2012 SOUTHEASTERN NATURALIST 11(2):279–286 Tardigrades of North America: Archechiniscus biscaynei, nov. sp. (Arthrotardigrada: Archechiniscidae), a Marine Tardigrade from Biscayne National Park, Florida William R. Miller1,*, Tiffany Clark1, and Cullen Miller1 Abstract - During the 2010 National Park Service BioBlitz at Biscayne National Park, Homestead, FL, barnacles were collected from a submerged mangrove log near the Park headquarters. From the washings of the crushed barnacles, Archechiniscus biscaynei nov. sp. was extracted. This new species can be distinguished by its lack of a median cirrus, its large peduncles at the base of the external claws, the rounded cephalic clava between the internal and external cephalic cirri, a club-shaped papilla above the base of leg IV, and bulbous papilla just above the claws on the anterior of legs I, II, and III. A table of comparison of characteristics among the four known species and a key to their identification are provided. Introduction In 1953, while examining washings of barnacles (Cirripedia) from El Salvador (Central America), Enrich Schultz found a single tardigrade with a unique claw pattern. The animal had the two external claws attached to the base of the leg like suborder Echiniscoidea and two internal claws extended on digits or toes like some Arthrotardigrada. Based on this unique toe-claw structure and the lack of a median cirrus, Schultz (1953) described the new sub-family Archechiniscinae in the family Oreellidae in the suborder Echiniscoidea, a new genus (Archechiniscus Schultz, 1953), and a new species (Archechiniscus marci Schultz, 1953). Ten years later, Renaud-Debyser (1963) reported the animal from Bimini in the Bahamas, and in a few years later, Schuster and Grigarick (1966) reported another single specimen from coral washings from tide pools on Santa Cruz Island in the Galapagos. While the next year, Renaud-Mornant (1967) collected specimens from the sandy bottom of Saint-Vincent Bay, New Caledonia. Ten years passed before Binda (1978) established the new family Archechiniscidae within the suborder Arthrotardigradea and transferred the new family with genus Archechiniscus from its previous classification in suborder Echiniscioidea, family Oreellidae because the internal claws were extended on digits (toes). Another ten years passed before a second species—Archechiniscus minutus Grimaldi de Zio and D’Addabbo Gallo, 1987—was described from depths up to 35 m in course coralliferous debris in both the Adriatic and Ionian Seas around southern Italy. A decade later, a third species—Archechiniscus symbalanus Chang and Rho, 1998— was described from barnacles from the Andaman Sea on the coast of Thailand. Recently, a species of Archechiniscus that was included in a molecular phylogenetic investigation suggested that Archechiniscus might not be a halechiniscid, but rather represents a distinct taxon (Jørgensen et al. 2010). This development has led 1Department of Biology, Baker University, Baldwin City, KS 66006. *Corresponding author - William.Miller@BakerU.edu. 280 Southeastern Naturalist Vol. 11, No. 2 Degma et al. (2011) to place Archechiniscus within Archechniscidae as suggested by Binda (1978). Since another decade has again passed, it seems timely to describe the fourth species of this rarely reported genus. The new species, like A. marci and A. symbalanus, was extracted from between the plates of barnacles in the intertidal zone, not the deeper water where A. minutus is found. Materials and Methods In early 2010, we were invited by the National Park Service and the National Geographic Society to attend the fourth annual United States Park Service BioBlitz held at Biscayne National Park, FL. We collected gravel, sand, and barnacles from the intertidal zone. Barnacles were scraped with a putty knife from rocks, concrete pilings, and a submerged mangrove log in the tidal water of Biscayne Bay and placed into sandwich- sized plastic bags. The barnacles were crushed in the bag, which punctured the plastic. Then 50 ml of bottled fresh water were added and the contents washed into a new bag. This bag was allowed to stand for two hours before sub-sampling. Five, 2-ml sub-samples of debris from the bottom of the bag were siphoned with a disposable pipette and examined in a 25- x 50-mm plastic tray with the bottom painted black. A 20x dissecting microscope was used to search the sub-samples. Two high-intensity LED lights were arranged, one at 90 degrees and another at 45 degrees to the dish, to give good color and shadow essential to seeing the nearly transparent marine tardigrades. The osmotically shocked specimens were extracted with an Irwin loop and placed directly into a drop of polyvinyl alcohol on a glass slide and covered with a glass cover slip. After drying for several days, the cover slip was sealed with clear fingernail polish. The key in Ramazotti and Maucci (1983) was used to identify the genus, and the checklist of Degma et al. (2011) was used to confirm that three species had been described. The original papers for each species were used to compare to the new species. Results Two specimens of Echinsicoides sigismundi sigismundi (M. Schultz, 1865) were found in five samples collected from barnacles attached to rocks and pilings collected from the visitors harbor on Elliot Key. Three specimens of the Archechiniscus biscaynei nov. sp. were found in two samples of barnacles removed from a dead mangrove log submerged near the Visitors Center at Convey Point. No tardigrades were found in the five sand or gravel samples examined. Phylum: Tardigrada Class: Heterotardigrada Marcus, 1927 Order: Arthrotardigrada Marcus, 1927 Family: Archechiniscidae Binda, 1978 Genus: Archechiniscus Schultz, 1953 2012 W.R. Miller, T. Clark, and C. Miller 281 Archechiniscus biscaynei nov. sp. (Figures 1, 2; Tables 1, 2) Diagnosis: Archechiniscus with smooth cuticle, cephalic clava a mounded circle between the small internal and external cirri, large peduncle under external claws, club-shaped papilla at the base of leg IV, bulbous papilla on anterior surface of each of legs I, II, 7 III, median cirrus absent, and a short cirri at E. Description: Measurements of holotype, (paratype 1, paratype 2). Cylindrical body shape, rounded posterior and narrower rounded anterior end with protrudable mouth cone. Body length 246.6 μm (198.5 μm, 207.8 μm), width 93.3 μm (74.5 μm, 75.5 μm), eye spots absent. Body segments not apparent, cuticle colorless and smooth. Internal cirrus 6.5 μm (5.6 μm, 6.7 μm) with annular refracting organelle (annulus) in expanded base. External cirrus 8.5 μm (7.8 μm, 7.0 μm) with annulus and expanded base. Cephalic clava is low, rounded dome 8.4 μm diameter (6.8 μm, 6.8 μm) with annulus and low protuberance above cuticle. Secondary clava located equal distance between and below (ventral) internal and external cirri. Median cirrus absent. Cirri A short 8.3 μm (8.3 μm, 10.3 μm), half the length of expanded base. Primary clava 5.9 μm (5.7 μm, 5.0 μm) club-shaped bulb with annulus arising from same base as cirrus A. Protrudable mouth cone, terminal to sub-terminal. Buccal tube, stylets, and pharynx not visible on holotype or paratypes. Cirrus E short, 10.3 μm including 4 μm base (12.9 μm, 10.8 μm). Club-shaped papilla with annular refracting organelle on dorsal lateral surface at base of leg IV, 4.5 μm (4.3 μm). Stubby, non-telescopic legs, sensory spine on leg I absent. Bulbous papilla just above claws on anterior of legs I, II, and III. Internal claws unbranched, sickle shaped uncus 6.3 μm long (6.6 μm, 6.0 μm) with thin or fine accessory points on 6.0 μm digits or toes. External claws, 7.5 μm (6.1 μm, 6.8 μm) with stout basal spur, 2.4 μm (2.1 μm, 2.1 μm) with large rectangular peduncles at base. Thin membranous sheaths on internal claws visible. Female gonopore with six folds located about 10 μm anterior to anus, male gonopore round 3 μm opening only 3 μm from anus. Slit like anus, 6 μm, surrounded by fan of folds on sides and posterior end. Seminal receptacles long, thin (2 μm wide) tubes exiting ventro-laterally anterior to legs IV. Holotype: Female, slide number B004515 under accession number BISC-271 at the South Florida Collections Management Center, Everglades National Park, Homestead, fl33034. Paratypes: Two males, slide numbers B004514 and B004516 under accession number BISC-271 at the South Florida Collections Management Center, Everglades National Park, Homestead, fl33034. Etymology: The proposed specific name, biscaynei was chosen to commemorate the location of discovery of the specimens, Biscayne National Park, Homestead, FL, USA. Type Locality: A submerged mangrove log in inter-tidal zone in front of the Dante Fascell Visitor Center, Convoy Point, Biscayne National Park, Homestead, FL, latitude 25.46503, longitude -80.33479. Differentiation: The new species is easily differentiated from other species of Archechiniscus by the lack of median cirrus, the peduncles at the base of the 282 Southeastern Naturalist Vol. 11, No. 2 external claws, the round cephalic clave equal distance between the cephalic cirri, the club-shaped papilla above leg IV, and the bulbous papilla on the front of each leg (Tables 1, 2). The new species differs from A. minutus by having Figure 1. Archechiniscus biscaynei nov. sp., A. Body, B. Legs IV, C. Claws leg IV, D. Leg II, E. Head segment. a = opening for seminal receptacle, b = bulbous leg papilla, c = gonopore, d = anus, e = peduncle, f = spur of external claw, g = accessory points of internal claw, h = lateral cirrus A, i = external cirrus, j = secondary clava, k = internal cirrus, l = primary clava, m = annular refracting organelle. 2012 W.R. Miller, T. Clark, and C. Miller 283 peduncles under the external claws, and a club-shaped posterior clava. It differs from A. symbalanus by the absence of the small median cirrus and the absence Figure 2. Archechiniscus biscaynei nov. sp., A. Lateral cirrus A and primary clava, B. secondary clava, C. cross section of cuticle, D. male gonopore and anus, E. seminal receptacle, F. female gonopore and anus, G. club-shaped papilla leg IV, H. lateral cirri E. fg = female gonopore, h = lateral cirri A, j = secondary clava, l = primary clava, m = annular refracting organelle, mg = male gonopore, o = anus, p = cuticular pillars, q = club papilla base leg IV. 284 Southeastern Naturalist Vol. 11, No. 2 of a sensory spine on Leg I. Archechiniscus marci exhibits nine transverse metameric folds that appear as plates and a cuticle with minute granulation, while A. biscaynei nov. sp. does not. Remarks: The holotype female was 22% larger than the paratype males and other features exhibit similar dimensional differences (see description). This differentiation might be sexual dimorphism or simply a function of age. Table 1. Comparison of species of Archechiniscus. NA = not applicable. Characteristic A. marci A. minutus A. symbalanus A. biscaynei Body Size (length x width) 198 μm 140 x 55 μm 209 x 64 μm 247 x 93 μm Eyes Red Present Black Absent Cuticle Fine dots Fine dots Fine dots Smooth Transverse metameric folds 9 7 Absent Absent Median cirrus Absent Absent 2.3 μm Absent Seminal receptacles NA Long S shaped Long Sensory spine leg I Absent Absent 2.3 μm Absent Internal cephalic cirrus 3.8 μm 5.5 μm 4.6 μm 6.5 μm External cephalic cirrus 4.5 μm 5.5 μm 5.0 μm 8.5 μm Cephalic clava shape Ring Round Oval Round Cephalic clava position Under ext cirri Under ext cirri Under ext cirri Between ext and int cirri Primary clava 4.5 μm 5 x 2.7 μm 3.8 x 3.2 μm 5.9 μm Cirrus A 6.4 μm 7.0 μm 5.8 μm 8.3 μm Buccal tube length NA 27 μm 38.1 x 2.3 μm NA Stylets Serrated Smooth 30 μm Smooth 28.5 μm NA Stylet supports Absent Absent Absent NA Stylet sheath Absent Absent 17.7 μm NA Furca Rounded Square Rounded NA Cirrus E 26 μm 8.25 μm 7.3 μm 10.3 μm Papilla base of leg IV Absent 3.3 μm Present Club 4.5 μm Anterior papilla, Legs I–III Absent Absent Present Bulbous Internal claws 6.5 μm 3 μm 5.0 μm 6.4 μm Internal claws: Accessory pts Absent Small Present Thin or fine Internal claw and toe (digit) 15.6 μm 8.5 μm 10.4 μm 12 μm Ext. claws w/ basal spur 7.0 μm 7 μm (3 μm) Small 7.5 μm Ext. claws w/ peduncles No 4.4 μm 3.5 x 1.9 μm 2 x 2 μm Retractable claws in sheaths Yes Yes No Yes Seminal receptacle NA U shaped S shaped S shaped Female gonopore NA NA 7.3 μm 5.0 μm Male gonopore NA NA NA 3.0 μm Table 2. Key to the species of Archechiniscus. 1 Median cirrus absent ..............................................................................................................2 1. Median cirrus present ........................................................................................A. symbalanus 2. Cephalic clava round, at base of external cirri ........................................................................3 2 Cephalic clava round, between and ventral to internal and external cirri .............. A. biscaynei 3. Papilla at base of leg IV, external claws without peduncles ........................................ A. marci 3. Lacking papilla at base of leg IV, external claws with peduncles ............................A. minutus 2012 W.R. Miller, T. Clark, and C. Miller 285 Discussion Archechiniscus biscaynei nov. sp. was transparent with bright green ingested algae in the digestive system. This coloration was in contrast to the bright red digestive system observed in Echinisoides sigismundi collected from other barnacles in the same area at the same time. The different colors suggest a noncompetitive food selection by these two barnacle inhabiting species. The barnacle habitat is exposed above tide for several hours twice per day. The compact oval body shape, small appendages, and short stubby legs may be adaptations that reduce the surface area and limit dehydration compared to animals with more elaborate body shapes, longer legs, and complex cuticles. However, this speculative observation warrants further study. It appears that this family/genus may be distributed in an equatorial pattern: El Salvador, Bahamas, Galapagos, New Caledonia, Italy, Thailand, and Florida. It also appears there may be two different habitats utilized by the genus: the intertidal barnacle and tide pool gravel and the sand and debris in deeper coastal water. Archechiniscus biscaynei nov. sp., like many marine tardigrades, has been described from only a few specimens. This underscores our lack of knowledge about both the diversity and ecology of marine tardigrades and points to the need and opportunity for further research. Acknowledgments We are grateful to the National Park Service for hosting the 2010 BioBlitz. We wish to acknowledge the organizational work of Tim Watkins of the National Park Service and John Francis of the National Geographic Society in making the BioBlitz happen and providing us the opportunity to explore this unique coastal habitat. The North American Tardigrade Project was funded under National Science Foundation DEB Grant 0640847. Finally, we appreciate the support and encouragement of our faculty colleagues Calvin Cink, Darcy Russell, Charmaine Henry, and Erin Morris at the Department of Biology, Baker University who make it possible for our undergraduates to participate in authentic field research. 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