External morphology, postcranial and appendicular osteology of three southwestern Atlantic flatfishes (Paralichthys, Paralichthyidae), and comparisons with other congeneric species

Comparisons of the external morphology and analysis of osteological features of the postcranial and appendicular skeletons of three southwestern Atlantic flatfish species of the genus Paralichthys (P. isosceles, P. orbignyanus and P. patagonicus) were carried out. Bones are described, and detailed morphological, morphometric and meristic characteristics of these flounders are given in order to provide information about the external and internal morphology of three species of Paralichthys occurring in the south-west Atlantic waters that add new information and will help regarding within the framework of a phylogenetic study of the group. Interspecific differences were found in the number of vertebrae and intermuscular bones, as well as in the morphology and morphometry of vertebrae, caudal skeletons, pectoral and pelvic girdle bones. Relationships between bones are discussed and bone characteristics compared with those found in other species of Paralichthys and in other pleuronectiform species. The position of Paralichthys isosceles within Paralichthys is discussed, along with other congeners such as P. triocellatus and P. oblongus.

Except for the Japanese flounder, Paralichthys olivaceus (Temminck, Schlegel, 1846), which occurs off Japan, Korea and China (Wu, 1932;Amaoka, 1969;Li, Wang, 1995;Munroe, 2015b), all other members of Paralichthys (ca. 25 nominal species) are distributed in temperate and tropical seas along the coasts and on the continental shelves of the American continent (Ginsburg, 1952;Hensley, 1995;Munroe, 2003).In the southwest Atlantic Ocean species of Paralichthys occur from northern Brazil just south of the Amazon river at ca. 1º N (Carvalho-Filho, 1999;Walsh et al., 2015) to central Patagonia, Argentina at ca. 47ºS (Díaz de Astarloa, Munroe, 1998).Throughout the southwest Atlantic region, species of Paralichthys occur in diverse habitats including coastal shallow-waters in areas containing muddy and silty substrata such as turbid estuaries, and also on sandy substrata in moderate depths on the continental shelf.In addition, some species of Paralichthys inhabit a variety of different substrata on the continental shelf, with some [P.isosceles Jordan, 1891;P. oblongus (Mitchill, 1815); P. triocellatus Miranda Ribeiro, 1903] also found on deep-water substrata located on the outer continental shelf.
In some demersal fish communities, especially those inhabiting soft-bottom habitats in the southwestern Atlantic Ocean (Walsh et al., 2015), paralichthyids can be abundant and may account for a significant portion of the fish biomass.For example, the Patagonian flounder (P.patagonicus) is widely fished on the continental shelf from Rio de Janeiro State to at least as far south as northern Patagonia (Díaz de Astarloa, Munroe, 1998).Although landing statistics of flatfishes for this region are aggregated, Patagonian flounder is the most abundant flatfish species in these landings, representing 61% of the total of flatfish species captured in Argentina (Rico, 2010).In southern Brazil, it is the main species of flatfish landed in the bottom trawl fisheries on the continental shelf and in coastal shallow-waters (Carneiro, 1995), representing 96.3% of the total flatfish landings (Haimovici, 1998).Other species of paralichthyids taken commercially include the Mud flounder (P.orbignyanus), a shallow-water flatfish occurring from Rio de Janeiro southward to San Matías Gulf, northern Patagonia, in Argentina, as well as the Isosceles flounder, P. isosceles.The Mud flounder represents 2.3% of the total flatfish landings in Rio Grande do Sul.At Mar del Plata harbor (Argentina), where almost 70% of the fishing fleet is located, the Mud and Patagonian flounders combined rank eighth in the total amount of kilograms of fish biomass sold.Paralichthys isosceles is marketed as small or moderate-sized flounder in Mar del Plata harbor, representing between 2.4% and 2.6% of the total amount of fish sold there (Fabré, Díaz de Astarloa, 2001).It is only occasionally caught in Brazilian waters (Haimovici, Mendonça, 1996).In Argentine waters (Díaz de Astarloa, 2002), three species of Paralichthys (P.isosceles, P. orbignyanus, and P. patagonicus) are the most important flatfishes in commercial fisheries conducted in those waters (Fig. 1a, b, c).Here, these fishes constitute the most abundant flounders in the commercial landings of Mar del Plata harbor (Cousseau, Fabré, 1990;Fabré, 1992;Rico, 2010) and they also play an important role in artisanal fisheries conducted in shallow waters of the Rio de la Plata estuary on the Uruguayan side and on the Atlantic coasts of Argentina and Uruguay, as well (Silva Junior et al., 2007).
Ginsburg´s (1952) study remains the most comprehensive systematic treatment of species of Paralichthys.Other taxonomic and systematic information is scattered among several works (Gutherz, 1967;Amaoka, 1969;Hensley, 1995;Díaz de Astarloa, Munroe, 1998;Figueiredo, Menezes, 2000;Munroe, 2003).However, Paralichthys is still not adequately defined based on shared derived morphological characters and even the species composition of this genus as presently known remains controversial.For example, some nominal species now included in Paralichthys were previously considered as members of the related paralichthyid Hippoglossina Steindachner, 1876 or Pseudorhombus Bleeker, 1862.Such is the case for the nominal species P. isosceles, which was placed in Pseudorhombus isosceles by Ginsburg (1952) but without further comment or investigation as to why Norman (1934) recognized it as a member of Paralichthys.More contemporary authors (Figueiredo, Menezes, 2000) also recognize this species as a member of Paralichthys.Placement of these nominal taxa into other paralichthyid genera has not always been based upon objective criteria supported by morphological features, but rather, placements or re-assignments have been based on authoritative opinions without substantiation supported by the possession of shared, derived characters among these taxa.Furthermore, none of the previous studies on Paralichthys has examined 3 e170164 [3] systematic relationships among members of this genus and our knowledge of the evolutionary relationships among these taxa remains at a basic level.
This paper provides new information about the external and internal morphology of the three most commercially important species of Paralichthys occurring in the Southwest Atlantic Ocean.This new information complements information provided in earlier studies by Cousseau, Díaz de Astarloa (1991), Díaz de Astarloa (1995a, 1996), Díaz de Astarloa, Munroe (1998), Figueiredo, Menezes (2000), Díaz de Astarloa ( 2005), and Díaz de Astarloa et al. (2006a).In addition, this new information will be helpful regarding studies in comparative anatomy, flatfish bone identification in archaeozoology or paleontology, and construction of a framework for phylogenetic study of the group.Also, comparisons of this new information with that of congeners will help in the assignment of species under study to the correct genus.These assignments will facilitate further investigation regarding the phylogenetic analyses of relationships among nominal species of Paralichthys.Finally, to assist other investigators interested in identifying and studying these fishes, we provide updated identification keys to the species occurring in each ocean based on new information derived from this study.

Material and Methods
A total of 553 specimens of Paralichthys were examined in the present study.The taxonomic analysis for the specimens occurring in the Southwest Atlantic waters was based on examination of both external (morphometrics and meristics) and internal (osteological) characters of 23 specimens of Paralichthys isosceles (130-370 mm SL), 35 specimens of P. patagonicus (250-480 mm SL) and 23 specimens of P. orbignyanus (390-1030 mm SL).The maximum length ranges correspond to the largest size of each species.All specimens were collected on the southwest Atlantic continental shelf between 34°30'S and 44°54'S, and between 40 and 100 m depth.
Thirteen morphometric characters on the ocular side were measured to the nearest 0.1 mm using dial calipers or a metal ruler.Measurements are expressed either as percentages of standard length (SL) or percentages of head length (HL) and are defined as follows (abbreviations in parentheses).Standard length -measured as a straight line from the anteriormost end of the lower lip to the caudalfin base (posterior end of hypural plate).Head and snout lengths (SNL) -measured from anteriormost end of lower lip to posterior end of opercular flap and to anterior fleshy margin of lower eye, respectively.Pectoral-fin length (PL) -measured as length of longest finray.Upper jaw length (UJL) -measured from anterior margin of upper lip to posterior end of maxilla.Eye diameter (ED) -measured as greatest fleshy diameter of the lower eye.Interorbital width (IW) -measured as least fleshy width between the orbits.Predorsal (PDL), prepelvic (PVL) , preanal (PAL) and prepectoral (PPL) lengths -all measured from anteriormost end of lower lip to bases of first finrays in the dorsal, ventral, anal and pectoral fins, respectively.Caudal-peduncle depth (CPD) -measured as least depth across caudal the peduncle.
Meristic data were also taken from the ocular side of each specimen.Numbers of perforated lateral-line scales were counted starting from the scale above the pectoral-fin base and ending with that located at the base of the caudal-fin rays.Counts of oblique rows of scales along the horizontal region of the lateral line were made from the point just posterior to the lateral-line arch above the pectoral-fin rays to bases of the caudal-fin rays.Vertebral numbers were obtained either from dissection of the fish, or from cleared and stained specimens, or were taken from radiographs of type and non-type specimens curated in institutions.The urostyle was included as one vertebra.Numbers of infraorbital bones, epineurals and ribs, premaxilla and dentary teeth were also counted.Abbreviations for meristic characters are: DO, dorsal-fin rays; AN, anal-fin rays; PE, pectoral-fin rays, CA, caudal-fin rays; GR, gill rakers on first arch; LL, lateral-line scales; VE, total vertebrae.
Regression analysis was performed to compare body proportions among the three species of Paralichthys.Standard length and morphometric relationships were calculated separately between sexes for each species, and between species.The null hypothesis of no difference between slopes of the linear regressions was tested with the Student´s t-test (Zar, 1984).Differences among species in meristic features were tested by a Kruskal-Wallis analysis of variance (ANOVA) (Hoaglin et al., 1991).Contrast comparisons between flatfish species were made using a Wilcoxon rank test with those characters that showed significant differences in the ANOVA.In all statistical tests, the probability of a type I error was set equal to 0.05.
Specimens examined included both fresh and alcoholpreserved fish that were dissected, and also cleared and stained specimens.Observations of anatomical features were made macroscopically and under a stereomicroscope.Drawings were made with the aid of a camera lucida attachment.Methods for preparing disarticulated skeletons followed Ossian (1970) and Mayden, Wiley (1984).Methods of clearing and staining for bone and cartilage followed Dingerkus, Uhler (1977), Potthoff (1984) and Kawamura, Hosoya (1991).Terminology for osteology mainly followed that of Cervigón (1985) and Hoshino, Amaoka (1998), except for intermuscular bones where Patterson, Johnson (1995) were followed, and terminology of Hoshino (2001) was used for elements of the caudal skeleton.Other osteological terms followed those in Rojo (1988).
Abbreviations used for names of bones are as follows, or are provided under individual illustrations.Museum acronyms follow those listed in Sabaj Pérez (2016).

Results
Morphology.Scale morphology.All species of Paralichthys examined herein have either ctenoid or cycloid scales on their ocular sides, but only P. isosceles has ctenoid scales on both the ocular side as well as on the blind side of the body.In addition, the species have accessory scales on their ocular sides with the exception of P. isosceles, P. triocellatus and P. oblongus, which lack accessory scales.
Osteology.Postcranial axial skeleton.This section is divided into three parts: vertebral column, ribs and epineurals, and caudal skeleton.
In all three Paralichthys of the SW Atlantic examined here the first precaudal vertebra is small with its corresponding neural spine fused to the centrum, also the neural spines of the first to the fourth vertebrae are stout and compressed.Starting from the third or fourth vertebra, the neural spines narrow posteriorly toward the hypural complex and they become more directed posteriorly, with this deflection becoming most conspicuous on the caudal vertebrae.Haemal spines are only present on the caudal vertebrae.On each successive anterior caudal vertebra, the neural spine is more elongate than that of the respective corresponding neural spine of the previous precaudal centrum.The haemal spine of the first caudal vertebra (Fig. 3b) is a very strong plate, supporting the first anal-fin pterygiophore (Amaoka´ s interhemal spine or Woolcott´ s abdominal rod).
All vertebral centra are amphicoelous, concave both anteriorly and posteriorly.In the three species of Paralichthys in our study, there are one or two small elliptical concavities present between the dorsal and ventral concave portions only on the last precaudal and the first caudal vertebrae.Beginning with the sixth or seventh and continuing on all subsequent precaudal vertebrae, paired parapophyses are present.On the sixth or seventh to the posteriormost precaudal vertebrae, the distal ends of the paired parapophyses unite, forming a closed haemal arch, which is referred to as haemapophyses by Amaoka (1969).These haemopophyses have a single non-bifurcated tip.Haemapophyses appear on the 7th to 10th precaudal vertebrae in P. patagonicus, on the 7th or 8th to 10th vertebrae in P. isosceles, and on the 6th or 7th to 10th vertebrae in P. orbignyanus.The preneural and postzygapophyses are well developed on all vertebrae of Paralichthys.On the precaudal vertebrae and on the first caudal vertebrae, the zygapophyses are tightly interlocked with each other, but in the posteriormost caudal vertebrae they are only slightly articulated, or are entirely free from each other.The prehaemal -and postzygapophyses are present only in P. orbignyanus, begining from the second caudal vertebra (Fig. 4).No haemal zygapophyses were found in the other two South Atlantic species of Paralichthys examined.Ribs and epineurals.In the three southwest Atlantic species examined, the first, or the first two, ribs are attached directly to the anterolateral side of the centrum, while the other ribs attach to the distal ends of the haemapophyses (Fig. 3).Ribs are directed downward following the joint line of the myosepta with the wall of the coeloma.In contrast, epineurals [epipleural (dorsal) ribs sensu Cervigón, 1980] extend outward from the vertebral centra following the horizontal myoseptum (Cervigón, 1980).
Of the three South Atlantic of interest to our study species, P. orbignyanus has the fewest ribs (6 pairs), P. patagonicus has the highest number of ribs (7-8 pairs), and P. isosceles has 7 pairs of ribs.
Epineurals occur on the 2nd to 10th precaudal vertebrae in the three species of Southwest Atlantic Paralichthys examined (Fig. 3).The first epineural bone is attached to the base of the neural prezygapophyses.The second epineural articulates slightly ventral to the prezygapophyses, and the third epineural is directly connected to the anterodorsal side of the vertebral centrum, where it shares the joint with the second epineural rib.The remaining epineurals are attached to the central portion of the parapophyses.Paralichthys patagonicus has the fewest number of epineurals (9 pairs), followed by that of P. isosceles [range (9-10); mean 9.6], whereas P. orbignyanus has the highest number [range (10-11); mean 10.3] of epineurals among the three species studied.Caudal skeleton.All three species of Paralichthys examined in this paper have the hypural complex pattern 6 (Hensley, Ahlstrom, 1984) which is characterized by two preural centra (pc 1-2); a detached autogenous parhypural (phy); hypurals 1 and 2 fused together forming a broad ventral hypural plate (vhp) articulated with the posteroventral surface of the first preural centrum (cp1); hypurals 3 and 4 fused together and fused to the tip of first preural centrum forming a broad dorsal hypural plate (dhp); an autogenous fifth hypural (H5) with its proximal end closest to the second epural; and with this caudal complex supporting a total number of 18 caudal-fin rays, of which 13 central-fin rays are branched (Fig. 5a).In the present study, we found two autogenous epurals in all cleared and stained specimens of P. isosceles and P. patagonicus, and in only 5 of 14 specimens of P. orbignyanus.In one specimen of P. patagonicus, we also noticed two unidentified bony elements, situated adjacent to the two epurals (Fig. 5b).Some scissures are present on distal margins of the parhypural and hypural elements.
Of the 18 caudal-fin rays, two segmented fin rays occur in the upper and lower distal parts of the fin, 13 branched fin rays are present in the middle section of the fin, and one unsegmented fin ray, also called "procurrent ray" (sensu Hoshino, 2001), is present at the upper and lower distal extremities of the fin.The uppermost fin ray is very small, covered by skin and musculature, and supported by the neural spine of preural centrum 2. The ventralmost fin ray is also very small and fused to its neighboring segmented ray (Fig. 5c).

Dorsal and anal fins.
Each fin ray is supported basally by a bipartite structure consisting of a small distal pterygiophore and an elongate proximal pterygiophore.The two small lentiform halves of the distal pterygiophore are located between the two halves of lepidotrichia.The upper extremity of the proximal pterygiophore is cup-shaped and bears two lamellar projections laterally.The proximal pterygiophores interdigitate by sutures with the neural or hemal spines (Fig. 3d).The anteriormost eight proximal pterygiophores of the dorsal fin articulate with the dorsal part of the supraoccipital.The anteriormost of these pterygiophores supports the first two dorsal-fin rays.Generally, two pterygiophores interdigitate between neural spines, but sometimes three pterygiophores insert in an interneural space (Fig. 3d).The first anal-fin pterygiophore is modified into a large, curved rod that together with the first haemal spine closes the abdominal cavity posteriorly.This pterygiophore supports three fin rays in P. isosceles.The following 11 proximal pterygiophores rest on the posterior face of the abdominal rod in P. patagonicus.In P. orbignyanus, 8 or 9 proximal pterygiophores rest on the abdominal rod (Figs 3b,d).
Appendicular skeleton.Pectoral girdle.The pectoral girdle consists of the girdle itself (cleithrum, coracoid and scapula), four radials and a chain of bones that connect the girdle to the cranium (supracleithrum and two postcleithra).Some authors also include the posttemporal in this series (Balart, 1985;Collette, Gillis, 1992).
Cleithrum-The cleithrum is a half moon-shaped bone with both dorsal and lower limbs sharply pointed.The inferior limb bears a medial groove on its lateral surface and a welldeveloped ridge on the inner face that delimits a deep furrow where the sternohioideus muscle is inserted.The upper limb of the cleithrum bears a shallow lateral depression into which fits the overlapping supracleithrum.At the posterior angle of the cleithrum where the upper and lower limbs join together, a lateral process expands from this bone to overlap and support, partly by suture, the scapula and the body of the coracoid.This lateral process also contacts the anterior tip of the first postcleithrum.The elongate anterior projections of the basipterygia are included between the lower limbs of the two cleithra (Fig. 6).In P. patagonicus and P. orbignyanus, the lateral process of the trailing edge of the cleithrum is more expanded, and has a more irregular border compared with that of P. isosceles (Figs.6b, c, d).Scapula-The scapula is a flattened bony plate with a slender caudal process.Anteriorly, the lateral surface fits into a depression on the medial face of the cleithrum.Ventrally, it is connected to the coracoid by means of a cartilaginous strip.Two of the four radials are attached posteriorly to the scapula.The other two arise from the posterior border of the scapula and do not have a facetal articulation (Fig. 6a).No differences were found among the scapulae in the three South Atlantic species of Paralichthys, examined in this study.Supracleithrum-This leaflike bone has its upper tip overlapped by the posttemporal to which it is joined by means of connective tissue.The lower part of the supracleithrum overlaps the anterior portion of the cleithrum (Fig. 6a).The maximum width of the supracleithrum varies from 15.0 to 23.0% of the total length of the bone in the three species.It is widest in P. isosceles (range 21.0-23.0%;mean 22.0%) and narrowest in P. orbignyanus (15.0-19.0%;16.7%), with intermediate values found in P. patagonicus (16.0-18.0%;17.2%).
Postcleithrum-Both upper and lower postcleithra are elongate, thin bones located posterior to the cleithrum where they are partially overlapped by the pectoral-fin rays (Fig. 6a).The upper (first) postcleithrum joins the inner face of the cleithrum.The bone is curved and elongate with its lower end pointed in P. isosceles.Both upper and lower ends are pointed, with lamellar expansions in P. patagonicus and P. orbignyanus.Pelvic girdle.The pelvic girdle consists of paired basipterygia each associated with six pelvic-fin rays (the first and second pelvic-fin rays being simple, and the remainder on each side branched).Each basipterygium is composed of an anterior process shaped like an elongate shaft obliquely directed into a space that is hidden by the lower limbs of the cleithra; a wide basal plate that posteroventrally supports the pelvic-fin rays; a strong posterior process; and a spiny process directed forward and located on the inner face of the basipterygium (Fig. 6).Both basipterygia meet in an extended sutural joint.The posterior process of the basipterygium is sharp in P. isosceles, wider and blunt in P. patagonicus, and slightly curved upwards in P. orbignyanus.Based on these findings, and for a comparison with all currently known species of Paralichthys occurring on both sides of America and the single species occurring in western Pacific Ocean, keys to the Atlantic and Pacific species of Paralichthys were created to assist in the identification of these species.tropicus in having a greater number of total gill-rakers and more lateral-line scales (Tabs 5,6).Paralichthys orbignyanus also has a smaller eye and a shorter pectoral fin compared with those of its congeners (Fig. 7a, b).Additionally, P. orbignyanus has a wider interorbital width compared to those of other species of Paralichthys (Fig. 8).Among species of Paralichthys, P. isosceles shares two features in common with both P. triocellatus and P. oblongus: one is the absence of accessory scales and the other is the fewest gill-rakers.Paralichthys isosceles differs from P. triocellatus in having more lateral-line scales (mean 75.9 vs.65.2) and pectoral-fi n rays (11.2 vs. 10.3).Paralichthys isosceles and P. triocellatus are very similar in external appearance in that both species have three conspicuous ocelli, and most of their meristic features, except those mentioned above, overlap (Bittencourt, 1982).However, these species are easily differentiated by the type of scales present on their blind sides (ctenoid in P. isosceles and cycloid in P. triocellatus), and also by the presence of a black spot on the ocular-side pelvic fi n of P. isosceles which is absent in P. triocellatus (Figueiredo, Menezes, 2000;Pers. obs.).Paralichthys isosceles differs from P. oblongus in having a greater number of dorsal-and anal-fi n rays (mean 84.3 vs. 77.4; and mean 67.0 vs. 62.4, respectively), and it has fewer pectoral-fi n rays, vertebrae and lateral-line scales (mean 11.2 vs. 11.7, 38.6 vs. 42.0 and 75.9 vs. 91.2, respectively).Also, P. isosceles has 10 precaudal vertebrae, while P. oblongus has either 11 or 12. Paralichthys isosceles differs from the other species of Paralichthys, except P. oblongus and P. triocellatus, in having a greater eye diameter and a longer ocular-side pectoral fi n (Fig. 9a, b).

Key to species of
Paralichthys patagonicus differs from P. brasiliensis in having greater numbers of lateral-line scales, vertebrae and dorsal and anal-fi n rays (mean 102.8 vs. 84.2;mean 38.0 vs. 35.0;mean 81.0 vs. 71.0 and mean 63.0 vs. 53.8,respectively).Paralichthys patagonicus has lower gill-raker counts than those of P. adspersus, P. aestuarius, P. dentatus, P. californicus, P. microps and P. olivaceus (mean 13.1 vs. 23.8; 27.5; 20.2; 29.2; 29.2 and 20.2, respectively).Also, it has fewer vertebrae than that found in P. dentatus and P. oblongus (mean 38.0 vs. 41.7 and 42.0, respectively).Paralichthys patagonicus also has a narrower interorbital width than that of P. adspersus, P. albigutta, P. brasiliensis, P. dentatus, P. microps and P. olivaceus (Fig. 10a), but this measurement is wider than that found in P. oblongus and P. triocellatus (Fig. 10b).Osteology.In the three southwest Atlantic species examined in the present paper, the fi rst neural spine is fused to the centrum.In other Pleuronectiformes, the fi rst neural spine is autogenous (Cervigón, 1985) or is lacking altogether (Amaoka, 1969).Although Amaoka (1969) reported that plate-like neural spines occurred on the fi rst to the third vertebrae in Pseudorhombus, versus on the fi rst to the fourth vertebrae in Paralichthys, we found that broad plate-like neural spines occurred only on the fi rst three vertebrae in P. isosceles, P. triocellatus and P. oblongus.Conversely, all other species of Paralichthys, including P. patagonicus and P. orbignyanus, have broad plate-like neural spines on their fi rst four vertebrae (Fig. 3).
Haemapophyses appear on the 7th to 10th precaudal vertebrae in P. patagonicus, on the 7th or 8th to 10th vertebrae in P. isosceles, and on the 6th or 7th to 10th vertebrae in P. orbignyanus.In comparison, Woolcott et al. (1968) reported that in P. albigutta, P. lethostigma and P. dentatus the haemal arch was completely formed beginning with the 7th precaudal vertebra.Ribs are attached directly to the anterolateral side of the centrum, while the other ribs attach to the distal ends of the haemapophyses.Ribs are present in all members of Paralichthyidae, but they purportedly are not present in species of the family Bothidae (Amaoka, e170164[17] 1969).However, Hensley, Ahlstrom (1984) concluded that those elements termed "abdominal hypomerals" by Amaoka (1969) are actually pleural ribs (Patterson, Johnson, 1995).Chanet et al. (2004) presented a table of the number of the anteriormost ribs occurring in members of different flatfish Families, including those of the Paralichthyidae and Bothidae.Amaoka (1969) noted the presence of ribs on the 3rd to 10th precaudal vertebrae in Paralichthys, however, we found differences and more variation from this arrangement among species of Paralichthys we examined.For example, we found ribs occurring only on the 3rd to 9th precaudal vertebrae in P. isosceles, and to the 10th vertebra (one specimen) in P. patagonicus, and on the 4th to 9th vertebrae (one case beginning on the 3rd vertebra) in P. orbignyanus.
The caudal fin and caudal skeleton of species of Paralichthys have been illustrated and discussed by several authors (Woolcott et al., 1968;Amaoka, 1969;Hensley, Ahlstrom, 1984;Balart, 1985;Díaz de Astarloa, 1991;Hoshino, 2001).The hypural complex of the Pleuronectiformes corresponds to the stegural acentral V-b2 type of Monod (1968).However, and according to several patterns of fusions among hypurals 1-4, Hensley, Ahlstrom (1984) situate Paralichthys in hypural pattern 6. Hoshino (2001), in contrast, regards the small spur on the ventralmost surface near the base of the first ventral caudal-fin ray, called the "splinter ray" by Hensley, Ahlstrom (1984), as an unsegmented ray fused to a segmented ray in the ventral part of the caudal fin (Fig. 3).Balart (1985) mentioned the presence of two epurals of contrasting size in P. olivaceus, with the anteriormost epural elongate whilst the posterior epural is very reduced in size compared to that of its counterpart.Woolcott et al. (1968) also described two autogenous epurals in species of Paralichthys they examined, but they erroneously pointed out hypurals 3, 4 and 5, and overlooked in their illustration the correct two epurals (illustrated in their Figs.3, 5).Díaz de Astarloa (1991) found only one autogenous epural in two nominal species of Paralichthys he examined.
Remarks.On the generic assignment of Paralichthys isosceles, P. triocellatus and P. oblongus.Paralichthys isosceles was originally described as a member of Paralichthys.Later, this species was transferred to Pseudorhombus by Ginsburg (1952) based primarily on the absence of accessory scales.Accessory scales are present in all other species currently assigned to Paralichthys, except P. triocellatus and P. oblongus (assigned to Hippoglossina by Ginsburg) which also lack accesory scales.Ginsburg stated that P. triocellatus is a species of doubtful relationship, and according to its diagnostic characteristics should be close to "Pseudorhombus" isosceles.Ginsburg (1952) did not examine the Holotype of P. triocellatus.Here, we examined the type of Miranda Ribeiro´s P. triocellatus and conclude that it is similar to, but can be clearly distinguished from, P. isosceles in having cycloid scales on the blind side and by other diagnostic features that were discussed above.Although P. isosceles and P. triocellatus may be closely related as hypothesized by Ginsburg (1952), they clearly do not belong to Pseudorhombus since these two species have 18 caudal-fin rays including a splinter ray on the ventralmost caudal-fin ray (vs.17 caudal-fin rays and no splinter ray in Pseudorhombus).This splinter ray is likely a remnant of a ray lost through fusion with an adjacent ray (Hensley, Ahlstrom, 1984) and this structure was observed in all species examined in the present paper that we consider belonging to Paralichthys.
Regarding P. oblongus, the generic placement of this species is problematic because it shares some features in common with those of members of both Paralichthys and Hippoglossina.During its history, this species has been placed in several different genera, and together with Hippoglossina tetrophthalma, it was even placed in a separate subgenus within Hippoglossina (see synonymy in Ginsburg, 1952).Some disagreement occurs within contemporary works (Munroe, 2003) regarding the correct generic assignment for this species.Paralichthys oblongus differs from members of Hippoglossina in placement of the origin of the dorsal fin, which in these species is well behind the posterior nostril of blind side and over the posterior part of the pupil.In P. oblongus, the dorsal fin commences just behind of the blind-side posterior nostril and the origin of the dorsal fin is located above the anterior margin of the upper eye.Additionally, members of Hippoglossina have 17 caudalfin rays and they lack a splinter ray in the caudal fin (vs.18 caudal-fin rays and splinter ray present in P. oblongus).

e170164[18]
Most contemporary works assign Paralichthys isosceles, P. triocellatus and P. oblongus to Paralichthys.However, several external and internal morphological features of these species differ from those found in other species currently assigned to Paralichthys.According to Amaoka (1969), broad plate-like neural spines occur on the first four vertebrae in Paralichthys, whereas in P. isosceles, P. oblongus and P. triocellatus, these broad-shaped neural spines are found only on the first to the third vertebrae, as is also observed in members of Pseudorhombus.This feature, along with the lack of accessory scales in these species, reinforces the hypothesis that these three nominal species currently assigned to Paralichthys should be reassigned to another genus or to other genera, and likely not Pseudorhombus or Hippoglossina, especially as currently defined.However, before a more definitive answer can be provided about the generic placement of these species, more investigation is needed to provide strong support for hypotheses regarding the relationships of these species.

Fig. 2 .
Fig. 2. Relationships of head length to standard length: a. interorbital width to head length, b. pectoral length to standard length, c. eye diameter to head length, d. caudal peduncle depth to standard length, e. among the following species: Paralichthys isosceles, P. orbignyanus and P. patagonicus.
Frequency comparison for counts of gill rakers in Paralichthys species.* Include types.Frequency comparison for counts of lateral-line scales in species of Paralichthys.* include types.
Tab. 3. Frequency comparison for counts of total vertebrae in species of Paralichthys.* include types.
Tab. 4. Frequency comparison for counts of pectoral-fin rays in Paralichthys species.*include types.