Habitat segregation and migration in tropical anguillid eels, Anguilla bengalensis bengalensis and A. bicolor bicolor.

Anguillid eels of the genus Anguilla, which have a unique catadromous life history, are widely distributed across many parts of the world. However, little research has been conducted on the behavioural mechanisms of habitat segregation between sympatric species in tropical anguillid eels. To understand the ecological and behavioural mechanisms involved in the life history and migration of tropical anguillid eels, strontium (Sr):calcium (Ca) ratios were examined in otoliths of A. bengalensis bengalensis (41 specimens) and A. bicolor bicolor (130 specimens) collected from ten rivers in northwestern Peninsular Malaysia. The otolith Sr:Ca ratios revealed different habitat use between the two species. The broad range of otolith Sr:Ca ratios and habitat shift found in A. bicolor bicolor suggested that its habitat utilization was opportunistic in environments of varying salinity. A. bicolor bicolor prefers to live in the midstream to downstream areas with tidal influences. A. bengalensis bengalensis, however, was found to only reside in freshwater environments throughout their continental growth. A. bengalensis bengalensis tends to live in upstream area with no tidal influence. Their habitat use, migratory history, and habitat distribution indicate that habitat segregation occurs between the two species, leading to the different habitat preferences in tropical river systems.

Prevalence of critically endangered European eel (Anguilla anguilla) in Hong Kong supermarkets.

European eel (Anguilla anguilla) is a critically endangered species requiring CITES permits for international trade. Despite the fact that no imports to Hong Kong were declared within the last 2 years, our study found that this species is still commonly sold in major supermarket chains across Hong Kong. In a COI barcoding survey of 49 retail vendors encompassing 13 brands, 9 of 13 carried A. anguilla, and 45% of all eel products available at retail outlets (n = 49) were unambiguously identified as A. anguilla. Considering the visual similarity of eel species and disproportionate amount of undeclared A. anguilla available for consumption, this finding raises urgent concerns regarding the enforcement of international CITES trade regulations. Furthermore, the prevalence of A. anguilla in supermarkets highlights how illicit wildlife products are not solely limited to specialized affluent buyers; some species have entered mainstream distribution networks for the average consumer.

Stable species boundaries despite ten million years of hybridization in tropical eels.

Genomic evidence is increasingly underpinning that hybridization between taxa is commonplace, challenging our views on the mechanisms that maintain their boundaries. Here, we focus on seven catadromous eel species (genus Anguilla) and use genome-wide sequence data from more than 450 individuals sampled across the tropical Indo-Pacific, morphological information, and three newly assembled draft genomes to compare contemporary patterns of hybridization with signatures of past introgression across a time-calibrated phylogeny. We show that the seven species have remained distinct for up to 10 million years and find that the current frequencies of hybridization across species pairs contrast with genomic signatures of past introgression. Based on near-complete asymmetry in the directionality of hybridization and decreasing frequencies of later-generation hybrids, we suggest cytonuclear incompatibilities, hybrid breakdown, and purifying selection as mechanisms that can support species cohesion even when hybridization has been pervasive throughout the evolutionary history of clades.

Surgical implantation of radio tags in three eel species (Anguilla spp.) in South Africa.

Studies have reported poor survival of surgically tagged freshwater fishes in warm African waters. This study aimed to assess the applicability of using radio telemetry (and surgical implantation of tags) for Anguilla spp. Nineteen yellow eels (Anguilla bengalensis, A. marmorata and A. mossambica) were surgically implanted with radio tags between October 2018 and January 2019 in the Thukela River, South Africa. Most eels were alive 6 months after tagging, and recaptured eels displayed advanced or complete healing at the incision site. Therefore, this method appears suitable for African freshwater eels.

Genetic variability and demographic history of Anguilla mossambica (Peters, 1852) from continental Africa and Madagascar.

In this study, population genetic and demographic parameters were inferred using sequence data from 151 individuals of Anguilla mossambica originating from continental south and south-east Africa and Madagascar. The analyses were based on a 532 bp segment of the mitochondrial cytochrome b gene. The absence of genetic structuring was observed thereby supporting the hypothesis of panmixia for the endemic A. mossambica. The overall nucleotide diversity π = 0.002 and the haplotype diversity reached h = 0.691. Significant negative values from several tests of neutrality and mismatch analysis pointed to fluctuating historical population sizes. Bayesian averaging resulted in higher support for population growth models vs. a constant population-size model. Population decline and subsequent growth most likely predated the last glacial and were probably related to extended periods of extreme drought followed by wetter and more stable hydroclimate between 150 and 75,000 years before present (kBP). According to this scenario the female effective population size has increased since 110 kBP by c. two orders of magnitude to a recent level of about 650,000 (219,317-2,292,000).

HaCeD-Seq: a Novel Method for Reliable and Easy Estimation About the Fish Population Using Haplotype Count from eDNA.

It is common to count the numbers of specified fish in the field after speciation of captured fish according to their morphology and to subject these counts to appropriate statistical analyses. In recent years, a non-invasive method to estimate the abundance of a particular fish species using environmental DNA (eDNA) has been developed. However, it is still difficult to determine accurate numbers of fish species using such method. We predict that the estimation of individuals of certain fish species in the field is more accurate and easier by using haplotypes of DNA in the fast evolutionary region. Therefore, we focused on the regulatory region (D-loop) in mitochondrial DNA, which is known to have a high genetic variation at the intraspecific level of the targeting eel. We investigated haplotype diversity in eel at first and then determined the number of D-loop haplotypes contained in their exfoliated cells in breeding water. Finally, we developed a novel analytical method, HaCeD-Seq, to estimate the number of individuals based on the abovementioned data.

New PCR primers for metabarcoding environmental DNA from freshwater eels, genus Anguilla.

Freshwater eels of the genus Anguilla comprise 16 species that include three subspecies and are characterized by their unique catadromous life cycles. Their life histories and nocturnal life styles make it difficult to observe them in freshwater and marine habitats. To investigate their distribution and ecology in aquatic environments, we developed new PCR primers for metabarcoding environmental DNA (eDNA) from Anguilla. The new primers (MiEel) were designed for two conserved regions of the mitochondrial ATP6 gene, which amplify a variable region with sufficient interspecific variations ranging from five to 22 nucleotide differences (one to three nucleotide differences between three subspecies pairs). We confirmed the versatility of the MiEel primers for all freshwater eels using tissue DNA extracts when analyzed separately. The metabarcoding combined with the MiEel primers using mock communities enabled simultaneous detection of Anguilla at the species level. Analysis of eDNA samples from aquarium tanks, a controlled pond and natural rivers demonstrated that the MiEel metabarcoding could successfully detect the correct Anguilla species from water samples. These results suggested that eDNA metabarcoding with MiEel primers would be useful for non-invasively monitoring the presence of the endangered anguillid eels in aquatic environments where sampling surveys are difficult.

Gelatinous plankton is important in the diet of European eel (Anguilla anguilla) larvae in the Sargasso Sea.

Limited insight into eel larvae feeding and diet prevents a holistic overview of the life-cycle of catadromous eels and an understanding of the ecological position of their early stages in marine waters. The present study evaluated the diet of larval European eel, Anguilla anguilla - a critically endangered species. Next-generation 18S rRNA gene sequencing data of Sargasso Sea eel larvae gut contents and marine snow aggregates was compared with a reference plankton database to assess the trophic relations of eel larvae. Gut contents of A. anguilla larvae were not well explained by the eukaryotic composition of marine snow aggregates; gut contents being dominated by gene sequences of Hydrozoa taxa (phylum Cnidaria), while snow aggregates were dominated by Crustacea taxa. Pronounced differences between gut contents and marine snow aggregates were also seen in the prokaryotic 16S rRNA gene composition. The findings, in concert with significant abundances of Hydrozoa in the study area, suggest that Hydrozoa plankton are important in the diet of A. anguilla larvae, and that consideration of these organisms would further our understanding of A. anguilla feeding strategies in the oligotrophic Sargasso Sea, which may be important for potential future rearing of A. anguilla larvae in captivity.

Anguillicola crassus infection affects mRNA expression levels in gas gland tissue of European yellow and silver eel.

Using Illumina sequencing, we investigated transcriptional changes caused by the nematode Anguillicola crassus within yellow and silver eels by comparing swimbladder samples of uninfected yellow with infected yellow eels, and uninfected silver with infected silver eels, respectively. In yellow eel gas gland, the infection caused a modification of steady state mRNA levels of 1675 genes, most of them being upregulated. Functional annotation analysis based on GO terms was used to categorize identified genes with regard to swimbladder metabolism or response to the infection. In yellow eels, the most prominent category was 'immune response', including various inflammatory components, complement proteins, and immunoglobulins. The elevated expression of several glucose and monocarboxylate transporters indicated an attempt to maintain the level of glucose metabolism, even in due to the infection thickened swimbladder tissue. In silver eel swimbladder tissue, on the contrary, the mRNA levels of only 291 genes were affected. Genes in the categories 'glucose metabolism' and 'ROS metabolism' barely responded to the infection and even the reaction of the immune system was much less pronounced compared to infected yellow eels. However, in the category 'extracellular matrix', the mRNA levels of several mucin genes were strongly elevated, suggesting increased mucus production as a defense reaction against the parasite. The present study revealed a strong reaction to an Anguillicola crassus infection on mRNA expression levels in swimbladder tissue of yellow eels, whereas in silver eels the changes ware almost negligible. A possible explanation for this difference is that the silvering process requires so much energy that there is not much scope to cope with the additional challenge of a nematode infection. Another possible explanation could be that gas-secreting activity of the silver eel swimbladder was largely reduced, which could coincide with a reduced responsiveness to other challenges, like a nematode infection.

Diversity, distribution and different habitat use among the tropical freshwater eels of genus Anguilla.

Along with the mysteries of their ecology, freshwater eels have fascinated biologists for centuries. However, information concerning species diversity, geographic distribution, and life histories of the tropical anguillid eels in the Indo-Pacific region are highly limited. Comprehensive research on the species composition, distribution and habitat use among tropical anguillid eels in the Peninsular Malaysia were conducted for four years. A total of 463 specimens were collected in the northwestern peninsular area. The dominant species was A. bicolor bicolor constituting of 88.1% of the total eels, the second one was A. bengalensis bengalensis at 11.7%, while A. marmorata was the least abundant at 0.2%. A. bicolor bicolor was widely distributed from upstream to downstream areas of the rivers. In comparison, A. bengalensis bengalensis preferred to reside from the upstream to midstream areas with no tidal zones, cooler water temperatures and higher elevation areas. The habitat preference might be different between sites due to inter-species interactions and intra-specific plasticity to local environmental conditions. These results suggest that habitat use in the tropical anguillid eels might be more influenced by ambient environmental factors, such as salinity, temperature, elevation, river size and carrying capacity, than ecological competition, such as interspecific competition.

Assessing pre- and post-zygotic barriers between North Atlantic eels (Anguilla anguilla and A. rostrata).

Elucidating barriers to gene flow is important for understanding the dynamics of speciation. Here we investigate pre- and post-zygotic mechanisms acting between the two hybridizing species of Atlantic eels: Anguilla anguilla and A. rostrata. Temporally varying hybridization was examined by analyzing 85 species-diagnostic single-nucleotide polymorphisms (SNPs; FST ⩾0.95) in eel larvae sampled in the spawning region in the Sargasso Sea in 2007 (N=92) and 2014 (N=460). We further investigated whether genotypes at these SNPs were nonrandomly distributed in post-F1 hybrids, indicating selection. Finally, we sequenced the mitochondrial ATP6 and nuclear ATP5c1 genes in 19 hybrids, identified using SNP and restriction site associated DNA (RAD) sequencing data, to test a previously proposed hypothesis of cytonuclear incompatibility leading to adenosine triphosphate (ATP) synthase dysfunction and selection against hybrids. No F1 hybrids but only later backcrosses were observed in the Sargasso Sea in 2007 and 2014. This suggests that interbreeding between the two species only occurs in some years, possibly controlled by environmental conditions at the spawning grounds, or that interbreeding has diminished through time as a result of a declining number of spawners. Moreover, potential selection was found at the nuclear and the cytonuclear levels. Nonetheless, one glass eel individual showed a mismatch, involving an American ATP6 haplotype and European ATP5c1 alleles. This contradicted the presence of cytonuclear incompatibility but may be explained by that (1) cytonuclear incompatibility is incomplete, (2) selection acts at a later life stage or (3) other genes are important for protein function. In total, the study demonstrates the utility of genomic data when examining pre- and post-zyotic barriers in natural hybrids.

RAD sequencing reveals within-generation polygenic selection in response to anthropogenic organic and metal contamination in North Atlantic Eels.

Measuring the effects of selection on the genome imposed by human-altered environment is currently a major goal in ecological genomics. Given the polygenic basis of most phenotypic traits, quantitative genetic theory predicts that selection is expected to cause subtle allelic changes among covarying loci rather than pronounced changes at few loci of large effects. The goal of this study was to test for the occurrence of polygenic selection in both North Atlantic eels (European Eel, Anguilla anguilla and American Eel, A. rostrata), using a method that searches for covariation among loci that would discriminate eels from 'control' vs. 'polluted' environments and be associated with specific contaminants acting as putative selective agents. RAD-seq libraries resulted in 23 659 and 14 755 filtered loci for the European and American Eels, respectively. A total of 142 and 141 covarying markers discriminating European and American Eels from 'control' vs. 'polluted' sampling localities were obtained using the Random Forest algorithm. Distance-based redundancy analyses (db-RDAs) were used to assess the relationships between these covarying markers and concentration of 34 contaminants measured for each individual eel. PCB153, 4'4'DDE and selenium were associated with covarying markers for both species, thus pointing to these contaminants as major selective agents in contaminated sites. Gene enrichment analyses suggested that sterol regulation plays an important role in the differential survival of eels in 'polluted' environment. This study illustrates the power of combining methods for detecting signals of polygenic selection and for associating variation of markers with putative selective agents in studies aiming at documenting the dynamics of selection at the genomic level and particularly so in human-altered environments.

Duplicated leptin receptors in two species of eel bring new insights into the evolution of the leptin system in vertebrates.

Since its discovery in mammals as a key-hormone in reproduction and metabolism, leptin has been identified in an increasing number of tetrapods and teleosts. Tetrapods possess only one leptin gene, while most teleosts possess two leptin genes, as a result of the teleost third whole genome duplication event (3R). Leptin acts through a specific receptor (LEPR). In the European and Japanese eels, we identified two leptin genes, and for the first time in vertebrates, two LEPR genes. Synteny analyses indicated that eel LEPRa and LEPRb result from teleost 3R. LEPRb seems to have been lost in the teleost lineage shortly after the elopomorph divergence. Quantitative PCRs revealed a wide distribution of leptins and LEPRs in the European eel, including tissues involved in metabolism and reproduction. Noticeably, leptin1 was expressed in fat tissue, while leptin2 in the liver, reflecting subfunctionalization. Four-month fasting had no impact on the expression of leptins and LEPRs in control European eels. This might be related to the remarkable adaptation of silver eel metabolism to long-term fasting throughout the reproductive oceanic migration. In contrast, sexual maturation induced differential increases in the expression of leptins and LEPRs in the BPG-liver axis. Leptin2 was strikingly upregulated in the liver, the central organ of the reproductive metabolic challenge in teleosts. LEPRs were differentially regulated during sexual maturation, which may have contributed to the conservation of the duplicated LEPRs in this species. This suggests an ancient and positive role of the leptin system in the vertebrate reproductive function. This study brings new insights on the evolutionary history of the leptin system in vertebrates. Among extant vertebrates, the eel represents a unique case of duplicated leptins and leptin receptors as a result of 3R.

Genomic footprints of speciation in Atlantic eels (Anguilla anguilla and A. rostrata).

The importance of speciation-with-geneflow scenarios is increasingly appreciated. However, the specific processes and the resulting genomic footprints of selection are subject to much discussion. We studied the genomics of speciation between the two panmictic, sympatrically spawning sister species; European (Anguilla anguilla) and American eel (A. rostrata). Divergence is assumed to have initiated more than 3 Ma, and although low gene flow still occurs, strong postzygotic barriers are present. Restriction-site-associated DNA (RAD) sequencing identified 328 300 SNPs for subsequent analysis. However, despite the presence of 3757 strongly differentiated SNPs (FST > 0.8), sliding window analyses of FST showed no larger genomic regions (i.e. hundreds of thousands to millions of bases) of elevated differentiation. Overall FST was 0.041, and linkage disequilibrium was virtually absent for SNPs separated by more than 1000 bp. We suggest this to reflect a case of genomic hitchhiking, where multiple regions are under directional selection between the species. However, low but biologically significant gene flow and high effective population sizes leading to very low genetic drift preclude accumulation of strong background differentiation. Genes containing candidate SNPs for positive selection showed significant enrichment for gene ontology (GO) terms relating to developmental processes and phosphorylation, which seems consistent with assumptions that differences in larval phase duration and migratory distances underlie speciation. Most SNPs under putative selection were found outside coding regions, lending support to emerging views that noncoding regions may be more functionally important than previously assumed. In total, the results demonstrate the necessity of interpreting genomic footprints of selection in the context of demographic parameters and life-history features of the studied species.

Speciation and demographic history of Atlantic eels (Anguilla anguilla and A. rostrata) revealed by mitogenome sequencing.

Processes leading to speciation in oceanic environments without obvious physical barriers remain poorly known. European and American eel (Anguilla anguilla and A. rostrata) spawn in partial sympatry in the Sargasso Sea. Larvae are advected by the Gulf Stream and other currents towards the European/North African and North American coasts, respectively. We analyzed 104 mitogenomes from the two species along with mitogenomes of other Anguilla and outgroup species. We estimated divergence time between the two species to identify major events involved in speciation. We also considered two previously stated hypotheses: one where the ancestral species was present in only one continent but was advected across the Atlantic by ocean current changes and another where population declines during Pleistocene glaciations led to increasing vicariance, facilitating speciation. Divergence time was estimated to ∼3.38 Mya, coinciding with the closure of the Panama Gateway that led to reinforcement of the Gulf Stream. This could have advected larvae towards European/North African coasts, in which case American eel would be expected to be the ancestral species. This scenario could, however, not be unequivocally confirmed by analyses of dN/dS, nucleotide diversity and effective population size estimates. Extended bayesian skyline plots showed fluctuations of effective population sizes and declines during glaciations, and thus also lending support to the importance of vicariance during speciation. There was evidence for positive selection at the ATP6 and possibly ND5 genes, indicating a role in speciation. The findings suggest an important role of ocean current changes in speciation of marine organisms.

Introgressive hybridization and latitudinal admixture clines in North Atlantic eels.

BACKGROUND: Hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies. Eels, both from North America and Europe migrate through the Atlantic to mate in a vast, overlapping area in the Sargasso Sea. Due to the lack of direct observation, it is unknown how these species remain reproductively isolated. The detection of inter-species hybrids in Iceland suggests on-going gene flow, but few studies to date have addressed the influence of introgression on genetic differentiation in North Atlantic eels. RESULTS: Here, we show that while mitochondrial lineages remain completely distinct on both sides of the Atlantic, limited hybridization is detectable with nuclear DNA markers. The nuclear hybridization signal peaks in the northern areas and decreases towards the southern range limits on both continents according to Bayesian assignment analyses. By simulating increasing proportions of both F1 hybrids and admixed individuals from the southern to the northern-most locations, we were able to generate highly significant isolation-by-distance patterns in both cases, reminiscent of previously published data for the European eel. Finally, fitting an isolation-with-migration model to our data supports the hypothesis of recent asymmetric introgression and refutes the alternative hypothesis of ancient polymorphism. CONCLUSIONS: Fluctuating degrees of introgressive hybridization between Atlantic eel species are sufficient to explain temporally varying correlations of geographic and genetic distances reported for populations of the European eel.

Conclusive evidence for panmixia in the American eel.

Eels are unique species in the biological world. The two North Atlantic eel species, the American eel (Anguilla rostrata) and the European eel (A. anguilla), occupy a broad range of habitats from the Caribbean to Greenland in the western Atlantic and from Morocco to Iceland in the eastern Atlantic, respectively. North Atlantic eels have a catadromous life cycle, spawning only in the Sargasso Sea and spending the majority of their lives in continental (fresh, brackish and coastal) waters. Despite such a wide distribution range, North Atlantic eels have been regarded as a textbook example of panmictic species. In contrast with the large amount of population genetic studies testing the panmixia hypothesis in the European eel, a relatively modest effort has been given to study the population structure of the American eel. In this issue of Molecular Ecology, C^ote et al. (2013) present the most comprehensive American eel data set to date, which includes samples of different life stages obtained throughout all its distribution range in North America. Results show a total lack of genetic differentiation among samples and provide decisive evidence for panmixia in the American eel.

Population genetics of the American eel (Anguilla rostrata): FST = 0 and North Atlantic Oscillation effects on demographic fluctuations of a panmictic species.

We performed population genetic analyses on the American eel (Anguilla rostrata) with three main objectives. First, we conducted the most comprehensive analysis of neutral genetic population structure to date to revisit the null hypothesis of panmixia in this species. Second, we used this data to provide the first estimates of contemporary effective population size (Ne ) and to document temporal variation in effective number of breeders (Nb ) in American eel. Third, we tested for statistical associations between temporal variation in the North Atlantic Oscillation (NAO), the effective number of breeders and two indices of recruit abundance. A total of 2142 eels from 32 sampling locations were genotyped with 18 microsatellite loci. All measures of differentiation were essentially zero, and no evidence for significant spatial or temporal genetic differentiation was found. The panmixia hypothesis should thus be accepted for this species. Nb estimates varied by a factor of 23 among 12 cohorts, from 473 to 10,999. The effective population size Ne was estimated at 10,532 (95% CI, 9312-11,752). This study also showed that genetically based demographic indices, namely Nb and allelic richness (Ar), can be used as surrogates for the abundance of breeders and recruits, which were both shown to be positively influenced by variation during high (positive) NAO phases. Thus, long-term genetic monitoring of American glass eels at several sites along the North American Atlantic coast would represent a powerful and efficient complement to census monitoring to track demographic fluctuations and better understand their causes.

Evolution of microsatellite Loci of tropical and temperate anguilla eels.

Anguilla eels are divided into temperate and tropical eels, based on their major distributions. The present study collected two temperate eels, Anguilla japonica and Anguilla anguilla, and two tropical eels, Anguilla marmorata and Anguilla bicolor pacifica, to examine two questions: do temperate and tropical Anguilla eels have different genetic polymorphic patterns?; and do temperate Anguilla japonica and Anguilla anguilla have a closer relationship to each other than to tropical eels? In total, 274 sequences were cloned and sequenced from six conserved microsatellite loci to examine polymorphic patterns of these four catadromous eels. Different mutational events, including substitutions, and repeat-unit deletions and insertions, appeared in major regions, while different point mutations were observed in flanking regions. The results implied that parallel patterns of microsatellite sequences occurred within both tropical and temperate freshwater eels. Consensus flanking sequences of six homologous loci from each of the four species were constructed. Genetic distances ranged from 0.044 (Anguilla bicolor pacifica vs. Anguilla marmorata) to 0.061 (Anguilla marmorata vs. Anguilla anguilla). The tree topology suggests the hypothesis of Anguilla japonica and Anguilla anguilla being a sister group must be rejected.

Behavioral migration diversity of the Yangtze River Japanese Eel, Anguilla japonica, based on otolith Sr/Ca ratios.

The Yangtze River estuary is the main production area of Anguilla japonica in China, as well as the only existing fishery area for adult eels. Japanese eels are distributed in the main rivers and many tributaries from the Yangtze River estuary to the upper Jinsha River, which extend to nearly 3 000 km. However, their migration behaviors remain relatively unknown. We analyzed the biological characteristics of 153 specimens of silver eels collected from the Jingjiang section of Yangtze River (31(o)30'N, 120(o)42'E) between September and November, 2008, and tested the sagittal Sr/Ca ratios of 27 specimens. Among the 153 specimens examined, 85 were female and 68 were male, which translated to a female-male ratio of 1 : 0.8. The ages of the female specimens ranged from 3 to 7 a (average 5.52) with an average total length (TL) of (669 ± 80) mm, average body weight (BW) of (555 ± 229)g, average condition factor of 1.77 ± 0.22, and average gonad somatic index (GSI) of 1.32 ± 0.31. The ages of the males ranged from 3 to 5 a (average:4.38) with an average TL of (518 ± 51) mm, average BW of (234 ± 76) g, average condition factor of 1.62 ± 0.18, and average GSI of 0.21 ± 0.11. All biological parameters of females were significantly larger than those of the male specimens (P<0.05). According to the average Sr/Ca ratio (7.99 ± 1.05) × 10(-3) of the elver mark of sagitta, 17 individuals (62.96%) were river eels and 10 individuals (37.04%) were estuarine eels. Of 16 females, 13 individuals (81.25%) were river eels and 3 were estuarine eels, while of 11 males, 36.36% were river eels and 63.64% were estuarine eels. The analysis on Sr/Ca ratios for every growth layer group (GLG) indicated there were no significant differences between second-age males and females. However, significant differences were observed between the third-age, fourth-age, and migration-age male and female specimens. This was likely related to the fact that second-age eels of both sexes stay in the same inhabitation waters; however, as they grow older, they move to different areas.