Microsatellite variation reveals weak genetic structure and retention of genetic variability in threatened Chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) within a Snake River watershed

Pacific salmon (Oncorhynchus spp.) have been central to the development of management concepts associated with evolutionarily significant units (ESUs), yet there are still relatively few studies of genetic diversity within threatened and endangered ESUs for salmon or other species. We analyzed genetic variation at 10 microsatellite loci to evaluate spatial population structure and genetic variability in indigenous Chinook salmon (Oncorhynchus tshawytscha) across a large wilderness basin within a Snake River ESU. Despite dramatic 20th century declines in abundance, these populations retained robust levels of genetic variability. No significant genetic bottlenecks were found, although the bottleneck metric (M ratio) was significantly correlated with average population size and variability. Weak but significant genetic structure existed among tributaries despite evidence of high levels of gene flow, with the strongest genetic differentiation mirroring the physical segregation of fish from two sub-basins. Despite the more recent colonization of one sub-basin and differences between sub-basins in the natural level of fragmentation, gene diversity and genetic differentiation were similar between sub-basins. Various factors, such as the (unknown) genetic contribution of precocial males, genetic compensation, lack of hatchery influence, and high levels of current gene flow may have contributed to the persistence of genetic variability in this system in spite of historical declines. This unique study of indigenous Chinook salmon underscores the importance of maintaining natural populations in interconnected and complex habitats to minimize losses of genetic diversity within ESUs.     

Population differentiation in the redshank (<Emphasis Type="Italic">Tringa totanus</Emphasis>) as revealed by mitochondrial DNA and amplified fragment length polymorphism markers

The redshank (Tringa totanus) is declining throughout Europe and to implement efficient conservation measures, it is important to obtain information about the population genetic structure. The aim of the present study was two-fold. First, we analysed the genetic variation within and between populations in the Baltic region in southern Scandinavia. Evidence of genetic structure would suggest that different populations might require separate management strategies. Second, in an attempt to study large-scale genetic structure we compared the Baltic populations with redshanks from northern Scandinavia and Iceland. This analysis could reveal insights into phylogeography and long-term population history. DNA samples were collected from six breeding sites in Scandinavia presumed to include two subspecies (totanus and britannica) and a further sample from Iceland (subspecies robusta). Two methods were used to study the population genetic structure. Domain II and III of the mitochondrial control region was analysed by DNA sequencing and nuclear DNA was analysed by screening amplified fragment length polymorphism (AFLP) markers. Mitochondrial DNA showed no variation between individuals in domain II. When analysing an 481 bp fragment of domain III seven haplotypes were found among birds. On the basis of mtDNA sequences, redshanks showed some evidence of a recent expansion from a bottlenecked refugial population. Bayesian analyses of AFLP data revealed a significant genetic differentiation between suggested subspecies but not between populations within the Baltic region. Our results indicate that populations of redshanks in Europe constitute at least three separate management units corresponding to the recognised subspecies.     

Genetic variation in populations of <Emphasis Type="Italic">Allothrombium pulvinum</Emphasis> (Acari: Trombidiidae) from Northern Iran revealed by mitochondrial <Emphasis Type="Italic">cox</Emphasis>I and nuclear rDNA <Emphasis Type="Italic">ITS</Emphasis>2 sequences

Allothrombium pulvinum Ewing is a common natural enemy of aphids and some other arthropods. So far, there are no studies that have addressed genetic variation of this predatory mite. We investigated genetic variation of A. pulvinum across its whole known range in Iran. A 410 bp portion of the mitochondrial cytochrome c oxidase subunit I gene (coxI) and 797–802 bp portion of the internal transcribed spacer 2 of rDNA (ITS2) were sequenced for 55 individuals from 11 populations, resulting in 12 and 26 haplotypes, respectively. In the coxI region, haplotype and nucleotide diversities varied among populations from 0.00 to 0.90 and from 0.0000 to 0.0110, respectively. In the ITS2 region they varied from 0.20 to 0.91 and from 0.0006 to 0.0023, respectively. For both gene regions the highest haplotype and nucleotide diversities were detected in population Mahmoud Abad from northern Iran. Statistically significant population differentiation (F _ST) was detected in most pair-wise population comparisons. The results of population differentiation for both gene regions were generally congruent indicating that A. pulvinum from Iran consists of genetically different populations. This suggests that A. pulvinum comprises at least two geographically distinct populations or even more than one species. This study is an initial step towards understanding genetic variation of A. pulvinum, a taxon for which little molecular information is available. More intensive sampling and analysis of additional DNA regions are necessary for more detailed classification of this taxon.     

Genetic variation in the endangered <Emphasis Type="Italic">Anisodus tanguticus</Emphasis> (Solanaceae), an alpine perennial endemic to the Qinghai-Tibetan Plateau

We used random amplified polymorphic DNA markers (RAPDs) to assess genetic variation between- and within-populations of Anisodus tanguticus (Solanaceae), an endangered perennial endemic to the Qinghai-Tibetan Plateau with important medicinal value. We recorded a total of 92 amplified bands, using 12 RAPD primers, 76 of which (P = 82.61%) were polymorphic, and calculated values of H_t and H_sp of 0.3015 and 0.4459, respectively, suggesting a remarkably high rate of genetic variation at the species level. The average within-population diversity also appeared to be high, with P, H_e and H_pop values of 55.11%, 0.1948 and 0.2918, respectively. Analyses of molecular variance (AMOVA) showed that among- and between-population genetic variation accounted for 67.02% and 32.98% of the total genetic variation, respectively. In addition, Nei’s coefficient of differentiation (G_ST) was found to be high (0.35), confirming the relatively high level of genetic differentiation among the populations. These differentiation coefficients are higher than mean corresponding coefficients for outbreeding species, but lower than reported coefficients for some rare species from this region. The genetic structure of A. tanguticus has probably been shaped by its breeding attributes, biogeographic history and human impact due to collection for medicinal purposes. The observed genetic variations suggest that as many populations as possible should be considered in any planned in situ or ex situ conservation programs for this species.     

Variation in the chloroplast DNA of Swiss stone pine (Pinus cembra L.) reflects contrasting post-glacial history of populations from the Carpathians and the Alps

Aim To characterize the genetic structure and diversity of Pinus cembra L. populations native to two disjunct geographical areas, the Alps and the Carpathians, and to evaluate the rate of genetic differentiation among populations. Location The Swiss Alps and the Carpathians. Methods We screened 28 populations at three paternally inherited chloroplast simple sequence repeats (cpSSRs) for length variation in their mononucleotide repeats. Statistical analysis assessed haplotypic variation and fixation indices. Hierarchical analysis of molecular variance (AMOVA), Mantel test, spatial analysis of molecular variance (SAMOVA) and barrier analyses were applied to evaluate the geographical partitioning of genetic diversity across the species’ range. Results Haplotypic diversity was generally high throughout the natural range of P. cembra, with the mean value substantially higher in the Carpathians (H = 0.53) than in the Alps (H = 0.35). The isolated Carpathian populations showed the highest haplotype diversity among the populations originating from the High Tatras (Velka Studena Dolina) and South Carpathians (Retezat Mountains). AMOVA revealed that only 3% of the total genetic variation derived from genetic differentiation between the two mountain ranges. Differentiation among Carpathian populations was higher (F_ST = 0.19) than among Alpine populations (F_ST = 0.04). Low, but significant, correlation was found between the geographical and genetic distances among pairs of populations (r = 0.286, P < 0.001). SAMOVA results revealed no evident geographical structure of populations. barrier analysis showed the strongest differentiation in the eastern part of the species’ range, i.e. in the Carpathians. Main conclusions The populations of P. cembra within the two parts of the species’ range still share many cpDNA haplotypes, suggesting a common gene pool conserved from a previously large, continuous distribution range. Carpathian populations have maintained high haplotypic variation, even higher than Alpine populations, despite their small population sizes and spatial isolation. Based on our results, we emphasize the importance of the Carpathian populations of Swiss stone pine for conservation. These populations comprise private haplotypes and they may represent a particular legacy of the species’ evolutionary history.     

Genetic structure of the rare and endangered moss <Emphasis Type="Italic">Campylopus oerstedianus</Emphasis> (Dicranaceae) in Europe

The genetic structure, diversity and phylogeography of the moss species Campylopus oerstedianus in Europe was studied, based on the ITS region of the nrDNA of nine selected European populations. Although this species is only known in sterile stage, long-distance dispersal and gene flow among populations seem to be present within Europe. High levels of genetic differentiation between the investigated Greek population and the western European populations indicate a long lasting isolation. The populations from France and Switzerland root together and supposedly have a common origin. From the French populations, the one from the Pyrenees is the most basal one and the populations in the Massif Central and the Vosges Mts can be derived from it. This indicates relatively recent dispersal of the species from the Mediterranean to the north, in spite of the lack of sporophytes and the fact that the type of the dispersed propagules and their vectors are not obvious.     

Understanding biological conservation strategies: a molecular-genetic approach to the case of myrtle (<Emphasis Type="Italic">Myrtus communis</Emphasis> L.) in two Italian regions: Sardinia and Calabria

Myrtle (Myrtus communis L.), a shrub widespread in the Mediterranean area, is the only species belonging to the Myrtaceae family growing in Europe. The pharmacological and aromatic properties of myrtle have caused a growing interest in this plant. The use of myrtle as an aromatic plant is traditionally established in the Italian regions of Sardinia and Calabria, where it is subjected to an ecological stress due to the large biomass removal for liquor production and environmental damage. The reduction in size and number of wild populations is often related to a loss of genetic variation and reproductive potential. In this study fluorescent amplified fragment length polymorphisms (fAFLPs) were employed to assess the genetic variation within and among natural populations of myrtle from Sardinia and Calabria to gain new insights into their fitness and survival potential. AMOVA analysis indicated that genetic variation was greater within populations (51.86%) than among populations (16.99%), as previously reported for outcrossing species. A significant amount of variation (31.15%) was attributed to variation between Sardinia and Calabria population groups, suggesting a genotypic differentiation between the myrtle populations of these two regions. Intra-population genetic variation, assessed by estimating expected unbiased heterozygosity (H _E), ranged from 0.0595 to 0.2595. These values resulted correlated with population extension (r = 0.918; P < 0.01) and with two reproductive parameters: seed germinability (r = 0.793; P < 0.01) and number of seeds per fruit (r = 0.631; P < 0.05). A moderate gene flow among Sardinia myrtle populations and Calabria populations (1.2719 and 1.0478, respectively) counteracts the low level of genetic variation observed in some␣populations and avoids their differentiation and isolation.     

Patterns of Allozyme Variation at Two Stages of the Life History in Wild Rice <Emphasis Type="Italic">Oryza rufipogon</Emphasis> and Conservation Genetic Implications

Oryza rufipogon Griff. occurs widely in aquatic ecosystem of tropics and subtropics of monsoon Asia as well as Southern China. It is a vital gene source for rice breeding programs. Many populations of the species, unfortunately, have drastically diminished because of the disappearance of aquatic habitats as a result of human disturbance. In order to determine patterns of genetic variation at two stages of the life-cycle in the wild rice species, we investigated allozyme variation of four natural populations in China. Two southern populations have significant asexual reproduction while two other northern marginal populations show a mixed reproduction in China. At 22 allozyme loci, a significantly lower genetic diversity was observed in the ratoons than in the seeds of the two southern populations, whereas a significantly higher genetic diversity was found in the ratoons than in the seeds of the two northern marginal populations. The results suggest that the variation of reproductive system is probably associated with their patterns of genetic variation in the species. Moreover, a significantly higher genetic differentiation among populations found in the ratoons than in the seeds may stem from pollen-mediated gene flow among them. Finally, we propose suggestions for conservation management of the endangered species.     

Hieracium vierhapperi (Asteraceae) a new species to the Carpathians, with some remarks on its origin

Hieracium vierhapperi (Zahn) Szeląg, known so far only from the Alps, was discovered in the Nízke Tatry Mts in Slovakia, as a new species to the Carpathians. Diagnostic characters, illustration and distribution as well as ecology of H. vierhapperi in the Nízke Tatry Mts are given. A key to distinguish H. vierhapperi from the morphologically related species is added. The Carpathian plants are tetraploid (2n = 36) and reproduce apomictically. The origin of H. sect. Cernua R. Uechtr in the Western Carpathians is briefly discussed.     

Genetic diversity,differentiation and conservation in <Emphasis Type="Italic">Araucaria bidwillii</Emphasis>(Araucariaceae), Australia's Bunya pine

Habitat fragmentation has resulted in many species becoming geographically restricted, as dispersal among subsequent isolates becomes compromised. This study investigated the effects of historical fragmentation on the genetic diversity and differentiation of Australia's Bunya Pine (Araucaria bidwillii) using random amplified DNA (RAPD) markers. High diversity characterises all Bunya populations sampled, regardless of population size or degree of isolation, which may be either due to similar effective population size among populations, or the lagging effects of the detection of contemporary genetic signal in long-lived conifer species. Large genetic differentiation characterises the northern and southern populations, although all sampled populations are significantly differentiated from each other. The northern population, at Mt Lewis, is responsible for the majority of variation detected among populations, and as such represents a significant genetic reservoir. The conservation of the genotypes of the Mt Lewis population may be imperative for the future of the species, given predicted models of accelerated climate change over the coming decades.     

Effects of population size on genetic diversity and seed production in the rare <Emphasis Type="Italic">Dictamnus albus</Emphasis>(Rutaceae) in central Germany

It is widely assumed that population size significantly affects the dynamics of plant populations. Smaller populations are threatened by genetic drift and inbreeding depression, both of which may result in a decrease of genetic variation and a resulting negative impact on plant fitness. In our study we analysed the patterns of random amplified polymorphic DNA (RAPD) variation among 10 Dictamnus albuspopulations of varying size. The aim was to examine local differentiation in relation to spatial isolation resulting from limited population size and geographical distancing between populations. Significant correlations were noted between population size and both percentage of polymorphic loci (P <0.01) and genetic diversity (P<0.01). The matrix correlation between genetic and geographical distances revealed that geographical differentiation was reflected in the RAPD profile (Mantel test: r²=0.34, P<0.001). We found the highest level of molecular variance of RAPD patterns among individuals within the populations (72.6%), whereas among-population variation accounted for only 21.6% of variation. These results were highly significant in that they indicated a restricted population differentiation, as would be expected from outcrossing species. An additional analysis of seed production showed that there was significant variation among populations in terms of mean seed number per flower and mean seed mass per population which could be attributed to differences in population size as well as levels of genetic variation.     

Threatened freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L. in NW Spain: low and very structured genetic variation in southern peripheral populations assessed using microsatellite markers

A genetic analysis of freshwater pearl mussel Margaritifera margaritifera populations from NW Spain, a peripheral area of its European distribution, was carried out using microsatellite markers. These populations were formerly reported as genetically differentiated on the basis of growth and longevity studies. Ten loci previously characterized in populations from central Europe were used to comparatively analyze the genetic variability at the southern edge of the species’ range. Iberian pearl mussel populations showed very low genetic variability and significant high genetic differentiation. Half of the total genetic diversity observed appeared to be distributed between populations, which suggested a highly structured adaptive potential in pearl mussel at the southern peripheral distribution of the species. Population distinctiveness was evidenced by assignment tests, which revealed a high accuracy of individual assignments to their population of origin. All data suggested low effective population size and major effects of genetic drift on population genetic structure. In order to avoid further loss of genetic variation in biologically distinctive populations from NW Spain, prioritization of genetic resources of this species is required for conservation and management.     

Towards a legal framework for systematic conservation: identification and development of allele-specific PCR markers for conspecific varieties of an endangered perennial herb <Emphasis Type="Italic">Primula kisoana</Emphasis> Miquel based on sequence variation of chloroplast DNA

Primula kisoana var. kisoana (Primulaceae) is a narrow endemic found only in extremely restricted areas of central Honshu Island, Japan. Although the species is included as ‘Critically Endangered (CR)’ in the latest Japanese Red List, it is not covered by legislation such as the ‘Law for the Conservation of Endangered Species of Wild Fauna and Flora, Japan’. This poor conservation status is due, largely, to the presence of another conspecific variety, var. shikokiana, which is regarded as less threatened than var. kisoana. In this study, we investigated the genetic dissimilarity between the two varieties by examining sequence variation in three noncoding regions of chloroplast DNA. Ten distinct haplotypes were detected, none of which were distributed across populations and varieties. The genetic differentiation between the two varieties revealed by an AMOVA and a minimum spanning network suggests that var. kisoana and var. shikokiana should be conserved and managed as separate units. Using the allele-specific PCR method, four primer pairs were developed at the point mutations and insertion/deletion loci that exhibited only inter-variety variation. These markers will facilitate reliable identification of var. kisoana, assisting in the implementation of restoration programs including the introduction of seedlings and construction of a legal framework for conservation.     

Genetic variation of <Emphasis Type="Italic">Picea jezoensis</Emphasis> populations in South Korea revealed by chloroplast,mitochondrial and nuclear DNA markers

Genetic variation associated with Picea jezoensis populations of South Korea was investigated using chloroplast (cp), mitochondrial (mt) and nuclear DNA markers. In South Korea, P. jezoensis is distributed across a very restricted area, being found on the summits of three mountains: Mts. Jiri, Dokyu and Gyebang. Examination of five region restriction enzyme combinations for mtDNA and four for cpDNA revealed haplotypes endemic to South Korea. The Gyebang population, the most northerly and most isolated, was genetically distinct from the other populations. Nuclear microsatellite markers indicated, overall, a low level of genetic diversity (H _e = 0.406) in South Korea; this could be attributed to genetic drift and/or founder effects associated with historical events. The Wilcoxon sign-rank test did not indicate a recent bottleneck in any of the populations irrespective of the model considered (infinite allele model, two-phased model of mutation, and stepwise mutation model). Microsatellite markers also demonstrated that the Gyebang population was distinct from the others. The results of this study could be used as the basis for conservation guidelines for the management of this species in South Korea.     

Conservation status of the White-Bellied Sea-Eagle <Emphasis Type="Italic">Haliaeetus leucogaster</Emphasis> in Australia determined using mtDNA control region sequence data

Considered to have a declining world population, concern has been expressed in recent years over the conservation status of the White-bellied Sea-Eagle Haliaeetus leucogaster (Gmelin, 1788) within Australia. We used mitochondrial (mtDNA) control region sequence data to investigate the current distribution of genetic variation in this species at the continental level and within and between specified regional units. We were specifically interested in identifying breaks in genetic connectivity between the west and east of the continent and between Tasmania and the Australian mainland. We also investigated the likelihood of a bottleneck at the time of colonisation, and propose hypotheses regarding colonisation history. Sequence data were obtained from 128 individuals describing 15 haplotypes. Overall, diversity was low and AMOVA results failed to provide any significant level of genetic subdivision between regions. We suggest that the population expanded from a bottleneck approximately 160,000 years ago during the late Pleistocene, and spread throughout the continent through a contiguous range expansion. There is insufficient evidence to suggest division of the population into different units for conservation management purposes based on the theoretical definition of the ‘evolutionary significant unit’. It is clear from the analysis that there are signatures of both historical and contemporary processes affecting the current distribution. Additional sampling and confirmation of the perceived pattern of population structure using a nuclear marker is recommended to validate conservation monitoring and management at a continental scale.     

Fine Spatial Structure of Atlantic Hake (<Emphasis Type="Italic">Merluccius merluccius</Emphasis>) Stocks Revealed by Variation at Microsatellite Loci

Genetic variation at 5 microsatellite loci was analyzed for European hake Merluccius merluccius sampled from 9 different regions in the Atlantic Ocean and the Mediterranean Sea. Significant genetic differentiation was found between samples, suggesting a fine subdivision of Atlantic and Mediterranean hake stocks. These results are discussed in the context of the decline of demersal fish species, probably due to overfishing.     

Patterns of genetic and morphometric differentiation in Melitaea (Mellicta) athalia (Lepidoptera: Nymphalidae)

Patterns of genetic and morphometric differentiation were surveyed in Melitaea (Mellicta) athalia populations of the Carpathian Basin. This species has a wide distribution and exists under a wide variety of ecological conditions. It has two ecotypes in Hungary: with either one or two broods per year. It is of particular interest to reveal the main factors driving differentiation patterns in this species. Samples in our study were obtained from five Hungarian and one Transylvanian (Romanian) regions. Enzyme polymorphism, wing characters and male external genitalia were analysed using traditional morphometric methods. Statistical methods were optimized to compare morphological and genetic data. The results of genetic surveys revealed a clear regional pattern of differentiation in M. athalia. Moreover, the results of principal component analysis, Bayesian clustering and the dendrogram all suggested that the regions can be classified into two groups corresponding to the East or West zones of the Carpathian Basin. In contrast, differentiation between the two ecotypes was less expressed in the genetic variation of M. athalia. Results of the analyses conducted on phenotypic variation also suggested a regional pattern for both sets of morphometric characters (wings and external genitalia). At the same time, neither East–West regional division nor ecotype differentiation was detected in the morphometric studies. In sum, our analyses confirmed that both genetic and phenotypic variations of M. athalia exhibit a regional pattern rather than the differentiation between the two ecotypes. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Morphological and genetic evidence for two evolutionarily significant units (ESUs) in the South American fur seal, <Emphasis Type="Italic">Arctocephalus gazella</Emphasis>

The South American fur seal (Arctocephalus australis) is widely distributed, occurring along both the Atlantic and the Pacific coasts of South America. Previous work suggests there may be more than one subspecies, highlighting the need for further study. Here, we combine traditional and geometric morphometric analysis of skull shape and size with genetic data to compare two populations of South American fur seals, one from Uruguay and one from Peru. As a control group we used material from the closely related species Arctocephalus gazella. Both techniques of morphometric analysis reveal pronounced geographic variation in size and shape of the skull, with Peruvian specimens (n = 102) being larger than Uruguayan skulls (n = 133) and significant shape differences concentrated in the rostral region. Similarly, seven highly polymorphic microsatellite loci reveal highly significant differences in allele frequency. Moreover, Bayesian analysis implemented using the program structure reveals two separate clusters corresponding perfectly to the two populations, with an assignment test correctly placing over 98% of specimens in their population of origin. This degree of differentiation for both genetic and morphological traits suggests complete and possibly prolonged isolation to the extent that we believe these populations should be considered distinct evolutionarily significant units.     

First Microsatellite Loci of Red Mullet (<Emphasis Type="Italic">Mullus barbatus</Emphasis>) and Their Application to Genetic Structure Analysis of Adriatic Shared Stock

In order to study the genetic structure of the Adriatic shared stock of red mullet (Mullus barbatus), we developed a set of dinucleotide microsatellite markers. A dinucleotide-enriched genomic library was obtained, and 6 polymorphic dinucleotide loci were successfully optimized. The markers showed high expected heterozygosity (from 0.68 to 0.92) and allele number (from 12 to 33); thus they appear to be suitable for detecting genetic differences in the population of red mullet. Four Adriatic samples were subsequently analyzed for microsatellite variation, and the results showed subtle but statistically significant genetic differentiation, indicating that the Adriatic red mullet may group into local, genetically isolated populations. No correlation between geographic distance and genetic differentiation was observed. In addition, the evidence of recent bottlenecks in the Adriatic samples indicates that the observed population subdivision might reflect random local allelic variations, generated by reproductive success, survival rates, or fishing pressure.     

Changes in morphological traits of the cabbage aphid (<Emphasis Type="Italic">Brevicoryne brassicae</Emphasis>) associated with the use of different host plants

The genetic and morphological differentiation of insect populations in relation to the use of different host plants is an important phenomenon that predates ecological specialisation and speciation in sympatric conditions. In this study, we describe the morphological variation of populations of Brevicoryne brassicae (Homoptera: Aphididae) associated with two host species, Brassica oleracea and Brassica campestris, which occur sympatrically in the highlands of Chiapas, Mexico. The study is aimed at obtaining evidence regarding phenotypic differentiation induced by, or associated with, the use of distinct but closely related host species. Seven morphological characters were measured in 696 wingless aphids collected from plants of the two host species at four localities. Morphological variation was summarised through principal components analysis (PCA). Sixty-two percent of morphological variation was explained by the first two PCs. The first component (PC1) was related to the general size of appendages, and PC2 was interpreted as the relationship between body size (body and leg size) and antenna length. Aphids growing on B. campestris were bigger than those collected from B. oleraceae. Significant differences between hosts were detected for PC1, whereas a significant effect of locality, host, and the interaction locality × host was detected for PC2. These results indicate that the average phenotype of B. brassicae individuals inhabiting different host-plant species differs as a consequence of the contrasting feeding environments the host species provide.