Chloroplast Intergenic Spacers Research Articles

Development of high-resolution chloroplast markers for intraspecific phylogeographic studies of Phaeocystis globosa

Phaeocystis globosa is an important harmful algal bloom causative species distributing widely in temperate and tropical coastal waters in the world. The morphological, physiological, and biochemical characteristics are different among geographic strains, which can not be distinguished with nuclear ribosomal DNA markers at present. Therefore, the genetic distance and phylogeographic relationships of nuclear 28S rDNA D1-D2 and ITS regions, and three chloroplast intergenic spacers (petN-trnS1, trnM1-psbA, and rbcS-rpl27) were analyzed and compared among 13 strains of P. globosa isolated from the Pacific Ocean and Atlantic Ocean in this study. In addition, the nucleotide polymorphisms of 28S rDNA D1-D2, ITS, and rbcS-rpl27 regions were evaluated in two P. globosa strains. The various levels of nucleotide polymorphism were in the nuclear 28S rDNA D1-D2 region and ITS region, but no polymorphism was in the chloroplast rbcS-rpl27 intergenic spacer. A reasonable intraspecific phylogeographic relationship was presented by rbcS-rpl27 intergenic spacer, which had the strongest distinction to geographic strains compared to those of 28S rDNA D1-D2 and ITS regions. In the phylogenetic tree of rbcS-rpl27 intergenic spacer, the two strains from the North Sea of the Atlantic Ocean were divided firstly from the species of P. globosa, and then formed an independent clade, while the other Atlantic strains and all of Pacific strains joined up to build the other clade. It was implied that at least two genetically distant populations of P. globosa existed in the Atlantic coastal regions. This study provided a high-resolution chloroplast marker to analyze intraspecific phylogeographic populations of P. globosa, and preliminarily clarified the genetic relationships of the Pacific and Atlantic strains of P. globosa.

Natural hybridization between Phyllagathis and Sporoxeia species produces a hybrid without reproductive organs.

Natural hybridization plays important roles in plant evolution and speciation. In this study, we sequenced ribosomal internal transcribed spacer (nrITS), four low-copy nuclear genes (Dbr1, SOS4a, SOS4b and PCRF1) and the chloroplast intergenic spacer trnV-trnM to test the hypothesis of hybridization between two species of Phyllagathis and Sporoxeia (Sonerileae/Dissochaeteae, Melastomataceae). Our results provided compelling evidence for the hybridization hypothesis. All hybrid individuals sampled were first-generation hybrids. The failure of flower production in the F1 hybrid individuals may work as the barrier preventing later-generation hybridization or backcross. Analysis of the chloroplast trnV-trnM sequences showed that the hybridization is bidirectional with S. petelotii as the major maternal parent. Several factors, such as sympatry, similar habitat preference, overlapping flowering season and shared pollinators, might have contributed to this hybridization event. The "intergeneric" hybridization reported in this study suggests close relationship between P. longicalcarata and S. petelotii.

What nature separated, and human joined together: About a spontaneous hybridization between two allopatric dogwood species (Cornus controversa and C. alternifolia).

In this study, possible hybridization between two allopatric species, Cornus controversa and Cornus alternifolia, was explored using molecular and morphological approaches. Scanning electron microscope analyses of the adaxial and the abaxial leaf surfaces yielded a few new not yet described characters typical for the particular species and intermediate for hybrids. With the use of 14 Random Amplified Polymorphic DNA and 5 Amplified Fragment Length Polymorphism primer combinations, 44 fragments species specific to C. controversa and 51 species specific to C. alternifolia were obtained. Most of these bands were also found in putative hybrids. All clustering analyses based on binary data combined from both methods confirmed a separate and intermediate status of the hybrids. Hybrid index estimates for hybrids C1-C5 indicated that all were the first generation of offspring (F1). Chloroplast intergenic spacers (trnF-trnL and psbC-trnS) were used to infer the hybridization direction. Based on the assumption of maternal inheritance of chloroplast DNA, C. controversa seems to be the maternal parent of the hybrid. Internal transcribed spacer sequences of the five hybrids analyzed here indicated higher similarity with the sequences of C. controversa (all shared the majority of its single nucleotide polymorphisms). Sequence analysis of PI-like genes fully confirmed the hybrid origin of C1-C5 hybrids. Our results also showed that two specimens in the C. alternifolia group, A1 and A3, are not free of introgression. They are probably repeated backcrosses toward C. alternifolia. Furthermore, molecular data seem to point not only to unidirectional introgression toward C. controversa (the presence of hybrids) but to bidirectional introgression as well, since the presence of markers specific for C. controversa in the profiles of C. alternifolia specimen A3 was observed.

Potential of Lemnoideae Species for Phytoremediation of Fresh Water with Elevated Manganese Concentration

Background . Wastewater treatment using physical, chemical, and biological methods is primary solution for the reduction of water pollution that reaching the critical thresholds. The members of subfamily Lemnoideae , commonly called duckweed, are considered the most efficient aquatic plants for wastewater remediation. Although properties of duckweed to survive in water with high concentration of heavy metal ions such as chromium, cobalt, lead, nickel and cuprum are well documented, the growth of duckweed in water with high concentrations of manganese and the efficiency of retention of manganese from water by these species has not been estimated. Objective . Four duckweed species ( Spirodela polyrhiza , Landoltia punctata , Lemna aequinoctialis , and L. turionifera ) were used for establishment of influence Mn on their vitality and growth and for studying their potential for phytoremediation of fresh water with elevated manganese concentration. Methods . Duckweed collected in Eastern China was introduced in tissue culture in vitro by surface sterilization. The identification of the collected duckweed species was determined by DNA barcoding using primers specific for chloroplast intergenic spacers atpF - atpH (ATP) and psbK - psbL (PSB). The experiments for establishment of influence Mn on duckweed growth carried out in aseptic condition. To determinate concentration of Mn, the samples of different water type (Hongze Lake, ponds around Hongze Lake, Huaian local municipal sewage plant and industrial sewage plant) were analyzed by the Inductively Coupled Plasma Optical Emission Spectrometry. Results . The most sensitive duckweed to Mn was S. polyzhiza , the first characteristic symptoms of toxicity like brown spots have appeared when concentration of Mn was 40 mg/L, the concentration 200 mg/L Mn resulted in chlorosis and death of fronds. L . aequinoctialis and L. turionifera had similar effects in SH medium supplemented with 650 mg/L and 975 mg/L Mn, respectively. L . punctata was the most tolerant duckweed to Mn plants continued to grow even at concentration 975 mg/L. Response of duckweed on Mn was dependent on availability of nitrogen in nutrient medium. Using four duckweed species for treatment of water containing 4.12 mg/L Mn allowed to reduce concentration until safe level of standard (0.1 mg/L Mn). Conclusions . All investigated duckweed species ( S. polyrhiza, L. punctata , L. aequinoctialis , and L. turionifera ) were characterized by a high level of resistance to manganese, especially L. punctata. Response of duckweed on Mn was dependent on availability of nitrogen in nutrient medium. The tested species of subfamily Lemnoideae were high effective for phytoremediation of water with elevated manganese concentration.

Phylogeny and biogeography of the Pleistocene Holarctic steppe and semi-desert goosefoot plant Krascheninnikovia ceratoides

Krascheninnikovia ceratoides (Chenopodiaceae) is a steppe and semi-desert plant with two subspecies, K. ceratoides subsp. ceratoides, which is widespread in Eurasia, and K. ceratoides subsp. lanata, which grows in western and central North America. A few disjunct populations of K. ceratoides subsp. ceratoides are found in Anatolia, Europe and North Africa to the west of its otherwise continuous Eurasian distribution. To understand the evolutionary history of this characteristic steppe and semi-desert plant, we analysed its phylogeny and biogeography. We sequenced several loci including ITS, ETS and the chloroplast intergenic spacer regions atpB-rbcL, rpl32-trnL and trnL-trnF to establish a time-calibrated phylogeny and reconstruct intraspecific relationships. Furthermore, we identified the ploidy level of individuals. While diploid, tetraploid and hexaploid individuals have been reported in the literature, we were only able to find diploids and tetraploids. The diploids were found in the east of Mongolia, Kazakhstan, Kyrgyzstan, Russia and the USA. The tetraploids were located in the west of Mongolia, Kazakhstan, Tajikistan, Russia, and Europe. Populations were uniformly di- or tetraploid. Our results indicate that the species may have spread from the area of Mongolia, northern China and Middle Asia (i.e., the Altai Mountains region) in two opposite directions – on the one hand, diploids migrated to the east, to eastern Asia and North America, and on the other hand diploids and tetraploids migrated to the west, to western Asia and Europe. Fossil-calibrated gene trees were used to estimate the age of the species. Diversification within the species is probably of Pleistocene age. Our dated analysis indicates that the first split among extant lineages of the species took place in the Early Pleistocene (Gelasian). The spread of the main lineages is likely related to major phases of steppe and semi-desert expansions during glacial periods of the Pleistocene.

A pre-Miocene Irano-Turanian cradle: Origin and diversification of the species-rich monocot genus Gagea (Liliaceae).

The Irano‐Turanian (IT) floristic region is considered an important center of origin for many taxa. However, there is a lack of studies dealing with typical IT genera that also occur in neighboring areas. The species‐rich monocot genus Gagea Salisb. shows a center of diversity in IT region and a distribution in adjacent regions, therefore representing a good study object to investigate spatial and temporal relationships among IT region and its neighboring areas (East Asia, Euro‐Siberia, Himalaya, and Mediterranean). We aimed at (a) testing the origin of the genus and of its major lineages in the IT region, (b) reconstructing divergence times, and (c) reconstructing colonization events. To address these problems, sequences of the ribosomal DNA internal transcribed spacer (ITS) region of 418 individuals and chloroplast intergenic spacers sequences (psbA‐trnH, trnL‐trnF) of 497 individuals, representing 116 species from all sections of the genus and nearly its entire distribution area were analyzed. Divergence times were estimated under a random molecular clock based on nrITS phylogeny, which was the most complete data set regarding the representation of species and distribution areas. Ancestral distribution ranges were estimated for the nrITS data set as well as for a combined data set, revealing that Gagea most likely originated in southwestern Asia. This genus first diversified there starting in the Early Miocene. In the Middle Miocene, Gagea migrated to the Mediterranean and to East Asia, while migration into Euro‐Siberia took place in the Late Miocene. During the Pleistocene, the Arctic was colonized and Gagea serotina, the most widespread species, reached North America. The Mediterranean basin was colonized multiple times from southwestern Asia or Euro‐Siberia. Most of the currently existing species originated during the last 3 Ma.

Molecular identification of natural hybridization between Melastoma malabathricum and Melastoama beccarianum in Sarawak, Malaysia.

Hybridization is very common in flowering plants and it plays a significant role in plant evolution and adaptation. Melastoma L. (Melastomataceae) comprises about 80–90 species in tropical Asia and Oceania, among which 41 species occur in Borneo. Natural hybridization is frequently reported in Melastoma in China, but so far there have been no confirmed cases of hybridization in Southeast Asia (including Borneo), where most species occur. Here, we identified a case of natural hybridization between Melastoma malabathricum L. and Melastoma beccarianum Cogn. in Sarawak, Malaysia, by using sequence data of three nuclear genes and one chloroplast intergenic spacer. Melastoma malabathricum is the most widespread species of this genus, occurring in almost the whole range of this genus, while M. beccarianum is a local species endemic to northern Borneo. Our results showed that natural hybridization and introgression occur between M. malabathricum and M. beccarianum, and the introgression was asymmetrical, mainly from M. malabathricum to M. beccarianum. As adaptive traits can be transferred by introgression, our study suggests that natural hybridization should be a significant mechanism for the evolution and adaptation of Melastoma in Southeast Asia. However, introgression from the common species M. malabathricum to the relatively rare species M. beccarianum may cause the decline of M. beccarianum, incurring conservation concern. With a large number of species of Melastoma and almost year‐around flowering in Southeast Asia, more cases of natural hybridization are expected to be found and identified in near future.

Phylogeography and Ecological Niche Modeling Reveal Reduced Genetic Diversity and Colonization Patterns of Skunk Cabbage (Symplocarpus foetidus; Araceae) From Glacial Refugia in Eastern North America.

Alternating glacial and interglacial periods during the Quaternary have dramatically affected the distribution and population genetic structure of plant and animal species throughout the northern hemisphere. Surprisingly, little is known about the post-glacial recolonization history of wetland herbaceous perennials that are widely distributed in the understory of deciduous or mixed deciduous-evergreen forests in eastern North America. In this study, we investigated infraspecific variation among 32 populations of skunk cabbage, Symplocarpus foetidus, to test the hypothesis that the extant species diversity of skunk cabbage is the result of a post-glacial range expansion from southern refugia during the Quaternary Ice Age. A total of 4041 base pairs (bp) of the chloroplast intergenic spacer region (cpDNA) was sequenced from 485 individuals sampled from glaciated (18 populations, 275 individuals) and unglaciated (14 populations, 210 individuals) regions east and west of the Appalachian Mountains. Haplotype number, haplotype diversity, and nucleotide diversity were calculated, and genetic variation within and among populations was assessed by analysis of molecular variance (AMOVA). The geographic pattern of genetic differentiation was further investigated with a spatial analysis of molecular variance (SAMOVA). A total of eight haplotypes and three genetic groups (SAMOVA) were recovered and a much higher haplotype number (eight haplotypes) and haplotype diversity (0.7425) was observed in unglaciated compared to glaciated populations (five haplotypes, haplotype diversity = 0.6099). All haplotypes found in glaciated regions represented a subset of haplotypes found in unglaciated regions. Haplotypes of S. foetidus likely diverged during the Tertiary (mid-Miocene and late Pliocene), predating the last glacial maximum (LGM). Predictions based on ecological niche modeling (ENM) suggested that there was considerably less suitable habitat for skunk cabbage during the LGM, and the habitat range was further south compared to the current distribution. Reduced variation and a subset of haplotypes in glaciated regions suggest a founder effect associated with range expansion via long-distance seed dispersal. Our results do not support the “Driftless Area” scenario for the northern refugium, rather the data suggest a “Northeastern” refugium near the southernmost extent of the LGM.

Phylogeny of the gymnosperm genus Cycas L. (Cycadaceae) as inferred from plastid and nuclear loci based on a large-scale sampling: Evolutionary relationships and taxonomical implications

The gymnosperm genus Cycas is the sole member of Cycadaceae, and is the largest genus of extant cycads. There are about 115 accepted Cycas species mainly distributed in the paleotropics. Based on morphology, the genus has been divided into six sections and eight subsections, but this taxonomy has not yet been tested in a molecular phylogenetic framework. Although the monophyly of Cycas is broadly accepted, the intrageneric relationships inferred from previous molecular phylogenetic analyses are unclear due to insufficient sampling or uninformative DNA sequence data. In this study, we reconstructed a phylogeny of Cycas using four chloroplast intergenic spacers and seven low-copy nuclear genes and sampling 90% of extant Cycas species. The maximum likelihood and Bayesian inference phylogenies suggest: (1) matrices of either concatenated cpDNA markers or of concatenated nDNA lack sufficient informative sites to resolve the phylogeny alone, however, the phylogeny from the combined cpDNA-nDNA dataset suggests the genus can be roughly divided into 13 clades and six sections that are in agreement with the current classification of the genus; (2) although with partial support, a clade combining sections Panzhihuaenses + Asiorientales is resolved as the earliest diverging branch; (3) section Stangerioides is not monophyletic because the species resolve as a grade; (4) section Indosinenses is not monophyletic as it includes Cycas macrocarpa and C. pranburiensis from section Cycas; (5) section Cycas is the most derived group and its subgroups correspond with geography.

Multi-locus Analyses Indicate that Melastoma dendrisetosum, an Endemic and Endangered Shrub in Hainan, is a Distinct Species

Melastoma dendrisetosum is an extremely narrowly distributed taxon endemic to the Diaoluo Mountain region in Hainan, China, and was recently taxonomically incorporated under a widespread species, M. sanguineum. Based on its distinct morphological traits, especially the indumentumon the hypanthium, we propose that it should represent a distinct species. In this study,we sequenced six partial nuclear genes and two chloroplast intergenic spacers of M. dendrisetosum and three other morphologically similar species, M. sanguineum, M. penicillatum, and M. normale, to clarify its taxonomic status. M. dendrisetosum exhibited an extremely low level of genetic diversity, and was well separated from the three other species with no shared haplotypes at all six nuclear genes, supporting the hypothesis that it is a distinct species. This finding was also supported by the Bayesian clustering-based STRUCTURE analysis, phylogenetic analysis, and Bayesian species delimitation analysis. We further propose that if a formal assessment were performed, M. dendrisetosum would be categorized as ‘Critically Endangered’ under the IUCN criteria, due to its extremely small population and declining habitat quality. We suggest that the conservation of M. dendrisetosum be prioritized, and road construction in its range should be avoided, to protect this distinct and critically endangered species.

Phylogeography of Eomecon chionantha in subtropical China: the dual roles of the Nanling Mountains as a glacial refugium and a dispersal corridor

BackgroundMountains have not only provided refuge for species, but also offered dispersal corridors during the Neogene and Quaternary global climate changes. Compared with a plethora of studies on the refuge role of China’s mountain ranges, their dispersal corridor role has received little attention in plant phylogeographic studies. Using phylogeographic data of Eomecon chionantha Hance (Papaveraceae), this study explicitly tested whether the Nanling Mountains, which spans from west to east for more than 1000 km in subtropical China, could have functioned as a dispersal corridor during the late Quaternary in addition to a glacial refugium.ResultsOur analyses revealed a range-wide lack of phylogeographic structure in E. chionantha across three kinds of molecular markers [two chloroplast intergenic spacers, nuclear ribosomal internal transcribed spacer (nrITS), and six nuclear microsatellite loci]. Demographic inferences based on chloroplast and nrITS sequences indicated that E. chionantha could have experienced a strong postglacial range expansion between 6000 and 1000 years ago. Species distribution modelling showed that the Nanling Mountains and the eastern Yungui Plateau were the glacial refugia of E. chionantha. Reconstruction of dispersal corridors indicated that the Nanling Mountains also have acted as a corridor of population connectivity for E. chionantha during the late Quaternary.ConclusionsOur results suggest that the Nanling Mountains may acted dual roles as a dispersal corridor in east-west direction and as a glacial refugium in subtropical China during the late Quaternary. The population connectivity mediated by the mountain range and a strong postglacial range expansion are the most likely reasons for the lack of phylogeographic structure in E. chionantha. The hypothesis of dual roles of the mountain range presented here sheds new insights into the phylogeographic patterns of organisms in subtropical China.

Distinguishing Orchid Species by DNA Barcoding: Increasing the Resolution of Population Studies in Plant Biology.

Increasing the resolution of population studies in plant biology is one of the leading frontiers for omics sciences. One of the most pervasive challenges in molecular phylogenetics is the incongruence between phylogenies obtained using different data sets such as individual genes [like ribulose bisphosphate carboxylase large chain (rbcL) and maturase K (matK)] and intergenic spacers (IGS) [like nuclear ribosomal internal transcribed spacer 1 (nrITS 1) and 2 (nrITS 2), and chloroplast IGS between transfer RNA for leucine and phenylalanine (cp trnL-trnF IGS)]. To solve this challenge, we have screened the four well-established candidate gene sequences (i.e., rbcL, matK, trnL-trnF IGS, and 18S-ITS1-5.8S-ITS2-28S nrDNA) of 65 Indian orchid species. We also have included 31 different species of Dendrobium to identify the suitable locus for resolving the phylogeny-related problem below the taxonomic rank of genus. The Consortium for the Barcode of Life has recommended the locus rbcL and matK for barcoding of all land plants, including orchids. However, in this study, matK and rbcL (species resolving capacity 52% and 48%, respectively) were found to work above the taxonomic limit of genus, and thus cannot be considered a suitable tool to resolve closely related species of Dendrobium, whereas, we found that the locus 18S-ITS1-5.8S-ITS2-28S nrDNA is the best choice with the highest species resolving ability (95.23%) and the highest mean Kimura 2-parameter distance (254 for intergeneric and 144 for intrageneric) for phylogeny construction, and thus have been taken as the most promising single-locus barcode for orchids.

Genetic variability of Eurasian Nuphar species unravels possible routes in which freshwater plants could fill their wide areas

Possible routes in which aquatic plants have spreaded across their vast ranges from ancestral areas and refugia still remain poorly known. We have chosen Eurasian yellow water-lilies (Nuphar Sm. sect. Nuphar, Nymphaeaceae) as a model group for a wide-scale phylogeographical study as they represent a common component of the continental freshwater flora. To elucidate the history of diversification of the section Nuphar, we sequenced nuclear ribosomal ITS and chloroplast intergenic spacers (trnH-psbA, trnL–trnF) from 117 localities from almost all the section area. Sequence data were analysed with a statistical parsimony approach using the network algorithm. We also incorporated data on morphological variation and pollen viability. The section Nuphar originated and diversified into several evolutionary lineages in Eastern Asia. Two lineages, represented by N. pumila and N. lutea, spread from the Russian Far East westwards to Siberia and further to Europe. Nuphar pumila also migrated from the Far East to North America (where it is referred to as N. microphylla). Nuphar lutea colonized deglaciated north-eastern Europe not only from the south, but also from the Siberian refugia. Nuphar lutea and N. pumila hybridize rarely in Eastern Europe and Asia and warrant species status. We provide the first evidence, based on a phylogeographical approach, for the origin and diversification of aquatic flowering plants in Eastern Asia. Our results indicate that phylogeographic analysis on the area-wide scale is one of the most efficient ways to deduce possible routes in which aquatic plants have spread across their total range from ancestral areas and refugia.

Phylogenic Analysis and Evaluation of Ephedra Plants and Aconites for Medicinal Use.

Although Kampo medicine is now fully integrated into the modern Japanese healthcare system, most Kampo formulations depend on imported crude drugs from limited foreign areas. To prepare for possible shortages of crude drugs in the future, a wider scope for the supply of medicinal plants is necessary. We conducted field research and collaborated with international laboratories for phylogenic analysis and evaluation of medicinal plant resources. Our research on ephedra plants from a wide region of Eurasia has, for example, confirmed their phylogenic structure: based on DNA sequencing analysis of nuclear ribosomal internal transcribed spacer region 1 (ITS1) as well as the chloroplast intergenic spacer between trnL and trnF (trnL-F), the 8 major Chinese species and related plants grown on the continent could be divided into 3 groups. Additionally, Ephredra sinica was found to be synonymous with Ephredra dahurica and was reduced to a subspecies of Ephredra distachya. Furthermore, Ephredra likiangensis and Ephredra gerardiana, which are grouped in separate phylogenic trees, would be good candidates for medicinal material. Aconites from Hokkaido, as an example of domestic plants reviewed, were collected for phylogenic and aconitine alkaloid content analysis. The phylogenic analysis of nr ITSs revealed that the majority of specimens were genetically similar. However, the aconitine alkaloid content of the tuberous roots demonstrated that specimens from different habitats had varying alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profiles of these genetically similar specimens to diversify.

Genetic diversity and population structure in the narrow endemic Chinese walnut Juglans hopeiensis Hu: implications for conservation

The conservation of narrow endemic species relies on accurate information regarding their population structure. Juglans hopeiensis Hu (Ma walnut), found only in Hebei province, Beijing, and Tianjin, China, is a threatened tree species valued commercially for its nut and wood. Sequences of two maternally inherited mitochondrial markers and two maternally inherited chloroplast intergenic spacers, three nuclear DNA sequences, and allele sizes from 11 microsatellites were obtained from 108 individuals of J. hopeiensis, Juglans regia, and Juglans mandshurica. Haplotype networks were constructed using NETWORK. Genetic diversity, population differentiation, and analysis of molecular variance (AMOVA) were used to determine genetic structure. MEGA was used to construct phylogenetic trees. Genetic diversity of J. hopeiensis was moderate based on nuclear DNA, but low based on uniparentally inherited mitochondrial DNA and chloroplast DNA. Haplotype networks showed that J. hopeiensis haplotypes were different than haplotypes found in J. regia and J. mandshurica. Allelic variants in nuclear genes that were shared among J. hopeiensis populations were not found in J. regia or J. mandshurica. Sampled populations of J. hopeiensis showed clear genetic structure. The maximum parsimony (MP) tree showed J. hopeiensis to be distinct from J. mandshurica but threatened by hybridization with J. regia and J. mandshurica. J. hopeiensis populations are strongly differentiated from sympatric Juglans species, but they are threatened by small population sizes and hybridization.

Similar Morphologies but Different Origins: Hybrid Status of Two More Semi-creeping Taxa of Melastoma.

Inferring the origins of hybrid taxa based on morphology alone is difficult because morphologically similar hybrids can arise from hybridization between different populations of the same parental species or be produced by hybridization of different parental species. In this study, we investigated the origins of two semi-creeping taxa in Melastoma, which are morphologically similar to a natural hybrid, M. intermedium, by sequencing a chloroplast intergenic spacer, nuclear ribosomal internal transcribed spacer and two low-copy nuclear genes (tpi and cam) in these taxa and their putative parental species. Our sequence analysis provides compelling evidence for the hybrid status of the two semi-creeping taxa: one originating from hybridization between M. dodecandrum and M. malabathricum, and the other between M. dodecandrum and M. normale. The origins of these hybrids are therefore clearly different from M. intermedium, and morphological similarity for the three hybrids is most likely due to their origins from hybridization between the same creeping species M. dodecandrum and a different erect species in each of the three cases. We also observed low rate of introgression from M. normale to M. dodecandrum, and genetic exchange between them may transfer adaptive traits to M. dodecandrum. Rare occurrence of these two hybrids may be due to small range overlaps between parental species in one case, and different flowering periods between parental species in the other.

Phylogeographic insights on the evolutionary breakdown of heterostyly.

The breakdown of heterostyly to homostyly is a classic system for the investigation of evolutionary transitions from outcrossing to selfing. Loss of sexual polymorphism is characterized by changes to population morph structure and floral morphology. Here, we used molecular phylogeography to investigate the geographical context for the breakdown process in Primula chungensis, a species with distylous and homostylous populations. We genotyped plants from 20 populations throughout the entire range in south-west China using the chloroplast intergenic spacer (trnL-trnF), nuclear internal transcribed spacer (ITS) and 10 nuclear microsatellite loci, and determined the genetic relationships among populations and the variation in floral traits associated with homostyle evolution. The marker data identified two multi-population lineages (Tibet and Sichuan) and one single-population lineage (Yunnan), a pattern consistent with at least two independent origins of homostyly. Evidence from flower and pollen size variation is consistent with the hypothesis that transitions to selfing have arisen by the same genetic mechanism involving recombination and/or mutation at the distyly linkage group. Nevertheless, flowers of homostylous lineages have followed divergent evolutionary trajectories following their origin, resulting in populations with both approach and reverse herkogamy. Our study illustrates a rare example of the near-complete replacement of sexual polymorphism by floral monomorphism in a heterostylous species.

Genetic structure and demographic history of Cycas chenii (Cycadaceae), an endangered species with extremely small populations

Geological activities and climate oscillations during the Quaternary period profoundly impacted the distribution of species in Southwest China. Some plant species may be harbored in refugia, such as the dry-hot valleys of Southwest China. Cycas chenii X. Gong & W. Zhou, a critically endangered cycad species, which grows under the canopy in subtropical evergreen broad-leaved forests along the upstream drainage area of the Red River, is endemic to this refugium. In this study, 60 individuals of C. chenii collected from six populations were analyzed by sequencing two chloroplast intergenic spacers (cpDNA: psbA-trnH and trnL-trnF) and two nuclear genes (PHYP and RBP-1). Results showed high genetic diversity at the species level, but low within-population genetic diversity and high interpopulation genetic differentiation. A Bayesian phylogenetic tree based on cpDNA showed that five chloroplast haplotypes were clustered into two clades, which corresponds to the division of the western and eastern bank of the Red River. These data indicate a possible role for the Red River as a geographic barrier to gene flow in C. chenii. Based on our findings, we propose appropriate in situ and ex situ conservation strategies for C. chenii.

Molecular Evidence for the Hybrid Origin of Ilex dabieshanensis (Aquifoliaceae).

Ilex, the largest genus of dioecious woody plants, is a good study system to assess the role of hybridization in speciation and evolution. Ilex dabieshanensis, a tree endemic to Dabieshan Mountains region, was initially described as a new species. Based on morphological intermediacy and sympatric distribution with its putative parental species, I. cornuta and I. latifolia, we proposed it as a natural hybrid between them. In this study, we sequenced one chloroplast intergenic spacer (trnH-psbA) and two nuclear genes (gapC and nepGS) in I. dabieshanensis and its putative parental species to test the hybrid origin hypothesis. Our results showed that there were one to two differentially fixed sequence differences between I. cornuta and I. latifolia at the two nuclear genes. Twelve of the 14 individuals of I. dabieshanensis exhibited additivity in chromatograms on these differentially fixed sites at both nuclear genes, and the remaining two exhibited additivity in chromatograms on the fixed site at only the nepGS gene. Except one haplotype of I. cornuta at the nepGS gene, all of the haplotypes of I. cornuta at the two nuclear genes were well separated from those of I. latifolia, and most haplotypes of I. dabieshanensis were shared with those of I. cornuta and I. latifolia. Phylogenetic analysis of these haplotypes was largely consistent with haplotype network analysis. I. cornuta and I. latifolia differed by two nucleotide substitutions in the chloroplast intergenic spacer, and 12 individuals of I. dabieshanensis had the same sequences as I. latifolia, while the remaining two were identical with I. cornuta. The molecular data provide convincing evidence for the hybrid origin of I. dabieshanensis and asymmetrical direction of hybridization. One haplotype of I. cornuta at the nepGS gene was nested with those of I. latifolia, indicating introgression to I. cornuta.

Genetic divergence between Mexican Opuntia accessions inferred by polymerase chain reaction-restriction fragment length polymorphism analysis.

Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico.