The limited spatial scale of dispersal in soil arthropods revealed with whole‐community haplotype‐level metabarcoding

Soil arthropod communities are highly diverse and critical for ecosystem functioning. However, our knowledge of spatial structure and the underlying processes of community assembly are scarce, hampered by limited empirical data on species diversity and turnover. We implement a high‐throughput sequencing approach to generate comparative data for thousands of arthropods at three hierarchical levels: genetic, species and supra‐specific lineages. A joint analysis of the spatial arrangement across these levels can reveal the predominant processes driving the variation in biological assemblages at the local scale. This multihierarchical approach was performed using haplotype‐level COI metabarcoding of entire communities of mites, springtails and beetles from three Iberian mountain regions. Tens of thousands of specimens were extracted from deep and superficial soil layers and produced comparative phylogeographic data for >1,000 codistributed species and nearly 3,000 haplotypes. Local assemblage composition differed greatly between grasslands and forests and, within each habitat, showed strong spatial structure and high endemicity. Distance decay was high at all levels, even at the scale of a few kilometres or less. The local distance decay patterns were self‐similar for the haplotypes and higher hierarchical entities, and this fractal structure was similar in all regions, suggesting that uniform processes of limited dispersal determine local‐scale community assembly. Our results from whole‐community metabarcoding provide insight into how dispersal limitations constrain mesofauna community structure within local spatial settings over evolutionary timescales. If generalized across wider areas, the high turnover and endemicity in the soil locally may indicate extremely high richness globally, challenging our current estimations of total arthropod diversity on Earth.This research was funded by Newton International Program, UK, to PA and the NHM Biodiversity Initiative to APV. PA was supported by postdoctoral grants from the Royal Society (Newton International Program, UK) and the Spanish Ministry of Economy and Competitiveness (MINECO, Spain) within the Juan de la Cierva Formación Program. CA was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Spain; CGL2015‐74178‐JIN MINECO/FEDER, UE).Peer reviewe

Biología floral y reproductiva de la planta carnívora Drosophyllum lusitanicum (L.) Link (Drosophyllaceae); una especie endémica, singular y amenazada

Drosophyllum lusitanicum es una especie amenazada y endémica del SO de la Península Ibérica y N de Marruecos, que se encuentra protegida por distintas legislaciones. Por ello, el estudio de su biología reproductiva es importante para establecer las medidas de conservación adecuadas. En este trabajo se estudia la importancia de los polinizadores y de la xenogamia en el éxito reproductivo en dos poblaciones centrales de esta especie, teniendo en cuenta que presenta un comportamiento fuertemente autógamo en poblaciones periféricas. Se realizaron experimentos de polinización, observando un aumento de la tasa de fructificación y de la producción de semillas tras la acción de polinizadores y tras cruces xenógamos, principalmente en poblaciones y plantas jóvenes. Asimismo, se analizaron las diferencias morfológicas entre clases de edad, obteniendo mayor separación flor-trampa en individuos de primer año reproductivo que en individuos de mayor edad. Drosophyllum es insectívora y polinizada por insectos, pudiendo sufrir un conflicto entre polinización y captura de presas. Se realizaron censos de polinizadores y de presas para estimar el solapamiento polinizador-presa y, a diferencia de estudios anteriores, la presencia de polinizadores fue importante, con predominancia de pequeños Coleoptera e Hymenoptera (abejas). Por otro lado, las principales presas fueron Diptera, Microlepidoptera e Hymenoptera (hormigas), mostrando así un escaso solapamiento polinizador-presa, que solamente afectó a dos especies (Enicopus sp. y Lasioglosossum sp.). Esto sugiere que Drosophyllum previene el posible conflicto mediante la separación flor-trampa y la presencia de flores llamativas y aparentemente seguras para los polinizadores.Drosophyllum lusitanicum is a threatened, redlisted species endemic to the SW Iberian Peninsula and N Morocco. So, the study about its floral and reproductive biology is important to help create suitable conservation strategies. In this thesis, the role of pollinators reproductive success have been studied in two central populations, taking into account that this species shows a strong autogamous behaviour in peripheral populations. Pollination experiments were carried out and it was observed both higher fruit set and seed set after pollinator activity and cross-pollinations, particularly in plants on its first reproductive year. Additionally, morphological differences between age class were analysed, showing higher flower-trap (leaf) separation in first reproductive-year plants that in mature ones. Drosophyllum is insectivorous and insect-pollinated, which might make it suffer a conflict between pollination and prey capture. Pollinator and prey censuses were carried out to estimate pollinator-prey overlap and, in contrast to previous studies, pollinators were important, particularly small Coleoptera and Hymenoptera (bees). On the other hand, the main prey insects were Diptera, Microlepidoptera and Hymenoptera (ants), showing a scarce pollinator-prey overlap, which only affected two insect species (Enicopus sp. and Lasioglosossum sp.). It is inferred that Drosophyllum avoids a possible pollinator-prey conflict with spatial flower-trap separation and flashy and apparently safe flowers for pollinators

Dispersal limitation: Evolutionary origins and consequences in arthropods

Niche and dispersal ability are key traits for explaining the geographical structuring of species into discrete populations, and its evolutionary significance. Beyond their individual effects, the interplay between species niche and its geographic limits, together with the evolutionary lability of dispersal ability, can underpin trait diversification and speciation when exposed to gradients of selection. In this issue of Molecular Ecology, two complementary papers demonstrate how evolutionary lability for dispersal ability linked to niche shift can drive such a model in a context that includes selection. Both papers investigate the evolution of dispersal limitation in arthropods across altitudinal gradients, but using taxa with contrasting ecologies. McCulloch et al. (2019) investigate the evolution of wing loss at higher altitudes in stoneflies, a taxon inhabiting freshwater systems. Suzuki et al. (2019) report a similar phenomenon, but involving wing reduction at higher altitudes in scorpionflies, a taxon associated with moist terrestrial habitats. Here, we compare and contrast the results of both studies to explore their broader implications for understanding diversification and speciation within arthropods.Peer Reviewe

Dispersal limitation: Evolutionary origins and consequences in arthropods

Niche and dispersal ability are key traits for explaining the geographical structuring of species into discrete populations, and its evolutionary significance. Beyond their individual effects, the interplay between species niche and its geographic limits, together with the evolutionary lability of dispersal ability, can underpin trait diversification and speciation when exposed to gradients of selection. In this issue of Molecular Ecology, two complementary papers demonstrate how evolutionary lability for dispersal ability linked to niche shift can drive such a model in a context that includes selection. Both papers investigate the evolution of dispersal limitation in arthropods across altitudinal gradients, but using taxa with contrasting ecologies. McCulloch et al. (2019) investigate the evolution of wing loss at higher altitudes in stoneflies, a taxon inhabiting freshwater systems. Suzuki et al. (2019) report a similar phenomenon, but involving wing reduction at higher altitudes in scorpionflies, a taxon associated with moist terrestrial habitats. Here, we compare and contrast the results of both studies to explore their broader implications for understanding diversification and speciation within arthropods.Peer Reviewe

Attract them anyway: Benefits of large, showy flowers in a highly autogamous, carnivorous plant species

© The Authors 2016Reproductive biology of carnivorous plants has largely been studied on species that rely on insects as pollinators and prey, creating potential conflicts. Autogamous pollination, although present in some carnivorous species, has received less attention. In angiosperms, autogamous self-fertilization is expected to lead to a reduction in flower size, thereby reducing resource allocation to structures that attract pollinators. A notable exception is the carnivorous pyrophyte Drosophyllum lusitanicum (Drosophyllaceae), which has been described as an autogamous selfing species but produces large, yellow flowers. Using a flower removal and a pollination experiment, we assessed, respectively, whether large flowers in this species may serve as an attracting device to prey insects or whether previously reported high selfing rates for this species in peripheral populations may be lower in more central, less isolated populations. We found no differences between flower-removed plants and intact, flowering plants in numbers of prey insects trapped. We also found no indication of reduced potential for autogamous reproduction, in terms of either seed set or seed size. However, our results showed significant increases in seed set of bagged, hand-pollinated flowers and unbagged flowers exposed to insect visitation compared with bagged, non-manipulated flowers that could only self-pollinate autonomously. Considering that the key life-history strategy of this pyrophytic species is to maintain a viable seed bank, any increase in seed set through insect pollinator activity would increase plant fitness. This in turn would explain the maintenance of large, conspicuous flowers in a highly autogamous, carnivorous plant.This study was supported by the Spanish Ministerio de Economía y Competitividad (project BREATHAL; Geographical barrier, habitat fragmentation and vulnerability of endemics: Biodiversity patterns of the Mediterranean heathland across the Strait of Gibraltar, CGL2011-28759).Peer Reviewe

Testing landscape effects on the evolution of island arthropod assemblages

Trabajo presentado en el II International Conference on Island Evolution, Ecology and Conservation, celebrado en Angra do Heroísmo, Islas Azores (Portugal) del 18 al 22 de julio de 2016.Geographic isolation is a recognised driver of speciation within oceanic island archipelagos, with the chance colonisation and establishment of species from one island to another being a cornerstone for the origin of new evolutionary lineages. The importance of geographic isolation within islands is less clear, as current distributions of closely related species may not be reflective of the geography of their shared ancestors. It may also be that physical geographic distance has been less important than ecological distance for the initiation of the speciation process within islands. We are addressing this information gap by testing for geographical and ecological isolation effects through the sampling of whole arthropod communities within insular forest biomes. We combine standardised ecological sampling with DNA sequencing and apply both traditional (FST-based) and novel (Bayesian MCMC) analyses of molecular data. In this presentation I will outline our study system and our methodological approach for the investigation of geographic effects on genetic differentiation among the Coleoptera fauna sampled within a topographically complex insular forest biome. I will illustrate our approach with some preliminary results and discuss their significance.Peer Reviewe

Metabarcoding for biodiversity inventory blind spots: A test case using the beetle fauna of an insular cloud forest

Soils harbour a rich arthropod fauna, but many species are still not formally described (Linnaean shortfall) and the distribution of those already described is poorly understood (Wallacean shortfall). Metabarcoding holds much promise to fill this gap, however, nuclear copies of mitochondrial genes, and other artefacts lead to taxonomic inflation, which compromise the reliability of biodiversity inventories. Here, we explore the potential of a bioinformatic approach to jointly “denoise” and filter nonauthentic mitochondrial sequences from metabarcode reads to obtain reliable soil beetle inventories and address open questions in soil biodiversity research, such as the scale of dispersal constraints in different soil layers. We sampled cloud forest arthropod communities from 49 sites in the Anaga peninsula of Tenerife (Canary Islands). We performed whole organism community DNA (wocDNA) metabarcoding, and built a local reference database with COI barcode sequences of 310 species of Coleoptera for filtering reads and the identification of metabarcoded species. This resulted in reliable haplotype data after considerably reducing nuclear mitochondrial copies and other artefacts. Comparing our results with previous beetle inventories, we found: (i) new species records, potentially representing undescribed species; (ii) new distribution records, and (iii) validated phylogeographic structure when compared with traditional sequencing approaches. Analyses also revealed evidence for higher dispersal constraint within deeper soil beetle communities, compared to those closer to the surface. The combined power of barcoding and metabarcoding contribute to mitigate the important shortfalls associated with soil arthropod diversity data, and thus address unresolved questions for this vast biodiversity fraction.We thank Paolo Audisio, Pedro Oromí, Antonio Machado, and Rafael García for their help in identifying beetle specimens, Víctor Noguerales for fruitful discussions, and reviewers and editor of this paper for their valuable comments and suggestions. We also thank the Cabildo Insular de Tenerife for allowing the field sample collection (AFF-107/17 [sigma no. 2017-00572]; AFF 137/21 [sigma no. 2021-01285]). This work was supported by projects CGL2015-74178-JIN (AEI, Spain/FEDER, EU) and SOILBIODIV (H2020-MSCA-IF-2015; award number: 705639) awarded to CA, CGL2017-85718-P and PID2020-116788GB-I00 (AEI, Spain/FEDER, EU) and 2017RCE03 (Fundación CajaCanarias/Obra Social “La Caixa”) awarded to BCE. PA was additionally funded through a Junior Leader Fellowship (LCF/BQ/PR21/11840006) by “la Caixa” Foundation (ID 100010434) and the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement 847648.Peer reviewe

Flightlessness in insects enhances diversification and determines assemblage structure across whole communities

Dispersal limitation has been recurrently suggested to shape both macroecological patterns and microevolutionary processes within invertebrates. However, because of potential interactions among biological, environmental, temporal, and spatial variables, causal links among flight-related traits, diversification and spatial patterns of community assembly remain elusive. Integrating genetic variation within species across whole insect assemblages, within a simplified spatial and environmental framework, can be used to reduce the impact of these potentially confounding variables. Here, we used standardized sampling and mitochondrial DNA sequencing for a whole-community characterization of the beetle fauna inhabiting a singular forested habitat (laurel forest) within an oceanic archipelago setting (Canary Islands). The spatial structure of species assemblages together with species-level genetic diversity was compared at the archipelago and island scales for 104 winged and 110 wingless beetle lineages. We found that wingless beetle lineages have: (i) smaller range sizes at the archipelago scale, (ii) lower representation in younger island communities, (iii) stronger population genetic structure, and (iv) greater spatial structuring of species assemblages between and within islands. Our results reveal that dispersal limitation is a fundamental trait driving diversity patterns at multiple hierarchical levels by promoting spatial diversification and affecting the spatial configuration of entire assemblages at both island and archipelago scales.Peer reviewe

Long-term cloud forest response to climate warming revealed by insect speciation history

Montane cloud forests are areas of high endemism, and are one of the more vulnerable terrestrial ecosystems to climate change. Thus, understanding how they both contribute to the generation of biodiversity, and will respond to ongoing climate change, are important and related challenges. The widely accepted model for montane cloud forest dynamics involves upslope forcing of their range limits with global climate warming. However, limited climate data provides some support for an alternative model, where range limits are forced downslope with climate warming. Testing between these two models is challenging, due to the inherent limitations of climate and pollen records. We overcome this with an alternative source of historical information, testing between competing model predictions using genomic data and demographic analyses for a species of beetle tightly associated to an oceanic island cloud forest. Results unequivocally support the alternative model: populations that were isolated at higher elevation peaks during the Last Glacial Maximum are now in contact and hybridizing at lower elevations. Our results suggest that genomic data are a rich source of information to further understand how montane cloud forest biodiversity originates, and how it is likely to be impacted by ongoing climate change

Validated removal of nuclear pseudogenes and sequencing artefacts from mitochondrial metabarcode data

Metabarcoding of Metazoa using mitochondrial genes may be confounded by both the accumulation of PCR and sequencing artefacts and the co-amplification of nuclear mitochondrial pseudogenes (NUMTs). The application of read abundance thresholds and denoising methods is efficient in reducing noise accompanying authentic mitochondrial amplicon sequence variants (ASVs). However, these procedures do not fully account for the complex nature of concomitant sequences and the highly variable DNA contribution of specimens in a metabarcoding sample. We propose, as a complement to denoising, the metabarcoding Multidimensional Abundance Threshold Evaluation (metaMATE) framework, a novel approach that allows comprehensive examination of multiple dimensions of abundance filtering and the evaluation of the prevalence of unwanted concomitant sequences in denoised metabarcoding datasets. metaMATE requires a denoised set of ASVs as input, and designates a subset of ASVs as being either authentic (mitochondrial DNA haplotypes) or nonauthentic ASVs (NUMTs and erroneous sequences) by comparison to external reference data and by analysing nucleotide substitution patterns. metaMATE (i) facilitates the application of read abundance filtering strategies, which are structured with regard to sequence library and phylogeny and applied for a range of increasing abundance threshold values, and (ii) evaluates their performance by quantifying the prevalence of nonauthentic ASVs and the collateral effects on the removal of authentic ASVs. The output from metaMATE facilitates decision-making about required filtering stringency and can be used to improve the reliability of intraspecific genetic information derived from metabarcode data. The framework is implemented in the metaMATE software (available at ).C. A. was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Spain) (CGL2015‐74178‐JIN) and Fundación CajaCanarias/Obra social “la Caixa.” B. C. E. was supported by the project CGL2017‐85718‐P (AEI, Spain/FEDER, EU). P. A., T. J. C., B. C. E. and A. P. V. were supported by the iBioGen project funded by the H2020 European Research Council, Grant/Award Number: 810729. We extend our gratitude to the regional governments of Andalucía and Canarias (Spain) for facilitating collecting of samples, to Jesús Arribas for assistance with field sampling, and Carlos Martínez for the mathematical advice.Peer reviewe