRE - 4._____+_ +__-4.. 4. . 4. -_.-. - 4_-_. 2700

CG BTA C OVI G CUN G HIN C MRE - __-4.4.+ _____+__-4.- 4. - 4. - 4. - 4. - 4. - 4. - 2800

FIG. 1. Aligned mtDNA sequences of B. taurus [BTA (19)], C. unicolor (CUN), H. inermis (HIN), M. reevesi (MRE), and 0. virginianus (OVI). Dashes refer to gaps, which have been included in the overall alignment to maximize homology (18). The B. taurus sequence is shown in its entirety, whereas only nucleotides at variable positions are presented for cervids. The locations of the mitochondrial tRNA and rRNA genes are indicated above the B. taurus sequence. the three representatives of antlered deer differ from the coileus alternatives, respectively (Fig. 2) (20). The Cervus/ outgroup (Hydropotes) by 7.7%. Muntiacus network is supported by 37 unique mutations Parsimony analysis of the three possible dichotomous representing 28 transitions (positions 71, 152, 192, 204, 388, solutions for the study group and outgroup demonstrates that 425, 431, 480, 513, 661, 662, 772, 812, 813, 933, 949, 961, 1103, the Cervus/Muntiacus arrangement is 17 and 21 mutations 1119, 1120, 1127, 1253, 1402, 1423, 1705, 1708, 2239, and shorter than the Cervus/Odocoileus and Muntiacus/Odo- 2665), six transversions (sites 57, 1526, 1671, 2091, 2605, and Downloaded by guest on October 2, 2021 6130 Evolution: Miyarnoto, et al. Proc. Natl. Acad. Sci. USA 87 (1990) Table 1. Pairwise comparisons of cervid mtDNA sequences cervid sequence file (Fig. 1) results in the same most- parsimonious arrangement for antlered deer, with Bosjoining Species Substitutions % Hydropotes. Further, this most-parsimonious solution, compared BP TS TV TS/TV Gaps divergence* which is at least 7 mutations shorter than its closest compet- CUN/HIN 2675 141 43 3.3 11 7.3 itors, supports the hypothesis that antlered deer constitute a CUN/MRE 2677 116 24 4.8 9 5.6 monophyletic group (1, 3, 5, 10, 11, 28). CUN/OVI 2673 141 41 3.4 12 7.2 The importance of using larger amounts of DNA sequence HIN/MRE 2673 142 51 2.8 14 7.7 data to resolve difficult systematic problems is illustrated by HIN/OVI 2672 156 47 3.3 13 8.0 the 12S and 16S rRNA orthologues of cervids (Figs. 1 and 2). MRE/OVI 2670 156 40 3.9 17 7.9 When analyzed separately, neither set of gene sequences BP, base positions shared by both sequences; TS, transitions; TV, provides significant support for the Cervus/Muntiacus solu- transversions; Gaps, insertions and deletions; CUN, C. unicolor; tion (sign test, 95% confidence intervals of 19.56-56.78% and HIN, H. inermis; MRE, M. reevesi; OVI, 0. virginianus. 21.93-57.47% for the 12S and 16S rRNA orthologues, re- *The percent sequence divergence was calculated as [(TS + TV + spectively). However, by combining the two sets of se- Gaps)/(BP + Gaps)] x 100%, where gaps are conservatively quences, statistically significant support is obtained for the counted as single differences, regardless of length. Cervus/Muntiacus clade (95% confidence interval of 22.73- 48.96%). Thus, longer sequences of orthologous DNA offer 2649), and three gap events (one deletion at site 1201 and two more phylogenetically informative characters and, thereby, insertions at positions 1608-1609 and 1728). In contrast, the better resolution of difficult phylogenetic questions. Cervus/Odocoileus and Muntiacus/Odocoileus alternatives Our phylogeny is robust for mtDNA sequence data. While are supported by 20 and 16 unique mutations [19 transitions extensive cladistic studies based on other character systems (positions 351, 398, 460, 776, 801, 936, 946, 963, 971, 1086, are uncommon, some other comparative data are congruent 1116, 1169, 1335, 1607, 1673, 1780, 2225, 2245, and 2664) and with the phylogenetic conclusions advanced here. Based on one deletion (site 380) versus 12 transitions (positions 208, morphological data, Groves and Grubb (10) have obtained the 986, 1571, 1599, 1670, 1681, 1688, 1785, 2319, 2327, 2341, and same cladogram for deer, although only one synapomorphy 2663) and four transversions (sites 815, 1674, 2651, and (plesiometacarpal condition of lateral metacarpals) serves to 2701)], respectively. diagnose the Cervus/Muntiacus clade. Similarly, an amino The reliability of the most-parsimonious phylogeny has acid replacement of serine for glycine at position 12 of been evaluated by several approaches (25). The total number fibrinopeptide A diagnoses this group (29). ofunique changes in favor of the Cervus/Muntiacus solution Our phylogeny, when coupled with paleontological data, is significantly greater than that for the Cervus/Odocoileus indicates that the tribe Dicrocerini cannot be closely related alternative (two-tailed sign test, a = 0.05, 95% confidence to living muntiacines. If these two groups were monophy- interval of 22.96-48.67%) (26). Consequently, the same con- letic, it would require that the splitting of cervines and clusion is reached for the Muntiacus/Odocoileus alternative. muntiacines antedate the oldest dicrocerines. That would The Cervus/Muntiacus arrangement is stable when the se- place the time of this split before 19-21 million years ago quences are resampled by bootstrapping (98.3% replication (MA), the oldest known age for fossil antlered deer (30, 31). out of 1000 trials) (27). Parsimony analysis of transversion However, recent muntiacines and cervines first appear in the differences indicates that the Cervus/Muntiacus phylogeny fossil record about 6-8 MA in the Late Miocene of Asia (with 11 unique substitutions) is shorter than the Cervusi and/or Europe (8, 32, 33). The same is true ofodocoileines (8, Odocoileus and Muntiacus/Odocoileus alternatives (with 0 34, 35), a lineage of even earlier ancestry according to our and 5 diagnostic changes) by 11 and 6 extra mutations, phylogeny. Thus, the paleontological data, in concert with respectively. When analyzed separately, the 12S and 16S our mtDNA phylogeny, falsify the hypothesis that extant rRNA gene sequences each join Cervus and Muntiacus muntiacines form a monophyletic group with dicrocerines. the B. taurus As such, the tribe Dicrocerini should be removed from the together once again. Finally, addition of to the subfamily Muntiacinae. This conclusion is not unexpected, CUN OVI CUN MRE MRE CUN given that the taxonomic assignments of early fossil deer have been based on primitive attributes rather than on shared derived similarities (4-6). MRE HIN OVI HIN OVI HIN The most-parsimonious solution (Fig. 2 Lower) indicates that rates of nucleotide change among antlered deer are 413 (37) 430 (20) 434 (16) similar (36). This pattern is apparent either when all changes or only unambiguous changes (e.g., those with one unequiv- ocal placement) are counted (24). By adopting a date of 6-8 65(43) Cervus unicolor (CUN) MA for the cervine/muntiacine split (8, 32, 33), the time of 44 (37) origin for extant antlered deer may be estimated from the Munti- 85(63) Muntiacus reevesi (MRE) relatively equal amounts of change leading to Cervus, acus, and Odocoileus (36). Such a clock calculation suggests 110 that odocoileines diverged from other antlered deer between (88) Odocoileus virginianus (OVI) 9.3 and 12.4 MA. This estimate agrees with a Late Miocene 109 (87) origin for the subfamily, as hypothesized from paleontolog- Hydropotes inermis (HIN) ical data (8, 34, 35). In the New World, all deer belong to the subfamily FIG. 2. (Upper) Tree lengths and total numbers of unique muta- Odocoileinae except for the cervine Cervus elaphus (6). In tions (in parentheses) for the three possible dichotomous arrange- the Old World, this subfamily is represented today by the ments of Cervus (CUN), Hydropotes (HIN), Muntiacus (MRE), and endemic as well as by Alces and Rangifer. The Odocoileus (OVI). (Lower) Most-parsimonious phylogeny rooted New are from North using Hydropotes as the outgroup. The parenthetical estimates of earliest fossils of World odocoileines branch length are based on unambiguous changes (e.g., those with America and are younger than those from Eurasia (Early only one most-parsimonious placement), whereas the other values Pliocene versus Late Miocene, respectively) (8, 34, 35, 37). are calculated from all mutations, including those with multiple The biogeographic history of odocoileines is more compli- assignments (see text). cated than that of other deer, which, except for C. elaphus, Downloaded by guest on October 2, 2021 Evolution: Miyarnoto et al. Proc. Natl. Acad. Sci. USA 87 (1990) 6131 are restricted to the Old World. Nevertheless, an Old World Classification of the Tetrapods: , ed. Benton, M. J. origin for Odocoileinae is suggested by both the geographic (Clarendon, Oxford), Vol. 2, pp. 273-282. distributions of other cervids, as well as by the relative 12. Brown, W. M., George, M., Jr., & Wilson, A. C. (1979) Proc. Natl. Acad. Sci. USA 76, 1967-1971. earliest ages ofits Eurasian versus North American fossils (8, 13. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 34). The most-parsimonious interpretation of the Old World Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold distributions of Capreolus [the sister group to all other living Spring Harbor, NY). odocoileines (10)], cervines, dicrocerines, muntiacines, and 14. Birnboim, H. D. & Doly, J. (1979) Nucleic Acids Res. 7, more distantly related cervids is that odocoileines originated 1513-1523. in Eurasia. Our mtDNA clock estimate places the time ofthis 15. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, origin in the Late Miocene at 9.3-12.4 MA. 499-560. 16. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. We thank J. L. Holloman (Fish and Wildlife Service) and P. E. 17. Chen, E. Y. & Seeburg, P. H. (1985) DNA 4, 165-170. Moler (Florida Game and Fresh Water Fish Commission), the 18. Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48, Zoological Society of San Diego, R. C. Rosen (Santa Fe Community 443-453. College Teaching Zoo), and P. E. Moler, respectively, for the tissue 19. Anderson, S., DeBruijn, M. H. L., Coulson, A. R., Eperon, samples ofCervus, Hydropotes, Muntiacus, and Odocoileus; S. Bell, E. C., Sanger, R. & Young, I. G. (1982) J. Mol. Biol. 156, M. C. McKenna, D. Penny, C. G. Sibley, M. R. Tennant, and S. D. 683-717. Webb for their recommendations and suggestions about the manu- 20. Swofford, D. L. (1985) PAup: Phylogenetic Analysis Using script; M. A. Burch and D. H. Sherman for their help in the Parsimony (Ill. Nat. Hist. Surv., Urbana). laboratory; and E. Chipouras for his assistance with the computer 21. Miyamoto, M. M. & Boyle, S. M. (1989) in The Hierarchy of analysis. This research was supported by grants to M.M.M. from the Life: Molecules andMorphology in Phylogenetic Analysis, eds. National Science Foundation (BSR-8717527 and BSR-8857264) and Fernholm, B., Bremer, K. & Jornvall, H. (Elsevier Science, from the Interdisciplinary Center for Biotechnology Research (UPN Amsterdam), pp. 437-450. 87030317) and Division of Sponsored Research (RDA-1-17 87-88), 22. Miyamoto, M. M., Tanhauser, S. M. & Laipis, P. J. (1989) University of Florida. Syst. Zool. 38, 342-349. 23. Felsenstein, J. (1989) PHYLIP: Phylogeny Inference Package 1. Scott, K. M. & Janis, C. M. (1987) in Biology and Management (Univ. Washington, Seattle). of the Cervidae, ed. Wemmer, C. (Smithsonian Inst., Wash- 24. Fitch, W. M. (1971) Syst. Zool. 20, 406-416. ington, DC), pp. 3-20. 25. Felsenstein, J. (1988) Annu. Rev. Genet. 22, 521-565. 2. Goss, R. J. (1983) Deer Antlers: Regeneration, Function, and 26. Templeton, A. R. (1983) Evolution 37, 221-244. Evolution (Academic, New York). 27. Felsenstein, J. (1985) Evolution 39, 783-791. 3. Janis, C. M. & Scott, K. M. (1987) Am. Mus. Novit. 2893, 28. Gentry, A. 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Polu- 9. Hennig, W. (1966) Phylogenetic Systematics (Univ. Illinois, ostrva 52, 399-407. Urbana). 36. Goodman, M. (1986) in Evolutionary Perspectives and the New 10. Groves, C. P. & Grubb, P. (1987) in Biology and Management Genetics, eds. Gershowitz, H., Rucknagel, D. L. & Tashian, ofthe Cervidae, ed. Wemmer, C. M. (Smithsonian Inst., Wash- R. E. (Liss, New York), pp. 121-132. ington, DC), pp. 21-59. 37. Gustafson, E. P. (1985) Trans. Nebr. Acad. Sci. Affil. Soc. 13, 11. Janis, C. M. & Scott, K. M. (1988) in The Phylogeny and 83-92. Downloaded by guest on October 2, 2021