Osteology of the African annual killifi sh genus Callopanchax (Teleostei: Cyprinodontiformes: Nothobranchiidae) and phylogenetic implications

The Aplocheiloidei is a diversifi ed clade of cyprinodontiform fi shes, with over 500 valid species occurring in freshwater biotopes of most tropical and subtropical areas of the world. Presently they are grouped into three families: the Aplocheilidae, endemic to southern Asia and Madagascar; the Nothobranchiidae, endemic to continental Africa; and, the Rivulidae, endemic to Americas (COSTA, 2004). All aplocheilids and several nothobranchiids and rivulids live in shallow streams and swampy areas, whereas other nothobranchiids and rivulids inhabit seasonal pools formed during rainy periods. The latter aplocheiloids are known as annual fi shes, since during the dry season all individuals die, but eggs undergo in diapause, buried in the bottom sediment for months, waiting for the next rainy season. Thus, every year an entire new generation arises when eggs emerge after the fi rst rains. Osteology has been an important source of morphological characters to erect hypotheses of relationships among aplocheiloid families (PARENTI, 1981; COSTA, 1998a, 2004) and among genera and species of the Rivulidae (e. g., COSTA, 1998b, 2005, 2006a, b). However, only recently osteological traits have been fully described and illustrated for some rivulid lineages (e. g., COSTA, 2005, 2006a, 2006b). On the other hand, systematics of the Nothobranchiidae is still incipient, with genera and subgenera poorly defi ned, and little is known about osteology (AMIET, 1987; AARN & SHEPHERD, 2001). However, a number of recent molecular studies provide hypotheses of nothobranchiid taxa relationships, corroborating monophyly of some previously proposed genera and subgenera (e. g., MURPHY & COLLIER, 1997, 1999; MURPHY et al., 1999; SONNENBERG et al., 2006). Molecular data support nothobranchiid clades presently not diagnosed by morphological synapomorphies, making clear the needing of improving the morphological data base for this group. Callopanchax MYERS is an annual fi sh genus, endemic to the region of western Africa encompassing Guinea, Sierra Leone and Liberia. It has a central position in the most controversial debates on taxonomy and phylogeny of nothobranchiids. The name CalOsteology of the African annual killifi sh genus Callopanchax (Teleostei: Cyprinodontiformes: Nothobranchiidae) and phylogenetic implications


Introduction
The Aplocheiloidei is a diversifi ed clade of cyprinodontiform fi shes, with over 500 valid species occurring in freshwater biotopes of most tropical and subtropical areas of the world.Presently they are grouped into three families: the Aplocheilidae, endemic to southern Asia and Madagascar; the Nothobranchiidae, endemic to continental Africa; and, the Rivulidae, endemic to Americas (COSTA, 2004).All aplocheilids and several nothobranchiids and rivulids live in shallow streams and swampy areas, whereas other nothobranchiids and rivulids inhabit seasonal pools formed during rainy periods.The latter aplocheiloids are known as annual fi shes, since during the dry season all individuals die, but eggs undergo in diapause, buried in the bottom sediment for months, waiting for the next rainy season.Thus, every year an entire new generation arises when eggs emerge after the fi rst rains.
Osteology has been an important source of morphological characters to erect hypotheses of relationships among aplocheiloid families (PARENTI, 1981;COSTA, 1998aCOSTA, , 2004) ) and among genera and species of the Rivulidae (e. g., COSTA, 1998bCOSTA, , 2005COSTA, , 2006a, b), b).However, only recently osteological traits have been fully described and illustrated for some rivulid lineages (e. g., COSTA, 2005COSTA, , 2006aCOSTA, , 2006b)).On the other hand, systematics of the Nothobranchiidae is still incipient, with genera and subgenera poorly defi ned, and little is known about osteology (AMIET, 1987;AARN & SHEPHERD, 2001).However, a number of recent molecular studies provide hypotheses of nothobranchiid taxa relationships, corroborating monophyly of some previously proposed genera and subgenera (e. g., MURPHY & COLLIER, 1997, 1999;MURPHY et al., 1999;SONNENBERG et al., 2006).Molecular data support nothobranchiid clades presently not diagnosed by morphological synapomorphies, making clear the needing of improving the morphological data base for this group.
Callopanchax MYERS is an annual fi sh genus, endemic to the region of western Africa encompassing Guinea, Sierra Leone and Liberia.It has a central position in the most controversial debates on taxonomy and phylogeny of nothobranchiids.The name Cal-lopanchax was erected by MYERS (1933), but all the three valid species were described after 1966.This was due to C. occidentalis (CLAUSEN), the type species of the genus, was known by aquarists since 1909 under an equivocal identifi cation, but recognized as undescribed species only by CLAUSEN (1966).Callopanchax also includes other two valid species, Callopanchax toddi (CLAUSEN) and Callopanchax monroviae (ROLOFF & LADIGES), and a fourth nominal species, Callopanchax huwaldi (BERKENKAMP & ETZEL), considered to be a synonym of C. occidentalis (e. g., WILDEKAMP, 1996).
Vertebrate Zoology ■ 59 (1) 2009   hobranchius included all African annual killifi shes.On the other hand, Archiaphyosemion and Scriptaphyosemion were considered as subgenera of Aphyosemion, which would be the sister group to the clade containing Fundulopanchax and Nothobranchius.However, most recent phylogenetic studies, based on molecular data, corroborate monophyly of clade comprising Callopanchax, Archiaphyosemion and Scriptaphyosemion, which were not closely related to the clade comprising Aphyosemion and Fundulopanchax (MURPHY & COLLIER, 1997, 1999;MURPHY et al., 1999).
The aim of the present study is to describe and illustrate the osteological traits of Callopanchax, comparing to other aplocheiloid species in order of searching for characters useful to test monophyly of Callopanchax and its phylogenetic position among nothobranchiids.

Figs. 1A -D
Nasal thin, scale-like.Lachrymal subtriangular, carrying distinctive lateral canal, anterior tip slightly twisted, posterior and ventral portions laminar.Dermosphenotic thin, ovoid, its outer surface gently concave.Frontal trapezoidal and thin.Parietal short, ovoid, without keels.Supraoccipital with short, paired posterior process.Epiotic small, without dorsal process.Sphenotic with prominent narrow lateral process.Vomer trapezoidal, with elongated posterior process; 4-5 teeth concentrated on anterocentral portion of vomer.Mesethmoid unossifi ed.Lateral ethmoid compact, its anterior portion overlapping lateral portion of vomer; medial margin separated by broad space from anterior process of parasphenoid.Parasphenoid cross-shaped; distal portion of anterior process narrow and dorsally overlapping posterior process of vomer, proximal portion widened; posterior process fi rmly and ventrally attached to basioccipital, constricted on proximal portion, abruptly widening towards distal region; short lateral processes ventrally attached to prootic.

Jaws, jaw suspensorium and opercular apparatus
Figs. 2A -B Premaxilla narrow, with teeth on medial half of anterior face; ascending process subtriangular, slightly curved distally to form slight concavity on posterior medial margin; subdistal portion of premaxilla with anteriorly directed, triangular process.Maxilla subcylindrical, proximal portion bifi d.Rostral cartilage large, round.Dentary robust, with teeth on distal two thirds of its anterodorsal surface.Angulo-articular triangular, with wide ventral process.Retro-articular small.Coronomeckelian cartilage elongate.Premaxillary and dentary teeth conical, with slightly curved tip; teeth arranged in irregular rows, largest teeth on most external row.
Autopalatine and ectopterygoid completely fused.Subdistal portion of autopalatine with small medial projection, supporting ligament connected to maxilla.Mesopterygoid thin, long, posteriorly abutting metapterygoid.Ventral portion of mesopterygoid overlapping dorsal portion of the quadrate.Quadrate approximately triangular, anterior and ventral margins approximately straight, dorso-posterior margin convex; posterior process of quadrate subcylindrical, short.Sympletic triangular, deep.Metapterygoid subrectangular, shorter than mesopterygoid.Hyomandibula compact, with prominent curved lateral crest between anterodorsal condyle and posteroventral tip of hyomandibula, separated from ventral condyle of hyomandibula by broad laminar extension; anterior margin of hyomandibula slightly expanded anteriorly.Opercle subtriangular, dorsal margin convex; posterior border of opercle and all borders of interopercle and subopercle membranous and poorly ossifi ed.Preopercle C-shaped, robust, with distinct vertical canal and expanded anteromedian fl ap; dorsal and anterior ends slightly pointed.

Hyoid and branchial arches
Figs. 3A -D Basihyal about pentagonal in dorsal view, fl attened; cartilaginous portion subrectangular, about twice longer than triangular osseous portion; basihyal width about 60 % of total basihyal length.Dorsal and ventral hypohyals ossifi ed.Anterior process of anterior ceratohyal cylindrical, abruptly narrowing posteriorly, with two narrow branchiostegal rays attached to ventral border; anterior ceratohyal compressed and deep in its posterior portion, rectangular in lateral view.Anterior and posterior ceratohyals separated by space of cartilage, supporting four branchiostegal rays gradually becoming wider posteriorly.Posterior ceratohyal compressed, subtriangular.Interhyal ossifi ed, cylindrical, attached to posterior end of posterior ceratohyal.Urohyal thin, deep, with short ventrolateral fl ap; anterodorsal process minute or absent.
Second pharyngobranchial approximately triangular, small, wider than long, lacking teeth.Third pha ryngo branchial wide, with well-developed dentigerous plate containing large conical, slightly curved teeth.Fourth pharyngobranchial tooth plate with numerous small conical teeth.Epibranchials 2-3 and inter arcual cartilage narrow and long, epibranchial 1 slightly shorter with proximal portion widened, fourth epibranchial longer and wider.Second epibranchial with pronounced triangular subdistal process.Third epi branchial with long uncinate process directly connected to fourth epibranchial by ligaments.Interarcual cartilage laterally attached to anterior tip of second pha ryngo branchial and to proximal portion of 1st epibranchial.

Vertebrae and caudal-fi n skeleton
Figs. 4A -D Neural spine of 1st vertebra with long anterior laminar extension, and short neural pre-and post-zygapophyses.Second neural spine wider than fi rst and second, longer than fi rst and so long as third; vertebrae posterior to third vertebra with rod-like neural spine.Neural prezygapophyses of vertebrae 2 and 3 long, neural postzygapophyses short.Pleural ribs on all precaudal vertebrae except the fi rst; epipleural ribs narrow, gradually widening and fl attening distally.Neural prezygapophyses of anterior caudal vertebrae long; neural postzygapophyses of caudal vertebrae short.Hemal spine of preural centrum 2 approximately so wide as hemal spines of anterior vertebrae.Hemal prezygapophysis of preural centrum 2 lengthened, directed ventrally.One or two pointed short processes on dorsal surface of compound caudal centrum.Epural and parhypural similar in shape, laminar, with proximal portion pointed.Hypurals ankylosed except for median horizontal gap to form two triangular plates without vestige of former hypural limits.Accessory caudal cartilages absent.

Figs. 5C -D
Dorsal-fi n origin between neural spines of vertebrae 13 and 15; anal-fi n origin between pleural ribs of vertebrae 14 and 15.Two rays associated to fi rst proximal radial of dorsal fi n, and two or three associated to fi rst proximal radial of anal fi n.Proximal radials narrow.Median and distal radials ossifi ed.

Phylogenetic analysis
Characters found to have informative variability to erect the phylogenetic hypothesis among nothobranchiids lineages are listed below.Distribution of character states among terminal taxa is presented in the data matrix (Table 1).The most parsimonious cladogram of phylogenetic relationships among 11 terminal nothobranchiid taxa is illustrated in Fig. 6.
Annualism is a unique style of life cycle among teleost fi shes, known to occur in African and South American aplocheiloids.The ability in to complete the entire life cycle in temporary pools implicates in the acquisition of a set of advanced characters, including behavior (bottom spawning), egg structure (thickened chorion), embryonic development (diapauses),

Discussion
The phylogenetic analysis of osteological characters support clades previously defi ned by molecular data (MURPHY & COLLIER, 1997, 1999;MURPHY et al., 1999), such as the clades comprising Callopanchax, Scriptaphyosemion and Archiaphyosemion, and Aphyosemion plus Fundulopanchax (Fig. 7).The clade comprising Callopanchax, Scriptaphyosemion and Archiaphyosemion, three sympatric genera endemic to western Africa, is here supported by the derived shape of the antero-proximal process of the fourth ceratobranchial, which is laterally displaced to form a wide lateral fl ap (Fig. 3C; character: 16.1).The clade comprising Aphyosemion and Fundulopanchax, two genera from central-western Africa, is corroborated by the expanded hypobranchials, which occupy most interspace areas (Fig. 3G; ch: 15.1).
The proposed sister group relationship between Callopanchax and Scriptaphyosemion (MURPHY & COLLIER, 1997;MURPHY et al., 1999) is highly supported by fi ve synapomorphies: subdistal portion of the posterior arm of the parasphenoid distinctively constricted (Fig. 1B; ch: 2.1), anterior margin of the hyomandibula with a distinct convexity (Fig. 2B; ch: 10.1), cartilaginous portion of the basihyal rectangu- and physiology (rapid growth rate), making annualism a complex life style (e. g., MURPHY & COLLIER, 1997;COSTA, 1998b).In a fi rst phylogenetic approach (PARENTI, 1981), annualism was considered as having a double origin from non-annual aplocheiloid ancestors, arising independently once in South American and once in African aplocheiloids.Evidence supporting two independent origins of annualism among South American aplocheiloids was found by COSTA (1998b) and HRBEK & LARSON (1999), but a third independent annual Neotropical lineage has been recently reported (COSTA, 2006c).
Parenti's African annual clade comprised both Nothobranchius, a strict annual fi sh genus, and Fundulopanchax (including Callopanchax as subgenus), which contained both typical annual and semi-annual fi shes (i.e. facultative annuals).However, under a new phylogenetic tree topology (MURPHY & COLLIER, 1997, 1999;MURPHY et al., 1999), two alternative explanations for the evolution of annualism in African aplocheiloids may be provided.It would be equally parsimonious to assume three independent origins in the three annual fi sh genera (i.e., Callopanchax, Nothobranchius and Fundulopanchax; DELTRAN option), or two independent origins, once in Callopanchax and once in the base of the clade Nothobranchius + Fundulopanchax + Aphyosemion, with a subsequent loss in Aphyosemion (ACCTRAN option, Fig. 7B).Among these two alternative hypotheses, the cladogram topology derived from the analysis of osteological features parsimoniously supports the hypothesis of a triple annualism origin, once in each annual fi sh genus (Fig. 7A).
Tab. 1. Matrix of 26 osteological characters for 13 aplocheiloid taxa.Character and character states are numbered according text.