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.

Feeding Ecology of the Cuvier's Gazelle (Gazella cuvieri, Ogilby, 1841) in the Sahara Desert.

Knowledge of the feeding ecology of ungulates in arid biomes offers an interesting model for understanding the drought resistance of large desert-adapted herbivores, a crucial issue in the face of increasing desertification due to climate change. To assess the feeding ecology of the endangered Cuvier's gazelle (Gazella cuvieri) in the Sahara desert, we used a multi-method approach combining faecal samples, direct observations, and the recording of indirect signs of feeding. We hypothesised that browser behaviour is the best foraging strategy for species living in hyper-arid environments, mainly due to long periods without grazing opportunities. Complementarily, we explored the effects of the main environmental descriptors (rainfalls and NDVI) on feeding patterns and diet quality. We found that Cuvier's diets are based mainly on acacias (Vachellia tortilis, V. flava) and occasionally on the annual forb Anastatica hierochuntica. In total, eighteen species (five trees, nine shrubs, three herbs, and one grass) belonging to fifteen families were recorded. Our result confirmed the browsers' characteristic of this species, reaffirming its ability to settle in a hostile environment. Acacias stand out as key species consumed at the southernmost limit of their range; hence, future conservation plans and strategies should take this into account for the survival of Cuvier's gazelle in desert environments.

Grazing influences biomass production and protein content of alpine meadows.

Alpine grasslands are essential for carbon sequestration and food supply for domestic and wild herbivores inhabiting mountainous areas worldwide. These biomes, however, are alternatively threatened by the abandonment of agricultural and livestock practices leading to a fast-growing shrubification process while other mountain grasslands are suffering from the impacts of overgrazing. The functioning of alpine meadow ecosystems is primarily driven by climatic conditions, land-use legacies and grazing. However, although it is critically important, the role of large herbivores on the aboveground biomass and protein content of palatable plants is poorly understood for most alpine meadows. In this work, we explore the effects of grazing on grassland vegetation at two different spatial and temporal scales in the Eastern Pyrenees, Spain. Remote sensing was used to assess the effect of high and moderate grazing (HG and MG respectively) on grass biomass using the leaf area index (LAI) at the meso-scale (patches between 2.3 and 38.7 ha). We also explored the impact of null (NG), overgrazing (MO, mimicked overgrazing) and high (HG) grazing intensities at local scale setting eighteen 1 m2 exclusion boxes in six meadows (three boxes each) commonly used by domestic and wild ungulates. Historical satellite data showed that LAI values are greater in high than in low grazed areas (HG, mean = 0.66, LG, mean = 0.55). Along the same lines, high and moderate grazing pressures improved biomass production at the local-scale (HG, mean = 590.3 g/m2, MO, mean = 389.3 g/m2 and NG, mean = 110.8 g/m2). Crude protein content reached higher values under MO pressure than under HG pressure. Our results confirm that grazing intensity exerts significant changes on the above-ground biomass production and the protein content of plants consumed by domestic (cattle and horses) and wild ungulates (Southern Chamois, Rupicapra pyrenaica). We can conclude that ungulates sustain biomass and nutritive values of grass exerting a negligible effect on biomass and protein content of woody vegetation. Our results will inform management guidelines to support profitable grazing activities and promote conservation of the open landscapes in the alpine ecosystems under the current global change scenario.

Insights Into the Evolutionary History of the Subfamily Orthotrichoideae (Orthotrichaceae, Bryophyta): New and Former Supra-Specific Taxa So Far Obscured by Prevailing Homoplasy.

Mosses of the subfamily Orthotrichoideae represent one of the main components of the cryptogam epiphytic communities in temperate areas. During the last two decades, this taxonomical group has undergone an extensive revision that has led to its rearrangement at the generic level. However, their phylogenetic relationships and inferences on the evolutionary patterns that have driven the present diversity have little advanced. In this study, we present a dated molecular phylogenetic reconstruction at the subfamily level, including 130 samples that represent the 12 genera currently recognized within the subfamily, and the analysis of four molecular markers: ITS2, rps4, trnG, and trnL-F. We also analyze 13 morphological characters of systematic value to infer their origin and diagnostic utility within the subfamily. The phylogenetic reconstruction yields three main clades within the subfamily, two of which correspond to the tribe Zygodonteae, and one to Orthotricheae. Within Zygodonteae, the genus Zygodon results to be a polyphyletic artificial assembly, and we propose to separate a new genus named Australoria. Conversely, our results do not support the separation of Pentastichella and Pleurorthotrichum at the genus level and we therefore propose to include Pleurorthotrichum in Pentastichella. Regarding Orthotricheae, our analyses clearly allow the distinction of two subtribes: Orthotrichinae and Lewinskyinae. Within the latter, Ulota results a polyphyletic entity, and therefore we propose the segregation of a separate new genus named Atlantichella. Dating analyses allow us to conclude that the split of the tribes within Orthotrichoideae dates from the Middle Jurassic, while the diversification of Orthotrichum and Zygodon probably started during the Late Cretaceous. However, most of the extant genera of this subfamily seem to be younger, and apparently its highest diversification burst took place during the Oligocene. Finally, the analysis of the morphological traits reveals that most of the characters previously used to separate genera and here tested are homoplastic, which has hindered the taxonomical and systematic proposals for decades. However, even if there are no exclusive characters, all of the genera can be defined by the combination of a few characters.

A new native plant in the neighborhood: effects on plant-pollinator networks, pollination, and plant reproductive success.

Ecological communities are dynamic entities subjected to extinction/colonization events. Because species are connected through complex interaction networks, the arrival of a new species is likely to affect various species across the community, as observed in plant biological invasions. However, plant invasions usually represent extreme scenarios in which the community is strongly dominated by the alien species, confounding the effects of a change in species composition with a massive increase in floral resource availability. Our study addresses changes in plant community composition involving native species, a common phenomenon under the current climate change scenario in which plants are modifying their distribution ranges. We experimentally manipulated patches of a natural scrubland community by introducing a native plant (henceforth colonizing plant). To avoid introducing a disproportionate amount of floral resources we adjusted the number of flowers of the colonizing plant to the amount of floral resources locally available in each patch. We had two objectives: (1) to analyse the effects of the arrival of a new plant on the pollinator community, the rearrangement of plant-pollinator interactions and the structure of the plant-pollinator network; (2) to evaluate potential consequences for pollination and the reproductive success of resident plant species. The colonizing plant acted as a magnet species, attracting bumble bees and facilitating interactions to other plants through spill-over. The introduction of the colonizing plant also affected the structure of plant-pollinator networks (colonized networks were more generalized and more nested than control networks) and modified the arrangement of plant and pollinator species into modules. Ultimately, these changes resulted in higher heterospecific (but not conspecific) pollen deposition and had contrasting effects on the reproductive success of two resident plant species (higher fruit set and lower seed set, respectively). Our study shows that relationships between plants and pollinators are rapidly rearranged in response to novel situations (even when the new plant is not overly dominant), with important functional consequences on pollination and plant reproductive success. Our study establishes a link between network structure and pollination and plant reproductive success, which may be mediated by differences among pollinator species in foraging behavior.

Comparing the accuracy of PCR-capillary electrophoresis and cuticle microhistological analysis for assessing diet composition in ungulates: A case study with Pyrenean chamois.

The study of diet composition is required to understand the interactions between animal and plant ecosystems. Different non-invasive techniques applied on faecal samples have commonly been used for such purposes, with cuticle microhistological analysis (CMA) and emerging DNA-based methods being the most relevant. In this work, we refined and optimized a qualitative DNA-based approach combining PCR amplification of long trnL(UAA) and ITS2 fragments and capillary electrophoresis (PCR-CE), instead of short trnL(UAA) fragments and massive sequencing technologies commonly reported. To do so, we developed a controlled diet assay using a stabled Pyrenean chamois specimen (Rupicapra pyrenaica pyrenaica), which included representative herbaceous and shrubby plant species. We also assessed the impact of sample freshness on the diet determination of this mountain caprinae by exposing faecal samples to the outdoor environment for three weeks. Faecal samples from both experiments were analysed by qualitative PCR-CE and semi-quantitative CMA in order to compare the pros and cons of both approaches. Our results show that all of the offered plant species were detected by both methodologies although CMA over-detected shrubs compared to herbaceous species. At the same time, sample degradation due to sustained climate exposure is a limiting factor for molecular analysis, but not for CMA. Taken all together, our results suggest that the qualitative information obtained by CMA and PCR-CE can be interchangeable when faecal samples are fresh (less than one week after deposition) but, afterwards, molecular analysis underestimates diet composition probably due to DNA degradation. CMA, however, can accurately be used at least three weeks after defecation. Moreover, by combining the results of simultaneous PCR amplification of two complementary genes, this optimized PCR-CE methodology provides a reliable, feasible and more affordable alternative for multiple and routine analyses of complex samples. Neither CMA nor PCR-CE seems to solve comprehensively the quatification of herbivore diets and thus further research needs to be done.

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.

Effects of boom and bust grazing management on vegetation and health of beef cattle used for wildfire prevention in a Mediterranean forest.

Humans and wildfires have historically driven landscape structure in the Mediterranean basin. The Iberian Peninsula is not an exception to that rule, and therefore, farmers, researchers, and governments seek alternative tools to minimize the loss of biodiversity and wildfire risks. Extensive livestock including beef cattle is currently promoted as a suitable management tool by European agro-environmental policies yet pieces of evidence exist regarding the reciprocal effects between cows and Mediterranean woody vegetation. In this work, we performed a field manipulation to evaluate whether free-ranging beef cattle without supplementary feeding, at high density (2 livestock units (LU)/ha) for a short period of time i.e. "boom and bust grazing" management, are able to adapt their grazing preferences to the Mediterranean woody vegetation without health impairment, and prevent from bush encroachment and wildfires. For our purposes, a native herd of 14 adult cows was kept captive without supplementary feeding in a 14 ha enclosure covered by Mediterranean vegetation for two months (April-June 2016). Plant and cattle fecal and blood samples were collected to assess diet composition (plant cuticle microhistological analysis), fecal nitrogen and protein contents of consumed plants, and the nutritional status (non-esterified fatty acids) of cattle. Our results showed that cattle adapted their feeding habits toward a more woody diet including potentially flammable taxa but with some detrimental effects on health status. Hence, cattle cannot control woody vegetation for long periods of time without supplementary feeding. Further research should be oriented to explore other alternative approaches to minimize the health impairment of cattle used for control flammable vegetation in Mediterranean regions.

Different effects of alpine woody plant expansion on domestic and wild ungulates.

Changes in land-use and climate affect the distribution and diversity of plant and animal species at different spatiotemporal scales. The extent to which species-specific phenotypic plasticity and biotic interactions mediate organismal adaptation to changing environments, however, remains poorly understood. Woody plant expansion is threatening the extent of alpine grasslands worldwide, and evaluating and predicting its effects on herbivores is of crucial importance. Here, we explore the impact of shrubification on the feeding efficiency of Pyrenean chamois (Rupicapra p. pyrenaica), as well as on the three most abundant coexisting domestic ungulate species: cattle, sheep and horses. We use observational diet composition from May to October and model different scenarios of vegetation availability where shrubland and woodland proliferate at the expense of grassland. We then predicted if the four ungulate species could efficiently utilize their food landscapes with their current dietary specificities measuring their niche breath in each scenario. We observed that the wild counterpart, due to a higher trophic plasticity, is less disturbed by shrubification compared to livestock, which rely primarily on herbaceous plants and will be affected 3.6 times more. Our results suggest that mixed feeders, such as chamois, could benefit from fallow landscapes, and that mountain farmers are at a growing economic risk worldwide due to changing land-use practices and climate conditions.

Seasonal diet composition of Pyrenean chamois is mainly shaped by primary production waves.

In alpine habitats, the seasonally marked climatic conditions generate seasonal and spatial differences in forage availability for herbivores. Vegetation availability and quality during the growing season are known to drive life history traits of mountain ungulates. However, little effort has been made to understand the association between plant phenology and changes in the foraging strategies of these mountain dwellers. Furthermore, this link can be affected by the seasonal presence of livestock in the same meadows. The objective of this work was to study the seasonal changes in diet composition of Pyrenean chamois (Rupicapra p. pyrenaica) and its relationship to primary production trends in a Mediterranean alpine environment. Moreover, diet composition in two populations with contrasting livestock pressure was compared in order to study the effect of sheep flocks on the feeding behaviour of chamois. From 2009 to 2012, monthly diet composition was estimated by cuticle microhistological analysis of chamois faeces collected in the eastern Pyrenees. The primary production cycle was assessed by remote sensing, using the Normalized Difference Vegetation Index. Additionally, the diet of sheep sharing seasonally the subalpine and alpine meadows with chamois was analysed. Diet selection of chamois and sheep and their overlap was also assessed. Our results show an intra-annual variation in the diet composition of Pyrenean chamois and demonstrate a strong relationship between plant consumption dynamics and phenology in alpine areas. In addition, Calluna vulgaris, Cytisus spp. and Festuca spp., as well as forbs in the summer, are found to be key forage species for Pyrenean chamois. Furthermore, this study couldn't detect differences between both chamois populations despite the presence of sheep flocks in only one area. However, the detection of a shift in the diet of chamois in both areas after the arrival of high densities of multi-specific livestock suggest a general livestock effect. In conclusion, Pyrenean chamois are well adapted to the variations in the seasonal availability of plants in alpine habitats but could be disturbed by the seasonal presence of livestock. Due to the key plants in their diet, we suggest that population management programmes should focus on the preservation of mixed grasslands composed of patches of shrubs and herbs. The effects of climate change and shrub expansion should be studied as they may potentially affect chamois population dynamics through changes in habitat composition and temporal shifts in forage availability.