Classification of the tribe Cardueae in natural subtribes has always been a challenge due to the lack of support of some critical branches in previous phylogenies based on traditional Sanger markers. With the aim to propose a new subtribal delimitation, we applied a Hyb-Seq approach to a set of 76 Cardueae species representing all the subtribes and informal groups defined in the tribe, targeting 1061 nuclear conserved orthology loci (COS) designed for Compositae and obtaining chloroplast coding regions as byproduct of off-target reads. For the extraction of target nuclear data, we used two strategies, PHYLUCE and HybPiper, and 776 and 1055 COS loci were recovered with each of them, respectively. Additionally, 87 chloroplast genes were assembled and annotated. With the three datasets, phylogenetic relationships within the tribe were reconstructed under approaches of concatenation (using supermatrices as input for maximum likelihood analysis with RAxML) and coalescence (species tree estimated with ASTRAL based on the individual gene trees of each COS locus). The phylogenetic analyses of the nuclear datasets fully resolved virtually all nodes with very high support. Although nuclear and plastid tree topologies are highly congruent, they still present some incongruences, which are shortly discussed. On the basis of the phylogenies obtained, we propose a new taxonomic scheme of 12 monophyletic and morphologically consistent subtribes: Carlininae, Cardopatiinae, Echinopsinae, Dipterocominae (new), Xerantheminae (new), Berardiinae (new), Staehelininae (new), Onopordinae (new), Carduinae (redelimited), Arctiinae (new), Saussureinae (new), and Centaureinae. Another main key result of the study was the high resolution recovered at the backbone of the Cardueae tree, which led to obtain better inter-subtribal relationships. Using as tree base the nuclear HybPiper phylogeny, we updated the temporal framework for the origin and diversification of the tribe and subtribes. Overall, the power of Hyb-Seq is demonstrated to solve relationships traditionally suggested by morphology but never recovered with support using Sanger sequencing of a few DNA markers.

Target enrichment is a cost-effective sequencing technique that holds promise for elucidating evolutionary relationships in fast-evolving lineages. However, potential biases and impact of bioinformatic sequence treatments in phylogenetic inference have not been thoroughly explored yet. Here, we investigate this issue with an ultimate goal to shed light into a highly diversified group of Compositae (Asteraceae) constituted by four main genera: Arctium, Cousinia, Saussurea, and Jurinea. Specifically, we compared sequence data extraction methods implemented in two easy-to-use workflows, PHYLUCE and HybPiper, and assessed the impact of two filtering practices intended to reduce phylogenetic noise. In addition, we compared two phylogenetic inference methods: (1) the concatenation approach, in which all loci were concatenated in a supermatrix; and (2) the coalescence approach, in which gene trees were produced independently and then used to construct a species tree under coalescence assumptions. Here we confirm the usefulness of the set of 1061 COS targets (a nuclear conserved orthology loci set developed for the Compositae) across a variety of taxonomic levels. Intergeneric relationships were completely resolved: there are two sister groups, Arctium-Cousinia and Saussurea-Jurinea, which are in agreement with a morphological hypothesis. Intrageneric relationships among species of Arctium, Cousinia, and Saussurea are also well defined. Conversely, conflicting species relationships remain for Jurinea. Methodological choices significantly affected phylogenies in terms of topology, branch length, and support. Across all analyses, the phylogeny obtained using HybPiper and the strictest scheme of removing fast-evolving sites was estimated as the optimal. Regarding methodological choices, we conclude that: (1) trees obtained under the coalescence approach are topologically more congruent between them than those inferred using the concatenation approach; (2) refining treatments only improved support values under the concatenation approach; and (3) branch support values are maximized when fast-evolving sites are removed in the concatenation approach, and when a higher number of loci is analyzed in the coalescence approach.

Molecular approaches have greatly increased the number of confirmed homoploid hybrids, which suggests that the frequency of this phenomenon was underestimated in the past because it was much more difficult to detect than allopolyploidy. Centaurea is a suitable model group for studying homoploid speciation, as hybridization events have been commonly reported for this genus. Based on this, here we study Centaurea × forsythiana, a naturally occurring homoploid hybrid between two Sardinian endemics, C. horrida and C. filiformis, using a molecular approach involving nuclear and plastid markers, to understand the underlying population dynamics between homoploid hybrids and their parents. Our results confirm that C. × forsythiana is a hybrid between the abovementioned species and define the roles of the parents. Plastid markers point towards C. horrida as the maternal progenitor, and nuclear markers reveal that the other parental species, C. filiformis, is itself an old, stabilized homoploid hybrid related to the C. paniculata complex from the Italian mainland. Homoploid hybrid speciation is discussed and C. × forsythiana and C. filiformis are compared with other similar examples. The study confirms the importance of introgression between parental species mediated by hybrids and its potential implications in conservation. Furthermore, it shows how hybridization studies become even more complex when the parents are themselves of probable hybrid origin.

In this paper we seek to elucidate the phylogeny of Centaurea sect. Jacea and sect. Phrygia (= sect. Lepteranthus nom. inval.) which have long been a source of controversy. A molecular phylogenetic approach is used based on nrDNA and plastid markers. The study confirmed incongruence between datasets, which can be explained both by hybridization and the occurrence of shared ancestral polymorphisms. Both factors are critical in limiting the resolution of phylogenetic trees. Despite this, we provide an interpretation of the current European distribution of sect. Phrygia, and suggest a probable eastern center of origin for the section. Our results support previous studies in Centaurea, suggesting on molecular grounds that sect. Phrygia and sect. Jacea can not be clearly separated, particularly when C. nigra s.l. or C. jacea s.l. coexist with other taxa of sect. Phrygia and sect. Jacea.