Plastid Regions Research Articles

Organelle Genome Inheritance in Deparia Ferns (Athyriaceae, Aspleniineae, Polypodiales).

Organelle genomes of land plants are predominately inherited maternally but in some cases can also be transmitted paternally or biparentally. Compared to seed plants (>83% genera of angiosperms and >12% genera of gymnosperms), plastid genome (plastome) inheritance has only been investigated in fewer than 2% of fern genera, and mitochondrial genome (mitogenome) from only one fern genus. We developed a new and efficient method to examine plastome and mitogenome inheritance in a fern species—Deparia lancea (Athyriaceae, Aspleniineae, Polypodiales), and found that plastid and mitochondrial DNAs were transmitted from only the maternal parentage to a next generation. To further examine whether both organelle genomes have the same manner of inheritance in other Deparia ferns, we sequenced both plastid and mitochondrial DNA regions of inter-species hybrids, and performed phylogenetic analyses to identify the origins of organellar DNA. Evidence from our experiments and phylogenetic analyses support that both organelle genomes in Deparia are uniparentally and maternally inherited. Most importantly, our study provides the first report of mitogenome inheritance in eupolypod ferns, and the second one among all ferns.

Plant macrofossils from lake sediment as the material to assess ancient genetic diversity: Did deforestation influence Norway spruce (Picea abies) in the South Carpathians?

How genetic diversity of populations reacts to neutral or adaptive processes such as population bottlenecks, immigration or local adaptation are central questions of population genetics. They may be directly answered through ancient DNA analysis, however such studies in plants are remarkably scarce, owing to the difficulty of gaining population-scale samples with good DNA preservation. Plant macrofossils are abundant in lake sediments, and here we assessed if they can be valuable material for population genetic studies. We extracted ancient DNA from early to late Holocene seed and needle remains of Norway spruce (Picea abies L. Karst) recovered from the sediment of a lake in the Retezat Mts. (South Carpathians). We aimed to reveal whether deforestations starting in the late Holocene and reaching maximum in the 19th and early 20th centuries have caused decreased genetic diversity in the modern Norway spruce population of the Retezat Mts. In a combined approach, we sequenced two plastid regions and generated detailed paleobotanical data to reconstruct the population history of Norway spruce. Our results show that genotyping success of the ancient material was high in samples younger than c. 2000-year-old, but was very low in early Holocene samples, and needles allowed genotyping with higher success compared to seeds. We discovered macrofossil samples that contained DNA from more than a single Norway spruce individual. This phenomenon, together with the possible multiple sampling of the same individual needs to be kept in mind when interpreting the genetic data obtained from macrofossils. Our results overall indicate a genetic diversity decrease during the late Holocene. We demonstrate that macrofossils preserved in sediments are invaluable material for population genetic studies.

Molecular phylogeny of Panicum s. str. (Poaceae, Panicoideae, Paniceae) and insights into its biogeography and evolution.

Panicum sensu stricto is a genus of grasses (Poaceae) with nearly, according to this study, 163 species distributed worldwide. This genus is included in the subtribe Panicinae together with Louisiella, the latter with 2 species. Panicum and subtribe Panicinae are characterized by including annual or perennial taxa with open and lax panicles, and spikelets with the lower glume reduced; all taxa also share a basic chromosome number of x = 9 and a Kranz leaf blade anatomy typical of the NAD-me subtype photosynthetic pathway. Nevertheless, the phylogenetic placements of many Panicum species, and the circumscription of the genus, remained untested. Therefore, phylogenetic analyses were conducted using sequence data from the ndhF plastid region, in an extensive worldwide sampling of Panicum and related genera, in order to infer evolutionary relationships and to provide a phylogenetic framework to review the classification of the genus. Diversification times, historical biogeography and evolutionary patterns of the life history (annual vs. perennial) in the subtribe and Panicum were also studied. Results obtained provide strong support for a monophyletic Panicum including 71 species and 7 sections, of which sections Arthragrostis and Yakirra are new in the genus; 7 new combinations are made here. Furthermore, 32 species traditionally assigned to Panicum were excluded from the genus, and discussed in other subtribes of Paniceae. Our study suggested that early diversification in subtribe Panicinae and Panicum occurred through the Early-Mid Miocene in the Neotropics, while the subsequent diversification of its sections mainly occurred in the Late Miocene-Pleistocene, involving multiple dispersals to all continents. Our analyses also showed that transition rates and changes between annual and perennial life history in Panicum were quite frequent, suggesting considerable lability of this trait. Changes of the life history, together with C4 photosynthesis, and the multiple dispersal events since the Mid Miocene, seem to have facilitated a widespread distribution of the genus. All these findings contribute to a better understanding of the systematics and evolution of Panicum.

Evolution of sexual systems and growth habit in Mussaenda (Rubiaceae): Insights into the evolutionary pathways of dioecy

Dioecy is a rare sexual system that is thought to represent an “evolutionary dead end”. While many studies have addressed the evolution of dioecy and/or its relationship with the evolution of the woody habit, few have explored the relationship between dioecy and climbing habit, and their effects on diversification rates. Here, we study the evolution of sexual systems and growth habit in Mussaenda (Rubiaceae) using a robust phylogeny of the genus based on eight plastid regions and a broad sampling of taxa (92 of the 132 species were sampled). A time-calibrated tree was constructed to estimate diversification rates in different clades and its correlates with focal characters. More specifically, we assess evolutionary correlations between dioecy and climbing habit and their respective influences on diversification rates. Ancestral character state reconstructions revealed that distyly is the most likely ancestral state in Mussaenda. Distyly has subsequently given rise to dioecy, short-styled floral monomorphism, and long-styled floral monomorphism. Dioecy has evolved independently at least four times from distyly, and has reversed to homostylous hermaphroditism at least twice, which does not support the “evolutionary dead end” hypothesis. A significant correlation between the evolution of dioecy and climbing growth form was found in Mussaenda. It is possible that a strong association between high net diversification rates and dioecy may exist in Mussaenda, but no association was found with climbing habit.

Phylogeny of Zehneria (Cucurbitaceae) with special focus on Asia

AbstractZehneria is a genus of small creepers and climbers from the Old–World Tropics and Subtropics. In its broadest circumscription, it comprises up to 94 species and its centre of diversity is Southeast Asia. Zehneria has been split into several smaller genera but this remained controversial mainly due to morphological plasticity of the species groups and lack of comprehensive molecular data. Here, we use one nuclear DNA (ITS1–5.8S–ITS2) and three plastid DNA regions (trnL–trnF, rpl20–rps12, matK) sequenced for about half of the species in Zehneria and its segregate genera Anangia, Neoachmandra, and Pilogyne to test the monophyly of the genus and analyse the evolutionary history of the group. We find that Zehneria s.l. is monophyletic and splitting is not supported by molecular, morphological or biogeographical evidence. Within Zehneria, we recover two strongly supported clades, one consisting mainly of species placed in Pilogyne, the other including Anangia, Neoachmandra and the type of Zehneria. Both clades comprise African, Asian, and Australian/Pacific Island species, which is evidence for high dispersal capacity in this young bird–dispersed group. For India, we accept the five species Z. bodinieri, Z. hookeriana, Z. maysorensis, Z. odorata, and Z. thwaitesii. For Myanmar, we increase the number of known species from four to eight, including three still unnamed species. The diversity hotspots for the genus, however, remain Indonesia and Thailand with together 25 species.

Phylogenetic relationships and generic reassessment of Proustia and allies (Compositae: Nassauvieae)

AbstractProustia is a small southern Andean genus of shrubs, vines and small trees, which are characteristic elements of Chilean and Argentine Andean forests, thickets and desert scrubs. Since Proustia possesses an unusual and characteristic morphology within the Nassauvieae, its circumscription as well as its phylogenetic placement is decisive in understanding the evolution of the tribe. Berylsimpsonia, from the Caribbean, was segregated from Proustia, which currently only includes three species. Lophopappus, another Andean genus, has been closely related to Proustia in sharing style and corolla features that are unusual in the Nassauvieae. The purpose of this work is to evaluate the placement of Proustia within the Nassauvieae, its relationships with Berylsimpsonia and Lophopappus, and the relationships of its species using molecular data and phylogenetic methods. This is the first comprehensive analysis of Proustia. We have included in our study the three currently accepted species of Proustia and seven of its ten infraspecific taxa. For each taxon of Proustia, one to seven accessions were analyzed. Species of Lophopappus and Berylsimpsonia, as well as 16 species belonging to 13 genera of Nassauvieae were also analyzed. We sequenced the nuclear ribosomal ITS and ETS, and the plastid regions ndhF, 5’ trnK–matK, trnL–trnF, and trnL(UAG)–rpl32. Unlike other previous studies, Proustia is not sister to the rest but nested in the tribe in our analyses. Berylsimpsonia vanillosma and P. ilicifolia are distantly related to P. pyrifolia, the type of the genus. According to nuclear DNA data, the infraspecific taxa of P. cuneifolia were recovered all in a well–supported clade, although based on the plastid data P. pyrifolia does not form a lineage separate from P. cuneifolia. Lophopappus is the genus most closely related to Proustia. Regarding the evolution of styles, our results suggest multiple origins of atypical stylar features in the Nassauvieae. As a result of our analyses we propose the segregation of Proustia ilicifolia into the new genus Spinoliva. Morphological data are consistent with the segregation of Proustia and Lophopappus as separate genera. Two species of Proustia, P. cuneifolia and P. pyrifolia are recognized. Three lectotypifications, one neotypification, two new combinations and five rank changes are proposed.

Species tree phylogeny, character evolution, and biogeography of the Patagonian genus Anarthrophyllum Benth. (Fabaceae)

Geologic events promoting the aridization of southern South America contributed to lineage divergences and species differentiation through geographic (allopatric divergence) and biotic and abiotic factors (ecological divergence). For the genus Anarthrophyllum, which is distributed in arid and semi-arid regions of Patagonia, we assessed how these factors affected species diversification and reconstructed its possible biogeographic history in South American arid environments. Sequences were obtained from two molecular markers: the ITS nuclear region and the trnS-trnG plastid region. Using Parsimony, Maximum likelihood and Bayesian inference individual gene trees were reconstructed, and a species tree was obtained using multi-species coalescent analysis. Divergence times among species were estimated using secondary calibrations. Flexible Bayesian models and stochastic character mapping were used to elucidate ancestral geographic distributions and the evolution of the floral and vegetative phenotypes in the genus. Gene trees and species tree analyses strongly support Anarthrophyllum as monophyletic; all analyses consistently retrieved three well-supported main clades: High Andean Clade, Patagonian Clade 1, and Patagonian Clade 2. Main diversification events occurred concomitant with the Andean uplift and steppe aridization; the Andean mountain range possibly acted as a species barrier for the High Andean Clade. Vegetative traits showed adaptations to harsh climates in some clades, while pollinator-related floral features were associated with independent diversification in bee- and bird-pollinated clades within both Patagonian Clades. In conclusion, evolutionary and biogeographic history of Anarthrophyllum resulted from the action of ecological, historical, and geographic factors that acted either alternatively or simultaneously.

Cryptic genetic diversity in Solanum elaeagnifolium (Solanaceae) from South America

Solanum elaeagnifolium is a global invasive weed that is polyploid in the South American range. Our aim was to establish the origin and distribution of these polyploid lineages, together with their relationship with the invasion of new habitats. Ploidy level was determined in 26 natural populations using chromosomal counts, and two non-coding plastid regions trnL-trnF and rpl32F-trnL were sequenced. Relationships among haplotypes were examined by a median-joining network, and groups of populations were identified by employing a spatial analysis of molecular variance. Phylogenetic relationships among haplotypes were reconstructed using Bayesian inference, and divergence times were estimated using BEAST. We found 21 haplotypes structured into three lineages: one with diploids in North America and invaded areas, and two in South America that developed independent polyploidisation events. The separation of lineages took place during the Pliocene with more recent divergences in the Early Pleistocene. The existence of three lineages can be attributed to the uplift of the Andes and the emergence of the Isthmus of Panama. Diversification within each lineage may be related to the recent cyclical glacial variation throughout the Pleistocene creating haplotype diversity and recurrent polyploidy. Thus, the South American populations of S. elaeagnifolium did not originate from a recent colonisation but are established ancient lineages.

New insights on the phylogeny of Tectaria (Tectariaceae), with special reference to Polydictyum as a distinct lineage

AbstractThe fern genus Tectaria (Tectariaceae) Cav. is morphologically diverse and difficult in terms of recognizing species and species groups. To infer the systematic positions of some species and identity‐unknown collections with special morphological characters, we undertook phylogenetic analyses based on sequences of five plastid regions (atpB, ndhF + ndhF‐trnL, rbcL, rps16‐matK + matK, and trnL‐F). Three analysis methods (maximum parsimony, maximum likelihood, and Bayesian inference) were used to reconstruct the phylogeny of Tectaria. The most surprising result is that T. menyanthidis (C. Presl) Copel., T. ternata (Baker) Copel., and T. variabilis Tardieu & Ching are revealed to represent a distinct lineage from Tectaria, which should be called Polydictyum C. Presl, and is supported as sister to Pteridrys C. Chr. & Ching. Other accessions of Tectaria are well resolved into four major clades, which is consistent with the results of previous studies. Of the four clades, Clade II (T. subtriphylla (Hook. & Arn.) Copel. group) is unpredictable, with morphologically very diverse species clustered there, and is supposed to be a minor evolutionary line within Tectaria in the Old World. In addition, the position of the climbing genus Arthropteris J. Sm. and the utility of molecular data in recognizing species of Tectaria are briefly discussed. As a conclusion, we formally reinstate the genus Polydictyum by providing diagnostic characters, key to species, nomenclature, and information of detailed distribution and habitat for the currently known three species.

Molecular phylogeny of the genus Bolusiella (Orchidaceae, Angraecinae)

Recent molecular studies have suggested the monophyly of Bolusiella, a small orchid genus comprising five species and one subspecies from Continental Africa, but sampling has been limited. Using the species delimitation presented in the recent taxonomic revision of the genus, this study aimed to confirm the monophyly of Bolusiella and assess the interspecific relationships using a comprehensive sampling and various analytical methods. DNA sequences of one nuclear spacer region (ITS-1) and five plastid regions (matK, rps16, trnL–trnF, trnC–petN, and ycf1) from 20 specimens representing all five species of the genus were analyzed using static homology, dynamic homology, and Bayesian methods. The monophyly of both the genus Bolusiella and each of its five species was confirmed, corroborating the previously published taxonomic revision. The use of dynamic homology methods was not conclusive for this particular group. The results of the total evidence analysis (combining all six sequence regions) using the dynamic homology approach yielded a slightly different hypothesis regarding interspecific relationships (namely the exchange of B. talbotii and Bolusiella iridifolia as the earliest diverging lineage), probably because the nodes in question are supported by a small subset of conflicting characters, compared to the hypotheses resulting from the static homology and Bayesian methods, which are congruent with the results of previous studies.

Testing the monophyly of Simaba (Simaroubaceae): Evidence from five molecular regions and morphology

Generic circumscriptions in the mostly pantropical family Simaroubaceae are somewhat controversial. Simaba is the largest genus, currently defined as exclusively neotropical, with around 25 species of trees and shrubs, but both its limits and infrageneric classification have been a matter of discussion and divergence. Traditionally, species of the genus have been treated in three sections: Simaba sect. Tenuiflorae, S. sect. Floribundae and S. sect. Grandiflorae, but a phylogenetic analysis suggested that the latter two may not be monophyletic. To test the monophyly of Simaba and its infrageneric classification, we used a molecular approach based on DNA sequence data from two nuclear ribosomal spacer regions (ITS and ETS) and three plastid regions (rps16 intron, and intergenic spacers psbA-trnH and trnL-trnF), including a comprehensive sampling of species from Simaba and closely related genera. We also performed ancestral character reconstructions to identify morphological characters that could serve as synapomorphies for major clades and to explore patterns of homoplasy in the morphological dataset. Our results show Simaba as traditionally circumscribed is not monophyletic, with taxa segregated into two strongly supported but distinct clades, one of which is more closely related to Simarouba. The three main clades that emerged in the phylogeny include a mostly Amazonian Simaba clade (which includes the type species of Simaba and the remaining species of S. sect. Tenuiflorae, here proposed to be recognized as Simaba sensu stricto), a mostly extra-Amazonian Simaba clade (a distinct lineage that will be recognized as Homalolepis, a genus currently treated in synonymy and equivalent to Simaba sections Grandiflorae and Floribundae), and the Simarouba clade (including all of its current species). These three clades are characterized by a combination of morphological characters, described in detail herein, some of which are novel features for Simaba not previously reported in the literature. Mapping character-states on the phylogenetic tree provides tests for evolutionary hypotheses. For example, our reconstruction of habit and geographic distribution suggests that the diversification of several shrubby species within the extra-Amazonian lineage in the South American cerrados probably occurred from ancestors inhabiting tropical forests, involving transitions in morphological and ecological traits.

A phylogenetic framework for the Hylocereeae (Cactaceae) and implications for the circumscription of the genera

The tribe Hylocereeae are represented by mainly Central American-Mexican epiphytic, hemi-epiphytic and climbing cacti. They are popular due to their spectacular nocturnal flowers and have some importance as crops grown for their edible fruits. We present the first comprehensive phylogenetic study of the Hylocereeae sampling 60 out of the 63 currently accepted species and 17 out of 19 infraspecific taxa. Based on four plastid regions (trnK/matK, the rpl16 intron, rps3-rpl16, and trnL-F) we find a highly supported core Hylocereeae clade that also includes Acanthocereus and Peniocereus p.p., while Strophocactus is depicted as polyphyletic and is resolved outside of the Hylocereeae tribe. The clades found within Hylocereeae agree, in general terms, with the currently accepted genera but none of the genera are entirely monophyletic in their current circumscription. A new concept for the Hylocereeae is presented to include the genera Acanthocereus (incl. Peniocereus p.p.), Aporocactus, Disocactus, Epiphyllum, Selenicereus (incl. Hylocereus and Weberocereus p.p.), Pseudorhipsalis, Kimnachia gen. nov., and Weberocereus. New nomenclatural combinations are provided to make these genera monophyletic. The genus Deamia is reinstated for Strophocactus testudo and S. chontalensis, while Strophocactus is newly circumscribed to include S. wittii, Pseudoacanthocereus brasiliensis, and P. sicariguensis. Both genera are excluded from Hylocereeae. A taxonomic synopsis of Hylocereeae is provided.

Origins of North American arid-land Verbenaceae: More than one way to skin a cat.

Verbenaceae originated and initially diversified in South America in wet forest habitats. They have diversified extensively in arid habitats in both South and North America. This study aims to understand the origin of the North American arid-land members of Verbenaceae. A phylogenetic approach is used to examine four genera (Aloysia, Citharexylum, Glandularia, Verbena) in three distinct clades with representatives in North American deserts and disjunct South and North American distributions. Phylogenetic analyses were conducted using maximum likelihood and Bayesian approaches. Analyses included both plastid and nuclear DNA regions and include the first study of Citharexylum and an expanded sampling of tribe Verbeneae (Glandularia and Verbena). Ancestral areas were reconstructed for each group. North American desert species of Aloysia and Glandularia were likely derived from ancestors in arid temperate South America, perhaps by long-distance dispersal. The pattern for Verbena was less clear, with evidence from plastid DNA implicating an Andean dispersal route to the North American clade, whereas nuclear data suggest that the Andean and North American species resulted from independent dispersals from southern South America. A previously unrecognized clade of Andean Verbeneae was discovered, raising the possibility of an Andean origin of Verbena or Verbena and Glandularia. North American desert species of Citharexylum represent multiple, independent origins from mesic habitat ancestors in Mesoamerica. North American arid-zone Verbenaceae are derived from South and Central American ancestors via multiple avenues, including long-distance, amphitropical dispersal, Andean migration corridors, and in situ evolution of desert-adapted species.

Molecular phylogenetics of cool-season grasses in the subtribes Agrostidinae, Anthoxanthinae, Aveninae, Brizinae, Calothecinae, Koeleriinae and Phalaridinae (Poaceae, Pooideae, Poeae, Poeae chloroplast group 1).

Circumscriptions of and relationships among many genera and suprageneric taxa of the diverse grass tribe Poeae remain controversial. In an attempt to clarify these, we conducted phylogenetic analyses of >2400 new DNA sequences from two nuclear ribosomal regions (ITS, including internal transcribed spacers 1 and 2 and the 5.8S gene, and the 3’-end of the external transcribed spacer (ETS)) and five plastid regions (matK, trnL–trnF, atpF–atpH, psbK–psbI, psbA–rps19–trnH), and of more than 1000 new and previously published ITS sequences, focused particularly on Poeae chloroplast group 1 and including broad and increased species sampling compared to previous studies. Deep branches in the combined plastid and combined ITS+ETS trees are generally well resolved, the trees are congruent in most aspects, branch support across the trees is stronger than in trees based on only ITS and fewer plastid regions, and there is evidence of conflict between data partitions in some taxa. In plastid trees, a strongly supported clade corresponds to Poeae chloroplast group 1 and includes Agrostidinae p.p., Anthoxanthinae, Aveninae s.str., Brizinae, Koeleriinae (sometimes included in Aveninae s.l.), Phalaridinae and Torreyochloinae. In the ITS+ETS tree, a supported clade includes these same tribes as well as Sesleriinae and Scolochloinae. Aveninae s.str. and Sesleriinae are sister taxa and form a clade with Koeleriinae in the ITS+ETS tree whereas Aveninae s.str. and Koeleriinae form a clade and Sesleriinae is part of Poeae chloroplast group 2 in the plastid tree. All species of Trisetum are part of Koeleriinae, but the genus is polyphyletic. Koeleriinae is divided into two major subclades: one comprises Avellinia, Gaudinia, Koeleria, Rostraria, Trisetaria and Trisetum subg. Trisetum, and the other Calamagrostis/Deyeuxia p.p. (multiple species from Mexico to South America), Peyritschia, Leptophyllochloa, Sphenopholis, Trisetopsis and Trisetum subg. Deschampsioidea. Graphephorum, Trisetum cernuum, T. irazuense and T. macbridei fall in different clades of Koeleriinae in plastid vs. nuclear ribosomal trees, and are likely of hybrid origin. ITS and matK trees identify a third lineage of Koeleriinae corresponding to Trisetum subsect. Sibirica, and affinities of Lagurus ovatus with respect to Aveninae s.str. and Koeleriinae are incongruent in nuclear ribosomal and plastid trees, supporting recognition of Lagurus in its own subtribe. A large clade comprises taxa of Agrostidinae, Brizinae and Calothecinae, but neither Agrostidinae nor Calothecinae are monophyletic as currently circumscribed and affinities of Brizinae differ in plastid and nuclear ribosomal trees. Within this clade, one newly identified lineage comprises Calamagrostis coarctata, Dichelachne, Echinopogon (Agrostidinae p.p.) and Relchela (Calothecinae p.p.), and another comprises Chascolytrum (Calothecinae p.p.) and Deyeuxia effusa (Agrostidinae p.p.). Within Agrostidinae p.p., the type species of Deyeuxia and Calamagrostis s.str. are closely related, supporting classification of Deyeuxia as a synonym of Calamagrostis s.str. Furthermore, the two species of Ammophila are not sister taxa and are nested among different groups of Calamagrostis s.str., supporting their classification in Calamagrostis. Agrostis, Lachnagrostis and Polypogon form a clade and species of each are variously intermixed in plastid and nuclear ribosomal trees. Additionally, all but one species from South America classified in Deyeuxia sect. Stylagrostis resolve in Holcinae p.p. (Deschampsia). The current phylogenetic results support recognition of the latter species in Deschampsia, and we also demonstrate Scribneria is part of this clade. Moreover, Holcinae is not monophyletic in its current circumscription because Deschampsia does not form a clade with Holcus and Vahlodea, which are sister taxa. The results support recognition of Deschampsia in its own subtribe Aristaveninae. Substantial further changes to the classification of these grasses will be needed to produce generic circumscriptions consistent with phylogenetic evidence. The following 15 new combinations are made: Calamagrostis × calammophila, C. breviligulata, C. breviligulata subsp. champlainensis, C. × don-hensonii, Deschampsia aurea, D. bolanderi, D. chrysantha, D. chrysantha var. phalaroides, D. eminens, D. eminens var. fulva, D. eminens var. inclusa, D. hackelii, D. ovata, and D. ovata var. nivalis. D. podophora; the new name Deschampsia parodiana is proposed; the new subtribe Lagurinae is described; and a second-step lectotype is designated for the name Deyeuxia phalaroides.

A rapid and efficient DNA extraction method suitable for marine macroalgae.

Macroalgae are a diverse group of organisms. Marine macroalgae, in particular, have numerous medicinal and industrial applications. Molecular studies of macroalgae require suitable concentrations of DNA free of contaminants. At present, numerous protocols exist for DNA extraction from macroalgae. However, they are either time consuming, expensive or work only with few species. The method described in this study is rapid and efficient and applicable to different types of marine macroalgae. This method yields an average of 3.85µg of DNA per 50mg of algal tissue, with an average purity of 1.88. The isolated DNA was suitable for PCR amplification of universal plastid region of macroalgae.

Towards resolving the evolutionary history of Caucasian pears (Pyrus, Rosaceae)—Phylogenetic relationships, divergence times and leaf trait evolution

AbstractWith approximately 25 endemic species, the genus Pyrus (pears) is highly diverse in the Caucasus ecoregion. The majority of Caucasian pears inhabit xerophytic open woodlands or similar habitats, to which they display morphological adaptations, such as narrow leaves. The other species, both Caucasian and non‐Caucasian taxa, mainly inhabit mesophytic forests and display broad leaves. Using a representative taxon sampling of Pyrus from the Caucasus, Europe and Asia, we reconstruct phylogenetic relationships in the genus based on multiple plastid regions. We also estimate the divergence times of major clades in Pyrus, reconstruct the evolution of leaf shapes, and discuss the emergence of xeromorphic leaf traits. Our results confirm the monophyly of Pyrus and the existence of two major clades: (a) an E Asian clade with a crown group age of 15.7 (24.02–8.37 95% HPD) My, and (b) a W Eurasian clade that comprises species from Europe, SW Asia and the Caucasus and that displays a slightly younger crown group of 12.38 (19.02–6.41 95% HPD) My. The existing infrageneric classification of Pyrus was found partially incongruent with the inferred phylogenetic trees. Several currently accepted species were not recovered as monophyletic, indicating that current species limits require re‐evaluation. Ancestral character state reconstructions revealed several independent transitions from broad‐ to narrow‐shaped leaves in Pyrus, probably via intermediate‐shaped leaves.

Phylogeographic and phylogenetic analysis for Tripterygium species delimitation.

Tripterygium wilfordii (Celastraceae) is a traditional Chinese medicine; and the dried root and rhizome constitute the main officinal parts. Tripterygium wilfordii has been identified as a potential candidate for the treatment of systemic lupus erythematosus, rheumatoid arthritis, nephritis, asthma, leprosy, and cancer. The phylogenetic relationships within the Tripterygium genus are ambiguous; thus, our aim is to clarify the relationships within this genus using phylogeographic and phylogenetic analyses. Here, we first sequenced three plastid DNA regions (i.e., psbA‐trnH, rpl32‐trnL, and trnL‐trnF) and found that Tripterygium hypoglaucum and T. wilfordii were clustered together based on the strength of the topology in the phylogenetic analysis: T. hypoglaucum is polyphyletic, and T. wilfordii is paraphyletic. A spatial analysis of molecular variance showed that the best group value is 4, and the groups were almost consistent with the topology of in the phylogenetic analysis. The Mantel analyses of Tripterygium using IBD web showed statistically significant relationships between genetic and geographical distance distributions (r = .3479, p < .0001). The molecular dating using Fossil calibration indicated that the divergence in Tripterygium was approximately 8.13 Ma. Furthermore, we also analyzed four DNA regions (i.e., ITS2, psbA‐trnH, matK, and rbcL) that were obtained from the NCBI nucleotide database; these results showed that T. wilfordii and T. hypoglaucum clustered together, while Tripterygium regelii represented a separate cluster. Tripterygium hypoglaucum and T. wilfordii were never distinct lineages, and the species circumscriptions are artificial. We propose that T. wilfordii and T. hypoglaucum are conspecific, while T. regelii likely constitutes a separate species.

Rapid classification of unknown biological material using a novel triplex assay

The methods employed for species-level identification of plant and animal material include DNA extraction, DNA quantification and Sanger sequencing of selected mitochondrial or plastid regions. In this study, samples without a clear origin (human/non-human/plant) are tested using a novel triplex PCR assay for rapid classification. The assay enables detection of animal/human and plant DNA as well as the presence of PCR inhibitors. PCR products are separated using agarose electrophoresis. In the case of positive detection of animal/human and/or plant material, the PCR product can be directly used for subsequent Sanger sequencing and species determination.

Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia.

DNA barcoding is used as a universal tool for delimiting species boundaries in taxonomically challenging groups, with different plastid and nuclear regions (rbcL, matK, ITS and psbA-trnH) being recommended as primary DNA barcodes for plants. We evaluated the feasibility of using these regions in the species-rich genus Terminalia, which exhibits various overlapping morphotypes with pantropical distribution, owing to its complex taxonomy. Terminalia bellerica and T. chebula are ingredients of the famous Ayurvedic Rasayana formulation Triphala, used for detoxification and rejuvenation. High demand for extracted phytochemicals as well as the high trade value of several species renders mandatory the need for the correct identification of traded plant material. Three different analytical methods with single and multilocus barcoding regions were tested to develop a DNA barcode reference library from 222 individuals representing 41 Terminalia species. All the single barcodes tested had a lower discriminatory power than the multilocus regions, and the combination of matK+ITS had the highest resolution rate (94.44%). The average intra-specific variations (0.0188±0.0019) were less than the distance to the nearest neighbour (0.106±0.009) with matK and ITS. Distance-based Neighbour Joining analysis outperformed the character-based Maximum Parsimony method in the identification of traded species such as T. arjuna, T. chebula and T. tomentosa, which are prone to adulteration. rbcL was shown to be a highly conservative region with only 3.45% variability between all of the sequences. The recommended barcode combination, rbcL+matK, failed to perform in the genus Terminalia. Considering the complexity of resolution observed with single regions, the present study proposes the combination of matK+ITS as the most successful barcode in Terminalia.

Taxonomic revision of the African genera Brieya and Piptostigma (Annonaceae)

Background and aims – Annonaceae are an important family in tropical rain forests of Africa. Here, we present a revision of the west-central African genus Piptostigma of the tribe Piptostigmateae. Previous molecular phylogenetic analyses of Piptostigmateae have revealed that the genus as currently circumscribed is paraphyletic. The aims of the article are: (1) to clarify the generic delimitation of the genus Piptostigma, especially in relation to its current polyphyletic delimitation; (2) to provide a new updated key to the genera of tribe Piptostigmateae; and (3) to provide for the first time in over 100 years a complete taxonomic revision of genera Brieya and Piptostigma. Material and methods – Normal practices of herbarium taxonomy were used for the morphological study of about 200 specimens from B, BR, BRLU, G, HBG, K, L, LBV, LISC, MO, P, U, US, WAG, YA and Z. We sequenced three plastid regions for 32 samples representing 26 ingroup (Piptostigmateae) and six outgroup species in order to explore the paraphyly of Piptostisma. The open source software QGis was used to generate the distribution maps of the species and their conservation status was obtained using the IUCN categories and criteria.Key results – Molecular and morphological evidence support the reinstatement of the genus Brieya De Wild. Three keys are presented, one to the genera of Piptostigmateae and the two others to the species of Brieya and Piptostigma. A total of thirteen species are described for Piptostigma and two for the genus Brieya. Four new species are presented for the genus Piptostigma: P. macrophyllum, P. mayndongtsaeanum, P. goslineanum and P. submontanum.