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.

Refugia are expected to preserve genetic variation of relict taxa, especially in polyploids, because high gene dosages could prevent genetic erosion in small isolated populations. However, other attributes linked to polyploidy, such as asexual reproduction, may strongly limit the levels of genetic variability in relict populations. Here, ploidy levels and patterns of genetic variation at nuclear microsatellite loci were analysed in Prunus lusitanica, a polyploid species with clonal reproduction that is considered a paradigmatic example of a Tertiary relict. Sampling in this study considered a total of 20 populations of three subspecies: mainland lusitanica (Iberian Peninsula and Morocco), and island azorica (Azores) and hixa (Canary Islands and Madeira). Flow cytometry results supported an octoploid genome for lusitanica and hixa, whereas a 16-ploid level was inferred for azorica. Fixed heterozygosity of a few allele variants at most microsatellite loci resulted in levels of allelic diversity much lower than those expected for a high-order polyploid. Islands as a whole did not contain higher levels of genetic variation (allelic or genotypic) than mainland refuges, but island populations displayed more private alleles and higher genotypic diversity in old volcanic areas. Patterns of microsatellite variation were compatible with the occurrence of clonal individuals in all but two island populations, and the incidence of clonality within populations negatively correlated with the estimated timing of colonization. Our results also suggest that gene flow has been very rare among populations, and thus population growth following founder events was apparently mediated by clonality rather than seed recruitment, especially in mainland areas. This study extends to clonal taxa the idea of oceanic islands as important refugia for biodiversity, since the conditions for generation and maintenance of clonal diversity (i.e. occasional events of sexual reproduction, mutation and/or seed immigration) appear to have been more frequent in these enclaves than in mainland areas.

Aim To investigate the ecophysiological traits allowing persistence of a subtropical relict tree (Prunus lusitanica L.) under a dry Mediterranean climate at the eroding edge of its range.Location A glasshouse for the study under controlled conditions and two marginal populations located in riparian forests of central Spain and exposed to summer drought, in contrast to subtropical populations that grow in mountain cloud forests.Methods Two experiments were conducted to assess tolerance to the abiotic conditions found in riparian habitats. In a glasshouse experiment, gas-exchange and light-use parameters were measured to evaluate seedling responses to a factorial combination of irradiance (60%, 10%, 2% and 0.5% full sun) and moisture (well watered control and drought stress). In a parallel field experiment, irradiance and soil moisture were measured as predictors of seedling survival at two sites in which half the seedlings were subjected to a threefold increase in summer precipitation by adding water every 10 days.Results Soil moisture strongly determined survival both in the glasshouse and in the two field sites. In the field, periodic addition of water failed to increase survival. Water-use efficiency (WUE) increased with drought. Seedlings did not tolerate deep shade (2-0.5%) and their performance and survival were better when exposed to moderate (10%) or high (60%) irradiance. The effect of water stress on seedling performance was stronger at moderate irradiance, moderate at high irradiance and negligible at very low light. Seedling size (height and number of leaves) attained after 1 month of experimental treatments had a positive effect on survival at the end of the summer, hence greater survival was not achieved at the expense of vertical growth.Main conclusions While studies in Macaronesia have shown that P. lusitanica occupies a wide range of moisture conditions along mountain slopes, it behaves as an obligate riparian species in dry peripheral populations. Intolerance to deep shade and tolerance to moderate and high irradiance allow the species to grow in small and moist gaps, or in treeless river sections. Despite the species' low range filling in marginal, drought-prone regions, long-term persistence might have been achieved through shifts to riparian habitats serving as local refuges.

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.

Understanding why host species differ so much in symbiont loads and how this depends on ecological host and symbiont traits is a major issue in the ecology of symbiosis. A first step in this inquiry is to know whether observed differences among host species are species-specific traits or more related with host-symbiont environmental conditions. Here we analysed the repeatability (R) of the intensity and the prevalence of feather mites to partition within- and among-host species variance components. We compiled the largest dataset so far available: 119 Paleartic passerine bird species, 75,944 individual birds, ca. 1.8 million mites, seven countries, 23 study years. Several analyses and approaches were made to estimate R and adjusted repeatability (Radj) after controlling for potential confounding factors (breeding period, weather, habitat, spatial autocorrelation and researcher identity). The prevalence of feather mites was moderately repeatable (R = 0.26–0.53; Radj = 0.32–0.57); smaller values were found for intensity (R = 0.19–0.30; Radj = 0.18–0.30). These moderate repeatabilities show that prevalence and intensity of feather mites differ among species, but also that the high variation within species leads to considerable overlap among bird species. Differences in the prevalence and intensity of feather mites within bird species were small among habitats, suggesting that local factors are playing a secondary role. However, effects of local climatic conditions were partially observed for intensity.