Comparative Analysis of the Sperm Ultrastructure of Three Species of 2 Sam Noble Oklahoma Museum of Natural Phyllomedusa (Anura, Hylidae)

Acta Zoologica (Stockholm) 85: 257–262 (October 2004)

ComparativeBlackwell Publishing, Ltd. analysis of the sperm ultrastructure of three species of Phyllomedusa (Anura, Hylidae) G. C. Costa,1 A. A. Garda,2 R. D. Teixeira,3 G. R. Colli4 and S. N. Báo5

Abstract 1Departamento de Ecologia, Universidade Costa, G.C., Garda, A.A, Teixeira, R.D., Colli, G.R. and Báo, S.N. 2004. de Brasília, 70919–970, Brasília, DF, Brazil; Comparative analysis of the sperm ultrastructure of three species of 2 Sam Noble Oklahoma Museum of Natural Phyllomedusa (Anura, Hylidae). — Acta Zoologica (Stockholm) 85: 257–262 History, Norman, Oklahoma, 73072–7029, USA; 3Departmento de Biologia, We describe the sperm ultrastructure of three species of frogs in the genus Universidade Católica de Brasília, EPCT Phyllomedusa. According to micrographs, total size of the spermatozoon of Q.S. 7, lote 1, Águas Claras, 72030–170, Phyllomedusa hypochondrialis is significantly smaller than those of Phyllomedusa 4 Taguatinga, DF. Brazil; Departamento de bicolor and Phyllomedusa tarsius. The acrosome complex consists of two conical Zoologia, Universidade de Brasília, 70919– structures covering the nucleus, the acrosome vesicle and the subacrosomal 970, Brasília, DF, Brazil; 5Departamento de Biologia Celular, Universidade de Brasília, cone. The subacrosomal cone of P. bicolor and P. tarsius is less electron-dense 70919–970, Brasília, DF, Brazil and appears more granular in transverse section than in P. h ypochondrialis. In P. bicolor and P. tarsius, the nuclear space is reduced and the subacrosomal cone Keywords: fills most of the space between the acrosome vesicle and nucleus. The anterior Hylidae, sperm, ultrastructure, region of the nucleus in the spermatozoa of P. bicolor and P. tarsius ends Phyllomedusa abruptly, while in P. hypochondrialis it is sharp-ended. In P. bicolor and P. tarsius, the axial fibre is much larger than in P. hypochondrialis. The sperm ultrastructure Accepted for publication: of Phyllomedusa appears conservative at the intrageneric level. Future studies on 16 December 2004 the sperm ultrastructure of hylids can provide new insights on the systematics of the group and a larger database for a cladistic analysis. Sônia Nair Báo, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, 70919–970 Brasília, DF, Brazil. E-mail: [email protected]

alternative data sets is necessary for a trustworthy phylo- Introduction genetic reconstruction of amphibians (Wake 1993). Several detailed studies on many taxa, including fishes Among amphibians, the first works on sperm ultrastructure (Jamieson 1991; Tanaka et al. 1995), reptiles (Jamieson 1995; described the peculiarities of the cell and made no reference Teixeira et al. 1999), invertebrates (Rouse and Jamieson to any aspect of species ecology or character evolution. Few 1987; Buckland-Nicks 1995) and amphibians (Jamieson recent studies have been conducted on the subject and et al. 1993; Lee and Jamieson 1993; Meyer et al. 1997; associates sperm morphology with fertilization biology Garda et al. 2002) revealed that sperm ultrastructure pro- and phylogeny (Meyer et al. 1997; Scheltinga et al. 2002a). vides an alternative source of useful characters for phyloge- Recently, detailed publications on amphibian sperm netic analysis. Teixeira et al. (1999) analysed the skewness ultrastructure of several families have provided careful of tree length distributions from lizard sperm ultrastructure descriptions such as Leiopelmatidae (Scheltinga et al. 2001), characters and concluded that the data set contained signi- Microhylidae (Scheltinga et al. 2002b) and Leptodactylidae ficant phylogenetic signal. (Amaral et al. 2000). However, there is still a lack of Phylogenetic relationships among anurans are still unclear information on several families and much to be investigated, even among higher taxa. This outcome derives from the especially in the Neotropical realm. Furthermore, little is limited number of characters traditionally employed in phy- know about the variation of sperm ultrastructure among logenetic analyses resulting from the relatively conserved closely related species. anuran bauplan (Inger 1967). Recently, analyses based on One of the best-studied groups of Anurans is the family other data sets such as mitochondrial DNA sequences have Hylidae, which consists of four subfamilies: Hemiphr- been profitably used in anuran phylogenetic reconstructions actinae, Hylinae (including pseudines, sensu Darst and (Hay et al. 1995; Darst and Cannatella 2004). The use of Cannatella 2004), Pelodryadinae and Phyllomedusinae. The

Sperm ultrastructure of Phyllomedusa • Costa et al. Acta Zoologica (Stockholm) 85: 257–262 (October 2004) most detailed works were on Australian hylids (Pelodryadi- nae) of the genera Litoria and Cyclorana (Lee and Jamieson 1993; Meyer et al. 1997; Scheltinga et al. 2002b). Few studies were made on Neotropical genera, including Pachymedusa (Rastogi et al. 1988) and Hyla (Pugin-Rios and Garrido 1981; Costa et al. 2004). These studies revealed several com- mon features of the hylid sperm as well as major differences, both among anuran families and within hylid genera. The genus Phyllomedusa (Phyllomedusinae) currently comprises 30 species ranging from Costa Rica to Argentina. There are few studies on the systematics and taxonomy of Phyllomedusa and not all species are assigned to species groups (Frost 2002). Herein, we provide detailed descriptions of sperm ultrastructure of Phyllomedusa hypochondrialis, Phyllomedusa bicolor, and Phyllomedusa tarsius, as well as a discussion on the variation of these characters within hylid genera and the use of sperm ultrastructure in systematics.

Materials and methods We obtained spermatozoa from reproductive adult individu- als of the following species: Phyllomedusa hypochondrialis (CHUNB 14001–2, 24067), collected at Luziânia, Goiás Fig. 1—Schematic reconstruction of the spermatozoon of Phyllomedusa. State, Brazil (16°25′S, 47°95′W), and at Minaçu, Goiás State (13°38′S, 048°15′W); P. bicolor (CHUNB 22046) and regions: head (acrosome complex and nucleus), midpiece, P. tarsius (CHUNB 22047) collected near Manaus, Brazil and tail. The head is slightly curved, and the tail presents an (02°04′S, 060°03′W). We deposited vouchers at the ‘Coleção axial rod and an undulating membrane (Figs 2H and 3G). Herpetológica da Universidade de Brasília’ (CHUNB). Total sperm length was 60.62 µm ± 5.12 µm (n = 13) for P. We euthanized frogs by rubbing xylocaine 5% on the hypochondrialis, 81.60 µm ± 6.02 µm (n = 10) for P. bicolor, abdominal skin, removed testes and placed them in a Petri and 85.54 µm ± 4.48 µm (n = 13) for P. tarsius. Sperm dish with phosphate buffer (PBS) pH 7.2, and cut them into length differed significantly among species (; F = small pieces. We fixed tissues in a solution containing 85.28; P < 0.0001). Tukey’s multiple comparison tests   2.5% glutaraldehyde, 5 m CaCl2, and 5% sucrose in 0.1 indicated that sperm of P. h ypochondrialis is shorter than sodium cacodylate buffer pH 7.2 at 4 °C overnight, postfixed P. bicolor and P. tarsius, while differences between P. bicolor for 1 h in 1% osmium tetroxide, 0.8% potassium ferricya- and P. tarsius were not significant.   nide, and 5 m CaCl2 in 0.1 sodium cacodylate buffer, pH 7.2. We dehydrated the material in a series of ascending Transmission electronic microscopy acetone (30–100%) and embedded tissues in Spurr’s epoxy resin. We stained ultra thin sections with uranyl acetate and The general structure of the sperm of Phyllomedusa is sche- lead citrate and made observations and photographed the matically shown in Fig. 1. The sperm ultrastructure of the material in a JEOL® 100C transmission electron micro- three species is similar and we therefore describe them scope. We made light microscopic observations of sper- together and indicate any differences. matozoa from 0.1  sodium cacodylate buffer pH 7.2 fixed smears under Normarsky contrast using a Zeiss® Axiophot Acrosome complex and nucleus microscope. We carried out statistical analyses using  version 10.2 for Windows with a significance level of 5% to The acrosome complex (acrosome vesicle and subacrosomal reject null hypotheses. Throughout the text, means are cone) is located at the anterior region of the head and covers followed by one standard deviation (SD). the anterior part of the nucleus. The acrosome vesicle covers the subacrosomal cone and consists of a single, narrow vesicle filled with homogeneous material of low electron-density Results (Figs 2A–D and 3A–C). The subacrosomal cone lies under the acrosome vesicle forming a conical cap (Figs 2A–D and Light microscopy 3A–C). The acrosome vesicle goes from the tip of the cell to The spermatozoa of the three species of Phyllomedusa are the anterior region of the nucleus, whereas the subacrosomal elongate and filiform, consisting of three conspicuous cone goes beyond (Figs 2A, 2E, 3A,D). The acrosome vesicle

 © 2005 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 85: 257–262 (October 2004) Costa et al. • Sperm ultrastructure of Phyllomedusa and the subacrosomal cone are conical in longitudinal Discussion section (Figs 2A and 3A) and circular in cross sections (Figs 2B–E and 3B–D). The subacrosomal cone of P. bicolor The ultrastructure of the spermatozoa of the three species of and P. tarsius is more granular and less electron-dense in Phyllomedusa is similar. The major differences are in the transverse sections and striated in longitudinal sections, acrosome and tail regions. The spermatozoa of P. bicolor and relative to P. hypochondrialis (Figs 2A–E and 3A–D). P. tarsius are also significantly larger than that of P. h ypo- A well-defined electron lucent region, the nuclear space, lies chondrialis. Usually, sperm ultrastructure is conservative between the subacrosomal cone and the nucleus (Figs 2A, at the intrageneric level, but other studies also reported 2C–D and 3A). This space may be formed in the process of intrageneric variation, for example in Litoria (Lee and Jamieson chromatin condensation, during the spermiogenesis (Báo 1993; Meyer et al. 1997) and Pseudis (Garda et al. 2004). et al. 1991). The nucleus is conical in longitudinal section Among the subfamilies of Hylidae, the sperm ultra- (Figs 2A and 3A) and circular in cross section (Figs 2D–F structure of Hemiphractinae remains unknown. Overall, and 3C–E), being filled with condensed chromatin (Fig. 2A, the sperm ultrastructure of Hylinae, Pelodryadinae, and 2D–F, 3A,C–E). In P. bicolor and P. tarsius, the nuclear space Phyllomedusinae is similar, exhibiting typical bufonoid is reduced and the subacrosomal cone fills most of the space structures, such as the mitochondrial collar and the shape between the acrosome vesicle and nucleus (Fig. 3A–D). In and structure of the subacrosomal cone (Rastogi et al. 1988; P. hypochondrialis there is a much wider nuclear space, which Meyer et al. 1997; Costa et al. 2004). The sperm of pseudids is seen above the subacrosomal cone throughout its exten- is different from other hylids because of the lack of auxiliary sion (Fig. 2A,C–D). The anterior region of the nucleus in fibers and axial sheath (Garda et al. 2004). This condition is P. bicolor and P. tarsius has an abrupt ending while in P. hypo- probably autapomorphic as all other members of this family chondrialis there is a sharper end (Figs 2A and 3A). and related families studied so far exhibit those tail elements. Several differences have been recorded among hylid genera, including the shape of the acrosome (Lee and Jamieson Midpiece 1993; Scheltinga et al. 2002b), the position and length of the The midpiece is the region connecting the head and the flag- mitochondrial collar (Meyer et al. 1997; Costa et al. 2004), ellum of the spermatozoon, and it consists of cytoplasm, the the number and shape of mitochondria (Costa et al. 2004), proximal and distal centrioles surrounded by pericentriolar and the arrangement of tail elements (Lee and Jamieson material, and the mitochondrial collar. At the anterior por- 1993; Meyer et al. 1997). Part of this variation may be related tion of this region, the proximal centriole lies in the nuclear to life-history attributes. For instance, the long acrosome in fossa. The axial rod is inserted in the nuclear fossa right next the sperm of Litoria longirostris has been interpreted as an to the proximal centriole (Figs 2G and 3F). The distal adaptation for penetration in a gelatinous layer that sur- centriole is located out of the nuclear fossa and oriented rounds the clutch of eggs (Scheltinga et al. 2002b). Detailed perpendicularly with respect to the proximal centriole, being studies on the reproductive mode and habitat use of Phyl- continuous with the axoneme (Figs 2G and 3F). The centrioles lomedusa could help understand part of the variation found are encircled by a dispersed, granular, and electron-dense in these closely related species. material, the pericentriolar material (Figs 2G and 3F). The The systematics of the family, Hylidae is in a state of flux. region from the posterior portion of the nucleus to the begin- Duellman (2001), based on da Silva (1998), placed pseud- ning of the axoneme is surrounded by numerous mitochondria, ines as a subfamily of Hylidae and, more recently, Darst and arranged inside a mitochondrial collar (Figs 2J and 3H–I). Cannatella (2004) in a study with molecular data found that pseudines are nested deep inside Hylinae. Cladistic analysis using different data sets did not support a close relationship Tail of Hemiphractinae with the other hylids (Hass 2003; Darst This region consists of the axoneme associated with a juxta- and Cannatella 2004). Previous investigations on the sperm axonemal fibre, an axial sheath and an axial fibre (Figs 2K structure of hylids have helped to solve taxonomic problems. and 3J). The axoneme is continuous with the distal centriole For example, Cyclorana alboguttata was formerly placed in showing a typical 9 + 2 microtubule arrangement (Figs 2G, the genus Litoria and, based on aspects of the biology and 2J–K, 3F,I–J). The axial sheath unites the axial fibre to the sperm ultrastructure, it was moved to the genus Cyclorana axoneme (Figs 2K and 3J). The axial fibre runs parallel to the (Meyer et al. 1997). Based on sperm morphology, the H. axoneme and is formed by electron-dense material (Figs 2G, rubra group was placed in the genus Ololygon (junior 2I–K, 3F,H–J). The juxta-axonemal fibre is located between synonym of Scinax) (Fouquette and Delahoussaye 1977). The the axial sheath and the axoneme, being closer to the latter spermatozoa of the species in the H. rubra group have two tail (Figs 2K and 3J). In P. bicolor and P. tarsius, the axial fibre filaments, rather than one as in other species of Hyla. Further is much larger than in P. hypochondrialis. This is clearly studies found osteological, larval, reproductive, and mor- observed when comparing the ratio axial fibre diameter/ phological features, which characterized the genus Scinax axoneme diameter (Figs 2K and 3J). (valid name for Ololygon) (Duellman and Wiens 1992).

Fig. 2—The spermatozoa of Phyllomedusa hypochondrialis. —A. longitudinal section of the acrosome complex. —B–D. cross sections of the acrosome complex. —E–F. cross section of the nucleus. —G. longitudinal section of the midpiece. —H. light microscopy. —I–J. cross section of the midpiece. —K. cross section of the tail. af, axial ﬁbre; as, axial sheath; av, acrosome vesicle; ax, axoneme; dc, distal centriole; h, head; jf, juxta-axonemal ﬁbre; m, mitochondria; mp, midpiece; n, nucleus; ns, nuclear space; sc, subacrosomal cone; t, tail; u, undulating membrane.

Fig. 3—The spermatozoa of Phyllomedusa bicolor and P. tarsius. Figures (A)–(E). (G) are from P. tarsius and Figs (F). (H)–(J) are from P. bicolor —A. longitudinal section of the acrosome complex. —B–D. cross sections of the acrosome complex. —E. cross section of the nucleus. —F. longitudinal section of the midpiece. —G. light microscopy. —H–I. cross section of the midpiece. —J. cross section of the flagellum. af, axial fibre; as, axial sheath; av, acrosome vesicle; ax, axoneme; dc, distal centriole; h, head; jf, juxta-axonemal fibre; mp, midpiece; n, nucleus; ns, nuclear space; sc, subacrosomal cone; t, tail; u, undulating membrane.

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