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

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. Received: January 14, 2001 / Revised: June 14, 2001 / Accepted: July 1, 2001     

Adaptive spatial utilization of host mussels by the Japanese rosy bitterling Rhodeus ocellatus kurumeus

The spatial pattern of resource utilization for oviposition in the Japanese rosy bitterling Rhodeus ocellatus kurumeus was investigated in two field experiments. The distribution of rosy bitterling eggs deposited in the four demibranchs of the gills of the test mussel species, Anodonta woodiana , differed with mussel reproductive state in pair spawnings, but not in group spawnings. In pair spawnings, female rosy bitterling may have had more time to select the site of oviposition in the gill in relation to the sex and reproductive state of the mussel, thereby maximizing embryo survival. Thus, the inner gill of female mussels may have been selected in preference to the outer gills to avoid mortalities of eggs due to the presence of the mussel's own embryos (glochidia) in the outer gill chambers. In male mussels, female rosy bitterling distributed their eggs equally among all parts of the mussel gill, thereby minimizing density-dependent mortality of embryos. During group spawnings, however, female rosy bitterling may have been more constrained in their decision making, ovipositing in the inner gills irrespective of mussel sex.     

Spermatozoa in triploids of the rosy bitterling Rhodeus ocellatus ocellatus

Artificially induced triploid male Rhodeus ocellatus ocellatus showed typical nuptial colorations, irrespective of spermiation. In milt from triploids, abnormal spermatozoa (malformation of the head and mitochondrion, excessive formation of the head, mitochondrion and flagellum, and no flagellum) occurred at 78°4% frequency. Spermatozoa with multiflagella were most common, often with a saccate-like organ. Many triploid spermatozoa moved actively as long as those of diploids (10·92±0·91 min=mean±S.D., P >0·05), but did not advance like diploids, spinning around until movement ceased. The sperm density in triploids was < 2% of that from diploids. In triploid testes, deformed and variously sized spermatids were often observed, and normal spermatids and spermatozoa were seldom recognized. The DNA content of triploid spermatozoa varied greatly, compared with that of diploids. Peak of sperm DNA content differed slightly between two triploid samples with two peaks at 1·5 n and 1·9 n ( P <0·0001 in both), respectively. Triploids had the greatest average sperm head diameter of 2·25±0·67 μm (mean±S.D.), while that of diploids was 1·83±0·15 μm ( P =0·002). In the fertilization test using the eggs of diploids ( n =1500, 30 trials), only one egg developed. The embryo chromosome number was 60 (2·5 n) and the ploidy of spermatozoa contributing to fertilization appears to be 1·5 n. The extremely low fertility of triploid R. o. ocellatus spermatozoa seems to be caused by the reduced motility and large head size of spermatozoa, and the low sperm density of the milt. The ploidy of spermatozoa that are successful in fertilization is likely to be related to the distribution pattern in the DNA content of cells.     

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.     

Factors affecting seasonal mortality of rosy bitterling (Rhodeus ocellatus kurumeus) embryos on the gills of their host mussel

I investigated the seasonal change in factors affecting embryonic mortality in the rosy bitterling, Rhodeus ocellatus kurumeus, a freshwater fish that spawns on the gills of living unionid mussels. Research was conducted in a small pond during 1999 and 2001 in which bitterling were provided with Anodonta sp. mussels for spawning. Bitterling spawned between April and July, peaking mid–late May. Seasonal survival rate of bitterling embryos in their mussel hosts was unimodal, with a peak between late April and mid May (about 70% of total spawnings). In mid April, survival was about 50%. The lowest survival was from late May to July (0%). Losses of bitterling embryos from mussels were identified by ejections from the mussel host. Ejections were categorized as either ejections of live embryos, or ejections of embryos that died in the mussel and were subsequently expelled from the mussel. Ejection rates of live embryos were higher in the earlier part of the spawning period (early–mid April) and dead embryo ejections in the later period (after June). The ejection rate of live embryos was higher among younger embryos earlier in the season, probably because of the incomplete development of morphological and behavioural traits associated with maintaining the embryo inside the mussel gill chambers, and as a consequence of a more protracted developmental period at low temperatures making them more susceptible to ejection. The ejection rate of dead embryos was higher in older embryos later in the season, and in larger mussels and at high embryo densities. The survival of embryos in mussels was probably related to oxygen availability, with mortalities probably caused by asphyxiation. Increased embryo mortalities may arise through competition among embryos, between embryos and mussel, and ambient dissolved oxygen levels. The optimal period for bitterling to spawn may represent a balance between two opposing factors; with positive and negative effects of a seasonal rise in temperature directly affecting embryonic growth rate and oxygen availability.An erratum to this article can be found at This revised version was published online January 2005 with the correction of the authors name.     

Seasonal change in the spatial utilization of host mussels in relation to ovipositor length by female rosy bitterling Rhodeus ocellatus kurumeus

The seasonal changes in ovipositor length and utilization patterns of mussels for oviposition in the rosy bitterling Rhodeus ocellatus kurumeus (Cyprinidae) were investigated in a field experiment and field surveys during the breeding period (April to August). The mean length of ovipositors at oviposition was short at the start (early April) and end (July) of the breeding period. Females with long ovipositor at oviposition were collected between mid‐April and June. Mark‐and‐recapture data showed that ovipositor length at oviposition changed rhythmically throughout the breeding period, shortening and lengthening as the female entered the spawning and resting phase. The density of rosy bitterling embryos in mussels increased between April and June, peaking in May, but decreasing in July. The position of eggs on mussel gills varied from close to the exhalant siphon to deeper inside the gill during April, and periodically thereafter. There was a positive correlation between ovipositor length at oviposition and the distance from exhalant siphon of mussels to eggs deposited by females, suggesting that ovipositor length at oviposition determined the position of eggs deposited on a mussel gill. Because dissolved oxygen in mussel gills decreased with the density of bitterling embryos, suitable positions for embryo survival in gills changed with embryo density. By changing ovipositor length at oviposition, females might be able to spawn their eggs in a position that maximizes embryo survival. Thus, plasticity in ovipositor length at oviposition may play an important role as an adaptation of rosy bitterling in utilizing mussels when their quality as a spawning substratum fluctuates seasonally.     

Genetic variation of the cytochrome b gene in the rosy bitterling, Rhodeus ocellatus (Cyprinidae) in Japan

According to conventional views, the rosy bitterling, Rhodeus ocellatus, comprises two subspecies, R. ocellatus kurumeus and R. ocellatus ocellatus, the former being native to Japan whereas the latter was introduced into Japan from China during World War II. To examine the genetic structure of Japanese R. ocellatus, part of the mitochondrial cytochrome b gene from 48 individuals collected from various locations in Japan was sequenced. Three major mitochondrial lineages were found. Based on historical evidence, two of these represent R. ocellatus ocellatus and the third R. ocellatus kurumeus. The existence of two distinct lineages of R. ocellatus ocellatus in Japan suggests at least two colonizations. Some local populations comprised purely R. ocellatus kurumeus, but those from Kashima and Ogori included both subspecies. Because the proportion of R. ocellatus ocellatus in Kashima increased from 1994 to 1995, invasion by R. ocellatus ocellatus into R. ocellatus kurumeus habitats is apparently in progress. Received: April 30, 1999 / Revised: March 22, 2000 / Accepted: December 20, 2000     

Genetic markers distinguishing between the two subspecies of the rosy bitterling,Rhodeus ocellatus (Cyprinidae)

Eleven populations of the rosy bitterling,Rhodeus ocellatus, from different localities in Japan, were genetically compared at 16 protein-coding loci using starch-gel electrophoresis. Two loci,Ldh-2 andPgdh, were demonstrated as diagnostic markers for the identification of two subspecies;R. ocellatus kurumeus, which is native to Japan, andR. ocellatus ocellatus, which was introduced from China. The two subspecies were distinguished by the complete substitution of different alleles between them. Population ofR. ocellatus kurumeus occurring in Yao City, Osaka, and in Kanzaki, Saga Prefecture were genetically closely related to each other (genetic distance: D=0.056) but distantly so toR. ocellatus ocellatus from Saitama Prefecture (D=0.202 or 0.265). Electrophoretic analyses also elucidated the existence of hybrid populations of the two subspecies. The populations ofR. ocellatus kurumeus in Yao City had lower genetic variability and a lower incidence of white coloration on the ventral fins than populations of the same in Saga Prefecture. The present study strongly implies that the introduction of the foreign freshwater fishes with subspecific differentiation, into the original range of indigenous subspecies, should be averted not to bring the genetic pollution.     

Timing of juvenile emergence from host mussels in the Japanese rosy bitterling, <Emphasis Type="Italic">Rhodeus ocellatus kurumeus</Emphasis>

The diel rhythm of juvenile emergence from host mussels in the Japanese rosy bitterling, Rhodeus ocellatus kurumeus, was investigated in a small pond in Osaka prefecture, Japan. The emergence of juvenile bitterling from host mussels occurred principally at night, with a peak in the early part of the night. The pattern of juvenile bitterling emergence was discussed in the context of an adaptive response to diurnal predation.     

A genetic interpretation of the variation in inbreeding depression

Inbreeding depression is expected to play an important but complicated role in evolution. If we are to understand the evolution of inbreeding depression (i.e., purging), we need quantitative genetic interpretations of its variation. We introduce an experimental design in which sires are mated to multiple dams, some of which are unrelated to the sire but others are genetically related owing to an arbitrary number of prior generations of selfing or sib-mating. In this way we introduce the concept of "inbreeding depression effect variance," a parameter more relevant to selection and the purging of inbreeding depression than previous measures. We develop an approach for interpreting the genetic basis of the variation in inbreeding depression by: (1) predicting the variation in inbreeding depression given arbitrary initial genetic variance and (2) estimating genetic variance components given half-sib covariances estimated by our experimental design. As quantitative predictions of selection depend upon understanding genetic variation, our approach reveals the important difference between how inbreeding depression is measured experimentally and how it is viewed by selection.     

Estimation of population fecundity of the bitterling,Rhodeus ocellatus,and ecological significance of its spawning habit into bivalves

Fluctuations of the population abundance of the rose bitterling,Rhodeus ocellatus (Kner) in a small pond, Shimizu-ike (700 m²), Osaka Prefecture, Japan, were estimated by the Petersen method from 1973 to 1977. The number of fish fluctuated between 12,600 and 46,700 during that period. In 1974, a large reproductive peak in May contributed mainly by 2- and 3-year-old spawners and a small peak in late July contributed by 1–2-year-old fish were observed. Average number of eggs laid in a bivalve,Anodonta woodiana Lea, in each month was estimated with field experiments from March to November, 1974. In total, 93,400 eggs were laid during the first reproductive peak, and 13,100 eggs during the second reproductive peak. The mortality of eggs and larvae incubated in the bivalve was less than 30%, and approximately 70 % of the larvae that had swum out from the host died in the following six months. Thus, it is estimated that approximately 20% of the eggs laid in the bivalves can survive and grow up to reach the first maturity. The high survival rate ofR. ocellatus among cyprinid fishes might be due to the fact that the eggs and larvae are protected from predation by being embedded in a bivalve, and to the fact that the larvae at the earliest free swimming stage have a good opportunity of surviving because they are much larger in size and more developed morphologically than those of other cyprinid fishes.     

Genetic variation in small,isolated fern populations

Abstract. Differences in genetic variability of several small, isolated populations of four fern species in a restricted area in the Swiss lowlands reflect differences in breeding system, population size, the degree of population fragmentation, and ecological requirements. The investigated populations of Asplenium septentrionale show only little genetic variability (isozyme variation) without gene flow among populations (based on the banding pattern of multi-locus phenotypes), and they persist for long periods despite the small population sizes. In Asplenium ruta-muraria, genetic variability is correlated with age. Young populations show no genetic variation, while old populations show some. All individuals of Polypodium vulgare investigated, either epiphytic or epilithic, share exactly the same enzyme phenotype. The results for these three species can be related to predominance of inbreeding, lack of inbreeding depression, polyploidy, long-distance dispersal, production of large amounts of diaspores, single-spore colonization, and perennial life cycles. Genetic variability in these three species does not seem to be absolutely necessary for the maintenance of their populations. Ecological and demographic factors are considered to be more important. An isolated, glacial relict population of diploid Asplenium viride shows high variability in two out of eight enzyme systems, which may be due to prevailing outbreeding. We discuss aspects of the importance of genetics and life history for conservation biology.     

The genetic status of two subspecies of Rhodeus atremius, an endangered bitterling in Japan

Rhodeus atremius is an endemic bitterling fish from Japan comprising two endangered subspecies, R. a. atremius and R. a. suigensis. The latter subspecies, which had dramatically declined in last decades, was reported to contain two distinct mtDNA lineages. In order to estimate the optimized units for conservation management, the genetic structure of R. atremius was inferred by analyzing mtDNA and microsatellite markers (MS). Allelic richness and heterozygosity of MS in R. a. suigensis was less than half that in R. a. atremius. In R. a. suigensis, not only within-population genetic diversity but also among-population genetic divergence was low, with the exception of population Ah1, while the diversity was high and the divergence showed isolation by distance in R. a. atremius. In mtDNA and MS, R. a. suigensis concordantly formed a single lineage, while R. a. atremius encompassed four mtDNA lineages, two of which were completely admixed into one group on the basis of MS. In population Ah1 a striking introgression between the two subspecies was suggested by a Bayesian-based assignment test, with the presence of mtDNA haplotype of R. a. atremius. Contrary to the prevailing theory, R. a. suigensis corresponds to a single conservation unit, while three units seem appropriate for R. a. atremius. In addition, low genetic diversity of R. a. suigensis might have arisen mainly as a result of recent bottlenecks before population fragmentation, followed by current anthropogenic effects. Genetic introgression in population Ah1 was probably the result of human transplantation of R. a. atremius.     

Microsatellite genetic variation in small and isolated populations of Magnolia sieboldii ssp. japonica

Magnolia sieboldii ssp. japonica, distributed mainly in western Japan, is restricted to high elevation areas (1000-2000 m above sea level) and usually forms small isolated populations. Four microsatellite loci were assayed for 19 populations from six regions spanning the range of distribution, and the levels and distribution of genetic variation were estimated. All four loci were variable, with a total of 39 alleles, but the overall level of microsatellite genetic variation was low, especially compared with a related species, M. obovata. Genetic structure in M. sieboldii was characterised by low intrapopulational genetic variation (A = 3.74 and H(o) = 0.366 on average) and high genetic differentiation even among regional populations. Highly significant isolation-by-distance (IBD) models at the short distance were detected. Genetic drift and limited gene flow was considered to be important in determining the genetic structure within regions. Total genetic differentiation was remarkably high (F(ST) = 0.488 and R(ST) = 0.538), suggesting genetic barriers among regions. Neighbour-joining dendrograms relating the 19 populations, and further analysis on the IBD models, revealed that a stepwise mutation model was more suited than an infinite allele model to explain the genetic differentiation among regions. It is suggested that mutation at microsatellite loci might be influential in generating the genetic differentiation among regions. These results showed the potential of hypervariable microsatellite loci to evaluate the effects of genetic drift and population isolation within regions, and to detect genetic distinctiveness, in spite of the loss of overall genetic variation in M. sieboldii.     

Dynamics of inbreeding depression due to deleterious mutations in small populations: mutation parameters and inbreeding rate

A multilocus stochastic model is developed to simulate the dynamics of mutational load in small populations of various sizes. Old mutations sampled from a large ancestral population at mutation-selection balance and new mutations arising each generation are considered jointly, using biologically plausible lethal and deleterious mutation parameters. The results show that inbreeding depression and the number of lethal equivalents due to partially recessive mutations can be partly purged from the population by inbreeding, and that this purging mainly involves lethals or detrimentals of large effect. However, fitness decreases continuously with inbreeding, due to increased fixation and homozygosity of mildly deleterious mutants, resulting in extinctions of very small populations with low reproductive rates. No optimum inbreeding rate or population size exists for purging with respect to fitness (viability) changes, but there is an optimum inbreeding rate at a given final level of inbreeding for reducing inbreeding depression or the number of lethal equivalents. The interaction between selection against partially recessive mutations and genetic drift in small populations also influences the rate of decay of neutral variation. Weak selection against mutants relative to genetic drift results in apparent overdominance and thus an increase in effective size (Ne) at neutral loci, and strong selection relative to drift leads to a decrease in Ne due to the increased variance in family size. The simulation results and their implications are discussed in the context of biological conservation and tests for purging.     

FISH-mapping of 18S ribosomal RNA genes and telomeric sequences in the Japanese bitterlings Rhodeus ocellatus kurumeus and Tanakia limbata (Pisces,Cyprinidae) reveals significant cytogenetic differences in morphologically similar karyotypes

The Japanese rose bitterling, Rhodeus ocellatus kurumeus, and the oily bitterling, Tanakia limbata, were cytogenetically studied by silver (Ag)- and chromomycin A₃ (CMA₃)-staining, by C-banding and by mapping of the 18S ribosomal genes and of the (TTAGGG) _n telomeric sequence. These two representative species of related genera of the subfamily Acheilognathinae show very similar chromosome complements. Nevertheless, significant differences in the chromosomal distribution of nucleolus organizer regions (NORs) and interstitial telomeric sequences were observed. Whereas R. ocellatus kurumeus shows a single NOR-bearing chromosome pair, T. limbata is characterized by a higher number of variable NORs. Multiple telomeric sequence sites were found at the pericentromeric regions of several chromosomes in the rose bitterling. No telomeric sequence sites were detected near centromeres, but they were found to be scattered along the NORs in the oily bitterling. Two karyoevolutive trends might have been identified in the subfamily.