The Auk 113(3):655-663, 1996
MITOCHONDRIAL
DNA SEQUENCE VARIATION AMONG
SUBSPECIES OF SARUS CRANE (GRUS ANTIGONE)
THE
TIMOTHY C. WOOD 1 AND CAREY KRAIEWSKI2
Department
of Zoology,
Southern
IllinoisUniversity,
Carbondale,
Illinois62901,USA
ABSTRACT.--We
examined DNA sequencevariation in an 1,831 base-pairsegmentof the
mitochondrialDNA genomefrom representativesof the three subspecies
of SarusCrane
(Grusantigone).The sequencesinclude the entire cytochrome-b,tRNA TM,tRNA Prø,and ND6
genes,as well as three short intergenic spacerregions.Nine distinct haplotypeswere identified in a sampleof nine individuals,three eachfrom the Indian (G. a. antigone),
Burmese
(G. a. sharpei),and Australian (G. a. gillae) subspecies.Phylogenetic analysisrevealed that
although SarusCrane haplotypesform a monophyleticassemblagerelative to Brolga (G.
rubicunda)
and White-napedCrane (G. vipio)outgroups,they cannotbe resolvedonto a dichotomouslybranchingtree. A minimum-length network for the SarusCrane haplotypes
revealsat least one instanceof direct ancestryand one hard polytomy, but showsno phylogeographicpartitioningof haplotypesamongsubspecies.
Net sequencedivergenceamong
subspeciesis not significantly different from zero. Estimatedsequencedivergence times,
neutral coalescenttimes, and data on the Quaternarygeologyof Australasiasuggestthat
SarusCranescolonizedAustraliaduring the late Pleistocene.Received
25 October1995,accepted
10 December 1995.
$ARUSCRANES
(Grusantigone)are among the
largestmembersin the cranefamily (Gruidae),
sometimesreaching an adult height of 1.8 m.
They are nonmigratoryand currently found in
matically reducedby human activity during the
pastcentury. Once widely distributed acrossIndia, Nepal, Bangladesh,and western Burma,the
Indian SarusCrane is now restrictedto portions
isolated areas of northwestern India, Southeast
of northwestern
Asia,and Australia.Blyth and Tegetmeier(1881)
of Nepal (Johnsgard1983).Until the late 1940s,
classified the Indian and Burmese Sarus Cranes
Burmese
as distinct specieson the basisof plumage and
body-sizecharacteristics
(the larger Indian Sarus Crane has a bright white neck ring and
white tertials), and this distinction persisted
through the classificationof Sharpe (1894).
China, eastern Burma, Philippines, the Malay
Blanford (1895), however, reduced the Indian
and BurmeseSarus Cranes to subspecies(G. a.
antigoneand G. a. sharpeLrespectively),and this
convention endured through all subsequentrevisions.
Sarus
Cranes
were
first
observed
in
Australia in 1966, and were then considered
membersof G. a. sharpei(Gill 1969, Archibald
1981). Schodde (1988) designated the Australian SarusCrane as a distinct subspecies(G. a.
gillae)on the basisof its darker plumage and
larger ear patch.
The range of the Sarus Crane has been dra-
India
and the Terai lowlands
Sarus Cranes were
found
in southern
Peninsula, Thailand, Cambodia, Laos, and Vi-
etnam (Delacour and Mayr 1946, Walkinshaw
1973, Meine and Archibald unpubl. report) but
have now been extirpated from all but the latter
three areas(Medway and Wells 1976, Madsen
1981, Yang 1991). The Australian Sarusis currently found in northern Queensland from
Normanton north to Aurukun, east to the Atherton Tableland, and west to Kunnunurra in
Western
Australia.
Although confidence in the anatomical and
geographicdistinctnessof SarusCrane subspecies has increased in recent years (Meine and
Archibald unpubl. report), questionshave arisen regarding their geneticdistinctnessand evolutionary history. Dessaueret al. (1992) documented a relatively low allozyme heterozygosity (H = 0.024) in a sample of nine Australian
• Presentaddress:Laboratoryof Medical Genetics, SarusCranes,but they did not assaythe other
University of Alabama,Birmingham,Alabama35294, subspecies.In contrast, Krajewski and Wood
USA.
(1995) identified a distinct mitochondrial hap2Addresscorrespondence
to this author. E-mail: lotype in eachof three individuals representing
[email protected]
each SarusCrane subspecies,with sequencedi655
656
wood ANDKRAJEWSKI
TABLEl.
Specimeninformationfor DNA samples.
Sample
no.'
Donor
institution
b
Donor
no. c
Grus vipio
1
ICF
ICF 10-2
G. rubicunda
8
247 a
ICF
Museum
77
222
252
ICF
St. Louis Zoo
Miami Metro Zoo
of Victoria
ICF 9-8
MV 790
G. antigone antigone
ICF
ICF
ICF
5
17
103
ICF
ICF
ICF
1'
2e
12 •
G. antigonegillae
ICF 8-28
ICF 8-39
ICF 8-31
' DNA sample catalognumbers used by Krajewski laboratoryat
Southern Illinois University.
b ICF, International Crane Foundation, Baraboo,Wisconsin; Museum
of Victoria, Melbourne, Australia; St. Louis Zoo, St. Louis, Missouri;
Miami Metro Zoo, Miami, Florida.
• Specimenidentification
numbersemployedby donorinstitutions.
a Partialcytochrome-b
sequencefrom this specimenwasreportedby
Leeton et al. (1995).
' Arbitrary numbersassignedto bloodsamplesfrom captivebirdsin
Thailand.
each subspecies.We employed sequencefrom two
Brolgas(G. rubicunda)
and one White-napedCrane(G.
vipio)as outgroups;previoussystematicstudies(Archibald 1976,Krajewski 1989, Krajewskiand Fetzner
1994) have shown that these speciesare the closest
relativesof the SarusCranes.Table 1 providesspecimen informationfor the sampleswe employed.
Technical
protocols.--DNAwasextractedfrom blood
samplesfollowing the methodoutlined by Sambrook
et al. (1989). Blood cells were diluted 1:10 with lysis
buffer (10 mM Tris, 100 mM NaC1, 100 mM EDTA,
ICF 8-45
A09023
A00225
antigone sharpei
55
56
66
[Auk, Vol. 113
0.5%SDS), digestedwith proteinaseK, and extracted
with phenol, phenol-chloroform,and chloroformisoamylalcohol.Eachsamplewasdigestedwith RNAse
A, redigestedwith proteinase,and re-extractedwith
phenol-chloroformand chloroform-isoamyl
alcohol.
Purified DNA wasprecipitatedwith cold, 95%ethanol, washedwith 70%ethanol, and rehydratedin a
storagebuffer (10 mM Tris, 0.1 mM EDTA).
Portionsof the cytochrome-b/ND6regionwere amplified via the polymerasechainreaction(PCR;Innis
and Gelfand1990)usingcombinations
of the primers
shownin Figure1. PCR reactionswere performedin
100 •L volumes using 1.5 mM MgC12,5 •M concentrationsof eachprimer, 200 •M dNTPs, one unit of
Taq polymerase,and 10-100 ng of template DNA.
Thermalcyclingbeganwith a three-minutedenaturation at 94øC,followed by 35 cyclesof denaturation
(94øC,1 min), annealing(50-55øC,1 min), and primer
extension (72øC, 1 min). A final extension for 7 min
No voucher data.
at 72øCwasincludedto minimize the numberof partial PCR products. Most PCR products were sequenced directly following asymmetricre-amplifivergencesof 0.7 to 1.5%.In this study, we ex- cationas describedby Krajewskiand Fetzner (1994).
amine mitochondrial DNA (mtDNA) sequence The 5' portion of ND6, however,includesregionsof
variation in a larger sampleof individualsto secondarystructurethat prevented extensionby the
assessthe genetic and phylogeographicdis- T7 polymerase.For these areas, we employed the
tinctness of the Sarus Crane subspecies.
CircumVentcyclesequencingsystem(New England
Biolabs).
Data analysis.--Sequences
were aligned manually
usingthe chickenmitochondrialgenome(Desjardins
Experimental
design.--Ourprevious study of se- and Morals 1990) as a reference.Basicsequencemaquence variation in cytochrome-b,tRNA TM, and nipulations (reverse complementation,conceptual
tRNAP'øgenesof SarusCranesrevealedlow levelsof translation,secondarystructureanalysis)were perhaplotypedivergence
(KrajewskiandWood1995).We formedwith Hitachi'sDNASIS software.Coding sethereforeextendedour datasetto includethe adjacent quencesof cytochromeband tRNA TMareon the heavy
and ND6 are on the light
NADH dehydrogenase
subunit6 (ND6) locus,sug- (H) strand,thoseof tRNAPrø
gestedto be appropriatefor recentdivergencesby (L) strand.
Moum and Johansen (1992). Our intention was to
To explore relative ratesof changeamong genes,
identify variablesitesin the sequences
from two in- divergencesamong haplotypeswere computedsepdividuals of each subspecies,then assayas many of arately for cytochrome-b,
spacerregionsand tRNAs,
thosesitesaspossiblewith restrictionendonucleases and ND6, using the proportion of mismatchedbases
for a largersampleof individuals.Preliminaryanal- (the p-valueof Nei 1987)as a metric. We alsotallied
ysis revealed,however, that divergencesremained the numbers of transitions and transversions for each
low even acrossthe four-gene alignment, and that region using the MEGA 1.0 package(Kumar et al.
evenan extensiverestriction-sitesurveywould detect 1993). To estimatemultilocusdivergences,we desless than one-half of the variable sites and fail to
ignated seven rate categoriesof sites:first, second,
diagnosehaplotypes.
We thereforeobtainedthe com- and third codon-positionsin each of cytochrome-b
plete,four-locussequencefroma third individualof and ND6 sequences,and tRNA sites(including spacMETHODS
Sarus Crane rntDNA Variation
July 1996]
L
1
H
ND5
3 4
2
10
5
11
6
12
Cytochromeb
7
657
8
13
14
Thr Pro
9
15
16
ND6
17
Glu
Fig. 1. Map eœgenesand primer locations.Scaleis approximate.L and H refer to light and heavystrands,
respectively,of mtDNA molecule.Regionssequencedin this study are shaded.Primersand sourcesare: (1)
L14851(Kornegayet al. 1993);(2) L14841(Kocheret al. 1989);(3) L15087(Edwardset al. 1991);(4) L15136
(Krajewskiet al. 1992);(5) L15418 (Krajewskiand Fetzner 1994);(6) L15615 (Krajewskiand Fetzner 1994);(7)
L16022(Krajewskiand Wood 1995);(8) L16152(Krajewskiand Wood 1995);(9) L16303(5'-AACCCCACATGAATAAAACA-3',this study);(10) H14954 (Thomaset al. 1989);(11) H15149(Kocheret al. 1989);(12)H15498
(Krajewskiet al. 1992);(13) H15767 (Edwardset al. 1991);(14) H15915 (Edwardset al. 1991);(15) H16208 (5'AGCTTGTACGAGGGTTGT-3',
this study);(16) H16621 (5'-ATCCTTCACCGTACTATGGG-3',
this study);(17)
H16744 (Krajewskiand Wood 1995).
ers).Relativeratesfor thesecategorieswere estimated
as their relativeproportionsof variablebases,standardized to the lowest value (cytochrome-bsecond
positions,seebelow).Pairwisedistances(dMLc)
were
computedusingFelsenstein's
(1992)DNAML model
in the DNADIST program of PHYLIP 3.5, with the
expectedtransition/transversion
ratio set to 6 (the
observedmeanvaluefor cytochrome-b
and ND6 comparisons)and empirical basefrequenciesemployed.
This strategyfor distanceestimationwasprompted
by the resultsof Krajewskiand King (1996),who
showedthat transitionbias,codon-position
rates,and
nonuniformbasecompositionwere the majordeparturesfrom randomsubstitutionin cranecytochromeb sequences.
Gene trees were estimatedusing additive distance,
pling using the DNAPARS programof PHYLIP 3.5.
A maximum-likelihoodestimateof haplotype relationshipswasobtainedwith the DNAML programof
PHYLIP 3.5, using the transitionand base-composition parametersdescribedabove(site-specificrate categoriescannot be employed in this version of the
program).Resolutionon the DNAML tree is approximated by parametricconfidenceintervals on internodal branchlengths.Gene treeswere estimatedfirst
separatelyfrom cytochrome-b,tRNA, and ND6 sequencesto check for congruenceof resolved nodes,
then from a complete alignment of the multilocus
data set.
All the phylogeny-estimationmethodsdescribed
above assumea strictly bifurcating tree, but such a
tree may not apply to recentlydivergedhaplotypes.
parsimony,and maximum-likelihoodmethods.Dis- In particular,the persistenceof an ancestralsequence
tanceanalysesemployedthe weightedleast-squares along with multiple descendantsequencesderived
(Fitch and Margoliash 1967) and neighbor-joining from it will result in an unresolvable (i.e. "hard")
(Saitou and Nei 1987) methods on dmc values. Res- polytomy.To assess
the influenceof this phenomeolution in distancetrees was assayedby bootstrap non, we manually constructedan unrooted, miniresamplingof sites.Because
ratecategories
cannotbe mum-lengthnetwork(Lansmanet al. 1983)from parresampledwith currently availablesoftware,boot- simony-informativesitesfor SarusCrane haplotypes.
strappeddistanceanalysesused DNAML distances On sucha network, haplotypesmay occupyinternal
computedwithoutratecategories
(dML).
Krajewskiand nodes,and polytomousbranchesmay occur.
We estimated the net number of nucleotide
differKing (1996)showedthat inclusionof rate categories
did not alter the precisionof distancetreesfor crane ences(dA)between subspeciesas
cytochrome-bsequences.Distance trees were obaA= axr - (ax + at)/2
(1)
tainedwith the FITCH and NEIGHBOR programsof
PHYLIP 3.5. Parsimonyanalysisemployedequally where dx, dr, and dxr are the average numbers of
weightedsitesandsubstitutions
with the branch-and- nucleotidesubstitutions
for a randomlychosenpair
boundsearchalgorithmDNAPENNY in PHYLIP 3.5. of haplotypesfrom subspecies
X, Y, and X and Y,
Given the small divergencesamong haplotypes,we respectively(Nei 1987:276-279).Variancesof each
did not employdifferentialweightingof positionsor d-value were computedfrom Nei's (1987) equations
d^valueswithinonestandard
deviation
transversions(Krajewskiand King 1996).Resolution 10.23210.26;
in parsimonytreeswas assayedby bootstrapresam- of zero were considerednonsignificant.
658
woor• ANDKRAJEWSKI
vipio-1
rubicunda-8
rubicunda-247
[Auk, Vol. 113
spacerpositions;and 4.6, 2.3, and 18.4 for ND6
codon positions.
Haplotypevariation.--Eachof the 12 individuals examined in this survey possesseda distinct multilocushaplotype,althoughseveralhad
identical single-locussequences.We designate
haplotypesby specificor subspecificepithet and
individual number (e.g. gillae-5 is the haplotype from G. a. gillaeindividual 5; seeTable 1).
Two pairs of haplotypes(sharpei-56and sharpei-66, gillae-17 and gillae-103) are identical at
all cytochrome-bsites, several have identical
tRNA sequences,and one pair (antigone-222
and antigone-252) is identical for all ND6 sites.
Single-locus
patterns.--Cytochrome-bdistances (dMLc)among SarusCrane haplotypesare 0
to 0.011, and those among speciesare 0.034 to
0.050; there are 36 informative sites for parsimony analysis.Only two nodes,thoseshowing
monophyly of Brolga and SarusCrane haplotypes,arerecoveredwith high resolution(> 90%
bootstrap)on cytochrome-btrees (not shown).
For tRNA and spacer regions, divergences
among SarusCrane haplotypesare 0 to 0.032,
and those between speciesare 0.018 to 0.052;
there are six parsimony-informativesites.Resolution is extremelylimited on tRNA trees(not
shown), but again monophyleticBrolgaand Sarus Crane groups are recovered. ND6 divergencesare 0 to 0.01 among SarusCrane haplotypes,and 0.049 to 0.064 among species;there
are 25 parsimony-informativesites.ND6 trees
(not shown) again resolve Brolga and Sarus
Crane groups, but reveal little structure among
SarusCrane haplotypes.There are no discordant patternsamongsingle-locustreesfor wellresolved groups. For weakly resolved groups
(50-90% bootstrap),only sharpei-55 and sharpei-66 show different affinities with different
antigone-252
sharpei-66
sharpei-56
antigone-222
ginae-10s
gi!lae-5
0
I
0.01
I
I
Fig.2. Treerecoveredby FITCH and NEIGHBOR
algorithmsfor dMLcdistancesfrom combinedcytochrome-b,spacer,tRNA, and ND6 sequences.
Branch
lengthscaleis substitution
per sitefromFITCHanalysis.Asterisks
belowbranchesindicatebootstrapresolution of >50% (*) and >90% (**), based on 200
resamplings.
RESULTS
Sequences.--Thecomplete sequence alignment is 1,831nucleotides,with specificregions
as follows (5' to 3' order on the L strand): cytochrome b (1,143 bases), spacer (4 bases),
tRNATM(70 bases),spacer(15-16 bases),tRNAPtø
(70 bases),spacer(6 bases),and ND6 (522bases).
The spacerregion betweentRNA loci was not
reportedin the chicken(Gallusgallus)sequence
genes.
Multilocustrees.--All phylogenetic analyses
of the 1,831 base-pairalignment show the expected monophyly of SarusCrane and Brolga
haplotypes.The multilocus distancetree (Fig.
2; based on dMLcvalues in Table 2) reveals some-
of Desjardinsand Morals(1990),but is found what more structure among Sarus Crane hapin all cranesand some other gruiforms (Kra-
lotypesthan that from single loci, but suggests
jewskiunpubl. data).All sequences
have been similar groupings. Gillae-5 appearsas an early
depositedin GenBankunderaccession
numbers branch, and there are consistentpairings of gilUl1060
to Ul1065,
U13622, and U43614 to
lae-17 with gillae-103, sharpei-55 with antigone-77, and sharpei-56with sharpei-66. Parthe authors. GenBank files contain corrections
simonyanalysisof all 67 informativesites(Tato previouslypublishedsequences.
Relativerates ble 3) yields three treesof 84 steps(consistency
for site categorieswere 2.6, 1.0, and 14.4 for index = 0.80), the strict consensusof which is
U43625;a completealignmentis availablefrom
cytochrome-b
codonpositions;
6.0for tRNA and
concordant
with
the distance trees at all nodes
July1996]
Sarus
Crane
mtDNAVariation
659
TABLE2. Sequencedistancesamong haplotypes.Values above diagonal are mismatchesper site (p-distances);
values below diagonal are substitutionsper site (dMLc).
1
1 vipio-1
--
2
3
5
6
7
8
9
10
11
12
0.037 0.038 0.038 0.040 0.040 0.040 0.040 0.040 0.038 0.042 0.038
2 rubicunda-8
3 rubicunda-247
0.043
0.044
-0.010
0.009
--
4
5
6
7
gillae-5
gillae-17
gillae-103
sharpei-55
0.045
0.048
0.048
0.048
0.040
0.042
0.042
0.044
sharpei-56
sharpei-66
antigone-77
antigone-222
antigone-252
0.048
0.048
0.045
0.050
0.045
0.045
0.043
0.042
0.046
0.041
8
9
10
11
12
4
0.034
0.035
0.039
0.038
0.035
0.034
0.041 -0.004 0.004 0.007 0.006 0.006 0.006
0.041 0.005 -0.002 0.007 0.004 0.004 0.006
0.040 0.004 0.002
-0.007 0.003 0.003 0.005
0.044 0.008 0.008 0.007
-0.006 0.006 0.006
0.007
0.005
0.004
0.009
0.003
0.002
0.002
0.005
0.040
0.042
0.044
0.044
0.039
0.006
0.006
0.007
-0.004
0.003
0.003
0.003
0.004
--
0.007
0.007
0.006
0.008
0.004
0.036
0.035
0.036
0.034
0.004
0.004
0.006
0.005
0.002
showing >50% boostrap values (Fig. 3). The
DNAML tree (Fig. 4) hasterminal clustersvery
similar to thoseon distanceand parsimonytrees,
but showsa basalpolytomy among Sarushap1otypes.
Parsimonynetwork.--Only 7 of the 31 variable
sites among Sarus Crane haplotypes are informative for parsimonyanalysis.These sitescan
be placedon a haplotype network with a minimum of 11 steps(Fig. 5). This network reveals
a potential hard polytomy corresponding to
nodesnot resolvableby phylogeneticanalysis.
Moreover, antigone-252is placedon an internal
node directly ancestralto at leastone other lineage.If this network reflectsthe actualmutation
history of SarusCrane haplotypes,it suggests
an almostcompleteabsenceof phylogeographic
structure.None of the possiblerootingswould
result in all haplotypes from any subspecies
forming a single lineage. However, a network
on which all Australian or Burmesehaplotypes
are proximal to one another requires only one
additional step (12 total), and a network with
0.004
0.004
0.005
0.005
0.002
0.038
0.038
0.006
0.006
0.006
0.010
0.005
0.038
0.034
-0.002
0.006
0.006
0.003
0.037
0.036
0.002
-0.005
0.006
0.003
0.036
0.037
0.006
0.005
-0.008
0.004
all three subspecieshaplotypes in proximal
groupsrequiresonly three additional steps(14
total). These topologies are not significantly
longer than the minimum-length network according to Templeton's(1983) test.
Net divergence.--Because each individual
sampledpossessed
a unique DNA sequence,we
used one third as the within-population frequencyof all haplotypes(Nei 1987:276).All estimated values of net divergences (d^) among
subspecies(Table 4) are lessthan one standard
deviation from zero. Thus, assumingthat the
nine haplotypesare representativeof eachsubspeciesand that they occur in roughly equal
frequencies, there is no evidence for genetic
differentiation of subspeciesat these loci.
DISCUSSION
Divergencerates of cytochrome
b and ND6.-Few studies since that of Moum and Johansen
(1992) have examined divergence of the ND6
sequencein birds, in contrastto the increasing
T^BLE3. Parsimony-informativesitesfor haplotypesexamined.Dots indicate a match to vipio-1 sequence.
Asterisksindicate sites occurring in spacerregions.
Haplotype
vipio-1
rubicunda-8
rubicunda-247
gillae-5
gillae-17
gillae-103
sharpei-55
sharpei-56
sharpei-66
antigone-77
antigone-222
antigone-252
Cytochrome b
ACCATTCAGATCCTTGCATTTTACACTCTTGCTTTT
......
TG...TT..ATG
.... GT..C.C.AA..C.
...TC.TG...TT..ATG
.... G...C.C.AAC.C.
.TT.C...AGC..CC...CCCC.TGT.T.CAACC.C
CTTTCC..AGC..CC...CCCC.TGT.T.CAACC.C
CTTTCC..AGC..CC...CCCC.TGT.T.CAACC.C
.TT.CC..AGC..CC...CCCC..GT.T.C.ACC.C
CTTTCC..AGC..CC...CCCC..GT.T.C.ACC.C
CTTTCC..AGC..CC...CCCC..GT.T.C.ACC.C
.TT.CC..AGC..CC...CCCC.TGT.T.C..CC.C
CTTTCC..AGC..CC...CCCC.TGT.T.C.ACC.C
.TTTCC..AGC..CC...CCCC.TGT.T.C.ACC.C
tRNA
ND6
CTGCAG
.G..G.
.G..G.
T.AT..
T.AT..
T.AT..
T.AT..
T.AT.T
T.AT.T
T.AT..
T.AT..
T.AT..
CCCAAATCCTGGCACACACCCATAC
T.T...CTT.AATG.G..T...CGG
T..G..CTT.AATG.G..T...CGG
.T..C ....
C.A..T.TG.TTG..A
.T..C .... C.A..T.TG.TTGC.A
.T..C .... C.A..T.TG.TTGC.A
.TT.CC...C.A..T.TG.TTGC.A
.T.GCC...C.A..T.TG.TTGC.A
.TT.C .... C.A..T.TG.TTGC.A
.TT.CT...C
....
T.TG.TTGC.A
.T..C .... C.A..T.TG.TTGC.A
.T..C ....
C.A..T.TG.TTGC.A
660
woor•ANDKRAJEWSKI
[Auk,Vol. 113
vipio-1
vipio-1
rubicunda-8
rubicunda-8
**• rubicunda-247
rubicunda-247
antigone-252
antigone-252
antigone-222
sharpei-66
sharpei-66
sharpei-56
• sharpei-
gi!lae-103
illae-17
iltae-103
antigone-222
e-l?
antigone-77
**
• antigone-77 •-- sharpei
sharpei-55
--
gi!!ae-5
Fig. 3. Strict consensusof three equally parsimonious trees for combined cytochrome-b,spacer,
tRNA, and ND6 sequences.Eachrequires84 stepsfor
67 characters.Labeling conventionsas for Figure 2.
popularity of cytochromeb among molecular
systematists.Mourn and Johansen(1992) suggestedthat ND6 incorporatessubstitutionsat a
sufficientlyrapid rate to be useful for intraspe-
0
I
gi!!ae-5
0.01
I
I
Fig. 4. Tree recoveredby DNAML for combined
cytochrome-b,spacer, tRNA, and ND6 sequences.
Branchlength scaleis substitutionsper site.
tion in cytochromeb.Severalauthors(e.g.Irwin
1991,Krajewskiand King 1996)haveidentified
but also reveal an interesting pattern relative constraintson cytochromeb evolution, but no
to cytochromeb. Within species,divergence comparableanalysisis availablefor ND6. Our
levels are comparablefor both genes(approx- resultssuggestthat ND6 and cytochromeb are
imately 0-1%). Among species,however, ND6 of similar utility for assayingintraspecificvariis distinctlymore divergent(4.9-6.4%)than cy- ation.
tochrome b (3.4-5.0%). This pattern might reSarus Crane phylogeography.--Theapparent
flectdifferentevolutionarydynamicsin the two absenceof phylogeographicstructurein Sarus
genes,with extremely labile sitesmutating at Crane haplotype trees implies that there has
roughly equal rates early in divergence, fol- been no long-term geographicisolationof sublowed by a more constrainedrate of substitu- species.Several caveatsare attachedto this concific studies. Our results are consistent with this,
TABLE4. Within-population (dx,diagonal) and net between-population(d,, below diagonal)divergences
(+SD) among SarusCrane subspecies
basedon mtDNA haplotypes.
G. a. antigone
G. a. sharpei
G. a. gillae
G. a. antigone
G. a. sharpei
G. a. gillae
0.00236 + 0.00155
0.00159 + 0.00510
0.00098 + 0.00398
0.00220 + 0.00200
0.00330 + 0.00377
0.00162 + 0.00106
July1996]
SarusCranemtDNAVariation
G17
S56
G103
2
1
A77
1
A252
/3
S66
661
1
4
16
G5
A222
Fig. 5. Minimum-length network for Sarushaplotypes.Haplotypes are indicated by single-letter codes
correspondingto subspeciesof origin (A = G. a. antigone;G = G. a. gillae;S = G. a. sharpei).Numbers along
branchesrepresentnucleotidesubstitutions.Network requires11 stepsfor 7 informative sites;autapomorphic
changesare alsoshown to distinguishhaplotypes.
clusion.Foremostis the problem of resolution.
Given the smalldivergencesand relatively high
levels of homoplasyamong SarusCrane hapiotypes,the gene tree cannotbe estimatedwith
high precision.Nevertheless,subspecies
monophyly is not the best estimateof relationships
for any optimality criterion employed(additive
distance,parsimony, and maximum likelihood)
or for any single-locuspartition of the data.
Another issueis sample size. It is unlikely that
we have identified all SarusCrane haplotypes
from a sampleof nine birds. Unsampled haplotypes might provide more structure to the
mtDNA genetree, perhapseven revealing clusters of closelyrelated geneswithin one or more
subspecies.
Nonetheless,the relationshipsof the
nine haplotypesassayedhere, to the extentthat
they are accurate,preclude a pattern of reciprocalmonophylyor paraphyly(Avise 1994)for
subspeciesmtDNAs. Unless new haplotypes
substantiallyalter the topology of Figure 5, at
leastone haplotypewithin eachsubspecies
will
remain mostcloselyrelatedto one or morehapiotypesfrom a different subspecies.
A third caveat is that mtDNA is a single linkage group
that revealsonly matrilineal relationships.It is
possiblethat other loci (e.g. from the nuclear
genome) would reveal a stronger geographic
pattern. Although the genetic basis for morphologicaldifferencesamong SarusCrane subspeciesis unknown, variation in genes influencing thesetraits may have a geographiccomponent. Thus, our conclusion is not that the
subspecies
are "invalid," but only that they are
not distinct
on the basis of mtDNA
variation.
Lack of long-term isolationis consistentwith
the historical
distributions
mese Sarus Cranes. Until
of Indian
and Bur-
the mid-1900s,
these
subspecieshad parapatric distributions bounded by the Godavari River in Bangladesh(Johnsgard 1983). Although nothing is known about
the extent of gene flow acrossthis boundary,
allopatry is a very recentphenomenon.The duration
of Sarus Crane
isolation
much less clear. There
of Australian
Sarus
in Australia
are no described
Cranes
and
is
fossils
no recorded
sightingsprior to 1966.Archibald (1981) speculated
that Sarus Cranes arrived
in Australia
as
recently asthe 1960s,a view seeminglyat odds
with the evolution of diagnosticmorphological
features.
An estimate
of the time
frame
of Sarus Crane
haplotype differentiation may be derived from
estimates
of coalescent
times
and times
to re-
ciprocal monophyly. There are currently no
more than about 23,000 Sarus Cranes in the wild
(Meine and Archibald unpubl. report). Applying demographicdata from Whooping Cranes
(Mirande et al. 1991) to Sarus Cranes, approximately 30% of all birds are breeding females
(Nf), so Nf = 6,900.The mean coalescenttime
for neutral mtDNA haplotypesis 2N• generations (Birky 1991), that is 2 x 6,900, or 13,800
generationsin SarusCranes. The average generation time in gruine cranesis 12.5 years (Krajewski and Wood 1995),giving a coalescenttime
for SarusCrane haplotypesof 13,800 x 12.5,or
172,500 years. The largest uncertainty in this
calculation involves using the current Sarus
Crane censusas an estimate of long-term population size. An alternative approachto dating
the coalescentis to root the network in Figure
662
WOODANDKRAJEWSKI
5 at its midpoint (i.e. either the node occupied
by antigone-252or the node at which gillae-5
joins the network) and compute the mean genetic distancebetween all pairs of haplotypes
at tips on oppositesidesof the root. This value
is 0.007 + 0.002 (dMLc,n = 15). Krajewskiand
King (1996) suggestedthat the maximum rate
of cytochrome-bdivergence in cranes is about
1.7% per million years (my). BecauseND6 appearsto evolve slightly fasterthan cytochrome
b, this rate may be applied to the SarusCrane
divergences.Thus, the elapsed time since the
commonancestorof all SarusCranehaplotypes
would be 0.7%divided by 1.7%/my, or 411,765
years.This suggeststhat the population size in
the coalescentcalculationis too smallby a factor
of 2.4.
A similar approachmay be usedestimatethe
duration
of Sarus Crane
isolation
in Australia.
Avise et al. (1984)showedthat populationshave
polyphyletic mtDNA haplotypes when they
have been isolatedfor fewer than Nf generations (assumingneutral haplotypesand a Poissonfemalefecunditymodelwith mean 1.0).The
current
census size of Australian
Sarus Cranes
is no more than 10,000 birds and is possibly
increasing (Meine and Archibald unpubl. report). Avise et al. (1984) demonstratedthat in
expandingpopulationsthe final value of N• is
a strongerdeterminant of lineage survivorship
than
the number
of founders.
It follows
Sarus Cranes have been isolated in Australia
fewer than
that
for
N• = (0.3)(10,000)= 3,000generations,
[Auk,Vol. 113
various phasesof this research,as well as the donor
institutions in Table 1. Our study was supported by
NSF grants BSR~9102482and DEB-9419907to C.K.
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