The complete chloroplast genome sequence of the Chinese endemic species Sorbus setschwanensis (Rosaceae) and its phylogenetic analysis

Sorbus setschwanensis Koehne is a pinnate-leaved Sorbus s.str. species endemic to China with narrow distribution and intriguing phylogeny that needs wider attention. In this paper, the complete chloroplast (cp) genome of S. setschwanensis is reported, and its phylogenetic position is analyzed. The complete cp genome of S. setschwanensis is 160 064 bp in size with 36.50% GC content. It has a typical quadripartite structure including a pair of inverted repeat regions (IRs) of 26 378 bp that separates a large single copy (LSC) region of 86 013 bp and a small single copy (SSC) region of 19 295 bp. The cp genome encodes 108 genes, comprising 76 protein-coding genes, 28 tRNA genes and 4 rRNA genes. Additionally, 52 simple sequence repeats (SSRs) and 43 dispersed repeats were identified. Comparison of the whole cp genome with those of other Sorbus species showed an overall high degree of sequence similarity, but there are six highly variable regions (trnR-atpA, petN-psbM, ndhC-trnV, trnE-trnT, trnT-trnL and rpl32-trnL) located in intergenic spacers that may be useful as molecular markers in future population genetic and phylogenetic studies in the genus. Phylogenetic analyses based on 108 coding genes from 25 species in Rosaceae revealed that S. setschwanensis is nested within Sorbus sect. Sorbus together with other pinnately leaved species, but does not form a sister lineage to S. rufopilosa belonging to the same series Multijugae. Thus, the systematic position of S. setschwanensis and relationships with other species in the genus needs to be further studied.     

Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.     

Analysis of DNA sequences of six chloroplast and nuclear genes suggests incongruence, introgression, and incomplete lineage sorting in the evolution of Lespedeza (Fabaceae)

The genus Lespedeza (Fabaceae) consists of 40 species disjunctively distributed in East Asia and eastern North America. Phylogenetic relationships of all Lespedeza species and closely related genera were reconstructed using maximum parsimony, maximum likelihood, and Bayesian analyses of sequence data from five chloroplast (rpl16, rpl32-trnL, rps16-trnQ, trnL-F, and trnK/matK) and one nuclear (ITS) DNA regions. All analyses yielded consistent relationships among major lineages. Our results suggested that Campylotropis, Kummerowia, and Lespedeza are monophyletic, respectively. Lespedeza is resolved as sister to Kummerowia and these two together are further sister to Campylotropis. Neither of the two subgenera, subgen. Lespedeza and subgen. Macrolespedeza, in Lespedeza based on morphological characters, is recovered as monophyletic. Within Lespedeza, the North American clade is retrieved as sister to the Asian clade. The nuclear and chloroplast markers showed incongruent phylogenetic signals at shallow-level phylogeny, which may point to either introgression or incomplete lineage sorting in Lespedeza. The divergence times within Lespedeza and among related genera were estimated using Bayesian approach with BEAST. It is assumed that following the divergence between Kummerowia and Lespedeza in Asia in the late Miocene, the ancestor of Lespedeza diverged into the North American and the Asian lineages. The North American ancestor quickly migrated to North America through the Bering land bridge in the late Miocene. The North American and Asian lineages started to diversify almost simultaneously in the late Miocene but resulted in biased numbers of species in two continents.     

A global plastid phylogeny of the fern genus Asplenium (Aspleniaceae)

The infrageneric relationships and taxonomy of the largest fern genus, Asplenium (Aspleniaceae), have remained poorly understood. Previous studies have focused mainly on specific species complexes involving a few or dozens of species only, or have achieved a large taxon sampling but only one plastid marker was used. In the present study, DNA sequences from six plastid markers (atpB, rbcL, rps4, rps4-trnS, trnL and trnL-F) of 1030 accessions (616 of them newly sequenced here) representing c. 420 species of Asplenium (60% of estimated species diversity), 16 species of Hymenasplenium, three Diplaziopsidaceae, and four Rhachidosoraceae were used to produce the largest genus-level phylogeny yet for ferns. Our major results include: (i) Asplenium as broadly circumscribed is monophyletic based on our inclusion of representatives of 32 of 38 named segregate genera; (ii) 11 major clades in Asplenium are identified, and their relationships are mostly well-resolved and strongly supported; (iii) numerous species, unsampled in previous studies, suggest new relationships and numerous cryptic species and species complexes in Asplenium; and (iv) the accrued molecular evidence provides an essential foundation for further investigations of complex patterns of geographical diversification, speciation and reticulate evolution in this family.     

Molecular phylogenetics of the juno irises,Iris subgenus Scorpiris (Iridaceae), based on six plastid markers

Relationships among the roughly 55 species of Iris subgenus Scorpiris have been studied. A matrix of six plastid DNA regions (matK, rpl14‐rps8 spacer, infA‐rpl36 spacer, trnE‐trnT spacer, trnL intron and trnL‐F spacer) was produced from 57 accessions (52 taxa) and analysed with both parsimony and Bayesian methods. Five major clades are identified, of which four have strong geographical correlations, whereas the fifth corresponds to Iris section Physocaulon. In our results, several species are placed with species not previously considered to be related, although, in some cases, there are morphological characters that suggest that these newly indicated relationships are reasonable. For some of the other oddly grouped species, we can only assume that remarkable parallelisms in morphology have occurred or hybridization is involved. Presently, with plastid DNA as our only comprehensive data resource, we are not able to evaluate more thoroughly these more puzzling associations of species. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167 , 281–300.     

The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia spp.)

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and improve knowledge about species distributions.     

A regional approach to plant DNA barcoding provides high species resolution of sedges (Carex and Kobresia, Cyperaceae) in the Canadian Arctic Archipelago

Previous research on barcoding sedges (Carex) suggested that basic searches within a global barcoding database would probably not resolve more than 60% of the world’s some 2000 species. In this study, we take an alternative approach and explore the performance of plant DNA barcoding in the Carex lineage from an explicitly regional perspective. We characterize the utility of a subset of the proposed protein-coding and noncoding plastid barcoding regions (matK, rpoB, rpoC1, rbcL, atpF-atpH, psbK-psbI) for distinguishing species of Carex and Kobresia in the Canadian Arctic Archipelago, a clearly defined eco-geographical region representing 1% of the Earth’s landmass. Our results show that matK resolves the greatest number of species of any single-locus (95%), and when combined in a two-locus barcode, it provides 100% species resolution in all but one combination (matK + atpFH) during unweighted pair-group method with arithmetic mean averages (UPGMA) analyses. Noncoding regions were equally or more variable than matK, but as single markers they resolve substantially fewer taxa than matK alone. When difficulties with sequencing and alignment due to microstructural variation in noncoding regions are also considered, our results support other studies in suggesting that protein-coding regions are more practical as barcoding markers. Plastid DNA barcodes are an effective identification tool for species of Carex and Kobresia in the Canadian Arctic Archipelago, a region where the number of co-existing closely related species is limited. We suggest that if a regional approach to plant DNA barcoding was applied on a global scale, it could provide a solution to the generally poor species resolution seen in previous barcoding studies.     

The Chloroplast trnT–trnF Region in the Seed Plant Lineage Gnetales

The trnT–trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL intron, a group I intron, and the trnT–trnL and trnL–trnF intergenic spacers. We analyzed the evolution of the region in the three genera of the gymnosperm lineage Gnetales (Gnetum, Welwitschia, and Ephedra), with especially dense sampling in Gnetum for which we sequenced 41 accessions, representing most of the 25–35 species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, while the spacers are so variable in length and composition that homology cannot be found even among the three genera. Palindromic sequences that form hairpin structures were detected in the trnL–trnF spacer, but neither spacer contained promoter elements for the tRNA genes. The absence of promoters, presence of hairpin structures in the trnL–trnF spacer, and high sequence variation in both spacers together suggest that trnT and trnF are independently transcribed. Our model for the expression and processing of the genes tRNA^Thr(UGU), tRNA^Leu(UAA), and tRNA^Phe (GAA) therefore attributes the seemingly neutral evolution of the two spacers to their escape from functional constraints. [Reviewing Editor: Debashish Bhattacharya]     

How Effective Are DNA Barcodes in the Identification of African Rainforest Trees?

Background

DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species.

Methodology/Principal Findings

We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95–100% success), but less for species identification (71–88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84–90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA.

Conclusions/Significance

Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements for barcoding applications.     

Species-Specific Identification from Incomplete Sampling: Applying DNA Barcodes to Monitoring Invasive Solanum Plants

Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling–through this, DNA barcoding will greatly benefit the current fields of its application.     

Taxonomic status and genetic differentiation of Hyrcanian Castanea based on noncoding chloroplast DNA sequences data

The taxonomy and phylogenetic relationships among Castanea species were investigated using sequence data from the chloroplast trnL-F and trnH-psbA intergenic spacer regions. Samples included Castanea specimens of uncertain taxonomic affinity that were collected in the Hyrcanian forest of northern Iran. The trnL-F data were more informative than trnH-psbA, having seven parsimony-informative sites. A low level of haplotype diversity was detected within Hyrcanian samples and the whole species of the genus Castanea. In the trnL-F dataset, Castanea sativa and Castanea mollissima have unique character states that differentiate them from other species of Castanea. The genus Castanea was recovered as a monophyletic with high to moderate support when inferred from combined trnH-psbA and trnL-F spacer data. Two main lineages received minimal support in the trnL-F analysis, whereas trnH-psbA could not distinguish different species of Castanea from each other. Finally, low levels of haplotype diversity was found within small remnant stands of Castanea in the Hyrcanian forest, indicating that genetic erosion may increase the extinction risk for these valuable trees.     

Parasitic flatworms infecting thorny skate,Amblyraja radiata: Infection by the monogeneans Acanthocotyle verrilli and Rajonchocotyle emarginata in Svalbard

Parasite diversity above the Arctic circle remains understudied even for commercially valuable host taxa. Thorny skate, Amblyraja radiata, is a common bycatch species with a growing commercial value. Its natural range covers both sides of the North Atlantic including the Arctic zone. Svalbard is a Norwegian archipelago located on the northwest corner of the Barents Shelf which sustains a spectacular species diversity. So far, several monogenean species have been reported infecting thorny skate across the Atlantic Ocean. In the present study, we intend to fill in the knowledge gap on monogenean parasites infecting thorny skate in the northern part of its range and thus indirectly assess the connectivity between the thorny skate populations off the Svalbard coast and from previously studied locations. 46 monogenean individuals were recovered from 11 specimens of thorny skate. Following morphological and molecular assessment, two species of monogeneans, Acanthocotyle verrilli and Rajonchocotyle emarginata, were identified. The results serve as the northernmost record for both parasite genera and the first record of monogenean species off Svalbard. Detailed morphometric evaluation revealed a relatively high level of morphological variation in A. verrilli compared to its congeners. Phylogenetic reconstruction placed A. verrilli in a well-supported clade with A. imo. Our study also suggests high diagnostic significance of sclerotised structures in the identification of Rajonchocotyle. Even though the occurrence of two directly transmitted parasite species supports the previously suggested long-distance migration of A. radiata, future studies employing highly variable genetic markers are needed to assess the ongoing and historical migration patterns.     

Identification of Scleractinian Coral Recruits Using Fluorescent Censusing and DNA Barcoding Techniques

The identification of coral recruits has been problematic due to a lack of definitive morphological characters being available for higher taxonomic resolution. In this study, we tested whether fluorescent detection of coral recruits used in combinations of different DNA-barcoding markers (cytochrome oxidase I gene [COI], open reading frame [ORF], and nuclear Pax-C intron [PaxC]) could be useful for increasing the resolution of coral spat identification in ecological studies. One hundred and fifty settlement plates were emplaced at nine sites on the fringing reefs of Kenting National Park in southern Taiwan between April 2011 and September 2012. A total of 248 living coral spats and juveniles (with basal areas ranging from 0.21 to 134.57 mm²) were detected on the plates with the aid of fluorescent light and collected for molecular analyses. Using the COI DNA barcoding technique, 90.3% (224/248) of coral spats were successfully identified into six genera, including Acropora, Isopora, Montipora, Pocillopora, Porites, and Pavona. PaxC further separated I. cuneata and I. palifera of Isopora from Acropora, and ORF successfully identified the species of Pocillopora (except P. meandrina and P. eydouxi). Moreover, other cnidarian species such as actinarians, zoanthids, and Millepora species were visually found using fluorescence and identified by COI DNA barcoding. This combination of existing approaches greatly improved the taxonomic resolution of early coral life stages, which to date has been mainly limited to the family level based on skeletal identification. Overall, this study suggests important improvements for the identification of coral recruits in ecological studies.     

Molecular,phytochemical and morphological characterization of the liverwort genus Radula in Portugal (mainland,Madeira, Azores)

Relationships of the eight species of the liverwort genus Radula occurring in Portugal (mainland, the Madeira and Azores archipelagos), including the Macaronesian endemics R. jonesii and R. wichurae, were evaluated based on molecular, phytochemical and morphological–anatomical data. Maximum parsimony and Bayesian analyses were performed with sequences from three plastid DNA markers (trnS _GGA-rps4 spacer, rps4 gene, trnL _UAA intron), volatile oil compounds, as well as qualitative morphological–anatomical characters. In addition, the molecular data were subjected to maximum likelihood analysis. The eight taxa, R. aquilegia, R. carringtonii, R. complanata, R. holtii, R. jonesii, R. lindenbergiana, R. nudicaulis and R. wichurae, can be clearly distinguished from each other, either by molecular data alone or by combination of characters from all three data sets. Radula aquilegia is monophyletic according to the molecular data, but shows considerable, yet undescribed intraspecific morphological and phytochemical variability. Recognition of R. complanata and R. lindenbergiana as separate species, previously based solely on the paroecious vs. dioecious sexual condition, is moderately supported by the molecular phylogenetic analyses and strongly supported by the phytochemical data. The Radula species, narrowly distributed in Macaronesia and Atlantic Europe, probably have two different origins. For Radula holtii and R. nudicaulis, connections with Radula species from the Neotropics are indicated. The other species, among them the two Macaronesian endemics, are closely related with the R. complanata/R. lindenbergiana complex, which is widely distributed in the northern hemisphere.     

Evaluation of suitable DNA regions for molecular identification of high value medicinal plants in genus Kaempferia

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Kaempferia (Zingiberaceae). Four primer pairs were evaluated (rbcL, rpoC, trnL and ITS1). It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Thus, the primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL primer pair gave the lowest resolution. Our Bar-HRM developed here would not only be useful for identification of Kaempferia plant specimens lacking essential parts for morphological identification but will be useful for authenticating products in powdered form of a high value medicinal species Kaempferia parviflora, in particular.     

Intra and interspecific sequence variation in closely related species of Cereus (CACTACEAE)

In comparative phylogenetic and population genetic studies, one of the most crucial steps is to select appropriate DNA markers, a decision based primarily on the estimated variation in markers in cross-taxonomic surveys. To assess whether genetic variation at the intraspecific level in one species predicts the variation in another closely related species we used two congeneric species of Cereus (Cactaceae: Cereeae). We screened and characterized eight noncoding plastid regions (trnS-trnG, atpI-atpH, trnT-trnL, psbD-trnT, petL-psbE, 3'rps16-5'trnk, trnG intron, and trnL intron), and one nuclear gene (PhyC) in Cereus fernambucensis and C. hildmannianus. A total of 40 individuals from 15 populations were characterized according to nucleotide diversity, number of haplotypes, and number of potentially informative characters. The results revealed that nucleotide substitutions and indels are the main source of variation, with the largest divergence between species found in trnS-trnG. The trnL intron and petL-psbE showed intraspecific variability in both species. The psbD-trnT, atpI-atpH, trnS-trnG, and trnT-trnL, which are the most variable regions in one species, showed no variation in the other. Finally, the nuclear gene PhyC showed more resolution between Cereus species than within species. We thus found considerable heterogeneity among widely used plastid markers, even between closely related species, and suggest the use of PhyC as a marker for phylogenetic inference in these species. These results reinforce the need of screening as a preliminary step to conduct phylogeographic or phylogenetic studies in face of unpredictable sequence variation of molecular markers in plants.     

Study of molecular genetic diversity and evolutionary history of medicinally important endangered genus Chlorophytum using DNA barcodes

This study was aimed to authenticate and present phylogenetic relationship among 19 species of genus Chlorophytum using DNA barcoding. In all, 107 accessions were analyzed with eight plastid (matK, rbcL, trnH-psbA, rpoC1, ycf5, rpoB, atp and psbK-psbI) and six nuclear (ITS) markers. The matK and rbcL were found to be ideal markers for identification and discrimination of Chlorophytum species. Phylogenetic analysis based on matK and rbcL sequences resolved the species in two major clades. All markers, except matK and rbcL, showed ambiguous reads and paralogy in analysis. DGGE analysis showed the presence of pseudogenes and/or co-amplification in these markers, which caused poor sequence quality. Phylogeny and probable evolution of genus Chlorophytum was proposed on the basis of cytological, morphological and genetic information.     

Cydippid ctenophores in the coastal waters of Svalbard: is it only Mertensia ovum?

Threat of potential arrival of non-indigenous species is increasing, particularly in the high Arctic, due to ecological shifts expected from climate change and increasing shipping traffic, facilitating long-distance transport of invaders. Hence, knowledge on the species present currently is crucial to assess the potential ecological impact of the non-indigenous species in the future. Ctenophores, and gelatinous zooplankton generally, are poorly known due to identification challenges and lack of systematic monitoring programmes. Yet, they are known to play important roles in the world’s ocean ecosystems and share physiological attributes making them exploit the changing environmental conditions better compared to most other zooplankton groups. Here, we report the co-occurrence of Mertensia ovum, Euplokamis sp. and an unidentified mertensiid-like species in the high Arctic, in the Svalbard archipelago region, based on a combination of morphological and molecular identification methods. This is a valuable first step toward establishing a baseline for future ecological studies, monitoring of climate impacts and assessing the threat of introduced species in the high Arctic.     

Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding

Background

At present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species.

Methodology/Principal Findings

We scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 genera were from GenBank and 3 genera were of our own. We identified nearly 5% of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected 23 loci that were present in at least three genera. The 23 loci included 4 coding regions, 2 introns, and 17 intergenic spacers. Of the 23 loci, the most variable (in order from highest variability to lowest) were intergenic regions ycf1-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnS^UGA-trnG^UCC, petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnS^UGA-trnG^UCC, trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (π values) across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species.

Significance/Conclusions

Chloroplast genome sequences contain regions that are highly variable. Such regions are the first consideration when screening the suitable loci to resolve closely related species or genera in phylogenetic analyses, and for DNA barcoding.