Anguilla marmorata (Giant Mottled ) Discovered in a New Location: Natural Range Expansion or Recent Human Introduction?1

Alex Handler2 and Shelley A. James3

Abstract: Freshwater in the family spend a majority of their adult life in freshwater but migrate to the ocean to and die. Because freshwater eels are believed to have a long larval period in the open ocean, it is unclear how the present global distribution of species arose. A stock of fresh- water eels of the family Anguillidae was found on Palmyra Atoll in the central Pacific Ocean, in June 2003. In October 2003, a single eel specimen was caught using a hand net from this small group of eels on Palmyra Atoll. Morphological and molecular characters (12S and 16S mitochondrial rRNA and cytochrome b mtDNA sequences) were used to identify the species as Anguilla marmorata Quoy & Gaimard. The discovery of these eels on Palmyra supports the hypoth- esis of natural range expansion from the Indo-Pacific eastward to the Gala´pagos through the Line Islands, but further analysis of oceanic currents and more vari- able genes are required to assess whether humans are involved in the recent spread of Anguilla marmorata to these new locations.

Eels in the genus Anguilla Shaw, 1803 have recently been documented in the Pa- (family Anguillidae) have been a favored hu- cific. McCosker et al. (2003) identified an eel man food item since ancient Roman times caught in the Gala´pagos in 1998 as a species (Smith 1989). Because of their commercial of Anguilla, most likely A. marmorata Quoy value, numerous Anguilla species have been & Gaimard, 1824. They hypothesized that introduced to locations outside their natural the presence of freshwater eels in the Gala´pa- range to boost local eel populations. Re- gos is a result of sporadic arrivals of larvae cently, isolated individuals or populations of carried by ocean currents that pass through a number of species of Anguilla have been dis- the Line Islands along the equator in the cen- covered in locations from which they were tral Pacific. They argued that the eels had not previously unknown (McDowall et al. 1998, been transported to the Gala´pagos by humans McCosker et al. 2003). It is unclear whether because they had been observed by island in- eels arrived in these new locations with or habitants long before air transportation could without human assistance. have enabled such transport. They did not New range expansions of Anguilla species address the possibility of live transport of eels by ship. The East Australian longfinned eel, 1 This work was funded by the NSF GK-12 grant DGE02-32016. Contribution No. 2004-019 to the Ha- Anguilla reinhardtii Steindachner, 1867, has wai‘i Biological Survey. Manuscript accepted 14 February recently been reported in northern New Zea- 2005. land (McDowall et al. 1998). Over eight con- 2 Center for Conservation Research and Training, secutive years, new classes of this species have Gilmore 409B, University of Hawai‘i at Ma¯noa, 3050 been recorded in the Waikato River, suggest- Maile Way, Honolulu, Hawai‘i 96822-2231 (e-mail: [email protected]). ing that recruitment is occurring on a regular 3 Pacific Center for Molecular Biodiversity, Bishop basis (Chrisnall 1996, Jellyman et al. 1996). Museum, 1525 Bernice Street, Honolulu, Hawai‘i 96817 Commercial eel fishers have noticed ‘‘unusual (e-mail: [email protected]). eels in some northern [New Zealand] rivers for perhaps 25 years,’’ but the formal identifi- Pacific Science (2006), vol. 60, no. 1:109–115 cation only more recently of A. reinhardtii : 2006 by University of Hawai‘i Press may have been because of its morphological All rights reserved similarity to New Zealand’s endemic fresh-

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Figure 1. Palmyra Atoll, Line Islands. water eel, A. diffenbachii Gray, 1846 (Mc- this small population on Cooper Island, Dowall et al. 1998:1). Palmyra Atoll. Historically, morphological Anguillid eels spend a majority of their characters have been used to identify Anguilla adult life in freshwater but migrate out to species (Ege 1939). Recent molecular data oceanic gyres to breed, spawn, and possibly suggest that identification based on mor- die (Smith 1989). Anguillidae are a homoge- phological characters may not be reliable neous group, relatively unspecialized mor- (Aoyama et al. 2001, Lin et al. 2001). There- phologically and highly adaptable (Smith fore, a combination of morphological and 1989). The single genus Anguilla consists of molecular characters was used to identify this 15 species and three subspecies (Watanabe specimen. 2003). The evolutionary history of the An- guillidae and the origins of the current global materials and methods distribution of species are still in question (Bastrop et al. 2000, Aoyama et al. 2001, Lin A freshwater eel was collected on 13 October et al. 2001). They occur in all temperate and 2003 on Palmyra Atoll (Figure 2) from an ar- tropical waters except the southern Atlantic tificial freshwater pond (former cesspool built and the east coast of the Pacific (Aoyama between 1941 and 1947) on the southeast et al. 2001). The larvae are transported away corner of Cooper Island, just off the airplane from spawning areas by oceanic currents runway (GPS latitude/longitude: 05 53.42 0 (Bastrop et al. 2000). N, 162 04.35 0 W). Morphological characters Anguilla species have not been recorded (Smith 1999) were used to obtain a prelimi- previously on Palmyra Atoll, Line Islands nary identification of the specimen. Its iden- (Figure 1). A stock of freshwater eels was dis- tity was confirmed by molecular analysis. covered on Cooper Island, Palmyra Atoll, A 427 base-pair fragment of the 12S mito- during a research trip in June 2003. In Octo- chondrial rRNA gene, a 628 base-pair frag- ber 2003, a freshwater eel was caught from ment of the 16S mitochondrial rRNA gene, Anguilla marmorata Discovered in New Location . Handler and James 111

Figure 2. Freshwater eel (Anguilla marmorata) collected on 13 October 2003 on Palmyra Atoll. and the complete mitochondrial cytochrome submitted to GenBank, under accession num- b gene were sequenced. Genomic DNA was bers AY735145–AY735147. extracted from muscle using the protocol Paup 4.0b10 (Swofford 2002) and Tree- of Sambrook and Russell (2001). Polymerase Rot.v2 (Sorenson 1999) were used to com- chain reaction (PCR) amplifications were pare the cytochrome b sequence of the performed in 50 ml of a solution containing Palmyra specimen with sequences of all other approximately 10 ng of genomic DNA, 400 Anguilla species in GenBank as well as with mM of each dNTP, 1.5 unit Taq Polymerase all GenBank sequences of A. marmorata (the (Promega), 2 mM MgCl2, each primer at 1 tentative identity of the specimen, based on mM, and buffer. Primer sequences for the morphological criteria) from other parts of mitochondrial 12S and 16S genes were from its range. All characters were unordered and Palumbi (1996) and for cytochrome b were unweighted. from Aoyama et al. (2001) and Palumbi et al. The specimen is deposited in the Bishop (1991). PCR cycling parameters (PTC 100, Museum ichthyology collection (bpbm 39291) MJ Research) for the initial double-stranded and voucher DNA is held at the Pacific Cen- amplification were 94 C for 1 min, 50 C ter for Molecular Biodiversity, Bishop Mu- for 1 min, and 72 C for 1 min, repeated for seum (PCMB B313). 45 cycles, with a final step of 72 C for 5 min. The PCR product was extracted using QIA- results Quick Gel Extraction Kit (Qiagen, Valencia, California) and visualized on an agarose gel. The specimen was identified as A. marmorata Cycle sequencing of 15–45 ng of the double- based on its mottled color and long dorsal fin, stranded PCR product was carried out with which differentiate it from other Anguilla each primer and BigDye terminators (v. 3.1, species (Smith 1999). The 12S, 16S, and ABI Biosystems) diluted to half concentration cytochrome b sequences confirmed the iden- using 2.5 buffer. The sequences have been tification. The 12S and 16S sequences were 112 PACIFIC SCIENCE . January 2006 identical to those of a specimen of A. Gala´pagos using the Equatorial Counter- marmorata from Kaupo¯ on the south coast Currents. of Maui, Hawai‘i, collected in 2002 (GenBank Multiple species of freshwater eels (A. an- accession numbers AY207028 and AY207029, guilla, A. japonica, and A. rostrata) have been respectively). This specimen from Maui intentionally moved around the world for hu- (bpbm 39092) was identified as A. marmorata man consumption and the aquarium trade on the basis of morphology; its voucher (Ringuet et al. 2002), suggesting that the eels DNA is also held in the Bishop Museum discovered on Palmyra may have arrived (PCMB B177). The 12S sequence of these there with the assistance of humans. Anguilla two specimens was also identical to those of marmorata is not usually caught for consump- individuals from South (GenBank tion, but occasionally it is illegally caught and accession number AF266485) and sold to restaurants (Lin et al. 2002). From the (GenBank accession number AB021890), and 1950s to the 1960s, Anguilla species were im- one base pair different from an individual ported into Hawai‘i, which is approximately from Taiwan (GenBank accession number 1,600 km north and slightly east of Palmyra, AF266484). The 16S sequences from the as live food and for the aquarium pet trade Maui and Palmyra individuals differed from (A. Suzumoto, pers. comm.). Currently their that of the Indonesian individual by one base import into Hawai‘i is prohibited. pair (GenBank accession number AB021760) An alternative hypothesis is that A. mar- and from those of four individuals from Tai- morata arrived in Palmyra naturally. There wan from zero to three base-pair differences is a natural freshwater lake on Washington (GenBank accession numbers AJ244816, Island, just south and east of Palmyra. This AJ244817, AJ244818, AJ244819). lake has not been investigated for the pres- Strong bootstrap support (100) and a de- ence of A. marmorata, but if it is present cay index of 17 of the maximum parsimony there, it may reflect expansion of its range tree derived from the cytochrome b data from the western Indo-Pacific eastward support monophyly of A. marmorata (Figure through the Line Islands and then to Palmyra 3). Maximum likelihood and distance-based when habitat became available, and to the (neighbor joining) trees based on the same Gala´pagos, as proposed by McCosker et al. data also support monophyly of A. marmorata (2003). (Maui Cytochrome b GenBank accession Multiple studies have found that A. mar- number AY738724). The other 14 species morata spawns along the western edge of the within the family Anguillidae served as out- North Equatorial Current in the Pacific groups for this particular test. Ocean where the North Equatorial Counter- Current splits from the North Equatorial discussion Current and begins its flow eastward toward Palmyra Atoll (Arai et al. 2001a,b, Miller et The discovery of a population of A. marmo- al. 2002). One of these studies also found A. rata on Palmyra Atoll provides possible new marmorata leptocephali in the westernmost insight into the spread of A. marmorata across edge of the South Equatorial Current and the Pacific Ocean, as proposed by McCosker South Equatorial Counter-Current, where it et al. (2003), who suggested that larvae could begins flowing eastward through the Pacific be transported in an easterly direction by Ocean (Arai et al. 2001a). either the Northern or Southern Equatorial The fact that A. marmorata takes approxi- Counter-Currents during El Nin˜ o/Southern mately 150 days to mature and migrate to a Oscillation (ENSO) events. Although otolith freshwater environment further supports the data from the specimen collected in the Gala´- possibility that this species of eel has the ca- pagos in 1997 (McCosker et al. 2003) do not pacity to travel long distances through the support this hypothesis that the larva was transport of the ocean currents mentioned transported during an ENSO event, it may here before having to move into a freshwater still be possible that larvae arrived in the habitat to live (Arai et al. 2001b). Historical Figure 3. Maximum parsimony consensus tree of cytochrome b mtDNA of family Anguillidae with bootstrap values above branches and decay indices below (A. marmorata: AF074863, South Africa; AF006705, Taiwan; AF006704, Taiwan; AB021778, Indonesia). Note that Anguilla marmorata forms a monophyletic group. 114 PACIFIC SCIENCE . January 2006 movements and range expansions of A. of tropical eels Anguilla spp. and implica- marmorata are complicated by the fact that tions for the evolution of oceanic migra- this species has the widest distribution of tion in the genus Anguilla. Mar. Ecol. any freshwater eel (Smith 1989). It exists in Prog. Ser. 216:253–264. and neighboring islands, Indone- Bastrop, R., B. Stehlow, K. Jurss, and C. sia, the Philippines, Taiwan, and eastward Sturmbauer. 2000. A new molecular phy- through the western Pacific to the Marquesas. logenetic hypothesis for the evolution of There is also a single record from South Af- freshwater eels. Mol. Phylogenet. Evol. rica (Smith 1989). Anguilliform fossils date 14:250–258. back to the Cretaceous about 100 million yr Chisnall, B. L. 1996. Aussie invasion. Seafood ago (Bastrop et al. 2000). N.Z. 4 (7): 81. This study did not permit a conclusive de- Ege, V. 1939. A revision of the genus Anguilla termination of the role humans have had in Shaw: A systematic, phylogenetic and transporting A. marmorata to new locations geographical study. Dana-Rep. Carlsberg in the central and western Pacific. Eels have Found. 16:1–256. begun to be noticed in new locations, possibly Jellyman, D. J., L. H. Dijkstra, B. W. Chis- also a result of recent shifts in oceanic cur- nall, and J. A. T. Boubee. 1996. The first rents (McDowell et al. 1998, McCosker et al. record of the Australian longfinned eel, 2003). Looking more closely at particular Anguilla reinhardtii, in New Zealand. Mar. oceanic currents in the Pacific and determin- Freshwater Res. 47:1037–1040. ing if freshwater eels occur on Washington Lin, Y., Y. Poh, and C. Tzeng. 2001. 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