A new Delphinium subsp. firom Formentera (Balearic Islands)

A new subspecies, Delphinium pentagynum subsp. formsnteranum N. Torres, L. Sáez, Rosselló & C. Blanché, is described from the island of Formentera (pytiusic Islands, Balearic archipelago). The new plant differs typical D. pentagynum by the narrower sepals and smaller size of the corolla, spur, and follicles. Both D. pentagynum subsp. pentagynum and D. pentagynum subsp. formenteranum have 2n= 16 chromosomes and share similar karyotypes. The new taxon is regarded as a schizoendemic and dry island vicariant of Iberian and North African populations of D. pentagynum subsp. pentagynum which probably evolved under insular isolation. After revision of its conservation status, it should be considered as critically endangered (CR) following the IUCN criteria.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

中国毛茛科翠雀属的分类学研究(八):短尾翠雀花的名实订正

发现近来根据云南西北部丽江标本描述的毛茛科短尾翠雀花(Delphinium brachyurum W. T. Wang)与此前根据该地标本描述的丽江翠雀花(D. likiangense Franch.)属于同一种植物,故将前者处理为后者的异名。     

中国毛茛科翠雀属的分类学研究(十三):狭序翠雀花的名实订正

发现根据甘肃南部标本描述的毛茛科狭序翠雀花（Delphinium angustipaniculatum W.T.Wang）与此前记载分布于甘肃南部和四川北部的松潘翠雀花（D.sutchuenense Franch.）属于同一种植物,故将前者处理为后者的异名。讨论了松潘翠雀花与其可能的近缘种秦岭翠雀花（D.giraldii Diels）和疏花翠雀花（D.sparsiflorum Maxim.）的形态区别。     

Genetic diversity in the two endangered endemic species Kirengeshoma koreana (Hydrangeaceae) and Parasenecio pseudotaimingasa (Asteraceae) from Korea: Insights into population history and implications for conservation

As mountains have been hypothesized as harboring important Quaternary refugia for plants, one would expect high within-population genetic variation and low or moderate among-population differentiation for those species occurring there. We selected Kirengeshoma koreana and Parasenecio pseudotaimingasa, endemic to mountains of Korea to test the scenario that the main mountain ranges of the Peninsula would also have acted as refugia. We undertook a population genetic analysis using 15 and 12 putative allozyme loci of K. koreana and P. pseudotaimingasa, respectively. We found contrasting levels of genetic diversity in the two species; extremely low within-population genetic variation and high among-population divergence in K. koreana and high within-population genetic variation and moderate among-population divergence in P. pseudotaimingasa. We can conclude that, whereas Korean mountains would have provided large refugial areas (i.e., ‘macrorefugia’) for P. pseudotaimingasa, K. koreana would have endured the Quaternary climate oscillations in small, isolated populations (i.e., ‘microrefugia’). Given the lack of genetic diversity and the extremely low number of populations and individuals of K. koreana, a comprehensive conservation program is urgently needed for this rare and critically endangered species.     

Sperm cryopreservation of the critically endangered olive barb (Sarpunti) Puntiussarana (Hamilton, 1822)

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg⁻¹. Sperm were activated with distilled water (24 mOsmol kg⁻¹) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ?287 mOsmol kg⁻¹. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 °C to −80 °C at 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at −196 °C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.     

Genetic diversity and mating system of the threatened plant <Emphasis Type="Italic">Kirengeshoma palmata</Emphasis> (Saxifragaceae) in Korea

The endangered herb Kirengeshoma palmata, from eastern Asia, has had its population severely reduced in number through habitat loss and fragmentation. All of the individuals within five subgroups at Mt. Baek-un-san, in the southern part of Korea, were genetically surveyed by allozyme analysis. Genetic diversity levels within subgroups were relatively high, and a consistently high outcrossing rate as well as a negligible biparental mating rate were confirmed by this study. Several groups of visibly connected ramets were observed in a clustered distribution which suggested cloning. Absence of mating partner rather than pollinators decreased seed production in small mating groups. The present genetic structure of the five subgroups was probably the result of local extinction of intervening populations. Because K. palmata may be a self-incompatible species, populations with few genets face lowered seed set due to mate scarcity. Thus, this type of population may be at an increased risk of extinction as a result of inbreeding depression, loss of genetic variability, and reduced sexual reproduction. The small, genetically depauperate subgroups may need an input of seeds or plants from other populations in China or Japan in order to regenerate, but the possibility of outcrossing depression leads us to recommend outbreeding among the local subgroups of Mt. Baek-un-san to restore genetic variability.     

Genetic diversity in <Emphasis Type="Italic">Delphinium staphisagria</Emphasis> (Ranunculaceae), a rare Mediterranean dysploid larkspur with medicinal uses

Delphinium staphisagria is an endemic annual or biennial herb from the Mediterranean Basin, widely distributed in isolated populations of variable size. We evaluated the allozyme diversity of 31 populations along its distribution range via starch gel electrophoresis, assaying 12 enzyme systems and scoring 17 loci. The low levels of genetic variability detected (A = 11.8, A _p = 1.6, H _o = 0.026, H _e = 0.057), are discussed in relation to the life-history traits of the species, such as short life-span, selfing or gravity seed dispersion. Other factors influencing genetic diversity, such as evolutionary history and spreading are also considered. Due to its historical medicinal uses, this plant has probably become widespread in the Mediterranean area. Human-mediated distribution could have promoted few migrant genotypes, recent founder events and long distance dispersal. These events would explain the genetic homogeneity found within and among populations, as well as the absence of a clear biogeographic structure. The limited genetic variability, the high genetic similarity among populations and the dysploidy of this species make it worthy of conservation. Management strategies are proposed mainly to preserve its genetic pool.     

Spatial distribution patterns and implications for conservation ofScrophularia takesimensis (Scrophulariaceae), an endangered endemic species on Ulleung Island, Korea

As part of an on-going effort to conserve endangered and endemicScrophularia takesimensis (Scrophulariaceae), we analyzed its spatial distribution patterns by applying an index of dispersion, plant-to-all-plant distances, and the varying quadrat size method. Three indices — Dispersion, Morisita, and Standardized Morisita — all revealed clumping with small aggregates, and distances between aggregates were more or less regular. The asymptote level occurred at a distance of 20 to 30 m; the distance showing 90% of cumulative frequency coincided with 20 to 21 m; 95%, 24 to 25 m; and 99%, 31 to 32 m. The 20 m× 20 m and 40 m×40 m quadrats contained 25 and 40 plants, respectively. We conclude that this number of individuals and size of area are the minimum required for the conservation of this species.     

Genetic diversity analysis and conservation of the endangered Chinese endemic herb Dendrobium officinale Kimura et Migo (Orchidaceae) based on AFLP

Dendrobium officinale is a critically endangered perennial herb endemic to China. Determining the levels of genetic diversity and patterns of population genetic structure of this species would assist in its conservation and management. Data of 12 populations were used to assess its genetic diversity and population structure, employing the method of amplified fragment length polymorphism (AFLP). A high level of genetic diversity was detected (H (E) = 0.269) with POPGENE. As revealed by AMOVA analysis, there was moderate variation between pairs of populations with Phi(ST) values ranging from 0.047 to 0.578 and on average 26.97% of the genetic variation occurred among populations. Three main clusters were shown in UPGMA dendrogram using TFPGA, which is consistent with the result of principal coordinate ananlysis (PCO) using NTSYS. Keeping a stable environment is critical for the in situ conservation and management of this rare and endangered plant, and for ex situ conservation it is important to design an integrated germplasm bank.     

On the verge of extinction: genetics of the critically endangered Iberian plant species, Borderea chouardii (Dioscoreaceae) and implications for conservation management

Borderea chouardii is a relictual and dioecious, strictly sexually reproducing, long-living geophyte of the Dioscoreaceae family. Previous biological and demographic studies have indicated the existence of a uniformly distributed panmictic population of this taxon at the southernmost Spanish pre-Pyrenean mountain ranges where it occurs in rather inaccessible crevices of a single limestone cliff. However, individuals of B. chouardii are spatially subdivided into two subpopulations located, respectively, on the upper and lower parts of the cliff, and vertically separated 150 m. Because of its extreme rarity, B. chouardii was the first Iberian taxon to have a specific conservation plan and has been included in several red lists under the category of critically endangered (CR). However, no previous attempts have been conducted to analyse the fine scale evolutionary mechanisms involved in its present microspatial distribution. Genetic diversity and population structure have been investigated through the analysis of neutral hypervariable markers such as simple sequence repeats (SSRs) and randomly amplified polymorphic DNAs (RAPDs) to unravel the impact of life history traits in the differentiation of the two subpopulations. Both types of molecular markers were unequivocal in distinguishing two genetically distinct groups of individuals corresponding to their spatial separation. However, SSRs detected a higher level of subpopulation differentiation (F(ST) = 0.35, R(ST) = 0.32) than RAPDs (F(ST) = 0.21). SSR data indicated significant deviation from random dispersal of genes and genotypes between the two groups, suggesting that mating occurs mainly among individuals within subpopulations, thus, favouring the divergence between the two groups. This microevolutionary differentiation scenario might have been caused by a coupled effect of past genetic drift and reproductive isolation, as a result of strong glacial age bottlenecks and inefficient dispersal system of pollen and seeds, respectively. The identification of such genetic structure in this narrow endemic prompts a modification of the management strategies of its single extant population.     

Habitat distribution modelling,under the present climatic scenario,of the threatened endemic Iberian Delphinium fissum subsp. sordidum (Ranunculaceae) and implications for its conservation

We modelled the potential habitat of a threatened species D. fissum subsp. sordidum, an endemic hemicryptophyte with a disjunct distribution in the Iberian Peninsula. Maxent was used to predict the subspecies habitat suitability by relating field sample-based distributional information with environmental and topographic variables. Our results suggest that the model performed well, predicting with high accuracy the current distribution of the species. The variables that most contributed to the model were Mean Temperature of Wettest Quarter (MTWtQ), Precipitation of Warmest Quarter (PWmQ), Temperature Annual Range (TAR) and Slope (Slo). These variables are biological significant for the taxon, as they have decisive influence in the critical stages of germination and fruiting. The current and potential distributional areas identified by the model fall mainly in regions with some degree of environmental protection, with some exceptions. A recovery plan for the species should be considered. Species Distribution Modelling cannot substitute long-term monitoring programmes, yet it is a useful tool for identifying appropriate areas of taxon occurrence, and thus allow for efficient use of the economic and human resources.     

Relationships between heterozygosity and fitness in the Iberian threatened larkspur Delphinium bolosii (Ranunculaceae)

The relationship between individual heretozygosity and fitness was explored in the perennial larkspur Delphinium bolosii (Ranunculaceae), an endangered species endemic to Catalonia (North-Eastern Spain), as a part of several studies prior to designing a programme for the reintroduction of this species in a locality where it has been extinct for approximately one century. Allozyme electrophoresis was used to quantify the levels of heterozygosity at nearly to one hundred surveyed individuals in two extant populations. At the same time, eight parameters of vegetative and reproductive fitness were measured. A principal component analysis reduced the original fitness variables to three uncorrelated principal components, two associated with the maternal plant and the other one with the offspring. However, none of the components were significantly correlated with heterozygosity. The low number of variable allozyme markers and the likely influence of ecological factors could be responsible for the lack of correlation between individual heterozygosity and fitness.     

Genetic variation and population structure in Korean endemic species: III.Hosta minor (Liliaceae)

Hosta minor, an insect-pollinated and rhizomatous herbaceous perennial, occurs in eastern and southern Korean Peninsula. AlthoughH. minor is a Korean endemic species and most populations of the species are scattered, the species maintains considerably high levels of genetic variation within the species, with a moderate level of variation (14%) found among populations. Nineteen of the 29 putative loci resolved (66%) were polymorphic within the species, the mean number of alleles per locus was 2.10 across all populations and averaged 1.68 within populations. In addition, genetic diversity was a considerably higher (for species and population level, mean estimates of genetic diversity were 0.275 and 0.230, respectively) than the average for other long-lived herbaceous perennials. Indirect estimate of the number of migrants per generation (Nm=1.03, calculated from mean G_ST) was moderate. Factors contributing to the high levels of genetic diversity found within populations ofH. minor include population maintenance via sexual and asexual modes of reproduction, primarily outcrossing breeding system, long generation time, probable ancient polyploid origin of the species, and moderate levels of seed dispersal by wind. Human disturbance in South Korea such as road and apartment constructions appears to be the major threat to this genetically diverse species.     

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.     

Taxonomical investigations onHelleborus viridis s. l. (Ranunculaceae) in Northern Italy

The taxon commonly namedHelleborus viridis in Lombardy (NW. Italy) differs from both, subsp.viridis and subsp.occidentalis, and is shown to fall within the variation range ofH. odorus subsp.laxus which has been reported so far for NE. Italy only. TrueH. viridis within Italy grows only in the Maritime Alps.     

Genetic diversity of an endangered plant, <Emphasis Type="Italic">Cypripedium macranthos</Emphasis> var. <Emphasis Type="Italic">rebunense</Emphasis> (Orchidaceae): background genetic research for future conservation

Cypripedium macranthos var. rebunense is an endangered plant endemic to Rebun Island, Japan. A proper understanding of genetic diversity is needed when conducting conservation programs for rare and endangered species. We therefore examined the genetic diversity of C. macranthos var. rebunense using allozyme markers with a view to future conservation. Our study revealed that C. macranthos var. rebunense has relatively high genetic diversity (P was 0.62, n _a and n _e were 1.85 and 1.28 respectively, and H _o and H _e were 0.163 and 0.187, respectively) when compared with other plant taxa. The natural habitats of C. macranthos var. rebunense are geographically separated into northern and the southern populations. Disappearance of alleles and increase in homozygosity expected as a result of the bottleneck effect were observed, particularly in the southern populations composed of a small number of plants. As additional negative effects (inbreeding depression and further genetic drift) due to fragmentation are predicted in these populations, the southern populations may show deterioration of genetic diversity in the near future.     

Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Chinese medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae)

Magnolia officinalis subsp. biloba, a traditional Chinese medicinal plant, experienced severe declines in the number of populations and the number of individuals in the late 20th century due to the widespread harvest of the subspecies. A large-scale cultivation program was initiated and cultivated populations rapidly recovered the loss in individual plant numbers, but wild populations remained small as a consequence of cutting. In this study, the levels of genetic variation and genetic structure of seven wild populations and five domestic populations of M. officinalis subsp. biloba were estimated employing an AFLP methodology. The plant exhibited a relatively high level of intra-population genetic diversity (h = 0.208 and H _j = 0.268). The cultivated populations maintained approximately 95% of the variation exhibited in wild populations, indicating a slight genetic bottleneck in the cultivated populations. The analysis of genetic differentiation revealed that most of the AFLP diversity resided within populations both for the wild group (78.22%) and the cultivated group (85.92%). Genetic differentiation among populations in the wild group was significant (F _ST = 0.1092, P < 0.005), suggesting wild population level genetic structure. Principal coordinates analysis (PCO) did not discern among wild and cultivated populations, indicating that alleles from the wild population were maintained in the cultivated gene pool. Results from the present study provide important baseline data for effectively conserving the genetic resources of this medicinal subspecies.     

Molecular confirmation of the hybrid origin of the critically endangered western Mediterranean endemic <Emphasis Type="Italic">Sonchus pustulatus</Emphasis> (Asteraceae: Sonchinae)

The critically endangered composite Sonchus pustulatus Willk. despite being known from fewer than ten locations in southern Spain and northern Africa, has never been characterized in robust phylogenetic context. Here, we report molecular evidence that strongly supports a hybrid origin for S. pustulatus. Although parentage cannot be identified with certainty, analysis of DNA sequence variation from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) supports a phylogenetic placement of S. pustulatus close to other species in the poorly known section Pustulati, whereas examination of chloroplast DNA (cpDNA) places S. pustulatus most closely with species from the sections Sonchus and Asperi. This is one of several instances of topological non-concordance reported for the genus Sonchus. Monophyly of S. pustulatus in both gene genealogies supports the null hypothesis of a single origin, and the relatively large amount of nucleotide substitutions is indicative of an origin in the range of millions of years. A hypothesis of a northern African origin of S. pustulatus followed by dispersal to the Iberian Peninsula during the Messinian salinity crisis/later Quaternary glaciations is proposed on the basis of biogeographic patterns and calibrated estimations of molecular evolution.