Sex after all: high levels of diversity detected in the arctic clonal plant Saxifraga cernua using RAPD markers

Arctic plants in general and arctic clonal plants in particular have often been assumed to contain low levels of genetic diversity. We used RAPDs (random amplified polymorphic DNAs) to investigate genetic diversity in the arctic-alpine Saxifraga cernua , which mainly reproduces clonally via bulbils, at three spatial scales in Svalbard: (i) 'macroscale', between two sites 11 km apart; (ii) 'mesoscale', along two crossing transects at each site; and (iii) 'microscale', within a 3 × 3 m square at each site. Thirteen putative clones (RAPD phenotypes) were distinguished among 93 ramets based on 38 RAPD markers. The genetic diversity ( D ; mean 0.52, range 0.10–0.81) and evenness ( E ; mean 0.42, range 0.00–0.82) were at the same level as in clonal plants in general. However, the diversity strongly depended on site and spatial scale. Several clones were highly divergent and clustered independently of site in UPGMA and PCO analyses. In an analysis of molecular variance ( AMOVA ), most of the variation (59%) was found within sites. Mantel tests revealed no correlation between spatial and genetic distance within sites. Our results suggest that occasional sexual reproduction as well as clonal migration via bulbil dispersal play a significant role in the treeless arctic environment, where S. cernua is widespread and locally very abundant. In contrast, Bauert et al. ( Molecular Ecology 7, 1519–1527) found no genetic variation within populations or regions of the Alps, where the species has highly isolated occurrences.     

Genetic enrichment of the arctic clonal plant Saxifraga cernua at its southern periphery via the alpine sexual Saxifraga sibirica

Isolation of populations at the margins of a species range may lead to decreasing genetic diversity via genetic drift and inbreeding. Hybridization between peripheral populations of two species can, however, counteract genetic impoverishment. The mainly clonal, polyploid plant Saxifraga cernua has a wide arctic distribution but also extends southwards into alpine sites. In the Ural Mountains, its peripheral distribution overlaps with that of its sexually reproducing, diploid relative Saxifraga sibirica, and fertile polyploids of more or less intermediate appearance are found in this overlap zone. We used amplified fragment length polymorphism (AFLP) analysis to address the potential impact of interspecific gene flow on genetic diversity in the peripheral populations. A total of 149 plants from 17 populations along a 1650 km south-north gradient were analysed for 253 markers. The results suggest that three Middle Ural populations containing fertile and morphologically more or less intermediate plants have been affected by hybridization. All of these plants formed a strongly supported (100%) group with S. cernua in a neighbour-joining tree, but their AFLP phenotypes assigned either to S. cernua or to artificial (simulated) F(1) hybrids between S. cernua and S. sibirica in multilocus assignment tests. The three populations were highly diverse with virtually every plant representing a distinct AFLP phenotype, providing additional evidence for formation of later-generation hybrids and/or backcrossing to S. cernua. In contrast, other peripheral populations of S. cernua were typically monoclonal, suggesting that hybridization with S. sibirica can increase genetic diversity in S. cernua at its southern periphery.     

The relationship between respiration and temperature in leaves of the arctic plant Saxifraga cernua

Abstract Saxifraga cernua, a perennial herb distributed throughout the arctic and subarctic regions, shows high levels of dark respiration. The amount of respiration exhibited by leaves and whole plants at any temperature is influenced by the pretreatment temperature. Plants grown at 10°C typically show higher dark respiration rates than plants grown at 20°C. The levels of alternative-pathway respiration (or cyanide-insensitive respiration) in leaves of S. cernua grown at high and low temperatures were assessed by treating leaf discs with 0.25 mol m^?3 salicylhydroxamic acid during measurements of oxygen consumption. Alternative pathway respiration accounted for up to 75% of the total respiration. Tissues from 20°C-grown plants yielded a Q₁₀ of 3.37 for normal respiration, and of 0.97 for alternative-pathway respiration. Tissues from 10°C-grown plants yielded a Q₁₀ of 2.55 for normal respiration, and of 0.79 for alternative-pathway respiration. The alternative pathway does not appear to be as temperature sensitive as the normal cytochrome pathway. A simple energy model was used to predict the temperature gain expected from these high rates of alternative-pathway respiration. The model shows that less than 0.02°C can be gained by leaves experiencing these high respiration rates.     

No genetic variation detected within isolated relict populations of Saxifraga cernua in the Alps using RAPD markers

Genetic variation in seven relict populations of Saxifraga cernua from three regions of the Alps was investigated using RAPD (random amplified polymorphic DNA) markers. No variation, either within the populations or within the regions, could be demonstrated. Nevertheless, each alpine region was characterized by a unique RAPD phenotype. Absence of genetic variation in these relict populations is attributed to population bottlenecks and founder effects during or following the ice ages. Contrasting hypotheses about the history of these populations, either as survivors of the glacial period or as products of postglacial immigration, are discussed in the light of the data presented.     

Low temperature acclimation of guard cell chloroplasts by the arctic plant Saxifraga cernua L.

Abstract. The potential for thermal acclimation of photosynthetic electron transport by guard cell chloropiasts (GC ch) was assessed in epidermal peels taken from the abaxial side of Saxifraga cernua leaves grown at 20°C and 10°C. Chlorophyll a fluorescence induction kinetics measured in pairs of guard cells in individual stomata from tissue grown at 10 °C demonstrated a rise in the fluorescence to a maximum and a larger amplitude in variable fluorescence when measured at temperatures below 18°C than was seen in GC ch from tissue grown at 20°C. The rates of fluorescence quenching in 10°C-grown tissue were also faster than in 20°C-grown tissue when measured at temperatures below 18°C. State 1-State 2 transitions by GC ch were measured at selected temperatures between 5 and 25 °C as changes in the magnitude of the fluorescence emission maxima at 685, 695 and 730nm (F685, F695 and F730) measured at 77K. At measuring temperatures of 5 and 10°C, GC ch in tissue grown at 10 °C showed a greater transition to State 2 (a larger F730/F695 ratio) than did GC ch in tissue grown at 20 °C. At measuring temperatures of 20 and 25 °C, there was no difference in either the kinetics or the magnitude of the State 1 to State 2 transition in the two tissues. The ultrastructure of GC ch from tissues grown at 10 and 20 °C was also examined using transmission electron microscopy. Less than half (48%) of the grana from the higher temperature grown tissue had more than nine thylakoids/grana. Grana in GC ch which had developed at 10 °C showed a dramatic reduction in stacking, such that 85% of the grana contained no more than two thylakoids. The reduction in grana stacking was also accompanied by a decrease in the degree of appression of thylakoid membranes. The results demonstrate a capacity for thermal acclimation of GC ch function to low temperatures. This acclimation is associated with alterations in the chloroplast ultrastructure.     

Molecules and morphology in concert: tests of some hypotheses in arctic Potentilla (Rosaceae)

We developed a combined molecular and morphological approach to unravel complex variation at low taxonomic levels, exemplified by some arctic members of Potentilla. Twenty-one populations from Svalbard were analyzed for random amplified polymorphic DNAs (RAPDs) and 64 morphological characters to test the hypotheses that (1) the P. nivea complex (section Niveae) consists of three taxa (P. chamissonis, P. insularis, and P. nivea), (2) three "eco-morphotypes" in P. pulchella (section Multifidae) should be considered different taxa, and (3) P. insularis originated as an intersectional hybrid (Niveae × Multifidae). Twenty-two RAPD multilocus phenotypes were observed in the 136 plants analyzed based on 35 markers. Three fairly distinct groups of RAPD phenotypes were identified in the P. nivea complex based on multivariate analyses and an analysis of molecular variance (AMOVA; 77.6% among-group variation). The variation within the P. nivea complex was more or less continuous in multivariate analyses of the morphological data. We identified, however, several individual morphological characters that separated unambiguously among the three groups of RAPD phenotypes, revealing that these groups correspond to the previously hypothesized taxa. Many identical RAPD multilocus phenotypes were observed in the "eco-morphotypes" of P. pulchella, suggesting that its conspicuous morphological variation is caused by plasticity or by genetic variation at a small number of loci. The hypothesis of the hybrid origin of P. insularis was not supported by the RAPD data. Overall, very little RAPD variation was observed within populations of the four taxa (2.1-16.7% in AMOVA analyses; average genotypic diversity, D, was 0.10-0.30). We conclude that detailed, concerted analysis of molecules and morphology is a powerful tool in low-level taxonomy.     

Conservation genetics of an arctic species, Saxifraga rivularis L., in Britain

Genetic diversity in British populations of Saxifraga rivularis L. (Saxifragaceae) was assessed using isozymes, RAPDs, inter-SSRs and RFLPs of two non-coding chloroplast DNA regions. Low levels of variation were detected: only five molecular phenotypes were recovered, polymorphic for a single RAPD band and Pgd enzyme phenotype. The problem of drawing conservation recommendations from studies that show low levels of marker variation is discussed, and the importance of recognizing the limits of molecular genetic data in conservation biology is emphasized.     

The effect of photoperiod length on respiration in leaves of Saxifraga cernua L., an arctic herb

Abstract Rates of oxygen uptake were measured in leaves of Saxifraga cernua which had been exposed to an 18-h photoperiod. These rates were compared to those in plants which had been exposed to continuous light. Rates of total dark respiration and alternative pathway respiration measured at the end of the photoperiod gradually decreased over the initial 3 d of exposure to an 18-h photoperiod. Thereafter, respiratory rates were constant. Rates of total dark respiration and alternative pathway respiration decreased during the 6h dark period. Rates of normal and alternative pathway respiration are equally affected during the dark period. The respiratory rates had reached a new minimum level 3 d after the initiation of a dark period. These results suggest that respiration rates in arctic plants are high because of the long photoperiod in the arctic. The kinetics of photoperiod induced changes in respiration are slow enough to suggest the involvement of the biological clock in setting respiration rates. Indeed, total dark respiration and alternative pathway respiration show a definite circadian rhythm. Free-running experiments show that normal respiration changes much less (has a smaller amplitude of variation) than alternative pathway respiration and that alternative pathway respiration accounts for most of the rhythmicity of respiration.     

Amplified fragment length polymorphism versus random amplified polymorphic DNA markers: clonal diversity in Saxifraga cernua

Random amplified polymorphic DNA (RAPD) markers are sensitive to changes in reaction conditions and may express polymorphisms of nongenetic origin. Taxa with variable chromosome numbers are particularly challenging cases, as differences in DNA content may also influence marker reproducibility. We addressed these problems by comparing RAPD and amplified fragment length polymorphism (AFLP) analyses of clonal identity and relationships in a chromosomally variable arctic plant, the polyploid Saxifraga cernua, which has been thought to be monoclonal over large geographical distances. Fifty-seven plants from four Greenland populations were analysed using a conservative scoring approach. In total, 26 AFLP and 32 RAPD multilocus phenotypes (putative clones) were identified, of which 21 were identical and each of the remaining five AFLP clones was split into two to three very similar RAPD clones. This minor difference can be explained by sampling error and stochastic variation. The pattern observed in Greenland corroborates our previous results from Svalbard, suggesting that rare sexual events in S. cernua are sufficient to maintain high levels of clonal diversity even at small spatial scales. We conclude that although AFLP analysis is superior in terms of efficiency, RAPDs may still be used as reliable markers in small low-tech laboratories.     

The importance of sexual and asexual reproduction in the recent evolution of Allium vineale

In the weedy plant species Allium vineale (wild garlic), individuals may simultaneously produce sexually and asexually derived offspring, by seed and bulbils, respectively. In this study, genetic and genotypic diversity was determined in samples from 14 European A. vineale populations using nuclear (RAPD) and cytoplasmic (PCR-RFLP of cpDNA) markers to investigate the importance of the different reproductive modes. In the whole sample, 77 nuclear multilocus genotypes and four chloroplast haplotypes (chlorotypes) were found. Populations exhibited a high degree of subdivision for nuclear and cytoplasmic markers as estimated from hierarchical F-statistics; at the same time, identical chlorotypes could be found in populations separated by large distances. Genotypic diversity was significantly lower than expected under free recombination in almost all populations, indicating that recruitment into populations is mostly by asexually produced offspring. Nevertheless, within each chlorotype, the distribution of markers from pairs of nuclear loci was incompatible with a purely clonal structure, suggesting that many multilocus genotypes have originated by sexual recombination rather than by mutation within asexual lineages. It is argued that the weedy habit of A. vineale is likely to have favored bulbil reproduction, whereas sexually generated genotypes may have facilitated local adaptation during the species' expansion across Europe.     

Molecular evidence for polyploid origins in Saxifraga (Saxifragaceae): the narrow arctic endemic S. svalbardensis and its widespread allies

The recently described polyploid Saxifraga svalbardensis is endemic to the arctic archipelago of Svalbard. We investigated relationships among four closely related species of Saxifraga in Svalbard and tested three previously proposed hypotheses for the origin of S. svalbardensis: (1) differentiation from the morphologically and chromosomally variable polyploid S. cernua; (2) hybridization between the diploid S. hyperborea and S. cernua; and (3) hybridization between the tetraploid S. rivularis and S. cernua. Fifteen populations were analyzed using random amplified polymorphic DNAs (RAPDs) and nucleotide sequences of the chloroplast gene matK and the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). RAPD and matK data suggest that S. svalbardensis has originated from a hybrid with S. rivularis as the maternal parent and S. cernua as the paternal parent, possibly a single time, whereas ITS data could not be used to discriminate among the hypotheses. The data also suggest that the diploid S. hyperborea is a progenitor of the tetraploid S. rivularis. The four populations examined of S. svalbardensis were virtually identical for RAPD and ITS markers, whereas S. cernua showed high levels of variation, suggesting that the latter polyploid either has formed recurrently or has undergone considerable differentiation since its origin.     

Promiscuity in populations of the cushion plant Saxifraga oppositifolia in the Swiss Alps as inferred from random amplified polymorphic DNA (RAPD)

Overviews on patterns of genetic variation within and among plant populations show that widespread, outcrossing species should have a high proportion of the total genetic variation within populations and a low proportion among populations, which results in little population differentiation. However, in Alpine areas, large–scale distribution barriers as well as small-scale habitat heterogeneity could lead to geographical and temporal isolation, respectively. We investigated the genetic variation of Saxifraga oppositifolia from 10 populations of the Alps in southeastern Switzerland using random amplified polymorphic DNA (RAPD). Based on the banding patterns of four RAPD primers, 84 polymorphic markers identified all 189 sampled individuals as being genetically different. The genetic variation was mainly found within populations (95%), whereas less than 5% was found among populations and among regions. Analyses of molecular variance ( AMOVA ) suggested that population differentiation was highly significant. However, grouping populations differently into regions did not appear to result in a clear correspondence of genetic and geographical relatedness. Genetic variation did not significantly differ between populations of two elevational levels. This coincides with results of former pollination experiments that revealed a breeding system of S. oppositifolia which remains the same irrespective of the elevation. We assume that the high outcrossing rate, rare clonal reproduction, and some long-distance dispersal even among topographically separated populations are the crucial determinants for the pattern of genetic variation found in the investigated area.     

Molecular analysis of the Pleistocene history of Saxifraga oppositifolia in the Alps

A recent circumpolar survey of chloroplast DNA (cpDNA) haplotypes identified Pleistocene glacial refugia for the Arctic-Alpine Saxifraga oppositifolia in the Arctic and, potentially, at more southern latitudes. However, evidence for glacial refugia within the ice sheet covering northern Europe during the last glacial period was not detected either with cpDNA or in another study of S. oppositifolia that surveyed random amplified polymorphic DNA (RAPD) variation. If any genotypes survived in such refugia, they must have been swamped by massive postglacial immigration of periglacial genotypes. The present study tested whether it is possible to reconstruct the Pleistocene history of S. oppositifolia in the European Alps using molecular methods. Restriction fragment length polymorphism (RFLP) analysis of cpDNA of S. oppositifolia, partly sampled from potential nunatak areas, detected two common European haplotypes throughout the Alps, while three populations harboured two additional, rare haplotypes. RAPD analysis confirmed the results of former studies on S. oppositifolia; high within, but low among population genetic variation and no particular geographical patterning. Some Alpine populations were not perfectly nested in this common gene pool and contained private RAPD markers, high molecular variance or rare cpDNA haplotypes, indicating that the species could possibly have survived on ice-free mountain tops (nunataks) in some parts of the Alps during the last glaciation. However, the overall lack of a geographical genetic pattern suggests that there was massive immigration of cpDNA and RAPD genotypes by seed and pollen flow during postglacial times. Thus, the glacial history of S. oppositifolia in the Alps appears to resemble closely that suggested previously for the species in northern Europe.     

A review on the relationships between plant genetic diversity and ecosystem functioning

全球变化和人类活动导致物种生境的萎缩, 造成很多植物种群数量缩减, 遗传多样性快速丧失。对于物种多样性低的生态系统, 优势种的遗传多样性可能比物种多样性对生态系统功能产生更大的影响。因此, 了解遗传多样性和生态系统功能的关系(GD-EF)及其机制对生物多样性保护、应对环境变化和生态修复具有指导意义。该文综述了植物遗传多样性对生态系统结构(高营养级生物群落结构)和生态系统功能(初级生产力、养分循环和稳定性)的影响及机制、功能多样性对GD-EF的影响、遗传多样性效应和物种多样性效应的比较, 以及GD-EF在生态修复等实际应用的研究进展。最后指出当前研究的不足之处, 以期为后续研究提供参考: 1)还需深入研究GD-EF机制; 2)未评估遗传多样性对生态系统多功能性的影响; 3)不同遗传多样性测度对生态系统功能的影响不明确; 4)缺少长期的和多空间尺度结合的GD-EF实验; 5)遗传多样性效应相对于其他因子的作用不清楚。     

On the occurrence of sexual reproduction in Saxifraga cernua and S. foliolosa (Saxifragaceae)

In all contemporary floras, the two circumpolar arctic species Saxifraga cernua and S. foliolosa are claimed to reproduce only by vegetative means (bulbils) and to have abandoned sexual reproduction. Scattered records in the literature indicate that this is erroneous, and that seed is set irregularly and at a low rate in both species. An inventory of herbarium material supports this view. Field studies at Latnjajaure, N Swedish Lapland, demonstrate that both species have an androdioecious mating system, a phenomenon which is extremely rare in natural plant populations. The two gender classes within each species are maintained by self-incompatibility in hermaphorodites and a higher rate of vegetative propagation in female-sterile plants. Hermaphrodites are rare, especially in S. cernua , and most populations consist exclusively of female-sterile plants. The sparsity of fruiting material in herbaria is regarded to be, in part, a result of low frequency of collecting, due to the in-conspicuousness of the plants in this stage. Scanning electron micrographs of seeds of the two species are provided.
Flora Nordica Notes no. 5.     

Evidence for massive clonal growth in the invasive weed Fallopia japonic a (Japanese Knotweed)

Clonal growth in introduced populations of Japanese Knotweed ( Fallopia juponica ) in Britain was assessed using RAPDs (Randomly Amplified Polymorphic DNA). A total of 150 British samples was analysed for genetic variation using ten arbitrary decamer primers, and compared with data from 16 samples of other introduced populations from Europe and the U.S.A. All samples produced an identical multi-primer RAPD profile. Accepting that RAPD profile identity need not equate to genet identity, based on the sensitivity of these markers for detecting genetic diversity in related taxa and on the absence of male fertile individuals of this species in Britain, we interpret this result as consistent with the presence of a single, exceptionally widespread clone. This clone must represent one of the world's largest vascular plants.     

植物遗传多样性与生态系统功能关系的研究进展

全球变化和人类活动导致物种生境的萎缩, 造成很多植物种群数量缩减, 遗传多样性快速丧失。对于物种多样性低的生态系统, 优势种的遗传多样性可能比物种多样性对生态系统功能产生更大的影响。因此, 了解遗传多样性和生态系统功能的关系(GD-EF)及其机制对生物多样性保护、应对环境变化和生态修复具有指导意义。该文综述了植物遗传多样性对生态系统结构(高营养级生物群落结构)和生态系统功能(初级生产力、养分循环和稳定性)的影响及机制、功能多样性对GD-EF的影响、遗传多样性效应和物种多样性效应的比较, 以及GD-EF在生态修复等实际应用的研究进展。最后指出当前研究的不足之处, 以期为后续研究提供参考: 1)还需深入研究GD-EF机制; 2)未评估遗传多样性对生态系统多功能性的影响; 3)不同遗传多样性测度对生态系统功能的影响不明确; 4)缺少长期的和多空间尺度结合的GD-EF实验; 5)遗传多样性效应相对于其他因子的作用不清楚。     

Reduced recombination in gynodioecious populations of a facultative apomictic orchid

Many plants combine sexual reproduction with some form of asexual reproduction to different degrees, and lower genetic diversity is expected with asexuality. Moreover, the ratios of sexual morphs in species with gender dimorphism are expected to vary in proportion to the reproductive success of the sexual process. Hence, sex ratios can directly influence the genetic structure and diversity of a population. We investigated genotypic diversity in 23 populations of a facultative, apomictic gynodioecious orchid, Satyrium ciliatum, to examine the effect on genotypic diversity of variation in the frequency of females and in the amount of sexual reproduction. The study involved one pure female, seven gynodioecious (both females and hermaphrodites present) and 15 hermaphroditic populations. Pollinia receipt was higher in hermaphroditic than in gynodioecious populations. Analyses of variation in ISSRs demonstrated that genotypic diversity was high in all populations and was not significantly different between hermaphroditic and gynodioecious populations. We used character compatibility analysis to determine the extent to which recombination by sexual reproduction contributed to genotypic diversity. The results indicate that the contribution of recombination to genotypic diversity is higher in hermaphroditic than in gynodioecious populations, consistent with the finding that hermaphroditic populations received higher amounts of pollinia. Our finding of reduced recombination in gynodioecious populations suggests that maintenance of sex in hermaphrodites plays an important role in generating genotypic diversity in this apomictic orchid.     

Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii

Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection.