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.

Inferring the ecological and evolutionary determinants of community genetic diversity.

Understanding the relative contributions of ecological and evolutionary processes to the structuring of ecological communities is needed to improve our ability to predict how communities may respond to future changes in an increasingly human-modified world. Metabarcoding methods make it possible to gather population genetic data for all species within a community, unlocking a new axis of data to potentially unveil the origins and maintenance of biodiversity at local scales. Here, we present a new eco-evolutionary simulation model for investigating community assembly dynamics using metabarcoding data. The model makes joint predictions of species abundance, genetic variation, trait distributions and phylogenetic relationships under a wide range of parameter settings (e.g. high speciation/low dispersal or vice versa) and across a range of community states, from pristine and unmodified to heavily disturbed. We first demonstrate that parameters governing metacommunity and local community processes leave detectable signatures in simulated biodiversity data axes. Next, using a simulation-based machine learning approach we show that neutral and non-neutral models are distinguishable and that reasonable estimates of several model parameters within the local community can be obtained using only community-scale genetic data, while phylogenetic information is required to estimate those describing metacommunity dynamics. Finally, we apply the model to soil microarthropod metabarcoding data from the Troodos mountains of Cyprus, where we find that communities in widespread forest habitats are structured by neutral processes, while high-elevation and isolated habitats act as an abiotic filter generating non-neutral community structure. We implement our model within the ibiogen R package, a package dedicated to the investigation of island, and more generally community-scale, biodiversity using community-scale genetic data.

Community metabarcoding reveals the relative role of environmental filtering and spatial processes in metacommunity dynamics of soil microarthropods across a mosaic of montane forests.

Disentangling the relative role of environmental filtering and spatial processes in driving metacommunity structure across mountainous regions remains challenging, as the way we quantify spatial connectivity in topographically and environmentally heterogeneous landscapes can influence our perception of which process predominates. More empirical data sets are required to account for taxon- and context-dependency, but relevant research in understudied areas is often compromised by the taxonomic impediment. Here we used haplotype-level community DNA metabarcoding, enabled by stringent filtering of amplicon sequence variants (ASVs), to characterize metacommunity structure of soil microarthropod assemblages across a mosaic of five forest habitats on the Troodos mountain range in Cyprus. We found similar ß diversity patterns at ASV and species (OTU, operational taxonomic unit) levels, which pointed to a primary role of habitat filtering resulting in the existence of largely distinct metacommunities linked to different forest types. Within-habitat turnover was correlated to topoclimatic heterogeneity, again emphasizing the role of environmental filtering. However, when integrating landscape matrix information for the highly fragmented Quercus alnifolia habitat, we also detected a major role of spatial isolation determined by patch connectivity, indicating that stochastic and niche-based processes synergistically govern community assembly. Alpha diversity patterns varied between ASV and OTU levels, with OTU richness decreasing with elevation and ASV richness following a longitudinal gradient, potentially reflecting a decline of genetic diversity eastwards due to historical pressures. Our study demonstrates the utility of haplotype-level community metabarcoding for characterizing metacommunity structure of complex assemblages and improving our understanding of biodiversity dynamics across mountainous landscapes worldwide.

Collective and harmonized high throughput barcoding of insular arthropod biodiversity: Toward a Genomic Observatories Network for islands.

Current understanding of ecological and evolutionary processes underlying island biodiversity is heavily shaped by empirical data from plants and birds, although arthropods comprise the overwhelming majority of known animal species, and as such can provide key insights into processes governing biodiversity. Novel high throughput sequencing (HTS) approaches are now emerging as powerful tools to overcome limitations in the availability of arthropod biodiversity data, and hence provide insights into these processes. Here, we explored how these tools might be most effectively exploited for comprehensive and comparable inventory and monitoring of insular arthropod biodiversity. We first reviewed the strengths, limitations and potential synergies among existing approaches of high throughput barcode sequencing. We considered how this could be complemented with deep learning approaches applied to image analysis to study arthropod biodiversity. We then explored how these approaches could be implemented within the framework of an island Genomic Observatories Network (iGON) for the advancement of fundamental and applied understanding of island biodiversity. To this end, we identified seven island biology themes at the interface of ecology, evolution and conservation biology, within which collective and harmonized efforts in HTS arthropod inventory could yield significant advances in island biodiversity research.

Dispersal ability and its consequences for population genetic differentiation and diversification.

Dispersal ability is known to influence geographical structuring of genetic variation within species, with a direct relationship between low vagility and population genetic structure, which can potentially give rise to allopatric speciation. However, our general understanding of the relationship between dispersal ability, population differentiation and lineage diversification is limited. To address this issue, we sampled mitochondrial DNA variation within lineages of beetles and spiders across the Canary Islands to explore the relationships between dispersal ability, differentiation within lineages and diversification. We found positive relationships between population genetic structure and diversification for both beetles and spiders. Comparisons between dispersive and non-dispersive lineages revealed significant differences for both lineage differentiation and diversification. For both taxa, non-dispersive lineages had stronger population genetic structure. Genus-level endemic species richness and proxies for diversification rate within genera were higher in non-dispersive taxa for both beetles and spiders. Comparisons of average and maximum node divergences within genera suggest that species turnover may be higher in non-dispersive genera. Our results reveal a model where dispersal limitation may shape the diversity of lineages across evolutionary timescales by positively influencing intraspecific and species diversity, moderated by higher extinction rates compared to more dispersive lineages.

Community assembly and metaphylogeography of soil biodiversity: Insights from haplotype-level community DNA metabarcoding within an oceanic island.

Most of our understanding of island diversity comes from the study of aboveground systems, while the patterns and processes of diversification and community assembly for belowground biotas remain poorly understood. Here, we take advantage of a relatively young and dynamic oceanic island to advance our understanding of ecoevolutionary processes driving community assembly within soil mesofauna. Using whole organism community DNA (wocDNA) metabarcoding and the recently developed metaMATE pipeline, we have generated spatially explicit and reliable haplotype-level DNA sequence data for soil mesofaunal assemblages sampled across the four main habitats within the island of Tenerife. Community ecological and metaphylogeographic analyses have been performed at multiple levels of genetic similarity, from haplotypes to species and supraspecific groupings. Broadly consistent patterns of local-scale species richness across different insular habitats have been found, whereas local insular richness is lower than in continental settings. Our results reveal an important role for niche conservatism as a driver of insular community assembly of soil mesofauna, with only limited evidence for habitat shifts promoting diversification. Furthermore, support is found for a fundamental role of habitat in the assembly of soil mesofauna, where habitat specialism is mainly due to colonization and the establishment of preadapted species. Hierarchical patterns of distance decay at the community level and metaphylogeographical analyses support a pattern of geographic structuring over limited spatial scales, from the level of haplotypes through to species and lineages, as expected for taxa with strong dispersal limitations. Our results demonstrate the potential for wocDNA metabarcoding to advance our understanding of biodiversity.

Mitochondrial Metagenomics Reveals the Ancient Origin and Phylodiversity of Soil Mites and Provides a Phylogeny of the Acari.

High-throughput DNA methods hold great promise for phylogenetic analysis of lineages that are difficult to study with conventional molecular and morphological approaches. The mites (Acari), and in particular the highly diverse soil-dwelling lineages, are among the least known branches of the metazoan Tree-of-Life. We extracted numerous minute mites from soils in an area of mixed forest and grassland in southern Iberia. Selected specimens representing the full morphological diversity were shotgun sequenced in bulk, followed by genome assembly of short reads from the mixture, which produced >100 mitochondrial genomes representing diverse acarine lineages. Phylogenetic analyses in combination with taxonomically limited mitogenomes available publicly resulted in plausible trees defining basal relationships of the Acari. Several critical nodes were supported by ancestral-state reconstructions of mitochondrial gene rearrangements. Molecular calibration placed the minimum age for the common ancestor of the superorder Acariformes, which includes most soil-dwelling mites, to the Cambrian-Ordovician (likely within 455-552 Ma), whereas the origin of the superorder Parasitiformes was placed later in the Carboniferous-Permian. Most family-level taxa within the Acariformes were dated to the Jurassic and Triassic. The ancient origin of Acariformes and the early diversification of major extant lineages linked to the soil are consistent with a pioneering role for mites in building the earliest terrestrial ecosystems.

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.

Dispersal limitations and long-term persistence drive differentiation from haplotypes to communities within a tropical sky-island: Evidence from community metabarcoding.

Neutral theory proposes that dispersal stochasticity is one of the main drivers of local diversity. Haplotypes-level genetic variation can now be efficiently sampled from across whole communities, thus making it possible to test neutral predictions from the genetic to species-level diversity, and higher. However, empirical data is still limited, with the few studies to date coming from temperate latitudes. Here, we focus on a tropical mountain within the Transmexican Volcanic Belt to evaluate spatially fine-scale patterns of arthropod community assembly to understand the role of dispersal limitation and landscape features as drivers of diversity. We sampled whole-communities of arthropods for eight orders at a spatial scale ranging from 50 m to 19 km, using whole community metabarcoding. We explored multiple hierarchical levels, from individual haplotypes to lineages at 0.5, 1.5, 3, 5, and 7.5% similarity thresholds, to evaluate patterns of richness, turnover, and distance decay of similarity with isolation-by-distance and isolation-by-resistance (costs to dispersal given by landscape features) approaches. Our results showed that distance and altitude influence distance decay of similarity at all hierarchical levels. This holds for arthropod groups of contrasting dispersal abilities, but with different strength depending on the spatial scale. Our results support a model where local-scale differentiation mediated by dispersal constraints, combined with long-term persistence of lineages, is an important driver of diversity within tropical sky islands.

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.

Connecting high-throughput biodiversity inventories: Opportunities for a site-based genomic framework for global integration and synthesis.

High-throughput sequencing (HTS) is increasingly being used for the characterization and monitoring of biodiversity. If applied in a structured way, across broad geographical scales, it offers the potential for a much deeper understanding of global biodiversity through the integration of massive quantities of molecular inventory data generated independently at local, regional and global scales. The universality, reliability and efficiency of HTS data can potentially facilitate the seamless linking of data among species assemblages from different sites, at different hierarchical levels of diversity, for any taxonomic group and regardless of prior taxonomic knowledge. However, collective international efforts are required to optimally exploit the potential of site-based HTS data for global integration and synthesis, efforts that at present are limited to the microbial domain. To contribute to the development of an analogous strategy for the nonmicrobial terrestrial domain, an international symposium entitled "Next Generation Biodiversity Monitoring" was held in November 2019 in Nicosia (Cyprus). The symposium brought together evolutionary geneticists, ecologists and biodiversity scientists involved in diverse regional and global initiatives using HTS as a core tool for biodiversity assessment. In this review, we summarize the consensus that emerged from the 3-day symposium. We converged on the opinion that an effective terrestrial Genomic Observatories network for global biodiversity integration and synthesis should be spatially led and strategically united under the umbrella of the metabarcoding approach. Subsequently, we outline an HTS-based strategy to collectively build an integrative framework for site-based biodiversity data generation.

Climate drives community-wide divergence within species over a limited spatial scale: evidence from an oceanic island.

Geographic isolation substantially contributes to species endemism on oceanic islands when speciation involves the colonisation of a new island. However, less is understood about the drivers of speciation within islands. What is lacking is a general understanding of the geographic scale of gene flow limitation within islands, and thus the spatial scale and drivers of geographical speciation within insular contexts. Using a community of beetle species, we show that when dispersal ability and climate tolerance are restricted, microclimatic variation over distances of only a few kilometres can maintain strong geographic isolation extending back several millions of years. Further to this, we demonstrate congruent diversification with gene flow across species, mediated by Quaternary climate oscillations that have facilitated a dynamic of isolation and secondary contact. The unprecedented scale of parallel species responses to a common environmental driver for evolutionary change has profound consequences for understanding past and future species responses to climate variation.

Irreversible habitat specialization does not constrain diversification in hypersaline water beetles.

Specialization to extreme environments is often considered an evolutionary dead-end, leading to irreversible adaptations and reduced evolvability. There is, however, mixed evidence for this macroevolutionary pattern, and limited data from speciose lineages. Here, we tested the effect of habitat specialization to hypersaline waters in the diversification rates of aquatic beetles of the genus Ochthebius (Coleoptera, Hydraenidae), using a molecular phylogeny with more than 50% of the 546 recognized species, including representatives of all but one of the nine recognized subgenera and 17 species groups. Phylogenies were built combining mitochondrial and nuclear genes, with the addition of 42 mitochondrial genomes. Using Bayesian methods of character reconstruction, we show that hypersaline tolerance is an irreversible ecological specialization that arose multiple times. Two lineages of Ochthebius experienced a significant increase in diversification rates, one of them inhabiting hypersaline waters, but there was no overall correlation with habitat or any significant decrease in diversification rates despite the irreversibility of hypersaline tolerance. Our study tested for the first time the impact of hypersaline habitat specialization on diversification rates, finding no support for it to be an evolutionary dead-end. On the contrary, multiple and ancient lineages fully adapted to these extreme osmotic conditions have persisted and diversified over a long evolutionary timescale.

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.

Why the COI barcode should be the community DNA metabarcode for the metazoa.

Metabarcoding of complex metazoan communities is increasingly being used to measure biodiversity in terrestrial, freshwater and marine ecosystems, revolutionizing our ability to observe patterns and infer processes regarding the origin and conservation of biodiversity. A fundamentally important question is which genetic marker to amplify, and although the mitochondrial cytochrome oxidase subunit I (COI) gene is one of the more widely used markers in metabarcoding for the Metazoa, doubts have recently been raised about its suitability. We argue that (a) the extensive coverage of reference sequence databases for COI; (b) the variation it presents; (c) the comparative advantages for denoising protein-coding genes; and (d) recent advances in DNA sequencing protocols argue in favour of standardizing for the use of COI for metazoan community samples. We also highlight where research efforts should focus to maximize the utility of metabarcoding.

Metabarcoding of freshwater invertebrates to detect the effects of a pesticide spill.

Biomonitoring underpins the environmental assessment of freshwater ecosystems and guides management and conservation. Current methodology for surveys of (macro)invertebrates uses coarse taxonomic identification where species-level resolution is difficult to obtain. Next-generation sequencing of entire assemblages (metabarcoding) provides a new approach for species detection, but requires further validation. We used metabarcoding of invertebrate assemblages with two fragments of the cox1 "barcode" and partial nuclear ribosomal (SSU) genes, to assess the effects of a pesticide spill in the River Kennet (southern England). Operational taxonomic unit (OTU) recovery was tested under 72 parameters (read denoising, filtering, pair merging and clustering). Similar taxonomic profiles were obtained under a broad range of parameters. The SSU marker recovered Platyhelminthes and Nematoda, missed by cox1, while Rotifera were only amplified with cox1. A reference set was created from all available barcode entries for Arthropoda in the BOLD database and clustered into OTUs. The River Kennet metabarcoding produced matches to 207 of these reference OTUs, five times the number of species recognized with morphological monitoring. The increase was due to the following: greater taxonomic resolution (e.g., splitting a single morphotaxon "Chironomidae" into 55 named OTUs); splitting of Linnaean binomials into multiple molecular OTUs; and the use of a filtration-flotation protocol for extraction of minute specimens (meiofauna). Community analyses revealed strong differences between "impacted" vs. "control" samples, detectable with each gene marker, for each major taxonomic group, and for meio- and macrofaunal samples separately. Thus, highly resolved taxonomic data can be extracted at a fraction of the time and cost of traditional nonmolecular methods, opening new avenues for freshwater invertebrate biodiversity monitoring and molecular ecology.

The contribution of mitochondrial metagenomics to large-scale data mining and phylogenetic analysis of Coleoptera.

A phylogenetic tree at the species level is still far off for highly diverse insect orders, including the Coleoptera, but the taxonomic breadth of public sequence databases is growing. In addition, new types of data may contribute to increasing taxon coverage, such as metagenomic shotgun sequencing for assembly of mitogenomes from bulk specimen samples. The current study explores the application of these techniques for large-scale efforts to build the tree of Coleoptera. We used shotgun data from 17 different ecological and taxonomic datasets (5 unpublished) to assemble a total of 1942 mitogenome contigs of >3000 bp. These sequences were combined into a single dataset together with all mitochondrial data available at GenBank, in addition to nuclear markers widely used in molecular phylogenetics. The resulting matrix of nearly 16,000 species with two or more loci produced trees (RAxML) showing overall congruence with the Linnaean taxonomy at hierarchical levels from suborders to genera. We tested the role of full-length mitogenomes in stabilizing the tree from GenBank data, as mitogenomes might link terminals with non-overlapping gene representation. However, the mitogenome data were only partly useful in this respect, presumably because of the purely automated approach to assembly and gene delimitation, but improvements in future may be possible by using multiple assemblers and manual curation. In conclusion, the combination of data mining and metagenomic sequencing of bulk samples provided the largest phylogenetic tree of Coleoptera to date, which represents a summary of existing phylogenetic knowledge and a defensible tree of great utility, in particular for studies at the intra-familial level, despite some shortcomings for resolving basal nodes.

Terra incognita of soil biodiversity: unseen invasions under our feet.

Whilst cartographers of the 19th century endeavoured to chart the last unknown lands, the great challenge for biologists in the 21st century is to fill the gaps on the biodiversity map of the Earth. And one of the largest gaps concerns the biodiversity of soils, a terra incognita right under our feet. The study of soil biodiversity, and particularly the complex communities of small invertebrates, has suffered from a severe 'taxonomic impediment' (Decaëns ) leading to great uncertainties about total species richness, phylogenetic diversity, geographical structure, temporal dynamics of soil organisms, and consequently about their role on ecosystem function (Bardgett & van der Putten ). However, the revolution in high-throughput sequencing is now revealing the hidden biodiversity of the soil with unprecedented detail (e.g. Arribas et al. ). In a noteworthy from the Cover article in this issue of Molecular Ecology, Cicconardi et al. () apply these new tools to study soil communities of Collembola in three distant oceanic islands of volcanic origin, obtaining a striking result: only 38 of 70 species (54%) are exclusively found in a single island, with the remaining shared among islands or with other distant regions, suggesting a massive recent introduction of soil species, whose impact is entirely unknown.

The chicken or the egg? Adaptation to desiccation and salinity tolerance in a lineage of water beetles.

Transitions from fresh to saline habitats are restricted to a handful of insect lineages, as the colonization of saline waters requires specialized mechanisms to deal with osmotic stress. Previous studies have suggested that tolerance to salinity and desiccation could be mechanistically and evolutionarily linked, but the temporal sequence of these adaptations is not well established for individual lineages. We combined molecular, physiological and ecological data to explore the evolution of desiccation resistance, hyporegulation ability (i.e., the ability to osmoregulate in hyperosmotic media) and habitat transitions in the water beetle genus Enochrus subgenus Lumetus (Hydrophilidae). We tested whether enhanced desiccation resistance evolved before increases in hyporegulation ability or vice versa, or whether the two mechanisms evolved in parallel. The most recent ancestor of Lumetus was inferred to have high desiccation resistance and moderate hyporegulation ability. There were repeated shifts between habitats with differing levels of salinity in the radiation of the group, those to the most saline habitats generally occurring more rapidly than those to less saline ones. Significant and accelerated changes in hyporegulation ability evolved in parallel with smaller and more progressive increases in desiccation resistance across the phylogeny, associated with the colonization of meso- and hypersaline waters during global aridification events. All species with high hyporegulation ability were also desiccation-resistant, but not vice versa. Overall, results are consistent with the hypothesis that desiccation resistance mechanisms evolved first and provided the physiological basis for the development of hyporegulation ability, allowing these insects to colonize and diversify across meso- and hypersaline habitats.

Speciation below ground: Tempo and mode of diversification in a radiation of endogean ground beetles.

Dispersal is a critical factor determining the spatial scale of speciation, which is constrained by the ecological characteristics and distribution of a species' habitat and the intrinsic traits of species. Endogean taxa are strongly affected by the unique qualities of the below-ground environment and its effect on dispersal, and contrasting reports indicate either high dispersal capabilities favoured by small body size and mediated by passive mechanisms, or low dispersal due to restricted movement and confinement inside the soil. We studied a species-rich endogean ground beetle lineage, Typhlocharina, including three genera and more than 60 species, as a model for the evolutionary biology of dispersal and speciation in the deep soil. A time-calibrated molecular phylogeny generated from >400 individuals was used to delimit candidate species, to study the accumulation of lineages through space and time by species-area-age relationships and to determine the geographical structure of the diversification using the relationship between phylogenetic and geographic distances across the phylogeny. Our results indicated a small spatial scale of speciation in Typhlocharina and low dispersal capacity combined with sporadic long distance, presumably passive dispersal events that fuelled the speciation process. Analysis of lineage growth within Typhlocharina revealed a richness plateau correlated with the range of distribution of lineages, suggesting a long-term species richness equilibrium mediated by density dependence through limits of habitat availability. The interplay of area- and age-dependent processes ruling the lineage diversification in Typhlocharina may serve as a general model for the evolution of high species diversity in endogean mesofauna.