Phylogeny and evolution of the epiphytic Rhipsalideae (Cactaceae)

Cactaceae are one of the major floristic components of the New World’s arid as well as seasonally moist tropical regions and at the same time one of the most popular plant families in horticulture. The taxonomic units (tribes, genera) and species limits in the Cactaceae have been difficult to define due to intergrading vegetative characters, pheno-typic plasticity and the largely uniform flower morphology. Molecular phylogenetic studies so far yielded largely unresolved trees, so relationships within Cactaceae remain in-sufficiently understood. This study focuses on the Rhipsalideae, a predominantly epiphytic Cactaceae tribe from the tropical rainforests of South -and Mesoamerica. The Rhipsalideae have not been subject of a phylogenetic study so far but are well-suited for this purpose: they are a comparatively small group and most of the taxa are well known morphologically and thus allow for the clear determination of Operational Taxonomic Units (OTUs). The Rhipsalideae are well represented in botanical collections so all but one species were available for this study. This study is therefore among the most comprehensive species-level-study for the Cactaceae. Aims: resolving species-level relationships, get better insights into species limits, find morphological characters synapomorphic or at least characteristic for the newly found clades. The phylogenetic relationships were addressed using sequence data from rapidly evolving plastid spacers (psbA-trnH, rps3-rpl16, trnS-trnG, trnQ-rps16), group II introns (trnK, rpl16, trnG) and the coding regions matK. The sequence data were analysed using Maximum Parsimony, Bayesian Inference and haplotype network construction. First, the position and circumscription of the genus Pfeiffera has been addressed. It had formerly been included in Lepismium abut there was already evidence that Pfeiffera is not part the Rhipsalideae. The phylogenetic analyses revealed Pfeiffera polyphyletic, comprising two unrelated lineages, both well resolved and highly supported. One clade includes the type species, P. ianthothele; the second contains two Pfeiffera and an erstwhile Lepismium species. The results justify generic status for this newly found clade. It includes the type species of the earlier-proposed monotypic genus Lyman¬bensonia and therefore its reinstatement is proposed in an amplified circumscription. The results further provide evidence that epiphytism evolved more frequently in Cactaceae than hitherto assumed. The taxonomic and nomen¬clatural consequences are the establishment of a separate tribe Lyman¬bensonieae, formally proposed here, to contain the genera Lyman¬bensonia and Calym¬manthium. To resolve phylogenetic relationships in the Rhipsalideae, a dataset of six fast evolving plastid regions was generated with c. 4200 nucleotides sequenced per sample for 120 accessions. The regions used were evaluated for their phylogenetic performance and species discrimination power for DNA based species recognition (DNA barcoding) based on beforehand defined operational taxonomic units (OTUs). The Rhipsalideae were found as monophyletic and contain five major clades that correspond to the genera Rhipsalis, Lepismium, Schlumbergera, Hatiora subg. Hatiora and Rhipsalidopsis. The relationships between the genera could not be clarified but otherwise the trees were well resolved down to species-level and well supported. Ninety-seven % of the OTUs could be successfully identified using c. 2500 nt. The rpl16 and trnK introns yielded the best phylogenetic signal and the best OTU identification potential. The phylogenetic performance of the markers was found to be not determined by the level of sequence variability. The species discrimination power of the markers did not always correlate with their phylogenetic performance. The reliable phylogenetic hypothesis for the Rhipsalideae provided a framework for a first detailed study of character evolution in a phylogenetic context. A matrix of 36 characters was compiled and ancestral states were reconstructed using a Bayesian approach. A focus was put on the characters associated with the epiphytic life form and the floral traits. The degree of homoplasy was found to high but many characters were homogenous within the clades. Rhipsalideae and all the highly supported clades found by the molecular phylogenetic analyses could also be defined morphologically. The distribution patterns of Rhipsalis baccifera, the most widespread cactus, were addressed using tree building methods and haplotype network algorithms based on the rps3-rpl16 spacer and the rpl16 intron. Two main groups of plastid haplotypes were found: a northern South American / Caribbean / Mesoamerican haplotype and an African haplotype. These results suggest a single dispersal of Rhipsalis baccifera to Africa and reveal high genetic diversity within its populations on both continents. To obtain further resolution among the populations, microsatellite markers for Rhipsalis baccifera have been deve-loped using 454 sequencing. The comprehensive taxon sampling and the usage of effective markers resulted in almost completely resolved species level trees which were hitherto hardly achieved in the Cactaceae. In order to resolve relationships between closely related species, com¬bined data sets of markers selected for their high phylo-genetic structure are needed. Morphological convergences are frequent in the Cactaceae. The level of homoplasy in morpho¬logical characters is high, especially regarding characters associated with an adaptation such as epiphytism. Generic classification based on single or few morphological characters consequently cannot predict actual relationships. For phylogenetic stu¬dies in the Cactaceae, the morphology-based taxonomic units consequently may be misleading to guide taxon sampling. The best solution therefore would be including all morphologically deviant groups and species in the given study. Nevertheless, the analysis of morphological characters in a phylogenetic context allowed finding synapomorphies or diagnostic characters for all the clades (genera, subgenera) found by the molecular phylogenetic analyses. An integrated approach using morphology and sequence data is therefore needed to establish sound generic limits in the Cactaceae

Development of Nuclear Microsatellites for the Arcto-Tertiary Tree Zelkova carpinifolia (Ulmaceae) Using 454 Pyrosequencing

Premise of the study: The current study aimed at developing nuclear microsatellite markers for the relict tree species Zelkova carpinifolia, which is threatened in its natural range in the South Caucasus. Methods and Results: Pyrosequencing of an enriched microsatellite library on the Roche FLX platform using the 454 Titanium kit produced 86,058 sequence reads, most of which contained short tandem repeats. Eighty microsatellite loci identified using the software package QDD version 1 were selected and tested for proper PCR amplification. Of these, 13 allowed proper amplification and were shown to be polymorphic among a sample of 25 Z. carpinifolia specimens from various geographic origins. Conclusions: The set of microsatellite markers will be suitable for the assessment of genetic diversity in Z. carpinifolia. They will allow for an examination of phylogeographic patterns as well as of population structure and gene flow within this species

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

Amborella trichopoda

Amborella trichopoda Baill., the most ancestral angiosperm, has been successfully cultivated in the Botanic Gardens of the University of Bonn in Germany (BG Bonn) for more than a decade. The distribution of this plant – limited to the South Pacific island of New Caledonia – and its cultivation has so far only been achieved in a few botanic gardens. This paper provides details about the cultivation and propagation of Amborella, and information on its cultivation in botanic gardens around the world. The authors propose that the collections of this plant in botanic gardens could be used to establish ex situ conservation collections

Cactaceae at Caryophyllales.org- A dynamic online species-level taxonomic backbone for the family

This data paper presents a largely phylogeny-based online taxonomic backbone for the Cactaceae compiled from literature and online sources using the tools of the EDIT Platform for Cybertaxonomy. The data will form a contribution of the Caryophyllales Network for the World Flora Online and serve as the base for further integration of research results from the systematic research community. The final aim is to treat all effectively published scientific names in the family. The checklist includes 150 accepted genera, 1851 accepted species, 91 hybrids, 746 infraspecific taxa (458 heterotypic, 288 with autonyms), 17,932 synonyms of accepted taxa, 16 definitely excluded names, 389 names of uncertain application, 672 unresolved names and 454 names belonging to (probably artificial) named hybrids, totalling 22,275 names. The process of compiling this database is described and further editorial rules for the compilation of the taxonomic backbone for the Caryophyllales Network are proposed. A checklist depicting the current state of the taxonomic backbone is provided as supplemental material. All results are also available online on the website of the Caryophyllales Network and will be constantly updated and expanded in the future. Citation: Korotkova N., Aquino D., Arias S., Eggli U., Franck A., Gómez-Hinostrosa C., Guerrero P. C., Hernández H. M., Kohlbecker A., Köhler M., Luther K., Majure L. C., Müller A., Metzing D., Nyffeler R., Sánchez D., Schlumpberger B. & Berendsohn W. G. 2021: Cactaceae at Caryophyllales.org- A dynamic online species-level taxonomic backbone for the family.-Willdenowia 51: 251-270. Version of record first published online on 31 August 2021 ahead of inclusion in August 2021 issue. Data published through: Http://caryophyllales.org/cactaceae/Checklis

A taxonomic backbone for the global synthesis of species diversity in the angiosperm order Caryophyllales

The Caryophyllales constitute a major lineage of flowering plants with approximately 12500 species in 39 families. A taxonomic backbone at the genus level is provided that reflects the current state of knowledge and accepts 749 genera for the order. A detailed review of the literature of the past two decades shows that enormous progress has been made in understanding overall phylogenetic relationships in Caryophyllales. The process of re-circumscribing families in order to be monophyletic appears to be largely complete and has led to the recognition of eight new families (Anacampserotaceae, Kewaceae, Limeaceae, Lophiocarpaceae, Macarthuriaceae, Microteaceae, Montiaceae and Talinaceae), while the phylogenetic evaluation of generic concepts is still well underway. As a result of this, the number of genera has increased by more than ten percent in comparison to the last complete treatments in the Families and genera of vascular plants” series. A checklist with all currently accepted genus names in Caryophyllales, as well as nomenclatural references, type names and synonymy is presented. Notes indicate how extensively the respective genera have been studied in a phylogenetic context. The most diverse families at the generic level are Cactaceae and Aizoaceae, but 28 families comprise only one to six genera. This synopsis represents a first step towards the aim of creating a global synthesis of the species diversity in the angiosperm order Caryophyllales integrating the work of numerous specialists around the world

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

Organizing and Maintaining a Taxonomic Expert Network: Lessons from the Caryophyllales TEN

The Caryophyllales TEN is one of the Taxonomic Expert Networks for the World Flora Online (WFO, worldfloraonline.org). It was initiated in 2011 with the aim to create a global taxonomic synthesys of the angiosperm order Caryophyllales (about 6% of the angiosperm species). The network currently includes about 170 scientists from 36 countries. The first priority is creating a dynamic taxonomic backbone for the order using the EDIT Platform for Cybertaxonomy (cybertaxonomy.org).One key product is an online portal (caryophyllales.org) that serves as a single entry point for expert-reviewed information. The EDIT Data Portal is one of the software components of the EDIT Platform (see the list of reference projects for further examples). Data entry, imports and editing is managed by the Taxonomic Editor component. Web services and data exchange via standardized formats will be used to connect to international biodiversity informatics initiatives such as the Global Biodiversity Information Facility and the International Plant Name Index. The data are available in a standardized format for open data exchange.Completed checklist projects with accompanying data papers so far consist of a genus-level checklist of the whole order (Hernández-Ledesma et al. 2015) and species-level treatments for Nepenthaceae (Berendsohn et al. 2018), Cactaceae (Korotkova et al. 2021) and Dianthus, Caryophyllaceae (Fassou et al. 2022). The entire Caryophyllaceae, Plumbaginaceae and the Amaranthaceae are under revision, Aizoaceae and most of the smaller families have been revised based on literature. These projects all aimed to network existing specialists for the respective families and to produce checklists that reflect the current state of research on the given group, where possible based on phylogenetic studies.The Caryophyllales TEN is coordinated by a scientist at the Botanic Garden Berlin but otherwise has no explicit governance or committees. The experiences from the aforementioned projects allow for some general insights as to organizing and maintaining a taxonomic expert network. Whereas a scientific network is usually a relatively loose connection between collaborators with varying levels of commitment over time, a strong expert network that works towards a specific product needs a firmer structure. According to our experience, five aspects are important.1. Clarity. A clearly defined framework which can include the written summary of the rationale and the main underlying principles, detailed editorial guidelines and guidelines for contributors, best-practice recommendations, a clear outline of different roles which experts can have in the project, and requirements for authorship on resulting publications. 2. Flexibility. The framework should be defined in a way that it still allows flexible approaches to the individual projects and the roles that can account for varying levels of commitment and needs of individual researchers.3. Community aspect. Bringing experts together regularly through in-person conferences or virtual meetings is essential to keep the network going. Participation should be open for everyone who is interested, regardless of career level and there should be different lowthreshold “entry points”. These can be participation in a conference, being invited to contribute to a checklist or seeking out other experts for collaboration. Already active experts can engage and further connect people to the network, thus creating a large and strong community.4. Credits. Our policy is that each source is referenced and each individual contribution is acknowledged in the data paper and online portal. This aims at increasing the visibility of research results and developing best-practice approaches within the community.5. Incentives and commitment. People will participate in such networks because they have a strong personal interest to invest their time and energy in a project that is valuable to them and aligns with their own goals. Expert networks need committed people and therefore, there must be strong incentives, for example being part of a widely known and highly visible initiative or co-authoring a key data paper

Development of Nuclear Microsatellites for the Arcto-Tertiary Tree <i>Zelkova carpinifolia</i> (Ulmaceae) Using 454 Pyrosequencing

Premise of the study: The current study aimed at developing nuclear microsatellite markers for the relict tree species , which is threatened in its natural range in the South Caucasus. Methods and Results: Pyrosequencing of an enriched microsatellite library on the Roche FLX platform using the 454 Titanium kit produced 86,058 sequence reads, most of which contained short tandem repeats. Eighty microsatellite loci identified using the software package QDD version 1 were selected and tested for proper PCR amplification. Of these, 13 allowed proper amplification and were shown to be polymorphic among a sample of 25 Z. carpinifolia specimens from various geographic origins. Conclusions: The set of microsatellite markers will be suitable for the assessment of genetic diversity in Z. carpinifolia. They will allow for an examination of phylogeographic patterns as well as of population structure and gene flow within this species