Parallel evolution of placental calcium transfer in the lizard Mabuya and eutherian mammals.

An exceptional case of parallel evolution between lizards and eutherian mammals occurs in the evolution of viviparity. In the lizard genus Mabuya, viviparity provided the environment for the evolution of yolk-reduced eggs and obligate placentotrophy. One major event that favored the evolution of placentation was the reduction of the eggshell. As with all oviparous reptiles, lizard embryos obtain calcium from both the eggshell and egg yolk. Therefore, the loss of the eggshell likely imposes a constraint for the conservation of the egg yolk, which can only be obviated by the evolution of alternative mechanisms for the transport of calcium directly from the mother. The molecular and cellular mechanisms employed to solve these constraints, in a lizard with only a rudimentary eggshell such as Mabuya, are poorly understood. Here, we used RT-qPCR on placental and uterine samples during different stages of gestation in Mabuya, and demonstrate that transcripts of the calcium transporters trpv6, cabp28k, cabp9k and pmca are expressed and gradually increase in abundance through pregnancy stages, reaching their maximum expression when bone mineralization occurs. Furthermore, CABP28K/9K proteins were studied by immunofluorescence, demonstrating expression in specific regions of the mature placenta. Our results indicate that the machinery for calcium transportation in the Mabuya placenta was co-opted from other tissues elsewhere in the vertebrate bodyplan. Thus, the calcium transportation machinery in the placenta of Mabuya evolved in parallel with the mammalian placenta by redeploying the expression of similar calcium transporter genes.

Plasma estradiol and progesterone concentrations during the female reproductive cycle in a highly placentotrophic viviparous lizard, Mabuya sp.

The neotropical genus Mabuya are obligate placentotrophic viviparous lizards, which have a short vitellogenesis that produces microlecithal oocytes and a prolonged time of gestation (9 to 10 months). The hormonal control of female reproductive activity during follicular growth and pregnancy has not been studied, although it is known that the corpus luteum can produce progesterone, but regresses early in pregnancy, being replaced in this function by the placenta. Through enzyme immunoassay (EIA) we measured the plasma concentrations of estradiol (E2) and progesterone (P4) in females of a population of Mabuya sp at different stages of their reproductive cycle. Previously, we confirmed the presence of P4 in plasma by high-performance liquid chromatography methods with diode-array detector ultraviolet (HPLC-DAD-UV). The average concentration values of E2 and P4 were compared among reproductive stages and their dynamics were related to what is known in other oviparous and viviparous amniotes. The plasma E2 concentrations of Mabuya sp. are below the levels found in other viviparous reptiles, probably related to the substantial reduction of its follicular growth phase. Its highest concentration was detected during vitellogenesis, related to its function in the growth and maturation of the ovarian follicles and oviduct preparation for pregnancy; lower levels were observed during pregnancy, but they increase at the end when a new vitellogenesis event begins and massive placental maternal-fetal nutrient transfer occurs. High concentrations of P4 were found during pregnancy, related to its function in the maintenance of the developing embryos within the oviduct. The highest levels of P4 were found at early gestation, then they descend from mid-gestation to the end of gestation. Although some characteristics of hormonal control related to the high level of placentotrophy were observed in this species, the changes in plasma sex steroid concentrations during the reproductive cycle in females of Mabuya sp. follow patterns seen in other viviparous amniotes.

An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard.

Syncytins are envelope genes from endogenous retroviruses that have been captured during evolution for a function in placentation. They have been found in all placental mammals in which they have been searched, including marsupials. Placental structures are not restricted to mammals but also emerged in some other vertebrates, most frequently in lizards, such as the viviparous Mabuya Scincidae. Here, we performed high-throughput RNA sequencing of a Mabuya placenta transcriptome and screened for the presence of retroviral env genes with a full-length ORF. We identified one such gene, which we named "syncytin-Mab1," that has all the characteristics expected for a syncytin gene. It encodes a membrane-bound envelope protein with fusogenic activity ex vivo, is expressed at the placental level as revealed by in situ hybridization and immunohistochemistry, and is conserved in all Mabuya species tested, spanning over 25 My of evolution. Its cognate receptor, required for its fusogenic activity, was searched for by a screening assay using the GeneBridge4 human/Chinese hamster radiation hybrid panel and found to be the MPZL1 gene, previously identified in mammals as a signal-transducing transmembrane protein involved in cell migration. Together, these results show that syncytin capture is not restricted to placental mammals, but can also take place in the rare nonmammalian vertebrates in which a viviparous placentotrophic mode of reproduction emerged. It suggests that similar molecular tools have been used for the convergent evolution of placentation in independently evolved and highly distant vertebrates.

Immunohistochemical localization of 3ß-Hydroxysteroid dehydrogenase and progesterone receptors in the ovary and placenta during gestation of the placentotrophic lizard Mabuya sp (Squamata: Scincidae).

In squamates, progesterone (P) plays a key role in the inhibition of uterine mobility during egg retention in oviparous species, and during gestation in viviparous species. The corpus luteum (CL) is the main organ responsible for the production of P; however, in some species, the CL degenerates early and the P needed for gestation maintenance should be produced in other tissues. Mabuya sp (Scincidae) is a viviparous lizard with a prolonged gestation, it produces microlecithal eggs and, consequently, has an obligate placentotrophy related with a highly complex placenta. Its CL degenerates at early stages of gestation and therefore, other sources of P should exist. The aim of this study was to determine and localize by immunohistochemistry the production of P by detection of the enzyme 3ß-Hydroxysteroid dehydrogenase (3ß-HSD) and P receptors (PR) during gestation in the ovary and placenta of Mabuya sp. Positive and negative control sections were used. The ovary of this species localizes 3ß-HSD and PR in the same tissues. The CL of the ovaries of females at early stages of gestation were positive for both molecules, whereas they did not localize from mid gestation to the end of pregnancy. Previtellogenic and vitellogenic follicles labelled for both molecules in the follicular epithelium and thecae. The placenta of Mabuya sp. demonstrated the potential for P production from mid gestation to the end of gestation in the uterine and chorionic tissues. PR were located in the uterine tissues throughout gestation, with a decrease towards its completion. Western blot analysis confirmed the presence of 3ß-HSD mainly in the ovary of early pregnant females and in the placental tissues at mid gestation stages. Therefore, the chorioallantoic placenta of Mabuya sp. has an endocrine function producing the P needed for gestation and replacing the CL from mid gestation to the end of pregnancy.

Proteomic Profile of Mabuya sp. (Squamata: Scincidae) Ovary and Placenta During Gestation.

Reptiles are one of the most diverse groups of vertebrates, providing an integrated system for comparative studies on metabolic, animal physiology, and developmental biology. However, the molecular data available are limited and only recently have started to call attention in the "omics" sciences. Mabuya sp. is a viviparous placentrotrophic skink with particular reproductive features, including microlecithal eggs, early luteolysis, prolonged gestation, and development of a highly specialized placenta. This placenta is responsible for respiratory exchange and the transference of all nutrients necessary for embryonic development. Our aim was to identify differentially expressed proteins in the ovary and placenta of Mabuya sp. during early, mid, and late gestation; their possible metabolic pathways; and biological processes. We carried out a comparative proteomic analysis during gestation in both tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization. Differential protein expression in both tissues (Student's t-test P < 0.05) was related to several processes such as cell structure, cell movement, and energy. Proteins found in ovary are mainly associated with follicular development and its regulation. In the placenta, particularly during mid and late gestation, protein expression is involved in nutrient metabolism, transport, protein synthesis, and embryonic development. This work provides new insights about the proteins expressed and their physiological mechanisms in Mabuya sp. placenta and ovary during gestation.

Molecular phylogenetics and biogeography of the Neotropical skink genus Mabuya Fitzinger (Squamata: Scincidae) with emphasis on Colombian populations.

Understanding the phylogenetic and geographical history of Neotropical lineages requires having adequate geographic and taxonomic sampling across the region. However, Colombia has remained a geographical gap in many studies of Neotropical diversity. Here we present a study of Neotropical skinks of the genus Mabuya, reptiles that are difficult to identify or delimit due to their conservative morphology. The goal of the present study is to propose phylogenetic and biogeographic hypotheses of Mabuya including samples from the previously under-studied territory of Colombia, and address relevant biogeographic and taxonomic issues. We combined molecular and morphological data sampled densely by us within Colombia with published data representing broad sampling across the Neotropical realm, including DNA sequence data from two mitochondrial (12S rRNA and cytochrome b) and three nuclear genes (Rag2, NGFB and R35). To evaluate species boundaries we employed a general mixed Yule-coalescent (GMYC) model applied to the mitochondrial data set. Our results suggest that the diversity of Mabuya within Colombia is higher than previously recognized, and includes lineages from Central America and from eastern and southern South America. The genus appears to have originated in eastern South America in the Early Miocene, with subsequent expansions into Central America and the Caribbean in the Late Miocene, including at least six oceanic dispersal events to Caribbean Islands. We identified at least four new candidate species for Colombia and two species that were not previously reported in Colombia. The populations of northeastern Colombia can be assigned to M. zuliae, while specimens from Orinoquia and the eastern foothills of the Cordillera Oriental of Colombia correspond to M. altamazonica. The validity of seven species of Mabuya sensu lato was not supported due to a combination of three factors: (1) non-monophyly, (2) <75% likelihood bootstrap support and <0.95 Bayesian posterior probability, and (3) GMYC analysis collapsing named species. Finally, we suggest that Mabuya sensu stricto may be regarded as a diverse monophyletic genus, widely distributed throughout the Neotropics.

Embryonic development of the skull of the Andean lizard Ptychoglossus bicolor (Squamata, Gymnophthalmidae).

The study of cranial design and development in Gymnophthalmidae is important to understand the ontogenetic processes behind the morphological diversity of the group and to examine the possible effects of microhabitat use and other ecological parameters, as well as phylogenetic constraints, on skull anatomy. Complete morphological descriptions of embryonic skull development within Gymnophthalmidae are non-existent. Likewise, very little is known about the complete chondrocranium of the family. Herein, the development of the skull of the semi-fossorial lizard Ptychoglossus bicolor is described along with an examination of the chondrocranium of other gymnophthalmid taxa and the teiid Cnemidophorus lemniscatus. Cranial chondrification begins with early condensations in the ethmoid, orbitotemporal and occipital regions of the chondrocranium as well as the viscerocranium. Ossification of the skull starts with elements of the dermatocranium (pterygoid, prefrontal, maxilla and jugal). The orbitosphenoid is the last chondral bone to appear. At birth, the skull is almost completely ossified and exhibits a large frontoparietal fontanelle. In general terms, the chondrocranium of the gymnophthalmids studied is characteristic of lacertiform terrestrial lizards, in spite of their life habits, and resembles the chondrocranium of C. lemniscatus in many aspects. However, the gymnophthalmids show great variation in the orbitosphenoid and a complex nasal capsule. The latter exhibits greater development of some nasal cartilages, which make it more complex than in C. lemniscatus. These characteristics might be related to microhabitat use and the well-developed olfactory and vomeronasal systems observed within this clade.

Morphology and histology of the male reproductive tract of Caecilia thompsoni (amphibia: Gymnophiona).

We studied the male reproductive tract of individuals of different body sizes of Caecilia thompsoni to describe morphological characteristics in comparison to other Gymnophiona. The reproductive tract consists of paired testes segmented into chains of primary and secondary lobes, sperm ducts that empty to Wolffian ducts, the cloaca that receives the Wolffian ducts and possesses a phallodeum. Müllerian ducts are present and develop into paired glands that empty into the cloacal urodeum. Testicular secondary lobes contain lobules with cysts of the entire germinal cell line, whereas primary lobes, in the terminal ends of the chains, only have spermagonia, Sertoli cells, and connective tissue. The smallest individual examined (21 cm body length) was immature and only possessed a few testicular primary lobes. Once the individuals reach sexual maturity, the morphological characteristics are quite consistent at macroscopic and histological level among males of very different body sizes. The histological features of the Wolffian and Müllerian glands suggest a complementary secretory role between the two ducts. In the cloaca we found the propulsor muscle, venous sinuses, and blind sacs in the phallodeum, which differentiate C. thompsoni from other species of the genus. Despite these slight differences, the general morphological characteristics, both macroscopic and microscopic, of the reproductive tracts of adult males of C. thompsoni follow the pattern known for the reproductively active males of Gymnophiona.

Hemipenial morphology of nine South American species of Mabuya (Scincidae: Lygosominae) with comments on the morphology of the family.

Species of the genus Mabuya are barely recognizable because of their highly conservative external morphology, resulting in unstable systematic and taxonomic status. Considering that hemipenial morphology has been extensively used as a source of systematic information for several groups of Squamata, we described the hemipenial morphology of nine species of Mabuya, searching for diagnostic features useful to taxonomic and systematic studies. To have comparative parameters within the family, we also presented the description of the hemipenes of two other Scincidae species (i.e., Trachylepis atlantica and Tiliqua rugosa). This information, together with the available literature, was analyzed in a comparative framework, allowing us to define some variation for the genus and family. The hemipenial morphology of the species of Mabuya studied is very conservative; features that show subtle variation were the shape of the protuberances present on the asulcate face of the hemipenial body and the presence and type of a lateral fold, on the body. Also, within the family, some variation was found in the shape and length of lobes, length of branches of the sulcus spermaticus, and in the kind of folds and protuberances in both faces of the lobes and hemipenial body. Although these features were not tested in a systematic context, this information suggested variations in hemipenial morphology that could contribute to taxonomic diagnostics and phylogenetic characters. The importance of obtaining proper preparations for gathering accurate information is emphasized, and the use of fresh-killed specimens that shows fewer difficulties for the procedures of preparation is highly recommended.

A morphological database for Colombian anuran species from conservation-priority ecosystems.

Species traits provide a strong link between an organism's fitness and processes at community and ecosystem levels. However, such data remain scarce for amphibians in the Neotropics. Colombia is the country with the highest number of threatened amphibians and the second greatest number of amphibian species worldwide. We present a data set containing eight morphological traits for 4,623 museum specimens of the seven largest collections in the country corresponding to 293 species of 14 families. The number of measured specimens per species ranged from 1 to 118 individuals with a median of 8 individuals per species. Overall, this database gathered morphological information for 37.6% of Colombian anuran diversity. Species measured were mainly distributed in the high Andean forest, the páramo, and wetland ecosystems, and was part of a national initiative led by the Instituto Alexander von Humboldt. The morphological traits were selected on the basis of their role in species' responses to environmental variability and their contributions to ecosystem processes. These traits were related to habitat use, (forearm length, tibia length, femur length, foot length, and foot webbing), predation and food chains (head width and mouth width), and nutrient recycling (snout-vent length). We expect this data set will be used in studies on functional diversity in amphibians and the development of conservation planning for these taxa. No copyright or proprietary restrictions are associated with the use of this data set other than citation of this Data Paper.

Clasper gland morphology and development in Potamotrygon magdalenae (Elasmobranchii: Potamotrygonidae).

Clasper gland morphology and development in Potamotrygon magdalenae and its relation with the acquisition of reproductive maturity is described in males of different developmental stages (embryos, neonates, juveniles, and reproductively active and resting adults). The glands are subcutaneous masses in the proximal base of each clasper. They are partially bilobate organs with a ventral groove that bears a row of papillae. Glands tend to be asymmetric, the left gland has a larger size, a trend that has been observed in other organs of elasmobranchs. Glands are formed by radially organized tubular secretory units lined with a simple columnar epithelium with basal nuclei and granular eosinophilic cytoplasm; vascularized loose connective tissue surrounds the gland units. The gland is covered by two layers of striated muscle tissue in circular and longitudinal arrangement. The clasper glands begin to develop in neonates and their secretory activity begins in juveniles. The active secretion of the clasper gland is observed in mature males, it includes glycoproteins and sulfated mucopolysaccharides. The size of the glands has a positive and direct relationship with body size, measured as disc width. Significant differences in clasper gland size were found between mature (active and resting) and immature (neonates and juveniles) males, suggesting that the acquisition of the sexual maturity involves the increase in the size of the gland due to a highly augmented secretory activity. Therefore, clasper glands are clearly associated with the reproductive activity of males and their secretion should have an endocrine control as other sexual secondary organs. J. Morphol. 278:369-379, 2017. © 2017 Wiley Periodicals, Inc.

Allantoplacental ultrastructure of an Andean population of Mabuya (Squamata, Scincidae).

Mabuya species are highly matrotrophic viviparous lizards with Type IV epitheliochorial allantoplacenta. The allantoplacenta of an Andean population of this genus, currently assigned to Mabuya sp., possesses specializations related to histotrophic nutrition at the embryonic hemisphere (placentome, paraplacentome, and chorionic areolas), while at the abembryonic hemisphere it has a mixed function: histotrophic transfer (absorptive plaques) and hemotrophic nutrition (gas exchange in respiratory segments). These placental specializations were studied using high-resolution light microscopy and transmission electron microscopy, and were compared with those found in other squamate reptiles and eutherian mammals. Cytological features of the placentome suggest that this is an important region for nutritional provision; the paraplacentome also shows characteristics for nutrient transfer, especially lipids. Chorionic areolas allow the absorption of glandular products, as well as uterine and chorionic cellular debris produced by lysis of some cells of both epithelia during areola formation. In the absorptive plaques both uterine and chorionic epithelia are firmly attached and their cellular apices exhibit electron-dense granules that could be related to autocrine and paracrine functions. The short interhemal distance found in the respiratory segments confirms their role in gas exchange. A common feature of all regional specializations in the Mabuya sp. allantoplacenta is the presence of lipids in the interacting chorionic and uterine epithelia, suggesting that lipids are transferred throughout the entire embryonic chamber; placental transfer of lipids may be the principal fetal energy and lipid source in this species. In spite of this feature, each one of the specialized areas of the allantoplacenta has different features suggesting particular functions in the transfer of nutrients (as ions, lipids, proteins, amino acids, sugar, water, and gases), and in the possible synthesis of hormones and proteins. The placental complexity observed in this species of Mabuya is greater than in any other reptile, and resembles that of eutherian mammals: Each one of these specializations of the placental membranes in Mabuya sp. is similar to those found among different eutherian mammals, indicating a very impressive evolutionary convergence at the histological and cytological levels between both clades. However, no eutherian mammal species simultaneously displays all of these specializations in the embryonic chamber as does Mabuya sp.

Morphology of the reproductive tract and acquisition of sexual maturity in males of Potamotrygon magdalenae (Elasmobranchii: Potamotrygonidae).

The purpose of this study was to describe the structure of the reproductive tract of males of Potamotrygon magdalenae before, during, and after they acquire sexual maturity, and to establish the first maturity scale for males within the family Potamotrygonidae. The male reproductive tract of P. magdalenae is composed of testes, efferent ducts, epididymides, deferent ducts, seminal vesicles, Leydig, alkaline, and clasper glands, and claspers, all of which are paired and functional. Four sexual maturity stages were established: immature, maturing, reproductively active, and resting. The degree of claspers calcification is also a good indicator of sexual maturity in this species. The testes are lobulated, each lobe contains numerous spermatocysts which are organized in zones and are displaced radially from germinal papillae to the spermatozoa zone where individual spermatozoa are conveyed to the efferent ducts. The epididymis can be regionalized in head, body, and tail; these regions are distinguished by external pigmentation and by the epithelium lining configuration. The tail of the epididymis is connected with the deferent duct and this, in turn, with the seminal vesicle. The spermatozoa are organized in spermatozeugmata which begin to form in the deferent duct; this latter organ is attached laterally at the Leydig gland that is composed by simple glandular units. Irregular and vesicular secretions can be found in the genital ducts. These secretions might be associated with the maturation of the spermatozoa and formation of spermatozeugmata. The male reproductive tract of P. magdalenae is similar to other elasmobranchs; however, two types of primary spermatogonia, an epididymis internally regionalized, and the presence and structure of spermatozeugmata are specific features not yet described in freshwater stingrays. Most of the year, the males were reproductively active, however, few resting adult males occurred during one of the months of the lowest waters.

Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae).

Topological and histological analyses of Mabuya mabouya embryos at different developmental stages showed an extraembryonic membrane sequence as follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula stages; mesodermal split and formation of an exocoelom in the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to form a chorioallantoic membrane at pharyngula stages; complete extension of the allantois into the exocoelom between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac placenta; chorioplacenta; allantoplacenta. All placentas are highly specialized for nutrient absorption from early developmental stages. The bistratified extraembryonic ectoderm possesses an external layer with cuboidal cells and a microvillar surface around the whole yolk sac, which absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of development. The dorsal absorptive plaque will constitute the placentome and paraplacentome during allantoplacental development. At late gastrula-early neurula stages some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and seem to have a specialized histotrophic and/or metabolic role. The extraembryonic mesoderm does not ingress into the yolk sac and neither an isolated yolk mass nor a yolk cleft are formed. This derived pattern of development may be related to the drastic reduction of the egg size and obligatory placentotrophy from early developmental stages. Our results show new specialized placentotrophic structures and a novel arrangement of extraembryonic membrane morphogenesis for Squamata.

Ovarian histology of the placentotrophic Mabuya mabouya (Squamata, Scincidae).

Ovarian structure and folliculogenesis of females at different reproductive stages are described for the viviparous placentotrophic lizard Mabuya mabouya. The small ovaries have a thin wall formed by the ovarian epithelium and a thin tunica albuginea. One to two germinal beds that contain numerous oogonia and developing primordial follicles are derived from the ovarian epithelium and are next to the ovarian hilum. The ovarian cortex contains follicles at different stages of development, corpora lutea, and atretic follicles. The yolk nucleus and Balbiani complex were not evident in the ooplasm of previtellogenic follicles. The follicular epithelium of these follicles is polymorphic, as in other species of Squamata, but the larger cells are spherical and monolayered rather than pyriform. The zona radiata of the preovulatory follicles is less developed than in lecithotrophic species. These features suggest a decrease in metabolic and absorptive processes during follicular growth. In preovulatory follicles (1.5-1.8 mm diameter), primordial yolk vacuoles and small cortical granules are deposited in the ooplasm instead of fatty yolk platelets, so that only one stage of vitellogenesis is observed. Polyovular atretic follicles occur in some females. Follicular atresia is minimal for preovulatory follicles, but is more frequent in follicles with polymorphic epithelia. In the corpus luteum, the luteal tissue is formed from granulosa cells and luteolysis occurs during the late gastrula -- late neurula embryonic stages. Thus, the maintenance of gestation from the pharyngula to preparturition stages seems to be related to secretion of extraluteal progesterone, possibly of placental origin. These observed ovarian features are related to the high degree of placental complexity of this species and show that the evolution of advanced placentotrophy in species of this lineage has been accompanied by concomitant changes in ovarian function.

Morphology of the ovary of Caiman crocodilus (Crocodylia: Alligatoridae).

We describe the ovarian histology and characterize the folliculogenesis of adult females of Caiman crocodilus in order to compare them with other species of Reptilia. The gonad has a thin irregular cortex and a well-developed reticular stroma, composed of vascularized chordae, drained by large lymphatic vessels and separated by extensive lacunae. Simple cuboidal to squamous epithelium, dense connective tissue with numerous elastin fibers, and smooth muscle constitute the ovarian wall. This morphology is similar to that of other Crocodylia, Aves and Testudines. Germinal nests are distributed in the ovarian cortex, some of them with oogonia. Oocytes leave the germinal nests with a single layered cuboidal granulosa, remaining simple during vitellogenesis, as in other Archosauria and Testudines and different from Squamata. As the oocyte grows, the theca is formed by numerous fibroblasts, collagen fibers, and smooth muscle fibers, becoming very thick, highly vascularized and developing muscle bands at advanced vitellogenesis. Folliculogenesis shares morphological features with other species of Reptilia. The corpus luteum is similar to that described for Archosauria; during luteolysis, the theca shrinks and the amount of luteinic cells decreases. The relationship of the corpus luteum to vitellogenesis and egg maintenance is unclear in Crocodylia. Follicular regression in Caiman crocodilus corresponds to an invasive atresia type. Histological features seen in the ovary of Caiman crocodilus are similar to those described in Alligator mississippiensis indicating that these features could be shared among Crocodylia. Also several of these morphological characteristics are observed in Aves suggesting a similar reproductive functional morphology in Archosauria.

Morphology of the male reproductive duct system of Caiman crocodilus (Crocodylia, Alligatoridae).

The male reproductive duct system of Caiman crocodilus in different reproductive stages was studied using light and electron microscopy, to determine whether shared morphological features exist between Crocodylia and Aves, in concurrence with the Archosauria hypothesis. The sexual duct system of Caiman crocodilus is constituted of the rete testis, ductuli efferentes, ductuli epididymides, ductus epididymidis, and ductus deferens. The morphology and histochemical properties of these ducts suggest their involvement in seminal fluid production and/or its modification. Three types of non-ciliated cells were identified along the duct system. 1. The noncliated cells of the ductuli efferentes contain electron dense worm-like structures and coated vesicles, both related to absorptive processes, as has been suggested in Aves. 2. The non-ciliated cells of the ductuli epididymides have apical electron dense granules suggesting a secretory role, and 3. The non-ciliated cells of the ductus epididymidis and ductus deferens did not exhibit conspicuous storage of secretory material, but have a prominent rough endoplasmic reticulum content indicating active protein production. The occurrence of abundant secretory granules in the epithelial cells of the distal ductus deferens during non-reproductive stages suggests its participation in the removal of luminal debris when the reproductive season ends. Additional ducts were observed running along the ductus deferens; they shared morphological characteristics with the ductuli epididymides. The maximum diameter and therefore the greatest sperm accumulation of the excurrent ducts were observed during the initial testicular regression. The comparative analysis suggests that the male reproductive system of the Crocodylia exhibits structural characteristics nearer to those of Aves than to Lepidosauria, Testudines, and Mammalia, additional data that support an Aves and Crocodylia sister group relationship.

Toxic, cytotoxic, and genotoxic effects of a glyphosate formulation (Roundup®SL-Cosmoflux®411F) in the direct-developing frog Eleutherodactylus johnstonei.

The aerial spraying of glyphosate formulations in Colombia to eradicate illegal crops has generated great concern about its possible impact on nontarget organisms, particularly amphibians. This study evaluated the toxic, cytotoxic, and genotoxic effects of a glyphosate formulation (Roundup®SL-Cosmoflux®411F) in the direct-developing frog Eleutherodactylus johnstonei by estimating the median lethal application rate (LC50 ), median hemolytic application rate (HD50 ), and extent of DNA damage using the in vitro and in vivo Comet assays. Toxicity results indicated that the application rate [37.4 µg acid equivalent (a.e.)/cm(2) ] equivalent to that used in aerial spraying (3.74 kg a.e./ha) is not lethal in male and female adult frogs, whereas neonates are highly sensitive. Glyphosate formulation at application rates above 5.4 µg a.e./cm(2) (in vivo) and concentrations above 95 µg a.e./mL (in vitro) showed clear evidence of cytotoxicity. In vivo and in vitro exposure of E. johnstonei erythrocytes to the glyphosate formulation induced DNA breaks in a dose-dependent manner with statistically significant values (P < 0.05) at all doses tested. DNA damage initially increased with the duration of exposure and then decreased, suggesting that DNA repair events were occurring during in vivo and in vitro exposures. These results are discussed from the perspective of possible ecotoxicological risks to anuran species from exposure to glyphosate formulation.

Ontogeny of the vertebral column of Eleutherodactylus johnstonei (Anura: Eleutherodactylidae) reveals heterochronies relative to metamorphic frogs.

Over the last century, the morphogenesis of the vertebral column has been considered as a highly conserved process among anurans. This statement is based on the study of few metamorphic taxa, ignoring the role of developmental mechanisms underlying the evolution of specialized life-histories. Direct development in anurans has been regarded as evolutionarily derived and involves developmental recapitulation and repatterning at different levels in all amphibian taxa studied so far. Herein, we analyze the vertebral column morphogenesis of the direct-developing frog Eleutherodactylus johnstonei, describing the sequence of chondrification and ossification, based on cleared and double-stained specimens from early stage embryos to adults. In general, our results show that the morphogenesis of the vertebral column in E. johnstonei recapitulates the ancestral tadpole-like pattern of development. However, the analysis of the sequence of events using heterochrony plots shows important heterocronies relative to metamorphic species, such as a delay in the chondrification of the vertebral centra and in osteogenesis. These ontogenetic peculiarities may represent derived traits in direct-developing frogs and are possibly correlated with its unusual life history. In addition, several features of the vertebral column of E. johnstonei are highly variable from its typical morphology. We report some malformations and small deviations, which do not seem to affect the survival of individuals. These anomalies have also been found in other frogs, and include many vertebral defects, such as vertebral fusion, and vertebral preclusion and/or induction.

Composición y distribución de hepáticas (Marchantiophyta) en un intervalo altitudinal en la Cordillera Oriental de Colombia / Composition and distribution of liverworts (Marchantiophyta) in a continuous altitudinal range on the Cordillera Oriental of Colombia

Resumen La composición de especies de hepáticas sobre los Andes del Norte varía con la altitud en relación con diferentes factores bióticos (vegetación) y abióticos (temperatura, humedad y luminosidad). Para probar esta afirmación se determinó la diversidad y distribución de las Marchantiophytas en un rango altitudinal entre los 2 400 y 3 400 m sobre la Cordillera Oriental colombiana, estableciendo cómo éstas varían entre altitudes y zonas de vida con respecto a los factores abióticos. Se realizaron muestreos cada 200 m y se registraron datos ambientales durante un año. Se encontraron 162 especies, la composición de hepáticas varió a lo largo del gradiente altitudinal con un gran número de especies únicas en cada altitud. Se encontró la máxima diversidad en los 3 000 m, con una dominancia del hábito folioso y de especies epifitas, mientras que la mayor riqueza de familias y géneros se presentó entre los 3 200 y 3 400 m. Anoplolejeunea conferta presentó la mayor cobertura en la zona así como el mayor valor del índice de valor de importancia (IVI) y distribución altitudinal más amplia, ubicándose desde los 2 400 hasta 3 000 m en cuatro diferentes sustratos. Las hepáticas se distribuyeron altitudinalmente de manera diferencial, con una diversidad beta alta (0.864) debido al recambio altitudinal de especies, con más del 30 % de disimilaría en la composición de especies cada 200 m altitudinales, dependiendo principalmente de cambios en la luminosidad. Consecuentemente, la temperatura, humedad y luminosidad y su relación con la vegetación son determinantes en la diversidad y distribución de las hepáticas en la Cordillera Oriental de los Andes colombianos.

Abstract The species composition of liverworts varies with altitude in the Northern part of the Andes due to the relationship of biotic (vegetation) and abiotic factors (temperature, humidity, and sunlight brightness). In order to test this affirmation we determined the diversity, species composition, abundance, and distribution of species of Marchantiophyta in an altitudinal range from 2 400 to 3 400 m on the Colombian Cordillera Oriental, and established how these vary with altitude and life-zones, regarding abiotic factors. Samples, taken every 200 m in the altitudinal range, and environmental data were registered during a year. We found 162 species, the composition of liverworts varied throughout the altitudinal gradient with a high number of unique species in each altitude. The maximum diversity was found at 3 000 m, along with a dominance of leafy habit and epiphytic species, while the higher richness of families and genera was found between 3 200 and 3 400 m. Anoplolejeunea conferta had the largest value of coverage in the zone, the highest value of importance value index (IVI), and the widest altitudinal distribution, from 2 400 to 3 000 m in four different substrates. The liverworts were distributed differently with altitude, we found a high beta diversity (0.864) due to the replacement of species, with more that 30 % of dissimilarity in species composition every 200 altitudinal meters, mainly depending on sunlight brightness variation. Accordingly, we found that abiotic factors like temperature, humidity, and sunlight brightness and its relationship vegetation are determinant in the diversity and altitudinal distribution of liverworts in the study area. Rev. Biol. Trop. 66(2): 559-570. Epub 2018 June 01.