Molecular phylogeny of the genus<Emphasis Type="Italic">Hypericum</Emphasis> (Hypericaceae) from Korea and Japan: evidence from nuclear rDNA ITS sequence data

As part of our ongoing phylogenetic study of genusHypericum, nuclear ribosomal DNA internal transcribed spacer sequences were analyzed for 36 species ofHypericum as ingroup and two species ofThornea as outgroup. This sampling included most of the previously described species from both Korea and Japan. The ITS phylogeny suggested that the surveyedHypericum species belong to a monophyletic section,Trigynobrathys, and a polyphyletic section,Hypericum. In addition, two monotypic sections,Sampsonia andRoscyna, were identified. Members of sectionHypericum occur in four different lineages worldwide, which imply at least four independent origins. The Korean and Japanese species of sectionHypericum form a monophyletic group, except forH. vulcanicum. Instead, that particular species belongs to a distinct monophyletic group withH. scoreri andH. formosa from other geographic areas, and is a sister to sectionTrigynobrathys. The Korean and Japanese species of sectionTrigynobrathys show a monophyletic origin.H. sampsonii is now recognized as a distinct section rather than being a member of sectionsHypericum orDrosocarpium, as had been indicated previously. Our results differ somewhat from those of recent morphological and cytological studies. The phylogenetic relationships among Korean and Japanese species have now been mostly resolved via ITS phylogeny.     

Molecular phylogenetics and morphological evolution of St. John’s wort (Hypericum; Hypericaceae)

Phylogenetic hypotheses for the large cosmopolitan genus Hypericum (St. John’s wort) have previously been based on morphology, and molecular studies have thus far included only a few species. In this study, we used 360 sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) for 206 species representing Hypericum (incl. Triadenum and Thornea) and three other genera of Hypericaceae to generate an explicit phylogenetic hypothesis for the genus using parsimony and model-based methods. The results indicate that the small genus Triadenum is nested in a clade within Hypericum containing most of the New World species. Sister to Hypericum is Thornea from Central America. Within Hypericum, three large clades and two smaller grades were found; these are based on their general morphology, especially characters used previously in taxonomy of the genus. Relative to the most recent classification, around 60% of the sections of Hypericum were monophyletic. We used a Bayesian approach to reconstruct ancestral states of selected morphological characters, which resulted in recognition of characters that support major clades within the genus and a revised interpretation of morphological evolution in Hypericum. The shrubby habit represents the plesiomorphic state from which herbs evolved several times. Arborescent species have radiated convergently in high-elevation habitats in tropical Africa and South America.     

New insights into diversity and selectivity of trentepohlialean lichen photobionts from the extratropics

Aerial green algae of Trentepohliaceae can form conspicuous free-living colonies, be parasites of plants or photobionts of lichen-forming ascomycetes. So far, their diversity in temperate regions is still poorly known as it has been mostly studied by phenotypic approaches only. We present new insights in the phylogenetic relationships of lichenized representatives from temperate and Mediterranean parts of Europe by analysis of 18S rRNA and rbcL gene fragments, and nuclear ITS sequence data. For this purpose we isolated the trentepohlialean photobionts from lichens representing different genera. Algal cultures from lichenized and free-living Trentepohliaceae were used to design new primers for amplification of the marker loci. We constructed a phylogenetic hypothesis to reveal the phylogenetic placements of lichenized lineages with 18S rRNA and rbcL sequences. ITS variation among the clades was substantial and did not allow including them in the general phylogenetic assessment, yet ITS appears to be a promising marker for DNA-barcoding approaches. Specific algae were found in particular lichen but the overall diversity of photobionts was limited. The multilocus tree does not support the current morphological classification of genera in Trentepohliaceae, suggesting that morphology is more variable than previously thought in this group of algae.     

Phylogeny of the Neotropical sages (<Emphasis Type="Italic">Salvia</Emphasis> subg. <Emphasis Type="Italic">Calosphace</Emphasis>; Lamiaceae) and insights into pollinator and area shifts

Salvia subg. Calosphace (Lamiaceae, Lamiales) is a highly diverse clade endemic to the New World. The phylogenetic relationships of Calosphace have been previously investigated using DNA sequences of nuclear ITS region and plastid psbA–trnH intergenic spacer, but the resulting trees lack resolution and support for many clades. The present paper reassesses the phylogenetic relationships of subgenus Calosphace, including a broader taxon sampling, with a special focus on representing previously unsampled sections, and using an additional plastid marker (trnL–trnF region). Our results show increased resolution and overall patterns of support, recovering ten main clades. Within core Calosphace, the most inclusive of these main clades, 17 new subclades were identified. Of the 42 sections for which more than one species was analysed, only 12 are monophyletic. Our biogeographical analysis identified at least twelve migrations to South America from Mexican and Central American lineages, in agreement with previous suggestions of multiple origins of South American Calosphace diversity. This analysis also confirmed a colonization of the Antilles by Andean lineages. The reconstruction of ancestral states of pollination syndromes showed multiple shifts to ornithophily from melittophily and one reversal to the latter.     

Molecular phylogenetics in 2D: ITS2 rRNA evolution and sequence-structure barcode from Veneridae to Bivalvia

In this study, we analyzed the nuclear ITS2 rRNA primary sequence and secondary structure in Veneridae and comparatively with 20 Bivalvia taxa to test the phylogenetic resolution of this marker and its suitability for molecular diagnosis at different taxonomic levels. Maximum likelihood and Bayesian trees based on primary sequences were congruent with (profile-) neighbor-joining trees based on a combined model of sequence-structure evolution. ITS2 showed higher resolution below the subfamily level, providing a phylogenetic signal comparable to (mitochondrial/nuclear) gene fragments 2-5 times longer. Structural elements of the ITS2 folding, such as specific mismatch pairing and compensatory base changes, provided further support for the monophyly of some groups and for their phylogenetic relationships. Veneridae ITS2 folding is structured in six domains (DI-VI) and shows five striking sequence-structure features. Two of them, the Basal and Apical STEMs, are common to Bivalvia, while the presence of both the Branched STEM and the Y/R stretches occurs in five superfamilies of the two Heterodonta orders Myoida and Veneroida, thus questioning their reciprocal monophyly. Our results validated the ITS2 as a suitable marker for venerids phylogenetics and taxonomy, and underlined the significance of including secondary structure information for both applications at several systematic levels within bivalves.     

The ITS region of groundwater amphipods: length,secondary structure and phylogenetic information content in Crangonyctoids and Niphargids

The phylogenetic analysis of groundwater amphipods is challenging due to the lack of suitable morphological characters. However, molecular phylogenies based on the 18S and 28S nuclear genes of two Crangonyctoidea species endemic to Iceland, Crymostygius thingvallensis and Crangonyx islandicus, support the taxonomy of these species on the basis of morphological characters. Molecular analyses suggest that the genus Crangonyx is paraphyletic, with the species that is found in Eurasia being highly divergent genetically from the species present in North America and Iceland. Studies of the phylogenetic relationships within the genus Niphargus also warrant further work. The nuclear ITS2 region has recently been proposed as a barcoding marker for plants and animals. In addition, ITS2 has been used to build phylogenies at high taxonomic levels by including its secondary structure. In this study, we want to evaluate the applicability of the ITS region for this group of species and describe its characteristics. The taxonomy of C. thingvallensis, as well as the paraphyly of the genus Crangonyx, is supported herein by phylogenies based on the ITS2 variation. The secondary structure and the length of the ITS2 sequences of the Crangonyctoidea and the Niphargidae species studied are highly variable and are characterized by duplications. The ITS2 sequence of Niphargus plateaui is the longest metazoan sequence deposited in the ITS2 database so far. Although saturation was observed in the nucleotide variation of this marker, the addition of the secondary structure information for the reconstruction of the phylogeny did not add support to the phylogenetic trees. The ITS1 region, which is known to be more variable than ITS2 and bears a large duplication within C. islandicus, was found to be less useful for phylogenetic reconstruction.     

Phylogeny of Campanuloideae (Campanulaceae) with Emphasis on the Utility of Nuclear Pentatricopeptide Repeat (PPR) Genes

Background

The Campanuloideae (Campanulaceae) are a highly diverse clade of angiosperms found mostly in the Northern Hemisphere, with the highest diversity in temperate areas of the Old World. Chloroplast markers have greatly improved our understanding of this clade but many relationships remain unclear primarily due to low levels of molecular evolution and recent and rapid divergence. Furthermore, focusing solely on maternally inherited markers such as those from the chloroplast genome may obscure processes such as hybridization. In this study we explore the phylogenetic utility of two low-copy nuclear loci from the pentatricopeptide repeat gene family (PPR). Rapidly evolving nuclear loci may provide increased phylogenetic resolution in clades containing recently diverged or closely related taxa. We present results based on both chloroplast and low-copy nuclear loci and discuss the utility of such markers to resolve evolutionary relationships and infer hybridization events within the Campanuloideae clade.

Results

The inclusion of low-copy nuclear genes into the analyses provides increased phylogenetic resolution in two species-rich clades containing recently diverged taxa. We also obtain support for the placement of two early diverging lineages (Jasione and Musschia-Gadellia clades) that have previously been unresolved. Furthermore, phylogenetic analyses of PPR loci revealed potential hybridization events for a number of taxa (e.g., Campanula pelviformis and Legousia species). These loci offer greater overall topological support than obtained with plastid DNA alone.

Conclusion

This study represents the first inclusion of low-copy nuclear genes for phylogenetic reconstruction in Campanuloideae. The two PPR loci were easy to sequence, required no cloning, and the sequence alignments were straightforward across the entire Campanuloideae clade. Although potentially complicated by incomplete lineage sorting, these markers proved useful for understanding the processes of reticulate evolution and resolving relationships at a wide range of phylogenetic levels. Our results stress the importance of including multiple, independent loci in phylogenetic analyses.     

Phylogenetic reconstruction of endophytic fungal isolates using internal transcribed spacer 2 (ITS2) region

Endophytic fungi are inhabitants of plants, living most part of their lifecycle asymptomatically which mainly confer protection and ecological advantages to the host plant. In this present study, 48 endophytic fungi were isolated from the leaves of three medicinal plants and characterized based on ITS2 sequence – secondary structure analysis. ITS2 secondary structures were elucidated with minimum free energy method (MFOLD version 3.1) and consensus structure of each genus was generated by 4SALE. ProfDistS was used to generate ITS2 sequence structure based phylogenetic tree respectively. Our elucidated isolates were belonging to Ascomycetes family, representing 5 orders and 6 genera. Colletotrichum/Glomerella spp., Diaporthae/Phomopsis spp., and Alternaria spp., were predominantly observed while Cochliobolus sp., Cladosporium sp., and Emericella sp., were represented by singletons. The constructed phylogenetic tree has well resolved monophyletic groups with >50% bootstrap value support. Secondary structures based fungal systematics improves not only the stability; it also increases the precision of phylogenetic inference. Above ITS2 based phylogenetic analysis was performed for our 48 isolates along with sequences of known ex-types taken from GenBank which confirms the efficiency of the proposed method. Further, we propose it as superlative marker for reconstructing phylogenetic relationships at different taxonomic levels due to their lesser length.     

Using a phylogenetic approach to selection of target plants in drug discovery of acetylcholinesterase inhibiting alkaloids in Amaryllidaceae tribe Galantheae

We present phylogenetic analyses of 32 taxa of Amaryllidaceae tribe Galantheae, 6 taxa of other Eurasian genera of Amaryllidaceae and Phaedranassa dubia as outgroup in order to provide a phylogenetic framework for selection of candidate plants for lead discovery in relation to Alzheimer’s disease. We used DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the plastid matK and trnL-F regions. Phylogenetic analyses using maximum parsimony and Bayesian inference strongly support a monophyletic tribe Galantheae in a narrow sense, including only Acis, Galanthus and Leucojum. Infrageneric relationships of Galanthus only partly support previous classifications. Alkaloid profiles and inhibition of acetylcholinesterase (AChE) were investigated for 18 taxa using gas chromatography–mass spectrometry (GC–MS) and an assay measuring inhibition of AChE activity. AChE inhibitory activity was found in all investigated clades and was correlated with alkaloid profiles of the plants. Lowest IC₅₀ values were expressed by extracts containing either galanthamine or lycorine type compounds. Evaluation of available chemistry and activity data in a phylogenetic framework could be used to select target species for further investigation.     

Application of the phylogenetic informativeness method to chloroplast markers: A test case of closely related species in tribe Hydrangeeae (Hydrangeaceae)

In evolutionary biology appropriate marker selection for the reconstruction of solid phylogenetic hypotheses is fundamental. One of the most challenging tasks addresses the appropriate choice of genomic regions in studies of closely related species. Robust phylogenetic frameworks are central to studies dealing with questions ranging from evolutionary and conservation biology, biogeography to plant breeding. Phylogenetic informativeness profiles provide a quantitative measure of the phylogenetic signal in markers and therefore a method for locus prioritization. The present work profiles phylogenetic informativeness of mostly non-coding chloroplast regions in an angiosperm lineage of closely related species: the popular ornamental tribe Hydrangeeae (Hydrangeaceae, Cornales, Asterids). A recent phylogenetic study denoted a case of resolution contrast between the two strongly supported clades within tribe Hydrangeeae. We evaluate the phylogenetic signal of 13 highly variable plastid markers for estimating relationships within and among the currently recognized monophyletic groups of this tribe. A selection of combined loci based on their phylogenetic informativeness retrieved more robust phylogenetic hypotheses than simply combining individual markers performing best with respect to resolution, nodal support and accuracy or those presenting the highest number of parsimony informative characters. We propose the rpl32–ndhF intergenic spacer (IGS), trnV–ndhC IGS, trnL–rpl32 IGS, psbT–petB region and ndhA intron as the best candidates for future phylogenetic studies in Hydrangeeae and potentially in other Asterids. We also contrasted the phylogenetic informativeness of coded indels against substitutions concluding that, despite their low phylogenetic informativeness, coded indels provide additional phylogenetic signal that is nearly free of noise. Phylogenetic relationships obtained from our total combined analyses showed improved resolution and nodal support with respect to recently published results.     

Phylogenetic relationships among members of the subfamily Sedoideae (Crassulaceae) inferred from the ITS region sequences of nuclear rDNA

Nucleotide sequences of the nuclear rDNA ITS regions were determined in 20 species of the subfamily Sedoideae (Crassulaceae). The phylogenetic relationships of these species with other members of the subfamily, occurring mainly in Southeast Asia, were analyzed. It was shown that the genus Orostachys was not monophyletic; its type subsection was significantly included into the clade of the genus Hylotelephium. Synapomorphic substitutions and indels, specific for the subsection Orostachys, were detected in ITS1. Sister relationships were established between clades Aziopsis and Phedimus, based on which they can be recognized as isolated genera.     

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.     

Sequence diversification of 45S rRNA ITS, trnH-psbA spacer, and matK genic regions in several Allium species

Allium is a very diverse genus with over 600 species distributed worldwide. Haplotype analyses of 45S rRNA ITS, trnH-psbA spacer, and matK gene sequences in 9 Allium species were carried out, subsequent to which phylogenetic relations of the nine species were also analyzed. Of the three genes, the nuclear 45S rRNA ITS sequences showed the highest variation with one haplotype in each species. The other two chloroplast genes revealed that more than one haplotype was present in each species, and each haplotype was present in several of the species. In the matK gene, EcoRI restriction revealed heteroplasmy in which the functional gene retains the EcoRI recognition site while the nonfunctional, pseudogene does not. Phylogenetic patterns were not consistent among the haplotypes of the 45 rRNA ITS, trnH-psbA spacer, and matK genic regions. This phylogenetic incongruency might be due to the presence of multiple haplotypes in each of the chloroplast genes. However, the inconsistency of the phylogenetic relationships, based on the 45S rRNA ITS sequences makes a strong case for further analysis.     

A Phylogenetic Analysis of Heterorhabditis (Nemata: Rhabditidae) Based on Internal Transcribed Spacer 1 DNA Sequence Data

Internal transcribed spacer 1 sequences were used to infer phylogenetic relationships among 8 of the 9 described species and one putative species of the entomopathogenic nematode genus Heterorhabditis. Sequences were aligned and optimized based on pairwise genetic distance and parsimony criteria and subjected to a variety of sequence alignment parameters. Phylogenetic trees were constructed with maximum parsimony, cladistic, distance, and maximum likelihood algorithms. Our results gave strong support for four pairs of sister species, while relationships between these pairs also were resolved but less well supported. The ITS1 region of the nuclear ribosomal repeat was a reliable source of homologous characters for resolving relationships between closely related taxa but provided more tenuous resolution among more divergent lineages. A high degree of sequence identity and lack of autapomorphic characters suggest that sister species pairs within three distinct lineages may be mutually conspecific. Application of these molecular data and current morphological knowledge to the delimitation of species is hindered by an incomplete understanding of their variability in natural populations.     

Non-coding nuclear DNA markers in phylogenetic reconstruction

Molecular DNA based data sets are the most important resource for phylogenetic reconstruction. Among the various marker systems, which were introduced and optimized within the last decade, coding sequences played an important role, especially when molecular clock approaches and multi-gene datasets were assembled. However, non-coding DNA sequences do not only play a quantitatively dominant role, as demonstrated by the two examples nuclear ITS (Internal transcribed spacer regions of nuclear ribosomal DNA) and plastidic trnL-F region, but there is also a wide range of different marker systems that can be applied in different ways. Herein, we review the application of several non-coding nuclear DNA marker systems for phylogenetic reconstructions and summarize valuable information for future research.     

Tribal assignment of <Emphasis Type="Italic">Heldreichia</Emphasis> Boiss. (Brassicaceae): evidence from nuclear ITS and plastidic <Emphasis Type="Italic">ndh</Emphasis>F markers

Heldreichia Boiss. is a monospecific genus mainly distributed in Anatolia and the Lebanon. Although morphological variation and infrageneric phylogenetic relationships were recently studied in detail, Heldreichia remained as one of the few orphan genera that have not yet been assigned to any tribe. In the current study, we used sequence data from the nuclear ITS and chloroplast ndhF regions of Heldreichia and representatives of main Brassicaceae lineages and tribes to determine its tribal affiliation. Bayesian-based phylogenetic analyses clearly show with high support that Heldreichia is a member of the recently expanded tribe Biscutelleae. Furthermore, we characterize the tribe Biscutelleae morphologically and provide a determination key for all its genera.     

Sequence variation in mitochondrial ATP synthase subunit 6 & 8 and nuclear genes ITS1 and ITS2 in 17 cyprinid species

Sequence variation for the mitochondrial ATP synthase subunit 6 & 8 and the nuclear ITS1 marker was investigated for 17 cyprinid fish species in order to increase the number of possible loci for character sampling. The mitochondrial locus provided appropriate information for further phylogenetic studies, which can be readily applied to other taxa; the nuclear ITS1, however, cannot be recommended for inference of relationships among different taxa. No congruence was obtained between the trees resulting from the mitochondrial and nuclear gene using Bayesian Inference.