Landscape genetic patterns of the rainbow darter Etheostoma caeruleum: a catchment analysis of mitochondrial DNA sequences and nuclear microsatellites

Catchment population structure and divergence patterns of the rainbow darter Etheostoma caeruleum (Percidae: Teleostei), an eastern North American benthic fish, are tested using a landscape genetics approach. Allelic variation at eight nuclear DNA microsatellite loci and two mitochondrial DNA regions [cytochrome (cyt) b gene and control region; 2056 aligned base pairs (bp)] is analysed from 89 individuals and six sites in the Lake Erie catchment (Blanchard, Chagrin, Cuyahoga and Grand Rivers) v. the Ohio River catchment (Big Darby Creek and Little Miami River). Genetic and geographic patterning is assessed using phylogenetic trees, pair‐wise F_ST analogues, AMOVA partitioning, Mantel regression, Bayesian assignment, 3D factorial correspondence and barrier analyses. Results identify 34 cyt b haplotypes, 22 control region haplotypes and 137 microsatellite alleles whose distributions demonstrate marked genetic divergence between populations from the Lake Erie and Ohio River catchments. Etheostoma caeruleum populations in the Lake Erie and Ohio River catchments diverged c. 1·6 mya during the Pleistocene glaciations. Greater genetic separations characterize the Ohio River populations, reflecting their older habitat age and less recent connectivity. Divergence levels within the Lake Erie catchment denote more recent post‐glacial origins. Notably, the western Lake Erie Blanchard River population markedly differs from the three central basin tributary samples, which are each genetically distinguishable using microsatellites. Overall relationships among the Lake Erie sites refute a genetic isolation by geographic distance hypothesis. Etheostoma caeruleum populations thus exchange few genes and have low migration among tributaries and catchments.     

Relationships among tributary length,census population size,and genetic variability of white-spotted charr, <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis>, in the Lake Biwa water system

The relationships between census population size and tributary length and between haplotype diversity of the mitochondrial DNA and census population size in ten white-spotted charr populations in the Lake Biwa water system and its adjacent basins were investigated. The census population size (number of fish with ≥100 mm in standard length) significantly increased with the tributary length. In the eastern part of the Lake Biwa water system, haplotype diversity increased with the census population size. On other hand, in the western part of the water system and adjacent basins, haplotype diversity was zero irrespective of the census population size. These results suggest that white-spotted charr populations in the eastern and western part of the Lake Biwa water system have undergone different levels of bottlenecks related to the habitat size in the postglacial warming.     

Genetic assessment of lake sturgeon (<Emphasis Type="Italic">Acipenser fulvescens</Emphasis>) population structure in the Ottawa River

Lake sturgeon (Acipenser fulvescens) are of conservation concern throughout their range. Many populations are dependent on fluvial habitats which have been increasingly impacted and fragmented by dams and human development. Although lake sturgeon were once abundant in the Ottawa River and its tributaries, historical commercial harvests and other anthropogenic factors caused severe declines and low contemporary numbers in lake sturgeon populations. Contemporary habitat fragmentation by dams may be increasing isolation among habitat patches and local rates of decline, raising concerns for persistence of local populations. We used microsatellite DNA markers to assess population structure and diversity of lake sturgeon in the Ottawa River, and analyzed samples from 10 sites that represent more than 500 km of riverine habitat. To test for evidence of anthropogenic fragmentation, patterns of genetic diversity and connectivity within and among river segments were tested for concordance with geographic location, separation by distance and obstacles to migration, considering both natural and artificial barriers as well as barrier age. Despite extensive habitat fragmentation throughout the Ottawa River, statistical analyses failed to refute panmixia of lake sturgeon in this system. Although the long generation time of lake sturgeon appears to have effectively guarded against the negative genetic impacts of habitat fragmentation and loss so far, evidence from demographic studies indicates that restoring connectivity among habitats is needed for the long-term conservation and management of this species throughout this river system.     

RAPTURE (RAD capture) panel facilitates analyses characterizing sea lamprey reproductive ecology and movement dynamics

Genomic tools are lacking for invasive and native populations of sea lamprey (Petromyzon marinus). Our objective was to discover single nucleotide polymorphism (SNP) loci to conduct pedigree analyses to quantify reproductive contributions of adult sea lampreys and dispersion of sibling larval sea lampreys of different ages in Great Lakes tributaries. Additional applications of data were explored using additional geographically expansive samples. We used restriction site‐associated DNA sequencing (RAD‐Seq) to discover genetic variation in Duffins Creek (DC), Ontario, Canada, and the St. Clair River (SCR), Michigan, USA. We subsequently developed RAD capture baits to genotype 3,446 RAD loci that contained 11,970 SNPs. Based on RAD capture assays, estimates of variance in SNP allele frequency among five Great Lakes tributary populations (mean F_ST 0.008; range 0.00–0.018) were concordant with previous microsatellite‐based studies; however, outlier loci were identified that contributed substantially to spatial population genetic structure. At finer scales within streams, simulations indicated that accuracy in genetic pedigree reconstruction was high when 200 or 500 independent loci were used, even in situations of high spawner abundance (e.g., 1,000 adults). Based on empirical collections of larval sea lamprey genotypes, we found that age‐1 and age‐2 families of full and half‐siblings were widely but nonrandomly distributed within stream reaches sampled. Using the genomic scale set of SNP loci developed in this study, biologists can rapidly genotype sea lamprey in non‐native and native ranges to investigate questions pertaining to population structuring and reproductive ecology at previously unattainable scales.     

Range expansion and lineage admixture of the Japanese evergreen tree Machilus thunbergii in central Japan

We investigated the range expansion histories of Machilus thunbergii populations in the Kinki region of central Japan on the basis of nuclear microsatellite data. In the Kinki region, M. thunbergii is typically found in the coastal area, with some fragmented populations inland, around Lake Biwa. Phylogenetic and Bayesian clustering analysis (STRUCTURE analysis) revealed that the inland populations have different genetic components between the west and east sides of Lake Biwa. The population located on the north side of the lake has an admixture of the two genetically differentiated lineages, contributing to an increase in the genetic diversity of the population. Populations around Lake Biwa had lost rare alleles and the F value obtained from STRUCTURE analysis was lower in the coastal populations than in the lake populations. These results suggest that populations around Lake Biwa experienced a bottleneck due to a founder effect during the initial migration to the lake and that glacial refugia of M. thunbergii in the Kinki region existed along the coast.     

Combining limnological and palaeolimnological approaches in assessing degradation of Lake Pskov

Historical spawning grounds for sea lampreys (Petromyzon marinus L.) in most Portuguese river basins are becoming inaccessible due to the construction of impassable dams and/or weirs. Studies like the one described in this paper are particularly important in areas like the Vouga river basin, where there is a considerable fishing effort from both professional fishermen and poachers. In fact, for management and conservation purposes, it is important to clarify several aspects of the sea lamprey spawning run in this particular watershed. Therefore, a total of 30 radio tagged, migrating sea lampreys were released in the River Vouga and some of its main tributaries during 2004 and 2005. Results from the tracking sessions were used to determine the effect of poaching on the spawners’ population and the characteristics of the resting sites used during the upstream movement. The rivers’ stretches were also characterized according to the type of substrate present in the riverbed and flow type, in order to determine its aptitude to constitute appropriate spawning habitats for sea lampreys. We have identified in the upstream stretches of River Vouga, and in the tributary River Caima, characteristics that are particularly suitable for the construction of nests by the spawners. Presence of larvae provided evidence that spawners migrated into the River Vouga’s upper reaches. However, abundance and age class diversity appeared to be higher downstream of Sernada and Carvoeiro weirs, corroborating the telemetry data which suggested difficulty in passing these obstacles during low precipitation years. In the River Caima, migration was most predictable, which might be related to daily water releases from a small hydropower dam at dusk, that stimulated the lampreys to resume migration. Poaching has had a considerable negative effect on the success of the tagged lampreys’ migration: 76% of the animals released during 2005 were captured. Electronic supplementary material  Supplementary material is available for this article at and accessible for authorized user     

Mitochondrial DNA population structure of the white-spotted Charr (Salvelinus leucomaenis) in the Lake Biwa water system

A phylogeographic analysis of mitochondrial DNA sequences was performed in order to elucidate the origin, dispersal process, and genetic structure of white-spotted charr in the Lake Biwa water system. Two haplotypes were most common in the Lake Biwa water system, and were also common in the adjacent inlet rivers of the Sea of Japan. These results suggest that in the glacial periods of the Pleistocene, white-spotted charr dispersed into the northern inlet rivers of Lake Biwa from adjacent inlet rivers of the Sea of Japan by watershed exchanges, colonizing the whole of the Lake Biwa water system. Mitochondrial DNA diversity contrasted sharply between the western and eastern parts of the system, suggesting that the populations in the western part might be more reduced than those in the eastern part in relation to the smaller habitat size. The high overall FST estimate (0.50), together with pairwise comparisons of FST, indicated significant genetic divergence between populations due to isolation and small population size. Hierarchical analysis (AMOVA) also showed that genetic variation was more pronounced among regions (28.39%) and among populations within regions (47.24%) than within populations (24.37%). This suggests that each population in and around the Lake Biwa water system should be treated as a significant unit for conservation and management.     

Movement of radio-tagged anadromous sea lamprey during the spawning migration in the River Mondego (Portugal)

The available spawning habitat for the anadromous sea lamprey, Petromyzon marinus L., population that enters the River Mondego has been drastically reduced in the last 20 years. The installation of a fish passage in the first impassable dam, the Açude-Ponte, would enable sea lamprey to recolonise the 34.6-km river stretch between the Açude-Ponte and Raiva dams. In order to assess the suitability of the upstream river stretches for this species, 10 radio-tagged sea lamprey were released upstream of the Açude-Ponte dam and tracked continuously throughout the entire migratory path. Lamprey were unable to pass over intact weirs that had been built for recreational purposes. Sea lamprey movements were more frequent during dusk and night than other periods. Increased river discharge at night, resulting from the operation of the Raiva power station, stimulated lamprey movements but reduced ground speeds recorded. After reaching a certain location, some of the animals maintained their position for several weeks, before undergoing a new movement. Two of the main tributaries of this river stretch were used by some sea lamprey, indicating that the animals were able to find these historical spawning grounds.     

The use of environmental DNA of fishes as an efficient method of determining habitat connectivity

This study demonstrated the use of environmental DNA (eDNA) to determine habitat connectivity for migration of fishes between the sea and river. Environmental DNA is DNA fragments released by fishes in water, which can be used as a species-specific marker of the presence/absence of the target species. A year-round water sampling regime at 15 sites on the Yodo River, Japan, was conducted to determine whether three major man-made barriers on the river inhibited the migration of fishes using species-specific detection of DNA fragments from three target migrant species, temperate seabass, Lateolabrax japonicus, flathead grey mullet, Mugil cephalus, and ayu, Plecoglossus altivelis altivelis. The presence/absence of eDNA from target species was consistent with known patterns of species’ seasonal migration. The detection of the DNA of temperate seabass and flathead grey mullet at sites upstream of the dam closest to the river mouth indicated successful upstream migration of these species via a fish ladder bypassing the dam. On the other hand, DNA of these two species was not detected from the upstream side of the two remaining dams, which are not equipped with fish ladders. Ayu is the only species among the three target species with a land-locked population in Lake Biwa located at the headwater of Yodo River. Ayu DNA was detected at most of the sites in the freshwater area during the warm months; however, in the coldest month of February, eDNA was only detected in the uppermost site of Yodo River at the southern tip of Lake Biwa. The eDNA we detected at this site suggests that it was derived from juvenile ayu spending their winter months in the lake. These results suggest that the eDNA analysis presented here can accurately track the seasonal migration of fishes in a river, demonstrating its application as an indicator of habitat connectivity for fishes in association with man-made barriers in a river. The sampling of eDNA involves merely scooping a tank full of water; therefore, it is a simple, rapid, and cost-effective method for long-term monitoring of habitat connectivity associated with the construction of barriers in a river.     

Genetic differentiation of piscivorous chub (genus Opsariichthys) in Japan,Korea and Russia

A comparison of allozyme variation, restriction fragment length polymorphisms in the mitochondrial DNA and partial sequences of the ND II gene (496 bp) was made among two lacustrine populations of the piscivorous chub (Opsariichthys uncirostris uncirostris) in Japan, four fluvial populations in Korea, one lacustrine population in Russia and one specimen from the Amur River (O. u. amurensis). All analyses indicate that the Japanese populations of piscivorous chub are separable from the Asian mainland populations of Korea and Russia. The latter populations were further divided into the Korean and Russian fish. Although opinions are divided on the phylogenetic position of the population in Lake Mikata, Japan, which shows unique morphological traits intermediate between those of the population in Lake Biwa and the mainland populations, the current analysis indicates a closer relationship to the population in Lake Biwa.     

Genome-wide SNPs detect fine-scale genetic structure in threatened populations of squirrel glider Petaurus norfolcensis

Australian arboreal mammals are experiencing significant population declines, particularly due to land clearing and resulting habitat fragmentation. The squirrel glider, Petaurus norfolcensis, is a threatened species in New South Wales, with a stronghold population in the Lake Macquarie Local Government Area (LGA) where fragmentation due to urbanization is an ongoing problem for the species conservation. Here we report on the use of squirrel glider mitochondrial (385 bp cytochrome b gene, 70 individuals) and nuclear DNA (6,834 SNPs, 87 individuals) markers to assess their population genetic structure and connectivity across 14 locations sampled in the Lake Macquarie LGA. The mitochondrial DNA sequences detected evidence of a historical genetic bottleneck, while the genome-wide SNPs detected significant population structure in the Lake Macquarie squirrel glider populations at scales as fine as one kilometer. There was no evidence of inbreeding within patches, however there were clear effects of habitat fragmentation and biogeographical barriers on gene flow. A least cost path analysis identified thin linear corridors that have high priority for conservation. These areas should be protected to avoid further isolation of squirrel glider populations and the loss of genetic diversity through genetic drift.

     

Multiple colonizations of Lake Biwa by Sarcocheilichthys fishes and their population history

Rapid adaptation and speciation have not been well documented for organisms in Lake Biwa, Japan, the oldest ancient lake in East Asia. To examine these processes, we focused on the divergence of Sarcocheilichthys gudgeons and conducted phylogeographic and population genetic analyses using mitochondrial DNA sequences and microsatellite data. No remarkable genetic differentiation was found between two species, S. biwaensis and S. variegatus microoculus, both endemic to Lake Biwa, whereas this species group, including S. v. variegatus, was genetically divided into three major regional groups: the Lake Biwa, Ise Bay, and western groups. Divergent mtDNA haplotypes were included within the Lake Biwa and western groups, strongly suggesting secondary contacts among allopatrically isolated populations. Dating for mtDNA phylogeny using a geological constraint suggested the colonization of Lake Biwa by multiple Sarcocheilichthys lineages that diverged from each other in the early–middle Pleistocene. Coalescent-based population analyses indicated that the local populations colonized the rocky bottom habitat in Lake Biwa from other habitats after the Last Glacial Maximum, likely reflecting past environmental changes in the lake, including the disappearance of rocky areas during the glacial climate. Divergent morphological adaptation in Sarcocheilichthys associated with substrate type may have rapidly proceeded along with environmental changes.     

Mitochondrial phylogeny reveals the artificial introduction of the pale chub Zacco platypus (Cyprinidae) in Taiwan

The presence of the pale chub Zacco platypus (Japanese name, Oikawa) in Taiwan has been suggested to be a result of its inadvertent introduction from Lake Biwa in Japan in the 1980s in conjunction with the Japanese Ayu, Plecoglossus altivelis altivelis, which was released several times into the Tamsui River to restock the extinct Ayu population of Taiwan. However, it is also possible that Z. platypus is native to Taiwan and has not been previously described for reasons of its narrow range. Knowledge of the colonizing history of Z. platypus is of considerable importance because it provides insight into the evolutionary process and, hence, impacts management decisions regarding this species in Taiwan. A portion of the mitochondrial D-loop was sequenced for 77 specimens from five populations of Z. platypus from Japan and Taiwan. A total of 22 haplotypes were identified, and nucleotide divergence among haplotypes ranged from 0.20% to 2.82%. Haplotype diversity was high in all populations examined, with a range from 0.718 in the Tagiri River population to 0.909 in the Lake Biwa population. Phylogenetic and statistical parsimony analyses of the molecular data revealed a close genetic relationship between Taiwanese and Japanese Z. platypus and supported the previous report that the Taiwanese Z. platypus originated in Lake Biwa in Japan.     

Inheritance of amplified fragment length polymorphism markers and their utility in population genetic analysis of Plecoglossus altivelis

The reproducibility, mode of inheritance and polymorphism of amplified fragment length polymorphism (AFLP) markers were examined in ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae). The AFLP markers were highly reproducible, their inheritance following Mendelian expectations. The number of fragments amplified (34–134), polymorphic ratio (0·15–0·78) and average heterozygosity (0·02–0·25) of the AFLP markers showed significant variation among six primer pairs and among ayu populations, including a landlocked Lake‐Biwa population, two amphidromous populations ( P. a. altivelis ) and two Ryukyu‐ayu populations ( P. a. ryukyuensis ). Although AFLP analysis provided similar results in intra‐population diversity and relationships among populations to those found by analyses of allozymes, microsatellites and mitochondrial DNA sequences, AFLPs showed higher polymorphisms and hence greater distinction between genetically close populations.     

Recent colonization by a coastal plant of inland habitats at an ancient freshwater lake,Lake Biwa: multilocus sequencing and a demographic history of Lathyrus japonicus (Fabaceae)

Ancient lakes have been recognized as “long‐term isolated islands” in terrestrial ecosystems. Lake Biwa, one of the few ancient lakes that formed around 4 million years ago, harbors many coastal species that commonly inhabit seashores. The beach pea, Lathyrus japonicus, is a typical coastal species of this freshwater lake, where morphological, physiological, and genetic differentiations have been reported between Biwa and coastal populations. Whether Biwa populations were isolated for long periods throughout Pleistocene climatic oscillations and subsequent range shifts is unclear. We assessed population genetic structure and demography of beach pea in this ancient freshwater lake using the sequences of eight nuclear loci. The results of STRUCTURE analyses showed evidence of admixture between Biwa and coastal populations, reflecting recent gene flow. The estimated demographic parameters implemented by the isolation with migration model (IM model) revealed a recent divergence (postglacial period) of Biwa populations, with some gene flow from Biwa to coastal populations. In addition, Biwa populations were significantly smaller in size than the ancestral or coastal populations. Our study suggests that a Holocene thermal maximum, when transgression could allow seeds from coastal plants to access Lake Biwa, was involved in the origin of the Biwa populations and their genetic divergence. Thus, coastal populations might have migrated to Lake Biwa relatively recently. Our study concluded that ancestral migrants in Lake Biwa were derived from small founding populations and accelerated genetic isolation of Biwa populations during short‐term isolation.     

Contrasting population genetic structure among freshwater‐resident and anadromous lampreys: the role of demographic history,differential dispersal and anthropogenic barriers to movement

The tendency of many species to abandon migration remains a poorly understood aspect of evolutionary biology that may play an important role in promoting species radiation by both allopatric and sympatric mechanisms. Anadromy inherently offers an opportunity for the colonization of freshwater environments, and the shift from an anadromous to a wholly freshwater life history has occurred in many families of fishes. Freshwater‐resident forms have arisen repeatedly among lampreys (within the Petromyzontidae and Mordaciidae), and there has been much debate as to whether anadromous lampreys, and their derived freshwater‐resident analogues, constitute distinct species or are divergent ecotypes of polymorphic species. Samples of 543 European river lamprey Lampetra fluviatilis (mostly from anadromous populations) and freshwater European brook lamprey Lampetra planeri from across 18 sites, primarily in the British Isles, were investigated for 13 polymorphic microsatellite DNA loci, and 108 samples from six of these sites were sequenced for 829 bp of mitochondrial DNA (mtDNA). We found contrasting patterns of population structure for mtDNA and microsatellite DNA markers, such that low diversity and little structure were seen for all populations for mtDNA (consistent with a recent founder expansion event), while fine‐scale structuring was evident for nuclear markers. Strong differentiation for microsatellite DNA loci was seen among freshwater‐resident L. planeri populations and between L. fluviatilis and L. planeri in most cases, but little structure was evident among anadromous L. fluviatilis populations. We conclude that postglacial colonization founded multiple freshwater‐resident populations with strong habitat fidelity and limited dispersal tendencies that became highly differentiated, a pattern that was likely intensified by anthropogenic barriers.     

Fragmentation by weirs in a riverine system: A study of genetic variation in time and space among populations of European grayling (Thymallus thymallus) in a Danish river system

Human induced habitat alterations affect the genetic structure of many fish populations. Weirs in particular have caused fragmented populations previously connected by gene-flow. We studied the effects of weirs on the distribution of genetic variation within European grayling (Thymallus thymallus) populations from the Skjern River, Denmark. We compared microsatellite data from DNA extracted from historic scale samples collected 60 years ago with data from contemporary samples. Pairwise multilocus F _ST estimates between all contemporary population samples were significant as well as exact tests for population differentiation. Assignment tests of individuals to a set of baseline samples showed correct assignment to the population of origin of between 54 and 79%. Assignment of individuals from recent samples to the historic population samples showed highly variable results (3–83%) of correct assignment suggesting different population histories. Pairwise multilocus F _ST estimates were significantly correlated with the number of intervening weirs but not with waterway distance. A simulation procedure was used to estimate differences in relative population sizes, which indicated that the main river population was approximately three times larger than those of the tributaries. There were no signs of any loss of genetic variation for the river system as a whole during these 60 years. The results show that weirs can be an important factor for creating the current distribution patterns of genetic variation among grayling populations, most likely by only allowing passive downstream drift of fry and obstructing active upstream migration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Parasitic Attachments by Overwintering Silver Lampreys, Ichthyomyzon unicuspis, and Chestnut Lampreys, Ichthyomyzon castaneus

We present evidence that at least some parasitic-phase silver lampreys, Ichthyomyzon unicuspis, and chestnut lampreys, I. castaneus, remain attached to host fish during the winter. Lake sturgeon, Acipenser fulvescens, harvested through the ice by spearfishers in the Lake Winnebago system in Wisconsin may bear silver lampreys or fresh lamprey wounds, and sturgeon with lamprey marks were significantly larger than sturgeon without them. Silver lampreys collected on paddlefish, Polyodon spathula, in the Wisconsin River in March were not significantly longer than silver lampreys collected previously in late October, but they were significantly heavier, an indication that they were feeding to at least some extent during the intervening period. Other large fish species, including northern pike, Esox lucius, and flathead catfish, Pylodictus olivaris, have been collected or observed during the winter with silver or chestnut lampreys attached. Although energy and nutrient intake by parasitic lampreys may be reduced during the winter, lampreys attached to hosts may also benefit from the hosts' mobility and ability to avoid potentially harmful situations.     

Size at maturity of fluvial white-spotted charr, <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis>, around the Lake Biwa water system varies with habitat size

Size at maturity of fluvial white-spotted charr, Salvelinus leucomaenis, was studied in small headwater tributaries of nine rivers around the Lake Biwa water system, Japan. Threshold size at maturity in both sexes showed significant positive relationships with water discharge, indicating that smaller threshold sizes at maturity of fluvial white-spotted charr occurred in smaller habitats. These results provide a link between size at maturity and habitat size and have important implications for the management of both habitats and white-spotted charr populations.     

Genetic diversity and population structure of white-spotted charr, Salvelinus leucomaenis, in the Lake Biwa water system inferred from AFLP analysis

The genetic variations—and the time dependence of such variations—of natural populations of the white-spotted charr ,Salvelinus leucomaenis, in the Lake Biwa water system as well as those of a hatchery-reared population were inferred from AFLP. Upon the application of principal coordinate analysis using 118 polymorphic AFLP fragments based on the Jaccard similarity index, specimens of each of six natural local populations from the inlet rivers of Lake Biwa grouped roughly together, suggesting that each local population was genetically differentiated. The hatchery-reared population was shown to be closely related to the local population in the Seri River, suggesting that the Seri River population originated from hatchery-reared charr due to extensive stocking. Furthermore, specimens of the Yasu River grouped in a somewhat different position from the other natural populations, agreeing well with its geographic distance from the other populations. The nucleotide diversities of six natural populations (Harihata River, Ishida River, two reaches of the Takatoki River, Ane River, and Yasu River) in 2002 or 2003 were relatively low (π = 0.067–0.146%) compared with that of the Seri River (0.278%) and the hatchery-reared charr (0.316%). The nucleotide diversity in the five local populations (Ishida River, two reaches of the Takatoki River, Ane River, and Yasu River) remained at a low level from 1994 to 2002/2003, but only the nucleotide diversity in the Harihata River actually decreased. From 1994 to 2002/2003, the nucleotide diversity in the Seri River remained at a higher level among the natural populations from 1994 to 2002/2003; it was enhanced by the artificial release of hatchery-reared charr before 1994. In order to conserve the genetic diversity of the white-spotted charr in the Lake Biwa water system, it is necessary to prevent the stocking of hatchery-reared charr in reaches where hatchery-reared charr have not previously been stocked.