Accepted on 6 March 2010 Ó 2010 Blackwell Verlag GmbH J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2010.00573.x 1Department of Biological Sciences, Southeastern Louisiana University, Hammond, LA; 2Department of Biology, Wittenberg University, Springfield, OH; 3Department of Biology, Saint Louis University, St Louis, MO, USA Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). VII. spermatozoon morphology and evolutionary trends of sperm characters in snakes Justin L. Rheubert1,Caleb D. McMahan1,David M. Sever1,Megan R. Bundy2,Dustin S. Siegel3 and Kevin M. Gribbins2 Abstract This study investigates the evolution of snake spermatozoa within a phylogenetic context, with the addition of a new ultrastructural description from the spermatozoa of the Black Swamp Snake, Seminatrix pygaea. Overall the spermatozoon of S. pygaea is similar to that described for other squamates, whereas some characters such as the electron lucent space separating the cortex and medulla of the acrosome complex may be a unique feature of S. pygaea spermatozoa. This preliminary analysis of sperm evolution within Serpentes leads to the hypothesis that some sperm characters are more plastic than others. Incongruencies are found between the molecular and the morphological topologies utilized, and the phylogeny derived from a morphological data set recovers more unequivocal ancestral states of snake sperm structure. Characters such as the beginning of the fibrous sheath at mitochondrial tier one unite the Colubroidea, whereas characters such as the presence of an acrosome vesicle subdivision, absent vacuity subdivision, and round ⁄ oval mitochondria in transverse section unite all squamates. However, from this analysis it is evident that more taxa need to be studied and taxa with current data need to be more thoroughly investigated to make more conclusive remarks regarding the evolution of sperm structure within snakes. Keywords: Evolution – snake – sperm – ultrastructure Introduction electron-dense structure inside the proximal centriole may be a Since the first ultrastructural description of a snake spermato- possible synapomorphy for the Colubroidae + Elapidae. zoon by Furieri (1965), multiple studies have been conducted to Although much attention has been paid to the ultrastructure determine the cytological similarities and differences of the of spermatozoa in the last decade, the number of taxa sampled spermatozoa within Serpentes (References in Cunha et al. 2008 within the Squamata, and certainly within Serpentes, needs to and Tavares-Bastos et al. 2008). Only a handful of these studies increase to more thoroughly study evolutionary trends. These have attempted to put these data into a phylogenetic context; spermatozoa characters along with developmental features Tavares-Bastos et al.Õs (2008) is the most recent study to map during spermiogenesis, which are also gaining much needed snake sperm traits (Jamieson and Koehler 1994; Harding et al. attention (Healy and Jamieson 1994; Gribbins et al. 2007, 1995; Jamieson 1995; Jamieson 1999; Oliver et al. 1996; 2010; Rheubert et al. in press), have been considered a rich Tourmente et al. 2006; Tavares-Bastos et al. 2007, 2008). source of non-traditional morphological characters that can be Tavares-Bastos et al. (2008) concluded that multiple characters utilized in phylogenetic analyses (Jamieson 1991; Newton and such as a dense collar in the neck region and mitochondria Trauth 1992; Jamieson et al. 1996; Teixeira et al. 1999; shape as sinuous tubes in oblique section are proposed Tavares-Bastos et al. 2002; Vieira et al. 2004; Wiens 2004). synapomorphies in Serpentes, and the ultrastructural data The purpose of this study is to provide a description of the provided by Cunha et al. (2008) and Tourmente et al. (2008) ultrastructure of the sperm of an additional snake, Seminatrix corroborated these findings. However, Jamieson et al. (1996) pygaea (Cope, 1871) and optimize snake sperm character states showed that sinuous mitochondria also occur in the sperma- to phylogenies reconstructed from morphological and molec- tozoa of Pygopodidae. As noted by Tourmente et al. (2008), the ular data sets to assess the possible ancestral states of sperm presence of extracellular microtubules proposed as a synapo- structures within the Serpentes. This is the seventh paper in a morphy among Serpentes (Tavares-Bastos et al. 2008) was not series that describes the reproductive morphological charac- observed in Bothrops, indicating the need for more taxonomic teristics of this natricine snake from the south-eastern United sampling to test this hypothesis. Furthermore, Jamieson et al. States. Previous papers have included female sperm storage (1996) showed that extracellular microtubules occur in, albeit location (Sever and Ryan 1999), the oviducal cycle (Sever et al. immature, teiid lizard sperm. Although Tavares-Bastos et al. 2000), renal sexual segment cycle (Sever et al. 2002), morphol- (2008) refer to some of these characters common among ogy of the ampulla ductus deferentis (Sever 2004), spermato- Serpentes as Ôputative synapomorph[ies]Õ, only nine species of genic cycle (Gribbins et al. 2005), and morphology of the the 3000+ extant species of snakes are included in their study. proximal efferent ducts (Sever 2010). This study aims to Within their study, they also report that the absence of an expand the information on snake spermatozoa ultrastructure as well as investigate evolutionary trends of spermatozoa characters among the Serpentes. Given the relatively few snake Corresponding author: Justin L. Rheubert ([email protected]) taxa for which ultrastructural sperm data have been published, Contributing authors: Caleb D. McMahan ([email protected]), David M. Sever ([email protected]), Megan R. Bundy (s10.mbundy@ the evolutionary trends elucidated here are intended to be wittenberg.edu), Dustin S. Siegel ([email protected]), Kevin preliminary, with the aim of stimulating dialogue and research M. Gribbins ([email protected]) on more snake taxa. 2 Rheubert, McMahan, Sever, Bundy, Siegel and Gribbins Materials and Methods cross-section (character 2), and character definitions and states are listed in the results. Sperm characters were coded in the same manner Tissue collection utilized by Tavares-Bastos et al. (2008) to maintain consistency Adult male Seminatrix pygaea were collected at Ellenton Bay on the between studies. Data were gathered from published electron micro- Department of EnergyÕs Savannah River Site in Aiken County, South graphs depicting the morphology of snake sperm cells by a single Carolina. Collections were made on 10 May 1998, 7 June 1998, 22–24 author (JLR) to ensure standardization. Table S1 shows the data July 1998, 29 September to 2 October 1998, and 17–22 March 1999. matrix developed for this study, as well as citations for references used Specimens were euthanized with a lethal dose of sodium pentobarbital to obtain ultrastructural sperm data. (3–5 ml injection of 10%; Abbott Laboratories, North Chicago, IL, Sperm character states were optimized onto two phylogenetic USA) in 70% ethanol as approved by the Animal Care and Use hypotheses of snake relationships under a parsimony framework using Committee of Saint MaryÕs College, Notre Dame, Indiana where the MacClade (version 4.08; Maddison and Maddison 2005). The specimens were euthanized. The reproductive tracts were removed morphological-based phylogeny of Lee et al. (2007) was used, given during gross dissection and placed in TrumpÕs solution for fixation that this is the most recent morphological study detailing snake (2.5% glutaraldehyde, and 2.5% formaldehyde in 0.1 M sodium evolutionary relationships at the family level. Eckstut et al. (2009) offer cacodylate buffer at pH 7.4; Electron Microscopy Sciences, Hatfield, a molecular phylogeny of squamates using the nuclear encoded C-mos PA, USA). gene. The Serpentes clade of this phylogeny was used as a molecular- based hypothesis in this study because it has the largest taxonomically sampled phylogeny of snakes to date and numerous studies have Tissue preparation clearly demonstrated the significance of comprehensive taxon sampling when doing phylogenetic studies (Zwickl and Hillis 2002; Hillis et al. Epididymal tissue in TrumpÕs solution was postfixed in 2% osmium 2003). There are some procedural and phylogenetic differences between tetroxide, dehydrated through a graded series of ethanol solutions, the morphological and the molecular-based studies of snake relation- cleared in propylene oxide, and embedded in epoxy resin (Embed 812; ships. Only a Varanus was used as an outgroup in the morphological Electron Microscopy Sciences, Hatfield, PA, USA). Sections were cut study (Fig. 1A), whereas both Iguana and Varanus were used as using an LKB automated ultramicrotome (LKB Produkter AB, outgroups in the molecular study (Fig. 1B). In the morphological Bromma, Sweden) at 90 nm with a DDK diamond knife (DDK, analysis, Liotyphlops was found to be sister to Typhlopidae (Fig. 1A), Wilmington, DE, USA) and placed on copper grids. Grids were whereas in the molecular analysis, Liotyphlops was found to be sister to stained with uranyl acetate and lead citrate and viewed using a Jeol all extant snakes (Fig. 1B). Another discordance between the two JEM-1200EX II transmission electron microscope (Jeol Inc., Peabody, topologies is within the derived Alethinophidians (Fig. 1A, B). MA, USA). Photographs were taken using a Gatan 785 Erlangshen Additionally, in the morphological