Research Article |
Corresponding author: Kirk S. Zigler ( kzigler@sewanee.edu ) Academic editor: Stefano Mammola
© 2024 Kathryn 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.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kennedy KA, Zigler KS, Cramphorn B, Harden CW, Helf K, Lewis JJ, Malabad TE, Milne MA, Niemiller ML, Stephen CDR (2024) Remarkably low genetic diversity in the widespread cave spider Phanetta subterranea (Araneae, Linyphiidae). Subterranean Biology 50: 105-118. https://doi.org/10.3897/subtbiol.50.135200
|
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.
Appalachians, genetic diversity, Interior Low Plateau, Linyphiidae, Phanetta subterranea
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 (
Spiders are a significant component of cave biodiversity, with more than 100 troglobiontic spiders known from the United States (
The linyphiid spider Phanetta subterranea (Emerton, 1875) (Fig.
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 (
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.
We surveyed the literature to compile a list of all known Phanetta subterranea occurrences. Resources consulted included
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.
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
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 |
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
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 |
|
LCO1490+M13R | CAGGAAACAGCTATGACCTAAACTTCAGGGTGACCAAAAAATCA |
|
PsHCO+M13F | GTAAAACGACGGCCAGTACAAATCATAAAGATATTGGAAGTTTG | This study |
PsLCO+M13R | CAGGAAACAGCTATGACCTTCAGGGTGACCAAAAAATCAAAATAA | This study |
We trimmed, assembled, edited, and aligned COI sequences using Geneious Prime (v. 2022.1.1). All sequences were submitted to GenBank (accession nos. PP815877–PP815923). We used MEGA11 (
Phanetta
is known from 669 caves across 12 states and 155 counties (Fig.
We sequenced 47 Phanetta individuals from 40 caves across seven states and 37 counties (Fig.
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 (
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.
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 |
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
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.
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 (
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 (
Phanetta
has never been reported from surface habitats, not even in a study of sinkholes within the range of the species (
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 (
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.
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.,
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.
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.