Long‐term isolation of the coastal plant Calystegia soldanella (Convolvulaceae) in ancient freshwater Lake Biwa,Japan

Lake Biwa is an ancient freshwater lake that was formed approximately 4 Mya and harbours many coastal plants that commonly inhabit the seashore. We used chloroplast DNA haplotype analysis using two spacer sequences and simple sequence repeat (SSR) analysis using eight nuclear microsatellite markers to detect genomic signatures indicating long‐term isolation of inland populations of Calystegia soldanella in Lake Biwa from coastal populations. We used 348 samples from 63 populations for haplotype analysis and 478 samples from 27 populations for SSR analysis covering the inland and coastal distribution of the species. We detected seven haplotypes, and the distribution pattern of these haplotypes was geographically highly structured between Lake Biwa and the coast. Nuclear SSR analysis also supported genetic differentiation between Lake Biwa and coastal populations (analyses of molecular variance, 43%), and the grouping of Lake Biwa and coastal populations by a Neighbour‐joining tree. In addition, genetic diversity of the inland populations (mean H_E = 0.153) was significantly lower than that of coastal populations (mean H_E = 0.328). These results suggested that inland populations at Lake Biwa have been isolated from coastal populations for a very long time. The inland populations most likely experienced a bottleneck effect, resulting in sufficient in situ genetic divergence to clearly distinguish them from coastal populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 51–66.     

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.     

Flow cytometric assessment of cytotype distributions within local populations of Phragmites australis (Poaceae) around Lake Biwa,the largest lake in Japan

Phragmites australis (Poaceae) is a clonal perennial that is an important component of wetland ecosystems worldwide. Using flow cytometry, we examined the cytotype distributions within five populations of P. australis located in the vicinity of Lake Biwa. As in previous reports, two ploidy levels, octoploid (2n = 8x = 96) and decaploid (2n = 10x = 120), were identified, which are assumed to be the main cytotypes around the lake. The coexistence of two cytotypes was detected in four of the five populations, suggesting the relatively common occurrence of mixed ploidy levels in the populations around Lake Biwa. Although intermingled cytotype distributions were observed in some populations, the 9x cytotype, that is, the expected outcome of inter‐cytotype crosses, was not observed, indicating limited gene flow between the two cytotypes.     

Introgression of Oncorhynchus masou subsp. (Biwa salmon) genome into lake-run O. m. ishikawae (Amago salmon) introduced into Lake Biwa, Japan

An investigation to determine the extent of introgression between Biwa salmon Oncorhynchus masou subsp. and lake-run Amago salmon O. m. ishikawae from Lake Biwa was conducted using mtDNA sequence and amplified fragment length polymorphism (AFLP) analysis. Hatchery-reared Amago salmon from the Samegai Trout Farm, the main source of stock released into the inflowing rivers of Lake Biwa, were also investigated. The extent of mtDNA introgression between these two subspecies was quite low: an anomalous haplotype was detected in just 1 of 55 Biwa salmon and 2 of 63 lake-run Amago salmon. Many of the mtDNA haplotypes found in lake-run Amago salmon were common to those in hatchery-reared Amago salmon. In the AFLP analysis, the nuclear genome of Amago salmon was hardly detected in Biwa salmon, but the Biwa salmon genome comprised 13.4% of the total amplified fragments from lake-run Amago salmon, being detected in different proportions in 28 of the 63 investigated individuals of the latter subspecies. These results suggest that introgression has occurred between Biwa salmon and lake-run Amago salmon descending from hatchery-reared Amago salmon from the Samegai Trout Farm.     

Isolation and characterisation of microsatellite loci in Calystegia soldanella (Convolvulaceae), an endangered coastal plant isolated in Lake Biwa,Japan

Eight microsatellite loci of Calystegia soldanella useful for comparisons of the genetic structure of isolated populations in the ancient Lake Biwa and coastal populations in Japan were isolated and characterised. The number of alleles ranged from 2 to 5. The expected (H _E) and observed (H _O) heterozygosities were 0.097–0.583 and 0.000–0.380, respectively, from 100 individuals from Lake Biwa and coastal populations. Seven of the eight loci exhibited significantly fewer heterozygotes than expected based on the Hardy–Weinberg equilibrium (P < 0.05). These primers amplifying microsatellites in C. soldanella may provide a population genetics tool useful in the establishment of a conservation strategy.     

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.     

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.     

Morphology and taxonomy of Stephanodiscus suzukii sp. nov. and Stephanodiscus pseudosuzukii sp. nov. (Bacillariophyceae) from Lake Biwa, Japan, and S. carconensis from North America

Two new, recent diatoms, Stephanodiscus suzukii Tuji et Kociolek and Stephanodiscus pseudosuzukii Tuji et Kociolek are described from Lake Biwa, Japan, where they had been identified previously as Stephanodiscus carconensis Grunow var. carconensis and S. carconensis var. pusillus Grunow, respectively. These forms are compared in terms of valve ultrastructure with populations of S. carconensis from North America. The Japanese species differ from the North American specimens in characteristics related to the central fultoportula and rimoportula.     

Observations on Aulacoseira nipponica from Lake Biwa, Japan, and Aulacoseira solida from North America (Bacillariophyceae)

A recent diatom, Aulacoseira nipponica (Skvortzow) Tuji comb. et stat. nov., is described from Lake Biwa, Japan, where they had been identified previously as Aulacoseira solida (Eulenstein) Krammer. These forms are compared with populations of A. solida from North America. The Japanese species differs from the North American specimens in characteristics related to the density of striae and form of the rimoportulae.     

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.     

Reproductive ecology of the Far Eastern catfish, Silurus asotus (Siluridae), with a comparison to its two congeners in Lake Biwa, Japan

Spawning activity of the catfish, Silurus asotus, takes place in temporary water (rice fields) from early evening to midnight, generally in connection with rainfall, from late April to late August. Spawning of the catfish was correlated with hydrographic parameters dependent on rainfall: daily precipitation, turbidity, water depth, and water temperature. The spawning habits of the catfish, and in particular the use of temporary waters such as rice fields that become submerged after rainfall or by irrigation, are presumed to be adaptations to the Asian monsoon climate with a pronounced rainy season. The apparent sex ratio of the catfish was extremely biased toward females. Intraspecific variation in the reproductive ecology, particularly mating behavior, of this species is observed among local populations. Factors that may have caused this variation are discussed in the context of a comparison of mating behavior, reproductive environment, and sex ratio between the Lake Biwa population of S. asotus and other conspecific populations, as well as two other species of silurid catfish that occur in the Lake Biwa drainage, S. biwaensis and S. lithophilus.     

Mesocyclops dissimilis n. sp. from Lake Biwa,Japan (Copepoda,Cyclopoida)

The status of the Mesocyclops from lake Biwa is reexamined and a new species, Mesocyclops dissimilis n. sp., distinct from M. thermocyclopoides Harada, 1931, is described.     

Geochemistry of Lake Biwa sediments revisited

Extensive chemical data for the Lake Biwa sediments were examined with statistical factor analysis to find any correlation patterns among elements and among samples. Based on those patterns, a series of x-y correlation plots of the concentrations of selected elements was constructed to see in detail how the concentrations of elements vary among different sample groups and explain the observed variation patterns.     

The effect of human activities on benthic macroinvertebrate diversity in tributary lagoons surrounding Lake Biwa

In aquatic ecosystems, tributaries play an important role in maintaining the populations and diversity of aquatic organisms throughout the drainage basin, but because of their small catchments, these ecosystems are often more susceptible to the effect of land-use changes and flow-regime alterations. Here, we examined anthropogenic effects on benthic macroinvertebrate diversity in the tributary lagoons, collectively called “Naiko,” surrounding Lake Biwa. We sampled macroinvertebrates and determined the environmental characteristics of 20 tributary lagoons. We identified the environmental factors determining the diversity of macroinvertebrates and found that turbidity significantly diminished species diversity. We assessed the anthropogenic stressors that contributed to the increase in turbidity and found that human population density and the proportion of paddy fields in the watershed area were positively correlated with turbidity, most likely caused by erosion of terrigenous organic matter from the paddy fields and urban areas. In addition, the presence of sluice gates and the lengths of channels connecting to the main lake were positively correlated with turbidity, suggesting that these factors lowered hydrologic connectivity and retained organic matter. We concluded that changes to the landscape and decreased hydrologic connectivity caused by human activity increased turbidity, which in turn decreased macroinvertebrate diversity. The identification of these factors in tributaries is vital for developing a strategy for habitat restoration to conserve the entire ecosystem of the Lake Biwa basin.     

Gytological studies of Anemone nemorosa L. (Ranunculaceae)

Investigations of mitosis in 10 populations of Anemone nemorosa L. all indicated that this species has a chromosome number of 2 n = 30. No B chromosomes were found. There were no significant differences in chromosome morphology between the populations. Meiosis in the anthers, studied in three populations, was found to be mostly regular, with 15 bivalents, although occasional irregularities such as bridges and pentads were observed. Pollen viability was tested in 12 populations and although fairly low, with micro pollen grains in four of the populations, was not low enough to prevent a high proportion of fertilized achenes.     

Morphological variation of Keratella cochlearis (Gosse) in Lake Biwa,Japan

The length of the lorica (LL) of Keratella cochlearis cochlearis and of K. cochlearis tecta and the length of posterior spine (PSL) of the latter morphotype were measured in the strongly eutrophic basin and also in the mesotrophic basin of Lake Biwa, Japan, from September to December, 1980. In the population from the mesotrophic basin, the individuals with longer PSL prevail and the tecta forms are extremely rare. The LL values of both morphotypes from one sample do not differ. In December the LL increased to 95 µm in both morphotypes from 80 µm observed in September, while the PSL values decreased abrubtly in both basins in the middle of this period. It is suggested that the observed increase of LL could be related to the thermic factor, i.e. a steady decrease of water temperature, and the changes of PSL are correlated with the increase of nannoplankton and detritus aggregates noted in November. In this month an increase in fecundity and in the total numbers of rotifers took place as well (Hillbricht-Ilkowska, in press).     

Sources of dissolved methane in Lake Biwa

The spatial distribution and seasonal variation in the concentration and carbon isotopic composition of dissolved methane in a river–lake ecosystem were studied in Lake Biwa, Japan, and its tributary rivers. Methane concentrations in all subsystems examined were supersaturated with respect to the atmosphere. The epilimnion showed higher concentrations of dissolved methane than the hypolimnion in the pelagic zone. Peak methane concentrations were observed at the thermocline. The largest amount of methane in the pelagic water column was recorded at the end of a stagnant period, at which the bottom water of the sublittoral zone (30m in depth) exhibited increased methane concentration. Transect observation of dissolved methane revealed three methane peaks at different water depths in the lake, and river water and the sediments in littoral and sublittoral zones were suggested to be the corresponding sources. Water at the river mouth was replete with dissolved oxygen but also contained a high concentration of methane. The present results suggest that river water and littoral sediment are potential sources of dissolved methane in Lake Biwa, and other sources, such as internal waves, are responsible for increased methane in the pelagic zone at the end of stagnant periods. Carbon stable isotope analysis indicated that there were different sources of dissolved methane, although it was difficult to identify the origins due to high variation of the isotopic composition of methane from different sources.     

Genetic Variation Within and Among Metal-Tolerant and Non-Tolerant Populations of Armeria maritima (Mill.) Willd. s.l. (Plumbaginaceae) in Central and Northeast Germany

Abstract: Six metal-tolerant populations and sub-populations of Armeria maritima ssp. halleri, ssp. hornburgensis, and ssp. bottendorfensis and two non-tolerant populations of ssp. elongata in Central and Northeast Germany have been analysed using RAPD markers. The populations show very strong genetic differentiation (Φ_ST = 0.46), corresponding gene flow between them is low (N_em = 0.29). A moderate positive correlation between the matrices of genetic and geographical distances was found between the seven populations and sub-populations of central Germany (r = 0.68, p < 0.001). Calculated parameters of genetic variability are molecular variance, percentage of heterozygosity and percentage of polymorphic loci. A significant correlation between population size and parameters of genetic variability was not recognisable. Genetic structure was investigated by an analysis of molecular variance (AMOVA). The studied populations show strong genetic differentiation. Genetic variation within populations ("normal" as well as metalliferous) is higher (53.9 %) than among them (46.1 %). Six hypotheses of possible genetic relatedness between the studied populations have been tested by AMOVA. A data set structure above the populational level is hardly recognisable. It was impossible to combine the populations to edaphic (tolerant and "non-tolerant") or taxonomic groups. A. maritima ssp. halleri of the north Harz mountains and ssp. hornburgensis are clearly separated from a geographical group containing all other populations (across taxonomic and edaphic boundaries). These results are a further indication for a polyphyletic origin of metal-tolerant populations of A. maritima s.l. by multiple colonizations of metalliferous sites from neighbouring populations on non-metalliferous soil.     

Predator-prey relationship between the landlocked dwarf ayu and planktonic Crustacea in Lake Biwa, Japan

The predator-prey relationship was investigated between the landlocked dwarf ayu (koayu; Plecoglossus altivelis) and three planktonic crustaceans, Daphnia galeata, Eodiaptomus japonicus, and Mesocyclops dissimilis, in Lake Biwa. The abundance of each prey species and daphniid size composition in the lake was compared with those in the stomachs of koayu collected at the same time and place. D. galeata was the main item of diet of koayu in spite of their low density, and the larger individuals were selectively preyed upon by the predator. In the stepwise multiple regression of the mean number of each prey species in the stomachs on prey densities in the lake and predator mass, the density of the prey itself was always selected as the predictor variable. The density of E. japonicus was also selected as the regressor of daphniid number in the stomachs with a negative slope. Multiple regression was also performed each for two size-classes of D. galeata, and a negative effect of large daphniids was detected for the consumption of small ones. The present results and additional information enable estimation of the collective predation rate by the koayu population on the prey populations.     

Low level of gene flow from cultivated beets (Beta vulgaris L. ssp. vulgaris) into Danish populations of sea beet (Beta vulgaris L. ssp. maritima (L.) Arcangeli)

Gene flow from sugar beets to sea beets occurs in the seed propagation areas in southern Europe. Some seed propagation also takes place in Denmark, but here the crop-wild gene flow has not been investigated. Hence, we studied gene flow to sea beet populations from sugar beet lines used in Danish seed propagation areas. A set of 12 Danish, two Swedish, one French, one Italian, one Dutch, and one Irish populations of sea beets, and four lines of sugar beet were analysed. To evaluate the genetic variation and gene flow, eight microsatellite loci were screened. This analysis revealed hybridization with cultivated beet in one of the sea beet populations from the centre of the Danish seed propagation area. Triploid hybrids found in this population were verified with flow cytometry. Possible hybrids or introgressed plants were also found in the French and Italian populations. However, individual assignment test using a Bayesian method provided 100% assignment success of diploid individuals into their correct subspecies of origin, and a Bayesian Markov chain Monte Carlo (MC MC) approach revealed clear distinction of individuals into groups according to their subspecies of origin, with a zero level of genetic admixture among subspecies. This underlines that introgression beyond the first hybridization is not extensive. The overall pattern of genetic distance and structure showed that Danish and Swedish sea beet populations were closely related to each other, and they are both more closely related to the population from Ireland than to the populations from France, the Netherlands, and Italy.