Northeastern Naturalist Vol. 23, No. 4 K. Shelton, P.G. Weaver, and B.W. Williams 2016 525 2016 NORTHEASTERN NATURALIST 23(4):525–531 New Records of Entocytherid Ostracods from New York and New Jersey Keri Shelton1, Patricia G. Weaver2, and Bronwyn W. Williams3,* Abstract - Ostracods of the family Entocytheridae are obligate ectosymbionts, primarily of crayfishes. Although the distribution and diversity of crayfishes is well known in the northeastern US, little is known about entocytherids in this region. In this study, we examined 5 species of crayfishes—Procambarus acutus (White River Crayfish), Orconectes limosus (Spinycheek Crayfish), Orconectes propinquus (Northern Clearwater Crayfish), Cambarus bartonii (Common Crayfish), and Cambarus robustus (Big Water Crayfish)—from 3 counties in New York (Saratoga, Orange, and Tompkins) and 8 counties in New Jersey (Essex, Sussex, Atlantic, Burlington, Camden, Cumberland, Gloucester, and Ocean). We recovered 2 species of entocytherid ostracods: Donnaldsoncythere cayugaensis and Donnaldsoncythere donnaldsonensis. Records from our study expand the known range of D. cayuagensis and fill in gaps in the known range of D. donnaldsonensis. Introduction Ostracods in the family Entocytheridae are obligate ectosymbionts of other crustaceans. They occur primarily on crayfishes, but are occasionally found on crabs, amphipods, and isopods (Danielopol 1971; Hart 1962; Hart and Hart 1967, 1974; Hart et al. 1967; Hobbs Jr. and Villalobos 1958; Marshall 1903). At present, the Entocytheridae is composed of 5 sub-families, 35 genera, and 220 species distributed in North and Central America, Europe, Asia, Australia, and Africa (Hart and Hart 1974, Mestre et al. 2014). The known distribution of entocytherids in North and Central America extends from Mexico north into the central Prairie Provinces of Canada (Hart and Hart 1974, Mestre et al. 2014, Williams et al 2011), generally coincident with the distribution of crayfishes; however, reports of entocytherids are sparse or lacking in several regions where potential host-crayfish species are known to occur, including the northeastern US. Although the paucity of records may reflect a true absence of entocytherid ostracods, results from a recent survey in the northern Interior Plains (Williams et al. 2011) suggest that these organisms are likely more widespread, but have gone unnoticed. The aim of this study was to document the diversity and distribution of entocytherid ostracods in a poorly sampled area of the northeastern US, with a specific focus on New York and New Jersey. 1Meredith College, 3800 Hillsborough St., Raleigh, NC 27607. 2North Carolina Museum of Natural Sciences, 11 West Jones Street, Raleigh, NC 27601-1029. 3North Carolina Museum of Natural Sciences, Research Laboratory, 1671 Gold Star Drive, Raleigh, NC 27699. Corresponding author - bronwyn.williams@naturalsciences.org. Manuscript Editor: David Yozzo Northeastern Naturalist 526 K. Shelton, P.G. Weaver, and B.W. Williams 2016 Vol. 23, No. 4 Methods We examined a total of 102 crayfishes housed in the North Carolina Museum of Natural Sciences (NCSM) Non-Molluscan Invertebrate Collection. These specimens included 80 Procambarus acutus (Girard) (White River Crayfish), 11 Orconectes limosus (Rafinesque) (Spinycheek Crayfish), 5 Cambarus robustus Girard (Big Water Crayfish), 4 Cambarus bartonii Fabricius (Common Crayfish), and 2 Orconectes propinquus (Girard) (Northern Clearwater Crayfish) from 14 sites in Saratoga, Orange, and Tompkins Counties in New York, and Essex, Sussex, Atlantic, Burlington, Camden, Cumberland, Gloucester, and Ocean Counties in New Jersey (Table 1, Fig. 1A). We employed a dissecting microscope to examine each crayfish for the presence of entocytherids, and used a dissecting needle and glass pipet to remove visible ostracods from the host. We subsequently aspirated each crayfish with 70% ethanol to remove any remaining symbionts. We collected dislodged entocytherids from the bottom of the examination container, as well as from detritus at the bottom of the jar in which the crayfishes were housed. We dehydrated entocytherids using step-wise solutions of 70%, 95%, and 100% ethanol, and cleared them using methyl salicylate, then slide-mounted the specimens in Canada balsam. We examined slides using a Nikon Eclipse Ni microscope with differential interference contrast and identified species based primarily on adult male morphology, using the keys in Hart and Hart (1974) and original species Figure 1. (A) Map of historical recorded locations of entocytherid ostracods in the Northeast (darkened circles) and the sites sampled during the current study (grey triangles). Historical data were retrieved from GBIF (Ecology Unit, Department of Microbiology and Ecology, University of Valencia 2016). (B) Map showing results of the present survey including sites where no entocytherids were observed (open circles) and where Donnaldsoncythere cayuagaensis (darkened stars) and D. donnaldsonensis (darkened polygons) were recovered. Light gray stars and polygons represent historical locations of D. cayugaensis and D. donnaldsonensis, respectively. Northeastern Naturalist Vol. 23, No. 4 K. Shelton, P.G. Weaver, and B.W. Williams 2016 527 descriptions cited therein. All slide mounts were deposited in the NCSM Non- Molluscan Invertebrate Collection. Results We recovered a total of 164 entocytherid ostracods, including 31 adult males. We identified 2 species belonging to the genus Donnaldsoncythere. We recovered Donnaldsoncythere cayugaensis (Hobbs and Walton) from C. robustus from 2 sites in central and eastern New York: Six-Mile Creek, Tompkins County, and Kayaderosseras Creek, Saratoga County (Table 1, Fig. 1B). We found Donnaldsoncythere donnaldsonensis (Klie) in samples from 2 sites in eastern and southeastern New York: Snook Kill, Saratoga County (C. robustus), and Clove Brook, Orange County (C. bartonii), and 2 sites in northern New Jersey: Quarryville Brook, Sussex County (C. bartonii), and Wigwam Brook, Essex County (C. bartonii) (Table 1, Fig. 1B). We did not recover entocytherids from any of the 80 P. acutus or the 2 Table 1. Locality and specimen information associated with entocytherid ostracods observed during this study. * indicates that coordinates were approximated from locality description.# = number of host individuals examined. Host genera: C. = Cambarus, O. = Orconectes, and P. = Procambarus. Entocytherid genus: D. = Donnaldsoncythere. Locality Latitude, longitude Host (#) Entocytherid species Clove Brook, Orange 41.3593°N, 74.6854°W C. bartonii (1) D. donnaldsonensis County, NY* Wigwam Brook, Essex 40.7819°N, 74.2101°W C. bartonii (1) D. donnaldsonensis County, NJ* Quarryville Brook, Sussex 41.2659°N, 74.5782°W C. bartonii (2) D. donnaldsonensis County, NJ* Kayaderosseras Creek, 43.1378°N, 73.8776°W C. robustus (2) D. cayugaensis Saratoga County, NY Snook Kill, Saratoga 43.1524°N, 73.7731°W C. robustus (2) D. donnaldsonensis County, NY Six Mile Creek, Tompkins 42.4307°N, 76.4820°W C. robustus (1), D. cayugaensis County, NY O. propinquus (2) Pond on Mashipacong(?) Creek, 41.3391°N, 74.7416°W O. limosus (11) Unknown (juveniles) Sussex County, NJ* Penny Pot Stream, Atlantic 39.5800°N, 74.8175°W P. acutus (3) None found County, NJ* Unnamed tributary of West 39.6681°N, 74.4376°W P. acutus (5) None found Branch Bass River, Burlington County, NJ* Clark Branch, Camden County, 39.7168°N, 74.7552°W P. acutus (2) None found NJ* Parvin Branch, Cumberland 39.4603°N, 75.0544°W P. acutus (31) None found County, NJ* Fourmile Branch, Gloucester 39.6965°N, 74.9399°W P. acutus (26) None found County, NJ* Spillpool of Lower Lake, Ocean 39.8359°N, 74.1959°W P. acutus (1) None found County, NJ* Swedes Run, Salem County, NJ* 39.5962°N, 75.3759°W P. acutus (12) None found Northeastern Naturalist 528 K. Shelton, P.G. Weaver, and B.W. Williams 2016 Vol. 23, No. 4 O. propinquus examined. Of the 11 specimens of O. limosus examined, only 1 harbored ostracod ectosymbionts, all of which were juveniles that we were not able to identify to species. When encountered on the host, we observed entocytherids on the ventral cephalothorax between the pereiopods, on the maxillipeds, and around the eyes of the crayfishes. We were unable to discern species-specific microhabitats. Discussion Our findings extend the known geographic distribution of D. cayugaensis, and help fill large gaps in the known distribution of D. donnaldsonensis and the genus Donnaldsoncythere. Consequently, our results expand understanding of the distribution and host associations of entocytherids in the northeastern US. The range expansion we observed is most likely not a result of entocytherid species moving in space over time or true absence, but rather the lack of previous targeted-survey efforts in this region. Prior to this study, D. cayugaensis had been reported to be narrowly distributed in Cayuga, Oswego, and Tompkins counties in central New York (Fig. 1B; Hart and Hart 1974, Hobbs and Walton 1966). Our observations of D. cayugaensis in Saratoga County in eastern NY suggest the species is more broadly distributed in this region. Hobbs and Peters (1977) synonymized Donnaldsoncythere hiwasseensis (Hobbs and Walton), Donnaldsoncythere humesi (Hoff), Donnaldsoncythere ileata (Hobbs and Walton), Donnaldsoncythere pennsylvanica (Hart), Donnaldsoncythere scalis (Hobbs and Walton), and Donnaldsoncythere tuberosa (Hart) into D. donnaldsonensis. Prior to Hobbs and Peters (1977), D. donnaldsonensis was only known from its type locality, Donaldson’s Cave, Lawrence County, IN (Klie 1931). This synonymization effectively extended the range of this species from a single site in the Mitchell Plateau of southern Indiana to widespread occurrence from northern Georgia to Indiana and northern Maine. In our study, we found D. donnaldsonensis in northern New Jersey and south-southeastern New York. These results help to fill in a large gap in the recorded geographic distribution of D. donnaldsonensis that had previously existed between southern Pennsylvania and northern Maine (Fig. 1B). As appears typical with many entocytherids (Hobbs 1968, Hobbs and Peters 1977, Hobbs et al. 1967, Mestre et al. 2014), we saw no host specificity in D. donnaldsonensis, which has been reported to be associated with 27 species of Cambarus, 6 species of Orconectes and 1 species of Procambarus (Hart and Hart 1974; Hobbs 1975; Hobbs and McClure 1983; Hobbs and Peters 1977, 1982, 1989, 1993; Hobbs and Walton 1976). We found D. donnaldsonensis on both C. robustus and C. bartonii, both of which were previously known hosts. Interestingly, D. cayugaensis remains associated with a single host species, C. robustus. Even though P. acutus is a known host for 6 different genera and 22 species of entocytherid ostracods (Hart and Hart 1974, Hobbs and Peters 1977), including D. donnaldsonensis (Hobbs and Peters 1977), we did not find any entocytherids on the 80 individuals we examined from Atlantic, Burlington, Camden, Cumberland, Northeastern Naturalist Vol. 23, No. 4 K. Shelton, P.G. Weaver, and B.W. Williams 2016 529 Gloucester, and Ocean counties, NJ (Fig. 1B). Procambarus acutus displays a disjunct distribution in North America, occurring from the Gulf coast through the Mississippi River Basin north to Wisconsin and Minnesota, and from northern Georgia northward along the Atlantic coastal plain into southern New England (Hobbs and Jass 1988, Loughman 2007). The northernmost record of entocytherid ostracod symbionts on P. acutus is of Ankylocythere copiosa (Hoff) in Henry County, northwestern IL (Hart and Hart 1974). No entocytherids have been reported from P. acutus north of southern Virginia along its coastal distribution (Hart and Hart 1974). It may be that the species does not host entocytherid symbionts in more eastern higher latitudes. Alternatively, the lack of observed symbionts on P. acutus examined during this study could be due to treatment during fixation and/or preservation (e.g., transfer of specimens into new preservative without retaining the detritus at the bottom of the original container). We also found no entocytherids on the 2 specimens of O. propinquus from Tompkins County, central NY. Orconectes propinquus is not historically known as a host for Donnaldsoncythere, but it is reported to harbor 5 other genera and 6 species of entocytherids from Illinois north and eastward through into Ontario, Canada (Hart and Hart 1974). These 2 O. propinquus specimens had been retained in their original fixative; thus, the absence of entocytherids is not due to post-collection handling. However, non-detection, particularly with small sample-sizes, does not mean true absence. It may be that additional surveys of O. propinquus in central New York will recover entocytherid associates. We found 2 juvenile entocytherid ostracods on one of 11 specimens of Orconectes limosus examined from Sussex County, NJ; though we were unable to identify these specimens to species, our findings confirm presence of the symbionts on this host species. Further targeted studies of entocytherids from the northeastern US are likely to expand the known geographic ranges and hosts for this region. Donnaldsoncythere has an extensive, if not somewhat discontinuous, geographic range from Georgia northwards to Maine and westward into West Virginia, Kentucky, Ohio, and Indiana (Hobbs and Peters 1977, 1993). It is the only entocytherid genus reported in the northeastern US north of Pennsylvania. Although not species rich, Donnaldsoncythere reflects a pattern of diversity indicative, in part, of the impacts of glacial advance and retreat. Three of the 5 currently accepted Donnaldsoncythere species appear restricted to the lower and mid-Appalachian region, suggesting a potential origin in this area. Donnaldsoncythere cayugaensis is known only from central and eastern New York, an area completely covered by the Laurentide Ice Sheet during the last glacial maximum (e.g., Mickelson and Colgan 2003). This distribution likely reflects post-glacial colonization from a historically ice-free refugium; thus, we expect that D. cayuagaensis will be recovered from yet-unsurveyed areas of northern and central Pennsylvania. Donnaldsoncythere donnaldsonensis displays the most widespread distribution of all species in the genus; indeed, the range of D. donnaldsonensis defines the distribution of Donnaldsoncythere in toto. Given the complex geologic history of the Appalachian region, it is unlikely that D. donnaldsonensis sensu Northeastern Naturalist 530 K. Shelton, P.G. Weaver, and B.W. Williams 2016 Vol. 23, No. 4 lato comprises a single species. Although the synonymization of 6 previously described species into D. donnaldsonensis was justified by Hobbs and Peters (1977), the high variation in morphology within this now widespread taxon underlies continued taxonomic uncertainty. The results of our study suggest that the paucity of entocytherid records in the northeastern US is likely a function of historic lack of targeted sampling in the region. Future studies in this area will help to discern true patterns of diversity from artifacts of historic selective sampling. Acknowledgments We thank Dr. Maria Pickering-Villa and Meredith College for providing this undergraduate research opportunity to Keri Shelton, and Janet Edgerton for bibliographic assistance. 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