Keywords Penguin · Spheniscidae · Phylogenetic tree · Major histocompatibility complex gene · Polymorphism Penguins belonging to the Speniscidae family have been placed in the superfamily Procellarioidae with other families, such as divers and albatrosses, by morpho- metric analyses identifying a suite of structural simi- larities. They are widely distributed in the southern hemisphere. Furthermore, based on morphological, behavioral, and ecological data, penguins have been classified into six genera which consist of 16 species and two subspecies (Hedges and Sibley 1994; Jouventin 1982). Previous comparative study of the amino acid se- quence of polymorphic proteins indicated that penguins were phylogenetically most closely related to petrels, loons, and albatroses (Ho et al. 1976). The ancestor of penguins is generally accepted to have originated from such flying oceanic birds, with flightlessness in penguins then evolving independently from the aquatic birds. The earliest known fossil penguins were collected from the lower to middle Eocene, dating from about 45–50 million years ago (MYA) (Williams 1995). Using a DNA-DNA hybridization technique, Sibley and co-workers (1988) estimated that the penguin lineage emerged and branched from the loon-tubenose clade about 47 MYA during the middle of the Eocene in the Tertiary period. This DNA dating is in good agreement with that obtained from the fossil evidence. The greatest diversity of penguin species arising from the ancient species is considered to have been generated on the mainland and islands of southern New Zealand. The Little penguin (Eudyptula minor) is considered to represent the most primitive genus. The Pygoscelis genus with three species, Adelie penguin (Pygoscelis adeliae), Chinstrap penguin (P. antarctica), and Gentoo penguin (Pygoscelis papua), constitutes a clear unity, restricted to the Antarctic and sub-Antarctic areas. We are interested in identifying genetic variations and investigating the genetic relationships among all penguin species. To attain this goal, we are systematically deter- mining the nucleotide sequences of the major histocom- patibility complex (MHC) in penguin species, because the MHC is known to display a high degree of polymorphism in other vertebrates. The aim of this study was to analyze the nucleotide sequences of the MHC class II B genes from the three penguin species in the Pygoscelis genus and the Little penguin (E. minor) in the Eudyptula genus, and to investigate the phylogenetic relationship among these species and to other birds using a molecular PCR- based sequencing technique. To our knowledge, this is the first report on nucleotide variation in the penguin MHC. In this study, 16 wild or captive penguins were used for sequencing analysis of the MHC (Table 1). Genomic The nucleotide sequence and/or amino acid sequence data reported in this paper have been submitted to the DDBJ data- base and have been assigned the accession numbers AB029994, AB029998, AB043556, AB043558-AB043559, AB043590– AB043591, AB043593–AB043595, AB043597–AB043601, AB043605, and AB060946–AB060949. A part of this paper was presented at the Fourth International Penguin Conference in Chile in September 2000 T.T. Tsuda · M. Tsuda · T. Naruse · H. Kawata · A. Ando T. Shiina · H. Inoko ( ✉ ) Department of Genetic Information, Division of Molecular Life Science, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan e-mail: hinoko@is.icc.u-tokai.ac.jp Tel.: +81-463-931121, Fax: +81-463-948884 M. Fukuda Tokyo Sea Life Park, Rinkai-cho, Edgawa-ku, Tokyo 134-0086, Japan M. Kurita Port of Nagoya Public Aquarium, Minato-machi, Minato-ku, Nagoya 455-0033, Japan I. LeMaho Centre d’Ecologie et de Physiologie Energetique, CNRS, 23 rue Becquerel, 67087 Strasbourg, France J.K. Kulski Centre for Bioinformatics and Biological Computing, School of Information Technology, Murdoch University, Murdoch, WA 6150, Australia Immunogenetics (2001) 53:712–716 DOI 10.1007/s002510100369 BRIEF COMMUNICATION Tomi T. Tsuda · Michio Tsuda · Taeko Naruse Hisako Kawata · Asako Ando · Takashi Shiina Michio Fukuda · Masanori Kurita · Ivon LeMaho Jerzy K. Kulski · Hidetoshi Inoko Phylogenetic analysis of penguin (Spheniscidae) species based on sequence variation in MHC class II genes Received: 1 June 2001 / Revised: 30 July 2001 / Published online: 6 November 2001 © Springer-Verlag 2001