Abstract
ABSTR^CT.--The Galapagos Darwin's finches (Geospizinae) have been classified as three major groups based on morphology and behavior: ground-finches, tree-finches, and the Warbler Finch (Certhidea olivacea). Little is known about the evolutionary relationships within and among these groups, which is partly due to the lack of a phylogeny based on molecular sequence data. We used mitochondrial sequence data to reconstruct a phylogeny of Darwin's finches. These data show that within the tree-fin&es, only one genus is conclusively monophyletic, and another is conclusively paraphyletic. It may be appropriate to uphold the classification of the tree-finches into two genera. The Warbler Finch complex is paraphyletic, as revealed by two divergent genetic lineages contained within this species. Stochastic lineage sorting within relatively recently diverged species and interspecific and intergeneric hybridization are the two most likely explanations for the sharing of haplotypes among taxa. The subfamily Geospizinae comprises 14 nominate species, 13 of which inhabit the Galapagos Archipelago. Darwin's finches have been divided into three groups based on morphology and behavior: ground-finches, treefinches, and the Warbler Finch (Certhidea olivacea; Table 1 ). The ground-finches (Geospiza) comprise one genus and six species that are finch-like in appearance, particularly with respect to their bills, and spend much of their time foraging on the ground. The tree-finches ( Camarhynchus, Platyspiza, and Cactospiza) comprise six species, but the number of genera osciliates between one and three (see below). These six species have bills intermediate to those of ground-finches and the Warbler Finch, and although they occasionally forage on the ground in a manner similar to ground-finches, they spend much of their time in foliage and vegetation exhibiting behavior similar to that 577

sight into a number of aspects of evolution and speciation, including adaptive radiation.
The subfamily Geospizinae comprises 14 nominate species, 13 of which inhabit the Galapagos Archipelago. Darwin's finches have been divided into three groups based on morphology and behavior: ground-finches, treefinches, and the Warbler Finch (Certhidea olivacea; Table 1 ). The ground-finches (Geospiza) comprise one genus and six species that are finch-like in appearance, particularly with respect to their bills, and spend much of their time foraging on the ground. The tree-finches ( Camarhynchus, Platyspiza, and Cactospiza) comprise six species, but the number of genera osciliates between one and three (see below). These six species have bills intermediate to those of ground-finches and the Warbler Finch, and although they occasionally forage on the ground in a manner similar to ground-finches, they spend much of their time in foliage and vegetation exhibiting behavior similar to that First, we address evolutionary relationships of the three groups. Existing phylogenies consistently treat the Warbler Finch as the basal taxon, but the positions of the tree-finches and ground-finches remain equivocal. The latter groups generally are treated as monophyletic sister groups (Lack 1947, Schluter 1984); however, it has also been suggested that the treefinches are ancestral to the ground-finches (Stern and Grant 1996) , and under this scenario it is possible that the tree-finches are a paraphyletic group. Prior to investigating evolutionary relationships within the tree-finches, we wished to ascertain whether taxonomic separation of the tree-finches and ground-finches, based on morphological data, was reflected by their DNA sequences. Because Darwin's finches underwent adaptive radiation relatively recently (Yang and Patton 1981), there may have been insufficient time for complete lineage sorting to have occurred following speciation, which could result in the sharing of haplotypes among species (Neigel and Avise 1986). In addition, hybridization has been documented in Darwin's finches (Grant 1986), and this may lead to introgression of mitochondrial haplotypes from one species to another (Tegelstr6m 1987). We reconstructed a phylogeny of the three groups using mitochondrial sequence data, which allowed us to infer relative levels of genetic relatedness within and among the tree-finches, ground-finches, and the Warbler Finch.
The second question we address pertains to the taxonomy of the tree-finches. Using morphological and behavioral data, Lack (1947) placed all tree-finches in Camarhynchus, although he later modified this and placed Platyspiza crassirostris (Vegetarian Tree-Finch) in a monospecific genus (Lack 1969). This classification has been upheld by Schluter (1984) , again based on morphological data. More common in recent literature is the division of the tree-finches into the genera Camarhynchus, Platyspiza, and Cactospiza, as shown in Table 1 . All six species appear to be closely related, as evidenced by the fact that allozyme data were not sufficiently differentiated to resolve relationships at either the genus or species level (Yang and Patton 1981). We used sequence data to clarify classification of the tree-finches.
The third question is directed at the Warbler Finch, which possibly is the most enigmatic Sequence alignment and phylogenetic analysis.--Gels were scored manually, and sequences were aligned using GeneWorks (IntelliGenetics, Inc.). Few insertions/deletions (gaps) occurred, but the gene alignment program never had to insert more than one gap at any given site to achieve a plausible alignment. Sequence divergence was calculated using the number of nucleotide differences between two sequences, including gaps. Sequence divergence equals the number of nucleotide differences divided by the total number of nucleotides in the sequence, expressed as a percentage.
Sequence alignments from GeneWorks were imported into PAUP (Swofford 1993 ) and PHYLIP (Felsenstein 1993). Transitions and transversions were equally weighted because owing to the low frequency of transversions (see Results), differential weighting did not affect tree topologies. Because the control region and 16S rRNA sequences are not protein-coding, we did not differentiate between synonymous versus nonsynonymous substitutions when analyzing these sequences. To circumvent the dilemma of differential weighting of gaps, we did two analyses that led to inferred maximum-parsimony trees: (1) one with all gaps treated as informative sites, and (2) one with all gaps treated as missing data. Maximumparsimony trees were generated in PAUP (Swofford, 1993) . Neighbor-joining (using the Kimura two-parameter distance measure) and maximum-likelihood (using empirical base frequencies and a single substitution rate category) trees were generated in PHY-LIP (Felsenstein, 1993).
RESULTS
The first data set provided 23 phylogenetically informative sites (18 excluding the outgroups) and 44 variable sites (22 excluding the outgroups). Twenty percent of the nucleotide substitutions were transversions, but only 5% of these involved comparisons within the Darwin's finches. Some taxa had a single deletion.
Only two different sequences were found in all six species of ground-finches. The sequence divergence was 0.2 to 2.4% within the Darwin's finches and 2.6 to 3.8% between the Darwin's finches and the outgroups ( Table 2 ). The relatively close relationship among the outgroups and the Geospizinae justifies the choice of outgroup. Maximum-parsimony (Fig. 1) , maximum-likelihood (not shown), and neighborjoining (not shown) trees showed the same topology. In addition, the same topology resulted when gaps were treated as either informative or missing sites, and when the transition/transversion ratio was set to either 1.0 or 4.0.
The phylogeny shows that the Warbler Finches are the sister group to the other Darwin's finches. The ground-finches form a well-supported clade within the cluster of tree-finches and ground-finches, demonstrating that treefinches are more closely related to one another than they are to ground-finches. Therefore, we can investigate the relationships among treefinches in isolation from ground-finches. We also have demonstrated the suitability of using the Warbler Finch as an outgroup versus the ground-finches or tree-finches.
The second data set yielded 15 control-region haplotypes, which in conjunction with the outgroup had 35 variable sites, 18 of which were phylogenetically informative (Table 3) . These values were 21 and 17, respectively, when the outgroup was not included. The amount of sequence divergence among haplotypes, including the outgroup, ranged from 0.25 to 3.6%. All nucleotide substitutions among the Warbler Finch and tree-finches were transitions. Two of the Warbler Finch haplotypes had a gap at one or two of the sites. The maximum-parsimony tree (Fig. 2) , maximum-likelihood analysis (not shown), and neighbor-joining analysis (not shown) recovered the same topology. Treating the gaps as either missing data or as informative sites did not affect the topology.
The tree-finches are presented as a monophyletic group relative to the Warbler Finch, regardless of whether the Bananaquit or a Warbler Finch subspecies is designated as the outgroup. Platyspiza crassirostris is the only species of tree-finch that appears to be monophyletic. Camarhynchus psittacula contains one individual that is allied with Cactospiza pallida and one individual that has the same haplotype as Camarhynchus parvulus. These levels of cohesion are reflected in a comparison of within-and among-species sequence divergences. Only P. crassirostris, the monophyletic assemblage, has conspecific sequence deviations that are absolutely lower than the heterospecific sequence deviations (Table 4) . Overall, the tree-finches are a very closely related group of species. (Fig. 1) . We found no evidence to support Stern and Grant's (1996) proposal that the ground-finches arose from the tree-finches, although as previously stated, these findings must be considered preliminary (Avise 1994). Platyspiza crassirostris does not appear to be older than the Camarhynchus-Cactospiza lineage, because the minimum divergence between P. crassirostris and the other treefinch species is 0.98%. This is considerably lower than the maximum interspecific divergence (1.8%) within the Cactospiza-Camarhynchus lineage (Table 4) is extremely difficult with existing data to differentiate between the effects of lineage sorting and hybridization. Based on the mtDNA available sequence data, no basis exists for dividing tree-finches into three genera. The classification of P. crassirostris into a monotypic genus is not disputed by our data, but the division of the remaining species into two genera is dubious. Although molecular evidence seldom is considered to be the sole criterion for identifying spe-cies and genera, it is reasonable to expect two genera to be genetically distinguishable. It is possible that the Camarhynchus psittacuIa individual that is allied with the Cactospiza paiiida individuals is an anomaly, although given our small sample size, probability dictates that this is unlikely to be the case. Although more individuals must be sequenced before firm conclusions can be drawn, the available genetic data tend to support Lack's (1969) and Schluter's (1984) classification of the tree-finches into two genera (Camarhynchus and PIatyspiza).
The Warbler Finch is divided into two distinct lineages according to the islands that each inhabits (Fig
The Warbler Finch subspecies.--Not all of the Warbler Finch subspecies are genetically distinct (Fig. 2, Table 3 Freeland and E Boag unpubl. data). Therefore, it seems unlikely that more sequencing would clarify phylogenetic relationships. We suggest that future work focus on developing more incisive molecular markers that will show greater differentiation among genera, species, and populations.

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