Molecular Genetic Divergence between Avian Sibling Species: King and Clapper Rails, Long-Billed and Short-Billed Dowitchers, Boat-Tailed and Great-Tailed Grackles, and Tufted and Black-Crested Titmice

-Surveys of electrophoretic variation in proteins, and restriction site variation in mitochondrial DNA (mtDNA), were conducted to assess the resolving power of these molecular genetic techniques to distinguish four pairs of avian sibling taxa. Samples of rails (Rallus elegans and R. longirostris), dowitchers (Limnodromus scolopaceus and L. griseus), grackles (Quiscalus major and Q. mexicanus), and titmice (Parus bicolor bicolor and P. b. atricristatus) were assayed for allozymes encoded by 34-37 nuclear loci, and for an average of 77 mtDNA restriction sites per individual by 19 endonucleases. MtDNA's of the two rail species showed large-scale size polymorphism and individual heteroplasmy, the first such findings of these molecular features in an avian species. Genetic distances based on allozyme comparisons were small for all assayed taxa (Nei's D c 0.063). The mtDNA assays offered consistently greater resolving power, providing at least five fixed restriction site differences for samples of any taxon pair. The Long-billed and Short-billed dowitchers were especially divergent, differing by at least 24 assayed mtDNA restriction sites and an estimated nucleotide sequence divergence of p = 0.082. We compared these results to previous reports of genetic distances within and among closely related bird species. The mtDNA divergence among dowitchers is near the high end of the scale of such estimates for avian congeners. The mtDNA distances between the pairs of rails (p = 0.006), titmice (p = 0.004), and grackles (p = 0.016) were typical for extremely closely related species, and overlap maximum values reported for some avian conspecifics. Received 13 November 1987, accepted 21 March 1988. VARIOUS laboratory techniques in molecular evolution offer differing resolution of genetic differentiation and phylogeny along a taxonomic hierarchy (Avise 1986a). For example, methods of nuclear DNA/DNA hybridization (Sibley and Ahlquist 1986) or of protein immunology such as microcomplement fixation (Wilson 1985) are usually best applied to higher taxonomic levels, because the observed genetic differences within and between closely related species are typically negligible. Conversely, multilocus protein electrophoresis (Ayala 1976) or restriction enzyme analysis of mitochondrial DNA (mtDNA; Avise 1986b) have proved best suited for phylogenetic comparisons within and among closely related species, because beyond the genus or family level, the maximum genetic distance observable by these techniques typically has been reached (see, however, Lanyon and Zink 1987). By the criterion of focus alone, it should not be claimed that any technique is necessarily "better" than another for systematics purposes. Rather, the situation can be likened to microscopy, where use of different lenses can bring into focus distinct and important aspects of structure at multiple tiers. As a general rule, birds at all taxonomic levels commonly exhibit less genetic divergence than do many of their counterparts in other vertebrate classes (Avise and Aquadro 1982; Barrowclough et al. 1985; Prager et al. 1974). Although this situation has facilitated molecular analysis of major groupings across the class Aves (Prager and Wilson 1980; Sibley and Ahlquist 1983), it has also exacerbated attempts genetically to differentiate and study entities at the lower end of the avian taxonomic scale. For example, many conspecific populations and congeneric avian species appear difficult to distinguish even when assayed by the sensitive and commonly employed protein electrophoretic methodology (e.g. Braun and Robbins 1986; Zink 1986). We characterized genetic divergence in each of 4 pairs of avian sibling "species" with two of the powerful molecular approaches of allozymes and mtDNA analysis. The taxa (Rallus 516 The Auk 105: 516-528. July 1988 This content downloaded from 207.46.13.21 on Tue, 27 Sep 2016 04:45:47 UTC All use subject to http://about.jstor.org/terms July 1988] Genetic Divergence Between Sibling Species 517 elegans and R. longirostris [Rallidae], Limnodromus scolopaceus and L. griseus [Scolopacidae], Quiscalus major and Q. mexicanus [Emberizidae], and Parus bicolor bicolor and P. b. atricristatus [Paridae]) include non passerines as well as passerines, and involve systematically controversial taxonomic pairs that have all been considered conspecific and specifically distinct at one time or another. We were interested in the relative sensitivities of allozyme and mtDNA assay techniques to disclose genetic markers that may distinguish avian sibling species, and what the genetic results reveal about the particular evolutionary histories of the taxa. MATERIALS AND METHODS Specimens were collected in Louisiana and Texas (precise locales available from RMZ on request) during the winter and spring of 1987. Voucher specimens are deposited in the Museum of Zoology, LSU. Allozyme data for titmice were obtained from Braun et al. (1984). For mtDNA analyses, samples of liver, heart, and kidney were immersed in MSB-Ca-EDTA buffer (Lansman et al. 1981), and transported on wet ice to the laboratory within 7 days. For protein electrophoresis, samples of liver and muscle were frozen until time of assay. Protein electrophoresis followed standard protocols (Selander et al. 1971, Johnson et al. 1984). Between 34 and 37 loci were scored for each species pair. Sixteen of these loci are listed in Table 1; the remainder (which proved monomorphic and incapable of discriminating members of any species pair in which they were assayed) are ethanol dehydrogenase (Enzyme Commission number 1.1.1.1), a-glycerophosphate dehydrogenase (1.1.1.8), lactate dehydrogenase-1 and 2 (1.1.1.27), malate dehydrogenases-1 and 2 (1.1.1.37), malic enzyme (1.1.1.40), isocitrate dehydrogenase-2 (1.1.1.42), glutamate dehydrogenase (1.1.1.47), glutathione reductase (1.6.4.2), superoxide dismutase (1.15.1.1), glutamic-pyruvic transaminase2 (2.6.1.2), creatine kinases-1 and 2 (2.7.3.2), acid phosphatases-1 and 2 (3.1.3.2), leucineamino peptidase (3.4.11), fumarate hydratase (4.2.1.2), aconitate hydratase (4.2.1.3), and non-enzymatic proteins-i and 2. Mean observed heterozygosity in each species was calculated from genotypic counts. These counts were also used to determine allele frequencies, from which genetic distances were computed by Nei's (1978) method. MtDNA in closed-circular form was isolated from pooled tissues of each specimen by ultracentrifugation in cesium chloride gradients. Following dialysis, the purified mtDNA was digested by each of 19 restriction endonucleases. Fifteen of these enzymes are listed in Table 2; the remaining 4 (which proved not to distinguish any of the sibling species assayed) are KpnI (recognition site GGTACC), PstI (CTGCAG), SstII (CCGCGG), and XbaI (TCTAGA). MtDNA fragments were end-labeled with 35S-radionuclides, separated by molecular weight through 1.0, 1.2, or 1.8% agarose gels, and revealed by autoradiography as bands in gel profiles (Brown 1980; Lansman et al. 1981; Maniatis et al. 1982). Fragment sizes were compared against a 1-kilobase ladder standard purchased from Bethesda Research Labs. MtDNA genetic distances in terms of estimated base substitutions per nucleotide were calculated by the fragment method of Nei and Li (1979), which involves appropriate weighting of distance estimates calculated separately for endonucleases recognizing 4-, 5-, and 6-base sites. Many of the mtDNA fragment profile differences between sibling taxa were attributable to gains (or losses, depending on which state was ancestral) of single restriction sites. Such instances are recognized when two digestion profiles (A and B) are identical at all except three fragments, and in which the sum of the molecular weights of the two unique fragments in A equals the weight of the third unique fragment in B (for a detailed description of the gel scoring procedure, see Fig. 2 in Avise et al. 1987). Thus, although sites were not formally mapped in this study, we were often able to interpret gel patterns with respect to restriction site as well as restriction fragment changes. We observed a total of more than 100 distinct fragment profiles. (Avise will gladly supply diagrams of any gel patterns upon request.)