Evolutionary history of Ash Meadows pupfish (genus <Emphasis Type="Italic">Cyprinodon</Emphasis>) populations inferred using microsatellite markers

Pupfish (genus Cyprinodon) persist in a series of isolated warm springs in Death Valley. Here we describe an analysis of microsatellite variation at six loci for nine populations encompassing three distinct taxa. Levels of genetic variation within populations and the pattern of relatedness among populations are best explained by spring elevation. Springs at higher elevations harbored less variation and exhibited greater among population divergence than lower elevation springs. This pattern reflects regional paleohydrological history showing a declining water table over the last 20,000 years. Continuing decline of the water table, a trend accelerated by local ground water mining, portends a future of increasing isolation and declining within-population variation.     

Seasonal races of chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> and their interrelations in Kamchatka Rivers

Dates of migration and spawning, size-age structure, and fecundity of seasonal races of chum salmon Oncorhynchus keta from the Kol River are reviewed, those of early summer, late summer, and autumn races, as well as localization and structure of their spawning grounds, bed-sediment particle size in the redds, hydrological and thermal conditions on spawning grounds, and meristic characters of races. Special traits of reproduction of seasonal races within the species range and in Kamchatka in particular are discussed. The number of seasonal races in rivers depends on the presence of various habitats suitable for spawning. In rivers with complicated hydrogeomorphological structure, the number of seasonal races increases. Variations of types of seasonal races in a particular river and in the whole species area are epigenetic, i.e., they depend on the hereditary genetic program and presence of conditions providing its realization via various channels.     

A search for null alleles at the microsatellite locus of chum salmon (<Emphasis Type="Italic">Oncorhynchus keta</Emphasis> Walbaum)

Population studies with the use of microsatellite markers face a problem of null alleles, i.e., the absence of a PCR product, caused by the mutations in the microsatellite flanking regions, which serve as the sites of primer hybridization. In this case, the microsatellite primer associated with such mutation is not amplified, leading to false homozygosity in heterozygous individuals. This, in turn, results in biased population genetic estimates, including the excess of homozygotes at microsatellite loci. Analysis of the population structure of a Pacific salmon species, chum salmon (Oncorhynchus keta Walbaum), revealed the presence of null alleles at the Oke3 microsatellite locus in the population samples, in which an excess of homozygotes was observed. The analysis was performed using different combinations of modified primers chosen to match the Oke3 locus. The use of these primers enabled identification of true heterozygotes among those individuals, which were previously diagnosed as homozygotes with the use of standard primers. Removal of null alleles eliminated the excess homozygotes in the chum salmon samples described. In addition to the exclusion of false homozygosity, the use of modified primers makes it possible to introduce polymorphic primer variants associated with certain microsatellite alleles into population studies.     

The genetic structure of chum salmon (<Emphasis Type="Italic">Oncorhynchus keta</Emphasis> Walbaum) populations inferred from the nucleotide variation of the mitochondrial DNA cytochrome <Emphasis Type="Italic">b</Emphasis> gene

The nucleotide sequences of a fragment of the mitochondrial DNA cytochrome b gene were determined in 12 chum salmon populations from the Russian Far East. The level of genetic diversity in the chum salmon populations from the Iturup Island, northern coast of the Sea of Okhotsk, and Anadyr’ River was found to be higher than in the populations from Kamchatka and Sakhalin, which may be related to the history of their origin and dispersal. The proportions of intrapopulation genetic variability (F _CT) and interpopulation genetic variability within the groups (F _SC) account for 90.87 and 0.9%, respectively, and the intergroup component (F _ST) comprises 8.23%. The predominance of one haplotype, B1, which is common for all populations studied, and a low share of intergroup variability suggest the beginning of colonization by the species of the given region from a common source (group of founders) and a relatively recent time of divergence of the chum salmon populations from the region examined.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

Development of expressed sequence tag-derived microsatellite markers for <Emphasis Type="Italic">Saccharina</Emphasis> (<Emphasis Type="Italic">Laminaria</Emphasis>) <Emphasis Type="Italic">japonica</Emphasis>

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.     

Genetic diversity of Asian chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> (Salmonidae,Salmoniformes) and the dynamics of gene pools in Sakhalin populations under artificial reproduction

The estimates of genetic diversity in populations of chum salmon Oncorhynchus keta from different regions of Sakhalin Island, Iturup Island, and the Anadyr’ River were obtained on the basis of analysis of allozyme variability. These estimates together with our published earlier data on chum salmon from the Amur River basin and the rivers of the northern coast of the Sea of Okhotsk demonstrate pronounced regional genetic differentiation in the Asian part of the fish range. The intraregional level of interpopulation genetic diversity was maximum on Sakhalin Island (G _ST = 6.6%) and was small on Iturup Island (G _ST = 0.9%) and the northern coast of the Sea of Okhotsk (G _ST = 0.6%). Interpopulation genetic diversity of Sakhalin chum salmon was almost commensurable to the diversity of the whole pool of studied Asian populations (G _ST = 7.6%) and would be presented more completely in baselines assigned for genetic identification of mixed stocks. It was demonstrated that the character and degree of genetic differentiation between populations of chum salmon from the main hatcheries situated in different regions of the Sakhalin oblast and connected to one another by numerous transplantations of fertilized eggs did not change significantly during an approximately 20-year period of our observation, and this fact suggests low efficiency of such transplantations.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Dinucleotide microsatellite markers in the genus <Emphasis Type="Italic">Caulerpa</Emphasis>

Caulerpa spp. are clonal green marine algae which often act as invasive species when growing outside their native biogeographical borders. Over the two past decades, Caulerpa taxifolia has spread along the Mediterranean coast, presently occurring at 70 sites and covering nearly 3,000 ha of subtidal area. New genetic markers (microsatellites) have been developed to assess clonal structure and genetic diversity of recently established populations of the invasive species C. taxifolia and Caulerpa racemosa in comparison with populations of the native Caulerpa prolifera in the Mediterranean. Our results show that nine polymorphic markers have been developed for C. prolifera, seven for C. taxifolia, and three for C. racemosa. Genetic diversity in Caulerpa was assessed in two geographical scales: one at a population scale where 40 thalli units were collected from C. prolifera in Cala d’Or, Mallorca, Spain, and another at a species scale, where 30 sample units were analyzed for C. prolifera, 24 for C. taxifolia, and 24 for C. racemosa from different sites in the Mediterranean, Atlantic, and Pacific Ocean. Number of alleles, expected heterozygosity, and marker amplification success are provided in each case.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

Genetic analysis of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> susceptibility in<Emphasis Type="Italic"> Brassica oleracea</Emphasis>

The genetic control and heritability of Agrobacterium tumefaciens susceptibility was investigated using a doubled haploid (DH) mapping population of Brassica oleracea and the associated RFLP map. Preliminary studies were carried out by analysis of an 8×8 diallel, for which the parental lines were selected to include a range of susceptibilities to A. tumefaciens. The variation observed within the diallel was attributed to both additive and dominant gene effects, with additive gene effects being more important. A broad sense heritability value of 0.95 suggested that 95% of the observed variation was due to genetic effects, with just 5% attributed to non-genetic or environmental effects. A high narrow-sense heritibility value of 0.79 suggested that 79% of this trait was controlled by additive gene effects and, therefore, the potential to introduce this trait into breeding material is high. Fifty-nine DH lines from the mapping population were screened for susceptibility towards A. tumefaciens. Variation in susceptibility was observed across the population. The results of the DH screen were entered into the mapping programme MAPQTL and a highly significant quantitative trait loci (QTL) associated with susceptibility to A. tumefaciens was identified on linkage group 09. The use of substitution lines covering this region confirmed the location of this QTL. This work shows that susceptibility to A. tumefaciens is a heritable trait, and the transfer of susceptibility into resistant lines is demonstrated. These findings may help to overcome genotype restrictions to genetic transformation.Communicated by G. Wenzel     

Genetic variation in island populations of tuatara (<Emphasis Type="Italic">Sphenodon</Emphasis> spp) inferred from microsatellite markers

Tuatara (Sphenodon spp) populations are restricted to 35 offshore islands in the Hauraki Gulf, Bay of Plenty and Cook Strait of New Zealand. Low levels of genetic variation have previously been revealed by allozyme and mtDNA analyses. In this new study, we show that six polymorphic microsatellite loci display high levels of genetic variation in 14 populations across the geographic range of tuatara. These populations are characterised by disjunct allele frequency spectra with high numbers of private alleles. High F _ST (0.26) values indicate marked population structure and assignment tests allocate 96% of all individuals to their source populations. These genetic data confirm that islands support genetically distinct populations. Principal component analysis and allelic sequence data supplied information about genetic relationships between populations. Low numbers of rare alleles and low allelic richness identified populations with reduced genetic diversity. Little Barrier Island has very low numbers of old tuatara which have retained some relictual diversity. North Brother Island’s tuatara population is inbred with fixed alleles at 5 of the 6 loci.     

Genetic Typing of <Emphasis Type="Italic">Corallium rubrum</Emphasis>

Corallium rubrum taxonomy is based on morphologic criteria; little is known about its genome. We set up a rapid, easy method based on amplified fragment length polymorphism to characterize the genetic patterns of C. rubrum in an attempt to understand better the evolutionary relations between species from diverse geographic areas and to help define migration patterns. Applying this procedure to C. rubrum specimens from Spain and Italy, we identified 6 AFLP amplification fragments common to the 4 coral populations studied and 4 fragments that differentiated between these populations. Using this characterization we were able to plot a genetic identity card of this commercially harvested species, which is also a marker of pollution.     

Sex ratio control in pink salmon (<Emphasis Type="Italic">Oncorhynchus gorbuscha</Emphasis> and chum salmon (<Emphasis Type="Italic">O. keta</Emphasis>) populations: The possible causes and mechanisms of changes in the sex ratio

Long-term changes in the sex ratio have been studied in pink salmon (Oncorhynchus gorbuscha and chum salmon (O. keta) populations of Kamchatka and Sakhalin. It has been demonstrated that these changes are an adaptation to population dynamics: an increase in the population size is accompanied by a shift towards a higher proportion of males; a decrease in population size, by a shift towards a higher proportion of females. The correspondence between morphological and molecular characters in populations of the two species has been analyzed in order to determine the mechanism of sex ratio control. In some pink salmon and chum salmon populations, there is a discrepancy between sex identifications based on morphological characters and molecular markers. This discrepancy is assumed to be accounted for by sex inversion mechanisms, which may be population-or region-specific. In two cases, it has been found that the sex ratio discrepancy in populations is related to the numbers of fish in subsequent generations. These findings suggest that sex inversion may be related to population size control.     

Genetic differentiation in sympatric wood ants, <Emphasis Type="Italic">Formica rufa</Emphasis> and <Emphasis Type="Italic">F. polyctena</Emphasis>

Summary Direct observations have suggested that the closely related wood ants Formica polyctena and F. rufa represent different social organizations, with high queen number in F. polyctena and a high frequency of monogynous nests in F. rufa. We examined social organization and genetic population structure in a setup where populations of the two species are sympatric and gene flow between the species is possible. Our aim was to compare social organization in the species, and study evolutionary relationships between them. The observed relatedness among colony workers suggested that the difference in the level of polygyny is quantitative rather than qualitative, with a higher queen number in F. polyctena. The observed difference in polygyny was not accompanied by a difference in spatial genetic differentiation which was weak in both species. The genetic distance between the species is consistent with limited interspecific gene flow. Identification of a few possible F. rufa migrants in F. polyctena populations suggests potential interspecific gene flow. Thus, reproductive isolation of the species may not be complete when they are sympatric.Received 14 March 2003; revised 10 October 2003; accepted 20 October 2003.     

Genetic diversity and population structure of<Emphasis Type="Italic">Kalopanax pictus</Emphasis> (Araliaceae)

Kalopanax pictus (Araliaceae) is a long-lived woody species primarily distributed throughout East Asia. This species is regarded as medically and ecologically important in Korea. We used starch gel electrophoresis to investigate its allozyme variation and genetic structure in samples from Korean populations (both natural and cultivated). Our analysis of 10 enzymes revealed 18 loci, of which 10 were polymorphic (35.6%). Genetic diversity at the species and population levels were 0.200 and 0.149, respectively, with the mean for cultivated populations (0.124) being lower than for natural (wild) populations (0.181). Asexual and sexual reproduction modes, perennial habitat, and longevity all were possible factors contributing to such high diversity. An indirect estimate of the number of migrants per generation (N _m = 1.08) indicated that gene flow was not extensive among these Korean populations. Therefore, we suggest that geographical distance as well as reproductive isolation between wild and cultivated plants may play roles in shaping the population structure of this species.     

Allozyme variations in six natural populations of scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis>) in Turkey

Genetic variation in six natural populations of Scots pine (Pinus sylvestris L.) was determined with isoenzyme analyses. For this purpose, haploid female gametophytes of seeds and horizontal starch gel electrophoresis technique were used. A total of 17 loci and 58 alleles were observed in studying 10 enzyme systems. The average proportion of polymorphic loci for populations ranged from 58.8% to 70.6%. The average number of alleles per locus per population was 2.65. The mean estimated expected heterozygosity (He) of populations was 0.294. A rather high proportion of genetic diversity (96.4%) was due to within-population variation and the remaining (3.6%) was due to variation among populations. The level of gene flow (N_em) was found to be 6.69 per generation. Nei’s genetic distance coefficient ranged from 0.006 to 0.027 (mean 0.017) among all possible population pairs. The mean value of Nei’s genetic distance is similar to the values reported for other European Scots pine populations. The low mean value of Nei’s genetic distance among populations is enough to explain low interpopulation variation. According to genetic variation parameters, three out of six populations (Akdagmadeni-Yozgat, Refahiye-Erzincan and Vezirkopru-Samsun) appear to be preferable populations for genetic conservation and forest tree breeding programs.     

Genetic structure of the Russian populations of <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis>, determined by using microsatellite markers

The population genetic structure of plant pathogenic fungus Pyrenophora tritici-repentis was examined using microsatellite (SSR) markers. According to the geographical origin of the pathogen populations, they were designated as North Caucasian (S, 33 isolates), northwest (Nw, 39), and Omsk (Om, 43). The populations were analyzed at the nine most polymorphic SSR loci, at which 75 alleles were identified. To characterize the genetic variation within and between populations, the AMOVA algorithm as implemented in the Arlequin v. 3.5 software program was used. The number of alleles per locus ranged from 5 to 12 and their sizes varied within the range from 180 to 400 bp. The mean gene diversity at SSR loci was high for all populations (H = 0.58–0.75). The populations were considerably different in the frequencies of individual alleles of the SSR loci. Most isolates in the populations were represented by unique haplotypes. The within-population variation of the isolates at molecular markers was 86.4%; among the populations, 13.6%. Substantial interpopulation differences were found between the Om and S (F_st = 0.16) and between the Om and Nw (F_st = 0.20) populations, whereas between the S and Nw populations, these differences were small (F_st = 0.05). Thus, it was demonstrated that the population of P. tritici-repentis from Omsk oblast had the independent status of the geographical population; northwest and North Caucasian populations differed in the allelic diversity of SSR loci, and despite the low F_st value (0.05), they also belonged to independent geographical populations.     

Geographic variation and temporal population differentiation of chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> from some regions of the Russian Far East

Allozyme variation of chum salmon Oncorhynchus keta was examined in the populations from nine rivers of the Russian Far East. A total of 12 loci were tested, and eight of these were shown to be polymorphic. The greatest contribution to the samples differentiation was made by the EST-D1* and sIDHP-2* loci. Most of the allele diversity was distributed among the individuals within the samples. The among-sample differences within races were statistically significant and accounted for slightly more than 7% of total variation. The differences between the races constituted 0.1% and were statistically nonsignificant. Genetic similarity of seasonal races within single locality allowed suggestion on their secondary formation after settling of founder population into a certain region.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.