Phylogenetic Utility of Avian Ovomucoid Intron G: A Comparison of Nuclear and Mitochondrial Phylogenies in Galliformes

—A novel nuclear marker, the avian ovomucoid intron G (OVOG) was sequenced from 19 galliform taxa. Results of the phylogenetic analyses using OVOG were compared to those obtained using the mitochondrial cytochrome b (cytb) gene to determine the phylogenetic utility of OVOG. OVOG appeared to have strong phylogenetic signal for reconstructing relationships among genera and families, and the only difference between OVOG and cytb was in the placement of the New World quail (Odontophoridae). Genetic distances estimated using OVOG are approximately half of those estimated using cytb, although that relationship was not linear. OVOG exhibited patterns of nucleotide substitution very different from cytb, with OVOG having little base compositional 4 Address correspondence to this author. E-mail: [email protected] bias, a relatively low transition–transversion ratio, and little among-site rate heterogeneity. Mitochondrial DNA (mtDNA) sequences are commonly used to estimate vertebrate phylogenies. MtDNA markers evolve rapidly, making investigation among closely related species possible, yet also contain enough slowly evolving sites to resolve deeper relationships. Although mtDNA phylogenies are likely to be correct in many cases (Moore 1995), use of mtDNA sequences can be problematic. MtDNA rarely undergoes recombination (Wolstenholme 1992), so problems due to lineage sorting or introgression cannot be detected. Nuclear pseudogenes of mtDNA sequences can also confound phylogenetic estimation (Sorenson and Quinn 1998). Therefore, it is useful to compare mtDNA phylogenies with nuclear gene phylogenies to control for such problems. 800 [Auk, Vol. 118 Short Communications TABLE 1. Species examined and accession number of sequence data. Accession numbers available from GenBank. Group Speciesa Common name cytb OVOG Cracids Crax pauxi Ortalis vetula Helmeted Currassow Plain Chachalaca AF068190 L08384 AF170973 AF170974 New World quail Cyrtonyx montezumae Oreortyx pictus Montezuma Quail Mountain Quail AF068192 AF252860 AF170976 AF170977 Guineafowl Turkeys Numida meleagris Meleagris gallopavo Helmeted Guineafowl Wild Turkey L08383 L08381 AF170975 AF170984 Grouse Falcipennis canadensis Tympanchus phasianellus Spruce Grouse Sharp-tailed Grouse AF170992 AF068191 AF170986 AF170985 Partridges Alectoris chukar Alectoris rufa Bambusicola thoracica Perdix perdix Chukar Red-legged Partridge Chinese Bamboo Partridge Gray Partridge L08378 Z48775 AF028790 AF028791 AF170987 AF170988 AF170978 AF170982 Pheasants Afropavo congensis Catreus wallichi Crossoptilon crossoptilon Gallus gallus Congo Peafowl Cheer Pheasant White-eared Pheasant Red Junglefowl AF13760 AF028792 AF028794 AF028795 AF170991 AF170980 AF170981 AF170979 Pavo cristatus Pavo muticus Pucrasia macrolopha Common Peafowl Green Peafowl Koklass Pheasant L08379 AF013763 AF028800 AF170990 AF170989 AF170983 a Samples were obtained from aviaries and zoos that maintain captive-reared stock of legal and documented origin, except for Falcipennis (collected by J. Hagelin in Alaska) and Tympanchus (collected by H.B. Tordoff in North Dakota). Nuclear intron sequences appear to evolve more rapidly than nuclear exons due to the relative lack of constraint upon their sequence (Li 1997). That rapid evolution makes nuclear introns attractive candidates for comparison with mtDNA phylogenies. However, relatively few phylogenies using nuclear intron data have been published in birds (but see Prychitko and Moore 1997, 2000; Johnson and Clayton 2000; all of which use b-fibrinogen intron 7). Ovomucoid is a single-copy gene that acts as a storage protein and protease inhibitor (Laskowski et al. 1987a), and it was one of the earliest genes for which intron and exon structure was determined (Stein et al. 1980). A large set of partial amino acid sequences exists for avian ovomucoids (Laskowski et al. 1987b, 1990; Apostol et al. 1993), providing data that can be used to develop primers for phylogenetic studies of birds. Here we compare the molecular evolution of intron G in domain III of ovomucoid (OVOG) with that of the mitochondrial cytochromeb (cytb) gene in Galliformes. These comparisons indicate that OVOG is useful for avian phylogenetics. Methods. OVOG primers were designed by locating conserved amino acid sequences surrounding the intron (using Laskowski et al. 1987b, 1990; Apostol et al. 1993) and back translating from those sequences. Nucleotide data from the chicken ovomucoid (Stein et al. 1980) was used to establish the nucleotides at 3rd codon positions; one site near the 39 end of each primer was left degenerate to ensure template matching (OVOG Forward: 59-CAAGACATACGGCAACAARTG-39; OVOG Reverse: 59GGCTTAAAGTGAGAGTCCCRTT-39). DNA was extracted as described elsewhere (Kimball et al. 1997), and most samples were identical to those used in our previous research (Kimball et al. 1997, 1999). OVOG was amplified by PCR using standard protocols, and the products cleaned using QIAquickTM PCR Purification columns (Qiagen, Inc., Valencia, California). Sequencing reactions (onequarter or one-half volume) were performed using Thermosequenase dye, terminator (Amersham Life Science, Piscataway, New Jersey), or BigDyeTM Terminator (Perkin-Elmer Applied Biosystems, Forest City, California) chemistry and the primers used for PCR amplification. Sequence reactions were analyzed using an ABI 310 or 377 automated DNA sequencer. Sequence chromatographs were edited manually and assembled into double-stranded contigs. A few ambiguous sites, probably reflecting heterozygosity, were evident, and those sites were coded using the proper IUPAC symbols. Cytb sequences were published or sequenced for this study as described previously (Kimball et al. 1999). Species and sequence accession numbers are in Ta-