cpDNA Fragments Research Articles

Phylogeography of a herbal Pinellia ternata reveals repeated range expansions and inter/postglacial recolonization routes on the fragmented distribution pattern in China.

Most plant phylogeographic studies in China have focused on the importance of genetic divergence and where should the shelter be located. Little attention has been paid to range expansion and recolonization routes in this region. In this study, two cpDNA fragments (psbK-psbI and trnL-F), two pairs of nuclear gene sequences (ITS and ETS), and nine pairs of SSR molecular markers were used, combined with Bayesian Skyline Plot method, gene barrier analysis, and species distribution models to explore the phylogeographical pattern, potential expansion routes and population dynamic history of Pinellia ternata from 22 population. The results showed that phylogeograhical pattern and genetic structure for P. ternata are effected by environmental heterogeneity and climate fluctuation, and it can be divided into two groups (Southwest group, Central and Eastern group) and thus there are at least two glacial refugia in China. Three expanding routes within groups were explored to contribute to the phylogeogrephic pattern of P. ternata based on the geographical distribution and network analysis of haplotypes. In a word, our study reveals repeated range expansions and inter/postglacial recolonization routes on the fragmented distribution pattern in China and resolves the refugia distributing in China and has also certain reference value for the protection of the medicinal plant P. ternata.

Phylogeography of Morella nana: The Wumeng Mountains as a natural geographical isolation boundary on the Yunnan-Guizhou Plateau.

The Yunnan-Guizhou Plateau (YGP) is characterized by the distinctive isolated habitat of the limestone Karst Islands and features the Wumeng Mountains, which divide the YGP into the two Plateaus of Yunnan and Guizhou. This study aimed to assess the effects of geographic isolation and past climate fluctuation on the distribution of flora in the YGP. To achieve this, we carried out the phylogeographical pattern and genetic structure based on chloroplast and nuclear ribosomal DNA sequence in relation to past (Last Glacial Maximum) and present distributions based on ecological niche modeling for Morella nana, an important wild plant resource and endemic to the YGP once considered a vulnerable species. The results suggest that the genetic and chlorotype network structures of M. nana are divided into at least two groups: cpDNA chlorotype H2 (or dominant nrDNA haplotypes h1 and h2), distributed primarily to the east of the Wumeng Mountains, and cpDNA chlorotypes H1 and H3-H10 (or dominant nrDNA haplotype h2 and h3), distributed to the west of the Wumeng Mountains. A deep genetic split was noted within the two groups to reach 25 steps, especially for the cpDNA fragment variation. This east-west divergence reveals the existence of a natural geographical isolation boundary in the form of the Wumeng Mountains, and supports the existence of at least two glacial refuges during the Quaternary glacial period, along with two genetic diversity center, and at least two large geographic protection units for the important species of M. nana. This study indicates that the phylogeographical pattern of M. nana can be attributed to geographic/environmental isolation caused by the Wumeng Mountains and climate fluctuation during the last glacial maximum, and proposes an effective strategy to protecting this important plant resource.

Phylogeographic Structure and Population Dynamics of Baoxing Osmanthus (Osmanthus serrulatus), an Endemic Species from the Southwest Sichuan Basin, China.

The mountainous regions of southwest China are recognized as pivotal centers for the origin and evolution of Osmanthus species. Baoxing Osmanthus (Osmanthus serrulatus Rehder), a rare and endemic species known for its spring blooms, is sparsely distributed within the high altitude evergreen broad-leaved forests surrounding the southwestern Sichuan Basin. However, persistent anthropogenic disturbances and habitat fragmentation have precipitated a significant decline in its natural population size, leading to the erosion of genetic resources. To assess the genetic status of O. serrulatus and formulate effective conservation strategies, we conducted sampling across ten wild populations, totaling 148 individuals in their natural habitats. We employed two cpDNA fragments (matK and trnS-trnG) to elucidate the phylogeographic structure and historical population dynamics. The results revealed low species-level genetic diversity, alongside pronounced regional differentiation among populations (FST = 0.812, p < 0.05) and a notable phylogeographic structure (NST = 0.698 > GST = 0.396, p < 0.05). Notably, genetic variation was predominantly observed among populations (81.23%), with no evidence of recent demographic expansion across the O. serrulatus distribution range. Furthermore, divergence dating indicated a timeline of approximately 4.85 Mya, corresponding to the late Miocene to early Pleistocene. This temporal correlation coincided with localized uplift events in the southwestern mountains and heightened Asian monsoons, suggesting pivotal roles for these factors in shaping the current phylogeographic pattern of O. serrulatus. These findings support the effective conservation of O. serrulatus germplasm and offer insights into the impact of Quaternary climate oscillations on companion species within evergreen broad-leaved forests. They also enhance our understanding of the origin and evolution of these forests in the southwestern mountains, aiding biodiversity conservation efforts in the region.

Demographic patterns of two related desert shrubs with overlapping distributions in response to past climate changes.

Numerous studies have revealed that past geological events and climatic fluctuations had profoundly affected the genetic structure and demographic patterns of species. However, related species with overlapping ranges may have responded to such environmental changes in different ways. In this study, we compared the genetic structure and population dynamics of two typical desert shrubs with overlapping distributions in northern China, Nitraria tangutorum and Nitraria sphaerocarpa, based on chloroplast DNA (cpDNA) variations and species distribution models. We sequenced two cpDNA fragments (trnH-trnA and atpH-atpI) in 633 individuals sampled from 52 natural populations. Twenty-four chlorotypes, including eight rare chlorotypes, were identified, and a single dominant haplotype (H4) widely occurred in the entire geographical ranges of the two species. There were also a few distinctive chlorotypes fixed in different geographical regions. Population structure analyses suggested that the two species had significantly different levels of total genetic diversity and interpopulation differentiation, which was highly likely correlated with the special habitat preferences of the two species. A clear phylogeographic structure was identified to exist among populations of N. sphaerocarpa, but not exist for N. tangutorum. The neutral tests, together with the distribution of pairwise differences revealed that N. tangutorum experienced a sudden demographic expansion, and its expansion approximately occurred between 21 and 7 Kya before present, while a rapid range expansion was not identified for N. sphaerocarpa. The ecological niche modeling (ENM) analysis indicated that the potential ranges of two species apparently fluctuated during the past and present periods, with obvious contraction in the Last Glacial Maximum (LGM) and recolonization in the present, respectively, comparing to the Last Interglacial (LIG). These findings suggest that the two species extensively occurred in the Northwest of China before the Quaternary, and the current populations of them originated from a few separated glacial refugia following their habitat fragmentation in the Quarternary. Our results provide new insights on the impact of past geological and climatic fluctuations on the population dynamics of desert plants in northwestern China, and further enforce the hypothesis that there were several independent glacial refugia for these species during the Quaternary glaciations.

A novel in-situ-process technique constructs whole circular cpDNA library

BackgroundThe chloroplast genome (cp genome) is directly related to the study and analysis of molecular phylogeny and evolution of plants in the phylogenomics era. The cp genome, whereas, is highly plastic and exists as a heterogeneous mixture of sizes and physical conformations. It is advantageous to purify/enrich the circular chloroplast DNA (cpDNA) to reduce sequence complexity in cp genome research. Large-insert, ordered DNA libraries are more practical for genomics research than conventional, unordered ones. From this, a technique of constructing the ordered BAC library with the goal-insert cpDNA fragment is developed in this paper.ResultsThis novel in-situ-process technique will efficiently extract circular cpDNA from crops and construct a high-quality cpDNA library. The protocol combines the in-situ chloroplast lysis for the high-purity circular cpDNA with the in-situ substitute/ligation for the high-quality cpDNA library. Individually, a series of original buffers/solutions and optimized procedures for chloroplast lysis in-situ is different than bacterial lysis in-situ; the in-situ substitute/ligation that reacts on the MCE membrane is suitable for constructing the goal-insert, ordered cpDNA library while preventing the large-insert cpDNA fragment breakage. The goal-insert, ordered cpDNA library is arrayed on the microtiter plate by three colonies with the definite cpDNA fragment that are homologous-corresponds to the whole circular cpDNA of the chloroplast.ConclusionThe novel in-situ-process technique amply furtherance of research in genome-wide functional analysis and characterization of chloroplasts, such as genome sequencing, bioinformatics analysis, cloning, physical mapping, molecular phylogeny and evolution.

Evaluation of Four Commonly Used DNA Barcoding Loci for Ardisia Species Identification.

Ardisia plants have been used as medicinal plants for a long time in China. Traditional techniques such as morphological, microscopic, and chemical identification methods all have limitations in the species identification of Ardisia. For the sake of drug safety, four DNA barcodes (psbA-trnH, ITS, rbcL, and matK) were assessed for Chinese Ardisia plants using a total of 121 individuals from 33 species. Four criteria (The success rates of PCR amplification, DNA barcoding gap, DNA sequence similarity analysis and NJ tree clustering analysis) were used to evaluate the species identification ability of these four DNA barcodes. The results show that ITS had the highest efficiency in terms of PCR and sequencing and exhibited the most apparent inter- and intra-specific divergences and the highest species identification efficiency. There was no significant increase in species identification after combining the three cpDNA fragments with the ITS fragment. Considering the cost and experimental effectiveness, we recommend ITS as the core barcode for identifying Chinese Ardisia plants.

Juniperus excelsa s. str. in crimea – differentiation and history inferred from genetic and morphological markers

Abstract Juniperus excelsa s. str. (Greek juniper) in Crimea is a relic species on the limits of its range, and represents the Mediterranean flora in the Sub-Mediterranean part of the peninsula. Its origin and history in this area remains unresolved. We aimed to analyze phylogeny and potential demographic expansion of the juniper in the Crimea as well as to study its morphological differentiation. We analyzed plant material from 59 trees inhabiting eight populations. Genetic variation assessments were based on the four non-coding chloroplast DNA (cpDNA) fragments and the nuclear internal transcribed spacer region ITS1-5,8S-ITS2 (ITS). To examine the morphological differentiation, eight measured/counted traits of cones, seeds, and shoots were chosen and eight ratios were calculated. Morphological parameters were compared using ANOVA, Student’s t test, discrimination analysis and Kruskal-Wallis and U Mann-Whitney tests. Two cpDNA fragments were polymorphic and, in total, 10 cpDNA haplotypes were found. Haplotype diversity (Hd) ranged from 0.0 to 0.9. Based on both cpDNA and ITS sequences variation, phylogenetic analyses revealed a close relationship of the Crimean junipers to the individuals from other parts of the species range. In general, our molecular results confirmed the low level of genetic differentiation of J. excelsa individuals inhabiting different parts of the species range, likely resulting from a common ancestry. Only slight morphological differences were found between populations with different geographic location or habitat. The analyzes showed the distinctness of the populations from the southern part of the coast. Some unique morphological and molecular features of southern coastal populations imply that they are remnants of Late Pleistocene abundant forests. We suggest that the recent fragmentation of the Juniperus populations in the Crimean Peninsula could have arisen during the Atlantic period of the Holocene.

Out of the Qinghai-Tibetan Plateau and rapid radiation across Eurasia for Allium section Daghestanica (Amaryllidaceae).

The disjunctive distribution (Europe–Caucasus–Asia) and species diversification across Eurasia for the genus Allium sect. Daghestanica has fascinating attractions for researchers aiming to understanding the development and history of modern Eurasia flora. However, no any studies have been carried out to address the evolutionary history of this section. Based on the nrITS and cpDNA fragments (trnL–trnF and rpl32–trnL), the evolutionary history of the third evolutionary line (EL3) of the genus Allium was reconstructed and we further elucidated the evolutionary line of sect. Daghestanica under this background. Our molecular phylogeny recovered two highly supported clades in sect. Daghestanica: the Clade I includes Caucasian–European species and Asian A. maowenense, A. xinlongense and A. carolinianum collected in Qinghai; the Clade II comprises Asian yellowish tepal species, A. chrysanthum, A. chrysocephalum, A. herderianum, A. rude and A. xichuanense. The divergence time estimation and biogeography inference indicated that Asian ancestor located in the Qinghai–Tibetan Plateau (QTP) and the adjacent region could have migrated to Caucasus and Europe distributions around the Late Miocene and resulted in further divergence and speciation; Asian ancestor underwent the rapid radiation in the QTP and the adjacent region most likely due to the heterogeneous ecology of the QTP resulted from the orogeneses around 4–3 million years ago (Mya). Our study provides a picture to understand the origin and species diversification across Eurasia for sect. Daghestanica.

Identification of &amp;lt;i&amp;gt;Indica-Japonica&amp;lt;/i&amp;gt; Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous &amp;lt;i&amp;gt;gos&amp;lt;/i&amp;gt; Gene

Asia cultivated rice are classified into two subspecies, indica and japonica rice (hsien and keng rice called in China, separately). It is necessary to establish a convenient and effective method to identify two subspecies because of the different quality characteristics between the rice products of them, which lead to different processing uses and commodity values. The identification method of PCR based on the difference of chloroplast DNA is convenient and effective, to be attempted to establish. Based on 69 bp fragment deletion of chloroplast DNA (cpDNA) found in ORF100 region nucleotides within the cpDNA Pst I-12 fragment in indica rice but not in japonica, the primer pair and a probe located on the specific cpDNA fragment in japonica rice were designed to identify indica and japonica rice. Another primer pair and probe used to detect endogenous gene gos in rice were combined with above primer pair and probe for detecting cpDNA to establish duplex fluorescence PCR to amplify cpDNA and gos gene for improving detection accuracy, to avoid the false negative results caused by DNA extraction error. The duplex fluorescence PCR detection method was established using typical japonica rice (pearl rice) and typical indica rice (Taixian 11). The accuracy of the method was validated with 547 samples including 177 samples of rice seed and eaten rice known as conventional japonica varieties and japonica type hybrid combinations and 370 samples of rice seed and eaten rice known as conventional indica rice varieties and indica type hybrid combinations. In 177 japonica samples, 170 samples with both positive results of cpDNA marker and gos gene, a coincidence rate of 96.05% agreement japonica rice, and 7 samples with positive results of gos gene and negative of cpDNA marker, not agreement japonica rice with non-coincidence rate of 3.95%, were detected. Of the 370 samples of indica rice seed and eaten rice, with positive results of gos gene and negative of cpDNA marker were detected in 340 samples, the rate of coincidence to indica rice was 91.89%. Other 30 samples with both positive results of cpDNA marker and gos gene, were detected, and the non-coincidence rate with indica rice was 8.11%. These identification results were in good agreement with known indica and japonica varieties, and the coincidence rate of japonica varieties was higher than indica. This method can be used to identify indica and japonica subspecies rice, especially suitable to identify conventional rice varieties.

Characterization of variability of the intergenic spacers cpDNA trnH–psbA, trnY–trnT AND rpoB–trnC in representatives of Pisum L. (Tribe Fabeae)

Background. Plant chloroplast genome have conservative structure, but its nucleotide sequence is polymorphous due to which cpDNA fragments are often used in taxonomic and phylogenetic studies. Despite the widespread distribution and use of Fabeae species, mainly peas (Pisum), data on the intraspecific diversity of cpDNA fragments are almost absent. The aim of the work was to analyze the intraspecific variability of three cpDNA spacers in Pisum.&#x0D; Materials and methods. As a result of the work, intergenic spacers trnYtrnT, trnHpsbA and rpoBtrnC in 38 accessions of the Pisum and related Fabeae species were sequenced. Despite the fact that the selected chloroplast fragments are generally considered to be sufficiently variable in plants and are often used for phylogenetic studies, Pisum accessions have been found to have no intraspecific differences in two of the three spacers sequences analyzed.&#x0D; Results and conclusion. A total 97 SNPs were detected in Pisum accessions, seven of them distinguished P. sativum from P. fulvum. The most variable of the analyzed fragments was the intergenic spacer rpoBtrnC. Based on rpoBtrnC sequence 17 haplotypes in P. sativum and four haplotypes in P. fulvum were revealed. The cpDNA sequencing data were used for a phylogenetic analysis. On the obtained tree Vavilovia formosa accession formed a separate branch from pea accessions. All Pisum accessions fall in one cluster, split into distinct P. sativum and P. fulvum subclusters (BI = 99%).

Historical biogeography of Acer L. (Sapindaceae): genetic evidence for Out-of-Asia hypothesis with multiple dispersals to North America and Europe

Biogeography is the study of where, when, and how modern species evolved and diversified. Acer L. (maple) is one of the most diverse and widespread genera in the Northern Hemisphere. It comprises 124–156 species in the world, approximately 80% species of Acer are native in Asia. The current diversity center of Acer is not congruent with the distribution of the oldest fossils of the genus. Therefore, we herein used 84 species and subspecies to reconstruct the phylogeny and investigate the biogeographic history of Acer using nuclear ITS and three cpDNA fragments (psbA-trnH spacer, rpl16 intron, and trnL-trnF spacer) with maximum likelihood, maximum parsimony, and Bayesian inference methods. The analyses showed that the current diversity center and the origin center of Acer is Asia. Additionally, the North American and Euro-Mediterranean species originated from multiple sources from Asia via the North Atlantic Land Bridge and the Bering Land Bridge, and intercontinental migration has mainly occurred since the Miocene. This study not only provides a novel insight of the origin and dispersal routes of Acer but also exemplifies how past climatic changes affect the diversification-rates of Northern Hemisphere forest trees.

Molecular Phylogeography and Evolutionary History of the Endemic Species Corydalis hendersonii (Papaveraceae) on the Tibetan Plateau Inferred From Chloroplast DNA and ITS Sequence Variation

In response to past climatic changes, the species with different habits or adaptive traits likely have experienced very different evolutionary histories, especially for species that restricted to high mountain areas. In order to trace how Quaternary climatic oscillations affected range distributions and intraspecific divergence of such alpine plants on the Tibetan Plateau, here, we investigated maternally inherited chloroplast DNA (cpDNA) markers and biparentally inherited nuclear ribosomal internal transcribed spacer (ITS) DNA variations and aimed to explore the phylogeographic history of the endemic alpine species Corydalis hendersonii Hemsl. (Papaveraceae). We sequenced four cpDNA fragments (trnS-trnG, trnT-trnL, atpH-atpI, and psbE-petL) and also the nuclear (ITS) region in 368 individuals from 30 populations across the species' range. The network and phylogenetic analysis based on cpDNA variations identified 15 chlorotypes that cluster into three distinct clades. However, our nuclear DNA results demonstrated that there were four genetic/geographical groups within C. hendersonii. Some common and highly divergent cpDNA and ITS haplotypes were distributed in the populations of central and northeastern Tibetan Plateau, and the highest nucleotide diversity and genetic differentiation were detected in the central region. Demographic tests further indicated that the populations of southwestern and western Tibet may have experienced recent range expansion, which most likely occurred during the last glacial maximum (LGM) and continued its expansion after the beginning of the Holocene. These two different groups of this species may have derived from potential refugia that existed in the central and/or northeastern regions of Tibet during recent interglacial periods. In addition, our AMOVA analyses detected high genetic differentiation along with the whole sampling range. Also, distinct phylogeographic structures were detected among populations of C. hendersonii based on both of cpDNA and ITS variation. These findings shed new light on the importance of climatic oscillations during Quaternary and complex local topography as causes of intraspecific diversification and demographic changes within cold-tolerant herbs in the Tibetan Plateau biodiversity hotspot.

Phylogeography and population dynamics of an endemic oak (Quercus fabri Hance) in subtropical China revealed by molecular data and ecological niche modeling

High numbers of endemic plants exist in subtropical China, but there have been few investigations of the phylogeography and dynamic history for endemic tree species in this region. In this study, we explored the phylogeographic pattern of Quercus fabri, an endemic oak species in subtropical China, to assess the effects of past geographical and climatic changes on the evolutionary history of this deciduous species using three chloroplast DNA (cpDNA) fragments and the internal transcribed spacer region (ITS) and ecological niche modeling (ENM). The cpDNA fragments differentiated three phylogeographical groups among Q. fabri populations. A BEAST-derived chronogram based on the cpDNA haplotypes indicated that the initial divergence of Q. fabri probably occurred during the late Miocene (12.33 Ma), whereas haplotype divergence time could date back to the late Pliocene to early Pleistocene. ENM showed that the suitable areas contracted during the last glacial maximum (LGM) and expanded in the warm period during the Pleistocene. Consistently, both the cpDNA and ITS datasets suggested that population expansion occurred after the LGM period (ca. 13–14 kyr) for Q. fabri. Demographical analyses based on multiple genetic datasets and a coalescent-based method also suggested a shrinkage–expansion trend for the effective population sizes of Q. fabri during the glacial–interglacial periods in the Quaternary. Our results suggest that the Neogene and Pleistocene were important periods for triggering species differentiation and intraspecific diversification in Q. fabri, thereby providing insights into the effects of environmental changes in the Quaternary on the population dynamics of endemic tree species in subtropical China.

Relict populations and Central European glacial refugia: The case of Rhododendron ferrugineum (Ericaceae)

AbstractAimTo assess the origin and genetic relationship of the northernmost population of Rhododendron ferrugineum in the Karkonosze Mts. (the Sudetes), located 350 km north of the previously acknowledged species limit, in the context of the whole species range; to discuss, based on this case study, the glacial history of the Central European mountain flora and importance of rare, relict populations for biogeographical inference and diversity conservation.LocationEuropean mountains: Sudetes, Alps, Pyrenees.MethodsWe sampled 90 individuals from 26 R. ferrugineum populations spanning the whole species’ range. To infer genetic structure, diversity and relationships among populations and isolated parts of the geographical range, we applied genome‐wide amplified fragment length polymorphism (AFLP) genotyping and sequencing of four cpDNA fragments and nrDNA ITS region.ResultsOur AFLP analysis revealed four distinct genetic groups in R. ferrugineum, one formed by the northernmost population from (a) the Karkonosze Mts. (the Sudetes) and the other three comprising populations from the (b) Western Alps and Pyrenees, (c) South‐Westernmost Alps and (d) Eastern Alps. Genetic isolation of the Karkonosze population was further corroborated by a repetitive pattern differentiation in plastid DNA sequences, otherwise almost monomorphic across the range. Population from the Karkonosze was most closely related to those from the Western Alps and not from the geographically closest Eastern Alps.Main conclusionsWe show that the population of R. ferrugineum in the Karkonosze Mts. is a glacial relict and not a recently established or introduced population. Its distinctiveness and high genetic diversity show that it represents a northern, previously undetected, genetic lineage of the species in Europe, which persisted through climatic changes in a small but stable microrefugium. Our case study highlights the importance of an adequate coverage of a species’ range in phylogeographical studies and the significance of peripheral parts of the range to reveal biogeographical history of lineages. It also supports the Karkonosze Mountains as an important refugium for the Central European mountain flora.

PLACEMENT OF Syzygium boerlagei (Merr.) Govaerts (MYRTACEAE) CONFIRMED WITH ATPB-RBCL INTERGENIC SPACER

A molecular analysis was conducted to determine whether Eugenia boerlagei Merr. (Myrtaceae) belongs to genus Eugenia or Syzygium based on sequences of cpDNA fragments namely atpB-rbcL intergenic spacer. The study used seven specimens of Syzygium sect. Jambosa, three of Syzygium sect. Syzygium, two of Eugenia s.s. and one of Eugenia boerlagei Merr. with Baeckea ovalifolia and B. tuberculata as the outgroup. The results show that Eugenia boerlagei is appropriately placed under the genus Syzygium.

Phylogeography of two closely related species of Allium endemic to East Asia: Population evolution in response to climate oscillations.

This study investigated the effects of climate oscillations on the evolution of two closely related Allium species, A. neriniflorum and A. tubiflorum. We sequenced three cp DNA (cpDNA) fragments (rps16, rpl32‐trnL, and trnD‐trnT, together approximately 2,500 bp in length) of two closely related Allium species, with samples from 367 individuals in 47 populations distributed across the total range of these species. The interspecific and intraspecific divergence times of the two species were in the Quaternary glaciation. The population divergence was high for the cpDNA variation, suggesting a significant phylogeographic structure (NST = 0.844, GST = 0.798, p < 0.05). Remarkable ecological differentiation was also revealed by Niche models and statistical analyses. Our results suggest the speciation event of the two species was triggered by violent climatic changes during the Quaternary glaciation.

Whole-Genome Comparison Reveals Heterogeneous Divergence and Mutation Hotspots in Chloroplast Genome of Eucommia ulmoides Oliver.

Eucommia ulmoides (E. ulmoides), the sole species of Eucommiaceae with high importance of medicinal and industrial values, is a Tertiary relic plant that is endemic to China. However, the population genetics study of E. ulmoides lags far behind largely due to the scarcity of genomic data. In this study, one complete chloroplast (cp) genome of E. ulmoides was generated via the genome skimming approach and compared to another available E. ulmoides cp genome comprehensively at the genome scale. We found that the structure of the cp genome in E. ulmoides was highly consistent with genome size variation which might result from DNA repeat variations in the two E. ulmoides cp genomes. Heterogeneous sequence divergence patterns were revealed in different regions of the E. ulmoides cp genomes, with most (59 out of 75) of the detected SNPs (single nucleotide polymorphisms) located in the gene regions, whereas most (50 out of 80) of the indels (insertions/deletions) were distributed in the intergenic spacers. In addition, we also found that all the 40 putative coding-region-located SNPs were synonymous mutations. A total of 71 polymorphic cpDNA fragments were further identified, among which 20 loci were selected as potential molecular markers for subsequent population genetics studies of E. ulmoides. Moreover, eight polymorphic cpSSR loci were also developed. The sister relationship between E. ulmoides and Aucuba japonica in Garryales was also confirmed based on the cp phylogenomic analyses. Overall, this study will shed new light on the conservation genomics of this endangered plant in the future.

The impact of phylogenetic uncertainty on the metrics of community phylogenetic structure

Phylogeny has been widely used to quantify community phylogenetic structure and to infer the underlying mechanism. Many studies, however, neglected phylogeny uncertainty and its potential impact on community phylogenetic structure. In this study, we explored the potential impact of phylogenetic uncertainty among 150 species in a 20 hm2 plot in Tiantong, Zhejiang. One consensus tree and 999 phylogenetic trees representing the phylogenetic uncertainty were estimated based on two cpDNA fragments (rbcL and matK). Combined with the species distribution data, community phylogenetic structure was quantified by two common indices (NRI and NTI) and their significances were tested by the independent swap null model. Our results showed that tree topology and node age showed a large uncertainty. The uncertainty was larger for young species and significantly increased with mean phylogenetic distance. Phylogenetic uncertainty increased the variation of both standardized NRI and NTI in each quadrat. These impacts were independent between both indices in either spatial pattern or the degree of impact. NRI was more sensitive than NTI to the uncertainty. At community scale, phylogenetic uncertainty also affected the variation of the mean standardized NRI and NTI of all quadrats, with mean standard deviation of 0.37 and 0.077, respectively. Such a result suggests that mean standardized NRI at community level was more vulnerable to the phylogenetic uncertainty, which is consistent with the result at the sample level. Our findings showed that phylogenetic uncertainty could add different variation into the NRI and NTI series indices and might increase biases in the quantification of community phylogenetic structure and its underlying ecological processes. Our results implied that non-random community phylogenetic structure was probably overestimated in the previous studies which ignored phylogenetic uncertainty.

Study on chloroplast DNA diversity of cultivated and wild pears (Pyrus L.) in Northern China

Eight pairs of chloroplast DNA (cpDNA) universal primers selected from 34 pairs were used to assess the genetic diversity of 132 pear accessions in Northern China. Among them, six amplified cpDNA fragments showed genetic diversity. A total of 24 variable sites, including 1 singleton variable site and 23 parsimony informative sites, as well as 21 insertion-deletion fragments, were obtained from the combined cpDNA sequences (5309–5535 bp). Two trnL-trnF-487 haplotypes, five trnL-trnF-413 haplotypes, five rbcL haplotypes, six trnS-psbC haplotypes, eight accD-psaI haplotypes and 12 rps16-trnQ haplotypes were identified among the individuals. Twenty-one haplotypes were identified based on the combined fragments. The values of nucleotide diversity (Pi), average number of nucleotide differences (k) and haplotype diversity (Hd) were 0.00070, 3.56408 and 0.7960, respectively. No statistical significance was detected in Tajima’s D test. Remarkably, the important cpDNA haplotypes and their representing accessions were identified clearly in this study. H_19 was considered as one of the ancient haplotypes and was a divergent centre. H_16 was the most common haplotype of the wild accessions. H_2 was the haplotype representing the most pear germplasm resources (46 cultivars and two wild Ussurian Pear accessions), followed by haplotype H_5 (30 cultivars, two wild Ussurian Pear accessions and four sand pears in outgroups) representing the cultivars ‘Dangshan Suli’ and ‘Yali’, which harbour the largest and the second largest cultivation areas in China. More importantly, this study demonstrated, for the first time, the supposed evolution routes of Pyrus based on cpDNA divergence in the background of pear phylogeny in Northern China.

Genetic and morphological variability analysis revealed a complex network in South-Eastern Sicilian Helichrysum occurrences

The Helichrysum occurrences spread over Hyblaean area (Sicily) are characterized by an unusual richness of morphotype variants which ancestral origin is still unclear. Morphological and genetic variability of seven populations attributable to six taxonomic entities (Helichrysum archimedeum, H. stoechas subsp. conglobatum, H. stoechas subsp. barrelieri, H. hyblaeum, H. pendulum and H. nebrodense) was examined, in order to highlight their relationships within the Helichrysum sect. Stoechadina. Morphological analysis showed a clear isolation of populations referred to H. pendulum, H. nebrodense and H. stoechas subsp. conglobatum. Whereas, genetic structure profiling, with 21 ISSRs, displayed a strong genetic relationship within H. stoechas and H. archimedeum accessions, where the latter exhibited evident genetic similarity with H. hyblaeum. Analysis of rbcL cpDNA fragment allowed to identify a shared haplotype between H. stoechas and H. rupestre species, while, through the construction of haplotype network based on trnL-F/rpl32 region, the sampled accessions were properly differentiated as confirmed by the cpDNA consensus network. These results confirmed the high affinity of H. archimedeum with other H. stoechas populations and the closer relationship of H. hyblaeum to H. stoechas respect to H. rupestre.