A Cladistic Reassessment

BULLETIN OF MARINE SCIENCE, 52(1): 516-540,1993

PLEURONECTIFORM RELATIONSHIPS: A CLADISTIC REASSESSMENT

Franqois Chapleau

ABSTRACT Flatfish monophyly, interrelationships and intrarelationships are examined. The order Pleuronectiformes is monophyletic on the basis ofthree synapomorphies: cranial asymmetry associated with ocular migration, advanced position of the dorsal fin over the cranium and presence of a recessus orbitalis. Characters used to postulate a relationship between percoids and flatfishes are found to be plesiomorphic and the sister group of flatfishes remains unknown. The monophyletic status of all suprageneric taxa of Pleuronectiformes is reexamined and a cladistic analysis is performed using a character state matrix of 39 polarized morphological (mainly osteological) characters, Eighteen equally parsimonious trees were generated. A consensus tree was constructed and discussed in detail. It is concluded that phylogenetic knowledge within flatfishes remains very incomplete. The monophyletic status of speciose taxa such as the Pleuronectinae and Paralichthyidae will have to be reassessed before any fruitful statement of relationships can be formulated.

Hensley and Ahlstrom (1984) and Ahlstrom et al. (1984) provided the most recent synthesis on flatfish classification (Table 1), phylogeny (Fig. 1) and larval morphology. Hensley and Ahlstrom (1984) reexamined the homology of characters traditionally used to define higher flatfish taxa (i.e., suborders, families and subfamilies). They also introduced new characters and suggested the formation of a "bothoid group" made up of an array of dextral and sinistral flatfishes. The monophyletic status of all higher taxa within flatfishes was examined, but no attempts were made to build a cladogram depicting flatfish intrarelationships. This article reviews studies on flatfish phylogeny since the synthesis of Hensley and Ahlstrom (1984) and addresses the following issues: (1) Are flatfishes a monophyletic group? (2) In light of recent advances in percoid phylogeny, what can be said of Regan's (1910) and Norman's (1934) assertions that Psettodes, the most primitive flatfish, is an asymmetrical percoid that could almost be placed in the family Serranidae? (3) What is the monophyletic status of higher taxa (suborders, families and subfamilies) within the Pleuronectiformes and what do we know of their interrelationships? To visualize current knowledge on flatfish relationships, a cladistic analysis of familial and subfamilial relationships is performed using available ordered and polarized morphological characters. Except for the cladogram of Lauder and Liem (1983) based on a more limited data set, this cladistic analysis represents the first attempt to incorporate all available information to build a cladogram of intrarelationships within the Pleuronectiformes.

MATERIALS AND METHODS

The following taxa were used in the cladistic analysis (see below for justification): Psettodidae, Lepidoblepharon, Brachypleura, Citharoides, Citharus, Scophthalmidae, Paralichthyidae, Bothidae, Pleuronectinae, Poecilopsettinae, Rhombosoleinae, Samarinae, Achiridae, Soleidae, and Cynoglos- sidae. All polarized characters used in the analysis are listed in the appendix. The character matrix is presented in Table 2. Characters examined in Chapleau (1988a, 1988b) and Chapleau and Keast (I988) that depicted the intra- and interrelationships of the Soleidae, Archiridae and Cynoglossidae with the Pleuronectoidei are part of the analysis except for the 27 characters used to build the hypothesis of subfamilial

516 CHAPLEAU: PLEURONECfIFORM RELATIONSHIPS 517

Table I. Classification of the Pleuronectiformes according to Ahlstrom et al. (1984)

Order Pleuronectiformes Suborder Psettodoidei Family Psettodidae Suborder Pleuronectoidei Family Citharidae Subfamily Brachypleurinae Subfamily Citharinae Family Scophthalmidae Family Paralichthyidae Family Bothidae Subfamily Taeniopsettinae Subfamily Bothinae Family Pleuronectidae Subfamily Pleuronectinae Subfamily Poecilopsettinae Subfamily Paralichthodinae Subfamily Samarinae Subfamily Rhombosoleinae Suborder Soleoidei Family Soleidae Subfamily Soleinae Subfamily Achirinae Family Cynoglossidae Subfamily Symphurinae Subfamily Cynoglossinae

relationships within the cynoglossids (Chapleau, 1988a). Autapomorphies of taxa are not incorporated in the data matrix as they do not help in clarifying interrelationships. Morphological data from several studies were used to construct the character matrix. Sakamoto (1984) observed the distribution of 78 characters for 77 species of Pleuronectidae. Amaoka (1969) noted the distribution of 48 characters for 41 species belonging to the Psettodidae, Citharidae, Par- alichthyidae and Bothidae. In Amaoka (1969), the following regions of the body were examined: cranium, orbital bones (infraorbitals), branchial arches, urohyal, vertebrae and accessory bones, and the caudal skeleton and fin. Features of phylogenetic importance, with known character state distribution in the Pleuronectidae, Achiridae, Soleidae and Cynoglossidae and not found in Amaoka (1969), were examined in some cleared and stained specimens (see list of material in Chapleau, 1986). Osteological data on Brachypleura were extracted from Amaoka (1972). Additional osteological and morphological data were taken from Hubbs (1945) and Hensley and Ahlstrom (1984). . The level of universality adopted in the present study is subfamilies and families. Only characters with the appropriate level of generality were included in the analysis. Because monophyly remains a problem at the familial and subfamiliallevel (see below), a set of conditions was defined to include a character in the analysis. The following types of characters were not included in the analysis: (I) Characters with an apomorphic state restricted to a few species and genera within a large taxonomic unit. These characters will eventually be useful in defining relationships at the genus and tribe levels but they do not add information on relationships at higher levels of universality. (2) Characters with two states (plesiomorphic and apomorphic) found in members of a family or subfamily but with one state found only in a few species. The more common (apomorphic or plesiomorphic) character state was attributed to the taxonomic unit. Exceptions to character state assignments are all listed in the character list (see appendix). This criterion was necessary because some families and subfamilies included in the analysis are obviously nonmonophyletic and will have to be redefined in the future. (3) Characters for which the polarity could not be established without a reasonable doubt. This was the case for the position and degree of asymmetry of pelvic fin bases, the ordering and polarity of ocular asymmetry within the flatfishes, and the type of optic chiasma (see Hensley and Ahlstrom (1984) for discussion of these characters). The character state matrix was analyzed using Hennig86 (version 1.5). All trees of equal length were found (procedure ie) and a Nelson consensus tree (procedure nelsen) was obtained. Psettodes, the most plesiomorphic flatfish, was used as the primary outgroup to define interrelationships within the Pleu- 518 BULLETINOFMARINESCIENCE,VOL.52, NO.1, 1993

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Figure 1. Hypothesis of interrelationships of pleuronectiform fishes presented in Hensley and Ahl- strom (1984). It is based on Norman (1934, 1966), Hubbs (1945) and Amaoka (1969). ronectoidei. Basal percoids (Johnson, 1980, 1984) and Beryciformes (Zehren, 1979; Keene and Tighe, 1984) were used as secondary outgroups. A hypothetical ancestor with plesiomorphic states for all characters was incorporated in the analysis. This consensus tree is discussed in detail. As already indicated, some taxa used in this analysis are, admittedly, not monophyletic and will have to be redefined in the future. However, because all the information on characters, their ordering and their polarity is presented, it is suggested that the hypothesis found in this study provides a good summary of our current knowledge of relationships. List of Materials. - The museum codes follow Leviton et al. (1985). Severa] X-rays were made from specimens preserved in alcoholic solutions. Some cleared and stained specimens were also examined. PSETTODIDAE.Psettodes ANSP 145394, cleared and stained (CS), 61 mm SL, 25°II'N 66°20'E. ANSP 145397, 5 spec., 112-154 mm SL, 13°24'S 48°42'E. ANSP 145396,132 mm SL, 13°24'30"S 48°39'30"E. ANSP 145399,6,109-170 mm SL, 15°08'N 94°54'E. ANSP 145395, 104 mm SL, 15°08'N 95°54'E. USNM 286356, 2, 111-132 mm SL, Ghana, Tema-Teshie Bay. CITHARIDAE.Citharus linguatula USNM 236123, 13, 100-1]9 mm SL, 05°30'30"N 09°44'30"W. Citharoides cf. macrolepidotus AMS 1.25801-016, 6, 85-156 mm SL, Australia, Queensland, just N of Townsville. Lepidoblepharon sp. AMS 1.20118-012, 6, 103-166 mm SL, Australia, New South Wales, E of Wooli. Brachypleura novaezeelandiae USNM 236122, 33, 74-94 mm SL, Philippines, purchased at Manila fish market. SCOPHTHALMIDAE.Scophthalmus aquosus ANSP 150131, I CS out of65, 75 mm SL, USA, New Jersey. ANSP 166257,6 X-rays, 70-93 mm SL, USA, New Jersey. Scophthalmus maeoticus ANSP 100071, 3 X-rays, 103-130 mm SL, Rumania, Black Sea at Sulina. Lepidorhombus boscii USNM 236124, CS, 113 mm SL, Mediterranean Sea, olfTunisia. Phrynorhombus unimaculatus ANSP 872932, 4 X-rays, 65-97 mm SL, Italy (Bonaparte Collection). Zeugopterus punctatus USNM 17361, X-ray, 157 mm SL, Norway, Bergen.

Are F1atfishes Monophyletic? Regan (1910), Norman (1934), Hubbs (1945), Li (1981), Lauder and Liem (1983) and Hensley and Ahlstrom (1984) considered the Pleuronectiformes a monophyletic group. Kyle (1921), Chabanaud (1949) and Amaoka (1969) believed, for different reasons, that the order was polyphyletic, having originated from different groups of symmetrical fishes. CHAPLEAU: PLEURONECTIFORM RELATIONSHIPS 519

Table 2. Matrix of character states (list of characters in appendix)

Character

Taxon 1-10 11-20 21-30 31-39 Psettodidae 1110000000 0000001000 0100000000 000000000 Lepidoblepharon 1111111111 1010000000 0010000000 000000000 Citharus 1111111111 1000000000 0010000000 000110000 Citharoides 1111111111 1010000000 0010000000 000010000 Brachypleura 1111111111 1000001100 0000011100 000110000 Scophtha1 midae 1111111111 1000010000 0111011100 000110000 Paralichthyidae 1111111111 1010010000 0011011100 000111000 Bothidae 1111111111 1010010100 0011111100 000111000 Pleuronectinae 1111111111 1010010000 0111011100 000111000 Poeci1opsettinae 1111111111 1010001100 0111200000 000111000 Rhomboso1einae 1111111111 1110100100 0111200000 000111000 Samarinae 1111111111 1011111111 1011200000 000111000 Achiridae 1111111111 1110101111 1111200000 000011111 Soleidae 1111111111 1111101111 2111200011 111111121 Cynoglossidae 1111111111 1111001111 2111200011 111111121

Hensley and Ahlstrom (1984) did not address this issue in detail but indicated that a hypothesis of monophyly was still the most parsimonious explanation for the origin of flatfishes. They added, however, that the monophyly of flatfishes was still an open question. They also recognized the loosely-defined "percomorphs" (sensu Rosen and Patterson, 1969; Rosen, 1973) as the sister group of the flatfishes. Kyle (1921) and Chabanaud (1949) shared similar views on flatfish relationships. They considered the most plesiomorphic flatfish, Psettodes, as the descen- dant of a recent percoid ancestor and as the most recent flatfish. They suggested that highly specialized flatfishes, such as cynoglossids, should be viewed as ancient groups within the order. Cynoglossids would have evolved from an ancestral group at the origin of taxa such as the Macrouridae and the Trachypteridae (Kyle, 1921) or from a "hypothetical preperciform" group (Chabanaud, 1949). Chabanaud (1949) agreed with Kyle (1921) and pushed the reasoning a step further by con- sidering all major flatfish taxa as independent offshoots of an evolving "preperciform" lineage (Fig. 2). These polyphyletic concepts are based on the assumption that because Psettodes shares several similarities with recent "perciforms," it must also share a unique and common ancestor with a recent representative of this group. As for highly specialized groups, such as the Cynoglossidae, their level of morphological spe- cialization is such that only a common origin with a very primitive or ancient "preperciform group" would have allowed sufficient time for the substantial morphological modifications to occur. This reasoning implies that independent offshoots of flatfishes would share unique features with different groups of symmetrical fishes. Neither Chabanaud (1949) nor Kyle (1921) provide tangible evidence to this effect. Amaoka (1969) considered Psettodes as the most "primitive" flatfish, but pro- posed in a polyphyletic origin of the order from an ancestral percoid stem. But, as did Chabanaud (1949), Amaoka does not define clearly the "percoid stem." He argued, as did Chabanaud (1949), that the Bothidae might be an independent lineage of flatfishes because ofthe presence of a complex system of intermuscular bones shared with primitive teleosts. Chabanaud (1949) provided a similar ar- gument for the pleuronectid Samarinae (Samaridae in his paper). Hensley (1977) 520 BULLETIN OF MARINE SCIENCE, VOL. 52, NO.1, 1993

...ACNYIflQf'TIJRfG1AN ... SCOPHTHALMIDAE·" PARALICHTHYIDAE .. POECll-OPSEITIDAE .. RHOMBOSOLEIDAE .. "ACHIRIDAE PLEURONECTIDAE . --SOLEIDAE INTERMUSCULAR BONES ...... ABSENT--..... BOTHIDAE .. ---CYNOGLOSSIDAE

SAMARIDAE - .. SYMPHURIDAE

Figure 2. Modified diagram taken from Chabanaud (1949) illustrating a polyphyletic origin for flatfishes. It suggests the independent origin of flatfish groups from an evolving preperciform lineage. Groups on the left have a monomorphic optic chiasma; groups on the right have a dimorphic type of optic chiasma. and Hensley and Ahlstrom (1984) have reexamined the intermuscular bone complex and have convincingly refuted Amaoka's contention. The important point is that no hypotheses of relationship based on the distribution of derived character states between monophyletic groups of flatfishes and monophyletic groups of symmetrical fishes have been formulated. Until such relationships are clearly established or until the homology of asymmetry within the flatfishes is refuted, it is unjustified to consider flatfishes as being polyphyletic. Moreover, three characters support the concept of a monophyletic Pleuronec- tiformes: i) Ontogeny Characterized by Migration of One Eye. - This process results in the typical asymmetrical cranium of adult flatfishes. The homology of this character was substantiated by Brewster (1987) when she indicated that flatfish asymmetry is always accompanied by the relocation of the anterior blind side frontal to the ocular side and by an enlargement of the blind side lateral ethmoid. This structural consistency is universal in flatfishes (see illustrations in Chapleau and Keast, 1988; Chapleau, 1988a; Sakamoto, 1984; Amaoka, 1969). ii) Anterior Position of the Origin of the Dorsal Fin. -All flatfishes have a dorsal fin that overlaps the neurocranium and sometimes extends in front of the jaws. Psettodes is the only flatfish with an anterior spinous portion (9-11 spines, N = 15) to the dorsal fin. The spinous portion is the first fin structure to form in larvae and is made of long spines that are lost during metamorphosis (Leis and Rennis, CHAPLEAU: PLEURONECTIFORM RELATIONSHIPS 521

dorsal

pterygiophore

proximal Pterygiophore of dorsal fin

neural spine of pre caudal vertebra 1

Figure 3. Eyed-side lateral view of the anterior portion of the dorsal fin in Psettodes erumei. The neural spine of the first precaudal vertebra is closely adjoined to the occipital region of the cranium. Scale = 3 mm.

1983). The posterior soft-rayed portion has 39-43 rays (N = 15) and is continuous with the spinous portion. The anteriormost proximal pterygiophore (Fig. 3) has its ventral aspect situated just posterior to the neural spine of the first precaudal vertebra, which is tightly attached to the occipital region of the cranium. Since the first pterygiophore is at an angle with the cranium, the first spine has its origin over the cranium. There are five to seven proximal pterygiophores in the inter- neural space between the first and second neural spines. The first proximal pterygiophore is enlarged and supports a supernumerary spine. No supraneurals (sensu Mabee, 1988) were found in any flatfishes. Johnson (1984) indicated that, in percoids, a compound first proximal pterygiophore with two supernumerary spines is frequently retained in anterior shifts of the dorsal fins. He suggested that forward shifts are probably the result of anterior displacements of proximal pterygiophores instead of the result of trans- formations of pre-existing supraneurals. If we consider the "percoids" (sensu Johnson, 1984) as a possible sister group of flatfishes, it seems reasonable to postulate that the forward shift of the dorsal fin observed in Psettodes resulting in the grouping of at least five proximal pterygiophores anterior to the second neural spine and the probable disappearance of supraneurals is apomorphic and has led to the apomorphic state found in all Pleuronectiformes. Consequently, the advanced position of the dorsal fin is a synapomorphy for the order. Two hypotheses can explain the disappearance of spines in the dorsal fin of flatfishes(except Psettodes): (a) a "regression" hypothesis where the spinous portion of the fin regressed and is followed by an anterior shift of the soft-ray portion of the fin and (b) a "transformation" hypothesis where spines transformed into soft rays before or during the anterior shift of the dorsal fin. Because most flatfishes show two soft rays on the first enlarged proximal pterygiophore of the dorsal fin, the "transformation" hypothesis is more plausible. iii) Presence of a Recessus Orbitalis. -Flatfishes can protrude their eyes above the body surface, allowing them to see while buried in the substrate. Holt (1894), Cole and Johnstone (1902) and Norman (1934) mentioned the presence of a recessus orbitalis, an accessory organ associated with the eyes, which is assumed to be possessed by all flatfishes, including Psettodes. The structure is described in detail in Cole and Johnstone (1902). It is a muscular sac-like evagination situated 522 BULLETIN OF MARINE SCIENCE, VOL. 52, NO. I, 1993 in the membranous wall of the orbit. It can be filled with fluid making the eyes protrude above the surface of the head. Retraction of the eye is accomplished by ocular muscles. The presence of this recessus orbitalis is a synapomorphy for the order. A detailed survey for this structure amongst flatfishes is needed to confirm its inferred widespread distribution.

Is Psettodes an Asymmetrical Percoid That Could Almost Be Placed in the Serranidae? Regan (1910, 1929), Norman (1934), Hubbs (1945) and Amaoka (1969) listed characters indicating a relationship between Psettodes and the Perciformes. These statements of a relationship are complicated by the lack of traits common to all families of Perciformes (Gosline, 1971 ; Johnson, 1984). Although Regan (1910) in his review ofpleuronectiforms mentioned that: "Pset- todes is simply an asymmetrical percoid" (p. 486), the Percoidei are now viewed as a regrouping of generalized perciforms that cannot be placed in any other perciform suborders (Johnson, 1984). The traits that have been used in the literature to postulate a relationship between Psettodes and the Percoidei are: presence of spines in dorsal (about 10 spines) and anal fins, thoracic position and the 1-5 fin-ray formula for pelvic fin, lateral and vertical pectoral fin base, 17 principal caudal fin rays, upper jaw bordered by the premaxillary, presence of a supramaxilla, presence of vomerine and palatine teeth, a dimorphic optic chiasma, 24 vertebrae, basihyal with tooth plate, lack of orbitosphenoid and mesocoracoid, two postcleithra, 7 branchiostegals and an autogenous hemal arch on the second preural vertebra. These traits are not only found in Percoidei, but also in Berycifor- mes (see Zehren, 1979) and sometimes in the Protacanthopterygii (sensu Nelson, 1984). Norman (1934) indicated that Psettodes, because of its striking similarities with Epinephelus, could almost be placed in the perciform family Serranidae. However, it is only recently that the monophyly of the Serranidae has been established (Johnson, 1983). It is based on four characters: (a) absence ofa posterior uroneural, (b) absence of a procurrent spur in caudal fin, (c) absence of a cartilage at the third preural pterygiophore, (d) presence of three opercular spines. Psettodes pos- sesses two of these characters: the absence of the procurrent spur and the absence of the third preural cartilage. It lacks three opercular spines and has two uroneurals in the caudal skeleton (serranids have only one). The two characters shared with Psettodes are reductive features that may have been subject to independent losses (Johnson, 1983) within the Perciformes. As a consequence, there is little evidence linking Psettodes and other flatfishes to the Serranidae. More research is needed to find the sister group of flatfishes. The survey will have to be extended to the Beryciformes and to other groups of "basal" percomorphs.

Monophyly and Interrelationships of Higher Taxa Within the Pleuronectiformes The classification of Ahlstrom eta!. (1984), modified by the findings of Chapleau and Keast (1988) is followed (i.e., subfamilies of the Soleidae (Achirinae and Soleinae) raised to the family level). Suborder Psettodoidei: Psettodidae. -Hensley and Ahlstrom (1984) indicated that no autapomorphies had been found to characterize Psettodes. However, the presence of an autogenous bone referred to as the pseudomesial bar (Amaoka, 1969) CHAPLEAU: PLEURONECTIFORM RELATIONSHIPS 523

e.$. lateral ethmoid e.s. frontal A

parasphenoid

Figure 4. Dorsal view (A) and blind side view (B) of the cranium in Psettodes erumei. Scale = 3 mm. or the "azygoste" (Chabanaud, 1934) is certainly a synapomorphy of the genus (Figs. 4, 5). This bone is situated between the blind-side lateral ethmoid and the blind-side frontal and bears along its ventral margin three pores of the suborbital canal. Chabanaud (1934, 1936) indicated that the autogenous pseudo mesial bar of Psettodes (=azygoste) was a hypertrophied suborbital bone, more precisely the blind-side "dermosphenoid" (or suborbital 6). In Psettodes, the supraorbital canal, which extends along the ventral margin of the bone, opens anteriorly just posterior to the articulation with the blind-side lateral ethmoid. This canal extends pos- teriorly on the sphenotic, frontal and pterotic. In other flatfishes, the suborbital canal is found along the ventral or lateral margin of the frontal bone and stops short of the articulation to the lateral ethmoid. From this, it is suggested that the 524 BULLETIN OF MARINE SCIENCE, VOL. 52, NO.1, 1993

A supraoccipital

ethmoid

pterosphenoid

Figure 5. Eyed-side lateral view (A) and ventral view (B) of the cranium in Psettodes erumei. Scale = 3mm. pseudo mesial bar is really an anterior extension of the blind-side frontal bearing a portion of the suborbital canal and not solely an hypertrophied suborbital canal as indicated in Chabanaud (1936). The fact that the bone is autogenous might indicate that the blind-side frontal has two sites of ossification or that the splitting of the bone occurs late in ontogeny. Patterson (1975) mentions only one site of ossification for the frontal bone in acanthopterygians. Developmental data are needed to verify these hypotheses. Suborder Pleuronectoidei. - The Pleuronectoidei is the largest flatfish suborder with more than 400 species (Nelson, 1984). Ahlstrom et al. (1984) and Hensley and Ahlstrom (1984) recognized five families: Citharidae (subfs. Citharinae and CHAPLEAU: PLEURONECfIFORM RELATIONSHIPS 525

Brachypleurinae), Scophthalmidae, Paralichthyidae, Bothidae (subfs. Taeniop- settinae and Bothinae) and Pleuronectidae (subfs. Pleuronectinae, Poecilopsettin- ae, Paralichthodinae, Samarinae and Rhombosoleinae). The loss of a truly dimorphic optic chiasma has been suggested, with caution, as the only synapomorphy of the Pleuronectoidei (Chapleau and Keast, 1988; Hensley and Ahlstrom, 1984). The caution, a serious one, is associated with the fact that no adequate survey of this character was ever done (Hensley and Ahl- strom, 1984). Morphological and osteological information regarding the suborder Pleuronec- toidei can be found in various publications. The most important ones dealing with phylogenetic relationships and osteology are: Hubbs (1945) and Amaoka (1969, 1972) for the Citharidae; Amaoka (1969, 1972), Futch (1977), and Hensley (1977) for the Bothidae and Paralichthyidae; and Kim (1973) and Sakamoto (1984) for the Pleuronectidae. A detailed osteological study of the Scophthalmidae is still needed. Except for the synthesis of Hensley and Ahlstrom (1984) (see also Lauder and Liem, 1983), no attempts have been made to integrate the anatomical information provided by the aforementioned studies. The important study of Sakamoto (1984) was published after the synthesis of Hensley and Ahlstrom (1984). It includes an assessment of relationships for 77 species of Pleuronectidae based on a detailed osteological survey. It is important to mention that Sakamoto's (1984) interpre- tation of the relationships within the Pleuronectidae is strictly phenetic. Because the osteological study covers a broad spectrum of Pleuronectidae, it is possible to reinterpret the osteological data in a cladistic analysis. Amaoka's (1969) study of the sinistral flounders of Japan relies on an osteological study of numerous species of Citharidae (including the dextral species Lepidoblepharon macrolepidotus), Paralichthyidae and Bothidae. To decipher intergeneric relationships, an eclectic approach combining phenetic and phyletic concepts was used. As in Sakamoto's study, the osteological information is precise and can be reinterpreted cladistically. Hensley and Ahlstrom (1984) indicated the presence of a bothoid group including the Pleuronectinae, Paralichthyidae (except Tephrinectes and Thysanop- setta), Scophthalmidae, Bothidae (except Mancopsetta) and the citharid Brachy- pleura. This assessment is based on the recognition ofa specific structural pattern in the caudal fin skeleton (Fig. 6). The bothoid group has the fused hypurals 1 and 2 closely articulated to the posteroventral surface of PU 1, and has fused hypurals 3 and 4 fused to the terminal half centrum. This caudal pattern, which is considered here to be a mosaic of 3 characters, is unique among flatfishes. The fusion of hypurals 3 and 4 to the centrum of PU 1 is a synapomorphy for the bothoid group and for the citharid Citharoides. The type of articulation ofhypurals 1 and 2 to the preural centrum is a second synapomorphy. In addition, the simultaneous presence of fused hypurals I and 2 and fused hypurals 3 and 4 is the third synapomorphy. In the analysis, these three advanced character states will be assumed to be independent from each other. Although this assessment could be inadequate, there are no a priori reasons to believe that these three characters are functionally and evolutionarily correlated. Chapleau and Keast (1988) have hypothesized that the suborder Pleuronectoidei of Hensley and Ahlstrom (1984) is paraphyletic. To become monophyletic, the Pleuronectoidei should also include all soleoid taxa.

Citharidae. -Hubbs (1945) erected a new family, the Citharidae, by regrouping two bothid and two pleuronectid genera. He subdivided the Citharidae in two 526 BULLETIN OF MARINE SCIENCE, VOL. 52, NO. I, 1993

epural

c hvpural 5 + epural

Figure 6. Left lateral view of the caudal skeleton in: (A) Brachypleura novaezeelandiae (USNM 236122,94 mm SL, blind side view), (B) Scophthalmus aquosus (Scophthalmidae) (USNM 118227, 51 mm SL, eyed-side view) and (C) Citharichthys arenaceus (Paralichthyidae) (USNM 203510, 68 mm SL, eyed-side view). Scale = 3 mm. subfamilies on the basis of the ocular position in adults: the dextral Brachypleu- rinae (Brachypleura and Lepidoblepharon) and the sinistral Citharinae (Citharus and Citharoides). A third genus (Paracitharus), incorporated in the Citharinae, is a synonym of Citharoides. By creating this new family, Hubbs (1945) filled what he believed to be an evolutionary or morphological gap between the Psettodoidei and the Pleuronec- CHAPLEAU: PLEURONECTIFORM RELATIONSHIPS 527 toidei. Hensley and Ahlstrom (1984) doubted the monophyly of the group. Most of the features used by Hubbs (1945) to define the Citharidae or to define its relationships with Psettodes are (1) plesiomorphic for the order and/or (2) not found in all genera. Of all the characters discussed in Hubbs' paper, one possible derived character for the family was indicated by Hensley and Ahlstrom (1984): deflection of the anus on the eye side of the body (other flatfishes have it on the mid-ventral margin or on the blind side). A study of this character for the four citharid genera is provided here. The only genus for which the eyed-side deflection was unequivocal was Citharus (N = 13). In Citharoides (N = 6), the anus is situated on the ventral midline of the body with a very slight tendency towards the eyed side in some specimens. The same pattern was observed in Lepidoblepharon (N = 6). In Brachypleura (N = 33), the anus is slightly deflected towards the eyed side. However, I have observed three specimens which had the anus directly in line with the anterior tip of the anal fin. Consequently, as suggested in Hensley and Ahlstrom (1984), this character cannot be considered an indicator of monophyly for the group. The distinctly blind-side position of the anus in Citharus should be considered an autapomorphy of the genus. Aboussouan (1972, 1988) has examined larvae of Eucitharus macrolepidotus (=Citharoides macrolepidotus). He suggested (Aboussouan, 1988) that the het- erogeneity within the Citharidae required phylogenetic considerations, i.e., the possible erection of a (new) suborder Citharoidei, a transitional step from which the Pleuronectoid and soleoid "branches" would have emerged. Lepidoblepharon, Citharoides and Citharus would make the transition to the Soleoidei. Brachypleura would make the transition towards the Pleuronectoidei. This hypothesis will be discussed after the cladistic analysis. Although the idea of transitional groups to the Soleioidei and to the Pleuronectoidei is verifiable, the subsequent identification of a suborder regrouping the citharid species means the formation of an undesir- able para phyletic taxa. Scophthalmidae. - The family Scophthalmidae comprises four genera (Scophthal- mus, Lepidorhombus, Phrynorhombus, Zeugopterus) distributed in the North At- lantic and the Mediterranean. A detailed osteological study of the family is want- ing. Some osteological data can be found in Traquair (1865) and Brewster (1987). All taxa examined share two elongated pelvic fin bases (slightly asymmetrical) extending anteriorly to the urohyal (Hensley and Ahlstrom, 1984). An elongated supraoccipital process forming a bridge with the dorsal margin of the blind side frontal was observed in several species (although sometimes difficult to see on X-rays. These characters are here considered as autapomorphies ofthe Scophthal- midae. Paralichthyidae. - Hensley and Ahlstrom (1984) provided a thorough review of the taxonomic changes in this taxon since Norman (1934). They suggested a few more changes (i.e., removal of Tephrinectes and Thysanopsetta from the family). They also recognized three subgroups within the family. The polarity of some of the characters is based on the acceptance of the bothoid lineage as a monophyletic group. The Cyclopsetta group is composed of the genera Cyclopsetta, Syacium, Cith- arichthys and Etropus. This group shares a urinary papilla oriented towards the blind side and an ocular pelvic fin based on the mid-ventral line of the body. The pelvic fin base of the blind side is anterior to that of the ocular side. All these taxa have a caudal fin with 17 rays, none of which are supported by preural, neural or hemal spines. They also have hypural 5 fused with the epural (Fig. 6C). 528 BULLETIN OF MARINE SCIENCE, VOL. 52, NO.1, 1993

Hensley and Ahlstrom (1984) also indicated a possible close relationship for the Cyclopsetta group with the both ids on the basis of the absence of a first neural spine and the presence of vertebral apophyses. A Pseudorhombus group with Pseudorhombus, Tarphops and Cephalopsetta is also suggested. The group was hypothesized to be monophyletic on the basis of 17 caudal rays, epural and hypural 5 fused to form a plate and the lack of a splinter ray (i.e., remnant of a ray partly fused with an adjacent ray). None of these characters are unique to this group within the flatfishes. The remaining Paralichthyidae contain 7 genera: Ancylopsetta, Gastropsetta, Hippoglossina, Lioglossina, Paralichthys, Verecundum, and Xystreurys. There is no evidence of monophyly for this group. The Paralichthyidae, as defined here, are not monophyletic. However, because the definition of monophyletic units within the family awaits detailed phylogenetic studies, the Paralichthyidae will be considered as a taxon in the cladistic analysis. Bothidae, - Hensley and Ahlstrom (1984) provide a good historical overview of the taxonomic history of this family. After reviewing characters indicated in Amaoka (1969), Hensley and Ahlstrom (1984) discussed the monophyletic status of the Bothidae using other bothoid taxa as outgroups. They indicated three derived states for the adult bothids: (a) asymmetrical states of ventral fin morphology (an elongated eyed side fin situated on the mid-ventral line of the body), (b) loss of blind side preorbital and (c) presence of myorhabdoi (type of intermuscular bones). They mentioned five more autapomorphies based on larval features: large size at metamorphosis (15-120 mm), eye migration below the dorsal fin, dorsal fin origin anterior to eyes just prior to metamorphosis, elongated, early-forming second dorsal fin ray and lack of preopercu1ar spines. The acceptance of these three adult autapomorphies for the Bothidae is based on the fact that these character states are unique when compared to states found in other bothoid taxa. However, some of these character states are also found in taxa outside the bothoid lineage. In the broader perspective of flatfish familial and subfamilial relationships they cannot be considered a priori to be unique to the Bothidae. An elongated eyed-side pelvic fin base on the mid-ventral line, as found in the Bothinae, is also found in the Rhombosoleinae and the Scophthalmidae. In this last family, however, the blind side fin is also very long. In the Rhombosoleinae, the number of rays varies interspecifically and intergenerically; 6-13 on the ocular side and 4-6 on the blind-side. In the Bothidae, there are 6 rays on each side of the body, a probable autapomorphy for the family. The absence ofthe blind-side preorbital (lachrymal) is not unique to the Bothidae but is also observed in the Poecilopsettinae and the Achiridae. Amaoka (1969) defined two subfamilies in the Bothidae: the Taeniopsettinae (Engyophrys, Perissias, Taeniopsetta, and Trichopsetta) and the Bothinae (all other both ids). The monophyletic status of Taeniopsettinae is doubtful (Hensley and Ahlstrom, 1984). Evseenko (1984) suggested the creation of a new family, the Achiropsettidae, that would include Mancopsetta, Achiropsetta and Neoachiropsetta. Hensley (1986) and Hensley and Ahlstrom (1984) indicated that the genus Mancopsetta (with Achiropsetta Norman, 1930, Apterygopectus Ojeda, 1978 and Neoachiropsetta Kotlyar, 1978, as synonyms) did not show the bothoid caudal pattern and should be removed from the Bothidae and united with the Rhombosoleinae. Until detailed phylogenetic studies of the Rhombosoleinae and of the" Achiropsettidae" CHAPLEAU: PLEURONECfIFORM RELATIONSHIPS 529 are made, it is premature to make any strong statement about the status of these taxa. In conclusion, the Bothidae are monophyletic but the status of the subfamilies remains to be better substantiated. Only the family will be included in the cladistic analysis. Lack of osteological data on the Achiropsettidae makes it impossible to include this taxa in the analysis.

Pleuronectidae. - The family Pleuronectidae was restricted to the right-eyed flounders by Regan (1910). He divided the group into three subfamilies: Pleuronectinae, Samarinae and Rhombosoleinae. Regan (1929) removed the South African genus Paralichthodes from the Samarinae and placed it in a separate subfamily, the Paralichthodinae. Norman (1934) created the Poecilopsettinae. The main diag- nostic features of the Pleuronectidae (Regan, 1910; Norman, 1934) is their dextrality and the absence of oil globules in the egg. The latter character is no longer considered informative as it is far more variable than first predicted (Ahlstrom et al., 1984). The first character is not restricted to this group within the order. In a phenetic study of 77 species of Pleuronectidae, Sakamoto (1984) defines the family on the basis of (1) their dextrality, (2) the monomorphic type of optic chiasma, (3) the free margin of one preopercle and (4) the absence of spines in the fins. All these character states are plesiomorphic for the above taxa. Conse- quently, there is no evidence that the family, as currently defined, is monophyletic. The discussion will therefore be centered on each subfamily, except for the genus Paralichthodes (Paralichthodinae) which will be discussed after the completion of the analysis.

Pleuronectinae. - The Pleuronectinae represent a large and heterogeneous group of flatfishes (26 genera and about 60 species) containing a great number of com- mercially important species. They are confined to northern seas and the Arctic. As a result of his phenetic analysis, Sakamoto (1984) lumped under Pleuronectes all the species of the following genera: Isopsetta, Parophrys, Lepidopsetta, Limanda, Pseudopleuronectes, Pleuronectes and Liopsetta. As well, he regrouped Eopsetta and Lyopsetta and Cleisthenes and Hippoglossoides. He recognized Kareius as a valid genus. Clyptocephalus zachirus became Errex zachirus, a monotypic genus. No attempts were made to check original descriptions or to redefine the genera. Sakamoto (1984) included Paralichthodes algoensis in the Pleuronectinae. Sakamoto (1984) characterized the Pleuronectinae by the presence of a neural arch on the first precaudal vertebra, a plesiomorphic feature for the order. Norman (1934, 1966) used two characters to define the Pleuronectinae: (1) presence of a well-developed lateral line on both sides of the body, and (2) olfactory lamellae nearly always parallel, without rachis. The distribution and the homology of the latter character is not well-known while the first character is plesiomorphic for the order (Hensley and Ahlstrom, 1984). The currently defined Pleuronectinae regroups right-eyed pleuronectid genera that cannot be placed in the other subfamilies. Nelson (1984), who relied on Li (1981), describes two tribes in the Pleuronectinae. The first one, the Hippoglossini contains 10 genera and about 18 species. It is characterized by a series of plesiomorphic features: mouth large and symmetrical, maxillae extending to or be- hind the pupil of the eyes, and teeth well-developed on both sides of the jaws. The second tribe, the Pleuronectini are characterized by a smaller asymmetrical mouth, the maxillae not extending to the pupil of the eye, and teeth chiefly on the blind side. 530 BULLETIN OF MARINE SCIENCE, VOL. 52, NO, 1, 1993

More research is needed to define intergeneric relationships within this group. Consequently, it is with reluctance, but without any other alternatives for the moment, that the pleuronectines are considered as a taxon in the cladistic analysis. Poeci!opsettinae, - This subfamily, defined by Norman (1934), comprises three genera (Poecilopsetta, Nematops and Marleyella) with approximately 14 species. The species are found in the Indo-Pacific region except for two species of Poe- ci!opsetta that occur in the western North Atlantic Ocean off the coast of New England, and in the Gulf of Mexico. Norman (1934) defined this group on the basis of the rudimentary or scarcely apparent lateral line on the blind side and on the structure of the olfactory laminae. As mentioned previously, the distribution of the second character is unknown. The first character is also found in some bothid, samarine and cynoglossine genera. Sakamoto (1984) characterized the Poecilopsettinae by the absence ofIachrymal (preorbital) and the fact that both lateral ethmoids are attached to each other on the lower part of the anterior portion of the frontal of the ocular side. The former character is also found in the Bothidae and the Achiridae. The latter might be the first autapomorphy for the subfamily since a contact between lateral ethmoids is found only in two other groups: the Samarinae and four genera of the Bothidae. However, in the Samarinae, the contact between the lateral ethmoids is on the upper part of the anterior portion of the eyed-side frontal. In the four bothid genera (Engyprosopon, Taeniopsetta, Parabothus and Tosarhombus), the contact is also dorsal (Amaoka, 1969). Hensley and Ahlstrom (1984) do not provide any evidence regarding the monophyly of the Poecilopsettinae. They mention a series of primitive features related to the caudal fin skeleton and the presence of a rudimentary hemal arch on the end of the parhypural. Sakamoto (1984) did not observe the latter feature in five species of Poecilopsettinae. Consequently, the only feature corroborating the monophyletic status of the Poecilopsettinae is the position of the contact zone between the lateral ethmoids. Samarinae. - The Samarinae contains three genera (Samaris, Samariscus and Plagiopsetta) and approximately twenty species confined to the tropical and sub- tropical Indo-Pacific. Norman (1934) characterized Samaris and Samariscus including Plagiopsetta by the elongated pelvic fin bases, small mouth, absence of pectoral fin on the blind side, straight lateral line, short gill-rakers and small scales. Of these features only the absence of a blind-side pectoral fin is unique within the Pleuronectidae. However, it is commonly observed in the Soleinae, Achirinae and Cynoglossidae. The elongate pelvic fin bases are also found in the Scophthal- midae. However, in this last group, they are slightly asymmetrical. All other features are plesiomorphic. Sakamoto (1984) listed 10 features characterizing the Samarinae. They will be discussed individually. (1) The lateral ethmoids are attached to each other on the dorsal part of the anterior portion of the eyed-side frontal. Four genera of the Bothidae (Taeniop- setta, Parabothus, Engyprosopon and Tosarhombus) have this character. However, the cranial structure in these bothid genera is so different from the samarine cranium that it is unlikely that this character state is homologous for both groups. (2) The blind-side lateral ethmoid is attached to the eyed-side frontal in the middle portion of the dorsal cavity of the migrated eye. This character state was observed by Amaoka (1969) in four genera of the Bothidae (Both us, Crossorhom- bus, Engyprosopon and Tosarhombus). Again, it is unlikely that this state is homologous for both groups. CHAPLEAU: PLEURONECTIFORM RELATIONSHIPS 531

(3) The eyed-side frontal is broadly attached to the parasphenoid in the interorbital region. This character is quite variable in flatfishes and, in samarines, it is probably associated with the lack of a pterosphenoid. The connection between the eyed-side frontal and the parasphenoid is also found in some genera of Pleu- ronectinae, Poecilopsettinae, and Rhombosoleinae and in other groups of flatfishes. However, the articulation in the Samarinae seems to be associated with the interorbital complex, not with the neurocranium. This character is probably an autapomorphy for the Samarinae. (4) The metapterygoid is small. This is an autapomorphy of the Samarinae. (5-6) The pectoral fin (character 5), coracoid and scapula (char. 6) are absent on the blind side. The general reduction of the pectoral fin and girdle was discussed previously and is found in many genera of the Achirinae, Soleinae and Cynoglossi- dae. It is also observed in the bothid genera Monolene and Mancopsetta. (7) The pleural and epipleural ribs are absent. The absence of pleural ribs is not unique to the Samarinae, it is also found in the Bothidae, Achiridae, Soleidae and Cynoglossidae. The absence of epipleural bones is shared with the Bothidae, the Achiridae, some Soleidae, and the Cynoglossidae. Consequently, these are considered as two different characters. Hensley and Ahlstrom (1984) indicated that, possibly, two of the five series of intermuscular bones indicated in Amaoka (1969) might be homologous with pleural and epipleural bones. (8) The epicentrum and hypomeral series are present. These intermuscular bones are also found in the Bothidae. (9) The first preural centrum is fused with the second, third and fourth hypural plates. This fusion is unique amongst flatfishes, except for some Achirinae: Tri- nectes fimbriatus, Soleonasus, Apionichthys. (10) The second, third and fourth hypurals are fused while hypurals 1 and 5 are autogenous. Apionichthys (Achiridae) has hypurals 2,3, and 4 fused. The fact that hypurals 1 and 5 are autogenous is a plesiomorphic condition. The rhombosoleine Psammodiscus shows this hypural fusion pattern. The monophyly of the Samarinae is a well-corroborated hypothesis. Rhombosoleinae. - This subfamily is composed of 8 genera and 21 species and is endemic to Southem Australia and New Zealand, except for a South American monotypic genus (Oncopterus darwinii). The Rhombosoleinae has been recognized by Regan (1910) and Sakamoto (1984) as a subfamily mainly on the basis of the pelvic fin asymmetry. In this group, as in the Bothidae, the ocular side fin is usually long compared to the blind side one, and situated along the mid-ventral line of the body. The fin shows a variable number of fin rays (6-13) on the eyed side. Norman (1934) indicated other characters for the Rhombosoleinae: no radials associated with the pectoral fins, anterior position of the dorsal fin, absence of hem apophyses on the precaudal vertebrae, coracoids reduced in size, the lateral lines equally developed and the olfactory lamina with or without a rachis. A survey ofthese characters shows that they are variable inside the subfamily or found in other flatfishes. Except for the peculiar placement of the bases of the pelvic fins, there is no evidence regarding the monophyly of the group. Although this group will be considered as a taxonomic entity in the following analysis, it is quite clear that more research is needed to corroborate its monophyly. Soleoidei. - In the traditional classification (Greenwood et aI., 1966; Nelson, 1984; Hensley and Ahlstrom, 1984), this suborder was composed of two families: the dextral Soleidae (subfamilies Achirinae and Soleinae) and the sinistral Cynoglos- sidae (subfamilies Symphurinae and Cynoglossinae). 532 BULLETIN OF MARINE SCIENCE, VOL. 52, NO. I, 1993

Norman (1934) suggested a Psettodes-like ancestor for the Soleoidei by em- phasizing the following shared features: dimorphic optic chiasma and presence of symmetrical nasal organs. These characters are plesiomorphic for flatfishes when compared to symmetrical outgroups and should not be used to infer relationships. Chapleau (1988a, 1988b) and Chapleau and Keast (1988) have found the So- leoidei to be monophyletic on the basis ofthree autapomorphies: (i) skin oflower jaw and interopercle continuous ventrally, covering isthmus and branchiostegals, (ii) blind side preopercular canal (where present) terminating near ventral margin ofpreopercle and, (iii) blind-side lateral ethmoid forming most of the upper orbit. Chapleau and Keast (1988), following an osteological study of several species, have refuted the monophyletic status of the Soleidae which was based mainly on the dextrality of all its species. Chapleau and Keast (1988) have indicated 7 synapomorphies shared by the Soleinae and Cynoglossidae: (i) edge of preopercle completely concealed by scales and skin, (ii) eyed-side mesopterygoid absent, (iii) opercular series (except preopercle) deeply fimbriated, (iv) portion of the blind- side dentary anterior to anguloarticular being convex, shorter and deeper than the eye side dentary, (v) first proximal pterygiophore of dorsal fin with long anterior process, (vi) fusion of proximal tip of four well-differentiated hypural plates to PUI and (vii) blind-side lateral ethmoid forming the entire margin of upper orbit. Consequently, they have suggested raising the Soleinae and Achirinae to the family level (Soles: Soleidae, Achiridae, Cynoglossidae). Achiridae and Soleidae. -Chapleau and Keast (1988) defined the Soleidae and Achiridae as monophyletic groups on the basis of 5 and 6 autapomorphies, re- spectively. Characters are listed in Chapleau and Keast (1988). Chapleau (1989) discussed characters linked with the supracranial portion of the dorsal fin that will help in elucidating intrarelationships of the Soleidae. Cynoglossidae. -Chapleau (1988a) did an osteological survey of taxa belonging to the two subfamilies Symphurinae (Symphurus) and Cynoglossinae (Cynoglos- sus, Paraplagusia) of this family. The Cynoglossidae were found to be monophyletic on the basis of 27 characters. The Symphurinae (Symphurus) were found to be monophyletic on the basis of 6 autapomorphies while 9 autapomorphies corroborated the monophyly of the Cynoglossinae. Characters are listed in Chapleau (1988a).

RESULTS The analysis yielded 18 equally parsimonious tree topologies (62 steps). The consistency index is 0.67 and the retention index is 0.81. The Nelson consensus tree is presented in Figure 7. Characters are mapped on the tree. Seven resolved branching points (lineages I to VII) are indicated on the tree with defining characters (rectangles). Because of the great number of unresolved branching points, which indicate several possibilities of relationships, characters that showed more than one reversal or one convergence on the consensus tree have their states indicated for each taxon (squares). Characters 1-3 corroborate the monophyly of the order Pleuronectiformes. Flatfish lineage I is made up of the Psettodidae (suborder Psettodoidei). The monophyletic status of this lineage was discussed in the previous section and is based on the presence of a pseudomesial bar. Flatfish lineage II (suborder Pleuronectoidei) share 8 advanced characters (char. 4-11, see appendix). In addition, taxa within this group (reversal in Brachypleura) CHAPLEAU: PLEURONECTIFORM RELATIONSHIPS 533

I:: ~ ~ ~ ~ e -c:l -c:l ~ .5 ~ ~ i: .~ II> -c:l ~ ;::s ·s ';>" .§ '0 .•... -c:l & ~ Cd .s ~ II> ....• ~ c.. ~ O ~ ~ 0 :s II> .£ '-& i: u ~ 0 ~ 0 ..c:: ij u 0 ~ 0..'" ...;j G G !:l:l v:l 0.. C!l ii: 0.. P:::: v:l « v:l U

16 15 VII

Figure 7. Consensus tree representing flatfish interrelationships based on 18 equally parsimonious trees calculated from a matrix of 39 ordered and polarized character states. Rectangles represent characters used to define branching points: black rectangles represent uniquely derived character states, shaded rectangles represent derived character states with one reversal. Squares represent polarized and ordered character states showing several reversals or convergences and/or different tree topologies: empty squares are plesiomorphic states, black squares are first apomorphic states, dotted squares are second apomorphic states. Roman numerals indicate lineages. Decimal numbers indicate order of apomorphic states. share an elongated anteriormost proximal pterygiophore on the anal fin (char. 23). The trichotomy at the base of this lineage indicates that several relationships are possible. The most common one is Citharoides as the sister group of lineage II on the basis of a parhypural free from the second preural centrum (char. 35). The relationships of the four genera previously placed in the Citharidae will have to be defined to clarify relationships at this level. Flatfishes in lineage III share a hemal arch fused with the centrum of the second preural vertebra (reversal in the Achiridae) (char. 34). The polytomy of lineage III is an indication of our poor knowledge of relationships amongst taxa currently 534 BULLETIN OF MARINE SCIENCE, VOL. 52, NO. I, 1993 included in the Pleuronectoidei. The bothoid group (Brachypleura, Scophthal- midae, Paralichthyidae, Bothidae and Pleuronectinae) of Hensley and Ahlstrom (1984) was observed in one tree (apomorphies char. 26-28) but, globally, there were too many conflicts with other advanced character states to support the concept of a monophyletic entity (Fig. 7) (see all other characters (except for char. 12, 15 and 35 which do not affect the bothoid group) represented by squares that are mapped on the tree). An alternative to the bothoid group could be, for example, the Paralichthyidae, Bothidae, Pleuronectinae and flatfishes from lineage IV being monophyletic based on medially fused branchiostegal membranes (char. 36) and the absence of vomerine teeth (char. 13 with convergence Lepidoblepharon and Citharoides). Flatfishes in lineage IV share an absence of a neural arch (and spine) on the first precaudal vertebra (char. 25). The Rhombosoleinae and lineage V (except Cynoglossidae) have the epioccipitals forming the dorsal margin of the foramen magnum (char. 15) and an absence of teeth on the eyed-side maxillary (char. 12) (except for the Samarinae). The Poecilopsettinae and lineage V share a blind-side infraorbital canal (including lachrymal) reduced to one bone or entirely absent (char. 17). This last character would have converged in Brachypleura and the Bothidae. Lineage V is characterized by three uniquely derived character states (char. 19, 20,21.1). These are: a long posterodorsal process of the hyomandibula articulating with the pterotic and, occasionally, with the intercalar (char. 19), absence of postcleithra and coraco-scapular complex (or reduced to small cartilaginous plates) on blind-side pectoral fin (char. 20) and first proximal pterygiophore running parallel anteriorly to the orbital region (char. 21.1). Another derived character would be the absence of pterosphenoids (char. 14) but a reversal is observed in the Achiridae. Flatfishes in lineage VI (Soleidae, Achiridae and Cynoglossidae) are monophyletic on the basis of three characters (char. 37, 38.1, 39). The characters are discussed in the section dealing with the monophyletic status of the Soleoidei. Lineage VII (Soleidae and Cynoglossidae) is monophyletic on the basis of 7 autapomorphies (char. 29-33, 21.2, 38.2). These are: fusion of proximal tips of hypural plates to the first preural centrum (char. 29), edge of preopercle entirely concealed by scales and skin (char. 30), eyed-side mesopterygoid absent (char. 31), deep fimbriation pattern of opercular bones (char. 32), portion of blind-side dentary, anterior to anguloarticular, is convex and is shorter and deeper than eyed- side dentary (char. 33), first proximal pterygiophore extending anteriorly relative to the attachment of the first fin rays or extending well in front of orbital and rostral regions (char. 21.2), and the blind side frontal not part of the bony orbit of the upper eye (char. 38.2). Status of the Genus Paralichthodes.-Paralichthodes algoensis was first included in the Samarinae by Regan (1910). Later, Regan (1929) recognized this South African genus as a subfamily within the Pleuronectidae. The subfamily was retained in Norman (1934) and Hubbs (1945). Nelson (1984) transferred the genus in the Samarinae again. It was raised to the subfamilial level by Regan (1910) due to the extension of the dorsal fin in front of the eyes. This is a primitive feature found in other genera of flatfishes. Norman (1934) believed that the genus Paralichthodes (Paralichthodinae) and the Samarinae were closely related on the basis of the advanced origin of the dorsal fin. Nelson (1984) regrouped Paralichthv- des with the Samarinae, on the basis of the origin of the dorsal fin in front of the eyes, a lateral line well-developed and symmetrical pelvic fins. The latter two CHAPLEAU: PLEURONECTIFORM RELATIONSHIPS 535 features are plesiomorphic for the Pleuronectiformes, while the first is commonly found in other groups of flatfishes. Sakamoto (1984) examined the osteology of Paralichthodes as part of his phenetic study of the family Pleuronectidae. He placed the genus within the Pleu- ronectinae on the basis of overall osteological similarity. However, it differs from the Pleuronectinae by the absence of the bothoid type of caudal skeleton (Hensley and Ahlstrom, 1984) and probably does not belong in this subfamily. In Paralichthodes, there is no fusion between hypural plates (primitive condition). The basioccipital does not form the ventral margin of the foramen magnum (plesiomorphic state of char. 16) and the epioccipitals do not form the dorsal margin of the foramen magnum (plesiomorphic state, char. 15). The relationships of this genus will have to await more comparative research within such groups as the Rhombosoleinae, Poecilopsettinae and Samarinae. General Phylogenetic Interpretations. - This study suggests that (i) convergence, reversal and parallelism are an important component ofthe evolution of flatfishes and (ii) the lack of detailed phylogenetic studies for several pleuronectoid taxa hinders our understanding of flatfish relationships even at the familial level. Other important points emerging from this study are: (I) Flatfishes are a monophyletic group based on three derived characters. To have any scientific value, polyphyletic hypotheses will have to be based on apomorphies shared between monophyletic flatfish groups and non-flatfish groups. This has not been the case. (2) The sister group of flatfishes remains unknown. Statements about a percoid sister group (Norman, 1934) are based on plesiomorphic character states. Also, statements about one or several hypothetical "preperciform" ancestors (Li, 1981; Amaoka, 1969; Chabanaud, 1949) are not supported by evidence. (3) The classification of flatfishes as found in Norman (1966) and Nelson (1984) and as reported in Hensley and Ahlstrom (1984) will probably have to be modified to accommodate new phylogenetic knowledge. To classify flatfishes as "flounders" (i.e., pleuronectoidei) and "soles" (i.e., soleoids) and further into "right-eyed flounders" (Pleuronectidae, Brachypleurinae), "left-eyed flounders" (Bothidae, Paralichthyidae, Scophthalmidae, Citharinae), "right-eyed soles" (Soleidae) or "left-eyed soles" (Cynoglossidae) might be intuitively practical but is, in fact, simplistic and phylogenetically misleading. As shown in this study, flatfish phylogeny remains a complex problem and the monophyletic taxa of large groups that were traditionally based on few obvious characters (ocular orientation, monomorphic or dimorphic type of optic chiasma, etc.) are not corroborated by the analysis (i.e., Pleuronectoidei, Pleuronectidae). Changes to flatfish classification must await a well-corroborated cladogram for these taxa. The only portions of the cladogram that are well-corroborated in this study are lineage II (suborder Pleuronectoidei) and lineages V, VI and VII dealing with the relationships of the Samarinae, Soleidae, Achiridae and Cynoglossidae. (4) I concur with Hensley and Ahlstrom (1984): the Citharidae (Hubbs, 1945) do not form a monophyletic group. A study of the only known autapomorphy of the family (deflection of anus on eyed-side) indicates that it is too variable to be a valid character. Detailed comparative studies of larval and adult specimens of these taxa with other pleuro- nectoids (sensu novo) will have to be done before any new hypotheses of relationships can be constructed. (5) Ifwe regroup the three families ofSoleoidei within the Pleuronectoidei, as suggested in Chapleau and Keast (1988), only two suborders remain in the Pleuronectiformes: Psettodoidei and Pleuronectoidei. Abous- souan's (1988) suggestion of a suborder Citharoidei is not corroborated by the present analysis. Hensley and Ahlstrom's (1984) bothoid lineage was observed in 536 BULLETIN OF MARINE SCIENCE, VOL. 52, NO.1, 1993 one of the 18 equally parsimonious trees but more congruent derived characters are needed to corroborate the monophyletic status of this taxon. (6) Chapleau and Keast (1988) suggested raising the Soleinae and Achirinae to the rank of family. Robins et al. (1991) suggested an expanded Soleidae that would include three subfamilies (Achirinae, Soleinae and Cynoglossinae). This implies that the Cy- noglossinae would be made up of two tribes: Symphurini and Cynoglossini. Be- cause some of these taxa are speciose (e.g., approximately 70 nominal species were found in the Symphurinae (Munro, 1990)), the former classificatory scheme (all families), would give a wider ranking flexibility to systematists interested in defining relationships within these taxa. 7. This study agrees with Hensley and Ahlstrom's (1984) assessment of the Pleuronectidae: it is a non monophyletic group. A recommendation to the effect that the Pleuronectinae, Poecilopsettinae, Rhombosoleinae, and Samarinae be raised to the familial ranking to reflect their phylogenetic relationships was made in Chapleau and Keast (1988). However, more research is needed on each of these subfamilies to define their monophyletic status and relationships.

ACKNOWLEDGMENTS

I would like to thank G. D. Johnson for providing me the opportunity to contribute to this important symposium. W. Smith-Yaniz and W. Saul of the Academy of Natural Sciences of Philadelphia (ANSP), J. Paxton and M. Mcgrouther of The Australian Museum (AMS), and S. Jewett ofthe National Museum of Natural History (USNM) provided the specimens indicated in the material section. J. A. Cooper, A. Murray and E. Gagnon used the equipment of the Canadian Museum of Nature to take several X-rays. R. Mooi, A. Harold and D. Hensley provided very insightful and thorough reviews of the submitted versions of this manuscript. L. Ridgway and C. Renaud read and criticized a latter version of this manuscript. K. Gannon provided technical assistance with the figures. All these scientists and institutions are gratefully acknowledged for their help. This research was financially supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada.

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DATEACCEPTED: May 27,1992.

ADDRESS:Ottawa- Carleton Institute of Biology, Department of Biology, University of Ottawa. Ottawa. Ontario KI N 6N5, Canada.

ApPENDIX

List of Characters. - This section includes a list o( characters for which the polarity was established using outgroup comparison (see materials and methods). The various states were determined by looking at the osteology of Psettodes and basal percomorphs (see references in text). The distribution of character states for each character amongst taxonomic units is summarized in Table 2. In the following description, for each character, the plesiomorphic condition (0) is indicated first, the apomorphic condition (I or 2) is indicated second and, thirdly, the distribution of the apomorphic condition among taxonomic units is indicated. Finally, exceptions to character state assignments are indicated. I. Adult cranium. Symmetrical cranium with one eye on each side of body (0). Asymmetrical with both eyes on one side of head (I) (see text for discussion). Found in all flatfishes. 2. Epicranial section of dorsalfin. Dorsal fin without epicranial section (0). Presence of an epicranial section (I) (see text for discussion). Found in all flatfishes. 3. Recessus orbitalis. Absent (0). Present (I). Found in all flatfishes. 4. Palatine teeth. Present (0). Absent (I). All flatfishes, except for Psettodidae. 5. Toothed plates on basihyal. Present (0). Absent (I). All flatfishes, except for Psettodidae. 6. Basisphenoid. Present (0). Absent (I). All flatfishes, except for Psettodidae. 7. Spines in median fins. Present (0). Absent (I). All flatfishes, except for Psettodidae. 8. Sciatic portion ofurohyal. Absent or not well developed (0). Distinct and often elongated sciatic portion of urohyal (I). All flatfishes, except for Psettodidae. 9. Uroneurall. Present (0). Reduction and losses ofuroneurals (I). All flatfishes, except for Pset- todidae. 10. Shape of second infrapharyngobranchial. Elongated second infrapharyngobranchial(O). Elliptical or rounded infrapharyngobranchial of various sizes (sometimes very small) (I). All flatfishes, except for Psettodidae. An autapomorphy of Cynoglossidae, second infrapharynogobranchial absent. II. Supramaxilla. Large supramaxilla (0). Vestigial or absent (I). All flatfishes, except for Psetto- didae. 12. Teeth on eyed side premaxilla. Present (0). Absent (I). Rhombosoleinae (except Oncopterus, Psammodiscus, Azygopus), Achiridae, Soleidae, and Cynoglossidae. Noted but not encoded for pleuronectine Hypsopsetta, Pleuronichthys, and two species of Pleuronectes and Microstomus and in the bothid Laeops. 13. Vomerine teeth. Present (0). Absent (I). Lepidoblepharon, Citharoides. Paralichthyidae, Both- idae, Pleuronectinae, Poecilopsettinae, Rhombosoleinae, Samarinae, Achiridae, Soleidae and Cyno- glossidae. CHAPLEAU: PLEURONECfIFORM RELATIONSHIPS 539

14. Pterosphenoid. Present (0). Absent (I). Samarinae, Soleidae, Cynogiossidae. Absent, but not encoded for rhombosoleine Azygopus and two species of bothid Laeops. 15. Epioccipitals andforamen magnum. Exoccipitals forming dorsal margin of foramen magnum (0). Epioccipitals forming dorsal margin of foramen magnum (I). Rhombosoleinae (except Psam- modiscus), Samarinae, Achiridae and Soleidae. 16. Ventral margin offoramen magnum. Exoccipitals forming ventral margin offoramen magnum (0). Basioccipital forming part of ventral margin offoramen magnum (I). Scophthalmidae, Paralichthy- idae, Bothidae, Pleuronectinae and Samarinae. 17. Blind side infraorbitals (including lachrymal). Present (0). Reduction to one bone or absent (I). Psettodidae, Brachypleura, Poecilopsettinae, Samarinae, Archiridae, Soleidae and Cynogiossidae. 18. Eyed-side infraorbitals. Present (0). Infraorbitals reduced to one or two small bones (I). Bra- chypleura, Bothidae, Poecilopsettinae, Rhombosoleinae, Samarinae, Achiridae, Soleidae and Cyno- glossidae. Observed but not encoded in Pleuronectine Embassichthys (Sakamoto, 1984) and Cyclop- setta group of Hensley and Ahlstrom (1984). 19. Articulation of hyomandibula to cranium. Hyomandibular articulates to cranium along its posterodorsal margin (sometimes in the form of a short process) into an articulatory facet formed by prootic and pterotic (0). Long posterodorsal process (definitively longer than anterodorsal one) articulating with pterotic and sometimes with intercalar (prootic not involved in articulation) (I). Sama- rinae, Soleidae, Achiridae and Cynoglossidae. 20. Postcleithra and coraco-scapular complex on blind-side pectoralfin. Large coraco-scapular complex with elongated coracoid, some radials and postcleithra (0). Postcleithra missing and entire coraco- scapular complex sometimes absent or reduced to small cartilaginous plates (I). Samarinae, Achiridae, Soleidae and Cynoglossidae. 21. Shape and orientation offirst proximal pterygiophore of dorsalfin. First proximal pterygiophore of dorsal fin without long anterior process (0). First apomorphic state is a first proximal pterygiophore oriented almost in parallel fashion over orbital region (I). Second apomorphic condition has same pterygiophore with long anterior process extending anteriorly relative to point of attachment of anteriormost fin rays or extending well in front of orbital and rostral regions (2). Apomorphic state (I) was observed in Samarinae and Achiridae. Apomorphic state (2) was observed in Soleidae and Cy- noglossidae 22. Teeth on third epibranchial. Present (0). Absent (I). Psettodidae, Scophthalmidae, Pleuronec- tinae, Poecilopsettinae, Rhombosoleinae, Achiridae, Soleidae and Cynoglossidae. Plesiomorphic condition noted (in Sakamoto, 1984) but not encoded for pleuronectine Atherestes, Reinhardtius and Hippoglossus stenolepis. 23. Shape of anteriormost proximal pterygiophore of anal fin. Short proximal pterygiophores with one fin ray (0). Anteriormost pterygiophore is elongated (reaches hemal spine of corresponding vertebrae and supports two anal fin rays (I). All flatfishes, except for Psettodidae and Brachypleura (drawing in Amaoka, 1972). 24. Spine in pelvic fin. Present (0). Absent (1). Scophthalmidae, Paralichthyidae, Bothidae, Pleu- ronectinae, Poecilopsettinae, Rhombosoleinae, Samarinae, Achiridae, Soleidae and Cynoglossidae. 25. Neural arch and spine onfirst precaudal vertebra. Neural arch and neural spine on first precaudal centrum (0). Absence of neural spine on neural arch (I). Incomplete (or absent) neural arch (2). (I) found in Bothidae (also found in Cyclopsetta group (see discussion on Paralichthyidae) but not encoded). (2) observed in Poecilopsettinae, Rhombosoleinae, Samarinae, Achirinae and Soleinae. Aut- apomorphy ofCynogiossidae (Chapleau, 1988): first precaudal centrum missing (lost or fused to second precaudal vertebrae). 26. Fusion of hypural plates. No fusion ofhypural plates (0). Hypurals 3 and 4 fused and hypurals I and 2 fused (I). Brachypleura, Scophthalmidae, Paralichthyidae (except Thysanopsetta and Tephri- nectes), Bothidae (except Mancopsetta), and Pleuronectinae. Other apomorphic state noted only for Samarinae (see text) is hypurals 2-3-4 fused. Archirines Trinectes fimbriata (Hensley and Ahlstrom, 1984) and Apionichthys unicolor (Chapleau, unpubl. data) and rhombosoleine Psammodiscus show samarine type of caudal skeleton (not encoded). Ontogenetic data and outgroup comparisons indicate that apomorphic states were acquired independently. Data corroborating independent origin of apomorphic character states are in Hensley and Ahlstrom (1984) for bothid Engyophrys senta and Achir- idae (Chapleau, 1986). In latter case, state within plesiomorphic achirids (Achirus) is presence of 5 distinct hypural plates. 27. Articulation ofhypurals 1 and 2 to PUI. Plesiomorphic condition is to have hypurall and 2 in (or without) contact with PU I but without a large articulatory surface with PU I. Apomorphic state is to have a tight and wide articulatory surface between hypural plate and PUI, resembling a baIl- and-socket arrangement (Hensley, pers. comm.). Brachypleura, Scophthalmidae, Paralichthyidae, Bothidae, Pleuronectinae. 28. Relation ofhypural plates with PUI. Absence of fusion (0). Fusion ofhypurals 3 and 4 to the centrum of PUI is apomorphic (I). Brachypleura. Scophthalmidae, Paralichthyidae (except Tephri- nectes and Thysanopsetta), Bothidae (except Mancopsetta) and Pleuronectinae. Hensley and Ahlstrom 540 BULLETIN OF MARINE SCIENCE. VOL. 52, NO. I, 1993

(1984) indicated that Citharoides had hypurals 3 and 4 fused to centrum of PU I but Chapleau and Keast (1988) observed closely adjoined but un fused hypurals 3 and 4 to centrum of PUI. 29. Fusion oj proximal tip oj hypurals to PU 1. Four hypurals articulating but not fused to PU I (0). Fusion of the proximal tip ofhypural plates to PUI (I). Soleidae and Cynoglossidae (except Symphurus australis). Apomorphic condition was observed but not encoded for two rhombosoleine genera (Pel- torha mph us, Rhombosolea). 30. Edge oj preopercle (see text for discussion of this character). Posterior and ventral edge of preopercle partly visible (0). Posterior and ventral edge of preopercle entirely concealed by scales and skin (I). Soleidae and Cynoglossidae. 31. Eyed-side mesopterygoid. Present (0). Absent (I). Soleidae (except Pardachirus) and Cynoglos- sidae. 32. Indentation oj opercular series. Absent (0). Deep fimbriation pattern of opercular bones (except preopercle) (I). Soleidae and Cynoglossidae. 33. Shape oj blind-side dentary. Portion of blind-side dentary anterior to anguloarticular not markedly convex and nearly equal in length and deepness on both sides of the head (0). Portion of blind side dentary anterior to anguloarticular is convex and is shorter and deeper than the eyed-side dentary (I). Soleidae and Cynoglossidae. 34. Hemal arch on PU2. Hemal arch not fused to centrum of PU2 (0). Hemal arch fused to the centrum (I). All flatfishes, except for Psettodidae, Lepidoblepharon, Citharoides and Achiridae. 35. Parhypural and centrum of PUl. Parhypural articulating with centrum of PUI. Parhypural forms a plate totally free from PUI (I). All flatfishes, except the Psettodidae and Lepidoblepharon. 36. Branchiostegal membranes. Branchiostegal membranes separate (0). Membranes fused medially, with anteriormost branchiostegals attached to each other (I). Paralichthyidae, Bothidae, Pleuronec- tinae, Poecilopsettinae, Rhombosoleinae, Samarinae, Achiridae, Soleidae and Cynoglossidae. 37. Blind-side preopercular canal. Preopercular canal opening at anteroventral tip of preopercle (0). Opening (where present) along ventral margin of bone (I). Achiridae, Soleidae (except Heteromycteris) and Cynoglossidae. Achirid Gymnachirus and cynoglossid Symphurus lack a preopercular canal. 38. Contribution oj blind-side lateral ethmoid and blind-side Jrontal to margin oj upper orbit. Important contribution (40% or more) of blind-side frontal forming blind side bony orbit. Blind side lateral ethmoid forming most of the blind side margin of the upper orbit (blind side frontal contribution being minimal (I) or nil (2). (I) observed in Achiridae. (2) was found in Soleidae (except Heteromycteris and Typhlachirus) and Cynoglossidae. 39. Skin on lowerjaw and interopercle. Skin covering not continuous ventrally (0). Skin is continuous and covering isthmus and branchiostegals (I). Soleidae, Achiridae and Cynoglossidae.