著者

村田 倫子

出版者

麻布大学

巻号頁・発行日

2014

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The African penguin (Spheniscus demersus), which is endemic to southern Africa, is one of the world's most endangered seabirds. While wild African penguin populations continue to decrease, properly maintained captive populations are steadily increasing each year. To avoid close inbreeding and to maintain genetic diversity, the Japanese Association of Zoos and Aquariums keeps studbooks, which it uses to promote long-term breeding plans. However, genetic data have not been collected on either wild or captive African penguins in Japan to date.This study addresses the genetic characterization of captive African penguins in Japan, and is organized into four chapters. The first and second chapters describe the genetic diversity and phylogenetic relationships among African penguins based on mitochondrial and microsatellite DNA. The third chapter characterizes DNA markers isolated from African penguins. The fourth chapter includes an analysis of the karyotype and nucleolus organizer region of the African penguin. Chapter 1 Mitochondrial DNA analysis of captive African penguins in Japan According to the 2011 Japanese regional studbook for the African penguin, they were first introduced to Japan in 1935, and 156 additional founders were introduced from 1973 to 2011. The captive African penguin populations in Japan comprise 485 individuals belonging to an estimated 87 different founder lineages. In this study, 236 African penguin samples derived from 62 founder lineages were analyzed based on two mitochondrial DNA (mtDNA) regions. 1) Analysis of the control region Multiple sequence alignments of the 433-bp partial control region showed 39 polymorphic sites and a total of 30 distinct haplotypes. Neighbor-joining (NJ) phylogenetic analysis using the sequences revealed that the captive African penguins clustered into two clades (A and B) supported by high bootstrap values. The divergence between African penguin clades A and B (d = 3.39%) observed in the present study may reflect geographical isolation, the existence of undefined subspecies, or both, although it must be noted that our data focused on captive-bred individuals.2) Analysis of the cytochrome b gene The complete 1140-bp sequence of the cytochrome b gene was obtained from 54 captive African penguins in Japan. We detected 8 haplotypes defined by 11 variable sites. NJ phylogenetic analysis using the cytochrome b sequences identified two clades similar to those observed using the control region. These mtDNA analyses suggest that captive African penguins in Japan are derived from two distinct maternal lines.Chapter 2 Genetic population structure of captive African penguins in Japan based on microsatellite DNA analysis Eight microsatellite loci (Sh1Ca12, Sh1Ca16, Sh2Ca21, PNN01, PNN03, PNN06, PNN09, and PNN12) were examined to estimate the genetic variability and relationships among 178 captive African penguins derived from 58 founder lineages. Deviation from Hardy–Weinberg equilibrium and linkage disequilibrium were not observed for any of the markers. Mean HE (expected heterozygosity) and HO (observed heterozygosity) values ranged from 0.45 to 0.72 and from 0.45 to 0.71, respectively. These heterozygosity values for captive African penguins were higher than those of a previously described wild population of yellow-eyed penguins (Megadyptes antipodes). A Bayesian clustering method was used to characterize genetic differentiation among populations, and three subpopulations of captive African penguins were inferred.Chapter 3 Isolation and characterization of novel DNA markers from the African penguin Four methods were used to isolate genetic markers specific to the African penguin.1) Isolation of a satellite DNA fragmentA 190-bp satellite DNA fragment (a type of repetitive DNA) was isolated by digesting African penguin genomic DNA with the resection enzyme BmeT110. PCR analysis with newly designed primers based on the sequence showed that the repetitive DNA sequence was shared among spheniscid species. Southern blot hybridization analysis was performed using the satellite DNA fragment as a probe. Hybridization with genomic DNA from the African penguin, Magellanic penguin, and Humboldt penguin, which all belong to genus Spheniscus, generated ladder signals of tandem repeats, whereas non-tandem repetitive signals were found in genera Pygoscelis and Aptenodytes.2) DNA markers obtained by randomly amplified polymorphic DNA Randomly amplified polymorphic DNA PCR techniques were used to identify a 778-bp band that differentiates the African penguin from the Humboldt penguin in addition to several common bands. Cloning and sequence analysis of the unique band and band-specific PCR analysis showed that the fragment was common to spheniscid species.3) DNA markers obtained by mini/microsatellite-associated sequence amplification analysis Mini/microsatellite-associated sequence amplification (MASA) techniques were used to generate a prominent 540-bp band that differentiates the African penguin from the Humboldt penguin. Cloning and sequence analysis of the unique band, and subsequent band-specific PCR analysis showed that this fragment distinguished genera Aptenodytes and Pygoscelis from genera Spheniscus and Eudyptes.4) Representational difference analysis Three series of representational difference analysis were performed using a combination of African penguin amplicons as testers and Gentoo penguin amplicons as drivers. One informative polymorphic marker, present exclusively in Spheniscus and Eudyptes, was obtained. No polymorphic DNA fragments were isolated when amplicons prepared from the Humboldt penguin were subtracted from those prepared from the African penguin.Chapter 4 Karyotype of the African penguin The African penguin karyotype was analyzed. To obtain metaphases, the direct culture technique was used for peripheral blood lymphocytes. The chromosome number of the diploid African penguin was, for the first time, determined to be 76 (2n = 76), where 7 pairs of autosomes and a pair of sex chromosomes were considered macrochromosomes, and the remaining 30 pairs (60 chromosomes) were microchromosomes. According to several previous studies, the diploid chromosomal numbers of the Magellanic penguin and the Humboldt penguin were 68 and 78, respectively. While the number of macrochromosomes was constant among species of genus Spheniscus, the number of microchromosomes varied. Taken together, we demonstrated the existence of two divergent clades of captive African penguins with moderate genetic distance based on mtDNA sequence analyses. Next, we showed three different subpopulations within the African penguin. The population of captive African penguins in Japan was derived from multiple genetic origins, resulting in genetic diversity. Moreover, we isolated DNA markers shared among family Spheniscidae, but did not detect genetic markers specific to the African penguins. This finding suggests that penguin species in Spheniscus, including the African penguin, have a high level of genetic homogeneity. In addition, the African penguin karyotype was determined for the first time. These molecular analyses should be useful to Japanese zoos and aquariums for future management decisions and the implementation of breeding programs.