osteological analysis of the killifish genus cynolebias (cyprinodontiformes: rivulidae)

Osteological Analysis of the Killifish Genus Cynolebias(Cyprinodontiformes: Rivulidae)

MARCELO LOUREIRO* AND RAFAEL O. DE SADepartment of Biology, University of Richmond, Richmond, Virginia

ABSTRACT Relationships among the species of the annual fish Cynolebiasare unclear. An analysis of the variation and utility of osteological charactersfor phylogenetic analysis was done using cleared and double-stained speci-mens representing 21 species of Cynolebias. This analysis showed that someof the characters previously used to diagnose this genus and some of thespecies are polymorphic. Osteologically, Cynolebias can be diagnosed by thefollowing synapomorphies: (1) triangular-shaped parietal, (2) vomer posi-tioned ventral to the parasphenoid, (3) long ventral process of the dentary, (4)teeth on fourth ceratobranchial, and (5) teeth on first epibranchial. In addi-tion, characters that help define some of the currently recognized speciescomplexes were identified. Species in the ‘‘antenori complex’’ share at leastfive synapomorphies, such as ossified medial radials of dorsal and anal fins,four pectoral radials, ventral process of the maxillae enlarged, mesopterygoidlong relative to the autopalatine, and proportion of cartilage in the basihyal.The ‘‘bellottii complex’’ is characterized by having a reduce basihyal and a deepurohyal, whereas species in the ‘‘elongatus complex’’possess a caudal fin supportedby four vertebrae and have unique modifications of jaw bones. The followingosteological features are useful as diagnostic characters at the specific level: (1) Twovertebrae supporting the caudal fin (C. nigripinnis); (2) cartilaginous pelvic bones(C. notatus); (3) short third postcleithrum and broad lateral process of the sphen-otic (C. wolterstorffi); (4) thick third postcleithrum (C. gymnoventris); (5) crest onthe parietal and reduced in the upper portion of the lacrimal (C. whitei); (6)anteriorly curved lacrimal (C. cheradophilus); (7) second dermosphenotic (C. bellot-tii); and (8) expansion of the ventral tip of the maxillae and long basyhial(C. constanciae). J. Morphol. 238:245–262, 1998. r 1998 Wiley-Liss, Inc.

KEY WORDS: Cynolebias; synapomorphies; species complexes

The genus Cynolebias belongs to the fam-ily Rivulidae (Order Cyprinodontiformes)and possesses a characteristic annual lifecycle. This life cycle is shared by most of thespecies in the family Rivulidae and withsome species of the African family Aplochei-lidae. This unique life cycle for vertebratesincludes drought-resistant eggs laid by theadults in the substrate of the temporaryponds they inhabit. Embryos develop andsurvive dry periods between wet seasons byundergoing diapause (Wourms, ’72). Subse-quently, larvae will complete developmentand hatch after the ponds are filled by heavyrains. Juveniles can reach maturity as earlyas 2 months after hatching.

The first phylogenetic and biogeographicanalysis of the order Cyprinodontiformes was

done by Parenti (’81), who provided uniquelyderived characters to define the family Rivu-lidae. In addition, she provided two synapo-morphies for Cynolebias: (1) caudal fin notscaled, and (2) preopercular canal closed.Furthermore, she included Cynopoecilus,Leptolebias, Simpsonichthys, Campellole-bias, and Terranatos in the synonymy ofCynolebias. A more comprehensive study ofthe family Rivulidae was done by Costa (’90),who recognized Cynopoecilus, Leptolebias,

Contract grant sponsor: Graduate Research Committee, Uni-versity of Richmond.

M. Loureiro is currently at the Facultad de Ciencias, SeccionVertebrados, Igua 4225, Montevideo 11400, Uruguay.

*Correspondence to: Marcelo Loureiro, Facultad de Ciencias,Seccion Vertebrados, Igua 4225, Montevideo 11400, Uruguay.

JOURNAL OF MORPHOLOGY 238:245–262 (1998)

r 1998 WILEY-LISS, INC.

Campellolebias, Terranatos, ’’Cynolebias’’ (5Plesiolebias, Costa ’91), and Cynolebias asmonophyletic lineages and grouped theminto a monophyletic subfamily, Cynolebiati-nae. In the analysis of Cynolebias, Costa(’90) proposed five generic synapomorphies:(1) males with more rays in the dorsal finthan females; (2) juveniles with a dark spoton each side in the middle of the body; (3) atleast 16 neuromast in the supraorbital se-ries; (4) preanal length representing 55% ofthe standard length; and (5) postero-lowerprocess of dentary elongated. A phylogeneticanalysis of the subfamily was done by Costa(’95), in which he proposed three autapomor-phies for Cynolebias: (1) male with moredorsal fin rays than female; (2) female withblack blotch on center of body sides; and (3)anal fin base of males enlarged. Species ofCynolebias have been clustered in ‘‘speciesgroups’’ by Amato (’86) and in ‘‘species com-plexes’’ by Costa and Brasil (’90, ’93) (Table

1), whereas interspecific relationships wereanalyzed by Costa (’95).

Reports on the characteristics of isolatedbones for various species are available in theliterature; however, the complete osteologyhas not been previously described for anyspecies of Cynolebias. The present study hasthree goals: first, to provide a complete osteo-logical description of Cynolebias luteoflamu-latus that could serve as baseline for com-parative purposes; second, to describe someof the inter- and intraspecific osteologicalvariation present in Cynolebias; and third,to analyze the utility of osteological charac-ters for phylogenetic analyses of the genusCynolebias as was defined by Costa (1995).

MATERIALS AND METHODS

Specimens of Cynolebias used in this studywere in part collected in the field in Uruguayand in part borrowed from Facultad de Cien-cias, Montevideo, Uruguay, with the restobtained in the pet trade (see the Appendix).

For osteological analysis specimens werecleared and double-stained for cartilage andbone following Dingerkus and Uhler (’77).Specimens were observed under a dissectingmicroscope (Leica Wild M3C), and drawingswere made with a camera lucida attached tothe microscope. Bone and cartilage terminol-ogy follows that of Parenti (’81) and Costa(’90).

RESULTSOsteological description

of Cynolebias luteoflamulatusVertebrae

The number of vertebrae varied between27 and 32 (x 5 30, sd 5 1, m 5 30). The firstvertebra lacks neural prezygapophyses andpleural ribs but has a laterally compressedneural spine (Fig. 1A). The second vertebraalso has a laterally compressed neural spine.The first pleural rib originates from the sec-ond vertebra. Neural prezygapophyses arepresent on the abdominal vertebrae. Thesestructures are larger on the most anteriorvertebrae, decreasing posteriorly. The neu-ral prezygapophysis is one fourth the heightof the neural spine. The caudal vertebraehave reduced prezygapophyses (Fig. 1C).

Caudal finThe caudal fin is homocercal and rounded.

The skeleton of the caudal fin is almostperfectly symmetrical due to the fusion ofthe hypural plates into a single fan-shapedstructure flanked by a dorsal free epural and

TABLE 1. Species complexes of Cynolebias(Costa and Brasil, ’90, ’93)

Plesiomorphic species Antenori complexC. constanciae1

C. whitei 1

C. myersi 1

C. izecksoni

C. fulminantisC. antenoriC. flavicaudatus1

C. flammeus1

C. magnificusSpecies related

C. stellatusC. alternatusC. notatus1

C. bokermanni 1

C. boitonei 1

C. zonatusC. chacoensis1

C. hellneriC. santanae

Bellottii complex Elongatus complexC. affinis1,2

C. alexandri 1,2

C. cyaenus1,2

C. gymnoventris1,2

C. luteoflamulatus1,2

C. nigripinnis1,2

C. adloffi 1,3

C. bellottii 1,3

C. viarius1,3

C. cinereus3

C. carvalhoi 3

Species relatedC. melanoorusC. vazferreiraiC. nonoiuliensisC. costaiC. duraznensis1

C. elongatusC. prognathusC. cheradophilus1

C. wolterstorffi 1

C. holmbergiPorosus complex

C. porosusC. albipunctatusC. griseusC. leptocephalusC. perforatus

Unknown relationsC. trilineatus

1Species used in this analysis.2Species of luteoflamulatus group.3Species of adloffi group (Amato, ’86). Species related accordingto Wildekamp (’95).

246 M. LOUREIRO AND R.O. DE SA

a ventral free parhypural. The last threevertebrae, and their corresponding neuraland hemal spines articulate with the finrays participating in the support of the cau-dal fin (Fig. 1L).

Dorsal and anal finThe proximal radials of dorsal and anal

fins are ossified and elongated, except for

their ventral tips, which are cartilaginous(Fig. 1E,I), whereas the medial radials areshort and cartilaginous. Each radial sup-ports one ray, except the first and the lastray of both fins that support two rays each.The first and second proximal radials of analand dorsal fins are partially or completelyfused into a single element. The distal radi-als of both fins are small cartilaginous ele-

Fig. 1. Lateral view of first and second vertebrae(ribs have been removed) of (A) Cynolebias luteoflamula-tus; (B) Cynolebias viarius. Lateral view of caudal verte-brae of (C) Cynolebias luteoflamulatus; (D) Cynolebiasbokermanni. Lateral view of first and second radials ofdorsal fin of Cynolebias luteoflamulatus (E). Lateralview of second radials of dorsal fin of (F) Cynolebiasconstanciae; (G) Cynolebias flavicaudatus; (H) Cyno-lebias bokermanni. Lateral view of first and secondradials of anal fin of (I) Cynolebias luteoflamulatus;

(J) Cynolebias notatus; (K) Cynolebias chacoensis. Lat-eral view of caudal skeleton of Cynolebias luteoflamula-tus (L). da, distal radial of anal fin; dd, distal radial ofdorsal fin; eh, epural; hp, hypural plate; hs, hemalspines of vertebrae; ma, medial radial of anal fin; md,medial radial of dorsal fin; ns, neural spine; pa, proximalradial of anal fin; ph, parhypural; poz, neural postzygapo-phisis; pz, neural prezygapophisis; 1pd, first proximalradial of dorsal fin; 2pd, second proximal radial of dorsalfin. Shaded area represents cartilage. Scale bars 5 1mm.

OSTEOLOGY OF CYNOLEBIAS 247

ments found at the base of the fin rays. Theproximal radials and the rays of the anal finare thicker in females than in males andtherefore appear closer to each other in fe-males. Females have reduced medial anddistal radials.

Pectoral fin and pectoral girdle (Fig. 2A)The pectoral girdle articulates with the

skull through the supracleithrum and thepost-temporal bones. The post-temporal ar-ticulates with the epiotic in the skull. Thepost-temporal bone is thin, elongated, andpartially fused to a flattened supraclei-

thrum; these two bones are about equal inlength. The post-temporal has a ventral pro-cess that points anteriorly. The first andsecond postcleithral bones are absent. Be-tween the girdle and the first pleural rib,there is a thin, elongated, and free thirdpostcleithrum bone. The third postcleithraeis slightly curved on its ventral end and is asthick as the first pleural rib. The main bodyof the girdle is formed by the cleithrum,scapula, coracoid, and pectoral radials. Thecleithrum is curved and tall, extending fromthe ventral region of the body to the origin ofthe neural spine of the second vertebra. The

Fig. 2. Lateral view of pectoral girdle of Cynolebiasluteoflamulatus (A). Lateral view of posttemporal andsupracleithrum of (B) Cynolebias bellottii, (C) Cynole-bias wolterstorffi. Anal fin ray of Cynolebias cheradophi-lus (D). Pectoral fin ray of Cynolebias luteoflamulatus(E). Ventral view of pelvic bones of Cynolebias luteofla-

mulatus (F). ap, autopterotic; ch, cleithrum; co, coracoid;e, epiotic; es, scapula; fr, first pleural rib; pt, posttempo-ral; r, pectoral radials; sl, supracleithrum; tpc, thirdpostcleithrum. Symbols: big arrow, posttemporal spine;small arrow, ray protuberances. Shaded area representscartilage. Scale bar 5 1mm.


ventral tip of the cleithrum extends belowthe level of the ventral edge of the coracoid.The cleithrum has anterior and posteriorflat expansions. The scapula is rounded andhas a notch on its anterior margin. Thecoracoid is also rounded with a posteriorspine-like projection. There are threerounded pectoral radials. However, in oneindividual examined, there are four pectoralradials. The pectoral rays of males havesmall and ossified projections arising fromthe internal side of the pectoral fin (Fig. 2E).Those projections are on rays 1–5. Thescapula and radials articulate with the clei-thrum and coracoid through a cartilaginousplate. Right and left cleithra articulate with

each other ventrally. The main axis of thepost-temporal–supracleithrum complex formsa 45° angle with the main axis of the dorsaledge of the cleithrum.

Pelvic fin and pelvic girdleThe two halves of the pelvic girdle do not

overlap due to reduction of the medial pro-cess (Fig. 2F). Each bone is posteriorly ex-panded and lacks the ischial process. Rayssupporting the pelvic fins articulate directlywith each one of these bones.

NeurocraniumDorsal view (Fig. 3A). The nasals are

paired broad bones, forming an approxi-

Fig. 3. Dorsal view of the neurocranium of Cynole-bias luteoflamulatus (jaws have been removed) (A). Dor-sal view of supraoccipital of (B) Cynolebias luteoflamula-tus, (C) Cynolebias whitei, (D) Cynolebias bellottii.Lateral view of the hyomandibular-skull articulation of(E) Cynolebias luteoflamulatus, (F) Cynolebias whitei,(G) Cynolebias wolterstorffi, (H) Cynolebias bellottii. ap,

autopterotic; dm, dermosphenotic; e, epiotic; ep, eth-moid process; ex, exoccipitals; f, frontal; h, hymandibu-lar; n, nasal; p, parietal; pk, parietal crest; pt, posttempo-ral; s, supraoccipital; sd, ‘‘second dermosphenotic’’; sh,sphenotic; shp, sphenotic process; sx, suboccipital. Sym-bol: arrow, supraoccipital spine. Shaded area representscartilage. Scale bars 5 1mm.


mately 45° angle with the horizontal plane.Nasals and frontals do not contact each otherand are separated by cartilaginous ethmoidprocesses. The upper tip of this process istriangular in shape and is placed anteriorand lateral to the frontals. The frontals arebroad rectangular bones. They are trun-cated anteriorly and extend from the nasalto the otic region. Laterally, the frontals foldinward into the orbital region and form ir-regular-shaped lateral projections. The su-

praoccipital is a single triangular shapedbone found posterior of the frontals. Thesupraoccipital has an anterior triangular-shaped projection that underlies the fron-tals, and a dorsal posterior process. Theposterior process, the supraoccipital spine,is formed by two parallel ‘‘wings’’ (Figs. 3A,B,4A). They are connected to each other by atransverse bony bridge.

The sphenotic (Figs. 3A,E, 4A) articulateswith the anterior arm of the hyomandibular.

Fig. 4. Lateral view of the skull Cynolebias luteofla-mulatus (A). Lateral view of the lacrimal of (B) Cynole-bias luteoflamulatus, (C) Cynolebias whitei, (D) Cynole-bias constanciae, (E) Cynolebias cheradophilus. Ventralview of vomer and parasphenoid of (F) Cynolebias luteo-flamulatus, (G) Cynolebias cyaenus, (H) Cynolebiaswolterstorffii. Ventral view of the neurocranium ofCynolebias luteoflamulatus (I). a, autopalatine; aa, angu-loarticular; ap, autopterotic; av, anterior process of thevomer; b, basihyal; c, ceratohyal; d, dentary; e, epiotic;ep, ethmoid process; ex, exoccipital; f, frontal; h, hyoman-dibular; io, interopercular; l, lacrimal; le, lateral eth-

moid; m, maxilla; ms, mesopterygoyd; mt, metaptery-goyd, n, nasal; o, opercular; p, parietal; pc, prootic; ph,parasphenoid; pm, premaxilla; po, preopercular; pt, post-temporal; pv, posterior process of the vomer; q, quad-rate; r, retroarticular; s, supraoccipital; sn, sphenotic;snn, sphenotic process; so, subopercular; ss, supraoccipi-tal spine; sx, suboccipital; sy, symplectic; v, vomer; vt,vomer tooth. Symbols: big arrow, orbital artery foramen;small arrow, trigeminofascialis recess; thick arrow, pro-cessus ascendens of the parasphenoid. Shaded area rep-resents cartilage. Scale bars 5 1mm.


The lateral process of the sphenotic pro-trudes from the skull, directed ventrally andposteriorly. The parietal is variably present;if present, it is triangular shaped (Figs. 3A,E,4A) and partially covers the frontal, thesphenotic, the autopterotic, and the epiotic.Cynolebias luteoflamulatus lacked parietalsin two of the examined individuals, whilethe parietals in one specimen were reduced.The autopterotic articulates with the poste-rior arm of the hyomandibular and with theepiotic. The back of the skull comprises theexoccipitals, which articulate with the firstvertebra through two occipital condyles, andthe basioccipital, which articulates with thecentrum of the first vertebra.

Ventral view (Fig. 4I). In the ventral view,the skeletal elements on the midline axis(from anterior to posterior) are: vomer, ros-tral cartilage, paired lateral ethmoids, para-sphenoid, and basioccipital. The vomer isoverall rhomboid shaped with posterior andanterior processes. The anterior edge of thevomer is thick (Fig. 4F,I). A single tooth islocated on the anterior process of the vomerin four of the individuals examined (Fig. 4F).The posterior process of the vomer lies ven-tral of the anterior arm of the parasphenoid.The lateral ethmoid expands anteriorly, ar-ticulating with the vomer and with the ante-rior arm of the parasphenoids. It also has anexpanded dorsal process that articulateswith the autopalatine anteriorly. The para-sphenoid is long, extending from the nasalregion to the back of the skull, and has twolateral projections on its medial region (pro-cessus ascendens of the parasphenoid), whichare triangular shaped. Medially the para-sphenoid is expanded laterally (Fig. 4F, I).

Otic region (Fig. 4I). Lateral to the me-dial series are the following bones: prootic,sphenotic, autopterotic, and exoccipital (allpaired bones). The prootic articulates later-ally with the sphenotic (anteriorly) and theautopterotic (posteriorly); posteriorly withthe basioccipital, and medially with the para-sphenoid. Two pairs of fenestrae are foundin the prootic, the larger one carries theorbital artery and the smaller one the tri-geminofascialis recess.

Orbital rimThe dorsal bones of the orbital rim are

attached to the skull. The infraorbital bonesare represented by the lacrimal, a free bonelocated anteriorly. The lacrimal overlaps the

medial portion of the maxilla laterally. Thelacrimal is a narrow and elongate bone, itsupper portion twisted in one direction andventrally on the opposite direction, the ven-tral tip of this bone is rounded (Fig. 4A,B).The dermosphenotic is absent.

Jaw structureThree bones are considered dorsal ele-

ments: premaxillae (paired), maxillae(paired), and rostral cartilage (single). Thepremaxillae have an ascending process thatextends dorsally and posteriorly. This pro-cess is flat and broad and covers the rostralcartilage in its anterior portion (Fig. 5A).The alveolar arm of the premaxilla is anteri-orly expanded. The maxillae are thin andperpendicular to the main body axis (Fig.4A). Anteriorly, the maxillae have dorsaland ventral processes (Fig. 5E). The ventralprocess is concave posteriorly. It is locatedbelow the ascending process of the premaxil-lae and above the anterior portion of therostral cartilage. The rostral cartilage has adiscoidal shape (Fig. 5A). In large adults, itmay be elongate and constrained medially.The rostral cartilage covers the anterior re-gion of the vomer and it is free, allowing forthe protrusion of the upper jaw.

There are four ventral elements: dentary,Meckel’s cartilage, anguloarticular, and ret-roarticular (all paired). The dentary pos-sesses an elongated ventral process and ar-ticulates posteriorly with the anguloarticular(Fig. 5I). The anguloarticular bone has twoanterior processes: a medial (large) and aventral (small). The retroarticular is small,located below and posteriorly to the angulo-articular. Anguloarticular and retroarticu-lar articulate with the quadrate. Meckel’scartilage runs between the dentary and theanguloarticular along the length of the man-dible.

Jaw suspensorium (Fig. 5L)The jaw suspensorium consists of the auto-

palatine, mesopterygoid, quadrate, symplec-tic, metapterygoid, and hyomandibular. Theautopalatine is elongate and covers part ofthe mesopterygoid; the latter is slender andcurved. The autopalatine and mesoptery-goid articulate with the quadrate ventrally.The mesopterygoid is about the same size ofthe autopalatine (Figs. 4A, 5L). The quad-rate is triangular shaped and has a smallanterior process that articulates with theanguloarticular and the retroarticular. The


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quadrate possesses a thick, elongate, poste-rior process that articulates with the sym-plectic, which is partially overlapped by thequadrate. The length of the posterior pro-cess of the quadrate is equal to the boneitself without the process. Metapterygoid andmesopterygoid are separated by cartilage.The metapterygoid bone is elongated andslightly curved and contacts the symplecticventrally. The symplectic is long, partiallycovers the metapterygoid, and has a poste-rior cartilaginous articulation to the hyoman-dibular. The hyomandibular is a Y-shapedbone; the upper arms articulate with thesphenotic anteriorly and with the autopter-otic posteriorly. The ventral arm of the hyo-mandibular articulates with the symplecticanteriorly and with the interhyal posteri-orly.

Opercular series (Fig. 4A)The opercular is triangular shaped and

articulates with the hyomandibular anteri-orly. In the place of articulation the opercu-lar has a small spine. The upper portionarticulates with the hyomandibular and theopercular bones. The horizontal segment islaterally compressed and partially coversthe interopercular. The interopercular isrounded and has a thick dorsal margin(Fig. 5L).

Gill archesVentral components (Fig. 6A). The inter-

hyal is cartilaginous. The basihyal is trap-ezoidal or triangular shaped. Laterally, thebasihyal articulates with the ventral and

dorsal hypohyals. Ventrally, it articulateswith the urohyal. Posteriorly, it articulateswith the first basibranchial. The ratio basi-hyal length/ventral hypohyal length is threeto one (Fig. 6B). The thickness of the basi-hyal is about 50% of its length. The basihyalconsists of two parts, a posterior ossifiedcomponent and a cartilaginous anterior one.The proportion of cartilage and bone is ap-proximately one to two.

There are three ossified basibranchials.The hypobranchials are square shaped.There are five ceratobranchials, all bearingteeth. The first, second, and third have tworows of teeth, the fourth has one row, andthe fifth is fully covered with teeth. The firstceratobranchial articulates with the first epi-branchial and the interarcual cartilage dor-sally. The second, third, and fourth cerato-branchials articulate with the second, third,and fourth epibranchials, respectively. Thefourth ceratobranchial articulates with a tri-angular-shaped cartilage ventrally. The lat-ter is located posteriorly to the third basi-branchial. The fifth ceratobranchial istriangular shaped, has a ventral process,and does not articulate with any epibran-chial. The urohyal bone is laterally com-pressed, is found ventral to the basihyal,and has an anterior and dorsally directedprocess. The height of the urohyal is 25% ofits length (Fig. 6H). Ventral and dorsal hypo-hyals articulate with a long ceratohyal. Theceratohyal is divided in two (Fig. 6N). Theanterior half is slender and has a posterior,expanded, and deep process, while the poste-rior half is deep in all its length. The ante-rior half supports two branchiostegal rays in50% of the specimens examined, whereas inthe other 50% it supports three branchioste-gal rays. The deep process of the anteriorhalf supports two rays. The posterior half ofthe ceratohyal supports one branchiostegalray, and articulates posterodorsally with acartilaginous interhyal. The processes of theanterior half and the posterior half of theceratohyal are embedded in a cartilaginousplate. This plate supports another branchio-stegal ray between the two portions.

Dorsal components (Fig. 6O). The firstepibranchial is the only element of this se-ries that possesses teeth. There are two teethon this element. The interarcual cartilageand the second epibranchial articulate medi-ally with pharyngobranchial 2. The thirdepibranchial articulates with pharyngobran-chial 3 and the fourth epibranchial articu-

Fig. 5. Dorsal view of the premaxillae and rostralcartilage of (A) Cynolebias luteoflamulatus, (B) Cynole-bias myersi, (C) Cynolebias chacoensis, (D) Cynolebiaswolterstorffi. Dorsal view of maxillae of (E) Cynolebiasluteoflamulatus; (F) Cynolebias myersi; (G) Cynolebiascheradophilus; (H) Cynolebias chacoensis. Lateral viewof lower jaw of (I) Cynolebias luteoflamulatus, (J) Cynole-bias constanciae, (K) Cynolebias cheradophilus. Lateralview of jaw suspensorium of Cynolebias luteoflamulatus(L). Lateral view of interopercular of Cynolebias boker-manni (M). Lateral view of anterior jaw suspensorium ofCynolebias wolterstorffi (N). a, autopalatine; aa, alveolararm of the premaxilla; aae, expansion of the alveolararm; am, ascending process of the premaxillae; ao, angu-loarticular; d, dentary; da, dorsal process of the angulo-articular; dm, dorsal process of the maxilla; h, hyoman-dibular; io, interopercular; ma, medial process of theanguloarticular; ms, mesopterygoid; mt, metaptery-goyd; q, quadrate; rc, rostral cartilage; sy, symplectic;va, ventral process of the anguloarticular; vd, ventralprocess of the dentary; vm, ventral process of the max-illa. Shaded area represents cartilage. Scale bar 5 1 mm.


lates with pharyngobranchial 4. Third andfourth epibranchials also articulate witheach other medially. The fourth epibranchialis the thickest. The pharyngobranchials ar-ticulate with each other and are covered

with teeth ventrally. The first pharyngobran-chial is absent. The third pharyngobran-chial is the largest and lies dorsal to thesecond and the fourth pharyngobranchials,covering them partially.

Fig. 6. Dorsal view of ventral gill arch of Cynolebiasluteoflamulatus (A). Ventral view of basihyal of (B)Cynolebias luteoflamulatus, (C) Cynolebias constanciae,(D) Cynolebias wolterstorffi, (E) Cynolebias cheradophi-lus, (F) Cynolebias whitei, (G) Cynolebias affinis. Lat-eral view of urohyal of (H) Cynolebias luteoflamulatus,(I) Cynolebias constanciae, (J) Cynolebias whitei, (K)Cynolebias nigripinnis, (L) Cynolebias cheradophilus,(M) Cynolebias bokermanni. Lateral view of ceratohyalof Cynolebias luteoflamulatus (N). Dorsal view of dorsalgill arch of Cynolebias luteoflamulatus (O). ac, anterior

ceratohyal; b, basihyal; bb, ossified basihyal; bc, basi-branchials; br, branchiostegal rays; c, ceratobranchials1, 2, 3, 4, 5; cb, cartilaginous basihyal; dhh, dorsalhypohyal; eb, epibranchials 1, 2, 3, 4; et, epibranchialteeth; h, hypobranchials; ic, interarcual cartilage; ii,interhyal; pc, posterior ceratohyal; ph, pharyngobranchi-als 2, 3, 4; vhh, ventral hypohyals. Symbols: big arrow,interhyal; small arrow, indentation of urohyal; thickarrow, anterior process of urohyal. Shaded area repre-sents cartilage. Scale bars 5 1mm.


TeethTeeth are present on the premaxillae and

the dentaries. The premaxillae has four rowsof conical teeth. These teeth are all equal insize, except those on the first row, which hasfour larger, medial teeth. The dentaries alsohave four rows of conical teeth, all equal insize, except for four teeth that are located atthe front and are three times the size ofothers. The gill arches teeth are also conical.The teeth of the ceratobranchials are largeron the first ceratobranchial, gradually de-creasing posteriorly to the fourth ceratobran-chial. Then, on the fifth ceratobranchial, theteeth increase in size again. The vomer tooth,when present, it is oriented anteriorly(Fig. 4B).

Osteological comparisons among speciesof Cynolebias

VertebraeNeural prezygapophyses are present in

the abdominal vertebrae in most species ex-amined; however, they are absent in Cynole-bias cheradophilus, C. myersi, and C. wolter-storffi. Abdominal vertebrae of C. bellottii,C. boitonei, and C. viarius have well-devel-oped neural prezygapophyses that are one-half the height of the neural spines (Fig. 1B).In other species, the neural prezygapophysisare only one-fourth the height of the neuralspines (e.g., C. luteoflamulatus, Fig. 1A).Neural prezygapophyses of the caudal verte-brae are absent in C. bokermanni, C. cher-adophilus, C. constanciae, C. myersi, andC. wolterstorffi (Fig. 1D). Other species pre-sent reduced neural prezygapophyses in thecaudal vertebrae.

Caudal finMost species have a caudal fin similar to

that described for Cynolebias luteoflamula-tus. In C. myersi and C. nigripinnis, thecaudal fin is supported only by the two lastvertebrae, whereas in C. cheradophilus andC. wolterstorffi, the support is given by thelast four vertebrae.

Dorsal finThe proximal radials have lateral flat

flanges in Cynolebias constanciae (Fig. 1F).The medial radials are short and variablyossified. In C. chacoensis and C. constanciae,they are completely ossified, whereas inC. flavicaudatus, C. notatus, and C. whitei,only the central region of the medial radialsis ossified (Fig. 1G). In C. bokermanni and

C. myersi, the medial radials remain carti-laginous only in their ventral tip (Fig. 5H).In other species examined the medial radi-als are similar to those of C. luteoflamula-tus.

Anal finThe medial radials are completely carti-

laginous in most species, except in Cynole-bias bokermanni, C. constanciae, and C. no-tatus, in which a center of ossification isfound at the center of the bone (Fig. 1J), andin C. chacoensis where the medial radialsare completely ossified (Fig. 1K). Fin rays ofmales of C. cheradophilus have small, spine-like, ossified projections (Fig. 2D).

The posterior rays of anal and dorsal finsof males of Cynolebias boitonei, C. boker-manni, C. constanciae, C. chacoensis, C. fla-vicaudatus, and C. whitei are elongated, withtheir distal fourth cartilaginous. Cynolebiaschacoensis has a single, elongated, anal raythat extends beyond the posterior margin ofthe caudal fin.

Pectoral fin and pectoral girdlePost-temporal and supracleithrum bones

are of the same length in all species exceptin Cynolebias boitonei, in which the post-temporal is shorter. In C. bellottii, C. boker-manni, C. duraznensis, C. flavicaudatus,C. viarius, and Cynolebias sp., the post-temporal ventral process is long and reachesthe back of the skull (Fig. 2B), whereas inC. boitonei, C. myersi, C. whitei, andC. wolterstorffi, this process is absent (Fig.2C). In other species, the process is similarto C. luteoflamulatus. Cynolebias wolter-storffi has the shortest third postcleithrum,less than one-half the height of the clei-thrum. In other species, the third postclei-thrum is about 75% of the cleithrum height.In C. gymnoventris, he postcleithrum isthicker than the rib, whereas in the rest ofthe species postcleithrum and rib have thesame thickness. The dorsal tip of the postclei-thrum of C. bellottii can be fused to the firstpleural rib (fused in 15 of the examinedspecimens). The number of pectoral radialsis variable. Cynolebias boitonei, C. boker-manni, C. chacoensis, C. constanciae, C. fla-vicaudatus, C. myersi, C. notatus, and C.whitei have four rounded, pectoral radialswhereas C. cheradophilus, C. gymnoventris,C. wolterstorffi, and Cynolebias sp. havethree, rounded, pectoral radials. The latterspecies have lost the upper radial. In the


other species, the number of radials variedbetween three and four among individuals.The percentage of individuals examinedhaving four radials was: C. alexandri 5 10%,C. affinis 5 20%, C. bellottii and C. nig-ripinnis 5 45%, C. cyaenus and C. duraz-nensis 5 25%. In these species with a vari-able number of radials, the radials arerounded as in the species with three pecto-rals. Like C. luteoflamulatus, the pectoralrays of males of C. boitonei, C. cheradophi-lus, and C. whitei have small, ossified projec-tions on the internal side of the pectoral fin.Similar structures were found on the analfin of C. cheradophilus. The arrangement ofthese projections is species specific. InC. boitonei, they are present on rays 1, 2,and 3 (counting dorsal to ventral), inC. whitei on rays 1–6, and in C. cheradophi-lus on rays 1 through 11. Species with fourpectoral radials have a more curved clei-thrum, with a well-developed posterior pro-jection that covers the dorsal half of thescapula. In these species, the scapula is leastrounded.

Pelvic fin and pelvic girdleMost species of Cynolebias have ossified

pelvic bones, but the posterior three-fourthsof the bone are cartilaginous in C. wolter-storffi, and in C. notatus the bones are com-pletely cartilaginous. Cynolebias boitonei isunique among Cynolebias species because itlacks pelvic girdle and pelvic fins.

NeurocraniumDorsal view. The ‘‘wings’’ of the supraoc-

cipital spine can be completely separatedfrom each other or they can be dorsally freebut joined at their origin. They are free inCynolebias myersi and C. whitei (Fig. 3C).They are joined at the origin in C. boitonei,C. bellottii, C. cheradophilus, C. viarius, andC. wolterstorffi (Fig. 3D). Other species ex-hibit a condition similar to that of C. luteofla-mulatus.

The lateral process of the sphenotic issimilar to the condition in Cynolebias luteo-flamulatus in most species examined; how-ever, in C. bellottii, C. cheradophilus, andC. viarius, the process points ventrally andanteriorly, and in C. wolterstorffi the processhas a broad edge (Fig. 3G). The parietal ofC. affinis is fused to the autopterotic, whereasin C. bokermanni it fuses with the epiotic.Cynolebias whitei has a parietal crest thatruns from the anterior to the ventral apicesof this bone (Fig. 3F). The parietal presents

two states in C. viarius; it is reduced in fiveof the individuals and not reduced in therest. Cynolebias wolterstorffi has a reducedand circular-shaped parietal in the onlyspecimen examined (Fig. 3G). In other spe-cies the parietal is similar to that ofC. luteoflamulatus. Parietals are absent inC. cheradophilus and C. constanciae. Thebones of the posterior and dorsal region ofthe skull are mostly separated from eachother in Cynolebias; however, in C. boitonei,C. bokermanni, C. chacoensis, C. flavicauda-tus, and C. notatus, these bones contact witheach other.

Ventral view. The posterior process of thevomer is thin and long in Cynolebias chacoen-sis, C. cyaenus, and C. gymnoventris. In thesespecies, the length of the posterior process isthree times the width of the bone (Fig. 4G).If present, the anterior process is rounded,but it is absent in C. boitonei and C. flavicau-datus. A single tooth may be present on theanterior process of the vomer. The tooth wasobserved in C. nigripinnis (4.4%) andC. sp.(6.7%). One specimen of C. viarius(2.6%) has two teeth on the vomer. InC. wolterstorffi, the processus ascendens ofthe parasphenoid is long and thin (Fig. 4H),while in other species examined it is similarto that of C. luteoflamulatus. In C. luteofla-mulatus, C. cheradophilus, C. bellottii, andC. viarius, the parasphenoid expands later-ally in its medial region; in other species,this bone is slender (Fig. 4G, H).

Orbital rimIn most species, the lacrimal has the same

characteristics described for Cynolebias lu-teoflamulatus. However, in C. whitei, theupper portion of the lacrimal is reduced (Fig.4C), in C. bokermanni and C. constanciae,the ventral portion curves posteriorly (Fig.4D), while in C. cheradophilus, it is curvedanteriorly (Fig. 4E). The dermosphenotic,when present, is a small, rounded, free bonenext to the sphenotic (Fig. 3F). Species witha dermosphenotic are C. whitei, C. constan-ciae, C. bokermanni, C. myersi, C. chacoen-sis, and C. bellottii. Cynolebias bellottii has asecond ossification similar and dorsal to thedermosphenotic (Fig. 3H).

Jaw structureThe rostral cartilage is discoidal in all

species examined in this study. The ascend-ing processes of left and right premaxillaeare fused into a single structure in Cynole-


bias myersi (Fig. 5B). In C. chacoensis, thepremaxillae overlap medially but do not fuse(Fig. 5C). Most species have the alveolararm of the premaxilla anteriorly expanded(Fig. 5A,B,C). This anterior expansion is re-duced in C. cheradophilus and C. wolter-storffi (Fig. 5D). Cynolebias boitonei, C. boker-manni, C. chacoensis, C. constanciae,C. flavicaudatus, C. myersi C. notatus, andC. whitei have the ventral process of themaxilla enlarged anteriorly (Fig. 5F),whereas in C. cheradophilus and C. wolter-storffi it is thin and straight (Fig. 5G). Thedorsal process of the maxilla is oriented dor-sally, but it is reduced to a rounde protuber-ance in C. chacoensis (Fig. 5H). The ventraltip of the maxilla is expanded in C. constan-ciae.

The ventral process of the dentary is re-duced in Cynolebias constanciae (Fig. 5J).Cynolebias cheradophilus and C. wolter-storffi also have a dorsal process on the angu-loarticular bone (Fig. 5K) that it is not foundin any other species.

Jaw suspensoriumThe mesopterygoid is longer than the auto-

palatine in Cynolebias boitonei, C. boker-manni, C. chacoensis, C. constanciae, C. fla-vicaudatus, C. notatus, and C. whitei. InC. cheradophilus and C. wolterstorffi, themesopterygoid is reduced (Fig. 5N), whereasin other species, the two bones are about thesame size as described for C. luteoflamula-tus (Fig. 5L).

Opercular seriesThe opercular bones of all species are as

those described for Cynolebias luteoflamula-tus. Cynolebias bokermanni is unique in hav-ing the thicker segment of the interopercu-lar extending beyond the anterior end of thebone like an anterior spine (Fig. 5M).

Gill archesVentral components. The basihyal is of

variable length. In Cynolebias constanciae itis long, representing about five times thelength of the ventral hypohyal (Fig. 6C). InC. wolterstorffi the relation is four to one(Fig. 6D), and in most other species, therelation is three to one (Fig. 6B). Cynolebiascheradophilus has a reduced basihyal. Itslength is about the same as that of thehypohyal (Fig. 6E). The basihyal is of vari-able thickness. It is thickest in C. cherado-philus, being twice its length. It is about

25% of the length in C. constanciae, 75% ofthe length in C. boitonei, C. bokermanni,C. chacoensis, C. myersi, C. notatus, and inC. whitei (Fig. 6F), and the thickness isabout the same as the length in other spe-cies. The proportion of cartilage and bonealso varies among the species. In C. boitonei,C. bokermanni, C. chacoensis, C. constan-ciae, C. flavicaudatus, C. myersi, C. notatus,and C. whitei, the cartilaginous componentrepresents 25% of the total length (Fig. 6C,F).In C. nigripinnis and Cynolebias sp., 33% iscartilaginous, as it occurs in C. luteoflamula-tus (Fig. 6B). In C. affinis, C. alexandri,C. bellottii, C. cyaenus, C. duraznensis, andC. gymnoventris, 50% is cartilaginous (Fig.6G), and in C. viarius, and C. wolterstorffithe cartilaginous component reaches 65% ofthe total length. Cynolebias wolterstorffi alsohas the cartilaginous segment widened ante-riorly (Fig. 6D).

The height of the urohyal represents 20%of its length in Cynolebias chacoensis,C. constanciae, C. flavicaudatus, C. myersi,C. whitei, and C. wolterstorffi (Fig. 6I). It is25% in other species (Fig. 6H). In C. whitei,the posterior part of the bone splits in two(Fig. 6L). The urohyal of C. affinis, C. alexan-dri, C. boitonei, C. bokermanni, C. duraznen-sis, C. nigripinnis, and C. notatus has anindentation next to the anterior process (Fig.6I). Cynolebias cheradophilus lacks the ante-rior process (Fig. 6K), while in C. boker-manni this process points anteriorly (Fig.6M). The anterior half of the ceratohyal sup-ports two branchiostegal rays in all species,except in C. notatus and C. wolterstorffi,where it supports three branchiostegal rays.In C. bokermanni, it supports only one ray.

Dorsal components. The first epibran-chial is the only element of this series thatpossesses teeth. The number of teeth is vari-able among species, Cynolebias chacoensisand C. flavicaudatus have four teeth. Cynole-bias boitonei, C. bokermanni, C. viarius, andC. whitei have three teeth. Cynolebias gym-noventris, C. luteoflamulatus, C. myersi, andC. wolterstorffi have two teeth. In the otherspecies, the number of epibranchial teeth isintraspecifically variable from two to three(C. affinis, C. alexandri, C. constanciae,C. cyaenus, C. duraznensis, C. nigripinnis,and C. notatus), three to four (C. bellottiiand C. cheradophilus), and two to four(Cynolebias sp.).


TeethThe characteristics of the teeth of all spe-

cies examined are similar to those describedfor Cynolebias luteoflamulatus. In C. myersi,where the ascending processes of the pre-maxillae are fused, there are only threelarger teeth, one of them located medially(Fig. 5B).

DISCUSSION

Overall, the osteological characteristics re-ported here for Cynolebias agree with thefew and isolated observations previously re-ported for the genus (Vaz-Ferreira and Si-erra, ’73; Parenti, ’81; Costa ’90, ’94). How-ever, there are noteworthy deviations fromthe general pattern and characteristics foundhere that have not been previously reported.

The absence of neural prezygapophysis onthe first vertebra has been suggested as aderived condition for the family Rivulidae(Costa, ’90), whereas the presence of pleuralrib on the second vertebrae was considered aunique characteristic uniting all Cyprino-dontiformes (Parenti, ’81). In the speciesstudied here, these characters agree withprevious reports. The lack of neural prezyg-apophyses on the caudal vertebrae has beenconsidered as a synapomorphy for Cynolebia-tini (Costa, ’90, ’94). However, these struc-tures although reduced, were present intwelve of the twenty species studied here.Thus, the presence of neural prezygapophy-ses on the caudal vertebrae in Cynolebiasmay be a plesiomorphic state for the genus.Otherwise the reduction, instead of the ab-sence of neural prezygapophyses, could beconsidered a derived state for Cynolebias.

The caudal fin of Cynolebias cheradophi-lus and C. wolterstorffi is supported by fourvertebrae. This character could be diagnos-tic for the ‘‘elongatus complex,’’ as it was notfound in any other species examined. Cynole-bias nigripinnis has two vertebrae support-ing the caudal fin, differentiating it from thesimilar C. affinis, which has three vertebraesupporting the caudal fin. These two specieswere differentiated by Amato (’86) using ex-ternal morphology.

The ossified medial radials of dorsal andanal fins have not been previously reportedfor Rivulidae, and represent a derived condi-tion among Cynolebias. This condition wasfound in species of the ‘‘antenori complex’’and in C. constanciae, C. myersi, andC. whitei, species previously suggested to be

related to the ‘‘antenori complex’’ (Costa andBrasil, ’93). Furthermore, it distinguishesthe ‘‘antenori complex’’ species from othergroups. Cartilaginous radials in C. boitonei,a species related to the ‘‘antenori complex,’’could be interpreted as an autapomorphy(secondary loss/reversal). The ossified me-dial radials of C. bokermanni and C. notatuscould be interpreted either as relating themto the ‘‘antenori complex’’ or as a conver-gence among the species. The small bonyprojections on the rays of the anal fin ofmales of C. cheradophilus are a characteris-tic that needs to be examined further inother species of the ‘‘elongatus complex.’’Similar structures are also in the pectoralfin of this species and could have a sensoryfunction in courtship (see below).

The loss of pelvic fins in Cynolebias boito-nei was used by Costa and Brasil (’90) torelate this species to C. zonatus, which hasreduced pelvic fins. Cartilaginous pelvicbones were found only in C. notatus, a char-acter diagnostic of the species.

The short third postcleithrum of Cynole-bias wolterstorffi and the thick third postclei-thrum of C. gymnoventris are diagnosticcharacteristics for those species respectively.The absence/reduction of the upper pectoralradial has been suggested as a synapomor-phy for the ‘‘bellottii complex’’ (Costa andBrasil, ’93). However, five species of thiscomplex—C. affinis, C. alexandri, C. bellot-tii, C. luteoflamulatus, and C. nigripinnis—show unreduced upper radials. Small struc-tures on the pectoral fin-rays of males havebeen described as tactile organs in Cynole-bias whitei (Carvalho, ’57) and in other gen-era of killifishes (Foster, ’67). These struc-tures include nerve endings and couldfunction during reproductive behavior (Fos-ter, ’67). The presence of these structures inC. whitei has been defined by Costa (’95) asautopomorphic; however, these structureswere also found in C. boitonei, C. cheradophi-lus, and C. luteoflamulatus. The four specieswere placed in different clades by Costa,(’95), so its acquisition is considered indepen-dent.

The supraoccipital spines are highly vari-able, having the same configuration in differ-ent species groups. However, the state whereboth wings of the spine are free is sharedonly by Cynolebias whitei and C. myersi.This character could suggest a close relation-ship between these two species. Among stud-


ied species, C. wolterstorffi is diagnosed by abroad lateral process of the sphenotic and aunique shape of the processus ascendens ofthe parasphenoid. The parietal bone is notalways present among Cyprinodontiformes.When present, its long axis extends obliquelyand forward from the supraoccipital (Rosen,’64). Costa (’94) illustrated the parietal reach-ing and partially covering the supraoccipi-tal, and extending between the supraoccipi-tal and the epiotic in the tribe Plesiolebiatini(sister group of Cynolebiatini). In Cynole-bias, the parietal is overall triangular shapedand does not resemble the parietal of otherCyprinodontiformes.Atriangular shaped pa-rietal is a synapomorphy of Cynolebias. Theparietal fusion with the autopterotics inC. affinis and with the epiotics in C. boker-manni is independently derived in those spe-cies. A crest on the parietal in C. whitei is anautapomorphy for the species. Reduction ofthe parietal is seen in less than 6% amongthe specimens of only two species within the‘‘bellottii complex,’’ C. luteoflamulatus andC. viarius . If Amato’s (’86) placement ofthese species in different subgroups of the‘‘bellottii complex’’ is correct, the parietalreduction is independent in both species.Cynolebias wolterstorffi also possesses re-duced parietals. All species with a compactskull, i.e., no spaces between bones, belongto the ‘‘antenori complex’’ or are related to it.

The absence of teeth on the vomer is asynapomorphy for the tribe Cynolebiatini(Costa, ’94). The presence of a tooth found onthis bone in Cynolebias luteoflamulatus,C. nigripinnis, and C. viarius is derivedwithin the genus. In the suborder Aplochei-loidei, the vomer is dorsal to the anteriorarm of the parasphenoid (Parenti, ’81); how-ever, in Cynolebias the vomer is ventral tothe parasphenoid. Thus, this condition is asynapomorphy for Cynolebias.

The shape and torsion of the lacrimal area synapomorphy for Aplocheiloidei (Parenti,’81). A lacrimal with reduced torsion andwith a wide upper portion is a synapomor-phy of the tribe Cynolebiatini (Costa, 90,’94).However, the lacrimal of the species exam-ined herein showed no reduction in torsion,resembling the typical lacrimal of Rivulinae(Costa, ’90). The lacrimal shows characteris-tics that can help to diagnose some species.For example, the reduction in the upperportion of the lacrimal is diagnostic of Cynole-bias whitei and an anteriorly curved lacri-mal diagnoses C. cheradophilus. The lacri-

mals of C. bokermanni and C. constanciaeare curved posteriorly and this conditioncould reflect a close relation between thesetwo species. Costa (’90) reported the lack of adermosphenotic in all Cynolebias exceptsome large species. A dermosphenotic wasnot found in the large species examinedherein, C. cheradophilus and C. wolter-storffi, but it was present in several of the‘‘small’’ species, C. bokermanni, C. chacoen-sis, C. constanciae, C. myersi, and C. whitei.All species with a dermosphenotic are in the‘‘antenori complex’’ or are related to thiscomplex. The ‘‘small’’ C. bellottii, not onlyhas a dermosphenotic but also another ossi-fication in the same region. This second ossi-fication has not been reported previously forRivulidae. The homology of this bone is un-clear. It could represent (1) a new ossifica-tion of a ‘‘second dermosphenotic,’’ (2) an-other bone of the orbital rim, or (3) thedivision of the original dermosphenotic intotwo separate centers of ossification. Theanalysis of a developmental series in thisspecies may help to understand the homol-ogy of this ossification.

Three jaw characteristics not previouslyreported were found in Cynolebias cherado-philus and C. wolterstorffi: (1) the reductionof the expanded process of the alveolar armof the premaxillae, (2) a thin and straightventral process of the maxillae, and (3) adorsal process of the anguloarticular. Thesethree characteristics could be derived condi-tions within the ‘‘elongatus complex.’’ Themodifications of jaw structure in C. cherado-philus and C. wolterstorffi could reflect differ-ences in feeding behavior with other Cynole-bias species or could be the consequence ofallometric growth since these species areamong the largest in the genus.

An enlarged ventral process of the maxil-lae is shared by all species of the ‘‘antenoricomplex’’ and the plesiomorphic species. Along ventral dentary process is a synapomor-phy for Cynolebias (Costa, ’90). The reduc-tion of this process and the expansion of theventral tip of the maxillae found in C. con-stanciae are autopomorphies diagnostic ofthe species. As suggested for the ‘‘elongatuscomplex’’ species, these characters could re-flect a change in the feeding habits ofC. constanciae in relation to other Cynole-bias.

A small mesopterygoid, relative to the au-topalatine, has been considered as a synapo-


morphy for Cynolebiatini (Costa, ’90). How-ever, the mesopterygoid is longer than theautopalatine in the ‘‘antenori complex’’ andrelated species. The only species examinedwith a reduced mesopterygoid are Cynole-bias cheradophilus and C. wolterstorffi.Species of the ‘‘bellottii complex’’ have a me-sopterygoid about equal in size to the auto-palatine, this state being the most commonamong cyprinodontiforms (Costa, ’90).

Cynolebias myersi has the ascending pro-cesses of the parasphenoids fused into asingle structure. The existence of possiblevariation in this character could not be testedbecause only one specimen was available.

A long basihyal is diagnostic of Cynolebiasconstanciae. This character is also consid-ered a synapomorphy of the tribe Plesiolebia-tini (Costa, ’94). Reduction of the bony partof the basihyal and a deep uruhyal wereconsidered synapomorphies for the ‘‘bellottiicomplex’’ (Costa and Brasil, ’93). This analy-sis agrees with that interpretation. The lackof teeth on the fourth ceratobranchial wasconsidered a synapomorphy for Cynolebia-tini (Costa, ’90). A row of teeth in the speciesstudied here suggests that the presence ofteeth could be a derived character within thetribe and a synapomorphy for Cynolebias.However, the presence of teeth on this boneis common in other genera of the family, sotheir presence could also be interpreted asprimitive and the absence in other Cynole-biatini as the derived condition. The pres-ence of teeth on the first epibranchial hasnot been previously reported for Cyprinodon-tiformes. This character can be considered asynapomorphy for Cynolebias.

Costa’s phylogeny of the genus Cynolebiasshows a common ancestry of the ‘‘bellottiicomplex,’’ the ‘‘antenori complex,’’ C. boito-nei, C. zonatus, and C. constanciae based onone character, i.e., the elongation of the up-per portion of the cleithrum. This study foundat least five characters, e.g., ossification ofdorsal fin medial radials, four pectoral radi-als, ventral process of the maxillae enlarged,mesopterygoyd bigger than autopalatine,and proportion of cartilage in the basihyal,that relate the ‘‘antenori complex,’’C. boitone,and C. constanciae to the clade comprised ofC. bokermanni, C. myersi and C. whitei andseparates all of them from the ‘‘bellottii com-plex.’’

The present study found that several osteo-logical characters, including some of the

characters previously use as diagnostic, ei-ther for the genus or the species, are polymor-phic, e.g., shape of the parietal bone, num-ber of pectoral radials, epibranchial teeth,and vomerine teeth. Polymorphisms havenot been taken into account in most phyloge-netic studies and their proper use couldchange some of the relations among taxa(Wiens, ’95; Rannala, ’95). Previous studiesin Rivulidae have been based on very fewspecimens per species and consequently poly-morphisms have been overlooked. Further-more, Parenti and Tigano (’93), reported poly-morphism in the rostral cartilage in an OldWorld cyprinodontiform. Polymorphic char-acters seem not to be infrequent in thisorder and should be taken into consider-ation in any phylogenetic reconstruction.Herein we have shown that the osteologicalcharacters can be used to diagnose speciesas well as address phylogenetic relation-ships among species of Cynolebias.

ACKNOWLEDGMENTS

The present paper represents a portion ofthesis presented in partial fullfillment of therequirements for the M.S. degree at the Uni-versity of Richmond. The original manu-script benefited from the critical readings ofJ. Hayden and G. Radice whose commentsare greatly appreciated. We are thankful tothe Graduate School of Arts and Sciences,University of Richmond for the financial sup-port provided to M.L. Supplies and materi-als for this project were purchased with fundsfrom several grants supported by the Gradu-ate Research Committee, University of Rich-mond. We are also thankful to all the peopleat the Limnology Section of Facultad deCiencias (Uruguay).

LITERATURE CITED

Amato, L.H. (1986) Seis especies nuevas del generoCynolebias Steindachner, 1876, de Uruguay y Para-guay (Cyprinodontiformes, Rivulidae). Com. Zool. Mus.Hist. Nat. Montevideo 11:1–27.

Carvalho, A.L. (1957) Notas para o conhecimento dabiologia dos peixes anuais. Rev. Bras. Biol. 17:459–466.

Costa, W.J.E.M. (1989) Redescricao do genero Cynole-bias (Cyprinodontiformes, Rivulidae), com a descricaode uma nova especie da bacia do Rio Tocantis. Comun.Mus. Cienc. PUCRS, Ser. zool. 2:181–190.

Costa, W.J.E.M. (1990) Analise filogenetica da famıliaRivulidae (Cyprinodontiformes, Aplocheiloidei). Rev.Bras. Biol. 50:65–82.

Costa, W.J.E.M. (1991) Systematics and distribution ofthe neotropical annual fish genus Plesiolebias (Cypri-nodontiformes, Rivulidae), with a description of a newspecies. Ichthyol. Explor. Freshwaters 1:369–378.


Costa, W.J.E.M. (1994) Two new genera and two newspecies of the neotropical annual fishes Plesiolebiatini(Cyprinodontiformes, Rivulidae), with studies on therelationships of the tribe. Rev. fr. Aquariol. 21:65–74.

Costa, W.J.E.M. (1995) Pearl Killifishes. The Cynolebi-atinae. Systematics and Biogeography of a Neotropi-calAnnual Fish Subfamily (Cyprinodontiformes: Rivu-lidae). Neptune City, T.F.H. Publications.

Costa, W.J.E.M., and G.C. Brasil (1990) Description oftwo new annual fishes of the genus Cynolebias (Cypri-nodontiformes: Rivulidae) from the Sıo Francisco ba-sin, Brazil. Ichthyol. Explor. Freshwaters 1:15–22.

Costa, W.J.E.M., and G.C. Brasil (1993) Two new speciesof Cynolebias (Cyprinodontiformes: Rivulidae) fromthe Sıo Francisco basin, Brazil, with notes on phylog-eny and biogeography of annual fishes. Ichthyol. Ex-plor. Freshwaters 4:193–200.

Dingerkus, G., and L.D. Uhler (1977) Differential stain-ing of bone and cartilage in cleared and stained fishusing alcian blue to stain cartilage and enzymes forclearing flesh. Stain Technol. 52:229–232.

Foster, N.R. (1967) Trends in the evolution of reproduc-tive behavior in killifishes. Studies Trop. Ocean. 5:549–566.

Parenti, L.R. (1981) A phylogenetic and biogeographicanalysis of cyprinodontiform fishes (Teleostei:Atherinomorpha). Bull. Amer. Mus. Nat. Hist. 168:341–557.

Parenti, L.R., and C. Tigano (1993) Polymorphic skeletalcharacters in Aphanius fasciatus (Telostei: Cypri-nodontiformes). Copeia 4:1132–1137.

Rannala, B. (1995) Polymorphic characters and phyloge-netic analysis: A statistical perspective. Syst. Biol.44:421–429.

Rosen, D.E. (1964) The relationships and taxonomicposition of the halfbeaks, killifishes, silversides, andtheir relatives. Bull. Amer. Mus. Nat. Hist. 127:217–268.

Vaz-Ferreira, R., and B. Sierra (1973) El genero Cynole-bias Steindachner, 1876 (Atheriniformes, Cyprinodon-tidae): caracteres, especies y distribucion. Trab. VCongr. Latinoam. Zool. 1:245–260.

Weitzman, S.H., and J.P. Wourms (1967) South Ameri-can cyprinodont fishes allied to Cynolebias with thedescription of a new species of Austrofundulus fromVenezuela. Copeia 1:89–100.

Wiens, J.J. (1995) Polymorphic characters in phyloge-netic systematics. Syst. Biol. 44:482–500.

Wildekamp, R.H. (1995) A World of Killies. Atlas ofOviparous Cyprinodontiform Fishes of the World. Vol.II. Mishawaka, Indiana. American Killifish Associa-tion, 383 pp.

Wourms, J.P. (1972) The developmental biology of an-nual fishes. III. Pre-embryonic and embryonic dia-pauses of variable duration in the eggs of annualfishes. J. Exp. Zool. 182:389–414.

APPENDIX

List of species used in this study. Part ofthe material examined in this study will bedeposited in the U.S. National Museum(Smithsonian Institution). The remainder be-long to Facultad de Ciencias, Depto. Zoolo-gia de Vertebrados, Montevideo (ZVC.P). Thematerial is listed below; number of speci-mens and their localities are specified.

Cynolebias affinis Amato—Pet trade, 25specimens examined, 13 cleared and stained.

Cynolebias alexandri Castello and Lopez—Uruguay: Salto: Parque Indıgena, 30 speci-mens examined, all cleared and stained.

Cynolebias bellottii Steindachner—Uru-guay: Salto: Banado Verocay, 37 specimensexamined, all cleared and stained. ZVC.P876; Uruguay: Colonia: Carmelo, 61 speci-mens examined, 19 cleared and stained.

Cynolebias boitonei Carvalho—Pet trade,1 specimen examined, cleared and stained.

Cynolebias bokermanni Carvalho andCruz—Pet trade, 2 specimens examined, allcleared and stained.

Cynolebias chacoensis Amato—Pet trade,2 specimens examined, all cleared andstained.

Cynolebias cheradophilus Vaz-Ferreira, Si-erra and Scaglia—Uruguay: Rocha: Ruta 10,Arroyo Valizas, 17 specimens examined, allcleared and stained.

Cynolebias constanciae Myers—Pet trade,45 specimens examined, 18 cleared andstained.

Cynolebias cyaenus Amato—Pet trade, 4specimens examined, all cleared and stained

Cynolebias duraznensis Reichert—Uru-guay: Durazno: Durazno, 41 specimens ex-amined, 26 cleared and stained. Uruguay:Durazno: Sarandi del Yi, 17 specimens exam-ined, 4 cleared and stained.

Cynolebias flavicaudatus Costa and Bra-sil—Pet trade, 3 specimens examined, allcleared and stained.

Cynolebias gymnoventris Amato—Uru-guay: Rocha: Velazquez, 7 specimens exam-ined, 5 cleared and stained.

Cynolebias luteoflamulatus Vaz-Ferreira—Uruguay: Rocha: Camino de los botes, 17specimens examined, all cleared and stained.Uruguay: Rocha: Ruta 16, 26 specimens ex-amined, all cleared and stained. Uruguay:Rocha: Ruta 9 km 200, 4 specimens exam-ined, all cleared and stained.

Cynolebias myersi Carvalho—Pet trade, 1specimen examined, cleared and stained.

Cynolebias nigripinnis Regan—Uruguay:Colonia: Higueritas, 36 specimens examinedall cleared and stained. ZVC.P 848; Uru-guay: Colonia: Higueritas, 44 specimens ex-amined, 4 cleared and stained.

Cynolebias notatus Costa and Brasil—Pettrade, 2 specimens examined, all clearedand stained.

Cynolebias viarius Vaz-Ferreira, Sierraand Scaglia—Uruguay: Rocha: Ruta 10 y


Ruta a Aguas Dulces, 25 specimens exam-ined, all cleared and stained. ZVC.P 525;Uruguay: Rocha: Palmeras gemelas, 50specimens examined, 8 cleared and stained.ZVC.P 596; Uruguay: Rocha: Totora, 14 speci-mens examined, 2 cleared and stained. Uru-guay: Rocha: Ruta 10, 7 specimens exam-ined, 2 cleared and stained.

Cynolebias whitei Myers—Pet trade, 55specimens examined, 18 cleared and stained.

Cynolebias wolterstorffi Ahl—Pet trade, 1specimen examined, cleared and stained.

Cynolebias sp.—Uruguay: Rocha: Ruta 16km 26, 26 specimens examined, 6 specimenscleared and stained. Uruguay: Rocha: Chuy, 19specimens examined, 5 cleared and stained.Uruguay: Treinta y Tres: Charqueada, 8 speci-mens examined, 2 cleared and stained. Uru-guay: Rocha: Arroyo San Miguel, 24 specimensexamined, 6 cleared and stained.


osteological analysis of the killifish genus cynolebias (cyprinodontiformes: rivulidae)

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