Duplication and Variation in the Major Histocompatibility Complex Genes in Blakiston's Fish Owl, Bubo blakistoni.

The major histocompatibility complex (MHC) includes many genes that are essential for the adaptive immune system, and variation in the antigen binding site (ABS) is related to resistance against pathogens. In the present study, quantitative real-time PCR indicated a larger number of MHC gene copies in the endangered population of Blakiston's fish owl (Bubo blakistoni) than in five other owl species, and massively parallel pyrosequencing detected more MHC class IIß per individual alleles in B. blakistoni than in the other species. A chromosomal fluorescence in situ hybridization (FISH) analysis showed that the MHC class I and class IIß loci are closely linked on a single pair of microchromosomes, indicating that the MHC genes were tandemly duplicated in a limited chromosomal region. Because B. blakistoni has twice as many MHC genes as its sister species, the tawny fish owl (Bubo flavipes), the duplication of MHC genes occurred after these species diverged by speciation. A Bayesian molecular phylogenetic analysis showed that the DAB1 and DAB2 lineages of MHC class IIß alleles from various strigid species each formed a separate clade, indicating that the two allelic lineages preceded the radiation of Strigidae and evolved as paralogs. By contrast, the ABS sequences did not form distinct clades between DAB1 and DAB2 alleles but were intermixed, presumably due to gene conversion. Despite the low diversity of alleles per locus, B. blakistoni had many lineages of MHC class IIß alleles. Gene duplication increases variation in the MHC genes in this species, and could have facilitated adaptation in small populations.

Spatial and temporal variation at major histocompatibility complex class IIB genes in the endangered Blakiston's fish owl.

INTRODUCTION: Quantifying intraspecific genetic variation in functionally important genes, such as those of the major histocompatibility complex (MHC), is important in the establishment of conservation plans for endangered species. The MHC genes play a crucial role in the vertebrate immune system and generally show high levels of diversity, which is likely due to pathogen-driven balancing selection. The endangered Blakiston's fish owl (Bubo blakistoni) has suffered marked population declines on Hokkaido Island, Japan, during the past several decades due to human-induced habitat loss and fragmentation. We investigated the spatial and temporal patterns of genetic diversity in MHC class IIß genes in Blakiston's fish owl, using massively parallel pyrosequencing. RESULTS: We found that the Blakiston's fish owl genome contains at least eight MHC class IIß loci, indicating recent gene duplications. An analysis of sequence polymorphism provided evidence that balancing selection acted in the past. The level of MHC variation, however, was low in the current fish owl populations in Hokkaido: only 19 alleles were identified from 174 individuals. We detected considerable spatial differences in MHC diversity among the geographically isolated populations. We also detected a decline of MHC diversity in some local populations during the past decades. CONCLUSIONS: Our study demonstrated that the current spatial patterns of MHC variation in Blakiston's fish owl populations have been shaped by loss of variation due to the decline and fragmentation of populations, and that the short-term effects of genetic drift have counteracted the long-term effects of balancing selection.

Recent fragmentation of the endangered Blakiston's fish owl (Bubo blakistoni) population on Hokkaido Island, Northern Japan, Revealed by Mitochondrial DNA and Microsatellite Analyses.

INTRODUCTION: Blakiston's fish owl (Bubo blakistoni) was previously widespread on Hokkaido Island, Japan, but is now distributed only in limited forest areas. The population size on Hokkaido decreased during the 20th century due to reduction and fragmentation of the owl's habitat. To elucidate temporal and spatial changes in population structure and genetic diversity, we analyzed 439 individuals collected over the last 100 years. RESULTS: We detected a population bottleneck and fragmentation event indicated by mitochondrial DNA (mtDNA) haplotype and microsatellite analyses. The lowest value for effective population size, which was estimated by moment and temporal methods from microsatellite data, occurred in the 1980s. Five haplotypes were found in the mtDNA control region; most haplotypes were previously widespread across Hokkaido, but have become fixed in separate areas after the bottleneck period. Genetic differentiation among local populations, as indicated by both mtDNA and microsatellite data, likely arose through population fragmentation. CONCLUSIONS: The owl population may have been divided into limited areas due to loss of habitats via human activities, and have lost genetic variability within the local populations through inbreeding. Our mtDNA and microsatellite data show that genetic diversity decreased in local populations, indicating the importance of individuals moving between areas for conservation of this species on Hokkaido.

Limited phylogenetic distribution of a long tandem-repeat cluster in the mitochondrial control region in Bubo (Aves, Strigidae) and cluster variation in Blakiston's fish owl (Bubo blakistoni).

To investigate the phylogenetic position of Blakiston's fish owl (Bubo blakistoni), we sequenced the mitochondrial (mt) DNA control region and cytochrome b (cyt b) for nine Bubo species. Maximum-likelihood analyses of combined control region and cyt b sequences, and cyt b sequences alone, showed that species formerly placed in genus Ketupa comprise a monophyletic group. Unexpectedly, we discovered a long cluster of 20-25 tandem repeat units 77 or 78bp long in the third control region domain in four of the nine Bubo species for which the control region was sequenced (B. blakistoni, B. flavipes, and B. ketupu in the Ketupa clade; B. lacteus), leading to overall control region lengths of 3.0-3.8kpb estimated from agarose gel electrophoresis. The control region in B. lacteus is the longest (3.8kbp) reported to date in vertebrates. Sequencing of eight repeat units at each end of the cluster in 20 B. blakistoni individuals detected several types of repeat units 77 or 78bp long, and six patterns in the order of unit types. The occurrence of a repeat cluster in all three species examined in the Ketupa clade suggests their common ancestor also had a cluster, whereas a maximum parsimony tree showed repeat-unit types grouping by species, rather than by paralog groups, suggesting independent origins of the clusters. We reconcile these results with a turnover model, in which the range in cluster-length variation and unit types at the 5' end are hypothetically functionally constrained by the protein-binding function of the control region, but otherwise there is a continual turnover of units in evolutionary time, with new unit types arising through mutations, proliferating by duplication of single and double repeat blocks, and being lost through deletion. Estimated free energies for reconstructed secondary structures of single and especially pairs of repeat units were higher than for homologous single-unit blocks in species lacking a repeat cluster, supporting slipped-strand mispairing as the mechanism of cluster turnover.

Temporal changes of genetic population structure and diversity in the endangered Blakiston's fish owl (Bubo blakistoni) on Hokkaido Island, Japan, revealed by microsatellite analysis.

The Blakiston's fish owl (Bubo blakistoni) population on Hokkaido Island, Japan, decreased to less than one hundred individuals over the last century due to habitat disruption by human activity. Although the ongoing conservation management has slightly restored the population, it remains endangered. In order to assess the genetic variation and population structure of the Blakiston's fish owl in Hokkaido, we genotyped eight microsatellite loci on 120 individuals sampled over the past three decades. The genotype data set showed low levels of genetic variation and gene flow among the geographically isolated five subpopulations. Comparative analysis of past and current populations indicated that some alleles shared by past individuals had been lost, and that genetic variation had declined over the last three decades. The result suggests that the genetic decline may have resulted from inbreeding and/or genetic drift due to bottlenecks in the Hokkaido population. The present study provides invaluable genetic information for the conservation and management of the endangered Blakiston's fish owl in Hokkaido.