Gateways to the underworld: molecular phylogenetic analyses identify patterns of groundwater fauna and two new species of hypogean Hyalella Smith, 1874 (Amphipoda, Hyalellidae) from the northern Mojave Desert

Gateways to the underworld: molecular phylogenetic analyses identify patterns of groundwater fauna and two new species of hypogean Hyalella Smith, 1874 (Amphipoda, Hyalellidae) from the northern Mojave Desert Andrew G. Cannizzaro1, Corey J. Lange2, David J. Berg3 1Department of Biology, Miami University, Oxford, OH 45056, USA 2US Bureau of Land Management, Southern Nevada District Office, Las Vegas, NV 89130, USA 3Department of Biology, Miami University, Hamilton, OH 45011, USA Corresponding author: Andrew G. Cannizzaro ([email protected]) Academic editor : Denis Copilas-Ciocianu|Received 14 August 2025|Accepted 10 October 2025|Published 21 November 2025 https://zoobank.org/730DE74C-1DF1-4E65-B6A9-7B1104D72A01 Citation: Cannizzaro AG, Lange CJ, Berg DJ (2025) Gateways to the underworld: molecular phylogenetic analyses identify patterns of groundwater fauna and two new species of hypogean Hyalella Smith, 1874 (Amphipoda, Hyalellidae) from the northern Mojave Desert. Subterranean Biology 54: 35–68. https://doi.org/10.3897/subtbiol.54.168846 Abstract In North America, the genus Hyalella (Crustacea: Amphipoda) is primarily epigean, with only two described species found in groundwater. In contrast, Stygobromus is an amphipod genus occupying subterranean habitats throughout most of the Nearctic. Using both molecular and morphological traits, we describe two new species of stygobitic amphipods, Hyalella plutonia sp. nov. and Hyalella keepuikantun sp. nov. from groundwater habitats in Nye County, Nevada, USA. These species form a monophyletic group that also contains the similar H. cretae from Ash Meadows National Wildlife Refuge. Members of this “H. cretae” complex are similar in terms of overall morphology but can be distinguished through examination of taxonomically relevant characters within the genus such as the third uropod, gnathopods, and pereopod 7. The description of these species highlights the unexpected diversity of stygobitic Hyalella within the northern Mojave Desert, where four such species comprise two independent lineages. Stygobromus is absent from these locations. This diversity of Hyalella is hypothesized to be due in part to the evolutionary history the genus and the ecological/climatic conditions of the region itself, which may exclude other more wide-ranging Nearctic stygobites like Stygobromus. Keywords Devils Hole, Hyalella muerta, Hyalella cretae, Nevada Subterranean Biology 54: 35–68 (2025) doi: 10.3897/subtbiol.54.168846 https://subtbiol.pensoft.net Copyright Andrew G. Cannizzaro et al. 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. RESEARCH ARTICLE Subterranean Biology Published by The International Society for Subterranean Biology A peer-reviewed open-access journal Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 36 Introduction While environments such as ponds, lakes, and streams are most commonly envisioned when considering freshwater on Earth, these habitats make up less than one percent of the Earth’s freshwater; nearly 70% is contained in glaciers, permafrost, and ice caps, and the remaining ~30% exists as groundwater (Gleick 1993). Similar trends follow the fauna associated with freshwaters: common surface inhabitants of familiar, accessible, systems come to mind (sportfish, insects and insect larvae such as the “EPT taxa”, etc.) when considering “freshwater fauna”, but a significant number of taxa occupy groundwater systems as well. Many of the latter—including vertebrates such as salamanders and fishes, and invertebrates including Mollusca, Arthropoda, Annelida, Platyhelminthes, and other phyla—have been recorded in hypogean environments; in fact, these taxa often display remarkably high species richness (Stoch 1995; Danielpol et al. 2000; Maurice and Bloomfield 2012). Such diversity within these environments is often promoted by their nature, as predicted by the “adaptive zone model” (Stoch 1995). This model highlights the high potential for colonization to occur in hypogean systems which can then lead to speciation and often radiation; this may, in part, explain the high species diversity observed among crustacean taxa in several groundwater systems (Stoch 1995; Danielpol et al. 2000). In the Nearctic, many taxa have been recorded from hypogean systems, but perhaps some of the highest such biodiversity can be observed within the Crustacea Brünnich, 1772, especially the abundant and species-rich members of the peracarid order Amphipoda Latreille, 1816 (Stoch 1995). To date, over 200 species from 19 genera and 8 families of amphipods have been identified from groundwater habitats in the Nearctic (Horton et al. 2025). A majority of these species are contained within families/genera which could be considered groundwater-obligates, such as the genus Stygobromus Cope, 1872 and most genera of the family Hadziidae S. Karaman, 1943 which do not possess surface representatives (Holsinger 1986; Horton et al. 2025). These taxa typically display highly stygomorphic facies and often represent older, often relictual, lineages (Holsinger 1986). Conversely, a smaller subset of taxa possesses both epigean and hypogean representatives; such a distribution is likely due to more recent introductions to groundwater. Examples of such taxa are found in the genera Gammarus Fabricius, 1775 and Hyalella Smith, 1874 (Holsinger 1986; Cannizzaro et al. 2023; Horton et al. 2025). Hyalella is a primarily epigean genus endemic to the New World, with over 100 species currently described (Horton et al. 2025). The center of diversity for the genus is in the Neotropical realm, where an overwhelming majority (~80%) of species occur (De Los Ríos et al. 2012; Horton et al. 2025). Here, species have been recorded from a wide range of epigean (springs, swamps, lakes, streams, etc.) and hypogean (caves, springs, hyporheic habitats) habitats. While a number of Hyalella spp. have been recorded from a variety of groundwater habitats in the Neotropics, hypogean Hyalella are only known from spring habitats in the Great Basin of California and Nevada within the Nearctic. Two species have recently been described: Hyalella muerta Baldinger, Shepard & Theroff, 2000 from Inyo County, California and Hyalella cretae Cannizzaro, Lange, & Berg, 2023 from Nye County, Nevada. The presence of Hyalella in some Nearctic groundwa- Two new hypogean Hyalella from Nevada 37 ters itself is curious, as these habitats are mostly occupied by members of the genus Stygobromus (Holsinger 1986). While Stygobromus spp. are present in the Great Basin, they are not sympatric with Hyalella spp. and are absent throughout the Mojave Basin and Range and most of the Sonoran Desert. Furthermore, molecular phylogenetic analyses of H. muerta and H. cretae suggest while similar morphologically and separated by an overland distance of ~45 km, the two species are not sister taxa and belong to different lineages within the genus, likely colonizing groundwater habitats independently (Cannizzaro et al. 2023). The presence of multiple Hyalella lineages in groundwater within the region and the absence of Stygobromus spp. suggest that Hyalella were able to exploit groundwater habitats here that are not occupied by other amphipod taxa (Cannizzaro et al. 2023). As a result, it is likely that within the Mojave Basin and Range Hyalella spp. would follow diversification trends similar to those of other groundwater taxa such as Stygobromus, where different localities would contain isolated, endemic species. Collections from Nye County, Nevada, USA have revealed the presence of additional populations of stygobitic Hyalella (Fig. 1). Here we examine these populations and similar species using both morphological analyses and molecular genetic comparisons of three genes commonly assessed for amphipod molecular phylogenetics: nuclear 18S ribosomal DNA (Cannizzaro et al. 2023; Cannizzaro et al. 2025b), nuclear 28S ribosomal DNA (Witt et al. 2006; Hou et al. 2007; Flot et al. 2010; Cannizzaro et al. 2023; Cannizzaro et al. 2025a, 2025b), and mitochondrial 16S rDNA (Macdonald et Figure 1. Updated distributions of hypogean Hyalella spp. in Nye County, Nevada and Inyo County, California, USA. Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 38 al. 2005; Hou et al. 2007; Cannizzaro et al. 2023; Cannizzaro et al. 2025a, 2025b). The mitochondrial cytochrome c oxidase subunit I (COI) gene which is also often assessed was excluded from these analyses due to issues with pseudogenes. Materials and methods Collection of specimens Individuals were collected live, using hand nets or small strainers, from both an unnamed spring, ~0.7 km north of the Highway 95 and Oleo Rd intersection near Beatty, and Devils Hole, Nye County, Nevada (Fig. 1). Collected specimens were preserved in 95% ethanol and stored at -20 °C. Specimens examined here were deposited at the National Museum of Natural History, Smithsonian Institution (USNM). Morphological analyses To aid in morphological analysis, four specimens of each population were cleared and stained prior to dissection. These individuals were digested in a mixture of 400 μL of Molecular Grade Water, 360 μL of 2× Digestion Buffer (Zymo Research, Irvine, CA, USA), and 40 μL of Proteinase K; samples were incubated in this mixture at 37 °C for 2 hours and then at room temperature overnight. Once sufficiently digested, specimens were transferred to a solution of Lignin Pink for 2–3 hours; after specimens were sufficiently stained, they were transferred to glycerin for dissection using a Nikon SMZ-8000 stereomicroscope. The appendages were mounted on temporary glycerin slides to facilitate examination using a Nikon Alphaphot-2 YS2 compound microscope with attached drawing tube. Plates were prepared using Adobe Illustrator CC®. Body length measurements were taken by measuring the distance from the rostrum to the base of the pleotelson, following the contour of the body using ImageJ software (Abràmoff et al. 2004). The term “defining angle” refers to the posterior margin of the palm and the distal-most point of the posterior margin of the propodus, the area where the tip of the dactylus closes on the propodus. Nomenclature for the terms “seta(e)” and “robust seta(e)” follows published guidelines (Watling 1989), as does the nomenclature of setal types (Zimmer et al. 2009). The term “seta(e)” refers to “simple seta(e)” unless otherwise specified. DNA extraction and sequencing Genomic DNA (gDNA) was extracted from the digested material prepared during the specimen clearing process. Extractions were performed using Tissue & Insect DNA MiniPrep kits (Zymo Research, Irvine, CA, USA), utilizing a modified protocol (Cannizzaro et. al., 2020). Extracted DNA was stored at -20 °C and quantified using a Qubit fluorometer (Invitrogen, Waltham, MA, USA). Polymerase chain reactions (PCR) using primer pairs 18SF/18S1000F and 18S700F/18S1250R (Englisch and Koenemann 2001) amplified ~1000 base pairs (bp) Two new hypogean Hyalella from Nevada 39 Table 1. GenBank accession numbers generated as a part of this study. Original catalog # Museum # Species Locality Coll Date Collector(s) Latitude, Longitude 18S Accession # 28S Accession # 16S Accession # AGC-1143.1 USNM 1694150 Hyalella plutonia sp. nov. Spring, ~11 km NNE of Beatty, Nye County, NV, USA Dec-142023 Corey J. Lange 37.00512, -116.72684 PX560105 PX560112 PX560116 AGC-1143.2 USNM 1694151 Hyalella plutonia sp. nov. Spring, ~11 km NNE of Beatty, Nye County, NV, USA Dec-142023 Corey J. Lange 37.00512, -116.72684 PX560106 PX560113 PX560117 AGC-1124.1 USNM 1694155 Hyalella keepuikantun sp. nov. Devils Hole II, Nye County, NV, USA Jan-082020 Ambre Chaudoin, Randall Paylor 36.42714, -116.29117 PX560103 PX560110 PX560114 AGC-1125.1 USNM 1694154 Hyalella keepuikantun sp. nov. Devils Hole II, Nye County, NV, USA Feb-072017 Jeffrey Goldstein, Olin Feuerbacher 36.42714, -116.29117 PX560104 PX560111 PX560115 of nuclear 18S rDNA; 28S_lev2 and 28S_des1 (Verovnik et al. 2005) amplified a ~730bp segment of the nuclear 28S rDNA gene; and the primer pairs 16StF (Macdonald et al. 2005) and 16SBR (Palumbi et al. 1991) amplified ~350 bp of the mitochondrial 16S rDNA gene. Total PCR volumes of 35 μl contained 30–70 ng of extracted gDNA. PCR master mix contained 17.5 μl GoTaq Master mix (Promega Corporation, Madison, WI, USA), 1 μl of each 10 μM primer, and 13.5 μl of molecular grade water. PCR was performed on a Bio-Rad T100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). A negative control lacking only gDNA was included for all sets of PCR reactions to rule out contamination. The following thermal cycler protocols were followed: 18S: 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 68.5 °C for 30 s, and 72 °C for 2 min, ending with a 10 min final extension at 72 °C; 28S: 95 °C for 5 minutes, followed by 37 cycles of 94 °C for 45 seconds, 45–53 °C for 30 seconds, and 72 °C for 50 seconds, ending with a 5-minute final extension at 72 °C; 16S: 95 °C for 2 minutes, followed by 37 cycles of 95 °C for 40 seconds, 42–45 °C for 40 seconds, and 72 °C for 1 minute, ending with a 8-minute final extension at 72 °C. PCR products were prepared for sequencing using Exo-CIP Rapid PCR Cleanup (New England Biolabs, Ipswich, MA, USA). Sanger sequencing of purified PCR products was carried out at Eurofins Genomics (Louisville, KY, USA) using industry-standard Sanger sequencing methodology. All sequences generated as a part of this study were submitted to GenBank (Table 1). Phylogenetic analyses Pairwise sequence alignment was conducted using MAFFT (Katoh et al. 2002; Katoh and Standley 2013) in GENEIOUS PRIME (www.geneious.com) and checked further by eye. Multiple sequence alignments were performed separately for each gene, accomplished under the same parameters as the pairwise alignments. All markers were concatenated into a final alignment using SEQUENCE MATRIX v.1.8 (Vaidya et al. 2011). We reconstructed phylogenetic relationships with maximum likelihood (ML) analyses using IQTREE v.2.3.6 (Minh et al. 2020). The IQTREE search was run using the MODELFINDER algorithm to select best-fitting substitution Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 40 models for individual partitions, which were analyzed under an edge-linked model. For both the 18S and 28S rDNA loci, a TIM model with equal/empirical base frequencies, and a FreeRate model with 2 categories (TIM+F+R2) was selected; for the 16S rDNA, an HKY model with equal/empirical base frequencies, and a gamma distribution with four categories (HKY+F+G4) was selected. Statistical support was estimated using 1,000 ultrafast bootstrap replicates (Minh et al. 2013) and the Shimodaria-Hasegawa approximate likelihood ratio test (Shimodaria and Hasegawa 1999; Guindon et al. 2010). Accession numbers of all individuals included in the phylogenetic analyses are provided in Suppl. material 1. Results Molecular phylogenetic analyses Phylogenetic trees reconstructed here recovered individuals of Hyalella from Devils Hole II and the unnamed spring near Beatty as a monophyletic group which also included H. cretae (Fig. 2). These populations differed from H. cretae at levels similar to those observed in other amphipod taxa (~98–99% similarity in nuclear genes and ~90% in the 16S rDNA), especially in the 18S rDNA, which has demonstrated great utility in identifying species in several amphipod taxa (White 2011; Sisco and Sawicki 2023; Cannizzaro et al. 2025b). Morphological analyses Individuals of Hyalella examined from the unnamed spring near Beatty and Devils Hole II differed from the other stygomorphic Hyalella species in the Nearctic (H. muerta and H. cretae) in several key morphological characteristics including the shape of the gnathopods, proportions and setation of the basis of pereopod 6, and the armament of the telson and uropod 3 (Table 2). Based on our morphological and molecular results, we describe each of these populations as a new species. Table 2. Diagnostic morphological characteristics among stygobitic Hyalella in the Nearctic. Abbreviations: GN = gnathopod; CPL = carpus posterior lobe. Character H. cretae H. muerta H. plutonia sp. nov. H. keepuikantun sp. nov. Maxilliped inner plate apical robust setae 3 3 2 4 Gn2 (♂) propodus dactylus vertical alignment well beyond CPL beyond CPL beyond CPL well beyond CPL Gn2 (♂) palmar margin anterior notch distinct, posterior angle distinct, posterior angle distinct, angled weak Pereopod 7 basis posterior margin setae 8 8 6 3 Pereopod 7 basis posterior margin distal-most setae robust small/simple small/simple small/simple Pereopod basis projection past ischium 1.5× 1.5× 2.0× 1.1× Pereopod basis vs. ischium-dactylus 33% 33% 33% 25% Uropod 3 ramus apex setae longest robust longest simple longest simple longest robust Telson apical setae 4 4 2 4 Gn1/2 (♀) palm concave straight/convex straight straight Two new hypogean Hyalella from Nevada 41 Systematics Order Amphipoda Latreille, 1816 Suborder Senticaudata Lowry & Myers, 2013 Infraorder Talitrida Rafinesque, 1815 Parvorder Talitridira Rafinesque, 1815 Superfamily Hyaloidea Bulyčeva, 1957 Family Hyalellidae Bulyčeva, 1957 Genus Hyalella Smith, 1874 Figure 2. Maximum-likelihood phylogeny reconstructed from the concatenated dataset (18S rDNA, 28S rDNA, 16S rDNA) using IQTREE. Node support for two methodologies is indicated by shaded circles placed on nodes; weakly supported nodes (< 0.40) not marked. Members of the H. cretae complex are highlighted by colored gradients. A Hyalella plutonia sp. nov. female, 3.43 mm, unnamed spring, ~0.7 km north of Hwy 95 and Oleo Rd, Nye County, Nevada, USA B Hyalella keepuikantun sp. nov. male, 3.15 mm, Devils Hole II, Nye County, Nevada, USA. UFBS, ultrafast bootstrap, SHalRT, Shimodaira-Hasegawa approximate likelihood ratio test. White scale bar represents 1 mm. Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 42 Hyalella plutonia Cannizzaro, Lange & Berg, sp. nov. https://zoobank.org/D855A0A5-1777-4CEF-8658-5A513EC4728E Figs 3A, B, 4–10 Material examined. • Holotype, male 4.20 mm: Unnamed spring, ~0.7 km north of Highway 95 and Oleo Rd intersection, Nye County, Nevada, USA (37.005118, -116.726843); collector: Corey J. Lange, 14 December 2023; USNM 1694150. • Allotype, female 3.25 mm: same locality and collection data as holotype; USNM 1694151. • Paratype males, n = 2, same locality and collection data as holotype; USNM 1694153. • Paratype females, n = 2, same locality and collection data as holotype; USNM 1694152. Type locality. Unnamed spring, ~0.7 km north of Highway 95 and Oleo Road intersection (on west side of highway), Oasis Valley, Nye County, Nevada, USA (37.005118, -116.726843). Etymology. The specific epithet plutonia is the feminine form of the Latin Pluto and the Ancient Greek Πλούτων (Pluton), the god of the Underworld. Pluto’s Gates or ploutonion were shrines erected to Pluto; one of the most famous is the shrine at Hierapolis, commonly called the “Gateway to the Underworld.” This epithet is given both in reference to the hypogean habitat of the species and to play off the nickname of the nearby town of Beatty: “Gateway to Death Valley.” Figure 3. Habitus photographs A Hyalella plutonia sp. nov. holotype male, unnamed spring, ~0.7 km north of Hwy 95 and Oleo Rd, Nye County, Nevada, USA (USNM 1694150), 4.20 mm B Hyalella plutonia sp. nov., allotype female, unnamed spring, ~0.7 km north of Hwy 95 and Oleo Rd, Nye County, Nevada, USA (USNM 1694151), 3.25 mm C Hyalella keepuikantun sp. nov., holotype male, Devils Hole II, Nye County, Nevada, USA (USNM 1694154), 3.48 mm D Hyalella keepuikantun sp. nov., allotype female, Devils Hole II, Nye County, Nevada, USA (USNM 1694155), 3.62 mm. Two new hypogean Hyalella from Nevada 43 Diagnosis. Small-sized, stygomorphic species distinguished from congeners— except members of the H. muerta and H. cretae complex—by the combination of the following characters: eyes absent; antenna 1 longer than antenna 2; pleonites lacking dorsoposterior carinae; maxilla 1 inner plate with 2 apical pappose setae; male uropod 1 inner ramus lacking long curved setae. Differentiated from H. muerta and other members of the H. cretae complex by the combination of the following characters: maxilliped inner plate with 2 apical robust setae; male gnathopod 2 palmar margin with distinct anterior notch; pereopod 7 basis projecting > 1.5 × the length of the ischium, with > 5 posterior setae, all simple, length > 25% remainder of the pereopod segments; uropod 3 ramus with simple setae; telson with 2 long apical setae. Description. Holotype male (Figs 3A, 4–8): 4.20 mm in length. Eyes and integumentary pigment absent (Fig. 3A). Antennae. Antenna 1 (Fig. 4A): Approximately 1.2 × length of antenna 2; peduncle segment 1 lacking posterodistal robust setae; primary flagellum with 12 segments, aesthetascs on distal segments, aesthetascs shorter than respective segments, unpaired. Antenna 2 (Fig. 4B): approximately 30% body length, gland cone distinct; peduncular segment 5 1.2 × length of segment 4; flagellum with 10 segments. Mouthparts. Mandibles: left mandible (Fig. 4C): incisor 9-dentate, lacinia mobilis 5-dentate, accessory setal row with 2 plumose setae; molar process well-developed, cylindrical, triturative, lacking seta. Right mandible (Fig. 4D): incisor 5-dentate, lacinia mobilis bifurcate, lobes with numerous fine dentations; accessory setae row with 2 plumose setae; molar similar in form to left mandible. Upper lip (Fig. 5A): rounded, apical margin of labrum with numerous setules. Lower lip (Fig. 5B): inner lobes indistinct, outer margin of inner and outer lobes covered in setules; face of lip covered in pubescent setules. Maxilla 1 (Fig. 5C): inner plate shorter than outer plate, with 2 apical pappose setae, and fine pubescent setules covering the entire plate; outer plate with 9 apical serrate setae, pubescent setules sparse and decreasing laterally and proximally; palp one-segmented, vestigial, 20% length of outer plate, tapering distally, with pubescent setules, palp emerges at slightly over half the length of the outer plate. Maxilla 2 (Fig. 5D): both inner and outer plates covered in pubescent setules; outer plate subequal in length to inner plate, with numerous apical setae; inner plate narrowing slightly distally with numerous apical setae and 2 pappose facial setae. Maxilliped (Fig. 5E): inner plate shorter than outer plate with 2 unarmed cuspidate setae along apical margin, surface of plate covered in pubescent setules; outer plate with numerous setae, lacking pubescent setules; palp 4–segmented, second and third segments with numerous marginal, and submarginal setae; dactylus surface lacking comb-scales, nail not elongate. Gnathopods. Gnathopod 1 (Fig. 6A): coxal plate with 5 short apical setae; basis narrowing proximally, distoposterior surface of segment lacking comb-scales; ischium with a posterior seta; merus with 3 posterior setae, distoposterior surface of segment with sparse comb-scales; carpus subequal in length to propodus, with cluster Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 50 Figure 8. Hyalella plutonia sp. nov. holotype male, unnamed spring, ~0.7 km north of Hwy 95 and Oleo Rd, Nye County, Nevada, USA (USNM 1694150), 4.20 mm A epimera B pleopod 1 (coupling spine enlarged) C uropod 1 D uropod 2 E uropod 3 F telson. Scale bars: 0.25 mm (A, B); 0.1 mm (C–E). Nevada Department of Transportation (NDOT) and lies 45 m from the edge of Hwy 95. The Federal Highway Administration (FHA) and NDOT are planning to expand and redesignate Hwy 95 to Interstate 11 through this area, which will involve updating the two-lane highway to a separated four-lane interstate. Additionally, groundwater Two new hypogean Hyalella from Nevada 51 Figure 9. Hyalella plutonia sp. nov. allotype female, unnamed spring, ~0.7 km north of Hwy 95 and Oleo Rd, Nye County, Nevada, USA (USNM 1694151), 3.25 mm A gnathopod 1 (palmar margin and dactylus enlarged) B gnathopod 2 (palmar margin and dactylus enlarged). Scale bar: 0.25 mm. withdrawal for the town of Beatty, and proposed and current gold and lithium mines in the immediate area, pose a substantial risk to the continued existence of the type locality spring and H. plutonia sp. nov. The type locality spring occurs near the edge of private property where a couple of additional springs occur on the west side of Hwy 95; Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 52 Figure 10. Hyalella plutonia sp. nov. allotype female, unnamed spring, ~0.7 km north of Hwy 95 and Oleo Rd, Nye County, Nevada, USA (USNM 1694151), 3.25 mm A uropod 1 B uropod 2 C uropod 3 D telson. Scale bars: 0.1 mm. however, surveys were not conducted on this private property. The second author has surveyed over 20 additional springs in Oasis Valley, on both private and public land, but no other populations of stygobitic hyalellid amphipods have been found; only numerous populations of epigean hyalellid amphipods have been found. Hyalella plutonia sp. nov. co-occurs with the endemic Oasis Valley springsnail (Pyrgulopsis micrococcus) and Amargosa toad (Anaxyrus nelsoni), which were found during a survey of the spring. Two new hypogean Hyalella from Nevada 53 Hyalella keepuikantun Cannizzaro, Lange & Berg, sp. nov. https://zoobank.org/E2FC2F71-5446-4BF2-9FE6-10B80600D83A Figs 11–17 Material examined. • Holotype, male 3.48 mm: Devils Hole II, Nye County, Nevada, USA (36.42714, -116.29117); collectors: Jeffrey Goldstein / Olin Feuerbacher, 08 February 2017; USNM 1694154. • Allotype, female 3.62 mm: Devils Hole II, Nye County, Nevada, USA (36.42714, -116.29117); collectors: Jeffrey Goldstein / Olin Feuerbacher, 08 January 2020; USNM 1694155. • Paratype, male, same locality and collection information as holotype; USNM 1694157. • Paratype male, same locality and collection information as allotype; USNM 1694156. Type locality. Devils Hole II, Death Valley National Park, Nye County, Nevada, USA (36.42714, -116.29117). Etymology. The specific epithet keepuikantun means “blind” in Timbisha Shoshone (literally “no eye having”, kee(no/negative)pui(eye)katün(possession or having)) is given in reference to the lack of eyes and the location of Devils Hole II within Death Valley, the homeland of the Timbisha Shoshone Tribe. This name was selected in consultation with the Timbisha Shoshone Tribal Historic Preservation Office. Diagnosis. Small sized, stygomorphic species distinguished from congeners— except members of the H. muerta and H. cretae complex—by the combination of the following characters: eyes absent; antenna 1 longer than antenna 2; pleonites lacking dorsoposterior carinae; maxilla 1 inner plate with 2 apical pappose setae; male uropod 1 inner ramus lacking long curved setae. Differentiated from H. muerta and other members of the H. cretae complex by the combination of the following characters: maxilliped inner plate with 4 apical robust setae; male gnathopod 2 palmar margin with weak anterior notch; pereopod 7 basis projecting < 1.2 × the length of the ischium, with < 5 posterior setae, all simple, length 25% remainder of the pereopod segments; uropod 3 ramus with robust setae; telson with 4 apical setae. Description. Holotype male (Figs 3C, 11–15): 3.48 mm in length. Eyes and integumentary pigment absent (Fig. 3C). Antennae. Antenna 1 (Fig. 11A): approximately 1.4 × length of antenna 2; peduncle segment 1 with posterodistal robust seta; primary flagellum with 9 segments, Table 3. Morphological variation observed among individuals of H. plutonia sp. nov. and H keepuikantun sp. nov. Character Variability (H. plutonia sp. nov.) Variability (H. keepuikantun sp. nov.) Body length ~2–4 mm ~2–4 mm Antenna 1 flagellar segments 9–12 9–10 Antenna 2 flagellar segments 9–11 8–9 Antenna 2 length to body length 1.1–1.3× 1.1–1.5× Pereopod 7 basis post. setae 6–8 4–5 Pereopod 7 basis post lobe vs ischium length 1.8–2.3× 1.1–1.3× Uropod 1/2 peduncle inner setae robust or simple robust or simple Uropod 3 ramus setal length short / long short / long Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 54 aesthetascs on distal segments, aesthetascs shorter than respective segments, antepenultimate 3 segments with paired aesthetascs. Antenna 2 (Fig. 11B): approximately 25% body length, gland cone distinct; peduncular segment 5 subequal in length to segment 4; flagellum with 8 segments. Mouthparts. Mandibles: left mandible (Fig. 11C): incisor 8-dentate, lacinia mobilis 5-dentate, accessory setal row with 3 plumose setae; molar process well-developed, cylindrical triturative, with plumose seta. Figure 11. Hyalella keepuikantun sp. nov., holotype male, Devils Hole II, Nye County, Nevada, USA (USNM 1694154), 3.48 mm A antenna 1 (single aesthetasc enlarged) B antenna 2 C left mandible D right mandible (lacinia mobilis enlarged). Scale bars: 0.25 mm (A, B); 0.1 mm (C, D). Two new hypogean Hyalella from Nevada 55 Right mandible (Fig. 11D): incisor 7-dentate, lacinia mobilis trifid, lobes with numerous fine dentations; accessory setae row with 2 plumose setae; molar similar in form to left mandible. Upper lip (Fig. 12A): rounded, apical margin of labrum with numerous setules. Lower lip (Fig. 12B): inner lobes indistinct, outer margin of inner and outer lobes covered in setules; face of lip covered in pubescent setules. Figure 12. Hyalella keepuikantun sp. nov., holotype male, Devils Hole II, Nye County, Nevada, USA (USNM 1694154), 3.48 mm A upper lip B lower lip C maxilla 1 (outer plate serrate setae enlarged) D maxilla 2 E maxilliped (apical margin of inner plate enlarged). Scale bars: 0.1 mm. Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 56 Maxilla 1 (Fig. 12C): inner plate shorter than outer plate, with 2 apical pappose setae, and fine pubescent setules covering the entire plate; outer plate with 9 apical serrate setae, pubescent setules sparse decreasing laterally and proximally; palp onesegmented, vestigial, 20% length of outer plate, tapering strongly distally, with sparse pubescent setules, palp emerges at approximately 70% the length of the outer plate. Maxilla 2 (Fig. 12D): both inner and outer plates covered in pubescent setules; outer plate subequal in length to inner plate, with numerous apical setae; inner plate narrowing slightly distally with numerous apical setae and 2 pappose facial setae. Maxilliped (Fig. 12E): inner plate shorter than outer plate with 4 unarmed cuspidate setae along apical margin, surface of plate covered in pubescent setules; outer plate with numerous setae, lacking pubescent setules; palp 4-segmented, second and third segments with numerous marginal, submarginal setae; dactylus surface lacking comb-scales, nail not elongate. Gnathopods. Gnathopod 1 (Fig. 13A): coxal plate with 4 short apical setae; basis narrowing proximally, distoposterior surface of segment lacking comb-scales; ischium with a posterior seta; merus with 3 posterior setae, distoposterior surface of segment lacking comb-scales; carpus subequal in length to propodus, with cluster of posterior serrulate setae; propodus 1.3 × longer than broad with 5 inner medial serrulate setae, anterodistal corner with tuft of setae, sparse comb-scales present along posterior margin; palm straight, with 5–9 setae on inner/outer margins; defining angle straight with 2 robust setae; dactylus with plumose seta on outer margin and 3 inner setae, surface lacking comb-scales. Gnathopod 2 (Fig. 13B): coxal plate with 3 short apical setae; basis narrowing weakly proximally; ischium with a posterior seta; merus with 3 posterior setae, distoposterior surface of segment lacking comb-scales; carpus 40% length of propodus, posterior margin lobate, 33% breadth of segment, lacking comb-scales; propodus enlarged, powerful, 1.1 × longer than broad, with tuft of anterodistal setae, posterior setae absent; palm straight, irregular with weak anterior notch, palm with 9 inner and 11 outer robust setae; defining angle weak, projecting slightly past carpus posterior lobe, with 2 robust setae arranged around notch where tip of dactylus rests, comb-scales present apically; dactylus with plumose seta on outer margin placed proximally and 3 inner setae. Pereopods. Pereopod 3 (Fig. 14A): coxal plate with 5 short apical setae, plate not excavated posteroproximally; basis much longer than coxal plate lacking posterior marginal setae; merus subequal in length to carpus; carpus 80% length of propodus; dactylus 50% length of propodus, with plumose seta placed proximally on outer margin and 2 setae inserted adjacent to base of nail. Pereopod 4 (Fig. 14B): coxal plate with 4 short apical setae, plate excavated posteroproximally; basis much longer than coxal plate lacking posterior marginal setae; merus subequal to carpus in length; carpus subequal in length to propodus; dactylus 50% length of propodus, setation as in pereopod 3. Pereopod 5 (Fig. 14C): coxal plate large, bilobate, anterior/posterior lobes both with a seta; basis posterior margin weakly convex, with 4 short setae, posterodistal corner convex, anterior margin with 4 robust setae; merus subequal in length to carpus; Two new hypogean Hyalella from Nevada 57 carpus 70% length of propodus, dactylus 40% length of propodus, with plumose seta placed proximally on posterior margin and 2 setae inserted adjacent to base of nail. Pereopod 6 (Fig. 14D): coxal plate large, bilobate, with produced posterior lobe; basis posterior margin straight to convex, with 3 short setae, posterodistal corner straight, anterior margin with 2 robust setae; merus subequal in length to carpus; carpus 85% length of propodus; dactylus 40% length of propodus, setation as in pereopod 5. Figure 13. Hyalella keepuikantun sp. nov., holotype male, Devils Hole II, Nye County, Nevada, USA (USNM 1694154), 3.48 mm A gnathopod 1 (palmar margin and dactylus enlarged) B gnathopod 2 (palmar margin and dactylus enlarged). Scale bar: 0.25 mm. Andrew G. Cannizzaro et al. / Subterranean Biology 54: 35–68 (2025) 58 Figure 14. Hyalella keepuikantun sp. nov., holotype male, Devils Hole II, Nye County, Nevada, USA (USNM 1694154), 3.48 mm A pereopod 3 B pereopod 4 C pereopod 5 D pereopod 6 E pereopod 7. Scale bar: 0.25 mm. Pereopod 7 (Fig. 14E): coxal plate diminutive, lobes indistinct; basis posterior margin weakly convex, projecting 1.1 × past the ischium, with 4 short setae extending to start of posterodistal corner, robust setae absent, posterodistal corner rounded, anterior margin with 2 robust setae, basis length 25% remainder of pereopod segments; Two new hypogean Hyalella from Nevada 59 merus 70% length of carpus; carpus 90% length of propodus; dactylus 50% length of propodus, setation like pereopods 5 and 6. Gills. Coxal gills present on somites 2–7, sternal gills present on somites 3–6 (Figs 13B, 14A-E). Pleon. Pleosome somites lacking dorsoposterior carinae. Epimera (Fig. 15A): first epimeron ventral margin unarmed, distoposterior corner not produced, posterior Figure 15. Hyalella keepuikantun sp. nov., holotype male, Devils Hole II, Nye County, Nevada, USA (USNM 1694154), 3.48 mm A epimera B pleopod 1 (coupling spine enlarged) C uropod 1 D uropod 2 E uropod 3 F telson. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/subtbiol.54.168846.suppl1