A taxonomic backbone for the Plumbaginaceae (Caryophyllales).

A taxonomic backbone of the Plumbaginaceae is presented and the current state of knowledge on phylogenetic relationships and taxon limits is reviewed as a basis for the accepted taxon concepts. In total, 4,476 scientific names and designations are treated of which 30 are not in the family Plumbaginaceae. The Plumbaginaceae are subdivided in three tribes with 26 genera and 1,179 accepted species. Two subgenera, 17 sections, two subsections and 187 infraspecific taxa are accepted. At the species and infraspecific level 2,782 synonyms were assigned to accepted taxa, whereas 194 names were excluded from the core checklist (i.e., unplaced taxa, infrageneric subdivisions with still uncertain application, names of verified uncertain application, invalid horticultural names, excluded names from other families, other excluded designations, and unresolved names). The EDIT Platform for Cybertaxonomy was utilized as the tool to compile and manage the names and further taxonomic data under explicit taxon concepts. Secundum references are given in case taxon concepts were taken from the literature, whereas this study serves as reference for newly circumscribed taxa. The family's division into the tribes Aegialitideae, Limonieae, and Plumbagineae departs from earlier two-subfamily classifications, prompted by recent phylogenetic findings that challenge the subfamilial affinity of Aegialitis. The genus Acantholimon was extended to include Gladiolimon, as currently available phylogenetic and morphological data support this merger. In Limonium, all accepted species could be assigned to sections and subsections or the "Mediterranean lineage", respectively, making use of the phylogenetic distribution of their morphological characters and states. A new combination and/or status is proposed for Dyerophytumsocotranum, Limoniumthymoides, Limonium×fraternum, Limonium×rossmaessleri, and Limoniumsect.Jovibarba. Special attention is given to nomenclatural issues, particularly for Staticenomenambiguum to resolve the names under accepted names. The use of artificial groupings like "aggregates", "complexes" and "species groups" in alpha-taxonomic treatments is discussed. The taxonomic backbone will receive continued updates and through the Caryophyllales Taxonomic Expert Network, it contributes the treatment of the Plumbaginaceae for the World Flora Online.

On the importance of sequence alignment inspections in plastid phylogenomics - an example from revisiting the relationships of the water-lilies.

The water-lily clade represents the second earliest-diverging branch of angiosperms. Most of its species belong to Nymphaeaceae, of which the "core Nymphaeaceae"-comprising the genera Euryale, Nymphaea and Victoria-is the most diverse clade. Despite previous molecular phylogenetic studies on the core Nymphaeaceae, various aspects of their evolutionary relationships have remained unresolved. The length-variable introns and intergenic spacers are known to contain most of the sequence variability within the water-lily plastomes. Despite the challenges with multiple sequence alignment, any new molecular phylogenetic investigation on the core Nymphaeaceae should focus on these noncoding plastome regions. For example, a new plastid phylogenomic study on the core Nymphaeaceae should generate DNA sequence alignments of all plastid introns and intergenic spacers based on the principle of conserved sequence motifs. In this investigation, we revisit the phylogenetic history of the core Nymphaeaceae by employing such an approach. Specifically, we use a plastid phylogenomic analysis strategy in which all coding and noncoding partitions are separated and then undergo software-driven DNA sequence alignment, followed by a motif-based alignment inspection and adjustment. This approach allows us to increase the reliability of the character base compared to the default practice of aligning complete plastomes through software algorithms alone. Our approach produces significantly different phylogenetic tree reconstructions for several of the plastome regions under study. The results of these reconstructions underscore that Nymphaea is paraphyletic in its current circumscription, that each of the five subgenera of Nymphaea is monophyletic, and that the subgenus Nymphaea is sister to all other subgenera of Nymphaea. Our results also clarify many evolutionary relationships within the Nymphaea subgenera Brachyceras, Hydrocallis and Nymphaea. In closing, we discuss whether the phylogenetic reconstructions obtained through our motif-based alignment adjustments are in line with morphological evidence on water-lily evolution.

Generic concepts and species diversity within the Gynoxyoid clade (Senecioneae, Compositae).

The Gynoxyoid clade of the Senecioneae (Asteraceae) until now included the five genera Aequatorium, Gynoxys, Nordenstamia, Paracalia and Paragynoxys as diagnosed using selected morphological characters. In their pre-phylogenetic circumscription, the genera Aequatorium and Paragynoxys were considered to inhabit the northern Andes in contrast to Nordenstamia and Paracalia that occur in the central Andes. The most species-rich genus, Gynoxys, was believed to be distributed throughout the Andes. We use a recently established plastid phylogenomic framework that rendered Gynoxys paraphyletic to further evaluate the delimitation of genera in the Gynoxyoid clade. We examine the morphological variation of all members of the Gynoxyoid to identify characters potentially informative at genus level. This results in a matrix of eleven, mostly multistate characters, including those originally used to diagnose these genera. The ancestral character state inference displays a high level of homoplasy, but nevertheless supports the recognition of four genera. Aequatorium is characterised by white radiate capitula. Paracalia and Paragynoxys share white flowers and floral characteristics, such as flower opening and length of disc flowers lobes, as plesiomorphic states, but differ in habit (scandent shrubs vs. trees). Paracalia also retained white flowers, but its two species are characterised by the absence of outer phyllaries. The genera Gynoxys and Nordenstamia comprise species with yellow capitula which appear to be a derived feature in the Gynoxyoids. The genus Nordenstamia, with eight species, is synonymised under Gynoxys since molecular evidence shows its species nested within various parts of the Gynoxys subclade and the morphological variation of Nordenstamia falls well within that of Gynoxys. With the goal to assign all species to four genera (Aequatorium, Gynoxys, Paracalia and Paragynoxys), we assess the states for the eleven characters for all members of the Gynoxyoids and generate new ETS and ITS sequences for 171 specimens belonging to 49 species to further support their generic placement. We provide a taxonomic treatment for the four genera recognised here including amended diagnoses and morphological descriptions. Furthermore, a species-level taxonomic backbone is elaborated for all genera using electronic tools that list 158 currently accepted names and synonyms (209 names in total) with the respective protologue and type information, as well as notes on the current understanding of species limits. Eleven names are newly synonymised, two are lectotypified and eight are newly transferred to other genera.

The diversification of Caribbean Buxus in time and space: elevated speciation rates in lineages that accumulate nickel and spreading to other islands from Cuba in non-obligate ultramafic species.

BACKGROUND AND AIMS: The genus Buxus has high levels of endemism in the Caribbean flora, with ~50 taxa. In Cuba, 82 % grow on ultramafic substrates and 59 % are nickel (Ni) accumulators or Ni hyperaccumulators. Hence it is an ideal model group to study if this diversification could be related to adaptation to ultramafic substrates and to Ni hyperaccumulation. METHODS: We generated a well-resolved molecular phylogeny, including nearly all of the Neotropical and Caribbean Buxus taxa. To obtain robust divergence times we tested for the effects of different calibration scenarios, and we reconstructed ancestral areas and ancestral character states. Phylogenetic trees were examined for trait-independent shifts in diversification rates and we used multi-state models to test for state-dependent speciation and extinction rates. Storms could have contributed to Cuba acting as a species pump and to Buxus reaching other Caribbean islands and northern South America'. KEY RESULTS: We found a Caribbean Buxus clade with Mexican ancestors, encompassing three major subclades, which started to radiate during the middle Miocene (13.25 Mya). Other Caribbean islands and northern South America were reached from ~3 Mya onwards. CONCLUSIONS: An evolutionary scenario is evident in which Buxus plants able to grow on ultramafic substrates by exaptation became ultramafic substrate endemics and evolved stepwise from Ni tolerance through Ni accumulation to Ni hyperaccumulation, which has triggered species diversification of Buxus in Cuba. Storms could have contributed to Cuba acting as a species pump and to Buxus reaching other Caribbean islands and northern South America'.

Nested singletons in molecular trees: Utility of adding morphological and geographical data from digitized herbarium specimens to test taxon concepts at species level in the case of Casearia (Salicaceae).

Using the genus Casearia, we assessed the status of nested singletons: individual specimens corresponding to accepted species but in molecular trees appearing nested within clades of closely related species. Normally, such cases would be left undecided, while on the other hand, timely taxonomic decisions are required. We argue that morphological, chorological, and ecological data can be informative to illuminate patterns of speciation. Their use can provide a first step in testing taxon concepts at species level. We focused on five cases of nested singletons in trees of the genus Casearia. We employed PCA and cluster analysis to assess phenotypic differentiation. Using geocoordinates, we calculated niche space differentiation based on 19 bioclim variables, by means of PCA and niche equivalency and similarity tests and generated dot maps. We found that the singletons were morphologically distinctive in two of the five cases (Casearia selloana and C. manausensis), relatively distinctive in two other cases (C. zizyphoides and C. mariquitensis), and partially overlapping in the last case (C. grandiflora). For two cases (C. mariquitensis and C. selloana), ecological niche space was broadly overlapping, in two cases it was found broadly nested (C. grandiflora and C. zizyphoides), and in one case narrowly nested (C. manausensis), but in no case niche differentiation was observed. Niche overlap, similarity and equivalency showed corresponding patterns. Given these data, one would interpret C. selloana and C. manausensis as presumably well-distinguished taxa, their narrow distribution ranges suggesting recently emerging lineages. The other three cases are not clearcut. Morphological data would suggest particularly C. grandiflora conspecific with C. arborea, but differences in the distribution are intriguing. Our approach would reject the notion of potential synonymy based on nested phylogenetic placement for at least two of the five cases. The other case also shows no complete lack of differentiation which would support synonymy.

Software Choice and Sequencing Coverage Can Impact Plastid Genome Assembly-A Case Study in the Narrow Endemic Calligonum bakuense.

Most plastid genome sequences are assembled from short-read whole-genome sequencing data, yet the impact that sequencing coverage and the choice of assembly software can have on the accuracy of the resulting assemblies is poorly understood. In this study, we test the impact of both factors on plastid genome assembly in the threatened and rare endemic shrub Calligonum bakuense. We aim to characterize the differences across plastid genome assemblies generated by different assembly software tools and levels of sequencing coverage and to determine if these differences are large enough to affect the phylogenetic position inferred for C. bakuense compared to congeners. Four assembly software tools (FastPlast, GetOrganelle, IOGA, and NOVOPlasty) and seven levels of sequencing coverage across the plastid genome (original sequencing depth, 2,000x, 1,000x, 500x, 250x, 100x, and 50x) are compared in our analyses. The resulting assemblies are evaluated with regard to reproducibility, contig number, gene complement, inverted repeat length, and computation time; the impact of sequence differences on phylogenetic reconstruction is assessed. Our results show that software choice can have a considerable impact on the accuracy and reproducibility of plastid genome assembly and that GetOrganelle produces the most consistent assemblies for C. bakuense. Moreover, we demonstrate that a sequencing coverage between 500x and 100x can reduce both the sequence variability across assembly contigs and computation time. When comparing the most reliable plastid genome assemblies of C. bakuense, a sequence difference in only three nucleotide positions is detected, which is less than the difference potentially introduced through software choice.

Contributions to the biodiversity of Vietnam - Results of VIETBIO inventory work and field training in Cuc Phuong National Park.

VIETBIO [Innovative approaches to biodiversity discovery and characterisation in Vietnam] is a bilateral German-Vietnamese research and capacity building project focusing on the development and transfer of new methods and technology towards an integrated biodiversity discovery and monitoring system for Vietnam. Dedicated field training and testing of innovative methodologies were undertaken in Cuc Phuong National Park as part and with support of the project, which led to the new biodiversity data and records made available in this article collection. VIETBIO is a collaboration between the Museum für Naturkunde Berlin - Leibniz Institute for Evolution and Biodiversity Science (MfN), the Botanic Garden and Botanical Museum, Freie Universität Berlin (BGBM) and the Vietnam National Museum of Nature (VNMN), the Institute of Ecology and Biological Resources (IEBR), the Southern Institute of Ecology (SIE), as well as the Institute of Tropical Biology (ITB); all Vietnamese institutions belong to the Vietnam Academy of Science and Technology (VAST). The article collection "VIETBIO" (https://doi.org/10.3897/bdj.coll.63) reports original results of recent biodiversity recording and survey work undertaken in Cuc Phuong National Park, northern Vietnam, under the framework of the VIETBIO project. The collection consist of this "main" cover paper - characterising the study area, the general project approaches and activities, while also giving an extensive overview on previous studies from this area - followed by individual papers for higher taxa as studied during the project. The main purpose is to make primary biodiversity records openly available, including several new and interesting findings for this biodiversity-rich conservation area. All individual data papers with their respective primary records are expected to provide useful baselines for further taxonomic, phylogenetic, ecological and conservation-related studies on the respective taxa and, thus, will be maintained as separate datasets, including separate GUIDs also for further updating.

Taxonomy of Dianthus (Caryophyllaceae) - overall phylogenetic relationships and assessment of species diversity based on a first comprehensive checklist of the genus.

In this study, we present an overall phylogenetic framework for Dianthus using four plastid regions (matK-trnK-psbA, rpl32-trnL, trnQ-rps16) and nuclear ITS and a species-level checklist for the genus developed by using all available databases and the literature. The trees from the plastid dataset depict a clade of Dianthus that also includes Velezia and a few taxa of Petrorhagia. New combinations in Dianthus are provided for these species. The checklist of Dianthus in this new delimitation covers 1781 names, with 384 accepted species, 150 subspecies, 12 heterotypic varieties and two forms (not counting autonyms), 1050 synonyms, 22 hybrid names and 172 unresolved names, 3 names were excluded. Implications for the evolution of flower characters, life forms, biogeography, as well as sectional classification are discussed based on the phylogenetic framework.

Plastid phylogenomics of the Gynoxoid group (Senecioneae, Asteraceae) highlights the importance of motif-based sequence alignment amid low genetic distances.

PREMISE: The genus Gynoxys and relatives form a species-rich lineage of Andean shrubs and trees with low genetic distances within the sunflower subtribe Tussilaginineae. Previous molecular phylogenetic investigations of the Tussilaginineae have included few, if any, representatives of this Gynoxoid group or reconstructed ambiguous patterns of relationships for it. METHODS: We sequenced complete plastid genomes of 21 species of the Gynoxoid group and related Tussilaginineae and conducted detailed comparisons of the phylogenetic relationships supported by the gene, intron, and intergenic spacer partitions of these genomes. We also evaluated the impact of manual, motif-based adjustments of automatic DNA sequence alignments on phylogenetic tree inference. RESULTS: Our results indicate that the inclusion of all plastid genome partitions is needed to infer well-supported phylogenetic trees of the Gynoxoid group. Whole plastome-based tree inference suggests that the genera Gynoxys and Nordenstamia are polyphyletic and form the core clade of the Gynoxoid group. This clade is sister to a clade of Aequatorium and Paragynoxys and also includes some but not all representatives of Paracalia. CONCLUSIONS: The concatenation and combined analysis of all plastid genome partitions and the construction of manually-curated, motif-based DNA sequence alignments are found to be instrumental in the recovery of well-supported relationships of the Gynoxoid group. We demonstrate that the correct assessment of homology in genome-level plastid sequence data sets is crucial for subsequent phylogeny reconstruction and that the manual post-processing of multiple sequence alignments improves the reliability of such reconstructions amid low genetic distances between taxa.

Seeing the wood despite the trees: Exploring human disturbance impact on plant diversity, community structure, and standing biomass in fragmented high Andean forests.

High Andean forests harbor a remarkably high biodiversity and play a key role in providing vital ecosystem services for neighboring cities and settlements. However, they are among the most fragmented and threatened ecosystems in the neotropics. To preserve their unique biodiversity, a deeper understanding of the effects of anthropogenic perturbations on them is urgently needed. Here, we characterized the plant communities of high Andean forest remnants in the hinterland of Bogotá in 32 0.04 ha plots. We assessed the woody vegetation and sampled the understory and epiphytic cover. We gathered data on compositional and structural parameters and compiled a broad array of variables related to anthropogenic disturbance, ranging from local to landscape-wide metrics. We also assessed phylogenetic diversity and functional diversity. We employed nonmetric multidimensional scaling (NMDS) to select meaningful variables in a first step of the analysis. Then, we performed partial redundancy analysis (pRDA) and generalized linear models (GLMs) in order to test how selected environmental and anthropogenic variables are affecting the composition, diversity, and aboveground biomass of these forests. Identified woody vegetation and understory layer communities were characterized by differences in elevation, temperature, and relative humidity, but were also related to different levels of human influence. We found that the increase of human-related disturbance resulted in less phylogenetic diversity and in the phylogenetic clustering of the woody vegetation and in lower aboveground biomass (AGB) values. As to the understory, disturbance was associated with a higher diversity, jointly with a higher phylogenetic dispersion. The most relevant disturbance predictors identified here were as follows: edge effect, proximity of cattle, minimum fragment age, and median patch size. Interestingly, AGB was efficiently predicted by the proportion of late successional species. We therefore recommend the use of AGB and abundance of late successional species as indicators of human disturbance on high Andean forests.

Phylogeny and diversification history of the large Neotropical genus Philodendron (Araceae): Accelerated speciation in a lineage dominated by epiphytes.

PREMISE OF THE STUDY: Philodendron is a large genus of ~560 species and among the most conspicuous epiphytic components of Neotropical forests, yet its phylogenetic relationships, timing of divergence, and diversification history have remained unclear. We present a comprehensive phylogenetic study for Philodendron and investigate its diversification, including divergence-time estimates and diversification rate shift analyses. METHODS: We performed the largest phylogenetic reconstruction for Philodendron to date, including 125 taxa with a combined dataset of three plastid regions (petD, rpl16, and trnK/matK). We estimated divergence times using Bayesian evolutionary analysis sampling trees and inferred shifts in diversification rates using Bayesian analysis of macroevolutionary mixtures. KEY RESULTS: We found that Philodendron, its three subgenera, and the closely related genus Adelonema are monophyletic. Within Philodendron subgenus Philodendron, 12 statistically well-supported clades are recognized. The genus Philodendron originated ~25 mya and a diversification rate upshift was detected at the origin of subgenus Philodendron ~12 mya. CONCLUSIONS: Philodendron is a species-rich Neotropical lineage that diverged from Adelonema during the late Oligocene. Within Philodendron, the three subgenera currently accepted are recovered in two lineages: one contains the subgenera Meconostigma and Pteromischum and the other contains subgenus Philodendron. The lineage containing subgenera Meconostigma and Pteromischum underwent a consistent diversification rate. By contrast, a diversification rate upshift occurred within subgenus Philodendron ~12 mya. This diversification rate upshift is associated with the species radiation of the most speciose subgenus within Philodendron. The sections accepted within subgenus Philodendron are not congruent with the clades recovered. Instead, the clades are geographically defined.

Bioinformatic Workflows for Generating Complete Plastid Genome Sequences-An Example from Cabomba (Cabombaceae) in the Context of the Phylogenomic Analysis of the Water-Lily Clade.

The sequencing and comparison of plastid genomes are becoming a standard method in plant genomics, and many researchers are using this approach to infer plant phylogenetic relationships. Due to the widespread availability of next-generation sequencing, plastid genome sequences are being generated at breakneck pace. This trend towards massive sequencing of plastid genomes highlights the need for standardized bioinformatic workflows. In particular, documentation and dissemination of the details of genome assembly, annotation, alignment and phylogenetic tree inference are needed, as these processes are highly sensitive to the choice of software and the precise settings used. Here, we present the procedure and results of sequencing, assembling, annotating and quality-checking of three complete plastid genomes of the aquatic plant genus Cabomba as well as subsequent gene alignment and phylogenetic tree inference. We accompany our findings by a detailed description of the bioinformatic workflow employed. Importantly, we share a total of eleven software scripts for each of these bioinformatic processes, enabling other researchers to evaluate and replicate our analyses step by step. The results of our analyses illustrate that the plastid genomes of Cabomba are highly conserved in both structure and gene content.

Evolutionary diversification of the African achyranthoid clade (Amaranthaceae) in the context of sterile flower evolution and epizoochory.

Background and Aims: Many African genera of the Amaranthaceae exhibit unique inflorescences that include sterile flowers modified to hooks or spines. Considering that the abundance of large terrestrial herbivores increased on the African continent with the expansion of grassland and savannah ecosystems, modified sterile flowers could have been an innovation that boosted the diversification of an African achyranthoid clade of Amaranthaceae, with large animals serving dispersal. Methods: We generated an extensively sampled phylogeny comprising 26 of the 31 achyranthoid genera as well as representatives of all other lineages of Amaranthaceae. Phylogenetic tree inference employed four genomic regions, using parsimony, likelihood and Bayesian inference methods. We estimated divergence times, evaluated trait-dependant changes and species diversification rates using state-dependent speciation and extinction models, and reconstructed ancestral character states for modified sterile flowers. Key Results: The achyranthoids were found to be a major clade of the Amaranthaceae, comprising mostly African members. Phylogenetic relationships within this clade were well resolved and supported two main subclades. Several genera were found to be polyphyletic. Our results indicate that the achyranthoids started to diversify ~28 million years ago, and that modified sterile flowers evolved multiple times. An asymmetry in transition rates towards the gain of sterile flowers was observed, whereas no trait-dependent increase in species diversification rates was detected. Bayesian rate heterogeneity analyses indicated that the achyranthoids diversified without significant rate shifts. Conclusions: The accumulation of modified sterile flowers within achyranthoids appears to result from the higher transition rates in favour of modified sterile flowers. Multiple gains suggest an adaptive value for this trait. However, epizoochory does not appear to fuel species diversification, possibly due to extensive gene flow through regularly migrating mammals, which limits the possibility of speciation by isolation.

When Homoplasy Is Not Homoplasy: Dissecting Trait Evolution by Contrasting Composite and Reductive Coding.

The conceptualization and coding of characters is a difficult issue in phylogenetic systematics, no matter which inference method is used when reconstructing phylogenetic trees or if the characters are just mapped onto a specific tree. Complex characters are groups of features that can be divided into simpler hierarchical characters (reductive coding), although the implied hierarchical relational information may change depending on the type of coding (composite vs. reductive). Up to now, there is no common agreement to either code characters as complex or simple. Phylogeneticists have discussed which coding method is best but have not incorporated the heuristic process of reciprocal illumination to evaluate the coding. Composite coding allows to test whether 1) several characters were linked resulting in a structure described as a complex character or trait or 2) independently evolving characters resulted in the configuration incorrectly interpreted as a complex character. We propose that complex characters or character states should be decomposed iteratively into simpler characters when the original homology hypothesis is not corroborated by a phylogenetic analysis, and the character or character state is retrieved as homoplastic. We tested this approach using the case of fruit types within subfamily Cinchonoideae (Rubiaceae). The iterative reductive coding of characters associated with drupes allowed us to unthread fruit evolution within Cinchonoideae. Our results show that drupes and berries are not homologous. As a consequence, a more precise ontology for the Cinchonoideae drupes is required.

Dynamic diversification history with rate upshifts in Holarctic bell-flowers (Campanula and allies).

Campanula s.l. is one of the most speciose flowering plant lineages of the Holarctic (ca. 600 species). In the present study we sequenced three regions of the plastid genome (petD, rpl16 and trnK/matK) across a broad sample of Campanula s.l., which markedly improved phylogenetic resolution and statistical support compared to previous studies. Based on this robust phylogenetic hypothesis we estimated divergence times using BEAST, diversification rate shifts using Bayesian Analysis of Macroevolutionary Mixture (BAMM) and TreePar, and ancestral ranges using Biogeography with Bayesian (and likelihood) Evolutionary Analyses in R. Campanula s.l. is estimated to have originated during the Early Eocene but the major diversification events occurred between the Late Oligocene and Middle Miocene. Two upward diversification rate shifts were revealed by BAMM, specific to the crown nodes of two Campanula clades: CAM17, a mostly South European-SW Asian lineage originating during the Middle Miocene and containing nearly half of all known Campanula species; and CAM15B, a SW Asian-Sino-Himalayan lineage of nine species originating in the early Pleistocene. The dynamic diversification history of Campanula and the inferred rate shifts are discussed in a geo-historical context.

Development of microsatellite markers for Crepis mollis (Asteraceae).

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed for the threatened species Crepis mollis (Asteraceae) to investigate population and conservation genetics. METHODS AND RESULTS: Illumina sequencing was conducted on pooled genomic DNA from 10 individuals of two populations. Ten polymorphic and 10 monomorphic microsatellite loci with di-, tri-, tetra-, penta-, and hexanucleotide repeat motifs were developed and characterized in C. mollis. In the polymorphic markers, up to 17 alleles per locus were detected with an observed and expected heterozygosity ranging from 0.120 to 0.780 and 0.102 to 0.834, respectively. Furthermore, the polymorphic markers were tested for cross-amplification in three congeneric species (C. biennis, C. foetida, and C. sancta) and amplified in up to three loci. CONCLUSIONS: The markers developed in this study are the first microsatellites tested on C. mollis and will be useful for performing population and conservation genetic studies in this threatened species.

Sample data processing in an additive and reproducible taxonomic workflow by using character data persistently linked to preserved individual specimens.

We present the model and implementation of a workflow that blazes a trail in systematic biology for the re-usability of character data (data on any kind of characters of pheno- and genotypes of organisms) and their additivity from specimen to taxon level. We take into account that any taxon characterization is based on a limited set of sampled individuals and characters, and that consequently any new individual and any new character may affect the recognition of biological entities and/or the subsequent delimitation and characterization of a taxon. Taxon concepts thus frequently change during the knowledge generation process in systematic biology. Structured character data are therefore not only needed for the knowledge generation process but also for easily adapting characterizations of taxa. We aim to facilitate the construction and reproducibility of taxon characterizations from structured character data of changing sample sets by establishing a stable and unambiguous association between each sampled individual and the data processed from it. Our workflow implementation uses the European Distributed Institute of Taxonomy Platform, a comprehensive taxonomic data management and publication environment to: (i) establish a reproducible connection between sampled individuals and all samples derived from them; (ii) stably link sample-based character data with the metadata of the respective samples; (iii) record and store structured specimen-based character data in formats allowing data exchange; (iv) reversibly assign sample metadata and character datasets to taxa in an editable classification and display them and (v) organize data exchange via standard exchange formats and enable the link between the character datasets and samples in research collections, ensuring high visibility and instant re-usability of the data. The workflow implemented will contribute to organizing the interface between phylogenetic analysis and revisionary taxonomic or monographic work. DATABASE URL: http://campanula.e-taxonomy.net/.

Analysis of nuclear microsatellites reveals limited differentiation between Colchic and Hyrcanian populations of the wind-pollinated relict tree Zelkova carpinifolia (Ulmaceae).

UNLABELLED: • PREMISE OF THE STUDY: The Caucasus represents one of the world's biodiversity hotspots and includes the climatic refugia Hyrcan on the southern coast of the Caspian Sea and Colchis on the eastern coast of the Black Sea, where different species survived during the Quaternary climatic oscillations. We evaluated the genetic diversity of the relict tree Zelkova carpinifolia shared between the two refugia and distributed throughout the Caucasus and adjacent areas.• METHODS: Specimens were collected from 30 geographical sites in Azerbaijan, Georgia, Iran, and Turkey and screened for variability at eight nuclear microsatellite loci. The genetic diversity among and within populations was assessed using a set of statistical measures.• KEY RESULTS: We detected 379 different genotypes from a total of 495 individuals with varying degrees of clonal reproduction at the different sites. Low to intermediate levels of genetic diversity were observed at all sites, and strong differentiation between sampling sites was absent. In addition, we observed no clear genetic differentiation between the Colchis and Hyrcan. Bayesian clustering of the genotypes revealed three populations with high levels of admixture between the sampling sites.• CONCLUSIONS: The lack of strong genetic structure of studied populations of Z. carpinifolia contrasts with a previous study based on chloroplast markers and suggests that long-distance pollen dispersal is an important factor of gene flow among populations of Z. carpinifolia. The present study does not reveal any particular site with particularly isolated genotypes that would deserve more attention for conservation purposes than others, although some sites should be considered for further investigation.

Fourteen polymorphic microsatellite markers for the threatened Arnica montana (Asteraceae).

PREMISE OF THE STUDY: Microsatellite markers were developed to investigate population genetic structure in the threatened species Arnica montana. • METHODS AND RESULTS: Fourteen microsatellite markers with di-, tetra-, and hexanucleotide repeat motifs were developed for A. montana using 454 pyrosequencing without and with library-enrichment methods, resulting in 56,545 sequence reads and 14,467 sequence reads, respectively. All loci showed a high level of polymorphism, with allele numbers ranging from four to 11 in five individuals from five populations (25 samples) and an expected heterozygosity ranging from 0.192 to 0.648 across the loci. • CONCLUSIONS: This set of microsatellite markers is the first one described for A. montana and will facilitate conservation genetic applications as well as the understanding of phylogeographic patterns in this species.

Variability among the most rapidly evolving plastid genomic regions is lineage-specific: implications of pairwise genome comparisons in Pyrus (Rosaceae) and other angiosperms for marker choice.

Plastid genomes exhibit different levels of variability in their sequences, depending on the respective kinds of genomic regions. Genes are usually more conserved while noncoding introns and spacers evolve at a faster pace. While a set of about thirty maximum variable noncoding genomic regions has been suggested to provide universally promising phylogenetic markers throughout angiosperms, applications often require several regions to be sequenced for many individuals. Our project aims to illuminate evolutionary relationships and species-limits in the genus Pyrus (Rosaceae)-a typical case with very low genetic distances between taxa. In this study, we have sequenced the plastid genome of Pyrus spinosa and aligned it to the already available P. pyrifolia sequence. The overall p-distance of the two Pyrus genomes was 0.00145. The intergenic spacers between ndhC-trnV, trnR-atpA, ndhF-rpl32, psbM-trnD, and trnQ-rps16 were the most variable regions, also comprising the highest total numbers of substitutions, indels and inversions (potentially informative characters). Our comparative analysis of further plastid genome pairs with similar low p-distances from Oenothera (representing another rosid), Olea (asterids) and Cymbidium (monocots) showed in each case a different ranking of genomic regions in terms of variability and potentially informative characters. Only two intergenic spacers (ndhF-rpl32 and trnK-rps16) were consistently found among the 30 top-ranked regions. We have mapped the occurrence of substitutions and microstructural mutations in the four genome pairs. High AT content in specific sequence elements seems to foster frequent mutations. We conclude that the variability among the fastest evolving plastid genomic regions is lineage-specific and thus cannot be precisely predicted across angiosperms. The often lineage-specific occurrence of stem-loop elements in the sequences of introns and spacers also governs lineage-specific mutations. Sequencing whole plastid genomes to find markers for evolutionary analyses is therefore particularly useful when overall genetic distances are low.