Southeastern Naturalist 197 E.J. Burge and S.D. King 22001155 SOUTHEASTERN NATURALIST 1V4o(1l.) :1149,7 N–2o1. 21 Parasites of the Carolina Bay Lake-Endemic Fundulus waccamensis (Waccamaw Killifish) Erin J. Burge1,* and Stanley D. King2 Abstract - Lake Waccamaw, NC, is an unusual Carolina bay home to endemic and rare species. Parasites of the lake-endemic Fundulus waccamensis (Waccamaw Killifish) have not been described previously. In 2011, we collected Waccamaw Killifish (n = 101) by seining and dip netting from 3 sites over 3 seasons (spring, summer, and fall) to investigate the identity, prevalence, and intensity of the parasite-component community. We found 13 taxa of parasites, all of which were new host records, and total prevalence of parasitism = 95%. Infected hosts contained 2.3 ± 1.0 (mean ± SD) parasite species, with the component community composed of 6 ecto- and 7 endoparasites. The most prevalent (70.3%) and highest mean intensity (17.3 metacercariae per host) infections were associated with the generalist trematode Posthodiplostomum minimum. Introduction Lake Waccamaw, Columbus County, NC, is the largest extant, permanently flooded Carolina bay lake (Frey 1949, 1950). Carolina bays are elliptical geological features arranged on a northwest–southeast axis along the Atlantic coastal plain of the US from Delaware to northern Florida. They are widely recognized as ecologically important and biodiverse habitats (Sharitz 2003). The modern Lake Waccamaw is estimated to have formed between 15,000 and 32,000 years BP (Stager and Cahoon 1987) and comprises the headwaters of the Waccamaw River, which drains into the Atlantic Ocean at Winyah Bay, SC. Lake Waccamaw has an unusual water chemistry that diverges from most other Carolina bay lakes in having near-neutral pH, and high alkalinity, calcium concentration, and primary productivity (Cahoon et al. 1990, Frey 1949, Stager and Cahoon 1987). The lake is the only one of its type that supports a rich endemic fauna of invertebrates and fishes (Casterlin et al. 1984, Dillon et al. 2013, Frey, 1951, Fuller 1977, Glover and Floyd 2004, Hubbs and Raney 1946, Porter 1985, Stager and Cahoon 1987). During the late 20th century, Lake Waccamaw experienced significant increases in nutrient concentrations attributed to lakeside development and changing land-use patterns within the watershed, leading to increased eutrophication and raising concerns about the sustainability of its endemic biota (Casterlin et al. 1984, Heise and Jones 2010, Lindquist and Yarbrough 1982, Shute et al. 2000). Biotic changes in recent years include the invasion and establishment of Labidesthes sicculus (Cope) (Brook Silverside; Moser et al. 1998), the introduction of the noxious submersed aquatic plant Hydrilla verticillata (L.f.) Royle (Indian 1Department of Marine Science, Coastal Carolina University, Marine Science, PO Box 261954, Conway, SC 29528-6054. 2Department of Biology, Dalhousie University, Department of Biology, 1355 Oxford Street, Halifax, NS, Canada, B3H 4J1. *Corresponding author - eburge@coastal.edu. Manuscript Editor: Hayden Mattingly Southeastern Naturalist E.J. Burge and S.D. King 2015 Vol. 14, No. 1 198 Stargrass; Heise and Jones 2013), and population declines of endemic mussels (Heise and Jones 2010). Lake Waccamaw is home to several endemic invertebrates, including the caddisfly Nectopsyche waccamawensis Glover & Floyd (Waccamaw White Miller; see Glover and Floyd 2004), an undescribed Floridobia sp. gastropod, (Waccamaw Siltsnail; see Dillon et al. 2013), and the 2 endemic unionid freshwater mussels, Elliptio waccamawensis (Lea) (Waccamaw Spike) and Lampsilis fullerkati (Johnson) (Waccamaw Fatmucket) (Porter 1985). Like nearly all unionids, these mussels produce glochidia, a parasitic larval stage that infects skin and gill tissues of host fishes (Kat 1984). The fishes hosting glochidia of endemic unionids have not yet been identified (Porter and Horn 1980). Three endemic fishes are also described from Lake Waccamaw: Fundulus waccamensis Hubbs & Raney (Waccamaw Killifish), Etheostoma perlongum (Hubbs & Raney) (Waccamaw Darter), and Menidia extensa Hubbs & Raney (Waccamaw Silverside) (Hubbs and Raney 1946). Additionally, Lake Waccamaw is home to a population of an undescribed Noturus sp. (broadtail madtom) (Bennetts et al. 1999, Rohde et al. 2009) and a slightly divergent morphotype of Notropis petersoni Fowler (Coastal Shiner; Krabbenhoft et al. 2009a)—a cyprinid originally described as Notropis waccamanus (Hubbs and Raney 1946). A genetically distinct population of Elassoma boehlkei Rohde & Arndt (Carolina Pygmy Sunfish) has sporadic, low-density occurrence in the canals and creeks adjacent to Lake Waccamaw and in a few other localities in the Waccamaw and Santee river basins of South Carolina (Quattro et al. 2001, Rohde and Arndt 1987). A lacustrine origin of Waccamaw Killifish and the other lake-endemic fishes (Waccamaw Darter and Waccamaw Silverside) was hypothesized by Hubbs and Raney (1946) to have resulted by differentiation from the geographically widespread sister species Fundulus diaphanus (Lesueur) (Banded Killifish), Etheostoma olmstedi Storer (Tesselated Darter), and Menidia beryllina (Cope) (Inland Silverside), respectively. These 3 progenitors are all found in the vicinity of Lake Waccamaw, but not in the lake itself (Hubbs and Raney 1946, Shute et al. 1981). In their original descriptions of the endemic fish, Hubbs and Raney (1946) remarked that the 3 species were “decidedly slenderer and more terete” compared to their widely distributed cognates and that they differed in head morphology and meristic characters, including higher lateral-scale counts and more vertebrae. Geometric morphometric analyses were used by Krabbenhoft et al. (2009a) to conclude that all 3 endemics have elongated and streamlined morphologies relative to their sister species. They speculated that convergent evolution for a stream-lined morphology may be an adaptation for higher sustained swimming speeds in Fundulus and Menidia, and a “less-benthic” lifestyle in Etheostoma selected for in the clear, open waters of Lake Waccamaw. The Waccamaw Killifish is considered vulnerable–endangered (IUCN 2013, Krabbenhoft et al. 2009b) and a Federal species of concern (LeGrand et al. 2012) because of its restricted distribution in Lake Waccamaw, proximate portions of the lake-inlet creek and canal system, and in the immediate headwaters of the Waccamaw River (Hubbs and Raney 1946, Shute et al. 1981). Its habitat is indirectly protected because of its Southeastern Naturalist 199 E.J. Burge and S.D. King 2015 Vol. 14, No. 1 sympatry with M. extensa which is federally listed as threatened and also restricted to Lake Waccamaw (USFWS 1987). Parasites have been shown to pose major threats to species at risk (McCallum and Dobson 1995). Some estimates suggest that parasites may outnumber freeliving species (reviewed in Dobson et al. 2008) and that they are an understudied and underappreciated component of global biodiversity (Brooks and Hoberg 2000). The roles of parasites in aquatic ecosystems through impacting food webs (Kuris et al. 2008, Lafferty et al. 2006, Sukhdeo 2012), modifying host behavior (Barber et al. 2000), and providing of ecosystem services (Dobson et al. 2008, Lafferty 2008) are important factors in aquatic community ecology. Until this study, there had been no published research or surveys on the parasites of any endemic fish of Lake Waccamaw. This information gap is somewhat surprising given the unique nature of the lake, its degree of endemism in fishes and invertebrates, and the rich literature on the parasite-component communities of other local littoral-zone fishes (e.g., Fundulus spp.). In their review of parasites of 9 east-coast fundulids, Harris and Vogelbein (2006) reported a speciose parasite fauna comprised of at least 93 taxa infecting F. heteroclitus (L.) (Mummichog). Banded Killifish, the allopatric progenitor of Waccamaw Killifish, also has a rich parasite fauna representing at least 3 protozoan and 60 metazoan parasite species (Harris and Vogelbein 2006). King (2009) extended the host records for both Mummichog and Banded Killifish to include 2 additional gyrodactylids from the Canadian range: Fundulotrema foxi (Rawson) (see King 2009) and the new species Fundulotrema porterensis King & Cone (see King and Cone 2009). Reported taxa of metazoan parasites from Fundulus spp. include representatives from nearly every phylum and those with both one-host (monoxenous) and multi-host (heteroxenous) life cycles. Fundulus spp. commonly host endoparasites including nematodes, acanthocephalans, myxozoa, cestodes, and digenean trematodes and ectoparasites from the monogenoid, ciliophoran, annelid, and crustacean (copepods and brachiurids) groups. Gyrodactylidae is a speciose group within the Monogenea that are common ectoparasites of teleosts, with a few exceptional species infecting amphibians and cephalopods (Bakke et al. 1992). Most gyrodactylids exhibit high host specificity wherein species often infect only a single host or host genus. Fundulotrema, for example, contains a group of 6 described species, 5 of which have only been reported from species of Fundulus, and the 6th from a closely related cyprinodontid, Lucania goodei Jordan (Bluefin Killifish). It has been suggested that gyrodactylids and other highly host-specific parasites are more likely to co-speciate with their host due to their phylogenetic conservatism in host choice (Huyse et al. 2003, Rohde 1993). Considering this possibility, we might expect to find new species of parasites when studying a recently diverged host that has yet to be examined for parasites. In the current study, we undertook the first survey of the parasite-component community of the Waccamaw Killifish. Our goals were to increase our understanding of the biology and ecology of this endemic fish and to evaluate whether the unique conditions in Lake Waccamaw have concealed undescribed species of parasites. Our data will also serve as a baseline for future comparisons of biodiversity in light of concerns regarding water quality and on-going biotic changes within the lake. Southeastern Naturalist E.J. Burge and S.D. King 2015 Vol. 14, No. 1 200 Methods Field collections We collected specimens from 3 different shoreline sites of Lake Waccamaw, Columbus County, NC. We chose sites based on historical occurrences of Waccamaw Killifish (Heise and Jones 2010; Shute et al. 1981, 1983, 2000), ease of accessibility, and preliminary collections that indicated this species was abundant at these locations. Characteristics of collection sites were largely representative of the entire periphery of Lake Waccamaw and included a north-shore beach (34°19'01"N, 78°31'30"W; site 1a in Shute et al. 1981), the southwest corner of the lake at the spillway (34°15'39"N, 78°31'22"W; site 1g in Shute et al. 1981), and a sandy beach area on the south shore in Lake Waccamaw State Park (34°15'28"N, 78°30'57"W; site 1f in Shute et al. 1981). More detailed site descriptions are available in Shute et al. (1981). For background on the physical and chemical characteristics and biota of Lake Waccamaw see Casterlin et al. (1984) and Frey (1949, 1950). We collected specimens retained for parasitological examination (n = 101) in 2011 on 16 March (n = 4), 22 April (n = 22), 12 July (n = 20), 26 August (n = 10), 6 October (n = 15), 27 October (n = 15), and 17 November (n = 15). Typically, we only retained adults >45 mm (n = 93) for parasite dissections but in some months with limited sample sizes, we used smaller individuals. We employed small seines (3.0 m × 0.91 m with 3-mm mesh and 6.1 m × 0.91 m with 6-mm mesh) and dip nets to capture fish in open water. Immediately after capture, we euthanized fish with a fatal overdose of tricaine methanesulfonate (MS-222; Sigma-Aldrich Company, St. Louis, MO). Each specimen was identified to sex, measured for total length (TL) to the nearest 0.1 mm with calipers, and transferred into 10% neutral-buffered formalin in individual glass vials to ensure retention of loosely associated or motile ectoparasites. Collections were permitted under the following licenses: North Carolina Wildlife Resources Commission (NCWRC) Endangered Species Permit 10-ES00306, NCWRC Scientific Fish Collecting License No. 1207, and North Carolina Division of Parks and Recreation Scientific Research and Collecting Permit R10-50. We submitted data on all fish collected to the NCWRC as required by 10-ES00306. Parasite surveys We used stereo and compound microscopes to aid our necropsies of Waccamaw Killifish, which included examination of the fins, body, buccal cavity and musculature of the head, gills, eyes, brain, ureters, intestine, liver, heart, gallbladder, gonads, air bladder, spleen, and mesenteries of preserved fishes following the procedures of Harris and Vogelbein (2006) and King (2009). We examined the fixative and sediment in each vial for dislodged ectoparasites. We prepared any parasites encountered as temporary wet-mounts and identified each to species or the lowest taxonomic level possible by comparing their morphological characters to taxonomic keys (Hoffman 1999), original species descriptions, and in some cases, the type material. We identified gyrodactylids by staining the haptoral bars of representative specimens with Gomori’s trichrome (Kritsky et al. 1978) and examined all sclerotized features of the opisthaptor (dorsal and ventral bars, anchors, and marginal hooks) and male copulatory organ using bright-field, phase, and differential Southeastern Naturalist 201 E.J. Burge and S.D. King 2015 Vol. 14, No. 1 interference-contrast optics (Malmberg 1970). We prepared gyrodactylid voucher specimens by dehydration in absolute ethanol, clearing in xylene, and re-mounting in Canada balsam all stained samples. Stephen S. Curran (Department of Coastal Sciences, The University of Southern Mississippi, Hattiesburg, MS) and Eugene M. Burreson (Department of Environmental and Aquatic Animal Health, Virginia Institute of Marine Science, Gloucester Point, VA) provided supplemental and confirmatory identifications of trematodes and nematodes, and annelids, respectively. We deposited voucher specimens for the following species at the Harold W. Manter Laboratory of Parasitology (University of Nebraska State Museum, Lincoln, NE) under the accession number P-2014-034 and the following catalog numbers: Ergasilus lizae Krøyer (HWML #64652 and 64653), Fundulotrema porterensis (HWML #74999 and 75001), Fundulotrema prolongis (Hargis) (HWML #74998 and 75002), Gyrodactylus stephanus Müller (HWML #75000 and 75003), Myzobdella lugubris Leidy (HWML #64654), Pomphorhynchus bulbocolli Linkins (HWML # 64655), and Posthodiplostomum minimum (MacCallum) (HWML #64656). Data analysis Prevalence and mean intensity as defined by Bush et al. (1997) with 95% confidence intervals were calculated with the parasitology statistical software QPweb 1.0.6 (Rózsa et al. 2000). Confidence intervals for prevalence were calculated by the Clopper-Pearson method, and those for mean intensity used bootstrap methods with 2000 permutations (Rózsa et al. 2000). Because the data presented on the parasite component community represent a relatively small number of sampled hosts of this threatened species (n = 101), additional analyses beyond describing the community are relatively limited. We include them for illustrative purposes and as suggestions for future research. To calculate a total parasite burden and examine seasonality and sex as factors affecting the parasite infracommunities of hosts, an individual parasitization index (IPI) was calculated for each host following the formula of Kalbe et al. (2002) and included all parasite taxa. The correlation of IPI and host TL was compared with the nonparametric Spearman’s rank correlation. Parasite burdens as IPI were compared between sex and season using two-way analysis of variance on square-root transformed IPI after confirming that the data were normally distributed (Shapiro-Wilk test) and of equal variance (SigmaPlot v. 12.3, Systat Software, Inc.). Individual fish were grouped by season as spring (March–April, n = 26); summer (July–August, n = 30); and fall (October–November, n = 45). We used nonmetric multidimensional scaling (NMDS) to examine preliminary patterns of the parasite infracommunities by host sex and season in the dataset. Bray-Curtis similarity on 4th-root transformed parasite abundances was calculated in Plymouth Routines in Multivariate Ecological Research (PRIMER-E v. 5.2.9) (Clarke 1993). Rarefaction analysis was conducted in EstimateS v. 9.1.0 with 100 randomized resamplings. The species richness estimation (Sobs) was considered to be asymptotic if the 95% confidence interval of Sobs and the Chao2 parameter were equal to the observed number of species (S obs) (Colwell 2013). Southeastern Naturalist E.J. Burge and S.D. King 2015 Vol. 14, No. 1 202 Results We examined 101 Waccamaw Killifish (67 females and 34 males) from 3 sites and 3 seasons (spring, summer, and fall) for the presence of ecto- and endoparasites. Individual fish ranged in size from 25.9–92.2 mm TL, with a mean ± SD TL of 63.4 mm ± 14.3, and we found no significant difference in TL by sex (ANOVA: P = 0.18). Lengths of 8 individuals were less than the target size of 45 mm. Of the 101 Waccamaw Killifish examined, we found parasites infecting 96 individuals (overall parasite prevalence = 95%) and identified 13 parasite taxa (Table 1). We present parasite prevalence, mean intensity, life stage, location on or in the host, and taxa in Table 1. The parasite-component community consisted of 200 individuals of 6 ectoparasites (3 monogenoids, 1 unionid, 1 copepod, and 1 leech) and 1560 individuals of 7 endoparasites (3 digeneans, 1 cestode, unidentified nematodes, and 2 acanthocephalans). Parasite-species richness ranged from 1 to 5 per host individual, with a mean ± SD = 2.3 ± 1.0 species per infected host. Rarefaction analysis suggested that the sample size of hosts examined was sufficient to capture the richness of the parasite-component community of the Waccamaw Killifish (Fig. 1). Both the Chao2 parameter and extrapolated species richness (data not shown) converged on the observed species richness. Figure 1. Rarefaction-curve analysis of parasite-species richness assessed from Waccamaw Killifish (n = 101). Drop points on the line represent the estimated numbers of individual fish required to capture 50%, 75%, and 95% of the observed paras ite-species richness. Southeastern Naturalist 203 E.J. Burge and S.D. King 2015 Vol. 14, No. 1 Table 1. Biological information and descriptive statistics for the parasite-component community found infecting Fundulus waccamensis (n = 101). Terminology follows the definitions of Bush et al. (1997). Location on/in host refers to the parasite habitat as F = fins, G = gills, H = head (buccal cavity and musculature), I = intestine, L = liver, M = mesenteries, O = operculum, S = swim bladder, and V = ovary. Confidence intervals (95% CI) for prevalence were calculated by the Clopper-Pearson method and values for mean intensity are bootstrapped from 2000 permutations. All values were calculated as described in Rózsa et al. (2000) using QPWeb 1.0.6. Parasite Location % prevalence Mean intensity Parasite taxa life stage on/in host (95% CI) (95% CI) Phylum Platyhelminthes, Class Monogenea Gyrodactylus stephanus Müller Adult F, G, O 8.91 (4.2–16.2) 1.67 (1.11–2.44) Fundulotrema prolongis (Hargis) Adult F 14.90 (8.6–23.3) 6.40 (3.53–13.7) Fundulotrema porterensis King & Cone Adult F, G 2.97 (0.6–8.4) 1.00 Phylum Platyhelminthes, Class Trematoda Posthodiplostomum minimum MacCallum Metacercariae H, M, V 70.30 (60.4–79) 17.27 (13.9 - 21.5) Homalometron cf. pallidum Stafford Adult I 20.80 (13.4–30) 8.38 (4.14–17.2) Unidentified digenean (Allocreadiidae?) Adult I 2.97 (0.6–8.4) 1.67 (1–2.33) Phylum Platyhelminthes, Class Cestoidea Proteocephalus sp. Metacestode M 8.91 (4.2–16.2) 1.33 (1–1.56) Phylum Nematoda Unidentifed nematodes (Eustrongylides sp. and Spiroxys sp.?) Larvae L, M, S, V 50.50 (40.4–60.6) 2.41 (2–2.86) Phylum Acanthocephala, Class Eoacanthocephala Neoechinorhynchus sp. Cystacanth L, M 11.90 (6.3–19.8) 1.42 (1.08–1.58) Phylum Acanthocephala, Class Palaeacanthocephala Pomphorhynchus bulbocolli Linkins Adult I 1.00 (0–5.4) 1.00 Phylum Annelida, Class Hirudinea Myzobdella lugubris Leidy Adult F 2.97 (0.6–8.4) 1.00 Phylum Arthropoda, Class Maxillopoda Ergasilus lizae Krøyer Adult G 8.91 (4.2–16.2) 2.89 (2–4.11) Phylum Mollusca, Class Bivalvia Unionid spp. Glochidia G 16.80 (10.1–25.6) 3.35 (2.06–5.53) Southeastern Naturalist E.J. Burge and S.D. King 2015 Vol. 14, No. 1 204 There was a significant positive correlation (Spearman r = 0.238, P = 0.03) between host standard length and individual parasitization index (IPI) independent of sex, although the relationship was not particularly predictive. We also examined IPI with regards to sex and season and found no significant differences in total parasite burden (two-way ANOVA: season P = 0.15, sex P = 0.92). In contrast, analysis of Bray-Curtis similarity between individual hosts using nonmetric multidimensional scaling (NMDS) suggested that there may be seasonal differences in parasite infracommunities (Fig. 2); spring samples were largely spatially separate from summer and fall samples, which substantially overlapped. Spring samples were distinguishable from summer and fall samples by the prevalence of up to 3 gyrodactylid species (Gyrodactylus stephanus, Figure 2. Nonmetric multidimensional scaling (NMDS) ordination map of parasite burden in Fundulus waccamensis (Waccamaw Killifish) constructed from the Bray-Curtis similarity matrix of all host infracommunities. Bubbles are shaded by season of host collection and the sizes of points scaled by the individual parasitization index (IPI) for that individual. Stress of the ordination was 0.10 from 30 random restarts. Southeastern Naturalist 205 E.J. Burge and S.D. King 2015 Vol. 14, No. 1 Fundulotrema prolongis, Fundulotrema porterensis) and the lower intensity and prevalence of Posthodiplostomum minimum. The latter trematode species was the parasite found at highest prevalence and intensity in our samples. Gyrodactylids had a combined spring prevalence (95% confidence interval) of 65.4% (44.3– 82.8). Eight Waccamaw Killifish had co-infections of 2 species of gyrodactylids and 1 specimen was host to all 3, but none of the fish we collected during the summer or fall showed evidence of infection with this group of parasites. In contrast, in specimens collected in summer and fall, P. minimum was at high prevalence and high intensity—88.0% (78.4–94.4) and 18.2 (15–23.5) metacercariae per fish, respectively—compared to spring specimens (prevalence = 19.2% [6.6–39.4]), mean intensity = 4.6 (1–11.4). We identified parasites to the lowest taxonomic unit possible using morphological characteristics (Table 1). Known reported hosts and information about life cycles for the identified parasites are presented in Table 2. The unidentified parasites included an adult digenean similar to members of the Allocreadiidae and present in 3 hosts (3% prevalence). Larval nematodes were relatively common (51%) but could not be separated to species without DNA analyses. Likewise, metacestodes of Proteocephalus sp. (9%), cystacanths of Neoechinorhynchus sp. (12%), and unionid glochidia (17%) require further morphological and DNA-sequence data to identify to species. Table 2. Reported hosts and life-cycle information for parasites identified to species infecting Fundulus waccamensis (Waccamaw Killifish). Parasite information is from Hoffman (1999) except for AKing and Cone (2009). Reported hosts known from Lake Waccamaw reported in BShute et al. (1981), CDillon et al. (2013), and DCahoon (2005). For life-cycle information, intermediates are numbered and definitive hosts noted (Def). Parasite taxa Reported hosts Life cycle Gyrodactylus stephanus Müller Fundulus diaphanus, F. grandis, Direct, viviparous F. heteroclitus, F. majalis, Pungitius pungitius (L.) (Nine-spined Stickleback) Fundulotrema prolongis (Hargis) F. diaphanus, F. grandis, F. heteroclitus Direct, viviparous Fundulotrema porterensis King F. diaphanus, F. heteroclitusA Direct, viviparous & Cone Posthodiplostomum minimum FW fishes including Fundulus, especially 1: Physa snailsC, MacCallum centrarchids and cyprinidsB 2: FW fishes, Def: piscivorous birds Homalometron cf. pallidum F. diaphanus, F. heteroclitus, Aplodinotus 1: Hydrobiid snailsC, Stafford grunniens Rafinesque (Freshwater Drum), Gemma clams, Morone americanaB (Gmelin) (White Perch) Def: FW fishes Pomphorhynchus bulbocolli FW fishes including Fundulus 1: Gammarid Linkins amphipodsD Myzobdella lugubris Leidy FW fishes including Fundulus Direct, oviviparous Ergasilus lizae Krøyer FW and marine fishes, F. diaphanus, Direct, oviviparous F. grandis, F. heteroclitus, F. similis (Baird & Girard) (Longnose Killifish) Southeastern Naturalist E.J. Burge and S.D. King 2015 Vol. 14, No. 1 206 Discussion Here we report, for the first time, on the parasite-component community of the threatened, endemic Waccamaw Killifish. This species had a relatively depauperate parasite community—13 taxa—compared to reports of 65 described parasite species for its allopatric progenitor, Banded Killifish (Harris and Vogelbein 2006, King 2009). The high numbers of parasite taxa reported from Banded Killifish and Mummichog, also found locally, are certainly influenced by their wide geographic ranges and the sizeable amount of investigative effort devoted to describing their parasite faunas. Fundulid-parasite surveys from a single or several close localities more narrowly constrained in time (months to years) have reported a variety of parasite-species richness values: 27 species for Banded Killifish (protozoan and metazoan, n = 869 hosts; Wiles 1975), 22 species for Mummichogs (protozoan and metazoan, n = 150 hosts; Harris and Vogelbein 2006), 10 species for Fundulus luciae (Baird) (Spotfin Killifish; metazoan only, n = 341 hosts; Byrne 1978), and 44 species from F. grandis Baird & Girard (Gulf Killifish; metazoan only, n = 3200 hosts; Ruiz 2013). It remains to be seen whether the parasites of other fundulid sibling-species pairs with a common representative and a rare/endemic one such as F. parvipinnis Girard (California Killifish)–F. lima Vaillant (Baja California Killifish) (Reyes-Valdez et al. 2011) and F. olivaceus Storer (Blackspotted Topminnow)–F. euryzonus Suttkus & Cashner (Broadstripe Topminnow) (Schaefer et al. 2009) also demonstrate the apparent large disparity in parasite-species richness. It would be of interest to test this observation by examining the parasite community of the North Carolina allopatric Banded Killifish species complex that includes Waccamaw Killifish and a genetically distinct, undescribed species, F. sp. cf. diaphanus (“Lake Phelps” Killifish; Tracy et al. 2013). A statistically significant difference in the parasite infracommunities was not evident by sex or season, although the results of NMDS ordination (Fig. 2) suggest that seasonality may be a factor of importance for gyrodactylids and Posthodiplostomum minimum. Seasonality in gyrodactylid infection in Mummichog has been previously reported (Barse 1998). Both factors have been implicated as important in some host systems but no universal conclusions seem to fit the variety of patterns (or lack thereof) observed (Kennedy 2009; Poulin 1996, 2007a). Conversely, strong evidence supports the relationship between corresponding increases between hostfish size or age and parasite intensity (Poulin 2000, Wilson et al. 2002). All of the parasites reported from Waccamaw Killifish represent new host records for those species. Waccamaw Killifish is a lacustrine-derived sibling species of the Banded Killifish, but the two species inhabit quite different habitats in the southern portion of their ranges in the Carolinas. Waccamaw Killifish are restricted to Lake Waccamaw and found seasonally in adjacent canals, while Banded Killifish are considered residents of clear-flowing waters, including inland streams and the shallow, tidal portions of larger rivers like the Waccamaw and Cape Fear rivers. The Waccamaw River basin represents the southern limit of the range for Banded Killifish, where it has only rarely been collected in the downstream portions of the river (see Rohde et al. 2009, Shute et al. 1981), ~80 km from Lake Waccamaw. Southeastern Naturalist 207 E.J. Burge and S.D. King 2015 Vol. 14, No. 1 The observed parasite fauna of Waccamaw Killifish probably reflects that of the component community infecting the ancestral host at the time Lake Waccamaw was originally colonized. Once isolated, this host was likely buffered from acquiring infections of new parasites, except generalist parasites with complex life cycles that may have been added to the lake via motile intermediate or definitive hosts (see Table 2). The parasite community of Waccamaw Killifish may have also experienced a reduction in membership due to seasonal fluctuations in parasite populations and the inability for re-colonization after local extinctions (Dunn et al. 2009, Lafferty and Kuris 2009), further explaining the paucity of parasite species. This may, in part, explain the absence of several monoxenous taxa that we expected to be present on our samples because they are common as infections of Banded Killifish (Harris and Vogelbein 2006, Hoffman 1999, King 2009). These taxa include protozoan trichodinids, fungal microsporidians, the cnidarian myxozoa, and mongenean dactylogyrids, all of which commonly infect Banded Killifish over a broad geographic range. The parasite community of Waccamaw Killifish was strikingly similar to that of Banded Killifish. All of the taxa we documented on Waccamaw Killifish, except the unidentified digenean, had previously been reported from Banded Killifish (Harris and Vogelbein 2006, Hoffman 1999, King 2009, King and Cone 2009, Wiles 1975), thus supporting the probable shared evolutionary history of these 2 host species. Interestingly, the host record for Fundulotrema porterensis is the first report of this gyrodactylid species outside of 2 adjacent lakes in Nova Scotia (King and Cone 2009), and our observation suggests a wider distribution for this species than has been demonstrated. One of our research objectives was aimed at investigating if any undescribed species of gyrodactylid infected Waccamaw Killifish. The 2 proposed mechanisms responsible for speciation in the Gyrodactylidae are via host-switching and parasite/host co-speciation (Bakke et al. 2002, Huyse et al. 2003, Ziętara and Lumme 2002). Although speciation via host switching is suspected to play a larger role in the speciation of gyrodactylids (Ziętara and Lumme 2002), there have been examples of co-speciation reported (Huyse and Volckaert 2005, Huyse et al. 2003). If patterns of co-speciation persist, the phylogeny of the parasite should be congruent with the phylogeny of the host, as suggested by Fahrenholz’s rule (Poulin 2007b). The isolation of Lake Waccamaw provided a unique opportunity to examine the co-speciation potential in gyrodactylids. Lake Waccamaw is suspected to have been isolated no more than 32,000 years BP (Stager and Cahoon 1987), so we can estimate that Waccamaw Killifish has likely undergone a maximum of 32,000 generations (assuming first spawning at 1+ years old). Clearly there have been a sufficient number of generations to accrue visible morphological differences from Banded Killifish (Hubbs and Raney 1946, Krabbenhoft et al. 2009a). Comparatively, the reproductive rate of gyrodactylids is much higher (reviewed in Bakke et al. 2002), and if assumed to be similar to Gyrodactylus bullatarudis Turnbull, which infects another cyprinodontid host, the total number of generations for the same time period is estimated to be orders of magnitude larger (~5.8 million generations; Scott 1982). Yet, the gyrodactylids infecting Waccamaw Killifish are morphologically very similar if not identical to individuals infecting Ba nded Killifish. Southeastern Naturalist E.J. Burge and S.D. King 2015 Vol. 14, No. 1 208 It was hypothesized that undescribed gyrodactylid diversity may be present in Lake Waccamaw given the unique conditions of the lake and its documented endemicity for many taxa (reviewed in LeGrand et al. 2012), but this was not the case for the Waccamaw Killifish. However, these parasites are renowned for their morphological conservation, and there are multiple examples of cryptic species being identified only after molecular genotyping (Cunningham et al. 2001, Ziętara and Lumme 2003). The same cryptic diversity may be at play here, but molecular investigation is needed. In the absence of molecular data, this study provides anecdotal evidence that speciation of the host does not always facilitate speciation in gyrodactylids, at least over relatively short evolutionary temporal frames. Building on this baseline description of the parasite-component community of Waccamaw Killifish, future work should focus on how changes in land-use patterns, water quality, and biota within Lake Waccamaw affect population resiliency and parasitism of endemics. A large body of research has demonstrated that parasites often respond differently than their hosts to pollution and changes in water quality (reviewed in Blanar et al. 2009). Parasites are often keystone species (McCallum and Dobson 1995), and changes to the host or parasite community facilitated by deteriorating water quality may have profound effects on the lake as a whole. In future studies, it would also be of interest to elucidate the host-parasite relationships of rare and endemic Lake Waccamaw mussel glochidia, which would require molecular analyses. Lastly, the parasites of Carolinian populations of Banded Killifish and another lacustrine-derived sister species within the Banded Killifish-complex, the “Lake Phelps” Killifish, have not yet been investigated and may provide insights on community structure of parasites during allopatric speciation of their hosts. Acknowledgments We were assisted with field collections by R. Burge, P. Burge, and W. Burge; C. Hill (Coastal Carolina University, Department of Biology); Coastal Carolina University marine science students S. Dumoff, A. Ruis, T. Schacht, L. Scribner, and J. 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