Preliminary analysis of the genetic diversity and population structure of mortiño (Vaccinium floribundum Kunth)

Mortiño (Vaccinium floribundum Kunth) is a deciduous perennial shrub endemic to the high Andes of South America. Despite a rich ethnobotanic history among indigenous communities, mortiño remains a wild species vulnerable to extinction from the ongoing fragmentation of its natural habitat. This study assessed the degree of genetic diversity and population structure of Ecuadorian V. floribundum as a preliminary step towards the establishment of effective conservation and sustainable-agriculture strategies. Mortiño individuals (126 in total) sampled from 3 regions in the northern highlands of Ecuador were characterized using 11 heterologous SSR markers originally developed for Vaccinium corymbosum. Expected heterozygosity (He = 0.49) revealed a moderate degree of genetic diversity for Ecuadorian mortiño, and pairwise F statistics between sampling regions (0.019≤ Fst ≤0.041) demonstrated low-to-moderate population differentiation. Population structure analysis clustered mortiño germplasm into 3 groups, each representing the 3 distinct regions from where samples were collected. The geographic patterning of genetic diversity for V. floribundum could be explained by an isolation-by-distance model, where physical barriers along the Andean highlands reduce genetic exchange between distanced populations. To confirm the latter, this study should extend to a wider sampling range, covering other regions along the Andean alley where the species is found.     

Influence of native and alien Prunus species and light conditions on performance of the leaf beetle Gonioctena quinquepunctata

The polyphagous beetle Gonioctena quinquepunctata Fabricius (Coleoptera: Chrysomelidae) is a serious leaf pest of the native European bird cherry, Prunus padus L., and the invasive alien black cherry, Prunus serotina Ehrh. (Rosaceae). In the shade, leaf damage is extensive in both species, whereas in full light, it is extensive in P. padus, but very low in P. serotina. We determined the influence of Prunus species and light conditions on differences in performance of both sexes of this folivore. In a laboratory experiment in which larvae were fed with leaves of a single species grown under particular light conditions, we measured larval, pupal, and adult mass, efficiency of conversion of ingested food (ECI), duration of development, total food eaten, and relative growth rate. In the field, we observed differences in beetle mass on shrubs of the two species growing under various light conditions. From the field observations, we hypothesised that leaves of the invasive P. serotina are not an equally good food source as leaves of P. padus for G. quinquepunctata, and the preference of these beetles for shaded shrubs is most favourable for their growth and development. Under laboratory conditions, we found that the beetle growth rate was not affected significantly by Prunus species or light conditions, despite the significant effect of light condition on the structure and chemical composition of Prunus seedlings. The lower ECI value for larvae feeding on sunlit leaves was compensated for by their higher level of consumption. In the field, adult insect mass was higher on P. padus than on P. serotina, and higher on sunlit shrubs of both species than on shaded ones. Under natural conditions, the mass of adult insects is probably also affected by other factors, such as predators and competition among folivores.     

Wild Panax vietnamensis and Panax stipuleanatus markedly increase the genetic diversity of Panax notoginseng (Araliaceae) revealed by start codon targeted (SCoT) markers and ITS DNA barcode

In order to evaluate whether the two wild species, Panax vietnamensis (from Vietnam) and Panax stipuleanatus (from primeval forest, Yunan Province) could markedly increase the genetic diversity of cultivated Panax notoginseng (Wenshan, Yunnan Province), both start codon targeted (SCoT) markers and internal transcribed spacer (ITS) DNA barcode were firstly employed in this genus. A total of 173 amplification bands were generated by 16 selected SCoT primers, in which 153 (89.5%) were polymorphic. Nei's gene-diversity indicated that the genetic diversity of three species (h = 0.16 and I = 0.27) was obviously higher than that of P. notoginseng (h = 0.09). Similarly, 38 different ITS sites out of 639 (5.9%) were detected among three species, but only one was different within 22 samples of P. notoginseng. Analysis of molecular variance (AMOVA) showed a greater proportion of genetic diversity existed within (61.3%) rather than among (38.7%) groups at genus level. In addition, P. vietnamensis had a closer relationship with P. notoginseng than P. stipuleanatus. These results would be significant for increasing the genetic diversity of P. notoginseng population by hybridization with P. vietnamensis and P. stipuleanatus, thus obtaining more varieties for future cultivar breeding and germplasm resources management.     

Estimation of genetic diversity in some Iranian wild Prunus subgenus Cerasus accessions using inter-simple sequence repeat (ISSR) markers

As Iran is one of the main origins of Prunus germplasm. In this study, ISSR markers were used for genetic diversity evaluation of 39 accessions of subgenus Cerasus belonging to six species i.e. Prunus avium L., Prunus cerasus L., Prunus mahaleb L., Prunus incana Pall., Prunus microcarpa Boiss., and Prunus brachypetala Boiss.. With 12 ISSR primers, 151 polymorphic bands were detected with polymorphism ratio range of 81.8%–100%. The lowest similarity (0.04) was found between P. avium and P. microcarpa genotypes and the mean of similarity between all genotypes was 0.28. Cluster analysis separated improved cultivars from wild accessions. Improved cherry cultivars and rootstocks were placed closer to the P. avium than the other species. The principal coordinate analysis (PCoA) supported the cluster analysis results. The wild accessions were separated according to their species and collection sites. ISSR markers are useful techniques for genetic diversity evaluation in Prunus subgenus Cerasus.     

Identification,development, and application of 12 polymorphic EST-SSR markers for an endemic Chinese walnut (Juglans cathayensis L.) using next-generation sequencing technology

The Chinese walnut (Juglans cathayensis L.), valued for both its nut and wood, is an ecologically important tree species endemic temperate southern China. Investigation of the genetic diversity of Chinese walnut has been limited to natural population genetics and genetic germplasm resources. Here, we describe the development of 12 polymorphic microsatellite markers using next-generation sequencing to screen 96 Chinese walnut individuals collected from 11 natural populations. The number of alleles per locus ranged from 5 to 12. The observed heterozygosity (0.288–0.748) overlapped well with the expected heterozygosity (0.337–0.751). This species has high genetic diversity and gene flow among different populations (F_ST = 0.075, Nm = 3.088). These markers will be useful for future studies on population genetic structure, evolutionary ecology, and genetic breeding of this walnut tree or other Juglans species.     

Clonal Diversity and Fine‐scale Genetic Structure in a High Andean Treeline Population

Clonal propagation becomes more abundant with increasing altitudes as environmental conditions worsen. To date, little attention has been paid to the way in which clonal propagation affects genetic diversity and the fine‐scale spatial genetic structure (FSGS) of clonal alpine trees. An AFLP study was undertaken to quantify the clonal and genetic diversity and FSGS of the vulnerable treeline species Polylepis reticulata in Ecuador. We successfully genotyped 32 and 75 ramets within 4 m × 100 m (coarse scale) and 4 m × 4 m (fine scale) transects of one population, respectively. Higher genotypic diversity was detected at the coarse scale than at the fine scale, while lower genetic diversity was detected for P. reticulata than other Polylepis spp. at both scales. Significantly stronger FSGS was detected at the ramet level than the genet level for P. reticulata within a spatial distance of 3 m. The studied P. reticulata population showed pronounced FSGS (Sp = 0.012 at the genet level, a statistic reflecting declining pairwise kinship with distance) revealed restricted gene dispersal, which implies restricted seed dispersal for this population, assuming pollen flow is as extensive as that described for other wind‐pollinated tree species. Our results revealed that clonal diversity is a function of both sample size and the spatial scale of the sampling area. The findings highlights that clonal propagation has affected FSGS within a spatial distance of 3 m for this species.     

Population genetic structure and germplasm conservation of Stipa purpurea on Qinghai-Tibetan Plateau under grazing

At what intensity the grazing should be practiced on the Qinghai-Tibetan Plateau in China is always confusing farmers, scientists and policy makers for a long time owing to its geographical exception. In order to develop a strategy for sustainable grazing management, we used the dominant Stipa purpurea as a model species to detect genetic diversity and fine spatial structure under different grazing intensities. Intra-population neutral genetic diversity in non-grazed population was significantly higher than three grazed populations; however, the highest value among grazed populations appeared in the moderately grazed population. A relatively low degree of genetic differentiation among populations by AMOVA analysis and a high level of gene flow existed among populations (Gst = 0.2649, Nm = 1.3875 〉 1). The genetic ‘patch’ size increased from 4 m to 16 m and subpopulation number within the S. purpurea populations ranged from 7 to 5 with increasing grazing intensity, therefore grazing will be beneficial to reduce the fragmentation and to increase the population-level adaptation. Though enclosure or no grazing is the best protection of plant germplasm like wind-pollination S. purpurea, moderate grazing is better strategy for grassland use on the Qinghai-Tibetan Plateau.     

Light as a regulator of structural and chemical leaf defenses against insects in two Prunus species

Light is a key factor influencing competition between species, and the mechanisms by which trees overcome insect outbreaks can be associated with alternation of the leaves structure, which then prevent or promotes their susceptibility to herbivores. It was predicted that leaf tissue anatomy would likely be different in sun and shade leaves, with a gradual decline of leaves resistance coupled with reduction of accessible light. We quantified anatomical patterns and the distribution of defence compounds (phenols, total tannins, catechol tannins) within heavily grazed leaves of Prunus padus, native in Europe and Prunus serotina, an invasive to Central Europe. Both species were strongly attacked by folivorous insects when shrubs grew in the shade. In the sun, however only P. padus leaves were grazed, but P. serotina leaves were almost unaffected. We identified that anatomical characteristics are not linked to different P. padus and P. serotina leaf vulnerability to insects. Furthermore, the staining of defence compounds of P. serotina leaves grown in full sun revealed that the palisade mesophyll cells had a higher content of phenolic compounds and catechol tannins. Thus, our results indicate that a specific distribution of defence compounds, but not the anatomical relationships between palisade and spongy mesophyll, may be beneficial for P. serotina growth outside its natural range. The identified pattern of defence compounds distribution is linked to a lower susceptibility of P. serotina leaves to herbivores, and is associated with its invasiveness. This likely reflects that P. serotina is a stronger competitor than P. padus, especially at high sunlit sites i.e. gaps in the forest.     

Tracing the native ancestors of the modern Theobroma cacao L. population in Ecuador

The native Theobroma cacao L. population from Ecuador, known as Nacional, is famous for its fine cocoa flavour. From the beginning of the twentieth century, however, it has been subjected to genetic erosion due principally to successive introductions of foreign germplasm whose hybrid descendants gradually replaced the native plantations, implying a decrease in cocoa quality. We attempted to trace this native cacao within a wide pool of modern Ecuadorian cacao population. Three hundred and twenty-two cacao accessions collected from different geographical areas along the pacific coast of Ecuador and maintained in two living collections were analysed using 40 simple-sequence repeat markers. Most of Ecuadorian cacao accessions displayed a high diversity and heterozygosity level. A factorial analysis of correspondence (FAC) showed a continuous variation among them, with a few ones, grouped at an extreme side of the FAC cloud, showing higher levels of homozygosity and lower introgression level by foreign cacaos. A paternity analysis revealed that these highly homozygous individuals are the most probable ancestors of the modern Nacional hybrid pool. These particular accessions studied could represent the native Nacional cacao present in Ecuador before the foreign introductions. Their identification will help to conserve valuable genetic material and to improve cocoa quality in new cacao varieties.     

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.     

Determination of genetic diversity of the cuttlefish Sepiella japonica inhabiting Chinese coastal waters using the mitochondrial D-loop region: The valuable inspiration to artificial releasing project

The cuttlefish Sepiella japonica, was once one of the four major marine fisheries of China and has dramatically declined since the 1980s to near extinction. The artificial releasing project was launched for resource restocking since 2006. Aiming to provide some valuable information to the management of the releasing project, the mitochondrial DNA control region sequence was employed to assess the genetic variation of various geographical populations in present study. The genetic diversity of S. japonica was characterized with the low value of nucleotide diversity (0.00313 ± 0.070). Pairwise fixation index (Fst) analysis indicated insignificant genetic differentiation among southern populations (p > 0.05). However, the QD population showed the significant genetic differention with other populations (p < 0.05). The highest genetic diversity was detected in ND population and was recommended to be the germplasm resources for artificial releasing project. In summary, the presented results provided some new information for genetic assessment in this study that would be crucial for establishing effective releasing strategies for this species.     

Genetic variation and clonal diversity in populations of Nelumbo nucifera (Nelumbonaceae) in central China detected by ISSR markers

To obtain accurate estimates of population structure for purposes of conservation planning for wild lotus (Nelumbo nucifera Gaertn.) in central China, genetic diversity among and within six populations, and clonal diversity within another two populations of the species were analyzed. The genetic diversity was high (percentage of polymorphic bands, PPB = 90.0%; Shannon's information index, I = 0.383 ± 0.234) at the species level, but low within individual study populations (PPB = 35.8%; Shannon's information index I = 0.165 ± 0.241). The mean coefficient of gene differentiation (G_st) was 0.570, indicating that 43.0% of the genetic diversity resided within the population. Analysis of molecular variance (AMOVA) indicated that 50.47% of the genetic diversity among the study populations was attributed to geographical location while 12.3% was attributed to differences in their habitats. An overall value of mean estimated number of gene flow (N_m = 0.377) indicated that there was limited gene flow among the sampled populations. The level of clonal diversity found within the populations was considerably high (Simpson's diversity index, D = 0.985) indicating that clonal diversity contributes to a major extent to the overall genetic variation in the genetic structure of N. nucifera. On the basis of the high G_st and D values detected in this study we recommend that any future conservation plans for this species should be specifically designed to include those representative populations with the highest genetic variation for both in situ conservation and germplasm collection expeditions.     

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.     

Molecular Markers and Conservation of Plant Species in the Latin-America: The Case of Phaedranassa viridiflora (Amaryllidaceae)

Phaedranassa viridiflora (Amaryllidaceae) is an endemic and endangered plant restricted to the Northern Andes in Ecuador. It is known in three locations where it is sympatric with other Phaedranassa species. Phaedranassa viridiflora is the only species of the genus with yellow flowers. We analyzed 13 microsatellite loci to elucidate the genetic structure of the populations of P. viridiflora. Our results provided the first evidence of natural hybridization in the genus (between P. viridiflora and P. dubia in the Pululahua crater of northern Ecuador). The central and southern populations did not show hybridization. Genetic diversity was the highest in the Pululahua population. Central and southern populations have a higher proportion of clones than Pululahua. Bayesian and cluster analysis suggest that the yellow flower type evolved at least three times along the Ecuadorean Andes. In contrast to other Phaedranassa species, Phaedranassa viridiflora shows lower genetic diversity, which is likely related to a vegetative reproductive strategy.     

High genetic diversity and stable Pleistocene distributional ranges in the widespread Mexican red oak Quercus castanea Née (1801) (Fagaceae)

The Mexican highlands are areas of high biological complexity where taxa of Nearctic and Neotropical origin and different population histories are found. To gain a more detailed view of the evolution of the biota in these regions, it is necessary to evaluate the effects of historical tectonic and climate events on species. Here, we analyzed the phylogeographic structure, historical demographic processes, and the contemporary period, Last Glacial Maximum (LGM) and Last Interglacial (LIG) ecological niche models of Quercus castanea, to infer the historical population dynamics of this oak distributed in the Mexican highlands. A total of 36 populations of Q. castanea were genotyped with seven chloroplast microsatellite loci in four recognized biogeographic provinces of Mexico: the Sierra Madre Occidental (western mountain range), the Central Plateau, the Trans‐Mexican Volcanic Belt (TMVB, mountain range crossing central Mexico from west to east) and the Sierra Madre del Sur (SMS, southern mountain range). We obtained standard statistics of genetic diversity and structure and tested for signals of historical demographic expansions. A total of 90 haplotypes were identified, and 29 of these haplotypes were restricted to single populations. The within‐population genetic diversity was high (mean h_S = 0.72), and among‐population genetic differentiation showed a strong phylogeographic structure (N_ST = 0.630 > G_ST = 0.266; p < .001). Signals of demographic expansion were identified in the TMVB and the SMS. The ecological niche models suggested a considerable percentage of stable distribution area for the species during the LGM and connectivity between the TMVB and the SMS. High genetic diversity, strong phylogeographic structure, and ecological niche models suggest in situ permanence of Q. castanea populations with large effective population sizes. The complex geological and climatic histories of the TMVB help to explain the origin and maintenance of a large proportion of the genetic diversity in this oak species.     

Genetic structure of the threatened Phaedranassa schizantha (Amaryllidaceae)

Phaedranassa schizantha (Amaryllidaceae) is an endangered species endemic to Ecuador and two varieties have been described: P. schizantha var. schizantha and P. schizantha var. ignea. We assessed population genetic structure and demographic patterns in 11 populations across the range of the species using 13 microsatellite loci. Our data show that genetic diversity was generally lower in the southern part of the range and was especially low in populations closest to cities. We found significant population differentiation (F_ST = 0.14, D_EST = 0.34) and evidence of a genetic bottleneck. Genetic variation did not show isolation by distance. Instead, results suggest genetic barriers around two main cities. Bayesian analysis identified two genetic groups, neither of which represents either of the two varieties previously recognized. Coalescent analysis indicates a relatively recent colonization pattern between the two genetic groups (< 3000 generations). Conservation efforts need to be taken to facilitate genetic exchange between the groups, especially between locations that seem to be genetically isolated.     

Polyphyly of the Padus group of Prunus (Rosaceae) and the evolution of biogeographic disjunctions between eastern Asia and eastern North America

Prunus subgenus Padus is a group with a wide distribution in temperate eastern Asia and eastern North America with one species extending to Europe and one to Central America. Phylogenetic relationships of subgenus Padus were reconstructed using sequences of nuclear ribosomal ITS, and plastid ndhF gene, and rps16 intron and rpl16 intron. Prunus subgenus Padus is shown to be polyphyletic. Taxa of subgenus Padus and subgenus Laurocerasus are highly intermixed in both the ITS and the plastid trees. The results support two disjunctions between eastern North America and Eurasia within the Padus group. One disjunction is between Prunus virginiana of eastern North America and P. padus of Eurasia, estimated to have diverged at 2.99 (95 % HPD 0.59–6.15)–4.1 (95 % HPD 0.63–8.59) mya. The other disjunction is between P. serotina and its Asian relatives. The second disjunction may have occurred earlier than the former one, but the age estimate is difficult due to the unresolved phylogenetic position of the P. serotina complex.     

Genetic diversity of two endemic and endangered Plantago species

Plantago algarbiensis and Plantago almogravensis are two endangered and endemic species from Portugal. Due to the rarity and endangered nature of these species as well as the lack of molecular data, their genetic variation was evaluated using ISSR and RAPD markers. P. algarbiensis species showed higher genetic variability (73.9% of polymorphism) than P. almogravensis (61.2%). The two species revealed a high level of genetic diversity, with a Nei's genetic diversity of 0.1965 and 0.2309 and a Shannon's diversity index of 0.2975 and 0.3520, for P. almogravensis and P. algarbiensis, respectively. A low level of genetic differentiation was observed (Gst = 0.1873) among the species. However, the cluster and PCA analyses, based on genetic similarity, revealed two main, clearly separate clusters, which directly corresponded to the plants isolated from each species. In situ and ex situ measures should be applied in order to preserve both species but, based on these results, P. almogravensis population should be a priority for conservation.     

Genetic depletion at adaptive but not neutral loci in an endangered bird species

Many endangered species suffer from the loss of genetic diversity, but some populations may be able to thrive even if genetically depleted. To investigate the underlying genetic processes of population bottlenecks, we apply an innovative approach for assessing genetic diversity in the last known population of the endangered Pale‐headed Brushfinch (Atlapetes pallidiceps) in Ecuador. First, we measure genetic diversity at eleven neutral microsatellite loci and adaptive SNP variation in five Toll‐like receptor (TLR) immune system genes. Bottleneck tests confirm genetic drift as the main force shaping genetic diversity in this species and indicate a 99 % reduction in population size dating back several hundred years. Second, we compare contemporary microsatellite diversity with historic museum samples of A. pallidiceps, finding no change in genetic diversity. Third, we compare genetic diversity in the Pale‐headed Brushfinch with two co‐occurring‐related brushfinch species (Atlapetes latinuchus, Buarremon torquatus), finding a reduction of up to 91% diversity in the immune system genes but not in microsatellites. High TLR diversity is linked to decreased survival probabilities in A. pallidiceps. Low TLR diversity is thus probably an adaptation to the specific selection regime within its currently very restricted distribution (approximately 200 ha), but could severely restrict the adaptive potential of the species in the long run. Our study illustrates the importance of investigating both neutral and adaptive markers to assess the effect of population bottlenecks and for recommending specific management plans in endangered species.     

Ecuadorian novelties in Blakea and Topobea (Melastomataceae)

Three new species of Melastomataceae (Blakeeae) are described:Blakea subpanduriformis, Topobea adscendens, andT. bullata. All are climbers endemic to the montane forests of the eastern slopes of the Andes in southern Ecuador. In this region, 35% of the Ecuadorian species and 41% of the endemic Ecuadorian species of Melastomataceae occur. The discovery of these three new endemic species underlines the diversity of Melastomataceae occurring on the slopes of the Andes in southeastern Ecuador.