This study is focused on the Gynoxyoid group a species‐rich lineage with low genetic distances within the subtribe Tussilaginineae. The Andean clade within the Asteraceae family comprises four genera and ca. 160 species. The genus Paracalia represents the smallest genus within the Gynoxyoid comprising only two species, the genera Aequatorium and Paragynoxys include 12 and 13 species respectively, and the largest genus Gynoxys contains 131 species. This group includes shrubs to big trees and eventually scandent shrubs (meanly Paracalia) which are distributed from north to south Andes, representing important components of the Andean vegetation. Analyses on plastid and nuclear data have retrieved this group as monophyletic, nevertheless these studies include only some representatives of this clade as part of their investigations. In that sense, the phylogenetic relationships at inter- and infrageneric level remained largely unresolved. Likewise, the morphological studies within this group is limited to close-distributed group of species. The aim of this study is to elucidate the phylogenetic relationships of the Gynoxyoid clade and define genera and (for a reduced group) species limits within it. In that sense, 21 complete annotated chloroplast genomes from a representative subgroup of the Gynoxyoids and four related members of the Tussilaginineae were generated. Thereafter, phylogenetic analyses were performed under maximum likelihood and Bayesian approaches. In order to estimate the strength of the phylogenetic signal in each genome partition trees topologies supported by gene, intron, and intergenic spacer partitions were compared. In a second instance, the impact of indel coding as well as manual adjustments of multiple automatic DNA sequence on the reconstruction of phylogenetic tree was evaluated. Given the phylogenetic backbone retrieved in the first phase of this study, genera delimitation was evaluated. Morphological variation among genera was evaluated by selecting a representative group of members of the Gynoxyoids and searching for discontinuities. The set of characters contributing to the discontinuities and already stated diagnostic characters in prologues were evaluated with a character reconstruction analysis. A second evaluation of the potential set of morphological characters retrieved in the previous analysis was further extended to all remaining members of the Gynoxyoids. Consequently, a revision of the current genera and species circumscription is made and a checklist including all members of the clade is provided. When necessary, new circumscriptions were proposed. Finally, species delimitation was evaluated for a reduced number of species distributed exclusively in Bolivia. The Asteraceae checklist for Bolivia was the basis for the species selection. A morphological revision as described for the genera evaluation was carried out at species level. Based on the discontinuities, morpho-species were defined. Additionally, phylogenetic inferences were reconstructed on the nuclear markers ETS and ITS. The set of putative characters suitable for morpho-species was tested with a principal component analysis in order to test the clustering of morpho-species and phylogenetic clades. The results of all analyses resulted in the elaboration of a taxonomic treatment for all supported Bolivian species.
The phylogenetic results resolve the Gynoxyoid group as monophyletic. Phylogenetic trees on all three plastid genomic partitions retrieved well-supported clades. Nevertheless, incongruences in tree topologies were found among all three partitions. Moreover, significant differences were found among tree inferences before and after the manual curation of the alignments, meaning that the automatic multiple sequence alignment failed at the assessment of homology. Furthermore, the results show that a manual alignment correction is essential for phylogeny reconstruction, specially for closely related taxa. The phylogeny retrieved in this study is partially incongruent with the current generic classification, which was purely based on morphological data. The genus Aequatorium was represented by only one representative and its monophyly could not be tested, nevertheless, this unique specimen was retrieved as an independent clade. The genus Paragynoxys was retrieved as monophyletic. All members of the genus Nordenstamia were retrieved as being part of the Gynoxys clade, as well as one of the two members of the genus Paracalia. The second member of the genus Paracalia was retrieved as basal clade of the tree inference. In the second part of the study, the ancestral character reconstruction suggested a set of potential characters for genera delimitation. After testing the validity of this characters set in all members of the Gynoxyoids the delimitation and characterization of four genera was achieved. In that sense, the genus Aequatorium comprises all species with radiate white capitula, Paragynoxys includes trees with discoid white capitula distributed in Colombia and Venezuela, Paracalia characterizes by the absence of outer phyllaries and a central (Bolivia and Peru) distribution, and finally all members with yellow capitula are included in Gynoxys. The checklist resulted in a total of 158 species belonging to the four previously mentioned genera. The genus Nordenstamia was synonymized under Gynoxys and so all its members were newly synonymized in this study. Finally, although the phylogeny on the ETS and ITS markers of the Bolivian species retrieved incongruent topologies, it supported the circumscription of some species. The results of the principal component analysis supported most of the morpho-species, but vaguely the clades retrieved in the phylogenetic inference. Interestingly, both molecular and morphological analysis suggested the presence of at least one putative hybrid species. Based on these results, the taxonomic treatment for the Bolivian species of the Gynoxyoid clade resulted in 14 species belonging to Paracalia (1 sp) and Gynoxys (13 sp). This represented a reduction on the species number stated in the Asteraceae checklist for Bolivia as seven names were synonymized, and three names were excluded because of misidentifications. This investigation represents the first comprehensive study on the Gynoxyoid clade. It includes a phylogenetic backbone, character states reconstruction and a morphological analysis. The molecular datasets reveled closely relationships among species, suggesting rapid-radiating evolution, which was supported by morphological data. Furthermore, based on this analysis we were able to delimit genera and in a more reduced geographic scale, at species level. This study aims to contribute to the Flora de Bolivia which is part of the cooperation agreement between the Botanic Garden and Botanical Museum Berlin (Germany), the “Herbario Nacional de Bolivia” and the “Instituto de Ecología” de La Paz-Bolivia. That aims of this association are to collaborate in the formation of professionals in the area of botany for the subsequent application of knowledge.
