2009 SOUTHEASTERN NATURALIST 8(2):227–243 Genetic Assessment of Species Ranges in Fundulus heteroclitus and F. grandis in Northeastern Florida Salt Marshes Iara Gonzalez1, Michael Levin1, Sura Jermanus1, Brent Watson1, and Matthew R. Gilg1,* Abstract - The limits of species ranges can be determined by a number of biotic and abiotic interactions, and in areas where closely related species overlap, some degree of reproductive isolation must exist in order for them to remain distinct. Understanding these interactions is essential for understanding what limits species distributions or causes hybridization. Fundulus heteroclitus (Mummichog) and Fundulus grandis (Gulf Killifish) are two closely related species with similar morphologies and ecological niches. Both species have widespread distributions that overlap in northeastern Florida. In the present study, two highly divergent loci (one nuclear and one mitochondrial) were utilized to distinguish these fundulid species in order to identify their ranges and to detect hybrids. Analysis of specimens collected along a north to south gradient in northeastern Florida salt marshes established that a relatively sharp transition (≈38 km) from relatively pure Mummichog populations to relatively pure F. grandis populations existed south of Jacksonville, FL, centered near Flagler Beach, FL. Putative hybrid genotypes were detected at moderate frequencies within the contact zone, suggesting that successful hybridization is likely occurring between the two species, but is relatively uncommon. These results provide a stepping stone to investigate the types of reproductive barriers that are involved in maintaining species distinctions in this system and their effects on the species’ ranges and ecological interactions. Introduction Areas of range overlap between closely related species provide opportunities to understand the mechanisms responsible for speciation and the maintenance of diversity. Interspecific interactions in regions of overlap can affect the extent to which adaptation may occur at the edges of a species’ geographic range (Case and Taper 2000). The distribution of species is known to be strongly infl uenced by closely related species because they tend to share many resources resulting in strong competition (Anderson et al. 2002). Competition among sympatric species can lead to one species being excluded from an area by the other, an event known as competitive exclusion (Day and Young 2004, Zaret and Rand 1971). Several studies have documented that species sharing the same resources are able to coexist through resource partitioning (Hart 2003, Schluter and McPhail 1992, Weisberg 1986, Zaret and Rand 1971). Furthermore, in areas of sympatry, hybridization can be a key factor in defining species boundaries (Gow et al. 2006). Closely related species 1Department of Biology, University of North Florida, 1 UNF Drive, Jacksonville, FL 32224. *Corresponding author - mgilg@unf.edu. 228 Southeastern Naturalist Vol. 8, No. 2 that come into contact have the potential to interbreed, which can lead to a number of potential outcomes. These outcomes include genetic assimilation of the two species, increasing the reproductive isolation of the parental species due to low fitness of hybrids (reinforcement), adding genetic variation to parental populations, or production of a third hybrid species that is reproductively isolated from both parental forms (Arnold 1997). The role of hybridization in species diversification remains a central issue in evolutionary biology (Coyne and Orr 2004). Two closely related teleost species, Fundulus heteroclitus (L.) (Mummichog) and Fundulus grandis Baird and Girard (Gulf Killifish) provide an opportunity to investigate the genetic structure of divergent populations that overlap in range. The geographical distribution of the Mummichog ranges from southwestern Newfoundland to northeastern Florida (Able and Felley 1986), while the Gulf Killifish ranges from northeastern Florida southward to the coasts of Mexico (Duggins et al. 1989). These fundulids are found in salt marsh habitat year round and utilize the marsh surface to feed during high tide and to spawn according to the semi-lunar cycles (Greeley and MacGregor 1983, Hsiao and Meier 1989, Weisberg et al. 1981). Both species are considered highly abundant with very productive populations—likely a consequence of the two species being opportunistic omnivores (Kneib 1986, Lipcius and Subrahmanyam 1986). Their diets consist of small crustaceans, insects, snails, polychaetes, and detritus (James-Pirri et al. 2001, Lipcius and Subrahmanyam 1986). Both fundulids are also prey for a variety of predators such as crustaceans (Callinectes spp., Ucca spp.) and larger fishes, including commercially important species such as Morone americana (Gmelin) (White Perch), and Sciaenops ocellatus (L.) (Red Drum) (James-Pirri et al. 2001, Kneib 1986). Given the extent of ecological overlap between the two species and the fact that hybridization among several fundulid species has been widely reported (Atz 1986, Duggins et al. 1995, Duvernell et al. 2007, Hernández- Chávez and Turgeon 2007), it seems likely that the Gulf Killifish and the Mummichog hybridize in areas of overlap. Furthermore, previous genetic data from Duggins et al. (1989) showed that the two species have many allozyme alleles in common, suggesting hybridization. In order to investigate the type of interactions between the Mummichog and the Gulf Killifish, it is necessary to establish their region of overlap in northeast Florida. Whereas their ranges were known to meet along the northeastern Florida coast, the specific location of their zone of sympatry and its width were not known. Our study intended to establish the distribution of Mummichog and Gulf Killifish along salt marshes in northeastern Florida through the use of species-specific genetic markers. Morphologically, the Mummichog and the Gulf Killifish can be distinguished in their adult stage by the eight mandibular pores and longer ovipositor of the Mummichog versus the ten mandibular pores and shorter ovipositor of the Gulf Killifish (Duggins et al. 1989). Nonetheless, the morphological characteristics of the adults are often overlapping in variation, making misidentification a strong possibility, and young juveniles and larvae of the two species are virtually impossible to tell apart morphologically. Therefore, 2009 I. Gonzalez, M. Levin, S. Jermanus, B. Watson, and M.R. Gilg 229 identifying molecular markers that distinguish members of these two similar species would provide a reliable identification method that can be used for any age class. The possibility of hybridization between the Mummichog and the Gulf Killifish can also be verified by species-specific nuclear markers. Both the allopatric and sympatric ranges of the two species can be determined by collecting adults in tidal creeks along a latitudinal gradient in northeastern Florida. According to Duggins et al. (1989), the two species overlap in range in the vicinity of Marineland, located approximately 13 km south of St. Augustine, FL. Therefore, we hypothesized that there is a latitudinal cline, with Mummichog individuals being abundant in the northern sites and tapering off as Gulf Killifish specimens become more common in the southern sites. Materials and Methods Sample collection and DNA extraction Representatives from allopatric populations were sampled to identify genetic markers that differentiated the two species. Personal communication with Russ Brodie of the Florida Fish and Wildlife Commission (Fish and Wildlife Research Institute, Jacksonville, FL) suggested that the St. Mary’s River (SM) in Georgia could be considered reference site for populations of Mummichog, while samples from Cedar Key (CK) and Port St. Joe (PSJ) on the Gulf coast of Florida (Fig. 1) would include only Gulf Killifish (Hoese and Moore 1998). According to morphological characteristics, only the Mummichog is found as far north as SM and only the Gulf Killifish is found on the Gulf coast of Florida. Furthermore, previous genetic work by Duggins et al. (1989) suggests the above locations to be reliable reference sites for each species. To determine the possible area of overlap, adult Fundulus were collected during the spring and summer of 2005 and 2006 from eight sites spanning about 200 km of northeastern Florida (Fig. 1). The localities were chosen to extend the previous study by Duggins et al. (1989) describing genotypic distributions for the Mummichog and Gulf Killifish in the southeastern United States. The sites sampled along northeastern Florida were distributed from north to south: Atlantic Beach (AB), Vilano Beach (VB), Moses Creek (MC), Marineland (ML), Pellicer Creek (PC), Flagler Beach (FB), Tomoka Basin (TB), and Indian River (IR). All of the sites had similar physical characteristics, except PC had a maximum salinity of 17 parts per thousand (ppt) while the other areas had salinity ranges between 20–35 ppt. A total of 523 fishes were collected from all the sites (Table 1) utilizing minnow traps, beach seines, or cast nets. Specimens were placed in Whirl- Pak bags and labeled by collection site and date. These bags were placed on ice, transported to the laboratory, and stored at -80 ºC upon arrival. Adult specimens used for genetic analysis were measured (standard length), individually labeled, and preserved in 95% ethanol. A small piece of fin tissue was removed from each sample for genomic DNA extraction using the phenol/chloroform method (Sambrook and Russell 2001) or the QIAGEN DNeasy Blood and Tissue Kit. 230 Southeastern Naturalist Vol. 8, No. 2 Mitochondrial DNA Multiple published sequences of the mitochondrial gene cytochrome-b (cyt-b) were available on GenBank for both Mummichog and Gulf Killifish from Florida and Georgia (Accession numbers: L23772–L23775, F. heteroclitus; AF321852, U77124, and L31594, F. grandis). These sequences suggested that the restriction enzyme NsiI would distinguish the two species at this locus by cutting PCR products of the Gulf Killifish, but not the PCR products of the Mummichog. A ≈450 bp region of the cyt-b locus was Figure 1. Location of sampling sites in the southeastern United States. Reference sites for Gulf Killifish populations are Cedar Key and Port St. Joe in the Gulf coast while the St. Mary’s River in Georgia served as the reference site for Mummichog specimens. 2009 I. Gonzalez, M. Levin, S. Jermanus, B. Watson, and M.R. Gilg 231 amplified by polymerase chain reaction (PCR) using the universal primers GLUDG-5’ and CB3-3’ as described in Palumbi (1996). Amplified PCR products were electrophoresed on a 1% agarose gel at 125 V for 1 hour. When a single amplification did not result in quality PCR products, the samples underwent a second internal amplification using the CB2-3’ primer with GLUDG-5’. Successfully amplified products underwent a restriction digest using NsiI as directed in the manufacturers’ protocol. Digested products were electrophoresed on a 3% agarose gel at 70 V for 2 hours, and after visualization, samples were scored as cut or uncut. Sequencing of a subset (n = 14) of our samples was performed on a Beckman-Coulter CEQ 8000 using the same primers as above with the manufacturer’s cycle sequencing protocol. Sequencher 4.7 was used to edit sequences, and aligned sequences were analyzed by maximum parsimony using PAUP (4.0.3). Nuclear DNA Eight sequences of the 5’ untranslated region of lactate dehydrogenase- B (LB UTR) are published in GenBank for the two species (Accession numbers: U59837–U59844) and were assessed for differences in restrictionenzyme cut patterns. Since only a single sequence has been published for F. grandis, we constructed internal primers to amplify and sequence additional PCR products of both species to verify possible Restriction Fragment Length Poymorphism (RFLP) differences. Best results were produced by performing a pair of PCR amplifications using external, UTR and FLK, and internal, MRG-3 (5’-TTG TTT CAT GGG GTC TGA ACA C-3’) and MRG- 4 (5’- GGC ATT ACA ATC AGA CAA GTA GAG-3’), primers (Schulte et al. 1997). Amplified PCR products were electrophoresed on a 1% agarose Table 1. Summary of cyt-b haplotype and LB UTR genotype frequencies. The letters h and g denote F. heteroclitus (Mummichog)-specific and F. grandis (Gulf Killifish)-specific alleles, respectively. P represents the probability a locus is in Hardy-Weinberg equilibrium based on Fisher’s exact test, and F values are the result of Wright’s F -statistics. Site codes are as follows: SM = St. Mary’s River, AB = Atlantic Beach, VB = Vilano Beach, MC = Moses Creek, PC = Pellicer Creek, ML = Marineland, FB = Flagler Beach, TB = Tomoka Basin, IR = Indian River, CK = Cedar Key and PSJ = Port St. Joe., n = sample size. Cyt-b LB UTR Site n h g n h/h h/g g/g P F SM 79 1.00 0.00 73 0.89 0.08 0.03 0.027† 0.37 AB 57 0.91 0.09 57 0.86 0.05 0.09 2.5x10-5† 0.82 VB 36 1.00 0.00 37 0.95 0.05 0.00 0.98 0.08 MC 44 0.98 0.02 36 0.89 0.03 0.08 0.008† 0.24 PC 50 0.92 0.08 49 0.94 0.04 0.02 0.061 0.47 ML 41 1.00 0.00 41 0.83 0.07 0.10 4.5x10-4† 0.35 FB 51 0.40 0.60 50 0.20 0.06 0.74 3.2x10-8† 0.83 TB 8 0.13 0.88 8 0.00 0.125 0.88 1.0 -0.11 IR 63 0.00 1.00 60 0.00 0 1.00 1.0 0.00 CK 54 0.02 0.98 55 0.00 0 1.00 1.0 0.00 PSJ 38 0.03 0.97 38 0.00 0.03 0.97 0.76 0.00 † indicates statistical significance (P < 0.05). 232 Southeastern Naturalist Vol. 8, No. 2 gel at 125 V for 1 hour. According to the sequence data (Accession numbers: EU482176–EU482180), the restriction enzyme PstI was hypothesized to cut the PCR product of the Mummichog, while PCR products of the Gulf Killifish should remain uncut. The internal primers MRG-3 and MRG-4 produce an uncut product that is approximately 485 bp long in the Gulf Killifish only, while the cut products are typically 176 bp and 309 bp in length. Small differences in product length due to insertions and deletions were observed in some samples. As with the mitochondrial locus, a subset of 12 samples from the present study were sequenced on a Beckman-Coulter CEQ 8000 using primers MRG-3 and MRG-4 and analyzed as described above for cyt-b. Statistical analysis Haplotype and genotype frequencies of the two markers were estimated for each collection site and compared using an exact population homogeneity test (GenePop; Raymond and Rousset 1995). Fisher’s (1922) exact test was used to determine whether genotypic frequencies of LB UTR were in Hardy-Weinberg equilibrium (HWE) and Wright’s (1922) F parameter was calculated to determine whether deviations from Hardy-Weinberg expectations were due to a deficiency or an excess of heterozygotes. Individuals from all sites were assigned to parental populations using STRUCTURE (ver. 2.1; Pritchard et al. 2000). Finally, exact tests of cytonuclear disequilibrium were conducted using CNDd (Asmussen and Basten 1994, Basten and Asmussen 1997). Statistical significance of all tests was set at α = 0.05. Results Reference sites The specimens from reference populations verified near species specificity of both the mitochondrial and nuclear genetic markers. The cyt-b PCR product was uncut in all the samples from SM in Georgia, correctly identifying them as Mummichogs (Table 1). The CK and PSJ specimens were cut at frequencies of 0.98 and 0.97, respectively, correctly identifying the majority of individuals as Gulf Killifish (Table 1). Considering all the samples from the reference sites, the NsiI restriction digest of the cyt-b marker had a >98% probability of giving the correct identification at the reference sites. Allele and genotype frequencies of LB UTR were highly divergent between the reference sites of the two species and supported the results obtained by the mitochondrial marker, but were not completely speciesspecific. Cut alleles were found at a frequency of 0.93 at SM, but only at frequencies of 0.00 and 0.01 at the Gulf Coast reference sites of CK and PSJ, respectively, showing clear differences in allele frequencies among the reference sites for the two species. Essentially, cut alleles are predominantly found in Mummichogs, and uncut alleles are nearly fixed in Gulf Killifish populations. The frequency at which specimens from the SM population had LB UTR cut completely by PstI digest was 0.89, representing homozygous specimens for the allele most often encountered in Mummichogs (Table 1). The resolution of the nuclear marker was more precise for homozygous uncut individuals, with a genotype frequency of 1.00 at CK and 0.97 at PSJ. 2009 I. Gonzalez, M. Levin, S. Jermanus, B. Watson, and M.R. Gilg 233 When all samples from the three reference sites are considered at the nuclear marker, 95% of the specimens were identified as being homozygous for the correct species-specific alleles. Whereas it is not a fixed difference, the LB UTR marker is reliable for species identification. Since both markers were shown to be highly species-specific, we designated a Mummichog haplotype/ allele as “h” and a Gulf Killifish haplotype/ allele as “g.” Adult distributions: Cyt-b Haplotype frequencies followed the expected north-to-south gradient, with significant spatial variation among all collection sites (P < 0.0001, S.E. < 0.0001). All sites north of FB had frequencies of h haplotypes ≥0.91, while sites south of TB were devoid of h haplotypes (Table 1, Fig. 2). FB had intermediate frequencies of 0.40 for the h haplotype. The observed haplotype frequencies at cyt-b suggested the presence of three distinct regions: a northern region with high frequencies (>0.91) of h haplotypes; a zone of overlap exhibiting a mixture of h and g haplotypes; and a southern region lacking h haplotypes (Fig. 2). Sites north of FB (including the reference site SM) that had high frequencies for the h haplotype did, however, show significant spatial variation (P = 0.0072, S.E. = 0.0008) except when samples from AB and PC were removed (P = 0.602, S.E. = 0.0046). This finding suggests that most of these sites have relatively pure populations of Mummichogs. Similarly, no significant differences in haplotype frequencies were observed among the southern sites of TB and IR when compared to the reference sites of CK and PSJ, suggesting these sites represented Gulf Killifish populations (P = 0.0575, S.E. = 0.0018). The marginally insignificant P value of the aforementioned comparison is due to the inclusion of TB, which because of its sample size (n = 8), makes its inclusion unreliable. These comparisons helped Figure 2. F. heteroclitus (Mummichog)- specific haplotype (cyt-b) and allele (LB UTR) frequencies across all sites from north to south along the Atlantic coast. Only sites from Saint Mary’s to Indian River were included. 234 Southeastern Naturalist Vol. 8, No. 2 establish the area of range overlap of the two species as being bordered by the ML and TB sites, which are separated by a distance of about 38 km. Since several sites north of FB contain samples with g haplotypes, a subset of seven samples were sequenced to verify whether these were truly Gulf Killifish haplotypes or if they were simply Mummichog haplotypes that happened to contain an NsiI cut site. An unrooted maximum parsimony analysis including samples from the reference sites (Accession numbers: EU482166, EU 482167), samples of each species downloaded from Genbank (Accession numbers: L23772–L23775, AF312852, U77124, L31594), and samples with unexpected haplotypes from sites between SM and FB (Accession numbers: EU482165–EU482154) verified that, in most cases, the “cut” haplotypes in the northern region (AB 31, 38; PC 20, 21, 24, 37) were indeed Gulf Killifish haplotypes (Fig. 3). Only the sample AB 57 had a haplotype that was cut by NsiI and was within the Mummichog clade. Adult distributions: LB UTR The allele frequencies of LB UTR followed a similar pattern to that observed at cyt-b. All sites north of FB had h allele frequencies ranging from 0.87 to 0.97. A marked shift in allele frequencies was again detected at FB, with h allele frequencies decreasing to 0.23 (Fig. 2). The remaining populations exhibited a high incidence of g alleles with frequencies of 0.94 at the Tomoka Basin site and 1.0 at the Indian River site. Comparisons of allele frequencies across all sites yielded significant spatial variation (P < 0.0001, S.E. < 0.0001). Analysis of LB UTR allele frequencies suggested the presence of the same three regions observed at cyt-b (Fig. 2). Results at LB UTR were slightly different than at cyt-b, since a comparison of the six sites north of FB showed no significant variation among populations (P = 0.0704, S.E. = 0.0045 ). Allele frequencies at LB UTR differed significantly among TB, IR, and the Gulf Coast reference populations (P = 0.0266, S.E. = 0.0011), but removal of TB removed the significant spatial variation (P = 0.2500, S.E. = 0.0026). Therefore, the zone of sympatry established by LB UTR is similar to that found with cyt-b; the FB site has intermediate frequencies of both h and g alleles, with predominantly Mummichog populations to the north and Gulf Killifish populations to the south. The Gulf Killifish reference sites of CK and PSJ showed no significant deviations from Hardy-Weinberg equilibrium (HWE) at LB UTR. On the other hand, the Mummichog reference site, SM, was not in HWE (Table 1). Of the eight non-reference sites, four had genotype frequencies that differed significantly from HWE including AB, MC, ML, and FB. All sites that deviated significantly from HWE showed a deficit in heterozygotes (Table 1). Even sites that were found to be in HWE tended to have positive values of Wright’s F statistic (1922), except for the population at TB in which the negative F is likely explained by the small sample size. The presence of low but consistent numbers of g alleles at the LB UTR locus in populations of predominantly Mummichogs (including reference site SM) prompted the sequencing of individuals with g/g genotypes at LB UTR 2009 I. Gonzalez, M. Levin, S. Jermanus, B. Watson, and M.R. Gilg 235 to determine whether these g alleles were representative of Gulf Killifish alleles in predominantly Mummichog populations or if they were simply polymorphisms within Mummichogs that lacked the PstI restriction site. An unrooted maximum parsimony analysis including samples from the reference sites (Accession numbers: EU482176–EU482180), samples of each Figure 3. Maximum parsimony cladogram of cyt-b including samples from Genbank (noted with accession numbers) and various collection sites from northeastern Florida. Numbers at the nodes represent the results of a bootstrap analysis with 1000 replicates. Site codes are as follows: AB = Atlantic Beach, PC = Pellicer Creek, FB = Flagler Beach and CK = Cedar Key. 236 Southeastern Naturalist Vol. 8, No. 2 species downloaded from Genbank (Accession numbers: U59844, U59835 and U59834), and samples with unexpected genotypes from sites between SM and FB (Accession numbers: EU482187 and EU482186) showed that both of the g alleles sequenced from samples collected north of FB (AB 35, SM 75) actually grouped with the Mummichog clade (Fig. 4). The same was true of most of the samples from FB (see below). Therefore, at least some of Figure 4. Maximum parsimony cladogram of LB UTR including samples from Genbank (noted with accession numbers) and various collection sites from northeastern Florida. Numbers at the nodes represent the results of a bootstrap analysis with 1000 replicates. Site codes are as follows: SM = St. Mary’s River, AB = Atlantic Beach, FB = Flagler Beach and CK = Cedar Key. 2009 I. Gonzalez, M. Levin, S. Jermanus, B. Watson, and M.R. Gilg 237 the g alleles within the predominantly Mummichog populations are actually polymorphisms within Mummichogs and are not due to introgression or the presence of Gulf Killifish at those locations. Hybridization and cytonuclear disequilibrium Haplotype and genotype data from both loci were combined to investigate the overall population structure of these two species and to test for the existence of hybrids. The program STRUCTURE (Pritchard et al. 2000) was used to assign each individual to a population of ancestry based on its dilocus genotype. This assignment score (q) ranges from 0 (Gulf Killifish) to 1 (Mummichog), and 95% probability intervals for each q are estimated using a Markov Chain Monte Carlo method (500,000 iterations after a burn-in period of 50,000 iterations under a model of admixture with K = 2 populations). When the probability distributions of ancestry are plotted for each individual across all collection sites, the results are very consistent with the single locus data above (Fig. 5). Most individuals have assignment scores near 0 or 1. The predominantly Mummichog population appears to have a consistent but relatively low level of introgression from Gulf Killifish, since several individuals have very low values of q. None of the samples from these northern populations, however, have values of q as low as those found at IR, PSJ, or CK. This finding is consistent with the fact that none of the samples from any site between SM and ML had any individuals with g:g/g (cyt-b:LB UTR) genotypes. Introgression is much less noticeable in the predominantly Gulf Killifish populations. Furthermore, the transition from Mummichog to Gulf Killifish populations is very sharp, mostly consisting of the FB site. Samples from FB show considerable overlap of probability distributions, with both populations suggesting the presence of individuals of mixed ancestry. The dilocus genotype frequencies at FB reveal that the two species are likely able to hybridize (Table 2). Putative hybrid genotypes include individuals that are heterozygous for the species-specific alleles at the LB UTR locus (e.g., h:h/g and g:h/g) and also individuals that have the cyt-b Table 2: Observed dilocus genotype frequencies of samples from Flagler Beach and estimates of cytonuclear disequilibrium. For disequilibrium estimates, A represents the F. heteroclitus nuclear allele and M the F. heteroclitus mitochondrial haplotype. Lower case letters are alleles/ haplotypes of F. grandis. Cytonuclear disequilibrium Genotypes P DA DAM DAAM DAaM DaaM h:h/h 0.16 Estimators 0.1471 0.0880 0.0800 0.0160 0.0960 h:h/g 0.04 Norm. Est. 0.8306 0.6377 0.6667 0.4444 0.6154 h:g/g 0.20 Variances 0.0006 0.0009 0.0008 0.0002 0.0009 g:h/h 0.04 St. Error 0.0251 0.0306 0.0277 0.0165 0.0304 g:h/g 0.02 Pr (Sample) 3.2 e-8 0.0161 0.0155 0.6232 0.0068 g:g/g 0.54 Pr (Exact) NA 2.9 e-5 0.0088 0.5561 0.0027 Sample Size 90 19 60 65 595 53 Sample Size 50 6 23 23 203 19 238 Southeastern Naturalist Vol. 8, No. 2 Figure 5. STRUCTURE analysis of dilocus genotypes from all collection and reference sites. Individuals are ranked based on collection site with the highest rankings representing samples from SM and the lowest rankings representing samples from from CK. Samples from Flagler Beach (FB) are noted. Diamonds represent individual mean values of q and 95% posterior probabilities are shown. haplotype of one species and are homozygous for the other species at LB UTR (e.g.: h:g/g and g:h/h). Individuals that are heterozygous at LB UTR could result from either F1 or other hybrid crosses, while h:gg and g:hh genotypes are suggestive of backcrosses. All four putative hybrid genotypes were present at FB, even though most of these were at lower frequencies than the parental dilocus genotypes g:g/g and h:h/h (Table 2). It is important to note that each of the putative hybrid genotypes could also be produced by populations outside of the defined contact zone since neither marker is fixed in either species. Therefore, to determine if the putative hybrid genotypes were representative of actual hybridization, we sequenced six individuals with putative hybrid genotypes at either cyt-b (Accession number: EU482159), LB UTR (Accession number: EU482183) or both (Accession numbers: EU482184, EU482185, EU482181 and EU482182). Only individuals with h:g/g or g:h/h genotypes were used in sequencing 2009 I. Gonzalez, M. Levin, S. Jermanus, B. Watson, and M.R. Gilg 239 to minimize the identification issues inherent in sequencing heterozygotes. These six individuals refl ect half of the samples with mismatched cytonuclear genotypes at FB. The results of the maximum parsimony analysis are shown in Fig. 3 and Fig. 4. Two samples that were sequenced at both loci, FB 17 and FB 46, were definitely the result of hybridization since the two loci fell into alternative clades. FB 17 was shown to have a cyt-b haplotype of Mummichog and an LB UTR genotype of Gulf Killifish. Sample FB 46 was just the opposite. The other two samples sequenced at both loci, FB 19 and FB 22, were initially genotyped as h:g/g, but the sequence analysis grouped both samples with Mummichog at both loci. Sample FB 16 was initially genotyped as h:g/g, but its LB UTR sequence was actually within the Mummichog clade, suggesting it was probably not a hybrid. Sample FB 52 was genotyped as g:h/h, and the cyt-b sequence was confirmed as being Gulf Killifish, suggesting the initial genotype was probably correct and the sample was of mixed ancestry since h alleles at LB UTR are more species specific than the g alleles. The observed frequencies of the putative hybrid genotypes at FB suggest that hybridization between the two species may be directional. Most of the putative hybrid genotypes have an h cyt-b haplotype, indicative of a higher proportion of hybrid matings involving Mummichog females with Gulf Killifish males. To test for significant directionality of the matings, we estimated the cytonuclear disequilibrium at FB. The results (Table 2) show significant disequilibrium at both species-specific genotypes (AAM and aaM), but none at the hybrid genotypes (AaM). These data suggest that the two species are predominantly mating assortatively and that the small level of hybridization occurring is not directional. It should be noted that the sample sizes from FB are not sufficient to detect the observed level of disequilibrium at mixed cytonuclear genotypes with either 50% or 90% power (Table 2). Discussion A zone of sympatry was detected in northeastern Florida for mummichogs and Gulf Killifish between Marineland and the Tomoka Basin. The frequencies of both cyt-b haplotypes and LB UTR alleles displayed a steep cline, with Flagler Beach having intermediate frequencies of alleles from each species. The observed patterns established three genetically distinct regions. The first was a northern region from the St. Mary’s River in Georgia to Pellicer Creek in Florida, which consisted primarily of Mummichog individuals with low but relatively consistent levels of Gulf Killifish alleles at both loci. At least some of the g alleles at the LB UTR locus in these northern sites are due to an intraspecific polymorphism in Mummichog, but g cyt-b haplotypes appear to be mostly due to introgression from Gulf Killifish. The second region was the area of overlap, and possibly hybridization, centered on Flagler Beach and potentially including the Tomoka Basin. Finally, a southern region located south of Tomoka Basin and along the Gulf coast of Florida was dominated by Gulf Killifish. 240 Southeastern Naturalist Vol. 8, No. 2 A study by Duggins et al. (1989) provides similar results, with Mummichog samples being collected primarily from northern sites and Gulf Killifish being found on the southern Atlantic and Gulf coasts of Florida. Collections by Duggins et al. (1989) near ML contained both Gulf Killifish and Mummichog specimens, which is on the northern edge of the contact zone described here. Contradictory to our findings, Duggins et al. (1989) suggest that collections made near FB were Mummichogs. Our data, on the other hand, show that FB is in the middle of the contact zone, with higher frequencies of Gulf Killifish and hybrid genotypes than Mummichogs. Samples collected near FB by Duggins et al. (1989), however, tended to contain a mixture of genotypes typical of both species. Therefore, it is likely that this site represents a mixed population in both studies. It is intriguing that Duggins et al. (1989) found higher proportions of Mummichog-like genotypes than Gulf Killifish-like genotypes, while the opposite was true in the present study. This may be due to microhabitat differences in the sampling locations of the two studies or to temporal changes in the population genetic structure at that location. Another possibility is that some of the individuals used in the study by Duggins et al. (1989) were incorrectly identified to species using morphology, leading to a misrepresentation of the genetic data. The present study consistently encountered Gulf Killifish-specific alleles and haplotypes in the primarily Mummichog populations in the northern region, including the Saint Mary’s reference site. The presence of these alleles could either be due to: 1) polymorphisms at both cyt-b and LB UTR within the Mummichog population, 2) the presence of small numbers of Gulf Killifish, or 3) introgression of Gulf Killifish-specific alleles/haplotypes into Mummichog populations via hybridization. Sequence analysis of individuals with Gulf Killifish cyt-b haplotypes from the predominantly Mummichog sites verified that most of our initial analyses were correct and F. grandis haplotypes were present in these populations. On the other hand, sequence analysis of g/g individuals at the LB UTR locus suggested that most of these individuals are actually homozygous for Mummichog alleles that lack the PstI restriction site found in most previously published Mummichog sequences. The existence of this polymorphism combined with the fact that no individuals collected from the predominantly Mummichog sites had g:g/g genotypes, makes it unlikely that Gulf Killifish are present in any significant numbers north of FB. Therefore, the presence of Gulf Killifish-like alleles or haplotypes is due to both introgression and, especially with the LB UTR locus, a polymorphism within Mummichog populations. The observation that Gulf Killifish are apparently not present in populations north of ML is surprising given the fact that LB UTR genotype frequencies are rarely in Hardy-Weinberg equilibrium at the northern sites. The lack of heterozygotes at most of these sites would be expected if the samples contained a mixture of two species with primarily intraspecific matings. If the variation observed at the LB UTR locus is mostly due to an intraspecific polymorphism in Mummichogs, the alleles should meet Hardy- Weinberg expectations. This conclusion is especially true since the marker we are using is an untranslated region and is expected to be neutral. On the 2009 I. Gonzalez, M. Levin, S. Jermanus, B. Watson, and M.R. Gilg 241 other hand, Schulte et al. (1997) provided evidence for functional differentiation associated with structural differences in LB UTR alleles between populations of Mummichogs in Florida and Maine. Therefore, it is possible that the two alleles found within Mummichogs in the present study represent functionally different regulatory regions of lactate dehydrogenase b. Furthermore, lactate dehydrogenase b has been shown to be under selection in populations of Mummichogs which exhibit a latitudinally associated allele frequency cline between Pennsylvania and North Carolina (Powers et al. 1991). Therefore, it is also possible that the two LB UTR RFLP alleles observed in Mummichogs in the present study are linked to distinct alleles of lactate dehydrogenase b or other loci that are experiencing assortative mating or disruptive selection resulting in a heterozygote deficiency. The polymorphism within Mummichog populations at the LB UTR locus also likely plays a role in the perceived directionality of hybridization observed at FB. While there was no significant cytonuclear disequilibrium among the putative hybrid genotypes, most individuals with mixed genotypes had h:g/g dilocus genotypes. The lack of complete species-specificity of our LB UTR RFLP could result in true Mummichogs being mis-genotyped as h:g/g hybrids. Indeed, sequencing verified that this was true for at least some of the h:g/g individuals. Still, it should be mentioned that sequence analysis also verified the existence of h:g/g hybrids. Additionally, the observed frequency of h:g/g genotypes at FB is approximately five-fold higher than could be produced by a randomly mating population of non-hybridizing Mummichogs polymorphic at LB UTR at the frequencies observed in the northern populations. This conclusion can be illustrated with the following example: Assume a mixed population of Gulf Killifish and Mummichogs with relative abundances similar to the ratios of allele frequencies observed at FB (≈75% Gulf Killifish and ≈25% Mummichog ). If the frequencies of h and g alleles within the Mummichog population are the same as those observed at AB, where g alleles are at the highest frequency among the northern sites (Table 1), under a model of no hybridization and random association of cyt-b haplotypes and LB UTR alleles, the expected frequency of h:g/g genotypes (AB: [(h = 0.91)*(g/g = 0.09)](0.25) + CK/PSJ: [(h = 0.02)*(g/g = 0.99)](0.75)) would only be approximately 0.04. The observed frequencies at FB, on the other hand, are 0.2. This discrepancy suggests that many of the h:g/g genotypes at FB are truly the result of hybridization. This same model predicts lower expected frequencies of all putative hybrid genotypes than those observed. Still, the extensive cytonuclear disequilibria of pure species cytonuclear genotypes are suggestive of primarily intraspecific matings (Asmussen et al. 1987). It must be noted that an in-depth analysis of hybridization in the present study is stymied by the fact that the markers used, especially LB UTR, are not completely species-specific and the SM population in Georgia used as a reference population for Mummichogs contains mitochondrial haplotypes of Gulf Killifish. The lack of completely diagnostic markers, however, is typical in many studies of hybridization, and the introgression of heterospecific alleles into areas of allopatry is also relatively common (Arnold 1997, 242 Southeastern Naturalist Vol. 8, No. 2 Harrison 1993). Recent work by Knowles and Carstens (2007) describes the use of probabilistic models to delimit species when there is incomplete lineage sorting between them, as appears to be the case with the LB UTR RFLP marker utilized in the present study. In these cases, it is important to utilize multiple loci to help correct for mistakes in assessment of genetic identity. The fact that both markers used here are highly divergent between the species and both show similar geographical patterns suggest that they are reliable markers at the species level. Obviously, however, mistakes in the assessment of genetic identity will be made in some cases resulting in some incorrect reporting of hybrids. Additional nuclear markers that show greater species-specificity will be necessary for further studies of ecological and reproductive compatibility on these two species. The area of overlap found in the Mummichog and Gulf Killifish complex is consistent with other studies that have detected a habitat transition zone around FB (Avise 1992, Duggins et al. 1995). Avise (1992) describes that the Florida peninsula has a transitional zone that separates temperate and tropical adapted forms, with the southern ranges of many temperate species terminating in the Cape Canaveral region. Around this region an ecotone exists where the Juncus-Spartina marsh characteristic of the northern Atlantic Coast are replaced by mangrove marsh characteristic of southern peninsular Florida (Duggins et al. 1995). Allele and haplotype frequencies found in the present study were consistent with this pattern, where Mummichogs diminished in frequency as they entered the transition zone and were completely replaced by Gulf Killifish at IR, which lies near Cape Canaveral. The similar clinal location and width at the cyt-b and LB UTR loci also suggests that the hybrid zone between Mummichogs and Gulf Killifish is due to secondary contact. This conclusion would also be consistent with Avise’s (2000) hypothesis of historical vicariance between Gulf and Atlantic populations of many species in the southeastern United States. Investigation of the clinal patterns of additional species-specific markers and studies of habitat use and the factors limiting the ranges of these species could help determine whether FB truly represents an area of secondary contact and whether the current ranges are defined by the Juncus-Spartina ecotone. 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