Cork oak (Quercus suber) is an ecologically and economically important Western Mediterranean tree species in severe risk of decline due to aggravated tree mortality and lack of natural regeneration. In Q. suber, two distinct chloroplast lineages, one of them of trans-specific origin, occur in sympatry in the western half of its species distribution. We hypothesize that selection may drive the maintenance of the two lineages and investigate this hypothesis by sequencing chloroplast genomes of 259 Q. suber samples across 24 locations. Protein-coding chloroplast genes were scanned for selection signal using different codon-based methods. Selection signal was found at different sites and genes, and polymorphism in selected sites was shown to segregate between the two chloroplast lineages. We postulate that trans-specific chloroplast diversity in Q. suber is preserved by balancing selection, rather than resulting exclusively from ongoing introgression. These results correspond to an unusual case of balancing selection on whole plastid genomes in a long-lived woody plant species and have implications on conservation and management practices for Q. suber, which may benefit from taking into account genetic variation in plastid genomes as a possible source of increased adaptive potential for the species.

Background Squamatinia algharbica Mendes & Reboleira, 2012 is an iconic gigantic troglobiont silverfish, which was recently listed endangered in the Portuguese Red List of Invertebrates. It is endemic from caves of the Algarve, the southernmost part of Portugal - a karst area known as Barrocal. This area faces severe threats to cave biodiversity, such as pollution, groundwater overexploitation, land-use change at surface, intensive agriculture and aridity, associated with the lack of concrete protection measures for cave-adapted species. New information Biological prospection of caves in the Algarve karst massif (Portugal) increased the knowledge on the distribution of S. algharbica. We provide new distributional data that enlarge the area of occurrence and the first phylogeny for this species. Based on new molecular data, the genus Squamatinia appears with an isolated position within the Nicoletiidae family, highlighting Squamatinia algharbica as a biological relict with particularly high conservationist value. We assess the conservation status, evaluating the anthropogenic threats to its habitat and present the IUCN Red List profile, based on this new information.

Despite the popularity of canned tuna in Portugal, limited research has explored how tuna species vary across brands and seasons. Accurate species identification is crucial to preserve both the economy and ecology of global tuna fisheries, as mislabelling and the use of certain species can have far-reaching implications. Our study analysed the diversity of tuna species used by the Portuguese tuna canning industry, focusing on brand-specific and seasonal variations, using molecular barcoding methods. In this industry, the occurrence of different species was observed only in products using water as a preservation medium. Skipjack tuna was predominant across all preservation media and brands analysed. Nonetheless, other species like Thunnus obesus and T. albacares, or Auxis spp. (not considered true tuna) were also detected. The use of different species was limited to cans produced during the second quarter of the year, which could reflect differences in seasonal availability of different tuna species or in sourcing strategies/market preferences of each company. Finally, more than one species was detected inside the same container in four brands, violating current European legislation. These results provide the first broad assessment of species used in the Portuguese tuna canning industry and showed the inclusion of vulnerable species is limited.

Cork oak woodlands are socio-ecosystems recognized as biodiversity hotspots, a fundamental economic source for companies and local communities as well as an identitarian landscape for residents and visitors. Cork oak woodlands, however, are facing tree mortality and lack of regeneration. Considering the oak decline scenario, we present Iberian cork oak montado/dehesa as a socio-ecosystem facing climate change, management transformations, local knowledge crisis and social uncertainty. We review montados/dehesas research through time and by different scholar perspectives. We defend that from an interdisciplinary and transdisciplinary perspective, including the experience and evidence observed in terrain, debate should be stimulated, and novel conceptual approaches may contribute to finding solutions. We argue that the confluence of ecology, genetics, anthropology and local knowledge can be explored to unveil the complexities and the challenges of these socio-ecosystems and contribute to prevent and mitigate threats to it. We propose a methodological approach built together with scientists, managers and workers, that can explore oak translocations, socio-ecological interactions models, knowledge transfer and other ways to overcome Iberian cork oak woodlands socio-environmental crisis.

The development of management strategies for the promotion of sustainable fisheries relies on a deep knowledge of ecological and evolutionary processes driving the diversification and genetic variation of marine organisms. Sustainability strategies are especially relevant for marine species such as the European sardine (Sardina pilchardus), a small pelagic fish with high ecological and socioeconomic importance, especially in Southern Europe, whose stock has declined since 2006, possibly due to environmental factors. Here, we generated sequences for 139 mitochondrial genomes from individuals from 19 different geographical locations across most of the species distribution range, which was used to assess genetic diversity, diversification history and genomic signatures of selection. Our data supported an extensive gene flow in European sardine. However, phylogenetic analyses of mitogenomes revealed diversification patterns related to climate shifts in the late Miocene and Pliocene that may indicate past divergence related to rapid demographic expansion. Tests of selection showed a significant signature of purifying selection, but positive selection was also detected in different sites and specific mitochondrial lineages. Our results showed that European sardine diversification has been strongly driven by climate shifts, and rapid changes in marine environmental conditions are likely to strongly affect the distribution and stock size of this species.

Increasing exposure to unfavorable temperatures and water deficit imposes major constraints on most crops worldwide. Despite several studies regarding coffee responses to abiotic stresses, transcriptome modulation due to simultaneous stresses remains poorly understood. This study unravels transcriptomic responses under the combined action of drought and temperature in leaves from the two most traded species: Coffea canephora cv. Conilon Clone 153 (CL153) and C. arabica cv. Icatu. Substantial transcriptomic changes were found, especially in response to the combination of stresses that cannot be explained by an additive effect. A large number of genes were involved in stress responses, with photosynthesis and other physiologically related genes usually being negatively affected. In both genotypes, genes encoding for protective proteins, such as dehydrins and heat shock proteins, were positively regulated. Transcription factors (TFs), including MADS-box genes, were down-regulated, although responses were genotype-dependent. In contrast to Icatu, only a few drought- and heat-responsive DEGs were recorded in CL153, which also reacted more significantly in terms of the number of DEGs and enriched GO terms, suggesting a high ability to cope with stresses. This research provides novel insights into the molecular mechanisms underlying leaf Coffea responses to drought and heat, revealing their influence on gene expression.

We used a population genomic approach to unravel the population structure, genetic differentiation, and genetic diversity of three widespread wild bee species across the Iberian Peninsula, Andrena agilissima, Andrena flavipes and Lasioglossum malachurum. Our results demonstrated that genetic lineages in the Ebro River valley or near the Pyrenees mountains are different from the rest of Iberia. This relatively congruent pattern across species once more supports the hypothesis of "refugia within refugia" in the Iberian Peninsula. The results for A. flavipes and A. agilissima showed an unexpected pattern of genetic differentiation, with the generalist polylectic A. flavipes having lower levels of genetic diversity (Ho = 0.0807, He = 0.2883) and higher differentiation (F-ST = 0.5611), while the specialist oligolectic A. agilissima had higher genetic diversity (Ho = 0.2104, He = 0.3282) and lower differentiation values (F-ST = 0.0957). For L. malachurum, the smallest and the only social species showed the lowest inbreeding coefficient (F-IS = 0.1009) and the lowest differentiation level (F-ST = 0.0663). Overall, our results, suggest that this pattern of population structure and genetic diversity could be explained by the combined role of past climate changes and the life-history traits of the species (i.e., size, sociality and host-plant specialization), supporting the role of the Iberian refugia as a biodiversity hotspot.

The current distribution patterns of many Mediterranean species are often a consequence of large and impactful past geoclimatic events, such as the Messinian Salinity Crisis (MSC) and the Quaternary glacial cycles. Cicadas are flying insects with poor dispersal ability, which have experienced intense local differentiation in the Mediterranean, where the genus Tettigettalna has surfaced as a biogeographic model. The genus includes 10 species with species‐specific calling songs but identical morphology. All Tettigettalna species are restricted to Southern Iberia, with the exception of T. estrellae (northwest Iberia), the widespread T. argentata (mainly Iberia, France and Italy), and T. afroamissa (Morocco). With an expanded genetic dataset involving nuclear ( EF1α ) and mitochondrial (5′ and 3′ COI and ATP ) loci, we reconstructed the phylogeny of the genus and estimated divergence dates for Tettigettalna species under a Bayesian framework. Phylogeny with the new mitochondrial dataset was in agreement with previous studies, whereas the nuclear EF1α supported T. josei and T. afroamissa as monophyletic clades but lacked resolution to resolve the remaining taxa. Some sister taxa share mitochondrial haplotypes, hinting for incomplete lineage sorting. Estimates of divergence time settled T. josei as the earliest diverging lineage, likely as a pre‐ or early‐MSC event. As for the origin of T. afroamissa in Morocco, though time estimates could not entirely rule out post‐MSC dispersal, the most likely scenario points to isolation of African Tettigettalna after the reopening of the strait of Gibraltar. The Pleistocene glaciations that followed likely impacted on the diversification of the remaining species of the genus in southern Iberia refugia.

The comparison of closely related taxa is cornerstone in biology, as understanding mechanisms leading up to differentiation in relation to extant shared characters are powerful tools in interpreting the evolutionary process. Hotspots of biodiversity such as the west-Mediterranean, where many lineages meet are ideal grounds to study these processes. We set to explore the interesting example of Sooty Copper butterflies: widespread Eurasian Lycaena tityrus (Poda, 1761) comes into contact in Iberia with closely related and local endemic, L. bleusei (Oberthur, 1884), which hasn't always been considered a distinct species. An integrative analysis was designed, combining the use of extensive molecular data (five genes), geometric morphometrics analyses, verified and up-to-date distribution data, and environmental niche modelling, aimed at deciphering their true relationship, their placement within European Lycaena and trace their evolutionary history. We revealed several levels of differ-entiation: L. bleusei and L. tityrus appear to be reciprocally monophyletic independent gene-pools, distinct in all genes analysed, having mutually diverged 4.8 Ma ago. L. tityrus but not L. bleusei, further displays a genetic structure compatible with several glacial refugia, where populations assignable to infraspecific taxa surface. Conversely, L. bleusei shows a loss in mtDNA diversity in relation to nuDNA. Morphological analyses differentiate both species according to size and shape but also discriminate strong seasonal and sexual traits and a geographical phenotype segregation in L. tityrus. Finally, updated distribution and its modelling for current and glacial timeframes reveal both species respond differently to environmental variables, defining a mostly para-patric distribution and an overlapping belt where sympatry was recovered. During the last glacial maximum, a wider expansion in L. bleusei distribution explains current isolated populations. Our study highlights the importance of gathering several lines of evidence when deciphering the relationships between closely related populations in the fringe of cryptic species realm.

Background Sea-lavenders ( Limonium Mill., Plumbaginaceae) are a cosmopolitan group of diploid and polyploid plants often adapted to extreme saline environments, with a mostly Tethyan distribution, occurring in the Mediterranean, Irano-Turanian, Euro-Siberian and in the New World. The halophylic Limonium vulgare polyploid complex in particular, presents a large distribution throughout extreme salt-marsh habitats and shows little morphological but high taximetric variation, frequently blurring species delimitation. In this work we pursue three main goals: assert whether SNP data from polyploid individuals has the resolution to distinguish the seven sampled species, to better understand how genetically structured Limonium vulgare is, and attempt to identify specific molecular mechanisms for the differentiation between L. maritimum and L. vulgare . For this purpose, 95 individuals were genotyped using Genotyping by Sequencing (GBS), which were assembled as two independent datasets using ipyrad . All analyses performed downstream of assembly were fully automated. Phylogenetic inference, PCA, and admixture plots were used to infer answers to the study’s main goals. Results Close to 10,000 SNPs were obtained for each dataset. Phylogenetic analyses reveal that polyploid data can be used to infer species relationships. Population structure analyses suggest a genetically structured L. vulgare . A set of 34 SNPs were found to be fully segregated between L. vulgare and L. maritimum , two of which are potentially linked to proteins that might be involved in the speciation process. Conclusion Despite polyploid data analyses shortcomings, GBS generated SNPs have the resolution to discern all seven included species. Limonium vulgare revealed pronounced genetic structure along a geographical north-south cline. L. maritimum always appears as a distinct genetic entity. Segregated SNPs between L. vulgare and L. maritimum indicate salinity response and morphological trait control genes as potentially interesting to follow up for studying these species’ divergence process.

Chloroplast genome diversity in cork oak ( Quercus suber ) is characterised by the occurrence of haplotypes that are akin to those found in other Mediterranean oak species, particularly in Q. ilex and Q. rotundifolia , suggesting the possible presence of an introgressed chloroplast lineage. To further investigate this pattern, we reconstructed chloroplast haplotypes by sequencing four chloroplast markers (cpDNA), sampled across 181 individuals and 10 taxa. Our analyses resulted in the identification of two diversified chloroplast haplogroups in Q. suber , corresponding to a geographically widespread lineage and an Afro-Iberian lineage. Time-calibrated phylogenetic analyses of cpDNA point to a Miocene origin of the two haplogroups in Q. suber , suggesting that the Afro-Iberian lineage was present in cork oak before the onset of glaciation periods. The persistence of the two haplogroups in the western part of the species distribution range may be a consequence of either ancient introgression events or chloroplast lineage sorting, combined with different fixation in refugia through glaciation periods. Our results provide a comprehensive insight on the origins of chloroplast diversity in these ecologically and economically important Mediterranean oaks.

The genus Limonium Mill. (sea lavenders) includes species with sexual and apomixis reproductive strategies, although the genes involved in these processes are unknown. To explore the mechanisms beyond these reproduction modes, transcriptome profiling of sexual, male sterile, and facultative apomictic species was carried out using ovules from different developmental stages. In total, 15,166 unigenes were found to be differentially expressed with apomictic vs. sexual reproduction, of which 4275 were uniquely annotated using an Arabidopsis thaliana database, with different regulations according to each stage and/or species compared. Gene ontology (GO) enrichment analysis indicated that genes related to tubulin, actin, the ubiquitin degradation process, reactive oxygen species scavenging, hormone signaling such as the ethylene signaling pathway and gibberellic acid-dependent signal, and transcription factors were found among differentially expressed genes (DEGs) between apomictic and sexual plants. We found that 24% of uniquely annotated DEGs were likely to be implicated in flower development, male sterility, pollen formation, pollen-stigma interactions, and pollen tube formation. The present study identifies candidate genes that are highly associated with distinct reproductive modes and sheds light on the molecular mechanisms of apomixis expression in Limonium sp.

Drought is a major constraint to plant growth and productivity worldwide and will aggravate as water availability becomes scarcer. Although elevated air [CO2] might mitigate some of these effects in plants, the mechanisms underlying the involved responses are poorly understood in woody economically important crops such as Coffea. This study analyzed transcriptome changes in Coffea canephora cv. CL153 and C. arabica cv. Icatu exposed to moderate (MWD) or severe water deficits (SWD) and grown under ambient (aCO(2)) or elevated (eCO(2)) air [CO2]. We found that changes in expression levels and regulatory pathways were barely affected by MWD, while the SWD condition led to a down-regulation of most differentially expressed genes (DEGs). eCO(2) attenuated the impacts of drought in the transcripts of both genotypes but mostly in Icatu, in agreement with physiological and metabolic studies. A predominance of protective and reactive oxygen species (ROS)-scavenging-related genes, directly or indirectly associated with ABA signaling pathways, was found in Coffea responses, including genes involved in water deprivation and desiccation, such as protein phosphatases in Icatu, and aspartic proteases and dehydrins in CL153, whose expression was validated by qRT-PCR. The existence of a complex post-transcriptional regulatory mechanism appears to occur in Coffea explaining some apparent discrepancies between transcriptomic, proteomic, and physiological data in these genotypes.

Divergence in acoustic signals may have a crucial role in the speciation process of animals that rely on sound for intra-specific recognition and mate attraction. The acoustic adaptation hypothesis (AAH) postulates that signals should diverge according to the physical properties of the signalling environment. To be efficient, signals should maximize transmission and decrease degradation. To test which drivers of divergence exert the most influence in a speciose group of insects, we used a phylogenetic approach to the evolution of acoustic signals in the cicada genus Tettigettalna, investigating the relationship between acoustic traits (and their mode of evolution) and body size, climate and micro-/macro-habitat usage. Different traits showed different evolutionary paths. While acoustic divergence was generally independent of phylogenetic history, some temporal variables' divergence was associated with genetic drift. We found support for ecological adaptation at the temporal but not the spectral level. Temporal patterns are correlated with micro- and macro-habitat usage and temperature stochasticity in ways that run against the AAH predictions, degrading signals more easily. These traits are likely to have evolved as an anti-predator strategy in conspicuous environments and low-density populations. Our results support a role of ecological selection, not excluding a likely role of sexual selection in the evolution of Tettigettalna calling songs, which should be further investigated in an integrative approach.

Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N2-fixing actinorhizal trees constitute important elements of plant communities and have been successfully used in land reclamation at a global scale. In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO3+) or nodulated by Frankia (NOD+) were exposed to a gradient of salt concentrations (200, 400, and 600 mM NaCl) and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD+) to 314 (600 mM NaCl/KNO3+), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species.

The aquatic environment is a reservoir of antibiotics, pathogenic and native microorganisms, and antibiotic resistance genes (ARGs), constituting a key aspect of the One Health approach. Thus, the problem of antibiotic resistance is no longer restricted to bacterial pathogens but is a consequence of the interplay between distinct clinical and environmental drivers. In the project “ExplorAR—Exploring the Aquatic Resistome”, we investigated the putative role of cyanobacteria (CB) in the freshwater resistome, using a multidisciplinary approach based on phenotypic/genotypic/genomic tools. In this presentation, we disclose the main results of the project: (i) the development of a microdilution-based antibiotic susceptibility assay for CB; (ii) the establishment of an antibiotic susceptibility profile of CB genus/species; (iii) the identification of CB strains with reduced susceptibility to multiple antibiotic classes; (iv) the identification of ARGs in CB genomes; (v) the characterization of the antibiotic resistance profile of CB-associated bacteria; (vi) mapping the occurrence of CB and ARGs in surface freshwater reservoirs by high-throughput sequencing approaches. Overall, ExplorAR strongly supports the hypothesis that CB are environmental players in the emergence and dissemination of ARGs in water environments, contributing to the problem, and challenges, of antibiotic resistance.