Subterranean arthropods are important components of soils and contribute essential food-web functions and other ecosystem services, however, their diversity and community composition has scarcely been assessed. Subterranean pitfall traps are a commonly used method for sampling soil habitats in Europe but have never been widely implemented in the Americas. We used subterranean pitfall traps to sample previously unsurveyed arthropod communities in southwestern Virginia, U.S. Traps were placed in shallow subterranean habitats (SSHs), underground habitats close to the surface where light does not penetrate, and more specifically at the interface between the soil and underlying “milieu souterrain superficiel”—a microhabitat consisting of the air-filled interstitial spaces between rocks (abbreviated MSS). In total, 2,260 arthropod specimens were collected constituting 345 morphospecies from 8 classes, 33 orders, and 94 families. A region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified and sequenced, and objective sequence clustering of 3% was used to establish molecular operational taxonomic units (mOTUs) to infer observed species richness. In all, 272 COI barcodes representing 256 mOTUs were documented for rare soil-dwelling arthropod taxa and are published to build a molecular library for future research in this system. This work is the first taxonomically extensive survey of North American soil-dwelling arthropods greater than 10 cm below the soil surface.

Completeness, DNA, endogean, epigean, hypogean, milieu souterrain superficiel, MSS, shallow subterranean habitats, SSH, survey

The need to study and describe global biodiversity has never been more urgent. Anthropogenic habitat loss has been implicated as the major driver of the currently ongoing sixth great mass extinction event in geological history (Cowie et al. 2022). Biodiversity is at greatest risk in highly diverse regions known as biodiversity hotspots (Myers et al. 2000; Hamilton et al. 2022). The Appalachian Mountains of eastern North America constitute one such hotspot, and has experienced tremendous habitat loss from exploitative mineral extraction, timber harvesting, and other land conversion practices (Stein 2000).

According to a recent review assessing global declines of arthropod diversity and abundance due to habitat loss and other factors, twice as many species show long-term population declines as those exhibiting population increases (Sánchez‐Bayo and Wyckhuys 2020). Subterranean arthropods, and other low-mobility invertebrates, are at elevated risk of extinction due to high rates of endemism and physiological constraints on dispersal (Mammola et al. 2019). Arthropods constitute the most species-rich animal group on Earth, with an estimated 7 million species, of which 5.5 million belong to the class Insecta (Santos et al. 2020). The Catalog of Life (2022) documents 1,128,168 species of arthropods, making up only 16% of the species estimated to exist. As such, arthropods constitute a globally understudied portion of biodiversity at high risk of species loss and anonymous extinction, a process in which a species is lost before it is discovered and described (Lobl et al. 2024). In the face of global decline, assessing and understanding insect and arthropod diversity is of paramount importance.

Research of subterranean organisms in North America has been dominated by taxonomically narrow studies with singular focal species or groups and have yielded important discoveries that hint at a significant but yet hidden diversity (e.g. Derkarabetian et al. 2010; Harden et al. 2024a, 2024b). General sampling and taxonomic assessment of soil-dwelling arthropods in North America has rarely, if ever, been conducted greater than 10 cm below the soil surface. As a result, little to nothing is known about the diversity and taxonomic composition of the arthropod communities that occupy the shallow subterranean habitats (SSH) of North America, and especially those within the Appalachian biodiversity hotspot. Broadly defined, SSH are underground habitats close to the surface where light does not penetrate, and at a basic level include soil and underlying milieu souterrain superficiel (MSS), a microhabitat consisting of the air-filled interstitial spaces between rocks (Mammola et al. 2016). But more broadly SSH can include underground aquatic interstitial habitats (e.g. hyporheic and hypotelminorheic zones), lava tubes, epikarst, and calcrete aquifers, which are defined in Culver and Pipan (2014). These habitats (except for some lava tubes) are typically close to the surface and are variable in depth but on average extend 0.1–5 meters in depth and up to 10 m (Culver and Pipan 2014). The arthropods that live the SSH have morphological and physiological adaptations such as the lack of pigmentation and eyes, shortened legs and elongate flexible bodies (Marek et al. 2021). SSH can be considered ecotones as they serve as transitional zones between adjacent epigean (above ground) and hypogean habitats (Prous 2004; Novak 2014). The term “hypogean” is often used to describe taxa or habitats associated with the rocky substrate that lays below the MSS, including cavernous karst cavities and cave systems distant from the surface (Prous 2004). Hypogean taxa have partially overlapping morphological adaptations to those in the SSH such as pigment and eye reduction, but contrast in having elongated appendages (Deharveng et al. 2024). Establishing tangible distinctions between different subterranean habitats is difficult as they exist more as a gradient than as distinct zones, thereby further contributing to the challenges of characterizing their diversity (Mammola et al. 2017). For the purposes of this study, SSH is used to refer to aphotic soil and MSS habitats 10–67 cm below the soil surface, but not extending into the bedrock, bedrock MSS, or into cave systems.

Sampling subterranean habitats is physically challenging, contributing to our lack of taxonomic and ecological knowledge in these systems. Although the number of studies focusing on subterranean arthropod communities has increased in recent decades, subterranean taxa continue to remain underrepresented in most biodiversity surveys due to difficulties associated with sampling the vertically distinct and ecologically important soil and MSS layers. Most studies focusing on soil invertebrate biota have taken place in the tropical and subtropical areas of the world and have focused on sampling ants (Wong and Guénard 2017) while most MSS studies have taken place in Europe, Japan, and Australia (Mammola et al. 2016; Ledesma et al. 2019; Halse and Pearson 2014). To our knowledge, SSHs have never been generally sampled in North America using ad hoc subterranean sampling.

Due in part to this lack of study, North American subterranean arthropods are vastly understudied. Essential research on them such as species descriptions, identification, and the assessment of biodiversity currently suffer from taxonomic shortcomings in which time, labor, and specialized expertise are direly required (Hebert et al. 2003; Ball and Armstrong 2011; Meierotto et al. 2019). Consequently, they are underrepresented in faunistic and ecological studies, and in major genetic repositories such as the National Center for Biotechnology Information (NCBI) and the Barcode of Life Data System (BOLD). The use of DNA barcodes, when coupled with traditional, morphology-based taxonomy, may help to partially overcome these shortcomings by streamlining identification and biodiversity assessment (Blaxter 2004). However, the taxonomic potential of DNA barcoding cannot be reached for North American soil-dwelling arthropods without the fuller representation of NCBI with expertly identified vouchers representing these taxa.

The aim of this study was to survey the subterranean arthropod communities of previously unexplored SSH in southwestern Virginia, U.S. This work also sought to generate high quality DNA barcodes for the sampled taxa in order to expand a molecular foundation for future research in North American subterranean systems. The COI barcodes generated from this study will contribute to the representation of subterranean arthropod genetic data at NCBI, Ecdysis, Global Biodiversity Information Facility (GBIF) and other biodiversity data repositories, and will serve as a tool for future work in characterizing and understanding subterranean arthropod communities of Appalachia and North America more generally.

This study is the first taxonomically extensive survey of soil-dwelling arthropods in North America, and the first in the Appalachian Mountains, to employ subterranean pitfall traps capable of sampling to a depth of up to 67 cm below the soil surface. In all, 2,260 individual arthropod specimens were collected constituting 345 morphospecies and 257 molecularly distinct species (mOTUs) representing 8 classes, 33 orders, and 94 families. In total, 272 COI barcodes were sequenced and are published at NCBI. Of these, 102 constitute mOTUs that are new to the NCBI and BOLD databases. Many of the taxa recovered during the study represent new records and have not, or have only rarely, been documented in the region. We suspect that a number of the morphospecies recovered are undescribed.

Several morphospecies exhibited hypogean/SSH adaptations: i.e., depigmentation, reduction of eyes and appendage lengthening (hypogean taxa) or shortening (SSH taxa) suggesting that they may be obligate subterranean inhabitants (Deharveng and Bedos 2018; Deharveng et al. 2024). These morphospecies included several species of ants (Hymenoptera: Formicidae—6 out of 13 morphospecies), beetles (Coleoptera—15 of 53), flies (Diptera—18 of 62), springtails (Collembola—18 of 56), two-pronged bristletails (Diplura: Japygidae—6 of 7), spiders (Arachnida: Araneae—7 of 19), and mites (Arachnida: Mesostigmata, Sarcoptiformes, Trombidiformes—21 of 62). However, most individuals displayed morphology associated with epigean origin, which is consistent with the findings of similar studies outside North America that have shown that SSH are commonly dominated by epigean taxa (Coiffait 1958; Mammola et al. 2017). This dominance of epigean taxa might have contributed to the lower observed richness in winter as the richness and abundance of epigean arthropods, especially insects, in subterranean systems are significantly impacted by seasonality due to surface temperature influencing SSH temperature to some extent. This is not the case for hypogean taxa as climatic stability tends to increase as depth from the surface increases (Mammola et al. 2017). Alternatively, the lower observed richness in the winter may be associated with the decreased mobility of arthropods during cold and dry periods when many are in a winter diapause, or in immobile pupal stages.

The taxonomic composition of our samples is comparable to that of SSH studies in the Canary Islands (Pipan et al. 2011), Italy (Mammola et al. 2017), mainland Spain (Gilgado et al. 2014), Bulgaria (Langourov et al. 2014), and France (Juberthie 2000) with arthropods and annelids being the most abundant groups. Annelids were very abundant in our samples and were mainly represented by potworms (Enchytraeidae). Our findings differ in the detection of mollusks and crustaceans (Isopoda: Oniscidea). We only collected a single mollusk and only a few terrestrial isopods, while both groups were commonly encountered in the studies mentioned above. Our results regarding insect abundance by order align with those of Mammola et al. (2017) in that Diptera, Hymenoptera, and Coleoptera were among the most abundant groups across sites. Our findings are consistent with those of Moldenke and Lattin (1990) in that the families Phoridae, Cecidomyiidae, and Sciaridae were among the most common dipterans encountered. Diptera abundance was highly variable across sites due to, in part, the relatively large numbers of early instar Phoridae larvae observed in single traps at the Quarry and Fallam sites. Ants dominated specific traps at all sites which is consistent with the findings of Mammola et al. (2017). Where ants dominated a trap, relatively few other insects were found, and rarely were two or more ant species found in the same trap. Regarding Coleoptera richness by family, our findings are consistent with those of Moldovan (2005) who reported ground beetles (Carabidae; 14.3% of total abundance in our samples) to be less common than round fungus beetles (Leiodidae; 18.4%). This is in contrast with the study of Mammola et al. (2017), in which carabids were largely observed as larvae with only a single mature specimen collected. In contrast to the studies mentioned above, rove beetles (Staphylinidae; 24.5%) were the most species rich beetle group present in our samples. Hemipterans were rare, consistent with the findings of Langourov et al. (2014). This may be due to bait-bias as phytophagous hemipterans are likely less attracted to protein-based bait than their coleopteran equivalents. Bait-bias has been shown to influence the abundance and richness of taxa recovered in previous surveys of arthropods (Checa et al. 2019). Orthoptera was among the most abundant insect orders at the Starroot site, largely due to the relatively high abundance of the locally common Ceuthophilus guttulosus, F. Walker, 1869 (Orthoptera, Raphidophoridae).

Little overlap in molecularly distinct species was observed between the different sites with the Quarry and Fallam sites sharing two species (Hymenoptera: Stenamma schmittii Wheeler, 1903; Diptera: Triphleba aequalis Schmitz, 1919), and the Quarry and Starroot sites sharing two species (Hymenoptera: Stenamma schmittii Wheeler, 1903; Araneae: Cicurina pallida Keyserling, 1887). The ant species Stenamma schmittii Wheeler, 1903 was observed across all sites. This lack of pronounced species overlap is consistent with Lamoncha (1994) who recorded minimal overlap of oribatid mites between nearby subterranean sites in North and South Carolina, U.S. This trend is consistent with the notion that subterranean arthropod communities are often highly specific to the individual habitats they occupy (Menta and Remelli 2020). That said, these patterns may be a byproduct of insufficient sampling and further work is needed to support these trends, especially for sites with lower sampling effort.

The high sample coverage at the Starroot site suggests that future surveys of similar experimental design should employ ≥ 10 subterranean traps per site and ≥ 4 weeks total of baited-collection time. Further studies are needed to optimize the application of subterranean pitfall traps in North America. Subsequent investigations may also address other factors including presence or choice of baits, duration of sampling, and digging-in effects. Previous work has shown taxon bias associated with baits (Checa et al. 2019) and recency of placement (Mammola et al. 2017). Prior work has shown that baited subterranean traps preponderantly select for epigean taxa such as ants and phorid flies (Mammola et al. 2017). Similarly, epigean taxa may be more abundant in freshly excavated traps and these digging-in effects have been shown in aboveground pitfall traps but not demonstrated in subterranean traps. Future studies without baits and/or including a post excavation and installation settling period may reduce epigean bias and narrow sampling to hypogean (broadly defined) taxa.

Our findings suggest that the understudied SSH arthropod communities of the Appalachian Mountains, and North America, are highly diverse and warrant further study. This is consistent with the findings of several studies showing high arthropod diversity within subterranean assemblages both more broadly and specifically within particular groups such as beetles, spiders, and mites (Lamoncha 1994; Christman et al. 2005; Mammola et al. 2017; Ledesma et al. 2019). The close, but not quite-asymptotic nature of the sample completeness curves for each of our sites indicate that more species remain undocumented, and additional sampling is needed to better characterize subterranean arthropod diversity of southwestern Virginia and the Appalachian region. We still lack solid taxonomic, ecological, and molecular foundations for our understanding of the arthropod communities that inhabit the North American SSH. It is our hope that the primary taxonomic and barcode data generated through this work will serve as a foundation upon which future studies of North American subterranean systems may build upon.

This work was supported by a National Science Foundation grant to P. Marek (Division of Environmental Biology # 1916368). We thank Ruth Neumann, also known as the artist Starroot, of Floyd County, Virginia, for permission to sample arthropods at her home (https://starroot.com/). We thank Barry Keith for assistance and expertise with trap fabrication, and India Garden Restaurant (Blacksburg, VA) for providing deli cups for fabrication of extra collecting receptacles. Kayla Elahi provided helpful assistance with uploading specimen metadata to Ecdysis. We thank Lindi Fitzpatrick and Maggie Thomas for assistance with specimen collection and processing. We are grateful to Amrita Srivathsan for assistance with Python code for objective clustering, and Steph Benbow for assistance with field work and figure preparation for this manuscript. We appreciate the comments of reviewers Stefano Mammola and David Culver who improved previous versions of the manuscript.