Within-population genetic structure and clonal diversity of a threatened endemic metallophyte, Viola calaminaria (Violaceae)

We studied the within-population genetic structure and the clonality extent of Viola calaminaria, a rare endemic species of calamine soils, by means of RAPD markers in two populations (one recent and one ancient) with expected harsh and heterogeneous heavy-metal stress. At a very local scale (0.2-3 m), clonal propagation was detected in both populations, but the levels of clonal diversity were high (number of genets/number of ramets sampled = 0.9 [recent] and 0.76 [ancient]) and the maximal observed extension of the clones was 0.4 m. This indicated that clonality is not, for the species, an important mode of propagation and that clonal growth cannot be interpreted as a strategy for propagating or perpetuating adapted genotypes under harsh ecological constraints. Spatial autocorrelations revealed a significant (P < 0.001) negative value of correlogram slope in the two populations even when a single individual per clone was considered (i.e., analysis at the genet level). We conclude that spatial genetic structure at a very local scale reflects limited gene flow due to restricted seed dispersal rather than variation in clonal pattern in response to environmental heterogeneity. At a larger scale (2-30 m), spatial autocorrelations revealed a positive (P < 0.001) correlation at < 3 m and a random pattern at larger distances for the two populations. This suggested a patchy distribution of the genetically linked individuals associated with a disrupted pattern at a longer distance probably due to gene flow by pollen dispersal and a seed bank effect. The implications for the conservation of V. calaminaria are discussed.     

Intraspecific variation in life history traits of Viola brevistipulata (Violaceae) in Hokkaido

Intraspecific life‐history variations of Viola brevistipulata var. brevistipulata were examined with special reference to reproductive features based on quantitative and qualitative monitoring of chasmogamous (CH) and cleistogamous (CL) flowers. We selected 10 populations in southwestern Hokkaido in common deciduous forests, and marked 40–70 individual plants at each population. Marked individuals were monitored every 1–2 weeks. The expression of CH and CL flowers was distinct among the populations. That is, there were populations consisting of plants with only CH flowers, while other populations produced CL flowers after CH flowers with greater shoot longevity This phenomenon was observed even in populations located at the same site. The results suggest that V. brevistipulata var. brevistipulata shows distinct life‐history variations among the populations.     

Evolution, phylogeography, and taxonomy within the Viola alba complex (Violaceae)

 Taxa of the Viola alba complex were investigated using allozymes and morphometry. A taxonomic revision is presented. A wide delimitation of V. alba with only three morphological and geographical subspecies is suggested: (1) ssp. dehnhardtii distributed in the Mediterranean eastwards to Turkey; (2) ssp. alba flanking ssp. dehnhardtii in the north and east; and (3) ssp. cretica endemic to Crete. Ssp. cretica, up to now treated as a separate species, is particularly close to ssp. dehnhardtii. Viola cadevallii (NW Mediterranean) is included in the synonymy of ssp. dehnhardtii. Ssp. scotophylla (S Europe), ssp. thessala (Balkan), V. armena (Turkey), and V. besseri (Caucasus) are reduced to synonyms of V. alba ssp. alba. Viola pentelica (Greece) might represent transitional forms between ssp. alba and ssp. dehnhardtii. Glacial refugia for ssp. alba are suggested from the eastern Mediterranean via Turkey to the Caucasus, for ssp. dehnhardtii in the Mediterranean area in general, and for ssp. cretica in Crete. A key to the subspecies is provided. Taxonomic recombination: Viola alba Bess. ssp. cretica (Boiss. & Heldr.) Marcussen, comb. nov. Received June 17, 2002; accepted November 27, 2002 Published online: March 20, 2003     

Anatomical studies on selected species of Viola (Violaceae)

Cross‐sections of roots, stems, petioles and peduncles were investigated in 12 species of Viola distributed mainly in northern Iran: V. kitaibeliana, V. arvensis, V. occulta, V. tricolor (sect. Melanium), V. somchetica (sect. Plagiostigma), V. spathulata (sect. Spathulidium ined.), V. alba, V. odorata, V. sintenisii (sect. Viola subsect. Viola), V. caspia, V. reichenbachiana and V. rupestris (sect. Viola subsect. Rostratae). General anatomical features of Viola species are discussed. The following characters were found to be taxonomically informative: cross‐section shapes of the aerial stem, petiole and peduncle as well as the number of vascular bundles in the aerial stem. In taxa of sect. Melanium, stem cross‐sections are elliptic with two wings, but they are semi‐circular with two wings in taxa of sect. Viola subsect. Rostratae. Within the latter subsection, the number of vascular bundles in the stem can be used to delimit the species V. caspia, V. reichenbachiana and V. rupestris. Calcium oxalate crystals were observed in all vegetative organs of taxa belonging to sect. Viola. All examined taxa had a secondary structure with the exception of V. somchetica. Our results show that anatomical characters are useful for delimiting species, subsections, and sections within Viola.     

Successful micropropagation of the cadmium hyperaccumulator Viola baoshanensis (Violaceae)

Viola baoshanensis is one of the most rare cadmium (Cd) hyperaccumulators, however, it is hard to propagate. Micropropagation has been applied to solve the problems with propagation of a few heavy metal hyperaccumulators. Therefore there is a high likelihood that micropropagation may offer a suitable method for large-scale propagation of V. baoshanensis To test this hypothesis, three types of explants were used for shoot regeneration and various combinations of four plant growth regulators were used to improve shoot regeneration efficiency from leaflet of V. baoshanensis. Best shoot regeneration efficiency was obtained by incubating leaflet in a 1/2 MS medium supplemented with 2.5 oM BA + 2.5 microM IBA, therein shoot regeneration rate was 70.9% and the number of shoots formation per explant was 22.4. Rooting was achieved from almost all regenerated shoot growing on 1/2 MS medium without plant growth regulator. Micropropagated seedlings were acclimatized under greenhouse conditions and 95% of them survived and showed no visible morphological variation compared to their donor plant. Furthermore, there were no significant differences between regenerated and seed-germinated V. baoshanensis in Cd tolerance and accumulation. These results suggested that an efficient and rapid micropropogation system was successfully developed for V. baoshanensis.     

Evolutionary relationships, interisland biogeography, and molecular evolution in the Hawaiian violets (Viola: Violaceae)

The endemic Hawaiian flora offers remarkable opportunities to study the patterns of plant morphological and molecular evolution. The Hawaiian violets are a monophyletic lineage of nine taxa distributed across six main islands of the Hawaiian archipelago. To describe the evolutionary relationships, biogeography, and molecular evolution rates of the Hawaiian violets, we conducted a phylogenetic study using nuclear rDNA internal transcribed spacer sequences from specimens of each species. Parsimony, maximum likelihood (ML), and Bayesian inference reconstructions of island colonization and radiation strongly suggest that the Hawaiian violets first colonized the Maui Nui Complex, quickly radiated to Kaua'i and O'ahu, and recently dispersed to Hawai'i. The lineage consists of "wet" and "dry" clades restricted to distinct precipitation regimes. The ML and Bayesian inference reconstructions of shifts in habitat, habit, and leaf shape indicate that ecologically analogous taxa have undergone parallel evolution in leaf morphology and habit. This parallel evolution correlates with shifts to specialized habitats. Relative rate tests showed that woody and herbaceous sister species possess equal molecular evolution rates. The incongruity of molecular evolution rates in taxa on younger islands suggests that these rates may not be determined by growth form (or lifespan) alone, but may be influenced by complex dispersal events.     

Phenetic analysis shows conspecificity between Hispaniolan Viola domingensis Urban and North American Viola macloskeyi sensu lato (Violaceae)

Phenetic investigations were undertaken to evaluate differences between the cloud forest endemicViola domingensis of the Dominican Republic and the morphologically similarV. macloskeyi of continental North America. Cluster, principal components (PCA), and canonical variates analyses (CVA) of leaf characters and of ratios derived from them consistently separateV. macloskeyi subsp.macloskeyi of the Pacific states fromV. domingensis and transcontinentalV. macloskeyi subsp.pallens. However, all analyses portray the latter two as extensively intergradient extremes of a continuum, withV. domingensis plants tending to be smaller. Phenetic analyses of capsule and seed features, which are often divergent among other species of subsect.Stolonosae, showV. domingensis to be nested within a broaderV. macloskeyi subsp.pallens. Similarly, floral morphology does not provide observable differences between the pair. Results support the synonymy ofV. domingensis underV. macloskeyi subsp.pallens and reveal that it represents a disjunct outlier of an otherwise continental North American violet.     

Viola suavis, a new species in the Nordic flora, with analyses of the relation to other species in the subsection Viola (Violaceae)

Evidence from cytology, enzyme markers and morphometry suggest that a species of Viola (Violaceae) collected in southeastern Norway should be referred to V. suavis , although differing in some phenotypic traits. V. suavis is not previously known from the Nordic countries, but must now be regarded as an established member of the Norwegian flora. It was probably introduced more than a century ago, providing an interesting analogy to the situation of V. alba in Öland (Sweden). Thus, it seems like human activity have played a role for expanding the distribution areas of several of the species within Viola subsection Viola (V. alba, V. hirta, V. odorata and V. suavis ). Electrophoretic evidence showed that the basal chromosome number of the subsection is x=5. V. suavis (2n=40) is allo-octoploid, and closely related tetraploid taxa were easily recognized. However, it is difficult to trace its origin to lower-ploid ancestors, and it seems likely that the complex may have contained extinct taxa with other allelic combinations that better explain the origin of V. suavis .     

Viola stoloniflora (Violaceae), a new species from the Ryukyus

Viola stoloniflora Yokota et Higa (Violaceae) is newly described from Okinawa Island, the Ryukyus. This species is characterized by the procumbent stolons, the almost round leaf blades, the densely pilose petioles, the ebarbate white petals with violet veins, and the chromosome number of 2n=22. The present species belongs to Sect.Nomimium, Subsect.Serpentes and is closely related to theV. iwagawai group of Sect.Nomimium, Subsect.Adnatae.     

Relative importance of ballistic and ant dispersal in two diplochorous Viola species (Violaceae)

Viola is one of the diplochorous plant genera that disperse their seeds in two ways, ballistic and ant dispersal. We compared the seed dispersal of two major Viola species of northern Japan, V. selkirkii and V. verecunda. The mean weight of seed was less in V. verecunda (0.42 ± SD 0.03 mg) than in V. selkirkii (0.61 ± 0.12 mg). The elaiosome of V. selkirkii (0.02 ± 0.004 mg) was larger than in of V. verecunda (0.006 ± 0.0004 mg), whereas the lipid component of elaiosome was not remarkably different between the two species. In ballistic dispersal, the mean dispersal distance was 56.0 ± 17.5 cm in V. verecunda but only 38.3 ± 5.1 cm in V. selkirkii. In ant dispersal, the mean dispersal distance was 28.1 ± 24.9 cm in V. selkirkii and 36.1 ± 33.7 cm in V. verecunda; however, the seed removal frequency of V. selkirkii (15.5%) was much higher than that of V. verecunda (3.0%). These results suggest that V. selkirkii is more dependent on ant dispersal while V. verecunda is more dependent on ballistic dispersal. The effect of seed predation was very serious in both species. In the quadrat census, 99.0% of V. selkirkii seeds and 99.1% of V. verecunda seeds were damaged by ground beetles, spiders, ticks, and others which frequently devoured diaspores. An experiment with V. verecunda seeds demonstrated that the overdispersion of seeds on the forest floor enhanced the frequency of removal by ants and reduced seed damage by predators.     

The maintenance of mixed mating by cleistogamy in the perennial violet Viola septemloba (Violaceae)

The production of both potentially outcrossed (chasmogamous) and obligately self-fertilized (cleistogamous) flowers presents a clear exception to the prediction that the only evolutionarily stable mating systems are complete selfing and complete outcrossing. Although cleistogamy has evolved repeatedly, the reason for its stability is not known for any species. We tested the hypothesis that the production of cleistogamous and chasmogamous flowers by a perennial violet constitutes adaptive phenotypic plasticity. We manipulated the season of flowering for each flower type and determined fruit set and the germination percentage of seeds produced by cleistogamous and chasmogamous flowers to test the hypothesis that adaptive plastic response to seasonal environmental variation makes mixed mating stable. Cleistogamous flowers had greater fruit set in all seasons and produced seeds with germination percentages as great as or greater than those from chasmogamous flowers. The consistent advantage of cleistogamous flowers is clearly not consistent with a role of adaptive plastic response to seasonal variation. The biomass cost of seed production by chasmogamous flowers was nearly three times that for cleistogamous flowers. Explaining why chasmogamous flower have not been eliminated by natural selection requires that this difference be balanced by an advantage to chasmogamous flowers that has not yet been identified.     

Inferring species networks from gene trees in high-polyploid North American and Hawaiian violets (Viola, Violaceae)

The phylogenies of allopolyploids take the shape of networks and cannot be adequately represented as bifurcating trees. Especially for high polyploids (i.e., organisms with more than six sets of nuclear chromosomes), the signatures of gene homoeolog loss, deep coalescence, and polyploidy may become confounded, with the result that gene trees may be congruent with more than one species network. Herein, we obtained the most parsimonious species network by objective comparison of competing scenarios involving polyploidization and homoeolog loss in a high-polyploid lineage of violets (Viola, Violaceae) mostly or entirely restricted to North America, Central America, or Hawaii. We amplified homoeologs of the low-copy nuclear gene, glucose-6-phosphate isomerase (GPI), by single-molecule polymerase chain reaction (PCR) and the chloroplast trnL-F region by conventional PCR for 51 species and subspecies. Topological incongruence among GPI homoeolog subclades, owing to deep coalescence and two instances of putative loss (or lack of detection) of homoeologs, were reconciled by applying the maximum tree topology for each subclade. The most parsimonious species network and the fossil-based calibration of the homoeolog tree favored monophyly of the high polyploids, which has resulted from allodecaploidization 9-14 Ma, involving sympatric ancestors from the extant Viola sections Chamaemelanium (diploid), Plagiostigma (paleotetraploid), and Viola (paleotetraploid). Although two of the high-polyploid lineages (Boreali-Americanae, Pedatae) remained decaploid, recurrent polyploidization with tetraploids of section Plagiostigma within the last 5 Ma has resulted in two 14-ploid lineages (Mexicanae, Nosphinium) and one 18-ploid lineage (Langsdorffianae). This implies a more complex phylogenetic and biogeographic origin of the Hawaiian violets (Nosphinium) than that previously inferred from rDNA data and illustrates the necessity of considering polyploidy in phylogenetic and biogeographic reconstruction.     

Two new species of Viola (Violaceae) from the Intermountain West,U.S.A.

Viola lithion andV. franksmithii are rock-dwelling endemics that belong to the North American perennial, caulescent, simple-leaved, blue-, purple-, or white-flowered group of species.Viola lithion, from the White Pine and Pilot mountain ranges of eastern Nevada and adjacent Utah, belongs to sectionChamaemelanium subsectionCanadenses. It most closely resembles the reniform-leaved, Olympic MountainV. flettii, together with which it possibly shares an ancestry with the widespread and allopatricV. canadensis. Viola franksmithii, restricted to the Logan Canyon drainage of the Bear River Range in northern Utah, belongs to sectionRostellatae subsectionRosulantes. It comes closest morphologically toV. howellii of the Coast Range, but likely evolved from the widespreadV. adunca or a common ancestor.     

Viola lilliputana sp. nov. (Viola sect. Andinium, Violaceae), one of the world's smallest violets, from the Andes of Peru

A new violet species of Viola Sect. Andinium, Viola lilliputana, is described from a single dry puna locality on an extensive intermontane plateau southeast of Cerro Palla Palla in the high Andes of Ayacucho Department in southern Peru. This diminutive rosulate violet is evidently among the smallest in the world and probably one of the smallest terrestrial dicots. It belongs to a distinctive species group with pinnatifid leaves that is endemic to central and southern Peru, including V. hillii, V. membranacea and V. weibelii. The new species is similar to V. weibelii in its large, strongly adnate stipules, elongate leaf lobes and dilated unappendaged style with ventral stigmatic orifice. It differs conspicuously from all other members of the pinnatifid-leaved group in its conduplicate leaf blades, straight, mostly nonoverlapping, oblong-lanceolate to broadly elliptical lobes with obtuse to rounded apices, and large basally fused pedicel bractlets. Despite many new collections of vascular plants from the high Andes of Peru and northern Bolivia in recent decades, this distinctive new species is still known only from its type locality, collected on the Iltis-Ugent expedition from November 1962 to January 1963.     

Morphological Characters Useful for the Delimitation of Taxa Within Viola Subsect. Viola (Violaceae): A Morphometric Study from the West Carpathians

Forty-nine morphological characters were scored or measured on 44 populations (376 individuals) of Viola subsect. Viola from the West Carpathians and adjacent areas (Slovakia, Czech Republic, Austria and Hungary). The presence of six species, namely V. alba (represented by subsp. alba), V. ambigua, V. collina, V. hirta, V. odorata and V. suavis s.l. was revealed based on pollen fertility, cytological and morphometric analyses. The morphological characters traditionally used to delimit taxa within the subsection and those revealed by our study as most reliable are widely discussed. A key for identifying the taxa and most common hybrids of subsection Viola occurring in the West Carpathians is presented. Chromosome counting and flow cytometry were used to determine the ploidy levels of the populations studied. All individuals of V. alba subsp. alba, V. collina, V. hirta and V. odorata were tetraploid, while those of V. ambigua and V. suavis s.l. were octoploid.     

Molecular phylogenetic analysis of Violaceae (Malpighiales) based on plastid and nuclear DNA sequences

A phylogenetic analysis of Violaceae is presented using sequences from rbcL, atpB, matK and 18S rDNA from 39 species and 19 genera. The combined analysis of four molecular markers resulted in only one most parsimonious tree, and 33 of all 38 nodes within Violaceae are supported by a bootstrap proportion of more than 50%. Fusispermum is in a basal-most position and Rinorea, Decorsella, Rinoreocarpus and the other Violaceae are successively diverged. The monogeneric subfamily Fusispermoideae is supported, and it shares a number of plesiomorphies with Passifloraceae (a convolute petal aestivation, actinomorphic flowers and connate filaments). The other monogeneric subfamily Leonioideae is sunken within the subfamily Violoideae and is sister to Gloeospermum, sharing some seed morphological characteristics. The present molecular phylogenetic analysis suggests that the convolute, apotact and quincuncial petal aestivation is successively derived within the family. The evolutionary trends of the other morphological characteristics, such as a filament connation, the number of carpels and floral symmetry, are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic diversity of the narrow endemic Allium aaseae (Alliaceae)

Low levels of genetic variability are common for a number of geographically restricted plants: these data are consistent with theoretical expectations that small populations should be genetically depauperate. However, in some species, high levels of variability have been found in rare species. Allium aaseae is a rare, narrow endemic in the foothills of the Boise Front of southwestern Idaho. Genetic variation in the rare endemic A. aaseae, and nearby populations of the more common species, A. simillimum, was examined with randomly amplified polymorphic DNA (RAPD) data. Eight populations of A. aaseae and six populations of A. simillimum (three near the range of A. aaseae, and three distant populations) of 25 individuals each were examined for this analysis. Genetic diversity as determined with RAPD markers of both species examined in this study is largely found within and not among populations. Levels of genetic diversity are high, especially for a narrow endemic species such as A. aaseae. Proportion of RAPD loci polymorphic was high in both species, although slightly higher in the more common A. simillimum. Because these higher levels of genetic variability run counter to theory, alternative explanations beyond population size must be invoked to explain the levels of genetic diversity found in this study. Possible explanations are (1) A. aaseae is only recently derived from A. simillimum, (2) hybridization between A. aaseae and A. simillimum is occurring, (3) multiple origins of A. aaseae, (4) populations of A. simillimum included in the analysis are all A. aaseae, (5) A. aaseae and A. simillimum are conspecific, and (6) an artifact of RAPD data.     

Genetic diversity of the narrow endemic Astragalus oniciformis (Fabaceae)

Astragalus oniciformis Barneby is a narrow endemic xerophyte of the upper Snake River Plain of central Idaho, USA, where it inhabits stabilized, aeolian sand deposits and previously burned, sandy sites over Quaternary basalt flows. The objective of this study was to determine the levels and distribution of genetic differentiation within and among populations of A. oniciformis. Fifteen individuals from each of eight populations, chosen from throughout the range of the species, were selected based on accessibility, density of individuals, and large population size. Inter-simple sequence repeats were chosen as the marker to assess genetic differentiation. The two primers selected yielded 40 polymorphic loci in A. oniciformis. In an analysis of molecular variance, 88.69% of the variation was significantly attributed to variation within populations. High gene flow (N(m) = 3.91-3.93; SD = 0.01) and a low percentage deviation from Hardy-Weinberg equilibrium due to population subdivision (G(ST) = 0.113-0.1134; SD = 0.0002) were found among sampled populations. These results suggest that current threats to this species, including changing fire patterns and habitat loss from grazing disturbance, have not yet affected the genetic diversity of this species. Preservation of large populations and smaller, intervening, dispersed patches will help preserve the genetic integrity and the genetic diversity found in A. oniciformis.     

堇菜属组间的系统发育与亲缘关系：基于trnL-trnF、psbA-trnH、rpL16、ITS序列,细胞学以及形态学证据

以鼠鞭草（Hybanthus enneaspermus）、鳞隔堇（Scyphellandra pierrei）、雷诺木（Rinorea benga-lensis）作为外类群,对堇菜属（Viola）20个类群的trnL-trnF序列,17个类群的psbA-trnH、rpL16序列以及1个类群的nrDNAITS序列进行了测定,并从GenBank下载相应的序列,运用最大简约法以及贝叶斯推论法进行系统分析,构建系统发育树。结果表明：堇菜亚属（subgen. Viola）不是一个单系类群,并明确了堇菜属部分组间类群的亲缘关系。本文还结合形态与细胞学证据对堇菜属进行性状演化的推测。结果表明：1）直立茎较匍匐茎、莲座状茎（叶基生）原始;2）托叶边缘长流苏状与托叶1/2～3/4合生分别是鸟嘴柱头堇菜组（sect. Trigonocarpae）和合生托叶组（sect. Adnatae）演化路线的重要性状标志;3）花柱样式从柱头无喙演化至柱头有喙,并由柱头简单演化至柱头复杂,再趋向于柱头简化。