Hidden Morphological Support for the Phylogenetic Placement of Pseudoryx nghetinhensis with

24 Pages·2001·0.62 MB·English

Syst.Biol.49(3):515–538,2000
Hidden Morphological Support for thePhylogenetic Placement
of Pseudoryx nghetinhensis with BovineBovids: A Combined Analysis
of Gross Anatomical Evidenceand DNA Sequences from FiveGenes
JOHNGATESY1,2,3 ANDPETERARCTANDER2
1LaboratoryofMolecularSystematicsandEvolution,DepartmentofEcologyandEvolutionaryBiology,Universityof
Arizona,BiosciencesWest,Tucson,Arizona85721,USA
2DepartmentofPopulationBiology,UniversityofCopenhagen,Universitetsparken15,DK-2100,Copenhagen,Denmark
D
o
Abstract.—Thesaola, Pseudoryxnghetinhensis, wasunknowntoscienceuntilitsformaldescription w
n
in1993. ThisendangeredspeciesisamemberoftheruminantartiodactylfamilyBovidae(cattle, lo
a
sheep,goats,andantelopes). However,givenitspuzzlingcombinationofmorphologicaltraits,the d
e
speci(cid:142)caf(cid:142)nitiesofPseudoryxwithinBovidaearecontroversial. Apreliminarygeneticinvestigation d
suggestedthatPseudoryxshouldbeplacedinthesubfamilyBovinae(cattle,buffaloes,spiral-horned fro
antelopes,andnilgai),butarecentcladisticanalysisofskeletalanddentalcharactersalliedPseudoryx m
wanidthmcaoplercinuelabrohvyipdost(hsehseeespd,igffoeartsin,masussigknoinxgent,hgeosaatoalantteoloepitehse,raonfdthPeantwthoolompos)s.tTdihveermgoenrpthcloaldoegsicoafl https
Bovidae.Inthisreport,phylogeneticanalysesofDNAsequencesfrom(cid:142)vegenesareusedtotestthese ://a
alternatives.Proteincodingregions,introns,andribosomalDNAsfromthenuclearandmitochondrial c
a
genomesdiscountthehypothesisthatPseudoryxisacloserelativeofCaprinae. Instead,combined d
e
analysesoftheDNAdataandpublishedmorphologicalevidenceplacePseudoryxwithBovini(cattle m
andbuffaloes),asubcladeofBovinae. Inaseparateanalysis,thematrixofmorphologicalcharacters ic.o
linksPseudoryxwithcaprinebovids,butinthecontextofthemoleculardata,thegrossanatomical u
p
evidencestronglysupportsagroupingofPseudoryxwithBovinae. Surprisingly,themorphological .c
partitionprovidesthemostcharactersupportinthecombinedanalysis.Thisstrikingresultisobscured om
byseparateanalysesoftheindividualdatasetsandthetaxonomiccongruenceapproach.[Bovidae; /s
y
hiddensupport;Pseudoryx;saola;VuQuang.] s
b
io
/a
rtic
In 1992, a distinctive new bovid species, The saola is con(cid:142)ned to the Annamite le
-a
the saola, Pseudoryx nghetinhensis, was dis- RangeofSouthEastAsia.Theregionischar- b
s
coveredinthemontaneevergreenforests of acterizedby amixtureofrecentandancient tra
c
Vu Quang, Vietnam (Dung et al., 1993) and taxonomic elements that in combination t/4
wassubsequentlydescribedfrom neighbor- formasigni(cid:142)cantcenterofendemism(Giao 9
/3
ing areas in Laos (Schaller and Rabinowitz, etal.,1998;Surridgeetal.,1999;Grovesand /5
1
1995). Pseudoryx nghetinhensis is character- Schaller, 2000). In the past 5 years, several 5
/1
ized by a unique combination of mor- new species of hoofed mammals have been 7
1
phological traits, including long, smooth, discovered in the Annamites and adjacent 12
7
spindle-shaped horns, a large preorbital areas. The discovery of even a single new 4
b
fossa, domed nasals, an elongated premo- mammalian species of large body size in y
g
lar row, frontal hollowings that extend to the late twentieth century is a rare event. u
e
the base of the horn cores, and a striking The discovery of several large mammalian st o
pattern of white markings on a predom- species in such a small geographic area n
0
inantly chestnut and black pelage (Dung is even more remarkable. Included in the 7
A
etal.,1993,1994;Thomas,1994;Schallerand list of novel taxa from the region are sev- p
Rabinowitz, 1995). The new bovid does not eral muntiacine cervids (Tuoc et al., 1994; ril 2
0
de(cid:142)nitively (cid:142)t into any of the 13 traditional Schaller and Vrba, 1996; Bauer, 1997; Giao 1
9
bovidtribesofSimpson(1945),mostofwhich et al., 1998; Groves and Dawson, in prep;
can be traced back to the Miocene (Savage Amato et al., 2000); a suid, Sus bucculentus,
andRussell,1983;Vrba,1985);thereforePseu- that was recently “rediscovered” (Groves
doryx may be a phylogenetic relict with no et al., 1997); a large bovid that is, as yet,
closeextantrelatives. poorlydescribed(“Pseudonovibos”;Peterand
Feiler, 1994; Dioli, 1997; Nadler, 1997); and
Pseudoryx. Phylogenetic analyses of the
3Presentaddress(andaddressforcorrespondence):
1206-A Downey St. Laramie, Wyoming 82072, USA; unique elements of the Annamite fauna
E-mail: [email protected] shouldcontributetoaclearerbiogeographic
515
516 SYSTEMATICBIOLOGY VOL.49
rDNA—are used to determine whether
Pseudoryxismorecloselyrelatedtobovineor
caprine bovids. A simultaneous/combined
analysis(Kluge,1989;NixonandCarpenter,
1996) of the DNA data and the morpholog-
ical evidence (Gentry, 1992; Thomas, 1994)
ultimately is used to delineate the speci(cid:142)c
phylogeneticaf(cid:142)nitiesofPseudoryx. Thedis-
tribution of unambiguous synapomorphies
among data partitions, partitioned branch D
o
support (Baker and DeSalle, 1997), hidden w
n
branch support (Gatesy et al., 1999), and lo
a
successive data set removal (Olmstead and d
e
Sowraetieorne,a1n9d94c)onar(cid:143)eicutsaemdotnogmdeaatsausreetscoinrrothbe- d fro
m
simultaneousanalysis. h
ttp
s
MATERIALS ANDMETHODS ://ac
a
GenesandTaxa de
m
The mt DNA of bovids accumulates nu- ic
FIGURE1. Two previous hypotheses regarding the .o
cleotide substitutions at a rapid rate (Irwin u
phylogeneticrelationshipofPseudoryxtootherbovids: p
inferences from (a) DNA data (Dung et al., 1993) etal.,1991;Allardetal.,1992;Chikunietal., .co
and (b)dental+skeletaldata(Thomas, 1994). Shaded 1995; Honeycutt et al., 1995; Groves and m
circles=Caprinae+Pantholops. Shields, 1996). Because of extensive homo- /sy
s
b
plasyandtherapidradiationofbovidtribes io
understandingofthisregion(e.g.,Giaoetal., iqnuetnhceesMhiaovceenoeff,eraendaolynsleyswoefakmlytsuDpNpAortseed- /article
1998;GrovesandSchaller,2000). resolutionofintertribalrelationshipswithin -a
b
Intheinitialdescriptionofthesaola,Dung Bovidae(e.g.,Allardetal.,1992;Gatesyetal., stra
etal.(1993)presentedpreliminarymitochon- 1997). Thereforeinadditiontomtgenes,we c
drial(mt)cytochromebevidencethatplaced chose to sequence several nu loci. Relative t/49
Pseudoryx with Bovinae (Fig. 1a). This sub- to mt DNA, the nu genes offer fewer vari- /3/5
family of bovids includes cattle (Bovini), ablesitesbut(itishoped)lesssaturationfrom 1
5
buffaloes (Bovini), and some “antelope” multiple,overlappingsubstitutions. /1
7
tribes such as Tragelaphini and Boselaphini Comparisons of published DNA se- 11
2
(Simpson,1945).Despitesomecorroboration quences from bovids were used as ref- 7
4
fromgrossanatomicalevidence(Dungetal., erences in choosing genes with enough b
y
1994;SchallerandRabinowitz,1995;Groves informative sites for our analysis. Rela- g
u
and Schaller, 2000), the bovine af(cid:142)nities of tively rapidly evolving nu loci were cho- es
thesaolaarecontroversial. sen in addition to two well-characterized t o
n
Thomas (1994) scored Pseudoryx for the mt genes. The seven DNA fragments in 0
7
cranialanddentalcharactersofGentry(1992) this study are characterized by a variety of A
p
and concluded that the saola is the sis- evolutionary dynamics (Gatesy et al., 1994, ril 2
ter taxon of the bovid subfamily Caprinae 1996, 1997; Chikuni et al., 1995; Cronin 0
1
(goats, sheep, goat antelopes, and musk et al., 1996; Gatesy, 1998; Milinkovitch 9
oxen)plustheproblematicgenusPantholops. et al., 1998). These sequences include pro-
The results of Thomas (1994) thus indicate tein coding regions (nu b -casein exon 7
thatPseudoryxismorecloselyrelatedtogoats 450bases,nuj -caseinexon4 400 bases),
» »
andtheirkinthantocattleandtheirrelatives introns (nu j -casein intron 4 400 bases,
»
(Fig.1b). nu b -casein intron 7 450 bases, nu a -
»
In this report, phylogenetic analyses of lactalbuminintron1 450bases),andrDNAs
»
DNA sequences from (cid:142)ve genes—nuclear (12SmtrDNA 250basesand16SmtrDNA
»
(nu)b -casein,nuj -casein,nua -lactalbumin, 350bases).Mitochondrialcytochromebse-
»
12S mt ribosomal (r) DNA, and 16S mt quences(Dungetal.,1993)arenotincluded
2000 GATESYANDARCTANDER—PSEUDORYXPHYLOGENY 517
inthepresentanalysis.Apparentnucopiesof Kraus and Miyamoto, 1991; Allard et al.,
mtcytochromeb werediscoveredinatleast 1992; Gatesy et al., 1992, 1994, 1997) were
one member of Bovidae (Boselaphus trago- used. A region of 250 bp of 12S mtrDNA
»
camelus; Arctander and Friis, unpublished andan 350bpfragmentof16SrDNAwere
»
data).Giventhiscomplication,completemt ampli(cid:142)edbyPCRanddirectlysequencedas
cytochrome b sequence for Pseudoryx will inGatesyetal. (1992,1997). Thesesequences
be presented in a subsequent publication correspond to the regions surrounded by
(Arctanderetal.,inprep). the “universal” primers of Kocher et al.
Relationships among the 13 traditional (1989), Simon (1991), and Palumbi et al.
bovid tribes are not well resolved (e.g., (1991). Theprimers used were12SA850(50- D
o
Georgiadis et al., 1990; Allard et al., 1992; AAACTGGGATTAGATACCCCACTAT-30), w
n
Gentry,1992;Gatesyetal.,1997),butanearly 12SB1270 (50-GAGGGTGACGGGCGGTGT lo
a
splitbetweenthesubfamilyBovinaeandall GT-30), 16SA2290 (50-CGCCTGTTTACCAA de
omtehreoruesxsttaundtiebso(vKidinsgdisonco,n19s8is2t;eLntowweinthsteniun-, AGAAAACCATCT-A30G),AaTnCdA1C6GSBT2-386).0Th(5e0-nCuCmGbGerTsCiTn d fro
0 m
1986; Allard et al., 1992; Wall et al., 1992; thenamesrefertopositionsintheBostaurus h
Gatesyetal.,1997).Multiplerepresentatives mtDNAsequenceofAndersonetal.(1982). ttp
s
from each of these two basal clades were Anadditional2000nucleotidesofmtrDNA ://a
c
sampled. and intervening tRNAs were available for a
d
Allsevengenefragmentsweresequenced 11 bovids and three outgroup taxa (see em
for acoregroupof10 taxa:Pseudoryx nghet- Table 1; Anderson et al., 1982; Miyamoto ic
.o
inhensis (tribe indeterminate), Bos taurus et al., 1990; Kraus and Miyamoto, 1991; u
p
(Bovini), Bubalus depressicornis (Bovini), Allard et al., 1992). These data were added .c
o
Syncerus caffer (Bovini), Boselaphus trago- to the total mt rDNA data set (see Gatesy m
/s
camelus (Boselaphini), Taurotragus oryx etal.,1997). y
s
(Tragelaphini), Capra hircus (Caprini), Oryx a -Lactalbuminintron1.—Fourteennewse- bio
gazella (Hippotragini), Kobus ellipsiprymnus quencesandonepublishedsequence(Vilotte /a
(Reduncini), and the outgroup Antilocapra et al., 1987) were used. A double-stranded rticle
americana (family Antilocapridae). Addi- fragment of a -lactalbumin spanning in- -a
b
tional species were sequenced for some trons 1 and 2 was ampli(cid:142)ed with primers stra
galesnoese,nrainchdedputhbelisthaxedondomataicfrreopmresGenetnaBtiaonnk. L-3a0)cIaI.nFd L(a5c0I-CV.CRA(5A0-AGAATCGTACTAGCTCCACGTTTATGGTTCT ct/49
These data permitted a more (cid:142)ne-grained AATTC-3) from Milinkovitch et al. (1998). /3
0 /5
placement of Pseudoryx among the bovids. Given that pseudogenes of a -lactalbumin 1
5
Table1includesalistoftheDNAsequences have been detected in domesticated bovids /1
7
sampledfromthe76taxainthisstudy. (Soulier et al., 1989; Vilotte et al., 1991), 11
2
the design of the above primers avoids 7
4
the ampli(cid:142)cation of these pseudoge- b
y
DNAExtraction,PolymeraseChain nes (D. Irwin, pers. comm.). PCR condi- g
u
Reaction(PCR)Ampli(cid:142)cation,Sequencing, tions for double-stranded ampli(cid:142)cations es
andSequenceAlignment were 1 min at 94 C, 1 min at 55 C, and t o
± ± n
Total DNA was extracted from fresh tis- 1 min at 72 C, for 30 cycles. The double- 0
± 7
sue and blood samples by standard proto- stranded PCR product was then puri(cid:142)ed A
p
cols. DNAfromdriedskinsamplesofPseu- on alow-melting-point agarose gel. Single- ril 2
doryxwasextractedasinDungetal. (1993). stranded PCR product for intron 1 and 0
1
Primers, PCR conditions, and sequencing the 5 end of exon 2 was ampli(cid:142)ed from 9
0
methods for each sequence are described the double-stranded PCR product with
below. The number of taxa sequenced for asymmetric dilutions of primers LacI.R
this study and the number of published se- (5-CTCACTGTCACAGGAGATGT-3) and
0 0
quences are indicated. All sequence align- LacII.F from Milinkovitch et al. (1998).
ments are available on the Internet at the PCR conditions for single-stranded amp-
homepageforSystematicBiology. li(cid:142)cations were 1 min at 94 C, 1 min at
±
12S and 16S mt rDNA.—Two new se- 55 C, and 1 min at 72 C, for 35 cycles.
± ±
quences and 114 published sequences Single-strandedPCRproductswerecleaned
(Andersonetal.,1982;Miyamotoetal.,1990; in Millipore 30,000 NMWL (cid:142)lters and
518 SYSTEMATICBIOLOGY VOL.49
TABLE1. The71bovidand(cid:142)veoutgrouptaxaanalyzedinthisstudy.
Bovidtribe Species Datasetsa
Tragelaphini Tragelaphusimberbis RbRAb
Tragelaphusangasi Rb
Tragelaphusscriptusc Mb
Taurotragusoryxc RbABEBIKEKI
Bovini Bostaurusc RbAbBEbBIbKEbKIbRAb
Bisonbonasusc KEbMb
Bisonbisonbison KEb
D
Synceruscafferc RbABEBIKEKI o
w
Bubalusdepressicornisc RbABEBIKEKIMb n
Bubalusbubalis KEb lo
a
d
Boselaphini Boselaphustragocamelusc RbABEBIKEKIMbRAb e
d
Neotragini Madoquakirki RbMbRAb fro
Raphiceruscampestris RbBE m
Raphicerusmelanotis Mb h
Ourebiaourebi RbMb ttps
Antilopini Antidorcasmarsupialis RbABE ://a
Gazellathomsoni RbARAb ca
d
Gazellagranti ABE e
m
Gazelladorcas Mb ic
Gazellasubgutturosa Rb .o
Antilopecervicapra RbBEMb up
Procaprapicticaudata Rb .co
m
Alcelaphini Damaliscusdorcasphillipsi RbRAb /s
Damaliscusdorcasdorcas Rb ys
Damaliscuslunatusjimela RbMb bio
Damaliscuslunatuslunatus RbBEMb /a
Beatragushunteri Rb rtic
Alcelaphusbuselaphus Rb le
Alcelaphuscaama Rb -ab
AColcnenlaopchhuaestleischtateunrsinteuinsi RRbbBAEKI strac
Connochaetesgnou RbBEKE t/4
9
Hippotragini Oryxgazellagazellac RbKIRAb /3
Oryxgazellacallotisc RbABEBIKE /51
5
Oryxdammah Rb /1
Oryxleucoryx Rb 71
Addaxnasomaculatus Rb 12
Hippotragusniger RbBE 74
Hippotragusequinusc RbMb b
y
Caprini Caprahircusc RbABEbBIKEbKIRAb gu
Capraaegagrusc Mb es
Hemitragusjemlahicus Rb t o
Pseudoisnayaur Rb n 0
Ovisdalli RbKEb 7 A
Oviscanadensis Rb p
Ovisaries BEbKEb ril 2
Ovisorientalis Mb 0
1
Ovibovini Ovibosmoschatus RbBEKEbMb 9
Budorcastaxicolor Mb
Rupicaprini Nemorhaedusgoral RbKEbMb
Capricorniscrispus RbKEb
Capricornisswinhoei KEb
Capricornissumatraensis KEbMb
Rupicaprarupicapra KEbMb
Oreamnosamericanus RbBEKEb
Reduncini Kobusellipsiprymnusc RbABEBIKEKIRAb
Kobusmegaceros Rb
Kobusleche Rb
Kobuskobc RbMb
2000 GATESYANDARCTANDER—PSEUDORYXPHYLOGENY 519
TABLE1. Continued
Bovidtribe Species Datasetsa
Kobusvardoni Rb
Reduncafulvorufula RbBEBI
Reduncaarundinum Rb
Reduncaredunca Rb
Cephalophini Cephalophusmaxwelli RbBEBIRAb
Cephalophusdorsalis BE
Sylvicapragrimmia Mb
Indeterminate Pantholopshodgsoni RbBEMb Do
Pseudoryxnghetinhensisc RABEBIKEKIRb w
n
Peleacapreolus RbBEMb lo
a
Aepycerosmelampus RbABEMbRAb d
e
Saigatatarica RbBEKEbMb d
Outgroups Antilocapraamericanac RbABEbBIKEbKIRAb fro
m
Giraffacamelopardalis RbRAb h
Hydropotesinermis RbRAb ttp
MMousncthiuacsumsomscuhnitfejarkucs MMbb s://a
c
a
aR=12S+16SribosomalDNA;A=a -lactalbuminintron1;BE=b -caseinexon7;BI=b -caseinintron7;KE=j -caseinexon4; de
KI=j -caseinintron4;M=morphology;RA=additional2,000basesofmitochondrialDNA. m
bcCDoarteatparxeavaionuaslylyzepdubinlisthhiesds.tudy. ic.ou
p
.c
o
m
/s
sequenced in both directions according to (5-GTCTTCTTTGATGTCTCCT-3) or KCA y
0 0 s
standard dideoxy sequencing protocols SA+KBOVR1 (50-ACCAGCAAATGTAAG bio
(Sequenase;USBiochemicals). TTATAATTA-3) were used. For intron 4, /a
j -Caseinexon4.—Eightnewsequencesand primer pair K0BOVL (50-AGGAGACATCA rticle
16 published sequences (Alexander et al., AAGAAGAC-30)+KCASB was utilized. -ab
1988; Furet et al., 1990; Chikuni et al., 1995; Double-strandedPCRconditionswere1min stra
Cusreodn.inet al., 1996; Gatesy et al., 1996)were afotr943±0C–,315mciyncalets5.2–P5C5R±C,caonnddi1tiomnisnafotr72t±hCe, ct/49
j -Casein intron 4.—Ten new sequences ampli(cid:142)cation of single strands from the /3
/5
and one published sequence (Alexander double-strandedPCRproductwere1minat 1
5
et al., 1988) were used. For most samples, 94 C, 1 min at 53–55 C, and 1 min at 72 C, /1
± ± ± 7
an 850 bp fragment that spans most for35–38cycles.Puri(cid:142)cationandsequencing 11
» 2
of the fourth exon and fourth intron of ofsingle-strandedPCRproductswereasfor 7
4
j -casein was ampli(cid:142)ed with published a -lactalbuminintron1. b
y
primers (Pinder et al., 1991): KCASA b -Casein exon 7.—Twenty–four new se- g
u
(K5C0-GATSBGC(T5G0-GATGAYAGAGGGTTAGTCCACATCAAGA-3C0)ACaTnGd q(Buoennsciensgeatnadl.,1f9o8u8r;Gpautebslyisehteadl.,1s9e9q6u)ewnceeres est on
G-3). PCR conditions were 1 min at 94 C, used. Primers BCASA (5-AGGATGAACT 0
0 ± 0 7
1 min at 52–55±C, and 1.5 min at 72±C, for CCAGGATAAA-30) and BCASB (50-AGGC Ap
35 cycles. This PCR product was TA-cloned TCCTGGTACAGCAGAA-30) were used to ril 2
(Invitrogen), and two to three clones were amplify an 450 bp section of b -casein 0
sequenced as in Gatesy et al. (1996). Ad- exon 7. PCR c» onditions were1 min at 94 C, 19
±
ditionally, PCR products were directly 1min at 55 C, and 1minat 72 C, for 35 cy-
± ±
sequenced in one direction to eliminate cles.PCRproductswereTA-cloned,andtwo
ambiguities.Single-strandedampli(cid:142)cations, clonesforeachspeciesweresequencedonan
puri(cid:142)cation of PCR products, and sequenc- ABIautomatedsequencer.
ingwereasfora -lactalbuminintron1. b -Casein intron 7.—Eleven new sequen-
For taxa that were not TA-cloned, PCR ces and one published sequence (Bonsing
products were directly sequenced in both et al., 1988) were used. An 450 bp region
»
directions. For the direct sequencing of of b -casein intron 7 was ampli(cid:142)ed with
exon 4, primer pairs KCASA+KBOVR primers BCASIL (5-TTCCAAAATAAGCA
0
520 SYSTEMATICBIOLOGY VOL.49
TAGCTG-3) and CASBR3 (5-TGAAATCY The following data sets were analyzed:
0 0
TCTTAGACCTT-3). PCR conditions were 12S plus 16S mtrDNA for 58 taxa, j -casein
0
1 min at 94 C, 1 min at 53 C, and 1 min exon 4for 24 taxaplus j -casein intron4 for
± ±
at 72 C, for 35 cycles. Single-stranded tem- 11 taxa, b -casein exon 7 for 28 taxa plus b -
±
plate was produced with theDYNABEADS caseinintron7for12taxa,anda -lactalbumin
protocol (Dynal), a system that utilizes intron1for15taxa.Inadditiontotheabove
biotinylated primers. Puri(cid:142)cation and seq- analyses, searches were performed for the
uencingofsingle-strandedproductswereas 10coretaxacommontoalloftheDNAdata
fora -lactalbuminintron1. sets (Table 1). The following four data sets
All (cid:142)ve genes exhibited length variation were analyzed for the core taxa: 12S mt D
o
amongspecies.Orthologoussequenceswere rDNA(forthe 250positionscommontoall w
» n
alignedwiththeparsimony-basedalignment taxa) plus 16S mt rDNA (for the 350 po- lo
» a
program MALIGN (WheelerandGladstein, sitions common toall taxa), j -caseinexon 4 d
e
1w9e9r4e).exFeocuurtemdufoltripelaechseoqfutehnece(cid:142)vaelingunmDeNntAs pb l-ucassjei-ncaisnetirnonin7t,raonnd4a,-bla-cctaaslebiunmexinonint7ropnlu1s. d from
fragments. The cost for opening a gap, ex- For the combined molecular data base, h
tendingagap,andmakinganucleotidesub- other partitioning scenarios could easily be ttp
s
stitution was varied from 1.5:1:1 to 3:2:1 to justi(cid:142)ed.Forexample,thegeneticallylinked ://a
c
5:4:1 to 10:9:1. Other MALIGN parameters caseingenescouldbecombinedinonedata a
d
were these: score 3, contig, quick, atbr, and set, nu introns could be separated from nu em
iter. ThemtrDNAgenes werealigned as in exons, the linked mt rDNAs that encode ic
.o
Gatesy et al. (1997) at gap cost:nucleotide separate RNA products could be divided, u
p
substitutioncostratiosof2:1and3:1.Forthe thethreecodonpositionsofb -caseinand j - .c
o
threenuintronsandthetwomtrDNAs,(cid:142)nal casein could be separated, a distinction be- m
/s
alignmentswerechosenbytreelength;align- tween mt and nu data sets could be made, y
s
b
ments that implied the shortest trees (with and so forth. Given limitations in journal io
eoqputiamlaclh.aFroarcttehrewtweioghntuinegx)ownse,rethceonalsgidoreirtehd- snpoatcbee,etvhaelsueaatedddihteiorne.al data partitions will /article
mic alignments were adjusted by eye, us- Morphology.—Thomas (1994) scored -a
b
ing SeqApp 1.9a (Gilbert, 1992). Gaps were (cid:142)ve artiodactyl taxa including Pseudoryx stra
consolidatedintomultiplesofthreetomain- for the 112 morphological characters of c
tain reading frames in these protein coding Gentry (1992) and added two characters: t/49
regions. presence/absence of cranial appendages /3/5
andpresence/absenceofuppercanines.The 1
5
data of Thomas (1994) were combined with /1
7
PhylogeneticAnalyses thedataofGentry(1992)for 22 otherbovid 11
2
DNA.—Cladisticanalysesweredonewith species (Table 1). The PAUP 3.1.1search for 7
4
PAUP 3.1.1 (Swofford, 1993) or PAUP the total of 27 taxa and 114 characters was b
¤ y
4.0.0d64 (Swofford, unpubl.). Phylogenetic as above. Characters were unordered, and g
u
searches were branch-and-bound orheuris- all transformations were equally weighted. es
ticwith100replicatesofrandomtaxonaddi- Cladograms were rooted with Moschus t o
n
tion and TBR branch swapping (Swofford, (Moschidae)andMuntiacus (Cervidae)asin 0
7
1993). All character transformations were Thomas (1994). An additional analysis was A
p
weighted equally, and gaps were treated executed in which characters were ordered ril 2
as missing data. Nonbovid, pecoran artio- asinGentry(1992). 0
1
dactyls were used as outgroups (Janis and Three of the 10 core taxa (Table 1)— 9
Scott, 1987; Gentry and Hooker, 1988). Ad- Pseudoryx nghetinhensis, Bubalus depressi-
ditional analyses were executed in which cornis, and Boselaphus tragocamelus—were
gaps were coded as a (cid:142)fth character state. scored for morphological characters by
Thiscodingofgapsassignsweightstoindels Gentry(1992)andThomas(1994). Addition-
that are proportional to their lengths (e.g., ally,(cid:142)vetaxascoredformorphologicalchar-
Wheeler et al., 1993). Giribet and Wheeler actersarecloserelativesofcoretaxa(Table1):
(1999)havepresentedasimplejusticationfor Bison bonasus (tribe Bovini) Tragelaphus
scoringsinglenucleotidegapsasindividual scriptus (tribe Tragelaphini), Capra aega-
characterstates. grus (tribe Caprini), Hippotragus equinus
2000 GATESYANDARCTANDER—PSEUDORYXPHYLOGENY 521
(tribe Hippotragini), and Kobus kob (tribe combinedanalyses. Transitionsubstitutions
Reduncini). Aseparatephylogenetic search wereignored(i.e., transversionparsimony),
was performed for these eight taxa and orgapswerecodedasa(cid:142)fthcharacterstate,
the outgroup, Muntiacus muntjak (family orboth.
Cervidae). Characters were unordered and
thephylogeneticsearchwasasabove. BaseCompositionand
DNA + morphology.—Miyamoto (1985) Transition/TransversionBias
andKluge(1989)havepresentedarguments
For the 10 core taxa (Table 1), nucleotide
forcombiningmolecularandmorphological
base compositions of the seven DNA frag-
characters in simultaneous phylogenetic D
ments were calculated by using Mac- o
analyses (Nixon and Carpenter, 1996). w
Clade3.04(MaddisonandMaddison,1992). n
Simultaneous analyses permit scrutiny of lo
Estimatesofthetransition/transversionbias a
molecular and morphological evidence d
for each DNA fragment also were calcu- e
a(ec.cgo.,rdPianttgertsooind,e1n9t8ic2a)l. criteriafor homology laaltigednmweintth,nMucalceColtaiddees3u.b0s4t.itFuotironesacwherDeNopA- d from
The morphological data of Gentry (1992) h
and Thomas (1994) were combined with timizedontothetotalevidencetopologyfor ttp
DNAsequencedatafrom(cid:142)vegenes. Because coofrteratnasxiatiobnyapnadrstirmanosnvye,rasinodn tshuebsntiutumtbioenrss s://ac
of the uneven taxonomic sampling among a
were recorded. All most-parsimonious op- d
data sets, many characters were scored as timizations were incorporated into the esti- em
missinginthecombineddatamatrix. Some ic
mates by using the “equivocal cycling” op- .o
closely related species in the molecular and u
tion.Eachcharacteroptimizationwasgiven p
morphological data sets were equated to .c
equal weight in determinations of the rela- o
make composite terminal taxa. The mono- m
tive number of transitions versus transver- /s
phyly of these hybrid terminals was as- y
sions for a given data set (Maddison and sb
sotciuafctm,6es2weaditnt.ahxdTa1hT0eaB0nRPdArbaU5rna6dPn0oc23mh.1cs.h1twaa.rxasaopecnpateriancrsdhgd.wfoiAtarilsoltnhchheerauetropratilcasi---l MaddisonN,o1d9a9l2S)t.abilityandSupport io/article-a
b
ter transformations were weighted equally, Bootstraps and branch support.—The rela- stra
gaps intheDNAalignmentswerecoded as tivestabilityofnodeswasassessedbyboot- c
missingdata, and morphological characters strappercentages(BP;Felsenstein,1985)and t/49
wereunordered. branch support (BS; Bremer, 1988, 1994). In /3/5
Asecondcombinedanalysiswasdonefor eachbootstrapanalysis,informativecharac- 1
5
the10coretaxa. Thiscorematrixwascharac- terswereresampledwithreplacement from /1
7
terizedbyalowerpercentageofmissingdata theoriginaldataset,andbootstrapreplicate 11
2
(per taxon) relative to the combined matrix datasetsthesamesizeastheoriginalwereas- 7
4
of 62 taxa above. Data from the (cid:142)ve genes sembled(Felsenstein, 1985). Thenumberof b
y
and morphology were merged. Closely re- bootstrapiterationsrangedfrom100to1,000, g
u
latedtaxainthemolecularandmorphologi- depending on the number of equally parsi- es
calmatriceswereequated(Table1)—thatis, monioustopologiesforaparticulardataset. t o
n
the bovinecattle (Bos taurus and Bisonbona- Each bootstrap replicate involved a heuris- 0
7
sus),thetragelaphines(Taurotragusoryxand tic parsimony search with simple taxon ad- A
p
Tragelaphusscriptus),thehippotragines(Oryx ditionandTBRbranchswapping(Swofford, ril 2
gazellaand Hippotragusequinus), thecaprine 1993). 0
1
goats (Capra hircus and Capra aegagrus), the BSscores for selected nodes werealso es- 9
reduncines (Kobus ellipsiprymnus and Kobus timatedwithPAUP.Foraparticulardataset
kob),andtheoutgrouptaxa(Antilocapraamer- and a particular node, BS is the minimum
icanaand Muntiacusmuntjak). Thephyloge- number of character steps for that data set
neticsearchwasbranch-and-bound,allchar- ontheshortesttopologiesthatdonotcontain
actertransformationswereweightedequally, that node, minus the minimum number of
gaps in the DNA alignments were coded characterstepsforthatdatasetontheshort-
as missing data, and morphological char- esttopologiesthatdocontainthatnode.Note
acters were unordered. Additional searches that withthis de(cid:142)nition,BScan be positive,
were executed to test the stabilities of the zero, or negative (in contrast to Bremer,
522 SYSTEMATICBIOLOGY VOL.49
1994).Ifthenodeofinterestissupportedby areadditive,andLBSI=1.IfLBSforasetof
agivendataset,BSispositive.Ifthenodeis nodesisgreaterthanthesumofBSscoresfor
notsupportedbyagivendataset,BSisnega- those nodes, then the collapse of one node
tiveorzero(seeGatesyetal.,1999).BSwases- makesthesimultaneous collapseofanother
timatedbyusingthe“constraints”command nodemorecostly,thereisastabilizingeffect
ofPAUPandbranch-and-boundorheuristic inthecladogram,andtheLBSIis >1.
searches. Heuristic parsimony searches for For the combined analysis of 10 core
these analyses included at least 10 random taxa (Table 1), LBS scores and LBSIs were
taxon addition replicates with TBR branch determined for each pair of nodes sup-
swapping.Becauseofthecomplexityofsome ported by the combined analysis (gaps D
o
data sets, BS scores at some nodes may be treated as missing data, all characters un- w
n
overestimates. ordered, and all character transformations lo
a
Interdependence of stabilities for different givenequalweight). Minimum treelengths d
e
ntiocduelsa.r—nBoSdemteoasruelraexsattihoensotafbtihlietypaorfsiampoanry- fsourptpooprotelodgnieosdtehsawt seirmeudletatenremouinselydlwacitkhttwhoe d fro
m
criterion (Bremer, 1988, 1994; Davis, 1993). “constraints” and “(cid:142)lter” options of PAUP. h
BSscoresforindividualnodesarecalculated AllPAUPsearcheswerebranch-and-bound. ttp
s
independently,butthestabilitiesofdifferent Distribution of con(cid:143)ict and support among ://a
c
nodes in a minimum length topology may data sets.—For the 10 core taxa (Table 1), a
d
notbeadditive(seeFaithandBallard,1994). the incongruence length difference (ILD; em
BycalculatingBSforseveralnodessimulta- Mickevich and Farris, 1981) test was used ic
.o
neously, theinteractionofstabilities for dif- to assess the null hypothesis of congruence u
p
ferentnodescanbeassessed.Foragivendata amongdatasets(Farrisetal.,1994).SixILD .c
o
matrix, linkedbranchsupport(LBS;Gatesy, tests were performed. Each of the (cid:142)ve in- m
in press)for a set of supported nodes is the dividual data sets (12S rDNA+16S rDNA, /sy
s
lengthoftheshortesttopologythatlacksall b -casein, j -casein, a -lactalbumin, or mor- bio
ofthosenodes,minusthelengthoftheshort- phology) was compared with the sum of /a
esttopologythatcontainsallofthosenodes. thecharacters fromtheother fourdatasets. rticle
If there is no homoplasy in a matrix of bi- Also, a division of the combined data set -a
b
nary characters, BSscores are additive. LBS intothe(cid:142)veindividualdatasetswastested. stra
foreachsetofnodesisequaltothesumofBS Toestablishanulldistributionforeachtest, c
scores for nodes in that set. If homoplasy is 999randomdatapartitionsweregenerated, t/49
present in a data matrix, the distribution of andILDswerecalculatedforeachILDrepli- /3
/5
characterincongruencedetermineswhether cate with PAUP (Swofford, unpubl.). In all 1
¤ 5
LBSfor a set of nodes is less than, equal to, ILDtests,uninformativecharacterswereex- /1
7
ormorethanthesumofBSscoresfornodes cluded, and searches were heuristic with 11
2
inthatset(Gatesy,inpress). simpletaxonadditionandTBRbranchswap- 7
4
LBS can be scaled tothe amount of BS to ping. P =0.05wastakenasthethresholdfor b
y
make LBS scores for different sets of nodes signi(cid:142)cance. g
u
more comparable. The LBS index (LBSI; Corroboration among the (cid:142)ve individual es
Gatesy,inpress)forasetofsupportednodes data sets also was estimated for the core t o
n
is group of 10 taxa (Table 1). This was done 0
7
in four ways. First, taxonomic congruence A
p
(LBSforthosenodes)¡ (Nelson, 1979) was assessed; that is, strict ril 2
(thelargestBSscoreamongthosenodes) consensustreesderivedfromeachofthe(cid:142)ve 01
data sets were compared to quantify topo- 9
(sumofBSscoresforthosenodes) logicalsimilarity.Second,foreachnodesup-
¡
(thelargestBSscoreamongthosenodes). ported by thesimultaneous analysis ofcore
taxa,unambiguouslyoptimizedsynapomor-
IfLBSforasetofnodesislessthanthesum phiesderivedfromeachofthe(cid:142)vedatasets
ofBSscoresforthosenodes,thenthestabil- were noted. The “list of apomorphies” op-
ity to relaxation of the parsimony criterion tionofPAUP was usedtodiagnoseclades.
¤
is not additive for those nodes and LBSI is Third, partitioned branch support (PBS;
<1. If LBS for a set of nodes is equal to the Baker and DeSalle, 1997)was calculated for
sum of BS scores for those nodes, BS scores eachdatasetandeachnodesupportedbythe
2000 GATESYANDARCTANDER—PSEUDORYXPHYLOGENY 523
combined analysis of all (cid:142)ve data sets. For criticalfortheresolutionofparticularnodes
a particular combined data set, a particular (Gatesyetal.,1999). PBSandDRIswerede-
node, and a particular data partition, PBS terminedasinGatesyetal. (1999).
is the minimum number of character steps Hidden nodal supportand con(cid:143)ict.—Thein-
for that partition on the shortest topologies teraction of different data sets in simulta-
forthecombineddatasetthatdonotcontain neousanalysisoftenimplieshiddencharac-
that node, minus the minimum number of tersupportandcon(cid:143)icts(Barrettetal.,1991;
characterstepsforthatpartitionontheshort- ChippindaleandWiens,1994;Olmsteadand
esttopologiesforthecombineddatasetthat Sweere, 1994). For a particular set of data
do contain that node. If there are multiple partitions and a particular node, hidden D
o
equallyshorttopologies,thentreelengthsare charactersupportcanbede(cid:142)nedasincreased w
n
averaged(BakerandDeSalle,1997). supportforthenodeofinterestinthesimul- lo
a
PBS offers a simple means for assess- taneous analysis of all data partitions rela- d
e
iwnigthsiunpaposirmt ruelntadneeroedusbayndaliyffseirsenfrtamdaetwaoserkts. ttihveesteopathraetesuamnaolyfsseuspopfoeratcfhorpathrtaittinono.deFoinr d fro
m
Themethodpermitsthedetectionofhidden aparticularsetofdatapartitions andapar- h
con(cid:143)ictsandsupport(e.g.,Barrettetal.,1991) ticular node, hidden con(cid:143)ict can be de(cid:142)ned ttp
s
that arenot obvious from separateanalyses asdecreasedsupportforthenodeofinterest ://a
c
ofeachdataset.Furthermore,becausechar- inthesimultaneous analysis ofalldatapar- a
d
actersareallowedtointeractinsimultaneous titionsrelativetothesumofsupportforthat em
analysis,therelativeweightofevidencefrom nodeintheseparateanalysesofthevarious ic
.o
eachdatasetistakenintoaccount.Withina datapartitions. u
p
simultaneousanalysisframework,apositive Hiddensupportandcon(cid:143)ictscanbequan- .c
o
PBSscoreindicatesthatagivendatasetpro- ti(cid:142)ed with a variation of BS, hidden branch m
/s
vides net positive support for that particu- support(HBS;Gatesyetal.,1999).Forapar- y
s
b
larnodeoverthealternativerelationshipsin ticular combined data set and a particular io
the shortest tree(s) without the given node; node, HBS is the difference between BS for /a
a negative PBS score shows that a data set thatnodeinthesimultaneousanalysisofall rticle
favorstheshortesttree(s)without thegiven datapartitionsandthesumofBSscores for -a
b
node over the minimum length solution(s); that node from each data partition. For a stra
and a PBS score of zero indicates the in- particular combined data set and a particu- c
difference of a given data set at that node. larnode,apositiveHBSscoreindicatesthat t/49
The sum of PBS scores at a particular node more hidden support than hidden con(cid:143)ict /3
/5
equals BS at that node (Baker and DeSalle, emerges at that node in simultaneous anal- 1
5
1997). ysis. A negative HBS score indicates more /1
7
Fourth, the data set removal index (DRI; hiddencon(cid:143)ictthanhiddensupport. 11
2
Gatesy et al., 1999) was calculated for each HBScanbepartitionedamongthevarious 7
4
node supported by the combined analysis. datasetsinthecombinedanalysis. Forapar- b
y
The DRI is analogous to the clade stability ticularcombineddataset,aparticularnode, g
u
index ofDavis (1993). For aparticular node andaparticulardataset,partitionedhidden es
andaparticularcombineddataset,theDRI branchsupport(PHBS;Gatesyetal.,1999)is t o
n
istheminimumnumberofdatasetremovals the difference between PBS at that node for 0
7
requiredtocollapsethatnode. Anodethatis thatdatasetandBSatthatnodeforthatdata A
p
notsupportedbythecombineddatasethas set. For aparticularnode, thesumofPHBS ril 2
aDRIofzero. Anodethatcollapseswiththe scores for thevarious datapartitions equals 0
1
removalofonlyonedatasethasaDRIofone. theHBSatthatnode. 9
Foracombineddatasetinwhicheachcom- Hidden support can also be de(cid:142)ned in
ponentdatasetandeachcombinationofdata terms of synapomorphy. For a particular
sets support the node of interest, the DRIis combined data set and a given clade sup-
equaltothenumberofdatasetsthatcompose ported by that combined data set, hidden
thecombineddataset. AhighDRIindicates synapomorphy(HS;Gatesyetal.,1999)isthe
that character support for a particular node number of unambiguous synapomorphies
is distributed among many data sets. Cal- for that clade in the simultaneous analysis
culationoftheDRIalsoidenti(cid:142)esparticular ofthe combined data set, minus the sum of
datasetsorcombinationsofdatasetsthatare unambiguoussynapomorphiesforthatclade
524 SYSTEMATICBIOLOGY VOL.49
in the separate analyses of individual data TABLE2. Rangesofbasecompositionandthetransi-
partitions. The contribution of a particular tiontotransversionratio(TI/TV)foreachgenefragment
forcoretaxa.
datapartition toHS for agiven clade is the
number of unambiguous synapomorphies
Genefragment %A %C %G %T TI/TV
forthatcladefromthatpartitioninsimulta-
12SrDNA 35–38 21–25 16–19 22–25 5.5
neousanalysis,minusthenumberofunam-
16SrDNA 33–35 21–23 19–20 23–25 4.6
biguoussynapomorphiesforthatcladefrom a -Lactalbumin 17–24 23–26 13–14 37–45 2.2
that partition in separate analysis (Gatesy b -Caseinexon 21–23 34–36 16–18 25–26 2.1
etal.,1999). b -Caseinintron 32–34 15–17 16–17 34–35 1.5
Forthesimultaneousanalysisof(cid:142)vedata j -Caseinexon 32–34 27–30 15–17 22–24 1.6 D
j -Caseinintron 38–39 13–15 11–13 35–37 1.7 o
setsand10coretaxa,HBS,PHBS,HS,andthe w
n
distributionofHSamongdatasetswerecal- lo
a
culatedforeachnodeintheminimumlength d
e
topologyasinGatesyetal. (1999). tgherneeesnaulsogehnaevse(lroewsuelrtsennsoetmshbloewcno)n.sTishteenmcyt d from
indices (Kluge and Farris, 1969) and reten- h
RESULTSANDDISCUSSION tgieonneisndfoicretsh(eFacrorries,g1r9o8u9p)thoafn10antyaxoaf.tThheensue ttps://a
SeparateAnalysesofMolecularand c
differences in evolutionary dynamics could a
MorphologicalDataSets in(cid:143)uencephylogeneticresultsforthemtand dem
Miyamoto and Fitch (1995) argued that nudatasets. ic
.o
congruenttopologiesderivedfrommanyin- Perhaps not surprisingly, topologies de- u
p
dependent,uniquelyevolvinglocioffercom- rived from separate analyses of the differ- .c
o
pelling evidence for phylogenetic relation- ent DNAdatasetsshow avariety ofresults m
/s
ships. Unfortunately, distinctions between (Figs. 2–5). However, none of the molecular y
s
b
different DNA data sets are not always so matrices supports an especially close rela- io
calebaror-acduts(eKlelcutgioenanodfmWtoalfn,d19n9u3)g.eRneegsacrldelaerslsy, tTihonomshaipsb(1e9tw94e)ehnaPdsepurdoopryoxseadnd(FCiga.p1ribn)a.e,Aalsl /article
represents a better sampling of the genetic of the genes except a -lactalbumin intron 1 -a
b
material than information from any single (Figure5) areconsistentwiththeplacement stra
gene. of Pseudoryx within Bovinae (Figs. 2–4), as c
Inthisstudy,diverseDNAsequencesfrom suggestedbyDungetal.(1993)(Fig.1a).For t/49
both the nu and mt genomes were used each DNA sequence, the cost of assigning /3/5
to test previous hypotheses of bovid phy- thesaolatoBovinaeorCaprinae+Pantholops 1
5
logeny (Fig. 1a and b). Base composition is shown in Figure 6. From 2 to 18 extra /1
7
varieswidelyamongthedifferentsequences nucleotide substitutions per DNA data set 11
2
(Table2). Forexample, theaveragepercent- are required to group Pseudoryx with the 7
4
age of cytosine ranges from 0.14 to 0.36. caprines. b
y
ThetwomtrDNAgeneshavebroadlysimi- The precise phylogenetic position of g
u
lar base compositions. However, regions of Pseudoryx relative to other members of es
the tightly linked nu casein genes, j and Bovinae varies from data set to data set. t o
n
b (Threadgill and Womack, 1990), show a The mt rDNA data weakly support a 0
7
range of compositional bias: The b -casein Pseudoryx+Boselaphus (Boselaphini) clade A
p
erexmonai7nsineqguceansecienssaerqeuGen+ceCsraircehA,w+hTer–ebaiasstehde (tFhieg.ca2t)t;le,j -BcaosseitnaurgursouapnsdPBseisuodnory(Fxigw. i3t)h; ril 20
1
(Table2). and the strict consensus tree for b -casein 9
Transition/transversion ratios estimated (Fig. 4) shows an unresolved polytomy
by parsimony are greatest in the two mt among Pseudoryx and members of Bovini
genes(4.6–5.5).ThenuDNAdatasetsareless (Bos,Bubalus,andSyncerus).j -Caseinandb -
extreme, their ratios ranging from 1.5to 2.2 casein agree in placing Pseudoryx closer to
(Table2).Besideshavinggreatertransition/ Bovini than to other members of Bovinae
transversion ratios, the mt data evolve at a (tribesTragelaphiniandBoselaphini).
faster overall rate relative to the nu data. The dental and skeletal characters, how-
Moreover, the average rates of nucleotide ever, are more consistent with the conclu-
substitution, insertion, and deletion are all sionsofThomas(1994),showninFigure1b,
greater in the mt rDNA genes than in the than with the results of Dung et al. (1993),