Biflagellate spermatozoon of the poison-dart frogs Epipedobates femoralis and Colostethus sp. (Anura,Dendrobatidae)

This study describes the spermatozoa of the dendrobatids Epipedobates femoralis and Colostethus sp. using light and transmission electron microscopy. Both species possess a biflagellate spermatozoon, an unusual characteristic only previously reported in two anuran species belonging to the families Leptodactylidae and Racophoridae. The acrosomal complex of both species consists of a conical acrosomal vesicle and a subacrosomal cone, both of which cover the anterior portion of the nucleus, but to differing extents. In the midpiece, the centrioles are disposed parallel to each other and to the cell axis and give rise to two axonemes. Two paraxonemal rods were also seen entering the nuclear fossa. Both flagella are surrounded by a single mitochondrial collar. Each flagellum is formed by an axial fiber connected to the axoneme by an axial sheath; juxta-axonemal fibers are absent. Our data seem to support that Epipedobates femoralis should be placed in a separate clade possibly related to Colosthetus and that these two genera may not be monophyletic.     

Correlated evolution of conspicuous coloration and body size in poison frogs (Dendrobatidae)

Conspicuous coloration is often used in combination with chemical defenses to deter predators from attacking. Experimental studies have shown that the avoidance inducing effect of conspicuous prey coloration increases with increasing size of pattern elements and with increasing body size. Here we use a comparative approach to test the prediction from these findings, namely that conspicuous coloration will evolve in tandem with body size. In our analysis, we use a previously published mitochondrial DNA-based phylogeny and comparative analysis of independent contrasts to examine if evolutionary shifts in color pattern have been associated with evolutionary changes in body size in aposematic poison frogs (Anura: Dendrobatidae). Information on body size (snout to vent length) and coloration were obtained from the literature. Two different measures of conspicuousness were used, one based on rankings by human observers and the other based on computer analysis of digitized photographs. The results from comparative analyses using either measure of coloration indicated that avoidance inducing coloration and body size have evolved in concert in poison frogs. Results from reconstruction of character change further indicate that the correlated evolution of size and coloration has involved changes in both directions within each of the different clades of the phylogenetic tree. This finding is consistent with the hypothesis that selection imposed by visually guided predators has promoted the evolution of larger body size in species with conspicuous coloration, or enhanced evolution of conspicuous coloration in larger species.     

Chromosome analysis of five Brazilian species of poison frogs (Anura: Dendrobatidae)

Dendrobatid frogs have undergone an extensive systematic reorganization based on recent molecular findings. The present work describes karyotypes of the Brazilian species Adelphobates castaneoticus, A. quinquevittatus, Ameerega picta, A. galactonotus and Dendrobates tinctorius which were compared to each other and with previously described related species. All karyotypes consisted of 2n = 18 chromosomes, except for A. picta which had 2n = 24. The karyotypes of the Adelphobates and D. tinctorius species were highly similar to each other and to the other 2n = 18 previously studied species, revealing conserved karyotypic characteristics in both genera. In recent phylogenetic studies, all Adelphobates species were grouped in a clade separated from the Dendrobates species. Thus, we hypothesized that their common karyotypic traits may have a distinct origin by chromosome rearrangements and mutations. In A. picta, with 2n = 24, chromosome features of pairs from 1 to 8 are shared with other previously karyotyped species within this genus. Hence, the A. picta data reinforced that the C-banding pattern and the NOR location are species-specific traits in the genus Ameerega. Moreover, the Ameerega monophyletism proposed by previous phylogenetic studies indicates that the karyotypic differences among species in this genus result from a long divergence time.     

Evolution of parental care in dart poison frogs (Amphibia: Anura: Dendrobatidae)

Dendrobatid frogs perform a unique mode of parental care, ranging from egg attendance and tadpole transport to tadpole feeding. It is hypothesized that a behaviour in which the egg attending parent remains with the cluch is the most primitive condition. In more advanced forms or parental care, the male is able to attend several clutches of eggs. Tadpole attendance and feeding, finally, started as deceit; the male induced the female to lay eggs into a bromeliad leaf axil already occupied by a larva. Costs and benefits of the different modes of parental care and possible alternative reproductive tactics are discussed.     

Evolution of dietary specialization and chemical defense in poison frogs (Dendrobatidae): a comparative analysis

Defensive mechanisms, including noxious or toxic substances, are favored by predation-driven natural selection. The acquisition of noxious/toxic substances can be either endogenous, in which the substances are produced by the organism, or exogenous, in which the substances are produced by another organism and are sequestered. Evidence indicates that the defensive skin alkaloids of Neotropical poison frogs (Dendrobatidae) have an exogenous source: a diet of ants and other small alkaloid-containing arthropods, which we term the diet-toxicity hypothesis. A critical prediction of the diet-toxicity hypothesis is that independent origins of dietary specialization will be found to be correlated with independent origins of skin alkaloids. We tested this prediction in an integrated framework using comparative methods with new and published data on feeding ecology and chemical defense for 15 species of dendrobatids in five genera. We found a significant correlation between alkaloid profiles and degree of dietary specialization. This reveals a recurring association of dietary specialization and alkaloid sequestration in dendrobatids, which suggests parallel evolutionary trends in the origins of defensive mechanisms.     

Morphology of the exocrine glands of the frog skin

Frog skin contains three distinct types of exocrine glands: granular (poison), mucous, and seromucous. The granular gland forms a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. The mucous and seromucous glands are easily identifiable as distinct glands. The serous and mucous secretory cells are arranged in a semilunar configuration opposite the ductal end and are filled with granules. Within the acinus, located at the ductal pole of the gland, are distinct groups of cells with few or no granules in the cytoplasm. In both the mucous and seromucous gland there is a cell type with abundant mitochondria; the one in the mucous gland is located in the region adjacent to the secretory cells. The duct of these glands is two-layered, with the individual cells appearing morphologically similar to the layers of the skin epithelium as the duct traverses the skin. The duct appears to be patent throughout its length. The morphological heterogeneity and distinct distribution of the cell types within the gland acinus may be indicative of a functional heterogeneity that allows the production of distinctly different types of secretion from the same gland type, depending on the type of stimulus.     

Assortative mating in poison-dart frogs based on an ecologically important trait

The origin of new species can be influenced by both deterministic and stochastic factors. Mate choice and natural selection may be important deterministic causes of speciation (as opposed to the essentially stochastic factors of geographic isolation and genetic drift). Theoretical models predict that speciation is more likely when mate choice depends on an ecologically important trait that is subject to divergent natural selection, although many authors have considered such mating/ecology pleiotropy, or "magic-traits" to be unlikely. However, phenotypic signals are important in both mate choice and ecological processes such as avoiding predation. In chemically defended species, it may be that the phenotypic characteristics influencing mate choice are the same signals being used to transmit a warning to potential predators, although few studies have demonstrated this in wild populations. We tested for assortative mating between two color morphs of the Strawberry Poison-Dart Frog, Dendrobates pumilio, a group with striking geographic variation in aposematic color patterns. We found that females significantly prefer individuals of their own morph under two different light treatments, indicating strong assortative mating based on multiple coloration cues that are also important ecological signals. This study provides a rare example of one phenotypic trait affecting both ecological viability and nonrandom mating, indicating that mating/ecology pleiotropy is plausible in wild populations, particularly for organisms that are aposematically colored and visually orienting.     

Sperm ultrastructure of the Brazilian Amazon poison frogs Epipedobates trivittatus and Epipedobates hahneli (Anura, Dendrobatidae)

The sperm ultrastructure of Epipedobates trivittatus and E. hahneli is described. The spermatological characteristics are identical in both species, except for the extension of the acrosomal vesicle. The structure of the acrosomal complex is shared with species of the Bufonoidea neobatrachian lineage, which differs from the condition observed in species included in the ranoid lineage. The phylogenetic significance of the mitochondrial collar, also seen in Eubufonoidea groups, is still unclear. Also the significance of the alternative mitochondrial arrangement seen here is unclear. The perpendicular arrangement of the centrioles is also shared with the Bufonoidea and is considered a synapomorphic condition. An expanded undulating membrane and the absence of a juxtaxonemal fibre appear to be apomorphic characteristics of dendrobatid species, as also observed (homoplastically) for species of Litoria and Cyclorana. However, the notable expansion of the undulating membrane appears to be an autapomorphy of Dendrobatidae frogs. The ultrastructural data presented here do not support the proposed placement of E. trivittatus in a separate genus.     

Studies in the secretory glands of Hiptage sericea (Malpighiaceae)

Hiptage sericea is shown to possess both lipophilic glands and extrafloral nectaries. Both types of glands develop from a group of initials and show similarities in organisation of tissue systems and secretion. The nature of the secretory substances is however different. The occurrence and function of the glands are discussed.     

Experimental evidence for predator learning and Müllerian mimicry in Peruvian poison frogs (Ranitomeya,Dendrobatidae)

The evolution of mimicry is one of the most powerful examples of evolution driven by natural selection; however it is rare in non-insect taxa and thus is understudied. Ranitomeya imitator underwent a ‘mimetic radiation’ and now mimics three congeneric model species (R. fantastica, R. summersi, and two morphs of R. variabilis), creating geographically distinct populations of the species, including four allopatric mimetic morphs. These complexes are thought to represent a case of Müllerian mimicry, but no prior empirical studies on learned avoidance by predators support this claim. In this study we used young chickens (Gallus domesticus) as naïve predators to determine if a co-mimetic morph of R. imitator and R. variabilis contribute to reciprocal learned avoidance by predators—a key component of Müllerian mimicry. Chickens exposed to either stimulus species demonstrated reciprocal learned avoidance; thus our results indicate that this complex functions as a Müllerian mimicry system. This study provides novel empirical evidence supporting predictions of the Müllerian mimicry hypothesis in anurans. Our study shows no difference between learned avoidance in stimuli frogs and a ‘novel’ morph of R. imitator that differed in both color and pattern, indicating that learned avoidance by predators may be generalized in this system. Generalized learning provides a plausible mechanism for the maintenance of both polytypic mimicry and the maintenance of intrapopulation phenotypic heterogeneity.     

Seasonal changes in the skin glands of Roe deer (Capreolus capreolus)

Seasonal changes in the specialized skin glands of the forehead, metatarsal gland and interdigital regions of Roebucks are described and compared with glands of the general body surface. In both the forehead region and dorsal skin the sebaceous and apocrine glands enlarge as the testes enlarge and regress after the rut, but the glands of the forehead region are at least three times larger than those of the general body surface. The apocrine glands of the forehead region discharge by exocytosis except at the time of maximum secretion, which precedes total cell breakdown. In the metatarsal and interdigital regions the skin glands are enlarged throughout the year.     

Morphology of the salivary glands of Mastotermes darwiniensis Froggatt (Isoptera : Mastotermitidae)

The salivary glands of Mastotermes darwiniensis (Isoptera : Mastotermitidae) workers consist of acini made up of peripherally and centrally placed cells with storage cells. A network of small efferent ductules is associated with each acinus. Intracellular ductules of the central and peripheral cells are filled with protruding microvilli. Central cells contain an abundance of rough endoplasmic reticulum and vacuoles. Continua of rough endoplasmic reticulum and vacuoles are present. Pinocytosis does not appear to be involved in secretion by the central cells. Peripheral cells are compartmental, having electron-lucent vesicle accumulations away from intracellular ductules and the basal plasmalemma invaginations. Peripheral cell morphology is similar to cells which are associated with fluid movement. Storage cells contain a large vacuole and exhibit micropinocytosis. Two axon profile types are present near and on the acini and salivary ducts, one of which appears associated with neuronal end plates.     

Take time to smell the frogs: vocal sac glands of reed frogs (Anura: Hyperoliidae) contain species‐specific chemical cocktails

Males of all reed frog species (Anura: Hyperoliidae) have a prominent, often colourful, gular patch on their vocal sac, which is particularly conspicuous once the vocal sac is inflated. Although the presence, shape, and form of the gular patch are well‐known diagnostic characters for these frogs, its function remains unknown. By integrating biochemical and histological methods, we found strong evidence that the gular patch is a gland producing volatile compounds, which might be emitted while calling. Volatile compounds were confirmed by gas chromatography–mass spectrometry in the gular glands in 11 species of the hyperoliid genera Afrixalus, Heterixalus, Hyperolius, and Phlyctimantis. Comparing the gular gland contents of 17 specimens of four sympatric Hyperolius species yielded a large variety of 65 compounds in species‐specific combinations. We suggest that reed frogs might use a complex combination of at least acoustic and chemical signals in species recognition and mate choice. © 2013 The Authors. Biological Journal of the Linnean Society published by John Wiley & Sons Ltd on behalf of The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 828–838.     

Preparation and ultrastructure of spermatozoa from green poison frogs, Dendrobates auratus, following hormonal induced spermiation (Amphibia, Anura, Dendrobatidae)

Few ultrastructural studies have been performed on members of the Dendrobatidae, although such investigations can be useful for the understanding of reproductive patterns, as a diagnostic method for males in breeding programs for endangered amphibians and for phylogenetic analysis. The sperm ultrastructure of the Green Poison Frog, Dendrobates auratus, from Panama is described following induced spermiation in living animals. To date only testicular spermatozoa in other dendrobatid frogs have been analysed. Moreover, an electron microscopic preparation method (transmission and scanning electron microscopy) for dendrobatid sperm cells in low concentration is presented. Sperm cells from stimulated frogs (100 IU human chorionic gonadotropin, hCG, twice at an interval of 1h) were recovered via cloaca lavage using 600 microl isotonic phosphate-free amphibian saline (IPS). Centrifuged flushings (5 min, 173 x g) were deposited on microscopic slides. Adherent spermatozoa were treated with Karnovsky fixative (overnight, 4 degrees C). After postfixation (2h, 1% osmium tetroxide), samples were dehydrated in series of ascending acetones (30-100%). For transmission electron microscopy sperm cells were encapsulated using Epon and 1.5% 2,4,6-tris(dimethylaminomethyl)phenol (DMP 30). Ultrathin sections (70 nm) were cut and stained with uranyl acetate (30 min) and lead citrate (5 min). Sperm cells are filiform with a 21.1+/-2.7 microm long and arcuated head and a single tail (35.0+/-4.2 microm length). Their acrosomal complex is located at the anterior portion of the head and consists of the acrosomal vesicle which has low electron density, and the subjacent electron-dense subacrosomal cone. In transverse section, the nucleus is circular (1.9+/-0.2 microm diameter) and conical in longitudinal section. It is surrounded by several groups of mitochondria. The chromatin is highly condensed and electron-dense but shows numerous electron-lucent inclusions. A short midpiece has a mitochondrial collar with a proximal and a distal centriole. The latter gives rise to the axoneme which alone forms the flagellum. The sperm ultrastructure of D. auratus differs from that of other Dendrobatidae because of the absence of a nuclear space and the absence of the undulating membrane associated with an axial fibre. This tail conformation is found in the Ranoidea but not in the Bufonoidea. These results show that the spermatozoa of D. auratus are the first within the Dendrobatidae without accessory tail structures. Methods of using sperm samples from hormonal treated frogs for ultrastructural studies is not only reasonable to examine e.g. amphibian phylogeny without killing frogs threatened with extinction but allows investigations in the field of assisted reproduction and male fertility for example in conservation programs for endangered amphibians.