Description of males of Phallotorynus pankalos Lucinda , Rosa & Reis , 2005 and reappraisal of Phallotorynus species relationships ( Teleostei : Cyprinodontiformes : Poeciliidae )

Recently discovered male specimens of Phallotorynus pankalos are described and diagnosed from males of congener species on the basis of gonopodial morphology, color pattern and number of dorsal and anal-fin rays. The phylogenetic hypothesis of relationships among Phallotorynus species is reappraised with the inclusion of P. pankalos. Phallotorynus pankalos is hypothesized as sister to P. jucundus, a clade sister to P. victoriae or to a clade composed of P. dispilos and P. psittakos. Biogeography of Phallotorynus species is discussed, and it is suggested: (1) an early basal split between the rio Paraíba do Sul drainage and the Paraná-Paraguay system, separating P. fasciolatus from the ancestor of the remaining Phallotorynus species followed by a dispersal to rio Paraíba do Sul; (2) that the formation of the Sete Quedas falls may have isolated the ancestors of P. psittakos and P. dispilos. However, the sequence of events associated with the evolutionary history of Phallotorynus remains unclear and unsatisfactory.


Introduction
Phallotorynus Eigenmann, a genus of the cyprinodontiform Poeciliidae, is endemic to southern South American drainages of rio Paraíba do Sul, rio Paraná, and rio Paraguay.Lucinda et al. (2005) proposed Phallotorynus as monophyletic based on seven uniquely derived and unreversed synapomorphies of the cephalic sensory pore system, gonactinostal and gonopodium complexes.These authors recognized six valid Phallotorynus species describing three new species.The description of one of these species, Phallotorynus pankalos Lucinda, Rosa & Reis was based only on females because males were unavailable for study.For this reason, Lucinda & Reis (2005) did not include the species in their phylogenetic analysis of the subfamily Poeciliinae, which was based principally on male morphology.
Recent examination of specimens housed at Coleção Ictiológica do Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (NUP) yielded the discovery of male specimens of Phallotorynus pankalos.The aims of this paper are to formally describe male specimens of P. pankalos and to reappraise the phylogenetic hypothesis of relationships among Phallotorynus species with the inclusion of P. pankalos in the phylogenetic framework of Lucinda & Reis (2005).Lucinda et al. (2005) and are presented in Table 1.Specimens were cleared and stained following Taylor & van Dyke (1985) and are indicated by "cs".Nomenclature of the gonopodium follows Rosen & Gordon (1953) and Lucinda & Reis (2005).Numbers in parentheses following the counts indicate number of specimens for each count.Studied specimens of Phallotorynus pankalos are the ones listed under "Material Examined".Comparative material is as listed by Lucinda & Reis (2005) and Lucinda et al. (2005).
Phylogenetic procedures followed Lucinda & Reis (2005).A phylogenetic analysis of the Poeciliinae was performed in order to elucidate the phylogenetic position of P. pankalos.A phylogenetic analysis was run with Hennig86 software vers.1.5 (Farris, 1988), using the "ie*" command.This command generates trees by an exact searching algorithm (implicit enumeration) examining all trees, and retaining all best trees (most equally parsimonious trees).The implicit enumeration command "ie*" is guaranteed to find all the most parsimonious trees (Platnick, 1989).Character optimization followed accelerated transformation model (ACCTRAN) for it is more consistent with the concepts of homology and synapomorphy as discussed by de Pinna (1991).
Outgroups were chosen to be consistent with Lucinda & Reis (2005).Outgroups are representatives of taxa most closely related to Poeciliinae according to previous phylogenetic hypotheses of relationships among cyprinodontiform fishes (Parenti, 1981;Costa, 1998;Ghedotti, 2000).Outgroups are the following cyprinodontiform species: Fundulus heteroclitus (Linnaeus), Cyprinodon macularius Baird & Girard, Jenynsia unitaenia Ghedotti & Weitzman, Aplocheilichthys spilauchen (Duméril), Fluviphylax pygmaeus (Myers & Carvalho), and Procatopus gracilis Clausen.Outgroup topology was unconstrained as recommended by Nixon & Carpenter (1993) and the unrooted networks generated by the phylogeny reconstruction program were rooted on Profundulus labialis (Günther).All transformation series were unordered.Character state assignments in transformation series are based on those presented in the phylogenetic analysis of the Poeciliinae by Lucinda & Reis (2005).Data gathered from the new taxon were added to the phylogenetic data presented in Lucinda & Reis (2005).These data are shown in Table 2.All character states were evaluated for the new species and analyzed in the phylogenetic framework of Lucinda & Reis (2005).Diagnosis of male specimens.Male specimens of Phallotorynus pankalos can be distinguished from males of its congeners, except P. jucundus Ihering: (1) by the color pattern (six to nine dark brown round spots along lower half of flanks vs. one or two dark brown round to elliptical spots, respectively); ( 2) by the size of the lateral processes on the trowel-like appendix (large vs. small, respectively);

Phallotorynus pankalos
(3) and by left and right halves of the trowel-like appendix united along its whole extension (vs.halves not entirely united and separate by a gap along its extension).A densely pigmented dorsal fin also distinguishes male P. pankalos from the males of remaining species in the genus, except P. jucundus.Males of P. pankalos may be distinguished from males of P. jucundus by the number of dorsal-fin rays (8 vs. 9 or 10, respectively) and by the number of anal-fin rays (eight vs. nine, respectively).Furthermore, males of P. pankalos and P. jucundus may be differentiated by the size of the membranous tip anterior to R4 and R5 (large vs. small, respectively).
Gonopodial complex composed of three functional gonapophyses and 11 gonactinosts.Gonactinosts 2, 3, 4, and 5 fused.Gonactinost 5 with winglike expansions.Ligastyle present.Eight gonopodial rays.R1 and R2 unbranched and short.R3 with 31 segments.Segments 19 to 31 abruptly narrower than preceding segments.Segments 19 to 31 progressively narrower and shorter towards tip.Ventral V-shaped projection located at level of segment 17 and 18, and covered by a large membrane forming two conspicuous undulations.First (more basal) undulation approximately twice smaller the second (more distal) one.Last segment of R3 attached to bony style that supports trowel-like appendix.Bony style slightly bent dorsally.Terminal appendix long and narrow, with large lateral processes.Both halves of terminal appendix with straight lateral border and united along its whole extension.R4a with 31 segments.Last distal segment followed by cub-shaped membrane.Thirty segments on R4p; five paired serrae located near tip.Serrae anteriorly directed and gradually shorter towards tip of R4p.Distal half of R6 expanded.Tip of R6 and R7 ankylosed.R7 and R8 small and branched.
Color in alcohol (of male specimens).Eye pupil black.Ground color pale brown, darker on upper half.Border of scales and subjacent skin replete with brown chromatophores, conferring reticulate pattern to body sides, mainly on upper half.Brown chromatophores scattered over whole body, more concentrated on dorsal portion, mainly on head, snout, and ventral surface of lower jaw.Pale brown postorbital band.Dark brown mandibular stripe inconspicuous in some specimens.Dark brown line along predorsal surface.Dorsal-fin membrane black contrasting with hyaline rays.Black band on dorsal fin near its base.Pectoral-fin rays with brown chromatophores.Dark brown line along R3.Caudal-fin rays grayish brown.Six to nine (usually six) dark brown round to irregular spots along lower half of flanks, alternating with narrow dark brown bars that occasionally reach dorsal and ventral profiles.
Phylogenetic relationships.Analysis of the data set resulted in 288 most equally parsimonious trees (MEPT's) (length = 762, CI = 0.35, RI = 0.75) illustrating variation in topology of the ingroup (the Poeciliinae).Concerning the relationships within Phallotorynus, topologies obtained are congruent with those discussed in Lucinda & Reis (2005) and Lucinda et al. (2005) (q.v. fig. 3 andLucinda et al., 2005:   Distribution.Phallotorynus pankalos is known only from its type locality (córrego Sossego, rio Iguatemi drainage, upper portions of rio Paraná system) (Fig. 4).Phallotorynus pankalos has been reported as inhabiting the córrego Mirim and córrego Piraí by Súarez et al. (2009).We could not confirm this distribution.Ecological Notes.Despite the small number of specimens deposited in fish collections (only 28), Súarez et al. (2009) recorded 2,680 specimens (948 males and 1,732 females) during one year of sampling in three first-order streams in the rio Iguatemi basin (córrego Sossego, córrego Mirim and córrego Piraí).Unfortunately, Súarez et al. (2009)'s voucher specimens are restricted to only one holding with a few specimens (NUP 5839).As a result, most specimens studied by these authors were probably discarded and their identity cannot be confirmed.If the specimens studied by Súarez et al. (2009) are Phallotorynus pankalos, this is an abundant yet restricted species.Therefore, Phallotorynus pankalos is likely a threatened species, because it is known to exist at no more than three locations imperiled by decline of habitat quality, caused mainly by agricultural activities.Furthermore, Súarez et al. (2009) concluded that P. pankalos has small fecundity and higher mortality rate for females after first reproduction.Thus, conservational actions on sites of occurrence of P. pankalos should be considered.

Discussion
The character states exhibited by P. pankalos allow its classification as a member of the monophyletic Phallotorynus as diagnosed by Lucinda et al. (2005).However, P. pankalos was not included in Lucinda & Reis (2005) phylogenetic analysis (which was primarily based on gonopodial morphology) due to the lack of males.Judging from its distribution, Lucinda et al. (2005) speculated that P. pankalos was more closely related to the clade composed of P. victoriae, P. jucundus, P. dispilos, and P. psittakos, and therefore also originated in upper Paraná.Since males have now become available for study this assumption may be confirmed.Global parsimony of character states in the consensus tree supports P. pankalos as sister to P. jucundus, a clade sister to P. victoriae or to a clade composed of P. dispilos and P. psittakos.
An area cladogram (Fig. 5) (derived from the consensus tree is congruent with previous biogeographic hypothesis (Lucinda et al., 2005: fig. 12).These authors suggested an early basal split between the rio Paraíba do Sul drainage + upper rio Tietê and the Paraná-Paraguay system, separating P. fasciolatus from the ancestor of the remaining species followed by a dispersal to rio Paraíba do Sul, an event likely associated with stream capture of rio Tietê headwaters.Lucinda et al. (2005) also raised a hypothesis that the formation of the Sete Quedas falls may have isolated the ancestors of P. psittakos and P. dispilos.The generic phylogenetic relationships and the sequence of geological events associated with cladogeneses along the evolutionary history of Phallotorynus is still partially unclear and unsatisfactory given the polytomy (P.victoriae (P.dispilos, P. psittakos)(P.pankalos, P. jucundus)), yet the cladogram and areagram express the state of our knowledge of the relationships among Phallotorynus species.Discovery of new characters and/or species likely would help clarify and may shed light on our understanding of Phallotorynus systematics and biogeography.

Table 1 .
Maximum standard length: 19.2 mm SL.Body compressed.Body width in predorsal region uniform and about half body depth.Postdorsal region compressed near caudal peduncle.Dorsal profile of head slightly concave.Predorsal profile convex.Dorsal-fin base oblique.Postdorsal profile slightly concave.Preanal profile convex.Anal-fin base convex; postanal profile nearly straight.Dorsal fin with semi-elliptical border, located posterior to mid-body.Origin of dorsal fin posterior to vertical passing through origin of anal fin.Longest pectoral-fin ray slightly anterior to origin of pelvic fin.Pelvic fin small, almost reaching origin of gonopodium in adult males.Origin of anal fin closer to snout tip than to caudal peduncle.Mouth superior, almost aligned dorsally with base of dorsalmost pectoral-fin ray.Premaxillary and dentary teeth flattened and incisiform with long and narrow pedicel progressively wider towards distal end.Distal end of teeth curved towards oral cavity.Premaxillary teeth varying from pointed to truncate, superior border varying from acute to oblique.