utility of nuclear dna intron markers at lower taxonomic levels.pdf

Molecular Phylogenetics and Evolution 35 (2005) 624–636

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Utility of nuclear DNA intron markers at lower taxonomic levels: Phylogenetic resolution among nine Tragelaphus spp.

Sandi Willows-Munro ¤, Terence J. Robinson, Conrad A. Matthee

Evolutionary Genomics Group, Department of Zoology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa

Received 21 July 2004; revised 19 January 2005Available online 2 March 2005

Abstract

Phylogenetic relationships among the nine spiral-horn antelope species of the African bovid tribe Tragelaphini are controversial.In particular, mitochondrial DNA sequencing studies are not congruent with previous morphological investigations. To test the util-ity of nuclear DNA intron markers at lower taxonomic levels and to provide additional data pertinent to tragelaphid evolution, wesequenced four nuclear DNA segments (MGF, PRKCI, SPTBN, and THY) and combined these data with mitochondrial DNAsequences from three genes (cytochrome b, 12S rRNA, and 16S rRNA). Our molecular supermatrix comprised 4682 characters whichwere analyzed independently and in combination. Parsimony and model based phylogenetic analyses of the combined nuclear DNAdata are congruent with those derived from the analysis of mitochondrial gene sequences. The corroboration between nuclear andmtDNA gene trees reject the possibility that genetic processes such as lineage sorting, gene duplication/deletion and hybrid specia-tion account for the conXict evident in the previously published phylogenies. It suggests rather that the morphological charactersused to delimit the Tragelaphid species are subject to convergent evolution. Divergence times among species, calculated using arelaxed Bayesian molecular clock, are consistent with hypotheses proposing that climatic oscillations and their impact on habitatswere the major forces driving speciation in the tribe Tragelaphini. 2005 Elsevier Inc. All rights reserved.

Keywords: Bovidae; Tragelaphini; Systematics; Phylogeny; Molecular clock

1. Introduction

Congruence among multiple data sets is arguably themost reliable indicator of phylogenetic accuracy(Cracraft and Helm-Bychowski, 1991; Miyamoto andCracraft, 1991; Miyamoto and Fitch, 1995; SwoVord,1991). Current phylogenetic analyses thus often utilize amultigene approach and analyses frequently includemorphological and/or palaeontological data (seeFlores-Villela et al., 2000; Gatesy et al., 2003; Nylanderet al., 2004 among others). One group providing a case inpoint is the Cetartiodactyla. Initial investigations werebased mainly on morphological and palaeontological

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evidence (McKenna, 1975; Novacek, 1982; O’Leary andGeisler, 1999; Simpson, 1945; Van Valen, 1968) andthese phylogenetic hypotheses were subsequently testedby making use of mitochondrial (mtDNA) analyses(Graur and Higgins, 1994; Irwin and Arnason, 1994;Montgelard et al., 1997; Ursing and Arnason, 1998).ConXict between the mtDNA gene tree and morphologi-cal evidence (for example, the monophyly of Artiodac-tyla—Luckett and Hong, 1998; O’Leary and Geisler,1999) prompted the inclusion of independent nuclearDNA markers (Gatesy et al., 1996; Gatesy et al., 1999,2002; Matthee et al., 2001).

Although the phylogenetic utility of nuclear DNAdata are now well entrenched in the mammalian litera-ture (Gatesy and Arctander, 2000; Madsen et al., 2001;Matthee et al., 2001, 2004; Matthee and Davis, 2001;Murphy et al., 2001; Springer et al., 2001), one of the

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S. Willows-Munro et al. / Molecular Phylogenetics and Evolution 35 (2005) 624–636 625

caveats for employing these markers is the slow rate atwhich nucleotide changes accumulate over time. Forexample, comparisons among sequences revealed thatthe rates of nucleotide change among members of thefamily Bovidae are on average 3.2 times higher for cod-ing mtDNA comparisons than for non-coding nuclearDNA introns (Matthee and Davis, 2001). In manyinstances this slow rate of change in the nuclear DNAdata seems to suggest that inferences at lower taxonomiclevels (for example, among mammalian species belong-ing to the same genus) will not be resolved with highbootstrap and or statistical support. Limited empiricalexamples exist to support this hypothesis (but seeMoore, 1995; Springer et al., 2001).

In the present study, we ampliWed a set of fournuclear intron markers from the spiral-horned antelopeof the tribe Tragelaphini (family Bovidae). This groupforms a monophyletic lineage within the subfamily Bovi-nae (Essop et al., 1997; Gentry, 1992; Georgiadis et al.,1990; Hassanin and Douzery, 1999a; Matthee and Rob-inson, 1999) and is endemic to the African continent.Nine extant species are recognized, belonging to threegenera. These genera were conventionally identiWedbased on morphology and limited fossil evidence(Ansell, 1971): Tragelaphus (containing T. strepsiceros,T. imberbis, T. angasi, T. buxtoni, T. spekei, and T. scrip-tus), Taurotragus (containing T. oryx and T. derbianus)and Boocercus (for T. euryceros). Cranial similarity,however, suggests an sister taxon relationship betweenT. spekei and T. angasi (Kingdon, 1982; Roberts, 1952),and T. imberbis and T. strepsiceros (Alden et al., 1995;Kingdon, 1982), while the presence of horns in bothsexes of T. euryceros, T. oryx, and T. derbianus has beentaken to indicate that these three lineages form a derivedmonophyletic entity (Gentry, 1990). Likewise, generalbody conformation and pelt coloration unite the eland(T. derbianus and T. oryx) and kudu (T. strepsiceros andT. imberbis) lineages (Walker, 1964). A recent mtDNAinvestigation based on cytochrome b data (Matthee andRobinson, 1999) provided an alternative workinghypothesis for the evolution of the tribe that did not sup-port the recognition of the three genera (see also Essopet al., 1997; Gatesy et al., 1997; Georgiadis et al., 1990).In addition, the mtDNA data contradicted most of theabovementioned morphological associations withthe contrasting evolutionary associations suggested bythe mitochondrial and morphological phylogenies beingattributed to convergent evolution at the morphologicallevel (Matthee and Robinson, 1999; Hassanin andDouzery, 1999b). It is equally likely, however, that thegroup experienced a fairly rapid radiation during whichindependent lineage sorting could have resulted inincreased homoplasy obscuring the “correct” phyloge-netic aYnities in the mtDNA gene tree. In other words,the conXict between the gene tree and the morphologicaltree could equally be explained by shared mtDNA char-

acters becoming Wxed in only a portion of the descen-dents of a polymorphic ancestor (Hillis, 1999).

The aims of this study were Wrst to test the utility ofnuclear DNA markers in a group that experienced afairly rapid radiation during the last 15 My (Hassaninand Douzery, 2003; Matthee and Robinson, 1999; Vrba,1985). Second, resolution at the nuclear DNA level couldclarify the apparent conXict that exists between themtDNA and morphological topologies. Additionally itwas anticipated that a more comprehensive moleculardata set would allow for greater accuracy in determiningof the pattern and timing of episodes of speciationwithin the group, as well as providing further insights onthe factors thought to be driving speciation in these Afri-can antelope. This is particularly pertinent since sugges-tions that species inhabiting moist forest habitats (T.euryceros, T. spekei, and T. scriptus) are derived, andthose adapted to more arid environments (T. imberbis,T. angasi, T. strepsiceros, T. oryx, and T. derbianus) aremore basal, rest on a single, maternally inherited locus(Matthee and Robinson, 1999).

2. Materials and methods

2.1. Taxonomic representation

All nine recognized tragelaphid species wereincluded in our study (Table 1). Moreover, whereverpossible, multiple representatives drawn from diVerentgeographic areas were included. Unfortunately in somecases no exact locality data were available and only abroad geographical region from where the specimenwas collected could be obtained. The Indian nilgai(Boselaphus tragocamelus), domestic cow (Bos taurus),and African buValo (Syncerus caVer) were used asclosely related outgroup taxa in conjunction with theimpala (Aepyceros melampus), a member of Antilopi-nae (Kingdon, 1982, 1997; Bronner et al., 2003), as amore distantly related lineage (Matthee and Davis,2001).

2.2. DNA ampliWcation, sequencing, and alignment

DNA was extracted using standard laboratory proto-cols (Sambrook et al., 1989). Four nuclear DNA markers(thyrotropin—THY, protein-kinase CI—PRKCI, B-spectrin non-erythrocytic—SPTBN, and stem cellfactor—MGF) were ampliWed and sequenced using pub-lished primers and protocols (Matthee et al., 2001). Anadditional 24 mtDNA sequences were obtained fromthree mtDNA genes—cytochrome b, 12S rRNA, and16S rRNA (for mtDNA PCR and primer details seeMatthee and Davis, 2001). The remainder of themtDNA data were obtained from public databases (33sequences in total; Table 1).

626 S. Willows-Munro et al. / Molecular Phylogenetics and Evolution 35 (2005) 624–636

Cycle sequencing was performed using Big Dye chem-istry (Version 3.1, Applied Biosystems) with the resultingproducts analyzed on a 3100 ABI automated sequencer.Sequences were aligned using the default settings ofCLUSTAL X (Thompson et al., 1997) and optimizedmanually to ensure accuracy. Areas of ambiguouslyaligned sites were limited to a single gene and this regionwas excluded from all analysis (positions 149–159 inaligned MGF data set). Heterozygous sites were codedusing the IUB codes. The exon and intron boundarieswere deWned from previously published sequence datafor each DNA region (Matthee et al., 2001; Matthee andDavis, 2001). To further ensure the homology of theDNA sequences, exon regions were translated intoamino acids and screened for functionality. No stopcodons or insertions were present in the exonic regions.All new sequences were deposited in GenBank, accessionnumbers are shown in Table 1.

2.3. Data partitioning

Each DNA region was analyzed separately. Addition-ally, data sets were partitioned and analyzed accordingto origin of marker (i.e., mitochondrial or nuclear), andall characters and taxa were merged into a single data

matrix or “molecular supermatrix.” To explore inconsis-tencies among data partitions, pair-wise incongruencelength diVerences (ILD) were calculated in PAUP*4.0b10 (SwoVord, 2003) excluding uninformative charac-ters (Hipp et al., 2004). To further explore the homoge-neity of signal among data partitions the crossedShimodaira–Hasegawa tests (crossed SH tests, Shimoda-ira and Hasegawa, 1999) were performed in PAUP*4.0b10. In these tests the most likely topology obtainedby the mitochondrial, nuclear partitions and the superm-atrix were compared in pair-wise fashion.

2.4. Data analysis

Parsimony and maximum likelihood searches wereperformed in PAUP* 4.0b10 using the heuristic searchoption with 100 replicates of random taxon addition andTBR branch swapping. For the maximum likelihoodanalyses the optimal evolutionary model and modelparameters were estimated applying the Akaike informa-tion criteria (AIC, Akaike, 1974) using MODELTEST3.06 (Posada and Crandall, 1998). All character transfor-mations were equally weighted and alignment gapstreated as missing characters in parsimony. Nodal sup-port for parsimony and maximum likelihood trees was

Table 1Taxonomic sampling used in the study

Accession numbers with * indicates specimens sequenced in the present study.a Anderson et al. (1982).b Allard et al. (1992).c Gatesy et al. (1997).d Hassanin and Douzery (1999a, b).e Matthee and Robinson (1999).f De Donto et al. (2001).g Matthee et al. (2001).h Matthee and Davis (2001).

Species name (Vernacular name) DNA region

Mitochondrial DNA Nuclear DNA

12S rRNA 16S rRNA Cyt b MGF PRKCI SPTBN THY

Taurotragus derbianus (Giant Eland) AY667206* AY667193* AF022062e AY667177* AY667162* AY667232* AY667217*

Taurotragus oryx (Common Eland) AF091710d AY667194* AF036278d AY667178* AY667163* AY667233* AY667218*

Taurotragus oryx (Common Eland) AY667207* U87060c AF022057e AY667179* AY667164* AY667234* AY667219*

Tragelaphus strepsiceros (Greater Kudu) AY667208* AY667195* AF022063e AY667180* AY667165* AY667235* AY667220*

Tragelaphus strepsiceros (Greater Kudu) AF091696d AY667196* AF036280d AY667181* AY667166* AY667236* AY667221*

Tragelaphus imberbis (Lesser Kudu) AY667209* M86493b AF036279d AY667182* AY667167* AY667237* AY667222*

Tragelaphus angasi (Common Nyala) AF091698d AY667197* AF091633d AY667183* AY667168* AY667238* AY667223*

Tragelaphus angasi (Common Nyala) AY667210* AY667198* AF022066e AY667184* AY667169* AY667239* AY667224*

Tragelaphus buxtoni (Mountain Nyala) AY667211* AY667199* AY667215* AY667185* AY667170* AY667240* AY667225*

Tragelaphus buxtoni (Mountain Nyala) AY667212* AY667200* AY667216* AY667186* AY667171* AY667241* AY667226*

Tragelaphus euryceros (Bongo) AF091691d AY667201* AF036276d AY667187* AY667172* AY667242* AY667227*

Tragelaphus euryceros (Bongo) AY667213* AY667202* AF022065e AY667188* AY667173* AY667243* AY667228*

Tragelaphus spekei (Sitatunga) AF091692d AY667203* AJ222680d AY667189* AY667174* AY667244* AY667229*

Tragelaphus scriptus (Bushbuck) AY667214* AY667204* AF036277d AY667190* AY667175* AY667245* AY667230*

Tragelaphus scriptus (Bushbuck) AF091693d AY667205* AF022067e AY667191* AY667176* AY667246* AY667231*

Boselaphus tragocamelus (Indian Nilgai) U86963c U87013c AJ222679d AF165724g AF165725g AF165726g AF165729g

Bos taurus (Domestic Cow) NC001567a NC001567a NC001567a AF165716g AY029293f AF165718g AF65721g

Syncerus caVer (African BuValo) AF091688d U87061c AF036275d AY667192* AF210175h AF210197h AF210219h

Aepyceros melampus (Impala) U86964c U87014c AF036289d AF165780g AF165781g AF165782g AF165785g


assessed by 1000 bootstrap iterations as well as Bremersupport values (Bremer, 1988, 1994) using TreeRot v.2(Sorenson, 1999).

Bayesian analyses were performed using MrBayes3.0b3 (Huelsenbeck and Ronquest, 2001) with eachBayes run launched from a random starting tree and runfor 1 £ 106 generations. To conWrm that the analysis wasnot trapped at a local optima (Leaché and Reeder, 2002)all runs were performed three times. Trees were sampledevery 100th generation and burnin was determined usingthe sumt command in MrBayes 3.0b3. The priors wereset to the GTR model of evolution and the combineddata (mitochondrial, nuclear, and supermatrix parti-tions) were examined using the partition option. MrBa-yes 3.0b3 was also used to estimate the 95% posteriorcredibility intervals for each DNA region and combinedpartition.

Recent studies have suggested that the posteriorprobability values generated by Bayesian inference maybe inXated (Cummings et al., 2003; Suzuki et al., 2002;Waddell et al., 2001) but in some instances may be betterestimates of “true” phylogeny (Wilcox et al., 2002). Inthe present study, robust associations were identiWed bybootstrap values 770% and posterior probability values795%.

2.5. Distribution of support among the diVerent data partitions

An important aspect of any multigene analysis is theexamination of support contributed by the diVerent datasets in the “supermatrix” topology. First, followingKluge’s (1989) suggestion, we determined the number ofunambiguous synapomorphies for selected nodes usingMacClade 4.0 (Maddison and Maddison, 1992). Second,partitioned Bremer support values for selected nodeswere calculated for each DNA region using TreeRot v.2(PBS, e.g., Gatesy et al., 1999). The signiWcance of PBSvalues are highly data dependent (Lee, 2000) makingcomparisons between data sets problematic (Bremer,1988; Sanderson and Donoghue, 1996). For this reasonwe also followed a method proposed by Lee (2000) toassess nodal signiWcance. This methodology is similar tothat in conventional Bremer support analysis (Bremer,1988, 1994) but uses Templeton’s test (1983) in PAUP*4.0b10 to statistically compare individual nodes on alter-native trees.

2.6. Molecular clock calibration

Divergence times of the tragelaphids were estimatedusing the relaxed Bayesian molecular clock method formultigene data (Thorne and Kishino, 2002). All sevengenes were included and the supermatrix phylogeny wasused as reference tree. The F84 model was applied toeach gene fragment independently. The Markov chain

was sampled 10,000 times, every 100th generation. Theburnin was set at 10,000 generations. The following priordistributions were adapted: 20 Mya (SD D 20 My) forthe expected time between tip and root of the tree if thenodes are unconstrained (rttm), 0.008 (SD D 0.004) sub-stitutions per site per million years for the mutationalrate at the root of the tree (rtrate). These settings weredetermined by approximating the prior distribution oftimes and rates. First, arbitrary time settings for rttmand rttmsd were made followed by the estimation of theprior distributions of node times. When these approxi-mations resulted in node calibrations that were toorecent (for example in comparison to the fossil dating),the value of rttm was increased. This process was iter-ated until a time estimate was obtained approximatingcurrently accepted values. The values of rtrate andrtratesd were determined a priori. For example, the trag-elaphids are thought to have originated approximately15 Mya (Hill et al., 1985)—we therefore use this value incombination with the branch length estimates to deter-mine the rate of evolution. In our case, the amount ofevolution from each tip to the root for each gene wasdetermined, and a median amount of evolution amonggenes separating roots and tips was estimated. Themedian was calculated at 0.12 substitutions per site andthis value was divided by the 15 million years to equateto 0.008. The highest possible number of time unitsbetween the tip and the root of the tree was set at 50 Myand the prior on the rate autocorrelation was set at 0allowing for the rate of each gene fragment to vary. Fos-sil calibration points were available for two of the deepernodes and these were incorporated as upper and lowerconstraints. The Wrst of these is the split between Bovi-nae and Antilopinae (16.4–23.8 Mya) as used by Hassa-nin and Douzery (2003 and references therein). Oursecond calibration point is based on the emergence ofthe Bovinae tribes (11.5–19.7 Mya, Hill et al., 1985; alsosee Hassanin and Douzery, 2003). To explore possibleinconsistencies between the mitochondrial and nuclearpartitions, divergence times obtained using each parti-tion were compared to each other.

3. Results

3.1. Data description

The aligned supermatrix contained 7 DNA regionsand 4682 characters, 3149 bp non-protein coding and1533 bp protein coding characters. The diVerent DNAregions were characterized by variation in base composi-tion and modes of evolution (Table 2). As typicallyexpected the mitochondrial partition contained a higherproportion of variable characters (28%) when comparedto that of the nuclear partition (12.7%). As expectedgiven the higher proportion of variable characters in the


mtDNA partition, the consistency (CI) and retention(RI) index values of the mitochondrial markers (CI:0.4861–0.5547, RI: 0.5882–0.7311) were lower than thoseof the nuclear markers (CI: 0.7209–0.8261, RI: 0.8400–0.9149; Table 3) indicating higher levels of homoplasy inthe mtDNA data.

3.2. Distribution of support among the diVerent data partitions

In an attempt to compare how well each independentDNA region recovered the phylogeny, eight nodes ofinterest (labelled A–H in Figs. 1 and 2) were identiWed.These nodes elucidate associations among all extanttragelaphid species and were supported across diVerentmethods in the supermatrix phylogeny (Fig. 2).

Apart from the cytochrome b and to a lesser extendthe SPTBN data which performed reasonably well inrecovering the associations among the tragelaphid spe-cies, the separate analysis of individual DNA regionsresulted in several unresolved nodes (Table 3). The 95%posterior intervals estimated by the Bayesian analysis ofthe mitochondrial, nuclear partitions, and the superma-trix resulted in considerably fewer unique topologies rel-ative to those occurring within the same posteriorintervals of the individual DNA regions (Table 3). Thisdecrease in the number of topologies reXects the increasein resolution obtained from the combined analysis ofDNA regions (Buckley et al., 2002). Importantly, manyof the independent DNA regions, however, providedpositive character support for nodes when analyzed aspart of the supermatrix. For example, analysis of theMGF data set was only able to retrieve node A, but PBSvalues for this data set (Fig. 3) indicate that the superma-trix topology was not negatively inXuenced by the inclu-sion of these data. This is also supported by Templeton’stest suggesting that the potential “phylogenetic noise”from the inclusion of the MGF region to the superma-trix did not reduce support at any of the 8 selected nodes(Fig. 3). In fact, nodes A and G (which were signiWcantly

supported in the supermatrix analysis, P > 0.05) were notsigniWcantly supported once the MGF data set wasremoved, suggesting that this data set probably containshidden support for these nodes.

Nodes A, C, E, and G in the supermatrix topologywere statistically supported by Templeton’s test (Fig. 3).These nodes were also consistently retrieved in both themitochondrial and nuclear topologies (bootstrap values>75% and posterior probability values >0.95). The distri-bution of probability values retrieved by Templeton’stests also illustrates how diVerent DNA regions supportdiVerent nodes. For example, the removal of only 3 DNAregions resulted in non-signiWcant P-values at node Esuggesting that the phylogenetic signal in support of thisnode was contained within the cytochrome b, 16S rRNA,and SPTBN data sets (i.e., node E was unaVected by theremoval of 12S rRNA, MGF, PRKCI, and THY).

3.3. The phylogenetic utility of indels

Indels among ingroup taxa (insertions/deletionsinvolving two or more consecutive bp with clearlydeWned boundaries) were only present in the nuclearintron data. These were mapped onto the supermatrixtopology (Fig. 2) to determine the phylogenetic informa-tion content of these markers. Within the ingroup it wasfound that deletions outnumbered insertions 4 to 1 (Fig.2); the T. imberbis lineage was characterized by the great-est number of unique deletions. This is consistent withthe early divergence of this group from the other trage-laphid taxa (see below). At a deeper phylogenetic levelthe Bovinae tribes retrieved a deletion–insertion ratio of5:1 and in this instance a single indel was homoplasiousinvolving a 2 bp deletion at position 3990–3991 in theMGF data set (present in B. taurus and B. tragocamelus).

3.4. Congruence of diVerent data partitions

The topologies derived from the mtDNA and nuclearDNA partitions were largely congruent. Pair-wise ILD

Table 2Patterns of sequence variability among the independent DNA regions, mitochondrial partition, nuclear partition, and supermatrix

In the case of nuclear sequence data % protein coding refers to Xanking exon regions.

DNA partition

Total(bp)

% Protein coding

% Variable characters

Gamma distribution (� value)

Nucleotide frequencies

%A %T %C %G

12S rRNA 591 0 15.6 0.736 36.1 22.7 24.7 16.516S rRNA 348 0 23.9 0.489 34.9 24.6 21.3 19.2Cyt b 1140 100 35.8 2.023 31.9 25.9 28.9 13.4MGF 674 0 14.1 0.499 32.3 34.6 18.1 15.0PRKCI 667 11 9.60 NA 34.3 38.7 11.2 16.0SPTBN 765 16 16.1 0.505 23.2 27.2 24.8 24.8THY 667 34 9.90 0.139 30.1 32.6 19.3 18.1Mitochondrial 2079 55 28.0 0.799 34.9 23.7 28.0 13.4Nuclear 2603 15 12.7 0.275 29.8 32.3 18.8 19.1Supermatrix 4682 33 19.5 0.623 31.6 28.6 22.9 16.9

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Table 3Summary supermatrix

The numb x (RI) values are given for each parsimony tree, as well as, thenumber o 2, bootstrap values (>50) are given, X indicates where a nodewas not re

Data part No. nodes with 770%bootstrap or 95% posterior probability

D E F G H

12S rRNA X X X X X 0X X X X X 0X X X X X 0

16S rRNA X X 66 X X 1X X 70 X X 1X X 0.95 X X 2

Cyt b 7 X 95 X 90 58 49 X 94 X 93 70 6.00 X 1.00 X 1.00 0.79 4

MGF X X X X X 1X X X X X 2X X X X X 1

PRKCI X X X X X 1X X X X X 1X X X X 0.64 1

SPTBN 2 X 95 X X 97 45 X 96 X X 90 4.97 X 1.00 X X 1.00 4

THY X X X X X 1X X X X X 1X X X X X 1

Mitochon 8 X 94 X 96 50 57 X 94 X 100 83 6.00 X 1.00 X 1.00 0.90 5

Nuclear 5 96 65 58 X 93 44 94 86 75 X 87 6.00 1.00 1.00 1.00 X 1.00 6

Supermatr 5 81 99 62 100 100 79 75 100 76 98 100 7.00 1.00 1.00 1.00 1.00 1.00 8

of the topologies obtained from analysis of the independent DNA regions, mitochondrial partition, nuclear partition, and

er of parsimony informative characters, number of trees, optimal tree length, consistency index (CI), and retention indef topologies found within the 95% posterior intervals of each partition. Nodes A–H correspond to those in Figs. 1 andtrieved.

ition Method No. of pars. informative characters

No. of equallyparsimonious trees

Tree length CI RI No. of topologies in 95% posterior interval

Nodes

A B C

Pars. 51 94 142 0.5510 0.5882 51 X XML 51 X XBayesian 14,096 0.70 X XPars. 61 3 151 0.5547 0.7311 72 77 XML 73 63 XBayesian 2015 0.99 1.00 XPars. 290 3 904 0.4861 0.6346 81 86 6ML 100 88 7Bayesian 212 1.00 0.99 1Pars. 37 5 116 0.7547 0.8632 100 X XML 99 88 XBayesian 7299 1.00 X XPars. 17 104 52 0.8261 0.9149 96 X XML 93 X XBayesian 13,242 1.00 X XPars. 37 1 142 0.7736 0.8667 89 X 9ML 80 57 8Bayesian 517 1.00 X 0Pars. 31 24 75 0.7209 0.8400 100 X XML 100 X XBayesian 10,078 1.00 X X

drial Pars. 550 1 1699 0.4753 0.5527 98 95 7ML 98 95 8Bayesian 22 1.00 1.00 1Pars. 145 6 491 0.7162 0.8215 100 X 8ML 100 X 9Bayesian 18 1.00 X 1

ix Pars. 700 2 2203 0.5043 0.5938 100 93 9ML 100 71 9Bayesian 5 1.00 0.97 1


tests showed that there was no signiWcant conXictbetween the mitochondrial and nuclear partitions(P D 0.70). The crossed SH tests indicated that the mito-chondrial and nuclear topologies were signiWcantlydiVerent (P D 0.018 and 0.004) and closer inspection ofthe topologies revealed that the only striking diVerencewas in the placement of the bushbuck, T. angasi (Fig. 1).The supermatrix topology was not, however, rejected byeither the mitochondrial or the nuclear partitions, under-scoring the utility of the supermatrix for phylogeneticinference. The crossed SH tests can allow for the identiW-

cation of areas of topological conXict for example thenuclear topology was rejected by the supermatrix dataset due to the association between the two basal species,T. angasi and T. imberbis. The exclusion of thesetwo basal taxa resulted in congruence between thesupermatrix and nuclear topologies (P D 0.112).

3.5. The tragelaphid phylogeny

Given the overall stability of the supermatrix analysis,the lower number of trees found in the 95% credibilityinterval (Table 3) and the absence of any major conXictbetween this topology and the mtDNA and nuclear

DNA partitions (see above), this phylogeny was selectedas best reXecting the evolutionary relationships of thetragelaphids (Fig. 2). Our molecular data conWrmed Wnd-ings of previous studies by retrieving a single origin forthe subfamily Bovidae (Essop et al., 1997; Gentry, 1992;Georgiadis et al., 1990; Matthee and Robinson, 1999;Hassanin and Douzery, 1999a) and no genetic supportfor the recognition of three Tragelaphid genera (Mat-thee and Robinson, 1999). The tribe Tragelaphini wasrecovered as a monophyletic entity, with T. imberbisbasal in the phylogeny with the next most basal lineagerepresented by T. angasi. The close but old evolutionaryrelationship of these taxa is further supported by thelack of a unique 31 bp deletion in the SPTBN DNAregion which is a synapomorphy uniting the remainingseven species. Moreover, T. buxtoni, T. euryceros, T. spe-kei, and T. scriptus (all species adapted to closed forestliving) formed a well-supported monophyletic clade inall the analyses. Within this “closed forest” group the sis-ter taxon relationship between T. spekei and T. eurycerossuggested by earlier mtDNA data (Matthee and Robin-son, 1999) was similarly retrieved by our extended DNAdata set. Tragelaphus scriptus was placed as a sisterlineage to the T. spekei and T. euryceros clade with

Fig. 1. Topologies retrieved by (A) the combined mitochondrial DNA data and (B) the combined nuclear DNA data. Values given above thebranches represent the molecular clock estimates of divergence times for nodes labelled A–H with standard deviations in parenthesis. Values belowthe branches represent Bayesian posterior probabilities, maximum likelihood bootstrap values, and parsimony bootstrap values.


T. buxtoni (not included in previous analyses) as themost basal lineage within the closed forest group. Anassociation between T. derbianus, T. oryx, and T. strep-siceros, which are united by physiological adaptationsto an arid savannah environment (Alden et al., 1995;Kingdon, 1982) was not consistently retrieved in allmethods of analysis. The morphologically similar T.derbianus and T. oryx were grouped as sister taxa,while the supermatrix suggests a more derived positionfor T. strepsiceros. It should be noted, however, thatonly two data sets contain unambiguous synapomor-phies (cytochrome b and MGF) in support of an asso-ciation between T. strepsiceros and the closed forestgroup (node D). Pair-wise sequence divergence valuesamong individuals of the same species, ranged from0.02% within the geographically isolated T. buxtoni

lineage, to 0.65% within the geographically widespreadT. scriptus lineage.

3.6. Molecular clock

Prior speciWcation of the mutational rate could onlybe made with reference to the sequence data thereby vio-lating the principles underpinning the Bayesian frame-work (Yang and Yoder, 2003). Although a priordistribution of 0.008 (SD D 0.004) substitutions per siteper million years was estimated and used for the rate atthe root, we found that large changes (i.e., 0.3–0.003) tothis prior distribution, and also to that of the root ageprior, had little or no impact on the resulting divergencetimes. Additionally, independent analysis of the mito-chondrial and nuclear partitions produced divergence

Fig. 2. Topology retrieved by the combined analysis of all molecular data. Values given above the branches represent the molecular clock estimates ofdivergence times for nodes labelled A–H with standard deviations in parenthesis. These divergence values represent the age of the most recent com-mon ancestor of the taxa contained within that clade. Values below the branches represent Bayesian posterior probabilities, maximum likelihoodbootstrap values, parsimony bootstrap values. Solid bars indicate deletion events and stippled bars represent insertion events. The single homoplas-ious deletion is highlighted with *.


times in the same range as those obtained when using alldata (Figs. 1 and 2).

The divergence of the Tragelaphini from the otherbovid tribes was estimated at approximately 14.08 Mya(SD D 2.60, Fig. 2) while that between the basal bushlandspecies, T. angasi and T. imberbis from the other trage-laphid antelope was estimated to have occurred approxi-mately 10.89 Mya (SD D 2.87). This was followed by aperiod of rapid ecological specialization which gave riseto those species conWned to moist forest environments(T. buxtoni, T. euryceros, T. spekei, and T. scriptus), andthose adapted to a more arid savannah environment (T.derbianus, T.oryx, and T. strepsiceros).

4. Discussion

4.1. Nuclear intron markers

The nuclear introns selected in the present investiga-tion have been applied successfully to explain evolution-ary relationships among the Cetartiodactyla (Matthee

et al., 2001), Leporidae (Matthee et al., 2004), and also theBovidae (Matthee and Davis, 2001) permitting a rigorousassessment of intron markers in mammalian systematics.Several broad conclusions are evident from these studies.

First, it is clear that phylogenetic resolution is posi-tively correlated with the number of nuclear DNA char-acters employed. All four studies revealed an increase inphylogenetic resolution when genes are combined. Thisis contrary to most of the results obtained from previousmammalian mtDNA endeavors where increasing thenumber of mtDNA genes (characters) had little eVect onrobust resolution (for examples see Irwin et al., 1991 andCorneli, 2003). Even recent investigations using Bayes-ian methods on complete mtDNA genomes (Reyes et al.,2004) reveal weak support for most of the well-sup-ported mammalian inter-ordinal associations as sug-gested by nuclear DNA studies (Madsen et al., 2001;Murphy et al., 2001).

Second, the low levels of homoplasy present in thenuclear DNA introns make them particularly wellsuited for retrieving information on rapid radiationevents such as those typically observed in the Pecora

Fig. 3. The number of unambiguous synapomorphies, partitioned Bremer support and nodal signiWcance values for each DNA region at nodes A–H.Positive PBS values indicate that a particular gene region contributes phylogenetic signal at that node, while a negative value (shown in black) indi-cates that a data set conXicts with the supermatrix at a particular node.


(Matthee et al., 2001), Bovidae (Matthee and Davis,2001), and Leporidae (Matthee et al., 2004). This is inmarked contrast to several mtDNA studies on the samegroups (Gatesy et al., 1997; Halanych et al., 1999; Mat-thee and Robinson, 1999).

Third, given the relatively conservative nature of thenuclear DNA intron markers, the number of charactersneeded to obtain phylogenetic resolution seems to bedirectly linked with the age of the speciation events (aswith most other loci). Our tragelaphid data, however,also suggests a link between the phylogenetic resolu-tion and the rapidity at which these evolutionary radia-tions occurred. For older radiations (among theCetartiodactyla) all individual nuclear DNA fragments(with low numbers of characters) resulted in well-resolved phylogenies (Matthee et al., 2001), whereas theresults are often equivocal in species characterized bymore recent radiations (such as the Tragelaphini). Forexample, in the present study only the combinednuclear DNA tree produced robust results for the Tra-gelaphini—a group that are thought to have evolvedapproximately 14 Mya. In fact, the best resolution ontragelaphid relationships from any single gene was thatobtained from the mtDNA cytochrome b data (Table3). This is contrary to the published literature wherecytochrome b failed to provide phylogenetic resolutionin the Bovidae and Leporidae (Halanych et al., 1999;Matthee and Robinson, 1999). This seems likely toreXect diVerences in the modes of speciation events—both the Leporidae and Bovidae are thought to haveexperienced rapid radiations between 14–10 (Lepori-dae) and 20–14 million years ago (Bovidae) (Mattheeand Davis, 2001 and Matthee et al., 2004). In theseinstances, reverse mutations could have randomizedthe limited phylogenetic signal that accumulated dur-ing the rapid radiation. In the Tragelaphini, the diversi-Wcation probably also started approximately 14 Myabut speciation was more gradual occurring—up till 5million years ago, which provided more time for theaccumulation of synapomorphies, and less time for themtDNA signal to be erased. We are of the opinion thatthis accounts for the cytochrome b gene’s usefulness inresolving phylogenetic relationships within the spiral-horned antelope.

4.2. Tragelaphid systematics: molecules versus morphology

The interspeciWc and intraspeciWc (Haltenorth,1963) variability in many of the morphological featurespreviously used to delimit the group makes the track-ing of species turn-over from the fossil record diYcult.For example, Gentry (1978) suggested that T. strepsic-eros and T. imberbis diverged approximately 3 Myawhich is clearly in conXict with the molecular phylog-eny presented herein (Fig. 2). Given that our investiga-

tion is based on full taxonomic representation,corroborating nuclear and mtDNA evidence, and isunaVected by method of phylogenetic constructionused, we consider our supermatrix phylogeny to beparticularly stable. This is especially true for the mono-phyletic status of the tribe, the monophyly of the fourclosed forest species (T. buxtoni, T. euryceros, T. spekei,and T. scriptus), and the basal position of T. imberbisand T. angasi. These associations, however, are inmarked disagreement with the morphological data.Although we cannot exclude the possibility that inde-pendent lineage sorting, gene duplication/deletion and/or hybrid speciation could have contributed to the con-Xicting placement of T. angasi and T. strepsiceros (Fig.1), the remaining evolutionary relationships are wellsupported in both data sets (nuclear and mtDNA).Based on the molecular supermatrix topology it seemsreasonable to suggest that many of the morphologicalsimilarities among taxa—i.e., the cranial similaritybetween T. spekei and T. angasi (Kingdon, 1982; Rob-erts, 1952), and T. imberbis and T. strepsiceros (Aldenet al., 1995; Kingdon, 1982), the presence of horns inboth sexes of T. euryceros, T. oryx, and T. derbianus(Gentry, 1990), and the similarity in general body formand pelt coloration uniting T. strepsiceros and T.imberbis (Walker, 1964)—are due to convergence andnot recent common ancestry.

4.3. Speciation within Tragelaphus

Although the timing of divergence among tragelap-hid species may be estimated using a molecular clock,the factors driving speciation can only be examined bycomparing the tragelaphid phylogeny with paleocli-matic changes coupled to vicariance in Africa. Ourresults suggest that the global cooling trend whichpeaked 14 Mya (Cerling et al., 1997; Zachos et al., 2001)coincided with the diversiWcation among speciesbelonging to this bovid tribe. During the mid-Miocene,declining global temperatures saw an increase in sea-sonality leading to the expansion of savannah grass-lands and the contraction of humid forests intogeographically isolated forest “islands” surrounded bydrier bushland and savannah (Lindsay, 1998). The mostprimitive members of the group, T. imberbis and T. ang-asi both occur in dense woodland, with the former gen-erally found at higher altitudes (Nowak, 1999). It seemsprobable that the fragmentation of dense forest habitatsresulted in the isolation of T. imberbis and T. angasi.The expansion of savannah grassland prompted thedevelopment of open savannah specialists such as T.strepsiceros and the two eland species (T. derbianus andT. oryx) between 14.08 and 10.89 Mya (Fig. 2). The sub-sequent diversiWcation of species adapted to a moretropical/wet environment (T. buxtoni, T. euryceros, T.spekei, and T. scriptus) at approximately 7.31 Mya is,


however, less clear. The latter period coincides with avegetational shift leading to the global expansion of C4ecosystems (Cerling et al., 1997). It seems likely there-fore that the diversiWcation within the closed forestgroup was driven by the continued fragmentation offorest habitats, as this speciation was not accompaniedby a shift in diet. In summary, we propose that the rapiddiversiWcation of the tragelaphids was likely to havebeen due to a combination of factors including paleocli-matic shifts, vicariance, and the sudden availability ofopen niches on the African continent.

Acknowledgments

We acknowledge E. Harley (University of CapeTown), M. Harman (Powell Cotton Museum), J. Sakwa(University of Pretoria), and the Pretoria ZoologicalGardens for providing samples. John Gatesy and threeanonymous reviewers are also thanked for their usefulcomments on the manuscript. Financial assistance wasprovided by the National Research Foundation ofSouth Africa (GUN 2053662).

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nuclear dna intron markers

phylogenetic resolution

current phylogenetic

nuclear dna segments

nuclear andmtdna gene

combined nuclear dnadata

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gene duplicationdeletion