Species discrimination of novel chloroplast DNA barcodes and their application for identification of Panax (Aralioideae, Araliaceae)

Certain species within the genus Panax L. (Araliaceae) contain pharmacological precious ginsenosides, also known as ginseng saponins. Species containing these compounds are of high commercial value and are thus of particular urgency for conservation. However, within this genus, identifying the particular species that contain these compounds by morphological means is challenging. DNA barcoding is one method that is considered promising for species level identification. However, in an evolutionarily complex genus such as Panax, commonly used DNA barcodes such as nrITS, matK, psbA-trnH, rbcL do not provide species-level resolution. A recent in silico study proposed a set of novel chloroplast markers, trnQ-rps16, trnS-trnG, petB, and trnE-trnT for species level identification within Panax. In the current study, the discriminatory efficiency of these molecular markers is assessed and validated using 91 reference barcoding sequences and 38 complete chloroplast genomes for seven species, one unidentified species and one sub-species of Panax, and two outgroup species of Aralia L. along with empirical data of Panax taxa present in Vietnam via both distance-based and tree-based methods. The obtained results show that trnQ-rps16 can classify with species level resolution every clade tested here, including the highly valuable Panax vietnamensis Ha et Grushv. We thus propose that this molecular marker to be used for identification of the species within Panax to support both its conservation and commercial trade.

Phylogenetic position of Phlojodicarpus villosus (Apiaceae) based on ITS and trnH-psbA nucleotide sequences

The aim of our work was to obtain chloroplast (trnH-psbA) and nuclear (ITS1-ITS2) DNA nucleotide sequences and identify the phylogenetic position of Phlojodicarpus villosus (Apiaceae). This species of vascular plants is represented in the Urals by isolated relic populations and is included in the regional Red Data Books. There is no data on P. villosus nucleotide sequences in the international open genetic databases. We studied two herbarium specimens of P. villosus, one collected from the Ural part of its range in the Komi Republic (Northern Urals) and the second collected from the main part of its range in the Magadan Region (Kolyma Highlands). Combining nuclear and chloroplast markers made it possible to reliably determine phylogenetic position of P. villosus within the tribe Selineae (subfamily Apioideae, family Apiaceae). We found ITS1-ITS2 and trnH-psbA nucleotide sequences to be sufficiently informative to identify specimens of this genus. High polymorphism of P. villosus sequences obtained from different parts of its range (Northern Urals and Kolyma Highlands) and the presence of evolutionary events (deletions) require more detail study of P. villosus and other Phlojodicarpus taxa by DNA barcoding methods.

Adiantum japonicum, a new species of the Adiantum pedatum complex (Pteridaceae) from Japan

The Adiantum pedatum complex is disjunctly distributed in North America and eastern Asia. In this study, we carried out a detailed morphological study based on 137 specimens representing the biogeographic diversity of this complex. The sequences of eight chloroplast markers of 35 samples were analyzed with maximum likelihood and Bayesian inference. The morphological and phylogenetic analyses support the recognition of a new species of the A. pedatum complex from Japan. We propose the new species as A. japonicum based on the examinations of specimens from Japan and the comparative analyses of the whole complex. This new species can be distinguished from A. pedatum by the erect rhizome and shows significant molecular differences from other species in this complex. A taxonomic description with detailed morphological characters of the new species is presented.

Hyoscyamus labiatus sp. nov. (Solanaceae: Hyoscyameae) from Henan, China: implications for the delimitation of Hyoscyamus

Background Hyoscyamus, the largest genus in the tribe Hyoscyameae, harbors more than 20 species. Although the monophyly of Hyoscyamus is supported by both morphological and molecular phylogenetic studies, the delimitation of Hyoscyamus has been hotly debated in different classifications. Here, we report a new species of Hyoscyamus (Solanaceae) from Henan Province, China, and discuss the delimitation of Hyoscyamus. Results This species is morphologically similar to the known species of Hyoscyamus and its close related genus Archihyoscyamus, but can be distinguished by corolla 2-lipped, adaxial lip 3-lobed and much longer than abaxial lips, tube slender at base, and stamens 5, inserted on inner side of disk, free, obviously unequal, and exceeding corolla. Phylogenetic analysis based on four chloroplast markers including rbcL, ndhF, trnC-psbM and trnL-trnF, strongly suggested that the new species was sister to a monophyletic group containing all species of Hyoscyamus and Archihyoscyamus previously described. Conclusions Both the morphological observations and molecular phylogenetic analyses support the recognition of Hyoscyamus labiatus as a new species. Our study also showed that Archihyoscyamus should be a synonym of Hyoscyamus. The delimitation of Hyoscyamus is thus revised in our study.

A Reassessment of the Little-Known Amazonian Fern Diplazium praestans Based on Molecular and Morphological Evidence

Abstract— Family- and genus-level circumscription of ferns in the suborder Aspleniineae (eupolypods II) has long been controversial, due in part to confusion about the relationship among the families Aspleniaceae and Athyriaceae. Recent studies have demonstrated that character states traditionally used to infer a close relationship between these two families are either symplesiomorphic or homoplastic, and re-examination of numerous taxa has led to the recircumscription of several clades, and the description of several new families and genera. In light of these findings, we re-evaluated the taxonomic affinities of Diplazium praestans, a little-known fern from western Amazonia that is morphologically disparate to the remainder of Neotropical Diplazium. Using sequence data from three chloroplast markers and analysis of eight morphological characters, we demonstrate that Diplazium praestans was erroneously placed in that genus and instead is a Hymenasplenium. We place it in a phylogenetic context, reassess its morphology in light of our findings, evaluate its conservation status under IUCN criteria, and provide a new combination: Hymenasplenium praestans. We also provide an updated key to the Neotropical species of Hymenasplenium and discuss unresolved taxonomic problems in the genus.

Analysis of Latvian and Belarusian Oak (Quercus robur L.) Population Provenance and Genetic Structure Using Chloroplast Markers

In this study, oak specific chloroplast simple sequence repeat (SSR) markers were used to analyse Latvian and Belarusian oak (Quercus robur L.) population provenance and genetic structure. Chloroplast haplotypes were compared between Latvian and Belarusian pedunculate oak, and several common haplotypes were identified. The SSR haplotypes were compared to previously reported PCR-RFLP haplotypes, and haplotypes from the A (eastern European) and C (central European) lineages were identified. C lineage haplotypes were only found in the western region of Latvia. Haplotypes shared between Latvia and Belarus were all from the A lineage. Despite the much smaller population size of oak in Latvia in comparison to Belarus, the level of genetic diversity identified using the chloroplast SSR markers is similar. Provenance trials will need to be established in order to determine the suitability of Belarusian oak reproductive material for deployment in Latvia. Based on the results from this comparison of chloroplast haplotypes, as well as climatic similarities, it is probable that Belarusian oak material will be better suited to the eastern regions of Latvia compared to the western regions of Latvia.

Genetic differentiation of indigenous (Quercus robur L.) and late flushing oak stands (Q. robur L. subsp. slavonica (Gáyer) Mátyás) in western Germany (North Rhine-Westphalia)

Slavonian oaks (Quercus robur subsp. slavonica (Gáyer) Mátyás) originating from Croatia have been cultivated in Germany mainly in the Münsterland region of North Rhine-Westphalia since the second half of the nineteenth century. Compared to indigenous pedunculate oak stands in Germany, they are characterised by their late bud burst, but also by their excellent bole shape and faster height growth. Previously, Slavonian pedunculate oaks (= late flushing oaks) were mainly studied at chloroplast (cp) DNA markers in order to determine their geographical origin. The origin of the material is probably the Sava lowland between Zagreb and Belgrade. In the present study, the aim was to genetically differentiate between indigenous Quercus robur and Slavonian oak stands using nuclear DNA markers. For this purpose, we used 20 nuclear Simple Sequence Repeats (nSSRs). A total of 37 pedunculate oak stands (mean: 18.6 samples per population with an age of 95 to 210 years) were examined, of which 21 were characterized as Slavonian late flushing oaks and three stands for which the Slavonian origin was not clear. Maternally inherited chloroplast markers were analysed earlier in all 37 stands to validate their geographic origin. We found that the stands of native pedunculate oaks and Slavonian pedunculate oaks are represented by two genetic clusters which are weakly differentiated. Slavonian oaks (Na = 9.85, Ar = 8.689, Ho = 0.490, He = 0.540) showed similar levels of genetic variation as native oak stands (Na = 7.850, Ar = 7.846, Ho = 0.484, He = 0.526). Differences in growth and phenology and low but consistent genetic differentiation between groups suggest that both taxa represent different ecotypes with specific local adaptations, which are perhaps separated by less overlapping flowering phenologies. The nuclear microsatellite markers in combination with the cpDNA markers are suitable to differentiate between Slavonian and local oak stands.

Detection of Peanut Allergen by Real-Time PCR: Looking for a Suitable Detection Marker as Affected by Processing

Peanut (Arachis hypogaea) contains allergenic proteins, which make it harmful to the sensitised population. The presence of peanut in foods must be indicated on label, to prevent accidental consumption by allergic population. In this work, we use chloroplast markers for specific detection of peanut by real-time PCR (Polymerase Chain Reaction), in order to increase the assay sensitivity. Binary mixtures of raw and processed peanut flour in wheat were performed at concentrations ranging from 100,000 to 0.1 mg/kg. DNA isolation from peanut, mixtures, and other legumes was carried out following three protocols for obtaining genomic and chloroplast-enrich DNA. Quantity and quality of DNA were evaluated, obtaining better results for protocol 2. Specificity and sensitivity of the method has been assayed with specific primers for three chloroplast markers (mat k, rpl16, and trnH-psbA) and Ara h 6 peanut allergen-coding region was selected as nuclear low-copy target and TaqMan probes. Efficiency and linear correlation of calibration curves were within the adequate ranges. Mat k chloroplast marker yielded the most sensitive and efficient detection for peanut. Moreover, detection of mat K in binary mixtures of processed samples was possible for up to 10 mg/kg even after boiling, and autoclave 121 °C 15 min, with acceptable efficiency and linear correlation. Applicability of the method has been assayed in several commercial food products.

Geographic Patterns of Genetic Variation among Cacao (Theobroma cacao L.) Populations Based on Chloroplast Markers

The cacao tree (Theobroma cacao L.) is native to the Amazon basin and widely cultivated in the tropics to produce seeds, the valuable raw material for the chocolate industry. Conservation of cacao genetic resources and their availability for breeding and production programs are vital for securing cacao supply. However, relatively little is still known about the phylogeographic structure of natural cacao populations. We studied the geographic distribution of cpDNA variation in different populations representing natural cacao stands, cacao farms in Ecuador, and breeding populations. We used six earlier published cacao chloroplast microsatellite markers to genotype 233 cacao samples. In total, 23 chloroplast haplotypes were identified. The highest variation of haplotypes was observed in western Amazonia including geographically restricted haplotypes. Two observed haplotypes were widespread across the Amazon basin suggesting long distance seed dispersal from west to east in Amazonia. Most cacao genetic groups identified earlier using nuclear SSRs are associated with specific chloroplast haplotypes. A single haplotype was common in selections representing cacao plantations in west Ecuador and reference Trinitario accessions. Our results can be used to determine the chloroplast diversity of accessions and in combination with phenotypic assessments can help to select geographically distinctive varieties for cacao breeding programs.