Jpn. J. Ornithol. 48: 205-218, 2000 A Study on Genetic Differentiation and Phylogenetic Relationships among East Asian Titmice (Family Paridae) and Relatives Noriko OHTA1,2,Seiji KUSUHARA1and Ryozo KAKIZAWA2 1 Graduate School of Science and Technology, Niigata University. Ninomachi 8050,Igarashi, Niigata-shi, Niigata 950-2181,Japan 2 Yamashina Institute for Ornithology, Konoyama 115, Abiko-shi, Chiba 270-1145,Japan By means of protein electrophoresis on 23 loci (15 enzymes), genetic variation and differentiation among eight East Asian Titmice (family Paridae, genus Parus), two long-tailed tits (family Aegithalidae, genus Aegithalos), a nuthatch (family Sittidae, Sitta) and Bearded Tit (family Paradoxornithidae, genus Panurus) were analyzed. A genealogic dendrogram is constructed by the UPGMA method, using genetic distances among the species. The results of the present study suggests that the East Asian Parus species investigated can be divided into four groups, i.e., groups consisting of (1) Parus montanus, (2) P. ater, P. venustulus and P. varius, (3) P. major and P. monticolus, and (4) P. spilonotus and P. holsti. P. spilonotus seems to have a close relationship to P. holsti, but distant relationships to P. major and P. monticolus. Aegithalos, Sitta and Panurus are relatively closely related to each other, whereas the Parus species are shown to have a family level genetic distance in the Passeriformes. In this study, we could not get enough samples of some species (espe- cially P. montanus and P. spilonotus). And since we obtained samples from dealers, there are some possibility that samples were collected from only one population of each species. The data of other samples from several populations and that of other species are needed to determine the phylogeny of the East Asian Parus species. Key Words: East Asian Titmice, Isozyme, Paridae, Parus, Phylogeny Titmice (family Paridae) are small birds which inhabit the forests of Africa, Europe, Asia and North America. They are very acrobatic in clinging to branches by their feet and have relatively short beaks and stout feet. Snow (1967) classified 46 species of the Paridae into three genera without recognizing subgenera: Parus (44 species), Melanochlora (1 species), and Sylviparus (1 species). His classifica- tion has been accepted widely at the present, whereas Thielcke (1968) classified 43 species of Paruts into 10 subgenera. We follow Thielcke in recognition of subgenera of Parus. Fourteen Parus species that belong to 8 subgenera are distributed in East Asia (Mongolia, China, the Korean Peninsula, Taiwan and Japan). In earlier studies, Vaurie (1950, 1957) discussed the taxonomic problems and relationships of Palearctic species of the Paridae using morphological characters, but the phylogenetic relationships of Asian titmice are still poorly known. 206 Noriko OHTA,Seiji KUSUHARAand Ryozo KAKIZAWA [Jpn.Ornithol.Vol.48 No. 3 Recently there have been systematic studies of the Paridae in Europe and North America based on isozymes and DNA analysis (Gill et al. 1989, Sheldon et al. 1992, Kvist et al. 1996, Slikas et al. 1996). However, there are no comparable studies on Asian species, which makes a study of Asian titmice desirable. The Aegithalidae, Sittidae and Paradoxornithidae (Panurus) are considered to be closely related to the Paridae. Aegithalos and a few related genera have been treated as a separate family, the Aegithalidae (Snow, 1967), but they are often considered as genera or a subfamily of the Paridae (e.g., Hellmayr 1903, Mayr & Amandon 1951). Panurus has been classified in the subfamily Panurinae (=Paradoxornithinae) of the family Muscicapidae (s. lat.) (Deignan 1964). In addition, the Sittidae have sometimes been considered as a subfamily of the Paridae (Vaurie 1959). This study attempts to reveal the phylogenetic relationships among the little studied members of the Paridae of East Asia and their relationships to Aegithalos, Sitta and Panurus. These species were examined by electrophoretic analysis on 23 loci (15 enzymes) with tissues (liver, muscle and heart) taken from 59 individual birds. MATERIALS AND METHODS We analyzed 59 individual birds belonging to 6 subgenera and 8 species of the Paridae, one species of the Paradoxornithinae, two species of the Aegithalidae, and one species of the Sittidae. They were obtained from pet dealers between March 1986 and May 1997, so detailed locality and population number of the samples are not known. The species, sample sizes and localities of the samples used in this study are given in Table 1. The liver, muscles and heart were removed from half- Table 1. Studied species, sample size and locality. Order of the species follow Snow (1967), Deignan (1964) and Thielcke (1968). The subgenera of Parus after Thielcke (1968) are given in parenthesis. February 2000] Relationships among East Asian Titmice and Relatives 207 defrosted birds, homogenized, and centrifuged at 15,000*g for 20 minutes at 2*C. The supernatant was then divided into aliquots and stored in capillary glass tubes at -80*C. The extracts of the tissues were analyzed by starch gel electrophoresis to determine the allelic composition of each protein locus. The combinations of tis- sues and buffer systems for enzyme detection are shown in Table 2. The staining Table 2. Combinations of tissues and buffer systems for detection of enzymes. * Tris-HCl, Poulik: Selander et al. (1971), Tris-citrate: Numachi et al. (1983). method for enzymes were essentially the same as those described by Bell et al. (1982), Shaw & Prasad (1970), Selander et al. (1971), Hopkinson et al. (1973) and Numachi et al. (1979). These enzymes and proteins are assumed to be coded by 23 gene loci. Allozymes representing alleles for individual loci are designated alpha- betically in decreasing order of mobility. Isozyme systems under the control of distinct loci were identified numerically, counting from the anodal side. Genetic differentiations in populations, both the proportion of polymorphic loci and heterozygosities, were calculated as follows. The proportion of polymorphic loci were identified as the proportion of genetic loci that showed variations. Heterozygosities were calculated in two ways: observed heterozygosities were calcu- lated by averaging the proportion of individual heterozygous birds observed per locus, and expected heterozygosities were calculated as the proportion of heterozygosities expected from allele frequencies, assuming Hardy-Weinberg equilibria. Genetic identities and genetic distances between species were calculated using Nei' s (1972) formula, and the values of genetic distances were clustered into a dendrogram using the unweighted pair-group method of analysis with arithmetic means (UPGMA) (Sneath & Sokal 1973). 208 Noriko OHTA,Seiji KUSUHARAand Ryozo KAKIZAWA Jpn.J.Ornithol.Vo1.48No.3 February 2000] Relationships among East Asian Titmice and Relatives 209 210 Noriko OHTA, Seiji KUSUHAHAand Ryozo KAKIZAWA Vol.48No.3 RESULTS 1) Genetic variation Alleles detected at each locus and the frequency of allele occurrences are shown in Table 3. The number of birds investigated for each species ranged from one to twelve, with intraspecific genetic variations found in all species of Parus studied except P. rnontanus, of which there was only one specimen. No intraspecific genetic variation was detected in both species of Aegithalos and Panurus tested (Sitta was represented by one specimen). The species with the highest genetic variation was P. venustulus and P. varius, which showed variations at 5 loci. Fourteen loci showed genetic variations within a species: SOD (variation found in four species), 6PGD (three species), CK-2, DIA, ESD-2, GOT-1, IDH-1, PGM-1 and PGM-2 (two species), CK-1, ESD-1, GOT-2, MDH-2, and PEP (one species). The largest number of alleles found at any one locus was eight at 6PGD. The proportions of polymorphic loci and average heterozygosities (observed and expected) of the species studied are shown in Table 4 and those data for other avian groups are shown in Table 5. The mean proportion of polymorphic loci of the 8 species of Parus was 0.125 (range 0 - 0.217), which is relatively lower than Table 4. Proportion of polymorphic loci and heterozygosities (observed and expected) of each species. February 2000 Relationships among East Asian Titmice and Relatives 211 Table 5. Proportion of polymorphic loci and expected heterozygosities in various groups of birds. for the Passeriformes (0.145). Of the 23 loci tested, inter-specific variations were found in 19 loci (CK-1, CK-2, DIA, ESD-1, ESD-2, GOT-1, GOT-2, *-GPDH, GPI, IDH-1, LDH-1, LDH-2, MDH-2, PEP, 6PGD, PGM-1, PGM-2, SDH and SOD) in the 8 species of Parus. LDH-1 showed a single allele except in P. major. Inter-specific variations of two Aegithalos species were found in 7 loci (GOT-2, GPI, IDH-1, PEP, 6PGD, PGM-2 and SOD). Genetic variations between Parus, Aegithalos, Sitta and Panurus were found in 21 loci (CK-1, CK-2, DIA, ESD-1, ESD-2, GOT-1, GOT-2, *-GPDH, GPI, IDH-1, IDH-2, LDH-I, LDH-2, MDH-2, PEP, 6PGD, PGM-1, PGM-2, PT-1, SDH and SOD). IDH-2 showed a single allele in Parus and Panurus and PT-1 showed a single allele in Aegithalos, Sitta and Panurus. 2) Genetic differentiation The genetic identity and distances were calculated from the data given in Table 6 according to Nei's (1972) formula. The results obtained from the analysis of the 23 loci showed that genetic variation was found among all the species of Parus, Aegithalos, Sitta and Panurus examined. The lowest genetic distance was found between P. major and P. monticolus (0.0892), and the second lowest one between P. venustulus and P. varius (0.1012). These values probably indicate relatively low 212 Nonko OHTA, Seiji KUSUHARA and Ryozo KAKIZAWA [Jpn.J.Ornithol.Vol.48No.3 February 2000] Relationships among East Asian Titmice and Relatives 213 genetic differentiation between the two species, respectively. The highest genetic distance (1.4088) was found between P.