Kouichi Kawamura

Origin and dispersal of bluegill sunfish, Lepomis macrochirus , in Japan and Korea

The bluegill sunfish, Lepomis macrochirus, is a notorious exotic species in many freshwater ecosystems, currently expanding its distribution worldwide. In 1960, a small group of bluegills captured in the Mississippi River at Guttenberg in Iowa were imported to Japan as a gift from the mayor of Chicago to the Japanese government. The offspring of these fish were released into the wild in Japan and also in Korea. Over 40 years after this first introduction, L. macrochirus now occupies all the freshwater ecosystems of both countries. We compared invading populations of L. macrochirus in Japan and Korea with native populations in the USA, using PCR–RFLP (polymerase chain reaction–restricted fragment length polymorphism) analyses of mitochondrial DNA, to estimate the origin and dispersal of L. macrochirus in Japan and Korea. Five haplotypes of mitochondrial DNA detected in Japanese and Korean populations completely coincided with the haplotypes of the Guttenberg population. Haplotype diversity of invading populations was shown to be highest in populations established in the 1960s, while genetic variability was lower in more recently established populations. Our results suggest that all L. macrochirus in Japan and Korea have originated from the 15 fish first introduced in 1960. Low haplotype diversity in newly established populations is probably due to genetic drift arising from repeated population bottlenecks, while the high similarity of haplotypes among neighbouring populations is considered to reflect the history of transplantation by humans.

HERMAPHRODITIC FRESHWATER CLAMS IN THE GENUS CORBICULA PRODUCE NON-REDUCTIONAL SPERMATOZOA WITH SOMATIC DNA CONTENT

Hermaphroditic freshwater clams in the genus Corbicula produce non-reductional spermatozoa. The DNA content of spermatozoa was almost identical with that of somatic cells in C. leana from Mie Prefecture, Japan. Hermaphroditic C. aff. fluminea from Saga Prefecture and C. fluminea from Taiwan also produce non-reductional spermatozoa. On the other hand, spermatozoa of the dioecious C. sandai had half the DNA found in somatic cells. Analysis of chromosome numbers suggests that C. leana (3n = 54 in somatic cells and 18 in meiotic cells) from Mie Prefecture and C. aff. fluminea (2n = 36 in gills and 18 bivalents in meiotic cells) from Saga Prefecture are triploids and diploids, respectively. C. leana, C. aff. fluminea, and C. fluminea may lack either first or second meiosis, resulting in non-reductional spermatozoa. We assume that gynogenetic reproduction occurs in both species; maternal chromosomes are also nonreductional, and spermatozoa activate development of the eggs, but do not contribute to the offspring.

Sperm mitochondrial DNA transmission to both male and female offspring in the blue mussel Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (M type) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, it has been reported that female mussels generally have only maternal mtDNA (F type). In this study, we examined the mode of mtDNA transmission in Mytilus galloprovincialis using M and F type‐specific primer sets. The ratio of M and F types were measured in each sample by SNaPshot. The M type was detected in the adductor muscle and female gonad of all females. In unfertilized eggs spawned by 84.6% of females (22/26), M type was also detected. The F type was more abundant than the M type in all females. Although the ratio of M type in females was very low, all females contained the M type. From these results, we propose a new possibility about DUI inheritance. The presence of M type in unfertilized eggs indicates that the M type of eggs may also contribute to M type inheritance.

Genetic Introgression by the Rose Bitterling, Rhodeus ocellatus ocellatus, into the Japanese Rose Bitterling, R. o. kurumeus (Teleostei: Cyprinidae)

Abstract The Japanese rose bitterling, Rhodeus ocellatus kurumeus, is an endangered cyprinid species. The main reason of drastic diminution of R. o. kurumeus has been suggested to be due to hybridization between this subspecies and R. o. ocellatus introduced from China. Both RFLP analysis of mitochondrial DNA and RAPD-PCR analysis of genomic DNA were performed to elucidate the genetic introgression of R. o. ocellatus into R. o. kurumeus. The two subspecies were distinguished in the D-loop region of mtDNA by six restriction endonucleases. Rhodeus o. kurumeus had eleven subspecies-specific RAPD markers and R. o. ocellatus had two. Except some R. o. kurumeus populations, most of the populations of R. ocellatus in Japan were hybrids, equipped with subspecies-specific RAPD markers for the two subspecies, respectively. The genetic constitution of these markers in hybrids, however, greatly differed among populations. The R. o. ocellatus mtDNA was predominantly observed in hybrid populations, except two populations with mtDNAs of the two subspecies. Judging from the genetic dominance of morphological and physiological characters of R. o. ocellatus against R. o. kurumeus, hybrids probably have the same ecological dominance as R. o. ocellatus against R. o. kurumeus. Therefore, it is considered that R. o. kurumeus not only has its genetic property spoiled by hybridization with R. o. ocellatus, but also is expelled by R. o. ocellatus and hybrids. The replacement of mtDNA and genomes of R. o. kurumeus with those of R. o. ocellatus in hybridization might be accelerated by the backcross between hybrids and R. o. ocellatus.

A peculiar relationship between genetic diversity and adaptability in invasive exotic species: bluegill sunfish as a model species

A peculiar relationship exists between population genetics and invasion biology. Introduced populations often suffer a depletion of genetic variation, but they can persist and adapt to new environments. Here, we show that this relationship is observed in bluegill sunfish (Lepomis macrochirus), an invasive exotic fish in Japan. Genetic analysis using selectively neutral genetic markers reconfirmed that the bluegill introduced into Japan from the United States in 1960 had a single origin with only 15 founders. The analysis also shows that in the process of range expansion, the introduced bluegills experienced severe depletion of genetic diversity due to the founder effect and/or genetic drift. Despite such a decline in genetic diversity, the bluegill populations exhibited a divergent feeding morphology in response to the colonized environments. Such a morphological divergence can facilitate prey exploitation, thereby causing a greater negative impact on native prey resources. Further, in a trophically polymorphic bluegill population in Lake Biwa, physiological characteristics and genetic structures of the intestinal bacterial communities were associated with the difference in diet among the trophic morphs in the host bluegill population. This empirical evidence suggests that despite the severe decline in genetic diversity, the introduced bluegill populations rapidly adapted to the new environment and formed diverse functional relationships with the native bacterial community. Thus, these findings suggest that genetic variation at selectively neutral markers does not always predict adaptability and invasiveness in introduced populations.

Androgenetic Reproduction in a Freshwater Diploid Clam Corbicula fluminea (Bivalvia: Corbiculidae)

Abstract Two shell color types of the exotic bivalve Corbicula fluminea were collected in Kyoto city, Japan. DNA microfluorometry revealed that both types were diploids with non-reductional spermatozoa. Maternal chromosomes were found to be extruded as two polar bodies at the first meiosis, and the second meiosis could not be observed. Only the male pronucleus was present in the egg cytoplasm and became metaphase chromosomes at the first mitosis. The present study indicates that the diploid C. fluminea in Japan has the same mode of androgenetic reproduction as the triploid C. leana.

Sex determination system of the rosy bitterling, Rhodeus ocellatus ocellatus

The rosy bitterling, Rhodeus ocellatus ocellatus, is the only fish species known in which artificial triploids are always male, regardless of the kind of polyploidization technique used. In order to elucidate the genetic sex determination system of the rosy bitterling, two kinds of gynogenesis were carried out: retention of the second polar body (GRSPB) and suppression of the first cleavage (GSFC). The sex ratio of progeny was nearly 7:1 (♂:♀) for both GRSPB and GSFC, while those of control and parental fish were almost 1:1. In backcrosses of female progeny by GRSPB and normal diploid males, male progeny were observed at low frequency (one or two individuals in each experiment), except in one experiment where the appearance rate of males was about 50%. From results of gynogenesis and backcrosses, the following conclusions can be made. The genetic sex determination system of the rosy bitterling is a heterogametic female system (ZW). Survival rate of superfemales (WW), produced by gynogenesis, is much lower than that of males (ZW).There is a possibility that crossovers between sex determining genes and a centromere occur in the first meiosis. With repect to the mechanism of unisexuality (male) of artificial triploids of the rosy bitterling, only males (ZZZ and ZZW) are presumed viable, while females (ZWW) are probably inviable.

Inheritance of two M type mitochondrial DNA from sperm and unfertilized eggs to offspring in Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (mtDNA) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, sperm mtDNA (M type) is inherited only by males. Female mussels receive maternal mtDNA (F type). However, in our previous study, we showed female and unfertilized eggs have both F and M types. We hypothesized that the two M types both from sperm and unfertilized eggs were transmitted to offspring. To test the hypothesis, we examined the number of M type haplotypes in mature M. galloprovincialis. The M type in larvae was compared with those of the parents. Cross experiments were carried out to test the inheritance of M type. In six of 20 mature mussels, two M types were detected by sequence analysis and polymerase chain reaction‐restriction fragment length polymorphism. In cross experiments of larval samples from five of 12 crosses, double peak wave was observed by single nucleotide polymorphisms analysis. In these larval samples, the higher peak wave was identical to the parental M type. Larvae received much more paternal M type than the maternal ones. We demonstrated that two M types from sperm and unfertilized eggs were transmitted to offspring in M. galloprovincialis.

Low Genetic Variation and Inbreeding Depression in Small Isolated Populations of the Japanese Rosy Bitterling, Rhodeus ocellatus kurumeus

Abstract The Japanese rosy bitterling, Rhodeus ocellatus kurumeus, has been affected not only by the invasion of another subspecies, R. o. ocellatus, from China, but also by habitat fragmentation. In this study, the effects of habitat fragmentation on the fitness of R. o. kurumeus were investigated. Owing to exclusion by R. o. ocellatus, R. o. kurumeus in Honshu and Shikoku has disappeared entirely, except for small populations in isolated man-made ponds in Osaka and Kagawa. In Kyushu it still occupies open water systems, into which R. o. ocellatus has only recently invaded. Meristic and genetic data show that the diversity of R. o. kurumeus is significantly lower in the isolated Osaka and Kagawa populations than the non-isolated Fukuoka population. The Osaka population is inferior to the Fukuoka population in terms of viability and growth. The viability of reciprocal inter-population hybrids between the Osaka and Fukuoka populations was, however, as high as that of the Fukuoka population. In addition to the high scores of band sharing index (BSI) in RAPD-PCR analysis, acceptance of transplanted scales among individuals, irrespective of natal pond, indicates that the Osaka population forms a highly inbred line. These results suggest that low genetic variation is associated with inbreeding depression in the small isolated Osaka populations. Consequently, the management of ponds, including the free movement of individuals, in addition to measures to prevent the invasion of R. o. ocellatus, is necessary for the conservation of R. o. kurumeus.

Genetic diversity in the Japanese rosy bitterling, Rhodeus ocellatus kurumeus (Cyprinidae)

Abstract Comparison of meristic characters (pored lateral line scales, vertebrae, and fin rays), and PCR-RFLP analysis in the D-loop and ND1 regions of mitochondrial DNA were performed to estimate the genetic diversity in local populations of the Japanese rosy bitterling, Rhodeus ocellatus kurumeus. In terms of meristic characters, the Fukuoka population was the largest in both range and variance of the number of pored lateral line scales and vertebrae (abdominal and caudal), and Osaka was the second, whereas the Kagawa population showed the smallest range and variance in these characters. In PCR-RFLP analysis, 11 haplotypes (3 in Fukuoka, 2 in Okayama, 2 in Kagawa, and 4 in Osaka) were observed, and nucleotide sequence divergence (NSD) was approximately two times larger in ND1 (mean, 0.61%) than in D-loop (mean, 0.31%). In the neighbor-joining (NJ) tree, based upon the NSD value in ND1, haplotypes were arranged into four clades, which corresponded to the locality of each haplotype. The Fukuoka population was conspicuously apart from the other populations (mean, 0.90% in NSD), but the remaining three showed a similar genetic distance with each other (mean, 0.48%–0.52% in NSD). In haplotype diversity of mtDNA, half the stations in Osaka and all in Kagawa were monomorphic. Especially, two haplotypes endemic to Kagawa were randomly distributed, irrespective of drainages. Rhodeus o. kurumeus in Fukuoka inhabits small rivers and creeks (open water systems), while that in Kagawa and Osaka lives in small ponds (closed water systems). Taking the information of morphology, mtDNA, and habitat into consideration, the low genetic diversity in Kagawa and Osaka populations of R. o. kurumeus is thought to be mainly the result of the isolation of their habitat.