Morphological and molecular characterization of two species of genus Ageratum.

BACKGROUND: The species of genus Ageratum (family Asteraceae) are distributed in various parts of the world. Ageratum conyzoides and A. houstonianum are the most commonly occurring species in India. These species are quite similar in their morphology thus creating a challenge in identification during the field survey and taxonomic validation. The accurate identification of the species is highly significant especially when those are of medicinal interest. To overcome the barriers in morphological based identification, DNA barcoding has been employed during the present investigation. METHODS AND RESULTS: Morphological and DNA barcodes matK and ITS genes, were employed to differentiate between Ageratum conyzoides and A. houstonianum. The obtained matK and ITS gene sequences were submitted to GenBank and BOLD system to obtain accession numbers. The DNA sequences were aligned with database sequences using BLAST and phylogenetic trees were constructed through neighbor-joining algorithm in MEGA 11 software. The distinguish features of A. conyzoides include ovate to elliptic-oblong leaves with a cuneate base and inflorescence heads forming domed to flat-topped clusters. However, A. houstonianum has triangular to ovate leaves with a cordate to truncate base, cymose clusters in the inflorescence and stipulate glandular involucre bracts. The matK gene has shown the highest identity percentages (100%) for A. houstonianum and 99.87% for A. conyzoides. The phylogenetic tree analysis has demonstrated a close association of A. conyzoides and A. houstonianum with their respective species, supported by bootstrap values in the matK and ITS trees. CONCLUSION: This study revealed that morphological and molecular data can be successfully utilized in the identification of A. conyzoides and A. houstonianum. The matK and ITS barcodes provide promising results in the identification of Ageratum species, with their phylogeny supporting classification within the family asteraceae.

Extreme plastomes in holoparasitic Balanophoraceae are not the norm.

BACKGROUND: Balanophoraceae plastomes are known for their highly condensed and re-arranged nature alongside the most extreme nucleotide compositional bias known to date, culminating in two independent reconfigurations of their genetic code. Currently, a large portion of the Balanophoraceae diversity remains unexplored, hindering, among others, evolutionary pattern recognition. Here, we explored newly sequenced plastomes of Sarcophyte sanguinea and Thonningia sanguinea. The reconstructed plastomes were analyzed using various methods of comparative genomics based on a representative taxon sampling. RESULTS: Sarcophyte, recovered sister to the other sampled Balanophoraceae s. str., has plastomes up to 50% larger than those currently published. Its gene set contains five genes lost in any other species, including matK. Five cis-spliced introns are maintained. In contrast, the Thonningia plastome is similarly reduced to published Balanophoraceae and retains only a single cis-spliced intron. Its protein-coding genes show a more biased codon usage compared to Sarcophyte, with an accumulation of in-frame TAG stop codons. Structural plastome comparison revealed multiple, previously unknown, structural rearrangements within Balanophoraceae. CONCLUSIONS: For the "minimal plastomes" of Thonningia, we propose a genetic code change identical to sister genus Balanophora. Sarcophyte however differs drastically from our current understanding on Balanophoraceae plastomes. With a less-extreme nucleotide composition, there is no evidence for an altered genetic code. Using comparative genomics, we identified a hotspot for plastome reconfiguration in Balanophoraceae. Based on previously published and newly identified structural reconfigurations, we propose an updated model of evolutionary plastome trajectories for Balanophoraceae, illustrating a much greater plastome diversity than previously known.

Phylogenetic analysis based on the ITS, matK and rbcL DNA barcodes and comparison of chemical contents of twelve Paeonia taxa in Türkiye.

BACKGROUD: Twelve taxa of herbaceous Paeonia species were recorded in Türkiye. All definitions were performed morphologically and/or anatomically and there is no study based on DNA barcode sequences. Three barcode regions were sequenced to determine the phylogenetic relationships of Turkish Paeonia taxa. The chemical comparison of roots was also investigated. METHODS AND RESULTS: The taxons were collected between May and June 2021 from nine cities. Leaf materials were used for DNA isolation and ITS, matK and rbcL regions were amplified and sequenced. There was no difference among taxa in terms of rbcL sequences. But the ITS and matK regions distinguished 12 taxa and structured them in two groups. ITS region distinguished P. peregrina, P. arietina, and P. tenuifolia from other taxa, while matK region distinguished P. arietina and P. witmanniana from other taxa. Both barcode sequences actually showed that the registration of P. mascula subsp. arasicola was actually 100% similar to P. arietina. ITS was the most polymorphic region (n = 54) followed by matK (n = 9). These sequences could successfully discriminate Paoenia species from each other and diploid P. tenuifolia. The methanolic root (100 gr) extracts were examined for total phenolic and flavonoid content, and antioxidant activities. Significant variation was found for polyphenolic content, and antioxidant properties (TPC from 204.23 to 2343.89 mg, TFC from 7.73 to 66.16 mg, and FRAP from 523.81 to 4338.62 mg). SC50 values of ABTS and DPPH were ranged from 115.08 to 1115.52 µg/ml and 73.83 to 963.59 µg/ml, respectively. CONCLUSION: It was concluded that 11 of 12 taxa had differences in terms of ITS and matK sequences and these region must be used for the correct identification of Turkish Paeonia.

Gene loss, genome rearrangement, and accelerated substitution rates in plastid genome of Hypericum ascyron (Hypericaceae).

BACKGROUND: Comparative genomic analysis exhibits dynamic evolution of plastid genome (plastome) in the clusioid clade of Malpighiales, which comprise five families, including multiple inversions and gene losses. Little is known about the plastome evolution in Hypericaceae, a large family in the clade. Only the plastome of one species, Cratoxylum cochinchinense, has been published. RESULTS: We generated a complete plastome sequence for Hypericum ascyron, providing the first complete plastome from the tribe Hypericeae (Hypericaceae). The H. ascyron plastome exhibits dynamic changes in gene and intron content, structure, and sequence divergence compared to the C. cochinchinense plastome from the tribe Cratoxyleae (Hypericaceae). Transcriptome data determined the evolutionary fate of the missing plastid genes infA, rps7, rps16, rpl23, and rpl32 in H. ascyron. Putative functional transfers of infA, rps7, and rpl32 were detected to the nucleus, whereas rps16 and rpl23 were substituted by nuclear-encoded homologs. The plastid rpl32 was integrated into the nuclear-encoded SODcp gene. Our findings suggested that the transferred rpl32 had undergone subfunctionalization by duplication rather than alternative splicing. The H. ascyron plastome rearrangements involved seven inversions, at least three inverted repeat (IR) boundary shifts, which generated gene relocations and duplications. Accelerated substitution rates of plastid genes were observed in the H. ascyron plastome compared with that of C. cochinchinense plastid genes. The higher substitution rates in the accD and clpP were correlated with structural change, including a large insertion of amino acids and losses of two introns, respectively. In addition, we found evidence of positive selection of the clpP, matK, and rps3 genes in the three branches related to H. ascyron. In particular, the matK gene was repeatedly under selection within the family Hypericaceae. Selective pressure in the H. ascyron matK gene was associated with the loss of trnK-UUU and relocation into the IR region. CONCLUSIONS: The Hypericum ascyron plastome sequence provides valuable information for improving the understanding of plastome evolution among the clusioid of the Malpighiales. Evidence for intracellular gene transfer from the plastid to the nucleus was detected in the nuclear transcriptome, providing insight into the evolutionary fate of plastid genes in Hypericaceae.

Sodium hyperaccumulators in the Caryophyllales are characterized by both abnormally large shoot sodium concentrations and [Na]shoot/[Na]root quotients greater than unity.

BACKGROUND AND AIMS: Some Caryophyllales species accumulate abnormally large shoot sodium (Na) concentrations in non-saline environments. It is not known whether this is a consequence of altered Na partitioning between roots and shoots. This paper tests the hypotheses (1) that Na concentrations in shoots ([Na]shoot) and in roots ([Na]root) are positively correlated among Caryophyllales, and (2) that shoot Na hyperaccumulation is correlated with [Na]shoot/[Na]root quotients. METHODS: Fifty two genotypes, representing 45 Caryophyllales species and 4 species from other angiosperm orders, were grown hydroponically in a non-saline, complete nutrient solution. Concentrations of Na in shoots and in roots were determined using inductively coupled plasma mass spectrometry (ICP-MS). KEY RESULTS: Sodium concentrations in shoots and roots were not correlated among Caryophyllales species with normal [Na]shoot, but were positively correlated among Caryophyllales species with abnormally large [Na]shoot. In addition, Caryophyllales species with abnormally large [Na]shoot had greater [Na]shoot/[Na]root than Caryophyllales species with normal [Na]shoot. CONCLUSIONS: Sodium hyperaccumulators in the Caryophyllales are characterized by abnormally large [Na]shoot, a positive correlation between [Na]shoot and [Na]root, and [Na]shoot/[Na]root quotients greater than unity.

DNA barcoding of some medicinally important plant species of Lamiaceae family in India.

Several species of the Lamiaceae family are the primary source of bioactive aromatic oils and secondary metabolites, having broader applications in the cosmetics, pharmaceuticals, food, confectionery and liquor industries. Due to the scarcity of raw materials and high costs of this family's economically vital species, its products often adulterated to cater to the market's high demand. The present study provides a DNA based approach for identifying different species of this family. Henceforth, the performance of three already proposed barcode loci (matK, trnH-psbA and trnL) was examined for their PCR amplification and species recognition efficacy on various Lamiaceae species and cultivars using three different approaches such as pairwise genetic distance method, BLASTn and phylogenetic tree based on maximum likelihood (ML) analysis. Results illustrate that among all the DNA barcoding loci, matK locus can accurately and efficiently distinguish all the studied species followed by trnH-psbA and trnL. Present investigation may help diminish the illegal trade and events of adulteration of medicinally important plants species in genus Mentha, Ocimum and Plectranthus. This investigation will also help fulfil the scarcity of sequences of barcode loci of these species in the NCBI database. Apart from providing a molecular level reference for identifying processed herbal products, this technique also offers a convenient method for species identification and germplasm conservation of the Lamiaceae family.

Identification of three seagrass species in coral reef ecosystem by using multiple genes of DNA barcoding.

Seagrasses constitute a significant part of coral reef ecosystems, representing high primary productivity and one of the most important coastal habitats in marine ecosystems. Though seagrasses possess irreplaceable ecological services to the marine environment, taxonomical ambiguity still exists due to similar morphological characters and phenotypic plasticity. As an emerging technology, DNA barcoding can effectively identify cryptic species using a short orthologous DNA region. In this study, we collected samples from five different locations (Daya Bay, Xincun Bay, Sanya Bay, Xisha Islands, and Nansha Islands), and three seagrass species Cymodocea rotundata, Thalassia hemprichii and Halophila ovalis was evaluated. Moreover, ITS, matK and rbcL genes were used as DNA barcodes. The results indicated that single ITS and concatenated ITS/matK/rbcL both conducted better species resolution than single matK and rbcL. Nevertheless, single ITS was more convenient. Furthermore, in all the four topology trees, three species resolved as 3 clusters as well H. ovalis and T. hemprichii grouped as sister clade. In the meantime, differentiation lay in intra-species based on the result of single ITS and three-locus analysis. Within H. ovalis and T. hemprichii separately, individuals from Xisha Islands first group together, then grouped with individuals from Nansha Islands and/or Xincun Bay and/or Sanya Bay and/or Daya Bay, which indicated that geographical distribution influenced population evolution. However, intra-species differentiation did not emerge in the tree of matK or rbcL.

Exploring phylogeny of the microsoroid ferns (Polypodiaceae) based on six plastid DNA markers.

The microsoroid ferns are one of the largest subfamilies of the Polypodiaceae with over 180 species mainly found in the humid forests of tropical Australasia. The phylogenetic relationships are still unclear, especially the delimitation of the genus Microsorum which has been recognized to be non-monophyletic. We analysed the microsoroid ferns using six chloroplast DNA regions (rbcL, rps4+rps4-trnS, trnL+trnL-trnF, atpA, atpB and matK) in order to present a robust hypothesis of their phylogeny. Our results suggest that they comprise up to 17 genera; of them, 12 agree with a previously accepted generic classification. Five tribes are proposed based on the phylogenetic relationships. Most of the species traditionally included in the genus Microsorum are found in six genera belonging to two tribes. In addition to the commonly used DNA markers, the additional atpA and matK are helpful to provide information about the phylogenetic relationships of the microsoroid ferns.

DNA barcoding and genetic fidelity assessment of micropropagated Aenhenrya rotundifolia (Blatt.) C.S. Kumar and F.N. Rasm.: a critically endangered jewel orchid.

Aenhenrya rotundifolia is a critically endangered terrestrial jewel orchid. It is monotypic and endemic to evergreen forests of southern western ghats of India. In the present study, identification of this plant species is validated with DNA barcoding using matK and rbcL chloroplast markers. Further, germ-free juvenile axillary bud explants were cultured on Mitra medium supplemented with different kinds of cytokinins like 6-benzyladenine, 6-furfurylaminopurine, N6-(Δ2-isopentyl) adenine, thidiazuron, zeatin and meta-topolin as well as auxins such as α-naphthaleneacetic acid, indole-3-acetic acid and indole-3-butyric acid at different concentrations and combinations for successful proliferation and establishment in vitro. After 12 weeks of culture, axillary bud explants produced an average of 30.12 ± 0.71 shoots per explant, 3.87 ± 0.06 cm shoot length, 1671 ± 2.82 mg fresh mass of proliferated shoots with a proliferation frequency of 100% on Mitra medium supplemented with 6.20 µM meta-topolin and 2.25 µM thidiazuron. No root formation was observed in in vitro proliferated microshoots. However, tiny hair like projections were observed in some elongated shoots on Mitra medium pertaining to 5.37 µM NAA. The tiny hair like structure bearing plantlets were hardened and acclimatized with 100% survival rate in the polytunnel chamber. After 8-10 months of establishment ex vitro, flowering was observed. Additionally, the genetic fidelity of in vitro derived plants was tested with ISSR and SCoT marker profiling. The test results revealed that the plants derived from the protocol has 99% genetic similarity to that of the donor mother plant. This study can be applied in forensic interventions of this species, describes the maintenance of germplasm in vitro and establishment of new viable population in its original habitats by restoring existing sites of this critically endangered jewel orchid.

Two new genera of Podostemaceae from northern Central Laos: saltational evolution and enigmatic morphology.

Podostemaceae, the river-weeds, are characterized by remarkable differences between species and genera, which resulted from saltational evolution. This paper presents additional cases of such two genera, which are described here from the Phou Khao Khouay National Protected Area in northern Central Laos. Molecular phylogenetic data show that Ctenobryum mangkonense (gen. & sp. nov.) is sister to Hydrodiscus koyamae, while Laosia ramosa (gen. & sp. nov.) is isolated from all Asian genera of subfamily Podostemoideae. Ctenobryum mangkonense is distinct from Hydrodiscus koyamae in the crustose roots (versus rootless in the latter), scattered flowers on the root (versus alternate on the shoot), and pectinate bracts (versus simple, sheath-like). Laosia ramosa is distinct from all the genera in the columnar, endogenously branched axes and single style-stigma complex. The axis is an enigmatic organ with combined characteristics of root, stem and leaf, pending further study. Laos, together with Thailand, is a center of diversity of the Southeast and East Asian Podostemoideae. The three monotypic genera, i.e., Ctenobryum, Hydrodiscus and Laosia, occur in neighboring and closely similar aquatic habitats within the Area. The new taxa are formally described.

Genetic diversity of leafy amaranth (Amaranthus tricolor L.) resources in Vietnam.

Leafy amaranths, which are consumed as traditional food in Asia and Africa, are now considered among the most promising vegetables. In Vietnam, leafy amaranths, particularly Amaranthus tricolor L., are important summer vegetables due to their excellent nutritional values and high tolerance to biotic and abiotic stresses. However, this species has not been subjected to systematic breeding. Here we describe species identification and evaluation of the genetic diversity of Vietnamese amaranth collection by using matK and simple sequence repeats (SSR) markers. Our phylogenetic analysis based on the matK marker classified the species of 68% of the accessions, of which 120 belonged to A. tricolor. We developed 21 SSR markers, which amplified a total of 153 alleles in 294 A. tricolor accessions originating from Vietnam and overseas, with a mean allelic richness of 7.29 per marker, observed heterozygosity of 0.14, expected heterozygosity of 0.38, and polymorphic information content of 0.35. The STRUCTURE and FST analysis indicated a positive relationship between geographic distance and genetic differentiation among most of the overseas groups and the Vietnamese collection, but not among geographic groups within the Vietnamese collection. Vietnamese amaranths could be divided into two major types, one common in East Asia and the other one unique to Vietnam.

Employing barcoding markers to authenticate selected endangered medicinal plants traded in Indian markets.

The high demand of medicinal plants and their unrestricted collection have rendered many of these as rare or endangered. The restrictions imposed on their collection and trade are difficult to implement because of the inability to identify them in fragmented form. The rarity of these plants in nature and lack of their cultivation raise doubt about the authenticity of the herbals sold in markets. Therefore, in the present investigation, ITS/ITS2, matK, rbcL and rpoC1 sequences of fourteen species of important medicinal plants, some of which are endangered, were generated and checked for their species-specificity (sequences having maximum similarity only with their own) by BLAST1 and/or BOLD identifications. ITS sequences of 12 species were species-specific. However, ITS2 of only 10 of these 12 species were species-specific. As for the chloroplast loci, rbcL and rpoC1 sequences of all 14 species could be obtained, while matK sequences of only 10 of these could be generated. Of the retrieved sequences, rbcL, rpoC1 and matK sequences of 7, 11 and 7 species, respectively, were species-specific. The sequences of the targeted loci from the herbal samples of these species were difficult to retrieve because of failure in the amplification or sequencing. Nevertheless, based on ITS2 and/or one or more of the chloroplast loci targeted, the botanical identities of 22 herbal market samples were checked by phylogenetic tree, BLAST1 and BOLD identification methods. Of these 22 samples, only one of each of Rauvolfia serpentina and Picrorhiza kurroa were found to be authentic.

Molecular Identification of Three Aquilaria (Thymelaeaceae) Species through DNA Barcoding.

Aquilaria LAM. is an endangered tropical tree that produces agarwood, a common ingredient in medicine, perfumes and incense. The species endemic to China, Aquilaria yunnanensis, is often misidentified as the two valuable species, Aquilaria sinensis and Aquilaria crassna. In present study, three DNA barcodes (internal transcribed spacer (ITS), maturase K gene (matK) and trnL-trnF) were used to evaluate whether these genes can be used to discriminate the three species, and evaluate the phylogenetic relationship between the three Aquilaria species. For accurate identification of the three Aquilaria species, a total of 26 nucleotide variations were detected when comparing the three DNA barcodes. We found that A. sinensis is closely related to A. crassna based on combination of nuclear and chloroplast DNA barcodes, and is closely related to A. yunnanensis based on chloroplast DNA barcodes. Taken together, we suggest that the combination of ITS+matK and ITS+trnL-trnF are suitable for identifying these three Aquilaria species.

Complete Chloroplast Genome Sequences of Four Meliaceae Species and Comparative Analyses.

The Meliaceae family mainly consists of trees and shrubs with a pantropical distribution. In this study, the complete chloroplast genomes of four Meliaceae species were sequenced and compared with each other and with the previously published Azadirachta indica plastome. The five plastomes are circular and exhibit a quadripartite structure with high conservation of gene content and order. They include 130 genes encoding 85 proteins, 37 tRNAs and 8 rRNAs. Inverted repeat expansion resulted in a duplication of rps19 in the five Meliaceae species, which is consistent with that in many other Sapindales, but different from many other rosids. Compared to Azadirachta indica, the four newly sequenced Meliaceae individuals share several large deletions, which mainly contribute to the decreased genome sizes. A whole-plastome phylogeny supports previous findings that the four species form a monophyletic sister clade to Azadirachta indica within the Meliaceae. SNPs and indels identified in all complete Meliaceae plastomes might be suitable targets for the future development of genetic markers at different taxonomic levels. The extended analysis of SNPs in the matK gene led to the identification of four potential Meliaceae-specific SNPs as a basis for future validation and marker development.

[DNA barcoding identification of Dendrobium huoshanense and its adulterants].

This research preliminarily discusses the relations of Dendrobium system growth through chloroplast gene rbcL, matK and the nuclear genome ITS2. The DNA barcoding universal sequence for authentication of the Dendrobium medical plants was slected and the possibility concerning utilizing the DNA barcoding to distinguish the D. huoshanenseand its adulterants was analyzed. Using the universal primer pair of ITS2, rbcL and matK, series of extended sequencing in the Dendrobium were conducted. Meanwhile, considering the different index about amplification and sequencing success rate of each sequence, the intraspecific and interspecific aberrance, the employment of BioEdit and MEGA 5.0 software were applied to establish the systematic tree of the NJ molecular and evaluate the diversified authentication capability of various sequences. The consequence demonstrates that the sequence of ITS2 is not only the largest one both in the intraspecific and interspecific aberrance of the Dendrobium but also has obvious barcoding gap. Considering the few overlap between the intraspecific and interspecific aberrance and the highest percentage regarding the formation of unilateral branch in diverse Dendrobium which have different ITS2 sequences, it can differentiate the species of Dendrobium. Furthermore, due to the inferior success rate of the rbcL and thematK and the lower reliability of NJ systematic tree, the percentage of the unilateral species which are generated by the systematic tree of rbcL and matK sequences is deficient. Therefore, the sequence of ITS2 can serves as DNA barcoding to distinguish the D. huoshanense, the D. moniliform and the D. officinale.

Morphological description and DNA barcoding study of sand rice (Agriophyllum squarrosum, Chenopodiaceae) collected in Kazakhstan.

BACKGROUND: Sand rice (Agriophyllum squarrosum (L.) Moq.) is an annual shrub-like plant adapted to the mobile sand dunes in desert and semi-desert regions of Asia. It has a balanced nutrient composition with relatively high concentration of lipids and proteins, which results in its nutrition being similar to legumes. Sand rice's proteins contain the full range of essential amino acids. However, calories content is more similar to wheat. These features together with desert stress resistance make sand rice a potential food crop resilient to ongoing climate change. It is also an important fodder crop (on young stages of growth) for cattle in arid regions of Kazakhstan. In our work, sand rice samples were collected from two distant regions of Kazakhstan as a part of the nation-wide project to determine genetic variation of the native flora. RESULTS: Samples were collected in western and southeastern parts of Kazakhstan separated by distances of up to 1300 km. Sequences of the nuclear ribosomal DNA ITS1-5.8S-ITS2 region and the chloroplast matK gene confirmed the identity of species defined by morphological traits. Comparison with GenBank sequences revealed polymorphic sequence positions among Kazakh populations and GenBank references, and suggested a distinction among local populations of sand rice. The phylogenetic analysis of nucleotide sequences showed a clear partition of A. squarrosum (L.) Moq. from Agriophyllum minus Fisch. & C.A. Mey, which grows in the same sand dunes environment. CONCLUSIONS: DNA barcoding analyses of ITS and matK sequences showed a segregation of A. squarrosum from A. minus into separate clades in Maximum-Likelhood dendrograms. ITS analysis can be successfully used to characterize A. squarrosum populations growing quite distant from each other. The data obtained in this work provide the basis for further investigations on A. squarrosum population structure and may play a role in the screening of sand rice plants growing in desert and semi-desert environments of Central Asia and China.

Taxonomic assessment of Allium species from Kazakhstan based on ITS and matK markers.

BACKGROUND: As part of nation-wide project to infer the genetic variation of the native flora in Kazakhstan, a study was attempted to assess phylogenetic relationships of endemic and rare Allium species. In total, 20 Allium species were collected in field trips in five different regions of Kazakhstan during 2015-2016. Most species (9) were collected in the southern part of the country along of Karatau mountains, followed by Altai mountains (5) in eastern Kazakhstan. The ITS and matK DNA regions were applied in order to assess the taxonomic relationships among species. The major goal of the study was to assess the taxonomic position of five endemic and rare species from Allium subgenus Reticulatobulbosa collected in Karatau mountains of Southern Kazakhstan. RESULTS: The 20 collected Allium species were assessed using morphological traits and a DNA barcoding approach. The morphological analyses of four different species in subgenus Reticulatobulbosa inferred similarities of A. inconspicuum and A. barszchewskii (both from section Companulata) that were separated from A. oreoscordum and A. oreoprasoides (section Nigrimontana) by several traits, including form of bulbs and leaves, presence of bracts, shape of perianth lobes and style. The Neighbor-Joining method was applied to generate ITS and matK phylogenetic trees for two groups of populations: 1) 20 Allium species collected within the project, and 2) 50 Allium worldwide species. CONCLUSIONS: The analyses of nucleotide sequences of ITS and matK robustly confirmed the monophyletic origin of the Allium species. The variability in 20 local Allium species in ITS was 6.6 higher than in matK, therefore the topology of the ITS tree was better resolved. The taxonomy of Allium species largely coincided with a recent classification of this genus. Analyses of both ITS and matK suggest that A. oreoscordum is genetically close to A. oreoprasoides in section Nigrimontana of subgenus Reticulatobulbosa. This result was also confirmed using morphological description of individual plants of four species in subgenus Reticulatobulbosa. The study is another contribution to taxonomy clarification in Allium.

Phylogeny of Helieae (Gentianaceae): Resolving taxonomic chaos in a Neotropical clade.

The monophyletic and Neotropical tribe Helieae of the worldwide family Gentianaceae (Gentianales, Asterids, Angiospermae) is well known for its problematic generic classifications. An initial phylogenetic analysis of Helieae shed light onto the relationships between genera, and indicated that traditional generic limits did not correspond to monophyletic groups. In order to obtain a more thorough understanding of generic relationships within the group, we enhanced sampling within the so-called Symbolanthus clade and performed phylogenetic analyses from DNA sequences from one plastid region (matK) and two nuclear regions (ITS and 5S-NTS), plus 112 morphological characters, which were analyzed separately and in combination, using parsimony and Bayesian approaches. A total of 83 individuals representing 20 genera and 51 species of Helieae were sampled; 13 species were included in this study solely based on their morphological characters. Ancestral character reconstructions were performed to identify potential synapomorphies of clades and patterns of homoplasy in the morphological dataset. Our results demonstrate that Prepusa is sister to the remainder of Helieae. Furthermore, the Macrocarpaea clade, the Irlbachia clade and the Symbolanthus clade were also recovered. Within the Symbolanthus clade, our results confirm that Calolisianthus and Chelonanthus are not monophyletic, and also contest the monophyly of Irlbachia as currently circumscribed. Specifically, two species of Calolisianthus group with the type species of Chelonanthus, while the other Calolisianthus species are more closely related to Tetrapollinia and Symbolanthus. Moreover, the green-white-flowered Chelonanthus species and Adenolisianthus are undoubtedly related to Helia and several analyses support Irlbachia pratensis as more closely related to the lineage including the type species of Chelonanthus described above The addition of new characters and taxa led to higher confidence in the relative position of some clades, as well as provided further support for a new generic circumscription of Calolisianthus, Chelonanthus, and Helia. Even though several morphological characters traditionally used in the taxonomy of the group were shown to be homoplasious, most clades can be diagnosed by a combination of morphological character states.

Occult recurrence of monomorphic epitheliotropic intestinal T-cell lymphoma and the role of MATK gene expression in diagnosis.

Monomorphic epitheliotropic intestinal T-cell lymphomas (MEITL), formerly Type II enteropathy-associated T-cell lymphomas (EATL), are rare peripheral T-cell lymphomas. They are associated with poor survival outcomes, in part because of their late diagnosis. Although MEITLs may be reliably diagnosed based on histological and immunophenotypic findings, overlaps with other NK/T and T-cell lymphomas may confound the diagnosis. The distinctive high-level nuclear staining of the novel marker Megakaryocyte-associated tyrosine kinase (MATK) in MEITLs is an invaluable tool in distinguishing MEITL from classical EATL and other NK/T or T-cell lymphomas. 18-Fluorine-2-fluorodeoxyglucose positron emission tomography (18 F-FDG PET) has been shown to be a useful tool in the staging and follow-up of aggressive lymphomas. Herein, we describe an unusual case of occult hepatic recurrence of MEITL that was non-avid on 18 F-FDG PET, in which diagnosis was confirmed based on the expression of MATK in tumour cells. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluating five different loci (rbcL, rpoB, rpoC1, matK, and ITS) for DNA barcoding of Indian orchids.

Orchidaceae, one of the largest families of angiosperms, is represented in India by 1600 species distributed in diverse habitats. Orchids are in high demand owing to their beautiful flowers and therapeutic properties. Overexploitation and habitat destruction have made many orchid species endangered. In the absence of effective identification methods, illicit trade of orchids continues unabated. Considering DNA barcoding as a potential identification tool, species discrimination capability of five loci, ITS, matK, rbcL, rpoB, and rpoC1, was tested in 393 accessions of 94 Indian orchid species belonging to 47 genera, including one listed in Appendix I of CITES and 26 medicinal species. ITS provided the highest species discrimination rate of 94.9%. While, among the chloroplast loci, matK provided the highest species discrimination rate of 85.7%. None of the tested loci individually discriminated 100% of the species. Therefore, multi-locus combinations of up to five loci were tested for their species resolution capability. Among two-locus combinations, the maximum species resolution (86.7%) was provided by ITS+matK. ITS and matK sequences of the medicinal orchids were species specific, thus providing unique molecular identification tags for their identification and detection. These observations emphasize the need for the inclusion of ITS in the core barcode for plants, whenever required and available.