The Auk 111(4):970-978, 1994
MITOCHONDRIAL
ANALYSIS
OF GENE
FLOW
BETWEEN
NEW ZEALAND MALLARDS (ANAS PLATYRHYNCHOS) AND
GREY DUCKS (A. SUPERCILIOSA)
JUDITH M. RHYMER,1'3MURRAY J. WILLIAMS,2 AND MICHAEL J. BRAUN1
XLaboratory
of MolecularSystematics,
Smithsonian
Institution,
MRC 534, Washington,
D.C. 20560, USA; and
2Directorate
of Science
andResearch,
Department
of Conservation,
P.O. Box 10-420, Wellington,New Zealand
ABSTP,•CT.--Oneof the more well-known examplesof hybridization in birds is the frequently documentedoccurrencebetween sexually dimorphic Mallards (Anasplatyrhynchos)
and severalcloselyrelated nondimorphic speciesin the mallard complex.In New Zealand,
the Grey Duck (Anassuperciliosa
superciliosa)
is the indigenous, nondimorphic Mallardlike
species,and extensivehybridization with introduced Mallards has been implicated in the
populationdecline of Grey Ducks.Individuals from throughoutthe countrywere classified
phenotypically as parentals or hybrids based on variation in plumage, bill color, and leg
color.We confirmedspecies-specific
mitochondrialDNA haplotypesby comparingrestrictionenzymefragmentpatternsin Grey Ducksand New ZealandMallardsto thoseof PacificBlack
Ducks (A. superciliosa
rogersi)from Australiaand Mallards from North America, respectively.
Our dataindicatethat hybridizationhasled not only to introgressionof Grey Duck mtDNA
into Mallard populations(the predicteddirection of gene flow), but also to significantintrogressionof Mallard mtDNA into Grey Duck populations.Thus, the contentionthat hybridization between Mallards and nondimorphic speciesinvolves primarily Mallard males
with femalesof the other speciesis not upheld for this examplefrom New Zealand.The
speciationprocessappearsto be undergoingreversal.Received
I April 1993,accepted
2 July
1993.
THE INCIDENCE
OF interspecificand intergeneric hybridization in the order Anseriformes
is higher than in any other orderof birds,reaching 30 to 40% by some estimates(Grant and
Grant 1992). In addition, a substantial propor-
Mallard/Mottled Duck(A. fulvigula)hybridsalso
are being reportedin someareasof Florida (Mazourek and Gray 1994).In fact, the AOU (1983)
declared the Mexican Duck to be conspecific
with the Mallard becauseof extensivehybrid-
tion of interspecifichybrids(20%)in this order ization between them.
have been reported to be fertile (Scherer and
In New Zealand, the Grey Duck (A. superciHilsberg 1982),so there is potential for exten- liosa superciliosa)is the indigenous, nondisive gene flow and introgressionbetweensome morphic, Mallardlike species.Grey Ducks are
species.
thoughtto have colonizedfrom Australia,like
Amongthe morewell-knownexamplesis the much of New Zealand's avifauna (Baker 1991),
frequentlydocumentedincidenceof hybridiza- and the PacificBlackDuck (A. superciliosa
rogersi)
tion betweensexuallydimorphicMallards(Anas in Australiais virtually identicalphenotypicalplatyrhynchos)
and severalcloselyrelated, non- ly to the New ZealandGreyDuck(Frith 1982).
dimorphic species. For instance, in North Mallards were introduced by the Otago AccliAmerica,hybridizationwith Mallardshasbeen matization Societyinto the southern region of
implicated as one factor in the population de- South Island, New Zealand in the mid-1800s
cline of American Black Ducks (A. rubripes; from Europeangame-farmstockand into North
Johnsgard1967,Heusmann1974,Ankney et al. Island by the Auckland AcclimatizationSociety
1987), Hawaiian Ducks (A. wyvilliana;Griffin et in the 1930s from North America (Williams
al. 1989), and Mexican Ducks (A. platyrhynchos 1981).
diazi; Hubbard 1977). Increasing numbers of
Over the last few decades,Mallard populations have increaseddramatically,while Grey
3Presentaddress:Departmentof BiologicalSci- Duck populationshave declined. Extensivehyences,ClemsonUniversity,Clemson,SouthCarolina bridization with Mallards, facilitatedby the loss
29634, USA.
of natural habitatsto agriculture,hasbeen im970
October
1994]
Mallard
andGreyDuckGene
Flow
971
plicatedin the populationdeclineof GreyDucks
in a situationanalogousto thosein North America (Williams 1981,Gillespie1985).By the early
1980s,levels of hybridization estimated from
plumagevariation had increasedto over 50%in
populations on South Island near Dunedin,
while the proportionof pure Grey Duckshad
declined to lessthan 5% (Gillespie 1985).This
hasled to concernthat the GreyDuckmayeventually disappearasa separatespeciesfrom New
Zealand (Weller 1980,Gillespie 1985),as it has
this study,we soughtspecies-specific
markers
for Mallardsand Grey Ducksusing restrictionenzymeanalysisof mtDNA, a techniquethat is
generallymoresensitivethan allozymeelectrophoresis.Our study focusedon the analysisof
pure Grey Ducksand Mallards togetherwith
Lemke 1994).
tanabe et al. 1985).
several
individuals
that had been classified
as
hybrids on the basisof their morphological
characteristics.
We also used the results to infer
the direc-
tionality of hybridization.If hybrid matingsocfrom the Marianas Islands. The so-called Marcur primarily between colorful Mallard males
ianaMallard (A. oustaleti
Salvadori)is generally and Grey Duck females,then the majority of
consideredto be a hybrid betweenstrayMal- hybrid individualsshould have a Grey Duck
lard DucksandA. superciliosa
(DelacourandMayr mitochondrial haplotype, since avian mtDNA
1945, Yamashina 1948; but see Reichel and
is maternally inherited (Giles et al. 1980, Wa-
Although assortativemating appearsto prevail
between
Mallards
and
American
Black
METHODS
Ducks in North America (E. Morton unpubl.
data),and betweenMallards and A. superciliosa Samplecollection.--We
sampledMallard, Grey Duck,
in Australia (Braithwaite and Miller 1975) and
andhybridindividualsfromNorth andSouthIslands
of New Zealand. We also analyzed Mallards from
is some experimental evidence that more col- North America(aspartof a largerstudyof the mallard
orful Mallard males outcompetedull-colored complexof waterfowl) and PacificBlackDucksfrom
malesfor mates(Brodskyet al. 1988). Ankney Australiato ensurethat we couldunequivocallyidenmtDNA haplotypesof the paet al. (1987)alsosuggested
that the majorityof tify the species-specific
New Zealand (Hitchmough et al. 1990), there
hybrid matingsin North Americamay result rental species.
Birdsshot by hunterswere collectedin May 1991
from forcedcopulationsbetweenMallard males from localitiesthroughoutthe country,as part of a
and BlackDuck femalesduring renesting.
largerstudyby M.J.W.to assess
the extentof hybridSeveral studies have tried to estimate
the ex-
tent of hybridization and introgressionbetween Mallards
and one or another
of the non-
dimorphicspeciesby qualitativeassessments
of
phenotypic variation (Braithwaite and Miller
1975, Gillespie 1985, Heusmann 1974; Kirby,
U.S. Fishand Wildlife Serviceunpubl. report),
but it is difficult to distinguishhybrids morphologicallyafter more than one generationof
backcrossing
to a parentalspecies(Williamsand
Roderick1973, Rhymer unpubl. data). In addition to the use of phenotypiccharactersto
estimatelevels of gene flow, somebiochemical
techniqueshave been applied to the Mallard
group. However, no species-specificmarkers
have been found to distinguishMallards from
American BlackDucksusingallozyme electrophoresis(Ankney et al. 1986) or restriction-enzyme analysisof mitochondrialDNA (mtDNA;
Avise et al. 1990).
ization in New Zealand. Samplesfrom six areas on
North Island(LakeWhangape,Cambridge,Ohakune,
Wairoa, Wanganui, Lake Wairarapa)and one area on
South Island (Lake Ellesmere) were selectedfor anal-
ysisin thisinitial study(Fig.1).Samples
wereselected
on the basisof phenotypiccharactersto include 12
Grey Ducks,9 Mallards,8 Grey Duck-like hybrids,
and 14 Mallardlike hybrids.It hasbeen observedthat
hybridstend to be Grey Duck-like or Mallardlike in
appearance(Yamashina1948,Weller 1980).
Heart
tissue from
these birds was stored in 70%
ethanol and shipped at room temperature. We also
analyzedheart and liver tissuefrom additionalbirds
that had been collected
between
1986 and 1991 and
storedat -80øC. Thesesamplesincluded sevenGrey
Ducks(PohanginaRiver;Apiti, Orua River), six Mallards(Apiti), and two Grey Duck-like hybrids (Rongotea;Apiti), all from North Island.We alsoanalyzed
tissueand blood samplesof North AmericanMallards
and blood samplesof 10 Pacific Black Ducks. The
Pacific Black Ducks were chosen from an area near
Canberra in eastern Australia (where no Mallards are
Allozyme studiesalso have been unsuccess- found) to comparewith our pure Grey Duck samples.
ful in separatingMallards from A. superciliosa Phenotypic
scoring.--Variationin some phenotypic
both in Australia and New Zealand (Braithwaite
charactersof each bird was quantified, using criteria
and Miller 1975, Hitchmough et al. 1990). In
similar to those outlined by Gillespie (1985) and
972
R•M•,
WILLIAMS,.•_NDBP,•UN
NORTH
ISLAND
[Auk, Vol. 111
TASLE
1. Phenotypiccriteriausedto scorehead,wing
and leg characteristics
of Grey Ducksand Mallards.
Face
Cambridge
Dhakune
Wanganui
Rongotea --
Apiti
Pohangina R.
e Wairarapa
SOUTH
ISLAND
(0) Two black stripes,the upper superciliarystripe
of uniform width and extending well beyond eye;
lower stripe tapering from gape to below eye and
giving way to mottled cheek;cleancreamstripe between eye and crown, throat and face cream.
(1) Two blackstripes,upperstripeasdescribedabove,
lower stripe merging with mottled face midway between bill and eye, clean creamstripe between eye
and crown; face and cheek mottled cream but with
obviouscream patch at baseof bill between the two
black stripes.
(2) Superciliarystripe obviousand extending from
bill to well beyondeye,lower stripeabsentbut small
patch of black feathersat bill baseat gape;mottled
creamstripebetweeneye and crown, faceand cheek
entirely mottled black on cream background,throat
cream.
Lake Eriesmere
0
300
km
(3) Entire face mottled black on fawn background,
superciliarystripeindistinctand narrow,smallpatch
at gape more heavily mottled to appeardarker than
rest of face, small fawn patch at bill base between
gapeand bill top,throatcleanto lightly mottledfawn.
(4) Dark mottled face and throat, black on fawn
Fig. 1. Collection
sites on North
and South Is-
lands, New Zealand.
backgroundin female, blackish-greenon dark fawn
in male; no obviouseye stripe, no fawn patch at bill
base.
(5) Predominantlydark green head,face,and throat
(males only).
Legs
Braithwaiteand Miller (1975). Theseincluded plum-
(l) Dark olive greenish-brown;(2) khaki; (3) yelagevariationon the headand wing, and differences low-orange to dull orange;(4) bright orange;(5) red
in legandbill color(detailsin Table1).Eachcharacter orange.
wasassigneda scoreranging from 0-1 up to 4-6, with
Bill
Grey Duck charactersscoredat the low end of the
(0)
Uniformly
black;
(1)
blackwith very dark green
scale(lowest overall cumulative scorepossiblewas 1)
particularly at baseand along edge of upper mandiand Mallard charactersat the upper end (highest pos- ble;(2) predominantlyblack/ darkgreen,someyellow
sible cumulative score was 25 for a Mallard male).
or brown at tip; (3) black and brown/yellow; (4) yelCumulativescoreswere designedsuchthat Mallard low-green; (5) green or a bluish shade (much more
malestended to havethe highestscores.Intermediate commonin New Zealandthan yellow or orangecolor
found in North American Mallards).
scoresare indicative of hybrid characteristics.
Laboratorymethods.--Totalgenomic DNA was isoSpeculum and upper alar bar
latedfrom eachsample.ProteinaseK (200/•g//•1)and
(0) Green, no discernible bar; (l) green, obvious
SDS (0.5%) were added to 50/•1 of blood in TNE ex- thin and narrow whitish/brown line; (2) green, bar
traction buffer (10 mM Tris, 100 mM NaC1, 100 mM
distinct but narrow and mottled fawn color; (3) purEDTA pH 8.0) or 0.5 to 0.7 g of tissuehomogenized ple, bar distinctbut narrow and mottledfawn color;
in ice-coldTNE. Following an overnight incubation (4) purple, bar distinct,wide, mottled fawn; (5) purat 55øC, the NaC1 concentration was raised to 200 mM
ple, bar narrow, pure white; (6) purple, bar wide, pure
and RNase was added to a concentrationof 100/•g/
mi. Samples were incubated at 37øCfor ! h and extracted with equal volumesof phenol:chloroform:
isoamylalcohol(25:24:1)and chloroform:isoamylalcohol (24:1) for 20 rain each at 55øC. DNA in each
samplewas precipitatedin ice-coldethanol and resuspendedin TLE (10 mM Tris, 0.1 mM EDTA pH
8.0) to a concentrationof 0.5 to 1.0/•g//•l. The concentrationand purity of eachDNA samplewere estimated spectrophotometrically.
DNA from each individual was digestedwith 16
white.
Posterior border to speculum
(0) Black only; (l) black followed by thin (! ram)
white line; (2) black followed by narrow (1-2 ram)
white line; (3) black followed by conspicuous(3-4
ram) white bar; (4) blackfollowed by wide (>4 ram)
white
bar.
October
1994]
MallardandGreyDuckGene
Flow
973
TAnLE
2. MitochondrialDNA haplotypesfor PacificBlackDucks,New ZealandGreyDucks,and Mallards.
Sequenceof restrictionenzymesis ApaI, AvaI, BamHI, BclI, BglI, BstEII,DraI, HincII, HindIII, Hinfi, PstI,
PvuII, RsaI,Sau3AI,StyI, and TaqI. Number of parentalsand hybridswith eachhaplotypelisted.
Hybrids
Haplotypes
Parentals
GrayDuck-like
Mallardlike
Pacific Black Duck
1 AGEGLDAECAECBCDE
2 ........
G ....
$ B .......
G ....
4
3
3
B-C
B-½
New Zealand Grey Duck
1 B ...........
AA-B
2 -D ....
3B .......
B-
-B .....
0 ....
i
2
3
I-CF-CDCC
- - - CFCDCC
- K- CFCDCC
2
½
8a
8
B-C
Mallard
-D-DFC- D - DFC
- D- DFC
7
6•
1
One
was
"•ure"
Mallard.
One was "pure" Grey Duck.
different restrictionendonucleases(2 •g per digest)
under conditionsrecommendedby the enzyme supplier. The enzymesincluded 4 enzymesthat recognize four-basesequences,2 that recognize five-base
sequences,and 10 that recognizesix-basesequences.
Fragmentsin digestedsampleswere separatedon 0.7
to 1.2%agarosegels and transferred to nylon membranes(MSI Magnagraph)via Southern(1975) blotting. Purified mtDNA, isolatedfrom fresh Mallard
heart tissue in a cesium chloride gradient, was labelled with 32pby random priming (Feinberg and
Vogelstein1983)and usedto probethe totalgenomic
DNA on each blot. A molecular size standard (•bX174
(unmapped) fragment observedfor all 16 enzymes.
This methodhasthe advantagethat it includesall of
our data, but suffers from the fact that some characters
(fragments)are not independentof oneanother.Second, we inferred the presenceor absenceof restriction sitesfor all enzymeswhere this waspossibleand
deleted thoseenzymeswhere it was not (Hinfi, StyI,
RsaI, Sau3AI, and TaqI).
RESULTS
We scoredeach individual for an averageof
Hae III plus lambda/HindIII) wasusedto determine 120 restriction fragments.In the circular mtfragmentsizeson autoradiograms.
The fragmentpro- DNA molecule, this correspondsto 120 restricfile for eachendonucleasewas assigneda letter using
tion sites,representingabout 600 basepairs of
the letter designationsof Kesslerand Avise (1984), recognitionsequence.To ensurethat we had
Avise et al. (1990) and Avise (pers.comm.)as a base-
identified pure Grey Duck and Mallard haplo-
line for consistency
amongstudies.Thus,eachindi- typesbefore attemptingto characterizehybrid
vidual's mtDNA haplotypeis codedas a seriesof 16
individuals, we compared New Zealand Grey
letters.Fragmentsizesfor eachprofile are available
Duck and New Zealand Mallard mtDNA hapfrom the senior author upon request.
Statisticalanalysis.--Geneticdistances were esti-
matedfor eachhaplotypepair accordingto the Nei
lotype profilesto thoseof PacificBlackDucks
and North American Mallards, respectively.
Nine mtDNA haplotypeswere observedalto-
and Li (1979) method for unmapped fragment data
(see Nei 1987:106-107) using the analysis package gether,threeeachin New ZealandGreyDucks,
RESTSITE(version 1.1;Nei and Miller 1990).The re- Pacific Black Ducks, and New Zealand Mallards
sulting distancematrix was then pheneticallyclus- (Table 2). We found no fixed differences betered by the unweighted pair-group method with
arithmeticaverages(UPGMA; Sneathand Sokal1973),
using mtDNA haplotypesas OTUs. Net divergence
between subspeciesand specieswas calculated,correctingfor within-taxonhaplotypevariation(Nei and
Miller 1990). We used PAUP 3.1.1 (Swofford 1991) to
estimatea phylogeny for the haplotypesusing different codingschemesfor the restriction-digest
data.
First, we evaluated the presenceor absenceof each
tween
Pacific
Black Ducks
and New
Zealand
Grey Ducksor betweenNew ZealandMallards
and North American Mallards. Grey Duck haplotype 3 and Pacific Black Duck haplotype 3
were identical, and New Zealand Mallard hap-
lotype 2 was identical to that of one North
AmericanMallard haplotype(Rhymerunpubl.
data, Kessler and Avise 1984).
974
RHYMER,
WILLIAMS,ANDBRAUN
[Auk, Vol. 111
TABLE
3. Nucleotidedivergence
amongmtDNA haplotypes
of PacificBlackDucksfromAustralia(AU), New
Zealand(NZ) Grey Ducks,and Mallardsestimatedfrom restriction-fragment
data(methodof Nei 1987).
Haplotype
1
2
3
4
0.0047
0.0066
0.0014
0.0027
0.0086
0.0000
0.0090
0.0027
0.016!
0.0160
0.0175
0.0179
0.0168
0.0180
0.0180
0.0!70
0.0184
! AU Black Duck 1
2 AU Black Duck 2
3 AU Black Duck 3
0.0026
0.0040
0.0014
4 NZ Grey Duck !
5 NZ Grey Duck 2
6 NZ Grey Duck 3
0.0047
0.0048
0.0040
7 Mallard
8 Mallard
9 Mallard
0.0!!9
0.0!08
0.0!20
1
2
3
The mtDNA of Grey Ducks and PacificBlack
Ducksare highly similar with an estimatednucleotidesequencedivergence(after correction
for within speciesvariation) of only 0.09%,a
reflection
of their close historical
affiliation.
We
5
0.0086
0.0108
0.0108
0.013!
6
7
0.0179
0.0168
0.0180
0.0013
0.0013
8
0.0018
lotype 2. Thirteen of 14 Mallardlike hybridshad
Mallard haplotypes(five had haplotype 1, six
had 2, two had 3), and one had Grey Duck 3,
while 5 of 10 Grey Duck-like hybridshad Grey
Duck haplotypes(onehad haplotype2 and four
had 3) (Table 2, Fig. 3). Of the five Grey Ducklike hybrids that had Mallard haplotypes,two
had Mallard haplotype 1 and three had Mal-
found nine species-specific
markersbetweenA.
platyrhynchos
and A. superciliosa,
however.Grey
Ducks and PacificBlackDucks have diverged
from Mallards by 1.40 and 1.43%,respectively. lard 2.
All A. superciliosa
haplotypes(New Zealandand
The proportionof hybridswith Mallard and
Australia combined)were very similar (genetic Grey Duck haplotypes was compared to evaldivergencerangingfrom 0.00to 0.90%),aswere uate whether hybrid matingsare primarily bethe three haplotypeswithin New ZealandMal- tween Mallard males and Grey Duck females.
lards (0.13 to 0.18%; Table 3).
Thishypothesispredictsa predominanceof Grey
Figure 2 showsthe strict consensusof five Duck haplotypesin hybrid individuals. There
equally-parsimonious
trees derived from par- was, however, a significanttendency for hysimonyanalysisof the unmappedfragmentdata. bridsto have Mallard (n = 19) rather than Grey
Key featuresof this tree includeseparationof Duck (n = 7) haplotypes(X2 = 6.54,P < 0.025).
A. platyrhynchos
and A. superciliosa
haplotypes
intotwo distinctgroups,clusteringof GreyDuck
DISCUSSION
and PacificBlackDuck haplotypesinto a single
mitochondrialDNA haplogroupthat doesnot subdividealongsubspecies Species-specific
lines, and the basal branching of Grey Duck typeswere distinguishedfor Mallardsand Grey
haplotype2 from the Grey Duck/BlackDuck Ducks in New Zealand. The genetic distance
group.All of thesefeaturesalsowere apparent (correctedfor within speciesvariation) is 1.4%,
in the UPGMA analysis,in the parsimonyanal- considerablymorethan the 0.7%divergencebeysisusingonly inferred restrictionsitechanges, tween the two mtDNA lineagesof North Amerandin a high proportionof treesresultingfrom ican Mallards (one of which containsAmerican
Black Ducks; Avise et al. 1990) and between
bootstrappingof either parsimonydata set.
Phenotypicscoresrangedfrom 1 to 4 for Grey Mallards and Mottled Ducks (Kesslerand Avise
Ducks and 18 to 24 for Mallards, with Grey 1984).It is also higher than that for the most
Duck-like hybrids scoringbetween 5 and 10, divergent pair of A. superciliosa
haplotypes
and Mallardlikehybridsscoringbetween12and (0.9%).
Hybridization and introgressionappearto be
16. The boundarybetweenGrey Duck-like and
Mallardlike hybridsis somewhatarbitrary.Hy- extensivein New Zealand, despitethe relativebrids were found at virtually all collection sites. ly large geneticdistancebetween Grey Ducks
One of the 19 individuals characterized phe- and Mallards (for the Mallard complexof spenotypicallyasa "pure"GreyDuckhadthe Mal- cies;Rhymer unpubl. data). Individuals charlard haplotype2 and one of the 15 phenotyp- acterizedby hybrid phenotypic traits are presically "pure" Mallardshad the Grey Duck hap- ent throughout the country on both islands.
October
1994]
Mallard
andGreyDuck
Gene
Flow
--
1
NZ Mallard
1
975
25
ß
ß Mallard mtDNA haplotype
O Grey DuckmtDNAhaplotype
ß
20
NZ Mallard
3
NZ Mallard
2
o
ß
F
M
ß
15
ß
10
NA Mallard
5
NZ Grey Duck 1
0
F
24 (100%)
16 (100%)
NZ Grey Duck 3
12 (100%)
M
F
M
Grey Duck Mallardlike
Grey Ducks
-like
Mallards
Hybrids
AU Black Duck 1
5 (100%)
AU Black Duck 2
AU Black Duck 3
NZ Grey Duck 2
Fig. 3. Comparison of phenotypic scores and
mtDNA haplotypesfor birdscategorizedaspure Grey
Ducks,Mallards,as Grey Duck-like hybrids,or Mallardlike hybrids.
1983,Carr et al. 1986,Tegelstrom1987,Lehman
et al. 1991], amphibians [Spolskyand Uzzell
1984, Lamb and Avise 1986],and fish [Avise and
Fig. 2. Strict consensusof five most-parsimonious Saunders 1984]).
treesrelating mtDNA haplotypesof Mallards, New
The contention that hybridization between
Zealand (NZ) Grey Ducks and Pacific Black Ducks Mallards and closelyrelated nondimorphic spefrom Australia(AU) basedon unmappedrestrictionciesinvolves primarily Mallard males with fefragment data for 16 enzymes (one North American
[NA] Mallard haplotype included for comparison). malesof the other speciesis not upheld in New
Each fully resolvedtree was 69 steps long (con- Zealand.The higher proportionof hybridshavsistencyindex excludinguninformativecharacters= ing Mallard mtDNA as opposedto Grey Duck
0.920, retention index = 0.971). Same topology ob- mtDNA indicates that crosses between Mallard
tained from analysisof reduced data set consistingof femalesand Grey Duck males must occur freinferred restrictionsitesfor 11 enzymes.In latter case, quently. Hybrid femalesapparentlymate sucthere were six most-parsimonioustrees,eachof length cessfully with either Mallard or Grey Duck
34 (consistencyindex excluding uninformative char- males.The fate of hybrid males is lesspredictacters = 0.912, retention index = 0.955). Branch-and-
bound algorithm employed for all tree searches.
Branch lengths that were invariant in all equallyparsimonioustreesgiven both asnumber of inferred
fragment changes(above)and as number of inferred
site changes(below). Numbers in parenthesesindicatepercentageof times that node appearedin analysesof 1,000bootstrappedpseudoreplicatedata sets.
able. If male plumage quality is an important
factor in their selection as mates, the femalelike
plumageof many hybrid malesmayreducetheir
chancesof successfullyattracting (and backcrossingwith) females of either species.They
could, presumably,be successfulin obtaining
forcedcopulationswith any renestingfemales.
Our study provides only a conservativeestimate of the degree of introgressivehybridization becauseit is basedon phenotypic characters and uniparentally inherited genetic
markers.Analysisof phenotypictraits of birds
from controlledbreedingcrosses
betweenMallards and Grey Ducks has shown that phenotypic charactersbecomelessreliable indicators
of hybridization, as the level of introgression
Our geneticdatafor theseindividuals indicate
that hybridizationhasled not only to introgression of Grey Duck mtDNA into Mallard populations(the predicteddirectionalityof hybridization), but alsoto significantintrogressionof
Mallard mtDNA into Grey Duck populations.
Thisaddsto the increasingnumberof examples
of mitochondrial gene exchangebetween spe- increases (Williams and Roderick 1973). Alcies in other taxa (e.g. mammals[Ferris et al. though we were able to identify two "hidden"
976
RHYming,
WILLIAMS,
ANDBP.•UN
hybrids,completecharacterizationof all hybrid
individuals will require species-specific
nuclear
DNA markers,which are biparentally inherited.
The genetic similarity of Mallards in New
Zealand
to those in North
America
is not sur-
prising given their intentional introduction
from North America in this century. Despite
the geographic isolation of Australia and New
[Auk, Vol. 111
tegrity of A. superciliosa
superciliosa
as a separate
speciesis doubtful (Williams 1981). There are
few other examplesof suchgeographicallyextensive introgressionfollowing the purposeful
introduction of one speciesinto the range of
another (see also Echelle and Connor 1989).
Nondimorphic speciesin the Mallard com-
plex often are presumedto have evolved from
the Mallard (Johnsgard1961),although it is posZealand,however,there is no phylogeneticdis- sible that they evolved from a nondimorphic
continuity of mtDNA haplotypes between A. "proto-Mallard." Regardlessof the evolutionsuperciliosa
rogersiin Australia and A. superciliosa ary history of the group, it appears that the
superciliosa
in New Zealand. Baker (1991) sug- speciationprocessis undergoingreversalin New
gestedthat post-Gondwanalanddispersalacross Zealand. Anas platyrhynchos
and A. superciliosa
the Tasman Sea via west wind drift was the
couldhavedivergedaslong agoas700,000years
principal route of colonization of avifauna from (based on clock estimation), but it is clear that
Australia
to New Zealand.
He discussed the idea
no pre- or postzygotic mechanismshave arisen
that New Zealand subspecies
of Australian spe- to prevent extensive introgression. Evidence
cies are about 20,000 years old. One might at- from captivestudiesindicatesthat hybrids surtempt to estimatethe time of divergenceusing vive and are asreproductivelysuccessfulasthe
a molecular-clockapproach.Basedon our data, parentalspecies
(Haddon1984).Our geneticdata
the sequencedivergencebetweenA. superciliosa now lend supportto the phenotypicevidence
subspecies(correcting for within subspecies that points to the eventual loss of identity of
variation) was 0.09%.Using a clock calibration the Grey Duck as a separate speciesin New
of 2% mtDNA sequenceevolution per million Zealand, and the subsequentdominance of a
years (Shields and Wilson 1987), we estimate hybrid swarm akin to the "Mariana Mallard."
that the two subspeciesdiverged about 45,000 Thus, the lossof natural Grey Duck habitat to
yearsago. Given the expectederror rate of such agriculturehasbeen compoundedby successful
estimates (Sheldon and Bledsoe 1993), this date introductionsof the highly adaptableMallard
does not conflict with Baker's suggestionof and the subsequenteffectsof interspecifichybridization.
20,000 years ago.
However,
this molecular-clock
estimate
is
probablyoverly simplistic.Limited samplesizes
and limited geographicsamplingof PacificBlack
Ducksmake our sequence-divergence
estimates
somewhat uncertain. Also, the mtDNA• haplotypes of the two subspeciesdo not resolve into
separate clades. Some haplotype pairs (both
amongand within subspecies)
areabout10 times
asdivergent as the weighted estimate(Table 3).
Using the sameclock calibration, we would be
forced to conclude that these haplotypesdiverged 330,000 to 450,000 years ago. Yet, one
of the haplotypes was identical between subspecies,yielding an estimated time of divergence of zero. This sharedhaplotype might be
due to more recentcolonizationor continuing
gene exchangebetween subspecies.
In fact,birds
may be moving in both directionsbetweenpopulations;a Grey Duck banded in New Zealand
in the 1950s was recovered in Australia (Williams 1981).
Given the extensivegene flow between Mallards and Grey Ducks in New Zealand, the in-
ACKNOWLEDGMENTS
We thank C. Krajewski and G. Shields for helpful
commentson a previousdraft of the manuscript,D.
Swofford for analytic advice, J. Avise for providing
mtDNA fragment-size profiles from previous studies
of ducks, and P. Fullagar and C. Davey for sending
PacificBlack Duck blood samplesfrom Australia. Assistanceto M.J.W. for the collectionof specimenswas
provided by the New Zealand Lottery ScienceBoard.
Support for J.M.R. was provided by a Smithsonian
Institution
Molecular
Evolution
Postdoctoral
Fellow-
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