Biflagellate Spermatozoon of the Poison-Dart Frogs Epipedobates

JOURNALOFMORPHOLOGY255:114–121(2003)

BiﬂagellateSpermatozoonofthePoison-DartFrogs EpipedobatesfemoralisandColostethussp.(Anura, Dendrobatidae)

OdairAguiar–Jr.,1 AdrianA.Garda,1,4 AlbertinaP.Lima,2 GuarinoR.Colli,3 SoˆniaN.Ba´o,4 and ShirleiM.Recco-Pimentel1*

1DepartamentodeBiologiaCelular,InstitutodeBiologia,UniversidadeEstadualdeCampinas,(UNICAMP), 13084-971,Campinas,SP,Brasil 2CoordenadoriadePesquisasemEcologia,InstitutoNacionaldePesquisasdaAmazoˆnia,(INPA),69011-970, Manaus,AM,Brasil 3DepartamentodeZoologiaand 4DepartamentodeBiologiaCelular,InstitutodeCieˆnciasBiolo´gicas,Universidade deBrası´lia,(UnB),70919-970,Bras´liaı ,DF,Brasil

ABSTRACTThisstudydescribesthespermatozoaofthe Manophryne,Phobobates,andAllobates(Myers, dendrobatidsEpipedobatesfemoralisandColostethussp. 1987;ZimmermanandZimmermann,1988;La usinglightandtransmissionelectronmicroscopy.Both Marca,1992,1994). speciespossessabiflagellatespermatozoon,anunusual Colostethusisthelargestdendrobatidgenus,with characteristiconlypreviouslyreportedintwoanuranspe- approximately100describedspecies(Frost,2000). ciesbelongingtothefamiliesLeptodactylidaeand AccordingtoGrantetal.(1997),mostspeciesgroups Racophoridae.Theacrosomalcomplexofbothspeciescon- sistsofaconicalacrosomalvesicleandasubacrosomal ofColostethusarenotfirmlyestablishedbecause cone,bothofwhichcovertheanteriorportionofthenu- theyaredefinedbyacombinationofcharacterstates cleus,buttodifferingextents.Inthemidpiece,thecen- widespreadinothergroups,ratherthanbyunam- triolesaredisposedparalleltoeachotherandtothecell biguoussynapomorphies.SincethegenusCo- axisandgiverisetotwoaxonemes.Twoparaxonemalrods lostethusisdefinedbysymplesiomorphiccharacters, werealsoseenenteringthenuclearfossa.Bothflagella someregarditasparaphyletic(Lynch,1982). aresurroundedbyasinglemitochondrialcollar.Each Epipedobatesisdefinedmainlybyplesiomorphic flagellumisformedbyanaxialfiberconnectedtothe charactersandmayormaynotbeanaturalgroup, axonemebyanaxialsheath;juxta-axonemalfibersare asarguedbyMyers(1987),whopartitionedthe absent.OurdataseemtosupportthatEpipedobatesfemo- genusDendrobates(sensuSilverstone,1975)into ralisshouldbeplacedinaseparatecladepossiblyrelated fournewgenera,includingEpipedobates.Most toColosthetusandthatthesetwogeneramaynotbe speciesofEpipedobateswereformerlyclassified monophyletic.J.Morphol.255:114–121,2003. intoothergenerasuchasDendrobates,Phyllo- ©2002Wiley-Liss,Inc. batesandColostethus(Frost,2000),thusreflect- KEYWORDS:spermultrastructure;biflagellarity;Anura; ingthedifficultyinthetaxonomicassignmentof Dendrobatidae;Epipedobates;Colostethus thesedendrobatids. Variousdatasetshavebeenusedtoinvestigate dendrobatidsystematicrelationships,includingcy- togeneticandmolecularmarkers(Morescalchi, Thesystematicrelationshipsofpoison-dartfrogs 1973;Rasottoetal.,1987;Bogart,1991;Hillisetal., (FamilyDendrobatidae)havebeenstudiedinten- 1993;Hayetal.,1995;Summersetal.,1999;Clough sively(reviewedinFord,1993;Burton,1998).De- andSummers,2000;Vencesetal.,2000)andalso spitethewell-acceptedmonophylyofdendrobatids, theirintergenericaffinitiesremainobscure(Myers etal.,1991;FordandCannatella,1993;Venceset al.,2000).AccordingtoCloughandSummers(2000), Contractgrantsponsor:FAPESP;Contractgrantnumber:Proc. 99/05602-0;Contractgrantsponsor:CNPq;Contractgrantnumber: somemembersofthisfamilycannotbeclassifiedin Proc.300965/00;Contractgrantsponsor:CAPES. anyparticulargenusbecauseoftheabsenceofdiag- nosticcharacters.Theseproblematicspecieshave *Correspondenceto:Dr.ShirleiM.Recco-Pimentel,Departamento undergoneseveraltaxonomicrevisionsthathave deBiologiaCelular,IB,Unicamp,Campinas,SP,Brasil,13084-971. resultedindifferentgenericarrangementsandthe E-mail:[email protected]. creationofnewgenera.Thiswasthecaseofsome Publishedonline00Month2002in speciesformerlyplacedinColostethusandEpipe- WileyInterScience(www.interscience.wiley.com) dobates, but now allocated in Nephelobates, DOI:10.1002/jmor.10052

Fig. 1. A: The spermatozoa of Epipedobates femoralis showing the presence of two independent flagella, both with an undulating membrane. LM. B: A spermatocyst of Colostethus sp. where several pairs of flagella surrounded by mitochondrial collars can be seen. TEM. a, acrosome; f, flagellum; LM, light microscopy; mc, mitochondrial collar; n, nucleus; TEM, transmission electron microscopy. Scale bars: A: 10 ␮m; B: 3 ␮m; behavioral characteristics (Zimmermann and Zim- MATERIALS AND METHODS mermann, 1988; Toft, 1995). Such analyses have Two adult male Epipedobates femoralis were collected by Al- been useful in introducing new parameters to com- bertina P. Lima in a 10,000-ha plot of tropical rainforest in the plement the classic morphological traits (Noble, Reserva Florestal Adolfo Ducke, 25 km northeast of Manaus, 1931; Lynch, 1971, 1973; Duellman and Trueb, Amazonas, Brazil (03°08Ј S, 60°04Ј W). Two specimens of Co- 1986; Ford, 1993; Ford and Cannatella, 1993; Bur- lostethus sp. were collected by Janalee P. Caldwell, Laurie J. Vitt, and Robson A. Souza in Guajara´-Mirim, Rondoˆnia, Brazil (10° 19Ј ton, 1998), which can be misleading because of their S, 64° 33Ј W). The specimens of E. femoralis were deposited in the limited number and the ambiguous and erroneous Museu de Histo´ria Natural “Prof. Adaõ Jose´ Cardoso,” Univer- reporting of some character states (Ford, 1993). sidade Estadual de Campinas, Brazil (ZUEC 11750, 11753), More recently, sperm ultrastructure has been whereas the specimens of Colostethus sp. were deposited in the Sam Noble Oklahoma Museum of Natural History, University of used in phylogenetic analyses of various taxa, in- Oklahoma, USA (OMNH 37001, 37002). cluding fishes (Mattei, 1991; Jamieson and Leung, For light microscopic observations, spermatozoa from 1991), reptiles (Jamieson, 1995; Teixeira et al., glutaraldehyde–paraformaldehyde-fixed smears were stained 1999a,b), platyhelminths (Justine, 1991), and in- with Giemsa (10% pH 6.8) and examined with an Olympus BX60 microscope. For transmission electron microscopy, fragments of sects (Jamieson et al., 1999). Several studies have testes from Epipedobates femoralis and Colostethus sp. were pre- also shown that sperm ultrastructure is also useful fixed in a solution of 2% paraformaldehyde, 2% glutaraldehyde, for phylogenetic inference in anurans (Lee and 3% sucrose, and 5 mM CaCl2 in 0.1 M sodium cacodylate buffer, Jamieson, 1992, 1993; Jamieson et al., 1993; Kwon pH 7.2, at 4°C overnight. The samples were then postfixed for 60 and Lee, 1995; Meyer et al., 1997; Scheltinga et al., min in 1% osmium tetroxide, 0.8% potassium ferricyanide, and 5 mM CaCl2 in 0.1 M sodium cacodylate buffer before staining en 2001). bloc with 0.5% uranyl acetate for 2 h. After dehydration in an In this article we describe the ultrastructure of ascending series of acetone, the samples of E. femoralis were testicular spermatozoa from Epipedobates femoralis embedded in Epon and polymerized at 60°C for 48 h. Alterna- (ϭAllobates femoralis, sensu Zimmermann and Zim- tively, the material of Colostethus sp. was embedded in Spurr resin after the same process of fixation. Ultrathin sections were mermann, 1988) and Colostethus sp. We report the stained with uranyl acetate (3% in water) for 30 min and then for presence of two complete flagella in the spermato- 8 min in lead citrate following Reynolds (1963), with three rinses zoon of these species, a feature not previously re- in distilled water after each solution. Grids were examined with ported for other anuran species, although two sim- a LEO 906 or JEOL 100C electron microscopes at 60kV or 80 kV. ple flagella have already been described to one leptodactylid and one racophorid species (Pugin- RESULTS Rios and Garrido, 1981; Wilson et al., 1991; Jamie- son, 1999). Finally, in light of the new data, we also Spermatozoa of Epipedobates femoralis and Co- discuss the affinities of E. femoralis with the genus lostethus sp. contain two flagella that are inserted Colostethus. into a short midpiece (Fig. 1A,B). The nucleus and 116 O. AGUIAR–JR. ET AL.

Fig. 2. Spermatozoa of Co- lostethus sp. A: Longitudinal section of the acrosome showing the end of the acrosomal vesicle (arrowheads). Also note the canal throughout the subacrosomal cone’s length and the nuclear space. B–E: Transverse sections of the headpiece showing reduction of the acrosomal vesicle and subacrosomal cone and progressive enlargement of the nucleus. F: In- sertion of both paraxonemal rods independently in the nuclear fossa of a spermatid. G,H: Implantation of both axonemes in the nuclear fossa and the corresponding parallel centrioles in the nuclear fossa. I: Insertion of the auxiliary fibers in the nuclear fossa showing the coalescence of the fibers forming the paraxonemal rod near the insertion. The paraxonemal rod penetrates the nuclear fossa only slightly (arrowhead). J-L: Trans- verse sections of the tail showing the presence of the mitochondrial collar proximally and its subse- quent disappearance, as well as the reduction in the length of the undulating membrane. Note the constant curved shape of the axial fiber. a, acrosomal vesicle; af, axial fiber; as, axial sheath; ax, axoneme; c, centriole; mc, mitochondrial collar; n, nucleus; nf, nuclear fossa; ns, nuclear space; p, paraxonemal rod; s, subacrosomal canal; sc, subacrosomal cone; u, undulating membrane. Scale bars: A–E,G,L: 0.2 ␮m; F,H,I–K: 0.5 ␮m. tail of E. femoralis are approximately 19.5 ␮m and portion of the head and extends to the beginning of 27.0 ␮m long, respectively. The paired flagella and the nucleus, where a large nuclear space can be seen mitochondrial collars are particularly evident in (Fig. 2A–C). Below the acrosomal vesicle, the sub- transverse sections through a spermatocyst (Fig. acrosomal cone ensheathes the nucleus (Fig. 2A,D). 1B). Above the nuclear membrane, the subacrosomal cone is traversed throughout its length by a canal, Colostethus sp. referred to here as the subacrosomal canal (Fig. 2A,B). In transverse section, at the anteriormost The acrosomal tip is blunt in longitudinal section portion of the acrosome, the subacrosomal canal is and a conical acrosomal vesicle covers the anterior surrounded by the subacrosomal cone and the de- BIFLAGELLARITY IN TWO DENDROBATED FROGS 117 tached acrosomal vesicle (Fig. 2B). More posteriorly, DISCUSSION the nuclear space is surrounded by the subacrosomal cone that adheres closely to the nuclear mem- The basic structure of the sperm of Epipedobates brane (Fig. 2C). Subsequently, the nucleus is sur- femoralis is very similar to that of Colostethus sp. rounded by the subacrosomal cone alone and, The structure of the acrosomal complex of the two eventually, only by the plasma membrane (Fig. species is similar to that observed in most neobatra- 2D,E). The transition from the tip of the acrosome to chians, including Bufo arenarum (Burgos and Faw- the end of the subacrosomal cone is smooth, with no cett, 1956), Batrachyla spp. (Garrido et al., 1989), nuclear shoulders. Odontophrynus cultripes (Baó et al., 1991), Litoria In the midpiece, two paraxonemal rods enter the spp. and Cyclorana spp. (Meyer et al., 1997), Phys- shallow nuclear fossa (Fig. 2F–I). The centrioles are alaemus spp. (Amaral et al., 1999), and Pseudopalu- disposed parallel to the longitudinal axis, with an dicola falcipes (Amaral et al., 2000). The conical- axoneme showing the typical 9ϩ2 pattern of micro- shaped acrosomal vesicle is shared by several tubules emerging from each centriole (Fig. 2G,H). anuran species (Kwon and Lee, 1995) and is a ple- The axial sheath (sensu Garda et al., in press) and siomorphic trait of sarcopterygians (Jamieson, axial fiber coalesce near the point of insertion into 1999). The homogeneous nature of the subacrosomal the nuclear fossa to form the paraxonemal rod that cone differs from the longitudinal bundles of fibers projects slightly into the nuclear fossa (Fig. 2I). Sev- observed in the so-called “conical perforatorium” of eral mitochondria are arranged around both flagella bufonoid anurans (Jamieson et al., 1993). In agree- within the mitochondrial collar (Fig. 2J). ment with Garda et al. (in press), we assumed that The tail is composed of a comma-shaped axial fiber the subacrosomal cone is homologous with the con- connected to the axoneme through an axial sheath; ical perforatorium of bufonoids. The comma-shaped the plasma membrane is detached from both the axial fiber and the absence of a juxta-axonemal fiber axial sheath and axial fiber and no juxta-axonemal appear to be common features of dendrobatids. fiber is present (Fig. 2J). Further back, the axial The placement of the Dendrobatidae has shifted sheath shortens, the undulating membrane disap- between the neobatrachian groups Ranoidea and pears, and then only the axial fiber is observed (Fig. Bufonoidea (Ford and Cannatella, 1993; Hillis et al., 2K,L). Finally, the axial fiber vanishes and the ax- 1993; Ruvinsky and Maxson, 1996; Emerson et al., oneme continues alone for a short distance. 2000). Besides the similarities in the acrosomal complex, which differ from that observed in ranoid groups (Kwon and Lee, 1995), the mitochondrial Epipedobates femoralis collar surrounding the axonemes in the species stud- The acrosomal vesicle consists of a thin, narrow, ied here is characteristic of most bufonoids, for membrane-bound vesicle containing homogeneous which it is considered a synapomorphic characteris- material of moderate electron-density and covers tic (Lee and Jamieson, 1992). However, the presence approximately one-third of the nucleus (Fig. 3A). of this characteristic does not necessarily imply af- The subacrosomal cone extends beyond the limits of finities between bufonoids and dendrobatids be- the acrosomal vesicle and consists of low-density cause Scheltinga et al. (2001) reassessed such char- material (Fig. 3A–C). A space containing very low- acteristics and argued that there is no arrangement density material (the nuclear space) is delimited by of mitochondria that can be convincingly regarded the nuclear membrane and chromatin (Fig. 3A,B). as plesiomorphic for the Anura. Even within Epipe- The chromatin is not wholly compact but is con- dobates, an alternative pattern was found by Garda densed into large lumps (Fig. 3A). Transverse sec- et al. (in press) in E. flavopictus sperm, in which the tions of the posterior end of the nucleus show that mitochondria are randomly distributed within the the chromatin is more compact (Fig. 3D). The nu- undulating membrane. clear fossa is occupied throughout its length by the Biflagellate spermatozoa have been described in two centrioles that are arranged parallel to each several animal groups. In fishes, for example, bi- other and give rise to two independent axonemes flagellate sperm have evolved at least six times, (Fig. 3E). The paraxonemal rod (sensu Jamieson et without any link to phylogeny or internal fertili- al., 1993) extends from the origin of the axoneme, as zation (Mattei, 1988; Jamieson and Leung, 1991). shown by transverse sections of the midpiece (Fig. The presence of two free flagella, widespread in 3F). Turbellaria, has been considered a plesiomorphic As in Colostethus sp., each flagellum consists of an characteristic of platyhelminths with a tendency axoneme with the typical 9ϩ2 pattern, an undulat- towards mono- and aflagellate conditions (Hendel- ing membrane, and an axial fiber. The latter is a berg, 1969; Justine, 1991). Franzeń (1982) de- comma-shaped structure connected to the axoneme scribed the only case of biflagellarity in Annelida through the axial sheath (Fig. 3G,H). In the proxi- (the polychaete Tomopteris helgolandica). Among mal region of the tails the mitochondria form a molluscs, biflagellate spermatozoa are known in sheath around the axoneme, axial fiber, and undu- two bivalves, Corbiculla flamae and C. leana, lating membrane (Fig. 3G). where they are considered a modification of the 118 O. AGUIAR–JR. ET AL.

Fig. 3. Spermatozoa of Epipe- dobates femoralis. A: Longitudinal section of the acrosomal region. Note the absence of the subacrosomal canal. B–D: Progressive enlargement of the nucleus and reduction of the acrosomal vesicle and subacrosomal cone. E: Inser- tion of both axonemes into the nuclear fossa. F: Sagittal section through the insertion of the axonemes showing a centriole and an early axoneme with the adjacent auxiliary fiber. G,H: Transversal sections of the tails showing the mitochondrial collar and its subse- quent disappearance. a, acrosomal vesicle; af, axial fiber; as, axial sheath; ax, axoneme; c, centriole; m, mitochondria; n, nucleus; nf, nuclear fossa; ns, nuclear space; sc, subacrosomal cone; u, undulating membrane. Scale bars: A,D,F,G: 0.5 ␮m; B: 0.25␮m; C,H: 0.2 ␮m; E: 1 ␮m. primitive monoflagellar condition related to the much so that later Selmi et al. (1997), through an specialized mode of reproduction found in these ultrastructural study of salamandrids sperm, em- species (Komaru and Konishi, 1996). In insects, phasized that sperm ultrastructure of Sirenidae spe- where sperm structure is highly diversified, a bi- cies was still unknown. In anurans, a number of flagellate condition is observed in some Hemiptera studies have reported the existence of biflagellarity. (Jamieson et al., 1999). In the Chilean leptodactylid Telmatobufo australis, Within the Caudata of Lissamphibia, Austin and the presence of two flagella was inferred to be a Baker (1964) suggested the presence of two flagella primitive characteristic related to the fertilization and two undulating membranes in Pseudobranchus environment, although the exact mode of fertiliza- striatus axanthus, a sirenid salamander. However, tion of this species was unknown (Pugin-Rios and such analysis was restricted to light microscopy and Garrido, 1981). Mainoya (1981) indicated that an- the figures shown by these authors are not convinc- urans may have sperm tails with either one or two ing as to the presence of such a peculiar tail. So flagella and erroneously cited Nicander (1970), who BIFLAGELLARITY IN TWO DENDROBATED FROGS 119 mentioned no case of biflagellarity in anurans. Lee of E. femoralis—also a nontoxic dendrobatid, as and Jamieson (1993) argued for a general trend noted by Caldwell (1996)—contrasts with the condi- towards the simplification of sperm among the tion found in other congeneric species, such as E. Anura (see also Jamieson et al., 1993) and suggested flavopictus (Garda et al., in press), E. trivittatus and that the biflagellarity in Rhacophorus (Rhacophori- E. hahneli (Aguiar–Jr. et al., unpubl. data). Hence, dae), Scaphiophus (Pelobatidae), and Telmatobufo our results also seem to suggest that these two spe- (Leptodactylidae) was apomorphic. This view was ciose genera (Epipedobates and Colostethus)may reinforced by Jamieson (1999) for Chiromantis, an- not be monophyletic in agreement with that pro- other racophorid genus. Although Mainoya (1981) posed by Vences et al. (2000) through molecular asserted that sperm cells of Chiromantis xer- data. Further ultrastructural studies of other spe- ampelina had only one tail flagellum with two axial cies of Epipedobates should confirm whether close filaments, Jamieson (1999), based on the report of relatives of E. femoralis are to be retained in the Wilson et al. (1991), emphasized that a pair of free genus, as noted by Myers et al. (1991). In addition, flagella comprise the tail piece in this species. How- an analysis of Aromobates nocturnus—the pre- ever, Rhacophorus (Mizuhira et al., 1986) actually sumed sister taxon of all other dendrobatids (Myers have two axonemes embedded in a matrix of hun- et al., 1991)—may shed light on the significance of dreds of microtubules, forming a single tail rather biflagellarity in the Dendrobatidae. Also, we suggest than two free flagella. Likewise, the pelobatids that as many as possible genera need to be studied Scaphiophus holbrookii (James, 1970) and before the taxonomic significance of this character Megophrys montana (Asa and Phillips, 1988) have a can be stated, considering the scarcity of sperm ul- single tail with two axonemes. Waggener and Carrol trastructure data of dendrobatids. (1998) also reported a biaxonemal condition in Lepi- dobatrachus laevis, but this conclusion was appar- ently based on a misinterpretation of micrographs: ACKNOWLEDGMENT the presence of two axonemes united by membrane The authors thank Dr. Lino Neto for technical (fig. 5A–F in their report) resulted, in fact, from the suggestions and valuable comments. same axoneme being hit twice by the same cut in the undulating tail; their figure 5G corroborates the common pattern found in neobatrachians, where the LITERATURE CITED tail is formed by the axoneme, juxta-axonemal fiber, Amaral MJLV, Fernandes AP, Baó SN, Recco-Pimentel SM. 1999. axial sheath, and axial fiber. We propose that true An ultrastructural study of spermiogenesis in three species of biflagellarity is a condition in which the two axo- Physalaemus (Anura, Leptodactylidae). Biocell 23:211–221. nemes are independent and isolated by the plasma Amaral MJLV, Fernandes AP, Baó SN, Recco-Pimentel SM. 2000. membrane. 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