Genetic diversity and structure in fragmented populations of the endangered species Ranunculus cabrerensis (Ranunculaceae): implications for conservation

Ranunculus cabrerensis is an endemic and endangered species of the Northwestern Iberian Peninsula. The molecular markers AFLP and ISSR were used to investigate the genetic diversity and population structure of four populations across its known distribution. Fifteen selective primer combinations of AFLP and seventeen ISSR primer combinations produced a total of 2830 and 103 unambiguously repeatable fragments respectively, of which 97.57 and 81.38% were polymorphic for both markers. The genetic diversity of R. cabrerensis at species level was high (H _E = 0.294 by ISSR and H _E = 0.191 by AFLP) and differentiation between sampled locations was also relatively high (G _ST = 0.316 and 0.158 by ISSR and AFLP analysis respectively) compared to other studies of endangered and rare species using the same techniques. The analysis of molecular variance (AMOVA) indicated that the main genetic variation was within sampled locations (73% by AFLP; 52% by ISSR), even though the variation among locations was also significant. Principal Coordinates, NeighborNet and Bayesian analyses revealed a weak but significant relationship between the genetic structures of different populations in R. cabrerensis, with gene flow acting as a homogenizing force that prevents stronger differentiation of populations. Finally, suggestions for conservation strategies to preserve the genetic resources of this species are outlined.     

Genetic diversity in fragmented populations of Berchemiella wilsonii var. pubipetiolata (Rhamnaceae)

BACKGROUD AND AIMS: Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is distributed in fragmented habitat patches in eastern China. It is highly endangered because of severe disturbance by anthropogenic activities. Information on genetic variation and structure is critical for developing successful conservation strategies for this species. METHODS: Allozyme variation of population genetic diversity and structure was investigated for a total of 98 individuals sampled from four extant populations using isoelectric focusing in thin-layer polyacrylamide slab gels. KEY RESULTS: Based on 20 loci scored from the nine enzymes examined, a high genetic diversity was detected at both the species and population level, while there was a loss of low frequency alleles (<0.1) in all populations. Most loci showed deviation from Hardy-Weinberg equilibrium due to excess of heterozygotes in all populations, suggesting that selection for heterozygotes has occurred in this species. The genetic diversity was mainly found within populations with a moderate genetic differentiation (F(ST) = 0.13), but the two geographically discontinuous population groups showed significant differences, with F-statistic values of 0.078 for the Zhejiang populations and 0.014 for the Anhui populations, respectively. CONCLUSIONS: It appears most likely that this species has experienced a recent decrease in population size, and genetic drift in small populations has resulted in a loss of alleles occurring at low frequency. The differentiation into two population groups reflects a population genetic consequence that has been influenced by the different land-use in the two regions. Some conservation concerns are discussed together with possible strategies for implementing in situ and ex situ conservation.     

Genetic diversity in fragmented populations of the critically endangered spider orchid Caladenia huegelii: implications for conservation

The Orchidaceae is characterised by a diverse range of life histories, reproductive strategies and geographic distribution, reflected in a variety of patterns in the population genetic structure of different species. In this study, the genetic diversity and structure was assessed within and among remnant populations of the critically endangered sexually deceptive orchid, Caladenia huegelii. This species has experienced severe recent habitat loss in a landscape marked by ancient patterns of population fragmentation within the Southwest Australian Floristic Region, a global biodiversity hotspot. Using seven polymorphic microsatellite loci, high levels of within-population diversity (mean alleles/locus = 6.73; mean H _E = 0.690), weak genetic structuring among 13 remnant populations (F _ST = 0.047) and a consistent deficit of heterozygotes from Hardy–Weinberg expectation were found across all populations (mean F _IS = 0.22). Positive inbreeding coefficients are most likely due to Wahlund effects and/or inbreeding effects from highly correlated paternity and typically low fruit set. Indirect estimates of gene flow (Nm = 5.09 using F _ST; Nm = 3.12 using the private alleles method) among populations reflects a historical capacity for gene flow through long distance pollen dispersal by sexually deceived wasp pollinators and/or long range dispersal of dust-like orchid seed. However, current levels of gene flow may be impacted by habitat destruction, fragmentation and reduced population size. A genetically divergent population was identified, which should be a high priority for conservation managers. Very weak genetic differentiation indicates that the movement and mixing of seeds from different populations for reintroduction programs should result in minimal negative genetic effects.     

Phylogeography of Camellia taliensis (Theaceae) inferred from chloroplast and nuclear DNA: insights into evolutionary history and conservation

ABSTRACT: BACKGROUND: As one of the most important but seriously endangered wild relatives of the cultivated tea, Camellia taliensis harbors valuable gene resources for tea tree improvement in the future. The knowledge of genetic variation and population structure may provide insights into evolutionary history and germplasm conservation of the species. RESULTS: Here, we sampled 21 natural populations from the species' range in China and performed the phylogeography of C. taliensis by using the nuclear PAL gene fragment and chloroplast rpl32-trnL intergenic spacer. Levels of haplotype diversity and nucleotide diversity detected at rpl32-trnL (h = 0.841; pi = 0.00314) were almost as high as at PAL (h = 0.836; pi = 0.00417). Significant chloroplast DNA population subdivision was detected (GST = 0.988; NST = 0.989), suggesting fairly high genetic differentiation and low levels of recurrent gene flow through seeds among populations. Nested clade phylogeographic analysis of chlorotypes suggests that population genetic structure in C. taliensis has been affected by habitat fragmentation in the past. However, the detection of a moderate nrDNA population subdivision (GST = 0.222; NST = 0.301) provided the evidence of efficient pollen-mediated gene flow among populations and significant phylogeographical structure (NST > GST; P < 0.01). The analysis of PAL haplotypes indicates that phylogeographical pattern of nrDNA haplotypes might be caused by restricted gene flow with isolation by distance, which was also supported by Mantel's test of nrDNA haplotypes (r = 0.234, P < 0.001). We found that chlorotype C1 was fixed in seven populations of Lancang River Region, implying that the Lancang River might have provided a corridor for the long-distance dispersal of the species. CONCLUSIONS: We found that C. taliensis showed fairly high genetic differentiation resulting from restricted gene flow and habitat fragmentation. This phylogeographical study gives us deep insights into population structure of the species and conservation strategies for germplasm sampling and developing in situ conservation of natural populations.     

Genetic structure of the endangered Leucomeris decora (Asteraceae) in China inferred from chloroplast and nuclear DNA markers

The genetic variation and structure of Leucomeris decora, an endangered species in China were investigated. Analyses of three chloroplast DNA (cpDNA) regions (the rpl16 intron, trnQ-5′rps16 intergenic spacer and rpl32-trnL intergenic spacer) and one nuclear gene (GAPDH: encoding glyceraldehyde 3-phosphate dehydrogenase) were conducted on 11 L. decora populations. Low levels of cpDNA genetic diversity were found in this species and within populations, with the identification of 2 haplotypes in a total of 2,745 bp, while the level of genetic diversity revealed by the nuclear gene GAPDH was relatively high, indicating that random losses of genetic polymorphisms from populations may have occurred recently. High levels of genetic differentiation among populations for both markers were detected in L. decora, which could be a consequence of the limited gene flow caused by geographic isolation among populations. An analysis of molecular variance revealed at the nuclear locus suggested the presence of geographic structure within the haplotype distribution possibly due to geographical barriers among populations. The haplotype network and mismatch distribution analyses did not detect the signal for a recent population expansion in L. decora. L. decora may persist in situ during climatic oscillations. Based on the genetic diversity and uniqueness of the populations, conservation strategies are discussed for this endangered species.     

中国板栗自然居群微卫星（SSR）遗传多样性

采用8对微卫星分子标记对中国板栗(Castanea mollissima)的28个自然居群进行了遗传多样性与遗传结构分析。在849个个体上扩增得到128个等位基因, 每位点平均等位基因数(A)为16。中国板栗居群的平均预期杂合度(H_E)为0.678, 平均观察杂合度(H_O)为0.590。华中地区的中国板栗居群遗传多样性最高(A = 8.112, H_E = 0.705, H_O = 0.618), 其次为西北地区和华东地区, 而西南地区遗传多样性最低(A = 6.611, H_E = 0.640, H_O = 0.559)。基于无限等位基因模型(IAM)和基于逐步突变模型(SMM)的遗传分化系数分别为F_ST = 0.120和R_ST = 0.208。分子方差分析(AMOVA)结果表明中国板栗野生居群的遗传变异主要存在于居群内(87.16%)。Mantel检测揭示遗传距离与地理距离之间无显著相关性, 表明基因流不是主导中国板栗居群遗传结构的关键因素。华中地区(尤其是神农架及其周边地区)是中国板栗遗传多样性的现代分布中心, 因而应该得到优先保护, 同时该区域的野生板栗居群可优先作为栽培板栗遗传育种的材料和基因库。     

Phylogenetic relationships in Ephedra (Ephedraceae) inferred from chloroplast and nuclear DNA sequences

Sequences of the nuclear ribosomal DNA internal transcribed spacer region 1 and the chloroplast-encoded genes maturase K and ribulose-1,5 biphosphate carboxylase large subunit were obtained from species of Ephedra (Ephedraceae) representing the geographic range and morphological diversity of the genus. Phylogenetic analyses of the DNA data indicate that relationships within the genus are better predicted by geographic region of origin than by ovulate cone characters. The sampled species with dry, winged (versus fleshy) ovulate cone bracts or single-seeded cones do not form monophyletic groups and therefore the previous classification systems of Ephedra based on these aspects of bract morphology appear to be largely unnatural. Three groups were identified among the Old World species studied, one comprising European and Mediterranean species and two including only Asian species. The sequence data suggest a possible early divergence of a New World clade of Ephedra from among the Old World groups. The South American species form a distinct clade apparently related to one of two groups of North American species, which accords with a frequent floristic pattern of close relationships between species groups in western South America and southwestern North America.     

Preliminary phylogeny of Valerianaceae (Dipsacales) inferred from nuclear and chloroplast DNA sequence data

Valerianaceae is a relatively small (ca. 350 species), but morphologically diverse angiosperm clade. Sequence data from the entire ndhF gene, the trnL-F intergenic spacer region, the trnL intron, the matK region, the rbcL-atpB intergenic spacer region and internal transcribed spacer (ITS) region of nuclear ribosomal DNA were collected for 21 taxa within Dipsacaceae and Valerianaceae (1 and 20, respectively). These data were included in several phylogenetic analyses with previously published sequences from Dipsacales. Results from these analyses (maximum parsimony, maximum likelihood, and Bayesian analysis) are in strong agreement with many of the conclusions from previous studies, most importantly: (1) Valerianaceae is sister to Dipsacaceae; (2) Triplostegia is more closely related to species of Dipsacaceae than to Valerianaceae; and (3) Valeriana appears not to be monophyletic, with Valeriana celtica falling outside the remainder of the species of Valeriana sampled here (with very strong support). With the exception of V. celtica, these data support two major clades within Valeriana; one that is exclusively New World and another that is distributed in both the Old and New World. Although the species of Valerianaceae and its sister group Dipsacaceae plus Triplostegia, are widely distributed in the Northern Hemisphere, and the data imply that Valerianaceae diversified initially in Asia (the Himalayan Patrinia and Nardostachys falling at the base of the clade), the center of modern species diversity for the group is in the Andes of South America with as many as 175 species restricted to that region. Although the exclusively South American taxa form a clade in the chloroplast and combined ITS and chloroplast analyses, support values tend to be low. Future studies will need to include additional data, in the form of both characters and taxa, before any strong conclusions about the character evolution, diversification, and biogeography of the South American valerians can be made.     

Genetic population structure of the alpine species Rhododendron pseudochrysanthum sensu lato (Ericaceae) inferred from chloroplast and nuclear DNA

Background  

A complex of incipient species with different degrees of morphological or ecological differentiation provides an ideal model for studying species divergence. We examined the phylogeography and the evolutionary history of the Rhododendron pseudochrysanthum s. l.     

根据叶绿体ndhF和核核糖体DNA序列推断梓属(紫葳科)的系统发育

Phylogenetics of Chilopsis and Catalpa (Bignoniaceae) was elucidated based on sequences of chloroplast ndhF and the nrDNA ITS region. In Bignoniaceae, Chilopsis and Catalpa are most closely related as sister genera. Our data supported section Macrocatalpa of the West Indies and section Catalpa of eastern Asian and North American continents. Within section Catalpa, Catalpa ovata of eastern Asia form a clade with North American species, C. speciosa and C. bignonioides, while the other eastern Asian species comprise a clade where C. duclouxii is sister to the clade of C. bungei and C. fargesii. The Caribbean species of Catalpa diverged early from the continental species. More studies are needed to test whether the phylogenetic pattern is common in eastern Asian-North American disjunct genera with species in the West Indies.     

Phylogenetics and diversification of Cotyledon (Crassulaceae) inferred from nuclear and chloroplast DNA sequence data

Crassulaceae includes approximately 35 genera and 1500 species of leaf and stem succulent flowering plants. The family is nearly cosmopolitan in distribution, but is particularly diverse in southern Africa, where five genera comprising approximately 325 species are found. One of these genera, Cotyledon, includes 10 species that are largely confined to South Africa, where they are commonly found on rocky hillsides, coastal flats, and cliff faces. Species of Cotyledon are characterized by five-parted, pendulous, sympetalous flowers, but the genus is highly diverse in growth form, flower color and size, and leaf morphology. One particularly variable species, C. orbiculata, has been divided into five varieties based on leaf morphology and biogeography; however, the monophyly of this species as well as the relationships among the varieties have not previously been investigated. Parsimony analyses of a combined data set of DNA sequences from chloroplast and nuclear genome provided the first estimate of phylogeny for Cotyledon, and resulted in two minimum-length trees and a fully resolved phylogeny for the genus. Results indicate that C. orbiculata is not monophyletic and suggest the need for additional studies and a revised classification within the genus.     

Genetic diversity analysis and conservation implications for the Iberian threatened populations of the irano-turanian relict Krascheninnikovia ceratoides (Chenopodiaceae)

Krascheninnikovia ceratoides (L.) Gueldenst. (Chenopodiaceae) is an irano‐turanian steppe plant with a striking geographical vicariance between the western Mediterranean and the central Asian regions. Its westernmost populations are located in the north‐east part of the Iberian Peninsula, in the narrow middle Ebro and Alfambra valleys, where they are threatened and have been catalogued as ‘vulnerable’. Genetic diversity and structure was studied in 150 individuals from five populations of these two valleys using inter‐simple sequence repeats (ISSR). A total of 121 informative bands allowed the identification of 150 genotypes. Nei's genetic diversity found in all natural populations (h = 0.448) was higher than that of other geographical restricted species. Analysis of molecular variance and spatial correlations analyses showed a strong genetic differentiation among populations (35.88%) and among valleys (22.33%). The unweighted pair‐group method of arithmetical averages and Neighbour‐joining clusterings further demonstrated a substructure of two population cores within the Ebro valley. Our genetic data suggest that the high levels of genetic diversity and the strong genetic structure found among the northern Iberian populations and ranges of K. ceratoides might result from the summed effects of its tetraploidy, its outbreeding nature, and its success in colonizing newly altered areas. However, the ISSR data might also reflect the existence of an ancient wider distribution range of the plant in the Iberian Peninsula coupled with a more recent history of habitat fragmentation. Different management conservation guidelines are recommended for this plant after our genetic analysis: a microreserve of approximately 2 km² is proposed for the highly threatened population of Osera (Ebro valley), whereas seed collection strategies and occasional population reinforcements are suggested for the remaining populations. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92 , 419–429.     

Phylogeny of Rhaponticum (Asteraceae, Cardueae-Centaureinae) and related genera inferred from nuclear and chloroplast DNA sequence data: taxonomic and biogeographic implications

BACKGROUND AND AIMS: The precise generic delimitation of the Rhaponticum group is not totally resolved. The lack of knowledge of the relationships between the basal genera of Centaureinae could imply that genera whose position is as yet unresolved could belong to the Rhaponticum group. On the other hand, the affinities among the genera that are considered as members of this group are not well known. The aim of the study is to contribute to the phylogenetic and generic delineation of the Rhaponticum group on the basis of molecular data. METHODS: Parsimony and Bayesian analyses of the combined sequences of one plastid (trnL-trnF) and two nuclear (ITS region and ETS) molecular markers were carried out. The results of these analyses are discussed in the light of the biogeographic history. KEY RESULTS: The Rhaponticum group appears as monophyletic, and closely related to the genus Klasea. The results confirm the preliminary generic delimitation of the Rhaponticum group, with the new incorporation of the genus Centaurothamnus. Ochrocephala is supported as a separate genus from Rhaponticum and, contrary to this, Acroptilon and Leuzea appear as merged into the genus Rhaponticum. Several nomenclatural rearrangements are made in Klasea and Rhaponticum. CONCLUSIONS: The new molecular evidence is consistent with the morphological and karyological data, and suggests particularly coherent biogeographic routes of migration and speciation processes for the genus Rhaponticum. The biogeographic inference proposes a Near East and/or Caucasian origin for the genus. Furthermore, representatives of Rhaponticum could have reached Europe in two different ways: (1) expansion across central Asia to eastern Europe, and (2) expansion through the Near East, North Africa and then to the Iberian Peninsula and the Alps.     

Genetic diversity of the tropical tree Terminalia amazonia (Combretaceae) in naturally fragmented populations

The effect of long-term fragmentation on the genetic diversity of populations of the neotropical tree species, Terminalia amazonia, was studied using random amplified polymorphic DNA (RAPD) analysis. Leaf material from 104 trees was collected from three naturally fragmented gallery forest patches and three plots in nearby continuous forest in the Mountain Pine Ridge, Belize. In total, 30 RAPD bands generated by five decamer primers were used to compare the genetic diversity of the six populations in the two groups. Genetic variation within the populations (H0), as estimated by the Shannon diversity index, ranged from 0.32 to 0.38, with an overall diversity of 0.38 (Hspecies). Analysis of molecular variation revealed that most (94.4%, P<0.001) of the variation was attributable to differences among individuals within populations. Population differentiation was significantly (P=0.038) lower among the fragmented populations than among continuous forest populations. On average, the fragmented populations also had slightly, but statistically significant (P=0.046) lower levels of genetic diversity. However, one gallery forest site had a higher level of genetic diversity than two of the continuous forest sites. We suggest that the long-term effect of fragmentation on the genetic diversity of tropical trees will depend upon the amount of local forest cover in proximity to the fragmented populations.     

Phylogenetic patterns in the genus Manihot (Euphorbiaceae) inferred from analyses of nuclear and chloroplast DNA regions

From a phylogenetic perspective, the genus Manihot can be considered as an orphan group of plants, and the scientific knowledge acquired has been mainly related to cassava, one of the most important crops in poor tropical countries. The goal of the majority of evolutionary studies in the genus has been to decipher the domestication process and identify the closest relatives of cassava. Few investigations have focused on wild Manihot species, and the phylogeny of the genus is still unclear. In this study the DNA sequence variation from two chloroplast regions, the nuclear DNA gene G3pdh and two nuclear sequences derived from the 3'-end of two cassava ESTs, were used in order to infer the phylogenetic relationships among a subset of wild Manihot species, including two species from Cnidoscolus as out-groups. Maximum parsimony and Bayesian analyses were conducted for each data set and for a combined matrix due to the low variation of each region when analyzed independently. A penalized likelihood analysis of the chloroplast region trnL-trnF, calibrated with various age estimates for genera in the Euphorbiaceae extracted from the literature was used to determine the ages of origin and diversification of the genus. The two Mesoamerican species sampled form a well-defined clade. The South American species can be grouped into clades of varying size, but the relationships amongst them cannot be established with the data available. The age of the crown node of Manihot was estimated at 6.6 million years ago. Manihot esculenta varieties do not form a monophyletic group that is consistent with the possibility of multiple introgressions of genes from other wild species. The low levels of variation observed in the DNA regions sampled suggest a recent and explosive diversification of the genus, which is confirmed by our age estimates.     

Monophyly and relationships of the tribe Exaceae (Gentianaceae) inferred from nuclear ribosomal and chloroplast DNA sequences

Both chloroplast trnL (UAA) intron and nuclear ribosomal ITS sequences highly confirmed the monophyly of the tribes of the Gentianaceae defined by the recent classification, and revealed the tribe Exaceae as a basal clade just next to the basal-most lineage, the tribe Saccifolieae. Within the tribe Exaceae, Sebaea (except Sebaea madagascariensis) appeared as the most basal clade as the sister group to the rest of the tribe. The Madagascan endemic genera Gentianothamnus and Tachiadenus were very closely related to each other, together standing as sister to a clade comprising Sebaea madagascariensis, Ornichia, and Exacum. The saprophytic genus Cotylanthera nested deeply inside Exacum. Sebaea madagascariensis was shown closer to the Madagascan endemic genus Ornichia than to any other sampled Sebaea species. Exacum appeared as the most derived taxon within this tribe. The topology of the phylogenetic trees conform with the Gondwana vicariance hypothesis regarding the biogeography of Exaceae. However, no evidence for matching the older relationships within the family to the tectonic history could be corroborated with various divergence time analyses. Divergence dating estimated a post-Gondwana diverging of the Gentianaceae about 50 million years ago (MYA), and the tribe Exaceae as about 40 MYA. The Mozambique Channel land-bridge could have played an important role in the biogeographic history of the tribe Exaceae.     

Genetic diversity of Chinese water deer (Hydropotes inermis inermis): implications for conservation

The Chinese water deer (Hydropotes inermis inermis) is endemic to China. Historically, the species was widely distributed, but now, habitat loss and poaching have reduced its range and number drastically. In order to provide useful information for its conservation, we have investigated the genetic diversity and population structure of the Chinese water deer by analyzing the 403 bp fragment of the mitochondrial DNA (mtDNA) control region (D-loop). Eighteen different haplotypes were detected in 40 samples. Overall, Chinese water deer have a relatively high-genetic diversity compared to other rare cervid species, with a haplotype diversity of 0.923+/-0.025 and nucleotide diversity of 1.318 +/- 0.146%. No obvious phylogenetic structure among haplotypes was found for samples of different origin. An analysis of molecular variance (AMOVA) showed significant differentiation between the Zhoushan and the mainland population (F(ST)= 0.088, P < 0.001; Phi( ST ) = 0.075, P = 0.043), which suggests that exchanges of individuals between Zhoushan and the mainland should be avoided. We also recommend that a breeding center be set up for the mainland population.     

Genetic diversity of North American populations of Cristatella mucedo, inferred from microsatellite and mitochondrial DNA

Research over the past 20 years has shown, with the help of molecular markers, that the population genetics and distribution patterns of freshwater invertebrates in North America are often more complex than was previously believed. Here we extend this research to an, as yet, unstudied but widespread and common group, the freshwater bryozoans. Colonies of the bryozoan Cristatella mucedo were collected from a number of lakes across central North America, and were characterized genetically by analysis of microsatellite loci and mitochondrial DNA (mtDNA) cytochrome b sequences. The microsatellites illustrate a pattern of generally diverse and highly differentiated populations that contain little evidence of recent gene flow. The mtDNA sequences yielded highly variable levels of divergence, ranging from 0.0 to 8.8% within populations, and 0.0 to 9.8% among populations. The multiple divergent mtDNA lineages within populations provide evidence for repeated colonization events. The lack of clustering of haplotypes by site suggests that there has been widespread dispersal of multiple genetic lineages since the last ice age. While some of the haplotype lineages may have evolved in disjunct glacial refugia, the maximum levels of divergence predate the time since the last glacial-interglacial cycles. It is likely that multiple factors including vicariance events, patterns of dispersal, localized extinction, and an unusual life history, explain the unique phylogeographic patterns evident today in populations of C. mucedo.