Regions matK Research Articles

Application of DNA barcoding markers to the identification of Hopea species.

Hopea chinensis (synonym: H. hongayensis) (Dipterocarpaceae) is a threatened species found so far in only two locations: Quang Ninh (Vietnam) and Guangxi (China). The species shares many morphological characteristics with H. mollissima and the two species are often confused. To overcome this problem of identification and to investigate the genetic relationships of Hopea species with other Dipterocarp species, we sequenced three candidate DNA barcodes for the chloroplast markers rbcL, trnH-psbA, and matK. These markers were used separately and in different combinations to determine whether they could establish an accurate and effective identification system for H. chinensis in Quang Ninh (Vietnam). Our analyses indicated that two of the candidate DNA barcodes, matK and rbcL, performed best. We also generated a neighbor-joining phylogenetic tree and confirmed the presence of four Hopea species (H. odorata, H. hainanensis, H. mollissima, and H. chinensis) in nature reserves and natural parks of Vietnam. These species showed a close relationship with an average genetic distance of 0.0045; both matK and rbcL separated all species, but their use in combination gave higher bootstrap values. The matK region was found to provide the most reliable barcode for the identification of the most closely related Dipterocarp species. Our study provides a means to identify rare Hopea species non-ambiguously and to support the protection of this decreasing natural genetic resource.

Taxonomic Review of the Genus Echinochloa in Korea (I): Inferred from Sequences of cpDNA and nrDNA

The genus Echinochloa (L.) P. Beauv. comprised of approximately 30-40 species in the tropical and warm temperate regions of the world, including numerous interspecific and intraspecific types which make the genus difficult to identify. As an attempt to identify the species within the genus easier, the taxonomy of the genus Echinochloa, Poaceae in Korea was reviewed on the basis of sequencing data derived from nuclear ribosomal DNA internal transcribed spacer (ITS) and external transcribe spacer and chloroplast DNA trnL intron, trnL-F intergenic spacer and matK regions using a total of 46 accessions representing all the species in Korea. The results of maximum parsimony found separate lineage comprised of E. colona and E. frumentaceae which are not Korean species, but no resolution within Korean Echinochloa species, supporting the suggestion of Yamaguchi group that E. crus-galli, E. oryzoides, and E. esculenta should be considered to belong to the same species. However, the relationship between these three species and the other species, i.e. E. oryzicola should be better understood with more detail studies.

The use of an integrated molecular-, chemical- and biological-based approach for promoting the better use and conservation of medicinal species: A case study of Brazilian quinas

Ethnopharmacological relevanceQuina is a popular name originally attributed to Cinchona pubescens Vahl (=Cinchona succirubra) and Cinchona. calisaya Wedd., species native from Peru that have the antimalarial alkaloid quinine. In Brazil, bitter barks substitutes for the Peruvian species began to be used centuries ago, and they still are sold in popular markets. To assess the authenticity and the conditions on which samples of quinas have been commercialized, using the DNA barcode, chemical and biological assays. Materials and methodsStarting with 28 samples of barks acquired on a popular market, 23 had their DNA extracted successfully. The regions matK and rbcL were amplified and sequenced for 15 and 23 samples, respectively. Phytochemical analyses were performed by chromatographic methods, and biological essays were done by antimalarial tests in vitro. ResultsThe identified species belonged to six different families, many of them endangered or with no correlation with use in traditional medicine as a Brazilian quina. The absence of typical bitter chemical substances indicated that barks have been collected from other species or from very young trees. The results of biological essays confirm the lack of standardization of the sold materials. ConclusionThe integrated approaches proved to be efficient to evaluate medicinal plants sold in popular markets and can be useful for promoting their better use and conservation.

Floral Evolution in the Detarieae (Leguminosae): Phylogenetic Evidence for Labile Floral Development in an Early-Diverging Legume Lineage

Premise of research. We examine floral evolution and phylogenetic relationships in the monophyletic Detarieae and related lineages of Caesalpinioideae. Tribe Detarieae (82 genera) includes nearly half of the genera in subfamily Caesalpinioideae and represents some of the most diverse legumes with respect to floral morphology.Methodology. A total of 67 floral ontogenetic and morphological characters were combined with DNA sequences from the plastid trnL-F and matK regions of 34 Detarieae species and representatives of Cassieae, Cercideae, Caesalpinieae, Papilionoideae, and Mimosoideae, for which we have near-complete ontogenetic series. The morphological and ontogenetic characters were optimized onto the resulting most parsimonious phylogenetic trees and Bayesian topology to study character evolution.Pivotal results. Our study supports previously proposed relationships within the tribe Detarieae and among caesalpinioid lineages and indicates that certain features (bracteole and hypanthium characters, sepal initiation, anther position in bud, overlap in timing of initiation of organ whorls) are phylogenetically informative for particular clades whereas others (reductions in petal and stamen number, sepal and petal initiation patterns) have evolved multiple times in parallel in the Detarieae and other Caesalpinioideae. These analyses suggest that modifications that occur early in ontogeny can be good phylogenetic characters for distinguishing both major taxonomic groups and more closely related taxa and that morphological differences that differentiate species within genera can be caused by changes that occur at all stages of ontogeny. Phylogenetic distribution of character states and ontogenetic evidence suggest that in the Caesalpinioideae, loss or suppression of organs within a whorl, both of which are very common, usually does not affect development in subsequent whorls.Conclusions. Our analyses reveal several switches from zygomorphy to actinomorphy (and vice versa), but in Caesalpinioideae (contrary to Papilionoideae), zygomorphy is likely not clearly associated with higher diversification rates. This study suggests that floral initiation patterns are much more variable in Caesalpinioideae than in the other two subfamilies. Although particular patterns may be canalized in certain lineages of the subfamily (Cercideae, Caesalpinieae), in other clades (Detarieae, Dialiinae) floral development is more labile, explaining the high diversity in floral morphology encountered overall in the paraphyletic Caesalpinioideae.

Testing the utility of matK and ITS DNA regions for discrimination of Allium species

Molecular phylogenetic analysis of the genus Allium L. has been mainly based on the nucleotide sequences of the ITS region. In 2009, matK and rbcL were accepted as a 2-locus DNA barcode to classify plant species by the Consortium for the Barcode of Life Plant Working Group. The matK region was chosen as a DNA barcode because of its effective species discrimination power, high quality sequence recovery, and easy experimental procedures. Integration of matK sequences into Allium phylogeny could improve phylogenetic reconstruction of this genus. This study was carried out to test the utility of nucleotide sequences of matK for discrimination of Allium species and to compare topologies of the phylogenetic trees based on matK and ITS analyses. Topologies of the phylogenetic trees based on ITS and matK analyses were very similar but a few accessions were placed into distant phylogenetic groups. Neither ITS nor matK analyses were able to discriminate some closely related Allium species alone. However, we do not suggest the use of a concatenated data approach to increase resolving power of ITS and matK because of the presence of the paralogous sequences and different types of cytoplasm in different accessions of a species. Therefore, we can suggest use of the matK region as an additional tool for phylogenetic analysis in Allium because characterization of the nucleotide sequences of matK region was easier to recover and more cost-effective than those of the ITS region.

Molecular phylogenetic relationships and implications for the circumscription of Colchicaceae (Liliales)

Colchicaceae (Liliales) comprise c. 250 species and 15–21 genera of rhizomatous or cormous perennials and are distributed through temperate and tropical Africa, Europe, Asia and North America. They have been traditionally divided into two subfamilies, but this division is still unclear and the phylogenetic relationships among members of the family and the sister relationships of the family have been debated. Moreover, it is uncertain whether the expanded circumscription of Colchicum, Gloriosa and Wurmbea to include Androcymbium, Littonia and Onixotis plus Neodregea, respectively, is unavoidable. In this study, sequences of three plastid genes, atpB, matK and rbcL, were analysed to reconstruct phylogenetic relationships in Colchicaceae. The matK region was the most variable and provided the most potentially parsimony-informative sites. We also conducted molecular phylogenetic analyses using the combined data with sequences for three non-coding regions generated by Vinnersten and Reeves. Our results strongly support the monophyly of Colchicaceae and the redefined circumscription to include two subfamilies, Wurmbeoideae and Uvularioideae. The Uvularia-Disporum clade, i.e. Uvularioideae, is sister to the rest of Colchicaceae and differentiated from Burchardia, previously suggested to be sister to the rest of Colchicaceae. In our analysis, Burchardia is placed in Wurmbeoideae. A close relationship between Tripladenia and Schelhammera was strongly supported and these are transferred to Wurmbeoideae from Uvularioideae. The six tribes sensu Vinnersten and Manning were shown to be monophyletic. Our results support the expanded circumscription of Colchicum (including Androcymbium) and Gloriosa (including Littonia), but the circumscription of Wurmbea and related genera requires further study. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 255–269.

Identification of medical plants of 24 Ardisia species from China using the matK genetic marker

Background:Ardisia is a group of famous herbs in China, which has been used as medical plants for more than 900 years. However, the species from the genus are so analogous that it is difficult to discriminate them just by morphological characteristics. DNA barcoding is a new technique that uses a short and standard fragment of DNA sequences to identify species.Objective:Choose a suitable DNA marker to authenticate Ardisia species.Materials and Methods:Four markers (psbA-trnH, internal transcribed spacer 2 [ITS2], rbcL, matK) were tested on 54 samples of 24 species from genus Ardisia. The success rates of polymerase chain reaction amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and identification efficiency were used to evaluate the discrimination ability.Results:The results indicate that matK has the highest interspecific divergence and significant differences between inter- and intra-specific divergences, whereas psbA-trnH, ITS2 and rbcL have much lower divergence values. Matk possessed the highest species identification efficiency at 98.1% by basic local alignment search tool 1 [BLAST1], method and 91.7% by the nearest distance method.Conclusion:The matK region is a promising DNA barcode for the genus Ardisia.

English

Paeonia is a phylogenetically and taxonomically complex group. In this study, two chloroplast coding region of the matK and rbcL genes, two intergenic spacers, 5’ trnK-matKand matK-3’ trnK, the ribosomal RNA 18S and 5S genes, and the nuclear ribosomal DNA ITS gene were sequenced to study phylogenetic relationships of four Paeonia species. In the matK coding region, most nucleotide variations pointed at the divergence betweenPaeonia suffruticosa (MO) and other three species, strongly supporting the phylogenetic evolution of section Paeonia and section Moutan in the Paeonia genus; the variable sites ofPaeonia lactiflora CHA1 accession compared to CHA accession were all identical toPaeonia obovata SHAN accession, indicating that CHA1 might be a hybrid result of CHA and SHAN. Another chloroplast coding region, rbcL showed lower variation rate and less phylogenetic information than the matK region in all four Paeonia species investigated in this study, while the 5S rRNA gene and nrDNA ITS region showed higher variation rate. However, combined phylogenetic relationship of all four peony species is more clearer than any single-locus one. This study would provide more sequence sources of peony species and help further understanding of the phylogenetic relationship. Key words: Deoxyribonucleic acid (DNA) barcoding, matK, nuclear ribosomaldeoxyribonucleic acid internal transcribed spacer (nrDNA ITS), Paeonia, phylogeny, ribulose-bisphosphate carboxylase (rbcL), 5S ribosomal ribonucleic acid (rRNA).

Authentication of Angelica anomala Avé-Lall cultivars through DNA barcodes

Angelica anomala Avé-Lall (Chuanbaizhi in Chinese) is an important medicinal plant which can be used in traditional Chinese medicines; however, there are no authentic and universal methods to differentiate this Sichuan famous-region drug of A. anomala from a large number of non-famous-region and false drugs. It has been demonstrated that DNA barcoding is a molecular diagnostic method for species identification, which uses a single standardized DNA fragment. In this study, we tested five DNA barcoding candidates (matK, ITS, ITS2, rbcL, and psbA-trnH), and we found that ITS was the best candidate to authenticate the famous-region drug of A. anomala. Moreover, through comparative analysis of these five DNA barcodes between A. anomala and Angelica dahurica, we found that ITS had the most and ITS2 had more variable regions, but the psbA-trnH, rbcL, and matK regions were identical. Hence, we suggest ITS as the DNA barcoding to identify A. anomala and A. dahurica. Moreover, we are determined to adopt the A. anomala as the accurate Latin name of Chuanbaizhi.

Combined phylogenetic analyses reveal interfamilial relationships and patterns of floral evolution in the eudicot order Fabales.

Relationships between the four families placed in the angiosperm order Fabales (Leguminosae, Polygalaceae, Quillajaceae, Surianaceae) were hitherto poorly resolved. We combine published molecular data for the chloroplast regions matK and rbcL with 66 morphological characters surveyed for 73 ingroup and two outgroup species, and use Parsimony and Bayesian approaches to explore matrices with different missing data. All combined analyses using Parsimony recovered the topology Polygalaceae (Leguminosae (Quillajaceae + Surianaceae)). Bayesian analyses with matched morphological and molecular sampling recover the same topology, but analyses based on other data recover a different Bayesian topology: ((Polygalaceae + Leguminosae) (Quillajaceae + Surianaceae)). We explore the evolution of floral characters in the context of the more consistent topology: Polygalaceae (Leguminosae (Quillajaceae + Surianaceae)). This reveals synapomorphies for (Leguminosae (Quillajaceae + Surianaceae)) as the presence of free filaments and marginal/ventral placentation, for (Quillajaceae + Surianaceae) as pentamery and apocarpy, and for Leguminosae the presence of an abaxial median sepal and unicarpellate gynoecium. An octamerous androecium is synapomorphic for Polygalaceae. The development of papilionate flowers, and the evolutionary context in which these phenotypes appeared in Leguminosae and Polygalaceae, shows that the morphologies are convergent rather than synapomorphic within Fabales.

DNA Barcoding of <i>Ricinus communis</i> from Different Geographical Origin by Using Chloroplast <i>matK</i> and Internal Transcribed Spacers

Ricinus communis have attracted considerable attention because of its specific industrial and pharmacological activities. DNA barcodes can be used as reliable tools to facilitate the identification of medicinal plants for the safe use, quality control and forensic investigation. In this study, the differential identification of eight accessions of R. com-munis was investigated through DNA sequence analysis of two candidate DNA barcodes. The nucleotide sequence of internal transcribed spacers (ITS2) and chloroplast maturase gene (matK) have been determined to construct the phylogenetic tree. The phylogenetic relationships of accessions based on the nrITS2 region and partial matK region showed that all accessions in this study were related to three geographical origins. Based on sequence align-ment and phylogenetic analyses we concluded that the ITS2 sequences can distinguish R. communis accessions from different geographical distributions.

PTIGS-IdIt, a system for species identification by DNA sequences of the psbA-trnH intergenic spacer region.

BackgroundDNA barcoding technology, which uses a short piece of DNA sequence to identify species, has wide ranges of applications. Until today, a universal DNA barcode marker for plants remains elusive. The rbcL and matK regions have been proposed as the “core barcode” for plants and the ITS2 and psbA-trnH intergenic spacer (PTIGS) regions were later added as supplemental barcodes. The use of PTIGS region as a supplemental barcode has been limited by the lack of computational tools that can handle significant insertions and deletions in the PTIGS sequences. Here, we compared the most commonly used alignment-based and alignment-free methods and developed a web server to allow the biologists to carry out PTIGS-based DNA barcoding analyses.ResultsFirst, we compared several alignment-based methods such as BLAST and those calculating P distance and Edit distance, alignment-free methods Di-Nucleotide Frequency Profile (DNFP) and their combinations. We found that the DNFP and Edit-distance methods increased the identification success rate to ~80%, 20% higher than the most commonly used BLAST method. Second, the combined methods showed overall better success rate and performance. Last, we have developed a web server that allows (1) retrieving various sub-regions and the consensus sequences of PTIGS, (2) annotating novel PTIGS sequences, (3) determining species identity by PTIGS sequences using eight methods, and (4) examining identification efficiency and performance of the eight methods for various taxonomy groups.ConclusionsThe Edit distance and the DNFP methods have the highest discrimination powers. Hybrid methods can be used to achieve significant improvement in performance. These methods can be extended to applications using the core barcodes and the other supplemental DNA barcode ITS2. To our knowledge, the web server developed here is the only one that allows species determination based on PTIGS sequences. The web server can be accessed at http://psba-trnh-plantidit.dnsalias.org.

Molecular phylogeny of relict-endemic Liquidambar orientalis Mill based on sequence diversity of the chloroplast-encoded matK gene

The genetic diversity and evolutionary divergence in Liquidambar species and Liquidambar orientalis varieties were compared with respect to the matK gene. A total of 66 genotypes from 18 different populations were sampled in southwestern Turkey. The matK region, which is about 1,512 bp in length, was sequenced and studied. L. orientalis, L. styraciflua, and L. formosana had similar magnitude of nucleotide diversity, while L. styraciflua and L. acalycina possessed higher evolutionary divergence. The highest evolutionary divergence was found between L. styraciflua and eastern Asian Liquidambar species (0.0102). However, the evolutionary divergence between L. orientalis and other species was of a similar magnitude. The maximum-parsimony phylogenetic tree showed that L. styraciflua and L. orientalis formed a closer clade while East Asian species were in a separate clade. This suggests that the North Atlantic Land Bridge through southern Greenland may have facilitated continuous distribution of Liquidambar species from southeastern Europe to eastern North America in early Tertiary period. The maximum-parsimony tree with only 18 Oriental sweetgum populations indicated that there were two main clusters: one with mainly L. orientalis var. integriloba and the other with var. orientalis and undetermined populations. High nucleotide diversity (0.0028) and divergence (0.00072) were found in L. orientalis var. integriloba populations and Mugla-1 geographical region. This region could be considered as the major refugium and genetic diversity center for the species. The low genetic diversity and divergence at intraspecies level suggest that L. orientalis populations in Turkey share an ancestral polymorphism from which two varieties may have evolved.

Phylogenetic Analysis of the Wintergreen Group (Ericaceae) based on Six Genic Regions

Previous phylogenetic studies based on DNA sequence data from the genic regions ndhF, matK, atpB-rbeL (cpDNA) and ITS 2 (nrDNA) strongly support the monophyly of the wintergreen group (Ericaceae: Gaultherieae: Diplycosia, Gaultheria, Tepuia) and have assessed the relationships of its major clades. Other studies that include additional genic regions have been limited to two clades corresponding to various sections or series within Gaultheria. Here we expand both the number of species (from 42 to 104) and genic regions (chloroplast mat K, ndhF, rpl16, trnL-trnF, trnS-trnG, and the complete ITS region) to further assess phylogenetic relationships in the wintergreen group. With the additional data we detected several areas of incongruence between the trees from the nuclear and combined chloroplast analyses, including a topologically deep conflict involving G. procumbens. Such incongruence likely originated from reticulation events, long considered to have influenced the evolution of various lineages in Gaultheria. We also detected a duplication, possibly nuclear-encoded, of the matK region in one of the Australian/New Zealand lineages. A combined six-gene analysis, in which taxa involved in the conflicting topologies were excluded, yielded higher support values for several early-diverging clades. The monophyly of both Diplycosia and Tepuia is corroborated, as is the successive nesting of Tepuia, G. section Hispidulae (both placements of which have newfound strong support), G. ser. Gymnobotrys, and Diplycosia. Whereas several sections or series with more than one species in the most recent classification of Gaultheria are supported as circumscribed (i.e. G. section Amblyandra and series Hispidulae, Myrtilloideae, and Trichophyllae), others comprise species from two to several different clades.

Differentiation of Populus species using chloroplast single nucleotide polymorphism (SNP) markers – essential for comprehensible and reliable poplar breeding

Within the genus Populus several species belonging to different sections are cross-compatible. Hence, high numbers of interspecies hybrids occur naturally and, additionally, have been artificially produced in huge breeding programmes during the last 100 years. Therefore, determination of a single poplar species, used for the production of 'multi-species hybrids' is often difficult, and represents a great challenge for the use of molecular markers in species identification. Within this study, over 20 chloroplast regions, both intergenic spacers and coding regions, have been tested for their ability to differentiate different poplar species using 23 already published barcoding primer combinations and 17 newly designed primer combinations. About half of the published barcoding primers yielded amplification products, whereas the new primers designed on the basis of the total sequenced cpDNA genome of Populus trichocarpa Torr. & Gray yielded much higher amplification success. Intergenic spacers were found to be more variable than coding regions within the genus Populus. The highest discrimination power of Populus species was found in the combination of two intergenic spacers (trnG-psbK, psbK-psbl) and the coding region rpoC. In barcoding projects, the coding regions matK and rbcL are often recommended, but within the genus Populus they only show moderate variability and are not efficient in species discrimination.

DNA barcoding of the recently evolved genus Holcoglossum (Orchidaceae: Aeridinae): a test of DNA barcode candidates

Orchidaceae is one of the largest families of flowering plants. Many species of orchid are endangered, and all species are included in Conventions on International Trade of Endangered Species of Fauna and Flora (CITES) I and II, but it is very difficult to identify orchid species, even those with fertile parts. The genus Holcoglossum (Orchidaceae: Aeridinae) has long been problematic in taxonomy. It consists of both long-evolved and radiated species and is an excellent case to use for testing DNA barcodes for Orchidaceae. We investigated the power of a subset of proposed plant barcoding loci [rbcL, matK, atpF-atpH, psbK-psbI, trnH-psbA and internal transcribed spacer (ITS)] to discriminate between species in this genus. Our results showed that all these DNA regions, except psbK-psbI and atpF-atpH, can be amplified easily from Holcoglossum and sequenced with established primers. The DNA regions matK and ITS had the highest variability. Among the six loci, matK resolved eight of the 12 Holcoglossum species and had the highest discriminatory ability. However, the combination of matK and ITS showed a greater ability to identify species than matK alone. Single or combined DNA markers discriminated between Holcoglossum species distributed in tropical areas effectively, but had less ability to identify radiated species from the temperate Hengduan Mountains of China. In the study, matK proved to be a useful DNA barcode for the genus Holcoglossum; however, complementary DNA regions are still required to accelerate the investigation and preservation of radiated species of orchid.

Testing four barcoding markers for species identification of Potamogetonaceae

Abstract The pondweeds (Potamogetonaceae) are among the most important plant groups in the aquatic environment. Owing to their high morphological and ecological diversity, species identification of this aquatic family remains problematic. DNA barcoding involves sequencing a standard DNA region and has been shown to be a powerful tool for species identification. In the present study, we tested four barcoding markers (rbcL, matK, internal transcribed spacer (ITS), and trnH–psbA) in 15 Potamogeton species and two Stuckenia species, representing most species of the Potamogetonaceae in China. The results show that all four regions can distinguish and support the newly proposed genera of Stuckenia from Potamogeton. Using ITS and trnH–psbA, significant interspecific genetic variability was shown. However, intraspecific genetic variability of trnH–psbA is high and so it is not suitable for barcoding in Potamogetonaceae. The ITS and matK regions showed good discrimination. However, matK was not easy to sequence using universal primers. The best performing single locus was ITS, making it a potentially useful DNA barcode in Potamogetonaceae.

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable.

A single Mid-Pleistocene long-distance dispersal by a bird can explain the extreme bipolar disjunction in crowberries (Empetrum).

The proposed age of the striking biogeographic disjunction between the Arctic and southernmost South America varies from more than 65 million to a few thousand years, but no estimates based on explicit models and molecular data are available. Here we address the origin of bipolarity in crowberries (Empetrum), which are heath-forming dwarf shrubs with animal-dispersed fruits. We apply a fossil-calibrated relaxed molecular clock to model sequence evolution in two nuclear low-copy and two plastid DNA regions from 41 individual plants (420 clones for the nuclear regions) representing the entire geographic distribution of crowberries. The plastid region matK and four fossil calibration points were used to infer the ages of the crowberry stem and crown groups. All analyses resolved three major crowberry clades (A-C). Clade A contained sequences from the eastern Canadian pink-fruited crowberry (E. eamesii) as sister to clades B and C, which both contained sequences from the black-fruited northern hemisphere crowberry (E. nigrum). Clade B also contained a subclade with all sequences from the red-fruited southern hemisphere crowberry, which is often referred to as a distinct species, E. rubrum. Its closest relatives were consistently identified as black-fruited plants from northwestern North America. The median time to the most recent common ancestor for northern and southern hemisphere crowberries was estimated to 0.56-0.93 Ma, and 0.26-0.59 Ma for the southern plants only. We conclude that a single dispersal by a bird from northwestern North America to southernmost South America, taking place in the Mid-Pleistocene, is sufficient to explain the disjunction in crowberries.

Identification of Fabaceae Plants Using the DNA BarcodematK

In this study, we tested the applicability of the core DNA barcode MATK for identifying species within the Fabaceae family. Based on an evaluation of genetic variation, DNA barcoding gaps, and species discrimination power, MATK is a useful barcode for Fabaceae species. Of 1355 plant samples collected from 1079 species belonging to 409 diverse genera, MATK precisely identified approximately 80 % and 96 % of them at the species and genus levels, respectively. Therefore, our research indicates that the MATK region is a valuable marker for plant species within Fabaceae.