Descriptive morphology and phylogenetic relationships among species of the Neotropical annual killifish genera Nematolebias and Simpsonichthys ( Cyprinodontiformes : Aplocheiloidei : Rivulidae )

Structures of the external morphology and skeleton of the annual fish genera Nematolebias and Simpsonichthys are described and illustrated. Phylogenetic relationships among all included species of both genera and other cynolebiatins are estimated, based on 116 morphological characters in a total of 50 terminal taxa. Nematolebias is hypothesized to be the sister group of a clade including Simpsonichthys, Austrolebias, Megalebias, and Cynolebias, which is diagnosed by ventral portion of mesopterygoid not overlapping quadrate, third neural spine approximately as wide as fourth neural spine, ventral condyle of coracoid narrow, scales slightly extending over anal-fin base, loss of vomerine teeth, loss of neuromast anterior to infraorbital series, ventral process of the angulo-articular short and narrow, and dorsal portion of cleithrum elongated. Simpsonichthys is divided into five subgenera, including Spectrolebias, previously considered a separate genus, Simpsonichthys, and three new subgenera, Xenurolebias, Ophthalmolebias, and Hypsolebias. Monophyly of Simpsonichthys is supported by a small pointed dorsal process on the palatine, unbranched fin rays on the tip of the dorsal and anal fins in males, and frontal E-scales overlapped.


Introduction
Nematolebias comprises two species endemic to the coastal plains of southeastern Brazil (Costa, 2002a). Simpsonichthys contains 43 valid species, constituting the most speciose genus of aplocheiloid annual fishes; it occupies a vast geographic range in northeastern, central and eastern South America (e.g., Costa, 2003a). The greatest diversity of Simpsonichthys is concentrated in the area drained by the middle and upper Tocantins-Araguaia, middle São Francisco, and upper Paraná river basins, to where a total of 31 species are endemic. Species of Simpsonichthys are also present in the Madeira basin, Bolivia; Paraguay basin, Paraguay; Jaguaribe, Pardo, Jequitinhonha, and Xingu basins, and smaller coastal basins of northeastern and eastern Brazil. Two phylogenetic studies, mainly based on color patterns and some osseous structures, involved a meaningful number of species of the genera Nematolebias and Simpsonichthys (Costa, 1996(Costa, , 2003. The name Simpsonichthys was first proposed by de Carvalho (1959), but it was considered as a synonym of Cynolebias Steindachner (Parenti, 1981). Subsequently, based upon phylogenetic studies on rivulids, Cynolebias was divided (e.g., Costa, , 1996. New genera, including Spectrolebias Costa & Nielsen, Nematolebias Costa, Austrolebias Costa, and Megalebias Costa were then erected (Costa & Nielsen, 1997;, and Simpsonichthys was resurrected (Costa, 1996). However, gradual addition of new taxa and new characters in most recent studies generated some impact in previous phylogenetic hypotheses. Nematolebias was first considered to be the sister group to Simpsonichthys, and Spectrolebias, to be the sister group to an assemblage including Nematolebias, Simpsonichthys, Austrolebias, Cynolebias, and Megalebias , but according to a more recent hypothesis (Costa, 2003b), Spectrolebias would be the sister group to a clade including only Nematolebias and Simpsonichthys. Two independent molecular phylogenies of rivulids (Murphy et al., 1999;Hrbek & Larson, 1999), including respectively three and four species of Simpsonichthys and Nematolebias, hypothesized some species of Simpsonichthys is more closely related to Austrolebias than to Nematolebias.
Until recent years, morphological traits were the only or the main source of characters to erect phylogenies and classifications of actinopterygian fishes. Different kinds of morphological characters have been progressively employed in killifish systematics, but Parenti (1981) first used them in a cladistic context. Subsequently, a variety of phylogenetically informative morphological traits, such as osseous features, scale and neuromast arrangement patterns, color patterns, besides data on behavior and ecology, were used to establish rivulid clades and formally diagnose taxa (e.g., Costa, , 2004a. However, despite morphological characters being regularly listed in systematics studies on killifishes, morphological studies are usually restricted to selected features that were found to be phylogenetically informative, not making available the data considered uninformative (e.g., Parenti, 1981;Costa, , 2001. Consequently, many structures are still undescribed or poorly known. Loureiro & de Sá (1998) conducted a descriptive osteological study of a broad range of taxa including some species today placed in Austrolebias, Megalebias and Simpsonichthys, mostly obtained from aquarium material. Unfortunately their descriptions were brief and some structures misidentified.
Phylogenetic studies of killifish using DNA sequence data have become much more common. However, these studies often neglect or omit previously published, conflicting morphological characters (e.g., Hrbek & Larson, 1999). Much of the recent enthusiasm for molecular studies as a unique powerful source of phylogenetic information is due to the rapid production of data and the putatively greater number of informative characters that result, in comparison to morphological analyses. However, recent debates indicate that morphological characters are crucial for understanding phylogenetic relationships (Wahlberg & Nylin, 2003;Smith & Turner, 2005), molecular and morphological characters may have similar numbers of relevant characters (Jenner, 2004;Lee, 2004), and morphological phylogenies are important to detect com-mon errors in molecular analyses, such as long-branch attraction, deviations between gene and species tree, and contamination and misidentification of specimens (Jenner, 2004;Wiens, 2004). However, most authors are in agreement about the striking necessity in to improve morphological phylogenies (e.g., Dettai et al., 2004;Wiens, 2004).
The main objectives of the present study are: to provide accurate descriptions of morphological features, including external morphology of body, head and fins, squamation, neuromasts, contact organs and skeleton in Nematolebias and Simpsonichthys; to find additional informative characters in order to improve phylogenetic analysis; and, to revise characters included in former analyses (e.g., Costa, 1996Costa, , 2003 to eliminate subjectivities and to make them more accurate. and monophyly of the Cynolebiatini, were also included as terminal taxa Cynolebias griseus Costa, Lacerda & Brasil and Austrolebias carvalhoi (Myers), two basal species of the well-corroborated cynolebiatin clade Cynolebias Steindachner + Megalebias Costa + Austrolebias Costa. Most recent phylogenetic hypotheses for the Rivulidae genera (Costa, , 2004a is the baseline for selecting out-groups to polarize character states (Nixon & Carpenter, 1993). Out-groups include Leptolebias minimus (Myers), a basal species of the Cynopoecilini, the sister group to Cynolebiatini, Neofundulus paraguayensis (Eigenmann & Kennedy), a member of a basal annual rivulid clade, and Kryptolebias brasiliensis (Valenciennes), a basal species of the most basal lineage of the Rivulidae (Costa, 1995(Costa, , 2004a. All characters and character states used in the phylogenetic analysis are listed and coded in the Appendix II and are organized in the data matrix of Appendix IV. Characters with the derived state occurring in a single terminal taxon were not included in the data matrix. Characters of behavior (Belote & Costa, 2002) were not included in the phylogenetic analyses due to the scarcity of available data among the terminal taxa. Character states of multistate characters were treated as ordered whenever possible. Boundaries between states of quantitative characters were determined according to variations found among terminal taxa, in order to accommodate the real range observed for each terminal species included in that category (i. e., character state). These characters (20,21,25,35,43,44,62,63,81) may contain some subjectivity, and consequently were excluded from a second analysis, which produced similar trees (see phylogenetic analysis below). Most parsimonious cladograms, consistency indices (CI) and retention indices (RI) were obtained using the heuristic algorithm mhennig*;bb* of the program Hennig86 (Farris, 1988). TreeGardener 2.2.1 (Ramos, 1996) was used for optimization of character state changes in the strict consensus tree of most parsimonious cladograms, using ACCTRAN. Bootstrap analysis (Felsenstein, 1985) was used to establish nodal support through the simple heuristic algorithm of PAUP 4.0 (Swoford, 1998) with 1000 replicates.

Descriptive and comparative morphology
Descriptions and comparisons are primarily based on males, except in morphological traits typical for females. Descriptions are focused on N. papilliferus, referring to other species of Nematolebias and Simpsonichthys when differences were noted. In view of to simplify information about distribution of morphological features among species of Simpsonichthys, they are grouped following the classification presented at the end of this paper, in which Simpsonichthys is divided into five subgenera: Xenurolebias, Ophthalmolebias, Spectrolebias, Simpsonichthys, and Hypsolebias. Monophyletic subunits of the latter subgenus, the most speciose, are referred as the following species groups: S. magnificus species group, including S. hellneri, S. adornatus, S. fulminantis, S. carlettoi, S. magnificus, and S. picturatus; S. notatus species group, including S. stellatus, S. rufus, S. notatus, S. radiosus, S. similis, S. trilineatus, S. auratus, and S. nielseni sp.; S. antenori species group, including S. antenori, S. igneus, S. ghisolfii, S. flavicaudatus, and S. flagellatus; and, S. flammeus species group, including S. delucai, S. alternatus, S. multiradiatus, S. flammeus, and S. brunoi. Size. Species of Nematolebias and most species of Simpsonichthys are medium sized killifishes, reaching about 35-60 mm SL as the maximum adult size. However, S. parallelus, S. cholopteryx, S. costai, S. reticulatus, and S. semiocellatus are miniature species, among the smallest species of the Rivulidae. Their maximum size does not surpass 25 mm SL (Costa, 1996(Costa, , 2003. On the other hand, S. ghisolfii is clearly bigger than any other congener, reaching at least 73 mm SL (Costa, 2003b).
As in most aplocheiloid fishes, all species of Nematolebias and Simpsonichthys exhibit sexual dimorphism in size. Males are always bigger than females. Females approximately reach 80-85% of the size of males, but in S. ocellatus, females reach only about 65%.
General morphology of body. Species of Nematolebias differ from most other cynolebiatins by their slender body. The maximum body depth, which is always at the level of the pelvic-fin base, is 28% SL in larger males. Among species of Simpsonichthys, except S. semiocellatus that is a slender species like species of Nematolebias, all are deep-bodied, most reaching 32-40% SL. Exceptionally, in some species of the S. flavicaudatus group, males may reach 41-44% SL (Costa, 2003b).
Increasing in body depth is proportional to increasing in head depth, and therefore, whereas in S. semiocellatus the maximum head depth is about 90% of HL, in most Simpsonichthys the head depth is approximately equal or longer than the head length. The minimum caudal peduncle depth is 14-19% SL in larger males of Nematolebias and Simpsonichthys, except in S. semiocellatus, with 12% SL.
The body is compressed, its widest portion situated at the humeral region, which is slightly narrower than the widest portion of the head. Head width is about 65-80% HL in larger males of most species of Simpsonichthys and Nematolebias, but in S. chacoensis, S. costai, S. reticulatus and S. semiocellatus, the head is slightly more compressed, about 60% SL.
The dorsal profile of the head is slightly concave in juveniles, becoming approximately straight in adult specimens. Sometimes the dorsal profile of the head is gently convex in older males. Both the dorsal profile between nape and the end of dorsal-fin base and the ventral profile between the lower jaw and the end of anal-fin base are always convex. The ventral profile of the head is angular in S. semiocellatus and S. filamentosus, in which there is a prominent flap of thickened tissue at the level of the angulo-articular bone. The dorsal and ventral profiles of the caudal peduncle usually are straight, sometimes weakly concave. The caudal peduncle is short, about half length of the head, except in S. semiocellatus, in which the caudal peduncle is distinctively elongated.
Cephalic structures (Fig. 1). The eyes are large, dorsolaterally placed on the head, and the orbital rim is not free from the head rim. The eyes occupy 22-28% HL in larger males and 29-31% in juvenile males of species of Nematolebias. In Simpsonichthys, the eye diameter varies from 25 to 38% HL in adult males of median sized species, but in juveniles and adults of miniature species the orbital diameter is 34-42% HL. In the subgenus Ophthalmolebias, the eyes are slightly ventrally displaced, becoming more laterally positioned on the head (Figs. 2a-3a).
The mouth is subdorsal, with the upper jaw moderately protractile. The lower jaw is robust, presenting a prominent fold at the mouth corner, and short, usually 15-28% HL. The mouth cleft extends ventroposteriorly, reaching a vertical through the anterior portion of the orbit. The snout is short, usually 10-17% of HL. The anterior naris is tubular and ante- (c) ventral view. ais = anterior infraorbital series; an = anterior naris; arn = anterior rostral neuromast; lal = lateral line neuromasts; lms = lateral mandibular series; mas = mandibular series; mis = median infraorbital series; mon = median opercular series; nai = neuromast anterior to infraorbital series; ots = otic series; pan = parietal neuromast; pbs = preorbital series; pis = posterior infraorbital series; pmn = paramandibular neuromast; pn = posterior naris; pos = post-otic series; prn = posterior rostral neuromast; prs = preopercular series; sos = supraorbital series; stn = supra-temporal neuromast; vos = ventral opercular series; A-H = frontal scales A-H; in bold, frontal scale with all borders free. Urogenital papilla (Fig. 4). In males, the urogenital papilla is tubular, slightly visible in a lateral view of the venter. However, in Spectrolebias the male urogenital papilla may be elongated (Fig. 4d). In females of all species, the urogenital opening is placed in a prominent pocket-like structure that usually slightly overlaps the anterior portion of the anal fin (Fig. 4C).
Fins. In males of both species of Nematolebias and most species of Simpsonichthys, the dorsal and the anal fins have a similar morphology . Their distal extremity terminates in a long pointed tip, producing an approximately triangular shape. The tip of the dorsal and anal fins bears some short filamentous rays. In females, both fins are shorter than in males, the dorsal fin is rounded to slightly pointed, the anal fin is always rounded, and both fins lack filamentous rays on their distal border. The anal fin is hardened in females, often having a distinctive thickened tissue on its anterodistal portion.
Several species of Simpsonichthys exhibit the dorsal and anal fins morphologically similar to the fins in Nematolebias (Fig. 3a), although often not so elongated in males. In species of the S. flammeus group, the anal fin is elongate in females, acquiring a spatula-like shape. In some species of Simpsonichthys, both the dorsal and the anal fins are rounded in males (S. costai, S. magnificus, S. picturatus, S. delucai) (Fig. 3c), or only the dorsal fin is rounded (S. reticulatus), or only the anal fin is rounded (S. semiocellatus, S. alternatus, S. suzarti) (Fig. 3d). In some species, the filaments are absent in both fins (S. costai, S. parallelus, S. cholopteryx, S. delucai, S. magnificus, S. picturatus) (Fig. 3c), or are absent only in the anal fin (S. reticulatus, S. alternatus, S. adornatus, S. semiocellatus) (Fig. 3d). In other species, filaments on the tip of the dorsal and the anal fins may be rather long, reaching or surpassing the posterior margin of the caudal fin (S. constanciae, S. izecksohni, S. filamentosus, S. semiocellatus, S. hellneri, S. flavicaudatus, S. flagellatus, S. igneus, S. flammeus, S. brunoi) (Figs. 2a,b). In S. chacoensis, dorsal filaments are short, but the anal-fin filaments are long. In S. flammeus and S. brunoi, instead of filaments being restricted to the tip of the dorsal fin as in other congeners, they are present along the entire extension of the distal border of the fin (Fig. 2c). Uniquely in S. myersi and S. izecksohni, there are short filaments on the posterior margin of the anal fin in females (Fig. 4e).
Depending on the number of fin rays, the length of the dorsal and anal-fin bases may change strongly. The dorsalfin base varies from about 30% SL in adult males of species with fewer rays, to about 60% in males of S. adornatus, the species with the greatest number of dorsal-fin rays (Costa, 2003b). Similarly, the anal-fin base ranges from 32% to 48% SL. The number of dorsal-fin rays also influences the predorsal length, which varies from 62% SL in adult males of species with fewer rays, to 28% in males of S. adornatus. 2003). In S. boitonei, S. parallelus, and S. cholopteryx the pelvic fin is absent (Costa, 2003b). In larger males of S. marginatus (above 35 mm SL) the pelvic fin is long, its length reaching 16-25% SL. The pelvic-fin bases are medially in contact or their bases are slightly united.
Body squamation. The scales are large and cycloid, with numerous radii. The body and the head are entirely scaled, except on the anterior portion of the ventral surface of head. The body squamation slightly extends over caudal-fin base. In Nematolebias the scales do not extend over base of dorsal and anal fins, but in Simpsonichthys, a few scales extend on the median portion of the anal-fin base in males. The number of scales along the main longitudinal series is [28][29][30][31] Simpsonichthys. The number of scales in the longest transverse series is 8-11 in Nematolebias and Simpsonichthys, except in the S. antenori group, with 12-14. There are 12-16 scales around the caudal peduncle.

Frontal squamation.
A circular frontal squamation pattern, unique among cyprinodontiforms, was considered a synapomorphy for rivulids (Parenti, 1981). This pattern consists of a central A-scale, with all borders overlapped by adjacent scales. However, although present in basal rivulid lineages (Costa, 2004a), a circular pattern does never occur in any cynolebiatin . The A-scale always has a free posterior margin in all species of Nematolebias and Simpsonichthys (Figs. 1,5).
In Nematolebias and in most species of Simpsonichthys, the frontal squamation is composed of five to 12 large scales between supraorbital series of neuromasts, of which three to eight scales are placed anterior to E-scales. Usually, there are two or one overlapped small scales, between supraorbital series of neuromasts and eye, but sometimes these supraorbital scales are absent. The paired E-scales are not overlapped in Nematolebias (Fig. 1b), except in a few specimens with Escales slightly overlapped. In all species of Simpsonichthys, the E-scales are always overlapped (Fig. 5).
In Nematolebias and in the great majority of species of Simpsonichthys, the frontal squamation is E-patterned (i.e., the E-scale has all margins free). Exceptions occur in the S. notatus group, in which all species are A-patterned, and in S. semiocellatus, that is F-patterned ( Fig. 5a, b).
Laterosensory system (Fig. 1). In all rivulids the laterosensory system is represented by rows of exposed neuromasts, and among cynolebiatins, cephalic neuromast are numerous (Parenti, 1981;. On the dorsal surface of the head, just posterior to the median portion of the snout, there are two neuromasts, the anterior and the posterior rostral neuromast (Costa, 2001). The anterior supraorbital series of neuromasts is longitudinally arranged and is united to the posterior supraorbital series, forming a single continuous series (Costa, , 1998. Sometimes, the anteriormost supraorbital neuromasts may be bigger than the posterior ones. In Ophthalmolebias, however, the anterior and the posterior Almost all species have the caudal fin rounded to subtruncate. An exception is the subgenus Xenurolebias, in which the caudal fin is asymmetrically lanceolate, the ventral portion being slightly expanded. The pectoral fin is elliptical and long. Its maximum length is 24-33% SL in adult males. The pelvic fin is usually short. Its length varies between 9 and 13% SL in adult males. In S. zonatus and S. santanae, the pelvic fin is very small, reaching only 3-5% SL (Costa, 1996, supraorbital series are separated by an interspace (Fig. 5c). In S. parallelus and S. cholopteryx, the two anteriormost supraorbital neuromasts are separated from the remaining posterior neuromasts (Fig. 5d). The total number of supraorbital neuromasts usually varies from 10 to 16, rarely 9 or 17-18. Posterior to the supraorbital series, there is a single parietal neuromast, or double as in S. costai.
The infraorbital series comprises three segments. A short anterior segment, usually with two and sometimes with a single neuromast, lays anterolateral to the posterior nostril. In Nematolebias there is an additional neuromast just anterior to the anterior infraorbital series (Fig. 1), a condition not found in other cynolebiatins, but present in basal rivulids. The median segment runs near the anterior and ventral margin of the orbit, comprising 12-22 neuromasts. The short posterior segment includes two to four neuromasts, dorsally connected to a short longitudinal otic series comprising one or two neuromasts. Often the median and posterior segments of the infraorbital series are continuous. Adjacent to the anterodorsal margin of the orbit, there is a pre-orbital series with two or three neuromasts. In S. parallelus and in S. cholopteryx, the pre-orbital series is connected to the median portion of the supraorbital series (Fig. 5d).
The opercle is dorsally bordered by a longitudinal postotic series composed of two or three neuromasts. Dorso-anterior to the post-otic series there is a supra-temporal neuromast. One median opercular neuromast lies on the central portion of the opercle, and two ventral opercular neuromasts below it. The preopercular series comprises 9-15 aligned neuromasts, besides some slightly displaced from the main line. In Nematolebias, the preopercular series is continuous to the mandibular series ( Fig. 1). The mandibular series includes 6-14 neuromasts. A lateral mandibular series comprises 3-5 aligned neuromasts. A single paramandibular neuromast is placed below the mouth cleft.
Contact organs. Dermal contact organs are common in atherinomorph fishes (Wiley & Collete, 1970). Possibly they are important to the maintenance of body contact between sexes during reproductive behavior. In rivulids, they are present on the scales and fin rays in males of some taxa, during all the adult stage.
Contact organs of flank scales of rivulids are minute dermal protuberances on the median portion of the posterior border of the scale. The flank scales bearing a contact organ have a superficial resemblance to a ctenoid scale, but this kind of scales is not present in atherinomorph fishes (Roberts, 1993). Scale contact organs are present in most species of Simpsonichthys, but are absent in S. constanciae, S. semiocellatus, S. chacoensis, S. reticulatus, S. costai, and in species of Nematolebias.  Contact organs of pectoral-fin rays are arranged in row along the inner surface of fin rays. They are small, papillate and restricted to the dorsalmost rays in Simpsonichthys, with the exception of S. constanciae, S. cholopteryx, S. costai, S. semiocellatus, and S. filamentosus, in which pectoral contact organs are absent. In S. flagellatus, the pectoral-fin contact organs are prominent and very conspicuous, in contrast to other congeners, in which the contact organs are hardly visible even under a dissecting microscope. In Nematolebias, the pectoral-fin contact organs are hypertrophied and dis-tributed over most pectoral-fin rays (de Carvalho, 1957;Costa, 2002a). They are tubular, the base supported by a bone extension derived from the outgrowth of the lepidotrichium, and possess a mesenchyme-filled axial space continuous to the space in the center of the fin ray (Wiley & Collete, 1970;Costa, 2002a) (Fig. 6).

Superficial dermal bones.
There are three ossified structures representing the superficial dermal bones in cyprinodontiform fishes. Among them, the nasal, present in all rivulids, is a scale-like, thin ossification on the anterodorsal portion of the head, with a short, longitudinal, ventral keel (Fig. 7).
The two other superficial dermal bones, the lachrymal and the dermosphenotic, correspond respectively to the first and sixty bones of the circumorbital series, whereas the second through fifth circumorbital bones are absent in all cyprinodontiforms. As in other rivulids, the lachrymal in Nematolebias and Simpsonichthys is a thin and twisted bone, adopting an almost cylindrical shape with the dorsal portion slightly widened and anteriorly directed (Parenti, 1981;Costa, 1998a, b) (Fig. 7). It is placed just anteriorly to the anterodorsal margin of the orbit. The dermosphenotic is very thin, ovoid, its outer surface gently concave to reach a shallow, open trough shape (Fig. 7). It is positioned just posteriorly to the posterodorsal margin of the orbit. It is always present in Nematolebias, but among species of Simpsonichthys, it is present only in the subgenera Xenurolebias and Ophthalmolebias . A minute dermosphenotic is also found in S. filamentosus. The remaining species of Simpsonichthys do not present any sign of ossification on the dermosphenotic region.  The neurocranium is depressed as in all aplocheiloid fishes (Costa, 1998b). Its dorsal surface is composed by poorly ossified structures with unclear limits, a condition present in all rivulids . The frontal is approximately rectangular and occupies the anterior two thirds of the neurocranium roof. It is extremely thin, with reduced lateral borders. The parietal is subtriangular, possessing a distinctive transverse keel (Fig. 8b). The posterodorsal region of the neurocranium is formed medially by the supraoccipital, which have a paired posterior process, and a small epiotic, placed posterolaterally to the supraoccipital. The supraoccipital process is usually short (Fig. 9a), but it is distinctively long in species of the S, flammeus group, its narrow tip lying in close proximity to the neural spine of the first vertebra (Costa, 2003b) (Fig. 9b). The posterolateral edge of the neurocranium is formed by the sphenotic, with a prominent lateral process, the pterotic and the exoccipital.
The limits of bones along the ventral surface of the neurocranium are conspicuous (Fig. 8a). On the anterior portion, there is a triangular vomer, with an elongated posterior process. In Nematolebias there are two to five, small, conical teeth on the anteromedian portion of the vomer. Vomerine teeth are absent in all species of Simpsonichthys. The mesethmoid is unossified. The lateral ethmoid is compact, bearing a distinctive anterior retrorse process. The anteromedian portion of the lateral ethmoid slightly overlaps dorsally the lateral portion of vomer, and the medial margin sometimes abuts the lateral surface of the anterior process of the parasphenoid. The parasphenoid comprises a crossshaped structure, in which the anterior process overlaps dorsally the posterior process of the vomer, the posterior process is firmly attached ventrally to the basioccipital, and each of their short lateral processes is ventrally attached to the prootic.
Jaws, jaw suspensorium and opercular apparatus. The jaws are connected to jaw suspensorium through the articulation between angulo-articular and quadrate, and through a ligamentous connection joining upper jaw and palatine (Fig. 10). The upper jaw is formed by the maxilla, the premaxilla and the rostral cartilage, which are joined by connective tissue. The premaxilla is an elongate bone with teeth on the medial half of its anterior face. It bears a posteriorly directed, rectangular flattened ascending process on its medial portion, and an anteriorly directed, trapezoidal process on its subdistal portion. The maxilla, which is placed dorsolaterally to the premaxilla, is elongate, bifid in its anterior medial portion, from  where a subtriangular process projects dorsally to the premaxillary ascending process, whereas another process ventrally overlaps that premaxillary process. The rostral cartilage is elongate to about round in older males of Nematolebias and Xenurolebias, but it is approximately pentagonal and longitudinally elongated in the remaining species of Simpsonichthys (Fig. 11). Its anterior portion lies ventral to the posterior portion of the premaxillary ascending process.
The lower jaw is formed by the dentary, angulo-articular, retro-articular, and the coronomeckelian cartilage. The dentary is the longer bone, occupying the most portion of the lower jaw, with teeth on the distal two thirds of its anterodorsal surface. Both in Nematolebias and Simpsonichthys the posteroventral part of dentary terminates in a prominent process, a feature also present in Cynolebias, Megalebias and Austrolebias . The angulo-articular is a triangular bone, with a ventral process, which is short in Nematolebias and Simpsonichthys, but wide in Nematolebias and moderately narrowed in Simpsonichthys (Fig. 12). In S. constanciae, the ventral angulo-articular process is narrower than in other congeners (Fig. 12c). Another smaller process is present on the posterior extremity of the angulo-articular, which supports the articulation with the quadrate. The retro-articular is a small bone placed posteroventrally to the anguloarticular. The coronomeckelian cartilage is elongate, attached medially along dentary and angulo-articular.
Both premaxillary and dentary teeth are arranged in irregular rows. A few distinctive larger teeth are placed on the most external row. All teeth are conical, with slightly curved tip, then directed to the inner of the mouth. The large tooth on the posterior portion of the dentary is slightly anteriorly directed in Nematolebias and strongly anteriorly directed in Simpsonichthys (Costa, 1996, except in the S. notatus species group, in which all teeth are directed to the mouth. In S. constanciae, there are some larger teeth of the external row of premaxilla and dentary with tips anteriorly directed (Fig. 12c).
The palatine and the ectopterygoid are completely fused, as in other aplocheiloids (Parenti, 1981;Costa, 1998b) (Fig.  10). In Simpsonichthys, except S. semiocellatus, the anterodorsal tip of the palatine bears a small, pointed process, supporting a thick ligament connected to the maxilla   (Fig. 13a-d). This process is longer in S. ocellatus (Fig. 13c) and may sometimes acquire a laminar shape in S. flagellatus, and it is absent in Nematolebias. In Nematolebias and in several species of Simpsonichthys, the medial surface of the palatine is approximately plain, but in  Ophthalmolebias and species of the S. antenori and S. flammeus groups, there is a medial semicircular crest, supporting a thick ligament connected to the lateral ethmoid. In Ophthalmolebias, the crest is more prominent, forming a ventral expansion (Fig. 13d).
The mesopterygoid is thin and posteriorly reduced, the posterior tip separated from metapterygoid by an interspace filled with cartilage. The ventral portion of the mesopterygoid slightly overlaps the dorsal portion of the quadrate in Nematolebias (Fig. 10). In Simpsonichthys, as well as in Cynolebias, Austrolebias and Megalebias, the ventral portion of the mesopterygoid only gently abuts the dorsal portion of the quadrate (Fig. 23a). In the subgenus Simpsonichthys, the mesopterygoid is shorter, its posterior end lying dorsal to the anterodorsal border of the quadrate (Fig. 13b).
The quadrate is approximately triangular, with the anterior and the ventral margins approximately straight, and the posterior margin rounded (Fig. 10). It bears a posterior short process. The angle formed between the anterior and the ventral margins is about 100º to 125º, but is variable within species, sometimes angle reaching about 80° or 90° in older males of deep bodied species (e.g., S. igneus). The posterior process is shorter than in most other rivulids, usually reaching between 34 and 47 % of the total longitudinal length of the quadrate. The medial surface of the posterior portion of the quadrate is firmly attached to the lateral surface of the anterior portion of the sympletic, which usually is a deep, triangular bone. In the subgenera Simpsonichthys and Spectrolebias, the sympletic is elongate (Fig. 13a-b).
The posterior portion of the sympletic and the ventral portion of the hyomandibula are dorsally bordered by the metapterygoid. In Nematolebias and in the subgenera Xenurolebias, Ophthalmolebias (except S. constanciae), and Simpsonichthys, the metapterygoid is narrow in its ventral portion, becoming abruptly wider in its dorsal portion (Fig.  14a). In Hypsolebias, the metapterygoid is approximately rectangular, with the dorsal portion only slightly wider than the ventral portion (Fig. 14b). In S. constanciae and in the subgenus Spectrolebias, the entire bone is narrow, assuming a rod shape (Fig. 14c).
The hyomandibula is a somewhat compact bone ( Fig. 14ab), but elongate in the subgenus Spectrolebias (Fig. 14c). It comprises two robust dorsal condyles, the anterior articulating with the sphenotic and the posterior with the pterotic, a ventral condyle attached to the sympletic, and a posterodorsal process supporting the articulation of the opercle. Running laterally from the anterodorsal condyle to the posteroventral tip of the hyomandibula, there is a prominent curved crest. Its posteroventral tip is separated from the ventral condyle of the hyomandibula by a broad laminar extension (Fig. 14a). The laminar extension is reduced In S. filamentosus, S. semiocellatus, and species of Hypsolebias (Fig. 14c), except in species of the S. antenori group (not including S. antenori), in which the laminar extension is vestigial (Fig. 14b).
All the bones of the opercular apparatus are thin. The dorsal and posterior borders of the opercle and all the borders of the interopercle and subopercle are membranous and poorly ossified. The preopercle is firmly attached to the hyomandibula and to the posterior process of the quadrate. The preopercle of all the cynolebiatins is thin, gently curved, with a reduced anteromedian flap (Fig. 15a)  . Its dorsal end is narrow and pointed, and the ventral portion wide. The anteromedian flap is narrower in species of the S. flammeus group (Fig. 15b), and vestigial or absent in species of the S. antenori group (Fig. 15c). The anteroventral portion of the preopercle is attenuated in species of the S. flammeus and S. antenori groups (Fig. 15b-c).
Hyoid and branchial arches. The basihyal is triangular and flattened, with an anterior broad cartilaginous portion, and a narrow posterior portion. The cartilage occupies 18-33% of the total longitudinal length of the basihyal in Nematolebias and in most species of Simpsonichthys (Fig. 16a), but reaches 38-45% in S. flammeus, S. brunoi and S. multiradiatus (Costa, 1996). The width of the basihyal may be variable among species, the greatest width between 30-80% of the basihyal length. However, in larger males of Nematolebias and in S. ghisolfii, this width reaches 90-100%.
Both the dorsal and the ventral hypohyals are small, but always ossified (Figs. 16a, c). The anterior ceratohyal is wide and cylindrical in its anterior portion, narrowed in the median portion, with two narrow branchiostegal rays attached to the ventral border, and compressed in the posterior portion (Fig.  16c). The anterior and the posterior ceratohyals are separated by a broad space of cartilage, supporting four branchiostegal rays, each gradually become wider posteriorly (Fig. 16c). The posterior ceratohyal is compressed, subtriangular (Fig. 16c). An oval and cartilaginous interhyal is attached to the posterior end of the posterior ceratohyal (Fig. 16c). The urohyal is thin, with short ventrolateral flap and an anterodorsal process (Fig. 16a).
The dorsal part of the branchial arches comprises a wide third pharyngobranchial with a well-developed dentigerous plate, anteriorly overlapped by the second pharyngobranchial and posteriorly by the small fourth pharyngobranchial tooth plate, three long epibranchials 1-3, a robust fourth epibranchial and a long interarcual cartilage (Fig. 16b). The second pharyngobranchial is approximately triangular, usually wider than long, but in Hypsolebias, it is clearly longer than wide (Fig. 17b). Often there are one to four teeth near the posterior edge of the second pharyngobranchial, but in the S. antenori species group there are five to ten teeth. In Nematolebias, Xenurolebias and in S. parallelus and S. cholopteryx, second pharyngobranchial teeth are always absent. In all species of Nematolebias and Simpsonichthys, except S. costai, the second pharyngobranchial possesses a small subdistal process (Fig. 16b). The third epibranchial bears a short uncinate process (Fig. 16b). The interarcual cartilage is laterally attached to the anterior tip of the second pharyngobranchial (Fig. 16b), but in S. trilineatus, S. auratus, S. magnificus, S. picturatus, S. carlettoi, and S. adornatus, the cartilage is attached to the medial margin of that bone (Fig. 17b).
The ventral part of the branchial arches comprises three ossified rectangular basibranchials, three rounded hypobranchials, four elongate ceratobranchials without teeth, and a fifth ceratobranchial with a well-developed dentigerous plate (Fig. 16a). The proximal edge of the first hypobranchial is rounded, without vestige of branching (Fig. 16a). The whole lateral border of the first hypobranchial is bordered by cartilage (Fig. 16a). The anterior portion of the second hypobranchial bears a small process anteroventrally directed. In S. magnificus, S. picturatus, S. carlettoi, and S. fulminantis there is an additional process directed to the second basibranchial (Fig. 17c). The proximal tip of the fourth ceratobranchial is clearly wider than the proximal tip of the first, second and third ceratobranchials (Fig. 16a), but in Spectrolebias, it is narrower (Fig. 17d). The fifth ceratobranchial is boomerang-shaped, its anterior portion on a longitudinal axis and the posterior portion slightly laterally displaced, on an axis forming angle of 125-135° with the longitudinal axis (Fig. 16a). The anterior portion is shorter than the posterior portion. In S. filamentosus and S. semiocellatus, the fifth ceratobranchial is slender and their anterior and posterior portions form an angle of 145° (Fig. 17d). In S. filamentosus, S. semiocellatus, and species of the S. flammeus and S. antenori groups, the anterior portion is longer that the posterior one (Fig. 17d).
Vertebrae and caudal skeleton. The first vertebra bears a neural spine, which has anterior and posterior laminar extensions (Fig. 18a). It lacks neural prezygapophyses, but there is a prominent neural postzygapophysis directed to the ventral surface of the neural prezygapophysis of the second vertebra. As in other cyprinodontiforms, the pleural rib is present in all precaudal vertebrae, except the first. All precaudal vertebrae possess a rod-like epipleural rib. In Nematolebias, the neural spines of the second and the third vertebrae are wider than the first neural spine (Fig. 18a), but in all species of Simpsonichthys, as well as in Cynolebias, Austrolebias and Megalebias, the second neural spine is slightly narrower than the first, and the third is about so narrow as all posterior neural spines (Fig. 18a). Also only in Nematolebias, the neural prezygapophysis of the second vertebra is directly attached to the first neural spine (Fig. 18a). In Simpsonichthys and most other rivulids, there is a ligament between the first neural spine and the neural prezygapophysis of the second vertebra. In caudal vertebrae, the neural prezygapophyses are vestigial or absent and the neural postzygapophyses are short (Fig. 18b).
The caudal skeleton comprises an epural and a parhypural with similar shape, in which the proximal portion is narrowed and slightly curved, with the tip anteriorly directed (Fig. 18b). All the hypurals are ankylosed, without any vestige of gaps (Fig. 18b). Accessory cartilages are absent. There are 20-31 caudal-fin rays.
Dorsal and anal fins. Males have more dorsal and anal-fin rays than females. The number of dorsal-fin rays and the  position of the dorsal-fin origin in males are extremely variable in species of Simpsonichthys. In Nematolebias, Xenurolebias, Ophthalmolebias, subgenus Simpsonichthys (except S. boitonei and S. marginatus), and S. semiocellatus and S. filamentosus, there are 15-21 rays and the dorsal-fin origin is between the neural spines of vertebrae 10 and 14; in S. brunoi, S. delucai, S. alternatus, S. trilineatus, and S. auratus, 18-22 rays, dorsal-fin origin between neural spines of vertebrae 7 and 9; in S. chacoensis, S. costai, S. reticulatus, S. boitonei, S. marginatus, S. flammeus, S. antenori species group, S. magnificus species group and S. notatus species group (except S. auratus and S. trilineatus), 20-25 rays, dorsal-fin origin between neural spines of vertebrae 6 and 9; in S. multiradiatus, 25-28 rays, dorsal-fin origin between neural spines of vertebrae 5 and 7; and, in S. adornatus, 28-32 rays, dorsal-fin origin between neural spines of vertebrae 3 and 4. In species of Nematolebias and Simpsonichthys, there are 17-25 anal-fin rays, and the anal-fin origin is placed between the pleural ribs of the vertebrae 5 and 9. In Nematolebias, except the two or three anteriormost rays that are unbranched, all other dorsal and anal-fin rays are branched. In Xenurolebias, most rays are also branched, but the longer rays forming the tip of the fin are unbranched, and in all other species of Simpsonichthys approximately all dorsal-fin rays are unbranched. In all species of Simpsonichthys, the rays on the tip of the anal fin are unbranched, except in S. constanciae, in which all anal-fin rays are unbranched.
Two rays are associated to the first proximal radial of the dorsal fin (Fig. 18c), and two or three associated to the first proximal radial of the anal fin (Fig. 18d). The first anal-fin proximal radials are widened (Fig. 18d). The dorsal-fin median and the distal radials are ossified (Fig. 18c). The anal-fin median radials are ossified, but the anal-fin distal radials are ossified only on the posterior portion of the fin; in the anterior portion of the fin, distal radials are absent or cartilaginous (Fig. 18d). In S. magnificus, S. picturatus, S. carlettoi, S. fulminantis, and S. adornatus, the median radials of the dorsal fin are longer than in other cynolebiatins (Fig. 19b). In all species of Cynolebias, Megalebias, and Austrolebias, the median radials of dorsal and anal fins are always cartilaginous.
Shoulder and pelvic girdles. The dorsal portion of the shoulder girdle is articulated with the neurocranium by an elon-  tical length between the dorsal edge of scapula and the ventral edge of coracoid (Fig. 20a). In all other species of Simpsonichthys, the dorsal portion of the cleithrum is distinctively longer (Fig. 21). In S. filamentosus, S. semiocellatus, and S. chacoensis, there is an anteriorly expanded flap on the anterior portion of the cleithrum (Fig. 21). The ventral tip of the cleithrum and the ventral tip of the coracoid are placed in closed proximity. The coracoid is about triangular and is ventrally separated from the rounded scapula by a small interspace filled with cartilage. In Nematolebias, the ventral portion of the coracoid possesses a narrow condyle, which is broad in all species of Simpsonichthys (Fig. 21). The proximal radials of the pectoral fin are rounded and thin, the ventralmost radial lying in close proximity to the posteroventral margin of the coracoid (Fig. 20c). Medial to the main axis of the shoulder girdle, there is a rod-like and elongate third postcleithrum. The first and the second postcleithra are absent.
The pelvic girdle comprises a narrow pubic bone without a distinct ischial process (Fig. 20c). The pelvic girdle and the pelvic fin are vestigial in S. zonatus and S. santanae, and are absent in S. boitonei, S. parallelus, and S. cholopteryx. There are 11-16 pectoral-fin rays, and 5-7 pelvic-fin rays. gated and forked posttemporal. The ventral process of the posttemporal is absent in Nematolebias and in S. costai, S. reticulatus, S. parallelus, and S. cholopteryx (Fig. 20a). An elongate supracleithrum is attached between the posttemporal and the cleithrum.
The dorsal portion of the cleithrum is about triangular, lacking a distinctive posterior flange present in basal rivulids (i.e., Kryptolebias Costa) (Costa, 2004a). This dorsal portion of the cleithrum is short in Nematolebias, Xenurolebias and S. semiocellatus, its length shorter or about equal to the ver-

Phylogenetic analysis
This phylogenetic analysis is based on both a re-examination of all previously reported phylogenetically informative morphological characters and a series of morphological characters not identified in other studies. This study comprises a total of 116 characters (see Appendix II), that are included in the data matrix (Appendix IV).
Six most parsimonious trees (tree length = 292, CI = 54%, RI = 79%) were found. Conflicts are restricted to different topologies involving species of the S. notatus group (clade F'). Other polytomies correspond to unsolved nodes present in all most parsimonious tress. The strict consensus tree (tree length = 297, CI = 53%, RI = 79%) and node supports are presented in Fig. 22. Synapomorphies for each clade are presented in Appendix III. A second analysis was performed excluding quantitative characters, which are suspect to contain some degree of subjectivity. The same trees were found (consensus tree with length = 266, CI = 53%, RI = 79%), demonstrating that these characters had a lower impact on the analysis.

Diagnosis.
Differs from all other members of the Cynolebiatini by the following unambiguous synapomorphies: widened ventral process of angulo-articular (vs. narrow), neural spine of the first vertebra directly attached to the neural prezygapophysis of the second vertebra (vs. connected by thin ligaments), hypertrophied tubular contact organs on the medial surface of the dorsal half of the pectoral fin in males, its basal portion ossified, forming a cup-shaped osseous structure (vs. papillate contact organs, when contact organs are present), and a subdistal orange stripe with transverse golden lines contrasting with a black zone dorsal to the orange stripe on the anal fin in males (vs. never a similar color pattern). Some synapomorphies of Nematolebias, homoplastically occurring in other cynolebiatins, are: widened basihyal in older males (also occurring in the S. antenori species group) (vs. narrow basihyal), absence of second pharyngobranchial teeth (also occurring in Simpsonichthys, subgenera Xenurolebias and Simpsonichthys) (vs. presence), preopercular and mandibular series of neuromasts continuous (also occurring in Cynolebias) (vs. preopercular and mandibular series of neuromasts separated), absence contact organs on the flank (also occurring in Ophthalmolebias, Spectrolebias and in the S. antenori species group) (vs. contact organs present), iridescent line on subdistal zone of caudal fin in males (also occurring in the subgenus Simpsonichthys) (vs. subdistal line absent), and pectoral-fin reddish brown with iridescent spots in males (reddish brown also in the S. notatus group, and iridescent spots also in the subgenus Simpsonichthys) (vs. never a similar color pattern). Costa and N. whitei (Myers).   Distribution. Brazilian eastern river basins, between rio Cachoeira, Estado da Bahia, and rio São João, Estado do Rio de Janeiro.

Included species. Nematolebias papilliferus
Etymology. From the Greek ophthamus (eye) and lebias (small fish, also a name used to compose generic names of rivulids), referring to the large eyes laterally placed on head. Gender masculine.  Distribution. Central and northeastern Brazil, including the upper and middle rio Tocantins basin, middle rio São Francisco basin, lower rio Jaguaribe basin and adjacent coastal basins, and middle rio Jequitinhonha.
Phylogenetic position of Nematolebias. It is hypothesized that Nematolebias is the sister group of the clade including Simpsonichthys, Cynolebias, Austrolebias, and Megalebias. This hypothesis differs from previous studies based on morphological characters (Costa, 1996(Costa, , 2003, but is congruent with studies based on molecular characters (Murphy et al., 1999;Hrbek & Larson, 1999), although the latter studies included only a few cynolebiatin species as terminal taxa.
The divergence between the present study and previous morphological studies is due to the introduction of five characters discovered during the course of this study, supporting the monophyly of the group comprising Simpsonichthys, Cynolebias, Austrolebias, and Megalebias: ventral portion of mesopterygoid not overlapping quadrate, third neural spine about as wide as fourth neural spine, a narrow ventral condyle of coracoid, loss of a neuromast anterior to the infraorbital series, and scales extending over anal-fin base. Other synapomorphies supporting the monophyly of the group including Simpsonichthys, Cynolebias, Austrolebias, and Megalebias were already in part reported in previous studies: loss of vomerine teeth (reversed in a group of Cynolebias, Costa, 2001), a short and narrow ventral process of the anguloarticular, and an elongated dorsal portion of cleithrum.
Derived conditions uniquely shared by Nematolebias and Simpsonichthys are: posterior outer dentary tooth anteriorly directed; anal-fin origin anteriorly placed, between pleural ribs of vertebrae 5 and 9; and, pelvic-fin bases medially in contact or united. These conditions are parsimoniously interpreted as having originated at the base of the Cynolebiatini, with a reversal in the clade comprising Cynolebias, Megalebias, and Austrolebias.
Phylogenetic position of Xenurolebias. Xenurolebias is herein erected to include two species, S. myersi and S. izecksohni, as a subgenus of Simpsonichthys. The two species share some synapomorphies, such as the presence of filamentous rays on the posterior border of the anal fin in females and an asymmetrical and lanceolate caudal fin in males.
Simpsonichthys myersi was considered to be closely related to Nematolebias both in morphological (Costa, 1996(Costa, , 1998) and molecular (Hrbek & Larson, 1999) analyses. However, Xenurolebias is here hypothesized to be closer related to other species assemblages of Simpsonichthys than to Nematolebias. The hypothesis of Xenurolebias as a clade within Simpsonichthys is supported by all species of Xenurolebias possessing the four synapomorphies defining the genus Simpsonichthys: a distinctive, small pointed dorsal process on the palatine; unbranched fin rays on the tip of dorsal fin in males; unbranched fin rays on the tip of anal fin in males; and, frontal E-scales overlapped. The hypothesis of Xenurolebias as a member of the clade that includes Simpsonichthys, Cynolebias, Megalebias, and Austrolebias, but precludes Nematolebias, is corroborated by seven synapomorphies: ventral portion of mesopterygoid not overlapping quadrate; third neural spine about as wide as fourth neural spine; a narrow ventral condyle of coracoid; scales slightly extending over anal-fin base; loss of vomerine teeth (reversed in a group of Cynolebias, Costa, 2001); loss of neuromast anterior to infraorbital series; and, a short and narrow ventral process of the angulo-articular. The hypothesis of Xenurolebias as a basal clade within the genus Simpsonichthys is supported by all species of Simpsonichthys, except those placed in Xenurolebias, sharing two synapomorphies: most dorsal-fin rays unbranched in males, and loss of black spots over the posterior portion of the caudal peduncle in females (reversed in the S. antenori species group).
There are two derived conditions shared by Nematolebias and Xenurolebias: a widened rostral cartilage, and the loss of second pharyngobranchial teeth. However, all species of Cynolebias also have a widened rostral cartilage (Costa, 2001), and consequently the condition is parsimoniously interpreted as having been originated at the base of the Cynolebiatini, with reversals in Simpsonichthys and Austrolebias. The loss of second pharyngobranchial teeth is considered to be homoplastic.

Monophyly and phylogenetic position of Ophthalmolebias.
The assemblage comprising S. constanciae, S. bokermanni, S. perpendicularis, and S. rosaceus was hypothesized to be a monophyletic group by Costa (2003). Subsequently, another species of this clade, S. suzarti, was described and the clade was named S. constanciae species group (Costa, 2004b). This clade is here corroborated and is considered as a subgenus of Simpsonichthys, named Ophthalmolebias. Monophyly of Ophthalmolebias is supported by five synapomorphies: a medial crest on the palatine, with a prominent ventral expansion; eye laterally placed on the head; anterior and posterior sections of the supraorbital series of neuromasts separated by interspace; metallic blue bars on the flank in females; and, anal fin pink in females.
Species of Ophthalmolebias were considered to be closely related to Xenurolebias and Nematolebias (Costa, 2003b), by all having pelvic-fin bases medially in contact or united, and by the putative common possession of pink anal fin in females. However, the condition of pelvic-fin bases medially in contact or united occurs in most species of Simpsonichthys, not constituting evidence of close relationships between Ophthalmolebias, Xenurolebias, and Nematolebias, and the character state anal fin pink in females is not present in Xenurolebias and in Nematolebias, except in a few specimens of N. whitei, in which the anal fin is pinkish hyaline.
Monophyly and phylogenetic position of Spectrolebias. Costa & Nielsen (1997) described Spectrolebias to include a new miniature species from central Brazil, S. semiocellatus, hypothesized to be the sister group of the clade comprising Nematolebias, Simpsonichthys, Cynolebias, Megalebias, and Austrolebias . Costa (2003) found evidence supporting a close relationship between S. semiocellatus and Simpsonichthys filamentosus, then transferring the latter species to Spectrolebias, which was considered to be the sister group to a clade including Nematolebias and Simpsonichthys.
Both species previously placed in Spectrolebias, S. semiocellatus and S. filamentosus, are considered closely related to S. chacoensis, S. costai, and S. reticulatus. Spectrolebias is redefined as a subgenus of Simpsonichthys to include S. semiocellatus, S. filamentosus, S. chacoensis, S. costai, and S. reticulatus by four synapomorphies: a narrowed metapterygoid (also occurring in S. constanciae); a long hyomandibula; a narrowed proximal tip of the fourth ceratobranchial; and, loss of organs of contact on the flank in males (reversed in S. filamentosus). Spectrolebias is parsimoniously considered to be the sister group of the subgenus Simpsonichthys by both sharing the following synapomorphies: an elongate sympletic, and four features of the color pattern of the dorsal and anal fins in males, including the presence of distal dark stripe and subdistal bright blue subdistal stripe in each fin, all reversed in some species of both Simpsonichthys and Spectrolebias. However, the black and blue distal spot on the posterior portion of the anal fin in S. semiocellatus (Costa & Nielsen, 1997) and also on the anterior portion of the dorsal fin of S. reticulatus (Costa & Nielsen, 2003) may be vestiges of those color patterns.
The hypothesized sister group of Spectrolebias is the monophyletic group including S. boitonei, the type species of the genus Simpsonichthys, and S. marginatus, S. zonatus, S. santanae, S. parallelus, and S. cholopteryx. It is herein established as the subgenus Simpsonichthys, weakly diagnosed by a flank red with bright blue bars on the anterior portion and vertical rows of blue dots on the posterior portion in males, a condition modified in S. cholopteryx, in which there blue bars on the entire flank. However, a clade within the subgenus Simpsonichthys including all species except S. marginatus is supported by three unambiguous synapomorphies: a shortened mesopterygoid, its posterior tip in a vertical through the anterior portion of the quadrate; pelvic fin and girdle vestigial or absent; and, side of head with alternating red and bright blue bars in males.
Monophyly and phylogenetic position of Hypsolebias. It is hypothesized that the sister group of the clade comprising the subgenera Spectrolebias and Simpsonichthys is a diversified monophyletic group, herein formally referred as Hypsolebias, a new subgenus. Hypsolebias shares two synapomorphies with Simpsonichthys and Spectrolebias: loss of dermosphenotic (also occurring in Cynolebias, Austrolebias, and Megalebias) and dorsal fin anteriorly positioned in males, between neural spines of vertebrae 3 and 9 (reversed in some species of Spectrolebias and Simpsonichthys). Monophyly of Hypsolebias is supported by two synapomorphies: a shortened lateroventral process of hyomandibula (also occurring in some species of Spectrolebias); and, second pharyngobranchial longer than wide. Hypsolebias is the most speciose subgenus of Simpsonichthys, including a total of 25 species.