Applying phylogenomics (HybSeq) to elucidate diversification, systematics and evolution in the tribe Cichorieae
The nuclear in contrast to the plastid genome, has always been the bottleneck for molecular phylogenetic analyses because of the difficulty to employ suitable loci that provide phylogenetic signal without orthology-paralogy conflation. To overcome this problem, a particularly promising approach of next generation or high throughput sequencing (HTS) is the target enrichment of the nuclear genome, which enables access to hundreds of orthologous loci at once. In this approach, only a small subset of the genome is targeted and the target sequences in the samples are captured by hybridisation of nucleotide baits (probes) to DNA fragments containing complementary sequences, followed by the subsequent separation and PCR enrichment of these targets.
The suitable target sequences are taxon specific. Mandel & al. (2014) designed sequence capture probes for the flowering plant family Asteraceae (or Compositae, sunflower family), targeting a set of 1061 conserved orthologous sequences (COS), which were identified from expressed sequence tags (ESTs). Combining hybrid capture and target sequence enrichment with genome skimming (shallow sequencing), this approach is also known as "Hyb-Seq" (Weitemier & al. 2014). It works with fresh material as well as with samples from herbarium specimens. Basically, DNA is extracted from the samples and sheared, and after size selection of the fragments barcoded sequencing libraries are prepared and enriched by PCR amplification. After pooling (multiplexing) a number of barcoded sample libraries, they are denaturated in a hybrid capture reaction and allowed to hybridise with baits for the target sequences. The resulting hybrids of the baits with the target sequences of the libraries are selected, the target sequences separated, PCR enriched and finally Illuminia-sequenced.
Participating in an international collaboration network (Mandel & al. 2017), we have established this approach at the Berlin Center for Genomics in Biodiversity Research (BeGenDiv), a consortium of six institutions including the Botanic Garden and Botanical Museum Berlin (BGBM). In the frame of this collaboration, we aim to investigate the phylogeny and diversification of the mega-diverse sunflower family by focusing on the tribe Cichorieae.
Establishing the Asteraceae COS Hyb-Seq in Berlin by Katy Jones was funded by the Forschungskommission of the Freie Universität Berlin in 2017/2018 using Lactuca (lettuce) as a pilot. Further development and optimization of the approach using the Scorzonerinae as a second pilot has been funded in 2018/2019 by the German Research Foundation (DFG) in a project by Katy Jones (PI) and Norbert Kilian. An empirical assessment of the hybrid capture locus set has been published recently.
Phylogeny, evolution, historical biogeography and systematics of the Lactuca alliance (Asteraceae, Cichorieae, subtribe Lactucinae)
Lettuce (Lactuca sativa L.) is the economically most important crop of the tribe Cichorieae, and Lactuca one of its widest known genera. Lactuca is also the namegiving member of one of the larger groups of the tribe, the Lactuca alliance, which is treated today as the subtribe Lactucinae. It comprises about 230 species with a preference of montane habitats, distributed in Europe, Africa, Asia and North America. The subtribe has two centres of current diversity, one in the Mediterranean-SW Asian region, the other in China and the adjacent Himalayan region (Jones & al. 2018; Kilian & al. 2017; Wang & al. 2013).
The classification of no other alliance of the tribe has faced so many controversies over the last 200 years than that of Lactuca and its presumed allies, and only towards the end of the 20th century, the Lactuca alliance was recognised as a separate subtribe, based on morphological and molecular phylogenetic analyses. With studies on its taxonomy, reconstructions of its phylogeny and biogeography (applying phylogenetics as well as phylogenomics), we aim at an understanding of the evolution of the diversity of this lineage.
The results of these studies, moreover, form the foundation for monographic systematic treatments of the Lactucinae genera finally recognised. These treatments are, at the same time, planned to explore a consistently matrix-based, integrative taxonomic workflow and dynamic monographic publication, hence as an approach to facilitate both the production and updating of as well as data re-use from taxonomic monographs.
This project, lead-managed by the Asterales Research Group, is conducted in partnership with researchers from Gießen (Birgit Gemeinholzer), Helsinki & St Petersburg (Alexander Sennikov), Kunming (Peng Hua, Wang Zehuan, Zhang Jianwen), Munich & S. Miguel, Acores (Hanno Schäfer, Mónica Maria Tavares Moura, Luís Filipe Dias e Silva & Elisabete F. Dias) and Tehran (Hossein Akhani). Lab work in Berlin has been funded by the Verein der Freunde des Botanischen Gartens und Botanischen Museums Berlin and the Dahlem Centre of Plant Sciences (DCPS), and the Forschungskommission of the the Freie Universität Berlin.
Phylogeny and historical biogeography of the Campanulaceae subfamily Campanuloideae
Preceded by a first broadly sampled molecular phylogenetic analysis across the Campanulaceae with a focus of Campanula s.l. (Borsch & al. 2009), we published in 2012 the largest phylogeny of the subfamily Campanuloideae so far, encompassing some 70–80 % of the described diversity (Mansion & al. in PLOS ONE 7(11). 2012). Based on a genomic region with high phylogenetic signal (petD region of the group II intron), we were able to resolve 17 well-supported clades, outlining avenues for further research. We also tested the effects of mass sampling versus lower taxon representation on several phylogenetic estimates including tree shape, branch robustness, and node ages calculation.
Subsequently, we achieved a robust phylogenetic hypothesis for Campanula s.l. with a combination of three plastid DNA markers and estimated on this basis divergence times, diversification rate shifts and ancestral ranges. We provided evidence (Jones & al. 2017) that Campanula s.l. originated in SW Asia during the Early Eocene but the major diversification events occurred between the Late Oligocene and Middle Miocene. Upward diversification rate shifts were revealed for the largest, chiefly S European-SW Asian clade (CAM17) and the small SW Asian/Sino/Himalayan clade (CAM15B).
A current project focusses on the diversification within the large CAM17 clade, taking Campanula in the Caucasus range as an example. A detailed study of the Campanula Scapiflorae group is subject of the doctorate thesis by Nana Silakadze at the Freie Universität Berlin under the supervision of Prof. Thomas Borsch. This project is founded by the VolkswagenStiftung in the frame of a larger Caucasus project at the BGBM.
Phylogeny and taxonomy of the Andean Gynoxys group (Asteraceae, Senecioneae, Tussilagininae)
Molecular phylogenetic analyses have shown that the genera Gynoxys (> 100 species), Aequatorium (22 species), Nordenstamia (15 species), Paracalia (3 species), Paragynoxys (11 species) form a well supported clade of shrubs and small trees distributed exclusively in the Andes of South America. The phylogeny of the member of this clade is largely unknown and its taxonomy is still in its infancy. Reconstruction of a phylogenetic backbone of this clade, assessing generic classification and clayfiying the taxonomy of its members in Bolivia are the subjects of the doctorate thesis by Andrea Belen Escobari Vargas at the Freie Universität Berlin under the supervision of Prof. Thomas Borsch and in cooperation with the Herbario National de Bolivia.
Achieving additivity of structured taxonomic character data by persistently linking them to preserved individual specimens
In a precursor project funded from 2012 to 2015 by the German Research Foundation (DFG), see Kilian & al. (2015 in Database : bav094), we have implemented a workflow on the EDIT (European Distributed Institute of Taxonomy) Platform for Cybertaxonomy, a comprehensive taxonomic data management and publication environment. The aim was to organise the links between (a) investigated samples of individual organisms, (b) research data obtained from them, (c) specimens of these individuals deposited in research collections, and (d) taxon assignments (“identifications”) of the investigated individuals. On this basis the proposed project will optimise the taxonomic research process with respect to delimitation and characterisation (“description”) of taxa. The angiosperm order Caryophyllales will provide exemplar use cases through cooperation within the Global Caryophyllales Initiative. The workflow for sample data handling will be extended to include two functionalities connected with each other: First, recording and storing character data (data on genoptypic and phenotypic characters of any type, here focusing on morphology) in structured form in character and character state matrices for individual specimens instead for taxa, shall allow to generate taxon characterisations by aggregating the data sets for the individual specimens included. Semantic web technology will be used to establish and continuously elaborate expert community-coordinated exemplar vocabularies with term ontologies and explanations for characters and states. Second, persistent linking of specimen-based character data sets with the individual specimens preserved in research collections via standard exchange formats shall guarantee a high visibility and instant re-usability of these character data. Taking into account that taxon concepts frequently change during the knowledge generation process in systematic biology, additivity of character data from specimen to taxon level therefore greatly facilitates the construction, extension as well as the reproducibility of taxon characterisations from changing specimen and character data sets. See also Henning & al. (2018)
The project is funded from 2017 to 2019 by the German Research Foundation (DFG) within the Scientific Library Services and Information Systems (LIS) programme and done jointly by the Research Groups Asterales and Biodiversity Informatics.
Consequences of polyploidy: phylogeny, phyloecology, and expression of duplicated genes in Leucanthemum (Compositae, Anthemideae)
Polyploidy (whole genome duplication via auto- or allopolyploidy) is an important process in angiosperm evolution. The genus Leucanthemum, centred in the western Mediterranean, of the Anthemideae subtribe Leucantheminae provides a striking example with an uninterrupted series of diploid to dodecaploid chromosome numbers among its 42 species. We use this genus as a model to investigate the genetic, genomic, biogeographical and ecological consequences of polyploidy. In a top-down approach we envisage (a) to prepare a hypothesis of the reticulate phylogeny of the whole genus using sequencing of nuclear and chloroplast markers and AFLP fingerprinting, along with an up-to-date taxonomic revision of the genus, and (b) to model eco-climatic niches of all species based on geo-referenced herbarium specimens. Phylogenetic information and reconstructions of actual and potential eco-climatic niches will be then used to circumscribe the temporal, spatial and ecological diversification of the polyploidy complex. In a bottom-up approach, we use the phylogentically distinct L. pluriflorum clan comprising diploid, tetraploid, and hexaploid species from Iberian coastal and inland habitats (a) to study the phylogeography of this group and (b) to assess the fate of duplicated candidate genes connected with salt tolerance by sequence comparisons and expression studies.
This is a project funded from 2011 to 2014 by the German Research Foundation (DFG) in a long-term cooperation between the BGBM (Robert Vogt) and the Institute for Botany, University of Regensburg (Christoph Oberprieler) dedicated to the phylogeny, evolution and systematics of the Anthemidae subtribe Leucantheminae.
Permanent online resources
In the workpackage "Unifying revisionary taxonomy" of the European Distributed Institute of Taxonomy (EDIT) project the Cichorieae exemplar group at the BGBM created an exemplar site using the EDIT Platform for Cybertaxonomy. The Cichorieae Portal is being established as a convenient access to the existing knowledge about this difficult group of plants, based on authoritative taxonomic information. It is designed to serve both the specialist and the occasional visitor, facilitating cooperation among workers of the group as well as an undelayed integration of new data and research results, and finally aiming at an encyclopedic knowledge base of entire tribe.
This Cichorieae Portal of the International Cichorieae network is sustained and further developed by the BGBM’s Asterales Research Group in cooperation with the BGBM's Research and Development Group Biodiversity Informatics.
We are using Campanula s.l. (bell flower), which is one of the species-richest flowering plant genera, in this portal as an exemplar (1) to display work in progress on the systematics of this genus, and (2) to investigate convenient ways to display the linking of metadata and descriptive data for collection items with taxonomic information as being developed in the frame of our projects on a workflow for comprehensive taxonomic data management.
The Campanula Portal is maintained and further developed by the BGBM’s Asterales Research Group and its Research and Development Group Biodiversity Informatics.