Oligocene killifishes ( Teleostei : Cyprinodontiformes ) from southern France : relationships , taxonomic position , and evidence of internal fertilization

Phylogenetic relationships of five species of Prolebias from the Oligocene of southern France (P. aymardi, P. cephalotes, P. delphinensis, P. meridionalis, and P. stenoura) were examined under an analysis comprising 36 terminal taxa, representing all the main lineages of the order Cyprinodontiformes. It indicated Prolebias as a polyphyletic group, supporting Prolebias stenoura, the type species of the genus, as a valenciid monotypic lineage. Prolebias aymardi and P. delphinensis comprise a new valenciid genus, Francolebias, gen. nov., diagnosed by a unique morphology of pelvic bone, anterior proximal radials of the dorsal fin and anal fins, and hemal spines in putative males. The apomorphic specialized morphology of the anal fin and adjacent vertebrae is interpreted as evidence of an internal fertilization reproductive mode. Prolebias meridionalis is designated as the type species of a new monotypic cyprinodontid genus, Eurolebias, gen. nov., hypothesized to be the sister group to a clade comprising all other cyprinodontids, which is diagnosed by the apomorphic morphology of jaws, parhy­ pural, and absence of parietals. Close relationships between P. cephalotes, P. egeranus from the lower Miocene of Czech Republic, and recent species of the poeciliid genus Pantanodon are corroborated on the basis of the apomorphic morphology of the dentary, pharyngobranchials, pelvic bone, and pelvic-fin rays, justifying the transference of those two species to the latter genus. The present study indicates that the European Oligocene fauna of cyprinodontiform fishes was greatly diversi­ fied when compared to its present poor fauna, comprising lineages now extinct or restricted to other continents.


Introduction
Contrasting with the great diversity of living cyprino dontiform species (about 1,120 species in 125 gen era and ten families), the fossil record of the order Cyprinodontiformes is poor, mainly concentrated in Europe (Costa, 2012).The oldest records are from the Lower Oligocene, all belonging to the European ge nus Prolebias sauvage, 1874, known until the middle Miocene (e.g., gaudant, 2003).
sauvage (1874) first considered Prolebias as a member of the Fundulina sensu günther (1866), which then included species presently distributed among all cyprinodontiform families excluding the Cy pri no dontidae.WoodWard (1901) restricted Pro le bias relationships to the North American fundulid genera Lucania and Fundulus, the latter at that time also including species today placed in the European valenciid genus Valencia.However, Parenti (1981) in cluded Prolebias in the Cyprinodontidae without justi fication, a placement followed by subsequent authors without criticisms (e.g., obrhelová, 1985;rei Chen baCher & gaudant, 2003;gaudant, 2009).gau dant (1988) considered Prolebias possibly related to the Andean cyprinodontid genus Orestias, but several new species have been described without checking charac ters supporting its inclusion in that family and objec tive comparisons to recent cyprinodon ti forms were never made.However, théobald & Piton (1937) had formerly noted that species placed in Pro le bias had uncertain relationships among the several distinct re cent cyprinodontiform genera, additionally suggesting that Prolebias goreti sauvage, 1880 is closely related to Fundulus gardneri (= Fun du lo pan chax gardneri) and Fundulus guentheri (= No tho bran chius guen theri), two species today placed in the African family Nothobranchiidae.More recently, Costa (2012) re corded a great morphological diversity among some species of Prolebias, suggesting that the genus is poly phyletic, probably comprising species related to differ ent cyprinodontiform families.
gaudant (2003) listed 15 valid species in Pro lebias, of which six are only known from the Oligocene of southern France.The southern France species as semblage, besides exhibiting a great morphological variability, contrasts with other taxa by being richly represented in fossil collections deposited in easily accessible European museums.In addition, among these species is Prolebias stenoura sauvage, 1874, the type species of the genus, providing unique possibility of testing monophyly and the taxonomic position of the genus.Thus, the present paper primarily focuses on five species of Prolebias from the Oligocene of southern France (the type species of the genus and P. aymardi (sauvage, 1869), P. cephalotes (agassiz, 1839), P. delphinensis gaudant, 1989, and P. me ridionalis gaudant, 1978), for which there are avail able representative collections adequate for searching characters currently employed in phylogenetic studies on cyprinodontiforms (Parenti, 1981;Costa, 1997Costa, , 1998)), with the objective of elucidating their phyloge netic position among cyprinodontiform lineages.

Material and methods
The phylogenetic analysis was based on a data matrix including five species of Prolebias from the Oligocene of southern France (P.aymardi, P. cephalotes, P. del phinensis, P. meridionalis, and P. stenoura) as terminal taxa.To test the phylogenetic position of those species among cyprinodontiforms, the data matrix also includ ed 27 recent species representing all the main lineages of the order Cyprinodontiformes.Due to previous evi dence supporting Prolebias in the Cyprinodontoidei clade (Costa, 2012), taxon sample was more con centrated in the seven families of the latter clade and proportional to the diversity found in each family (two anablepids, eight cyprinodontids, two fundulids, four goodeids, five poeciliids, one profundulid, and two valenciids).The taxon sample also particularly focused on species endemic to Europe, thus includ ing the only two species belonging to the European family Valenciidae, and two species of Aphanius, the only recent cyprinodontid genus occurring in Europe.In order to test the proposed close relationships be tween Prolebias and the South American cyprinodon tid Orestias (gaudant, 1988), between P. cephalotes and Prolebias egeranus from the upper Miocene of the Cheb basin (gaudant, 2009), and between those spe cies and the recent African poeciliid genus Pantanodon Myers, 1955 suggested by Costa (2012), two spe cies of Orestias, P. egeranus, and Pantanodon stuhl manni (ahl, 1924), the type species of Pantanodon, were included in the analysis.Additional outgroup taxa were Oryzias matanensis and Oryzias sarasino rum representing the Adrianichthyidae, the most ba sal family of the Beloniformes, the sister group of the Cyprinodontiformes; and, Melanotaenia affinis, a rep resentative of the Atheriniformes, the sister group of Beloniformes plus Cyprinodontiformes.Comparative material, including that used in the phylogenetic ana lysis, is listed in Costa (2012).Osteological data from extant species was obtained from material prepared according to the clearing and staining techniques de scribed by taylor & van dyke (1985).Characters, mainly extracted from Costa (1998) and listed in Appendix 1, focused on bone structure, excluding characters involving cartilages and superficial ossi fications, which show high levels of subjectivity in character state delimitation among the present taxon sample.Character statements were formulated fol lowing sereno (2007).Distribution of character states among taxa appears in the data matrix of Appendix 2. All characters were treated as unordered.The search for most parsimonious trees (using 'traditional' search and setting random taxon-addition replicates to 1,000, tree bisection-reconnection branch swapping, mul titrees in effect, collapsing branches of zero-length, characters equally weighted, and a maximum of 1,000 trees saved in each replicate), bootstrap analysis (1,000 replicates) and Bremer-support indices were performed with TNT 1.1 (goloboff et al., 2008).

Relationships of Prolebias stenoura
The analysis supports Prolebias stenoura (Fig. 2A) as a member of the Valenciidae.This family is endemic to Europe and presently comprises only two extant species in a single genus, Valencia.Parenti (1981) diagnosed the Valenciidae on the basis of the unique presence of an elongate and attenuate dorsal process of the maxilla, but Costa (1998)   ened (Fig. 3D), whereas in other specimens with simi lar size and sharing all other morphological characters that widening is not present (Fig. 3E).This putative sexual dimorphism character is easily observable in specimens about 30 -35 mm SL, whereas in smaller specimens of about 20 -25 mm SL, this condition is present but much less conspicuous, as occurring in F. aymardi, a smaller species, with examined specimens not surpassing 22 mm SL.Interestingly, only in those individuals with widened hemal spines, the first analfin rays are robust, distinctively broader than subse quent rays and the proximal radials are proportionally wider (Fig. 3D, E).Internal fertilization is relatively common among living cyprinodontoids, occurring in numerous spe cies of three families, which corresponds to three in dependent evolutionary events -in the clade compris ing the American genera Anableps and Jenynsia of the Anablepidae, in the Mexican subfamily Goodeinae of the Goodeidae, and in the American subfamily Poeciliinae of the Poeciliidae (Parenti, 1981;Meyer & lydeard, 1993).Even with internal fertilization being independently acquired in those three cyprinodontoid families, all evolutionary acquisitions involve strik ing modifications in the anal fin structure (rosen & gordon, 1953;rosen & bailey, 1963;Parenti, 1981;ghedotti, 1998).These bone modifications, which are much more pronounced in males, are always associ ated with anal fin mobility during sperm transference (e.g., rosen & gordon, 1953).On the other hand, no modification is found in externally fertilizating species of anablepid, goodeid and poeciliid lineages, thus in dicating a high level of dependence between internal fertilization and modified anal fin structure.
Strong sexually dimorphic modifications in the structure of the hemal arches above the anal fin as sociated with robust anal-fin rays and correspond ing proximal radials, analogous to that occurring in Francolebias, are only present in internal inseminating taxa of the Anablepidae and Poeciliidae (e.g., rosen & bailey, 1963;Parenti, 1981;ghedotti, 1998), as well as in species of the internal inseminating rivulid genus Campellolebias vazferreira & sierra, 1974 (Costa, 1995).Since sexually dimorphic modifications of the anal-fin structure in living cyprinodontiforms are al ways related to morphological specializations associ ated to internal fertilization, the presence of such mod ifications in Oligocene valenciid cyprinodontiforms highly supports an internal fertilization mode.

Eurolebias and other cyprinodontid fossils
The placement of the taxon Eurolebias meridionalis (Fig. 2D) among cyprinodontids is supported by the presence of a reduced jaw dentition, consisting of an branchiids, and rivulids, rudimentary in fundulids, go odeids, profundulids, and some poeciliids, and well developed but widened in anablepids, cyprinodontids, and most poeciliids.The present analysis supports the last condition as plesiomorphic for cyprinodontoids.A long and narrow dorsal process of the maxilla is vis ible in some specimens of P. stenoura (Fig. 3A), as well as in species of Francolebias (see bellow).
Unique derived conditions supporting monophyly of the Valenciidae, herein first reported, are the greatly widened neural spine of preural vertebra 2, its width about three to four times the width of neural spine of preural vertebra 4 (Fig. 2B), and anal-fin rays distinc tively thicker than dorsal-fin rays (Fig. 2A), which are not found elsewhere among cyprinodontiforms.The analysis also indicates that no other examined species of Prolebias is closely related to P. stenoura.Since P. stenoura is the type species of the genus, Prolebias is herein considered as a monotypic genus.

Relationships of Francolebias, evidence of sexual dimorphism and putative internal fertilization
The presence of a greatly widened neural spine of preural vertebra 2 and anal-fin rays distinctively thicker than dorsal-fin rays supports inclusion of Francolebias among valenciids, as well as the pres ence of a broad pelvic bone, coalesced anterior proxi mal radials of the dorsal fin, and long anterior proxi mal radials of the anal fin with tip at the level of the basal portion of the adjacent hemal spines unambigu ously distinguish Francolebias from all other fossil killifish taxa.gaudant (1988,1989) already noted the peculiar morphology of the pelvic bone in F. delphin ensis and F. aymardi, suggesting that the species are closely related.In those two species, the greatest width of the pelvic bone is about 70 % of the bone length (Fig. 3B), in contrast to never more than 50 % in other cyprinodontiforms, besides the anterior portion being rounded, instead of pointed as in other cyprinodonti form taxa.The coalesced anterior proximal radials of the dorsal fin (Fig. 2C) and the long anterior proximal radials of the anal fin (Fig. 3D, E), although not previ ously reported, are conspicuous in all specimens stud ied.Among cyprinodontiforms, similar conditions to those were, respectively, only found in the Goodeinae clade of Goodeidae and in the anablepid clade com prising Anableps and Jenynsia, parsimoniously sup ported as homoplastic.
As already reported by gaudant (1989), in some specimens of F. delphinensis, the distal portion of the hemal spines adjacent to the anal fin are strongly wid almost rectangular ascending process of the premaxil la, parietals and a long parhypural overlapping preural centrum 1, as well as the absence of a distinct groove on the dorsal process of the maxilla, and a ventral ex pansion on the dentary in E. meridionalis, which are plesiomorphic conditions among cyprinodontoids, in dicates its basal position among the Cyprinodontidae.
Costa (2012) suggested inclusion of E. meridi onalis among cyprinodontids on the basis of charac ters of the caudal skeleton, such as the presence of a constriction on the basal portion of the hemal spine of the preural centrum 2 and a short proximal part of the parhypural.However, re-examination of the entire type series, including several specimens with wellpreserved caudal fin skeletons, confirmed that con striction on the hemal spine of the preural centrum 2, but also revealed that the parhypural is not so short, but slightly overlaps the preural centrum 1.
Abundant fossil material from the Miocene of Europe and Middle East have been assigned to the cyprinodontid genus Aphanius (e.g., gaudant, 1979Aphanius (e.g., gaudant, , 1993Aphanius (e.g., gaudant, , 2009Aphanius (e.g., gaudant, , 2011)), which also comprises numerous recent species (e.g., hrbek & Meyer, 2003).Both recent and fossil species of Aphanius share a pro nounced hook-shaped process on the ventral portion outer tooth row followed by a few smaller teeth near the symphysis of the premaxilla and dentary (Fig. 3C).In other cyprinodontiforms, there are multiple series of small teeth adjacent to a single outer row of larger teeth, except in the Recent North American fundulid genus Lucania, in which a similar reduction is con vergently found.In addition, although head bones are highly fragmented in the material of E. meridiona lis, it is possible to note that the anterior axis of the jaw suspensorium, comprising the autopalatine, and the posterior portion, comprising the hyomandibula, have their main vertical axes nearly parallel (not il lustrated), a condition only recorded in cyprinodontids among cyprinodontiforms.Also supporting inclusion of E. meridionalis in the Cyprinodontidae is the broad dorsal process of the maxilla (Fig. 2E), a condition occurring only in cyprinodontids, poeciliids and ana blepids.However, some derived conditions shared by all the cyprinodontids do not occur in E. meridionalis -presence of a distinct groove on the dorsal process of the maxilla; a short, sharp, almost triangular ascend ing process of the premaxilla; dentary with a ventral expansion; proximal part of the parhypural short, not overlapping preural centrum 1; and, parietals absent (Parenti, 1981;Costa, 1998).The presence of a long greatly enlarged second pharyngobranchial toothplate, a condition unique among cyprinodontiforms (rosen, 1965;Parenti, 1981).In addition, the presence of spine-like contact organs on the extremities of the an terior rays of the dorsal and anal fins rays is herein first recorded for some specimens of P. cephalotes (Fig. 3I).Contact organs on fin rays and body scales are common in males of several cyprinodontiform lin eages, particularly in aplocheiloids, which is usually related to elaborate courtship behaviour (e.g., Costa, 2006).

Incertae sedis species of Prolebias
The material of Prolebias goreti from the lower Oligocene of Céreste, France available for study did not provide useful evidence about its phylogenetic po sition.Details on jaw and caudal skeleton morphology were not clearly visible, and no data about other struc tures were seen.Although the premaxilla morphology and tooth arrangement of P. goreti are to those in Prolebias stenoura, the thin anal-fin rays and the dorsal-fin origin anteriorly positioned relative to the anal-fin origin indicate that the taxa are not congener ic.Thus, P. goreti is herein considered as an incertae sedis taxon.
Other well established nominal species of Pro le bias, including P. catalaunicus gaudant, 1982 from the lower Oligocene of Sarreal, Spain, P. euskadien sis gaudant, 2003 from the upper Oligocene -lower Mio cene of Izarra, Spain, P. hungaricus gaudant, 1991 from the middle Miocene of Szurdokpüspöki, Hun gary, and P. rhenanus gaudant, 1981 from the lower Oligocene of Kleinkems, Germany, which were not available for the present study and are still known only from a limited set of characters provided in the literature (gaudant, 1981, 1982, 1991, 2003), are presently considered as incertae sedis taxa.

Other European killifish fossil genera
The monotypic genus Cryptolebias gaudant, 1978, was based on a single specimen of Cryptolebias seno galliensis (CoCChi, 1859) from the Messinian marls of Senigallia, Italy (gaudant, 1978b).The genus was then placed in the Cyprinodontiformes on the basis of the typical structure of the caudal skeleton, but the poorly preserved specimen does not provide evi dence supporting its inclusion in any cyprinodontiform fam ily.Cryptolebias senogalliensis differs from all other Cenozoic killifish taxa by the anal-fin origin positioned through a vertical through the posterior half of the dor sal-fin base and a very slender body, depth about 12 % standard length.
of the dentary (Parenti, 1981;Costa, 1997), which is not present in E. meridionalis.The fossil cyprino dontid record is also represented in South America by the Miocene Andean taxon Carrionellus diumortuus, which is closely related to the recent genus Orestias, and may be distinguished from E. meridionalis by the presence of large tricuspidate teeth on jaws (White, 1927;Costa, 2011).

Poeciliid fossils
Costa (2012) recently recorded some morphologi cal evidence supporting close relationships between Prolebias cephalotes and P. egeranus, and the Recent African poeciliid genus Pantanodon, which is con firmed in the present study.Prolebias cephalotes and P. egeranus have the general morphology typi cal among poeciliids, such as dorsal-fin origin poste rior to anal-fin origin, pectoral-fin base high on flank, pelvic-fin girdle placed just posterior to shoulder gir dle, besides a small size, barely surpassing 30 mm SL (Costa, 2012) (Fig. 2E).
Pantanodon has been known from two small and rare species inhabiting brackish waters of East Africa and Madagascar (P.stuhlmanni and P. mada gascariensis).Their peculiar morphology inspired two papers focusing on osteology (Whitehead, 1962;rosen, 1965), the first one proposing inclusion in their own subfamily Pantonodontinae (Whitehead, 1962), which has not been followed by recent authors (e.g., Parenti, 1981).Some apomorphic features shared by Prolebias cephalotes, P. egeranus and recent spe cies of Pantanodon, not occurring in other poeciliids and other cyprinodontiforms, support monophyly of a group comprising all these taxa: presence of an expan sion on the postero-dorsal border of the dentary, which terminates posteriorly as a long narrow prolongation (Fig. 2F); third and fourth pharyngobranchial tooth plates coalesced; pelvic bone long and narrow, and extremity of pelvic-fin rays thickened in males, often with curved tips (Fig. 2H).
The presence of transverse rows of minute teeth on the fifth ceratobranchial and pharyngobranchials, as well as the wide, drop-shaped fifth ceratobranchi al, with two lateral processes (Fig. 2G), are particu larly remarkable, being identical in P. cephalotes, P. egeranus and in both recent species of Pantanodon.This morphology strongly contrasts with the slender, boomerang-shaped fifth ceratobranchial, with a single terminal process and large teeth irregularly arranged, which is present in all other cyprinodontiforms except in some New World poeciliids.
Pantanodon stuhlmanni and P. madagascariensis are clearly more closely related to one another than to congeneric fossil species by the two former sharing a or exchange of comparative material.The manuscript benefited from the valuable criticisms and suggestions provided by a. Murray.The Willi Hennig Society generously made available free access to TNT.This study was funded by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico -Ministério de Ciência e Tecnologia).Germany (reiChenbaCher & gaudant, 2003), which had been previously placed in Prolebias since sauvage (1874).Material of this taxon was not available for the present study and the few characters presented in the description do not permit an accurate character analy sis.However, Aphanolebias meyeri does not present the derived character states of the two new genera herein described, as well it does not present the di agnostic conditions of Pantanodon.Some character states of A. meyeri, including the dorsal-fin origin posterior to anal-fin origin, pelvic-fin base slightly closer to anal-fin origin than to pectoral-fin base, jaw teeth conical and arranged in multiple series and hy pural plates partially separated, suggest a basal posi tion among cyprinodontoids, but an analysis based on examination of material is necessary to establish hypotheses of relationships.Several other species of Aphanolebias have been uniquely proposed on the basis of otolith morphology (e.g., reiChenbaCher & koWalke, 2009), which does not provide evidence of relationships among cyprinodontoids.
Francolebias nov.gen.by having slender pelvic bone, greatest width about 50 % of length (vs.widened, about 70 %); hemal spines of caudal vertebrae above anal fin unmodified, similar to subsequent hemal spines (vs.distinctively widened in putative males); anterior proximal radials of dorsal fin separated (vs.coalesced); and, anterior proximal radials of anal fin short, reaching vertical through dis tal portion of adjacent hemal spines (vs.long, close to basal portion of adjacent hemal spines).Only the type spe cies, P. stenoura, from the lower Oligocene of Corent, Puy-de-Dôme, southern France (see Discussion be low).