First record of harpacticoid copepods from Lake Tahoe, United States: two new species of Attheyella (Harpacticoida, Canthocamptidae)

Abstract Benthic harpacticoids were collected for the first time at Lake Tahoe, California-Nevada, United States. Two species were identified as members of the genus Attheyella Brady, 1880. The genus Attheyella comprises about 150 species within six subgenera, but only twelve species have previously been reported from North American freshwater habitats. The two new species of Attheyella described here have a 3-segmented endopod on P1 and 2-segmented P2–P4 endopods, the distal segment of exopod of P2–P4 has three outer spines, and the P5 has five setae on the exopod and six setae on the baseoendopod. Attheyella (Attheyella) tahoensis sp. n. most closely resembles Attheyella (Attheyella) idahoensis (Marsh, 1903) from Idaho, Montana, and Alaska (United States) and Attheyella (Attheyella) namkungi Kim, Soh & Lee, 2005 from Gosu Cave in South Korea. They differ mainly by the number of setae on the distal endopodal segment of P2–P4. In addition, intraspecific variation has been observed on the caudal rami. Attheyella (Neomrazekiella) tessiae sp. n. is characterized by the extension of P5 baseoendopod, 2-segmented endopod of female P2–P3, and naked third seta of male P5 exopod. The two new species are likely endemic to Lake Tahoe, an isolated alpine lake within the Great Basin watershed in the western United States.


Introduction
Lake Tahoe is a large freshwater lake in the Sierra Nevada of the United States. At a surface elevation of 1,897 m, it is located along the border between California and Nevada. Lake Tahoe is the largest alpine lake in North America. Its depth is 501 m, making it the deepest in the United States after Crater Lake (593 m). Lake Tahoe is one of the largest lakes by volume (1.5 × 1011 m 3 ) in the United States, only being exceeded by the Great Lakes. While, some basic ecological investigations have occurred in Lake Tahoe (Flint and Goldman 1975, Frantz and Cordone 1996, Vander Zanden et al. 2003, most recent investigations are focused on studies of water quality (Jassby et al. 2003), invasive species (Denton et al. 2012, Wittmann et al. 2012, and loss of native biodiversity (Caires et al. 2013). Meiobenthic (especially harpacticoid copepods) diversity has not been studied in Lake Tahoe until recently, and the ecological role of meiobenthos in Lake Tahoe remains unstudied.
During the past century, fresh water harpacticoid copepods in North America have been reported by several researchers, with checklists provided by: Wilson CB (1932; Woods Hole, Massachusetts), Coker (1934;Illinois, North Carolina and Minnesota), Reid (1996;Washington, D.C.), Hudson et al. (1998;the Great Lakes), Suárez-Morales and  the Yucatan peninsula), Bruno et al. (2005;Florida), and Reid and Hribar (2006;the Florida Keys). Moreover, Wilson and Yeatman (1959) and Reid and Williamson (2009) provided key to species in North American freshwater harpacticoid copepods.

Material and methods
Samples were collected from the Lakeside Beach littoral zone, 5 m water depth, on south shore of Lake Tahoe near the California-Nevada border in July 2013. Sediment samples were collected manually by SCUBA divers. Sediments were sampled with 2.9 cm inner diameter core tubes and were fixed with 70% ethanol and stained with Rose Bengal. Meiofauna was extracted from sediments by Ludox isopycnic centrifugation (Burgess 2001). Due to the coarseness of sand typical of Lake Tahoe, vortexing was replaced with gentle agitation during the Ludox extraction procedure to prevent mechanical damage to animals. Harpacticoids were sorted and enumerated under a Leica S8APO dissecting microscope, and stored in 70% ethanol.
Specimens were dissected in lactic acid and the dissected parts were mounted on slides in lactophenol mounting medium. Preparations were sealed with transparent nail varnish. All drawings have been prepared using a camera lucida on a Leica DM 2500 differential interference contrast microscope. Specimens were deposited at the Smithsonian National Museum of Natural History.

Order
Genital somite and first abdominal somite partly fused forming double-somite, wider than long. Genital field as in Fig. 4B. Genital apertures located anteriorly, closely set together. Copulatory pore located anteriorly between genital apertures. Seminal receptacle well developed on each side. P6 with small protuberance bearing 2 pinnate setae.
Caudal rami (Fig. 5D) slightly more elongated than female, about 3.3 times as long as wide, seta III bare, seta IV pinnate. Inner margin of each ramus with lateral concavity.
Antennule (Fig. 6A) 10-segmented; subchirocer with geniculation between segments 5 and 6, and between segment 7 and 8. Segment 1 with a row of spinules along anterior margin. Segment 4 and 6 represented by a small sclerite. Segment 5 swollen with large bump along posterior margin. Segment 7 and 8 with 3 spinular processes from modified setae on each segment. Armature formula: Apical acrothek consisting of a small aesthetasc fused basally to 2 bare setae. P3 (Fig. 6B). Exopod as in female, except for outer spine on first and second exopodal segment of P3 proportionately stronger. Endopod modified, 3-segmented; enp-1 shortest with inner pinnate seta; enp-2 with well-developed inner apophysis; enp-3 with 1 pinnate apical seta and 1 long bare seta. P4 (Fig. 6C). Setae and spines on exopod modified, outer spine on first and second exopodal segment proportionally stronger than female. Endopod 2 with 1 pinnate seta and 2 pinnate spines and longer than those in female.
Fifth pair of legs (P5) (Fig. 5B) smaller and much shorter than female and fused medially, with no spinules. Baseoendopod with outer setophore bearing the basal seta. Endopodal lobe with 2 distal pinnate setae with large pore on anterior surface. Exopod shorter than in female, as long as wide, with 1 inner bare seta, 2 distal pinnate setae and 1 pinnate spine, and 2 outer pinnate spines. P6 (Fig. 5C) asymmetrical, bearing 2 naked seta on a cylindrical process. On left side a lobe with two setae, on the right side a small plate with two setae.
Variability. Intraspecific variability was observed in the shape of caudal rami of female (about 20%; 6/30 observed individuals). Caudal rami (Fig. 4D-F) lamelliform and elongate, about twice as long as wide, laterally compressed, inner margin of each ramus with lateral concavity. Each ramus with 7 setae: seta III bare, seta IV bipinnate, seta V extremely reduced, seta VI bare and longer than normal.
Etymology. The species name refers to the type locality, Lake Tahoe. This is one of the largest alpine lakes in the world is known for its pristine waters and aesthetic beauty.  Description. Female. Total body length 660 μm (n=5; range: 635-694 μm), measured from anterior margin of rostrum to posterior margin of caudal rami. Largest width measured at posterior margin of cephalic shield: 176 μm. Urosome narrower than prosome (Fig. 7A).
Cephalothorax (Fig. 7A) bell-shaped, with oval dorsal integumental window and some scattering sensillae on dorsal surface and along lateral margin. Rostrum (Fig. 7B) small and triangular, completely fused to cephalothorax and with pair of sensilla and pore near anterior margin.
Genital double-somite as wide as long. Original segmentation marked by discontinuous internal chitinous rib laterally, completely fused ventrally. A row of spinules present on lateral side of genital field. Genital field located far anteriorly (Fig. 7C). Genital apertures paired, closely set together. Copulatory pore located anteriorly between genital apertures. Seminal receptacle well developed on each side. P6 with small protuberance bearing 2 bare unequal setae.
Anal somite (Fig. 7D-E) with well-developed rounded operculum. Surface ornamentation consisting of a pair of sensilla dorsally and a pair of pores ventrally, posterior margin with spinules ventrally and dorsally, with triangular process dorsally, anal opening with a fringe of fine setules. Caudal rami short, as long as wide, each ramus with 7 setae: setae I-II small, closely set, seta III bare, seta IV pinnate, seta V bipinnate and longest, seta VI bare, seta VII tri-articulate at base. Antennule (Fig. 7B)     Antenna, mandible, maxillule, maxilla, and maxilliped as in A. (A.) tahoensis sp. n. P1 (Fig. 8A) with well-developed coxa with spinular row on anterior surface. Basis with setules along outer margin and anterior surface, with pinnate outer and inner spines. Endopod 3-segmented, 1.3 times as long as exopod; enp-1 longer than enp-2, with one inner pinnate seta; enp-2 with 1 bipinnate inner seta; enp-3 with a small inner seta, and 1 long geniculate seta and 1 pinnate seta distally. Exopod 3-segmented, reaching end of enp-2, exopodal segments with strong spinules along outer margin and outer distal corner; exp-3 with 2 geniculate distal setae and 2 strong spinulose outer spines. P2 (Fig. 8B) with small triangular praecoxa, with row of spinules on anterior surface along distal margin. Coxa nearly 1.5 times as wide as long, ornamented with row of long spinules along outer margin; Basis with outer pinnate spine; P2 enp-2 more than twice as long as enp-1; with 1 short, pinnate inner seta; enp-2 with 1 inner pinnate seta, and 2 distal bipinnate setae and 1 bipinnate outer seta; exopod 3-segmented; each segment with row of spinules along outer margins; third segment about 2.3 times as long as second segment with 3 bipinnate outer spines, 1 apical pinnate spines and 1 plumose seta, and 1 inner long bipinnate seta. P3 (Fig. 8C) with small praecoxa. Coxa nearly 1.5 times as wide as long, with row of spinules along outer margin. Basis with outer pinnate seta and spinular row along outer margin. Endopod-1 with 1 bare inner seta; enp-2 with 2 inner naked setae, and 1 pinnate seta and short distal spine; exp-3 about twice as long as second segment with 3 bipinnate outer spines, 2 apical pinnate spines and 2 long bipinnate inner setae. P4 (Fig. 8D) with small and triangular praecoxa. Coxa with spinular row along outer margin. Enp-2 with 2 inner pinnate setae, and 3 pinnate setae, the innermost longest; exp-3 with 3 bipinnate outer spines, 2 apical pinnate setae and 2 long bipinnate inner setae. Spine and setal formulae as follows: P5 (Fig. 8E). Baseoendopod forming short, outer setophore bearing the basal seta. Endopodal lobe trapezoidal, with 2 pinnate inner setae, 2 distal setae, and 2 pinnate outer seta; all setae of different length and apical outermost is the longest. A secretory pore on anterior surface. Exopod twice as long as wide, with one short inner, 2 distal (innermost longest) and 2 outer setae, all pinnate.
Etymology. The species name refers to Tahoe Tessie, a cryptozoological creature which supposedly resides in Lake Tahoe. While some claim to have seen the mythical Tahoe Tessie, none until now have seen these non-mythical microscopic creatures of the sand.

Discussion
The family Canthocamptidae is the largest family of Harpacticoida found in freshwater habitats, and can be found in rivers, streams, ponds, lakes, and wetland, and even in hot springs, glacial melt water and damp moss (Boxshall and Halsey 2004). Canthocamptidae is in need of revision because several genera have high species diversity and many species exhibit wide variation, the widest variability recorded for freshwater Harpacticoida. Additionally, many species are incompletely described, often with major features such as A2 exopod setation and the setal formula for P2-P4 being unknown (Wells 2007).
The genus Attheyella Brady, 1880 is a genus of Canthocamptidae, and is cosmopolitan in distribution at the generic level (Boxshall and Halsey 2004). Attheyella is highly diverse, with more than 150 species, and is divided into six subgenera: Attheyella, Canthosella, Chappuisiella, Delachauxiella, Neomrazekiella, and Ryloviella. The genus Attheyella is also in need of revision because the species boundaries are not clear due to high variability in number of characters (Reid and Williamson 2009).
Both new species are placed in the genus Attheyella because of the following diagnostic features: small rostrum, P1 with 3-segmented rami, 2-segmented endopod of P2-P3 in female, and 3-segmented P3 in males.
In 1876, Forbes S.A. reported the freshwater harpacticoid copepod Canthocamptus illinoisensis from Normal, Illinois, which is represented as Attheyella (Neomrazekiella) il-linoisensis. Since then, several new species were added, and the genus Attheyella in North America currently includes 3 subgenera (Attheyella, Neomrazekiella, Ryloviella) and 12 species. Wilson and Yeatman (1959) and Reid and Williamson (2009)  Morphological variation and abnormality are common in harpacticoid copepods. In the present study, intraspecific variation of A. (A.) tahoensis sp. n. has been recorded, with some specimens having variations on the shape and armature of the caudal rami (about 20% of total observed specimens). In the most commonly observed condition, caudal rami are about 2.5 times as long as wide, bottle-shaped, strongly tapering distally. In the less common morphological variant, caudal rami are about twice as long as wide, laterally compressed, with the inner margin of each ramus having lateral concavity.
In numerous instances, the morphological variation or deformity occurred due to environmental factors such as water pollution. However, some studies suggest that variation in caudal rami may be caused by interspecific competition. For example, Ishida (1994) reported high proportions of caudal rami variation in Attheyella (Attheyella) nakaii (Chappuis, 1927), and suggested that it played a role in sexual segregation due to sympatric distribution with Attheyella (Attheyella) yesoensis Ishida, 1993. Certainly, morphological variation in other harpacticoid species has led to discoveries of concomitant genetic differences and presumed cryptic speciation (Garlitska et al. 2012).
Attheyella (Neomrazekiella) tessiae sp. n. is placed in the subgenus Neomrazekiella on account of the following combination of characters: prosome with smooth posterior margins, female P5 endopodal lobe triangular, basal expansion wider than exopod, with six setae, male P5 not produced into narrow prolongation, P3 spine of exopod 2 not greatly enlarged in male. A. (N.) tessiae sp. n. can be clearly distinguished from other members of the subgenus Neomrazekiella by the 2-segmented endopods of female P2-P3, P5 baseoendopod produced to middle of exopod segment in female, and naked third seta of the male P5 exopod .
This investigation marks the first record of meiobenthos, and more specifically, of harpacticoid copepods, in Lake Tahoe. The newly described A. (A.) tahoensis sp. n. and A. (N.) tessiae sp. n. are likely endemic to Lake Tahoe. A total of 10 endemic macrobenthos have previously been identified in Lake Tahoe (summarized by Caires et al. 2013), so it is reasonable to hypothesize that several species of meiobenthos also evolved in this ecosystem. Expanded sampling in the western United States, and beyond, will be necessary to validate the endemism of these species.
Together with newly described A. (A.) tahoensis sp. n. and A. (N.) tessiae sp. n., the three subgenera and fourteen species currently recognized as valid in the genus Attheyella from North America can be identified with the specific key given below. It is amended from Wilson and Yeatman (1959) and Reid and Williamson (2009).