Phylogenetics of Darwin's Finches: Paraphyly in the Tree-Finches, and Two Divergent Lineages in the Warbler Finch

The Auk A Quarterly Journalof Ornithology Vol. 116 No. 3 July 1999

The Auk 116(3):577-588, 1999

PHYLOGENETICS OF DARWIN'S FINCHES: PARAPHYLY IN THE TREE-FINCHES, AND TWO DIVERGENT LINEAGES IN THE WARBLER FINCH

JOANNAR. FREELAND• AND PETERT. BOAG Departmentof Biology,Queen• University, Kingston, Ontario K7L 3N6, Canada

ABSTR^CT.--TheGalapagos Darwin's finches (Geospizinae) have been classifiedas three majorgroups based on morphology and behavior: ground-finches, tree-finches, and the War- blerFinch (Certhidea olivacea). Little is knownabout the evolutionary relationships within and amongthese groups, which is partly due to the lack of a phylogenybased on molecular sequencedata. We used mitochondrial sequence data to reconstructa phylogenyof Darwin's finches.These data show that within the tree-fin&es,only one genus is conclusivelymono- phyletic,and another is conclusivelyparaphyletic. It maybe appropriateto upholdthe classificationof the tree-finchesinto two genera.The WarblerFinch complex is paraphyletic,as revealedby two divergentgenetic lineages contained within this species.Stochastic lineage sortingwithin relativelyrecently diverged species and interspecificand intergenerichy- bridizationare the two mostlikely explanationsfor the sharingof haplotypesamong taxa. Received3 March 1998,accepted 6 November1998.

ADAPTIVERADIATION refers to the processin sightinto a numberof aspectsof evolutionand which one speciesevolves into numerousspe- speciation,including adaptive radiation. ciesover a relativelyshort period of time. The The subfamily Geospizinaecomprises 14 questionof how this occursis fundamentalto nominatespecies, 13 of whichinhabit the Gal- studies of evolution and speciationand has apagos Archipelago.Darwin's finches have been at the heart of considerableresearch (e.g. been divided into three groupsbased on mor- DeSalleet al. 1987,Sang et al. 1994,Losos 1995, phology and behavior:ground-finches, tree- Tarr and Fleischer 1995, Cameron et al. 1996, finches,and the WarblerFinch (Certhideaoliva- Radtkey1996, Shaw 1996). Perhaps the mostfa- cea;Table 1). The ground-finches(Geospiza) mous ongoingstudy of adaptiveradiation in- compriseone genusand six speciesthat are volves Darwin's finches(Geospizinae). More finch-likein appearance,particularly with re- than 100years of researchhave been conducted spectto their bills, and spend much of their on Darwin'sfinches, yet, manyquestions about time foragingon the ground.The tree-finches theirevolutionary history remain unanswered. (Camarhynchus, Platyspiza, and Cactospiza)com- A glaringomission in this field of studyis the prise six species,but the numberof generaos- lack of a sequence-basedphylogeny. A molec- ciliates between one and three (see below). ular dataset for the groupwill yield furtherin- These six specieshave bills intermediate to thoseof ground-finchesand the WarblerFinch, • Present address: School of Animal and Microbial and althoughthey occasionallyforage on the Sciences,P.O. Box 228, Universityof Reading,Read- groundin a mannersimilar to ground-finches, ing RG6 6AJ,United Kingdom. they spendmuch of their time in foliage and E-mail:j.r. [email protected] vegetationexhibiting behavior similar to that

577 578 FREELANDAND BOAG [Auk, Vol. 116

TABLE1. Darwin'sfinch species, taken from Lack (1947), Bowman (1961), and Grant(1986). Authors for speciesnames are given in Lack(1947), although some generic names of tree-finchesdiffer from this ref- erence.

Scientificname Englishname Ground-Finches Geospizamagnirostris Gould • LargeGround-Finch Geospizafortis Goulda Medium Ground-Finch Geospizafuliginosa Gould • Small Ground-Finch Geospizadifficilis Sharpe a Sharp-beakedGround-Finch Geospizascandens (Gould) • Cactus Ground-Finch Geospizaconirostris Ridgway a LargeCactus Ground-Finch Tree-Finches Camarhynchusparvulus (Gould) a Small Tree-Finch Camarhynchuspauper Ridgway Medium Tree-Finch Camarhynchuspsittacula Gould • LargeTree-Finch Platyspizacrassirostris Gould • VegetarianFinch Cactospizapallida (Sclater and Salvin)• WoodpeckerFinch Cactospizaheliobates (Snodgrass and Heller) MangroveFinch Warbler Finch Certhidea o. olivaceaGould a WarblerFinch (Santiago, Santa Cruz, Isabela, Ferdinand,Rabida, Seymour,Pinzon islands) C. o. bifasciata WarblerFinch (Santa Fe Island) C. o.fusca a WarblerFinch (Pinta and Marchenaislands) C. o. luteola Warbler Finch (FloreanaIsland) C. o. ridgwayi Warbler Finch (San Cristobal Island) C. o. becki Warbler Finch (Wolf and Darwin islands) C. o. mentalis a WarblerFinch (Genovesa Island) C. o. cinerascens • WarblerFinch (Espafiola Island) • Taxaused in this study. of the Warbler Finch. The Warbler Finch is a taxon,but the positionsof the tree-finchesand monotypicgenus consisting of eight subspe- ground-finchesremain equivocal.The latter cies.True to its name, this speciesclosely re- groupsgenerally are treated as monophyletic semblesa warblerwith respectto its smallsize, sistergroups (Lack 1947, Schluter 1984); how- slenderbill, andhabit of gleaninganimal food ever,it has alsobeen suggested that the tree- from foliage. finches are ancestralto the ground-finches Morphological,behavioral, and allozyme (Sternand Grant 1996),and under this scenario data(Lack 1947, Yang and Patton1981, Schluter it is possiblethat the tree-finchesare a para- 1984) all agreewith the divisionof the Gala- phyleticgroup. Prior to investigatingevolution- pagosfinches into the three groupsoutlined ary relationshipswithin the tree-finches,we above.However, little is known aboutthe phy- wished to ascertainwhether taxonomicsepa- logeneticassociations among the groups,and ration of the tree-finchesand ground-finches, even less is known about the relationshipsof basedon morphologicaldata, wasreflected by genera,species, and subspecieswithin each their DNA sequences.Because Darwin's finches group.The only publishedgenetic studies of underwent adaptive radiation relatively re- Darwin's finchesare basedon allozymedata cently(Yang and Patton1981), there may have (Fordet al. 1974,Yang and Patton1981, Polans been insufficient time for complete lineage 1983)that lack the level of resolutionnecessary sortingto have occurredfollowing speciation, to infer many phylogeneticrelationships. In whichcould result in the sharingof haplotypes this paper,we usemitochondrial sequence data amongspecies (Neigel and Avise 1986).In ad- to clarify unansweredquestions pertaining to dition, hybridizationhas been documentedin the phylogenyof Darwin'sfinches. Darwin's finches(Grant 1986), and this may First, we addressevolutionary relationships lead to introgressionof mitochondrialhaplo- of the threegroups. Existing phylogenies con- typesfrom onespecies to another(Tegelstr6m sistentlytreat the WarblerFinch as the basal 1987). We reconstructeda phylogenyof the July1999] Phylogenyof Darwin'sFinches 579 three groups using mitochondrialsequence populationsfrom SantaCruz, Marchena,Es- data, which allowed us to infer relative levels pafiola, and Genovesahad divergedfrom one of geneticrelatedness within and among the another(Yang and Patton1981). All of these tree-finches,ground-finches, and the Warbler conclusionsremain equivocal becausesample Finch. sizeswere very small.Our goal was to ascer- The secondquestion we addresspertains to tain the phylogeneticrelationships of the sub- the taxonomyof the tree-finches.Using mor- species. phologicaland behavioraldata, Lack (1947) placed all tree-finchesin Camarhynchus,al- METHODS thoughhe later modifiedthis and placedPla- tyspizacrassirostris (Vegetarian Tree-Finch) in a The phylogenyof the three groupswas recon- monospecificgenus (Lack 1969).This classifi- structed from a combination of 16S rRNA and con- cation has been upheld by Schluter(1984), trol-regionsequences from representativesof the ground-finch,tree-finch, and WarblerFinch groups. againbased on morphologicaldata. More com- We also includedtwo mainlandspecies, the Black- mon in recent literature is the division of the faced Grassquit(Tiaris bicolor) and the Bananaquit tree-finchesinto the generaCamarhynchus, Pla- (Coerebafiaveola), that weredesignated as outgroups. tyspiza,and Cactospiza,as shown in Table1. All These outgroupswere chosenbecause they have six speciesappear to be closelyrelated, as ev- beensuggested as closerelatives of the Geospizines idencedby the factthat allozymedata were not (Harris 1973,Bowman 1983, Baptista and Trail 1988). sufficientlydifferentiated to resolve relation- For our secondphylogeny, we amplified and se- shipsat eitherthe genusor specieslevel (Yang quenceda portionof thecontrol region from 20 tree- finchand WarblerFinch species and subspecies,and and Patton 1981). We used sequencedata to reconstructeda phylogenyof theseindividuals us- clarify classificationof the tree-finches. ing the Bananaquitas the outgroup. The third questionis directedat the Warbler Amplification.--DNAwas extractedfrom approxi- Finch, which possiblyis the most enigmatic mately10 •L of bloodusing 500 •L of 5% Chelex100 speciesin terms of its historical classification. (BioRad,Hercules) in ddH20, following the manu- Gould(1837) included the WarblerFinch in the facturer'sprotocol. All polymerasechain reactions first comprehensivedescription of Darwin's were donein a PerkinElmer 9600 thermocycler, us- finches, but Darwin and other taxonomists ing 3 •L of extractedDNA in a total volumeof 100 questionedthe validity of classifyingit as a •L, with 0.5 U Taq polymeraseand 1X react buffer finch.Subsequent to Gould'streatise, Certhidea (GibcoBRL), 2 mM MgC12,200 •M dNTPs, and 1 •M was reclassified as a member of various other of eachprimer. Amplification primers were GSL Glu (5'-TTGGTTGTAACTTCAGGAAC-3')and 12 sr (5'- families, including the wood-warbler family AAGGTTAGGACTAAGTCTTT-3')for the controlre- Parulidae (formerly the Mniotiltidae; Darwin gion (H. Gelter unpubl. data), and 16SL (5'- 1841, Ridgway 1897, Snodgrassand Heller CGCCTGTTTATCAAAAACAT-3') and 16SH (5'- 1904). Sincethe turn of the century,most tax- CGGTCTGAACTCAGATCACGT-3') for 16S rRNA onomistshave agreedwith the placementof (Palumbiet al. 1991).The parameterswere one cycle Certhideain the Geospizinae(e.g. Lowe 1936, of 94øCfor 2 min; 35 cyclesof 94øCfor 1 min, 50øC Lack 1947,Tordoff 1954, Yang and Patton1981, for 1 min, and 72øCfor 1 min; and one cycleof 72øC Schluter1984). for 10 min. The amplificationproducts were precip- itated with 250 •L of 95%ethanol and 20 •L of linear The WarblerFinch inhabits all majorislands polyacrylamideand then resuspendedin 15 •L and a few of theminor islands of theGalapagos ddH20. The resuspendedsamples were harvested (Lack 1947, Harris 1973, Grant and Schluter from a 0.8% agarosegel with 1X TBEbuffer usinga 1984,Grant 1986).The eight subspeciesare dif- QIAEX (QIAGEN) kit and eluted in 24 •L of ddH20. ferentiatedlargely on the basisof plumagecol- DNA sequencing.--Allsequencing was done fol- or (Lack 1947, Bowman 1961, Lack 1969). The lowing a double-strandeddideoxy sequencingpro- geneticrelationships among the Certhideasub- tocol, using primers GSL 148 (5'-CCCTATTCTCAT- specieshave not been adequatelyinvestigated. TATTTTCGGC-3'), GSL 248 (5'-TATGAATCCCCT- AACACCCAG-3'), and CR 367 (5'-TAGTGTAATG- Two biochemicalstudies have tentativelycon- GTTGCCGGAC-3') for the control region and 16SL, cludedthat the SantaCruz populationdiffers 16SH (sequencesshown above), and 16SL2(5'-TC- from a groupcontaining the Marchena,Espafi- TCTTACAGGCAATCGGTG-3') for 16S rRNA. A ola, and Genovesapopulations (Ford et al. Sequenase2.0 kit (UnitedStates Biochemical) and 1974, Polans1983). A third study found that deoxyadenosine-5'-triphosphate[alpha-S35] were 580 FREELANDAND BOAG [Auk, Vol. 116 employedwith the followingdeviations from theSe- rameter distancemeasure) and maximum-likelihood quenaseUSB protocol:(1) the primer and template (usingempirical base frequencies and a singlesub- annealingreaction included 1 p•Lof 5% NP40, 1 stitutionrate category)trees were generatedin PHY- DMSO, 1 p•Lof 10 p•Mprimer, 7 p•LDNA, and 2 LIP (Felsenstein,1993). 5X Sequenasereaction buffer per sample;(2) during the primer and templateannealing step, the samples RESULTS were boiled for 3 min, placedin liquid nitrogenfor 3 min, and then allowedto warm up to 15øC;(3) the The first data set provided23 phylogeneti- labelingreaction included 1.3 p•LddH20, 1 p•LMn cally informativesites (18 excludingthe out- buffer, and 1 p•L5% NP40; and (4) the termination reaction was incubated for 4 min at 42 to 44øC. After groups)and 44 variablesites (22 excludingthe sequencing,the sampleswere run on a 35 x 45 cm outgroups).Twenty percentof the nucleotide 6% polyacrylamidegel for 2 and4.5 h at 60 watts.We substitutionswere transversions,but only 5% dried the gelsin a BioRadModel 583 gel dryer and of theseinvolved comparisons within the Dar- thenexposed the sequencesto KodakBiomax film. win'sfinches. Some taxa had a singledeletion. Datacollected for the phylogenyof thethree major Only two different sequenceswere found in all groupscomprised a combinationof 361 basepairs six speciesof ground-finches.The sequence di- (bp) of 16SrRNA and 304 bp of controlregion (665 vergencewas 0.2 to 2.4% within the Darwin's bp total) from two populationsof WarblerFinch (Cer- finches and 2.6 to 3.8% between the Darwin's thideao. fusca and C. o. cinerascens),six ground-finch- finchesand the outgroups(Table 2). The rela- es (Geospizadifficilis, G. scandens,G. magnirostris,G. fortis,G. fuliginosa, and G.conirostris), four tree-finch- tively closerelationship among the outgroups es (Camarhynchusparvulus, C. psittacula,Cactospiza and the Geospizinaejustifies the choiceof out- pallida,and Platyspizacrassirostris), the Black-faced group. Maximum-parsimony (Fig. 1), maxi- Grassquit,and the Bananaquit.The datafor thephy- mum-likelihood(not shown), and neighbor- logenyof thetree-finch genera and theWarbler Finch joining(not shown)trees showed the sameto- subspeciescomprised 385 bp of controlregion from pology.In addition,the sametopology result- 20 tree-finches and Warbler Finches and a Banana- ed when gaps were treated as either quit (Table1, Appendix1). Sequences from each spe- informativeor missingsites, and when the cieswere deposited in GenBank (accessionnumbers transition/transversion ratio was set to either AF089768 to AF089795) 1.0 or 4.0. Sequencealignment and phylogeneticanalysis.--Gels were scoredmanually, and sequenceswere aligned Thephylogeny shows that the Warbler Finch- using GeneWorks(IntelliGenetics, Inc.). Few inser- es are the sistergroup to the other Darwin's tions/deletions(gaps) occurred, but the genealign- finches.The ground-finchesform a well-sup- mentprogram never had to insertmore than one gap ported cladewithin the clusterof tree-finches at any givensite to achievea plausiblealignment. Se- and ground-finches,demonstrating that tree- quencedivergence was calculatedusing the number finchesare moreclosely related to oneanother of nucleotidedifferences between two sequences,in- than theyare to ground-finches.Therefore, we cludinggaps. Sequence divergence equals the num- can investigatethe relationshipsamong tree- ber of nucleotidedifferences divided by the total finchesin isolationfrom ground-finches.We numberof nucleotidesin the sequence,expressed as alsohave demonstrated the suitabilityof using a percentage. Sequencealignments from GeneWorkswere im- the WarblerFinch as an outgroupversus the ported into PAUP(Swofford 1993) and PHYLIP (Fel- ground-finchesor tree-finches. senstein 1993). Transitions and transversions were The seconddata set yielded 15 control-region equallyweighted because owing to the low frequen- haplotypes,which in conjunctionwith the out- cy of transversions(see Results), differential weight- grouphad 35 variablesites, 18 of whichwere ing did not affecttree topologies. Because the control phylogeneticallyinformative (Table 3). These region and 16SrRNA sequencesare not protein-cod- valueswere 21 and 17, respectively,when the ing, we did not differentiate between synonymous outgroupwas not included.The amountof se- versusnonsynonymous substitutions when analyz- quencedivergence among haplotypes, including thesesequences. To circumventthe dilemma of differentialweighting of gaps,we did two analyses ing theoutgroup, ranged from 0.25to 3.6%.All that led to inferred maximum-parsimonytrees: (1) nucleotidesubstitutions among the Warbler Finch and tree-finches were transitions. Two of onewith all gapstreated as informative sites, and (2) onewith all gapstreated as missing data. Maximum- the WarblerFinch haplotypes had a gap at one parsimonytrees were generatedin PAUP(Swofford, or two of the sites.The maximum-parsimony 1993).Neighbor-joining (using the Kimura two-pa- tree (Fig.2), maximum-likelihoodanalysis (not July1999] PhylogenyofDarwin's Finches 581

shown), and neighbor-joininganalysis (not shown)recovered the sametopology. Treating the gapsas eithermissing data or as informative sitesdid not affectthe topology. The tree-finchesare presentedas a monophyleticgroup relative to theWarbler Finch, regardlessof whetherthe Bananaquitor a War- bler Finchsubspecies is designatedas the outgroup.Platyspiza crassirostris is the only species of tree-finchthat appearsto be monophyletic. Camarhynchuspsittacula contains one individual that is allied with Cactospizapallida and one individual that has the samehaplotype as Ca- marhynchusparvulus. These levels of cohesion are reflectedin a comparisonof within- and among-speciessequence divergences. Only P. crassirostris,the monophyleticassemblage, has conspecificsequence deviations that are abso- lutely lower than the heterospecificsequence deviations(Table 4). Overall, the tree-finches are a very closelyrelated group of species. The Warbler Finch is divided into two dis- tinctlineages according to theislands that each inhabits(Fig. 3): (1) the SantaCruz clade(C. 0. olivacea)and (2) the Marchena,Espafiola, and Genovesa(M-E-G) islands clade (C. o.fusca, C. o. mentalis,and C. o. cinerascens).The control-re- gionsequence divergence within each of these two Warbler Finch clades is 0 to 0.7%, whereas the divergencebetween clades is 2.0 to 2.7%. The WarblerFinches are presentedas a para- phyleticgroup regardlessof whetherthe outgroup comprisesonly the Bananaquit,or the Bananaquitplus the WarblerFinches.

DISCUSSION

Relationshipsof the threegroups.•Our results do not disagreewith the traditionalview (Lack 1947) of the Warbler Finch as the basal taxon and the tree-finchesand ground-finchesas monophyleticsister groups (Fig. 1). We found no evidenceto supportStern and Grant's(1996) proposalthat the ground-finchesarose from the tree-finches,although as previouslystated, thesefindings must be consideredpreliminary because the inclusion of alternate or additional individualsmay alter the resultingphylogeny. These data do not support Yang and Patton's (1981)suggestion that the tree-finchesdiffer- entiatedmore recentlythan the ground-finches, becausethe tree-finch and ground-finch mtDNA sequencedivergence from the basal 582 FREELANDAND BOAG [Auk,Vol. 116

Tiaris bicolor 01

Platyspizacrassirostris 01

Camarhynchusparvulus 08

86 Cactospizapallida 01

64 [__Camarhynchus psittacula 01

74 Geospizadifficilis 02, G. magnirostris02, G. fortis 73, G. conirostris01 80 [__Geospiza scandens 24, G. fulig7nosa26

Certhidea olivacea 01

100 Certhidea olivacea 09

Coerebafiaveola 01 FIG.1. Maximum-parsimonytree showingthe relationshipsamong selected Darwin's finches, based on 665bp of controlregion and 16S rRNA sequence. Tiaris bicolor and C. fiaveola are outgroups. Bootstrap values areshown above branches (2,000 bootstrap replicates). Tree length = 53, CI = 0.811.See Appendix I for specimeninformation.

Warbler Finches is 0.9 to 2.6% and 1.1 to 2.3%, a monotypicgenus, which is consistentwith respectively.This geneticphylogeny was re- our results(Fig. 2). In contrast,Camarhynchus quiredto investigatepossible instances of mor- parvulusdoes not form a monophyleticgroup, phologicalconvergence or parallelism,because and the statusof C. psittacularemains equivo- thesetwo phenomenaare commonin passer- cal. The sharingof mitochondrialhaplotypes ines (Bledsoe1988) and can distort reconstruct- between species may result from ancestral ed phylogeniesthat are basedsolely on mor- polymorphismand incomplete lineage sorting, phologicaland behavioraldata. or hybridizationand introgression(Takahata Classificationof the tree-finches.--Withinthe and Slatkin 1984, Neigel and Avise 1986, Te- tree-finches,Platyspiza is currentlyclassified as gelstr6m1987). When two populationsor spe-

TABLE3. Nucleotidedifferences between the 15 tree-finches and Warbler Finch mtDNA control-region hap- lotypesand one Coerebafiaveola (Cfa) haplotype, based on 385 bp. See Figure 2 forhaplotype species iden- tities;"*" denotesan insertion/deletion,and "-" indicatesidentity with the first sequence.

Haplotype

H1 G C C AAC GAG TTTAAT T T G C T * * C C C T C G G TC T C AC H2 H3 T .... GA .... C- - T AT--C ...... H4 A- T G G - G A ...... C - A- - - A T ...... TGT H5 A - T G - A GA ..... C-A---A T ...... GT H6 A - T - G GA ..... C-A---A T ...... TGT H7 A - T G - A GA- --C-A-- A T ...... TGT H8 A - - G - - GA ...... C A --A T--C ..... T-T H9 A - - - G - - GA ...... C A --A T - - C - C T - T - T H10 AT- - G - - GA ...... AT--A T - - C ..... T- - H11 AT - G - - GA ...... AT AT - - C ..... T - T H12 AT .... G A ...... AT - - AT - - C ...... T - T H13 AT - G - - GA ...... A- AT - - C .... T - T H14 A - - - G ? GA ...... C - A AT - - C ...... H15 A- - G - - GA - - - C ..... AT - C ..... T - T Cfa - - - G G A AC GAT T G C A- - * T * TT T T C T - - C - July1999] PhylogenyofDarwin's Finches 583

Coerebafla•eola 01

Cactospizapallida 01 (H 10)

82 C. pallida 02 (HI 1)

C.pallida O3(H12)

Camarhynchuspsittacula01 (HI 3)

Platyspizacrassirostris 01 (H4)

-- P. crassirostris12 (H7)

-- P. crasslrostris03 (H5)

7081 I 55 P. crassirostris04 (H6)

Carnarhynchusparvulus08 (H9)

86 Carnarhynchuspstittacula 02 (H 14) C. parvulus03, C. parvulus07, C. psittacula04 (HS)

C. psittacula05 (H15) Certhideaolivacea 19, C. olivacea20, C. olivacea25 (SantaCruz) (H3)

94 -- C. olivacea27 (Genovesa)(H2) C. olivacea01 (Marchena), C. olivacea09 (Espanola)(H1)

FIG.2. Maximum-parsimonytree of the tree-finchand WarblerFinch control region haplotypes 1 to 15 (H1 to H15), with C.fiaveola as the outgroup.Tree length= 81, CI = 0.864.Bootstrap values written above branches(2,000 bootstrap replicates). See Appendix 1 for specimeninformation. ciesare first separated,their haplotypes are ex- gel and Avise1986, Pamilo and Nei 1988).As a pectedto be polyphyleticwith respectto one result,certain haplotypes will be maintained another,based simply on the chanceof certain in a population,and others will goextinct. Bar- ancestralhaplotypes occurring in more than ring selection,this is generallya randompro- one populationor species.Stochastic lineage cess.If populationsare sampledduring the sortingresults in a progressionfrom polyphyly stagesof polyphylyor paraphyly,then shared to paraphylyto monophyly(Tajima 1983, Nei- haplotypesmay be theresult of incompletelineage sorting (Avise et al. 1983, Moran and Kornfield 1993, P6rez-Sufirez et al. 1994). TABLE4. Comparisonof conspecificand heterospe- Giventhe closegenetic relationships of the cific sequencedivergences among four speciesof tree-finches. tree-finches,it is possiblethat some individuals in the Camarhynchusand Cactospizagenera Sequencedivergences (%) sharehaplotypes as a resultof incompletelin- Within Among eagesorting. Platyspiza is the only tree-finch species species genusthat appears monophyletic in thisstudy, Platyspizacrassirostris 0.26 to 0.78 1.00 to 2.3 and this seemsunlikely to haveresulted from Camarhynchusparvulus 0.00 to 0.78 0.00 to 2.3 eitherrelatively greater age or relativelysmall- Camarhynchuspsittacula 0.26 to 1.00 0.26 to 2.1 er populationsize in this species,two factors 0.26 to 0.52 0.26 to 2.6 Cactospizapallida that can acceleratehaplotype lineage sorting 584 FREELANDAND BOAG [Auk, Vol. 116

Pinta• • Genovesa Marchena Isabela

• Salvador

Fernandina $ •ltra ß • SantaCruz

San Cristobal 1 -

0 30 60 90km • SantaMaria 91 90 • Espafiola i i i i i

FIG3. Map of the GalapagosArchipelago. Warbler finch subspecies were sampled from the fourislands labeledin boldtype (Genovesa,Marchena, Espafiola, and SantaCruz).

(Avise 1994). Platyspizacrassirostris does not has played a role in the adaptiveradiation of appearto be older than the Camarhynchus-Cac-that group(Grant 1993, 1994; J. Freeland and P. tospizalineage, because the minimum diver- Boag unpubl. data). Unfortunately,although gencebetween P. crassirostris and the othertree- hybridizationamong the tree-fincheshas been finchspecies is 0.98%.This is considerablylow- documented (Lack 1947, Bowman 1983, Grant er than the maximuminterspecific divergence 1986), neither the frequencyof hybridization (1.8%)within the Cactospiza-Camarhynchuslin- nor the relativefitness of hybridsis known.The eage(Table 4). Furthermore,Platyspiza, Camar- tree-finchesand ground-fincheshave many hynchus,and Cactospizalikely had similarlong- similarities,including a high overallsequence term populationsizes during their evolution- similaritywithin eachgroup, a similarage, the ary histories,because population bottlenecks same number of species,and interspecifichy- presumably occurred on numerousoccasions bridization(Grant 1986,Yang and Patton1981, during the colonizationof new islands.In ad- J.Freeland and E Boagunpubl. data). Although dition, oncethe islandsare colonized,ecologi- furtherresearch is warranted,we suggestthat, cal conditionsgreatly deplete the sizes of es- like in ground-finches,hybridization played a tablishedpopulations in someyears (Boag and role in the adaptiveradiation of tree-finches. Grant 1981,Grant and Grant 1992).If we accept The extent of this role remains unclear, and it that similar evolutionarydemographic condi- is extremelydifficult with existingdata to dif- tions are likely to have prevailedfor all tree- ferentiatebetween the effects of lineagesorting finch genera,then the identificationof both and hybridization.Based on the mtDNA avail- monophyleticand paraphyleticgenera may be ablesequence data, no basisexists for dividing attributedto chanceand smallsample sizes. tree-finchesinto threegenera. The classification An alternateexplanation for the paraphylyof of P. crassirostrisinto a monotypicgenus is not Camarhynchusis historic and/or ongoinghy- disputedby our data,but the divisionof the re- bridization between C. parvulus,C. psittacula, maining speciesinto two generais dubious.Al- and Cactospizapallida. This phenomenonis thoughmolecular evidence seldom is consid- knownto occuramong the ground-finchesand eredto be the solecriterion for identifyingspe- July1999] Phylogenyof Darwin'sFinches 585

ciesand genera,it is reasonableto expecttwo and Espafiolafor up to 1 millionyears. In ad- generato be geneticallydistinguishable. It is dition, the distinction between the Santa Cruz possiblethat the CamarhynchuspsittacuIa indi- andthe M-E-G Warbler Finches agrees with the vidualthat is alliedwith the Cactospizapaiiida tentativefindings of Ford et al. (1974)and Po- individualsis an anomaly,although given our lans (1983),suggesting that the patternof mi- smallsample size, probability dictates that this tochondrialhaplotype sharing is similarto the is unlikelyto be the case.Although more indi- patternof nucleardifferentiation among these vidualsmust be sequencedbefore firm conclu- four Warbler Finch subspecies.These facts, sionscan be drawn,the availablegenetic data combinedwith the regularbottlenecks (Boag tend to support Lack's (1969) and Schluter's and Grant 1981, Grant and Grant 1992) that (1984)classification of the tree-finchesinto two wouldhave accelerated stochastic lineage sort- genera(Camarhynchus and PIatyspiza). ing, suggestthat at somepoint in their evolu- TheWarbler Finch subspecies.--Not all of the tionaryhistory, gene flow playeda role in the WarblerFinch subspecies are geneticallydis- maintenanceof genetichomogeneity among tinct (Fig. 2, Table3). The Marchenaand Es- the M-E-GWarbler Finch populations. pafiolapopulations share the samehaplotype, The M-E-G WarblerFinches lineage is con- and their sequencedivergence from the Gen- siderablyolder than the Santa Cruz Warbler ovesapopulation is 0.7%.This may result from Finchlineage. Both Espafiola and SantaCruz eitherretention of an ancestralhaplotype, or areamong the oldest islands in thearchipelago ongoinggene flow betweenthe Marchenaand (Cox1983), and the M-E-G clademay be a relic EspafiolaWarbler Finches. Once again, we can- of one of the first colonizations. The Santa Cruz not differentiatebetween the two processesWarbler Finch population is geneticallyinter- mediate to the tree-finches and the M-E-G War- with certainty,but theprobability of theformer is inverselyproportional to the time sincecol- blerFinch clade, and the Warbler Finch subspe- onizationand to the frequencyand durationof cies compriseanother paraphyleticspecies. Whereas a number of different scenarios could bottlenecks. Estimates of the time since the tree-finchand Warbler Finch lineages diverged explainthis, the mostintriguing possibility is will vary,depending on whichpopulations are thatthe original founder population on the Gal- apagosIslands bore a morphologicalresem- compared.The tree-finch/SantaCruz Warbler blance to the Warbler Finch. Future work on the Finchsequence divergence is 1.3to 2.3%,com- comparisonof the Geospizinaeto mainland pared with 2.3 to 3.9% for tree-finch/M-E-G specieswill allow elaborationon this idea. WarblerFinch divergence. The control-region We acknowledgethat the paucityof infor- sequenceused in this study evolvesapproxi- mative characters limits our conclusions. How- mately2.5 timesfaster than the rate of cyto- ever,a preliminaryinvestigation into the ge- chrome-b and 16S rRNA evolution in Darwin's neticdivergence of Darwin'sfinches using 16S finches(Freeland 1997), or 5% per million rDNA, cytochromeb, and control-regionse- years.If this is true,then the Warbler Finch and quencesdemonstrated that on the whole the tree-finchlineages split about 750,000years Geospizinaedo not comprisea geneticallydi- ago.Although this must be treated as a veryap- vergent group of birds (Freeland 1997). In all proximatefigure, it fallswithin the geological likelihood,genetic differentiation is limitedbe- agesof theislands, which range from 4 million causeof recencyof speciation(Yang and Patton yearsto lessthan 500,000years (Bailey 1976, 1981) and hybridization(Grant 1986, J. Free- Cox1983). This age also is closeto theestimate land and E Boagunpubl. data). Therefore, it of 570,000years since the divergenceof Certhi- seemsunlikely that more sequencingwould deafrom the otherGeospizines based on allo- clarify phylogeneticrelationships. We suggest zymedata (Yangand Patton1981). thatfuture work focus on developingmore in- If theWarbler Finch lineage is between0.5 to cisivemolecular markers that will showgreater 1 millionyears old, it seemssurprising that the differentiationamong genera, species,and sharingof haplotypesbetween two popula- populations. tionswould be the result of incompletelineage sorting,because this would meanthat the rel- ACKNOWLEDGMENTS ativelyrapidly evolving control region would We thank P. Grant, G. Seutin, Louisiana State Mu- haveremained unchanged in both Marchena seum, and Florida Museum of Natural History for 586 FREELANDAND BOAG [Auk, Vol. 116 providingblood samples.H. Gelter, S. Lougheed, FREELAND,J. R. 1997.The geneticevolutionary his- andD. Michaudprovided help and advicein thelab, tory of Darwin'sfinches (Aves: Geospizinae). and K. Conradand V. Friesensupplied useful com- Ph.D. dissertation,Queen's University, King- mentson the manuscript.Financial assistance came ston, Ontario. from NSERC (PTB) and Queen'sUniversity (JRF). GOULD,J. 1837.Description of new speciesof finches collectedby Darwin in the Galapagos.Proceed- LITERATURE CITED ingsof the ZoologicalSociety of London5:4-7. GRANT,P. R. 1986.Ecology and evolutionof Darwin's AVlSE,J. C. 1994.Molecular markers, natural history, finches.Princeton University Press, Princeton, andevolution. Chapman and Hall, New York. New Jersey. AVISE, J. C., J. E SHAPIRA,S. W. DANIEL, C. F. AQUAD- GRANT,P. R. 1993.Hybridization of Darwin'sfinches RO, AND g. g. LANSMAN. 1983. Mitochondrial on IslaDaphne Major, Galapagos. 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APPENDIX1. Speciesidentification, showing location and collectoror sourcefor eachsample.

Species Site collected• Collector/source• Coerebafiaveola 01 Peru LSU (B5168) Certhidea olivacea 01 Marchena G. Seutin Certhidea olivacea 09 Espafiola G. Seutin Certhidea olivacea 19 Santa Cruz (CDRS) P. Boag Certhidea olivacea 20 Santa Cruz (CDRS) P. Boag Certhidea olivacea 25 Santa Cruz (CDRS P. Boag Certhidea olivacea 27 Genovesa P. Boag Cactospizapallida 01 Santa Cruz (CDRS P. Boag Cactospizapallida 02 Santa Cruz (CDRS P. Boag Cactospizapallida 03 Santa Cruz (highlands) P. Boag Camarhynchusparvulus 03 Santa Cruz E Boag Camarhynchusparvulus 07 Santa Cruz (CDRS) P. Boag Camarhynchusparvulus 08 Santa Cruz (CDRS) E Boag Camarhynchuspsittacula 01 Marchena G. Seutin Camarhynchuspsittacula 02 Marchena G. Seutin Camarhynchuspsittacula 04 Santa Cruz P. Boag Camarhynchuspsittacula 01 Santa Cruz (highlands) P. Boag Geospizaconirostris 01 Espafiola G. Seutin Geospizadifficilis 02 Genovesa P. Boag Geospizafuliginosa 06 Santa Cruz P. Boag Geospizafortis 73 Daphne P. Boag Geospizamagnirostris 02 Marchena G. Seutin Geospizascandens 24 Santa Cruz P. Grant Platyspizacrassirostris 01 Santa Cruz P. Grant Platyspizacrassirostris 03 Marchena G. Seutin Platyspizacrassirostris 04 Marchena G. Seutin Platyspizacrassirostris 12 Santa Cruz E Boag Tiaris bicolor 01 Unknown FLMNH (331105) • LSU = Louisiana State Museum of Natural Sciences; CDRS = Charles Darwin Research Station; FLMNH = Florida Museum of Natural History.