African Mountain Thistles: Three New Genera in the Carduus-Cirsium Group.

The floras on the highest mountains in tropical eastern Africa are among the most unique floras in the world. Despite the exceptionally high concentration of endemic species, these floras remain understudied from an evolutionary point of view. In this study, we focus on the Carduus-Cirsium group (subtribe Carduinae) to unravel the evolutionary relationships of the species endemic to the tropical Afromontane and Afroalpine floras, aiming to improve the systematics of the group. We applied the Hyb-Seq approach using the Compositae1061 probe set on 190 samples (159 species), encompassing representatives of all genera of Carduinae. We used two recently developed pipelines that enabled the processing of raw sequence reads, identification of paralogous sequences and segregation into orthologous alignments. After the implementation of a missing data filter, we retained sequences from 986 nuclear loci and 177 plastid regions. Phylogenomic analyses were conducted using both concatenated and summary-coalescence methods. The resulting phylogenies were highly resolved and revealed three distinct evolutionary lineages consisting of the African species traditionally referred to as Carduus and Cirsium. Consequently, we propose the three new genera Afrocarduus, Afrocirsium and Nuriaea; the latter did notably not belong to the Carduus-Cirsium group. We detected some incongruences between the phylogenies based on concatenation vs. coalescence and on nuclear vs. plastid datasets, likely attributable to incomplete lineage sorting and/or hybridization.

Impact of the climatic changes in the Pliocene-Pleistocene transition on Irano-Turanian species. The radiation of genus Jurinea (Compositae).

The Irano-Turanian region is one of the world's richest floristic regions and the centre of diversity for numerous xerophytic plant lineages. However, we still have limited knowledge on the timing of evolution and biogeographic history of its flora, and potential drivers of diversification remain underexplored. To fill this knowledge gap, we focus on the Eurasian genus Jurinea (ca. 200 species), one of the largest plant radiations that diversified in the region. We applied a macroevolutionary integrative approach to explicitly test diversification hypotheses and investigate the relative roles of geography vs. ecology and niche conservatism vs. niche lability in speciation processes. To do so, we gathered a sample comprising 77% of total genus richness and obtained data about (1) its phylogenetic history, recovering 502 nuclear loci sequences; (2) growth forms; (3) ecological niche, compiling data of 21 variables for more than 2500 occurrences; and (4) paleoclimatic conditions, to estimate climatic stability. Our results revealed that climate was a key factor in the evolutionary dynamics of Jurinea. The main diversification and biogeographic events that occurred during past climate changes, which led to colder and drier conditions, are the following: (1) the origin of the genus (10.7 Ma); (2) long-distance dispersals from the Iranian Plateau to adjacent regions (∼7-4 Ma); and (3) the diversification shift during Pliocene-Pleistocene Transition (ca. 3 Ma), when net diversification rate almost doubled. Our results supported the pre-adaptation hypothesis, i.e., the evolutionary success of Jurinea was linked to the retention of the ancestral niche adapted to aridity. Interestingly, the paleoclimatic analyses revealed that in the Iranian Plateau long-term climatic stability favoured old-lineage persistence, resulting in current high species richness of semi-arid and cold adapted clades; whereas moderate climate oscillations stimulated allopatric diversification in the lineages distributed in the Circumboreal region. In contrast, growth form lability and high niche disparity among closely related species in the Central Asian clade suggest adaptive radiation to mountain habitats. In sum, the radiation of Jurinea is the result of both adaptive and non-adaptive processes influenced by climatic, orogenic and ecological factors.

Phylogenomic and population genomic analyses reveal the spatial-temporal dynamics of diversification of the Nigella arvensis complex (Ranunculaceae) in the Aegean archipelago.

The continental-shelf islands of the Aegean Sea provide an ideal geographical setting for evolutionary-biogeographical studies but disentangling the relationships between palaeogeographical history and the times, orders of modes of taxon divergence is not straightforward. Here, we used phylogenomic and population genomic approaches, based on orthologous gene sequences and transcriptome-derived SNP data, to reconstruct the spatial-temporal evolution of the Aegean Nigella arvensis complex (Ranunculaceae; 11 out of 12 taxa). The group's early diversification in the Early/Mid-Pliocene (c. 3.77 Mya) resulted in three main lineages (Greek mainland vs. central Aegean + Turkish mainland/eastern Aegean islands), while all extant taxa are of Late Plio-/Early Pleistocene origin (c. 3.30-1.59 Mya). Demographic modelling of the outcrossing taxa uncovered disparate modes of (sub)speciation, including divergence with gene flow on the Greek mainland, para- or peripatric diversification across eastern Aegean islands, and a 'mixing-isolation-mixing (MIM)' mode of subspeciation in the Cyclades. The two selfing species (N. stricta, N. doerfleri) evolved independently from the outcrossers. Present-day island configurations are clearly insufficient to explain the spatial-temporal history of lineage diversification and modes of (sub)speciation in Aegean Nigella. Moreover, our identification of positively selected genes in almost all taxa calls into question that this plant group represents a case of 'non-adaptive' radiation. Our study revealed an episodic diversification history of the N. arvensis complex, giving new insight into the modes and drivers of island speciation and adaption across multiple spatiotemporal scales.

Genetic diversity and structure of the narrow endemic Seseli farrenyi (Apiaceae): implications for translocation.

Seseli farrenyi (Apiaceae) is an extremely narrow endemic plant, which is considered as one of the species of most conservation concern in Catalonia (NW Mediterranean Basin). Given the accelerated fragmentation and reduction of population size (of over 90%), the environmental agency of Catalonia is currently preparing a recovery plan that includes reinforcements of the extant populations. The present study is aimed at providing the necessary knowledge to carry out genetically-informed translocations, by using microsatellites as genetic markers. Fourteen microsatellites have been specifically developed for S. farrenyi, of which nine have been used. Besides the extant natural populations, the three ex situ collections that are known to exist of this species have also been studied, as they would be the donor sources for translocation activities. Our main finding is that levels of genetic diversity in the natural populations of S. farrenyi are still high (H e = 0.605), most likely as a result of a predominantly outcrossing mating system in combination with the limited time elapsed since the population decline. However, population fragmentation is showing the first genetic signs, as the values of genetic differentiation are relatively high, and two well-differentiated genetic lineages have been found even in such a narrow geographic range. These genetic results provide important information when designing conservation management measures.

Nuclear and plastid DNA phylogeny of tribe Cardueae (Compositae) with Hyb-Seq data: A new subtribal classification and a temporal diversification framework.

Classification of 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 subtribes and informal groups defined in the tribe, targeting 1061 nuclear conserved orthology loci (COS) designed for Compositae and obtaining chloroplast coding regions as by-product of off-target reads. For the extraction of the 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 three datasets, phylogenetic relationships were reconstructed using both concatenation and coalescent approaches. Phylogenetic analyses of the nuclear datasets fully resolved virtually all nodes with very high support. Nuclear and plastid tree topologies are mostly congruent with a very limited number of incongruent nodes. Based on the well-solved 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. In addition, we further updated the temporal framework for origin and diversification of these subtribes. Our results highlight the power of Hyb-Seq over Sanger sequencing of a few DNA markers in solving phylogenetic relationships of traditionally difficult groups.

Influence of the Quaternary Glacial Cycles and the Mountains on the Reticulations in the Subsection Willkommia of the Genus Centaurea.

Late Neogene and Quaternary climatic oscillations have greatly shaped the genetic structure of the Mediterranean Basin flora, with mountain plant species tracking warm interglacials/cold glacials by means of altitudinal shifts instead of broad latitudinal ones. Such dynamics may have enhanced population divergence but also secondary contacts. In this paper, we use a case example of subsection Willkommia of Centaurea (comprising three narrowly distributed endemic species, Centaurea gadorensis, C. pulvinata, and C. sagredoi) to test for reticulate evolution and recurrent hybridizations between nearby populations. For this, we combine analyses of genetic diversity and structuring, gene flow and spatial correlation, and ecological niche modeling. Our results support the contention that the current genetic structure of the three species is the result of historical gene flow at sites of secondary contact during the glacial periods, followed by isolation after the retraction of populations to the middle-upper areas of the mountains during the interglacial periods. The extent and direction of the gene flow was determined largely by the location of the populations on mountainsides oriented toward the same valley or toward different valleys, suggesting the intermountain valleys as the areas where secondary contacts occurred.

Reinterpretation of an endangered taxon based on integrative taxonomy: The case of Cynara baetica (Compositae).

The Strait of Gibraltar, the gateway between the Atlantic Ocean and the Mediterranean Sea, has a convulsive geological history, with recurring closing and opening events since the late Miocene. As a consequence, this region has played a major role in the evolutionary history of many species. Cynara baetica (Compositae) is a diploid perennial herb distributed in both sides of this strait. It is currently subdivided into two subspecies: C. baetica subsp. baetica for the Spanish populations, and C. baetica subsp. maroccana for the Moroccan ones. Following three different approximations of species delimitation, including phylogenetic and population genetic analyses (based on three AFLP primer combinations and two intergenic spacers of cpDNA), ecological niche modeling (ENM) and morphological studies, this taxon is investigated and reinterpreted. The results obtained showed a clear genetic, morphological and ecological differentiation between the two taxa and the important role played by the Strait of Gibraltar as a geographical barrier. Based on this evidence, the current taxonomic treatment is modified (both taxa should recover their specific rank) and specific conservation guidelines are proposed for the newly delimited taxa.

Population genetic dynamics of Himalayan-Hengduan tree peonies, Paeonia subsect. Delavayanae.

According to the present taxonomical treatment, Paeonia subsect. Delavayanae consists of only two species (P. delavayi and P. ludlowii) endemic to the Himalayan-Hengduan Mountains. Although P. ludlowii can be distinguished from P. delavayi on the basis of a series of morphological characters, the species delimitation remains controversial because the more widespread one, P. delavayi, exhibits considerable morphological diversity. Both chloroplast DNA markers and nuclear microsatellites or simple sequence repeats (nSSR) are used herein to reveal genetic diversity and relationships of the two taxa included in this subsection, and ecological niche modeling (ENM) is employed to get insights into their paleodistribution. Our results show that genetic boundaries between the two currently recognized species are unclear, probably due to recent divergence. Paeonia ludlowii is budding from P. delavayi, probably by genetic isolation but also by shifting its niche to the harsher upland Tibetan conditions. Paeonia delavayi itself would be, however, under active speciation, showing significant genetic differentiation and morphological diversity. Whereas P. ludlowii would have endured the Pleistocene glacial periods by in situ persistence in local, small refugia, a 'dual' model seems to apply for P. delavayi (in situ persistence and retreat to refugia). The rarity of P. ludlowii and high evolutionary potential of P. delavayi imply high priority for in situ conservation of both taxa. The Himalayan-Hengduan Mountains are an ideal arena for differentiation within subsect. Delavayanae of Paeonia, by means of expansions/contractions/displacements, vertical migrations, and local survival/extinctions in response to the Neogene climate fluctuations and geological changes.

Natural epigenetic variation within and among six subspecies of the house sparrow, Passer domesticus.

Epigenetic modifications can respond rapidly to environmental changes and can shape phenotypic variation in accordance with environmental stimuli. One of the most studied epigenetic marks is DNA methylation. In the present study, we used the methylation-sensitive amplified polymorphism (MSAP) technique to investigate the natural variation in DNA methylation within and among subspecies of the house sparrow, Passer domesticus We focused on five subspecies from the Middle East because they show great variation in many ecological traits and because this region is the probable origin for the house sparrow's commensal relationship with humans. We analysed house sparrows from Spain as an outgroup. The level of variation in DNA methylation was similar among the five house sparrow subspecies from the Middle East despite high phenotypic and environmental variation, but the non-commensal subspecies was differentiated from the other four (commensal) Middle Eastern subspecies. Further, the European subspecies was differentiated from all other subspecies in DNA methylation. Our results indicate that variation in DNA methylation does not strictly follow subspecies designations. We detected a correlation between methylation level and some morphological traits, such as standardized bill length, and we suggest that part of the high morphological variation in the native populations of the house sparrow is influenced by differentially methylated regions in specific loci throughout the genome. We also detected 10 differentially methylated loci among subspecies and three loci that differentiated between commensal or non-commensal status. Therefore, the MSAP technique detected larger scale differences among the European and non-commensal subspecies, but did not detect finer scale differences among the other Middle Eastern subspecies.

Phylogeny of the Centaurea group (Centaurea, Compositae) - geography is a better predictor than morphology.

The Centaurea group is part of the Circum-Mediterranean Clade (CMC) of genus Centaurea subgenus Centaurea, a mainly Mediterranean plant group with more than 200 described species. The group is traditionally split on morphological basis into three sections: Centaurea, Phalolepis and Willkommia. This division, however, is doubtful, especially in light of molecular approaches. In this study we try to resolve this phylogenetic problem and to consolidate the circumscription and delimitation of the entire group against other closely related groups. We analyzed nuclear (internal transcribed spacer of the ribosomal genes) and chloroplast (rpl32-trnL intergenic spacer) DNA regions for most of the described species of the Centaurea group using phylogenetic and network approaches, and we checked the data for recombination. Phylogeny was used to reconstruct the evolution of the lacerate-membranaceous bract appendages using parsimony. The magnitude of incomplete lineage sorting was tested estimating the effective population sizes. Molecular dating was performed using a Bayesian approach, and the ancestral area reconstruction was conducted using the Dispersal-Extinction-Cladogenesis method. Monophyly of the Centaurea group is confirmed if a few species are removed. Our results do not support the traditional sectional division. There is a high incongruence between the two markers and between genetic data and morphology. However, there is a clear relation between geography and the structure of the molecular data. Diversification in the Centaurea group mainly took place during the Pliocene and Pleistocene. The ancestral area infered for the Circum-Mediterranean Clade of Centaurea is the Eastern Mediterranean, whereas for the Centaurea group it is most likely NW-Africa. The large incongruencies, which hamper phylogenetic reconstruction, are probably the result of introgression, even though the presence of incomplete lineage sorting as an additional factor cannot be ruled out. Convergent evolution of morphological traits may have led to incongruence between morphology-based, traditional systematics and molecular results. Our results also cast major doubts about current species delimitation.

Miocene-Pliocene speciation, introgression, and migration of Patis and Ptilagrostis (Poaceae: Stipeae).

Genetic interchange between American and Eurasian species is fundamental to our understanding of the biogeographical patterns, and we make a first attempt to reconstruct the evolutionary events in East Asia that lead to the origin and dispersal of two genera, Patis and Ptilagrostis. We conducted a molecular phylogenetic study of 78 species in the tribe Stipeae using four plastid DNA sequences (ndhF, rpl32-trnL, rps16-trnK, and rps16 intron) and two nuclear DNA sequences (ITS and At103). We use single copy nDNA gene At103 for the first time in the grasses to elucidate the evolutionary history among members of the Stipeae. Ampelodesmos, Hesperostipa, Oryzopsis, Pappostipa, Patis, and Stipa are found to be of multiple origins. Our phylograms reveal conflicting positions for Ptilagrostis alpina and Pt. porteri that form a clade with Patis coreana, P. obtusa, and P. racemosa in the combined plastid tree but are aligned with other members of Ptilagrostis in the ITS tree. We hypothesize that Ptilagrostis still retains the nucleotype of an extinct genus which transited the Bering land bridge from American origins in the late Miocene (minimum 7.35-6.37 mya) followed by hybridization and two plastid capture events with a Trikeraia-like taxon (7.96 mya) and para-Patis (between 5.32 and 3.76 mya). Ptilagrostis porteri and Patis racemosa then migrated to continental North America 1.7-2.9 mya and 4.3-5.3 mya, respectively.

Reduced seed predation after invasion supports enemy release in a broad biogeographical survey.

The Enemy Release (ER) hypothesis predicts an increase in the plant invasive capacity after being released from their associated herbivores or pathogens in their area of origin. Despite the large number of studies on biological invasions addressing this hypothesis, tests evaluating changes in herbivory on native and introduced populations and their effects on plant reproductive potential at a biogeographical level are relatively rare. Here, we tested the ER hypothesis on the South African species Senecio pterophorus (Asteraceae), which is native to the Eastern Cape, has expanded into the Western Cape, and was introduced into Australia (>70-100 years ago) and Europe (>30 years ago). Insect seed predation was evaluated to determine whether plants in the introduced areas were released from herbivores compared to plants from the native range. In South Africa, 25 % of the seedheads of sampled plants were damaged. Plants from the introduced populations suffered lower seed predation compared to those from the native populations, as expected under the ER hypothesis, and this release was more pronounced in the region with the most recent introduction (Europe 0.2 % vs. Australia 15 %). The insect communities feeding on S. pterophorus in Australia and Europe differed from those found in South Africa, suggesting that the plants were released from their associated fauna after invasion and later established new associations with local herbivore communities in the novel habitats. Our study is the first to provide strong evidence of enemy release in a biogeographical survey across the entire known distribution of a species.

Reconstructing the evolution and biogeographic history of tribe Cardueae (Compositae).

PREMISE OF THE STUDY: Tribe Cardueae (thistles) forms one of the largest tribes in the family Compositae (2400 species), with representatives in almost every continent. The greatest species richness of Cardueae occurs in the Mediterranean region where it forms an important element of its flora. New fossil evidence and a nearly resolved phylogeny of Cardueae are used here to reconstruct the spatiotemporal evolution of this group. • METHODS: We performed maximum parsimony and Bayesian phylogenetic inference based on nuclear ribosomal DNA and chloroplast DNA markers. Divergence times and ancestral area reconstructions for main lineages were estimated using penalized likelihood and dispersal-vicariance analyses, respectively, and integrated over the posterior distribution of the phylogeny from the Bayesian Markov chain Monte Carlo analysis to accommodate uncertainty in phylogenetic relationships. • KEY RESULTS: The phylogeny shows that subtribe Cardopatiinae is sister to the remaining subtribes, and subtribes Carlininae and Echinopsinae appear as consecutive sister-clades to the Carduinae/Centaureinae. Tribe Cardueae is inferred to have originated around the Mid Eocene in West Asia, which is also the ancestral area of most subtribes within Cardueae. Diversification within each subtribe began during the Oligocene-Miocene period. • CONCLUSIONS: Most diversification events within Cardueae are related to the continuous cycles of area connection and division between the Anatolian microplate and the western Mediterranean Basin during the Oligocene-Miocene and with the uplift of the Himalayan range from the Miocene onward. From these two regions, thistles dispersed and colonized the rest of the continents (e.g., the New World, Africa, and Australia), most likely during the colder Pliocene-Pleistocene period.

The polyploid series of Centaurea toletana: glacial migrations and introgression revealed by nrDNA and cpDNA sequence analyzes.

The polyploid series of Centaurea toletana comprises diploid, tetraploid, and hexaploid cytotypes. Previous studies suggested that the tetraploid was an autopolyploid, while the hexaploid was an allopolyploid and should be considered a different species, C. argecillensis. Sequencing of the ITS and rps4-trnT-trnL, ycf3-trnS, and rpL16 regions, and extensive cloning and sequencing of the ETS region have revealed that many diploid individuals and populations show different ribotypes, likely resulting from ancient hybridization events. Ribotypes found in the diploid populations are also present in tetraploid populations. The extreme difficulties in classifying the tetraploid as auto- or allopolyploid are discussed. The hexaploid C. argecillensis also shows many different ribotypes, including a ribotype not found in the diploids and making an autopolyploid origin unlikely. The pattern of introgression and gene flow implicates several species from the Iberian Peninsula and the High Atlas Mountains in Morocco as genetic donors in ancient hybridization events. This long-reaching network of hybridization may trace its origin to the climatic history of the western Mediterranean during the Neogene.

Evolution and biogeography of Centaurea section Acrocentron inferred from nuclear and plastid DNA sequence analyses.

BACKGROUND AND AIMS: Section Acrocentron of the genus Centaurea is one of the largest sections of Centaurea with approx. 100 species. The geographic distribution, centred in the Mediterranean, makes it an excellent example for studies of the biogeographic history of this biodiversity-rich region. METHODS: Plastid (trnH-psbA) and nuclear (ITS and ETS) DNA sequence analysis was used for phylogenetic reconstruction. Ancestral biogeographic patterns were inferred by dispersal-vicariance analysis (DIVA). KEY RESULTS: The resulting phylogeny has implications for the sectional classification of Acrocentron and confirms merging sect. Chamaecyanus into Acrocentron as a subsection. Previous suggestions of an eastern Mediterranean origin of the group are confirmed. The main centres of diversification established in previous studies are now strongly supported. Expansion of the group in two different radiations that followed patently diverse paths is inferred. CONCLUSIONS: Radiation followed two waves, widely separated in time scale. The oldest one, from Turkey to Greece and the northern Balkans and then to North Africa and Iberia, should be dated at the end of the Miocene in the Messinian period. It reached the Iberian Peninsula from the south, following a route that is landmarked by several relictic taxa in Sicily and North Africa. A later radiation during the Holocene interglacial periods followed, involving species from the north of the Balkan Peninsula, along a Eurasian pathway running from Central Iberia to the steppes of Kazakhstan. A generalized pattern of reticulation is also evident from the results, indicating past contacts between presently separated species. Molecular data also confirmed the extent of hybridization within Acrocentron and were successful in reconstructing the paleogeography of the section.

Molecular evidence for hybrid origins of the invasive polyploids Carthamus creticus and C. turkestanicus (Cardueae, Asteraceae).

The hexaploids Carthamus creticus and C. turkestanicus are noxious weeds with wide but non-overlapping Mediterranean distributions, and C. creticus, together with another polyploid, C. lanatus, have also invaded similar climatic regions in North and South America, South Africa and Australia. Here we infer their origins using sequences of the plastid intergenic spacer trnH-psbA and the intron trnK and three introns of nuclear low-copy genes of the RNA Polymerase family (RPD2 and the duplicated RPC2), as well as RAPD markers (random amplified polymorphic DNA). Phylogenetic analyses of the nuclear introns and additivity analysis of the RAPD markers support the hypotheses that the two hexaploids are allopolyploids sharing a tetraploid progenitor lineage represented by the broadly Mediterranean C. lanatus, combined with different diploid progenitor lineages consistent with the different geographic distributions of the hexaploids. Whereas C. leucocaulos from the south-eastern Greek Islands represents the diploid progenitor lineage of the western C. creticus, the Irano-Turanian C. glaucus represents the diploid progenitor lineage of the eastern C. turkestanicus. The plastid data suggest that the diploid lineages served as the maternal progenitors of the hexaploids.

Genome size variation in the genus Carthamus (Asteraceae, Cardueae): systematic implications and additive changes during allopolyploidization.

BACKGROUND AND AIMS: Plant genome size is an important biological characteristic, with relationships to systematics, ecology and distribution. Currently, there is no information regarding nuclear DNA content for any Carthamus species. In addition to improving the knowledge base, this research focuses on interspecific variation and its implications for the infrageneric classification of this genus. Genome size variation in the process of allopolyploid formation is also addressed. METHODS: Nuclear DNA samples from 34 populations of 16 species of the genus Carthamus were assessed by flow cytometry using propidium iodide. KEY RESULTS: The 2C values ranged from 2.26 pg for C. leucocaulos to 7.46 pg for C. turkestanicus, and monoploid genome size (1Cx-value) ranged from 1.13 pg in C. leucocaulos to 1.53 pg in C. alexandrinus. Mean genome sizes differed significantly, based on sectional classification. Both allopolyploid species (C. creticus and C. turkestanicus) exhibited nuclear DNA contents in accordance with the sum of the putative parental C-values (in one case with a slight reduction, frequent in polyploids), supporting their hybrid origin. CONCLUSIONS: Genome size represents a useful tool in elucidating systematic relationships between closely related species. A considerable reduction in monoploid genome size, possibly due to the hybrid formation, is also reported within these taxa.

Tribal and subtribal delimitation and phylogeny of the Cardueae (Asteraceae): a combined nuclear and chloroplast DNA analysis.

Tribal delimitation of Cardueae is controversial, and the traditional classification in four subtribes (Echinopsidinae, Carlininae, Carduinae, and Centaureinae) has fluctuated widely. Most of the problems are centered in subtribes Echinopsidinae and Carlininae, often segregated with tribal rank. We therefore analyzed DNA sequences of the internal transcribed spaces (ITS) of the nuclear ribosomal DNA genes and the matK gene of the chloroplast DNA of a broad representation of the tribe to examine (1) the phylogeny of the tribe, (2) the position of Echinopsidinae and Carlininae, (3) the circumscription of the subtribes and the position of some conflicting genera, and (4) the delimitation of some generic complexes in the Carduinae. Phylogenetic analysis of ITS and matK sequence variation, both separate and combined, strongly support the monophyly of Cardueae including Carlininae and Echinopsidinae. The combination of both genomes suggest that Xeranthemum and its allies should be included among the Echinopsidinae rather than the Carlininae, which implies that the capitulum of Xeranthemum could be interpreted as a syncephaly. The subtribe Centaureinae forms a well-supported clade, and their sister clades contain the genera Arctium, Cousinia, Jurinea, and Saussurea from the Carduinae. However, some problems persist: Carduinae are a paraphyletic assemblage, and the subtribal placement of Berardia, Cardopatium, Cousiniopsis, and Staehelina remains unresolved. Our results also indicate that present classification in four subtribes is unsatisfactory, but it is still the only practical approach.