Early Ontogeny and Systematics of Bothidae, Pleuronectoidei

BULLETIN OF MARINE SCIENCE, 60(1): 192-212,1997

EARLY ONTOGENY AND SYSTEMATICS OF BOTHIDAE, PLEURONECTOIDEI

Atsushi Fukui

ABSTRACT Of the 20 bothid genera, larvae of the following 16 genera are presently known: Taeniop- settinae, (1) Taeniopsella, (2) Engyophrys, (3) Trichopsetta; Bothinae, (4) Parabothus, (5) Tosarhombus, (6) Crossorhombus, (7) Engyprosopon, (8) Bothus, (9) Grammatobothus, (10) Asterorhombus, (11) Psellina, (12) Lophonectes, (13) Arnoglossus, (14) Monolene, (15) Laeops, (16) Chascanopsetta, This study reviews the early ontogeny and systematics of bothids of the world. A cladistic analysis of 16 characters results in 25 equally parsimonious trees. A strict consensus tree for these 25 trees is shown. It has resolved two monophyletic groups and unresolved polytomy at basal nodes. The two monophyletic groups indicate that (1) Asterorhombu,\' and Engyprosopon except sp.2 of the subfamily Bothinae are sister group for the subfamily Taeniopsettinae, (2) Arnoglossus and Engyprosopon is not monophyletic, and (3) Taeniopsetta, Engyophrys, and Trichopsetta of the subfamily Taeniopsettinae are monophyletic.

Fishes of the family Bothidae, suborder Pleuronectoidei, are very diverse, being comprised of 20 genera in two subfamilies, four in Taeniopsettinae and 16 in Bothinae (Amaoka, 1969; Ahlstrom et aI., 1984), The larvae of this family have a transparent, extremely compressed, body. The dorsal fin begins anterior to the eye, its second ray being elongated. The pelagic larvae are the largest among the pleuronectoid fishes, e.g., Chascanopsetta lugubris with 120-mm larvae, Many studies have been made on bothid larvae, e.g" Amaoka (1964, 1970, 1971a, 1972, 1973, 1974, 1976), Futch (1977), and Hensley (1977). Ahlstrom et aI. (1984) reviewed these studies. However, early development of bothids is still poorly understood, because the early and middle postlarvae are nearly unknown, After the publication by Ahlstrom et aI. (1984), Ozawa and Fukui (1986) made extensive studies on the early ontogeny of bothids based on numerous larvae collected in the western North Pacific, My present study reviews the early ontogeny and systematics of bothids of the world, and discusses phylogenetic information provided by larval characters.

METHODS

The larval period was divided into the following five stages: stage I, before flexion of notochord; stage II, until completion of notochord flexion or of caudal fin ray formation; stage IlIa, until disappearance of air bladder; stage IUb, until completion of right-eye migration to left side; stage IV, after metamorphosis, Metamorphosis is the period of right-eye migration. Morphometric measurements follow Ozawa and Fukui (1986). Definitions of the terms used in the present study are as follows: rostrum above snout, anterior projection of dorsal fin; interspine base of dorsal (anal) fin, along the dorsal (anal) edge of the epaxial (hypaxial) musculature; interspinous region of dorsal (anal) fin, region of dorsal (anal) fin pterygiophores.

GENERAL FEATURES OF THE DEVELOPMENT OF BOTHID LARVAE Of the 20 bothid genera, the larvae of 16 are known (Table lA, B; Figs. 1-6). Larvae of four of the genera, Parabothus, Tosarhombus, Grammatobothus, and Asterorhombus, were identified after Ahlstrom et al. (1984). Early larvae (stages I or II) are known for the following 12 genera: Engyophrys and Trichopsetta in

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Figure 1. Larvae of Bothidae. (A) Taeniopsetta ocel/ata, 59.0 mm SL, from Amaoka, 1970; (B) Trichopsetta ventralis, 21.9 rom SL, from Evseenko, 1982; (C) Engyophrys senta, 12.3 mm SL, from Hensley, 1977; (D) Asterorhombus sp. 1, 11.0 mm SL; (E) Asterorhombus sp. 2 7.80 mm SL; (F) Epiotic spines of Asterorhombus (left, A. sp. 1; right, A. sp. 2).

Taeniopsettinae; Crossorhombus, Engyprosopon, Bothus, GrammatobiJthus, As- terorhombus, Psettina, Amoglossus, Monolene, Laeops, and Chascanopsetta in Bothinae. General features of bothid larvae are summarized below. Bothid larvae have a body that is very laterally compressed and deep to fairly slender and long dorsal and anal fin bases with many slender rays. The dorsal fin origin is anterior to the eyes and its second ray becomes elongated in stages I ]96 BULLETIN 0 F MARINE SCIENCE , VOL ,60, NO, I, 1997

Figure 2 L . arvae of B th' fiI E ]4.] mm SL- 0 Idae. (A) En SL; (F) E, s~. ~~)9~Om;:~is:~~ma,, (G) Grammatobothus14.!:r::;:o:~~ lb~r;iSqUama,. sp. 1, 19.54.08mmmmSL'SL'(~)Bk . grandisquama sp., 10.5 mm SL; (H) G. sp., 27.1. sp. mm2, 9.54SL. m~ FUKUI: ONTOGENY AND SYSTEMATICS OF BOTHIDAE 197

Figure 3. Larvae of Bothidae. (A) Crossorhombus kobensis, 2.88 mm SL; (B) C. kobensis, 15.4 mm SL; (C) C. azureus (=kanekonis), 15.1 mm SL; (D) Lophonectes gallus, 18.5 mm SL, from Ahlstrom et al. 1984; (E) Psettina gigantea, 18.0 mm SL; (F) P. iijimae. 28.0 mm SL; (G) P. tosana, 3.12 mm SL; (H) P. tosana, 23.2 mm SL. 198 BULLETIN OF MARINE SCIENCE. VOL. 60. NO. I. 1997

Figure 4. Larvae of Bothidae. (A) Bothus myriaster, 8.90 mm SL; (B) B. myriaster 23.8 mm SL; (C) B. pantherinus, 32.0 mm SL; (D) B. mancus, 24.6 mm SL; (E) B. oceLlatus, 16.2 mm SL. from Evseenko. 1976; (F) Tosarhombus octoculatus, 26.7 mm SL.

and II. The mouth is small and the eyes are elliptical, generally small and sub- tended by a lunate choroid tissue. The urohyal is widely exposed and the posterior basipterygial process is slender and borders the lower part of the abdominal cavity. Most of the abdominal cavity is filled with the gut and liver. The pelvic fin is FUKUI: ONTOGENY AND SYSTEMATICS OF BOTHIDAE 199

Figure 5. Larvae of Bothidae. (A) Arnoglossus tenuis, 24.0 mm SL; (B) A. japonicus, 34.6 mm SL; (C) A. yamanakai, 8.00 mm SL; (D) A. yamanakai, 49.0 mm SL; (E) A. sp. 1, 39.2 mm SL; (F) A. sp. 2, 35.0 mm SL; (G) A. debilis, ca. 59 mm SL, from Ahlstrom et al. 1984; (H) A. [aterna, 16 mm SL, from Russell, 1976. supported by a triangular cartilaginous anterior basipterygial process between the urohyal and posterior basipterygial process. After pelvic fin rays develop, those in the left fin move anteriorly to the fixed adult position, the first ray of the right fin being opposite to the second one of left fin in Taeniopsettinae, and to third or fourth (rarely second) in Bothinae. In 10 genera, minute short spines are devel- 200 BULLETIN OF MARINE SCIENCE, VOL. 60, NO. I, 1997

Figure 6, Larvae of Bothidae. (A) Monolene sessilicauda, 14,3 mm SL, from Futch, 1971; (B) Laeops nigromaculatus, 56.6 mm SL; (C) L. kitaharae, 5,00 mm SL; (D) L. kitaharae, 64,5 mm SL; (E) Chascanopsetta lugubris lugubris, 8,20 mm SL; (F) C. lugubris lugubris, 66,8 mm SL; (G) C. micrognathus, ]0,8 mm SL; (H) Parabothus sp., 22,8 mm SL, from Tsukamoto et aI., 1991. FUKUI: ONTOGENY AND SYSTEMATICS OF BOTHIDAE 201

oped on the posterior basipterygial process, urohyal, cleithrum, and epiotic. Body melanophores are generally absent or small. The air bladder disappears during development. During metamorphosis, the right eye migrates through a slit or hole formed below the origin of the dorsal fin base. Size at metamorphosis ranges from 13 to 125 mm, being the largest in the Pleuronectiformes. Many changes occur rapidly during metamorphosis, differentiation of pecrtoral fin rays, degeneration of spines, and appearance of many melanophores, etc. (Ozawa and Fukui, 1986: 334). Among the above characters, the extremely compressed body, elongated second dorsal fin ray in stages I and II, elliptical eye with choroid tissue, and asymmetric pelvic fin placement are the diagnostic characters for bothid larvae. Larval characters of the Bothidae are summarized in Table 2. Features of GenUs.-TAENIOPSETT1NAE.In the taeniopsettine genera, spines are developed on the posterior basipterygial process, urohyal, cleithrum, and epiotic. References are shown in Table 1.

Taeniopsetta Gilbert Larvae of stages llIb and IV of T. ocellata and T. radula are known. Diagnosis.-Body oval; three epiotic spines; masses of melanophores present on left side of body after end of stage llIb (about 60 mm SL); elongated dorsal fin ray short; metamorphosis occurring at about 60 mm SL; vertebrae 10 + 32-33 = 42-43.

Engyophrys Jordan et Bollman The early ontogeny of E. senta and E. sancti-Iaurentii is known. Diagnosis.-Body oval in stages II-IIIb; three or four epiotic spines until 8 mm SL, four after 8 mm SL; melanophores present on head, urohyal, and ventral margin of intestinal coiling only in stage I; elongated dorsal fin ray becomes short after about 10 mm SL; metamorphosis occurring at about 20 mm SL; vertebrae 10 + 27-31 = 37-41.

Trichopsetta Gill The early ontogeny of T. ventralis is known. Diagnosis.-Body oval in stage IlIa; two or three epiotic spines until 16 mm SL, three after 16 mm SL; masses of melanophores present on interspinous regions of dorsal and anal fins, and on midlateral line after 17 mm SL; elongated dorsal fin ray remains until about 21 mm SL; largest collected larvae 29 mm SL; vertebrae 10-11 + 30-33 = 40-43.

Features of the Genus Bothinae

Parabothus Norman Larvae of stage IIIb referable either to P. coarctatus or P. kiensis are known. Diagnosis.-Body moderately slender; rostrum above snout developed; spines present on posterior basipterygial process and urohyal; melanophores absent; second to 11th dorsal fin rays elongated; largest collected larvae 23mm SL; vertebrae 10 + 30-36 = 40-46. 202 BULLETIN OF MARINE SCIENCE, VOL. 60, NO, I, 1997

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Tosarhombus Amaoka Larvae of stage IIIb of T. octoculatus are known. Diagnosis.-Body oval; spines absent; melanophores present along interspine base of dorsal fin; length of elongated dorsal fin ray about 30% SL; largest collected larvae 27 mm SL; vertebrae 10 + 28-32 = 38-42.

Crossorhombus Regan The early ontogeny of C. kobensis and C. azureus (as C. kanekonis) are known. Crossorhombus kanekonis is a junior synonym of C. azureus (Hensley and Rand- all, 1993). Diagnosis.-Body oval in stages II-IIIb, moderately slender in stage IV; serrated spines present on posterior basipterygial process after stage IIIa; melanophores absent on left side of body until stage ITIb; elongated dorsal fin ray becomes shorter at about 6 mm SL, at completion of notochord flexion; fin rays develop early, e.g., pelvic fin rays at about 5 mm SL, dorsal and anal fin rays at 6-7 mm SL; left pelvic fin rays fixed in position at 6-7 mm SL; disappearance of air bladder at about 14 mm SL; metamorphosis occurring at 15-20 mm SL; vertebrae 10 + 24-27 = 34-37.

Engyprosopon Gunther The early ontogeny of E. grandisquama and E. multisquama, and three unidentified types (as sp. 1, sp. 2, and sp. 3) are known. These types seem to be referable to Japanese species as follows: sp. 1 E. xystrias; sp. 2 either E. mac- roptera or E. longipelvis; sp. 3 E. longipelvis (Ozawa and Fukui, 1986). The larvae identified as E. grandisquama by Pertseva-Ostroumova (1965) and Liew (1986, 1989) are correct to the generic, but not the species level (Ozawa and Fukui, 1986: 348; Fukui, 1991: 35). Diagnosis.-Body oval or moderately slender in stages IIIa and IIIb, and moderately slender in stage IV; spines present on posterior basipterygial process and urohyal, present or absent on cleithrum; melanophores absent up to stage IIIb, except in sp. 3; eyes largest among bothid genera; elongated dorsal fin ray present until stage IIIb, length being less than 60% SL; fin rays develop early, e.g., caudal fin rays at about 6mm SL, dorsal and anal fin rays at 7-9 mm SL, pelvic fin rays at 8-10 mm SL; left pelvic fin rays fixed in position at 10 mm SL; exposed part of urohyal broad; disappearance of air bladder at about 14 mm SL; largest collected larvae 15-20 mm SL; smallest size at metamorphosis among bothids, being 13-15 mm SL in E. multisquama; vertebrae 10 + 23-27 = 33-37.

Bothus Rafinesque The early ontogeny is known fairly well: in the Pacific, B. myriaster, B. mancus, and B. pantherinus; in the western Atlantic, B. ocellatus; in the Mediterranean B. podas. Some morphometric differences are recognized between larvae from the Indo-Pacific and Atlantic and Mediterranean. Diagnosis.-Indo-Pacific species: Body rounded (oval for the Atlantic and Med- iterranean species) in stages IlIa and IIIb, and either oval or rounded in stage IV, depth 80-90% SL (65-70%) in stage IIIb; the maximum width of interspinous regions of dorsal and anal fins 60-85% (about 55%) head length at end of stage IIIb; posterior basipterygial process long (short) and arched in stages IlIa and 204 BULLETIN OF MARINE SCIENCE. VOL. 60. NO. \, 1997

IIIb; spines absent; melanophores present on origin of dorsal fin from stage I to 17 mm SL of stage IIIb, and on finfold at tail tip in stage I; elongated dorsal fin ray and air bladder persistent until about 13 mm SL; fin rays except pectorals complete at 8-9 mm SL; position of left pelvic fin rays fixed, at stage IV; metamorphosis occurring at 27-40 mm SL; vertebrae 10 + 25-32 = 35-42.

Grammatobothus Norman Larvae for the following species are known: G. sp. (10.5-27.1 mm SL) from the western North Pacific (Ozawa and Fukui, 1986), G. polyophthalmus (3.6-19.0 mm SL) and G. pennatus (5.80-10.90 mm SL) from Australia (Liew, 1986, 1989). Although G. sp. seems to be either G. polyophthalmus or G. krempji, identification is not confirmed because the dorsal and anal fin ray counts fall into the region of overlap between the two species. The larvae of G. sp. resemble G. polyophthalmus and G. pennatus from Australia in having several elongated dorsal fin rays, but it differs from them in lacking melanophores along the myosepta, and having spines on the posterior basipterygial process. It is possible that spines on the posterior basipterygial process do not develop until about 27 mm SL (Fig. 2G, H). Diagnosis.-Body oval; spines present on posterior basipterygial process, second to fourth or fifth dorsal fin rays elongated (length of the longest ray less than 60% SL), and small ctenoid scales with one spinule at end of stage IIIb (27 mm SL); masses of melanophores present on interspinous regions of dorsal and anal fins, on midlateral line, and on dorsal and anal fins; metamorphosis occurring at about 27 mm SL; vertebrae 10 + 27-28 = 37-38.

Asterorhombus Tanaka This genus was comprised of only one Indo-West Pacific species, A. intermedius. Hensley (1984, 1986) placed Engyprosopon jijiensis in Asterorhombus. At the same time, Ozawa and Fukui (1986) described larvae of two types of Aster- orhombus and pointed out the occurrence of an undescribed species in the western North Pacific. Recently, Amaoka et al. (1994) recorded A. jijiensis from around Okinawa Prefecture, Japan; this species had been known from northern Mozam- bique and the Fiji Islands. The two types of larvae can not be identified, because meristic counts between A. intermedius and A. jijiensis are almost identical. The larvae identified as A. intermedius by Liew (1986, 1989) differ from the two types of larvae described by Ozawa and Fukui (1986) in lacking spines on the posterior basipterygial process, cleithrum, and epiotic and in having melanophores on the body. The larvae of Liew (1986, 1989) are probably species of Engyprosopon, because they resemble E. sp. 3 in melanophore pattern. Diagnosis.-Body ovaloI' moderately slender; spines present on posterior basipterygial process, urohyal, cleithrum, and epiotic (two-three spines); melanophores absent; fin rays develop early, e.g., caudal fin rays at 6-7 mm SL, dorsal and anal fin rays at 8 mm, pelvic fin rays at 11 mm SL; left pelvic fin rays fixed in position at 11-13 mm SL; exposed part of urohyal broad; posterior basipterygial process long; largest collected larvae 20 mm SL; vertebrae 10 + 25-26 = 35-36.

Psettina Hubbs The early ontogeny of P. iijimae, P. gigantea, and P. tosana are known. The larvae of P. iijimae and P. hainanensis described by Pertseva-Ostroumova (1965) FUKUI: ONTOGENY AND SYSTEMATICS OF BOTHIDAE 205

appear to be P. gigantea. The larvae identified by Amaoka (1976) as P. iijimae, P. gigantea, and P. tosana are P. tosana (see Ozawa and Fukui, 1986: 380). Diagnosis.-Body oval or moderately slender; spines completely absent or present on posterior basipterygial process and urohyal; up to 10 mm SL, melanophores present either on posterior wall of abdominal cavity, air bladder, or pectoral fin base; beyond 10 mm SL, melanophores present either along myosepta or midlat- eralline and along interspine bases (or on interspinous regions) of dorsal and anal fins; length of elongated dorsal fin ray 10-60% SL (no second dorsal fin ray elongated at stage IIIb in P. iijimae); fin rays develop early, e.g., caudal fin rays at about 7 mm SL, dorsal and anal fin rays at 8-10 mm SL, pelvic fin rays at 8- 12 mm SL; left pelvic fin rays fixed in position at about 12 mm SL; posterior basipterygial process short; disappearance of air bladder at about ]3 mm SL; metamorphosis occurring at 20-28 mm SL; vertebrae 10 + 26-30 = 36-40.

Lophonectes GUnther This genus is endemic to the south eastern Australia, Tasmania, and New Zea- land and is monotypic (L. gallus). Ahlstrom et al. (1984) figured a larva of stage IIIb (18.5mm SL) of L. gallus. Diagnosis.-Body oval; spines present on posterior basipterygial process; melanophores absent; vertebrae 10 + 32-33 = 42-43.

Arnoglossus Bleeker Early ontogeny of this genus is fairly well known: in the Pacific, A. tenuis, A. japonicus, and A. yamanakai, and two unidentified types (sp. 1 and sp. 2); in the Atlantic and Mediterranean, five species and one unidentified type. Several differences in morphology are recognized between the larvae from the Indo-Pacific and ones from the Atlantic and Mediterranean. The larvae identified provisionally as P. iijimae by Uchida (1936) and regarded as A. japonicus by Ochiai and Amaoka (1963) and Amaoka (1973) are A. yamanakai (see Ozawa and Fukui, 1986: 384). The larvae of A. waitei identified by Liew (1986,1989) appear to be Psettina gigantea (see Fukui, 1991: 77). Diagnosis.-Indo-Pacific species: Body slender or moderately slender, body depth 33-45% SL (40-55% for Atlantic species) in stage IIIb; rostrum above snout developed except A. tenuis (undeveloped); elongated dorsal fin ray string (thread)- like in shape, and greatly elongated, length 70-160% SL (less than 70%); spines absent (scale cteni present on body); largest larvae collected 24-60 mm SL (23- 33 mm SL); vertebrae 10-12 + 30-38 = 40-48 (10 + 23-35 = 33-45); melanophores present on elongated dorsal fin ray; caudal fin rays complete at about 10 mm SL, dorsal and anal fin rays at 9-12 mm SL, pelvic fin rays at 24 mm SL; left pelvic fin rays fixed in position at about 24-32 mm SL; posterior basipterygial process short.

Monolene Goode Larvae of stages II and IIIa of M. sessilicauda and M. antillarum are known. Evseenko (1977) suggested that the larvae described by Futch (1971) as M. sessilicauda appear to be M. antillarum. Diagnosis.-Body moderately slender, rostrum above snout developed; spines absent; melanophores present on posterior wall of abdominal cavity, on right side of head, and on elongated dorsal fin ray; elongated dorsal fin ray string-like in 206 BULLETIN OF MARINE SCIENCE, VOL. 60, NO. I, 1997

shape, length 40-65% SL; caudal fin rays complete at 12-14 mm SL, dorsal and anal fin rays at about 14 mm SL; posterior basipterygial process short; largest larvae collected 30 mm SL; vertebrae 10 + 28-38 = 38-48.

Laeops Gunther Larvae of the Japanese L. kitaharae and L. nigromaculatus and the Indonesian L. parviceps are known. The larva of Parabothus thackwrayi described by Smith (1967) is Laeops (Hensley, 1984; Tsukamoto et aI., 1991). Diagnosis.-Body slender; gut protruding at more than 10 mm SL; rostrum above snout developed; spines absent; melanophores in small masses on ventral contour of caudal region, air bladder, and midlateral line up to stage IlIa; melanophores present on entire left side,. and three or four longitudinal rows of pigment on dorsal and anal fins in stage IIIb; eyes small in stage IIIb; elongated dorsal fin ray string-like in shape, long, length 60-130% SL; longest rays of dorsal and anal fins (except elongated dorsal ray) longer than head; caudal fin rays complete at about 14mm SL, dorsal, anal, and pelvic fin rays at 14-24 mm SL; left pelvic fin rays fixed in position at about 48 mm SL; length of posterior basipterygial process about 80% head length in stages II and IlIa, 150-260% in stage IIIb; size at metamorphosis 56-90 mm SL; vertebrae 11-12 + 37-41 = 48-53.

Chascanopsetta Alcock Early ontogeny of the Japanese C. lugubris lugubris and C. micrognathus are known. Diagnosis.-Body moderately slender and greatly thin; abdominal cavity spacious and gut broadly protruding in stage IIIb; rostrum above snout developed; spines absent; melanophores present on posterior wall of abdominal cavity, interspine bases of dorsal and anal fin rays and along midlateral line (the last ones being embedded in muscle beyond 15 mm SL); elongated dorsal fin ray string-like, length 10-80% SL; maximum lengths of dorsal and anal fin rays (except elongated dorsal ray), twice head length; formation of fin rays latest to complete in bothids, pelvic fin rays at 11 mm SL, dorsal, anal, and caudal fin rays at 11-17 mm SL; left pelvic fin rays fixed in position after metamorphosis; length of posterior basipterygial process 130-270% head length in stage lIIb, longest among bothids; metamorphosis occurring at 120-125 mm SL, pelagic larvae largest among bothids; vertebrae 15-18 + 34-44 = 50 + 61.

PHYLOGENETIC INFORMATION PROVIDED BY LARVAL CHARACTERS Intergeneric relationships for the family Bothidae based on larval characters are here examined using cladistic analysis in order to obtain cryptic information among adult systematics. As Hensley and Ahlstrom (1984) and Chapleau (1993) pointed out, the family Bothidae is monophyletic owing to its sharing the several synapomorphies: for adults, (1) loss of the preorbital on the blind side, (2) presence of myorhabdoi, and (3) asymmetrical states of pelvic-fin morphology; for larvae, (1) large size at metamorphosis, (2) eye migration below the dorsal fin, (3) dorsal fin origin anterior to eyes, (4) elongated second dorsal fin ray before completion of notochord flexion, and (5) lack of preopercular spines. On the basis of caudal skeleton morphology, Hensley and Ahlstrom (1984) indicated the presence of a bothoid group including Pleuronectinae, Paralichthyidae (except Te- phrinectes and Thysanopsetta), Scophthalmidae, one citharid (Brachypleura) and FUKUI: ONTOGENY AND SYSTEMATICS OF BOTHIDAE 207

Table 3. Matrix of character states. Character numbers correspond to the text; the letters "IP" and "AM" show Indo-Pacific and Atlantic and Mediterranean.

Character

2 4 6 7 9 10 II 12 13 14 15 16

Outgroup 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Taeniopsetta 0 0 0 0 0 1 1 0 0 0 0 1 I 1 0 0 Engyophrys 0 0 0 0 0 0 1 0 0 0 0 I 1 I 0 0 Trichopsetta 0 0 0 0 0 0 I 0 0 0 0 I I I 0 0 Parabothus 0 0 0 0 1 0 0 0 0 0 I 0 I 1 0 1 Tosarhombus 0 0 0 0 0 0 1 0 0 0 I 0 0 0 0 1 Crossorhombus 0 0 0 0 0 0 I 0 0 0 1 0 1 0 0 1 Engyprosopon except E. sp. 2 0 0 0 0 0 0 1 0 0 0 1 1 1 1 0 I Engyprosopon sp. 2 0 0 0 0 0 0 1 0 0 0 1 0 I I 0 I IP Bothus 1 0 1 0 0 0 I 0 0 0 I 0 0 0 0 I AM Bothus 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Grammatobothus 0 0 0 0 0 0 0 0 0 0 1 0 I 0 0 1 Asterorhombus 0 0 0 0 0 0 1 0 0 0 0 I I 1 0 I Psettina except P. gigantea 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Psettina gigantea 0 0 0 0 0 0 1 0 0 0 1 0 1 1 0 I Lophonectes 0 0 0 0 0 0 1 0 0 0 1 0 I 0 0 1 IP Arnoglossus tenuis 0 0 0 0 0 0 1 I 0 0 1 0 0 0 0 1 IP Arnoglossus except A. tenuis 0 0 0 0 1 I 1 I 0 0 1 0 0 0 0 1 AM Arnoglossus 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Monolene 0 0 0 0 1 0 I 1 0 0 1 0 0 0 0 0 Laeops 0 0 0 1 1 1 1 I 1 I 1 0 0 0 0 1 Chascanopsetta 0 1 0 1 1 1 1 I 1 1 1 0 0 0 I 1

Bothidae. I adopted the family Paralichthyidae as outgroup in the present analysis, because Chapleau (1993) showed that the Paralichthyidae shares many synapomorphies with the Bothidae than other bothoids. The larval characters of the outgroup followed Ahlstrom et al. (1984). The polarity of a character was deter- mined by outgroup comparison. Cladistic analysis was computed using the com- puter program, PAUP (Phylogenetic Analysis Using Parsimony) with accelerated transformation (ACCTRAN), delayed transformation (DELTRAN), and minimum F-value (MINF) version 3.1. Sixteen morphological differences for character analysis are recognized within the bothid larvae, and are briefly described below. In each, the plesiomorphic state (0) is indicated first, and the apomorphic state (1) and the distribution of ones among taxonomic units is indicated second and thirdly. Finally, the state of the outgroup is mentioned in brackets. The character matrix is presented in Table 3. 1. Body. Slender to oval, depth 30-75% SL (0). Rounded 80-90% SL (1). Indo- Pacific Bothus. [Body is slender to moderately slender, depth less than 50% SL.] 2. Body thickness. Thin (0). Greatly thin (1). Chascanopsetta. [Body is moderately thin.] 3. Maximum width of interspinous regions of dorsal and anal fins. Narrow (0). Broad (1). Indo-Pacific Bothus. [The width is narrow.] 4. Gut. Normal (0). Protruding (1). Laeops and Chascanopsetta. [Gut is normal, unprotruding.] 5. Rostrum above snout. Absent (0). Present (1). Parabothus, Indo-Pacific Ar- noglossus except A. tenuis, Monolene, Laeops, and Chascanopsetta. [The character is absent.] 6. Size at metamorphosis (or largest collected larvae). Less than 40 mm SL (0). 208 BULLETIN OF MARINE SCIENCE. VOL. 60. NO. I. 1997

More than 40 mm SL (1). Taeniopsetta, Indo-Pacific Arnoglossus except A. tenuis, Laeops, and Chascanopsetta. [Size at metamorphosis is 8 mm to 15 mm SL in most paralichthyids (rarely more than 30mm SL).] 7. Number of elongated dorsal fin rays. 3-10 (0). 1 (l). All bothid genera, except for Grammatobothus and Parabothus. [Most paralichthyids have several elongated dorsal fin rays, e.g., 5-8 in Syacium, 8-11 in Cyclopsetta.] 8. Shape of elongated dorsal fin rays. Thread (0). String (1). Indo-Pacific Ar- noglossus, Monolene, Laeops, and Chascanopsetta. [The character is thread.] 9. Lengths of dorsal and anal fin rays (except elongated dorsal fin rays). Less than head length (0). More than head length (1). Laeops and Chascanopsetta. [The lengths are short, less than head length.] 10. Length of posterior basipterygial process. Less than 130% head length (0). More than 130% head length (1). Laeops and Chascanopsetta. [The length is short, less than head length.] 11. Epiotic spines. Present (0). Absent (1). All bothid genera, except for taeniopsettine genera and Asterorhombus. [Paralichthys olivaceus develops one spine on the epiotic.] 12. Cleithral spines. Absent (0). Present (l). All taeniopsettine genera, Astero- rhombus and Engyprosopon except sp. 2. [The spines are absent.] 13. Posterior basipterygial spines. Absent (0). Present (1). All taeniopsettine genera, Asterorhombus, Crossorhombus, Engyprosopon, Lophonectes, Parabo- thus, Psettina gigantea and Grammatobothus. [The spines are absent.] 14. Urohyal spines. Absent (0). Present (1). All taeniopsettine genera, Astero- rhombus, Engyprosopon, Parabothus, and Psettina gigantea. [The spines are absent.] 15. Abdominal cavity. Narrow (0). Spacious (1). Chascanopsetta. [Abdominal cavity is narrow and norma!.] 16. Pelvic fin morphology. The origin of the left pelvic-fin base is adjacent to the cleithrum or slightly anterior to one (0). The origin of the left pelvic-fin base is on the urohyal, which is well in advance of the cleithrum (1). [The origin of the left pelvic-fin base is posterior to the cleithrum.]

A cladistic analysis of 16 characters results in 25 equally parsimonious trees (length 23; consistency index 0.696). A strict consensus tree for these 25 trees is shown in Figure 7. Character states using ACCTRAN, OELTRAN, and MINF optimizations are mapped on tree (they are common to these three results). The strict consensus tree indicates resolved two monophyletic groups (AI, taeniopsettine genera (Taeniopsetta + Engyophrys + Trichopsetta) + Asterorhombus + Engyprosopon except sp. 2; A2, Indo-Pacific Arnoglossus tenuis + Indo-Pacific Arnoglossus + Laeops + Chascanopsetta + Monolene) and unresolved polytomy at basal nodes. The monophyletic group Al shares two synapomorphies, c1eithral and urohyal spines (char. ]2 and 14). Branch Cl comprises taeniopsettine genera and Asterorhombus with a plesiomorphy, presence of epiotic spines (reversal, char. 11). Branch C2 comprises only Engyprosopon except sp. 2. Branch 01 comprises taeniopsettine genera with a plesiomorphy, slightly elongated left pelvic-fin base (reversal, char. 16). Branch 02 comprises only Asterorhombus. The monophyletic group A2 shares a synapomorphy, string-like elongated dorsal fin ray (char. 8). Branch Bl comprises only Indo-Pacific Arnoglossus tenuis. Branch B2 comprises Indo-Pacific Arnoglossus except A. tenuis, Laeops, Chascanopsetta, and Mono- [ene, and shares a synapomorphy, rostrum above snout (char. 5). Branch C3 comprises Indo-Pacific Arnoglossus, Laeops, and Chascanopsetta, and shares a synapomorphy, larger metamorphosis size (char. 6). Branch C4 comprises only Mono- FUKUI: ONTOGENY AND SYSTEMATICS OF BOTHIDAE 209

Paralichthyidae (OUTGROUP) 6 Taeniopsetta

Enavoohrvs, Trichopsetta 12 14 A1 Asterorhombus 02 5 7R EnayprosoDon (exceptsp.2) C2 Parabothus Tosarhombus, AM Bothus, Psettina (exceptP. aiaanteal, IAM Arnoalossus Crossorhombus, r Lophonectes Enavprosopon sp. 2, r Psettina aiaantea IP Bothus

Grammatobothus

IP Arnoalossus tenuis 03 IP Arnoalossus (exceptt,. tenuisl Laeops 5 ChascanoDsetta

82 ----- Monolene C4

Figure 7. Strict consensus tree of bothid based on larval characters. Character states indicate distribution optimized using PAUP with ACCTRAN, DELTRAN, and MINF options. Open boxes represent plesiomorphic states. Solid boxes represent apomorphic states. Numbers correspond to the text; the letter "R" indicates reversal character state transformation; the letters "IP" and "AM" show lndo- Pacific and Atlantic-Mediterranean. lene with a plesiomorphy, slightly elongated left pelvic-fin base (reversal, char. 16). Branch D3 comprises only Indo-Pacific Arnoglossus except A. tenuis. Branch D4 comprises Laeops and Chascanopsetta. It shares three synapomorphies, protruding gut, longer dorsal and anal fin rays and posterior basipterygial process (char. 4,9, and 10). On the basis of the present analysis, Asterorhombus and Engyprosopon except sp. 2 of the subfamily Bothinae are sister group for the subfamily Taeniopsettinae. Arnoglossus is not monophyletic, because Atlantic-Mediterranean Arnoglossus has not a synapomorphy, string-like elongated dorsal fin ray (char. 8) which Indo- Pacific Arnoglossus including A. tenuis shares, and monophyletic group A2 for Indo-Pacific Arnoglossus excludes Atlantic-Mediterranean Arnoglossus. A. tenuis and other species also are not monophyletic in Indo-Pacific Arnoglossus, because A. tenuis has not two synapomorphies, rostrum above snout and larger metamorphosis size (char. 5 and 6) which other Indo-Pacific species share. Amaoka (1969: 125) mentioned that Arnoglossus tenuis may represent a distinct genus owing to their adults having close-set and scarcely enlarged anterior teeth, gill rakers with- out serrations, and lower meristic counts (e.g., scales in lateral line 48-57) etc. Hensley (1986) also mentioned that Arnoglossus is not probably monophyletic on 210 BULLEHN OF MARINE SCIENCE, VOL. 60, NO.1, 1997 the basis of adult characters, although the reason was not given. Reexamination of adult systematics is necessary in Amoglossus. Simultaneously, more effort should be used to examine the larvae of Indo-Pacific Amoglossus species which are not known, because some species resemble A. tenuis in having the characters as shown above (e.g., A. macrolophus (as A. tapeinosoma) Norman, 1934; Amao- ka, 1971b; Amaoka et aI., 1992; Arai and Amaoka, 1966). Since Engyprosopon is a presence or absence apomorphy, larval cleithral spines (char. 12), it seems that this genus is not monophyletic. Hensley and Ahlstrom (1984) mentioned that the subfamily Taeniopsettinae may not be monophyletic on the basis of adult characters. However, it is shown that three genera of Taeniopsetta, Engyophrys, and Trichopsetta for which larvae are known are monophyletic in the present analysis. Amaoka (1969) present{:d a scheme of intergeneric relationships for Japanese bothids based on adults characters. Amaoka's analysis was eclectic, e.g., a com- bination of phenetic and cladistic methods and did not include Engyophrys, Tri- chopsetta, Grammatobothus, Lophonectes and Monolene for which larvae are known. I compare the two monophyletic groups based on larvae with Amaoka's scheme. As a result, some differences are recognized between them. For example, the sister group for Asterorhombus is taeniopsettine genera and the sister group for Indo-Pacific Amoglossus (except A. tenuis) is Laeops and Chascanopsetta in the present analysis. However, the sister group for Amoglossus is Psettina and the sister group for Amoglossus and Psettina is Asterorhombus in Amaoka's scheme. Those differences may indicate that Amaoka's scheme is incorrect owing to his analysis being eclectic. A cladistic analysis of bothid interrelationships based on adult characters should be awaited. Several derived characters are thought to be associated with the pelagic function in the bothid larvae, e.g., larger metamorphosis size, thread-like elongated dorsal fin ray, and longer dorsal and anal fin rays etc. Larger metamorphosis size (body elongation) correlated with high vertebral counts, may be related to an active pelagic existence where the larvae are not totally passive relative to the pelagic environment (relationship between maximum size of larvae (Y) and vertebral counts (X), Y = 3.6X-1l2.3, r = +0.888; Ozawa and Fukui, 1986: 329).

ACKNOWLEDGMENTS

I am very grateful to Dr. M. Okiyama, Ocean Research Institute, University of Tokyo, for providing me the opportunity to contribute to this important symposium and for his valuable advice and critical reading of the manuscript. I would like to thank Dr. T. Ozawa, Kagoshima University and Dr. K. Amaoka, Hokkaido University, for their valuable suggestions. I also wish to express to my gratitude Dr. S. Shirai, Seikai regional Fisheries Research Laboratory for his valuable advice and help. For reading drafts of the manuscript and providing me with very useful comments and suggestions, I thank anonymous reviewers. Finally, my thanks go to K. Kitano of Japan NUS Co. ltd., for his help.

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DATEACCEPTED: February 23, 1996.

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