Genetic diversity in Delphinium variegatum (Ranunculaceae): a comparison of two insular endemic subspecies and their widespread mainland relative

Delphinium variegatum is subdivided into three subspecies: D. v. variegatum is widespread in central and northern California, while D. v. kinkiense (an endangered taxon) and D. v. thornei are endemic to San Clemente Island off the coast of southern California. Electrophoretic data for 19 loci were collected from 7 populations of the mainland subspecies and all 24 known populations of the two insular endemic subspecies. Populations of the widespread mainland subspecies have more polymorphic loci (33.6% vs. 24.5%) and more alleles per polymorphic locus (2.61 vs. 2.15) than the insular endemic subspecies. However, observed heterozygosities are lower in the mainland subspecies (0.041 vs. 0.071), presumably due to lower levels of outcrossing (t = 0.464 vs. 0.895). Expected heterozygosities are similar (0.064 vs. 0.074) due to lower alternative allele frequencies in populations of the mainland subspecies (mean q = 0.075 vs. 0.190). Populations of the two insular subspecies are almost equivalent genetically (mean I = 0.997) regardless of taxonomic designation or geographic location. In contrast, one of the mainland populations is genetically well differentiated from the others. If this exceptional population is excluded, the mainland subspecies partitions genetic diversity similarly to the island subspecies, with most variation being found within populations (G(ST) = 0.073 vs. 0.030).     

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.     

Genetic variation within and among populations of the narrow endemic,delphinium viridescens (Ranunculaceae)

Delphinium viridescens Leiberg (Ranunculaceae) is a narrow endemic, known from only 17 populations in the Wenatchee Mountains of central Washington. Electrophoretic data for ten loci and approximately 50 plants from each of the 17 populations indicate high levels of variability both within and among populations. High levels of heterozygosity, coupled with a general conformance of genotype frequencies to Hardy-Weinberg expectations, suggest that D. viridescens is an outcrossing species, and outcrossing rates range from 0.391 to 1.454. Although all populations share a suite of common alleles, interpopulational differences in allele frequency were detected. Nei's genetic identities range from 0.883 to 1.000, with a mean of 0.964, suggesting only slight differentiation among populations and at least moderate levels of interpopulational gene flow. However, F_ST values indicate greater interpopulational heterogeneity. The data from this study are intended for use in developing a management plan for D. viridescens, to maintain the viability of this species, and to assess the need for its listing as threatened or endangered.     

Volatile leaf oil diversity in the narrow range endemic Eucalyptus argutifolia (Myrtaceae) and its widespread congener Eucalyptus obtusiflora

Variation in the qualitative composition of volatile leaf oil was examined in the rare Eucalyptus argutifolia and its widespread congener Eucalyptus obtusiflora . The results revealed that, consistent with the pattern seen with allozymes, E. argutifolia had less variation within populations than E. obtusiflora . Total leaf oil diversity was also significantly lower in the rare species. As found with allozymes, most leaf oil diversity was within populations, but there was also a significant proportion of the variation between populations (25.2% and 27.3% for E. argutifolia and E. obtusiflora , respectively). There were significant associations between phenotypic distance based on leaf oils and geographical distance and between phenotypic and genetic distance across all populations, but these associations were not evident within species. Factors leading to reduced variation in E. argutifolia appear to affect all types of variation, but the relationships between different types of variation within the species are less apparent.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 738–745.     

Limitations on reproductive success in endemic <Emphasis Type="Italic">Aquilegia viscosa</Emphasis> (Ranunculaceae) relative to its widespread congener <Emphasis Type="Italic">Aquilegia vulgaris</Emphasis>: the interplay of herbivory and pollination

Plant reproduction can be strongly affected by herbivory and different features of pollination ecology, such as pollinator visitation rates and capacity for self-pollination. The purpose of this study is to compare the relative impact of herbivory and pollination on maternal reproductive success in endemic Aquilegia viscosa and its widespread congener Aquilegia vulgaris. We conducted herbivore exclusion experiments in two populations of each species in 2 different years and showed that the maternal fertility of A. viscosa was significantly more limited by floral predation and pre-dispersal seed predation than its widespread congener. In the absence of herbivory, A. viscosa retained significantly lower maternal fertility than A. vulgaris. Experimental pollinations in an insect-free glasshouse showed that the two species have an equal seed/ovule ratio both in the absence of pollinators and in the presence of non-limiting outcross pollination. Pollinator visitation rates were significantly higher in populations of A. vulgaris than in populations of A. viscosa. In addition, path analyses showed that spur length, an important trait for pollinator attraction in Aquilegia, and, indirectly sepal and petal width, contribute positively to the seed/ovule ratio in A. vulgaris, but not in A. viscosa. These results indicate that maternal fertility of endemic A. viscosa is strongly reduced by flower and seed predation despite low rates of pollinator visitation, and that pollen or resource limitation in the wild may further reduce maternal fertility. Finally, floral trait variation appears to be decoupled from fertility variation in endemic A. viscosa, which possibly constrains the evolution of reproductive traits in this species.     

A butenolide atypical of the Ranunculaceae: aquilegiolide from Aquilegia atrata (var. Atroviolacea)

Roots of Aquilegia atrata have afforded 6β-hydroxy-2(6,7-dihydro-7aβH)-benzofuranone (aquilegiolide) and its 7aαH-isomer, or their enantiomers, two butenolides atypical of the Ranunculaceae. Hot aqueous 20 % sulphuric acid rapidly equilibrates the two isomers in a 1:4 ratio.     

Comparisons of genetic diversity in a narrow endemic, Impatiens hypophylla, and in its widespread congener, I. textori (Balsaminaceae)

Genetic diversity in two species of Impatiens in Korea was investigated using starch gel electrophoresis. Impatiens hypophylla is a rare species endemic to southwestern Japan and southeastern Korea, whereas I. rextori is widespread in East Asia. Impatiens textori has considerably higher genetic diversity than I. hypophylla . For example, mean values of genetic diversity within populations (He) of I. textori and I. hypophylla were 0.206 and 0.102, respectively. The high outcrossing rate (mean 1 = 0.89), abundance and widespread distribution, and large population size may be factors responsible for the maintenance of high levels of genetic variation in I. textori . Analyss of fixation indices indicated that considerable population substructuring within populations of I. hypophylla , partly resulting from inbreeding and/or genetic drift in small populations coupled with limited gene flow (mean F_IS= 0.699). However, the degree of population divergence observed in I. hypophylla (mean G_ST= 0.059) was considerably lower than that in other plants with similar life history traits. It is supposed that similar selection at populations of I. hypophylla examined might maintain similar gene frequencies in isolated populations.     

Experimental dissection of inbreeding and its adaptive significance in a flowering plant, Aquilegia canadensis (Ranunculaceae)

Inbreeding is a major component of the mating system in populations of many plants and animals, particularly hermaphroditic species. In flowering plants, inbreeding can occur through self-pollination within flowers (autogamy), self-pollination between flowers on the same plant (geitonogamy), or cross-pollination between closely related individuals (biparental inbreeding). We performed a floral emasculation experiment in 10 populations of Aquilegia canadensis (Ranunculaceae) and used allozyme markers to estimate the relative contribution of each mode of inbreeding to the mating system. We also examined how these modes of inbreeding were influenced by aspects of population structure and floral morphology and display predicted to affect the mating system. All populations engaged in substantial inbreeding. On average, only 25% of seed was produced by outcrossing (range among populations = 9-37%), which correlated positively with both population size (r = +0.61) and density (r = +0.64). Inbreeding occurred through autogamy and biparental inbreeding, and the relative contribution of each was highly variable among populations. Estimates of geitonogamy were not significantly greater than zero in any population. We detected substantial biparental inbreeding (mean = 14% of seeds, range = 4-24%) by estimating apparent selfing in emasculated plants with no opportunity for true selfing. This mode of inbreeding correlated negatively with population size (r = -0.87) and positively with canopy cover (r = +0.90), suggesting that population characteristics that increase outcross pollen transfer reduce biparental inbreeding. Autogamy was the largest component of the mating system in all populations (mean = 58%, range = 37-84%) and, as expected, was lowest in populations with the most herkogamous flowers (r = -0.59). Although autogamy provides reproductive assurance in natural populations of A. canadensis, it discounts ovules from making superior outcrossed seed. Hence, high autogamy in these populations seems disadvantageous, and therefore it is difficult to explain the extensive variation in herkogamy observed both among and especially within populations.     

Effects of population size and isolation on reproductive output in Aquilegia canadensis (Ranunculaceae)

It is generally expected that small, isolated populations will suffer reduced fitness due to inbreeding, yet few studies have investigated the relation between population characteristics, inbreeding and fitness. Among Ontario populations of the short‐lived, perennial plant Aquilegia canadensis, large populations (N>90 flowering plants) outcross twice as frequently as small populations (N=30–40), and inbreeding depression is extremely strong. We tested the prediction that reproductive output, a major component of population fitness, should be positively associated with population size. Data from a survey of 33 populations located on small islands in the St. Lawrence River, Canada and 23 populations on adjacent mainland areas supported this prediction. Population size correlated positively with reproductive output, measured as the number of seedlings produced per plant in 1995 (average r=+0.39 pooled P=0.019), and the number of fruits per plant in 1997 (r=+0.30, P=0.056). We also tested the prediction that fitness should decline with increasing spatial isolation between populations by measuring the distance separating all island populations. However, reproductive output did not correlate with isolation in either year. We compared island and mainland populations to test the prediction that reproductive output should be lower for populations on small islands than those occurring in more continuous mainland habitat. In contrast to our predictions, island populations exhibited, if anything, higher reproductive output than mainland populations. We also found no support for the prediction that the positive association between population size and reproductive output should be stronger for presumably isolated populations on small islands than for those on adjacent mainland areas. While the mechanisms underlying the association between population size and fitness are impossible to identify with correlations alone, our results are consistent with the hypothesis that inbreeding can significantly reduce the fitness of natural populations.     

Cross‐species amplification of microsatellite loci in Aquilegia and Semiaquilegia (Ranunculaceae)

We developed 16 microsatellite loci from an F₂ hybrid between Aquilegia formosa and Aquilegia pubescens. In samples of 28 individuals, we found an average of 14 alleles per locus from each parental species. We tested these loci for cross‐amplification in 10 additional species of Aquilegia and found that all 16 loci amplified in other North American species and 12 consistently amplified in European or Asian species. Nine loci amplified in the sister species to Aquilegia, Semiaquilegia adoxoides. The success of cross‐species amplification suggests that these microsatellites should prove useful for studies in a broad range of Aquilegia species.     

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions. Received: 12 January 1998 / Revision received: 10 October 1998 / Accepted: 28 October 1998     

Genetic variation,breeding system evolution,and conservation of the narrow sand dune endemic Stellaria arenicola and the widespread S. longipes (Caryophyllaceae)

Genetic variation was examined by electrophoresis in 14 populations of Stellaria arenicola, an endemic of the Athabasca sand dunes in northern Saskatchewan, Canada, and seven populations of S. longipes, its progenitor. Three of the 5. longipes populations were sympatric with the endemic. Populations of the endemic were found to have fewer alleles per polymorphic locus (2.21 vs. 2.37), fewer polymorphic loci (29.9 vs. 33.8), and lower genetic diversity (0.087 vs. 0.107) than populations of the progenitor. Genetic identities for all pairs of populations were high (0.932 to 1.000). The endemic had one novel allele and shared ten alleles with progenitor populations from the sand dunes that were not found in other populations of S. longipes. Populations of both species were found to partition most of their genetic variation within populations. An investigation of the multilocus outcrossing rates revealed that S. arenicola had higher rates of selling and biparental inbreeding than S. longipes. This study suggests that partial genetic isolation through a shift in the breeding system, in addition to previously reported strong directional selection, has been important in the sympatric evolution of the endemic S. arenicola. The close genetic relationship between populations of S. arenicola and S. longipes found on the Athabasca sand dunes supports the suggestion that the endemic evolved while sympatric to the gene pool of the progenitor species that is found presently in the region.     

Palaeopolyploidy, spatial structure and conservation genetics of the narrow steppe plant Vella pseudocytisus subsp. paui (Vellinae, Cruciferae)

BACKGROUND AND AIMS: Vella pseudocytisus subsp. paui (Cruciferae) is a narrow endemic plant to the Teruel province (eastern Spain), which is listed in the National Catalogue of Endangered Species. Two distinct ploidy levels (diploid, 2n = 34, and tetraploid, 2n = 68) have been reported for this taxon that belongs to the core subtribe Vellinae, a western Mediterranean group of shrubby taxa with a chromosome base number of x = 17. Allozyme and AFLP analyses were conducted (a) to test for the ploidy and putative palaeo-allopolyploid origin of this taxon, (b) to explore levels of genetic diversity and spatial structure of its populations, and (c) to address in-situ and ex-situ strategies for its conservation. METHODS: Six populations that covered the entire geographical range of this taxon were sampled and examined for 19 allozyme loci and three AFLP primer pair combinations. In addition, the gametic progenies of five individuals were analysed for two allozyme loci that showed fixed heterozygosity. KEY RESULTS: Multiple banded allozyme profiles for most of the surveyed loci indicated the polyploidy of this taxon. Co-inherited fixed heterozygous patterns were exhibited by the gametophytic tissues of the mother plants. Both allozyme and AFLP markers detected high levels of genetic diversity, and a strong micro-spatial genetic structure was recovered from AFLP phenetic analyses and Mantel correlograms. CONCLUSIONS: Allozyme data support the hypothesis of an allotetraploid origin of Vella pseudocytisus subsp. paui that could be representative of other taxa of the core Vellinae group. AFLP data distinguished three geographically distinct groups with no genetic interaction among them. Allotetraploidy and outcrossing reproduction have probably contributed to maintenance of high levels of genetic variability of the populations, whereas habitat fragmentation may have enhanced the high genetic isolation observed among groups. In-situ microgenetic reserves and a selective sampling of germplasm stocks for ex-situ conservation of this taxon are proposed.     

Relationship between floral longevity and sex allocation among flowers within inflorescences in Aquilegia buergeriana var. oxysepala (Ranunculaceae)

Understanding the fitness of plants with inflorescences requires examining variation in sex allocation among flowers within inflorescences. We examined whether differences in the duration of the male and female phases of flowering lead to variation in sex allocation and reproductive success among flowers within inflorescences. In 2002 and 2003, we quantified floral longevity, floral sex allocation, and reproductive success between the first and the second flowers within inflorescences in a protandrous species, Aquilegia buergeriana var. oxysepala. Floral longevity was greater in the first flowers than in the second ones in both years. The male phase lasted longer, and the initial number of pollen grains and the number of pollen grains removed were greater in the first flowers than in the second ones in both years. Within first flowers, the number of pollen grains removed was greater in flowers that had longer male phases, thus duration of the male phase may positively affect male reproductive success in the first flowers. The female phase lasted longer and the number of ovules was greater in the first flowers than in the second only in 2002. However, seed production per flower and female phase duration in both years were not significantly related. The variation in the number of pollen grains among flowers in this species may be caused by the variation in male phase duration.     

Genetic diversity in the tetraploid sand dune endemic Deschampsia mackenzieana and its widespread iploid progenitor D. Cespitosa (Poaceae)

Electrophoretic variation was examined in 14 populations of tetraploid Deschampsia mackenzieana, an endemic of the Athabasca sand dunes in northern Saskatchewan, Canada, and 20 populations of its geographically widespread diploid progenitor, D. cespitosa. Three of the D. cespitosa populations were sympatric with the endemic on the Athabasca sand dunes. Populations of the endemic were found to have fewer alleles per locus (1.22 vs. 1.52), fewer alleles per polymorphic locus (2.17 vs. 2.70), lower percent polymorphic loci (18.9 vs. 30.5), and lower heterozygosity (0.062 vs. 0.119) than progenitor populations. Species level genetic diversity parameters also indicated that D. mackenzieana was genetically depauperate relative to its progenitor D. cespitosa. Deschampsia mackenzieana had no novel alleles but did share one allele with sympatric progenitor populations that did not occur in populations of D. cespitosa from other habitats. Although both species were found to partition most of their genetic diversity within populations, D. mackenzieana did have more of its limited genetic diversity partitioned among populations than D. cespitosa. The close genetic relationship between D. mackenzieana and sympatric populations of D. cespitosa may suggest the endemic tetraploid evolved from the sympatric diploid gene pool in the Athabasca sand dune region. The low levels of genetic diversity in D. mackenzieana suggest a restricted origin with limited gene flow from the progenitor since speciation.     

Genetic variability of the narrow endemic Rhamnus persicifolia Moris (Rhamnaceae) and its implications for conservation

Rhamnus persicifolia Moris is an endemic small tree belonging to the Rhamnus cathartica group, growing along mountainous streams of Central-Eastern Sardinia (Italy). ISSR markers were used to detect the genetic diversity within and among six populations representative of the species distribution range. In spite of the limited distribution of this endemic taxon, fairly high levels of genetic diversity were detected. Percentage of polymorphic bands (PPB), gene diversity (H_S and H_T) and Shannon information measure (Sh) were calculated both at population (PPB = 30.70%, H_S = 0.1105, Sh = 0.1646) and at species level (PPB = 68.42%, H_T = 0.2066, Sh = 0.3139).The existence of a spatial distribution of genetic diversity in R. persicifolia was revealed by a low gene flow, a fairly high level of genetic differentiation (G_ST = 0.4583) among populations and a positive correlation between genetic and geographic distances (Mantel test, r = 0.71, p = 0.016). The spatial genetic structure was also confirmed with BAPS analysis. Our results show that a certain level of isolation by distance and sex-ratio bias may explain the distribution of genetic diversity among populations.Conservation measures are suggested on the basis of the genetic diversity detected, by implementing an integrated in situ and ex situ conservation program for each population, in order to ensure effective protection for this endemic species.     

Pollination ecology in the narrow endemic winter-flowering Primula allionii (Primulaceae)

Reduction of pollen flow can affect plant abundance and population viability and cause selection on plant mating system and floral traits. Little is known on the effect of this phenomenon in species naturally restricted to small and isolated habitats, that may have developed strategies to cope with long-term isolation and small population size. We investigated the pollination ecology of the endemic distylous winter-flowering P. allionii to verify the possible limitation of female fitness due to reduced pollinator visits. We recorded a higher production of pollen grains in long-styled morph, and a higher seed set in short-styled morph. The high intra-morph variability of sexual organ position may explain the hybridization phenomena allowing and easier intra-morph pollination. The fruit set is constant, although its winter-flowering period might decrease pollen transfer. Nevertheless, the lower competition for pollinators with neighbouring plants and the long-lasting anthesis may offset its reproductive success. Even if our results show no evidence of imminent threats, changes in plant–pollinator interactions might increase inbreeding, resulting in an increased extinction risk.