Comparative morphology and classification of South American cynopoeciline killifishes ( Cyprinodontiformes : Aplocheilidae ) , with notes on family-group names used for aplocheiloids

A comparative morphological study involving all cynopoeciline killifishes indicates that Leptolebias marmoratus is more closely related to species of the genera Campellolebias and Cynopoecilus than to other species of Leptolebias, and that Mucurilebias leitaoi is the sister group of a clade comprising all other cynopoecilines. Leptopanchax, new genus, is described to place species that share urogenital papilla in males separated from the anal-fin origin by broad interspace, basal portion of the urogenital papilla in males not forming a pronounced wide structure, absence of thickened tissue on the basal portion of the anal fin in males, caudal fin oval and symmetrical in males, anterior proximal radials of the anal fin not placed in close proximity in males, presence of a broad iridescent golden stripe on the distal zone of the dorsal fin in males, and caudal fin with vermiculate dark red marks in males. Poecilopanchax, new subgenus of Cynopoecilus, is diagnosed by having the basihyal narrow, about rectangular, the interarcual element of the dorsal branchial arch ossified, the uncinate process of the third epibranchial distinctively widened, a long filamentous ray on the tip of the anal fin in males, caudal fin lanceolate in males, dark bars on the flank in females, branchiostegal region with intense red pigmentation in males, a brownish red stripe on the basal portion of the dorsal fin in males, and dark red marks extending to the antero-distal margin of the dorsal fin in males. This study follows an old classificatory scheme in which all aplocheiloids are placed in a single family, the Aplocheilidae.


Introduction
The tribe Cynopoecilini comprises seasonal killifishes that inhabit temporary pools in tropical and subtropical areas of eastern and southern Brazil, mostly concentrated in the Atlantic Forest biodiversity hotspot, with one species reaching the adjacent plains of eastern Uruguay (Costa, 2002a(Costa, , 2008(Costa, , 2009)).Some cynopoecilines are unique among aplocheiloid killifishes by having specialised morphological structures for internal fertilization (Costa, 1995(Costa, , 1998a;;Costa et al., 2016a) and most species are threatened with extinction (e.g., Costa, 2012).
The taxonomic history of cynopoecilines begun with the description of Cynolebias melanotaenia regan, 1912 (regan, 1912a) from southern Brazil, which was immediately placed in a new genus, Cynopoecilus, by regan (1912b) after reading Berg's (1897) paper reporting sexual dimorphism in Cynolebias steindaChner, 1876.Cynopoecilus was then distinguished from the closely related genus Cynolebias by, in the latter genus, males having more rays in the dorsal and anal fins than females and a very distinctive colour pattern.In the following three decades, other cynopoecilines then described from south-eastern Brazil were placed in Cynopoecilus (Ladiges, 1934a, b; Faria & MuLLer, 1937;riBeiro, 1939).
The first of a series of classification changes was made by Myers (1942), who transferred all cynopoecilines to Cynolebias.Subsequently, Myers (1952) kept C. melanotaenia in Cynopoecilus as a monotypic subgenus of Cynolebias, and described another subgenus, Leptole bias, for the remaining cynopoecilines.However, Cyn olebias sense Myers (1942Myers ( , 1952) ) was not diagnosticable, making difficult to recognise generic limits among South American killifishes, as discussed by WeitzMan & WourMs (1967).
Vaz-Ferreira & sierra (1974) described Campellole bias brucei Vaz-Ferreira & sierra, 1974 as a new genus and species from southern Brazil, but Parenti (1981) placed Campellolebias and kept Cynopoecilus in the synonymy of Cynolebias.However, Cynolebias sense Parenti (1981) was polyphyletic, including in its synonymy the genus Terranatos taPhorn & thoMerson, 1978, later placed in a distinct subfamily (e.g., Costa, 1998;Mur-Phy et al., 1999).Costa (1990a, b) first recognised monophyly of the group comprising Campellolebias, Cynopo ecilus, and Leptolebias, which were then considered as valid genera.Eighteen years later, Costa (2008) found evidence indicating that Leptolebias sense Costa (1990b) was paraphyletic.Consequently, part of the species previously placed in Leptolebias were transferred to the genus Notholebias.Similarly, another phylogenetic analysis based on 73 morphological characters taken from previous studies but including a rare and possibly extinct species from north-eastern Brazil as terminal taxon, L. lei taoi (Cruz & Peixoto, 1991), indicated that this species is the sister group of a more inclusive cynopoeciline clade, making necessary to place it in a new genus, Mucurile bias (Costa, 2014).However, a recent larger set of morphological data, with a total of 115 characters, indicates that Leptolebias marmoratus (Ladiges, 1934), the type species of the genus, is more closely related to species of the genera Campellolebias and Cynopoecilus than to other species of Leptolebias, and that Mucurilebias leitaoi is the sister group of a clade comprising all other cynopoecilines (Costa, 2016).This set of unpublished morphological characters highly supports the new classification here provided for the Cynopoecilini.

Material and methods
List of examined material is given in Appendix 1. Comparative morphological characters included morphology of bones, urogenital papilla, fins, jaws, frontal squama-tion, latero-sensory system, contact organs, muscles of the anal-fin support, egg and colour patterns.Osteological characters were examined in specimens cleared and stained according to tayLor & Van dyke (1985).Terminology for osteological structures followed Costa (2006a), for muscles, WinterBottoM (1974), for frontal squamation, hoedeMan (1958), for cephalic neuromast series, Costa (2001), and for egg chorion morphology, Costa & LeaL (2009).Data on colour patterns were obtained from direct examination of live individuals during collections, and photographs of both sides of live individuals, at least two males and one female per collection, taken in aquarium between five and 24 hours after collection.Included species, distribution and habitat.Four species, N. cruzi (Costa, 1988), N. fractifasciatus (Costa, 1988), N. minimus (Myers, 1942) (Costa, 2008).L. opalescens (Myers, 1942), and L. splendens (Myers, 1942), between the Laguna de

Discussion
Classification of the Cynopoecilini.The comparative morphological analysis indicated relationships among the main lineages of the Cynopoecilini that highly support clades not delineated in other analyses and provided new diagnostic characters for clades already delineated in previous studies.This phylogenetic tree is based on a recent phylogenetic study (Costa, 2016) -D).All these character states differ from the plesiomorphic condition shared by all other cynolebiasines, including species of Leptopan chax (Fig. 2A).Interestingly, all those derived character states are present in L. marmoratus, a species having external fertilization as its only reproductive strategy, but lacking the specialized morphological features of the anal fin structure found in species of Campellolebias and Cynopoecilus, which adopted an internally fertilizing mode of reproduction (Costa et al., 2016a).In L. marmoratus, the anterior rays and proximal radials are placed in close proximity as in other species of the clade (Fig. 2A), but they are not elongated to support a specialized musculature as in species of Campellolebias and Cynopoecilus (Figs. 2C -D); and the urogenital papilla of L. marmoratus is robust and placed close to the anal fin as in other species of the clade (Fig. 2B), but lacking the internal muscular ejaculatory pump present in those species.The remaining two synapomorphies of the clade comprising Leptolebias, Campellolebias and Cynopoeci lus refer to derived colour patterns in males: a dark reddish chocolate brown to black stripe between pectoral-fin base and posterior portion of the anal-fin base [ch.90.1] and small round dark reddish brown spots on the dorsal fin [ch.112.0] (Fig. 1).
The forest-dweller taxon C. notabilis, besides highly differing from all other species of Cynopoecilus by several morphological features, exhibits nine autapomorphies, which justify its inclusion in the new subgenus Poecilo panchax.More remarkable is the ossification of the interarcual element of the dorsal branchial arch, resulting in an interarcual bone [ch.24.1; Fig. 5B).The presence of this bone, not reported in the recent description of the species (Ferrer et al., 2014), is unique among members of the order Cyprinodontiformes, in which that element is always cartilaginous (Fig. 5A The Cynopoecilini is further supported by a distinctive postero-ventral process on the autopalatine [ch.13.1] (Figs.4A -B), which is conspicuous in Notholebias, Lep topanchax, and Leptolebias (Fig. 4B), but minute in species of Mucurilebias, Campellolebias, and Cynopoecilus.This process does not occur in any other aplocheilid killifish (Fig. 4A).
Family-group names used for aplocheiloid killifishes.Aplocheiloid killifishes have been classified in three families: Aplocheilidae BLeeker, 1859, Nothobranchiidae garMan, 1895, and Rivulidae Myers, 1925 (Parenti, 1981;Costa, 2004).The first step for this classification was given by Parenti (1981) combining the morphological characters herein listed and molecular data taken from Costa et al. (2016a).Monophyly of Leptolebias as diagnosed by Costa (2008) is not corroborated.A clade including only L. marmoratus and all species of Campellolebias and Cy nopoecilus is supported by six synapomorphies, among which four are related to the genital region and adjacent part of the anal fin in males: anterior proximal radials of the anal fin in close proximity [ch.43.1], urogenital papilla in close proximity to the anal-fin origin [ch.63.1], basal portion of the urogenital papilla forming a pronounced wide structure [ch.64.1], and presence of a distinctively thickened tissue on the basal portion of the anal fin [ch.68.1] (Figs.2B

Fig. 1 .
Fig. 1.Phylogenetic relationships among cynopoeciline genera and subgenera supported by the comparative analysis of 115 morphological characters.
) or absent.Two autapomorphies of Poecilopanchax are also unique among members of the subfamily Cynolebiasinae: uncinate process of the third epibranchial distinctively widened [ch.27.1] (Fig 6B) and presence of a brownish red stripe on the basal portion of the dorsal fin in males [ch.109.1] (Fig. 1); and three are unique among cynopoecilines: a long filamentous ray on the tip of the anal fin in males [ch.70.1], caudal fin lanceolate in males [ch.73.3), and dark bars on the flank in females [ch.92.1] (Fig. 1).In other cynopoecilines, the uncinate process of the third epibranchial is narrow (Fig 6A), there is neither a red stripe on the basal portion of the dorsal fin nor a long filamentous ray on the tip of the anal fin, and the caudal fin is never lanceolate, although a small projection on the posterior part of the fin is present in Mucurilebias.The remaining three autapomorphies of Poecilopanchax are not unique among cynopoecilines: basihyal narrow, about rectangular [ch.20.1] (Fig 2B; independently acquired in Notholebias), branchiostegal region with intense red pigmentation in males [ch.102.1] (independently acquired in Mucurilebias), and dark red marks extending to antero-distal margin of dorsal fin in males [ch.111.0] (a reversal).
Leptolebias Myers, 1952: 140 (type species: Cynopoecilus mar moratus Ladiges, 1934, by original designation; first proposed as a subgenus of Cynolebias steindaChner, 1876).temporary pools in open vegetation areas, sometimes at semi-deciduous forest border.The study of material recently collected in the broad area between the type locality of C. fulgens and C. multipapillatus Costa, 2002 (see list of localities in Appendix 1), indicated that the latter species is a synonym of the former.Species of the subgenus Cynopoecilus occurs between the Rio Tubarão basin in southern Brazil and the Laguna dos Patos-Lagoa Mirim system in southern Brazil and adjacent areas of Uruguay (Costa, 2002a; Costa et al., 2016b).
Maricá systemin south-eCosta et al., 1988;iver basins draining into the Baía de Paranaguá, southern Brazil.Two species, L. citrinipinnis and L. opalescens are found in temporary pools within open vegetation formations, including coastal 'Restinga', whereas the remaining species of Leptopanchax are found in seasonal shallow channels within dense rain forest(Costa & LaCerda, 1988;Costa et al., 1988; Costa, 2008).Etymology.From the Greek leptos, meaning thin or elongate, and panchax, a name of uncertain origin, possibly referring to an Indian popular name for aplocheiloid killifishes, often used to compose generic names of the killifish order Cyprinodontiformes.Gender masculine.glandularonbasalportion of anal fin in males [ch.68.1, reversed in Campellolebias), dark reddish chocolate brown to black stripe between pectoral-fin base and posterior portion of anal-fin base in males [ch.90.1), small round dark reddish brown spots on dorsal fin in males [ch.112.0).Genus Leptolebias Myers, 1952Diagnosis.Posterior process of vomer distinctively longer than main portion of bone [ch.2.1], ventral process of angulo-articular short, well-visible in lateral view [ch.9.1], dorsal-fin origin anterior to anal-fin origin [ch.41.1], anterior proximal radials of anal fin rod-shaped in males [ch.42.1], anterior inclinatores and depressores anales hypertrophied in males [ch.49.1], presence of muscular ejaculatory pump on basal portion of urogenital region in males [ch.50.1], pelvic bones medially separated [ch.59.1], pelvic fin sub-triangular in males [ch.72.1], supraorbital series continuous in its anterior portion [ch.79.0], dark pigmentation present on flank in females, forming distinctive dark marks [ch. 91.1], dark reddish brown pigmentation extending between orbit and posterior limit of head in males [ch.100.1].spine with small process bearing ligament attached to anterior tip of epural [ch.36.1],anterior two rays of anal fin in males long, thickened, separated from the posterior part of the fin by a deep gap in the fin membrane just posterior to second ray to form an inseminating tube [ch.46.1], genital duct extending along anterior margin of anal fin, its opening near the tip of first anal-fin ray Genus Cynopoecilus Regan, 1912 Cynopoecilus regan, 1912b: 642 (type species: Cynolebias melanotaenia regan, 1912, type by original designation and monotypy).27.1], long filamentous ray on tip of anal fin in males [ch.70.1], caudal fin lanceolate in males [ch.73.3], dark bars on flank in females [ch.92.1], branchiostegal region with intense red pigmentation in males [ch.102.1](independentlyacquired in Mucurilebias), brownish red stripe on basal portion of dorsal fin in males [ch.109.1],darkEtymology.From the Greek poecilus, meaning multicoloured, and panchax, a name of uncertain origin, possibly referring to an Indian popular name for aplocheiloid killifishes, often used to compose generic names of the killifish order Cyprinodontiformes.Gender masculine.Remarks.The recently described taxon Cynopoecilus notabilis, although sharing all the nine diagnostic synapomorphies with species of Cynopoecilus (see above), exhibits a series of distinctive morphological features, including some apomorphic conditions unique among cynopoecilines (e.g., interacual element of dorsal branchial arches ossified, dorsal fin with numerous rays and anteriorly placed on body, long filamentous ray on tip of anal fin in males).This striking morphological gap between C. notabilis and the clade comprising all species of Cy nopoecilus, justifies recognition of the new subgenus Po ecilopanchax.SubgenusCynopoecilus Regan, 1912 Included species, distribution and habitat.Five species, C. feltrini Costa, aMoriM & Mattos, 2016, C. ful gens Costa, 2002, C. intimus Costa, 2002, C. melano taenia (regan, 1912), and C. nigrovittatus Costa, 2002, found in in recognising the suborder Aplocheiloidei containing two families, the Aplocheilidae, comprising Old World genera, and the Rivulidae, comprising New World genera.After molecular (MurPhy & CoLLier, 1997) and morphological evidence indicating Aplocheilidae sensu Parenti (1981) to be paraphyletic, Costa (2004) proposed a new classification also including Nothobranchiidae for continental Africa genera, and restricting Aplocheilidae to the genera endemic to Asia, Madagascar, and Seychelles.More recently, Van der Laan et.al. (2014) showed that the stem Rivul-used for