Biol. Bull. 204: 104-108. (February 2003)
© 2003 Marine Biological Laboratory
Short-Distance Spawning Migration of Tropical Freshwater Eels
Jun Aoyama1,*,
Sam Wouthuyzen2,
Michael J. Miller1,
Tadashi Inagaki1 and
Katsumi Tsukamoto1
1 Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan
2 Research Center for Oceanography, Indonesian Institute of Sciences, Anchor, Jakarta, Indonesia
* To whom correspondence should be addressed. E-mail: jaoyama{at}ori.u-tokyo.ac.jp
The freshwater eels have fascinated biologists because of their spectacular long-distance migrations between their freshwater habitats and their spawning areas far out in the ocean (1,2,3). Recent progress on the molecular phylogeny of freshwater eels suggests that they originated in the tropics (4), and information on the reproductive ecology and recruitment of tropical species will provide new insight into the evolution of the spawning migration of the freshwater eels (5,6). However, the larvae (leptocephali) of the many sympatric tropical species are morphologically similar (7), so they are impossible to identify, and their spawning areas are thus virtually unknown. Recently, however, we have collected small leptocephali from around Sulawesi, Indonesia, and have used species-specific genetic markers to identify them as larvae of Anguilla celebesensis and A. borneensis, which provides the first definitive information about the general spawning areas of these tropical eels. Moreover, the discovery of a spawning area of A. celebesensis in Tomini Bay and the presence of small specimens of two species in the Celebes Sea indicate that, in contrast to the long migrations made by temperate eels, tropical eels make much shorter migrations to spawn in areas near their freshwater habitats. This difference in migratory behavior may reflect an evolutionary cline among freshwater eels that extends from tropical to temperate regions.
Early in the last century, the Danish oceanographer Johannes Schmidt succeeded in collecting small anguillid leptocephali in the Sargasso Sea thousands of kilometers away from their growth habitats in Europe and North America, which indicated that the two species of Atlantic freshwater eels make remarkably long spawning migrations (1). After this finding, he and his colleagues shifted their efforts to search for the spawning areas of freshwater eels in the Indo-Pacific region where most of the species in the genus are found. They successfully collected more than 1400 leptocephali in the Indo-Pacific region (7) during the Carlsberg Foundation’s oceanographic expedition around the world from 1928 to 1930. However, most of these leptocephali were relatively large, and the overlap in their morphological characters made it difficult to identify them exactly (7,8). Since then, the spawning areas of the Indo-Pacific anguillid species have remained a mystery, except for the Japanese eel, Anguilla japonica, which was found to spawn to the west of the Mariana Islands in the western North Pacific (3).
Molecular phylogenetic research on the genus Anguilla has recently stimulated interest in the spawning migrations of tropical freshwater eel species by suggesting that the tropical eel, Anguilla borneensis, which is endemic to Borneo, is the most basal species, and that the genus radiated out from the tropics to colonize temperate regions (4). Because of these studies, species-specific genetic markers can be used to identify all species of anguillid leptocephali (9). Molecular techniques have, for the first time, allowed studies on the distribution of tropical anguillid leptocephali that can reveal the location of eel spawning areas and the nature of their migrations. In this study, we have collected the smallest tropical eel leptocephali ever reported and used molecular genetic methods to identify species of anguillid leptocephali from the Celebes Sea and Tomini Bay on the east side of Sulawesi. We provide the first definitive information on the spawning areas of tropical freshwater eels.
A cruise of the R/V Hakuho Maru (Ocean Research Institute, University of Tokyo) in the western Pacific, Celebes, and Sulu Seas was conducted from 14 January to 10 March 2000 (Fig. 1). A subsequent cruise of the R/V Baruna Jaya VII (Research Center for Oceanography, Indonesian Institute of Sciences), made in the waters around Sulawesi from 8 to 30 May 2001, partly overlapped the sampling area of the Hakuho Maru cruise (Fig. 1). Leptocephali were collected during both cruises using identical Isaacs-Kidd midwater trawls with 8.7-m2 mouth openings and 0.5-mm mesh. The collections usually consisted of 60-min oblique or step tows within the upper 300 m. Aboard ship, the leptocephali were tentatively identified on the basis of morphological characteristics (7,8), but these characteristics could not always indicate one species. Total length and other measurements were recorded, and the specimens were preserved in 95% ethanol. In the laboratory, the specimens were identified by comparing their mitochondrial 16S rRNA gene sequences with those of morphologically well-identified adult specimens, as has been previously reported (9). Briefly, total genomic DNA was extracted from each leptocephalus according to a standard protocol (9). A portion of the mitochondrial 16S ribosomal RNA gene (about 500 base pairs) was amplified by the polymerase chain reaction (PCR) using two oligonucleotide primers, H2510 and H3058 (9). Amplification parameters were 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 60 s. The PCR products were sequenced according to the manufacturer’s protocol (Applied Biosystems Inc.) on a 373A DNA sequencer (Applied Biosystems Inc.). Sequences were determined from the light strand only. The sequence data obtained from the leptocephali were directly compared, without any alignments, to homologous data for anguillids in the Pacific region (A. japonica, A. australis, A. borneensis, A. celebesensis, A. dieffenbachii, A. bicolor, A. megastoma, A. marmorata, A. reinhardtii, A. obscura, and A. interioris), deposited in DDBJ/EMBL/GenBank under accession numbers AB021748, AB021751–AB021754, AB021757, AB021758, AB021760–AB021762, AB021764. Within species the sequences were the same or had only one or two site differences, but among species the differences were more than threefold (6–74 sites). All of the sequences determined in the present study will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers: AB097700–AB097767, in sequence.
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Figure 1. The study area, showing the sampling stations and the locations where the various species of anguillid leptocephali were collected. Upper left: Stations during cruises of the R/V Hakuho Maru (circles) in February 2000 and the R/V Baruna Jaya VII (squares) in May 2001. Only a few station numbers are shown to indicate the order of sampling or to identify those mentioned in the text. Upper right: Solid symbols indicate sampling stations where A. celebesensis leptocephali were collected and open symbols indicate negative stations. Lower left: Collection locations of A. borneensis. Lower right: Collection locations of A. marmorata, A. bicolor, and A. interioris. Symbols same as for upper right panel.
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During the two research cruises, we collected 67 leptocephali and one glass eel (the transparent early juvenile stage of eels) of the genus Anguilla (15 leptocephali from the Hakuho Maru cruise and 53 from the Baruna Jaya VII cruise, which included the glass eel). Genetic species identification of the leptocephali clearly distinguished 12 A. marmorata (34.0–50.7 mm in total length and one glass eel of 47.8 mm), 41 A. celebesensis (13.0–47.8 mm), 3 A. borneensis (8.5, 13.0, 35.4 mm), 4 A. bicolor (42.6–49.2 mm), and 1 A. interioris (48.9 mm), all from the waters around Sulawesi (Fig. 1). Also identified were 3 A. bicolor (31.3–46.0 mm) from the waters to the north of New Guinea, and 2 A. marmorata (28.0, 36.5 mm), 1 A. obscura (36.7 mm), and 1 A. australis (47.0 mm) from the western South Pacific (their distributions are not shown).
This is the first description of the distribution of anguillid leptocephali identified using genetic markers from Indonesian waters, and these data suggest that as many as five species of the genus Anguilla may use the Indonesian waters as an area for spawning and larval development. Jespersen (7) reported on collections of relatively large anguillid leptocephali from many of these same areas, but could not make precise species identifications. Another more recent study used the same molecular genetic techniques used in this study to identify anguillid leptocephali as small as 16.3 mm in the western North and South Pacific, but did not make collections in the Indonesian Seas (9). Of particular interest in our study were the small leptocephali of A. borneensis that were 8.5 and 13.0 mm (indicating an age of about 16 and 26 days after hatching [10,11]), which were collected in the Celebes Sea to the east of Borneo, and the specimen (35.4 mm) that was collected to the south in Makassar Strait, where water from the Celebes Sea is transported (12). The freshwater growth habitat of A. borneensis is limited to the east-central part of Borneo (5,6), which strongly suggests that this species spawns in the Celebes Sea and then migrates back to its growth habitat adjacent to the spawning area (Table 1). Another tropical anguillid species, Anguilla celebesensis, has a wider distribution that extends from Luzon of the Philippines to across Sulawesi (5,6). Interestingly, the small leptocephali of this species collected about 25 days after hatching (10,11) were found in two different seasons and in two different areas separated by the northern arm of Sulawesi: a 12.3-mm specimen was found at Station 50 in the Celebes Sea in February, and a 13-mm specimen was found at Station 21 in Tomini Bay in May (Fig. 1). Further, the collection of nine A. celebesensis leptocephali at Station 21 in Tomini Bay ranged in total length from 13 to 48.9 mm (fully grown larval stage [10,11]). These facts indicate that individuals of A. celebesensis inhabiting the watershed of Tomini Bay spawn over a relatively long period and that their leptocephali are retained in Tomini Bay because it is semi-enclosed and its waters apparently do not mix much with those of other areas (13). Therefore, these eels are probably geographically isolated from those in the Celebes Sea.
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TABLE 1 Comparison of presumed spawning areas, ranges, and distance of migration of temperate and tropical eels. (genus Anguilla)
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The findings reported here indicate that freshwater eels living in tropical areas may have life-history characteristics that differ markedly from those of their temperate relatives, which have a single spawning site for each species, long spawning migrations in both the North Atlantic (1,2) and North Pacific Ocean (3), and distinct spawning seasons (Table 1). This distinction is supported by recent analyses of otolith microstructure which showed that tropical anguillids in the Indonesian region may spawn (14) and recruit to freshwater (15) throughout much of the year. Catadromous freshwater eels have been suggested to have originated in the tropical waters near present-day Indonesia sometime around the late-Cretaceous to Eocene, and the endemic tropical species A. borneensis has been found to be the most likely basal catadromous eel species (4). Various characteristics of the migrations of present-day anguillid species clearly show at least a partial geographic cline (Table 1). For example, the most likely basal species, A. borneensis, is distributed narrowly over about 7 degrees of latitude (Equator to 7 °N) and spawns nearby in the Celebes Sea at a distance of only 480–650 km; in contrast, the growth habitat of the European eel extends widely over 40 degrees of latitude, and the distance to its spawning area in the Sargasso Sea ranges from about 4000 to 8000 km (Table 1). This and the short spawning migration of A. celebesensis in Tomini Bay suggest that freshwater eels of the genus Anguilla originally had migrated only short distances to local spawning areas in the warm waters surrounding their freshwater growth habitats in the tropics. But following the passive, long-range dispersion of their leptocephalus stages by currents, freshwater eels may have had to evolve long-distance migrations to return from their temperate growth habitats to their tropical spawning grounds.
The Atlantic species of freshwater eels are often used, even in basic biological textbooks, as a classic example of a species with a spectacular long-distance migration. However, our findings provide the first evidence that this long-distance migration is an adaptation by eels that colonized temperate regions. Therefore, a new era of research on the ecology and behavior of tropical eels has begun, and it promises to unveil the mystery of the origin and evolution of the catadromous migrations of the genus Anguilla.
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Acknowledgments
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We thank all of the scientists who participated in the eel cruises and who worked together discussing the sampling design, operating the nets, and sorting samples. We wrote this paper on behalf of all the scientists aboard, and we also thank all the crew of the Hakuho Maru and the Baruna Jaya VII for their help during the cruise. This work was supported in part by Grants-in-Aid Numbers 07306022, 07556046, 08041139, 08456094, 10460081, and 11691177 from the Ministry of Education, Science, Sports and Culture, Japan, and by grant Numbers JSPS-RFTF 97L00901 from the "Research for the Future Program" of the Japan Society for the Promotion of Science. KT was supported by the Research Foundation "Touwa Shokuhin Shinkoukai" and the Eel Research Foundation "Noborika."
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Footnotes
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Received 20 June 2002; accepted 4 December 2002.
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