The role of pollinators in the evolution of corolla shape variation, disparity and integration in a highly diversified plant family with a conserved floral bauplan.

BACKGROUND AND AIMS: Brassicaceae is one of the most diversified families in the angiosperms. However, most species from this family exhibit a very similar floral bauplan. In this study, we explore the Brassicaceae floral morphospace, examining how corolla shape variation (an estimation of developmental robustness), integration and disparity vary among phylogenetically related species. Our aim is to check whether these floral attributes have evolved in this family despite its apparent morphological conservation, and to test the role of pollinators in driving this evolution. METHODS: Using geometric morphometric tools, we calculated the phenotypic variation, disparity and integration of the corolla shape of 111 Brassicaceae taxa. We subsequently inferred the phylogenetic relationships of these taxa and explored the evolutionary lability of corolla shape. Finally, we sampled the pollinator assemblages of every taxon included in this study, and determined their pollination niches using a modularity algorithm. We explore the relationship between pollination niche and the attributes of corolla shape. KEY RESULTS: Phylogenetic signal was weak for all corolla shape attributes. All taxa had generalized pollination systems. Nevertheless, they belong to different pollination niches. There were significant differences in corolla shape among pollination niches even after controlling for the phylogenetic relationship of the plant taxa. Corolla shape variation and disparity was significantly higher in those taxa visited mostly by nocturnal moths, indicating that this pollination niche is associated with a lack of developmental robustness. Corolla integration was higher in those taxa visited mostly by hovering long-tongued flies and long-tongued large bees. CONCLUSIONS: Corolla variation, integration and disparity were evolutionarily labile and evolved very recently in the evolutionary history of the Brassicaceae. These floral attributes were strongly related to the pollination niche. Even in a plant clade having a very generalized pollination system and exhibiting a conserved floral bauplan, pollinators can drive the evolution of important developmental attributes of corolla shape.

Evolution of pollination niches in a generalist plant clade.

It is widely assumed that floral diversification occurs by adaptive shifts between pollination niches. In contrast to specialized flowers, identifying pollination niches of generalist flowers is a challenge. Consequently, how generalist pollination niches evolve is largely unknown. We apply tools from network theory and comparative methods to investigate the evolution of pollination niches among generalist species belonging to the genus Erysimum. These species have similar flowers. We found that the studied species may be grouped in several multidimensional niches separated not by a shift of pollinators, but instead by quantitative variation in the relative abundance of pollinator functional groups. These pollination niches did not vary in generalization degree; we did not find any evolutionary trend toward specialization within the studied clade. Furthermore, the evolution of pollination niche fitted to a Brownian motion model without phylogenetic signal, and was characterized by frequent events of niche convergences and divergences. We presume that the evolution of Erysimum pollination niches has occurred mostly by recurrent shifts between slightly different generalized pollinator assemblages varying spatially as a mosaic and without any change in specialization degree. Most changes in pollination niches do not prompt floral divergence, a reason why adaptation to pollinators is uncommon in generalist plants.

Post-fire salvage logging alters species composition and reduces cover, richness, and diversity in Mediterranean plant communities.

An intense debate exists on the effects of post-fire salvage logging on plant community regeneration, but scant data are available derived from experimental studies. We analyzed the effects of salvage logging on plant community regeneration in terms of species richness, diversity, cover, and composition by experimentally managing a burnt forest on a Mediterranean mountain (Sierra Nevada, S Spain). In each of three plots located at different elevations, three replicates of three treatments were implemented seven months after the fire, differing in the degree of intervention: "Non-Intervention" (all trees left standing), "Partial Cut plus Lopping" (felling 90% of the trees, cutting the main branches, and leaving all the biomass in situ), and "Salvage Logging" (felling and piling the logs, and masticating the woody debris). Plant composition in each treatment was monitored two years after the fire in linear point transects. Post-fire salvage logging was associated with reduced species richness, Shannon diversity, and total plant cover. Moreover, salvaged sites hosted different species assemblages and 25% lower cover of seeder species (but equal cover of resprouters) compared to the other treatments. Cover of trees and shrubs was also lowest in Salvage Logging, which could suggest a potential slow-down of forest regeneration. Most of these results were consistent among the three plots despite plots hosting different plant communities. Concluding, our study suggests that salvage logging may reduce species richness and diversity, as well as the recruitment of woody species, which could delay the natural regeneration of the ecosystem.

Tracking Phenological Changes over 183 Years in Endemic Species of a Mediterranean Mountain (Sierra Nevada, SE Spain) Using Herbarium Specimens.

Phenological studies have a crucial role in the global change context. The Mediterranean basin constitutes a key study site since strong climate change impacts are expected, particularly in mountain areas such as Sierra Nevada, where we focus. Specifically, we delve into phenological changes in endemic vascular plants over time by analysing data at three scales: entire massif, altitudinal ranges, and particular species, seeking to contribute to stopping biodiversity loss. For this, we analysed 5262 samples of 2129 herbarium sheets from Sierra Nevada, dated from 1837 to 2019, including reproductive structure, complete collection date, and precise location. We found a generalized advancement in phenology at all scales, and particularly in flowering onset and flowering peak. Thus, plants flower on average 11 days earlier now than before the 1970s. Although similar trends have been confirmed for many territories and species, we address plants that have been studied little in the past regarding biotypes and distribution, and which are relevant for conservation. Thus, we analysed phenological changes in endemic plants, mostly threatened, from a crucial hotspot within the Mediterranean hotspot, which is particularly vulnerable to global warming. Our results highlight the urgency of phenological studies by species and of including ecological interactions and effects on their life cycles.

Soil filtration-sedimentation improves shelled protist recovery in eukaryotic eDNA surveys.

A large part of the soil protist diversity is missed in metabarcoding studies based on 0.25 g of soil environmental DNA (eDNA) and universal primers due to ca. 80% co-amplification of non-target plants, animals and fungi. To overcome this problem, enrichment of the substrate used for eDNA extraction is an easily implemented option but its effect has not yet been tested. In this study, we evaluated the effect of a 150 µm mesh size filtration and sedimentation method to improve the recovery of protist eDNA, while reducing the co-extraction of plant, animal and fungal eDNA, using a set of contrasted forest and alpine soils from La Réunion, Japan, Spain and Switzerland. Total eukaryotic diversity was estimated by V4 18S rRNA metabarcoding and classical amplicon sequence variant calling. A 2- to 3-fold enrichment in shelled protists (Euglyphida, Arcellinida and Chrysophyceae) was observed at the sample level with the proposed method, with, at the same time, a 2-fold depletion of Fungi and a 3-fold depletion of Embryophyceae. Protist alpha diversity was slightly lower in filtered samples due to reduced coverage in Variosea and Sarcomonadea, but significant differences were observed in only one region. Beta diversity varied mostly between regions and habitats, which explained the same proportion of variance in bulk soil and filtered samples. The increased resolution in soil protist diversity estimates provided by the filtration-sedimentation method is a strong argument in favour of including it in the standard protocol for soil protist eDNA metabarcoding studies.

Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mtDNA. Here, we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from 2 patients with MELAS who harbored the A3243G mutation. Both mitochondrial respiratory chain enzyme activities and coenzyme Q(10) (CoQ) levels were significantly decreased in MELAS fibroblasts. A similar decrease in mitochondrial membrane potential was found in intact MELAS fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and the activation of mitochondrial permeability transition (MPT), which triggered the degradation of impaired mitochondria. Furthermore, we found defective autophagosome elimination in MELAS fibroblasts. Electron and fluorescence microscopy studies confirmed a massive degradation of mitochondria and accumulation of autophagosomes, suggesting mitophagy activation and deficient autophagic flux. Transmitochondrial cybrids harboring the A3243G mutation also showed CoQ deficiency and increased autophagy activity. All these abnormalities were partially restored by CoQ supplementation. Autophagy in MELAS fibroblasts was also abolished by treatment with antioxidants or cyclosporine, suggesting that both reactive oxygen species and MPT participate in this process. Furthermore, prevention of autophagy in MELAS fibroblasts resulted in apoptotic cell death, suggesting a protective role of autophagy in MELAS fibroblasts.

Trade-Off between Facilitation and Interference of Allelopathic Compounds in Vegetation Recovery: The Case of Rosmarinus officinalis in Degraded Gypsum Habitats.

Rosmarinus officinalis advantageously competes with other species in restored gypsum outcrops, and further research is needed to understand the causes. Specifically, we focus on the potential allelopathic effects derived from its terpenes on the emergence of gypsum species. To this end, we established 120 circular subplots in a previously restored gypsum outcrop, and randomly applied four different treatments based on the presence/absence of rosemary plants and their leaves on the soil. Afterwards, we conducted an experimental sowing of native gypsophiles. All subplots were monitored to estimate seedling emergence, and soil and leaf samples were analysed for terpenes. The results show that the treatments had significant effects on the overall emergence of seedlings, and terpenes were found in rosemary leaves and soils, with no significant differences in terpene composition. In particular, we identified a clear negative effect in the treatment where rosemary plants were eliminated but its leaves were left along with allelopathy (2.57 ± 0.54 individuals/subplot). Unexpectedly, the presence of rosemary plants seems to facilitate the emergence of gypsum species (9.93 ± 1.61 individuals/subplot), counteracting the effects of the allelopathic substances in the soil. Consequently, we do not suggest removing rosemary plants in early stages to encourage the emergence of gypsum species in restored areas.

Using complementary techniques to distinguish cryptic species: a new Erysimum (Brassicaceae) species from North Africa.

PREMISE OF THE STUDY: Cryptic species are superficially morphologically indistinguishable and therefore erroneously classified under one single name. The identification and delimitation of these species is usually a difficult task. The main aim of this study is to provide an inclusive methodology that combines standard and new tools to allow accurate identification of cryptic species. We used Erysimum nervosum s.l. as a model system. METHODS: Four populations belonging to E. nervosum s.l. were sampled at their two distribution ranges in Morocco (the Atlas Mountains and the Rif Mountains). Fifteen individuals per population were collected to assess standard taxonomic traits. Additionally, corolla color and shape were quantified in 30 individuals per population using spectrophotometry and geometric morphometrics, respectively. Finally, we collected tissue samples from each population per species to study the phylogenetic relationships among them. KEY RESULTS: Using the standard taxonomic traits, we could not distinguish the four populations. Nonetheless, there were differences in corolla color and shape between plants from the two mountain ranges. The population differentiation based on quantitative morphological differences were confirmed and supported by the phylogenetic relationships obtained for these populations and the rest of the Moroccan Erysimum species. CONCLUSIONS: The joint use of the results obtained from standard taxonomic traits, quantitative analyses of plant phenotype, and molecular data suggests the occurrence of two species within E. nervosum s.l. in Morocco, one located in the Atlas Mountains (E. nervosum s.s.) and the other in the Rif Mountains (E. riphaeanum sp. nov.). Consequently, we suggest that combining quantitative and molecular approaches with standard taxonomy greatly benefits the identification of cryptic species.

Habitat fragmentation in arid zones: a case study of Linaria nigricans under land use changes (SE Spain).

Habitat fragmentation due to human activities is one of the most important causes of biodiversity loss. In Mediterranean areas the species have co-evolved with traditional farming, which has recently been replaced for more severe and aggressive practices. We use a methodological approach that enables the evaluation of the impact that agriculture and land use changes have for the conservation of sensitive species. As model species, we selected Linaria nigricans, a critically endangered plant from arid and semiarid ecosystems in south-eastern Spain. A chronosequence of the evolution of the suitable habitat for the species over more than 50 years has been reconstructed and several geometrical fragmentation indices have been calculated. A new index called fragmentation cadence (FC) is proposed to quantify the historical evolution of habitat fragmentation regardless of the habitat size. The application of this index has provided objective forecasting of the changes of each remnant population of L. nigricans. The results indicate that greenhouses and construction activities (mainly for tourist purposes) exert a strong impact on the populations of this endangered species. The habitat depletion showed peaks that constitute the destruction of 85% of the initial area in only 20 years for some populations of L. nigricans. According to the forecast established by the model, a rapid extinction could take place and some populations may disappear as early as the year 2030. Fragmentation-cadence analysis can help identify population units of primary concern for its conservation, by means of the adoption of improved management and regulatory measures.

Assessing effectiveness of exclusion fences in protecting threatened plants.

Overgrazing stands out as threat factors on biodiversity, being especially harmful in the Mediterranean, due to strong human pressure and an accelerated climate change acting synergistically. Fencing is a common tool used in conservation biology to tackle this problem. Advantages of fences are usually fast, intuitive, and easy to evaluate. However, disadvantages could also arise (increasing interspecific competition, disturbing habitat structure, limiting pollination, reducing dispersion). Together with management issues (maintenance, conflicts with stakeholders, and pulling effect). Effectiveness of fencing for conservation has been frequently assessed for animals, while it is almost a neglected topic in plants. We evaluated the outcome of fencing three threatened and narrow-endemic plants. Selected 5 populations were only partly fenced, which allowed comparing different variables inside and outside the fence. For evaluating the fencing effects, we sampled several habitats (vegetation cover, composition, density of target species), and target-species features (individual size, neighbouring species, and fruit-set). Fencing had strong effects on the habitat and on target-species individuals, showing contrasting responses at species and population level. Particularly, for Erodium cazorlanum, fence had a positive effect in one case, and negative in another. In Hormathophylla baetica effect was positive in all populations. Finally, fencing negatively affected Solenanthus reverchonii by increasing competition and limiting seed-dispersal. Fencing outcome was different in assessed species, highlighting the need to a case-by-case evaluation to determine the net balance (pros vs. cons), also its suitability and most favourable option (i.e. permanent vs. temporary fences).

FloraSNevada: a trait database of the vascular flora of Sierra Nevada, southeast Spain.

Providing a complete data set with species and trait information for a given area is essential for assessing plant conservation, management, and ecological restoration, for both local and global applications. Also, these data sets provide additional information for surveys or data collections, establishing the starting point for more detailed studies on plant evolution, vegetation dynamics, and vegetation responses to disturbance and management. This data base covers Sierra Nevada mountains (southeastern Spain), a recognized plant biodiversity hotspot within the Mediterranean context. According to previous available data (before this augmented compilation), these mountains host 7% of the 24,000 Mediterranean vascular plants, despite covering just 0.01% of its area. Another characteristic of the Sierra Nevada is the great singularity of its flora, with 95 taxa being endemic to the high-mountain area of Sierra Nevada and surroundings. From these endemic taxa, 70% are endangered by different threats, global warming being a leading cause. We seek to provide a complete and updated database of the flora of the Sierra Nevada mountains (southeast Spain). The goal of the present data set is to compile the names of all the vascular plant taxa inhabiting Sierra Nevada, together with relevant features including taxonomical, morphological-ecological traits, distribution, habitats, abundance, and conservation status. The data were compiled according to all the available information sources on taxonomy, ecology, and plant-species distribution. The resulting data set includes 2,348 taxa belonging to 1,937 species, 377 subspecies, and 34 hybrids, from a total of 756 genera and 146 families represented in the collection. For each taxa, together with taxonomical information (Phylum, Class, Family, Genus, Taxa), we compiled plant traits (life form, spinescence, flower symmetry, flower sexuality, plant gender, androecium:ginoecium ratio, flower color, perianth type, pollinator type, flowering, seed dispersal, and vegetative reproduction), and their environmental association (origin, endemic character, general distribution, substrate, elevation, habitat, local abundance, hygrophilous behavior, and conservation status). All these traits were compiled from all the available information sources, resulting in a complete and updated database for Sierra Nevada vascular flora. This data set provides valuable information on plant traits in an outstanding micro hotspot within the Mediterranean hotspot. This data set can be freely used for noncommercial purposes. This data set is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). When you use this data set, we request that you cite the data and this data paper.

Phenotypic variability in a Spanish family with a Caveolin-3 mutation.

UNLABELLED: We report a Spanish family affected from a late onset, hand-involved and autosomal dominant distal myopathy associated to Caveolin-3 mutation. Signs of muscle hyperexcitability and hyperckemia were observed in the youngest relatives but not motor symptoms. PATIENTS AND METHODS: Neurological examination was performed in all members of the family. Muscle biopsy sample was taken from the proband and DNA genomics was amplified for the two exons of Cav-3 by the polymerase chain reaction (PCR) in all the affected members and in three asymptomatic relatives. RESULTS: Signs of muscle hyperexcitability and hyperckemia were observed in the affected members from early ages. Cav-3 expression was greatly reduced in the sarcolemma of the proband's muscle. Genetic studies revealed a G --> A transition at nucleotide position 80 in exon 1 of the Cav-3 gene (c.80G>A), generating a Arg --> Gln change at codon 27 (p.R27Q) of the amino acid chain in heterozygous state, while no mutation was found in unaffected members. CONCLUSIONS: Signs of muscle hyperexcitability and hyperckemia at early ages may predict the development of a late onset autosomal dominant hand-involved myopathy associated to Cav-3 mutation in the family reported herein.

Genetic diversity and differentiation in narrow versus widespread taxa of Helianthemum (Cistaceae) in a hotspot: The role of geographic range, habitat, and reproductive traits.

Unraveling the relationships between ecological, functional traits and genetic diversity of narrow endemic plants provide opportunities for understanding how evolutionary processes operate over local spatial scales and ultimately how diversity is created and maintained. To explore these aspects in Sierra Nevada, the core of the Mediterranean Betic-Rifean hotspot, we have analyzed nuclear DNA microsatellite diversity and a set of biological and environmental factors (physicochemical soil parameters, floral traits, and community composition) in two strictly endemic taxa from dolomite outcrops of Sierra Nevada (Helianthemum pannosum and H. apenninum subsp. estevei) and two congeneric widespread taxa (H. cinereum subsp. rotundifolium and H. apenninum subsp. apenninum) that further belong to two different lineages (subgenera) of Helianthemum. We obtained rather unexpected results contrasting with the theory: (a) The narrow endemic taxa showed higher values of genetic diversity as well as higher average values of pollen production per flower and pollen-to-ovule ratio than their widespread relatives; and (b) the two taxa of subg. Helianthemum, with larger corollas, approach herkogamy and higher pollen production than the two taxa of subg. Plectolobum, displayed lower genetic diversity and higher values of inbreeding. Altogether, these results disclose how genetic diversity may be affected simultaneously by a large number of intrinsic and extrinsic factors, especially in Pleistocene glacial refugia in mountains where the spatial context harbors a great ecological heterogeneity. On the other hand, differences in mating system and the significant effect of the substrate profile, both being highly diverse in the genus Helianthemum, in the genetic variability illustrate about the importance of these two factors in the diversification and species differentiation of this paradigmatic genus in the Mediterranean and open the field to formulate and test new hypotheses of local adaptation, trait evolution, and habitat diversification.

Molecular phylogeny and evolutionary history of Moricandia DC (Brassicaceae).

BACKGROUND: The phylogeny of tribe Brassiceae (Brassicaceae) has not yet been resolved because of its complex evolutionary history. This tribe comprises economically relevant species, including the genus Moricandia DC. This genus is currently distributed in North Africa, Middle East, Central Asia and Southern Europe, where it is associated with arid and semi-arid environments. Although some species of Moricandia have been used in several phylogenetic studies, the phylogeny of this genus is not well established. METHODS: Here we present a phylogenetic analysis of the genus Moricandia using a nuclear (the internal transcribed spacers of the ribosomal DNA) and two plastidial regions (parts of the NADH dehydrogenase subunit F gene and the trnT-trnF region). We also included in the analyses members of their sister genus Rytidocarpus and from the close genus Eruca. RESULTS: The phylogenetic analyses showed a clear and robust phylogeny of the genus Moricandia. The Bayesian inference tree was concordant with the maximum likelihood and timing trees, with the plastidial and nuclear trees showing only minor discrepancies. The genus Moricandia appears to be formed by two main lineages: the Iberian clade including three species, and the African clade including the four species inhabiting the Southern Mediterranean regions plus M. arvensis. DISCUSSION: We dated the main evolutionary events of this genus, showing that the origin of the Iberian clade probably occurred after a range expansion during the Messinian period, between 7.25 and 5.33 Ma. In that period, an extensive African-Iberian floral and faunal interchange occurred due to the existence of land bridges between Africa and Europa in what is, at present-days, the Strait of Gibraltar. We have demonstrated that a Spanish population previously ascribed to Rytidocarpus moricandioides is indeed a Moricandia species, and we propose to name it as M. rytidocarpoides sp. nov. In addition, in all the phylogenetic analyses, M. foleyi appeared outside the Moricandia lineage but within the genus Eruca. Therefore, M. foleyi should be excluded from the genus Moricandia and be ascribed, at least provisionally, to the genus Eruca.

Rock climbing alters plant species composition, cover, and richness in Mediterranean limestone cliffs.

Rock climbing is among the outdoor activities that have undergone the highest growth since the second half of the 20th century. As a result, cliff habitats, historically one of the least disturbed by human colonization worldwide, are facing more intense human pressure than ever before. However, there is little data on the impact of this activity in plant-communities, and such information is indispensable for adequate manager decision-making. The goal of this study was to determine the impact of rock climbing on plant communities in terms of cover, richness, and composition in relation to climbing intensity on typical Mediterranean limestone cliffs. Three rock-climbing sites were selected in the Baetic range (SE Spain), corresponding to qualitative categories of climbing frequentation: i)"low" (low frequentation with intermittent climbing), ii)"medium" (high frequentation without overcrowding), and iii) "high" (high frequentation with overcrowding). Within each site, we selected climbing routes and adjacent areas free of climbing, then we carried out a photoplot-based sampling by rappelling. We analysed the images to calculate: richness, species cover, and total cover. This study shows that rock climbing negatively affected the cliff plant community at all three study sites. A significant decrease in plant cover, species richness and a shift in the community composition were recorded for climbed areas, the cover being the variable most sensitive to rock climbing. Impact observed proved to be related to the frequentation level. Low-frequentation sites, with usually more specialized climbers, underwent relatively mild damages, whereas at high frequentation sites the impact was severe and the conservation of the species, especially rare ones, became jeopardized. Our study is the first one available to investigate climbing impact on plant communities in Mediterranean areas, but more research on the impact of rock climbing is needed to assess the regulation of this activity. Regarding management guidelines, we propose a management guideline protocol to evaluate climbing routes and design: i) "Sites free of climbing", ii) "Strictly regulated climbing routes", iii) "Mildly regulated climbing routes", or iv) "Free climbing routes".

Designing conservation strategies to preserve the genetic diversity of Astragalus edulis Bunge, an endangered species from western Mediterranean region.

Astragalus edulis (Fabaceae) is an endangered annual species from the western Mediterranean region that colonized the SE Iberian Peninsula, NE and SW Morocco, and the easternmost Macaronesian islands (Lanzarote and Fuerteventura). Although in Spain some conservation measures have been adopted, it is still necessary to develop an appropriate management plan to preserve genetic diversity across the entire distribution area of the species. Our main objective was to use population genetics as well as ecological and phylogeographic data to select Relevant Genetic Units for Conservation (RGUCs) as the first step in designing conservation plans for A. edulis. We identified six RGUCs for in situ conservation, based on estimations of population genetic structure and probabilities of loss of rare alleles. Additionally, further population parameters, i.e. occupation area, population size, vulnerability, legal status of the population areas, and the historical haplotype distribution, were considered in order to establish which populations deserve conservation priority. Three populations from the Iberian Peninsula, two from Morocco, and one from the Canary Islands represent the total genetic diversity of the species and the rarest allelic variation. Ex situ conservation is recommended to complement the preservation of A. edulis, given that effective in situ population protection is not feasible in all cases. The consideration of complementary phylogeographic and ecological data is useful for management efforts to preserve the evolutionary potential of the species.

Secondary coenzyme Q10 deficiencies in oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders.

We evaluated the coenzyme Q10 (CoQ) levels in patients who were diagnosed with mitochondrial oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders (n=72). Data from the 72 cases in this study revealed that 44.4% of patients showed low CoQ concentrations in either their skeletal muscle or skin fibroblasts. Our findings suggest that secondary CoQ deficiency is a common finding in OXPHOS and non-OXPHOS disorders. We hypothesize that cases of CoQ deficiency associated with OXPHOS defects could be an adaptive mechanism to maintain a balanced OXPHOS, although the mechanisms explaining these deficiencies and the pathophysiological role of secondary CoQ deficiency deserves further investigation.