GENETIC-VARIATION AND DIFFERENTIATION OF THE 2 RIVER SCULPINS, COTTUS-NOZAWAE AND COTTUS-AMBLYSTOMOPSIS, DEDUCED FROM ALLOZYME AND RESTRICTION ENZYME-DIGESTED MTDNA FRAGMENT LENGTH POLYMORPHISM ANALYSES

Genetic differentiation of the two sibling species, Cottus nozawae and C. amblystomopsis, from the northern part of Japan (Hokkaido Island a nd the Tohoku District) was investigated using allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA. Altho ugh the two species are morphologically very similar, previously being thought to be a single species, they have different life-cycles; C. n ozawae has a fluvial life-cycle with a small number of large-sized egg s, whereas C. amblystomopsis is an amphidromous species with a large n umber of small-sized eggs. Four populations of C. amblystomopsis from Hokkaido Island and 24 populations of C. nozawae (22 from Hokkaido Isl and and 2 from the Tohoku District) were sampled and examined. Intrapo pulational differentiation in the two species was measured by examinin g several indexes, including proportion of polymorphic loci (P), mean heterozygosity (H) and nucleotide diversity (pi). All measurements wer e higher in the C. amblystomopsis populations, suggesting that intrapo pulational variation in C. nozawae was less than in C. amblystomopsis and reflecting the difference in effective population sizes between th em. Cluster analyses were performed using the UPGMA method, based on t he data matrices of genetic distance (D) and the net nucleotide differ ence (<(delta)over cap>) between populations. The C. nozawae and C. am blystomopsis populations from Hokkaido Island composed a large cluster (Hokkaido group), while the C. nozawae populations from the Tohoku Di strict composed a different cluster (Tohoku group). Bootstrap probabil ities deduced from 1000 bootstrap replications for presence or absence of restriction sites showed that the mtDNA haplotypes detected within the Tohoku Group occurred in 99.9% of the bootstrap replicates outsid e the mtDNA haplotypes of the Hokkaido group, while those within the H okkaido group occurred in 3.5-64.9% of bootstrap replicates. Consequen tly, the Hokkaido populations of the two species (Hokkaido group) were genetically close to each other, while C. nozawae from the Tohoku Dis trict (Tohoku group) were distant from the Hokkaido group. These resul ts suggest that the ancestral populations of the two species on Hokkai do Island shared the same gene pool, even after becoming geographicall y isolated fi-om the ancestral population of C. nozawae in the Tohoku District by the formation of the Tsugaru Straits.