<I>Megalops Cyprinoides</I> (Elopiformes, Megalopidae)

BULLETIN OF MARINE SCIENCE, 60(1): 23-36, 1997

METAMORPHOSIS OF THE PACIFIC TARPON, MEGALOPS CYPRINOIDES (ELOPIFORMES, MEGALOPIDAE) WITH REMARKS ON DEVELOPMENT PATTERNS IN THE ELOPOMORPHA

Y. Tsukamoto and M. Okiyama

ABSTRACT The metamorphic style of Pacific tarpon, Megalops cyprinoides, based on morphological observations, is described and the relationships of Elopomorpha are discussed. Generally, it is known that metamorphosis of leptocephali and body shrinkage are simultaneous, culmi- nating in greatest shrinkage in the adult-like body. However, the most shrunken Pacific tarpon remains as a whitebait form, like clupeiform larvae, and its main organismal developments (e,g., ossification, attainment of juvenile body proportion and pigmentation) are concentrated after the most shrunken stage, The metamorphic process of Pacific tarpon is divided into two aspects: 1) changes from leptocephalus to whitebait larva; and 2) substantial development as whitebait larvae. This metamorphic style of Pacific tarpon seems to be primitive among those of Elopomorpha.

The Elopomorpha, including four orders (Elopiformes, Albuliformes, Anguil- liformes and Saccopharyngiformes), pass through a leptocephalus stage in early ontogeny (Nelson, 1994). Many scientists have discussed relationships among those groups, including also the Clupeiformes and relatives (Greenwood et aI., 1966; Gosline, 1971; Forey, 1973; Greenwood, 1977; Patterson and Rosen, 1977; Smith, 1984; Hulet and Robins, 1989; review by Nelson, 1994). In recent years, the concept of Elopomorpha, based largely on the common occurrence of a leptocephalus larval stage, has obtained general approval. On the other hand, leptocephalus was compared with larvae of Clupeiformes and Gonorynchiformes, owing to similarity of their morphology (Uchida, 1966; akiyama, 1979; Mc- Gowan and Berry, 1984; Richards, 1984). Hulet and Robins (1989) viewed the possession of a leptocephalus larva as a primitive character with limited system- atic significance. Further consideration of the validity of the concept of Elopo- morpha is necessary. Pacific tarpon, Megalops cyprinoides (Elopiformes: Megalopidae), is known to have the smallest fully grown leptocephalus in the Elopomorpha, with metamorphosis starting at about one month after hatching (Wade, 1962; Tsukamoto and akiyama, 1993). After apparent shrinkage of the body during metamorphosis, this fish is almost stable in size for about 1 month ("sluggish growth phase"; see Fig. 1). This phase does not occur in the metamorphosis of other orders in Elopo- morpha. In this paper, we describe changes in the morphology of Pacific tarpon during the metamorphosis to clarify the pattern of metamorphosis of this species. Relationships of elopomorphs are then discussed by comparing their developing patterns.

MATERIALS AND METHODS

The series of Pacific tarpon used in this study were reared from fully grown leptocephali caught in the wild. Fully grown leptocephali of Pacific tarpon were collected by a dip net at Ohara harbor in Boso Peninsula, Japan. They were transported to Ocean Research Institute, University of Tokyo and reared in aquarium of the laboratory. Water temperature and salinity were controlled at 25°C and 25%0, respectively. About 200 specimens were fixed immediately after collection in 10% buffered sea water formalin for 8-12 h and then preserved in 70% ethanol. About 50 specimens were fixed in Bouin's

23 24 BULLETIN OF MARINE SCIENCE, VOL. 60, NO. I, 1997

fully grown leptocephali --E 40 E '-' ..c:.•... C) 30 c Q) most shrunken larvae "U roL.. 20 "U c .•...ro en 10 leptocephalus negative sluggish growth phase juvenile growth phase growth phase o 20 40 60 80 100 Age (days)

Figure 1. Early growth of Pacific tarpon, Megalops cyprinoides. Modified after Tsukamoto and Oki- yama (1993). solution (for histological observation), 2% paraformaldehyde-2% glutaraldehyde mixture in cagosdy- late buffer and post-fixed in 2% Os04 (for SEM observation). The specimens are deposited at the Department of Zoology, National Science Museum (Nat. Hist.), Tokyo (NSMT). External features (body proportion, fin development, squamation, and pigmentation), ossification, gill development, and internal body development were observed by the following methods. External features: measurements, counts, and other observations were made under a binocular microscope, Ossification: specimens were stained using the method of Dingerkus and Uhler (1977), Gill development: Specimens were dehydrated through graded ethanol series, critically dried. After opercles were removed from the specimens, the specimens were coated with gold by ion spatter. The gills were observed by scanning electron microscopy (SEM), Internal body development: histological methods for light microscopy were used. Specimens embedded in paraffin and sectioned at a thickness of 4 to 8 fJ-m, sections stained with Mayer's haematoxylin and eosin, Staging and Terminology.-The period of metamorphosis of leptocephali was defined as the interval between start of shrinkage and acquisition of juvenile or adult characters. Furthermore, the metamorphic process was divided into three phases (Fig. I) as follows: 1) leptocephalus negative growth phase: from the fully grown leptocephali to the most shrunken larvae, measuring approximately 32-16 mm SL; 2) sluggish growth phase: long period when length is stabile after leptocephalus negative growth phase, measuring under approximately 16 mm SL in leptocephalus and under approximately 20 mm SL in juvenile; and 3) juvenile growth phase: after sluggish growth phase, measuring approximately 20-40 mm SL. In addition, we also use following two terms to indicate the critical points during leptocephalus metamorphosis: 1) fully grown leptocephali: the most developed leptocephali just before metamorphosis, measuring over approximately 30 mm SL; 2) most shrunken larvae (leptocephali): the smallest larvae when leptocephalus negative growth phase transfers to sluggish growth phase, measuring under approximately 16 mm SL. In this paper, we term clupeoid-type-Iarvae (i.e., larvae of Clupeiform, Gonorynchiform and Os- meriformes, etc.) "whitebait," which corresponds to "leptocephalus" in Elopomorpha larvae. White- bait have small rounded head, extremely slender body shape, poorly pigmented body and straight gut, and anus located near the end of body.

RESULTS External Features and Pigmentation.- The fully grown leptocephali (FGL) have a small rounded head and strongly compressed body (Fig. 2A). Dorsal and caudal TSUKAMOTO AND OKIYAMO, METAMORPHOSIS OF PACIFIC TARPON 25

Figure 2. Series of photographs of Pacific tarpon, Mega/ops cyprinoides, during metamorphosis. A) fully grown leptocephalus, 30.5 mm SL; Band C) larvae in leptocephalus negative growth phase, 25.4 mm SL and 19.8 mm SL, respectively; D, E and F) larvae in sluggish growth phase, 16.9 mm SL, 18.9 mm SL AND 20.4 mm SL, respectively; G and H) juveniles in juvenile growth phase, 26.3 mm SL and 29.6 mm SL, respectively. fin rays have started to form. Branched melanophores are distributed as follows: under the eye, along the dorsal contour of abdominal cavity, on the dorsal surface of the swimbladder, and between myomeres of the tail. The pelvic fin bud ap- peared at the start of body shrinkage. During leptocephalus negative growth phase (NGP), the head region (i.e., eye and mouth) is well developed. The body remains transparent until the most shrunken larvae (MSL) (Fig. 2B, C). In sluggish growth 26 BULLETIN OF MARINE SCIENCE, VOL. 60. NO. I, 1997

A ------~

~---~

Figure 3. Sequence of development of the squamation of Pacific tarpon, A) 22.4 mm SL; B) 24.3 mm SL; C) 26.2 mm SL; D) 27.0 mm SL; E) 30.0 mm SL; F) 33.5 mm SL. All specimens are in juvenile growth phase. phase (SGP), body length is then stabilized but proportional changes of body occur, relative increase in head length, body depth, and width. All fin rays are well developed and dorsal and anal fin move anteriorly. Pigments such as melanophores, xanthophores, and erythrophores, appear and increase. Iridophores appear on the opercle and abdominal cavity. During juvenile growth phase (JGP), all the fin rays become well developed, the posteriormost soft ray of the dorsal fin elongates, and the dorsal and anal fins move anteriorly. Iridophores spread around the body. Most proportional change was completed in specimens of approximately 20 mm standard length (SL), which have the adult appearance except for the squamation. Squamation.-Squamation starts in JGP. Scales first appear on the mid-flank of the tail in specimens over approximately 22 mm SL (Fig. 3A), and spread anteriorly with growth. In juveniles of approximately 26 mm SL, scales develop further dorsally and ventrally. About half of the body surface is covered by scales in juvenile of approximately 28 mm SL. Except for the ventral surface of the trunk, squamation is nearly complete in juveniles of approximately 33 mm SL. Squamation of the ventral surface is achieved in juveniles over 35 mm SL. Body Section.-In FGL, the integument and muscle are very thin and the body is filled with mucinous material (Fig. 4A). This mucinous material decreases in volume with body shrinkage. In MSL, integument and muscle are well developed, and the area of mucinous material occupies nearly half of the body (Fig. 4B). In SGp, the volume of abdominal cavity increases and ventral mucinous materials almost disappear, whereas dorsally mucinous materials are left in the region of the dorsal aorta (Fig. 4C). These materials completely disappear at about 17 mm in the JGP (Fig. 4D). Ossification.-In FGL, all elements of the skull remain cartilaginous, except for the anterior part of the meckelian cartilage (Fig. 5A). Ossification progresses no further in NGP (Fig. 5B). In the most shrunken larvae of 15.0 mm SL, all elements of cartilage bone appear. The membrane bones of the jaws (premaxillary, max- TSUKAMOTO AND OKIYAMO: METAMORPHOSIS OF PACIFIC TARPON 27

500 I'm

Figure 4. Cross sections of trunk of Pacific tarpon. A) fully grown leptocephali, 32.2 mm SL; B) most shrunken larva, 15.6 mm SL; C) larva in sluggish growth phase, 16.] mm SL; D) juvenile in juvenile growth phase, 17.8 mm SL. MC: muscle; SK: skin; NC: notocord; as: mucinous material. 1 is whole section and 2 is enlarged photograph of muscle. 28 BULLETIN OF MARINE SCIENCE, VOL. 60, NO. I, 1997

A o

B E

.•.. -- - -- c F

Figure 5, Development of skuU of Pacific tarpon, A) fully grown leptocephali. 30,2 mm SL; B) leptocephalus negative growth phase. 26,0 mm SL; C) most shrunken larva; 15,0 mm SL; D) sluggish growth phase; 16,5 mm SL; E) juvenile growth phase. 18,0 mm SL; F) young, 58.4 mm SL. Ang: angular; Bra: branchiostegal; Cer: ceratohyal; De: supraethmoid; Den: dentary; Ect: ectopterygoid; End: endopterygoid; Eth: ethmoid; Fr: frontal; Hyo: hyomandibular; Le: lateral ethmoid; Ino: infra- orbital; lop: interopercle; La: lachrymal; Mc: Meckelian cartilage; Met: metapterygoid; Mx: maxillary; Na: nasal; Op: opercle; Pa: parit:tal; Pal: palatine; Pmx: premaxillary; Pop: preopercle; Qu: quadrate; Smx: supramaxillary: Sop: subopercle; Stt: supratemporal. White areas indicate cartilage, stipple indicates bone.

illary, dentary, and angular) ossify (Fig. 5C). Most elements of the oromandibular, hyoid and branchial regions are ossify in SGP (Fig. 5D, E). All skull elements, including the circumorbital region are ossified in juveniles of approximately 35 mm in JGP. The shape of all head bones become as in the adult at about 50 mm SL, but ethmoid remains cartilaginous (Fig. 5F). In FGL, the notochord is flexed completely, the caudal complex is poorly developed, and cartilaginous buds of the caudal skeleton appear (Fig. 6A). In NGp, uroneural 1 and the fifth and sixth hypurals ossify (Fig. 4B). Most of the caudal skeleton, except for ural (:entram and the epurals, ossify in SGP (Fig. 6C, D). All caudal elements are ossified at about 20 mm SL in JGP (Fig. 6D). Development of Digestive Tract.-In FGL, the gut is straight, and the esophagus and intestine are divided by a constriction located at anterior approximately 60% of length. The swimbladder connects just anterior to this constriction (Fig. 7A). In NGp, the length of the alimentary canal decreases, especially in the esophagus TSUKAMOTO AND OKIYAMO: METAMORPHOSIS OF PACIFIC TARPON 29

Hy1

Hy7

Figure 6. Development of the caudal complex of Pacific tarpon. A) fully grown leptocephalus, 30.2 mm SL; B) most shrunken larva, 15.0 mm SL; C) sluggish growth phase, 18.0 mm SL; D) juvenile growth phase, 20.0 mm SL. Cop: opisthoral cartilage; Ep: epural; Hs: hemal spine; Hy: hypural; Ph: parhypural; Npu: neural spine: Un: uroneural; Ur: ural centram. White areas indicate cartilage, stipple indicates bone. region. In MSL, the alimentary canal is still straight, but the thickness of the intestine increases and the stomach is developing (Fig. 7B). In SGp, the stomach coils (Fig. 7C), and then the intestine coils and pyloric caeca start to form until JGP. In JGp, the capacity of the stomach increases and the relative length of esophagus obviously decreases (Fig. 7D).

B~--~ o

Figure 7. Sequence of the development of digestive tract of Pacific tarpon. A) fully grown leptocephalus, 30.5 mm SL; B) most shrunken larva, 15.4 mm SL; C) larvae in sluggish growth phase, 15.9 mm SL; D) juvenile in juvenile growth phase, 28.5 mm SL. Arrows indicate the border between esophagus and intestine, before development of stomach. 30 BULLETIN OF MARINE SCIENCE, VOL. 60, NO.1, 1997

Figure 8. SEM photographs of gills of Pacific tarpon. A) fully grown leptocephalus, 31.6 mm SL; B) larva in negative growth phase, 22.3 mm SL; C) larva in sluggish growth phase, 17.5 mm SL; D) juvenile growth phase, 22.8 mm SL. GF: gill filament; SG: secondary gill lamellae; GL: gill raker. TSUKAMOTO AND OKIYAMO: METAMORPHOSIS OF PACIFIC TARPON 31

A B PECO ON IN IP G

Figure 9. Transverse section of eye in fully grown leptocephali of Pacific tarpon, 32.2 mm SL. A) photograph of left eye; B) enlarged photograph of retina region. CO: cone; G: ganglion layer; IP: inner plexiform layer; IN: inner nuclear layer; L: lens; ON: outer nuclear layer; PE: pigment epithelium layer.

Gills.-In FGL, the gill filaments are poorly developed and there are no secondary gill lamellae (Fig. 8A). In SGp, secondary gill lamellae develop with decrease of body length, and the basic components of the gill are formed in MSL (Fig. 8E). In SGp, the length of gill filaments and the number of secondary gill lamellae increase (Fig. 8e). In JGp, the number of secondary gill lamellae increases further and formation of the gills is completed in juveniles of approximately 20 mm SL (Fig. 80).

DISCUSSION Morphology of Fully Grown Leptocephali.-Even at the fully grown phase, leptocephali of Pacific tarpon reveal retarded organismal development, when compared with the fully grown leptocephali of other species such as Conger myriaster (Kubota, 1961) and Ariosoma balearicum (Hulet, 1978). Hulet and Robins (1989) wrote that vision and olfaction stand out as the only two functions represented by well-developed organ systems in the leptocephalus. However, development of the visual and olfactory systems of Pacific tarpon is poorer than those of A. balearicum. Sectioned eyes (Fig. 9) of Pacific tarpon indicate that optical function of this fish might exclude image formation, but light and shade could be detected, because the basic morphology of eye is completed whereas the retina is poorly developed. SEM photograph of the head of Pacific tarpon (Fig. 10) shows that formation of olfactory organ is delayed as compared with A. balearicum, because the nasal cavity is exposed and the anterior and posterior nostrils have not formed. The development of those organs in fully grown leptocephali of Pacific tarpon is similar to that in other marine fish larvae at 2-3 days after hatching, when the yolk-sac is absorbed and the eye pigmented. These morphological observations of the fully grown leptocephali of Pacific tarpon reveal that organismal development is still extremely retarded during the leptocephalus stage. Metamorphic Style of Pacific Tmpon.-Figure 11 summarizes the organismal developmental events in Pacific tarpon during metamorphosis. In leptocephalus negative growth phase (NGP), secondary gill lamellae develop conspicuously. The main organismal development is concentrated in sluggish growth phase (SGP) and finished in the early juvenile growth phase (JGP) when juveniles reach about 22 mm SL. Presence of this sluggish growth phase is considered to be critically important in the metamorphosis of this fish, because the body proportions, pigmentation, and organismal development of the most shrunken larvae of Pacific 32 BULLETIN OF MARINE SCIENCE, VOL. 60, NO. I, 1997

Figure 10. SEM photograph of head region of leptocephalus in leptocephalus negative growth phase, 29.5 mm SL. tarpon are similar to those of whitebait (Fig. 2D). Also development in Megalops during SGP and JGP can be regarded as the same pattern as in whitebait changing (metamorphosis) to juvenile or adult. Therefore, this metamorphic process of Pa- cific tarpon is divided into the following two aspects: 1) changes from leptocephalus to whitebait; and 2) substantial development as whitebait. Without leptocephalus stage (from yolk absorption to the end of leptocephalus negative growth phase), early ontogeny of Pacific tarpon seems to be similar to that of whitebait. Development Patterns and Relationships in Elopomorpha.-Elopiformes have been recognized as related to Anguilliformes primarily by sharing the leptocephalus larva (Greenwood et al., 1966). In this paper, relationship between members of Elopomorpha and taxa with whitebait type larval development is considered from the view point of development patterns. Regrettably, information on the metamorphosis of leptocephali is extremely limit because most leptocephali of Elopomorpha are available only by means of net collection, Despite incomplete knowledge of Elopomorpha, we wish to try to discuss the relationships of this group from their development patterns. Elopiformes: Two families (Megalopidae and Elopidae), two genera, and about eight species are included in this order (Nelson, 1994). In proportional changes and ossification, the development pattern of Elops hawaiiensis is similar to that of Pacific tarpon (Sato and Yasuda, 1980). The proportional changes of E. saurus (Gehringer, 1959) and M. atlanticus (Breder, 1944; Wade, 1962) are also similar to those of Pacific tarpon. It may safely be said that the metamorphic style is the same in all Elopiformes, Albuliformes: Three families (Albulidae, Halosauridae and Notacanthidae), eight genera, and about 29 species are included in this order (Nelson, 1994). Metamorphosis has been described only for the albulids. In the metamorphosis of Istieus (=Pterothrissus) gissu, proportional change and body shrinkage make simultaneous progress and the proportions of the most shrunken larvae are like TSUKAMOTO AND OKIYAMO: METAMORPHOSIS OF PACIFIC TARPON 33

shrinkage in length I change of body proportions Proportions Lwhitc bait fonn L juvenile proportions attained

appearnnce ofxanthophores and erythrophores j rappearance ofiridophores Pigmentation melanophores increase Ljuvenile pigmention attained

r pelvic fin mys fonned r pectoral fin mys formed Fins Lstart of movement of Lfinish of movement of dorsal and anal fin dOlSaland anal fin

start finiSh j r Squamation

Ossification cartilages appeare + ossification start full complement ossified ---, head region , cartilages appeare r ossification start r full complement ossified caudal complex ossification start j full complement ossified l vertebrae

stomach fonnation I ,- stomach coiled ,- intestine coiled, pyloric caeca fonned Digestive tract , t

secondary gill lamellae secondary gilllameUae formed j number increased Gill

leptocephalus negative growth phase j sluggish growth phase juvenile growth phase fullygrown leptocephali j r most shrinked larvae I I I Standard length (rnm) 32 15 20 I I I I I Days after hatching 30 50 80

Figure II. Summary of organismal development of Pacific tarpon. Bold lines indicate the period when the organs are well developed. those in adults (Matsubara, 1942). The development pattern of Alhula vulpes is similar to that of 1. gissu (Rasquin, 1955; Alexander, 1961). Though information on metamorphosis is available for only two genera, that knowledge suggests that metamorphosis and body shrinkage of Albuliformes take place almost simulta- neously. Anguilliformes: Fifteen families, 141 genera, and about 738 species are included in this order (Nelson, 1994), which comprises about 95% of elopomorpha species. However there is little information about metamorphosis, which has been described only in the families Anguillidae and Congridae. A series of photographs of Anguilla anguilla suggests that the most shrunken larvae (glass eel) represent the stage of almost complete change to adult body proportions and fin ray development (Schmidt, 1909). Metamorphosis in Conger eel, Conger myriaster, is known in detail because live fully grown leptocephali are readily available in 34 BULLETIN OF MARINE SCIENCE, VOL. 60, NO. I, 1997 coastal areas of Japan (Kubota, 1961; Asano et aI., 1978). The events of metamorphosis such as change in body proportions, ossification and organismal development occur during body shrinkage. This fish never passes through the sluggish growth phase observed in the metamorphosis of Pacific tarpon. Whitebait: Most of species in Clupeiformes, Gonorynchiformes, Osmeriformes and Aulopiformes pass through whitebait larval stage (McGowan and Berry, 1984; Richards, 1984; Hearne, 1984; akiyama, 1984). Those species have small rounded head, extremely slender body shape, poorly pigmented body and straight gut, and anus located near the end of body in their larval stage. In most taxa, metamorphic size of whitebait is relatively large, i.e., over 20 mm SL. Whitebait metamorphosis is finely defined by the gradual shifting of the gut and the dorsal and anal fins (akiyama, 1979, McGowan and Berry, 1984). The Japanese sardine, Sardinops melanostictus, has a typical whitebait-type larvae (Uchida, 1958). The body is elongate and transparent in larvae. The alimentary canal is straight and the anus is located near the posterior end of the body. Development of fin rays, except pectorals, is complete in larvae of 30 mm SL. However, the body is transparent and retains whitebait characters after completion of fin ray development. In larvae about 40 mm SL, the pectoral fin rays are formed and pigmentation is complete. Albuliformes and Anguilliformes almost complete their early development by the most shrunken larval stage, when they have acquired juvenile characteristics in body proportions, ossification, and other characteristics. On the other hand, development in the most shrunken larvae of Elopiformes is retarded, and resem- bles that of whitebait. Thereafter, Elopiformes pass through the same development pattern as whitebait. Whitebait-type and leptocephalus larvae are found in a variety of major taxa in the lower teleostei. Hulet and Robins (1989) suggested that possession of leptocephalus larvae is a primitive character, and Megalops is possibly in the process of losing the leptocephalus. However, some facts suggest that in the early ontogeny of Megalops comprising three steps (first whitebait, second leptocephalus, and third whitebait), the leptocephalus stage might be derived from the whitebait- type larva. Including our present findings, these are: 1) newly hatched larvae of Megalops and other Elopomorph species do not have the characteristics of the leptocephalus, but closely resemble newly hatched whitebait-type larvae (Breder, 1944; Mito, 1961); 2) organismal development of Megalops leptocephalus is still extremely retarded and organogenetically it can be considered the same stage as that of a whitebait-type larvae just after completion of yolk absorption; 3) the development pattern of Megalops after the most shrunken larval stage is the same as that of the whitebait-type larvae. For these reasons, we propose Elopiformes (Mega lops and Elops) is more primitive than other taxa of the Elopomorpha, none of which reveal clear steps in their early development. Leptocephali have specialized feeding, digestion and metabolism (PfeiJer, 1986; Otake et aI., 1993; Pfeiler and Govoni, 1993). These seem to be related to their adaptation to oligotrophic sub- and tropical oceanic conditions and such conditions might select for the evolution of the peculiar body form of the leptocephalus. Although available data are limited, the assumption that the whitebait-type larvae represents the most primitive state in the early development of the lower teleostei seems to provide a reasonable understanding of the evolution of its larval morphology.

ACKNOWLEDGMENTS The authors wish to thank Drs. K. Tsukamoto and T. Otake, Ocean Research Institute, University of Tokyo, for their helpful suggestions. Thanks are also due to Mr. J. Kojima, Marine Ecology Research TSUKAMOTOANDOKIYAMO:METAMORPHOSISOFPACIFICTARPON 35

Institute, for his advice in the course of collection and for the offer of specimens. This work was supported partially by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (No. 02954096).

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DATEACCEPTED: March 25, 1996.

ADDRESSES: (Y.T.) National Research Institute of Fisheries Science, Fukuura 2-12-4, Kanazawa-ku, Yokohama, Kanagawa 236, Japan; (M.O.) Ocean Research Institute, University of Tokyo, Minamidai 1-15-1, Nakano-ku, Tokyo 164, Japan.