Southeastern Naturalist K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 438 2018 SOUTHEASTERN NATURALIST 17(3):438–455 Health and Genetic Structure of the American Eel in Florida Kimberly I. Bonvechio1,*, Brandon Barthel2, and Jessica Carroll2 Abstract - We collected biological, health, and parasite-infection (e.g., Anguillicoloides crassus [a swim-bladder nematode]) data for 609 Anguilla rostrata (American Eel), and population- genetics data from a subset of 299 individuals captured throughout Florida from 2014 through 2016. We did not find evidence of genetic differentiation between groups and concluded that a single American Eel stock extends through the Atlantic-coast drainages into the Gulf of Mexico. We found spatial and seasonal differences in swim-bladder condition and in prevalence and incidence of A. crassus infection in Florida’s American Eels. Average values for the ratio of swim-bladder to length and health indices were similar between regions, among seasons, and between uninfected and infected American Eels. Information gathered by this study will be important in future conservation and management efforts. Introduction Anguilla rostrata Lesueur (American Eel) is a facultatively catadromous species (McCleave and Edeline 2009) with a large geographic distribution in the Western Atlantic Ocean, Caribbean Sea, and Gulf of Mexico drainages, stretching from Greenland (Møller et al. 2010) to Venezuela (Benchetrit and McCleave 2016). Based on a review of available data on larval American Eels, Miller et al. (2015) suggested that the American Eels spawn in the western Sargasso Sea, between longitudes 75°W and 60°W, from February through April. After hatching, the transparent, leaf-like leptocephali are transported by currents to areas throughout the species’ range in the northwestern Atlantic, Caribbean Sea, and Gulf of Mexico. Before they enter coastal estuaries, American Eels metamorphose into the glass-eel life stage, in which they transform to an eel-like morphology but still lack pigment. As they gain pigment and increase in size, the eels transform into elvers and immature yellow eels, which can remain in marine or estuarine waters or travel upstream into lakes and rivers (McCleave and Edeline 2009). It can take as long as 30 years for individuals to mature into silver eels, at which point they migrate back to the Sargasso Sea to spawn (Helfman et al. 1987, McCleave and Edeline 2009). Based on a restriction-fragment–length polymorphism analysis of the mitochondrial DNA (mtDNA) genome, Avise et al. (1986) reported that there was no genetic divergence between American Eels collected from a number of sites from Maine to Louisiana, including the Carabelle River from the Gulf coast of Florida. Côté et al. (2013) conducted a more expansive study, using 18 microsatellite markers to genotype American Eels from Canada to northeast Florida. Results from both 1Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, Eustis Fisheries Lab, Eustis, FL 32726. 2Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, St. Petersburg, FL 33701. *Corresponding author - Kim.Bonvechio@MyFWC.com. Manuscript Editor: Benjamin Keck Southeastern Naturalist 439 K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 studies were consistent with a panmixia hypothesis, which posited that a single breeding-stock spawned in the Sargasso Sea and the resulting larvae dispersed into drainages across its range. The more robust study by Côté et al. (2013), however, did not include tissue samples collected south of St. Augustine, FL, and excluded American Eels from Gulf of Mexico and Caribbean Sea drainages. While the mtDNA analysis conducted by Avise et al. (1986) would have been expected to identify multiple breeding-populations that had been isolated from one another for an extended period, it might not have had the resolution needed to detect finer-scale differentiation. Studies using microsatellite DNA markers are more likely to detect fine-scale genetic differences because they typically include multiple, independent nuclear loci that are inherited in a Mendelian fashion, while mtDNA surveys are restricted to a single, maternally transmitted DNA molecule. It is possible to imagine a scenario in which offspring of American Eels that spawned in one portion of the Sargasso Sea are more likely to settle in the Gulf of Mexico than in other areas. This situation could lead to fine-scale genetic differences that could be detected at a minority of the nuclear genetic loci included in a microsatellite-markers study, while there would continue to be sufficient gene flow to prevent mtDNA divergence between regions. Further, Miller et al. (2015) analyzed nearly 10,000 records of American Eel larvae collected from locations throughout the Atlantic basin from 1863 to 2007 and found that a small number (~0.2 %) had been collected in the Gulf of Mexico and western Caribbean Sea, which suggested that spawning might have occurred outside the Sargasso Sea. In Florida, American Eels inhabit drainages on both the Atlantic and Gulf of Mexico coasts and have been assumed to be part of the same breeding stock. But whether spatial structure exists between American Eels along both coasts of the state has not been evaluated using methods that are best suited to identify such a structure. The American Eel faces a myriad of threats, which include changing oceanic currents, habitat degradation, disease, and overfishing (ASMFC 2012). In 2006, approximately 24% of American Eels subsampled from the commercial fishery in the St. Johns River, FL, were found to be infected with the nonnative swim-bladder nematode Anguillicoloides crassus (Kuwahara, Niimi, & Hagaki; Florida Fish and Wildlife Conservation Commission [FWC], Eustis, FL, unpubl. data). Although not considered harmful to its native host, Anguilla japonica Temminck & Schlegel (Japanese Eel), this parasite is considered pathogenic in other anguillids (Kennedy 2007). It was first observed in an American Eel at an aquaculture facility in Texas and subsequently, in 1995, in a wild individual in South Carolina (Fries et al. 1996, Johnson et al. 1995). The nematode’s range extends north to Canada, where it was first observed in wild American Eels in 2007 (Rockwell 2009). An American Eel becomes infected by eating an intermediate or paratenic host of the larval parasite. The larval parasite travels to the swim bladder, where it completes its life cycle (De Charleroy et al. 1990), causing hemorrhaging, thickening of the swim-bladder wall, and increased stress to the American Eel (Kennedy 2007, van Banning and Haenen 1990). Laboratory studies have shown that the life cycle is completed in about 3 months at 20–22 °C (Moravec et al. 1994), but development time can vary based Southeastern Naturalist K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 440 on salinity (Kennedy and Fitch 1990, Kirk et al. 2000), temperature (Knopf et al. 1998), and availability of suitable hosts (Kirk et al. 2000). Nematode infections may affect the function of the swim bladder and impact the eel’s ability to withstand environmental stressors, such as low-oxygen situations, or to migrate to the Sargasso Sea to spawn (Lefebvre et al. 2007). Following both its 2012 American Eel Benchmark Stock Assessment and its 2017 Assessment Update, the Atlantic States Marine Fisheries Commission declared that the American Eel stock was in a state of decline (ASMFC 2012, 2017). The American Eel is managed as a single stock; thus, it is important to have information about the species throughout its range to develop effective conservation and management strategies. Unfortunately, little is known about the American Eel in Florida, particularly those in river systems that drain to the Gulf of Mexico. Collecting information on the health, growth, and genetic structure of American Eels in Florida’s freshwater systems would provide much-needed insight into how the species persists within its southern range. We developed a study that was intended to: (1) assess the overall health of the American Eel in different systems throughout Florida, with focus on the presence and severity of A. crassus infestation; (2) collect data on basic biological parameters, including age and growth of American Eels inhabiting Florida Waters; and (3) assess the population genetics of American Eels inhabiting Florida waters, with emphasis on comparing individuals between Gulf and Atlantic drainages. This study was expected to provide managers with critical information on the biology, life history, and status of the American Eel in Florida. Methods American Eels were collected from 72 bodies of water from February 2014 through May 2016. Most specimens were collected during electrofishing surveys for FWC’s Freshwater Long-term Monitoring Program, but other American Eels were collected incidentally during sampling for other projects, and 1 was caught by an angler. Biologists were instructed to freeze any American Eels that they encountered until the specimen(s) could be processed in the laboratory. For each eel, we recorded total length (nearest mm), swim-bladder length (nearest mm), total-body weight (nearest g), liver weight (nearest mg), and spleen weight (nearest mg). From these measurements, we calculated each individual’s length-ratio index (LRI; swim-bladder length/eel length; Palstra et al. 2007), hepato-somatic index (HSI; liver weight/eel weight), and spleen-somatic index (SSI; spleen weight/eel weight). We also assessed the condition and infection of the swim bladders using swim-bladder degenerative index (SDI) scoring for opacity, thickness, and presence of pigmentation and exudate (Lefebvre et al. 2002). We used sagittal otoliths to determine eel age. We removed cleaned, and dried both sagittal otoliths from each eel and stored them in vials. We processed the left otolith for age determination unless it was broken through the core or missing, in which case, the right was processed. We marked the core with a pencil, embedded the otolith in a 2-part epoxy, and mounted it on card stock with hot glue. We sectioned otoliths with a Buehler Isomet low-speed saw (Buehler, Lake Bluff, IL) Southeastern Naturalist 441 K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 equipped with 4 equally spaced diamond wafering-blades. One transverse cut with this multiblade technique yields three ~400-μm–thick sections that encompass both the core and the entire region surrounding the core (VanderKooy 2009). After processing, we mounted sections on glass slides with Flo-Texx® mounting medium. We employed at least 2 blind reads to examine sectioned otoliths on a stereomicroscope using transmitted light. These reads were conducted either by a single reader examining the otolith on separate occasions, or by 2 readers each examining the otolith once. We conducted a third read to resolve the discrepancy if age estimates differed between reads. For determinations of overall eel health and index of swim-bladder condition, we separated American Eels into 5 regions: St. Johns River, Other Northeast, West, Panhandle, and South (Fig. 1). The St. Johns River region included any American Eels collected from the main river and its tributaries and was included as a distinct region, separate from other systems in northeast Florida, because the commercial American Eel fishery in Florida currently only operates in the St. Johns River system. Unlike the first 2 regions where American Eels ingress from the Atlantic Ocean, the Panhandle and West regions refer to American Eels collected from Gulf of Mexico drainages. The West region extends from the Steinhatchee River south to Charlotte Harbor; the Panhandle extends west of the Steinhatchee River to the Perdido River (Florida/Alabama border). American Eels may enter south Florida systems, such as Lake Okeechobee and the Figure 1. Map depicting American Eel collection locations and regions. Southeastern Naturalist K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 442 Kissimmee River via rivers and canals that connect with either the Atlantic or the Gulf; thus, we considered this area separately. We calculated the average LRI, HSI, SSI, and number of swim-bladder nematodes and determined prevalence (% of eels with active infections) for all specimens by region and, when possible, by season (i.e., winter = January–March, spring = April–June, summer = July–September, fall = October–December) within regions. American Eel samples were collected incidentally from non-target surveys in a nonsystematic way, so many regions and seasons had low sample sizes that precluded formal statistical analysis. We genotyped a subset of the collected American Eels. For each specimen, we removed and placed into a vial of 95% ethanol a small piece of fin tissue. We extracted DNA using PUREGENE DNA purification kits (Gentra Systems, Minneapolis, MN) in accordance with the manufacturer’s directions. We adjusted recovered DNA to a concentration of 50 ng/μl with low tris-EDTA buffer and ran 6 polymerase chain reactions (PCR) to amplify 17 of the microsatellite markers Côté et al. (2013) used to study the American Eel. The PCR profile included initial denaturation at 95 °C for 5 min followed by 30 cycles of 30 s of denaturation at 94 °C, 90 s of annealing at 57 °C, and an extension of 60 s at 72 °C. There was a final extension of 30 min at 72 °C. The forward primers had 5' labels of FAM, HEX, or NED, and we used a Genescan 500 ROX-size standard run on an ABI 3130 genetic analyzer (Applied Biosystems, Foster City, CA) to visualize allele sizes. We conducted allele scoring with GENEMAPPER version 3.7 (Applied Biosystems). We excluded specimen genotypes from data analyses if they displayed evidence of DNA contamination (i.e., when we observed >2 alleles at a locus) or were genotyped at fewer than 13 loci. We calculated observed and expected heterozygosity (HO and HE, respectively) using the program ARLEQUIN (Excoffier et al. 2005) and estimated the inbreeding coefficient (FIS) with the program GENETIX (Belkhir et al. 1996–2004). We tested whether each locus conformed to Hardy–Weinberg expectations using the exact-test approach in ARLEQUIN. We determined statistical significance (α < 0.05) following sequential Bonferroni correction for all analyses involving multiple comparisons (Rice 1989). The primary objective of the genetic component of the study was to determine whether American Eels captured in Atlantic coast drainages were genetically different from those captured in streams that drain into the Gulf of Mexico. We pooled specimens from the 2 sets of drainages into different groups (Atlantic vs. Gulf regional groups of American Eels). A third regional group was composed of specimens captured in Lake Okeechobee and south Florida canals because these systems are hydrologically connected to both the Atlantic and Gulf coasts. In addition to the comparison of the pooled specimens, we evaluated whether there was evidence of differentiation between American Eels captured in the St. Johns River (an Atlantic drainage) versus the Apalachicola River (a Gulf drainage) because these systems had much larger sample sizes than any of the other drainages. Although we anticipated that this analysis would produce similar results to comparisons involving the Atlantic and Gulf coast pooled groups, it was possible that pooling American Southeastern Naturalist 443 K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 Eels from different drainages into groups may have obscured differences that could be identified only when specimens from the 2 river systems were considered separately. We employed the program GENEPOP (Raymond and Rousset 1995) to test for genetic differentiation (i.e., differences in allele frequency) between the groups and river systems at each individual locus. We evaluated multilocus genetic differentiation by estimating pairwise FST values between the groups and river systems using ARLEQUIN. We also estimated the effective population size, or number of American Eels contributing offspring to the next generation, assuming all the American Eels belong to a single population. We made the estimate using the linkage-disequilibrium estimator from the program NeESTIMATOR (Do et al. 2014); the critical value was set to 0.02, and we employed the jackknife method to calculate 95% confidence intervals. Results A total of 609 American Eels was collected from 72 systems throughout Florida (Fig. 1); the majority (78%) were collected from the St. Johns River (SJR) and Panhandle regions (Table 1). Sample size varied by season, with summer samples generally the most poorly represented and even lacking for some regions (Table 1). Broad variation in size (136–804 mm total length) and age (0–12 y) of American Eels were represented in the collection, encompassing the elver (immature) stage to the silvering (mature) stage (Figs. 2, 3; Table 1). The 5 silvering females varied in size from 606 mm to 800 mm TL and in age from 4–7 y. Two of these eels were collected in the South and West regions in December, and the remaining individuals were collected from the Panhandle region in October or November. Of the 589 aged individuals, the majority (69%) were aged 3–5 y (Fig. 3), and there was considerable variation in the size at age for most age classes (Fig. 4). The parasitic nematode A. crassus was present in as many as 78% of the American Eels in a single region’s sample and was most common in northeastern Florida (Table 2). The Panhandle American Eels had a low (13%) prevalence of infection, and all but 1 American Eel in the West and South regions were uninfected Table 1. Sample characteristics for American Eels collected from freshwater systems in 5 regions of Florida from 2014 through 2016. Characteristics include size variation, age variation, seasons when collections occurred (ALL = all seasons, WIN = January–March, SPR = April–June, FALL = October– December), and n = sample size. Continental Total length age min–max min–max Region (mm) (y) Seasons collected Total n Genetics n St. Johns River 147–669 1–9 ALL 270 110 Other Northeast 299–577 4–7 WIN, SPR, FALL 18 0 Panhandle 136–804 0–12 ALL 206 133 West 266–800 1–8 WIN, SPR, FALL 50 31 South 158–758 0–11 ALL 65 25 Overall 136–804 0–12 ALL 609 299 Southeastern Naturalist K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 444 (Table 2). Average intensity (number of parasites/eel) followed the same pattern as prevalence across regions (Table 2). In infested American Eels, the number of nematodes ranged from 1 to 52, with average intensity ± 1 SD higher for SJR (4.34 ± 6.32 nematodes/eel) and Other Northeast (6.14 ± 7.67 nematodes/eel) than the Panhandle (2.27 ± 1.91 nematodes/eel). In addition to regional differences, parasite intensity and prevalence may also vary seasonally. In particular, we detected a measurable drop in both parasite prevalence and intensity during the summer, but low sample size (n = 0–6) in the SJR region precluded formal analysis (Fig. 5). Figure 2. Length–frequency distribution of American Eels collected from Florida’s freshwaters during 2014–2016. Figure 3. Age distribution of American Eel collected from Florida’s freshwaters during 2014–2016. The continental age of the fish is represented here (ICES 2009). Southeastern Naturalist 445 K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 Table 2. Summary statistics for prevalence (% of fish with active infection) and intensity (number of parasites per fish) of A. crassus infection and swim-bladder condition (length ratio index [LRI] and swim-bladder degenerative index [SDI]) and fish health (hepato-somatic index; HSI and spleen somatic index; SSI) indices for American Eel collected from freshwater habitats within 5 different geographic regions of Florida. The averages (standard deviation) are presented for each index and intensity, % of fish for prevalence, and number of fish collected (n) for each region and for all fish combined. Intensity Prevalence Region LRI SDI HSI SSI (#/fish) (%) n Other Northeast 0.17 (0.05) 1.39 (0.92) 0.93 (0.16) 0.13 (0.06) 4.78 (7.21) 78 18 Panhandle 0.17 (0.04) 0.32 (0.66) 0.83 (0.41) 0.11 (0.07) 0.29 (1.01) 13 206 South 0.19 (0.03) 0.02 (0.12) 0.91 (0.19) 0.09 (0.04) 0.05 (0.37) 2 65 St. Johns River 0.18 (0.05) 1.31 (1.15) 1.04 (0.25) 0.17 (0.09) 2.21 (5.00) 51 270 West 0.16 (0.04) 0.04 (0.20) 0.94 (0.26) 0.09 (0.04) 0.00 (0.00) 0 50 All fish combined 0.18 (0.04) 0.73 (1.03) 0.94 (0.32) 0.13 (0.08) 1.22 (3.77) 29 609 Figure 4. Total length (mm) and continental age of American Eel specimens collected from 4 regions of Florida during 2014–2016. Data points above the dashed line at 400 mm are assumed to represent females, based on Harrell and Loyacano (1980). With an arbitrary birth date of 1 January, we adjusted age for time of year fish were collected (i.e., age + [days past 1 Jan/364]). Figure 5 (following page). Average swim-bladder condition (LRI, SDI) and overall health (HSI, SSI) indices for American Eels collected from 4 regions of Florida from 2014 through 2016. Values are calculated and presented by (winter = January–March, spring = April–June, summer = July–September, and fall = October–December). Sample sizes (n), prevalence (% of fish with active infection), and intensity (number of parasites per fish) of A. crassus are also presented. Southeastern Naturalist K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 446 Figure 5. [Caption on preceding page.] Southeastern Naturalist 447 K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 We calculated 2 swim-bladder–condition indices (SDI, LRI) and 2 health indices (HSI, SSI) to assess the overall American Eel health. Aside from the SDI, average index-values were similar across regions and between infected and uninfected American Eels within regions (Tables 2–4). Presumably due to damage to the swim bladder from present and past A. crassus infections, the average SDI was higher for areas where the nematode is known to exist (Table 2). The SDI has a maximum value of 6; the largest value observed for American Eels in this study was 5 (Fig. 6). Even when we did not observe nematodes, the SDI value was as high as 4, and in the South and West regions, all SDI scores were 0, with the exception of 1 infected individual that had a score of 1 (Fig. 6). In addition to spatial differences, we also observed seasonal effects in the average SDI for the Panhandle and SJR regions, Table 4. Summary statistics, by season, for overall and swim-bladder health indices for Panhandle American Eels with (present) and without (absent) active A. crassus infections. We include the mean ± 1 SD and sample size (n) for length-ratio index (LRI), swim-bladder degenerative index (SDI), hepato-somatic index (HSI), and spleen somatic index (SSI). Eels were collected from March 2014 through May 2016 and are grouped by season (winter = January–March, spring = April–June, summer = July–September, and fall = October–December). No infected eels were collected in summer. Infection Season Status n LRI SDI HSI SSI Winter Absent 41 0.16 ± 0.04 0.32 ± 0.61 0.95 ± 0.77 0.10 ± 0.08 Present 9 0.17 ± 0.04 1.78 ± 0.44 0.82 ± 0.42 0.10 ± 0.05 Spring Absent 14 0.18 ± 0.04 0.43 ± 0.65 1.07 ± 0.24 0.10 ± 0.03 Present 3 0.18 ± 0.02 1.67 ± 1.15 1.16 ± 0.46 0.08 ± 0.03 Summer Absent 48 0.17 ± 0.04 0.02 ± 0.14 0.62 ± 0.14 0.11 ± 0.09 Present 0 --- --- --- --- Fall Absent 76 0.17 ± 0.05 0.07 ± 0.25 0.83 ± 0.21 0.13 ± 0.05 Present 14 0.21 ± 0.04 1.43 ± 0.76 0.86 ± 0.20 0.12 ± 0.05 Table 3. Summary statistics, by season, for overall and swim-bladder health indices for St. Johns River American Eels with (present) and without (absent) active A. crassus infections. The means ± 1 SD and sample sizes (n) are presented for length-ratio index (LRI), swim-bladder degenerative index (SDI), hepato-somatic index (HSI), and spleen somatic index (SSI). Eels were collected from February 2014 through May 2016 and are grouped by season (winter = January–March, spring = April–June, summer = July–September, and fall = October–December). Infection Season Status n LRI SDI HSI SSI Winter Absent 59 0.16 ± 0.05 0.81 ± 1.21 1.08 ± 0.28 0.17 ± 0.06 Present 70 0.19 ± 0.04 2.13 ± 0.88 1.12 ± 0.25 0.20 ± 0.12 Spring Absent 42 0.16 ± 0.05 0.38 ± 0.62 0.91 ± 0.19 0.14 ± 0.06 Present 33 0.17 ± 0.04 1.94 ± 0.70 0.99 ± 0.23 0.17 ± 0.08 Summer Absent 4 0.19 ± 0.03 0.50 ± 0.58 1.05 ± 0.36 0.15 ± 0.08 Present 2 0.21 ± 0.01 0.50 ± 0.71 1.19 ± 0.34 0.11 ± 0.07 Fall Absent 27 0.19 ± 0.05 0.67 ± 1.14 0.94 ± 0.24 0.14 ± 0.06 Present 32 0.20 ± 0.04 1.66 ± 0.75 1.05 ± 0.23 0.16 ± 0.07 Southeastern Naturalist K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 448 where the majority (92%) of infected American Eels were collected (Tables 3, 4; Fig. 5). We noted a reduction in the average SDI values in the summer, as compared to other seasons of the year (Fig. 5). We determined genotypes for 299 American Eels collected from 16 watersheds. The levels of genetic variation were very similar to those reported by Côté et al. (2013; Table 5). FIS values indicated that most markers had more Table 5. Genetic-diversity estimates for American Eel specimens (n = 299) sampled from Florida freshwater systems during the period 2014–2016. The presented metrics include number of alleles (Na), observed heterozygosity (HO), expected heterozygosity (HE), inbreeding coefficient (FIS), Hardy– Weinberg equilibrium-test result (P), and the first reference for each locus. An asterisk (*) indicates a statistically significant difference. Locus Na HO HE FIS P Reference ARO-54 21 0.729 0.850 0.144 0.0004* Wirth and Bernatchez 2003 ANG-101 41 0.891 0.957 0.068 0.0000* Wirth and Bernatchez 2003 ANG-114 59 0.916 0.967 0.052 0.2057 Wirth and Bernatchez 2003 AjfaBP 18 0.569 0.600 0.052 0.3882 Als et al. 2011 AJMS-06 31 0.817 0.865 0.057 0.0312 Tseng et al. 2001 AJTR-24 58 0.834 0.955 0.129 0.0567 Ishikawa et al. 2001 AJTR-25 43 0.850 0.874 0.030 0.3565 Ishikawa et al. 2001 AJTR-37 22 0.869 0.886 0.021 0.2932 Ishikawa et al. 2001 AJTR-45 96 0.920 0.976 0.057 0.4023 Ishikawa et al. 2001 AAN-03 10 0.470 0.494 0.054 0.0679 Daemen et al. 1997 AAN-05 9 0.515 0.476 -0.081 0.7491 Daemen et al. 2001 AangCT53 23 0.636 0.635 0.001 0.4041 Wielgoss et al. 2008 AangCT68 25 0.895 0.917 0.023 0.7209 Wielgoss et al. 2008 AangCT76 38 0.887 0.936 0.051 0.2923 Wielgoss et al. 2008 AangCT82 21 0.515 0.539 0.048 0.3051 Wielgoss et al. 2008 AangCT87 59 0.911 0.955 0.045 0.0757 Wielgoss et al. 2008 AangCT89 36 0.926 0.941 0.015 0.0640 Wielgoss et al. 2008 Figure 6. Relationship between the swim-bladder degenerative index (SDI) and number of nematodes present in the swim bladders of American Eel specimens collected from freshwater habitats in Florida from 2014 through 2016. Southeastern Naturalist 449 K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 homozygotes than expected under Hardy–Weinberg equilibrium, but only2 loci had statistically significant heterozygote deficits after Bonferroni correction (ARO-54 and ANG-101). Although Côté et al. (2013) reported higher FIS values for both of these markers than those we report, those authors determined that the vales were not out of the HWE. We detected no differentiation between the Atlantic and Gulf Groups or between the Atlantic and South Florida groups for any of the 17 loci tested. One locus (AangCT76) was genetically differentiated between the Gulf and South Florida groups. The South Florida group, however, had a particularly small sample size, so sampling error may have affected allele frequencies collected for that group. The pairwise FST estimates were not significant for any of the comparisons (Atlantic versus Gulf FST = 0.00035, P = 0.1841; Atlantic versus south Florida FST = −0.00123, P = 0.7510; Gulf versus south Florida FST = −0.00021, P = 0.2305). We found no loci to be genetically differentiated in the direct comparison between American Eels from the St. Johns (n = 110) and Apalachicola (n = 107) rivers, and the pairwise FST estimate was not significant (F ST = 0.00008, P = 0.4078). The estimated effective-population size for the American Eels sampled in Florida was 7698 (95% CI, 2698–infinity). This result is similar to the estimate from Côté et al. (2013) for 2 life stages of American Eels captured from numerous locations between Florida and Newfoundland (Ne = 10,532, 95% CI = 9312–11,752). The confidence intervals from Côté et al. (2013) are likely narrower because the sample size in that study was much larger than in the present study. Discussion Little information is available about the American Eel in its southern range, particularly in regards to comparing those in drainages closest to the putative spawning location in the Sargasso Sea compared to those having access to the Gulf of Mexico. Thus, this project attempted to collect baseline data that could inform management decisions and direct future research efforts. Using non-target surveys, we gathered information on the basic biology and health, the intensity and prevalence of the nonnative swim-bladder nematode A. crassus, and the broad-scale patterns of genetic relationships in American Eels throughout Florida. We could not determine the sex of specimens that had been frozen due to artifacts in frozen tissue that make interpretation difficult. However, 69% of the more than 600 American Eels collected from water bodies during this study were 400 mm TL or larger, suggesting that most were female (Harrell and Loyacano 1980). This result agrees with those of other studies showing that the sex ratio of American Eels in upstream waters tends to be skewed towards slow-growing females (Côte et al. 2015, Davey and Jellyman 2005). Our female-skewed sample was expected because all but 1 American Eel was collected by electrofishing in upstream freshwater (less than 1–2 ppt), where American Eel density can be relatively low (Davey and Jellyman 2005, Krueger and Oliveira 1999) and to which slow-growing females are known to migrate and develop (Côte et al. 2015). We collected 5 silvering females, varying from 606 mm to 800 mm TL and from 4 to 7 y in age. Two of these American Eels were collected Southeastern Naturalist K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 450 in the South and West regions in December, the others from the Panhandle region in October and November. This finding coincides with observations of silver American Eel migration in Georgia, which begins in October (Facey and Helfman 1985). Additional efforts are needed to fully assess growth, sex ratio, and life history of Florida’s American Eel populations. We did not find compelling evidence of genetic differentiation between any geographically defined groups of American Eels evaluated in this study. All pairwise FST estimates were extremely small and statistically nonsignificant. While we found that American Eels from the Gulf of Mexico drainages and South Florida had different allele frequencies at 1 locus; the south Florida group consisted of a small number of American Eels and so may not have been representative of allelic distribution of the American Eels in this region. Additional samples should be collected and analyzed to acertain whether this finding was the result of population structure rather than small sample size. Our study evaluated much larger samples from the Gulf of Mexico and Atlantic regions and found that the American Eels from these 2 regions were part of the same genetic population. Côté et al. (2013) found that American Eels captured in Atlantic coastal drainages from Newfoundland to Florida were from a single, panmictic population. Our results suggest that this population extends beyond the Atlantic coast at least as far as the Gulf Coast drainages in the Florida panhandle. Citing the collection of small larval American Eels in the western Caribbean, Miller et al. (2015) noted the possibility of another spawning location outside the Sargasso Sea. However, our results indicate that larval American Eels from the Sargasso Sea are at least capable of reaching the eastern Gulf of Mexico, although the pathway of that travel remains unknown. Given that American Eels of every age class, from 0 to 12 y, were collected for this study, recruitment of American Eels is occurring annually into this region. After growing and developing, these American Eels must then either migrate back to the Sargasso Sea to spawn, or they represent a reproductive sink for the stock and do not contribute gametes to the next generation. Our results do not exclude the possibility that another spawning ground exists nor that these Sargasso Sea-spawned American Eels, when mature, would migrate back to an area other than the Sargasso Sea to spawn. Thus, additional samples from Gulf of Mexico drainages in other states would be required in order to determine whether the American Eel makes up a single panmictic population throughout its range. We found spatial differences in the prevalence and incidence of A. crassus infection in Florida American Eels, with the highest rates detected in Northeast Florida and the St. Johns River. Furthermore, the evidence of swim-bladder damage, as indicated by SDI scores up to 4 (out of a possible 6), for American Eels without active infections suggest that these and perhaps other eels in these areas may have had previous infections. Machut and Limburg (2008) posited that parasite-infection rates may be elevated in urbanized areas, but this suggestion fails to explain the lack of nematodes in other Florida American Eels. For example, the Southeast Florida region is highly urbanized and channelized, and none of the American Eels collected in human-made canals in this region were infected with the nematode Southeastern Naturalist 451 K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 or had nonzero SDI scores. Another possible explanation is that the nematode was transported by ballast water that contained infected hosts, such as copepods (De Charleroy et al. 1990). This scenario, too, fails to explain the distribution of nematode occurrence in Florida because the major Florida ports of Miami, Fort Lauderdale, and Tampa are all areas where the nematode has not been observed in wild American Eels. Kennedy (2007) suggested that the primary pathway of the nematode is movement of infected American Eels between areas for purposes such as stocking, aquaculture, and bait. To our knowledge, there has been no large-scale stocking or aquaculture of American Eels in Florida, but the 2 areas where the nematodes are known to exist in Florida (Panhandle and Northeast Florida regions) were home to the only historical commercial American Eel fisheries in Florida. Since 2000, most (≥80%) American Eels have been harvested for human consumption (as opposed for use as bait), but annual exports vary greatly, ranging from 0 to 100%. In years with high exports, out-of-state dealers picked up and transported live American Eels from various locations along the Atlantic coast states, including Florida, and may have accidentally released nematode larvae into state waters during water exchanges. Other avenues of introduction, including the importation of infected bait American Eels from other Atlantic coast drainages, may also have played a role in the establishment of this nonnative parasite in some areas. Additional efforts should be made to determine the extent of the nematode’s distribution in Florida waters. If it is limited to certain regions, future efforts to establish bait markets or aquaculture ventures should consider ways of reducing the possibility of spreading infection to unaffected populations. Salinity may play a role in the distribution of the nematode within a region. Under laboratory conditions, hatching of nematode eggs and survival of larval stages have been shown to be inhibited, although not completely, in high-salinity conditions (Kennedy and Fitch 1990). Field observations coincide with these results. For example, Neto et al. (2010) found that prevalence of the nematode in Anguilla anguilla L. (European Eel) followed the salinity gradient in the Tagus Estuary, Portugal, with decreasing prevalence as salinity increased. Denny et al. (2013) found similar results for American Eel in Bras d’Or Lakes, NS, Canada, with prevalence of the nematode higher in riverine areas. All but 1 of the American Eels included in our study were obtained by electrofishing, a gear that is limited to freshwater environments; therefore, we could not address the role of salinity in the distribution of the nematode in our samples. Temperature may also play a role in the A. crassus life cycle and ultimately how the nematode affects American Eels in Florida. Although we collected only a limited number of specimens in summer, our data suggest that average SDI, prevalence, and intensity of infection are reduced during the period from July to September, when waters are generally warmest in Florida. Thomas and Ollevier (1993) found the incubation time of nematode eggs decreased as temperature increased, from 5 °C to 30 °C. However, Schippers et al. (1991) found that 58–87% of nematodes died when water temperatures were raised to 36.5 °C. Thus, it is possible that high water-temperatures can impact the survival of A. crassus during the summer months in Florida and reduce their incidence in American Eels. Southeastern Naturalist K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 452 Alternatively, average SDI, prevalence, and intensity of infection may be reduced in summer months due to other factors, such as increased mortality of American Eels. Gollock et al. (2005) determined that high temperature alone would be unlikely to cause mortality in American Eels, but Lefebvre et al. (2002) attributed a drop in the swim-bladder index of European Eel to the death of individuals more severely infected by A. crassus during the warmest months. Lefebvre et al. (2007) showed that inducing oxygen stress led to increased mortality of American Eels that had swim-bladder damage. We did not observe differences in health between infected and uninfected American Eels. Average LRI, SSI, and HSI values were similar between regions, among seasons within regions, and between uninfected and infected American Eels in the Panhandle and St. Johns River regions, where most of the infected American Eels were collected. Furthermore, condition (weight as a function of length) was nearly identical between infected and uninfected American Eels in both the Panhandle and St. Johns River regions. This finding coincides with Machut and Limburg’s (2008) observations that condition of yellow eels was not related to nematode prevalence or intensity. Still, it remains unclear what effect the combination of localized stressors, including high temperature, low dissolved oxygen, and environmental toxins, might have on American Eels in Florida in summer or to their capacity to return to the Sargasso or another spawning area, should one exist. Published information on the American Eel in Florida is extremely limited. This study provides important baseline data about its basic health and biology, the distribution of A. crassus, and the population genetics of the American Eel in Florida. Given that A. crassus infections diminish swim-bladder function and may impair the ability of infected individuals to reach the Sargasso Sea to spawn (Kennedy 2007), additional efforts must be made to describe the distribution of the nematode in Florida waters and to limit its spread there and beyond. Furthermore, the genetic information produced by this study has implications for American Eel conservation and management, but additional population-genetics work is needed for Gulf of Mexico drainages in other states. Finally, for the future management and conservation of this species, it is critical that future efforts focus on describing basic population characteristics including abundance, sex-specific growth rates, and sex ratio, and life-history characteristics, such as age at maturation and timing of inland and outgoing migration, for Florida and other Gulf of Mexico American Eel populations. Acknowledgments We thank all of the personnel with the Florida Fish and Wildlife Conservation Commission who collected, stored, and transported samples for processing. We extend special appreciation those who helped with laboratory work, including T. Alfermann, M. Bakenhaster, J. Benton, G. DelPizzo, J. Feltz, N. Feltz, D. Gandy, S. Hamby, J. Hill, J. Holder, Y. Kiryu, E. Lundy, W. Porak, D. Richard, C. Steward, and A. Strickland. We are also grateful to N. Balk, M. Cantrell, B. Crowder, A. Strickland, and N. Trippel for their reviews of previous versions of this manuscript. This project was developed with financial assistance provided by the Fish and Wildlife Foundation of Florida, Inc., through the Conserve Wildlife Tag grant program (CWT 1516-06). Southeastern Naturalist 453 K.I. Bonvechio, B. Barthel, and J. Carroll 2018 Vol. 17, No. 3 Literature Cited Als, T.D., M.M. Hansen, G.E. Maes, M. Castonguay, L. Riemann, K. Aarestrup, P. Munk, H. Sparholt, R. Hanel, and L. 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