Journal of Crustacean Biology Advance Access published 19 April Crustacean Biology

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1 The Crustacean Society Journal of Crustacean Biology Advance Access published 19 April 2017 Journal of Crustacean Biology Journal of Crustacean Biology, 37(3), , doi: /jcbiol/rux031 Version of Record, first published online April 19, 2017, with fixed content and layout in compliance with Art ICZN. A new species of subterranean amphipod (Amphipoda: Gammaridae: Crangonyctidae) from Florida, with a genetic analysis of associated microbial mats Thomas R. Sawicki 1, John R. Holsinger 2, Eric A. Lazo-Wasem 3 and Richard A. Long 1 1 Department of Biological Sciences, Florida A & M University, Tallahassee, FL 32307, USA; 2 Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529, USA; and 3 Division of Invertebrate Zoology, Peabody Museum of Natural History, Yale University, New Haven, CT 06520, USA Correspondence; T.R. Sawicki; thomas.sawicki@famu.edu (Received 13 February 2017; accepted 8 March 2017) ABSTRACT Crangonyx sulphurium n. sp., a new species of stygobitic amphipod, is described from three springs in central Florida, U.S.A. The description of the new species brings the number of described stygobitic species of Crangonyx found in the Floridan aquifer to three. The specimens were collected in the spring basins of Wekiwa and Volusia springs and De Leon Springs Cave. Specimens from De Leon Springs Cave were found swimming in and around white microbial mats characteristic of sulfur oxidizing bacteria. Next generation sequencing of microbial mat samples, collected from the spring ten years after the collection of the new species, indicates a heterotrophic and autotrophic community dominated by five phylotypes. The ecology of these three caves may be unique when compared to other caves within the Floridan aquifer. Key Words: Floridan aquifer, Sporomusa, stygobitic species, sulfur oxidizing bacteria INTRODUCTION The water-filled caves of the Floridan aquifer form a complex, interconnected habitat inhabited by numerous animal species. At least 29 invertebrate and one vertebrate species have been described from Florida caves (Franz et al., 1994; Holsinger & Sawicki, 2016; Lewis & Sawicki, 2016). To date, out of this rich biodiversity, only three species of stygobitic amphipods have been formally described from the Floridan aquifer, Crangonyx hobbsi (Shoemaker, 1941), C. grandimanus (Bousfield, 1963), and Stygobromus floridanus (Holsinger & Sawicki, 2016). A new stygobitic species of Crangonyx (Bate, 1859) is described from three freshwater caves in central Florida (Fig. 1). These caves appear to have a unique ecology from that of most caves within the Floridan aquifer. At the time of collection of the new species, the walls of both Wekiwa and De Leon springs were covered by thick mats, microscopically characterized to consist of the chemoautotrophic sulfur oxidizing bacteria Thiothrix and Beggiatoa, along with filamentous iron bacteria (Franklin et al., 2005). Observations made during scuba dives in De Leon Springs Cave from July 2012 to February 2016 suggest changes in the microbial mat community as indicated by the deterioration of the mats. No new specimens of Crangonyx sulphurium n. sp. were found during these collection efforts. The Author Published by Oxford University Press on behalf of The Crustacean Society. All rights reserved. For permissions, please journals.permissions@oup.com Microbial mat samples were taken on 26 June 2015 from De Leon Springs Cave. These chemosynthetic microbes may be providing a carbon source for the fauna, including crayfish, isopods, and amphipods that were previously collected from these caves. MATERIALS AND METHODS Of the three caves from which specimens of the new species were collected, only De Leon Springs was easily accessible using scuba diving. The amphipods in De Leon Springs were moving in and around thick white microbial mats. Specimens in Wekiwa Springs State Park and Blue Spring State Park were collected from fissure cracks in rocks surrounding the spring entrances. Specimens at all locations were collected with 250 ml Nalgene bottles with plastic tubes inserted into the bottle openings, modified as one way valves for use as a suction device. After each dive specimens were fixed in 70% ethyl alcohol. Specimens were dissected using a Leica M125 stereomicroscope and appendages mounted on temporary glycerin slides. Appendages were analyzed and drawn using a Leica DM 750 compound microscope with camera lucida. Dissected parts were later permanently mounted on microscope slides using Canada Balsam, or placed in vials containing 70% ethanol. We used the guidelines suggested by Watling, (1989) for the terms seta(e) and robust seta(e) used in the description.

2 T. R. SAWICKI ET AL. Figure 1. Geographic distribution of Crangonyx sulphurium n. sp. (triangles) and the general localities of Crangonyx hobbsi (Shoemaker, 1941), and Crangonyx grandimanus (Bousfield, 1963) (circles). Nomenclature for setal patterns on segment 3 of the mandibular palp follows Stock, (1974). The term defining angle refers to the posterior margin of the palm and distal most point of the posterior margin of the propodus, the point at which the tip of the dactyl closes on the propodus. Triplicate mat samples were collected in 50 ml sterile centrifuge tubes on 26 June 2015 and stored on ice until processed in the laboratory. Mat material was allowed to settle, the water was decanted, and the mat was transferred into 1.7 ml microcentrifuge tubes and pelleted at 5,000 RCF for 1 min. The supernatant was pipetted off. Genomic DNA (gdna) was extracted using PowerBiofilm DNA Extraction Kit (Mo Bio, Carlsbad, CA, USA) per the manufacturers protocol with one modification: The lysis buffer (reagent 1) was initially added to the microcentrifuge tube with the pellet and vortexed to re-suspend the mat material, and then transferred into the extraction bead mix tube. Extracted gdna was stored at 80 C. The prokaryote community was characterized by NextGen DNA sequencing on an Ion Torrent PGM. The V4 hypervariable region of 16S rdna was targeted by the combination of PCR primers 515F and 806R. Equivalent amounts of gdna from three mat samples were pooled and amplified in a single-step 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, Germantown, MD, USA), under the following parameters: 94 C for 3 min, followed by 28 cycles (5 cycles used on PCR products) of 94 C for 30 s, 53 C for 40 s and 72 C for 60 s, with a final elongation step at 72 C for 5 min. Amplicon sequencing on an Ion Torrent PGM per the manufacturer s guidelines was conducted at MR DNA (Shallowater, TX, USA). Sequence data were initially processed using a proprietary analysis pipeline (MR DNA). Barcodes and primer sequence were removed, as were DNA sequences < 150 bp. Sequences were denoised and those with ambiguous base calls, homopolymer ( 6 bps) and chimeras were removed. Sequences were clustered into operational 286

3 NEW SPECIES OF SUBTERRANEAN AMPHIPOD taxonomic units (OTUs) at 97% similarity level. OTUs were taxonomically assigned using BLASTn against a reference database derived from Ribsomal Data Project II (Cole et al., 2014) and NCBI GenBank (Clark et al., 2016). The Fast.Q file was deposit at NCBI with accession number SRR The taxonomic work was undertaken by TRS and JRH. SYSTEMATICS Family Crangonyctidae Bousfield, 1973 (emended by Holsinger, 1977) Genus Crangonyx Bate, 1859 Crangonyx sulphurium n. sp. Sawicki & Holsinger (Figs. 2 8) Type material: Female holotype (10.0 mm; dissected), collected 11 April 2005, Terrance Tysall coll.; deposited in the National Museum of Natural History, Smithsonian Institution (USNM ). One female paratype (9.0 mm; dissected), same data as holotype, deposited at the Yale Peabody Museum of Natural History (YPM IZ ). One female (7.5 mm, dissected) deposited at the Yale Peabody Museum of Natural History (YPM IZ ), and one male (9.0 mm; dissected), paratypes, same data as holotype. Two male paratypes (6.7 mm; body intact except for pereopods 5, 6, and 7 from the right side of the body, dissected for DNA extraction; 8.0 mm; dissected), collected 11 April 2005, Terrence Tysall coll., Volusia Blue Spring, Blue Spring State Park, Florida (latitude: , longitude: ). Type locality: De Leon Springs, De Leon Springs State Park, Volusia County, Florida (latitude: , longitude: ; elevation 1 m; water temperature 22.7 C). Additional material examined: Two males and one female collected by Terrance Tysall, 21 January 2005, Wekiwa Springs, Wekiwa Springs State Park, Florida (latitude: , longitude ). Etymology: The specific epithet refers to the microbial mats, microscopically characterized to be largely sulfur oxidizers by Franklin et al., (2005), that were found within the caves in which this species was collected. Diagnosis: Medium-size troglomorphic stygobiont species easily distinguished from all congeners by highly setose mouthparts, particularly maxilla 1, maxilla 2. Male antenna 2 without calceoli. Propod of gnathopod 1 approximately twice longer than broad, 1.25 times longer, subequal in width to carpus, palm transverse; propod of gnathopod 2 approximately twice longer than broad, 1.3 times longer, 1.2 times wider than carpus, palm transverse. Male uropod 2 outer ramus without comb spines, not deflected laterally or curved. Figure 2. Crangonyx sulphurium n. sp. Paratype, Volusia Blue Spring, Blue Spring State Park, Orange City, Florida. Male (8.0 mm). Scale bar = 3 mm. 287 Description: Female holotype 10.0 mm (Figs. 3 7). Antenna 1 approximately 85% length of body, 1.8 times longer than antenna 2, primary flagellum with 21 or 22 segments, accessory flagellum 2 segmented, calceoli present on distal segments. Antenna 2 segments 4, 5 subequal, flagella with 8 segments. Right mandible molar process weak, with seta; lacinia mobilis bifid, both bifurcations with small serrate teeth, incisor 4 dentate, margin adjacent to lacinia with 6 robust setae, 4 additional small plumose setae; right palp with numerous A setae, 2 B setae, 2 C setae, 12 D setae appearing plumose or serrate distally, and 4 E setae. Left molar with seta; lacinia mobilis 4 dentate, incisor 5 dentate, margin adjacent to lacinia with up to 7 robust setae, up to 7 additional small plumose setae; left palp with numerous A setae, 2 B setae, 2 C setae, 12 D setae appearing plumose or serrate distally plus 4 E setae. Lower lip without inner lobes; outer lobes broadly covered with fine setae, especially medially, laterally, with few blade-like robust setae along apical margin. Maxilla 1 inner plate broadly covered with fine setae, with 5 large plumose apical setae; outer plate broadly covered with fine setae, particularly along medial margin, with 7 apical comb spines; palp 2-segmented, distal segment broadly covered with fine setae, apex with up to 8 plumose or serrate robust setae on outer margin, second row of 4 smooth robust setae just proximal to apex. Maxilla 2 inner plate broad medially, narrowing distally, broadly covered with fine setae, with oblique submarginal row of 5 large plumose setae, apical margin with up to 14 setae, with second row of 4 setae just proximal to apex; outer plate apical margin with 2 rows of 7 11 setae. Maxilliped inner plate broadly covered with fine setae, apical margin with 4 bladelike robust setae, 4 large plumose setae, inner margin with 3 large plumose setae; outer plate covered with fine setae along lateral margin, distal inner margin with 3 serrate robust setae, apical margin with plumose seta, 3 smooth robust setae; palp segment 3 pubescent distally, inner margin with numerous large setae. Gnathopod 1 propod 1.5 times longer than broad, 1.25 times longer, subequal in width to carpus. Palm transverse, approximately 80% length of posterior margin, with 6 setae, double row of 8 12 robust setae; defining angle with large robust seta, numerous bifid robust setae, with 2 long setae just proximal to defining angle; dactyl subequal in length to palm with 3 setae at base of nail; carpus posterior margin with approximately 16 long setae; merus posterior margin pubescent; basis posterior margin with 8 long setae, anterior margin with 4 long setae, medial margin with 4 long setae; coxa deeper than broad with 6 marginal setae. Gnathopod 2 propod approximately twice longer than broad, 1.3 times longer, 1.2 times wider than carpus. Palm transverse, approximately 80% length of posterior margin, with 3 setae, a double row of 10 robust setae; defining angle with 2 large bifid robust setae, numerous smaller bifid robust setae, with 2 large, distally serrate setae, and 3 small setae just proximal to defining angle, 9 singly inserted superior medial setae; dactyl subequal in length to palm bearing 3 setae at base of nail, 2 along posterior margin; carpus posterior margin with 6 groups of numerous long setae; merus distoposterior margin with 3 long setae; basis posterior margin with 6 long setae, anterior margin with 6 long setae, medial margin with long seta; coxa deeper than broad with 6 marginal setae. Pereopod 3 subequal in size to 4, coxal plate 2.6 times deeper than broad, lower margin rounded bearing 10 setae; basis not expanded, posterior margin with 5 long setae, short seta, anterior margin with 9 long, 4 short setae; dactyl proximal anterior margin with plumose seta, posterior margin with robust seta just proximal to base of nail. Pereopod 4 coxal plate 1.6 times deeper than broad, with 21 small setae along lower, posterior margins, excavate posteriorly; basis not expanded, posterior margin with 6 long setae, short seta, anterior margin with 4 long setae; dactyl proximal anterior margin with plumose seta, posterior margin with robust seta just proximal to base of nail. Pereopods 5, 7 approximately 80% length of body, pereopod 6 subequal to length of body; bases of pereopods 5 7

4 T. R. SAWICKI ET AL. Figure 3. Crangonyx sulphurium n. sp. Holotype, De Leon Springs, De Leon Springs State Park, De Leon Springs, Florida. Female (10.0 mm): A, antenna 1 (single calceolus on distal flagellar segment enlarged); B, antenna 2; C, lower lip; D, left mandible (lacinia mobilis and incisor enlarged); E, right mandible (lacinia mobilis and incisor enlarged). Scale bars: A, B = 1 mm; C = 0.25 mm; D, E = 0.5 mm; enlarged lacinia mobilis and incisor each 0.1 mm. not greatly expanded, distoposterior lobes weakly developed; dactyl of pereopod 5 approximately 31% length of corresponding propod, dactyls of pereopods 6, 7 approximately 30% length of corresponding propods; pereopods 5, 6 dactyl proximal anterior margin with plumose seta, posterior margin with robust seta just proximal to base of nail, pereopod 7 dactyl proximal anterior margin with 3 plumose setae, posterior margin with robust seta just proximal to base of nail. Coxal gills found on gnathopod 2, pereopods 3 7, ellipsoidal, with distinct peduncles; sternal gills lanceolate, present on pereonites 2, 3, 7; brood plates moderately setose, found on gnathopod 2, pereopods 3, 4, 5. Epimeral plates 1 3 posteriodistal margins each with small toothlike extension; plate 1 with small seta on posterior margin; plates 2, 3 with 3 robust setae along ventral margin, small seta on posterior margin. Uropod 1 outer ramus approximately 90% length of inner, 56% length of peduncle, with 4 apical robust setae, inner margin with 4 robust setae, outer margin with 5 robust setae; inner ramus 63% length of peduncle with 5 apical robust setae, inner margin with 3 robust setae, outer margin with 3 robust setae; peduncle with 7 robust setae, 6 on outer margin. Uropod 2 outer ramus approximately 83% length of inner ramus, 66% length of peduncle, with 4 apical robust setae, outer margin with 4 robust setae, inner margin with 4 smaller, robust setae; inner ramus 80% length of peduncle bearing 5 apical robust setae with plumose seta, outer margin with 3 robust setae, inner margin with 3 robust setae; peduncle with 7 robust setae, 3 on outer margin. Uropod 3 outer ramus 3.1 times longer than inner ramus, 1.6 times longer than peduncle, with 3 apical robust setae, smooth seta, outer margin with 6 robust setae in 3 sets, inner margin with 3 robust setae; inner ramus with subapical robust seta, robust seta along inner margin; peduncle with 4 robust setae. Telson 1.2 times broader than long at base, narrowing distally, notched approximately 30% to base, each lobe with 3 robust apical setae, small plumose seta, each lateral margin with set of 2 small plumose setae. Male paratypes (Fig. 8): 1 male paratype (De Leon Springs; 9.0 mm; YPM IZ ); 2 male paratypes (Volusia Blue Spring; 6.7 mm; YPM IZ ; 8.0 mm; YPM IZ ). Males differing from females as follows. Smaller than females (largest male 9.0 mm, largest female 10 mm). Antenna 1 approximately 60% length of body, 1.7 times longer than antenna 2. Telson length to width 288

5 NEW SPECIES OF SUBTERRANEAN AMPHIPOD Figure 4. Crangonyx sulphurium n. sp. Holotype, De Leon Springs, De Leon Springs State Park, De Leon Springs, Florida. Female (10.0 mm): A, maxilliped; B, maxilla 2; C, maxilla 1; D, gnathopod 1; E, enlarged distal end of propod and dactyl of gnathopod 2. Scale bars: A = 0.1 mm; B, C = 0.25 mm; D = 1 mm; E = 0.25 mm. subequal, notched 40 50% to base; females notched 28 35% to base. Outer ramus of uropod 2 as in the female, not reduced, deflected laterally, or curled backward in specimens from De Leon Springs and Volusia Blue Spring. Remarks: Two males, one 6.0 mm, one fragmented, from Wekiwa Spring showed a maxilla 1 with an inner plate having ten large plumose apical setae. In specimens from Volusia Blue Spring and De Leon Springs, the maxilla 1 inner plate have 4 or 5 plumose apical setae. The palm of gnathopod 2 is slightly more oblique than specimens from Volusia Blue Spring or De Leon Springs, and the uropod 3 has an outer ramus that is twice the length of the peduncle. The uropod 3 outer ramus of males from Volusia Blue Spring and De Leon Springs are times the length of the corresponding peduncle, females The maxillae 1 and 2 appear slightly less setose in specimens from Volusia Blue Spring and Wekiwa Spring relative to specimens from De Leon Springs, and the outer ramus of uropod 2 is weakly deflected laterally. Specimens from Wekiwa Spring were fragmented and in poor condition. It is unclear if the variation, particularly in the apparent deflection in the outer ramus of uropod 2, reflects the condition of the specimens, or an important distinction. It is possible that the Wekiwa Spring population may represent a separate species; however, without additional specimens to analyze both morphologically and genetically, we tentatively assign the Wekiwa population to Crangonyx sulphurium n. sp. Habitat: At the time of collection, areas of the cave surface in Wekiwa Spring were covered by white microbial mats that Franklin et al., (2005) identified to be Thiothrix, Beggiatoa, and a filamentous iron bacterium. At the time of collection, the surfaces of De Leon Springs appeared to be covered by these same types of bacterial mats 289

6 T. R. SAWICKI ET AL. Figure 5. Crangonyx sulphurium n. sp. Holotype, De Leon Springs, De Leon Springs State Park, De Leon Springs, Florida. Female (10.0 mm): A, gnathopod 2; B, enlarged distal end of propod and dactyl of gnathopod 2; C, brood plate and gill of gnathopod 2; D, pereopod 3, with epibionts. Scale bars: A and D = 1 mm, (C drawn at same scale as A); B = 0.25 mm. (T. Tysall, personal communication). The water flow coming from Volusia Blue Spring, 4.48 m 3 /s (Scott et al., 2004), prevents significant exploration of the cave passage, and it is not known if the walls of this cave are covered by filamentous bacteria as in De Leon and Wekiwa Springs; however, TRS has observed what appears to be bacterial filaments floating in the water in the cavern and spring basin. At Volusia Blue and Wekiwa springs, specimens were collected in small, cavern-like crevices located outside of the primary cave spring that extended horizontally into rock, approximately 10 m from open water. The amphipods were apparently seeking refuge in these dark passages after presumably being washed out of the cave. Specimens from De Leon Springs were collected swimming in and around white microbial mats within the cave (T. Tysall, personal communication). In July, 2012, one of us (TRS) began exploring these cave systems to obtain fresh specimens for genetic analysis and to augment the collection for morphological description. From July 2012 to February 2016, seven separate dives were made in De Leon Springs but no additional specimens were seen; however, over this period of time, changes in the biomass and apparent composition of the microbial mats were noted. The white microbial mats, which possessed filaments that were initially at least 10 cm in length and thickly covered the walls, ceilings, and even the sediments on the cave floor, appeared to thin out, exposing bare rock and sediments in places, and transitioned from white to brown in most cave regions. The white microbial mats became largely restricted to a single vent, located approximately 20 m from the cave entrance, through which water flows into the cave at a high velocity (see S1 Figure). The other main water source is a tunnel, approximately 40 m from the cave entrance that is restricted by a metal grate, preventing deeper penetration into the cave (see S2 Figure). 290

7 NEW SPECIES OF SUBTERRANEAN AMPHIPOD Figure 6. Crangonyx sulphurium n. sp. Holotype, De Leon Springs, De Leon Springs State Park, De Leon Springs, Florida. Female (10.0 mm): A, pereopod 4; B, pereopod 5, with epibiont; C, pereopod 6; D, pereopod 7. Scale bars: A D = 1 mm. Starting in January of 2014, a sonde was used to collect ph, conductivity, temperature, oxygen concentration, and depth during each dive. The sonde indicated that the vent and grate water are derived from separate water sources, each with different, although fluctuating, ph and conductivity values (Table 1). Oxygen concentration throughout the aphotic cave was anoxic, averaging less than 0.2 mg/l. Oxygen concentration data collected by the St. John s River Water Management District from the photic open water spring basin was hypoxic (mean oxygen reading 1.05 mg/l (0 mg/l to 1.88 mg/l, N = 39; 19 December 2011 and 13 April 2016; data collected at 0.5 m depth). Water temperature fluctuated between and C throughout the cave. Analysis of the white microbial mats by NextGen DNA sequencing of the V4 region of the 16S rdna revealed a complex prokaryotic community. There was a total of reads (high quality DNA sequences) greater than 200 bps that were binned into 2126 OTUs (Operational Taxonomic Units, > 97% similarity). These OTUs contain representatives from 400 genera, 378 families, and 28 phyla; nearly 80% of the reads were from three phyla, Firmicutes, Proteobacteria, and Plantomycetes (S3 Figure). Eight OTUs had abundances of >1%, while another 17 had > 0.5% abundance. Grouping these OTUs into their nearest phylogenetic groups results in the top five genera (Table 2), which represent a diversity of metabolic strategies. The dominant genus was Sporomusa, a spore forming heterotroph, both in terms of OTUs (114), and reads (22858, or 34%). Also in the top five were candidats Kuenenia and Brocadia (both only described as phylotypes), which are capable of anaerobic oxidation of ammonium (anammox); and Thermodesulfovibrio, a sulfate-reducer, and Acidithiobacillus, a sulfur-oxidizer. 291

8 T. R. SAWICKI ET AL. Figure 7. Crangonyx sulphurium n. sp. Holotype, De Leon Springs, De Leon Springs State Park, De Leon Springs, Florida. Female (10.0 mm): A, pleopod 1 (coupling spines enlarged); B, epimeral plates; C, uropod 1; D, uropod 2; E, uropod 3; F, telson 3. Scale bars: A, B, D, E = 1 mm; C= 0.25 mm; F = 0.5 mm. Franklin et al., (2005) microscopically identified the dominant prokaryotes in the white microbial mats from Wekiwa Springs as Thiothrix, Beggiatoa, and a filamentous iron bacterium. The molecular analysis of the more recently collected white microbial mats in De Leon Springs included the two sulfur oxidizers and the genera of numerous metal-oxidizing genera of bacteria, which were not the dominant phylotypes (Table 2). There appears to be a diverse prokaryotic community of potential sulfur oxidizers and potential sulfate-reducers within the mat. Potential sulfur oxidizers are represented by 50 genera or 12.5% of the detected genera. These accounted for 6.4% of the OTUs, and 6.9% of the reads (see S4 Figure). It is unclear if the apparent changes in microbial biomass and composition in De Leon Springs are temporary, or if they are indicative of a fundamental change in the cave s biogeochemistry and water quality. The mouthparts of Crangonyx sulphurium n. sp. (particularly the maxilla 1 and maxilla 2), which are broadly covered with fine setae, and the fact that all specimens were collected swimming in and around the microbial mats, suggest that the new species could feed directly off the microbial mats, possibly using their mouthparts to strain the filaments. Despite numerous attempts, the new species has not been collected since its discovery in 2005, which may be due to changes to the microbial mat coverage of the cave (as documented visually by TRS) and the apparent changes in these microbial communities. The setae of Crangonyx. sulphurium n. sp. appeared to be broadly covered with epibionts, which are partially visible in Figs. 5D (coxal plate lower margin), 6B (basis posterior margin), and 8B (basis anterior margin), note small ovate circles drawn at the tips of setae. Bauermeister et al., (2013), described Thiothrix ectosymbionts on species of Niphargus (Schiödte, 1849), from sulfiderich caves in central Italy. The epibionts on C. sulphurium n. sp. may reflect a similar possible symbiotic relationship. If so, the absence of C. sulphurium n. sp. from De Leon Springs Cave since 2005 may be correlated with the apparent change in microbial mat community from one dominated by sulfur oxidizers such as 292

9 NEW SPECIES OF SUBTERRANEAN AMPHIPOD Figure 8. Crangonyx sulphurium n. sp. Paratype, De Leon Springs, De Leon Springs State Park, De Leon Springs, Florida. Male (9.0 mm): A, gnathopod 1; B, gnathopod 2, with epibionts; C, uropod 2. Scale bars: A, B = 1 mm; C= 0.5 mm. Table 1. ph and specific conductivity in two locations within De Leon Springs Cave, Florida. Data was taken using a Hydrolab HL4 sonde. Date ph Specific conductivity µs/cm Vent Grate Vent Grate 2 Jan Jan Aug Feb Thiothrix (Franklin et al., 2005) to one dominated by heterotrophic bacteria, e.g., Sporomusa. Nomenclatural statement: A life science identifier (LSID) number was obtained for the new species: urn:lsid:zoobank. org:pub:0341f8d3-01fd-471d-a3b1-5caa0b3a881d. DISCUSSION Crangonyx sulphurium n. sp. is distinguished from all species in the genus Crangonyx by the presence of highly setose mouthparts, particularly in the maxilla 1 and maxilla 2. Based on morphological characteristics, Zhang & Holsinger, (2003) established six phylogenetic species groups within Crangonyx. C. sulphurium n. sp. aligns with the gracilis species group comprising the hobbsi, baculispina, and floridanus subgroups, with a total of 19 species. These and the new species share superior medial setae of gnathopod 2 propod being singly inserted. 293

10 T. R. SAWICKI ET AL. Table 2. Most abundant Operational Taxonomical Units (OTU) in the white microbial mat from De Leon Springs, Florida. Community composition was analyzed by the next generation sequence of the hypervariable region 4 of the 16S rdna. The top 5 of 400 genera are presented, also included is information on the genera Beggiatoa and Thiothrix, two previously identified genera in the white microbial mats from Wekiwa Spring, Florida. *, values within parenthesis indicate the % of total OTUs and high quality DNA sequence reads, respectively. Genus OTU* Reads* Sporomusa 114 (5.4) (34.3) Thermodesulfovibrio 90 (4.2) 3997 (6.0) Candidatus Kuenenia 42 (2.0) 3264 (4.9) Acidithiobacillus 24 (1.1) 1085 (1.6) Candidatus Brocadia 19 (0.9) 889 (1.3) Beggiatoa 6 (0.3) 245 (0.4) Thiothrix 4 (0.2) 38 (0.06) Crangonyx sulphurium n. sp. is distinguished from C. gracilis (Smith, 1871), C. floridanus (Bousfield, 1963), C. pseudogracilis (Bousfield, 1958), C. rivularis (Bousfield, 1958), C. acicularis (Zhang & Holsinger, 2003), C. aka (Zhang & Holsinger, 2003), C. baculispina, (Zhang & Holsinger, 2003), C. consimilis (Zhang & Holsinger, 2003), C. longidactylus (Zhang & Holsinger, 2003), C. montanus (Zhang & Holsinger, 2003), C. ohioensis (Zhang & Holsinger, 2003), C. palustris (Zhang & Holsinger, 2003), and C. stagnicolous, (Zhang & Holsinger, 2003) by the absence of eyes. The new species is also distinguished from C. floridanus, C. pseudogracilis, C. consimilus, and C. longitdactylus, by the absence of comb spines on the outer ramus of male uropod 2. The new species is further distinguished from all species within the gracilis group, except C. hobbsi (Shoemaker, 1941) and C. caecus (Zhang & Holsinger, 2003), by the male outer ramus of uropod 2 not being deflected laterally or curved (except possibly the Wekiwa population; see above), and the male antenna 2 not having calceoli. It is further distinguished from C. caecus by size, the largest female being 7.3 mm and largest male 5.7 mm in C. caecus, whereas the largest female is 10.0 mm and the largest male 9.0 mm in C. sulphurium n. sp. The new species is distinguished from C. islandicus (Svavarsson & Kristjánsson, 2006) by having four robust setae on the maxilliped inner plate, whereas C. islandicus has only one robust seta. The description of Crangonyx sulphurium n. sp. brings the number of stygobitic amphipod species in the Floridan aquifer to four together with, C. hobbsi (Shoemaker, 1941), C. grandimanus (Bousfield, 1963), and Stygobromus floridanus (Holsinger & Sawicki, 2016). The new species is distinguished from C. hobbsi by the carpus and propod of gnathopods 1 and 2 being significantly longer in C. hobbsi, with the carpus longer than the corresponding propod. The propods of gnathopods 1 and 2 of female C. sulphurium n. sp. are relatively robust, with the propod longer than the corresponding carpus. The new species is distinguished from C. grandimanus, which Zhang & Holsinger, (2003) aligns with the richmondensis group, by size, the largest females of C. grandimanus being 17 mm and the largest males 13 mm; the propod of gnathopods 1 and 2 of both sexes of C. grandimanus is strongly oblique, twice larger than the carpus, the palm almost twice larger than the posterior margin, and the gnathopod 2 superior medial setae inserted in groups of 3 or 4. Crangonyx is a species-rich freshwater genus, holarctic in distribution. A genetic analysis of the species in the genus is needed to provide evidence for the morphological phylogeny established by Zhang & Holsinger, (2003), an investigation that is underway (TRS). Unfortunately, given our inability to find additional specimens of C. sulphurium n. sp., the exact phylogenetic relationship of the new species to the other species of Crangonyx is not currently possible. 294 SUPPLEMENTARY MATERIAL Supplementary material is available at Journal of Crustacean Biology online. S1 Figure. Vent De Leon Springs. S2 Figure. Grate De Leon Springs. S3 Figure. Distribution of phyla of organisms within the De Leon Spring white microbial mat. S4 Figure. Phylogenetic distribution of sulfur oxidizing bacteria within De Leon Spring white microbial mat. ACKNOWLEDGEMENTS We thank Becky Buck and the staff at De Leon Springs, Blue Spring, and Wekiwa Springs state parks for their enthusiastic support of this research. We also thank Terrance Tysall, Amy Giannotti, Renee Power, Kris Shannon, Marissa Williams, and the other divers and support staff of the Cambrian Foundation for their knowledge and support during the dives. We are also grateful to Bonnie Stine, Casey McKinlay, and Steve Cox for their diving support. We are also grateful to Dr. Michael Stine and Mark Long for their support in diving and exceptional underwater photography. We gratefully thank two anonymous reviewers for their constructive feedback. Funding for this project was provided in part by the American Public University System Faculty Research Grant and by the State of Florida, Florida Fish and Wildlife Conservation Commission, State Wildlife Grants No , U.S Fish and Wildlife Service Federal Award No. FL-T-F15AF REFERENCES Bate, C.S On the genus Niphargus (Schiödte). Proceedings of the Dublin University Zoological and Botanical Association, 1: Bauermeister, J., Assig, K. & Dattagupta, S Exploring the sulfide tolerance of ectosymbiotic Niphargus amphipods from the Frasassi caves, central Italy. International Journal of Speleology, 42: Bousfield, E. L Fresh-water amphipod crustaceans of glaciated North America. 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