cpDNA Sequences Research Articles

Population genetic structure and demography of Magnolia kobus: variety borealis is not supported genetically

Species delimitations by morphological and by genetic markers are not always congruent. Magnolia kobus consists of two morphologically different varieties, kobus and borealis. The latter variety is characterized by larger leaves than the former. For the conservation of M. kobus genetic resources in natural forests, the relationships between morphological and genetic variation should be clarified. We investigated variations in nuclear microsatellites, chloroplast DNA (cpDNA) sequences and leaf morphological traits in 23 populations of M. kobus over the range of species. Two genetically divergent lineages, northern and southern were detected and their geographical boundary was estimated to be at 39°N. The northern lineage consisted of two genetic clusters and a single cpDNA haplotype, while the southern one had multiple genetic clusters and cpDNA haplotypes. The northern lineage showed significantly lower genetic diversity than the southern. Approximate Bayesian computation indicated that the northern and southern lineages had experienced, respectively, population expansion and long-term stable population size. The divergence time between the two lineages was estimated to be 565,000 years ago and no signature of migration between the two lineages after divergence was detected. Ecological niche modeling showed that the potential distribution area in northern Japan at the last glacial maximum was very small. It is thus considered that the two lineages have experienced different population histories over several glacial-inter-glacial cycles. Individuals of populations in the central to northern part of Honshu on the Sea of Japan side and in Hokkaido had large leaf width and area. These leaf characteristics corresponded with those of variety borealis. However, the delimitation of the northern and southern lineages detected by genetic markers (39°N) was not congruent with that detected by leaf morphologies (36°N). It is therefore suggested that variety borealis is not supported genetically and the northern and southern lineages should be considered separately when identifying conservation units based not on morphology but on genetic markers.

Molecular systematic study in the genus Linum (Linaceae) in Iran

The genus Linum L. is an important plant genus as it contains the species with economic values and particularly Linum usitatissimum L. that is source of fibre and linseed oil. This genus contains 230 species throughout the world and has about 22 species in Iran. Little is known about Linum species relationship and phylogeny. Therefore, the aim of present study was molecular phylogenetic investigation of the Linum species growing in Iran and to present data on their biogeography. We used both ITS and chloroplast DNA sequences (psbA-trnHGUG region) for inferring the species phylogeny and relationship. We also used cpDNA for inferring the species time of divergence and with ISSR markers to identify the path of species distribution in the country. The phylogenetic trees obtained for both ITS and cpDNA sequences were almost congruent. NeighborNet diagram and BEAST tree based on Bayesian method separated the outgroup species Hugonia and Anisadenia from the other species studied. The subspecies studied in Linum macronicum were placed close to each other and along with L. corymbulosum comprised a separate clade. The clades obtained showed divergence time between 5–20 mya. The present study revealed that the species of the sect. Linum are monophyletic, while members of the sections Linastrum and Syllinum are intermixed and seem to be paraphyletic.

Misidentifications of Echinochloa crus‐galli var. formosensis as Echinochloa oryzicola as a result of similar cpDNA sequences

Echinochloa crus‐galli (L.) Beauv. var. formosensis Ohwi (2n = 6x = 54, AABBCC genomes) and Echinochloa oryzicola (Vasinger) Vasinger (2n = 4x = 36, AABB) are major paddy weeds in East and Southeast Asia. E. oryzicola has been generally considered to be a paternal genome donor of E. crus‐galli s. l., which includes E. crus‐galli var. formosensis based on cpDNA sequences. Thus, molecular characterization using polymerase chain reaction‐restriction fragment length polymorphism analysis of cpDNA has been proposed as a reliable method for discriminating between the two species. In this study, we report that four accessions of E. crus‐galli var. formosensis from Okinawa, Nagasaki, Shizuoka and Tokyo had similar cpDNA sequences to E. oryzicola and had been misidentified as E. oryzicola using molecular methods. In addition, our results demonstrated that these accessions likely inherited their chloroplast genomes from E. oryzicola and not from an anonymous diploid species during polyploidization. Our findings provide new insights into the evolution of E. crus‐galli s. l. and suggest that identification using the cpDNA molecular method alone is not an appropriate approach to differentiate E. crus‐galli var. formosensis and E. oryzicola.

The complete chloroplast genome sequence of Pericampylus glaucus

The first complete chloroplast genome (cpDNA) sequence of Pericampylus glaucus was determined from Illumina HiSeq pair-end sequencing data in this study. The cpDNA is 162,450 bp in length, contains a large single-copy region (LSC) of 90,871 bp and a small single-copy region (SSC) of 21,137 bp, which were separated by a pair of inverted repeats (IR) regions of 25,221 bp. The genome contains 130 genes, including 85 protein-coding genes, 8 ribosomal RNA genes, and 37 transfer RNA genes. The overall GC content of the whole genome is 38.0%, and the corresponding values of the LSC, SSC, and IR regions are 36.2, 32.1, and 43.5%, respectively. Further, phylogenomic analysis showed that P. glaucus and Stephania japonica clustered in a clade in family Menispermaceae.

The complete chloroplast genome sequence of Dendrocalamus sinicus

Dendrocalamus sinicus is the world’s largest bamboo species. In this study, the complete chloroplast genome (cpDNA) sequence of D. sinicus was determined from Illumina HiSeq pair-end sequencing data. The cpDNA is 139,441 bp in length, contains a large single-copy region (LSC) of 82,777 bp and a small single-copy region (SSC) of 12,803 bp, which were separated by a pair of inverted repeat (IR) regions of 21,296 bp. The genome contains 136 genes, including 89 protein-coding genes, 8 ribosomal RNA genes, and 40 transfer RNA genes. The overall GC content of the whole genome is 38.9%, and the corresponding values of the LSC, SSC, and IR regions are 37%, 33.2%, and 44.2%, respectively. Further, phylogenomic analysis showed that D. sinicus clustered together with D. latiflorus.

The complete chloroplast genome sequence of Phlegmariurus carinatus

The first complete chloroplast genome (cpDNA) sequence of Phlegmariurus carinatus was determined from Illumina HiSeq pair-end sequencing data in this study. The cpDNA is 150,349 bp in length, contains a large single-copy region (LSC) of 100,582 bp and a small single-copy region (SSC) of 19,455 bp, which were separated by a pair of inverted repeat (IR) regions of 15,156 bp. The genome contains 120 genes, including 79 protein-coding genes, 8 ribosomal RNA genes, and 33 transfer RNA genes. The overall GC content of the whole genome is 34.0%, and the corresponding values of the LSC, SSC, and IR regions are 31.6%, 30.4%, and 44.2%, respectively. Further phylogenomic analysis showed that P. carinatus and Huperzia serrata clustered in a clade in family Lycopodiaceae.

Next generation sequencing technologies for the development of molecular markers and the analysis of genome diversity in Capsicum spp.

The accessibility and use of natural genetic variability in crops is essential for the management of genetic resources and the establishment of innovative breeding programs. Nuclear whole-genome sequences of several cultivated and wild peppers have been recently generated in order to provide crucial information on the evolution, domestication and divergence of pepper species (Capsicum spp.). Despite next generation sequencing (NGS) technologies providing a significant advance in generation of high throughput data, the analysis of a large-scale data and of individuals is still a challenging task. An alternative strategy to Whole Genome Sequencing is to generate a reduced representation of the genome using target enrichment strategies. Among them, genotyping-by-sequencing (GBS) provides a rapid, highly informative, cost-effective tool for exploring genetic diversity on a genome-wide scale. We applied GBS for SNP marker discovery in nuclear genomes and population structure analysis in a collection of cultivated and domesticated peppers. Information generated in this study represents a first step towards future genome-wide association mapping studies and marker-assisted selection programs. In addition, results from this analysis can be used to gain a better understanding of the genetic relationships within the Capsicum genus. In order to provide a further contribution to that issue, we also determined the complete chloroplast DNA (cpDNA) sequence of 11 Capsicum accessions for a total of 8 species generally included in the C. annuum (7), C. baccatum (3) or C. pubescens/eximium complex (1). We performed comparative analysis and identified potential molecular markers able to discriminate among Capsicum species.

PCR-based markers developed by comparison of complete chloroplast genome sequences discriminateSolanum chacoensefrom otherSolanumspecies

One of wild diploid Solanum species, Solanum chacoense, is one of the excellent resources for potato breeding because it is resistant to several important pathogens, but the species is not sexually compatible with potato (S. tuberosum) causing the limitation of sexual hybridization between S. tuberosum and S. chacoense. Therefore, diverse traits regarding resistance from the species can be introgressed into potato via somatic hybridization. After cell fusion, the identification of fusion products is crucial with molecular markers. In this study, S. chacoense specific markers were developed by comparing the chloroplast genome (cpDNA) sequence of S. chacoense obtained by NGS (next-generation sequencing) technology with those of five other Solanum species. A full length of the cpDNA sequence is 155,532 bp and its structure is similar to other Solanum species. Phylogenetic analysis resulted that S. chacoense is most closely located with S. commersonii. Sequence alignment with cpDNA sequences of six other Solanum species identified two InDels and 37 SNPs specific sequences in S. chacoense. Based on these InDels and SNPs regions, four markers for distingushing S. chacoense from other Solanum species were developed. These results obtained in this research could help breeders select breeding lines and facilitate breeding using S. chacoense in potato breeding.

Complete chloroplast genome of Camellia japonica genome structures, comparative and phylogenetic analysis.

Camellia is an economically, ecologically and phylogenetically valuable genus in the family Theaceae. The frequent interspecific hybridization and polyploidization makes this genus phylogenetically and taxonomically under controversial and require detailed investigation. Chloroplast (cp) genome sequences have been used for cpDNA marker development and genetic diversity evaluation. Our research newly sequenced the chloroplast genome of Camellia japonica using Illumina HiSeq X Ten platform, and retrieved five other chloroplast genomes of Camellia previously published for comparative analyses, thereby shedding lights on a deeper understanding of the applicability of chloroplast information. The chloroplast genome sizes ranged in length from 156,607 to 157,166 bp, and their gene structure resembled those of other higher plants. There were four categories of SSRs detected in six Camellia cpDNA sequences, with the lengths ranging from 10 to 17bp. The Camellia species exhibited different evolutionary routes that lhbA and orf188, followed by orf42 and psbZ, were readily lost during evolution. Obvious codon preferences were also shown in almost all protein-coding cpDNA and amino acid sequences. Selection pressure analysis revealed the influence of different environmental pressures on different Camellia chloroplast genomes during long-term evolution. All Camellia species, except C. crapnelliana, presented the identical rate of amplification in the IR region. The datasets obtained from the chloroplast genomes are highly supportive in inferring the phylogenetic relationships of the Camellia taxa, indicating that chloroplast genome can be used for classifying interspecific relationships in this genus.

Evolutionary shifts in fruit dispersal syndromes in Apiaceae tribe Scandiceae

Apiaceae tribe Scandiceae includes species with diverse fruits that depending upon their morphology are dispersed by gravity, carried away by wind, or transported attached to animal fur or feathers. This diversity is particularly evident in Scandiceae subtribe Daucinae, a group encompassing species with wings or spines developing on fruit secondary ribs. In this paper, we explore fruit evolution in 86 representatives of Scandiceae and outgroups to assess adaptive shifts related to the evolutionary switch between anemochory and epizoochory and to identify possible dispersal syndromes, i.e., patterns of covariation of morphological and life-history traits that are associated with a particular vector. We also assess the phylogenetic signal in fruit traits. Principal component analysis of 16 quantitative fruit characters and of plant height did not clearly separate species having different dispersal strategies as estimated based on fruit appendages. Only presumed anemochory was weakly associated with plant height and the flattening of mericarps with their accompanying anatomical changes. We conclude that in Scandiceae, there are no distinct dispersal syndromes, but a continuum of fruit morphologies relying on different dispersal vectors. Phylogenetic mapping of ten discrete fruit characters on trees inferred by nrDNA ITS and cpDNA sequence data revealed that all are homoplastic and of limited use for the delimitation of genera. Spines evolved from wings developing on secondary ribs. We hypothesize that spines cannot form on primary ribs because these contain vascular bundles that may constrain such a transformation. We describe a new subtribe for Artedia and propose three new combinations in Daucus.

Recent Trends in Research on the Genetic Diversity of Plants: Implications for Conservation

Genetic diversity and its distribution, both within and between populations, may be determined by micro-evolutionary processes, such as the demographic history of populations, natural selection, and gene flow. In plants, indices of genetic diversity (e.g., k, h and π) and structure (e.g., FST) are typically inferred from sequences of chloroplast markers. Given the recent advances and popularization of molecular techniques for research in population genetics, phylogenetics, phylogeography, and ecology, we adopted a scientometric approach to compile evidence on the recent trends in the use of cpDNA sequences as markers for the analysis of genetic diversity in botanical studies, over the years. We also used phylogenetic modeling to assess the relative contribution of relatedness or ecological and reproductive characters to the genetic diversity of plants. We postulated that genetic diversity could be defined not only by microevolutionary factors and life history traits, but also by relatedness, so that species more closely related phylogenetically would have similar genetic diversities. We found a clear tendency for an increase in the number of studies over time, confirming the hypothesis that the advances in the area of molecular genetics have supported the accumulation of data on the genetic diversity of plants. However, we found that the vast majority of these data have been produced by Chinese authors, and refer specifically to populations of Chinese plants. Most of the data on genetic diversity have been obtained for species in the International Union for Conservation of Nature (IUCN) category NE (Not Evaluated), which indicates a relative lack of attention on threatened species. In general, we observed very high FST values in the groups analyzed and, as we focused primarily on species that have not been evaluated by the IUCN, the number of plant species that are threatened with extinction may be much greater than that indicated by the listing of this organization. We also found that the number of haplotypes (k) was influenced by the type of geographic distribution of the plant, while haplotype diversity (h) was affected by the type of flower, and the fixation index (FST), by the type of habitat. The plant species most closely-related phylogenetically have similar levels of genetic diversity. Overall, then, it will important to consider phylogenetic dependence in future studies that evaluate the effects of life-history traits on plant genetic diversity.

Long-term survival and successful conservation? Low genetic diversity but no evidence for reduced reproductive success at the north-westernmost range edge of Poa badensis (Poaceae) in Central Europe

Many steppe species reach their (north)westernmost distribution limit in western Central Europe. This also applies to Poa badensis, a rare steppe plant of calcareous rock/sand vegetation. To explore potential differences in reproductive success and genetic composition of peripheral populations, we analysed the absolute (north)westernmost occurrences in Western Germany and populations at the western margin (Eastern Austria) and the centre (Central Hungary) of the Pannonicum, representing a part of the continuous range. Specifically, we discuss the genetic and reproductive constitution of the (north)westernmost exclave and draw conclusions on the species’ biogeographical and conservation history in this region. Therefore, we used two independent molecular marker systems (AFLPs, cpDNA sequences) and a set of performance parameters. Overall, lowest regional genetic diversity was found in Western Germany, which is mainly a result of the specific history of two populations. However, this low genetic diversity was not accompanied by reduced reproductive success. The Eastern Austrian populations showed reduced genetic diversity and predominantly reduced performance, interpreted as a consequence of small population sizes. Central Hungarian populations showed the overall highest genetic diversity and comparatively high performance values. We observed high admixture and haplotype sharing between Austrian and Hungarian populations, indicating gene flow among these regions. In contrast, we interpreted the increased population differentiation within, and the clear distinctiveness of the German exclave as a long-term isolation of these (north)westernmost occurrences. Our results, overall, prove the good constitution of these populations and, together with their particular biogeographical history, highlight their conservation value.

Intraspecific divergences and phylogeography of Panzerina lanata (Lamiaceae) in northwest China.

Climatic fluctuations during the Quaternary significantly affect many species in their intraspecific divergence and population structure across northwest China. In order to investigate the impact of climate change on herbaceous plants, we studied Panzerina lanata (Lamiaceae), a widely distributed species. Sequences of two chloroplast DNA (cpDNA) intergenic spacers (trnH-psbA and rpoB-trnC) and a nuclear ribosomal region (nrDNA, ITS) were generated from 27 populations of Panzerina lanata and resulted in the identification of seven chloroplast haplotypes and thirty-two nuclear haplotypes. We applied AMOVA, neutrality test and mismatch distribution analysis to estimate genetic differentiation and demographic characteristics. The divergence times of the seven cpDNA haplotypes were estimated using BEAST. Our results revealed high levels of genetic diversity (cpDNA: Hcp = 0.6691, HT = 0.673; nrDNA: Hnr = 0.5668, HT = 0.577). High level of genetic differentiation (GST = 0.950) among populations was observed in the cpDNA sequences, while the genetic differentiation values (GST = 0.348) were low in nuclear sequences. AMOVA results revealed major genetic variation among the three groups: northern, central, and eastern group. However, the genetic differentiation in ITS data was not found. The species distribution modeling and demographic analysis indicated that P. lanata had not experienced recent range expansion. The occurrence of divergence between seven cpDNA haplotypes, probably during Pleistocene, coincides with aridification and expansion of the desert across northwest China that resulted in species diversification and habitat fragmentation. In addition, we discovered that the deserts and the Helan Mountains acted as effective geographic barriers that promoting the intraspecific diversity of P. lanata.

Distinct phylogeographic structure of the halophyte Suaeda malacosperma (Chenopodiaceae/Amaranthaceae), endemic to Korea–Japan region, influenced by historical range shift dynamics

Wide-ranging changes to the coastline of East Asia during the glacial periods of the Quaternary might have significantly influenced the genetic structure of coastal plants. To verify its historical migration and potential breaks to gene flow, we investigated the genetic structure of the vulnerable halophyte Suaeda malacosperma, whose distribution is restricted to brackish tidal areas of Korea and Japan. Two chloroplast DNA (cpDNA) regions (rpl32-trnL, trnH-psbA) were used for five individuals each from 11 populations. SNP data obtained via multiplexed ISSR genotyping by sequencing (MIG-Seq) were used for 181 individuals from those populations. Ecological niche modeling (ENM) was combined with genetic analyses to compare current and past distributions. The cpDNA sequences and MIG-Seq data revealed largely congruent results indicating that S. malacosperma consists of three genetic clusters: western coast of Korea, southern coast of Korea, and Japan. The patterns produced through structure analysis and cpDNA haplotypes showed that the gene flow occurred at or after the last glaciation from Japan to the southern coast of Korea via the Korea/Tsushima Strait land bridge. Despite the coastal habitat of this species, ocean currents had less influence. Integrating genetic data with past distribution models by ENM provided insight into potential recolonization routes from refugia in each region. Those data suggested that, instead of contemporary gene flow, a historical range shift due to climate change affected the population structure. These results support the need for a conservation strategy and advance our understanding about the historical range dynamics of coastal plants in temperate East Asia.

The complete chloroplast genome sequence of Betula alnoides

Betula alnoides is a fast-growing hardwood species in South-east Asia and South China. In this study, the complete chloroplast genome (cpDNA) sequence of B. alnoides was determined from Illumina HiSeq pair-end sequencing data. The cpDNA is 161,022 bp in length, contains a large single-copy region (LSC) of 89,440 bp and a small single-copy region (SSC) of 19,535 bp, which were separated by a pair of inverted repeat (IR) regions of 26,022 bp. The genome contains 134 genes, including 86 protein-coding genes, 8 ribosomal RNA genes, and 41transfer RNA genes. The overall GC content of the whole genome is 36.0%, and the corresponding values of the LSC, SSC, and IR regions are 33.6%, 29.4%, and 42.5%, respectively. Further, phylogenomic analysis showed that B. alnoides clustered in a unique clade in genus Betula.

The complete chloroplast genome sequence of Calanthe delavayi (Orchidaceae), an endemic to China

Calanthe delavayi, an endemic terrestrial orchid distributed in southwestern China. In this study, the complete chloroplast genome (cpDNA) sequence of C. delavayi was determined from Illumina pair-end sequencing data. With a total length of 150,181 bp in length and includes two inverted repeat regions (IRs) of 23,721 bp each, which were separated by a large single copy region (LSC) 87,392 bp and a small single copy region (SSC) 18,693 bp. The chloroplast genome contained 135 genes, including protein-coding genes, tRNA genes, and eight rRNA genes. Phylogenetic analysis indicates that C. delavayi, C. davidii, and C. triplicata cluster together, placed them within tribe Collabieae.

Genetic structure of the endangered, relict shrub Amygdalus mongolica (Rosaceae) in arid northwest China

In the present study we selected the endangered species Amygdalus mongolica to investigate the influence of climatic fluctuations on the spatial genetic patterns and evolutionary history of organisms in arid northwest China. The intraspecific genetic variation and demographic history of A. mongolica were investigated using two cpDNA sequences (psbK-psbI and trnL-trnF) from 174 individuals in 15 populations across most of its covered range. Significant genetic differentiation among populations was identified, which resulted from fragmented populations coupled with the enhanced aridification and desertification of the Pleistocene. Twelve haplotypes clustered into two clades, corresponding to the Western region (northernmost Gansu, Hexi Corridor and Alxa Left Banner) and the Eastern region (Urad Houqi, Yinshan Mountains, Urad Zhongqi and Daqing Mountains) respectively. Five independent Pleistocene refugia in different mountainous areas were identified. Amygdalus mongolica has likely experienced westward shifts from refugia along the margins of the Ulanbuhe and Tengger deserts, and also postglacial re-colonisation northward along the Hexi Corridor.

Genetic fingerprinting of date palm (Pheonix dactylifera L.) by using ISSR and cpDNA sequences

The date palm (Phoenix dactylifera L.) is the most important fruit bearing crop in arid regions of the Middle East and North Africa. The knowledge on genetic structure of date palm is limited and understanding of genetic variability within date palms is important for the efficient utilization of the genetic resources in breeding. Therefore, in the present study, genetic analysis of 14 date palm cultivars collected from 28 localities in Iran was performed to provide prospective of genetic variability and population structure. In addition, publically available sequencing data of chloroplast genome available for 47 cultivars was also explored. The results showed a good level of genetic variability among the cultivars. Grouping of the cultivars was not related to their geographical distribution and no correlation was observed between genetic distance and geographical distance of date palms. Population structure analysis revealed the presence of a low degree of common shared alleles between date palms cultivars that indicate probable local adaptation in structuring the genetic content of date palms.

One more species in the genus Jungermannia (Marchantiophyta: Jungermanniaceae)

Jungermannia afoninae is described from the mountains of South Siberia based on a complex taxonomical approach exploring critical reinvestigation of morphological features and analyses of newly obtained trnL–trnF and trnG-intron cpDNA sequences from six Jungermannia specimens incorporated into a previously published dataset. Description and illustrations of the new species are provided with notes on its differentiation from allied species, ecology and distribution.

Molecular phylogenetic relationships among populations of Sagittaria aginashi Makino (Alismataceae) and endemic Chinese species

The aquatic plant Sagittaria aginashi, which is an endangered emergent species distributed in South Korea, Japan, and the Russian Far East, has an unclear phylogeny and genetic structure. Phylogenetic trees of Sagittaria were constructed based on one nuclear ribosomal DNA (nrDNA) region and on three chloroplast DNA (cpDNA) regions. S. aginashi was included in a clade comprising three endemic Chinese Sagitarria species characterized by the presence of small tubercles at the base of the axils. Furthermore, phylogenetic trees based on cpDNA sequences indicated that S. aginashi was more closely related to S. tengtsungensis than to two other species. S. aginashi exhibited little genetic variation, suggesting that this species had rapidly spread in these areas. With regard to factors associated with the rapid spread, we considered that multiple dispersal methods were involved, namely hydrochory via tubercles, endozoochorous long-distance dispersal, and anthropogenic impacts of rice farming on S. aginashi dispersal.