Response to simultaneous dehydration and thermal stress in three species of Puerto Rican frogs

Summary The response to simultaneous temperature and dehydration stress was examined in three species of Puerto Rican frogs.Eleutherodactylus antillensis is found primarily in hot, arid grasslands at low altitudes.Eleutherodactylus portoricensis is restricted to cool, forested montane habitats above 300 m.Eleutherodactylus coqui occurs in both the lowlands and the highlands. The physiological tolerance of the frogs to temperature was measured at 20, 25, and 30°C at various levels of dehydration using an index derived from jumping performance. Although jump distance of fully hydratedE. antillensis was unaffected by temperature, this species tolerated significantly more dehydration at high temperatures than low.Eleutherodactylus portoricensis died at 30°C, but in this species both distance jumped and dehydration tolerance were unaffected by lower temperatures. Distance jumped byE. coqui increased with increasing temperature, but tolerance of dehydration remained unchanged.Eleutherodactylus coqui was less tolerant of high temperatures thanE. antillensis but more tolerant thanE. portoricensis.Abbreviations DT dehydration tolerance - SVL snout vent length     

Molecular anatomy of the developing limb in the coquí frog,Eleutherodactylus coqui

The vertebrate limb demonstrates remarkable similarity in basic organization across phylogenetically disparate groups. To gain further insight into how this morphological similarity is maintained in different developmental contexts, we explored the molecular anatomy of size‐reduced embryos of the Puerto Rican coquí frog, Eleutherodactylus coqui. This animal demonstrates direct development, a life‐history strategy marked by rapid progression from egg to adult and absence of a free‐living, aquatic larva. Nonetheless, coquí exhibits a basal anuran limb structure, with four toes on the forelimb and five toes on the hind limb. We investigated the extent to which coquí limb bud development conforms to the model of limb development derived from amniote studies. Toward this end, we characterized dynamic patterns of expression for 13 critical patterning genes across three principle stages of limb development. As expected, most genes demonstrate expression patterns that are essentially unchanged compared to amniote species. For example, we identified an EcFgf8‐expression domain within the apical ectodermal ridge (AER). This expression pattern defines a putatively functional AER signaling domain, despite the absence of a morphological ridge in coquí embryos. However, two genes, EcMeis2 and EcAlx4, demonstrate altered domains of expression, which imply a potential shift in gene function between coquí frogs and amniote model systems. Unexpectedly, several genes thought to be critical for limb patterning in other systems, including EcFgf4, EcWnt3a, EcWnt7a, and EcGremlin, demonstrated no evident expression pattern in the limb at the three stages we analyzed. The absence of EcFgf4 and EcWnt3a expression during limb patterning is perhaps not surprising, given that neither gene is critical for proper limb development in the mouse, based on knockout and expression analyses. In contrast, absence of EcWnt7a and EcGremlin is surprising, given that expression of these molecules appears to be absolutely essential in all other model systems so far examined. Although this analysis substantiates the existence of a core set of ancient limb‐patterning molecules, which likely mediate identical functions across highly diverse vertebrate forms, it also reveals remarkable evolutionary flexibility in the genetic control of a conserved morphological pattern across evolutionary time.     

Parasite loss and introduced species: a comparison of the parasites of the Puerto Rican tree frog, (<Emphasis Type="Italic">Eleutherodactylus coqui</Emphasis>), in its native and introduced ranges

The Puerto Rican frog, Eleutherodactylus coqui has invaded Hawaii and reached densities far exceeding those in their native range. One possible explanation for the success of E. coqui in its introduced range is that it lost its co-evolved parasites in the process of the invasion. We compared the parasites of E. coqui in its native versus introduced range. We collected parasite data on 160 individual coqui frogs collected during January-April 2006 from eight populations in Puerto Rico and Hawaii. Puerto Rican coqui frogs had higher species richness of parasites than Hawaiian coqui frogs. Parasite prevalence and intensity were significantly higher in Hawaii, however this was likely a product of the life history of the dominant parasite and its minimal harm to the host. This suggests that the scarcity of parasites may be a factor contributing to the success of Eleutherodactylus coqui in Hawaii.     

Climate as a driver of tropical insular diversity: comparative phylogeography of two ecologically distinctive frogs in Puerto Rico

The effects of late Quaternary climate on distributions and evolutionary dynamics of insular species are poorly understood in most tropical archipelagoes. We used ecological niche models under past and current climate to derive hypotheses regarding how stable climatic conditions shaped genetic diversity in two ecologically distinctive frogs in Puerto Rico. Whereas the mountain coquí Eleutherodactylus portoricensis is restricted to montane forest in the Cayey and Luquillo Mountains, the red‐eyed coquí E. antillensis is a habitat generalist distributed across the entire Puerto Rican Bank (Puerto Rico and the Virgin Islands, excluding St Croix). To test our hypotheses, we conducted phylogeographic and population genetic analyses based on mitochondrial and nuclear loci of each species across their range in Puerto Rico. Patterns of population differentiation in E. portoricensis, but not in E. antillensis, supported our hypotheses. For E. portoricensis, these patterns include: individuals isolated by long‐term unsuitable climate in the Río Grande de Loíza Basin in eastern Puerto Rico belong to different genetic clusters; past and current climate strongly predicted genetic differentiation; and Cayey and Luquillo Mountains populations split prior to the last interglacial. For E. antillensis, these patterns include: genetic clusters did not fully correspond to predicted long‐term unsuitable climate; and past and current climate weakly predicted patterns of genetic differentiation. Genetic signatures in E. antillensis are consistent with a recent range expansion into western Puerto Rico, possibly resulting from climate change and anthropogenic influences. As predicted, regions with a large area of long‐term suitable climate were associated with higher genetic diversity in both species, suggesting larger and more stable populations. Finally, we discussed the implications of our findings for developing evidence‐based management decisions for E. portoricensis, a taxon of special concern. Our findings illustrate the role of persistent suitable climatic conditions in promoting the persistence and diversification of tropical island organisms.     

Invasive litter, not an invasive insectivore, determines invertebrate communities in Hawaiian forests

In Hawaii, invasive plants have the ability to alter litter-based food chains because they often have litter traits that differ from native species. Additionally, abundant invasive predators, especially those representing new trophic levels, can reduce prey. The relative importance of these two processes on the litter invertebrate community in Hawaii is important, because they could affect the large number of endemic and endangered invertebrates. We determined the relative importance of litter resources, represented by leaf litter of two trees, an invasive nitrogen-fixer, Falcataria moluccana, and a native tree, Metrosideros polymorpha, and predation of an invasive terrestrial frog, Eleutherodactylus coqui, on leaf litter invertebrate abundance and composition. Principle component analysis revealed that F. moluccana litter creates an invertebrate community that greatly differs from that found in M. polymorpha litter. We found that F. moluccana increased the abundance of non-native fragmenters (Amphipoda and Isopoda) by 400% and non-native predaceous ants (Hymenoptera: Formicidae) by 200%. E. coqui had less effect on the litter invertebrate community; it reduced microbivores by 40% in F. moluccana and non-native ants by 30% across litter types. E. coqui stomach contents were similar in abundance and composition in both litter treatments, despite dramatic differences in the invertebrate community. Additionally, our results suggest that invertebrate community differences between litter types did not cascade to influence E. coqui growth or survivorship. In conclusion, it appears that an invasive nitrogen-fixing tree species has a greater influence on litter invertebrate community abundance and composition than the invasive predator, E. coqui.     

An invasive frog, Eleutherodactylus coqui, increases new leaf production and leaf litter decomposition rates through nutrient cycling in Hawaii

A frog endemic to Puerto Rico, Eleutherodactylus coqui, invaded Hawaii in the late 1980s, where it can reach densities of 50,000 individuals ha⁻¹. Effects of this introduced insectivore on invertebrate communities and ecosystem processes, such as nutrient cycling, are largely unknown. In two study sites on the Island of Hawaii, we studied the top-down effects of E. coqui on aerial, herbivorous, and leaf litter invertebrates; herbivory, plant growth, and leaf litter decomposition rates; and leaf litter and throughfall chemistry over 6 months. We found that E. coqui reduced all invertebrate communities at one of the two study sites. Across sites, E. coqui lowered herbivory rates, increased NH₄⁺ and P concentrations in throughfall, increased Mg, N, P, and K in decomposing leaf litter, increased new leaf production of Psidium cattleianum, and increased leaf litter decomposition rates of Metrosideros polymorpha. In summary, E. coqui effects on invertebrates differed by site, but E. coqui effects on ecosystem processes were similar across sites. Path analyses suggest that E. coqui increased the number of new P. cattleianum leaves and leaf litter decomposition rates of M. polymorpha by making nutrients more available to plants and microbes rather than through changes in the invertebrate community. Results suggest that E. coqui in Hawaii has the potential to reduce endemic invertebrates and increase nutrient cycling rates, which may confer a competitive advantage to invasive plants in an ecosystem where native species have evolved in nutrient-poor conditions.     

Opercular development and ontogenetic re-organization in a direct-developing frog

The direct-developing frog, Eleutherodactylus coqui, has eliminated the tadpole stage from its ontogeny, and lacks many larval characters. We demonstrate that the dermal folds of E. coqui are homologous with the opercular folds of metamorphosing frogs. In both E. coqui and its metamorphic counterparts the opercular folds grow over the developing forelimb before perforating to free the entrapped limb. Opercular perforation in E. coqui occurs even in the absence of the forelimb but shows no signs of thyroid hormone dependence. The condensation of E. coqui development appears due to the excision of the extended larval period of developmental stasis. Analysis of opercular development, when viewed in conjunction with other developmental characters, suggests the ontogenetic period in the ancestral Eleutherodactylus life-history from which the tadpole was likely eliminated. Received: 7 October 1999 / Accepted: 17 January 2000     

Induced ovulation and egg deposition in the direct developing anuran <Emphasis Type="Italic">Eleutherodactylus coqui</Emphasis>

This study investigates ovulation and egg deposition behaviors in the anuran Eleutherodactylus coqui from Puerto Rico in response to stimulation with gonadotropin and gonadotropin releasing hormones. Five hormones were tested by injection over a range of doses, including mammalian LHRH, avian LHRH, fish LHRH, D-Ala6, des-Gly10 ethylamide LHRH and hCG. We report a low level of ovulation and egg deposition in response to all hormones, with the most complete and consistent results from the non-natural D-Ala6, des-Gly10 ethylamide LHRH derivative. To confirm the viability of eggs produced in this manner we performed in vitro fertilization experiments that resulted in the development of normal frogs. Reproductive behaviors in E. coqui are apparently not controlled by a mammalian form of LHRH as reported in other common laboratory anuran species. D-Ala6, des-Gly10 ethylamide LHRH induces ovulation and deposition of mature and fertilizable eggs in E. coqui.     

Cranial muscle development in frogs with different developmental modes: Direct development versus biphasic development

Normal development in anurans includes a free swimming larva that goes through metamorphosis to develop into the adult frog. We have investigated cranial muscle development and adult cranial muscle morphology in three different anuran species. Xenopus laevis is obligate aquatic throughout lifetime, Rana (Lithobates) pipiens has an aquatic larvae and a terrestrial adult form, and Eleutherodactylus coqui has direct developing juveniles that hatch from eggs deposited on leaves (terrestrial). The adult morphology shows hardly any differences between the investigated species. Cranial muscle development of E. coqui shows many similarities and only few differences to the development of Rana (Lithobates) and Xenopus. The differences are missing muscles of the branchial arches (which disappear during metamorphosis of biphasic anurans) and a few heterochronic changes. The development of the mandibular arch (adductor mandibulae) and hyoid arch (depressor mandibulae) muscles is similar to that observed in Xenopus and Rana (Lithobates), although the first appearance of these muscles displays a midmetamorphic pattern in E. coqui. We show that the mix of characters observed in E. coqui indicates that the larval stage is not completely lost even without a free swimming larval stage. Cryptic metamorphosis is the process in which morphological changes in the larva/embryo take place that are not as obvious as in normal metamorphosing anurans with a clear biphasic lifestyle. During cryptic metamorphosis, a normal adult frog develops, indicating that the majority of developmental mechanisms towards the functional adult cranial muscles are preserved. J. Morphol. 275:398–413, 2014. © 2013 Wiley Periodicals, Inc.     

Human-mediated Escalation of a Formerly Eradicable Problem: The Invasion of Caribbean Frogs in the Hawaiian Islands

Two species of neotropical frog, Eleutherodactylus coqui and E. planirostris, have been introduced into the State of Hawaii via the horticulture trade. E. coqui was introduced prior to 1988 and E. planirostris was first reported in 1994. Since these dates frog colonies have rapidly spread accidentally and intentionally and frog abundance within colonies has grown rapidly. Although these frogs were originally restricted to horticulture sites, they are now found in residential areas, resorts and hotels, and public lands. Due to the high potential biomass of introduced frogs there are realistic anthropogenic and ecological concerns associated with the spread of these frogs. Though there currently is a tool that can be used for localized control of frogs in limited circumstances, overall efforts by Federal, State, and County agencies to control the frog in Hawaii have been hampered by limited authorities and funds, disbelief in the threat, and the reluctance to act.     

Ectopic expression of Xenopus noggin RNA induces complete secondary body axes in embryos of the direct developing frog Eleutherodactylus coqui

We tested the effects of noggin RNA from Xenopus laevis on axis induction in embryos of a direct developing frog, Eleutherodactylus coqui. We microinjected noggin RNA into one blastomere of 4-cell embryos at the site close to the animal pole, and found that overexpression of noggin RNA is not only sufficient to induce additional axes but also induces heads with eyes. We also injected noggin RNA into 8-cell or 16-cell embryos in various sites, including the marginal zone, above the marginal zone, and the vegetal pole, and found the formation of a complete secondary axis in all three types of injection. These effects of X. laevis noggin RNA on the E. coqui embryo are remarkably different from those found in the X. laevis embryo itself. It has been shown previously that overexpression of noggin RNA on the ventral side of the normal X. laevis embryo induces only a partial axis, with no head structures. We show here that the failure of noggin induction of a complete axis when overexpressed on the ventral side of the X. laevis embryos is not due to an insufficient amount of RNA injected. Also, the failure is unlikely due to inhibition from the primary axis since noggin RNA can induce duplicated head structures on opposite sides of UV-irradiated X. laevis embryos. There appear to be fundamental differences in the responses of E. coqui and X. laevis embryos to exogenous noggin RNA. We propose that these differences stem from an alteration in cytoplasmic arrangements that occurred during evolution of this large egg. Received: 26 July 1999 / Accepted: 1 September 1999     

Expression of a cardiac myosin gene in non-heart tissues of developing frogs

Direct developing frogs, like Eleutherodactylus coqui, provide opportunities to investigate limb early development in anuran amphibians that are less available in species with tadpoles. We have found that myosin heavy chain 6 (myh6), a myosin gene usually considered heart-specific in Xenopus and other animals, is expressed in limbs of E. coqui embryos. The gene for microRNA(miR)-208 is contained in an intron of the E. coqui myh6 gene as in mammals, and miR -208 was detected as a microRNA, more highly expressed in a microarray of E. coqui limb buds, compared to Xenopus laevis limb buds. Myh6 is also expressed in several muscles of tadpoles and froglets of Xenopus tropicalis. These connections raise the possibility of an involvement of myh6 and miR-208 in the thyroid dependent metamorphosis of anurans.     

Taste organ growth and development in the direct developing frog Eleutherodactylus coqui (Lissamphibia: Eleutherodactylidae)

Previous research on amphibian taste organs concerned amphibians with a biphasic life history, that is, with larval period and metamorphosis. Direct developing frog species, such as Eleutherodactylus coqui, undergo a cryptic metamorphosis before hatching, and many larval‐specific features are vestigial or have been lost entirely from their ontogeny. Taste buds are present in larval stages of biphasically developing anurans and are replaced by taste discs during metamorphosis. One goal of this study was to characterize the ontogeny of taste buds and/or discs in E. coqui. The other goal was to examine correlations between body size and taste organ density and size in different regions of oral epithelium. The research reveals the presence of only one type of taste organ, characteristic of metamorphs of biphasic amphibians, namely taste disc. In addition, taste disc density and the area of the taste disc sensory zone change dramatically during growth.     

A Fungal Pathogen of Amphibians,Batrachochytrium dendrobatidis,Attenuates in Pathogenicity with In Vitro Passages

Laboratory investigations into the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), have accelerated recently, given the pathogen’s role in causing the global decline and extinction of amphibians. Studies in which host animals were exposed to Bd have largely assumed that lab-maintained pathogen cultures retained the infective and pathogenic properties of wild isolates. Attenuated pathogenicity is common in artificially maintained cultures of other pathogenic fungi, but to date, it is unknown whether, and to what degree, Bd might change in culture. We compared zoospore production over time in two samples of a single Bd isolate having different passage histories: one maintained in artificial media for more than six years (JEL427-P39), and one recently thawed from cryopreserved stock (JEL427-P9). In a common garden experiment, we then exposed two different amphibian species, Eleutherodactylus coqui and Atelopus zeteki, to both cultures to test whether Bd attenuates in pathogenicity with in vitro passages. The culture with the shorter passage history, JEL427-P9, had significantly greater zoospore densities over time compared to JEL427-P39. This difference in zoospore production was associated with a difference in pathogenicity for a susceptible amphibian species, indicating that fecundity may be an important virulence factor for Bd. In the 130-day experiment, Atelopus zeteki frogs exposed to the JEL427-P9 culture experienced higher average infection intensity and 100% mortality, compared with 60% mortality for frogs exposed to JEL427-P39. This effect was not observed with Eleutherodactylus coqui, which was able to clear infection. We hypothesize that the differences in phenotypic performance observed with Atelopus zeteki are rooted in changes of the Bd genome. Future investigations enabled by this study will focus on the underlying mechanisms of Bd pathogenicity.     

Secondary coverage of the yolk by the body wall in the direct developing frog, Eleutherodactylus coqui: an unusual process for amphibian embryos

 Eleutherodactylus coqui develops directly from a large 3.5-mm egg to a froglet, without an intervening tadpole stage. We have examined the development of the body wall, a structure whose behavior has been altered in this derived development. In an event that is unusual for amphibian embryos, the yolk mass is secondarily surrounded by the body wall, which originates near the embryo’s trunk. The epidermis of the body wall is marked by melanophores, and the rectus abdominis, which will form the ventral musculature, is near its leading edge. As the body wall expands, the epidermis, melanophores, and rectus abdominis all move from the dorsal side to close over the yolk at the ventral midline. The original ectoderm over the yolk undergoes apoptosis, as it is replaced by body wall epidermis. Intact muscles are not required for ventral closure of the body wall, despite their normal presence near the advancing edge. Comparative examination of embryos of Xenopus laevis and Rana pipiens suggests that ventral closure does not occur in species with tadpoles. The expansion of dorsal tissues over the yolk, as illustrated by E. coqui, may have been important in the origin of amniote embryos. Received: 23 April 1998 / Accepted: 28 June 1998     

Correlation of both respiratory and cutaneous water losses of lizards with habitat aridity

Summary Respiratory and cutaneous evaporative water losses were measured in dried air from 5 xantusiid lizard species to determine whether adaptations for water conservation were present in one or both components. These species represent the range of arid to mesic habitats occupied by the Xantusiidae. The respiratory proportion of evaporative water loss ranged from 20–50% and did not show consistent patterns of temperature dependence or interspecific differences. However, respiratory water loss expressed as mg H₂O per ml O₂ consumed and cutaneous water loss (mg H₂O· cm^–2·h^–1) exibited parallel correspondence to habitat aridity. Adaptations for reducing water loss from the skin involved an increased skin resistance to water flux while reduction of respiratory water loss was probably the result of reduced convection requirements for respiratory gas exchange.Abbreviations EWL evaporative water loss - RWL respiratory water loss - CWL cutaneous water loss     

Temperature variation,bacterial diversity and fungal infection dynamics in the amphibian skin

Host‐associated bacterial communities on the skin act as the first line of defence against invading pathogens. Yet, for most natural systems, we lack a clear understanding of how temperature variability affects structure and composition of skin bacterial communities and, in turn, promotes or limits the colonization of opportunistic pathogens. Here, we examine how natural temperature fluctuations might be related to changes in skin bacterial diversity over time in three amphibian populations infected by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Our focal host species (Eleutherodactylus coqui) is a direct‐developing frog that has suffered declines at some populations in the last 20 years, while others have not experienced any changes. We quantified skin bacterial alpha‐ and beta‐diversity at four sampling time points, a period encompassing two seasons and ample variation in natural infections and environmental conditions. Despite the different patterns of infection across populations, we detected an overall increase in bacterial diversity through time, characterized by the replacement of bacterial operational taxonomic units (OTUs). Increased frog body temperatures possibly allowed the colonization of bacteria as well as the recruitment of a subset of indicator OTUs, which could have promoted the observed changes in diversity patterns. Our results suggest that natural environmental fluctuations might be involved in creating opportunities for bacterial replacement, potentially attenuating pathogen transmission and thus contributing to host persistence in E. coqui populations.     

Physiological basis of habitat partitioning in Jamaican Eleutherodactylus

Summary Rate of evaporation, resistance to drying, temperature selection, and critical thermal maximum were measured for Jamaican Eleutherodactylus acclimated to 20° C and a 12:12 L:D photoperiod. Two introduced species, E. planirostris and E. johnstonei, selected higher temperatures and had higher CTMs than two native species, E. cundalli and E. gossei. The introduced species lost water as rapidly as the native species, but tolerated 30–73% greater water loss before losing their righting response. The physiological differences are reflected in the microhabitat selection and activity patterns of the four species.     

Interspecific Aggression between Lizard Competitors,Anolis cooki and Anolis cristatellus

Aggressive encounters were staged between two species of Puerto Rican lizards (Anolis cooki and Anolis cristatellus) which share the same microhabitat (syntopic). The intensity of these interspecific matches was as great as their respective conspecific matches. When the two species interacted with either “look alike” congeners (A. cooki with A. monensis and A. cristatellus with A. gundlachi) or with the quite different appearing A. evermanni, aggressive intensity was only 1/6 that of the cooki-cristatellus encounters. It seems that A. cooki and A. cristatellus recognize each other as competitors; it was improbable that this interspecific aggression was caused by the two species misidentifying each other as conspecifics. Because A. cristatellus eventually dominated A. cooki during staged encounters, it is predicted A. cristatellus should ecologically displace A. cooki in their syntopic zones of contact.