DNA metabarcoding, diversity partitioning and null models reveal mechanisms of seasonal trophic specialization in a Mediterranean warbler.

Optimal Foraging Theory (OFT) predicts that a population's trophic niche expansion should occur in periods of food scarcity as individuals begin to opportunistically exploit sub-optimal food items. However, the Niche Variation Hypothesis (NVH) posits that niche widening may result from increased among-individual differentiation due to food partitioning to avoid competition. We tested these hypotheses through a DNA metabarcoding study of the Sardinian Warbler (Curruca melanocephala) diet over a year. We used null models and the decomposition of beta diversity on among-individual dietary differentiation to infer the mechanisms driving the population's niche variation. Warblers fed frequently on berries, with a peak in late summer and, to a lesser extent, in autumn. Their diet also included a wide range of arthropods, with their prevalence varying among seasons. Consistent with OFT, the population's niche width was narrower in spring/summer when the population was strongly specialized in berries. In winter, the population's niche expanded, possibly reflecting seasonal declines in food abundance. As predicted by NVH, among-individual differentiation tended to be higher in winter, but this was mainly due to increased differences in dietary richness rather than to the partitioning of resources. Overall, our results suggest that within-individual niche does not increase in lean periods, and instead, individuals adopt either a more opportunistic or more specialized foraging strategy. Increased competition in periods of scarcity may help explain such patterns, but instead of showing increased food partitioning as expected from NVH, it may reflect OFT mechanisms on individuals with differential competitive ability to access better food resources.

Dietary metabarcoding reveals the simplification of bird-pest interaction networks across a gradient of agricultural cover.

Agriculture is vital for supporting human populations, but its intensification often leads to landscape homogenization and a decline in non-provisioning ecosystem services. Ecological intensification and multifunctional landscapes are suggested as nature-based alternatives to intensive agriculture, using ecological processes like natural pest regulation to maximize food production. Birds are recognized for their role in increasing crop yields by consuming invertebrate pests in several agroecosystems. However, the understanding of how bird species, their traits and agricultural land cover influence the structure of bird-pest interactions remains limited. We sampled bird-pest interactions monthly for 1 year, at four sites within a multifunctional landscape, following a gradient of increasing agricultural land cover. We analysed 2583 droppings of 55 bird species with DNA metabarcoding and detected 225 pest species in 1139 samples of 42 bird species. As expected, bird-pest interactions were highly variable across bird species. Dietary pest richness was lower in the fully agricultural site, while predation frequency remained consistent across the agricultural land cover gradient. Network analysis revealed a reduction in the complexity of bird-pest interactions as agricultural coverage increased. Bird species abundance affected the bird's contribution to the network structure more than any of the bird traits analysed (weight, phenology, invertebrate frequency in diet and foraging strata), with more common birds being more important to network structure. Overall, our results show that increasing agricultural land cover increases the homogenization of bird-pest interactions. This shows the importance of maintaining natural patches within agricultural landscapes for biodiversity conservation and enhanced biocontrol.

A agricultura é essencial para suportar a população humana, mas a sua intensificação geralmente leva à homogeneização da paisagem e à redução dos serviços do ecossistema que não sejam de provisão. A intensificação ecológica e paisagens multifuncionais são sugeridas como alternativas naturais à agricultura intensiva, utilizando processos ecológicos como a regulação natural de pragas para maximizar a produção de alimentos. As aves são conhecidas pelo seu papel no aumento da produtividade das culturas por consumirem pragas em diversos agroecossistemas. Contudo, o conhecimento de como as espécies de aves, as suas características e a cobertura agrícola influenciam as interações entre aves e pragas são limitados. Nós amostrámos estas interações mensalmente durante um ano, em quatro locais, numa paisagem multifuncional, ao longo um gradiente de aumento da cobertura agrícola. Analisamos 2583 dejetos de 55 espécies de aves com DNA metabarcoding e detetamos 225 espécies praga em 1139 amostras de 42 espécies de aves. Como esperado, as interações entre aves e pragas foram muito distintas entre as várias espécies de aves. A riqueza de pragas na dieta foi menor no local completamente dominado por área agrícola, enquanto a frequência de predação de pragas foi constante ao longo do gradiente de cobertura agrícola. A análise de redes demonstrou uma redução na complexidade das interações entre aves e pragas à medida que a cobertura agrícola aumenta. A abundância das espécies de aves influenciou mais a contribuição das aves para a estrutura da rede do que qualquer uma das características analisadas (peso, fenologia, frequência de invertebrados na dieta e estrato de alimentação), sendo as aves mais comuns as mais importantes na estrutura da rede. De forma geral, os nossos resultados indicam que o aumento da cobertura agrícola aumenta a homogeneização das interações entre aves e pragas. Isto demonstra a importância de preservar áreas naturais em paisagem agrícolas para a conservação de biodiversidade e melhor controlo biológico.

Crowding after sudden habitat loss affects demography and social structure in a bat population.

The sudden loss of habitats due to natural or anthropogenic disturbances causes displacement of mobile animals from affected areas to refuge habitats, where large but often transitory concentrations of individuals may occur. While these local density increases have been previously described, the hypothesis that crowding disrupts demographic processes remains largely untested. Here we used the sudden flooding of a river valley by a hydroelectric reservoir as a quasi-experiment to investigate the consequences of crowding on demography, fecundity and social structure in the European free-tailed bat Tadarida teniotis. We monitored bat populations at roosts near and far from the flooded area, before (2013-2014), during (2015) and after (2016) habitat flooding. We assessed population demographic parameters using Capture-Mark-Recapture (CMR) models (3,821 PIT-tagged individuals), and used genetic relatedness among individuals (1,407 individuals genotyped for 14 microsatellite markers) to infer changes in social structure. Habitat loss through flooding was associated with significant but transitory increases in the number of bats using nearby roosts. This may be related to the higher probability of individuals arriving at those roosts during flooding, together with increases in individual local residency through time, particularly among males. Individual apparent survival was highest during flooding and lowest in the following year, while the probability of leaving a roost safe from flooding was higher near the impact area than farther away. Crowding did not negatively affect fecundity, but the arrival of new individuals led to changes in social structure as revealed by lower genetic relatedness between individuals after disturbance at roosts near the flooding area, but not in those farther afield. Our study documents a clear example of crowding effects, suggesting that bats losing roosts due to a hydroelectric reservoir moved to alternative roosts, where local increases in population size and the arrival of new individuals reduced genetic relatedness and apparent survival, but not fecundity. These results support the hypothesis that crowding after habitat loss can disrupt population processes, even though effects may be subtle and short-lived. Also, they point out the need to duly consider crowding effects when assessing and mitigating anthropogenic impacts on animal populations.

Modelling technical and biological biases in macroinvertebrate community assessment from bulk preservative using multiple metabarcoding markers.

DNA metabarcoding from the ethanol used to store macroinvertebrate bulk samples is a convenient methodological option in molecular biodiversity assessment and biomonitoring of aquatic ecosystems, as it preserves specimens and reduces problems associated with sample sorting. However, this method may be affected by errors and biases, which need to be thoroughly quantified before it can be mainstreamed into biomonitoring programmes. Here, we used 80 unsorted macroinvertebrate samples collected in Portugal under a Water Framework Directive monitoring programme, to compare community diversity and taxonomic composition metrics estimated through morphotaxonomy versus metabarcoding from storage ethanol using three markers (COI-M19BR2, 16S-Inse01 and 18S-Euka02) and a multimarker approach. A preliminary in silico analysis showed that the three markers were adequate for the target taxa, with detection failures related primarily to the lack of adequate barcodes in public databases. Metabarcoding of ethanol samples retrieved far less taxa per site (alpha diversity) than morphotaxonomy, albeit with smaller differences for COI-M19BR2 and the multimarker approach, while estimates of taxa turnover (beta diversity) among sites were similar across methods. Using generalized linear mixed models, we found that after controlling for differences in read coverage across samples, the probability of detection of a taxon was positively related to its proportional abundance, and negatively so to the presence of heavily sclerotized exoskeleton (e.g., Coleoptera). Overall, using our experimental protocol with different template dilutions, the COI marker showed the best performance, but we recommend the use of a multimarker approach to detect a wider range of taxa in freshwater macroinvertebrate samples. Further methodological development and optimization efforts are needed to reduce biases associated with body armouring and rarity in some macroinvertebrate taxa.

Combining DNA metabarcoding and ecological networks to inform conservation biocontrol by small vertebrate predators.

In multifunctional landscapes, diverse communities of flying vertebrate predators provide vital services of insect pest control. In such landscapes, conservation biocontrol should benefit service-providing species to enhance the flow, stability and resilience of pest control services supporting the production of food and fiber. However, this would require identifying key service providers, which may be challenging when multiple predators interact with multiple pests. Here we provide a framework to identify the functional role of individual species to pest control in multifunctional landscapes. First, we used DNA metabarcoding to provide detailed data on pest species predation by diverse predator communities. Then, these data were fed into an extensive network analysis, in which information relevant for conservation biocontrol is gained from parameters describing network structure (e.g., modularity) and species roles in such network (e.g., centrality, specialization). We applied our framework to a Mediterranean landscape, where 19 bat species were found to feed on 132 insect pest species. Metabarcoding data revealed potentially important bats that consumed insect pest species in high frequency and/or diversity. Network analysis showed a modular structure, indicating sets of bat species that are required to regulate specific sets of insect pests. A few generalist bats had particularly important roles, either at network or module levels. Extinction simulations highlighted six bats, including species of conservation concern, which were sufficient to ensure that over three-quarters of the pest species had at least one bat predator. Combining DNA metabarcoding and ecological network analysis provides a valuable framework to identify individual species within diverse predator communities that might have a disproportionate contribution to pest control services in multifunctional landscapes. These species can be regarded as candidate targets for conservation biocontrol, although additional information is needed to evaluate their actual effectiveness in pest regulation.

Species traits, patch turnover and successional dynamics: when does intermediate disturbance favour metapopulation occupancy?

BACKGROUND: In fragmented landscapes, natural and anthropogenic disturbances coupled with successional processes result in the destruction and creation of habitat patches. Disturbances are expected to reduce metapopulation occupancy for species associated with stable habitats, but they may benefit species adapted to transitory habitats by maintaining a dynamic mosaic of successional stages. However, while early-successional species may be favoured by very frequent disturbances resetting successional dynamics, metapopulation occupancy may be highest at intermediate disturbance levels for species with mid-successional habitat preferences, though this may be conditional on species traits and patch network characteristics. Here we test this 'intermediate disturbance hypothesis' applied to metapopulations (MIDH), using stochastic patch occupancy simulation modelling to assess when does intermediate disturbance favour metapopulation occupancy. We focused on 54 virtual species varying in their habitat preferences, dispersal abilities and local extinction and colonization rates. Long-term metapopulation dynamics was estimated in landscapes with different habitat amounts and patch turnover rates (i.e. disturbance frequency). RESULTS: Equilibrium metapopulation occupancy by late-successional species strongly declined with increasing disturbance frequency, while occupancy by early-successional species increased with disturbance frequency at low disturbance levels and tended to level-off thereafter. Occupancy by mid-successional species tended to increase along with disturbance frequency at low disturbance levels and declining thereafter. Irrespective of habitat preferences, occupancy increased with the amount of habitat, and with species dispersal ability and colonisation efficiency. CONCLUSIONS: Our study suggests that MIDH is verified only for species associated with mid-successional habitats. These species may be particularly sensitive to land use changes causing either increases or decreases in disturbance frequency. This may be the case, for instance, of species associated with traditional agricultural and pastoral mosaic landscapes, where many species disappear either through intensification or abandonment processes that change disturbance frequency.

How much is enough? Effects of technical and biological replication on metabarcoding dietary analysis.

DNA metabarcoding is increasingly used in dietary studies to estimate diversity, composition and frequency of occurrence of prey items. However, few studies have assessed how technical and biological replication affect the accuracy of diet estimates. This study addresses these issues using the European free-tailed bat Tadarida teniotis, involving high-throughput sequencing of a small fragment of the COI gene in 15 separate faecal pellets and a 15-pellet pool per each of 20 bats. We investigated how diet descriptors were affected by variability among (a) individuals, (b) pellets of each individual and (c) PCRs of each pellet. In addition, we investigated the impact of (d) analysing separate pellets vs. pellet pools. We found that diet diversity estimates increased steadily with the number of pellets analysed per individual, with seven pellets required to detect ~80% of prey species. Most variation in diet composition was associated with differences among individual bats, followed by pellets per individual and PCRs per pellet. The accuracy of frequency of occurrence estimates increased with the number of pellets analysed per bat, with the highest error rates recorded for prey consumed infrequently by many individuals. Pools provided poor estimates of diet diversity and frequency of occurrence, which were comparable to analysing a single pellet per individual, and consistently missed the less common prey items. Overall, our results stress that maximizing biological replication is critical in dietary metabarcoding studies and emphasize that analysing several samples per individual rather than pooled samples produce more accurate results.

Unravelling population processes over the Late Pleistocene driving contemporary genetic divergence in Palearctic buzzards.

Population range expansions and contractions as a response to climate and habitat change throughout the Quaternary are known to have contributed to complex phylogenetic and population genetic events. Speciation patterns and processes in Palearctic buzzards (genus Buteo) are a long-standing example of morphological and genetic data incongruence, attributed to panmixia, habitat range shifts, contact zones, and climate change. Here we assess the systematics, phylogeography and population genetic structure of three nominal species of Palearctic buzzards, Buteo buteo (including B. b. vulpinus), B. rufinus (including B. r. cirtensis) and B. hemilasius. Phylogenetic analyses inferred from mitochondrial data recover B. hemilasius as sister to the sister clades B. r. rufinus and B. buteo complex (B. b. buteo, B. b. vulpinus, but also including B. r. cirtensis). In contrast, we find an unresolved genetic delimitation inferred from four nuclear loci, suggesting an ancestral genetic pool for all species. Time-trees suggest population contractions and expansions throughout the Pleistocene, which likely reflect habitat change and contrasting ecological niche requirements between species. Microsatellite-based extended Bayesian skyline plots reveal relatively constant population sizes for B. hemilasius, B. r. rufinus, and B. b. vulpinus, in contrast to a dramatic population expansion in B. r. cirtensis within the last 3 kya. Overall, our study illustrates how complex population processes over the Late Pleistocene have shaped the patterns of genetic divergence in Palearctic buzzards, due to the joint effects of shared ancestral polymorphisms, population expansions and contractions, with hybridization at contact zones leading to admixture and introgression.

Female dietary bias towards large migratory moths in the European free-tailed bat (Tadarida teniotis).

In bats, sexual segregation has been described in relation to differential use of roosting and foraging habitats. It is possible that variation may also exist between genders in the use of different prey types. However, until recently this idea was difficult to test owing to poorly resolved taxonomy of dietary studies. Here, we use high-throughput sequencing to describe gender-related variation in diet composition of the European free-tailed bat (Tadarida teniotis), while controlling for effects of age and season. We analysed guano pellets collected from 143 individuals mist-netted from April to October 2012 and 2013, in northeast Portugal. Moths (Lepidoptera; mainly Noctuidae and Geometridae) were by far the most frequently recorded prey, occurring in nearly all samples and accounting for 96 out of 115 prey taxa. There were significant dietary differences between males and females, irrespective of age and season. Compared to males, females tended to consume larger moths and more moths of migratory behaviour (e.g.Autographa gamma). Our study provides the first example of gender-related dietary variation in bats, illustrating the value of novel molecular tools for revealing intraspecific variation in food resource use in bats and other insectivores.

Hierarchical spatial segregation of two Mediterranean vole species: the role of patch-network structure and matrix composition.

According to ecological theory, the coexistence of competitors in patchy environments may be facilitated by hierarchical spatial segregation along axes of environmental variation, but empirical evidence is limited. Cabrera and water voles show a metapopulation-like structure in Mediterranean farmland, where they are known to segregate along space, habitat, and time axes within habitat patches. Here, we assess whether segregation also occurs among and within landscapes, and how this is influenced by patch-network and matrix composition. We surveyed 75 landscapes, each covering 78 ha, where we mapped all habitat patches potentially suitable for Cabrera and water voles, and the area effectively occupied by each species (extent of occupancy). The relatively large water vole tended to be the sole occupant of landscapes with high habitat amount but relatively low patch density (i.e., with a few large patches), and with a predominantly agricultural matrix, whereas landscapes with high patch density (i.e., many small patches) and low agricultural cover, tended to be occupied exclusively by the small Cabrera vole. The two species tended to co-occur in landscapes with intermediate patch-network and matrix characteristics, though their extents of occurrence were negatively correlated after controlling for environmental effects. In combination with our previous studies on the Cabrera-water vole system, these findings illustrated empirically the occurrence of hierarchical spatial segregation, ranging from within-patches to among-landscapes. Overall, our study suggests that recognizing the hierarchical nature of spatial segregation patterns and their major environmental drivers should enhance our understanding of species coexistence in patchy environments.

Filling gaps in a large reserve network to address freshwater conservation needs.

Freshwater ecosystems and biodiversity are among the most threatened at global scale, but efforts for their conservation have been mostly peripheral to terrestrial conservation. For example, Natura 2000, the world's largest network of protected areas, fails to cover adequately the distribution of rare and endangered aquatic species, and lacks of appropriate spatial design to make conservation for freshwater biodiversity effective. Here, we develop a framework to identify a complementary set of priority areas and enhance the conservation opportunities of Natura 2000 for freshwater biodiversity, using the Iberian Peninsula as a case study. We use a systematic planning approach to identify a minimum set of additional areas that would help i) adequately represent all freshwater fish, amphibians and aquatic reptiles at three different target levels, ii) account for key ecological processes derived from riverscape connectivity, and iii) minimize the impact of threats, both within protected areas and propagated from upstream unprotected areas. Addressing all these goals would need an increase in area between 7 and 46%, depending on the conservation target used and strength of connectivity required. These new priority areas correspond to subcatchments inhabited by endangered and range restricted species, as well as additional subcatchments required to improve connectivity among existing protected areas and to increase protection against upstream threats. Our study should help guide future revisions of the design of Natura 2000, while providing a framework to address deficiencies in reserve networks for adequately protecting freshwater biodiversity elsewhere.

Sampling effects on the identification of roadkill hotspots: Implications for survey design.

Although locating wildlife roadkill hotspots is essential to mitigate road impacts, the influence of study design on hotspot identification remains uncertain. We evaluated how sampling frequency affects the accuracy of hotspot identification, using a dataset of vertebrate roadkills (n = 4427) recorded over a year of daily surveys along 37 km of roads. "True" hotspots were identified using this baseline dataset, as the 500-m segments where the number of road-killed vertebrates exceeded the upper 95% confidence limit of the mean, assuming a Poisson distribution of road-kills per segment. "Estimated" hotspots were identified likewise, using datasets representing progressively lower sampling frequencies, which were produced by extracting data from the baseline dataset at appropriate time intervals (1-30 days). Overall, 24.3% of segments were "true" hotspots, concentrating 40.4% of roadkills. For different groups, "true" hotspots accounted from 6.8% (bats) to 29.7% (small birds) of road segments, concentrating from <40% (frogs and toads, snakes) to >60% (lizards, lagomorphs, carnivores) of roadkills. Spatial congruence between "true" and "estimated" hotspots declined rapidly with increasing time interval between surveys, due primarily to increasing false negatives (i.e., missing "true" hotspots). There were also false positives (i.e., wrong "estimated" hotspots), particularly at low sampling frequencies. Spatial accuracy decay with increasing time interval between surveys was higher for smaller-bodied (amphibians, reptiles, small birds, small mammals) than for larger-bodied species (birds of prey, hedgehogs, lagomorphs, carnivores). Results suggest that widely used surveys at weekly or longer intervals may produce poor estimates of roadkill hotspots, particularly for small-bodied species. Surveying daily or at two-day intervals may be required to achieve high accuracy in hotspot identification for multiple species.

The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from Portugal.

Background: The InBIO Barcoding Initiative (IBI) Orthoptera dataset contains records of 420 specimens covering all the eleven Orthoptera families occurring in Portugal. Specimens were collected in continental Portugal from 2005 to 2021 and were morphologically identified to species level by taxonomists. A total of 119 species were identified corresponding to about 77% of all the orthopteran species known from continental Portugal. New information: DNA barcodes of 54 taxa were made public for the first time at the Barcode of Life Data System (BOLD). Furthermore, the submitted sequences were found to cluster in 129 BINs (Barcode Index Numbers), 35 of which were new additions to the Barcode of Life Data System (BOLD). All specimens have their DNA barcodes publicly accessible through BOLD online database. Stenobothruslineatus is recorded for the first time for continental Portugal. This dataset greatly increases the knowledge on the DNA barcodes and distribution of Orthoptera from Portugal. All DNA extractions and most specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources.

The InBIO Barcoding Initiative Database: DNA barcodes of Iberian Bees.

Background: Bees are important actors in terrestrial ecosystems and are recognised for their prominent role as pollinators. In the Iberian Peninsula, approximately 1,100 bee species are known, with nearly 100 of these species being endemic to the Peninsula. A reference collection of DNA barcodes, based on morphologically identified bee specimens, representing 514 Iberian species, was constructed. The "InBIO Barcoding Initiative Database: DNA Barcodes of Iberian bees" dataset contains records of 1,059 sequenced specimens. The species of this dataset correspond to about 47% of Iberian bee species diversity and 21% of endemic species diversity. For peninsular Portugal only, the corresponding coverage is 71% and 50%. Specimens were collected between 2014 and 2022 and are deposited in the research collection of Thomas Wood (Naturalis Biodiversity Center, The Netherlands), in the FLOWer Lab collection at the University of Coimbra (Portugal), in the Andreia Penado collection at the Natural History and Science Museum of the University of Porto (MHNC-UP) (Portugal) and in the InBIO Barcoding Initiative (IBI) reference collection (Vairão, Portugal). New information: Of the 514 species sequenced, 75 species from five different families are new additions to the Barcode of Life Data System (BOLD) and 112 new BINs were added. Whilst the majority of species were assigned to a single BIN (94.9%), 27 nominal species were assigned to multiple BINs. Although the placement into multiple BINs may simply reflect genetic diversity and variation, it likely also represents currently unrecognised species-level diversity across diverse taxa, such as Amegillaalbigena Lepeletier, 1841, Andrenarussula Lepeletier, 1841, Lasioglossumleucozonium (Schrank, 1781), Nomadafemoralis Morawitz, 1869 and Sphecodesalternatus Smith, 1853. Further species pairs of Colletes, Hylaeus and Nomada were placed into the same BINs, emphasising the need for integrative taxonomy within Iberia and across the Mediterranean Basin more broadly. These data substantially contribute to our understanding of bee genetic diversity and DNA barcodes in Iberia and provide an important baseline for ongoing taxonomic revisions in the West Palaearctic biogeographical region.

The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese moths.

Background: The InBIO Barcoding Initiative (IBI) Dataset - DS-IBILP08 contains records of 2350 specimens of moths (Lepidoptera species that do not belong to the superfamily Papilionoidea). All specimens have been morphologically identified to species or subspecies level and represent 1158 species in total. The species of this dataset correspond to about 42% of mainland Portuguese Lepidoptera species. All specimens were collected in mainland Portugal between 2001 and 2022. All DNA extracts and over 96% of the specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. New information: The authors enabled "The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese moths" in order to release the majority of data of DNA barcodes of Portuguese moths within the InBIO Barcoding Initiative. This dataset increases the knowledge on the DNA barcodes of 1158 species from Portugal belonging to 51 families. There is an increase in DNA barcodes of 205% in Portuguese specimens publicly available. The dataset includes 61 new Barcode Index Numbers. All specimens have their DNA barcodes publicly accessible through BOLD online database and the distribution data can be accessed through the Global Biodiversity Information Facility (GBIF).

Illustrated keys and a DNA barcode reference library of the amphibians and terrestrial reptiles (Amphibia, Reptilia) of São Tomé and Príncipe (Gulf of Guinea, West Africa).

The herpetofauna of São Tomé and Príncipe consists of nine species of amphibians, all endemic, and 21 species of terrestrial reptiles, of which 17 are endemic. Our current knowledge regarding its natural history, ecology, and distribution is limited. Here two important tools are provided to support researchers, conservationists, and local authorities in the identification of the country's herpetofauna: an illustrated key to the herpetofauna of the two islands and surroundings islets and a DNA barcode reference library. The keys allow a rapid and unambiguous morphological identification of all occurring species. The DNA barcodes for the entire herpetofauna of the country were produced from 79 specimens, all of which are deposited in museum collections. The barcodes generated are available in online repositories and can be used to provide unambiguous molecular identification of most of the species. Future applications and use of these tools are briefly discussed.

The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of cuckoo wasps, with the description of new species from the Iberian Peninsula (Hymenoptera, Chrysididae).

Background: DNA barcoding technologies have provided a powerful tool for the fields of ecology and systematics. Here, we present a part of the InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of cuckoo wasps (Hymenoptera, Chrysididae) dataset representing 144 specimens and 103 species, covering approximately 44% of the Iberian and 21% of the European fauna. The InBIO Barcoding Initiative (IBI - DNA Barcoding Portuguese terrestrial invertebrate biodiversity) aims to fill the barcoding gap for the terrestrial invertebrate taxa. All DNA extractions are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources and specimens are deposited in the University of Mons collection (Belgium) and in the Natur-Museum in Lucerne (Switzerland). New information: This dataset increases the knowledge on the DNA barcodes and distribution of 102 species of cuckoo wasps. A total of 52 species, from 11 different genera, were new additions to the Barcode of Life Data System (BOLD), with DNA barcodes for another 44 species added from under-represented taxa in BOLD. All specimens have their DNA barcodes publicly accessible through the BOLD online database. Nine cuckoo wasp species are newly recorded for Portugal. Additionally, two new species for science are described: Chrysiscrossi Rosa, sp. nov. from southern Portugal and Hedychridiumcalcarium Rosa, sp. nov. from eastern Spain. Several taxonomic changes are proposed and Hedychrumrutilans Dahlbom, 1845 is found to consist of two different taxa that can be found in sympatry, Hedychrumrutilans s. str. and Hedychrumviridaureum Tournier, 1877 stat. nov. Stilbumwestermanni Dahlbom, 1845 stat. nov. is confirmed as distinct from Stilbumcalens (Fabricius, 1781), with the latter species not confirmed as present in Iberia; barcoded Stilbum material from Australia is distinct and represents Stilbumamethystium (Fabricius, 1775) sp. resurr.; Portuguese material identified as Hedychridiumchloropygum Buysson, 1888 actually belongs to Hedychridiumcaputaureum Trautmann & Trautmann, 1919, the first confirmed record of this species from Iberia. Philoctetesparvulus (Dahlbom, 1845) is confirmed to be a synonym of Philoctetespunctulatus (Dahlbom, 1845). Chrysislusitanica Bischoff, 1910 is confirmed as a valid species. Chrysishebraeica Linsenmaier, 1959 stat. nov. is raised to species status.

Arachnid Fauna (Araneae and Opiliones) from the Castro Verde Special Protection Area, southern Portugal.

Background: With the increasing recognition of the significance of arachnid conservation, it is crucial to allocate greater efforts towards implementing targeted monitoring programmes. Despite recent studies, our understanding of arachnid populations in Portugal remains limited. This study serves as the initial inventory of arachnids (Araneae and Opiliones) within the Castro Verde Special Protection Area (SPA) located in Beja, southern Portugal. The surveys were conducted during the spring of 2012 across 80 open grasslands that were grazed by cattle and sheep. New information: A total of 71 species of Araneae and two species of Opiliones have been identified. Notably, three spider species, namely Argennasubnigra, Civizelotesibericus and Walckenaeriacucullata, are documented for the first time in Portugal. Additionally, two harvestmen species (Dasylobusibericus and Homalenotusbuchneri) and 14 spider species (Cheiracanthiumpennatum, Haplodrassusrhodanicus, Marinarozelotesminutus, Tapinocybaalgirica, Agraecinalineata, Tibellusmacellus, Talaverapetrensis, Tetragnathaintermedia, Dipoenaumbratilis, Enoplognathadiversa, Neottiurauncinata, Ruborridionmusivum, Theridionpinastri and Xysticusgrallator) are recorded for the first time in the Beja District. The occurrence of each documented species within the SPA, including family and species details, is presented, underscoring the significance of the Castro Verde SPA for arachnid conservation. These findings contribute novel insights into the biodiversity of the Castro Verde SPA, emphasising the necessity of incorporating this area into arachnid diversity conservation efforts.

Identifying freshwater priority areas for cross-taxa interactions.

Information about biotic interactions (e.g. competition, predation, parasitism, diseases, mutualism, allelopathy) is fundamental to better understand species distribution and abundance, ecosystem functioning, and ultimately guide conservation efforts. However, conservation planning often overlooks these important interactions. Here, we aim to demonstrate a new framework to include biotic interactions into Marxan. For that, we use freshwater mussels and fish interaction (as mussels rely on fishes to complete their life cycle) in the Douro River basin (Iberian Peninsula) as a case study. While doing that, we also test the importance of including biotic interactions into conservation planning exercises, by running spatial prioritisation analysis considering either: 1) only the target species (freshwater mussels); 2) freshwater mussels and their obligatory hosts (freshwater fishes); 3) freshwater mussels, fishes and their interactions. With this framework we found that biotic interactions tend to be underrepresented when the data on both freshwater mussels and fishes is not simultaneously included in the spatial prioritisation. Overall, the priority areas selected across all scenarios are mostly located in the western part of the Douro River basin, where most freshwater mussels and fishes still occur. Given the low overlap of priority areas identified here and the current Natura 2000 network, our approach may be useful for establishing (or enlarging) protected areas, especially in light of the EU Biodiversity Strategy for 2030. Also, this work may provide guidance for future habitat restoration and management of main threats to freshwater biodiversity.

DNA Barcode library of the endemic-rich avifauna of the oceanic islands of the Gulf of Guinea.

Background: The BioSTP: DNA Barcoding of endemic birds from oceanic islands of the Gulf of Guinea dataset contains records of 155 bird specimens belonging to 56 species in 23 families, representing over 80% of the diversity of the breeding landbird community. All specimens were collected on Príncipe, São Tomé and Annobón Islands between 2002 and 2021 and morphologically identified to species or subspecies level by qualified ornithologists. The dataset includes all endemic species and 3/4 of the extant endemic subspecies of the islands. This dataset is the second release by BioSTP and it greatly increases the knowledge on the DNA barcodes of Gulf of Guinea birds. All DNA extractions are deposited at Associação BIOPOLIS - CIBIO, Research Center in Biodiversity and Genetic Resources. New information: The dataset includes DNA barcodes for all 29 endemic bird species and for 11 of the 15 extant endemic bird subspecies from the oceanic islands of the Gulf of Guinea. This is the first major DNA barcode set of African birds. The three endemic subspecies of Crithagrarufobrunnea, an island endemic with three allopatric populations within the Archipelago, are also represented. Additionally, we obtained DNA barcodes for 16 of the 21 non-endemic landbirds and for one vagrant (Sylviacommunis). In total, forty-one taxa were new additions to the Barcode of Life Data System (BOLD), with another 11 corresponding to under-represented taxa in BOLD. Furthermore, the submitted sequences were found to cluster in 55 Barcode Index Numbers (BINs), 37 of which were new to BOLD. All specimens have their DNA barcodes publicly accessible through BOLD online database and GenBank.