Nucleotide diversity and demographic history of Pinus bungeana,an endangered conifer species endemic in China

Orographic and climatic oscillations have played crucial roles in shaping the nucleotide diversity and evolutionary history of many species across the Northern Hemisphere. In this study, based on 10 nuclear loci and a chloroplast DNA marker, we analyzed the nucleotide polymorphisms and demographic history of the endangered conifer species Pinus bungeana in Northwest China and investigated the phylogenetic relationships between P. bungeana and two related species, that is, Pinus gerardiana and Pinus squamata. We found that P. bungeana exhibited an extremely low level of nucleotide diversity (π_sil = 0.00159). Demographic simulations based on DIYABC analysis showed that P. bungeana underwent demographic expansion and contraction during the Miocene. According to ecological niche modeling, we found that this species survived in situ during the glacial period and was not restricted to southern refugia. We speculate that P. bungeana may have experienced widespread population shrinkage from the Last Interglacial to the Last Glacial Maximum due to geological or climatic events. Isolation‐with‐migration analysis revealed that the divergence (~2.4–4.2 Ma) among P. bungeana and its related species was significantly associated with the Qinghai–Tibetan Plateau uplift events in the mid‐to‐late Tertiary period. Species tree analyses suggested that these three related Pinus species formed a monophyletic clade with high bootstrap support. These results suggest that the Miocene–Pliocene and Pleistocene geological and climatic fluctuations might have profoundly affected the nucleotide diversity and demography of this psychrotolerant conifer species in western China.     

Genetic diversity of a dominant species Stipa bungeana and its conservation strategy in the Loess Plateau of China

In order to investigate the genetic diversity and population structure of Stipa bungeana under the background of grassland utilization and grazing exclusion, ten S. bungeana populations were selected from different steppe type in the Loess Plateau of China. Sequence-related amplified polymorphism (SRAP) marker was used to assess the genetic diversity. Fifteen SRAP primer combinations generated a total of 482 amplification bands, 418 (86.72%) were polymorphic bands. A relatively high level of genetic diversity (PPB = 89.80%, h = 0.1972, H = 0.3154) was detected at the species level, but the genetic diversity was low at the population level (PPB = 17.01–33.33%, h = 0.0438–0.0967, H = 0.0361–0.1447). AMOVA analysis revealed a high level of genetic differentiation among populations (Φ_ST = 0.6757), and a limited among-population gene flow (N_m = 0.1200). There was no significant correlation between genetic distance and geographic distance by Mantel test (r = 0.1126, P = 0.204). Conservation implications were proposed for S. bungeana.     

High genetic similarity between Polish and North European Scots pine (Pinus sylvestris L.) populations at nuclear gene loci

Nucleotide polymorphisms in a set of 32 nuclear genes were studied in 19 mountain, peatbog and lowland populations of Scots pine representing known phenotypic races and populations of presumably relict character for the species in Poland. At 29 genes, the pattern of genetic variation was compared to 11 reference populations from Northern, Western and Southern Europe. Similar levels of nucleotide polymorphism and excess of low-frequency mutations were observed in Polish populations (π _tot?=?0.0055, D?=??0.308) and as compared to the reference samples (π _tot?=?0.0054, D?=??0.170). Bayesian assignment and conventional frequency-based statistics indicate that Polish populations share the same genetic background at the analysed nuclear gene markers. However, the populations showed a much closer genetic relationship with North European samples than other regional groups of populations. Across the very uniform genetic background of the populations, we identified several genes with outlier patterns of haplotype, polymorphism frequency variation and departures from compound neutrality tests. Our data indicate that the Central and North European parts of the Scots pine distribution seem particularly suitable for association genetic studies to link phenotypic and genetic variation at a large geographical scale.     

Genetic diversity and population structure of endangered endemic Paeonia jishanensis in China and conservation implications

Paeonia jishanensis, one of the most important ancestral species of cultivated tree peonies, is an endangered ornamental and medicinal plant endemic to China. A total of 236 individuals of P. jishanensis from 10 extant populations were analysed using 21 EST-SSR markers to assess their genetic diversity and population structure. Moderate genetic diversity levels (H_E = 0.340) and high genetic differentiation among populations (F_ST = 0.335) were revealed. Combining the results of STRUCTURE, PCoA and neighbour-joining analyses, the 10 populations were divided into four genetic groups that were significantly related to their geographical origins, which was further supported by hierarchical AMOVA showing the highest variation of 17.9% among groups. The Mantel test showed a significant positive correlation between genetic distance and geographic distance among populations (r = 0.873, P < 0.0001). The genetic structure of P. jishanensis may be due to limited gene flow hindered by vicariance and its breeding system: facultative vegetative reproduction. This study carries significant implications for the conservation and utilization of this endangered species.     

Genetic diversity and structure of Pinus dabeshanensis revealed by expressed sequence tag-simple sequence repeat (EST-SSR) markers

Assessing patterns of genetic variation in rare endangered species is critical for developing both in situ and ex situ conservation strategies. Pinus dabeshanensis Cheng et Law is an endangered species endemic to the Dabieshan Mountains of eastern China. To obtain fundamental information of genetic diversity, population history, effective population size, and gene flow in this species, we explored patterns of genetic variation of natural populations, in addition to an ex situ conserved population, using expressed sequence tag-simple sequence repeats (EST-SSR) markers. Our results revealed moderate levels of genetic diversity (e.g., H_E = 0.458 vs. H_E = 0.423) and a low level of genetic differentiation (F_ST = 0.028) among natural and conserved populations relative to other conifers. Both contemporary and historical migration rates among populations were high. Bayesian coalescent-based analyses suggested that 3 populations underwent reductions in population size ca. 10,000 yr ago, and that two populations may have experienced recent genetic bottlenecks under the TPM. Bayesian clustering revealed that individuals from the ex situ population were largely assigned to the ‘red’ cluster. Additionally, our results identified private alleles in the natural populations but not in the ex situ population, suggesting that the ex situ conserved population insufficiently represents the genetic diversity present in the species. Past decline in population size is likely to be due to Holocene climate change. Based on the genetic information obtained for P. dabeshanensis, we propose some suggestions for the conservation and efficient management of this endangered species.     

Patterns of genetic variation in the Chinese endemic Psilopeganum sinense (Rutaceae) as revealed by nuclear microsatellites and chloroplast microsatellites

Psilopeganum sinense is a perennial herb endemic to Three-Gorges Reservoir Area (TGRA) in China. Genetic diversity of this endangered species was assessed by using 11 nuclear microsatellites and three chloroplast microsatellite (cpSSR) markers. A total of 8 haplotypes were identified in a survey of 212 individuals sampled from nine populations encompassing most of the natural range of the species. A low level of genetic diversity was detected: H_E = 0.301 for SSR and H_E = 0.28 for cpSSR. Populations were highly differentiated from one another: an AMOVA analysis that showed that 56.3% and 68.2% genetic variation resided between populations based on SSR and cpSSR analysis, respectively, and F_ST and F_STc (0.467 for SSR and 0.644 for cpSSR, respectively) were high. Significant differences were found between estimates of haplotypic differentiation calculated by using unordered alleles (G_STc = 0.857) and ordered alleles (N_STc = 0.728), which indicated the existence of phylogeographical structure in P. sinense. The indirect ratio of pollen flow/seed flow derived from estimates of haplotypic and nuclear DNA differentiation indicated that gene flow via pollen is less efficient than via seed. Two distinct evolutionary lineages (evolutionary significant units, ESUs) were recognized for P. sinense on the basis of both the PCoA and NCA analysis. Sampling strategies for conserving this endangered plant were discussed.     

Genetic diversity of the endangered Chinese endemic plant Monimopetalum chinense revealed by amplified fragment length polymorphism (AFLP)

Monimopetalum chinense Rehd. is an endangered woody vine endemic to eastern China. Using amplified fragment length polymorphism (AFLP) markers, we examined levels of genetic variation within and among eleven populations located across the species’ distribution. Although modest levels of heterozygosity were detected, other measures of genetic diversity registered relatively high levels of variability, both at the species level (P = 91.0%, H_E = 0.232, I_S = 0.365) and at the population level (P = 53.0%, H_E = 0.155, I_S = 0.239). Populations also exhibited high levels of genetic differentiation (Nei’s genetic diversity analysis, G_ST = 0.330), corresponding to isolation-by-distance and hierarchical population structure. These results indicate that, despite low levels of gene flow, populations of M. chinense still harbor substantial amounts of genetic diversity. Management plans for the species should include measures that ensure genetic diversity remains high within and among extant populations.     

The value of small habitat islands for the conservation of genetic variability in a steppe grass species

The habitat loss and fragmentation due to agricultural land-conversion affected the steppe throughout its range. In Ukraine, 95% of steppe was destroyed in the last two centuries. Remaining populations are confined to few refuges, like nature reserves, loess ravines, and kurgans (small burial mounds), the latter being often subject to destruction by archeological excavations.Stipa capillata L. is a typical grass species of Eurasian steppes and extrazonal dry grasslands, that was previously used as a model species in studies on steppe ecology. The aim of our research was to assess genetic diversity of S. capillata populations within different types of steppe refuges (loess ravines, biosphere reserve, kurgan) and to evaluate the value of the latter group for the preservation of genetic diversity in the study species.We assessed genetic diversity of 266 individuals from 15 populations (nine from kurgans, three from loess ravines and three from Askania-Nova Biosphere Reserve) with eight Universal Rice Primers (URPs).Studied populations showed high intra-population variability (I: 0.262–0.419, PPB: 52.08–82.64%). Populations from kurgans showed higher genetic differentiation (Φ_ST = 0.247) than those from loess ravines (Φ_ST = 0.120) and the biosphere reserve (Φ_ST = 0.142). Although the diversity metrics were to a small extent lower for populations from kurgans than from larger refugia we conclude that all studied populations of the species still preserve high genetic variability and are valuable for protection. To what extent this pattern holds true under continuous fragmentation in the future must be carefully monitored.     

Population genetics and conservation of the Azorean tree Picconia azorica

Picconia azorica (Tutin) Knobl. (Oleaceae) is an endangered species, endemic to the Azores. Samples from 31 populations in 8 islands were genotyped using 8 newly developed nuclear microsatellite markers. From the amplified loci, 81% were polymorphic across all populations and the species showed a relatively high total genetic diversity (H_T = 0.7). Several populations were close to Hardy–Weinberg equilibrium while others presented positive F_IS values (0.02–0.2). The largest proportion of genetic variation (98%) occurred within populations and the level of differentiation between populations, was generally low, although 27% of the population pairwise comparisons showed relatively high differentiation values (0.25 ≤ R_ST ≤ 0.65). Relatively high levels of gene flow were also found among most populations. Using the Bayesian clustering method implemented in STRUCTURE we found a particular genetic pattern in Corvo samples, and also similarities between Santa Maria, São Miguel and Flores populations. Considerable levels of genetic admixture within P. azorica populations might have resulted from: (i) fruit dispersal by native birds; and/or (ii) human mediated dispersal between islands. Our results revealed the existence of some genetically depauperate populations needing specific conservation measures, and indicate that arbitrary translocation of individuals between islands should be avoided.     

Genetic diversity within and among populations of the endangered and endemic species Primula merrilliana in China

Primula merrilliana Schltr. is an endangered and narrowly-distributed endemic species of southern Anhui Province in China. In this study, the level of genetic variation and the pattern of genetic structure in six natural populations of P. merrilliana were assessed by using ISSR (inter-simple sequence repeats) markers. Based on ten primers, 137 clear and reproducible DNA fragments were generated, of which 109 were polymorphic. The statistical results indicated that there was a relatively low genetic diversity within populations, and a high genetic differentiation among populations (G_ST = 0.53, Φ_ST = 0.49). The level of population genetic diversity was correlated to habitat type and the gene flow (N_m) was low with only 0.45. The unexpected genetic structure of P. merrilliana may be explained by limited gene flow that was caused by habitat fragmentation and limited seeds and pollen dispersal ability, self-compatible breeding system and biennial life form.     

Highly differentiated populations of the narrow endemic and endangered species Primula cicutariifolia in China,revealed by ISSR and SSR

The perennial herb Primula cicutariifolia Pax is an endangered and endemic species with narrow distribution in eastern Anhui and Zhejiang provinces of China. In this study, the levels of genetic variation and the pattern of genetic structure in five natural populations of P. cicutariifolia were assessed by inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) markers. Both markers revealed that there was remarkably low genetic variation within populations (e.g., H_e = 0.19 and 0.18, for ISSR and SSR respectively) and high differentiation among populations (G_ST = 0.714 and 0.611; Φ_ST = 0.698 and 0.599, for ISSR and SSR respectively). The level of population genetic diversity was correlated to population size only detected by ISSR markers. The genetic structure of P. cicutariifolia may be explained by limited gene flow that was caused by habitat fragmentation and limited seeds and pollen dispersal ability, self breeding system and biennial life form. To protect and avoid disappearance of P. cicutariifolia, much more attentions should be paid to protect all the remnant populations and their habitats, and three management units, i.e. Tianmushan, Damodao, and Panan units, were proposed.     

Genetic diversity in endangered Notopterygium forbesii Boissieu based on intraspecies sequence variation of chloroplast DNA and implications for conservation

A thorough understanding of the levels and partitioning of genetic variation across populations and geographical regions of endangered species is a prerequisite to ensure effective conservation and/or restoration activities. Here, we examined chloroplast DNA (cpDNA) trnH-psbA intergenic spacer sequences variation within Notopterygium forbesii, an endangered and endemic perennial herb in China. Sequence data obtained from 141 individuals in 14 populations revealed twenty-two haplotypes. A high level of haplotype diversity (Hd = 0.81) and low level of nucleotide diversity (Pi = 0.0047) were detected. Low genetic differentiation among populations and also among regions was consistently indicated by both hierarchical analyses of molecular variance (AMOVA) and the structure of a neighbor-joining tree. Low level of population differentiation between populations or between regions in cpDNA sequences may be due to effects of the abundance of ancestral haplotype sharing and the high number of private haplotypes fixed for each population. Based on our results, we proposed some conservation strategies.     

Population genetic structure of Sagittaria natans (Alismataceae), an endangered species in China,revealed by nuclear SSR loci analyses

Sagittaria natans (Alismataceae) is an endangered aquatic herb in China. The nuclear microsatellite (SSR) loci variation of the 11 extant populations of S. natans was investigated in the present study. A relative high level of genetic diversity was found in this species. The Wilcoxon’s signed rank tests did not indicate a recent bottleneck in any of the populations. The Analysis of molecular variance (AMOVA) showed a low level of population differentiation (FST = 0.284) among the populations of S. natans. This result was supported by STRUCTURE analysis (K = 1), the NJ analyses and Mantel test (r = ?0.0221, p > 0.5). The genetic structure could be contributed to an earlier period of more pronounced gene flow when the species had a more continuous distribution. The results of this study could be used as the basis for conservation guidelines for the management of this endangered species in China.     

Nucleotide divergence and genetic relationships of Pseudoroegneria species

Nucleotide variation in 8 diploid Pseudoroegneria species was characterized using two single copy nuclear genes, the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor G (EF-G), and one chloroplast TrnD/T intergenic region. Among the Pseudoroegneria species studied, the estimates of nucleotide diversity (π) ranged from 0.04577 (RPB2) and 0.00183 (TrnD/T) for Pseudoroegneria tauri to 0.10667 (RPB2), 0.06174 (EF-G) and 0.03743 (TrnD/T) for Pseudoroegneria spicata. The highest nucleotide diversity of the RPB2 data set was found for P. spicata among the taxa analyzed. Our phylogenetic analyses separated the accessions of P. spicata into several groups, with 7 accessions of P. spicata forming a highly supported subclade (BS = 100%, PP = 1.00). The phylogenetic analysis also suggests that P. spicata, Pseudoroegneria gracillima and Pseudoroegneria stipifolia have a closer relationship than the other species within Pseudoroegneria. Pseudoroegneria libanotica and P. tauri were also found to exhibit a high level of sequence homology, however, only nuclear gene data (RPB2 and EF-G) clearly indicated the differentiation between the P. libanotica + P. tauri group with other St genome species.     

Migration patterns counteract seasonal isolation of <Emphasis Type="Italic">Squalius torgalensis</Emphasis>, a critically endangered freshwater fish inhabiting a typical Circum-Mediterranean small drainage

Freshwater species with small distribution ranges are vulnerable to extinction, especially when they exhibit small effective population sizes. Squalius torgalensis, Cyprinidae, is an endemic critically endangered fish species from southwestern Portugal, confined to the intermittent streams of the Mira drainage. Assessment of genetic diversity and population structure levels was conducted through the analysis of both mitochondrial (cytochrome b) and nine nuclear microsatellite markers. Remarkable low levels of genetic diversity for both markers were observed (He < 0.38, π < 0.001). These results may be related with the restricted distribution range and dispersion patterns of the species together with demographic fluctuations associated with the intermittent features of the river system. Population structure analyses suggest the existence of two incipient divergent populations; one comprising the tributaries located upstream and the other comprising downstream tributaries of the Mira River. Data also supports the occurrence of high gene flow mainly from downstream to upstream, failing to reflect isolation of populations due to seasonal drought. Obtained results should be incorporated in future management plans for the species. These should be centred around the current connection between populations and on the maintenance of suitable habitat patches, not only for reproduction, but also for summer refuges.     

Genetic diversity of the endangered Chinese endemic herb Dayaoshania cotinifolia (Gesneriaceae) revealed by simple sequence repeat (SSR) markers

Dayaoshania cotinifolia W.T. Wang is a critically endangered perennial herb endemic to southern China. Simple sequence repeat (SSR) markers were employed to assess genetic diversity in two populations of D. cotinifolia. Eight primer pairs generated a total of 36 alleles, with a mean of 4.5 alleles per locus. The expected heterozygosity (H_e = 0.416) and observed heterozygosity (H_o = 0.508) indicated a moderate level of genetic diversity, though the genetic differentiation between the populations was low (F_st = 0.014), a result that was supported by a higher gene flow (N_m = 18.000). No severe bottleneck effect was detected in the two populations. Thus, the endangered status of this species is most likely due to anthropologic effects rather than a lack of genetic diversity. In situ conservation strategies should be promoted, and the sizes of the populations should be increased through artificial breeding.     

High allozymic diversity in natural populations of Mycoheterotrophic Orchid Gastrodia elata,an endangered medicinal plant in China

Gastrodia elata Blume, a mycoheterotrophic orchid native to the Far East, is an endangered medicinal plant in China. Genetic variation among 19 natural populations of G. elata was examined in central China by using allozyme polymorphism (16 loci in six enzymatic systems). The species exhibited high level of genetic diversity (P = 56.3%, A = 2.2 and H_E = 0.221), which was mainly attributed to its perennial habit and mixed reproduction system (both sexual and asexual). Evident genetic differentiation in G. elata natural populations was suggested by F_ST = 0.241. AMOVA analysis showed 31.3% of the total molecular variation was attributed to inter-population differentiation. Obvious genetic structure and genetic depauperation of some populations indicate forest fragmentation and over-collection have affected genetic variation of G. elata. A conservation strategy, which is conserving populations with great genetic distinction or high level of genetic variation from four management units, is recommended.     

Twelve polymorphic expressed sequence tags‐derived markers for Plasmopara halstedii,the causal agent of sunflower downy mildew

Twelve expressed sequence tags‐derived markers were isolated from Plasmopara halstedii (Oomycetes), the causal agent of sunflower downy mildew. A total of 25 single nucleotide polymorphisms and five indels were detected by single‐strand conformation polymorphism analysis and developed for high‐throughput genotyping of 32 isolates. There was a high level of genetic diversity (H_E = 0.484). Observed heterozygosity ranged from 0 to 0.143 indicating that P. halstedii is probably a selfing species. These markers were also useful in detecting significant genetic variations among French populations (F_ST = 0.193) and between French and Russian populations (F_ST = 0.23). Cross‐amplification tests on three closely related species indicated that no loci amplified in other Oomycete species.     

High genetic diversity but limited gene flow among remnant and fragmented natural populations of Liriodendron chinense Sarg

As a relic species, Liriodendron chinense is now recognized as an endangered species. To better understand the genetic structure and differentiation among remnant populations of L. chinense, we determined the genotypes of 14 simple sequence repeats (SSRs) loci across 318 individuals from 12 natural populations and 750 seedlings from five offspring populations. We found that L. chinense maintained high genetic diversity (H_e = 0.7385) within populations but moderate genetic differentiation (F_st = 0.1956) and low gene flow (N_m = 1.0283) between populations. The genetic diversity was slightly lower for offspring populations than for their corresponding natural populations. Moreover, using a two-phased model of mutation (TPM), we demonstrated that significant bottlenecks had occurred in six populations. A Mantel test revealed a statistically significant correlation between the geographic distances and genetic distances between populations (r = 0.5011, P = 0.002). Hence, we presume that geographical isolation and habitat fragmentation might contribute jointly to current population structure of L. chinense. We suggest that populations from southern Yunnan can be regarded as a variety of L. chinense, given their large deviation from other populations. Our findings may be of value for the conservation and use of L. chinense.     

A rapid,efficient method for the mass production of pollen protoplasts from Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast.

An optimized protocol was established to isolate large numbers of mature living pollen protoplasts of Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast. Intact pollen grains of P. bungeana or pollen with short tubes were incubated with gentle agitation in a solution of 2% cellulase R-10, 1.5% macerozyme R-10, 15% sucrose, 0.01% H₃BO₃, and 0.01% CaCl₂. Intact pollen protoplasts with diameters of 40 μm were liberated, with an isolation rate of up to 70% after 6 h of enzymatic incubation. The optimal pH and temperature for the reaction were 5.8 and 24 °C, respectively, and the optimal enzymatic digestion conditions were 6 h of incubation in the above solution. The method for isolating pollen protoplasts from P. wilsonii was similar to that for P. bungeana, except that the incubation medium contained 12% rather than 15% sucrose and the optimal enzyme concentrations were 3% cellulase and 2% macerozyme. The isolated pollen protoplasts were demonstrated to be living by microscopy in a fluorochromatic reaction with fluorescein diacetate (FDA).