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We interpreted the results of nuclear DNA sequencing to be inconsistent with the recognition of California Gnatcatcher (Polioptila californica) subspecies. McCormack and Maley (2015) suggested that our data did support 2 taxa, one of which was P. c. californica, listed as Threatened under the Endangered Species Act (ESA). We summarize here how 2 sets of researchers with access to the same data reached different conclusions by including different analyses. We included the southern subspecies' boundary from the taxonomy of Atwood (1991), the taxonomic basis for the ESA listing, which resulted in an Analysis of Molecular Variance that provided no support for subspecies. In contrast, using a novel taxonomic hypothesis without precedent in the literature, McCormack and Maley (2015) found statistically significant FST values for 2 loci, which they suggested supports P. c. californica. We propose that our mitochondrial and nuclear data had sufficient power to capture geographical structure at either the...

An important step in conservation is to identify whether threatened populations are evolutionarily discrete and significant to the species. A prior mitochondrial DNA (mtDNA) phylogeographic study of the California Gnatcatcher (Polioptila californica) revealed no geographic structure and, thus, did not support the subspecies validity of the threatened coastal California Gnatcatcher (P. c. californica). The U.S. Fish and Wildlife Service concluded that mtDNA data alone were insufficient to test subspecies taxonomy. We sequenced eight nuclear loci to search for historically discrete groupings that might have been missed by the mtDNA study (which we confirmed with new ND2 sequences). Phylogenetic analyses of the nuclear loci revealed no historically significant groupings and a low level of divergence (G ST = 0.013). Sequence data suggested an older population increase in southern populations, consistent with niche modeling that suggested a northward range expansion following the Last Glacial Maximum...

Previous microsatellite analysis (Barr et al. 2008) of the endangered Black-capped Vireo (Vireo atricapilla) revealed a low (FST = 0.021) but statistically significant level of population differentiation with isolation by distance (IBD); most pairwise FST values among populations were significant. No clear geographic pattern was detected, although populations in Oklahoma were somewhat differentiated, as expected from the observed pattern of IBD. Because nuclear loci, including microsatellites, coalesce more slowly than mtDNA, we sequenced the mitochondrial ND2 gene for 108 individuals sampled from breeding populations in Oklahoma, Texas, and Mexico to determine whether there was a clearer geographic pattern than that resolved by the microsatellite allele-frequency data. We obtained an FST value of 0.024 (P = 0.08), confirmed IBD, but also did not detect geographic structuring in the haplotype network. The authors of the microsatellite study suggested that the significant FST value indicated that...

We sequenced the complete mitochondrial cytochrome-b gene (1,143 nucleotides) for representatives of each species in the cardinalid genera Passerina (6 species), Guiraca (1 species), and Cyanocompsa (3 species), and used a variety of phylogenetic methods to address relationships within and among genera. We determined that Passerina, as presently recognized, is paraphyletic. Lazuli Bunting (P. amoena) is sister to the much larger Blue Grosbeak (Guiraca caerulea). Indigo Bunting (P. cyanea) and Lazuli Bunting are not sister taxa as generally thought. In all weighted parsimony trees and for the gamma-corrected HKY tree, Indigo Bunting is the sister of two sister groups, a “blue” (Lazuli Bunting and Blue Grosbeak) and a “painted” (Rosita's Bunting [P. rositae], Orange-breasted Bunting [P. leclancherii], Varied Bunting [P. versicolor], and Painted Bunting [P. ciris]) clade. The latter two species form a highly supported sister pair of relatively more recent origin. Uncorrected (p) distances for ingroup (Passerina and Guiraca) taxa range from 3.0% (P. versicolor–P. ciris) to 7.6% (P. cyanea–P. leclancherii) and average 6.5% overall. Assuming a molecular clock, a bunting “radiation” between 4.1 and 7.3 Mya yielded four lineages. This timing is consistent with fossil evidence and coincides with a late-Miocene cooling during which a variety of western grassland habitats evolved. A reduction in size at that time may have allowed buntings to exploit that new food resource (grass seeds). We speculate that the Blue Grosbeak subsequently gained large size and widespread distribution as a result of ecological character displacement.

We examined patterns of variation in skeletal morphometrics (29 characters), allozymes (34 loci), mitochondrial DNA (mtDNA) restriction sites (n = 74) and fragments (n = 395), and mtDNA sequences (1,739 bp from cytochrome b, ND2, ND6, and the control region) among all species of Toxostoma. The phenetic pattern of variation in skeletal morphometrics generally matched traditional taxonomic groupings (based on plumage patterns) with the exceptions of T redivivum, which because of its large size clusters outside of its proper evolutionary group (lecontei), and T. occelatum, which did not cluster with T. curvirostre. Skull characters contributed highly to species discrimination, suggesting that unique feeding adaptations arose in different species groups. Although genetic variation was detected at isozyme loci (average heterozygosity = 3.6%), these data yielded little phylogenetic resolution. Similarly, mtDNA restriction sites were relatively uninformative; hence, phylogenetic conclusions were based on sequence data. Phylogenetic analyses confirmed the monophyly of these traditionally recognized assemblages: rufum group (T. rufum, T. longirostre, and T. guttatum), lecontei group (T lecontei, T crissale, and T. redivivum), and cinereum group (T. bendirei and T. cinereum). The cinereum and lecontei groups appear to be sister lineages. Monophyly of the curvirostre group (which also includes T. occelatum) was not confirmed. Sequence data suggest that T. occelatum and T. curvirostre, which differ by 7.7% sequence divergence, are probably most closely related to the rufum group. Toxostoma rufum and T. longirostre have similar external appearances and differ by 5.0%. Toxostoma guttatum is restricted to Cozumel Island and often is considered a subspecies of T. longirostre; it differs by more than 5% from the other two members of the rufum group and is a distinct species constituting the basal member of this group. The phenotypically distinctive T. bendirei and T. cinereum differ in sequence divergence by only 1.6%. Overall, mtDNA distances computed from coding genes (mean 8.5%) exceeded distances computed from the control region (mean 7.6%), contrary to expectation. Because neither allozymes nor mtDNA could unambiguously resolve the placement of T. occelatum and T. curvirostre, a scenario involving contemporaneous speciation is suggested. Application of a molecular clock suggested that most speciation occurred in the late Pliocene or early Pleistocene.

We obtained 1,040 bp of sequence from the mitochondrial DNA (mtDNA) genes cytochrome b (cyt b; 736 bp) and NADH-subunit 2 (ND2; 304 bp) to address phylo- genetic relationships among the four species in the Scaled Quail complex. California Quail (Callipepla californica) and Gambel's Quail (C. gambelii) were sister taxa, whereas the rela- tionships of the Elegant Quail (C. douglasii) and Scaled Quail (C. squamata) were unclear; they might be sister species, or Elegant Quail might be the sister to California plus Gambel's quails. A third, less-likely alternative predicts a contemporaneous origin of Elegant Quail, Scaled Quail, and the ancestor of California and Gambel's quail. The latter phylogenetic hy- pothesis, however, matches Hubbard's (1973) biogeographic model. Irrespective of which biogeographic hypothesis is correct, calibration of mtDNA genetic distances suggests that the speciation events are much older than the late Pleistocene dates given by Hubbard. Cal- ibration of the rate of mtDNA (cyt b, ND2) evolution based on dating of fossil remains of the extinct species Cyrtonyx cooki suggested a rate of 2% per million years. Northern Bobwhite (Colinus virginianus), Mountain Quail (Oreortyx pictus), and Montezuma Quail (Cyrtonyx montezumae) were successively more distantly related to the Scaled Quail complex. Phylo- genetic trees derived from allozymes (Gutierrez et al. 1983) and mtDNA sequences were topologically identical, suggesting that both types of gene trees recover the species tree. Re

Although many aspects of hummingbird biology have been studied, few recent analyses of higher-level systematic relationships exist. Based on morphology, it has been hypothesized that the Trochilidae includes six major clades. We used starch-gel electrophoresis to construct and test phylogenetic hypotheses for representatives of the six clades, using two species of swifts (Apodidae) as outgroups. Of 45 loci scored, 38 were polymorphic. The average Nei's genetic distance (D) among 14 hummingbird taxa was 0.625; D averaged 1.61 between the swifts and hummingbirds. These distances are large, and are consistent with other nonpasserine groups, suggesting that hummingbird taxa are relatively old. Phylogenetic analyses generally were consistent with the hypothesis that hermits are a sister group to all other trochilines. Within the Trochilinae, two broad groups are recognized, here called trochiline-A and B, which correspond to the morphologically determined "primitive" and "advanced" trochiline groups of Zusi and Bentz (1982). Androdon aequatorialis is genetically distinct but generally aligns with the trochiline-A group. Within the trochiline-B group, four radiations hypothesized by Zusi (pers. comm.), here called Bee, Amazilia ("Emeralds"), Andean, and High Andean, were corroborated by our analyses. Our distance analysis suggests a phylogenetic pattern consistent with that derived from Sibley and Ahlquists' (1990) and Bleiweiss et al.'s (1997) DNA-DNA hybridization studies. Received 31 October 1996, accepted 20 June 1997. THE HUMMINGBIRDS (TROCHILIDAE) form one of the largest bird families, with approximately 325 species. Although many aspects of hummingbird biology have been studied, few higher-level studies of systematic relationships exist. Early taxonomists (Gould 1861, Boucard 1895, Simon 1921, Peters 1945, Zimmer 19501953) provided species descriptions and general details of geographic variation in most species, but they did not explicitly identify systematic relationships. Recently, several approaches have been used to identify phylogenetic patterns within parts of this family: comparative analysis of vocalizations and mating behavior (Schuchmann unpubl. data), external morphology and ecology (Graves 1980, 1986; Stiles 1983, 1996; Hinklemann 1989), comparative myology (Zusi and Bentz 1982), protein electrophoresis (Gerwin and Zink 1989, Gill and Gerwin 1989 ), mitochondrial DNA sequences (Hernandez-Banos et al. unpubl. data), and 2 Present address: North Carolina State Museum of Natural Sciences, P.O. Box 29555, Raleigh, North Carolina 27626, USA. E-mail: ncs1416@interpath.com 3Address correspondence to this author. Present address: James Ford Bell Museum, University of Minnesota, 100 Ecology Building, St. Paul, Minnesota 55108, USA. E-mail: rzink@biosci.umn.edu DNA-DNA hybridization (Sibley and Ahlquist 1990; Bleiweiss et al. 1994, 1997). Most of these studies were not comprehensive at the level of the family. However, the study by Bleiweiss et al. (1997), which involved 26 species, provided a molecular phylogenetic hypothesis for the major clades in the family. We report an allozymic survey of major groups of hummingbirds designed to test phylogenetic hypotheses derived from previous morphological and DNA-DNA hybridization analyses. Our study was based in part on Zusi and Bentz's (1982) hypothesis of higher-level groups in the Trochilidae. They identified four major groupings: hermits (Phaethorninae), "primitive" trochilines, and two groups of "advanced" trochilines. The terms primitive and advanced are reserved for discussion of characters and are misleading without an in-depth phylogenetic analysis; we refer to the two groups as trochiline-A (Zusi and Bentz's [1982] primitive group) and trochiline-B (their advanced group). The widespread notion that the hermits represent the "primitive" hummingbirds is incorrect in one sense: if these are sister clades, each is by definition the same age. Phylogenetic analysis is required to show that the hermit clade retains a disproportionate number

We sequenced 432 base pairs (bp) of the mitochondrial DNA (mtDNA) cyto- chrome-b gene in all recognized biological species in the genera Zonotrichia, Passerella, and Melospiza, as well as Junco hyemalis and Pipilo chlorurus. Our goals were: to estimate the phylogenetic pattern within and among genera; to compare our estimate with previous estimates based on allozymes and mtDNA restriction sites; to map morphometric distances onto the phylogenetic hypothesis; and to determine the extent of geographic variation in two polytypic species, the Fox Sparrow (Passerella iliaca) and Song Sparrow (Melospiza melodia). There was no geographic pattern to cytochrome-b variation within the Song Sparrow, whereas four mtDNA lineages of Fox Sparrows were found; these results corroborate those obtained from mtDNA restriction-site data. Analysis of cytochrome b yielded 14 equally-parsimonious trees. Although mtDNA and allozyme trees were statistically congruent, they differed some- what, and the data were combined to estimate phylogeny; two equally-parsimonious trees resulted. The consensus tree indicated the following relationships: within Melospiza, the pattern is {Song Sparrow {Swamp Sparrow (M. georgiana), Lincoln's Sparrow (M. lincolnii)} }; Junco and Zonotrichia are sister genera; within Zonotrichia, the pattern is (Rufous-collared Sparrow (Z. capensis) {White-throated Sparrow (Z. albicollis) (Harris' Sparrow (Z. querula) (White- crowned Sparrow (Z. leucophrys), Golden-crowned Sparrow (Z. atricapilla)} }} }; the data could not reliably resolve relationships among the other genera. In general, restriction sites and cytochrome-b sequence data yielded congruent phylogenies. Morphometric distances mapped onto the phylogenetic hypothesis revealed instances in which molecular and phenotypic evolution proceeded at different rates, except within Melospiza, where the two data sets yielded

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We used 19 restriction endonucleases to analyze patterns of cleavage site variation in the mitochondrial DNA (mtDNA) of the five recognized species of Zonotrichia. Each species possesses a unique mtDNA profile. Relative to most congeneric avian comparisons, these species are closely related, with an average percent nucleotide divergence of 4.1%. The Golden-crowned Sparrow (Z. atricapilla) and White-crowned Sparrow (Z. leucophrys) differ by a single restriction site (out of 122), which yields an estimate of 0.11% sequence divergence. These species are sister species in all phylogenetic analyses (Wagner and Dollo parsimony, bootstrapping, distance analyses), and White-throated Sparrow (Z. albicollis) is the sister to these, followed by Harris' Sparrow (Z. querula) and Rufous-collared Sparrow (Z. capensis). The phylogeny based on mtDNA restriction sites differs from that based on allozymes (Zink 1982) in that Harris' and White-throated sparrows are reversed in this sequence. In general, however, the allozyme and mtDNA phylogenies are highly concordant. Similarities in song between White-throated and Golden-crowned sparrows can be interpreted as ancestral retentions. The song of the White-crowned Sparrow is derived relative to other north temperate congeners, and song dialects appear to have evolved independently in White-crowned and Rufous-collared sparrows. Based on the mtDNA data, patterns of hybridization are inconsistent with phylogenetic relationships, and we suggest that hybridization should not be used in taxonomic decisions. Received 4 September 1990, accepted 26 December 1990. A PHYLOGENY not only provides a trace of a lineage's evolutionary diversification, but it also serves as a framework for the interpretation of the evolution of characters and suites of characters. Molecular methods have been widely heralded because they provide a set of characters that have a simple, known genetic basis, and are genetically independent. Furthermore, it is likely that molecular characters are selectively neutral (e.g. Barrowclough et al. 1985), and thus their evolution can be predicted by explicit evolutionary models (Nei 1987) and can serve as an approximate molecular clock. Although molecular characters have disadvantages for phylogeny inference (Hillis 1987), they are used with increasing frequency as a sort of "null" phylogenetic hypothesis, both for estimation of evolutionary history and interpretation of character evolution. Whether molecular analyses actually produce superior estimates of phylogeny remains to be documented, and tests will likely involve comparison of independently derived phylogenies (including other molecular estimates) of common sets of organisms. In avian systematics, protein electrophoresis has been used to produce a number of phylogenetic estimates. In many interspecific comparisons of avian protein evolution (e.g. Avise et al. 1980, Johnson and Zink 1983), there was little variation partitioned among species. This does not inspire confidence in any pattern (without independent corroboration from other data sets). Other studies of allozyme variation that used "phylogenetic" and phenetic methods often produced results that surprised many taxonomists, including the biochemical systematists themselves (e.g. Johnson et al. 1988, Dittmann et al. 1989). However, few protein-based phylogenetic estimates for birds were tested for robustness or confidence with data-resampling techniques such as bootstrapping (Felsenstein 1985b). Therefore, the confidence in these protein-based branching diagrams as phylogenies is unknown. One method of assessing confidence is to compare phylogenetic estimates derived from independent data sets. Zink and Avise (1990) compared allozyme and mitochondrial DNA (mtDNA) evolution in the genus Ammodramus and found a high degree of congruence. Zink and Dittmann (1991) found a generally high correspondence between allozymes and mtDNA in towhees (Pipilo). In these examples, the phylogenetic signal appeared in both of the genetically independent data sets. However, in Ammodramus and Pipilo, unlike many avian studies, there was considerable allozymic dif578 The Auk 108: 578-584. July 1991 This content downloaded from 157.55.39.208 on Fri, 14 Oct 2016 04:21:30 UTC All use subject to http://about.jstor.org/terms July 1991] mtDNA Evolution in Zonotrichia 579 ferentiation, which rather strongly supported a particular phylogeny. Congruence of mtDNA and allozyme results implies confidence in phylogenetic conclusions, whereas confidence in a single data set is difficult to assess (Felsenstein 1985b). The genus Zonotrichia contains five species, several of which have been studied extensively by behaviorists and ecologists. For example, vocal dialects in the White-crowned Sparrow (Z. leucophrys) have been studied thoroughly (Kroodsma et al. 1985). Vocalizations and dialects have also been studied in the Rufous-collared Sparrow (Z. capensis; Handford and Nottebohm 1976). Interpretation of the evolution of vocal as well as morphological and ecological conditions in Zonotrichia requires an independently derived phylogeny (Felsenstein 1985a, Mickevich and Weller 1990). Zink (1982) used allozymes and morphometrics to assess the systematic status of species in Zonotrichia, and he concluded White-crowned and Golden-crowned (Z. atricapilla) sparrows were sister taxa, followed by Harris' Sparrow (Z. querula), the White-throated Sparrow (Z. albicollis), and the Rufous-collared Sparrow. This arrangement conflicted with previous opinions (Paynter 1964, Short and Simon 1965). For example, Mayr and Short (1970) stated that Golden-crowned and White-throated sparrows were sister species, and together with White-crowned Sparrows formed a species group. By default, Harris' and Rufous-collared sparrows would be outliers to this group. Evidence cited for the close relationship between White-throated and Golden-crowned sparrows was an essentially allopatric distribution; these species also exhibit similarities in male primary song. Mayr and Short (1970) suggested that hybridization between the White-throated Sparrow and Darkeyed Junco (Uunco hyemalis) revealed a close relationship between these taxa. For birds, mtDNA has been shown to provide more characters for systematic analysis than allozymes (Avise and Zink 1988, Zink 1991). This corresponds with the relatively fast rate of mtDNA evolution observed in birds (Shields and Helm-Bychowski 1988) and other vertebrates (Avise 1986, Moritz et al. 1987). We used restriction endonuclease analysis of purified mtDNA to provide information for the inference of phylogenetic relationships among species of Zonotrichia. In so doing, we test the allozyme-based tree of Zink (1982) and the taxonomic conclusions of others, provide data on evolution of this organellar piece of DNA, and comment on the evolution of behavioral and distributional characteristics in the genus.

Protein electrophoresis has been used frequently in the study of genetic variation within and among natural populations (Lewontin 1974, Avise and Aquadro 1982, Nevo et al. 1984). A goal of protein electrophoretic studies is to describe patterns of genetic variation and deduce historical processes (such as fragmentation of populations and gene flow) that have produced such patterns (Felsenstein 1982). A second, but relatively little-studied, aspect of the geography of genetic variation is the temporal stability of patterns of gene frequencies. There are both systematic and evolutionary implications of temporal variation in allozymic frequencies. If samples are taken from different sites in different years, temporal variation might confound systematic interpretation of geographic patterns (Zink 1983). Many investigators have implicated natural selection in the maintenance of enzyme polymorphisms (Mitton and Grant 1984, Mueller et al. 1985, Barker et al. 1986; but see Zink et al. 1985). A correlate of genetic polymorphisms influenced by selection is temporal change in gene frequencies at a site, resulting from directional natural selection. Thus, it is of interest to examine the temporal stability of gene frequencies. Previous studies of temporal variation in gene frequencies have either considered relatively few generations, limited geographic areas, or few loci (Baker and Fox 1978, Gyllensten 1985, Henderson 1977, McCauley et al. 1988, Redfield 1974). In this study, we assessed temporal and geographic variation in gene frequencies in population samples of the Fox Sparrow (Passerella iliaca). A previous allozymic survey (Zink 1986), based on samples taken in 1978-1980 in California, Oregon, and Nevada, revealed essentially no geographic differentiation in gene frequencies; the FST value (derived from 31 samples of individuals, 38 presumptive genetic loci, and a total of 619 individuals) was 0.0135 ? 0.0033 (SE). At some of these sites, Zink (1983) detected morphometric differentiation between samples taken in the mid-1920s and 19781980, a span of 50 years. We used protein electrophoresis to compare levels and patterns of genetic variation in seven samples (from California and Nevada) of Fox Sparrows taken in 1988 with those taken in 1978-1980, a span of 8-10 generations. In addition, we sampled two new sites (Table 1) geographically distant from the original sample sites to assess geographic differentiation on a larger scale. Approximately one half of our aqueous tissue extracts were obtained during preparation of tissues for mitochondrial DNA (mtDNA) analysis (see Avise and Zink 1988), and the others were prepared as outlined by Zink (1986). Methods for protein electrophoresis are given in Zink (1986). For the samples collected in 1988, we surveyed only the loci found to be polymorphic in the earlier study (Table 1). Because sample sizes for our recent samples are relatively small (1120) and because Zink (1986) showed that the same allele was the common allele at all sites for each locus, we compared only the frequencies of common alleles among sites and across generations; noncommon alleles were pooled. We performed 2 x 2 G-tests (Sokal and Rohlf 1981) for each locus and site by tallying the number of common and noncommon alleles in the "old" (1978-1980) and "new" (1988) samples. Such multiple testing requires adjustment of significance values. We followed Rice's (1989) suggestion and divided the alpha level of 0.05 by the number of G-tests (72) to get a significance level (0.0007) appropriate for our multiple G-tests. The 19 sites at which the common allele was fixed in samples taken in both years were not tested, which explains why we tested 72 values and not 91 (13 loci x 7 sites). Rice (1989) noted that, in a series of tests such as ours, if the smallest probability value is not significant, then all entries in the table are judged nonsignificant, regardless of the significance levels of individual tests. We recognize that this is a conservative test, and one that requires perhaps larger samples than we have to detect significant effects. Therefore, we computed a G-value by summing all 72 tests to test for a table-wide effect of temporal variation. We report the observed directcount heterozygosity value for each sample (see Zink 1986). We also computed separate FsT values (each corrected for sampling a finite number of individuals (Wright 1978)) for the old samples, new samples, and the new samples plus those from Wyoming and Colorado to evaluate temporal and geographic variation. Temporal variation.-G-tests revealed no significant values (at the corrected P-value) in the occurrence of common alleles between old and new samples (Table 1). We found that 7 of the 72 (10%) tests produced significant G-values at the 0.05 level when each test was evaluated independently; hence, correction for multiple testing influences interpretation (Rice 1989). We also tested for locus and site effects by summing G-values across loci and across sites, and found only one significant value, that for the locus Lgg (Table 1). We noted during our study that gel patterns at Lgg were difficult to interpret for individuals prepared for mtDNA analysis relative to standard preparations, an apparent artifact of the mtDNA buffer. It is unclear, therefore, if the marginally significant value for Lgg deserves special explanation, both because of possible