The Auk

A Quarterly Journal Ornithology of

Vol. 116 No. 3 July 1999

TheAuk 116(3):577-588, 1999

PHYLOGENETICS

OF DARWIN'S WARBLER

FINCHES: FINCH

PARAPHYLY

IN THE

TREE-FINCHES,

AND TWO DIVERGENT

LINEAGES IN THE

JOANNAR. FREELANDAND PETERT. BOAG

Department Biology, of QueenUniversity, Kingston, OntarioK7L 3N6, Canada

ABSTR^CT.--The Galapagos Darwin'sfinches (Geospizinae) havebeenclassified three as majorgroups based morphology behavior: on and ground-finches, tree-finches, theWarand blerFinch(Certhidea olivacea). Littleis knownabout evolutionary the relationships withinand amongthesegroups,which is partly due to the lack of a phylogeny basedon molecular sequence data.Weusedmitochondrial sequence to reconstruct phylogeny Darwin's data a of finches. These datashowthatwithin thetree-fin&es, onlyonegenus conclusively is monophyletic, another conclusively and is paraphyletic. maybeappropriate upholdtheclasIt to sification the tree-finches two genera. of into The Warbler Finchcomplex paraphyletic, is as revealed two divergent by genetic lineages contained within this species. Stochastic lineage sortingwithin relativelyrecentlydivergedspecies and interspecific intergeneric and hybridizationare the two mostlikely explanations the sharing haplotypes for of amongtaxa.
Received March 1998,accepted November 3 6 1998.

ADAPTIVE RADIATION refersto the process in which onespecies evolves into numerous speciesover a relativelyshortperiodof time.The questionof how this occursis fundamentalto studies of evolution and speciationand has been at the heart of considerable research (e.g. DeSalleet al. 1987,Sanget al. 1994,Losos 1995,

sightinto a numberof aspects evolution of and speciation, includingadaptiveradiation. The subfamily Geospizinaecomprises14 nominate species, of whichinhabitthe Gal13 apagos Archipelago.Darwin's finches have been divided into three groupsbasedon morphology and behavior:ground-finches, treeTarr and Fleischer 1995, Cameron et al. 1996, finches, and the WarblerFinch (Certhidea olivaRadtkey 1996,Shaw1996). Perhaps mostfa- cea;Table 1). The ground-finches the (Geospiza) mousongoingstudy of adaptiveradiationin- comprise one genusand six species that are volves Darwin's finches(Geospizinae). More finch-likein appearance, particularly with rethan 100yearsof research havebeenconducted spectto their bills, and spendmuch of their on Darwin'sfinches, manyquestions yet, about time foragingon the ground.The tree-finches theirevolutionary historyremainunanswered. (Camarhynchus, Platyspiza, Cactospiza) and comA glaringomission thisfield of studyis the prise six species, the numberof generaosin but lackof a sequence-based phylogeny. molec- ciliates between one and three (see below). A ular datasetfor the groupwill yieldfurtherin- These six specieshave bills intermediateto thoseof ground-finches the Warbler and Finch, and although they occasionally forageon the Present address: School of Animal and Microbial Sciences, Box228, Universityof Reading,Read- groundin a mannersimilarto ground-finches, P.O. they spendmuchof their time in foliageand ing RG6 6AJ,United Kingdom. E-mail:j.r.freeland@reading.ac.uk vegetation exhibitingbehaviorsimilar to that
577

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FREELAND BOAG AND

[Auk, Vol. 116

TABLE Darwin's 1. finchspecies, takenfromLack(1947), Bowman (1961), andGrant(1986). Authors for species names givenin Lack(1947), are although some generic names tree-finches of differfromthisreference.

Scientific name
Ground-Finches

English name

Geospiza magnirostris Gould Geospiza fortis Goulda Geospiza fuliginosa Gould

LargeGround-Finch
Medium Ground-Finch Small Ground-Finch

Geospiza difficilis Sharpe a

Geospiza scandens (Gould) Geospiza conirostris Ridgwaya

Sharp-beaked Ground-Finch
Cactus Ground-Finch

LargeCactus Ground-Finch
Tree-Finches

Camarhynchus parvulus (Gould)a Camarhynchus pauper Ridgway Camarhynchus psittacula Gould Platyspiza crassirostris Gould Cactospiza pallida(Sclaterand Salvin) Cactospiza heliobates (Snodgrass and Heller)
Warbler

Small Tree-Finch Medium Tree-Finch

LargeTree-Finch Vegetarian Finch Woodpecker Finch Mangrove Finch

Finch

Certhidea o. olivaceaGould

Warbler Finch(Santiago, Santa Cruz,Isabela,

Ferdinand,Rabida,Seymour,Pinzon islands) WarblerFinch(SantaFeIsland) WarblerFinch(Pintaand Marchena islands) WarblerFinch (FloreanaIsland)
Warbler Finch (San Cristobal Island) WarblerFinch (Wolf and Darwin islands)

C. o. bifasciata C. o.fusca a
C. o. luteola

C. o. ridgwayi
C. o. becki C. o. mentalis a C. o. cinerascens Taxaused in this study.

WarblerFinch(Genovesa Island) WarblerFinch(Espafiola Island)

of the Warbler

taxon,but the positions the tree-finches of and monotypicgenusconsisting eight subspe- ground-finches of remain equivocal. The latter cies.True to its name, this species closelyre- groupsgenerallyare treated as monophyletic sembles warblerwith respect its smallsize, sistergroups(Lack1947,Schluter a to 1984); howthat slender bill, andhabitof gleaning animalfood ever,it has alsobeensuggested the treefinches are ancestralto the ground-finches from foliage. Morphological,behavioral, and allozyme (Sternand Grant 1996),and under this scenario are data(Lack1947, YangandPatton 1981,Schluter it is possiblethat the tree-finches a para1984)all agreewith the divisionof the Gala- phyletic group. Priorto investigating evolutionwe pagosfinches into the three groupsoutlined ary relationshipswithin the tree-finches, above. However,little is known aboutthe phy- wished to ascertainwhether taxonomicsepalogenetic associations amongthe groups, and rationof the tree-finches ground-finches, and even lessis known about the relationships of basedon morphological data,wasreflected by Because Darwin's finches genera,species, and subspecies within each theirDNA sequences. group.The only publishedgeneticstudiesof underwent adaptive radiation relatively reDarwin's finchesare basedon allozymedata cently(Yangand Patton 1981),theremayhave (Fordet al. 1974,Yangand Patton1981,Polans been insufficient time for complete lineage 1983)that lackthe levelof resolution necessary sortingto haveoccurred followingspeciation, of to infer many phylogenetic relationships. whichcouldresultin the sharing haplotypes In thispaper,we usemitochondrial sequence data amongspecies (Neigel and Avise 1986).In adin to clarify unansweredquestions pertainingto dition, hybridizationhas been documented Darwin's finches(Grant 1986), and this may the phylogeny Darwin'sfinches. of First, we address evolutionary relationships lead to introgression mitochondrial of haploof the threegroups. Existing phylogenies con- typesfrom onespecies another to (Tegelstr6m sistentlytreat the WarblerFinch as the basal 1987).We reconstructed phylogenyof the a

Finch. The Warbler

Finch is a

July1999]

PhylogenyDarwin's of Finches

579

three groups using mitochondrialsequence populations from SantaCruz, Marchena, Esdata, which allowed us to infer relative levels pafiola, and Genovesa had divergedfrom one of geneticrelatedness within and among the another(Yangand Patton1981).All of these remain equivocalbecause sample tree-finches, ground-finches, the Warbler conclusions and Finch. sizeswere very small.Our goal was to ascerrelationships the subof The second question address we pertainsto tain the phylogenetic the taxonomyof the tree-finches. Using mor- species. phologicaland behavioraldata, Lack (1947) METHODS placed all tree-finchesin Camarhynchus, althoughhe later modifiedthis and placedPlaThe phylogenyof the three groupswas recontyspiza crassirostris (Vegetarian Tree-Finch) a in structed from a combination of 16S rRNA and conmonospecific genus(Lack 1969).This classififrom representatives the of cation has been upheld by Schluter(1984), trol-regionsequences ground-finch, tree-finch, and WarblerFinchgroups. againbasedonmorphological data.Morecom- We alsoincludedtwo mainlandspecies, Blackthe
mon in recent literature is the division of the

tree-finches the genera into Camarhynchus, Platyspiza, Cactospiza, shownin Table1. All and as six species appearto be closelyrelated,as evidenced thefactthat allozymedatawerenot by sufficientlydifferentiatedto resolverelationshipsat eitherthe genus species or level(Yang and Patton 1981). We used sequence data to clarify classification the tree-finches. of The third question directedat the Warbler is Finch,which possiblyis the most enigmatic speciesin terms of its historicalclassification. Gould(1837)included Warbler the Finchin the first comprehensive descriptionof Darwin's

facedGrassquit (Tiarisbicolor) and the Bananaquit (Coereba fiaveola), weredesignated outgroups. that as These outgroupswere chosenbecause they have beensuggested close as relatives the Geospizines of (Harris1973,Bowman1983,Baptista andTrail 1988). For our secondphylogeny, amplified and sewe quenced portion thecontrol a of region from20treefinchand WarblerFinchspecies subspecies, and and reconstructed phylogeny theseindividualsusa of ing the Bananaquit the outgroup. as
Amplification.--DNAwas extractedfrom approxi-

mately10 L of bloodusing500 L of 5% Chelex 100 (BioRad,Hercules)in ddH20, following the manufacturer's protocol. polymerase All chainreactions were donein a PerkinElmer9600thermocycler, usDNA in a total volumeof 100 finches, but Darwin and other taxonomists ing 3 L of extracted of eachprimer.Amplification primers wereGSLGlu
(5'-TTGGTTGTAACTTCAGGAAC-3')and 12 sr (5'-

questioned validity of classifying as a L, with 0.5 U Taqpolymeraseand 1X react buffer the it 200 finch.Subsequent Gould'streatise, to Certhidea (GibcoBRL),2 mM MgC12, M dNTPs, and 1 M
was reclassified as a member of various other

families, including the wood-warbler family Parulidae (formerly the Mniotiltidae; Darwin 1841, Ridgway 1897, Snodgrassand Heller 1904).Sincethe turn of the century,most taxonomists have agreedwith the placement of

AAGGTTAGGACTAAGTCTTT-3') the controlrefor gion (H. Gelter unpubl. data), and 16SL (5'CGCCTGTTTATCAAAAACAT-3') and 16SH (5'CGGTCTGAACTCAGATCACGT-3') for 16S rRNA

(Palumbi al. 1991). et The parameters wereonecycle for 10 min. The amplification products wereprecipitated with 250 L of 95%ethanoland 20 L of linear

Certhidea the Geospizinae in (e.g.Lowe 1936, of 94Cfor 2 min; 35 cyclesof 94Cfor 1 min, 50C Lack 1947,Tordoff1954,Yangand Patton1981, for 1 min, and 72Cfor 1 min; and one cycleof 72C
Schluter1984).

The WarblerFinchinhabitsall majorislands anda few of theminorislands theGalapagos of (Lack 1947, Harris 1973, Grant and Schluter 1984,Grant 1986).The eightsubspecies difare ferentiated largelyonthe basis plumage of color (Lack 1947, Bowman 1961, Lack 1969). The genetic relationships amongthe Certhidea subspecies havenot been adequately investigated. Two biochemical studieshavetentativelyconcludedthat the SantaCruz populationdiffers from a groupcontaining Marchena, the Espafiola, and Genovesapopulations(Ford et al. 1974, Polans1983).A third study found that

polyacrylamide and then resuspended 15 L in ddH20. The resuspended samples were harvested from a 0.8%agarose with 1X TBEbuffer usinga gel
QIAEX (QIAGEN) kit and eluted in 24 L of ddH20.

DNA sequencing.--All sequencing was done following a double-stranded dideoxy sequencing protocol, using primers GSL 148 (5'-CCCTATTCTCATTATTTTCGGC-3'), GSL 248 (5'-TATGAATCCCCTAACACCCAG-3'), and CR 367 (5'-TAGTGTAATGGTTGCCGGAC-3') for the controlregionand 16SL, 16SH (sequences shownabove),and 16SL2(5'-TCTCTTACAGGCAATCGGTG-3') for 16S rRNA. A

Sequenase kit (UnitedStates 2.0 Biochemical) and deoxyadenosine-5'-triphosphate [alpha-S35]were

580

FREELAND BOAG AND

[Auk, Vol. 116

employed with thefollowingdeviations from theSequenase USB protocol:(1) the primer and template annealing reaction included1 pLof 5% NP40, 1 DMSO, 1 pLof 10 pMprimer, 7 pLDNA, and 2 5X Sequenase reactionbuffer per sample;(2) during the primer and templateannealing step,the samples were boiled for 3 min, placedin liquid nitrogenfor 3 min, and then allowedto warm up to 15C; the (3) labelingreactionincluded1.3 pLddH20, 1 pLMn buffer, and 1 pL5% NP40; and (4) the termination

rameter distancemeasure)and maximum-likelihood

(usingempiricalbasefrequencies and a singlesubstitutionrate category)treeswere generated PHYin

LIP (Felsenstein,1993).
RESULTS

The first data set provided23 phylogenetically informative sites(18 excluding outthe groups) 44 variable and sites(22 excluding the reaction was incubated for 4 min at 42 to 44C. After outgroups). Twenty percentof the nucleotide sequencing, samples the wererun on a 35 x 45 cm
substitutions were transversions, only 5% but

6%polyacrylamide for 2 and4.5h at 60watts.We gel dried the gelsin a BioRadModel 583 gel dryer and thenexposed sequences KodakBiomax the to film. Datacollected thephylogeny thethreemajor for of groupscompriseda combination 361 basepairs of (bp) of 16SrRNA and 304 bp of controlregion(665 bp total)from two populations WarblerFinch(Cerof thidea fusca o. and C. o.cinerascens), ground-finchsix es (Geospiza difficilis, scandens, magnirostris, G. G. G. fortis,G.fuliginosa, G.conirostris), tree-finchand four

of these involvedcomparisons within theDarwin'sfinches. Some taxahad a singledeletion.

Only two different sequences were found in all

sixspecies ground-finches. sequence of The divergence was 0.2 to 2.4% within the Darwin's
finches and 2.6 to 3.8% between the Darwin's

finches the outgroups and (Table The rela2). tivelyclose relationship among outgroups the justifies choice outthe of es (Camarhynchus parvulus,C. psittacula, Cactospiza and the Geospizinae
pallida, and Platyspiza crassirostris), Black-faced the Grassquit, theBananaquit. datafor thephyand The logeny thetree-finch of genera theWarbler and Finch subspecies comprised bp of controlregionfrom 385

group. Maximum-parsimony (Fig. 1), maxi-

mum-likelihood (not shown),and neighborjoining(notshown) treesshowed sametothe pology.In addition, sametopology the result20 tree-finches and Warbler Finches and a Bananaed when gaps were treated as either quit (Table1, Appendix1).Sequences eachspe- informativeor missingsites, and when the from
cieswere depositedin GenBank(accession numbers transition/transversion AF089768 to AF089795) 1.0 or 4.0. Sequence alignment phylogenetic and analysis.--Gels

ratio was set to either

were scoredmanually,and sequences were aligned using GeneWorks(IntelliGenetics,Inc.). Few insertions/deletions (gaps)occurred, the genealignbut mentprogram never had to insertmorethanonegap at any givensiteto achieve plausible a alignment. Sequence divergence wascalculated usingthe number of nucleotidedifferences betweentwo sequences, includinggaps.Sequence divergence equalsthe number of nucleotidedifferencesdivided by the total numberof nucleotides the sequence, in expressed as
a percentage.

Sequencealignmentsfrom GeneWorkswere imported into PAUP(Swofford1993)and PHYLIP (Felsenstein 1993). Transitions and transversions were

equallyweighted because owingto thelow frequency of transversions Results), (see differentialweighting did not affecttreetopologies. Because control the
regionand 16SrRNA sequences not protein-codare ing, we did not differentiatebetween synonymous

versusnonsynonymous substitutions when analyzing thesesequences. circumvent dilemmaof To the differentialweightingof gaps,we did two analyses that led to inferredmaximum-parsimony trees:(1) Finch and tree-finches were transitions. Two of onewith all gapstreatedasinformative sites,and (2) Finchhaplotypes a gapat one had onewith all gapstreatedasmissing data.Maximum- theWarbler
parsimonytreeswere generated PAUP(Swofford, in

Thephylogeny shows theWarbler that Finches are the sistergroupto the otherDarwin's finches. The ground-finches form a well-supported cladewithin the clusterof tree-finches and ground-finches, demonstrating treethat finches moreclosely are relatedto oneanother thantheyare to ground-finches. Therefore, we can investigate relationships the amongtreefinches isolation in from ground-finches. We alsohavedemonstrated suitabilityof using the the Warbler Finchas an outgroup versus the ground-finches tree-finches. or Thesecond datasetyielded15 control-region haplotypes, whichin conjunction theoutwith grouphad 35 variable sites,18 of whichwere phylogenetically informative(Table3). These values were 21 and 17, respectively, whenthe outgroupwasnot included. The amountof sequence divergence among haplotypes, including theoutgroup, ranged from0.25to 3.6%.All nucleotidesubstitutions among the Warbler
or two of the sites.The maximum-parsimony

1993).Neighbor-joining (usingthe Kimura two-pa- tree (Fig.2), maximum-likelihood analysis (not

July1999]

PhylogenyDarwin's of Finches

581

shown), and neighbor-joining analysis (not shown)recovered sametopology. the Treating the gapsas eithermissingdataor as informative sitesdid not affectthe topology. The tree-finches presentedas a monoare phyletic grouprelative theWarbler to Finch, regardless whetherthe Bananaquit a Warof or bler Finchsubspecies designated theoutis as group. Platyspiza crassirostris onlyspecies is the of tree-finch that appearsto be monophyletic. Camarhynchus psittacula contains one individual that is allied with Cactospiza pallida and one individual that has the samehaplotypeas Camarhynchus parvulus. Theselevelsof cohesion are reflectedin a comparison within- and of among-species sequencedivergences. Only P. crassirostris, monophyletic the assemblage, has conspecific sequence deviations that are absolutely lower than the heterospecific sequence deviations(Table4). Overall, the tree-finches are a very closelyrelatedgroup of species.
The Warbler Finch is divided into two dis-

tinctlineages according theislands each to that inhabits(Fig. 3): (1) the SantaCruz clade(C. 0. olivacea) (2) the Marchena,Espafiola,and and
Genovesa (M-E-G) islandsclade (C. o.fusca,C. o. mentalis, C. o.cinerascens). control-reand The

gionsequence divergence withineach these of

two Warbler Finch clades is 0 to 0.7%, whereas

the divergence betweencladesis 2.0 to 2.7%. The WarblerFinches presented a paraare as phyleticgroupregardless whetherthe outof group comprises only the Bananaquit, the or Bananaquit plustheWarbler Finches.
DISCUSSION

Relationships thethreegroups.Our results of do not disagree with the traditional view (Lack 1947) of the WarblerFinch as the basal taxon and the tree-finches and ground-finches as monophyletic sistergroups(Fig. 1). We found no evidence supportSternandGrant's(1996) to proposalthat the ground-finches arosefrom the tree-finches, although previously as stated, thesefindingsmustbe considered preliminary
because the inclusion of alternate or additional

individualsmay alter the resulting phylogeny. These data do not support Yang and Patton's (1981)suggestion the tree-finches that differentiatedmore recentlythan the ground-finches, becausethe tree-finchand ground-finch mtDNA sequence divergencefrom the basal

582

FREELAND BOAG AND

Tiaris bicolor 01

[Auk,Vol.116

Platyspiza crassirostris 01

Camarhynchus parvulus08
86

Cactospiza pallida 01

64 [__
74

Camarhynchus psittacula 01
Geospiza difficilis02, G. magnirostris 02,

80 [__ 100

G. fortis 73, G. conirostris 01

Geospiza scandens G.fulig7nosa 24, 26

Certhidea olivacea 01
Certhidea olivacea 09

Coereba fiaveola 01

FIG.1. Maximum-parsimony showing relationships tree the amongselected Darwin'sfinches, basedon

665bp of control region 16S and rRNAsequence. bicolor C.fiaveola outgroups. Tiaris and are Bootstrap values areshown above branches (2,000 bootstrap replicates). length= 53, CI = 0.811. Appendix for Tree See I
specimeninformation.

Warbler Finches is 0.9 to 2.6% and 1.1 to 2.3%,

a monotypicgenus,which is consistent with

respectively. This genetic phylogeny was re- our results(Fig. 2). In contrast, Camarhynchus quiredto investigate possible instances mor- parvulus of doesnot form a monophyletic group, phological convergenceparallelism, or because and the status C. psittacula of remains equivothesetwo phenomena common passer- cal. The sharingof mitochondrial are in haplotypes
ines (Bledsoe1988)and can distort reconstruct- between species may result from ancestral

ed phylogenies are based that solelyon mor- polymorphism incomplete and lineage sorting, phological behavioral and data. or hybridizationand introgression (Takahata
Classification the tree-finches.--Within of the and Slatkin 1984, Neigel and Avise 1986, Tetree-finches, Platyspiza currently is classified gelstr6m1987).Whentwo populations speas or
TABLE Nucleotide 3. differences between 15tree-finches Warbler the and Finch mtDNAcontrol-region haplotypes oneCoerebafiaveola haplotype, and (Cfa) based 385bp.See on Figure forhaplotype 2 species identities;"*" denotes insertion/deletion, "-" indicates an and identitywith the firstsequence.
Haplotype
H1 H2
G C C AAC
T AA -

GAG
GA

TTTAAT
.... A ...... ..... .....
--C-A--

T
CC

G C
-

*
T

C C C T

C G G TC
.......

C AC

H3 H4 H5 H6 H7
H8 H9

.... G G
G

AT--C

T
T

G GA GA

A-

T
T

........
.......

TGT
GT

C-A---A C-A---A
A

A
A A

T
T -

G
G A -

T
T

.......
.......

TGT
TGT

GA-

GA GA GA

...... ...... .......

C C

A A AT--A

--A --A

T--C
T T C C C C ..... ......

.....
..... C T -

T-T
T TT T T T T

A AT-

G G

H10 H11 H12 H13 H14 H15 Cfa

AT AT
AT

....
-

G
G

GA G A
GA

...... ........
......

AT AT
A-

AT AT
AT

C
C C

....
...... .....

A
A-

G
G

?
-

GA
GA

......
-

C
C

.....

A * T

AT
AT

TT

G G

AC

GAT

G C A-

C T

July1999]

Phylogeny ofDarwin's Finches

Coereba flaeola 01

583

Cactospiza pallida 01 (H 10)

82

C.pallida 02 (HI 1)

C.pallidaO3(H12)
Camarhynchuspsittacula (HI 3) 01

Platyspiza crassirostris (H4) 01

--P. crassirostris (H7) 12 P. crasslrostris (H5) 03 P. crassirostris (H6) 04 Carnarhynchusparvulus (H9) 08
86

70 I 55 81

Carnarhynchus pstittacula02 (H 14) C. parvulus03, C.parvulus07, C. psittacula04 (HS) C.psittacula05 (H15)

Certhideaolivacea19, C. olivacea20, C. olivacea25 (SantaCruz) (H3)
94

--

C. olivacea (Genovesa) 27 (H2)

C. olivacea01 (Marchena), C. olivacea (Espanola) 09 (H1)

FIG.2. Maximum-parsimony of the tree-finch Warbler tree and Finchcontrol regionhaplotypes to 15 1 (H1 to H15), with C.fiaveola the outgroup. as Tree length= 81, CI = 0.864.Bootstrap values written above
branches (2,000bootstrapreplicates). Appendix 1 for specimen See information.

ciesarefirstseparated, theirhaplotypes exare pectedto be polyphyletic with respect one to another, based simplyon the chance certain of ancestral haplotypes occurring morethan in one population species. or Stochastic lineage sorting results a progression polyphyly in from to paraphyly monophyly to (Tajima1983,NeiTABLE Comparison conspecific heterospe4. of and cific sequence divergences amongfour species of
tree-finches.

gel andAvise1986,PamiloandNei 1988).As a result,certainhaplotypes will be maintained in a population, others and will goextinct. Barring selection, is generally randomprothis a cess. populations sampledduring the If are stages polyphyly paraphyly, shared of or then haplotypes be theresultof incomplete may lineage sorting (Avise et al. 1983, Moran and
Kornfield 1993, P6rez-Sufirez et al. 1994).

Sequence divergences (%)

Within
species

Giventhe close genetic relationships the of tree-finches, is possible some it that individuals in the Camarhynchus Cactospiza and genera sharehaplotypes a resultof incomplete as lin-

Among
species
1.00 0.00 0.26 0.26 to to to to 2.3 2.3 2.1 2.6

Platyspiza crassirostris Camarhynchus parvulus Camarhynchus psittacula Cactospiza pallida

0.26 0.00 0.26 0.26

to to to to

0.78 0.78 1.00 0.52

eagesorting. Platyspiza the only tree-finch is genus appears that monophyleticthisstudy, in and this seems unlikelyto haveresulted from
eitherrelativelygreater or relatively age smaller population size in this species, factors two that can accelerate haplotypelineagesorting

584

FREELAND BOAG AND

[Auk,Vol. 116

Pinta

Isabela

Genovesa

Marchena

Fernandina

Salvador
$ ltra
Santa Cruz
San Cristobal 1-

0
i

30 6091 km Santa 90 Maria 90

i i i

Espafiola
i

FIG3. Map of the Galapagos Archipelago. Warbler finchsubspecies sampled were fromthefourislands labeled boldtype(Genovesa, in Marchena, Espafiola, Santa and Cruz).

(Avise 1994). Platyspizacrassirostris does not has playeda role in the adaptiveradiationof appearto be olderthan the Camarhynchus-Cac- group(Grant1993,1994; Freeland that J. andP. tospiza lineage,becausethe minimum diver- Boag unpubl. data). Unfortunately,although gence betweenP.crassirostris theothertree- hybridization and amongthe tree-finches been has finchspecies 0.98%.Thisis considerably is low- documented (Lack 1947, Bowman 1983, Grant er than the maximuminterspecific divergence 1986), neither the frequencyof hybridization (1.8%)within the Cactospiza-Camarhynchus nor the relativefitness hybridsis known.The linof

eage(Table4). Furthermore, Platyspiza, Camar- tree-finches and ground-finches have many hynchus, Cactospiza and likely had similarlong- similarities, includinga high overallsequence term populationsizesduring their evolution- similarity within each group, similarage,the a ary histories,becausepopulationbottlenecks samenumber of species, and interspecific hypresumably occurred on numerousoccasions bridization(Grant 1986,Yangand Patton1981, during the colonization new islands.In ad- J.Freeland of andE Boag unpubl.data).Although dition, oncethe islandsare colonized, ecologi- furtherresearch warranted, suggest is we that, cal conditions greatly depletethe sizesof es- like in ground-finches, hybridization playeda tablished populations some in years(Boagand role in the adaptiveradiationof tree-finches. Grant 1981,Grant and Grant 1992).If we accept The extent of this role remains unclear, and it that similar evolutionary demographic condi- is extremely difficultwith existingdatato diftions are likely to have prevailedfor all tree- ferentiate between effects lineage the of sorting finch genera,then the identification both and hybridization.Basedon the mtDNA availof monophyletic paraphyletic and genera may be ablesequence data,no basisexists dividing for attributed chance to and smallsample sizes. tree-finches threegenera. classification into The An alternate explanation theparaphylyof of P.crassirostris a monotypicgenusis not for into Camarhynchushistoricand/or ongoinghy- disputedby our data,but the divisionof the reis bridization between C. parvulus, psittacula, maining species C. into two generais dubious.Aland Cactospiza pallida.This phenomenon is thoughmolecularevidence seldomis considknownto occuramongthe ground-finches and eredto bethesolecriterion identifying for spe-

July1999]

Phylogeny Darwin's of Finches

585

bottlenecks.

ciesand genera, is reasonable expect it to two and Espafiola up to 1 millionyears.In adfor genera be genetically to distinguishable. is dition, the distinction between the Santa Cruz It possible the Camarhynchus that psittacuIa indi- andtheM-E-GWarbler Finches agrees the with vidualthatis alliedwith theCactospiza paiiida tentativefindingsof Ford et al. (1974)and Pothat individuals an anomaly, is although givenour lans(1983),suggesting the patternof mismallsample size,probability dictates this tochondrial that haplotype sharing similar the is to differentiation among these is unlikelyto be the case. Although moreindi- patternof nuclear These facts, vidualsmustbe sequenced beforefirm conclu- four Warbler Finch subspecies. with the regularbottlenecks (Boag sions be drawn,the available can genetic data combined tend to support Lack's (1969) and Schluter's and Grant 1981, Grant and Grant 1992) that have accelerated stochastic lineage sort(1984)classification the tree-finches two would of into ing, suggest at some that point in their evolugenera (Camarhynchus PIatyspiza). and geneflow playeda rolein the TheWarbler Finchsubspecies.--Not of the tionaryhistory, all of among WarblerFinchsubspecies genetically are dis- maintenance genetichomogeneity Finch populations. tinct (Fig. 2, Table3). The Marchenaand Es- theM-E-GWarbler Finches lineage conis pafiolapopulations sharethe samehaplotype, The M-E-GWarbler siderablyolder than the Santa Cruz Warbler and their sequence divergence from the GenBothEspafiola SantaCruz and ovesa population 0.7%.Thismayresultfrom Finchlineage. is the islands thearchipelago in eitherretention an ancestral of haplotype, areamong oldest or andtheM-E-G clade maybe a relic ongoing geneflow between Marchena the and (Cox1983), of one of the first colonizations. The Santa Cruz Espafiola Warbler Finches. Onceagain,we canFinchpopulation genetically is not differentiate betweenthe two processesWarbler the tree-finches and the M-E-G intermediate to Warwith certainty, theprobability theformer but of blerFinch clade, theWarbler and Finch subspeis inversely proportional the time sincecolto cies compriseanother paraphyleticspecies. onization and to the frequency durationof Whereas a number of different scenarios could and

this,the mostintriguing possibility is tree-finch Warbler and Finch lineages diverged explain thattheoriginal founder population theGalon will vary,depending whichpopulations on are resemcompared. The tree-finch/SantaCruz Warbler apagosIslandsbore a morphological Finch sequence divergence 1.3to 2.3%,com- comparison the Geospizinaeto mainland is of
pared with 2.3 to 3.9% for tree-finch/M-E-G
blance to the Warbler Finch. Future work on the

Estimates of the time since the

WarblerFinchdivergence. control-region We acknowledge the paucityof inforThe that sequence usedin this studyevolves approxi- mative characters limits our conclusions. Howmately2.5 timesfasterthan the rate of cyto- ever,a preliminaryinvestigation into the gechrome-b and 16S rRNA evolution in Darwin's neticdivergence Darwin's of finches using16S finches(Freeland1997), or 5% per million rDNA, cytochrome and control-region b, seyears.If thisis true,thentheWarbler Finchand quencesdemonstratedthat on the whole the tree-finchlineagessplit about 750,000years Geospizinae not comprisea geneticallydido ago. Although must treated a veryap- vergent group of birds (Freeland 1997). In all this be as proximate figure,it fallswithinthe geological likelihood, genetic differentiation limitedbeis ages theislands, of which range from4 million cause recency speciation of of (Yang andPatton yearsto lessthan 500,000 years(Bailey1976, 1981) and hybridization(Grant 1986,J. FreeCox1983). Thisagealsois close theestimate land and E Boagunpubl.data).Therefore, to it of 570,000 years since divergence Certhi- seems the of unlikely that more sequencing would deafrom the otherGeospizines basedon allo- clarifyphylogenetic relationships. suggest We thatfutureworkfocus developing on moreinzymedata(Yang and Patton 1981). molecular markers will show that greater If theWarbler Finch lineage between to cisive is 0.5 1 millionyears it seems old, surprising the differentiationamong genera, species,and that sharingof haplotypes between two popula- populations. tions would theresult incomplete be of lineage ACKNOWLEDGMENTS sorting,because would meanthat the relthis ativelyrapidlyevolving control regionwould We thank P. Grant, G. Seutin, Louisiana State Muhaveremained unchanged both Marchena seum, and Florida Museumof Natural History for in

species will allow elaborationon this idea.

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providingblood samples. Gelter,S. Lougheed, FREELAND, 1997.The genetic H. J. R. evolutionary hisandD. Michaudprovidedhelpandadvicein thelab, tory of Darwin'sfinches(Aves:Geospizinae). and K. Conradand V. FriesensuppliedusefulcomPh.D. dissertation, Queen'sUniversity,Kingston, Ontario. mentson the manuscript. Financialassistance came from NSERC (PTB) and Queen's University (JRF). GOULD, 1837.Description new species finches J. of of collected Darwin in the Galapagos. by Proceedingsof the Zoological Society London of 5:4-7. LITERATURE CITED GRANT, R. 1986.Ecology P. andevolution Darwin's of AVlSE, C. 1994.Molecularmarkers, J. naturalhistory, finches. Princeton UniversityPress,Princeton, andevolution. Chapman Hall, New York. and New Jersey. AVISE,J. C., J. E SHAPIRA, W. DANIEL, C. F. AQUAD- GRANT, R. 1993. S. P. Hybridization Darwin's of finches RO, AND g. g. LANSMAN. 1983. Mitochondrial on IslaDaphne Major,Galapagos. Philosophical DNA differentiation during the speciation proTransactions theRoyalSociety London of of Secessin Peromyscus. Molecular Biology and Evolution 1:38-56. ries B 340:127-139.

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APPENDIX Species 1. identification, showing location and collector source eachsample. or for
Species Coereba fiaveola 01
Certhidea Certhidea Certhidea Certhidea Certhidea Certhidea olivacea olivacea olivacea olivacea olivacea olivacea 01 09 19 20 25 27
Peru

Site collected
Marchena

Collector/source
LSU (B5168)
G. Seutin G. Seutin

Espafiola
Santa Cruz Santa Cruz Santa Cruz Genovesa Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz

(CDRS) (CDRS) (CDRS

(CDRS (CDRS

Cactospiza pallida01 Cactospiza pallida02 Cactospiza pallida03 Camarhynchus parvulus 03 Camarhynchus parvulus 07 Camarhynchus parvulus 08 Camarhynchus psittacula 01 Camarhynchus psittacula 02 Camarhynchus psittacula 04 Camarhynchus psittacula 01 Geospiza conirostris 01 Geospiza difficilis 02 Geospiza fuliginosa 06 Geospiza fortis 73 Geospiza magnirostris 02 Geospiza scandens 24 Platyspiza crassirostris 01 Platyspiza crassirostris 03 Platyspiza crassirostris 04 Platyspiza crassirostris 12
Tiaris bicolor 01

(highlands)
(CDRS) (CDRS)

P. Boag P. Boag P. Boag P. Boag P. Boag P. Boag P. Boag E Boag P. Boag E Boag
G. Seutin

Marchena Marchena
Santa Cruz Santa Cruz

G. Seutin

(highlands)

P. Boag P. Boag
G. Seutin

Espafiola
Genovesa Santa Cruz

Daphne
Marchena
Santa Cruz Santa Cruz

P. Boag P. Boag P. Boag

G. Seutin P. Grant P. Grant G. Seutin G. Seutin

Marchena Marchena
Santa Cruz Unknown

E Boag FLMNH (331105)

= Florida Museum of Natural

LSU = Louisiana State Museum of Natural Sciences; CDRS = Charles Darwin Research Station; FLMNH

History.