The urogenital system of male squamates consists of the testis, testicular ducts, and sexual segment of the kidney. These structures undergo seasonal variation, and their morphology is variable across taxa. Despite multiple studies examining the morphology of the various components of the reproductive system, few studies have examined squamates in sympatry. The purpose of this study was to investigate the morphology and seasonal variation of the reproductive system in two taxa that are sympatric with one another, Anolis carolinensis and Sceloporus undulatus, from southeastern Louisiana. Specimens were collected monthly for an entire year and processed for histological and ultrastructural analysis. Both species have a single rete testis leading from the testis to the ductuli efferentes, which leads into the proximal portion of the epididymis. The rete testis consists of a simple squamous epithelium with irregularly shaped nuclei, the ductuli efferentes consists of a ciliated simple cuboidal epithelium, and the epididymis a pseudostratified columnar epithelium. No differences were observed in the morphology of the urogenital systems between taxa but phenological variation was observed in terms of the tissue development for reproduction. Anolis carolinensis has an extended active period and two separated periods of maximal activity evidenced by two peaks in the ductuli efferentes epithelial height and tubular diameter (March and July) whereas Sceloporus undulatus only had a single peak occurring in June. These results suggest that reproductive morphology may be conserved at taxonomic levels above family level, and reproductive activity may be constrained phylogenetically and is not solely dependent on environmental factors, at least in terms of geographic range, though it is acknowledged that microhabitats may play a major role in cyclicity of reproductive events. Future studies examining species at various taxonomic levels, as well as species in sympatry, will provide additional insight into hypotheses concerning abiotic factors and reproductive biology.

Gladwyn Kingsley Noble was the first investigator to collectively examine courtship glands and correlate their function to the tail-straddling walk in plethodontid salamanders. While mental glands and caudal courtship glands have received the majority of attention since Noble's seminal work, Noble described other glands from Eurycea bislineata that were putatively involved in courtship that have received little or no attention (e.g., glands at the temporal regions of the heads of males). Previous studies demonstrated that heads of males enlarge during the mating season and some studies indicated that the enlargement was because of Noble's previously described temporal glands; however, current consensus of male head enlargement is that skeletal muscle hypertrophy is the cause of the head growth. In this study, we examined male and female E. bislineata throughout the year to test Noble's hypothesis that males possess courtship glands in the integument of the temporal regions of their heads and to assess what underlying tissues are involved with male head enlargement during mating. We found that the temporal regions of male heads change dramatically from non-mating to mating months, exemplified by two-dimensional geometric morphometrics. This variation is a result of M. levator mandibulae externus hypertrophy rather than glandular activity underlying the integument. Although no glandular masses isolated at the temporal regions of the heads result in this dynamic shape change, simple alveolar glands that resemble stereotypical courtship glands are found in the integument of only males from the mating season. These putative courtship glands are scattered amongst mucous and serous glands within the integument around the eyelids to at least the posterior termination of the cranium. The function of these glands is unknown but is probably involved with pheromone production, similar to more thoroughly examined courtship glands.

The Eurycea bislineata complex ("two-lined salamanders") of eastern North America contains six described species, of which three have very similar morphologies and relatively broad geographic distributions, and three have more divergent morphologies with narrow geographic distributions. Recent molecular phylogenetic analyses found that four of the six species in the complex contain deep genetic structure, and that two of the species (E. cinigera and E. wilderae) are paraphyletic in mitochondrial and nuclear DNA, inferring that current taxonomy does not reflect actual species boundaries in the complex. The member of this complex in the Sandhilis physiographic region of south-central North Carolina, USA, is notable for its distinctive coloration and ecology, and a study on allozymic variation published over 30 years ago demonstrated that it is genetically distinct but clustered within the paraphyletic E. cirrigera. This study investigates the taxonomic status of the Sandhills population using morphology, mitochondria] DNA, and a 21-locus nuclear DNA data set to test if the Sandhills taxon represents a local ecomorph that is conspecific with adjacent populations of E. cirrigera, or if it represents a divergent evolutional), lineage that warrants taxonomic recognition. Mitochondria! and nuclear DNA revealed that the Sandhills taxon is genetically distinct and phylogenetically unrelated to adjacent populations of E. cirrigera. Principal components analysis of 316 adult specimens of the Sandhills taxon and the three morphologically similar species in the complex found considerable overlap among these fain but pairwise comparisons of heavily loading morphological characters showed that the Sandhills taxon usually has a shorter body, shorter tail, and narrower head. Based on these corroborated lines of evidence, the hypothesis that the Sandhills taxon represents only a local ecomorph is rejected and it is described as a new species. Clarification of the extent of its geographic range (including its possible presence in South Carolina), differentiation of larvae from other members of the complex, and verification of hypothesized narrow zones of hybridization with E. cirrigera at the peripheries of its geographic range are needed. The description of the Sandhills taxon brings the number of endemic salamander species in North Carolina to seven. Integrative taxonomic revisions of the E. bislineata complex, particularly E. cirrigera and E. wilderae, are needed to estimate species diversity and distributions of these salamanders more accurately.

Salamanders in the family Plethodontidae exhibit a unique tail-straddle walk during courtship that can include the use of sexually dimorphic mental and caudal courtship glands. This study presents novel histological and fine structure data on mental glands and caudal courtship glands in Plethodon mississippi, Desmognathus conanti and Eurycea quadridigitata using both light microscopy and scanning electron microscopy. This study represents the first use of scanning electron microscopy to observe these glands. Both mental and caudal courtship glands were observed to vary seasonally in gland diameter and histology according to the breeding season of each species. Morphological variation was observed across the three species studied in both clustering and relative size of the glands compared to neighbouring mucous and granular glands. Hypertrophied mental glands are larger than mucous or granular glands in all species, but relationships among caudal courtship glands and other skin glands vary among species. In E. quadridigitata, active caudal courtship glands are larger than mucous and granular glands, but in D. conanti, caudal courtship glands are similar in size to granular glands and larger than mucous glands. In P. mississippi, caudal courtship glands are scattered among significantly larger granular glands and are similar in size to mucous glands.

Amphibian skin is unique among vertebrate classes, containing a large number of multicellular exocrine glands that vary among species and have diverse functions. The secretions of skin glands contain a rich array of bioactive compounds including antimicrobial peptides (AMPs). Such compounds are important for amphibian innate immune responses and may protect some species from chytridiomycosis, a lethal skin disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). While the bioactivity of skin secretions against Bd has been assessed for many amphibian taxa, similar studies are lacking for Bsal, a chytrid fungus that is especially pathogenic for salamanders. We studied the skin glands and their potential functions in an aquatic salamander, the three-toed amphiuma (Amphiuma tridactylum). Skin secretions of captive adult salamanders were analyzed by RP-HPLC and tested against the growth of Bd and Bsal using in vitro assays. We found that compounds within collected skin secretions were similar between male and female salamanders and inhibited the growth of Bd and Bsal. Thus, skin secretions that protect against Bd may also provide protection against Bsal. Histological examination of the skin glands of preserved salamanders revealed the presence of enlarged granular glands concentrated within caudal body regions. A site of potential gland specialization was identified at the tail base and may indicate specialized granular glands related to courtship and communication.

Nasolacrimal ducts are a terrestrial vertebrate adaptation and appear to have co-evolved with orbital glands. Although plethodontid salamanders possess orbital glands, a recent study concluded that plethodontid salamanders lack nasolacrimal ducts. Functionally, the absence of nasolacrimal ducts closes the route for orbital gland secretion passage into the nasal and vomeronasal organ cavities. Orbital glands have been implicated in enhancement of vomeronasal function so loss could have important implications for communication. Multiple older studies depict or discuss nasolacrimal ducts in plethodontid salamanders. Interestingly, the only consensus between recent and older literature is that Desmognathus lacks nasolacrimal ducts. To determine if plethodontid salamanders truly lack nasolacrimal ducts, we sectioned plethodontid salamander heads for general histological examination of species from the majority of the plethodontid tribes. From our representative sample, we found only two species that completely lacked nasolacrimal ducts (Desmognathus fuscus and Eurycea tynerensis) and one species that possessed nasolacrimal ducts that ended blindly before reaching the nasal cavities (E. spelaea). Bayesian ancestral state reconstruction resulted in the presence of nasolacrimal ducts on the branch leading to Plethodontidae and both subfamilies within Plethodontidae, with two independent losses in Desmognathus and Eurycea. Anat Rec, 301:765-775, 2018. © 2017 Wiley Periodicals, Inc.

Previous studies demonstrated that variation in sperm morphology exists below the species level in a variety of organisms. However, most of the studies focus on invertebrates with only a few recent studies on vertebrates with a majority on birds and mammals. Understanding variation at various taxonomic levels is necessary for comparative studies. Therefore, to test the hypothesis that sperm shows little variation between and within closely related taxa, sperm morphology was analyzed in multiple populations of two sister species of lizards, Sceloporus consobrinus and Sceloporus undulatus. The ultrastructure of the sperm did not differ intraspecifically nor interspecifically, but differences were observed in sperm morphometrics between populations. The data gathered in this study show that the ultrastructure of the spermatozoa is consistent with other squamates, and although no variation was observed between the two taxa, slight variations were observed when compared to other members of Phrynosoma. The observed differences in sperm morphometrics show that sperm size differs despite variation in ultrastructure and there may be differences in selection on sperm size between the populations.

G. Kingsley Noble reported the possibility of orbital gland involvement in reproduction of spelerpine salamanders, particularly Eurycea, through rudimentary gross examination of the heads of male and female salamanders. In his report, Noble provided details from only one taxon of salamander, E. bislineata. To examine the variability of orbital glands with secondary sexual function in Eurycea, orbital glands of E. longicauda were assessed for sexual dimorphism and a distinct seasonal secretory cycle that mirrored the reproductive cycle. Orbital glands were found not to be sexually dimorphic in E. longicauda and possessed constant secretory activity in males and females throughout the year. Known secondary sexual glands (mental glands, caudal courtship glands, and cloacal glands) were sexually dimorphic between males and females and possessed a secretory cycle that could be correlated with events of the reproductive cycle, particularly mating activity. Thus, no evidence was found for orbital gland involvement in reproduction in E. longicauda. Orbital glands of both male and female E. longicauda are morphologically identical and produce copious lipoproteins throughout the year. Orbital gland secretions are potentially involved in lubrication of the eye and the prevention of fluid evaporation from the eye as described for lipoprotein secretions in other vertebrate lineages.

Little is known about spermatid development during spermiogenesis in snakes, as there is only one complete study in ophidians, which details the spermatid ultrastructure within the viperid, Agkistrodon piscivorus. Thus, the following study will add to our understanding of the ontogenic steps of spermiogenesis in snakes by examining spermatid maturation in the elapid, Pelamis platurus, which were collected in Costa Rica in 2009. The spermatids of P. platurus share many similar ultrastructural characteristics to that described for other squamates during spermiogenesis. Three notable differences between the spermatids of P. platurus and those of other snakes is a round and shorter epinuclear lucent zone, enlarged caudal nuclear shoulders, and more prominent 3 and 8 peripheral fibers in the principal and endpieces. Also, the midpiece is much longer in P. platurus and is similar to that reported for all snakes studied to date. Other features of chromatin condensation and morphology of the acrosome complex are similar to what has been observed in A. piscivorus and other squamates. Though the spermatids in P. platurus appear to be quite similar to other snakes and lizards studied to date, some differences in subcellular details are still observed. Analysis of developing spermatids in P. platurus and other snakes could reveals morphologically conserved traits between different species along with subtle changes that could help determine phylogenetic relationships once a suitable number of species have been examined for ophidians and other squamates.

The skin glands and cloacal morphology of the Korean crevice salamander, Karsenia koreana, were similar to those of other plethodontids. The skin contained mucous, granular, and modified granular glands in varying frequencies and sizes. Males had sexually dimorphic glands in the skin of the chin (mental glands) and the dorsal tail base (caudal courtship glands). On the ventral surface of the tail base, modified granular glands were sexually dimorphic in size, with male glands larger than those in females. The cloacal glands in males, as in other plethodontids, consisted of four eosinophilic gland clusters (dorsal pelvic glands, lateral pelvic glands, caudal pelvic glands, and vent glands) and three basophilic glands (anterior ventral glands, posterior ventral glands, and Kingsbury's glands). In females, the only cloacal gland was the spermatheca, which, as in other plethodontids, was a compound tubulo-alveolar gland in the roof of the cloaca.

Mental glands and their associated delivery behaviors during courtship are unique to the plethodontid salamanders. Because previous interpretations of the evolution of these features were conducted using older phylogenetic hypotheses, we reanalyzed these traits with newer courtship descriptions and contemporary phylogenetic methods. Using Bayesian ancestral state reconstruction methods that have been developed since the first phylogenetic analyses were conducted in the mid-1990s, we reconstructed mental gland and courtship behavior evolution on a Bayesian phylogeny of the nuclear gene Rag1. The most probable ancestral condition for plethodontids was resolved as presence of a mental gland. Loss of a mental gland occurred in each subfamily and was recovered as the most probable ancestral condition for the Spelerpinae. In contrast, parsimony reconstruction recovered the presence of a mental gland in the ancestor to Spelerpinae with multiple secondary losses. We hypothesize that that absence of a mental gland is possibly ancestral in some clades (i.e., Spelerpinae) and secondary in others (e.g., paedomorphic Eurycea). The most probable ancestral form of the mental gland is likely to be the large pad-type distributed extensively in Plethodontinae and Bolitoglossinae. Desmognathans have the most unique mental glands, occurring in an anterior protrusion or bifurcated form (in Desmognathus wrighti). Fan-shaped mental glands evolved independently in Eurycea and Oedipina. Small pads arose independently in Bolitoglossinae, Plethodontinae, and Spelerpinae. Head-rubbing behavior for mental gland delivery mode was recovered as the most probable and parsimonious ancestral state for the Plethodontidae, with independent losses of this behavior in Plethodontinae and Spelerpinae. Because head-rubbing was observed in outgroups, we hypothesize that head-rubbing behavior predated mental gland evolution. Pulling, snapping, slapping, and biting behaviors evolved independently in the Plethodontinae and Spelerpinae and are not homologous with head-rubbing. All hypotheses of mental gland and courtship evolution invoke homoplasy.

Several anuran species of the genus Phyllomedusa are known to possess specialized cutaneous glands producing lipids and exhibit a peculiar wiping behavior. This behavior is a stereotyped repertory of fore and hind limb movements distributing hydrophobic molecules onto the body surface and reducing evaporative water loss. No reports are presently available on the occurrence of lipid glands in other phyllomedusine genera, and data on the structure of the secretory units specialized for the production of cutaneous lipids are still unclear. The present report is aimed to answer both questions: it describes lipid glands of the Phyllomedusa type in Agalychnis callidryas and provides light and transmission electron microscope evidence of the syncytial structure of their secretory units, a typical feature of serous glands in anuran skin. This morphological trait supports the hypothesis that lipid glands are a specialized subset of the anuran serous glands, and underlines their flexible role in the skin adaption to sub-aerial environments. Anat Rec, 300:503-506, 2017. © 2016 Wiley Periodicals, Inc.

Studies on reptilian sperm morphology have shown that variation exists at various taxonomic levels but studies on the ontogeny of variation are rare. Sperm development follows a generalized bauplan that includes acrosome development, nuclear condensation and elongation, and flagellar development. However, minute differences can be observed such as the presence/absence of manchette microtubules, structural organization during nuclear condensation, and presence/absence of a nuclear lacuna. The purpose of this investigation was to examine sperm development within the Sceloporus genus. The process begins with the development of an acrosomal complex from Golgi vesicles followed by nuclear condensation and elongation, which results in the presence of a nuclear lacuna. As the acrosomal complex differentiates, flagellar development commences with elongation of the distal centriole. Spermatid development culminates in a mature spermatid with a highly differentiated acrosomal complex, a condensed nucleus with a nuclear lacuna, and a differentiated flagellum. Although the overall developmental pattern is consistent with other squamate species, minute differences are observed, even within the same genus. For example there is variation in the presence/absence of an endoplasmic reticulum complex during acrosome development, presence/absence of a nuclear lacuna, and presence/absence of manchette microtubules within the three species of Sceloporus studied to date. Future studies concerning sperm morphology in closely related species will aid in our understanding of variation in sperm development and may prove to be useful in testing phylogenetic and evolutionary hypotheses.

1 Department of Biology, Southeast Missouri State University, Cape Girardeau, MO 63701, USA. 2 CNRS-UMR5175 CEFE, Centre d’Ecologie Functionnelle et Evolutive, 1919 route de mende, 34293 Montpellier cedex 5. 3 College of Sciences, The University of Findlay, Findlay, Ohio 45840, USA. 4 Department of Biological Sciences, Southeastern Louisiana University, Hammond, LA 70402, USA. * Corresponding author 6

Recent studies detailed the spermatogenic cycle of the Western Cottonmouth Snake, Agkistrodon piscivorus and noted that spermatogenesis is bimodal, with active periods during March-June and August-October in southeastern Louisiana. However, only spermatogonia were present in September in the only specimen that was captured and the authors state that the individual "should have a high testis volume and also show spermiogenic activity." The specimen in their study was caught immediately following Hurricane Katrina outside of its normal habitat. Therefore, in order to verify their assumption, individuals were captured during September of 2008 and the testes were spermatogenically active with spermatogonia, spermatocytes, and mature spermatozoa being present in the seminiferous epithelium of the testes. These data indicate that Hurricane Katrina could have had an impact on the spermatogenic cycle in Cottonmouths, resulting in stress-induced testicular regression.

Data on cloacal anatomy were gathered for 128 species of salamanders, representing 48 genera and the nine extant families. Twenty-five characters were recognized, but after resolution of polymorphisms and elimination of autapomorphies, only 12 characters remained for use in phylogenetic analyses at the family level. Weighting the characters equally and using branch-andbound parsimony algorithms, 15 (PAUP) or 9 (Hennig86), equally parsimonious cladograms were revealed, each with 19 steps and a consistency index of 0.63. The variation in phylogenies is due to opposing hypotheses concerning convergence or reversal in two characters: the extent of epidermal lining in the cloaca, and the presence of primary and secondary folds in the cloacal tube. The strict consensus cladogram gives the topology: Sirenidae (Hynobiidae, Cryptobranchidae (Amphiumidae (Salamandridae (Ambystomatidae, Dicamptodontinae (Rhyacotritoninae, Plethodontidae, Proteidae))))). These results and consensus analyses with other cladograms based upon morphological data support the notion that the Salamandroidea, as defined by Duellman and Trueb (1986), is a monophyletic group and that internal fertilization evolved once, and is a synapomorphy for the

This paper represents the first ultrastructural descriptions of the cloacal glands of a male salamander. All male salamanders in the suborder Salamandroidea possess these glands, which are required for internal fertilization. These glands are responsible for producing spermatophores and, in some species, certain glands produce female-attracting pheromones. Both scanning and transmission electron microscopy provided higher resolution and magnification of the cytology of cloacal glands than possible in previous studies limited to light microscopy. The glands are hypertrophied in October and April samples, and much reduced in size and secretory activity in June and August samples. Each of the cloacal glands, when active, has secretory vacuoles that are unique in appearance and that secrete carbohydrates and/or proteins. Kingsbury's glands are modified mucous glands that have secretory vacuoles filled with a flocculent substance. Active pelvic glands have uniform electron-lucent secretory vacuoles that fill the cytoplasm and narrow the lumen. Ventral glands have squamous epithelium with moderately dense vacuoles scattered around the apical border, and a granular secretion that fills the wide lumen. In the breeding season, vent glands have biphasic secretory vacuoles characteristic of glycoproteins. All glands release their products by a merocrine process, and all have myoepithelial sheaths, which are best developed in ventral glands. Future research should focus on differences in cytology among the pheromone producing glands and comparisons with the ventral glands in salamander clades that do not produce spermatophores (e. g., Hynobiidae and Cryptobranchidae).

Previous investigators have described the spermatogenic cycles of numerous species of plethodontid salamanders. Most studies describe a fairly stereotypical cycle with meiotic divisions of spermatogenesis commencing in the spring/summer. However, many studies lack details obtainable from histological examination and/or testicular squashes and, instead, provide only mensural data from the testes. Studies that lacked microscopic evaluation often revealed spermatogenic cycles that varied greatly from that of the stereotypical cycle with meiotic divisions commencing in the fall/winter. Those studies hamper comparisons between the spermatogenic cycles of different species and their environments, as they do not provide a correlation between testicular size and any aspect of the spermatogenic cycle. In the following manuscript, we elucidate the spermatogenic cycle of Eurycea longicauda longicauda in an effort to outline an appropriate protocol for analyzing spermatogenesis in salamanders that will facilitate future comparative studies. Like many Nearctic plethodontids, E. l. longicauda exhibits a meiotic wave that travels through the testes during the summer; this process is followed by spermiogenesis, spermiation, and recrudescence in the fall, winter, and spring.