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Research Article
Remarkably low genetic diversity in the widespread cave spider Phanetta subterranea (Araneae, Linyphiidae)
expand article infoKathryn A. Kennedy, Kirk S. Zigler, Brendan Cramphorn§|, Curt W. Harden, Kurt Helf#, Julian J. Lewis¤, Thomas E. Malabad«, Marc A. Milne», Matthew L. Niemiller|, Charles D. R. Stephen˄
‡ University of the South, Sewanee, United States of America
§ Cornell University, Ithaca, United States of America
| The University of Alabama in Huntsville, Huntsville, United States of America
¶ Clemson University, Clemson, United States of America
# National Park Service, Mammoth Cave National Park, Mammoth Cave, United States of America
¤ Lewis and Associates, Cave, Karst and Groundwater Biological Consulting, Borden, United States of America
« Natural Heritage Program, Virginia Department of Conservation and Recreation, Richmond, United States of America
» University of Indianapolis, Indianapolis, United States of America
˄ Georgia State University, Atlanta, United States of America
Open Access

Abstract

Most cave-obligate species (troglobionts) have small ranges due to limited dispersal ability and the isolated nature of cave habitats. The troglobiontic linyphiid spider Phanetta subterranea (Emerton, 1875), the only member of its genus, is a notable exception to this pattern; it has been reported from more counties and caves than any other troglobiont in North America. As many troglobionts exhibit significant genetic differentiation between populations over even small geographic distances, it has been hypothesized that Phanetta may comprise multiple, genetically distinct lineages. To test this hypothesis, we examined genetic diversity in Phanetta across its range at the mitochondrial cytochrome c oxidase subunit I gene for 47 individuals from 40 caves, distributed across seven states and 37 counties. We found limited genetic differentiation across the species’ range with haplotypes shared by individuals collected up to 600 km apart. Intraspecific nucleotide diversity was 0.006 +/- 0.005 (mean +/- SD), and the maximum genetic p-distance observed between any two individuals was 0.022. These values are within the typical range observed for other spider species. Thus, we found no evidence of cryptic genetic diversity in Phanetta. Our observation of low genetic diversity across such a broad distribution raises the question of how these troglobiontic spiders have managed to disperse so widely.

Keywords

Appalachians, genetic diversity, Interior Low Plateau, Linyphiidae, Phanetta subterranea

Introduction

Caves are populated by a diverse community of organisms, with more than 1,300 cave-obligate species (i.e., troglobionts) known from the United States alone (Niemiller et al. 2019). Because caves provide ‘islands’ of habitat for cave-limited species, and because troglobionts typically have limited ability to disperse through surface habitats, most troglobionts have small, restricted distributions, and many are restricted to a single or few geographically clustered cave systems. For example, 31% (218/710) of troglobionts in the Appalachians and Interior Low Plateau karst regions in the eastern United States are known from a single cave, with many other species limited to just a handful of nearby caves (Christman et al. 2016). Only a select few species have even moderately broad ranges, with just nine troglobionts (three arachnids, three hexapods, and three crustaceans) reported from more than 30 counties (Christman and Culver 2001).

Spiders are a significant component of cave biodiversity, with more than 100 troglobiontic spiders known from the United States (Niemiller et al. 2019) and ~1,000 troglobiont spiders described worldwide (Mammola et al. 2017). The best studied cave spiders in the eastern United States are from the genus Nesticus, which has diversified into three dozen cave and surface species across the southern Appalachians (Hedin and Milne 2023). As is often the case for troglobionts, cave-limited Nesticus species are characterized by small ranges (three species are known from just a single cave, and many others from just a handful of caves) (Hedin and Milne 2023). In cases where a Nesticus species is known from multiple caves, they often exhibit high genetic divergence between caves, even over short distances (Hedin 1997; Snowman et al. 2010; Balogh et al. 2020; Zigler and Milne 2022; Hedin and Milne 2023).

The linyphiid spider Phanetta subterranea (Emerton, 1875) (Fig. 1), the only member of its genus, is a small (1.5–2 mm in total length) troglobiont. They are found in multiple cave habitats, from near entrances to deep cave zones, and are often quite common (Poulson, 1977, 1981). They are thought to feed on springtails (Poulson, 1977, 1981). Phanetta exhibit variation in the degree of eye formation; most individuals have eyes, but in some cases eyes are nearly absent (Millidge, 1984). Phanetta can grow from hatching to full size in about four months and have a lifespan of about one year (Poulson 1981). Clutch size ranges from three to 16 eggs that are ~0.6 mm in diameter, and a single spider can lay multiple clutches within a year (Poulson 1975). Its range extends across two karst regions (the Interior Low Plateau and the Appalachians (Niemiller et al. 2019)) spanning a dozen states, and the species is known from more counties and caves than any other troglobiont in North America (Christman and Culver 2001; Niemiller et al. 2013; Christman et al. 2016). Although widespread and common in caves of the eastern United States, Phanetta has never been reported from surface habitats.

Figure 1. 

Subterranean Sheetweb Spider (Phanetta subterranea). Photo by Matthew L. Niemiller.

Despite its remarkably broad range, nothing is known about genetic diversity in this species. It has been suggested that modern taxonomic study would result in the splitting of Phanetta into multiple species (Christman and Culver 2001). This scenario was observed in the Southern Cavefish (Typhlichthys subterraneus) species complex, which is known from the southern Interior Low Plateau, southern Appalachians, and Ozarks karst regions. Genetic analysis of T. subterraneus revealed nine genetically distinct lineages, including the identification of T. eigenmanni as a distinct species, and efforts to delineate and describe other lineages as distinct species are underway (Niemiller et al. 2012; Niemiller et al. 2013; Hart et al. 2023). Similar results have been reported for various troglobionts in other parts of the world (e.g., Lefébure et al. 2006; Zhang and Li 2014), including the cave beetle Darlingtonea kentuckensis from eastern Kentucky (Boyd et al. 2020).

In this study we investigated potential cryptic diversity in Phanetta across its broad distribution through genetic analysis of the mitochondrial cytochrome c oxidase subunit I gene (COI), a marker commonly employed in the study of genetic diversity in invertebrates. We sought to estimate genetic diversity and explore genetic structure within this spider while addressing the question of whether Phanetta represents a complex of morphologically similar but genetically distinct lineages, or a single genetic lineage connected through gene flow over broader spatial scales.

Methods

Geographic analysis

We surveyed the literature to compile a list of all known Phanetta subterranea occurrences. Resources consulted included Culver et al. (2000), Christman et al. (2016), and Zigler et al. (2020), as well as unpublished records from various cave biologists. We mapped the range of Phanetta, and our sampling sites (Fig. 2), using ArcGIS Online (https://www.arcgis.com/index.html). We calculated the range extent/extent of occurrence (EOO) for Phanetta using GeoCAT (https://geocat.iucnredlist.org/editor). Range extent/EOO is the area of a minimum convex polygon which contains all the sites of occurrence (Bachman et al. 2011).

Figure 2. 

Range and sampling map. The distribution of Phanetta subterranea in the eastern United States. State boundaries are indicated by grey lines and karst terrain as blue-grey shading. Sites where Phanetta has been reported are indicated by orange points. Sites sampled in this study in the Interior Low Plateau karst region are indicated by blue points, and sites sampled in the Appalachians karst region are indicated by yellow points. This map includes ~600 georeferenced Phanetta sites. The inset indicates the extent of the main map, and includes three additional Phanetta sites (one in northeast Ohio, one in northwest Illinois, and one in central Arkansas), each more than 200 km from any other known Phanetta site, that are not visible on the main map.

Sampling

Phanetta were collected by hand between 1998–2023 from 40 caves in 37 counties across seven states (Alabama, Georgia, Illinois, Indiana, Kentucky, Tennessee, and Virginia) (Table 1) and two karst regions (the Interior Low Plateau and the Appalachians) (Fig. 2). We aimed to sample as broadly as possible, so generally limited our sampling to one cave per county. Specimens were preserved in 95% ethanol and stored at -20 °C until DNA extraction. Individuals were identified to species under the microscope; mature Phanetta females are easily identified by their distinctive epigynum (Emerton, 1875). In most cases, one spider per cave was sequenced; however, we sequenced two spiders from seven different caves. Collections were permitted by a variety of agencies (see Acknowledgements). Voucher specimens from this study are accessioned at the Auburn University Museum of Natural History.

Table 1.

Sample sites for Phanetta subterranea.

State County Cave
Alabama Colbert Georgetown Cave
Alabama DeKalb Manitou Cave
Alabama Jackson Pseudo Lava Cave B
Alabama Madison Hering Cave
Alabama Marshall MacHardin Cave
Georgia Dade Howards Waterfall Cave
Illinois Monroe Danes Cave
Illinois Monroe Icebox Cave
Indiana Dubois Vowell Cave
Indiana Harrison Big Mouth Cave
Indiana Washington Twin Oaks Pit
Kentucky Monroe cave near Hestand, KY
Tennessee Bedford Fountain Cave
Tennessee Campbell New Mammoth Cave
Tennessee Campbell Norris Dam Cave
Tennessee Cannon Sycamore Creek
Tennessee Claiborne Obie Mill Cave
Tennessee Coffee Jernigan Cave
Tennessee Davidson Bull Run Cave
Tennessee Davidson Newsom Branch Cave
Tennessee DeKalb Indian Grave Point Cave
Tennessee Dickson Sinuous Stream Cave
Tennessee Franklin Tom Pack Cave
Tennessee Grundy Crystal Cave
Tennessee Hamilton Levi Cave
Tennessee Lincoln Kelso Saltpeter Cave
Tennessee Marion Pryor Cave Spring
Tennessee Meigs Sensabaugh Cave
Tennessee Montgomery Durham Cave
Tennessee Overton Mill Hollow Cave
Tennessee Pickett Frog Cave
Tennessee Smith New Salem Cave No. 1
Tennessee Wilson Spring Cave
Virginia Bland Repass Saltpeter Cave
Virginia Highland Five Springs Cave
Virginia Lee Grassy Springs Cave
Virginia Rockingham Massanutten Cave
Virginia Russell Bundys Cave No. 2
Virginia Scott Jesse Branch Cave
Virginia Shenandoah Flemmings Cave

Molecular techniques

We extracted DNA from specimens using the DNeasy Blood and Tissue Kit (Qiagen; Cat. No. 69504). We followed the manufacturer’s protocol for extractions from whole or partial spiders. Polymerase chain reactions (PCRs) were prepared using the DNA extractions as template, GoTaq G2 Green Master Mix (Promega; Cat. No. M7822), dH2O, and primers. Two different primer sets were employed to amplify a 651 base pair fragment of the mitochondrial COI locus. We initially used the primers HCO2198+M13F and LCO1490+M13R (modified from Folmer et al. (1994)), but we subsequently developed primers (PsHCO+M13F and PsLCO+M13R) that were more effective for amplifying Phanetta (Table 2). The PCR protocol was initial denaturation for 5 minutes at 95 °C, then 35 cycles of 15 seconds of denaturation at 95 °C, 30 seconds of primer annealing at 45 °C, and 60 seconds of extension at 72 °C. PCR products were visualized on 1% agarose gels. Successful PCRs were prepared for sequencing by treatment with Antarctic Phosphatase (New England Biolabs, Cat. No. M0289) and Exonuclease I (New England Biolabs, Cat. No. M0293). Samples were then sequenced on both strands using M13F and M13R primers on an Applied Biosystems 3730×l DNA Analyzer at the Keck DNA Sequencing Core of the Yale University School of Medicine (New Haven, CT).

Table 2.

Primer names and sequences. Primers used to amplify a 651 bp fragment of the mitochondrial cytochrome oxidase I gene in Phanetta subterranea.

Primer name Sequence (5’-3’) Reference
HCO2198+M13F TGTAAAACGACGGCCAGTCGGTCAACAAATCATAAAGATATTGG Folmer et al. (1994)
LCO1490+M13R CAGGAAACAGCTATGACCTAAACTTCAGGGTGACCAAAAAATCA Folmer et al. (1994)
PsHCO+M13F GTAAAACGACGGCCAGTACAAATCATAAAGATATTGGAAGTTTG This study
PsLCO+M13R CAGGAAACAGCTATGACCTTCAGGGTGACCAAAAAATCAAAATAA This study

Genetic analysis

We trimmed, assembled, edited, and aligned COI sequences using Geneious Prime (v. 2022.1.1). All sequences were submitted to GenBank (accession nos. PP815877PP815923). We used MEGA11 (Tamura et al. 2021) to calculate genetic distances between sequences. P-distance, the genetic distance measure used here, is the proportion of nucleotides that differ between any two sequences. We used POPART (Leigh and Bryant 2015) to build a median joining tree (Bandelt et al. 1999) from the COI sequences. We looked for a pattern of isolation by distance by comparing linear geographic distance between sites and COI p-distance between individuals from those sites.

Results

Phanetta is known from 669 caves across 12 states and 155 counties (Fig. 2). When calculating the species range extent, we excluded three sites (one in northeast Ohio, one in northwest Illinois, and one in central Arkansas) because each was more than 200 km from any other known Phanetta site, raising the possibility of identification errors, or vagrancy. Even after excluding those sites, the species’ EOO was 412,223 km2 (Table 3).

We sequenced 47 Phanetta individuals from 40 caves across seven states and 37 counties (Fig. 2, Table 1). Full-length (651 bp) sequences were obtained from all individuals, and no indels or stop codons were observed. Genetic distances between Phanetta samples were low. Nucleotide diversity (π) in Phanetta was 0.006 ± 0.005 (mean ± SD), with a minimum pairwise p-distance of 0.000 and a maximum pairwise p-distance of 0.022 (Table 3). Twenty-one haplotypes were observed, and seven of these were shared, ranging in frequency from two to 14 individuals. The most common haplotype was present in Phanetta from Indiana, Illinois, Tennessee, and Virginia.

In seven cases, we sampled two individuals from the same cave. In six of those cases, the two individuals had identical COI sequences, and in the seventh case there was a single nucleotide difference between the two individual sequences. We found a positive correlation between the genetic distance between Phanetta individuals and the linear geographic distance between their sample sites (d.f. = 779, R2 = 0.32, F = 373.7, significance F < 0.0001), indicating a pattern of isolation by distance, although the correlation was not particularly strong, and identical haplotypes were identified from sites as far as 600 km apart.

As one of the few troglobionts that is widespread across two major karst regions – the Appalachians and the Interior Low Plateau (Niemiller et al. 2019) (Fig. 2, Table 3) – Phanetta provided an opportunity to explore the effect of differing geologic history on genetic diversity within a single species. Haplotype diversity (h) was similar for the two karst regions (hAppalachians = 0.892, hInterior Low Plateau = 0.841) (Table 3). However, nucleotide diversity in Phanetta from the Appalachians (πAppalachians = 0.009 ± 0.006) was greater than in Phanetta from the Interior Low Plateau (πInterior Low Plateau = 0.003 ± 0.004) (Table 3). This pattern can be visualized in the haplotype network (Fig. 3) where haplotypes from the Interior Low Plateau are quite similar, mostly differing by just one or a handful of nucleotide differences. In contrast, haplotypes from the Appalachians typically differed from one another by multiple nucleotide differences (Fig. 3). Only one haplotype was shared by individuals from the Interior Low Plateau and the Appalachians (Fig. 3). The higher genetic divergence observed in the Appalachians may be related to the great linear distance (~850 km) across which samples were collected (Fig. 2), although Phanetta does range across a greater area in the Interior Low Plateau (Table 3).

Overall, we observed remarkably low genetic variation across the broad range of Phanetta, with individuals from the Interior Low Plateau being particularly genetically uniform. Phanetta from the Appalachians exhibited slight genetic divergence from those from the Interior Low Plateau, and were also relatively more divergent from each other, but the overall genetic distance between any two Phanetta individuals was low. There was no evidence of cryptic genetic diversity within Phanetta.

Table 3.

Distribution of and genetic diversity in Phanetta across karst regions. Range extent of Phanetta in the Interior Low Plateau and the Appalachians karst regions, and combined across the two regions, calculated as extent of occupancy (EOO). Measures of genetic diversity were calculated from all pairwise comparisons between individuals within the specified region. Based on cytochrome oxidase I sequences.

Karst region Combined
Interior Low Plateau Appalachians
Range extent (EOO) 214,418 km2 140,669 km2 412,223 km2
# of georeferenced sites 392 206 598
# of individuals sequenced 31 16 47
# of haplotypes 13 9 21
Haplotype diversity (h) 0.841 0.892 0.878
# of segregating sites 17 20 32
Nucleotide diversity (π) (+/- SD) 0.003 (+/- 0.004) 0.009 (+/- 0.006) 0.006 (+/- 0.005)
Maximum pairwise p-distance 0.015 0.020 0.022
Figure 3. 

Median joining haplotype network for all Phanetta sequences. Haplotypes are indicated by circles and nucleotide differences between haplotypes are indicated by hash marks. Haplotypes are colored by karst region of origin as in Figure 2. Circle size indicates the number of individuals sharing a haplotype. The multicolored circle indicates the single haplotype shared by individuals from the Interior Low Plateau and individuals from the Appalachians.

Discussion

Phanetta subterranea is known from more caves and more counties than any other North American troglobiont. We aimed to determine whether Phanetta comprised a complex of genetically distinct lineages, or if it was genetically uniform across its range. After sampling 47 Phanetta individuals from 37 counties across seven states in the eastern United States, we found no evidence of cryptic genetic diversity. Genetic distances between sites were low, and haplotypes were shared across significant geographic distances (up to 600 km). Phanetta from the Appalachians exhibited slight genetic differentiation from individuals from the Interior Low Plateau, as well as more genetic variation from each other (Fig. 3, Table 3). The higher nucleotide diversity observed in Appalachian Phanetta (Table 3) may be due to the highly faulted and fractured karst of the Appalachians causing greater isolation between Phanetta populations, whereas the lower nucleotide diversity observed in Interior Low Plateau Phanetta (Table 3) may reflect the more contiguous horizontal carbonate layers of this karst region, which could foster population connectivity.

We can compare our results to other spider species and to other troglobiont spiders from the eastern United States. A review of DNA barcoding efforts in spiders (Čandek and Kuntner 2015), using the same genetic marker (COI) that we employed in our study, provides a broad comparison. Summarizing results for 162 species, Čandek and Kuntner (2015) reported a mean intraspecific nucleotide diversity of 0.009, slightly higher than the 0.006 that we observed in Phanetta. Further, Domènech et al. (2022) used COI sequences to study genetic diversity in 371 spider species across a similarly-sized geographic region in Spain. They found a mean maximum intraspecific distance of 0.021, which is similar to the maximum intraspecific distance of 0.022 we observed for Phanetta. Clearly, the amount of genetic diversity we observed in Phanetta is not out of the ordinary range for a spider species.

In contrast, the Phanetta results are quite different from those observed in other troglobiont spiders for which genetic data are available. Nesticus spiders of the southern Appalachians exhibit high species diversity across a region smaller than the range extent of Phanetta, with many species having very small ranges (Hedin and Milne 2023). Multiple species of Nesticus are often found in close proximity, sometimes at sites just a few kilometers apart (Zigler and Milne 2022; Hedin and Milne 2023). Previous studies found considerable genetic diversity within species, even when those species ranges are very small. For example, Zigler and Milne (2022) reported COI genetic distances of 0.026 (in N. cressleri) and 0.031 (in N. lula) for cave populations less than 10 kilometers apart. As an additional example, Nesticus barri is known from around 60 caves on the southern Cumberland Plateau in Tennessee and Alabama. Genetic analysis of N. barri from a dozen caves found no haplotypes shared by individuals that were more than 12 km apart, and genetic distances (also for the COI locus) between individuals from different caves were as high as 0.045 (Snowman et al. 2010). These patterns strongly contrast with Phanetta, where haplotypes were shared by individuals as far as 600 km apart, and the maximum genetic distance (across a vastly larger geographic range) between individuals was 0.022.

Phanetta has never been reported from surface habitats, not even in a study of sinkholes within the range of the species (Lewis et al. 2020), and we have shown that populations across its range are genetically uniform. This raises the question as to how Phanetta has managed to colonize so many caves across such a broad area. We offer two, potentially complementary, hypotheses. First, as a tiny spider, it may be moving, undetected, through subterranean passageways such as caves and the interstitial spaces in shallow subterranean habitats (SSH) (Culver and Pipan 2019), including the epikarst and the “milieu souterrain superficiel” (MSS), a layer of fractured rock beneath an insulating soil layer (reviewed in Mammola et al. 2016). In deeper cave habitats, troglobiont spiders have been shown to traverse through historical cave connections (Marsh et al. 2023). However, the distances traveled by historical Phanetta populations to form its current distributions are magnitudes larger than that studied by Marsh et al. (2023) and it is unknown if subterranean dispersal could fully explain its current range. Spiders have been collected within the MSS, especially in Europe (e.g., Růžička 1990, 1996; Růžička and Thaler 2002). However, studies on spiders from the MSS in North America are non-existent (Mammola et al. 2016).

A second possibility is that Phanetta disperses via ballooning, where spiders use their silken threads to be carried by the wind from one place to another (Greenstone et al. 1987). Studies of the diversity of ballooning spiders in the United States and Europe indicate that members of the family Linyphiidae are the spiders most commonly observed (Dean and Sterling 1985; Plagens 1986; Greenstone et al. 1987; Blandenier 2009; Blandenier et al. 2014), so it is not unreasonable to suggest Phanetta, a linyphiid spider, may also disperse in that way. If this is occurring, ballooning would probably have to be paired with at least some subsequent surface movement of individuals post-landing, as caves and cave entrances are relatively rare on the surface. Ballooning, which would allow the spiders to disperse across great distances, could explain the species’ broad range, and the sharing of COI haplotypes between individuals collected as far as 600 km apart. However, the fact that Phanetta have never been observed on the surface weighs against the likelihood of ballooning as a method of dispersal.

This study could be extended in several ways. Further sampling of Phanetta from eastern Kentucky and from West Virginia would be valuable. We were unable to acquire samples from those areas. We also suggest searching for Phanetta from the three peripheral populations (Fig. 2) that we omitted from our estimation of range extent, as confirming or dismissing those observations would clarify the true range of the species. Two other linyphiid species – Porrhomma cavernicola and Anthrobia monmouthia – are wide-ranging troglobionts in eastern North America whose ranges overlap with Phanetta (Miller 2005a, 2005b). While neither is as common nor as wide-ranging as Phanetta, both are found across multiple states and karst regions, and genetic analyses of these species would provide an interesting comparison to the patterns we observed in Phanetta.

We also recommend exploring the possibility of ballooning in Phanetta. It might be possible to search directly for ballooning in Phanetta by setting aerial traps at the entrance of caves known to host Phanetta, aiming to catch any spiders leaving the cave by ballooning. Although some research on ballooning has been conducted in the United States (e.g., Dean and Sterling 1985; Plagens, 1986; Greenstone et al. 1987), none of these studies were done within the range of Phanetta. As a result, it remains unclear whether Phanetta, like many linyphiid species, disperses by ballooning. In addition, study of SSH within the range of Phanetta could clarify whether Phanetta are present in these habitats. In combination, studies of ballooning and SSH could support or reject our hypotheses for how Phanetta spread across such a large range.

In summary, we reject the suggestion that Phanetta subterranea contains cryptic genetic diversity and represents multiple species. Rather, it is a single, genetically uniform, species that has dispersed broadly across the caves of eastern North America. How it has managed to do this remains a mystery.

Acknowledgements

José Iriarte-Díaz and Deborah McGrath provided helpful comments on the manuscript. Christopher Van de Ven assisted with GIS analyses. This work was supported by The University of the South and by grants from several sources to MLN, including the Tennessee Wildlife Resources Agency (contract no. 32801-00526), U.S. Fish & Wildlife Service (no. F17AC00939), the National Speleological Society, the Cave Conservancy Foundation (no. A 14-0574), the University of Alabama in Huntsville, the Alabama Department of Conservation and Natural Resources, and the National Science Foundation (award no. 2047939). This work was permitted by Tennessee Wildlife Resources Agency scientific permit no. 1385, Alabama Department of Conservation and Natural Resources scientific permit nos. 2018035450068680, 2019060225068680, 2020083527668680, 2021091324068680, 2022096307868680, and 2023109374868680, Georgia Department of Natural Resources scientific permit no. 8394, Virginia Department of Game and Inland Fisheries scientific permit no. 058511, Virginia Department of Wildlife Resources scientific collection permit no. 3629089, and National Park Service scientific permits CUGA-2016-SCI-0011 and MACA-2023-SCI-0007.

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