Mammoth Cave: A Hotspot of Subterranean Biodiversity in the United States

: The Mammoth Cave System in the Interior Low Plateau karst region in central Kentucky, USA is a global hotspot of cave-limited biodiversity, particularly terrestrial species. We searched the literature, museum accessions, and database records to compile an updated list of troglobiotic and stygobiotic species for the Mammoth Cave System and compare our list with previously published checklists. Our list of cave-limited fauna totals 49 species, with 32 troglobionts and 17 stygobionts. Seven species are endemic to the Mammoth Cave System and other small caves in Mammoth Cave National Park. The Mammoth Cave System is the type locality for 33 cave-limited species. The exceptional diversity at Mammoth Cave is likely related to several factors, such as the high dispersal potential of cave fauna associated with expansive karst exposures, high surface productivity, and a long history of exploration and study. Nearly 80% of the cave-limited fauna is of conservation concern, many of which are at an elevated risk of extinction because of small ranges, few occurrences, and several potential threats.


Introduction
The Mammoth Cave System in central Kentucky, USA is the most extensive cave system in the world with over 663 km (412 miles) of mapped passaged, including 27 entrances and 10 significant caves that have been connected since explorations began in the late 1700s: Colossal, Crystal (=Floyd Collins' Crystal), Donkey, Hoover, Mammoth, Morrison, Proctor, Roppel, Salts, and Unknown caves. Colossal, Crystal, Salts, and Unknown caves comprise the 206 km (128 mile) Flint Ridge Cave system ( Figure 1). Mammoth Cave National Park was created in 1941 and includes two-thirds of the Mammoth Cave System [1]. The Mammoth Cave System was recognized as a UNESCO World Heritage Site in 1981 because of its uniqueness as the world's longest cave system as well as its extensive geological, mineral, and biological resources. The region was recognized as the core of an UNESCO Biosphere Reserve-Mammoth Cave Biosphere Region-in 1990.
The Mammoth Cave System is developed in three major limestone layers at the northwestern extent of the Pennyroyal Plateau, an expansive flat karst plain within the Interior Low Plateau physiographic province. The limestone layers include, from youngest to oldest, the Girkin Formation (40 m thick), Ste. Genevieve Limestone (35 m thick), and St. Louis Limestone (53-60 m thick) [2][3][4][5]. The Girkin Formation is capped by resistant sandstone and shale of the Big Clifty Formation that form the Mammoth Cave, Flint, Joppa, and Toohey Ridges. Most of the cave system is developed in the Ste. Genevieve Limestone and the upper 40 m of St. Louis Limestone [5]. The limestone strata gently slope from the southeast to the northwest. The Pennyroyal Plateau is exposed at the surface to the The Mammoth Cave System is characterized by a complex network of vadose and phreatic passages with at least five primary horizontal levels of passages (four fossil stream levels and the modern base level) representing distinct stages of development in association with past periods of water table stability and intervening periods of downcutting of the Green River valley through the resistant caprock into the soluble limestone layers below [1,6]. The evolution of the Mammoth Cave system is linked to the incision history of the Green River, drainage reorganizations, and significant climatic changes from the Pliocene through the Pleistocene, with the oldest upper-level passages dating to 3.2 Mya and the lower levels developing over the past 2 Mya [9].
Mammoth Cave has long been a focal region of study for North American subterranean biodiversity and for advancing our foundational knowledge of the ecology and evolution of cave fauna. Studies of the biodiversity in the Mammoth Cave System have an extensive history dating back to the 1820s (see [10]) when Constantin S. Rafinesque first visited Mammoth Cave [11]. Darwin [12] even mentions cave life from the Mammoth The Mammoth Cave System is characterized by a complex network of vadose and phreatic passages with at least five primary horizontal levels of passages (four fossil stream levels and the modern base level) representing distinct stages of development in association with past periods of water table stability and intervening periods of downcutting of the Green River valley through the resistant caprock into the soluble limestone layers below [1,6]. The evolution of the Mammoth Cave system is linked to the incision history of the Green River, drainage reorganizations, and significant climatic changes from the Pliocene through the Pleistocene, with the oldest upper-level passages dating to 3.2 Mya and the lower levels developing over the past 2 Mya [9].
Herein we present an updated list of terrestrial and aquatic cave obligate fauna (i.e., troglobionts and stygobionts, respectively) of the Mammoth Cave system. Our goal is not to duplicate recently published checklists by Culver and Hobbs [37] and Toomey et al. [1] but rather complement these works by including a comprehensive bibliography on the cave obligate fauna of Mammoth Cave. In addition, we compare our list with past checklists from Mammoth Cave and comment on the exceptional biodiversity of this North American and global hotspot of subterranean biodiversity.

Materials and Methods
We conducted a search of the scientific literature to compile an updated list of troglobiont and stygobiont species for the Mammoth Cave System. For an overview of taxa that are not cave-limited, we refer readers to Barr [10], Culver and Hobbs [37], Helf and Olson [63], and Poulson [62]. Scientific literature sources included journal articles, book chapters, books, conference proceedings, theses and dissertations, and government reports. Searches of literature sources included keyword queries of ISI Web of Science, Google Scholar, and Zoological Record. In addition, we also searched biodiversity databases including the Global Biodiversity Information Facility (GBIF; Available online: https://gbif.org (accessed on 28 June 2021)), VertNet (Available online: http://www.vertnet.org (accessed on 28 June 2021)), Symbiota Collections of Arthropods Network (SCAN; Available online: https://scan-bugs.org/portal/(accessed on 28 June 2021)), and InvertEBase (Available online: http://www.invertebase.org/portal/index.php (accessed on 28 June 2021)). The list of cave obligate fauna includes the scientific name, authority, and conservation status of each species. Taxonomic nomenclature followed primarily the Integrated Taxonomic Information System (ITIS; Available online: http://itis.gov (accessed on 28 June 2021)). For conservation status, we include the International Union for Conservation of Nature (IUCN) Red List of Threatened Species (Available online: http://www.iucnredlist.org (accessed on 28 June 2021)) and NatureServe (Available online: http://www.natureserve.org (accessed on 28 June 2021)) conservation statuses when available. The status of a species according to the United States list of threatened and endangered species under the U.S. Endangered Species Act is included (Available online: http://www.fws.gov/endangered (accessed on 28 June 2021)), as well its status (endangered, threatened, or of greatest conservation need) under the latest Kentucky State Wildlife Action Plan (Available online: https://fw.ky.gov/WAP/Pages/default.aspx (accessed on 28 June 2021)).

Results
Packard [36] summarized the North America cave fauna, which at that time was primarily limited to the fauna of Mammoth Cave. He reported 31 permanent cave species, 18 of which we recognize as cave-limited species today, including 12 troglobionts and six stygobionts (Table 1). Barr [10] reported 44 cave-limited species (28 troglobionts and 16 stygobionts). More recently, Culver and Hobbs [37] listed 48 species (32 troglobionts and 16 stygobionts, 11 of which (nine troglobionts and two stygobionts) are endemic to the Mammoth Cave System, while Toomey et al. [1] reported 50 cave-limited species (32 troglobionts and 18 stygobionts). The authors also included two springtails not yet identified to species (Willemia sp. and Onychiurus sp.) on their list of cave-limited taxa, which were also reported by Barr [10].   Figure 2). Both Culver and Hobbs [37] and Toomey et al. [1] included the snail Helicodiscus punctatellus and copepod Atteyella pilosa in their respective lists of cave-limited taxa. Helicodiscus punctatellus is known from surface collections [64]. Atteyella pilosa is a facultative associate of several species of surface and cave-limited crayfishes and is also known from surface collections [65]. Culver and Hobbs [37] did not include the isopod Caecidotea bicrenata, which was included in our list and that of Toomey et al. [1]. Lewis [66] reported several collections of C. bicrenata from the Mammoth Cave System where it predominately occurs in lower-level aquatic habitats. Toomey et al. [1] included the phorid fly Megaselia cavernicola in their list of cave-limited taxa. Megaselia cavernicola is a widely occurring species in caves on eastern North America that lacks obvious troglomorphic characters, is known from surface collections [67], and has been treated as a troglophile (i.e., non-obligate) by most past authors (e.g., [68,69] Figure 2). Both Culver and Hobbs [37] and Toomey et al. [1] included the snail Helicodiscus punctatellus and copepod Atteyella pilosa in their respective lists of cavelimited taxa. Helicodiscus punctatellus is known from surface collections [63]. Atteyella pilosa is a facultative associate of several species of surface and cave-limited crayfishes and is also known from surface collections [64]. Culver and Hobbs [37] did not include the isopod Caecidotea bicrenata, which was included in our list and that of Toomey et al. [1]. Lewis [65] reported several collections of C. bicrenata from the Mammoth Cave System where it predominately occurs in lower-level aquatic habitats. Toomey et al. [1] included the phorid fly Megaselia cavernicola in their list of cave-limited taxa. Megaselia cavernicola is a widely occurring species in caves on eastern North America that lacks obvious troglomorphic characters, is known from surface collections [66], and has been treated as a troglophile (i.e., non-obligate) by most past authors (e.g., [67,68]).  Mammoth Cave is the type locality for 33 cave-limited species (Table 1). Seven species are endemic to the Mammoth Cave system and other smaller caves in Mammoth Cave National Park (Table 1).

Terrestrial Fauna
Two troglobiotic snails have been documented in the Mammoth Cave System. Carychium stygium is found in association with cricket guano and is the most common of the two species [37]. Weigand et al. [70,71] suggest C. stygium may be an ecotype of the troglophile C. exile, as C. stygium shows limited mitochondrial COI sequence divergence from and is nested within a clade containing C. clappi and C. exile. However, this inference is based on a single locus and only two populations of C. stygium were included in analyses. Alternative hypotheses such as incomplete lineage sorting and mitochondrial introgression cannot be ruled out at present and warrant study. Regardless, these studies suggest that it is likely that C. stygium has recently colonized caves. Glyphyalinia specus is a wide-ranging snail known from 27 occurrences in five states [72]. Significant publications include Call [28], Hubricht [49,50,52,53], Barr [10], Poulson et al. [73], Dourson [74], Poulson [62], and Gladstone et al. [72].
Four troglobiotic pseudoscorpions occur in the Mammoth Cave System. Hesperochernes mirabilis is a widely distributed species most abundant near entrances. It is often observed in and near rodent (Neotoma and Peromyscus sp.) nests, which may facilitate phoretic dispersal. The other three species are thought to be associated with deep cave habitats. Kleptochthonius cerberus was described from White's Cave in Mammoth Cave National Park [90] and has to date, only been found there. Kleptochthonius hageni was described from Mammoth Dome in Mammoth Cave [91]. Kleptochthonius cereberus is thought to be endemic to Mammoth Cave National Park. Kleptochthonius hageni is reported to occur in the Mammoth Cave System and possibly some nearby caves not on the park (C.D.R. Stephen, pers. comm.). Tyrannochthonius hypogeus is a small, eyeless species with attenuated appendages first collected from log litter in Bruce Hollow [92]. Muchmore [92] considered this species to be cave adapted and associated with the Mammoth Cave fauna. Notable publications include Hubbard [83], Packard [36], Banks [93], Malcolm and Chamberlin [90], Muchmore [91,92], and Barr [10].
Although more than 10 species of collembolans (i.e., springtails) have been documented in the Mammoth Cave System [10], just two taxa are considered troglobionts and both are endemic to the cave system. Pygmarrhopalites altus was described by Christiansen [100] from Eyeless Fish Trail in the Unkown Cave section of Mammoth Cave. Pseudosinella espanita was described by Christiansen and Bellinger [101] from Styx River near Charon's Cascade in Mammoth Cave. Notably absent from the fauna of the Mammoth Cave System are P. hirsuta and Sinella cavernarum, which have broad distributions that include the Western Pennyroyal Karst of nearby Barren County, Kentucky [102]. Barr [10] reported two undescribed collembolans as potential troglobionts from Mammoth Cave: Willemia sp. have been collected from rotting boards in the Roaring River section. This genus includes several edaphic species, but no troglobionts are known to date and it is unlikely that this taxon represents a true troglobiont. Onychiurus sp. also have been collected from Mammoth Cave. Four described species in this genus are considered troglobionts in caves of the eastern United States. Additional study is needed on the collembolans of the Mammoth Cave System. Significant publications include Packard [36], Call [28], Christiansen [97,[103][104][105], Barr [10], Poulson and Culver [86], Christiansen and Bellinger [101], and Poulson [62].
The only other troglobiotic insect documented from Mammoth Cave is the dipteran Spelobia tenebrarum, a widely distributed species in caves of eastern North America [148]. Notable publications include Barr [10], Marshall and Peck [148], and Poulson [62].
A single groundwater snail has been documented in the Mammoth Cave System. Antroselates spiralis occurs in base-level streams in cave system. It was described from Echo River Spring, a major drain of the Mammoth Cave System. Notable publications include Hubricht [51], Barr [10], Barr and Kuehne [132], Hershler and Hubricht [155], Lewis [66], Pearson and Boston [154], and Helf and Olson [63].

Discussion
The Mammoth Cave obligate cave fauna is exceptionally rich with 49 troglobionts and stygobionts, making it one of the most diverse systems globally [37,225,226]. The terrestrial fauna is particularly diverse-tied for the third richest cave system in the world behind the Postojna Planina Cave System (36 species) in Slovenia and Cueva de Felipe Revention (34 species) in the Canary Islands [226]. With respect to stygobiotic fauna, the Mammoth Cave System ranks second in North America behind San Marcos Artesian Well in San Marcos, Texas (55 taxa, 39 described and 16 undescribed; [227]).
Several hypotheses have been proposed [10,61,127,224,228] to explain the high species richness in the Mammoth Cave System (recently reviewed in [37]). First, high species richness in the Mammoth Cave System may reflect the long history of more intensive sampling and study compared to other cave systems in the region [37]. While sampling intensity and bias may partially explain the high species richness at Mammoth Cave, several other biogeographical hypotheses warrant mention. The Mammoth Cave System is developed within a thick, continuous karst exposure over a large area in the Interior Low Plateau, which supports larger and more stable population sizes, more complex communities, and greater dispersal potential [113,129,130]. Moreover, the Mammoth Cave System is located at an intersection of hypothesized dispersal routes for cave-limited species from other karst areas, such as the Pennyroyal Karst Plain, Cumberland Saddle, and Bluegrass Region, and its cave fauna includes not only endemic species but also taxa also found in these adjacent regions [10,37,66,130]. The Mammoth Cave System lies within a hypothesized ridge of high troglobiont diversity found in temperate North America and Europe identified by Culver et al. [228]. This ridge corresponds to a general region of high surface primary productivity, which provides higher levels of allochthonous input into cave systems [228]. Mammoth Cave is noted for having high levels of allochthonous productivity but also chemoautotrophic productivity [37,63,229]. However, whether chemosynthesis subsidizes troglobiont communities or contributes significantly to the high troglobiont diversity found in the Mammoth Cave System remains speculative, as it is not well supported by empirical evidence.
The obligate fauna of the Mammoth Cave System is diverse and includes 39 cavelimited species (18 troglobionts an 11 stygobionts) of conservation concern, highlighted by the federally endangered cave shrimp Palaemonias ganteri. Most of these species are at an elevated risk of extinction due to their limited distributions and/or are known from few occurrences. For example, the cave pseudoscorpion Tyrannochthonius hypogeus is known from just two specimens collected from a single locality [92]. Cave-limited fauna face many threats, such as habitat loss and degradation, groundwater overexploitation and contamination, and climate change [230,231].
Although much of the Mammoth Cave System lies within the boundaries of Mammoth Cave National Park, the cave system is not immune to direct and indirect threats to its biodiversity, particularly those stressors that originate from outside of the park, such as industrial and tourism development, oil and gas drilling, runoff from agriculture, residential areas, and highways, and emergent diseases [63,[232][233][234][235][236]. For example, sewage from the town of Park City was previously known to drain into the headwaters of the Echo River basin potentially impacted the stygobiotic fauna [130], including Typhlichthys subterraneus, Amblyopsis spelaea, Palaemonias ganteri, Orconectes pellucidus, and Antroselates spiralis. A hydrocarbon spill along Interstate 65 was responsible for a significant die-off of aquatic cave life [232,236]. Flow reversals and back-flooding from the Green River into cave springs also may transport sediment, potential contaminants, pathogens, and invasive aquatic species into base level streams in the Mammoth Cave System [237][238][239].
Great potential still exists to discover new taxa and add to the list of obligate species at Mammoth Cave. Two potentially cave-limited springtails that we do not include in our checklist (Willemia sp. and Onychiurus sp.) are known from Mammoth Cave and have not been identified to species [1,10]. Terrestrial woodlice are notably absent from the troglobiotic fauna of Mammoth Cave and may be discovered in the future. Seven troglobiotic trichoniscids (Isopoda, family Trichoniscidae) are known caves of the Interior Low Plateau and Appalachians karst regions [240], including Miktoniscus barri known from several caves of Indiana and Kentucky [241]. A troglophilic species, Miktoniscus mammothensis, occurs in cave and surface habitats at MCNP [242]. Other taxonomic groups have not been particularly well studied in the Mammoth Cave System, such as flatworms, copepods, springtails, and mites. More intensive work on these groups may uncover additional taxa. With more than 651 km of passage, much of the Mammoth Cave System has not been comprehensively bioinventoried, and some habitats, such as epikarst, have been disproportionately under-sampled and may harbor undescribed taxa [37]. In addition, over 500 other caves occur in MCNP, including several biologically rich sites, such as White and Great Onyx caves. These cave systems also may harbor undocumented diversity. Finally, few genetic studies to date have incorporated samples from the Mammoth Cave System. Comprehensive sampling within the Mammoth Cave System has the potential to uncover cryptic diversity in some taxonomic groups, which is an increasingly common discovery of genetic and phylogenetic studies in cave-limited taxa [223,[243][244][245].