(Seminatrix Pygaea). VII

Accepted on 6 March 2010 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 study there was no discrimination digital camera (Gatan Inc., Warrendale, PA, USA) and analysed by between major lineages within the Colubroidea, thus weakening the making composite micrographs using Adobe Photoshop 7.0 (Adobe ability to assess ancestral states. Given the gaps in our data set for Systems, San Jose, CA, USA). certain taxa (e.g. Homalopsids, Xenopeltids, etc.), any descriptive statistics (consistency index, retention index, etc.) would be inconclu- sive for all major snake groups. For that reason we only addressed Character optimization preliminary trends in the evolution of sperm ultrastructure among Ultrastructural sperm characters were chosen a priori from Tavares- snake lineages (i.e. only character states that were determined as Bastos et al. (2008) with the addition of the acrosomal shape in unequivocal at internal nodes from the parsimony optimization).

Iguana iguana (a)Varanus gouldii (b) †Pachyrhachis Varanus gouldii †Haasiophis Liotyphlops beui †Madtsoiidae Typhlops reticulatus †Dinilysia Ramphotyphlops waitii Typhlops reticulatus Ramphotyphlops endoterus Ramphotyphlops endoterus Leptotyphlops koppesi Ramphotyphlops waitii Bolyeriidae Liotyphlops beui Leptotyphlops koppesi Aspidites melanocephalus Anomochilidae Loxocemidae Uropeltidae Xenopeltidae Cylindrophiidae Corallus hortulanus Aniliidae Epicrates cenchria Loxocemidae Boa constrictor occidentalis Xenopeltidae Boa constrictor amarali Calabaria Eryx jayakari Eryx jayakari Aspidites melanocephalus Uropeltidae Corallus hortulanus Aniliidae Epicrates cenchria Tropidophiidae Boa constrictor occidentalis Bothrops diporus Boa constrictor amarali Bothrops alternatus Ungaliophiinae Crotalus durissus Tropidophiidae Bolyeriidae Vipera aspis Bothrops diporus Nerodia sipedon Bothrops alternatus Elaphe scalaris Crotalus durissus Boiga irregularis Vipera aspis Stegonotus cucullatus Nerodia sipedon Seminatrix pygaea Elaphe scalaris Homalopsidae Boiga irregularis Stegonotus cucullatus Oxyuranus microlepidotus Seminatrix pygaea Pareatidae Oxyuranus microlepidotus Xenodermidae Acrochordidae Acrochordidae

Fig. 1. Phylogenetic trees used for the parsimony optimization of character states. (a) Morphological phylogeny adapted from Lee et al. 2007;. (b) Molecular phylogeny adapted from Eckstut et al. 2009 doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH Spermatozoon ultrastructure in Seminatrix pygaea 3

Results (a) Spermatozoon morphology of Seminatrix pygaea The acrosome vesicle (Figs 2A–C and 3; AV) caps the elongated nucleus (Fig. 3; Nu) of the mature spermatozoa. No unilateral ridge is present, and in transverse section (Fig. 3A–C) the acrosome complex is circular in shape. Two distinct layers within the apical portion of the acrosome vesicle can be observed based on electron density. The cortex

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(f) Fig. 3. Anterior views of a mature spermatozoon from Seminatrix pygaea. Left) Transverse view through the apical portion of a mature spermatozoon detailing the acrosome complex, acrosome vesicle (Av), perforatorium (Pf), perforatorial base plate (Pb), epinuclear lucent zone (Elz), white arrow, electron lucent band (white arrow), subacr- (f) osomal cone (Sc), nucleus (Nu). (a) Cross-sectional view through the apical portion of the acrosome complex showing the subdivision into cortex (Co) and medulla (Me) and the perforatorium (Pf) extending into the apical portion of the acrosome complex. Plasma membrane (black arrowhead). (b) Cross-sectional view through the medial portion of the acrosome complex detailing the acrosome vesicle (Av), subacrosomal cone (Sc), epinuclear lucent zone (Elz), electron lucent (g) band (white arrow) and plasma membrane (black arrowhead). (c) (g) Cross-sectional view through basal portion of the acrosome complex showing the acrosome vesicle (Av), subacrosomal cone (Sc), electron (h) lucent band (white arrow) rostrum of the nucleus (Nu), and plasma (h) membrane (black arrowhead). (d) Cross-section view through the nuclear body detailing the homogenous electron density of the nucleus Fig. 2. Schematic diagram of a mature spermatozoon from Seminatrix (Nu) and multilaminar membranes (Ml) pygaea. Showing spermatozoon in sagittal section and representative cross-sectional views of subsequent ﬁgures doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH 4 Rheubert, McMahan, Sever, Bundy, Siegel and Gribbins

(Fig. 3A; Co) is the outermost layer and is less electron dense triangular-shaped electron-dense structures making up the than the inner layer, the medulla (Fig. 3A; Me). The subacr- annulus (Fig. 4E; An) are found at the base of the midpiece. osomal cone (Fig. 3; Sc) is separated from the acrosomal The fibrous sheath continues into the principal piece (Fig. 4E; vesicle (Fig. 3; Av) by an electron lucent area (Fig. 3B,C; white Fs), and the enlarged peripheral fibres associated with micro- arrows) that extends to the base of the acrosome complex. The tubule doublets three and eight of the axoneme are lost. At the slightly electron lucent perforatorium (Fig. 3A; Pf) extends level of the principal piece (Fig. 2G) the multilaminar mem- from the apical portion of the subacrosomal cone (Fig. 3C; branes are not evident, but the extracellular microtubules SC) through the medulla (Fig. 3A; Me) of the acrosome (Fig. 4F; Mt) persist. The fibrous sheath continues through the vesicle. At the base of the perforatorium, a densification of the principal piece (Fig. 4F; black arrow) but ends at the begin- subacrosomal cone serves as the perforatorial base plate ning of the endpiece (Fig. 2H). The extracellular microtubules (Fig. 3; Pb) and is stopper-like in shape. Also within the are not observed, and the plasma membrane is in close subacrosomal cone a tapered portion of the nucleus (Fig. 3C; proximity to the axoneme with minimal cytoplasmic material Nu), the nuclear rostrum (Fig. 3C; Nu) can be seen capped associated with the endpiece. Throughout the endpiece, the with an electron lucent area, the epinuclear lucent zone axoneme continues to display the conserved 9 + 2 microtu- (Fig. 3B; Elz). The plasma membrane can be seen in juxtapo- bule arrangement (Fig. 4G; Ax). sition to the acrosome complex with minimal cytoplasmic material (Fig. 3A–C; black arrowhead). The nucleus (Fig. 2D) is homogenous in its electron density Comparative anatomy of snake sperm: a review from previous and does not contain nuclear lacunae (Fig. 3D; Nu). Apically literature the nucleus is tapered into the nuclear rostrum and caudally it Below are the differing states for sperm ultrastructural contains a slight indentation, the nuclear fossa, where the characters for all snakes previously investigated, plus the proximal centriole (Fig. 4A; Pc) is situated. Along the length of new data on Seminatrix pygaea. Two lizards were also used in the nuclear body, multiple circumferential multilaminar bodies the coding to act as outgroups for character polarization in (Fig. 3D; Ml) can be seen. The proximal centriole (Fig. 4A; Pc) subsequent evolutionary analysis. Some characters are con- lies within the nuclear fossa, contains no centralized electron- served throughout all taxa utilized in this study; however, we dense material, and shows a 9 + 3 microtubule arrangement also present some possible synapomorphies for recognized (Fig. 4B; Mt). The connecting piece (Figs 2E and 4A; white clades. Refer to Table S1 for the representative state of each arrow) between the proximal centriole and the distal centriole character for each taxon and a list of citations from which can be observed medial to the dense collar of the distal centriole. ultrastructural data were gathered. Two electron-dense laminar structures (Fig. 4A; black arrows) Character 1: The acrosome complex lateral ridge: Absent (0), extend bilaterally off the proximal centriole. Another electron- Present (1). The acrosome complex lateral ridge is present in all dense structure extends posteriorly along the lateral edges of the snakes investigated except Liotyphlops beui and is also absent distal centriole (Fig. 4; Dc) as a dense collar (Fig. 4A; black in Varnaus gouldii, but present in Iguana iguana. arrowheads). At the anterior portion of the distal centriole, the Character 2: Acrosome complex shape: Circular shaped (0), dense bodies make up the major and minor compartments of the Depressed (1). All snakes have a circular shaped acrosomal distal centriole, with the major compartment (right of Fig. 4C; complex in a cross-sectional view except Typhlops reticulatus, Vc) housing four peripheral fibres and the minor housing three in which it is depressed and more oval in shape. The acrosome (left of Fig. 4C; Vc), which can be observed in cross-section complex is also depressed in Iguana iguana and Varanus divided by a dorsal and ventral longitudinal column (Fig. 4C; gouldii. Vc). At this level the fibrous sheath is irregular in shape because Character 3: Acrosome vesicle subdivision: Absent (0), Present of the presence of the dense collar and connecting piece. The (1). The acrosome vesicle subdivision is absent in Boa peripheral fibres associated with microtubules three and eight constrictor amarali, Corallus hortulanus, and Epicrates cenchria are grossly enlarged (Fig. 4C; Pf) and both the multilaminar but present in all other species studied. membranes (Fig. 4C; Ml) and the extracellular microtubules Character 4: Subacrosomal cone: Paracrystalline (0), not (Fig. 4C; Mt) are observed at the level of the midpiece. paracrystalline (1). In all species utilized in this study the The beginning of the midpiece (Fig. 2F) is marked by the subacrosomal cone is paracrystalline. presence of mitochondria (Fig. 4A; Mi) and the fibrous sheath Character 5: Acrosomal vacuity subdivision: Present (0), Absent (Fig. 4D; Fs) beginning at mitochondrial tier 1 (Fig. 4A). (1). Only the Ramphotyphlops species contains an acrosomal Multiple solid dense bodies (Fig. 4; Db) can be seen among the vacuity subdivision that appears as electron lucent swirls mitochondria of the midpiece and are found in juxtaposition within the acrosome complex, whereas, in all other species (Fig. 4D; Db) to the fibrous sheath (Fig. 4D; Fs). In longitu- studied it is absent. dinal section the mitochondria (Fig. 4; Mi) appear slightly Character 6: Epinuclear lucent zone within the subacrosomal elongated with rounded ends and do not appear trapezoidal or cone: Absent (0), Present (1). The epinuclear lucent zone within columnar in shape. In transverse section the mitochondria the subacrosomal cone is present in all species studied except (Fig. 4D; Mi) are round or somewhat oval and contain linear Aspidites melancephalus and Boa constrictor occidentalis in cristae. The distal centriole displays the conserved 9 + 2 which it is absent. microtubule arrangement, and within the midpiece grossly Character 7: Perforatorium number: Multiple perforatoria (0), enlarged peripheral dense fibres (Fig. 4D; Pf) are observed in Single perforatorium (1). A single perforatorium is found in all association with microtubule doublets three and eight. At the species included here. level of the midpiece, the circum-cylindrical multilaminar Character 8: Perforatorium tip shape: Pointed (0), Rounded membranes (Fig. 4D; Ml) can be seen as well as extracellular (1). The perforatorium tip is rounded in all species of snakes microtubules (Fig. 4D; Mt). At the base of the midpiece the and Varanus gouldii but pointed in Bothrop alternatus and cytoplasm decreases and the mitochondrial tiers end. Two Iguana iguana. doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH Spermatozoon ultrastructure in Seminatrix pygaea 5

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Fig. 4. Posterior view of a mature spermatozoon from Seminatrix pygaea. Left) Transverse view of the posterior portion detailing the distal centriole (Dc), dense bodies (Db), mitochondria (Mi) of the midpiece, and the fibrous sheath (Fs). (a) Enlarged transverse view of the neck region detailing the proximal centriole (Pc) within the nuclear fossa of the nucleus (Nu), bilateral laminar projection (black arrows) extending from the proximal centriole, connecting piece to the distal centriole (white arrow) and dense collar (black arrowhead) extending distally lateral to the distal centriole. The distal centriole demonstrates the presence of the fibrous sheath (Fs) and mitochondria (Mi) within the midpiece. (b) Cross-sectional view of the proximal centriole showing the 9 + 3 microtubule (Mt) arrangement. (c) Cross-sectional view of the distal centriole at the proximal centriole junction detailing the major and minor compartments separated by the vertical column (Vc). The peripheral fibres (Pf) at microtubules 3 and 8 are grossly enlarged and multilaminar membranes (black arrow) and extracellular microtubules (Mt) surround the centriole. (d) Cross- sectional view of the midpiece detailing the mitochondria (Mi) and dense bodies (Db) surrounding the fibrous sheath (Fs), peripheral fibres (Pf) associated with doublets 3 and 8 of the axoneme, multilaminar membranes (Ml), and extracellular microtubules (Mt). (e) Detailed transverse view of the annulus (An) at the base of the midpiece and extension of the fibrous sheath (Fs) into the principal piece. (f) Cross-sectional view of the principal piece detailing the fibrous sheath (black arrow) and extracellular microtubules (Mt). (g) Cross-Sectional view of the endpiece detailing the axoneme (Ax)

Character 9: Perforatorial base plate: Absent (0), Present (1). Seminatrix pygaea, Stegonotus cuculatus, the Scolecophidians, The perforatorial base plate is present in Boa constrictor Iguana iguana and Varanus gouldii, but absent in all other amarali, Epicrates cenchria, Eryx jayakari, Crotaulus durissus, taxa. doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH 6 Rheubert, McMahan, Sever, Bundy, Siegel and Gribbins

Character 10: Perforatorial base plate morphology: Knob Before Mitochondrial tier 1 (4), Annulus level (5). The shaped (0), Stopper-like (1), n ⁄ a (2). When present, the beginning of the fibrous sheath within the midpiece occurs at perforatorial base plate is stopper like in all snake species mitochondrial tier 1 in the boas and pythons, Crotalus but knob shaped in Iguana iguana and Varanus gouldii. durissus, Seminatrix pygaea, and Stegonotus cucullatus, before Character 11: Nuclear lacunae: Absent (0), Present (1). Nuclear mitochondrial tier one in Boiga irregularis, Elaphe scalaris, lacunae are present in Crotalus durissus, Liotphylops beui, and Nerodia sipedon, Oxyuranus microlepidotus, and the Scoleco- Typhlops reticulatus but absent in all other species studied. phidians, and at mitochondrial tier 2 in Bothrops alternatus, Character 12: Stratified laminar structure within the neck region Iguana iguana, and Varanus gouldii. of the flagellum: Absent (0), Present (1). A stratified laminar Character 24: Annulus: Absent (0), Present (1). The annulus is structure within the neck region of the flagellum is absent in present in all species utilized here. Epicrates cenchria and Oxyuranus microlepidotus but present in Character 25: Annulus morphology: scythe shaped (0), Irreg- all other species studied. ularly shaped (1). The annulus is irregularly shaped in all Character 13: Projection of the laminar structure morphology: species utilized here. Unilateral (0), bilateral (1). The projection of the laminar Character 26: Anterior fibres associated with microtubule structure is unilateral in all species in which it is found. doublets three and eight within the principal piece: Absent (0), Character 14: Dense structure within the proximal centriole: Present (1). The anterior fibres associated with microtubule Absent (0), Present (1). A dense structure within the proximal doublets three and 8 within the principle piece are absent in all centriole is present in all boas and pythons (except Corallus species except Eryx jayakari, Crotalus durissus, Nerodia hortunlanus), Bothrops alternatus, Crotalus durissus, Nerodia Sipedon, Stegonotus cucullatus, and Iguana iguana. sipedon, Stegonotus cucullatus, Typhlops reticulatus, Iguana Character 27: Multilaminar membranes: Absent (0), Present iguana, and Varanus gouldii, but absent in all other species. (1). Multilaminar membranes are present in all species studied Character 15: Dense collar of the neck region: Absent (0), to date except Bothrops alternatus, Bothrops diporus, Iguana Present (1). The dense collar of the neck region is present in all iguana, and Varanus gouldii. species studied except Varanus gouldii. Character 28: Extracellular microtubules: Absent (0), Present Character 16: Enlarged dense fibres at microtubule doublets (1). Extracellular microtubules are absent in Boa constrictor three and eight of the axoneme: Present (0), Absent (1). All occidentalis, Bothrops alternatus, Bothrops diporus, Leptotyph- species utilized here contain enlarged dense fibres at microtu- lops koppesi, Iguana iguana, and Varanus gouldii, but present in bule doublets three and eight of the axoneme. all other taxa. Character 17: Linear mitochondrial cristae: Absent (0), Present (1). All species utilized here have linear mitochondrial cristae. Character 18: Mitochondria morphology in oblique section: Character optimization Columnar (0), trapezoidal (1), Sinuous (2). All species utilized Results are shown in Figs 5 and 6 with the formation character here have sinuous tubed mitochondria except Varanus gouldii, (character state). in which the tubes are columnar. Character 19: Mitochondria morphology in longitudinal section: Morphological phylogeny Trapezoidal (0), Rounded (1), Columnar (2). The mitochon- Results of character optimization on the morphological dria in longitudinal section are trapezoidal in Varanus gouldii, phylogeny show the beginning of the fibrous sheath in the rounded in Aspidites melancephalus, Corallus hortulanus, Epi- midpiece at mitochondrial tier 1 (Fig. 5, 23[3]) as the ancestral crates cenchria, Boiga irregularis, Bothrops alternatus, Bothrops condition for the Colubroidea and before mitochondrial tier diporus, Elaphe scalaris, Nerodia sipedon, Oxyuranus microlep- one for the Scolecophidia (Fig. 5, 23[4]). A circular acrosome idotus, Seminatrix pygaea, and the Scolecophidians, and are complex in cross-section, stopper-like perforatorial base plate, columnar in Boa constrictor amarali, Eryx jayakari, Crotalus present dense collar in the neck region, sinuous tubed durissus, Stegonotus cucullatus, and Iguana iguana. mitochondria in oblique section, rounded mitochondria in Character 20: Mitochondria morphology in transverse section: longitudinal section, solid dense bodies within the midpiece, Round ⁄ oval (0), Irregular shaped (1), Trapezoidal (2). The and presence of multilaminar membranes were recovered as shape of the mitochondria in transverse section is round ⁄ oval the ancestral character state for all snakes (Fig. 5, 2[0]; 10[1]; in all species except Leptotyphlops koppesi, where they are 15[1]; 18[2]; 19[1]; 21[0]; 27[1]). An absence of an acrosomal irregular, and in Iguana iguana, where they are trapezoidal. ridge, vacuity subdivision within the acrosome, knob-shaped Character 21: Morphology of dense bodies in the midpiece: Solid perforatorial base plate, round ⁄ oval mitochondria in trans- (0), Granular (1), Not applicable (because of the absence; 2). verse section, and the presence of an acrosome vesicle The dense bodies of the midpiece are absent in Eryx jayakari, subdivision were recovered as the ancestral state for all Bothrops alternatus, and Bothrops diporus, solid in all other squamates within this study (Fig. 5, 1[0]; 3[1]; 5[0]; 10[0]; 20[0]). snake species, and granular in both Iguana iguana and Varanus gouldii. Molecular phylogeny Character 22: Location of dense bodies in the midpiece: Optimization of character states on the molecular phylogeny Separated from the fibrous sheath (0), Juxtapositioned to the results in a non-applicable perforatorial base plate shape and fibrous sheath (1), Not applicable (because of the absence) (2). presence of extracellular microtubules as the ancestral state for The midpiece dense bodies are absent in Eryx jayakari, the Caenophidia (Fig. 6, 10[2]; 28[1]). A non-applicable Bothrops alternatus, and Bothrops diporus, but juxtapositioned perforatorial base plate shape, mitochondrial tier one as the to the fibrous sheath in all other species utilized here. beginning of the fibrous sheath, and presence of extracellular Character 23: Location of the beginning of the fibrous sheath microtubules were recovered as the ancestral state for the within the midpiece: Mitochondrial tier 4 (0), Mitochondrial Henophidia (Fig. 6, 10[2]; 23[3]; 28[1]). A stopper-like PBPS tier 3 (1), Mitochondrial tier two (2), Mitochondrial tier 1 (3), and beginning of the fibrous sheath before mitochondrial tier doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH Spermatozoon ultrastructure in Seminatrix pygaea 7

Fig. 5. Morphological phylogeny from Lee et al. 2007 with select ultrastructural sperm characters mapped one are the ancestral states for the Scolecophidia (minus analysed. The acrosome of S. pygaea contains no acrosomal Anomalepididae) (Fig. 6, 10[1], 23[4]). An absent acrosomal ridge, is circular in cross-sectional shape, and is highly ridge, rounded mitochondria in longitudinal section, and compartmentalized with an acrosome vesicle subdivision, presence of multilaminar membranes were recovered as subacrosomal cone, single perforatorium, and epinuclear ancestral states for all snakes excluding Liotyphlops beui electron lucent zone, which is consistent with all other snakes (Fig. 6, 1[0]; 19[1]; 27[1]). A circular acrosome complex in studied to date with the exception of single lineages suggesting cross-section and solid dense bodies within the midpiece were these may be conserved characters within the Serpentes recovered as the ancestral state for all Serpentes (Fig. 6 2[0]; (Jamieson 1991; Jamieson and Koehler 1994; Jamieson 1995; 21[0]). A present acrosomal subdivision, absent epinuclear Oliver et al. 1996; Cunha et al. 2008; Tavares-Bastos et al. zone, present perforatorial base plate, present dense collar 2008). Even though characters are highly conserved among within the neck region, and round ⁄ oval mitochondria in Serpentes, many appear to be ancestral among all squamates transverse section were recovered as the ancestral state for all (Jamieson 1991, 1995; Colli et al. 2007; Cunha et al. 2008; squamates studied to date (Fig. 6, 3[1]; 5[0]; 9[1]; 15[1]; 20[0]). Tavares-Bastos et al. 2008). The high compartmentalization of the acrosome of squamate sperm has been hypothesized to aid in the release of hydrolytic enzymes during fertilization (Talbot Discussion 1991). The nucleus of S. pygaea is electron dense and devoid of lacunae, which is thus far consistent among Serpentes with the Black swamp snake spermatozoon exception of Typhlops reticulatus (Tavares-Bastos et al. 2008). Because all snake sperm structures were optimized onto Surrounding the nucleus are multilaminar membranes, which phylogenies, we feel it is unnecessary to compare each have been seen in all snakes studied to date except Bothrops character of Seminatrix pygaea to other serpents; however, irregularis and Bothrops alternatus (Tourmente et al. 2008). we do observe some interesting character similarities ⁄ diﬀer- These multilaminar membranes have also been observed in ences between the spermatozoa of S. pygaea and other species pygopodids (Jamieson et al. 1996) and during spermiogenesis doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH 8 Rheubert, McMahan, Sever, Bundy, Siegel and Gribbins Aspidites melanocephalus Iguana iguana Ramphotyphlops waitii Ramphotyphlops endoterus Nerodia sipedon Elaphe scalaris Epicrates cenchria Eryx jayakuri Boiga irregularis Boa constrictor occidentalis Boa constrictor amarali Bothrops diporus Bothrops alternatus Liotyphlops beui Leptotyphlops koppesi Bolyeriidae* Loxocemidae* Xenopeltidae* Uropeltidae* Aniliidae* Tropidophiidae* Stegonotus cucullatus Seminatrix pygaea Homalopsidae* Oxyuranus microlepidotus Pareatidae* Xenodermidae* Acrochordidae* Crotalus durissus aspis Vipera Varanus gouldii Varanus reticulatus Typhlops Corallus hortulanus

10[2], 23[3], 28[1]

2[1], 10[0], 10[2], 28[1] 21[1], 23[2], 27[0], 28[0]

10[1], 23[4]

1[1], 19[?], 23[?], 1[0], 19[1], 27 [1] 27[?], 28[?] 2[0], 21[0]

3[1], 5[0], 9[1], 15[1], 20[0]

Fig. 6. Molecular phylogeny from Eckstut et al. 2009 with select ultrastructural sperm characters mapped in Anolis lineatopus (Rheubert et al. in press) resulting from only one to two lizard taxa examined in these analyses, vesicles originating from the endoplasmic reticula. character optimization across a phylogeny of Squamata will The flagellar structure of the S. pygaea spermatozoon has be necessary to determine whether some observed differences numerous similarities and differences to other snakes studied are truly distinct between snakes and lizards. Differences to date suggesting this may be a more variable portion of the found between Iguana and Varanus further show the need for mature sperm. However, surrounding the neck region and the a thorough examination of character evolution within the tail are extracellular microtubules that were observed in all Squamata. Thus far, character optimization on both the snakes studied to date and have been observed in immature morphological and the molecular phylogenies illustrates at spermatids during spermiogenesis (Gribbins et al. 2007, 2010) least a few apomorphic traits for some snake lineages. The and immature teiid sperm (Jamieson 1995). We assume that morphological tree provides more sperm ultrastructure traits the extracellular microtubules observed in association with the uniting snakes and lineages within, than does the molecular mature spermatozoa are remnants of the manchette not shed tree (see Fig. 1A,B). Thus, it appears that sperm ultrastruc- during spermiation. With previous reports of the microtubules ture may support phylogenies reconstructed from other of the manchette being involved in nuclear elongation during morphological data over the phylogeny reconstructed with spermiogenesis (Russell et al. 1990), the absence of these molecular data. However, the difference in the number of microtubules in non-scincids and non-serpents such as Anolis apomorphic character states between the two phylogenies lineatopus (Clark 1967; Healy and Jamieson 1994; Rheubert may be attributable to the number of outgroups utilized and et al. in press) may suggest that they serve another role besides the resolution within the Colubroidea clade. Only Varanus nuclear elongation during spermiogenesis, as sperm without was used as an outgroup in the morphological phylogeny of these tubules still acquire normal filiform shape. Lee et al. (2007), although Sanders and Lee (2008) and Eckstut et al. (2009) recovered Anguimorpha + Iguania as the sister lineage to snakes. Evolutionary trends Character optimization on the molecular phylogeny reveals Overall, it appears that some ultrastructural sperm traits may that the beginning of the fibrous sheath within the mid- be more conserved than others across the Serpentes. With piece, before miotchondrial tier 1, is synapomorphic for the doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH Spermatozoon ultrastructure in Seminatrix pygaea 9

Scolecophidia (minus Anomelepididae) and the beginning of the se tienen datos, en orden de poder hacer comentarios ma´s conclusivos fibrous sheath at mitochondrial tier 1 unites Boidae, Python- con respecto a la evolucioń de estructuras esperma´ticas dentro de las idae, and Colubroidea. No characters unite the boas and serpientes. pythons on the morphological tree, but three character states are synapomorphic for this clade on the molecular tree. Both References the morphological and the molecular trees show the presence *Al-Dokhi OA, Al-Onazee YZ, Mubarak M (2007) Fine structure of of an acrosome vesicle subdivision, absence of an acrosome epididymal sperm of the snake Eryx jayakari (Squamata, Reptilia). vesicle vacuity subdivision, and round ⁄ oval mitochondrial Int J Zoo Res 3:1–13. shape in transverse section uniting squamates, which has been *Camps JL, Bargallo´ R (1977) Espermatogeńesis de reptiles. I. proposed in previous studies (Jamieson 1991; Colli et al. 2007; Ultraestructura dose l espermatozoides de Elaphe scalaris (Schinz). Cunha et al. 2008; Tavares-Bastos et al. 2008). From this Bol R Soc Esp Hist Nat Secc Biol 75:439–446. study it appears that many of the characters associated with Clark AW (1967) Some aspects of spermiogenesis in a lizard. Am J Anat 121:369–400. the acrosome complex are conserved among snakes. However, Colli GR, Teixeira RD, Scheltinga DM, Mesquita DO, Wiederhec- the structures associated with the tail of the spermatozoon ker HC, Baó SN (2007) Comparative study of sperm ultrastruc- seem to be more variable (Hamilton and Fawcett 1968). ture of five species of teiid lizards (Teiidae, Squamata), and The main objective of this preliminary evolutionary anal- Cercosaura ocellata (Gymnophtalmidae, Squamata). Tissue Cell ysis of sperm ultrastructure data is to introduce hypotheses of 39:59–78. phylogenetic trends among snakes using sperm data, and in Cope ED (1871) Ninth contribution to the herpetology of tropical turn stimulate discussion and further research on this topic. America. Proc Acad Nat Sci Philadelphia 23:200–224. Cunha LD, Tavares-Bastos L, Baó SN (2008) Ultrastructural descrip- The preliminary trends elucidated thus far indicate that these tion and cytochemical study of the spermatozoon of Crotallus characters may prove useful as non-traditional morphological durissus (Squamata, Serpentes). Micron 39:915–925. characters in systematic analyses. One of the main hindrances Eckstut ME, Sever DM, White ME, Crother BI (2009) Phylogenetic in taking full advantage of sperm morphological data in analysis of sperm storage in female squamates. In: Danhof LT systematic analyses is taxonomic sampling. We hope that this ((ed.)), Animal Reproduction: New Research Developments. Nova study will encourage work on under-represented snake taxa Science Publishers Inc, Happauge, New York, pp 185–218. Furieri P (1965) Prime osservaioni al microscopio eletrronico sulla (e.g. homalopsids, tropidophiids, etc.), which will provide ultrastruttura dello spermatozoo di Viper aspis aspis L. Boll Soc Ital data crucial for investigating the systematic relationships Biol Sper 41:478–480. among squamates using sperm characters with more confi- Gribbins KM, Happ CS, Sever DM (2005) Ultrastructure of the dence. reproductive system of the black swamp snake (Seminatrix pygaea). V. The temporal germ cell development strategy of the testis. Acta Zool 86:223–230. Acknowledgements Gribbins KM, Mills EM, Sever DM (2007) Ultrastructural examination of spermiogenesis within the testis of the Ground Skink, The authors would like to thank Travis Ryan for providing the snakes Scincella laterale (Squamata, Sauria, Scincidae). J Morphol utilized in this research. We would also like to thank Diana Solis who 268:181–192. helped with the translation of the abstract for this manuscript. This Gribbins KM, Rheubert JL, Anzalone ML, Siegel DS, Sever DM study was funded by competitive research grants provided by (2010) Ultrastructure of spermiogenesis in the Cottonmouth, Wittenberg University (to JLR and KMG) and National Science Agkistrodon piscivorus (Squamata: Viperidae: Crotalinae). J Mor- Foundation Grant # DEB-0809831 (to DMS). A special thanks to two phol 271:293–304. anonymous reviewers in their helpful comments on this manuscript. Hamilton DW, Fawcett DW (1968) Unusual features of the neck and middlepiece of snake spermatozoa. J Ultrastruct Res 23:81–97. Harding RH, Aplin KP, Mazur M (1995) Ultrastructure of sperma- Resumen tozoa of Australian blindsnakes, Ramphotyphlops spp. (Typhlopi- Ultraestructura del Sistema Reproductivo de la Culebra Negra de dae, Squamata): first observations on the mature spermatozoon of Suampo (Seminatrix pygaea). VII. Morfologıá del Espermatozoo y Scolecophidian snakes. in: Jamieson BGM, Ausio J, Justine JL Tendencias Evolutivas de los Caracteres del Esperma en Serpientes (eds). Advances in Spermatozoal Phylogeny and Taxonomy, Me´m natn Hist nat Paris, Paris. 166:385–396. Este estudio investiga la evolucioń del espermatozoa de las serpientes Healy JM, Jamieson BGM (1994) The ultrastructure of spermatogen- en un contexto filogene´tico, con la adicioń de una nueva descripcioń esis and epididymal spermatozoa of the Tuatara Sphenodon punct- ultraestructural del espermatozoa de la culebra negra de suampo, atus (Sphenodontida, Amniota). Philos Trans R Soc Lond B Biol Sci Seminatrix pygaea. En general, el espermatozoo de S. pygaea es similar 344:187–199. al de los descritos para otros escamosos (Squamata), considerando que Hillis DM, Pollock DD, McGuire JA, Zwickl DJ (2003) Is sparse algunos caracteres como el espacio translucido del electroń, que separa taxon sampling a problem for phylogenetic inference? Syst Biol la corteza y la medula del complejo acroso´mico, puede que sea un 52:124–126. caraćter uńico para el espermatozoa de S. pygaea. El ana´lisis prelim- Jamieson BGM (1991) Fish Evolution and Systematics: Evidence from inar de la evolucioń esperma´tica dentro de Serpentes lleva a la Spermatozoa. Cambridge University Press, Cambridge, UK. hipo´tesis que algunos caracteres del esperma son ma´s pla´sticos que Jamieson BGM (1995) The ultrastructure of spermatozoa of the otros. Se encuentran incongruencias entre las topologıás moleculares y Squamata (Reptilia) with phylogenetic considerations. In: Jamieson morfolo´gicas utilizadas, y en la filogenia obtenida de un grupo de datos BGM, Ausio J, Justin JL (eds). Advances in Spermatozoal morfolo´gicos recupera ma´s caracterı´sticas ancestrales inequı´vocas de la Phylogeny and Taxonomy. Me´m natn Hist nat Paris, Paris, estructura del esperma de las serpientes. Caracteres tales como el 166:359–383. comienzo de la cubierta fibrosa en el nivel mitocondrial uno unen a Jamieson BGM (1999) Spermatozoal phylogeny of the Vertebrata. In: Colubroidea, mientras que caracteres como la presencia de una Cagnon C (ed.), The Male Gamete: From Basic Science to Clinical subdivisioń en la vesıćula acrosoma´tica, la ausencia de la subdivisioń Applications. Cache River Press, Vienna, USA, pp 303–331. de vacuidad, y una mitocondria circular ⁄ ovalada en seccioń transver- Jamieson BGM, Koehler L (1994) The ultrastructure of the sperma- sal unen a todos los escamosos. Aun ası´, gracias a este ana´lisis es tozoon of the Northern Water Snake, Nerodia sipedon (Colubridae, evidente que ma´s taxones necesitan ser estudiados y que se necesita Serpentes), with phylogenetic considerations. Can J Zool 72:1648– investigar ma´s minuciosamente los taxones para los cuales actualmente 1652.

doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH 10 Rheubert, McMahan, Sever, Bundy, Siegel and Gribbins

Jamieson BGM, Oliver SC, Scheltinga DM (1996) The ultrastructure Tavares-Bastos L, Teixeira RD, Colli GR, Baó SN (2002) Polymor- of the spermatozoa of Squamata- I. Scincidae, Gekkonidae, and phism in the sperm ultrastructure among four species of lizards Pygopodidae (Reptilia). Acta Zool 77:85–100. in the genus Tupinambis (Squamata: Teiidae). Acta Zool 83:297– Lee MSY, Hugall AF, Lawson R, Scanlon JD (2007) Phylogeny of 307. snakes (Serpentes): combining morphological and molecular data Tavares-Bastos L, Cunha LD, Colli GR, Baó SN (2007) Ultrastructure in likelihood Bayesian and parsimony analyses. Syst and Biod 5: of spermatozoa of scolecophidian snakes (Lepidosauria, Squamata). 371–389. Acta Zool 88:189–197. Maddison DR, Maddison WP (2005) MacClade Version 4.08. Sinauer Tavares-Bastos L, Colli GR, Baó SN (2008) The evolution of Associates, Inc, Sunderland MS, USA. sperm ultrastructure among Boidae (Serpentes). Zoomorphology 8: Newton WD, Trauth SE (1992) Ultrastructure of the spermatozoon of 62–68. the lizard Cnemidophorus sexlineatus (Sauria: Teiidae). Herpetolog- Teixeira RD, Vieira GHC, Colli GR, Baó SN (1999) Ultrastructural ica 48:330–343. study of spermatozoa of the neotropical lizards, Tropidurus semita- Oliver SC, Jamieson BGM, Scheltinga DM (1996) The ultrastructure eniatus and Tropidurus torquatus (Squamata, Tropiduridae). Tissue of spermatozoa of Squamata. II. Agamidae, Varanidae, Colubridae, Cell 31:308–317. Elapidae, and Boidae (Reptilia). Herpetologica 52:216–241. Tourmente M, Cardozo G, Bertona M, Guidobaldi A, Giojalas L, Rheubert JL, Wilson B, Wolf K, Gribbins KM (In press) Ultrastruc- Chiaraviglio M (2006) The ultrastructure of the spermatozoa of Boa tural morphology of spermiogenesis in the Jamaican Gray Anole, constrictor occidentalis, with considerations on its mating system and Anolis lineatopus (Reptilia: Polychrotidae). Acta Zool, doi: 10.111/ sperm competition theories. Acta Zool 87:25–32. j.1463-6395.2009.00446.x. Tourmente M, Giojalas L, Chiaraviglio M (2008) Sperm ultrastructure Russell LD, Ettlin RA, Hikim AMP, Cleff ED (1990) Histological and of Bothrops alternatus and Bothrops diporus (Viperidae, Serpentes), Histopathological Evaluation of The Testis. Cache River Press, and its possible relation to the reproductive features of the species. Clearwater, FL. Zoomorphology 127:241–248. Sanders KL, Lee MSY (2008) Molecular evidence for a rapid late- Vieira GHC, Colli GR, Baó SN (2004) The ultrastructure of the Miocene radiation of Australasian venomous snakes (Elapidae, spermatozoon of the lizard Iguana iguana (Reptilia, Squamata, Colubroidea). Molec Phylo Evol 46:1180–1188. Iguanidae) and the variability of sperm morphology among iguanian Sever DM (2004) Ultrastructure of the reproductive system of the lizards. J Anat 204:451–464. black swamp snake (Seminatrix pygaea). IV. Occurrence of an Wiens JJ (2004) The role of morphological data in phylogeny ampulla ductus deferentis. J Morphol 262:714–730. reconstruction. Syst Biol 53:653–661. Sever DM (2010) Ultrastructure of the reproductive system of the Zwickl DJ, Hillis DM (2002) Increased taxon sampling greatly reduces black swamp snake (Seminatrix pygaea). VI. Anterior testicular phylogenetic error. Syst Biol 51:588–598. ducts and their nomenclature. J Morphol 274:104–115. Sever DM, Ryan TJ (1999) Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea): Part I. Evidence for Supporting Information oviducal sperm storage. J Morphol 241:1–18. Sever DM, Ryan TJ, Morris T, Patton D, Swafford S (2000) Additional Supporting Information may be found in the online Ultrastructure of the reproductive system of the black swamp snake version of this article: (Seminatrix pygaea). II. Annual oviducal cycle. J Morphol 245:146– Table S1. Data matrix for ultrastructural sperm characters 160. among snakes. Characters are defined in Results section. Sever DM, Ryan TJ, Stephens R, Hamlett WC (2002) Ultrastructure References for electron micrographs are listed on the right. of the reproductive system of the black swamp snake (Seminatrix Please note: Wiley-Blackwell are not responsible for the pygaea). III. Sexual segment of the male kidney. J Morphol 252:238– content or functionality of any supporting materials supplied 254. Talbot P (1991) Compartmentalization in the acrosome. In: Bacetti B by the authors. Any queries (other than missing material) (ed), Comparative Spermatology – 20 Years After. Raven Press, should be directed to the corresponding author for the New York, pp 255–259. article.

doi: 10.1111/j.1439-0469.2010.00573.x 2010 Blackwell Verlag GmbH