Mitochondrial perspective on the phylogenetic relationships of the Parula Wood-warblers

Auk, The, Jan 2001 by Lovette, Irby J, Bermingham, Eldredge

Studies of intraspecific genetic variation in Neotropical resident birds have frequently revealed a high degree of phylogeographic differentiation (e.g. Capparella 1988, Hackett and Rosenberg 1990, Peterson et al. 1992, Bates and Zink 1994, Hackett 1996). That trend suggests tropical bird species are particularly likely to be subdivided geographically and that their constituent lineages tend to be evolutionarily old. Only a few counterexamples have been reported in which molecular surveys of Neotropical avian species did not reveal phylogeographic structure. Brawn et al. (1996) found no differences in mtDNA RFLP haplotypes between populations of three passerine species on the Perlas Islands in the Bay of Panama and conspecific populations on the nearby mainland. However, the geographic distances (

Here we explore the magnitude of mtDNA differentiation among geographically distant populations of the Tropical Parula (Parula pitiayumi), and we use mtDNA sequences to explore the relationship of P pitiayumi to the three other species currently placed in Parula (AOU 1998). Parula pitiayumi has a breeding distribution that extends from Northern Mexico south through much of South America and has been divided into 14 subspecies (Lowery and Monroe 1968). Based on its broad geographic distribution and high phenotypic diversity, our a priori expectation was that P pitiayumi would display evidence of a high degree of phylogeographic variation in mtDNA. Instead, we found surprisingly modest mitochondrial variation both within P pitiayumi and between that taxon and its congener P americana. As described below, our mtDNA-based phylogenetic reconstructions suggest that P. pitiayumi and P. americana are conspecific, and reconstructions that included the other two Parula species, as well as representatives of several other wood-warbler genera, suggest that the four species currently placed in the genus Parula (AOU 1998) do not form a monophyletic group.

Methods.-Accession numbers, sources, and tissue collecting localities are given in Table 1. Our study included 15 P pitiayumi individuals and two samples each of the other three currently recognized Parula species (P. americana, P gutteralis, and P superciliosa). Multiple representatives of two additional parulid genera (Vermivora ruficapilla, V peregrine, Dendroica adelaidae, D. tigrina) thought to be closely allied to Parula (see AOU 1998:533) and a Coereba flaveola sample were also included in some analyses. DNA extraction, amplification, and sequencing procedures followed standard laboratory protocols described elsewhere (Lovette et al. 1998, 1999). We obtained the complete sequence (842 nucleotides) of the overlapping mitochondrial ATPase 8 and ATPase 6 genes from all individuals. From one or two individuals per species, we also obtained an additional 2,797 nucleotides of mtDNA sequence representing the complete cytochrome b and ND2 genes and 613 nucleotides of the cytochrome oxidase I gene. Cytochrome b sequences from V peregrine were not included in analyses owing to the probable co-amplification of a nuclear-encoded pseudogene (Numt; I. Lovette unpubl. data) All sequences have been archived in GenBank (AF018097, 18207, 256468-256519).

We used PAUP* 4.0b2 (Swofford 1999) to estimate genetic distances among individuals and to generate phylogenetic hypotheses. In analyses that included distantly related taxa, distances were estimated using the Hasegawa-Kishino-Yamura (HKY) metric (Hasagawa et al. 1985) with the transition: transversion ratio set to 6 and the gamma parameter set to 0.12 with eight rate categories (see Lovette and Bermingham 1999). In ATPase-based comparisons of our focal taxa P pitiayumi and P americana, pairwise divergences among haplotypes were very low and we estimated distances based on the uncorrected % transition + transversion divergence across all sites. We employed two phylogenetic methods-maximum-likelihood (ML) and maximum-parsimony (MP)-to reconstruct phylogenetic relationships among mtDNA haplotypes using Paup*. ML analyses were conducted using the quartet puzzling search algorithm of Strimmer and von Haeseler (1996), with parameters set as described above. Exhaustive MP searches on the subset of taxa from which we obtained long mtDNA sequences were run using only transversion substitutions owing to the rapid saturation of mitochondrial transitions at large genetic distances. We weighted all changes equally in our MP heuristic searches of trees best representing the relationships of the P pitiayumi and P americana ATPase haplotypes. In both MP analyses, bootstrap values were based on 1,000 heuristic replications.

Results and discussion.-Phylogenetic reconstructions based upon long mitochondrial sequences indicated that the four species currently placed in Parula constitute two pairs of closely related taxa, P. gutteralis/P superciliosa and P pitiayumi/P americana. MtDNA sequence divergence between these two species pairs was high (9.4-10.0% HKY divergence). Rooted hypotheses of phylogenetic relationship (Fig. 1) that included representative Vermivora and Dendroica species suggested that those two Parula Glades are not each others' closest relatives, with gutteralis and superciliosa being allied to Vermivora and pitiayumi, and americana allied to Dendroica. We tested that inference by comparing observed trees to trees identified in searches where the four Parula species were constrained to be monophyletic. In transversion parsimony searches, the observed tree (249 steps) was considerably shorter than the shortest constraint tree (276 steps). Similarly, a likelihood ratio test (Kishino and Hasegawa 1989) based on those alternative topologies demonstrated that the observed HKY ML tree (-Ln 10,597) was a highly significant improvement over the constraint tree (-Ln 10,705; T = 5.49; P