De novo assembly and characterization of transcriptome using Illumina sequencing and development of twenty five microsatellite markers for an endemic tree Juglans hopeiensis Hu in China

The Chinese walnut (Juglans hopeiensis Hu) is an endemic temperate tree species and narrowly distributed in China. However, there are still few specific molecular markers for understanding genetic diversity of this walnut. In this study, more than 44 million sequencing reads were generated using Illumina sequencing technology. De novo assembly yielded 93,822 unigenes with an average length of 731 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.3%) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 15,903 (17.0%) unigenes. To contribute to its conservation and management, twenty five microsatellite markers were identified in J. hopeiensis were screened for polymorphism across 27 Chinese walnut tree individuals from two locations. The number of alleles ranged from two to nine, observed heterozygosity ranged from 0.016 to 0.933 (mean 0.468), and expected heterozygosity from 0.022 to 0.823 (mean 0.462). The polymorphic information content (PIC) ranged from 0.012 to 0.831 (mean 0.437). The development of these new microsatellite markers will be useful for studying population genetic structure, evolutionary ecology, conservation, and genetic breeding of this endemic walnut tree or other Juglans 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.     

Genetic diversity in Mentha cervina based on morphological traits,essential oils profile and ISSRs markers

Morphological, phytochemical and genetic differences were studied to evaluate the level and distribution of diversity in twelve populations of the Portuguese endangered medicinal plant Mentha cervina L. Morphological variation was correlated with ecological conditions at the site of origin. Pulegone was the major essential oils compound in all of the populations collected at full flowering (68–83%), in different growing conditions (51–82%), and for all the developmental stages studied (47–82%). Although clusters were defined, the analysis revealed a high chemical correlation among all populations (S_corr ≥ 0.95%). Inter-simple sequence repeats markers were used to assess the population structure and genetic variation. Populations exhibited a relatively low genetic diversity (PPB = 14.3–64.6%, H_e = 0.051–0.222, I = 0.076–0.332), with high structuring between them (G_ST = 0.51). However, the genetic diversity at species level was relatively high (PPB = 97.7%; H_e = 0.320). The levels and patterns of genetic diversity were assumed to result largely from a combination of evolutionary history and its unique biological traits, such as breeding system, clonal growth, low capacity of dispersion and habitat fragmentation. The relatively low genetic diversity in the populations analyzed indicates that the maintenance of their evolutionary potential is at risk if population sizes are maintained and if there is no protection of the habitats.     

Low genetic diversity and significant structuring in the endangered Mentha cervina populations and its implications for conservation

Eighteen populations of the endangered aromatic and medicinal plant Mentha cervina (Lamiaceae) were sampled across its natural range, in the western half of the Iberian Peninsula, and inter-simple sequence repeats (ISSRs) markers were used to assess genetic diversity and population structure. M. cervina populations exhibited a relatively low genetic diversity (percentage of polymorphic loci PPB = 14.2–58.3%, Nei's genetic diversity H_e = 0.135–0.205, Shannon's information index I = 0.08 − 0.33). However, the genetic diversity at species level was relatively high (PPB = 98.3%; H_e = 0.325; I = 0.23). The results of the analysis of molecular variance indicated very structured populations, with 50% of the variance within populations, 44% among populations and 6% between regions defined by hydrographic basins, in line with the gene differentiation coefficient (G_ST = 0.532). A Mantel test did not find significant correlation between genetic and geographic distance matrices (r = 0.064), indicating that isolation by distance is not shaping the present genetic structure. The levels and patterns of genetic diversity in M. cervina populations were assumed to result largely from a combination of evolutionary history and its unique biological traits, such as breeding system, low capacity of dispersion, small effective size and habitat fragmentation. The high genetic differentiation among populations indicates the necessity of conserving the maximum possible number of populations. The results also provide information to select sites for ex situ conservation. Optimal harvesting strategies, cultivation and tissue culture should also be developed as soon as possible to guarantee sustainable use of the species under study.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

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

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

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.     

Low genetic diversity and weak population differentiation in Firmiana danxiaensis,a tree species endemic to Danxia landform in northern Guangdong,China

Danxia landform, a unique type of petrographic geomorphology, sporadically occurs in southern China. There are about 400 rare or threatened plants and animals in this landform, but little information on genetic aspects of these species has been available. Of them, Firmiana danxiaensis is a dominant tree species endemic to Mount Danxia, a typical Danxia landform in northern Guangdong, China. In this study, we examined genetic diversity and population structure of F. danxiaensis, in comparison with two other narrowly distributed Firmiana species, Firmiana hainanensis and Firmiana kwangsiensis, which grow in non-Danxia-landform regions. Nine microsatellite markers were used to investigate genetic diversity and differentiation in a total of 256 individuals from nine populations of F. danxiaensis, three of F. hainanensis, and one of F. kwangsiensis. The results showed that F. danxiaensis had the lowest genetic diversity (H_E = 0.364 ± 0.019) among the three species, but weak population structure similar to another Firmiana species (F_ST = 0.042 and 0.020, respectively). This low genetic diversity observed in F. danxiaensis is possibly due to confined natural distribution range and strong selective pressure in soils of Danxia landform, which may also be true for other endemic species in Danxia landform. For the long term survival of these endemic species, habitat conservation and anthropogenic introduction to similar habitats such as the adjacent Danxia landform areas should be two major strategies.     

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.     

A molecular approach to understand the riddle of the invasive success of the tarantula,Brachypelma vagans,on Cozumel Island,Mexico

Invasive populations typically demonstrate genetic isolation which results in a loss of genetic diversity and a reduction in invasion success. This study focused on the genetic population of a successful invasive species of tarantula. Individuals were sampled in two mainland localities of the Yucatan Peninsula (Zoh-Laguna and Raudales), in addition to two island localities (El Cedral and Rancho Guadalupe on Cozumel Island). All populations present high genetic diversity (mean: H_e = 0.23, P = 99%), with significant differences between the Raudales and Rancho Guadalupe localities. The AMOVA analysis revealed a significant population structure (14.5% variation among populations), consistent with the gene differentiation coefficient (G_ST = 0.21), and spatial analysis of population structure. Our results suggested that the original introduced population did not suffer a loss of genetic diversity during establishment on the island, possibly a result of different biological conditions. Population structure analysis leads us to suggest that one island population is similar to the original genetic profile, whereas the genotypic profile of the other island population reflects recent introductions from the mainland. We identified a potential risk of extinction for one local mainland population, suggesting that this species may be a successful invader in a new environment but endangered in some parts of its natural area.     

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 of Astragalus nitidiflorus, a critically endangered endemic of SE Spain,and implications for its conservation

Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population structure in five populations of Astragalus nitidiflorus, a critically endangered species endemic to southeast Spain. Eight primers amplified 78 bands with 40 (51.3%) being polymorphic. Statistical results indicated a low genetic diversity at the population and species level, with percentages of polymorphic bands (PPB) ranging from 28.2 to 37.2% (an average of 31.8%), and means of gene diversity (H_E) of 0.129 and 0.171 respectively. The Shannon’s index (SI) ranged from 0.160 to 0.214 at the population level and was 0.260 at the species level. A low level of genetic differentiation among populations was detected, based on the Shannon’s information index (0.297), the coefficient of genetic differentiation between populations (G_ST = 0.2418) and AMOVA analysis (Φ_ST = 0.255). The estimated gene flow (Nm) was 0.789. The high genetic connectivity found among populations of A. nitidiflorus is an evidence of a recent habitat fragmentation. In addition, a bottleneck event in the past has been revealed, with a subsequent reduction of population size and a loss of genetic variation. Based on these results, the conservation strategy of A. nitidiflorus was proposed.     

Population structure and genetic diversity of Dysosma versipellis (Berberidaceae), a rare endemic from China

Dysosma versipellis (Berberidaceae) is an endangered and endemic species in China. To provide scientific foundation for formulating conservation strategies, we sampled six extant populations of this species and assessed the levels and patterns of genetic diversity using ISSR markers (11 primers). Of 144 bands detected 57.64% were polymorphic, but on average only 20.72% were polymorphic within populations. Our results revealed a low level of intraspecific genetic diversity (at population level: H_pop = 0.082, H_B = 0.177, SI = 0.1194; at species level: H_pop = 0.207, H_B = 0.378, SI = 0.3069). A high level of genetic differentiations among populations was detected based on Nei's genetic diversity analysis (60%), AMOVA analysis (65%), and Bayesian analysis (53%). The low levels of heterozygosity and high genetic differentiation observed in D. versipellis may be the consequence of low rate of natural recruitment, clonal growth, gene drift, and habitat fragmentation. Based on this, we suggest that in situ conservation be an important and practical measure for maintaining the genetic diversity of this species. Ex situ conservation should sample from different populations across the distribution range of the species to conserve high genetic diversity.     

Genetic diversity of natural populations of Larix principis-rupprechtii in Shanxi Province,China

Prince Rupprecht's Larch (Larix principis-rupprechtii Mayr.) is one of dominant components of middle and high elevation forests in North China. Shanxi Province is well known as “the Hometown of Prince Rupprecht's Larch” in China. In this study, six natural populations of this species across Shanxi were selected to investigate the genetic variation of the species using amplified fragment length polymorphism (AFLP) markers. Results showed that in comparison with some other species of Larix, higher genetic diversity was revealed at the species level for L. principis-rupprechtii (percentage of polymorphic loci PPL = 71.9%, Nei's gene diversity H_E = 0.225, Shannon information index I = 0.341). Most of genetic variation existed within populations (80.5%), while the genetic differentiation among populations was significant (p < 0.001) and higher (G_st = 0.194) than most other species of Larix. The differentiation can be attributed to the limited gene flow (N_m = 1.035) among populations, which could be due to the spatial isolation and habitat fragmentation. The six populations can be divided into three groups based on the Nei's genetic distances between populations (from 0.033 to 0.076). There was no significant correlation (r = 0.268, p > 0.05) between genetic distance and geographic distance among populations. The measures for in-situ or ex-situ conservation should be taken to preserve the genetic diversity of this species.     

High genetic differentiation and low genetic diversity in Incarvillea younghusbandii, an endemic plant of Qinghai-Tibetan Plateau,revealed by AFLP markers

Incarvillea younghusbandii Sprague (Bignoniaceae) is a perennial herbaceous plant endemic to Qinghai-Tibetan Plateau. As a species of medical and horticulture importance, I. younghusbandii is threatened by over exploitation and habitat fragmentation. In this study, we analyze the genetic diversity and population structure of I. younghusbandii using amplified fragment length polymorphism (AFLP) markers. Our data reveal very low levels of genetic diversity in seven natural populations across Tibet. Specifically, at population level, the average Nei's genetic diversity index (H_E) and Shannon's diversity index (I) were 0.063 and 0.096, respectively. In contrast, high genetic differentiation among populations (Gst = 0.6238, Φ_ST = 0.614) is detected. The results of Neighbor-joining cluster, PCO, and STRUCTURE assignment reveal consistent pattern, suggesting seven well-defined genetic groups that are concordant with their geographical origins. The possible mechanisms and implications of these findings for conservation are discussed.     

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.     

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.     

Effect of Yangtze River on population genetic structure of the relict plant Parrotia subaequalis in eastern China

Parrotia subaequalis (Hamamelidaceae) is a Tertiary relic species endemic in eastern China. We used inter‐simple sequence repeat (ISSR) markers to access genetic diversity and population genetic structure in natural five populations of P. subaequalis. The levels of genetic diversity were higher at species level (H = 0.2031) but lower at population level (H = 0.1096). The higher genetic diversity at species levels might be attributed to the accumulation of distinctive genotypes which adapted to the different habitats after Quaternary glaciations. Meanwhile, founder effects on the early stage, and subsequent bottleneck of population regeneration due to its biological characteristics, environmental features, and human activities, seemed to explain the low population levels of genetic diversity. The hierarchical AMOVA revealed high levels (42.60%) of among‐population genetic differentiation, which was in congruence with the high levels of Nei's genetic differentiation index (G_ST = 0.4629) and limited gene flow (N_m = 0.5801) among the studied populations. Mantel test showed a significant isolation‐by‐distance, indicating that geographic isolation has a significant effect on genetic structure in this species. Unweighted pair‐group method with arithmetic average clustering, PCoA, and Bayesian analyses uniformly recovered groups that matched the geographical distribution of this species. In particular, our results suggest that Yangtze River has served as a natural barrier to gene flow between populations occurred on both riversides. Concerning the management of P. subaequalis, the high genetic differentiation among populations indicates that preserving all five natural populations in situ and collecting enough individuals from these populations for ex situ conservation are necessary.     

Genetic diversity of SSR markers in wild populations of Tapiscia sinensis,an endangered tree species

Tapiscia sinensis is a Tertiary relict and endangered tree species with unique scientific research value and great economic value. In this study, we assessed the genetic diversity of five wild T. sinensis populations from different geographical regions using 10 polymorphic simple sequence repeat (SSR) markers. Our results reveal that the natural populations of T. sinensis have rich genetic diversity (PPL = 100%, He = 0.6904, I = 1.4368), with Shannon's index indicating that the T. sinensis populations are at a relatively stable stage. Of the genetic relationships among populations, the distance between the Hunan Yanling (YL) and Guizhou Xifeng (XF) populations is the smallest (0.4829); the genetic distance between the Shaanxi Ningshan (NS) and the Guizhou Xifeng (XF) populations is the largest (0.9821). A Mantel test shows that there is no correlation among the populations between geographic distance and genetic distance. AMOVA suggest that 33.3% of the genetic variation arose among the populations, while 66.7% of the variation arose within them. The moderate gene flow among populations (Nm = 0.7274) is not sufficient to counteract genetic drift within the populations and result in significant differentiation (Fst = 0.2987). Our results will benefit the conservation and exploitation of T. sinensis and provide a theoretical basis for further study of the evolution and phylogeography of the species.     

Genetic diversity of Lilium tsingtauense in China and Korea revealed by ISSR markers and morphological characters

To study the genetic diversity and population structure of Lilium tsingtauense Gilg (Qingdao Lily), we collected 648 samples from 12 sites in China and Korea, and analyzed their Inter-Simple Sequence Repeat (ISSR) molecular markers and morphological characters. ISSR data revealed a relatively high genetic diversity at the species level, with 72.31% polymorphic loci, effective numbers of alleles of 1.437, average expected heterozygosity of 0.231 and Shannon’s information index of 0.369. Considerable genetic differentiation among the natural populations (G_ST = 0.144) and the gene flow (N_m = 1.487) were detected. AMOVA analysis and UPGMA-dendrogram suggested a hierarchical regional structure among populations, and spatial autocorrelation analysis showed a micro-scaled spatial structure. Furthermore, there was a high correlation between morphological characters and genetic parameters obtained from ISSR parameters. There was only a low genetic differentiation among the different morphological types of L. tsingtauence in China. Based on these findings, we recommend in situ and ex situ conservation strategies for the preservation of L. tsingtauense.