MITOCHONDRIAL DNA PHYLOGENY AND SPECIATION IN THE TRAGOPANS

1003

The Auk 117(4):1003–1015, 2000

MITOCHONDRIAL DNA PHYLOGENY AND SPECIATIONIN THE TRAGOPANS

ETTORE RANDI,1,5 VITTORIO LUCCHINI,1 TARA ARMIJO-PREWITT,2 REBECCA T. KIMBALL,2,3

EDWARD L. BRAUN,2,4 AND J. DAVID LIGON2

1Istituto Nazionale per la Fauna Selvatica, Via Ca Fornacetta 9, 40064 Ozzano dell’Emilia (BO), Italy;2Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131, USA;

3Department of Evolution, Ecology and Organismal Biology, Ohio State University, Columbus, Ohio 43210, USA;and

4Department of Plant Biology, Ohio State University, Columbus, Ohio 43210, USA

ABSTRACT.—We sequenced mitochondrial DNA (mtDNA) from cytochrome b (cyt b) and thecontrol region (CR) for all five extant species in the genus Tragopan. We incorporated informationon comparative patterns and rates of molecular evolution into phylogenetic analyses, using botha single-gene and a combined data approach. Sequence variability was distributed heteroge-neously among the three domains of CR and the three codon positions of cyt b, but the two genesevolved at comparable rates, on average, and produced concordant topologies independent ofthe method used for phylogenetic reconstructions. Phylogenetic trees suggest that Tragopan in-cludes two main evolutionary lineages grouping caboti-temminckii (clade A), and blythii-satyra(clade B). A shorter CR sequence from one museum sample could not consistently resolve theposition of T. melanocephalus. The mtDNA phylogeny is better supported than alternative topol-ogies inferred from morphological and behavioral traits and is compatible with a mechanism ofallopatric speciation of Tragopan in two different episodes about 4 and 2 million years ago. Inthose periods, the vicariant events that might have fostered allopatric speciation of Tragopan arerepresented by landscape changes that affected the Indohimalayan region after the sudden risingof the Himalayas less then 8 million years ago, and by climatic fluctuations during the Pleistoceneless than 2 million years ago. Received 18 June 1999, accepted 30 May 2000.

THE FIVE EXTANT species in the genus Trago-pan (Phasianidae) are almost linearly distrib-uted across an arc that includes the Himalayanrange, northern Pakistan to Burma, the forestedmountains of central and southeastern Chinafrom Yunnan to Zhejiang, and northeasternBurma and northern Vietnam (Fig. 1). Distri-butions are allopatric except for limited contactzones between T. satyra and T. blythii moleswor-thi in the eastern Himalayas (Bhutan), and be-tween T. temminckii and T. caboti guangxiensis insouth-central China at Guangxi (Cheng 1980;Fig. 1).

Adult male tragopans have conspicuous andhighly diagnostic color patterns that allowstraightforward species identification (Johns-gard 1986, McGowan 1994). In particular,males have bright naked cheeks, bright throatand gular lappets, and two erectile horns thatcan be inflated and expanded during courtshipdisplays (Johnsgard 1986, McGowan 1994:469).The sexes are highly dimorphic (McGowan1994), and females and yearlings have incon-

5 E-mail: [email protected]

spicuous and similar plumages. Sexual dimor-phism and highly ritualized, complex court-ship displays are important signals in materecognition by tragopans, and these traitscould have had a role in speciation via sexualselection (Islam and Crawford 1996, 1998).

Allopatric distributions suggest that trago-pans speciated by vicariant fragmentation ofwidespread ancestral populations fostered bygeographic barriers to gene flow and divergentadaptation of the isolated populations (Mayr1970). However, owing to extreme sexual di-morphism and complex courtship behaviors inthe group, sexual selection (Møller and Cuervo1998) also may have had a role in segregatingpopulations in contact, thus producing a se-quential set of parapatric species in the absenceof geographic isolation. To date, an explicitphylogeny of tragopans has not been availableexcept for hypotheses based on qualitativeevaluation of plumage color (Johnsgard 1986)and comparative behavior (Islam and Craw-ford 1996, 1998).

In this paper, we analyze an alignment ofabout 2,341 nucleotides of mitochondrial DNA

1004 [Auk, Vol. 117RANDI ET AL.

FIG. 1. Geographic distributions (Johnsgard 1986) and summary of phylogenetic relationships from cyto-chrome-b (cyt b) and control-region (CR) sequence analyses of the five extant species of Tragopan (drawingsfrom McGowan [1994]). The phylogenetic tree summarizes concordant topologies obtained using maximum-parsimony, neighbor-joining, and maximum-likelihood methods. Values of internodal support for clades Aand B are in Table 4. The phylogenetic position of T. melanocephalus was not fully resolved using the shortCR sequence obtained by the available museum sample. The tree was rooted using cyt-b and CR sequencesfrom Pucrasia macrolopha, Ithaginis cruentus and Lophophorus impejanus (not shown).

(mtDNA) that were sequenced from represen-tatives of all five extant species of tragopansand from three phasianid outgroups. Phylo-genetic analyses of these data are used to (1)describe the comparative patterns and rates ofsequence evolution of the mtDNA control re-gion (CR) and cytochrome-b (cyt b) genes andevaluate their phylogenetic utility; (2) producea molecular phylogeny of the tragopans thatcan be used to assess the evolution of morpho-logical and behavioral traits; and (3) correlatethe inferred molecular phylogeny and extent ofinterspecific genetic divergence with Pliocene/Pleistocene biogeographical scenarios in theHimalayan region to suggest putative specia-tion patterns in Tragopan.

MATERIALS AND METHODS

DNA samples, PCR amplification, and sequencing.—We obtained samples (Table 1) from aviaries thatmaintain captive-reared stock of legal and docu-

mented geographic origins, except for T. melanoce-phalus, which is rare in nature and not reared in cap-tivity outside of India. This species is represented byonly one museum sample (A. Ghigi collection, INFS,Bologna).

Total DNA was extracted from 95% ethanol-pre-served muscles or feather roots, using guanidiniumthiocyanate and diatomaceous silica particles (Ger-loff et al. 1995, Randi and Lucchini 1998) and a Pur-egene DNA isolation kit (Gentra Systems, Inc.; Kim-ball et al. 1999). In four species, the entire mtDNA CRand cyt-b genes were PCR amplified and sequencedas described by Randi and Lucchini (1998) and Kim-ball et al. (1999). Partial CR sequences (domain I)from the museum sample of T. melanocephalus wereobtained by nested PCR. We used the primers PHDLand PH1H (Randi and Lucchini 1998) in the firstamplification and PHDL with HimpDS1 (59-TCA-TGGAGTACATTACGGGC-39) in the semi-nestedamplification. The museum sample was processed ina separate room dedicated only to ancient DNA. Theabsence of contamination was checked in all samplesusing negative controls during the extraction and

October 2000] 1005Phylogeny and Speciation in Tragopans

TABLE 1. Species names and GenBank accession numbers of Tragopan and outgroup samples used in thisstudy.

Species

GenBank numbers

Control region Cytochrome b

Tragopan caboti (Cabot’s Tragopan)T. temminckii (Temminck’s Tragopan)T. blythii (Blyth’s Tragopan)T. satyra (Satyr Tragopan)T. melanocephalus (Western Tragopan)Pucrasia macrolopha (Koklass Pheasant)Ithaginis cruentus (Blood Pheasant)Lophophorus impejanus (Himalayan Monal)

AF230301, AF230302AF230306, AF230307, AF230305AF230300 (two identical sequences)AF230303, AF230304AF230311 (CR-375 nt)AF230310AF230308AF230309

AF200723AF028802, AF229838AF200722AF200724, AF229837

—AF028800AF068193AF028796

successive PCR amplifications. Additional phasianidoutgroup sequences (Pucrasia macrolopha, Ithaginiscruentus, and Lophophorus impejanus) were obtainedusing the same PCR and sequencing methods.

Sequence analysis.—We aligned sequences withCLUSTAL W (Thompson et al. 1994) and editedalignments using Se-Al (A. Rambaut; ,http://evolve.zoo.ox.ac.uk/Se-Al/Se-Al.html.). Nucleotidecomposition, variability at different positions anddomains, and genetic distances were estimated us-ing PAUP* (Swofford 1998). We performed a likeli-hood test for heterogeneity of substitution ratesamong CR and cyt-b domains using PLATO 2.01 (N.Grassly and A. Rambaut; ,http://evolve.zoo.ox.ac.uk/Plato/Plato2.html.).

Sequences were analyzed both independently andcombined. Phylogenetic congruence of combinedcyt-b 1 CR sequences was assessed by a partition-homogeneity test (Farris et al. 1995) with 1,000 rep-licates, as implemented in PAUP*. Phylogenetic anal-yses were performed by PAUP* using (1) the neigh-bor-joining algorithm (NJ; Saitou and Nei 1987) withTamura and Nei’s (1993) DNA distance formula(TN93); (2) the exhaustive maximum-parsimonyprocedure (MP; Swofford 1998) with unordered andequally weighted characters, or with differentialweighting of transversions (tv) over transitions (ti);and (3) the heuristic maximum-likelihood (ML) pro-cedure (Felsenstein 1981). The best-fit ML model ofDNA substitution was selected by likelihood-ratiotests among a suite of models of increasing complex-ity performed according to the following procedure(Huelsenbeck and Crandall 1997). First, we obtainedthe exhaustive MP trees using the cyt-b and CR se-quences, either independently or combined; these to-pologies were identical and were used to computethe likelihood scores for substitution models of in-creasing complexity. Next, we assessed the best-fitmodels by the likelihood-ratio test:

D 5 2(log L1 2 log L0), (1)

where log L1 5 likelihood of the most complex modeland log L0 5 likelihood of the simpler model. Valuesof D are distributed accordingly to a chi-square dis-

tribution (Huelsenbeck and Crandall 1997) and areused to determine whether simpler models shouldbe rejected in favor of ones that are more complex.

We modeled substitution heterogeneity amongsites by the discrete-rate g distribution (Yang 1994).Values of the shape parameter a of the g distribu-tions, and ti/tv for cyt b, CR, and the combined se-quences, were estimated by PAUP* using maximumlikelihood with the best-fit substitution model andfour discrete-rate categories.

We assessed the robustness of the phylogenies bybootstrap percentages (BP; Felsenstein 1988) com-puted using 1,000 random resamplings with replace-ment by PAUP*. Statistical differences among alter-native phylogenetic trees were tested by Kishino andHasegawa’s (1989) likelihood and parsimony tests,and by Templeton’s (1983) Wilcoxon test, as imple-mented in PAUP*.

RESULTS

Comparative evolution of mtDNA CR and cyt-bgenes.—The average observed genetic distances(p-distances; Table 2) among the four species oftragopans were D 5 0.061 6 SD of 0.011 for thecyt b and D 5 0.056 6 0.010 for the CR. Intra-specific values of D ranged from 0.000 (be-tween identical CR sequences of T. blythii andcyt b of T. satyra) to 0.013 between two CR se-quences of T. satyra. Observed genetic distancesbetween tragopans and the outgroups were D5 0.122 6 0.018 for cyt b and D 5 0.132 6 0.007for the CR.

Plotting of genetic distances (either propor-tional or corrected) of cyt b versus the CR sug-gested that the two genes evolved approxi-mately at the same rate among tragopans andbetween tragopans and their outgroups (Fig.2A), with the cyt b accumulating comparativelymore ti but fewer tv than the CR (Fig. 2B). Cytb evolved mainly by substitutions at third-co-


TABLE 2. Uncorrected proportional genetic distances (p-distances) among the studied Tragopan and out-group species. Intraspecific CR/cyt-b distances are shown on the diagonal (not estimated in T. caboti, T.blythii, and the outgroups), interspecific CR distances are shown above the diagonal, and interspecific cyt-b distances are shown below the diagonal.

Species 1 2 3 4 5 6 7

1 T. caboti2 T. temminckii3 T. blythii4 T. satyra5 P. macrolopha6 I. cruentus7 L. impejanus

0.0008/—0.03650.06650.06740.11110.14870.1102

0.03890.0070/0.0035

0.06750.06470.11460.14350.1085

0.05920.0625

0.0000/—0.05860.11810.15140.1137

0.06310.06380.0507

0.0127/0.00000.11110.13730.1032

0.14100.13760.13800.1419

—0.13740.1067

0.13100.12930.13590.14240.1276

—0.1199

0.12800.13060.11670.12220.12070.1078

—

don positions (Figs. 3A and B, Table 3), inagreement with the well-known functionalconstraints of protein-coding genes (Irwin etal. 1991, Kumar 1996). The number of tv differ-ences at first and second positions of cyt-b se-quences was negligible in comparisons amongtragopans and also among tragopans and theoutgroups (Fig. 3B). Ti and tv accumulated atdifferent rates in the three CR domains, and, inparticular, ti leveled off faster in the hyper-variable CR-I (Figs. 3D,E). Ti might saturaterapidly, but the CR seems to accumulate moretv at greater evolutionary distances than the cytb (Figs. 3C,F).

Visual inspection of the sliding-window plotof the substitutions observed among tragopans(Fig. 4) suggested that sequence variability wasdistributed more homogeneously in the cyt bthan in the CR. In fact, PLATO did not detectany cyt-b region evolving at significantly dif-ferent rates based on a uniform substitutionmodel. On the contrary, a region of 24 nucleo-tides in the second part of domain I, corre-sponding to hypervariable CR-IB as defined inthe Alectoris partridges by Randi and Lucchini(1998), and a region of 214 nucleotides in theterminal part of domain III, evolved signifi-cantly faster than average in the CR (see alsothe proportions of variable sites in Table 3).

Quantitative data on nucleotide compositionand variability showed that (1) the GC propor-tion was similar among inner, transmembrane,and outer cyt-b domains (Zhang et al. 1998) andamong the three CR domains; (2) third-codonpositions and domain IB were most variable incyt b and CR, respectively; and (3) parsimony-informative sites were more frequent in cyt-bthird-codon positions and cyt-b transmem-brane, CR-IA, and CR-III domains (Table 3).

Testing models of DNA substitution.—For cyt b,

CR, and the combined sequences, the best-fitmodel was the general time-reversible model(GTR; Lanave et al. 1984). The discrete g dis-tribution significantly improved the likelihoodvalues over GTR with invariable sites (P ,0.01). Ti/tv ratios, estimated by ML with GTR1 g, were 6.22, 7.23, and 9.79 for cyt b, CR, andcyt b 1 CR sequences in Tragopan (without out-groups), respectively. These ratios were usedfor weighted maximum-parsimony analyses ofcyt b (tv 5 6 ti), CR (tv 5 7 ti), and cyt b 1 CR(tv 5 10 ti) alignments, respectively.

Phylogenetic relationships in tragopans.—Mo-lecular, morphological, and behavioral datasuggested that Pucrasia, Ithaginis, and Lophopho-rus are the most probable outgroups of Trago-pan (Kimball et al. 1999). Using these threephasianids as outgroups, the genus Tragopanwas clearly monophyletic (Fig. 5) despite thegene region and phylogenetic method used inphylogenetic analyses.

The phylogenetic trees computed by NJ, ML,and MP using nine individual tragopan CR se-quences were identical to the topology in Fig-ure 5 in showing a basal splitting of Tragopaninto two clades that join the sister species ca-boti-temminckii (clade A) and blythii-satyra(clade B). Bootstrap values (Table 4) suggestedthat clade A (caboti-temminckii) was stronglysupported independent of the method used inphylogenetic reconstruction, whereas clade B(blythii-satyra) received lower bootstrap sup-port, particularly from equally weighted MPanalyses of the CR (BP , 50%).

We obtained the same topology using thecyt-b sequences (one sequence from each spe-cies of Tragopan) or the combined cyt-b 1 CRalignment (the partition-homogeneity test wasnot significant; P 5 0.49). Bootstrap support ofclades A and B obtained with NJ, MP, and ML


FIG. 2. (A) Pairwise cytochrome-b (cyt b) versuscontrol-region (CR) TN93 DNA distances amongTragopan (lower left) and between Tragopan and theoutgroups (upper right). (B) Cyt b versus CR percentdifferences among Tragopan and between Tragopanand the outgroups. Filled dots 5 transversions (tv);empty dots 5 transitions (ti). Diagonal lines indicateexpected values given identical rates of substitutionsin cyt-b and CR genes.

methods, and using cyt b, CR, and combinedsequences (Table 4), indicated that (1) NJ anal-yses computed using TN93 or other distancemodels with both independent and combinedsequences identified the two clades; however,the exclusion of highly variable transitions atthird positions of cyt b disrupted the mono-phyly of the blythii-satyra clade (BP , 50%), andthe exclusion of highly variable CR-IB disrupt-ed the monophyly of the caboti-temminckii clade

A (BP , 50%); (2) in equally weighted MP anal-yses, cyt b and the combined genes supportedthe two clades, whereas the blythii-satyra cladewas not supported using the CR; (3) weightedMP analyses slightly improved the BP supportof tragopan clade B (BP 5 60%) using the CR;and (4) ML analyses performed with the GTRmodel and heterogenous substitution ratessupported clades A and B in analyses of com-bined and separate data.

The museum sample of T. melanocephalusyielded degraded DNA, and we obtained onlyshort sequences (375 nucleotides) of CR-I. Phy-logenetic analyses performed using this partialdata set associated melanocephalus with the bly-thii-satyra clade (Fig. 6A), although with lowbootstrap support; alternative relationships ofmelanocephalus to the caboti-temminckii clade orto a basal unresolved polytomy also were pos-sible (Figs. 6B,C). The topology shown in Fig-ure 6A was the best one in terms of likelihood(2log L 5 1,440.57, with GTR 1 g), but it didnot differ significantly from the alternativetrees (Figs. 6B,C) using the KH likelihood test(Table 5). This topology was shorter (L 5 223vs. 237 and 232) and significantly different (P, 0.05) from the alternatives using the KH par-simony and Templeton tests (Table 5). However,this topology had the same likelihood andlength of trees representing alternative phylo-genetic relationships among Tragopan (Johns-gard 1986; Islam and Crawford 1996, 1998;Figs. 6G,H). Thus, a better resolution of the re-lationships of T. melanocephalus will be possiblewhen fresh samples become available for ad-ditional DNA sequencing.

Testing alternative phylogenetic hypotheses.—Johnsgard (1986) suggested that tragopansconsisted of the superspecies blythii-caboti andmelanocephalus-satyra-temminckii based on bodysize and plumage coloration. In contrast, basedon quantitative analyses of vocalizations andqualitative comparisons of courtships displays,Islam and Crawford (1996, 1998) suggestedthat ‘‘the centrally distributed species of trag-opans (Satyr, Blyth’s and Temmink’s) retainedcharacteristics of the ancestral population. . . .’’and were closely related to each other, whereas‘‘the peripheral species (Western and Cabot’s)underwent vocal modifications. . . .’’ and wereseparated into two distinct groups. The differ-ent phylogenies underlying morphological, be-havioral, and mtDNA data are shown in Figure


FIG. 3. (A) Cyt-b transitions (ti) and (B) transversions (tv) versus uncorrected proportional DNA distances(p-distances); (C) cyt-b ti versus tv; (D) number of CR ti; (E) tv versus percent pairwise DNA distances; and(F) ti versus tv in Tragopan and their outgroups (in box). First, second, and third cyt-b codon positions are


TABLE 3. Nucleotide composition and sequence variability at first, second, and third cyt-b codon positions;at inner, transmembrane (trans) and outer cyt-b domains (Zhang et al. 1998); and at IA, IB, II, and III con-trol-region domains (Randi and Lucchini 1998).

Cytochrome b

1st 2nd 3rd Inner Trans Outer

Control region

IA IB II III

NucleotidesGC proportion% Variablea

% Parsimonyb

3810.500.050.22

3810.390.0030.00

3810.500.280.27

2280.430.130.17

5850.480.100.30

3300.470.110.26

1630.380.080.31

1540.400.280.23

4680.500.040.26

3970.300.110.40

a Proportion of total nucleotides that are variable.b Proportion of variable nucleotides that are parsimony informative.

←

indicated as 1, 2, and 3. The three CR domains are indicated as I, II, and III. Ti and tv numbers are notindependent from the estimated DNA distances, so fitted second-order polynomials are intended to showtrends of the plot and not to analyze their statistical properties.

6, and statistical significance of the topologieshas been analyzed using KH and Templetontests (Table 5). The mtDNA phylogeny (Fig. 6D)represents the best explanation of the data us-ing the total cyt-b 1 CR alignment without T.melanocephalus. The alternative topologies pro-posed by Islam and Crawford (Fig. 6E) andJohnsgard (Fig. 6F) are different from themtDNA phylogeny at P , 0.0001 (Table 5). Us-ing only the short alignment of 375 nucleotidesincluding T. melanocephalus, the alternative phy-logenies (Figs. 6G,H) were not significantly dif-ferent from the best mtDNA topology (Fig. 6A).

DISCUSSION

Evolution of CR and cyt-b genes, and phyloge-netic inference.—The evolution of mitochondrialgenes is conditioned by peculiar functionalconstraints that define the number of sites thatare free to vary and the kind of mutations thatcan be fixed, thus determining their total di-vergence rates (Zardoya and Meyer 1996). Onaverage, CR and cyt b evolved at similar ratesamong tragopans and their outgroups over ge-netic distances ranging from 4 to 16% (see Fig.2A, Table 2). These findings are in apparentcontrast with the expected evolution of CR,which should be faster at early and slower athigher divergence levels owing to an inherentlyfast rate of evolution and rampant saturation ofhypervariable sites and domains (Zhu et al.1994, Lyrholm et al. 1996). However, in trago-pans and the phasianid outgroups in this study,cyt b accumulated more ti but fewer tv differ-

ences than the CR (Fig. 2B). It is well knownthat most ti, particularly at third-codon posi-tions, are synonymous and can evolve and sat-urate rapidly (Irwin et al. 1991). Thus, cyt-bsites that are free to vary tend to accumulate ti,which contribute the bulk of divergence at ear-ly intraspecific evolutionary steps but mightsaturate rapidly (Fig. 3C). The CR evolves rath-er conservatively, on average, and hypervari-able sites are confined in two short regions atCR-IB and in the final part of CR-III domains(Fig. 4). The CR positions that are free to mu-tate can accept more tv than cyt b, thus con-tinuing to accumulate mutations at greater evo-lutionary distances (Fig. 3F). That is why, rath-er surprisingly, avian CRs express substantialphylogenetic signal and can be used for phy-logenetic inference in a relatively wide windowof evolutionary divergence (Kimball et al. 1997,1999; Kidd and Friesen 1998; Randi and Luc-chini 1998).

Variable rates of substitution at different sitesand domains require that complex models ofDNA evolution be used for phylogenetic infer-ence of mtDNA sequences (Tamura and Nei1993, Yang 1996). In this case, the best-fit max-imum-likelihood model had the most parame-ters, the GTR. Complex evolutionary modelsincrease the variance of the estimated param-eters and therefore require longer sequences toincrease the probability of obtaining well-sup-ported (although not necessarily correct) phy-logenies. Phylogenetic relationships amongtragopans can be reliably reconstructed with


FIG. 4. Variability at first-, second-, and third-codon positions in inner, transmembrane, and outer do-mains of cyt b (after Zhang et al. 1998) and in CR domains IA, IB, II, and III (after Randi and Lucchini 1998)plotted along a nonoverlapping sliding window of 50 nucleotides in Tragopan.

FIG. 5. Phylogenetic tree of nine individual CR sequences from four species of Tragopan and three phas-ianid outgroups. Tree computed by PAUP* using the heuristic maximum-likelihood procedure with the GTRmodel (log L 5 23,771.99). Values of ti/tv (2.15) and the shape parameter a (0.22) of a discrete g distributionwith four categories were estimated from the data. Identical topologies were obtained by NJ and MP methodsusing either the CR, cyt-b, or the combined cyt-b 1 CR sequences. Values of bootstrap support computedusing NJ (with TN93 genetic distances; other distance models produced identical results), weighted MP (withtv 5 7ti), and ML (with GTR 1 g model) are reported above internodes of tree. Other bootstrap values forclades A and B are reported in Table 4.

shorter sequences, but the combined cyt-b 1CR alignment consistently produced the stron-gest internodal support values (Table 4). De-spite the suspected increase of homoplasy athypervariable sites and domains, the signal ex-pressed by CR-IB and third-position ti of cyt bhelped to improve the resolution of the trago-pan phylogeny (see also Kimball et al. 1999).Although saturation plots are often used to de-termine rapidly evolving sites that are morelikely to be saturated, saturation in itself doesnot preclude phylogenetic information.

Phylogenetic relationships of tragopans and tax-onomic implications.—The cyt-b 1 CR sequencesidentified two unambiguous clades in Tragopan,the first joining temminckii and caboti (clade A)and the second one joining satyra and blythii(clade B). However, the partial CR-I melanoce-phalus sequence obtained from a feather sampledid not firmly resolve the relationships of thisspecies. The mtDNA phylogeny was not con-cordant with the two superspecies of Johnsgard(1986), i.e. melanocephalus-satyra-temminckii andblythii-caboti, nor with the central versus pe-


TABLE 4. Percent bootstrap support for Tragopanclade A (caboti-temminckii) and clade B (blythii-sa-tyra) from phylogenetic analyses using neighbor-joining with Tamura-Nei (TN93) genetic distances,equally weighted and weighted maximum-parsi-mony methods, and maximum-likelihood methodwith the GTR model and discrete g-distributedsubstitution rates (four categories). Weighted max-imum-parsimony analyses were performed usingtv 5 6ts, tv 5 7ts, and tv 5 10ts for cyt b, CR, andcombined sequences, respectively. Phylogeneticanalyses were performed with nine individualTragopan CR and four individual cyt b and com-bined Tragopan sequences.

Model Sequencea Clade A Clade B

Neighbor joiningTN93 cyt b

cyt b-3CRCR-IBcyt b 1 CR

10088

100,50100

74,50

916896

Maximum parsimonyEqually weighted

Weighted

cyt bCRcyt b 1 CRcyt bCRcyt b 1 CR

9795

100937195

78,50

82936093

Maximum likelihoodGTR g(4) cyt b

CRcyt b 1 CR

888796

686894

a Sequences: cyt b 5 cytochrome b; cyt b-3 5 cyt b excluding third-codon positions; CR 5 control region; CR-IB 5 CR excluding hyper-variable domain IB; cyt b 1 CR 5 cyt b 5 CR combined.

ripheral species groups, i.e. satyra-blythii-tem-minckii versus melanocephalus and caboti as in-dicated by analyses of vocalizations and behav-ior (Islam and Crawford 1996, 1998).

Biogeography and speciation in tragopans.—Thetopology of the molecular tree overlaps thepresent geographic distribution of tragopans(Fig. 1). The observed phylogenetic pattern iscompatible with a mechanism of allopatric spe-ciation and could have been generated either byfragmentation of a widespread ancestral pop-ulation, or by dispersal and divergence alongthe Himalayan and central Chinese mountainranges. Johnsgard (1986) postulated the exis-tence of a major center of origin of pheasant di-versity in the eastern Himalayas and acrossnorthern Burma and Yunnan. The hypotheticalancestral populations could have been locatedin this area and were then progressively frag-mented or dispersed, producing the westernand the eastern tragopan lineages that spread

toward the Himalayas and central and south-eastern China, respectively.

The two main clades of tragopans divergedat about 8% cyt-b genetic distance, which couldhave been generated in a time span of about 4million years, assuming the standard rate ofvertebrate mtDNA evolution of 2% per millionyears (Avise and Walker 1998). Thus, the firstevolutionary split could have been fostered bythe dramatic consequences of the sudden up-lifting of the Himalayas less than 8 millionyears ago (Harrison et al. 1992, Abdrakhmatovet al. 1996), which created a series of parallelmountain ranges running from east to west(and from north to south in Pakistan and Bur-ma), thus inducing the first basal split amongthe Himalayan and Chinese ancestors of extanttragopans. The Himalayas continued to risethroughout the Pliocene and Pleistocene andforced the pre-existing main fluvial watershedsto cut deep canyons through the mountain val-leys. Present distributions of Himalayan trag-opans are bounded by some of the major fluvialwatersheds of the area (Johnsgard 1986); e.g.the distribution of T. melanocephalus corre-sponds to the basin of the Indus River, T. satyrainhabits the entire watershed of the GangesRiver, and T. blythii occurs on the western (T. b.blythii) and eastern (T. b. molesworthi; knownonly from three specimens) sides of the Bra-hamaputra River (Fig. 1).

The rising of the Himalayas also had majorclimatic consequences (Cracraft 1973). For ex-ample, the warm tropical climate of the TibetanPlateau changed rapidly, and formerly uniformtropical forests shifted to the humid southernslopes and disappeared from the arid northernslopes and internal valleys. Habitat diversifi-cation followed an obvious vertical gradientand an east-west gradient, with the monsoonrains being heaviest on the eastern side of theHimalayas and the west being arid (Shaller1977). Further major habitat changes in theHimalayas have been produced by Pleistoceneglacial/interglacial cycles. Ice sheets expandedin the Himalayas during glacial periods withconsequent elevational shifting of steppes andforests and cyclic fluctuations in the distribu-tions of animals.

The cyt-b sequence divergence between sisterspecies of Tragopan is 3.6% (clade A) and 5.9%(clade B), corresponding to about 1.8 to 2.9 mil-lion years of evolutionary divergence. Thus,


FIG. 6. Alternative relationships of Tragopan melanocephalus resulting from phylogenetic analyses of 375nucleotides of CR-I sequences (A, B, and C). The first tree (A) is significantly better than the alternatives (Band C) using likelihood or parsimony testing (Table 5). Tree D represents the phylogeny of Tragopan as ob-tained in this study based on the entire cyt-b and CR sequences. Alternative phylogenies proposed by Islamand Crawford (1996, 1998) based of vocalizations and courtship displays, and Johnsgard (1986) based onexternal morphology, are represented in trees E and F, respectively. The molecular tree (D) is significantlybetter than the alternatives (E and F), using likelihood or parsimony testing (Table 5). Trees G and H cor-respond to Islam and Crawford’s and Johnsgard’s phylogenies and include T. melanocephalus. Using the shortCR-I sequence available for T. melanocephalus, trees G and H have the same likelihood (2log L 5 1,440.57)


TABLE 5. Results of the likelihood and parsimony testing of alternative relationships of Tragopan melano-cephalus (A, B, and C) estimated using 375 nucleotides of CR-I, and alternative phylogenies of Tragopan, asproposed in this study based on the entire mtDNA cyt-b and CR sequences (D), by Islam and Crawford(1996, 1998), and (E) by Johnsgard (1986) (F). The topologies of these trees are represented in Figure 6.Likelihood tests were performed using the GTR 1 g model, with parameters estimated from the data.Parsimony tests were performed using all characters unordered and equally weighted.

Trees

Likelihood test

2log L KH P

Parsimony test

Length KH P Templeton P

ABCDEF

1,440.571,450.621,450.967,035.207,089.757,089.75

Best0.080.07Best

,0.0001,0.0001

223237232845886891

Best,0.05,0.05Best

,0.0001,0.0001

Best,0.05,0.05

Best,0.0001,0.0001

←

and length (L 5 223) and are not statistically different from tree A. Tsa 5 T. satyra; Tme 5 T. melanocephalus ;Tte 5 T. temminckii; Tbl 5 T. blythii ; Tca 5 T. caboti. Trees were rooted using sequences from Pucrasia macro-lopha, Ithaginis cruentus, and Lophophorus impejanus (not shown).

speciation in Tragopan might have been fosteredby events in the Pliocene and Pleistocene,which split the Himalayan populations intowestern melanocephalus, central satyra, and east-ern blythii and split the Chinese populationsinto western temminckii and eastern caboti.

Sexual selection and speciation in tragopans.—Aphylogenetic tree similar to the one expectedfrom geographic speciation through vicariancealso could be produced by parapatric specia-tion driven by sexual selection. In this case, thephenotypic traits selected during parapatricspeciation owing to sexual selection should bephylogenetically informative (Brooks andMcLennan 1991).

However, this is not the case in tragopans.The lack of concordance between the phyloge-netic patterns and the distribution of phenotyp-ic traits likely to be subject to sexual selectionis illustrated by lappet morphology. All fivetragopans possess inflatable blue horns andlarge frontal lappets or bibs. In three species(satyra, temminckii, and melanocephalus) belong-ing to both major branches of the molecularphylogeny (Fig. 1), the primary colors adorningthe lappet are blue and red (Johnsgard 1986,McGowan 1994:469). In two others (blythii andcaboti), also occupying different phylogeneticbranches, the lappets are largely yellow or or-ange (although small amounts of blue colora-tion occur in each of these species). Given thedistribution of color patterns, it is likely that

blue and red evolved initially, and that orange/yellow coloration evolved independently ineach lineage.

This evolutionary model assumes that a lap-pet-like structure occurred in the commonancestor of the five species, and as each popu-lation became isolated, female preferences se-lected for unique color patterns that providedinformation about the quality of a given malerelative to other males. The lappets of maletragopans possess features that may signalmale health or quality. For example, if the yel-low and red on the lappets of all five tragopanspecies are based on carotenoid pigments, thenthe lappets have the potential to signal condi-tion, as seen in some other avian species (e.g.Ligon 1999). In addition, the patterns of four ofthe five species (all but blythii) exhibit bilateralsymmetry in color pattern. Bilateral symmetry,like carotenoids, is thought to provide a mech-anism for signaling quality (Ligon 1999). Thelappets of all tragopans except blythii appear tobe beautifully designed to emphasize symmet-ric development of color patterns.

The allopatric distributions and the patternof phylogenetic relationships among trago-pans, together with the idiosyncratic distribu-tion of phenotypic traits likely to be under sex-ual selection, provide evidence against sym-patric speciation. Taken together, molecularphylogeny, geographic distribution, and be-havior suggest that tragopans evolved in allop-


atry by vicariance owing to historical fragmen-tation and extrinsic barriers to gene flow. Otherspecies that evolved in the same periods andthat share similar habitats should have been af-fected by the same biogeographic factors pro-moting isolation and speciation. It is worth not-ing that closely related Lophophorus impejanusand L. sclateri and Lophura leucomelanos and L.nycthemera have distributions similar to cladesA and B of Tragopan (Johnsgard 1986, Mc-Gowan 1994), indicating that a main split be-tween Himalayan versus Chinese pheasantpopulations might have been generated bycommon biogeographic factors.

ACKNOWLEDGMENTS

This work was supported by the Istituto Nazionaleper la Fauna Selvatica (INFS) and by NSF through theRIMI molecular biology facility at the University ofNew Mexico. We thank A. Hennache (Parc Zoolo-gique de Cleres, France), M. Kaiser (Tierpark Berlin,Germany), R. Sumner (Triple S Game Farm, USA),and S. Latimer for kindly providing us with tissuesamples. F. H. Sheldon, T. Crowe, and an anonymousreviewer provided helpful comments that improvedan early draft of this paper. We deeply appreciate theeditorial help provided by J. S. Marks.

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Associate Editor: F. H. Sheldon

MITOCHONDRIAL DNA PHYLOGENY AND SPECIATION IN THE TRAGOPANS

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