DNA-DNA Hybridization Evidence of Phylogenetic Relationships among Major Lineages of Parus

Abstract

ABSTRACT.-DNA-DNA hybrids were formed among 2 outgroups and 12 taxa of titmice and chickadees to estimate the genealogical relationships of the main Parus lineages. A complete matrix of reciprocal comparisons among seven parids and the Verdin (Auriparus flaviceps) indicated that the Blue Tit (P. caeruleus) and Great Tit (P. major) together form the sister group of the rest of the genus, and that the Bridled Titmouse (P. wollweberi) is more closely related to North American titmice than to the Old World crested tits. The DNAhybridization data complement information from allozyme and mtDNA studies of closely related parids and provide historical insight into patterns of tit behavior. (Table 1). A complete matrix of pairwise measurements was made among seven of the Parus species and Auriparus (Table 2). The taxa included in the matrix were selected based on preliminary mtDNA restriction-fragment comparisons, which indicated that these seven species were members of the most diverged lineages within the genus and presented particularly interesting taxonomic problems (Gill and Slikas 1992; F. B. Gill, A. Mostrom, and A. L. Mack, unpubl. manuscript).Biochemistry.-The DNA-hybridization procedure we used is based on that of Sibley and Ahlquist (1990), with the following modifications. DNA was extracted from frozen tissues (mainly liver and heart), as opposed to alcohol-preserved tissues or erythrocytes. These were placed in liquid nitrogen or on dry ice in the field and stored at -80?C in the laboratory. In most cases, the nuclei were separated from the mitochondria before nuclear DNA was extracted. This separation was achieved by grinding the tissues in cold STE buffer (0.25 M sucrose, 0.03 M Tris, 0.1 M EDTA) and spinning the homogenate at 3,000 rpm (700 g) for 5 min to pellet the nuclei and large tissue fragments. The pellets were then resuspended in STE, treated with pronase, and extracted as usual with 173

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