山 階 鳥 研 報(J. Yamashina Inst. Ornithol.),22:114-23,1990 Genetic Divergence and Relationships in Fifteen Species of Procellariiformes Nagahisa Kuroda*, Ryozo Kakizawa* and Masayoshi Watada** Abstract The genetic analysis of 23 protein loci in 15 species of Procellariiformes was made The genetic distancesbetween the specieswas calculatedand a dendrogram was formulated of the group. The separation of Hydrobatidae from all other taxa including Diomedeidae agrees with other precedent works. The resultsof the present study support the basic Procellariidclassification system. However, two points stillneed further study. The firstpoint is that Fulmarus diverged earlier from the Procellariidsthan did the Diomedeidae. The second point is the position of Puffinuspacificus which appears more closely related to the Pterodroma petrels than to other Puffinus species. These points are discussed. Introduction The taxonomy of the Procellariiformes has been proposed from various approaches. The earliest study by Forbes (1882) was made by appendicular myology. Godman (1906) and Loomis (1918) studied this group from a morphological point of view. The taxonomy of the Procellariiformes by functional osteology and appendicular myology was studied by Kuroda (1954, 1983) and Klemm (1969), The results of the various studies agreed in proposing four families of Procellariiformes: Diomedeidae, Procellariidae, Hydrobatidae, and Pelecanoididae. They also pointed out that the Procellariidae was a heterogenous group among them. Timmermann (1958) found the parallel evolution of mallophaga and their hosts in Procellariiformes. Recently, electrophoretical studies have been made on the Procellariiformes. Harper (1978) found different patterns of the electromorph among the families. Bar- rowclough et al. (1981) studied genetic differentiation among 12 species of Procellari- iformes at 16 loci, and discussed the genetic distances among the taxa but with no consideration of their phylogenetic relationships. In the present study, we report on genetic differentiation and phylogenetic rela- tionships of Procellariiformes by means of electrophoresis of 23 loci, dealing with 15 Procellariid species, and similarities with the work of Barrowclough et al. (1981). Materials and Methods The sample of Procellariiformes used in the present study included 52 individuals of 15 species from three families. The species were as follows: 1) Diomedea nigripes, Received 15 December 1989, accepted 17 May 1990 * Yamashina Institute for Ornithology , Konoyama, Abiko, Chiba 270-11, Japan ** Department of Biology , Faculty of General Education, Ehime University, Matsuyama, Ehime 790, Japan 114 Genetic Divergence and Relationships in Fifteen Species of Procellariiformes 115 116 N. Kuroda, R. Kakizawa & M. Watada 2) D. immutabilis, 3) Fulmarus glacialis, 4) Pagodroma nivea, 5) Pterodroma externa, 6) P. hypoleuca, 7) Bulweria bulwerii, 8) Calonectris leucomelas, 9) Puffinus pacificus, 10) P. griseus, 11) P. tenuirostris, 12) P. lherminieri, 13) Oceanodroma castro, 14) O. leucorhoa and 15) O. tristrami. Diomedea and Oceanodroma belong to the Diomedeidae and Hy- drobatidae, respectively. All the other genera in this study were included in the Procel- lariidae. The sample size for each species is shown in Table 1. Genetic variations of the following enzymes and proteins were surveyed: creatine kinase (CK), diaphorase (DIA), esterase (EST), glucose oxzaloacetate transisomerase (GOT), a-glycerophosphate dehydrogenase (aGPD), glucose phosphate isomerase (GPI), isocitrate dehydrogenase (IDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), peptidase (PEP), 6-phosphate dehydrogenase (6PGD), phosphoglucomutase (PGM), general protein (PT), superoxide dismutase (SOD), and xylulose reductase (XR). Diomedea immutabilis was accidentally obtained as alive bird from which only a blood sample was available, and 12 protein loci were examined for the species. In the case of other species, 23 loci were studied using blood, liver and muscle. The treatment of samples and the methods of electrophoresis have already been described in Kakizawa & Watada (1985). The calculation of the genetic distances followed Nei (1972) and the phylogenetic dendrogram was made by UPGMA after Sneath and Socal (1973). Results Out of 23 protein loci examined, all taxa were fixed for common alleles at the fol- lowing eight loci: CK-1, DIA, GOT-1, GOT-2, IDH-1, MDH-1, MDH-2, and PT-2. These were excluded from Table 1, in which the alleles and the frequencies of the other 15 loci were shown for all 15 species of Procellariiformes. Most of the alleles given in Table 1 are common among species and/or genera, and only a few were species specific. Although the number of individuals examined was small within a range from 1 to 12, 23 protein loci were studied in the present study. There was no intraspecific genetic variation except at 6PGD locus where three species, Fulmarus glacialis, Oceanodroma castro and O. leucorhoa were highly polymorphic. Bulweria bulwerii was also polymor- phic at the locus with a low level of heterozygosity. The observed heterozygosities for the above four species were also low: 0.029 in Fulmarus glacialis, 0.004 in Bulweria bulwerii, and 0.043 in the two Oceanodroma species. The average observed heterozygosi- ty was 0.008 for Procellariiformes. In Diomedeidae, two species, D. nigripes and D. immutabilis, shared the same alleles in all loci except two, LDH-1 and 6PGD, which were highly variable, though electropho- retic data were lacking from 11 protein loci of D. immutabilis. Out of 23 loci examined, Diomedea had common alleles to other groups at 17 loci, but had its own peculiar alleles at 6. They were Pgm-1b common to the two Diomedea species, a-Gpdb, Ldh-1a, 6Pgdk, Pgm-2d and Xrb of D. nigripes, and Ldh-1b of D. immutabilis. At 6PGD locus, 6Pgd1 was observed in only D. immutabilis and four Puffinus species. Also Gpic, Ldh-2a and Pt-3b were common alleles to the Diomedeidae and most of the Procellariidae, but were not found in any Hydrobatidae. No similar alleles were observed only in Diomedeidae and Hydrobatidae. Genetic Divergence and Relationships in Fifteen Species of Procellariiformes 117 The Procellariidae is a large heterogenous group comprising Fulmarine (Fulmarus and Pagodroma), Petrodromine (Pterodroma and Bulweria) and Puffinine (Calonectris and Puffinus) species. Only Pgm-2a was common and restricted to this group. How- ever, Ldh-1d and Xrc were also observed to be restricted to the Procellariidae with a few exceptions. Ldh-1e was found only in Fulmarines, and Xre and Xrd were species specific alleles for Bulweria bulwerii and Calonectris leucomelas, respectively. The highly variable locus, 6PGD showed different alleles by in each species and genera . The other loci in Table 1 showed intermediate variation with species and genus specific alleles. aGpdd and Ldh-1e were restricted to Fulmarine, suggesting the peculiarity of this group amongst the Procellariidae. Puffinines also had aGpde as a specific allele of this sub- family. In SOD locus, Pterodromines and Fulmarines were connected by Sodc, but Puffinines had Soda and thus differed from the above two subfamilies. These results give genetical support to the heterogenous grouping of this family. In the Hydrobatidae, alleles specific for this family were Ldh-1c Ldh-2c, Sodb and Xra. Oceanodroma castro and O. leucorhoa had aGpda, but O. tristrami shared aGpdc with Pterodroma externa and P. hypoleuca. Similarly, O. castro and O. tristrami had a common allele, Gpib with Calonectris leucomelas. Idh-2a suggested a relationship between the Fulmarines and the Hydrobatidae. At the 6PGD loci, O . castro and O. leucorhoa were highly polymorphic, and had species specific alleles, 6Pgdd and 6Pgdb, respectively. In addition, Pgm-2c and Pt-3a were found only in O. castro. Genetic differentiation and phylogenetic relationships Based on the electrophoretic data of 23 loci shown in Table 1, genetic identities and genetic distances were calculated according to Nei's (1972) fomula. Among the 15 species examined, no genetic difference was observed between Puffinus griseus and P. tenuirostris and between Pterodroma externa and P. hypoleuca. The average genetic distance within genus was 0.137 which was larger than that of the Anatidae, and was close to 0.151 in the Alcidae (Numachi et al. 1983, Watada et al. 1987). The mean intergeneric and inter- familial genetic distances of the Procellariiformes were 0.350 and 0.554, respectively. These were somewhat lower than the values (0.435 for intergeneric and 0.683 for inter- familial) reported by Barrowclough et al. (1981). The largest genetic distance, 0.833, was found between Puffinus griseus (Procellariidae) and Oceanodroma tristrami (Hy- drobatidae). Based on the genetic distances in Table 2, a dendrogram was made for these 15 species of Procellariiformes using UPGMA (Fig. 1). Most congeneric species were clustered together by genera such as Diomedea, Pterodroma and Oceanodroma. In Puffinus, P. pacificus was grouped with Pterodroma apart from the three other congeneric species. Grouping within the Procellariidae was complicated , but the following are suggested: 1) Fulmarus and Pagodoroma (Fulmarini), 2) the puffinus group without P . pacificus and 3) a heterogenous group of Pterodroma, Bulweria, Calonectris and Puffinus pacificus. It is important to point out that two species of Diomedeidae were included in the large group of Procellariidae in Fig. 1, although their phylogenetic relationships were not close to each