Unexpected diversity and co-occurrence of phytotelmic frogs (Guibemantis) around Andasibe, one of the most intensively surveyed amphibian hotspots of Madagascar, and descriptions of three new species.

The area around the Malagasy village of Andasibe, which includes Analamazaotra-Mantadia National Park as well as other protected areas, is characterized by very species-rich and well-studied communities of animals and plants, but new species are still regularly discovered. Three species of phytotelmic frogs of the subgenus Pandanusicola in the genus Guibemantis are known from this area, G. flavobrunneus, G. liber, and G. pulcher. Further Pandanusicola frogs from this area have been provisionally assigned to G. bicalcaratus or G. albolineatus, pending detailed taxonomic review. During preliminary exploration of the ecology of these specialized frogs that live and reproduce in the leaf axils of Pandanus screw pines, we noticed the syntopic presence of two differently colored and differently sized Pandanusicola in Andasibe that could not be unambiguously assigned to any known species. A genetic screening revealed that these correspond to yet two further species in the area. Based on our data, seven species of Pandanusicola occur in Andasibe and nearby forests: (1) G. liber, the only non-phytotelmic species of the subgenus in the region; (2) G. flavobrunneus which is the largest species and characterized by a diagnostic yellowish brown dorsal pattern; (3) G. pulcher, characterized by translucent-green color with purplish brown spotting not observed in any other species in the area; (4) G. methueni, a brownish species usually lacking contrasted dorsolateral bands that differs from the other species in the area by emitting a characteristic trill-like advertisement call series (rather than clicks or chirps) and according to our data is widespread along Madagascars east coast; as well as three new species: (5) G. ambakoana sp. nov., a brownish species, typically with contrasted incomplete light dorsolateral bands and with single click-like advertisement calls; (6) G. vakoa sp. nov., a species that is equally brownish but lacks contrasted light dorsolateral bands and that has single click-like advertisement calls of very short duration; and (7) G. rianasoa sp. nov., a species that is smaller sized and has less distinct femoral glands than all the others, and emits a short series of soft chirp-like advertisement calls. All these species are genetically highly distinct, with >5% uncorrected pairwise distances in the mitochondrial 16S rRNA gene, and lack of haplotype sharing in two nuclear-encoded genes. The co-occurrence of seven Pandanusicola frogs in a relatively small geographic area is unprecedented in Madagascar and calls for in-depth studies of a possible differentiation in habitat use and life history.

Spatial Ecology of an Arboreal Iguana (Oplurus cyclurus) in a Treeless Landscape.

Understanding the spatial ecology of species has important implications for conservation, as it helps identify suitable habitats and minimum requirements for biodiversity monitoring and management. The spiny-tailed lizard Oplurus cyclurus is a widespread endemic iguanid occurring in dry areas of southern and western Madagascar. While the species is known to be mostly arboreal, populations of the Isalo sandstone massif suggest local adaptation to a less forested savannah and a more exposed habitat. We radio-tracked 19 spiny-tailed lizards to investigate the species' rock-dwelling behaviour and spatial ecology at Isalo National Park. Tracked individuals showed high site and burrow fidelity, and a basking behaviour mostly tied to the accessibility of their burrow, the time of day, and their life stage. Activity peaked during the sunniest hours, while juveniles were more active than adults with unfavourable weather conditions. Despite high burrow fidelity, lizards used shelters non-exclusively, regularly changing (approx. once a week) with neighbouring burrows (average distance between burrows = 13.6 m). However, there was no obvious relation between lizards' body and/or tail size and the width and depth of selected burrows. Dynamic Brownian Bridge Movement Models estimated frequented areas over 247.8 m2 (95% isopleth), where territorial overlap is common. Our results challenge the notion that burrow-site fidelity is the sole driving factor behind space utilization in the studied population. We argue that the apparently unusual saxicolous habits imposed by habitat features (the absence of trees) may lead to local behavioural adjustments influencing antipredatory and foraging strategies, as well as intraspecific interactions.

Growing as slow as a turtle: Unexpected maturational differences in a small, long-lived species.

Turtle body size is associated with demographic and other traits like mating success, reproductive output, maturity, and survival. As such, growth analyses are valuable for testing life history theory, demographic modeling, and conservation planning. Two important but unsettled research areas relate to growth after maturity and growth rate variation. If individuals exhibit indeterminate growth after maturity, older adults may have an advantage in fecundity, survival, or both over younger/smaller adults. Similarly, depending on how growth varies, a portion of the population may mature earlier, grow larger, or both. We used 23-years of capture-mark-recapture data to study growth and maturity in the Spotted Turtle (Clemmys guttata), a species suffering severe population declines and for which demographic data are needed for development of effective conservation and management strategies. There was strong support for models incorporating sex as a factor, with the interval growth model reparametrized for capture-mark-recapture data producing later mean maturation estimates than the age-based growth model. We found most individuals (94%) continued growing after maturity, but the instantaneous relative annual plastral growth rate was low. We recommend future studies examine the possible contribution of such slow, continued adult growth to fecundity and survival. Even seemingly negligible amounts of annual adult growth can have demographic consequences affecting the population vital rates for long-lived species.

Ring-tailed lemurs (Lemur catta) use olfaction to locate distant fruit.

OBJECTIVES: As many primates live in forests where visibility is limited, the ability to detect the aroma of distant fruit and navigate odor plumes would be highly adaptive. Our study is the first to investigate this ability with strepsirrhine primates. MATERIALS AND METHODS: We tested the ability of a group of ring-tailed lemurs to detect hidden fruit from afar using scent alone. We hid containers in the underbrush of a semi-natural forest, some baited with real cantaloupe and some with sham cantaloupe, 4-17 m from a path routinely used by the lemurs. Crucially, the containers were not visible from the path. Therefore, the lemurs had to use olfactory cues, but did not have to prioritize them to locate the bait. RESULTS: The lemurs found the real cantaloupe on days that the wind blew the scent of the fruit toward the trail. They did not find the sham cantaloupe. Upon detecting the odor of the bait, the lemurs sniffed the air at one or more locations as they moved toward the bait, a process of navigation known as klinotaxis. DISCUSSION: The traditional belief is that primates are unable to track odor plumes. The untrained lemurs in this study were able to detect the odor of the cantaloupe among the complex odors of the forest and navigate the odor plume to the fruit. The results indicate that olfaction may be used to respond to cues from distant sources. The ability to track odor plumes may be a critical foraging skill for strepsirrhines.

A new species of the Spinomantis bertini species complex (Anura: Mantellidae) from Pic d'Ivohibe Special Reserve (Madagascar).

We describe a new frog species of the Spinomantis bertini species complex based on congruent genetic evidence from mitochondrial and nuclear DNA sequences as well as minor morphological differences. A molecular phylogeny derived from the mitochondrial 16S rRNA gene revealed that Spinomantis mirus sp. nov. is sister to a clade containing the other two described species of the species complex, S. bertini and S. beckei, but strongly differentiated from these two species by 8.1-9.8% pairwise distances in this gene. DNA sequences from two nuclear genes revealed that S. mirus sp. nov. shares no alleles with these two species. Phenotypically, S. mirus sp. nov. differs from the other species of the complex by its larger size and some aspects of its coloration, including clear mottling on the flanks, tri-color banding on the legs, and distinct brown dots on the dorsum. The new species is so far only known from Pic d'Ivohibe Special Reserve. Its distribution thus appears to not overlap with those of the other two species, which as far as known are restricted to the Andohahela Massif in the extreme South-East of Madagascar, at a distance of about 250 km from Ivohibe.

Natural exposure to ultraviolet-B radiation in two species of chameleons from Madagascar.

Many reptiles require ultraviolet-B radiation between 290 and 315 nm (UV-B) to synthesize vitamin D3 and process dietary calcium. In captivity, exposure to too little or too much UV-B can result in health problems such as metabolic bone disease. While it is recognized that UV-B is necessary to successfully maintain many reptiles in captivity, the actual levels of UV-B that species are exposed to in nature is poorly known. We measured the UV-B exposure of two species of chameleon (Calumma brevicorne and C. nasutum) in the field in Madagascar over a period of four months. We found that both species were exposed to less UV radiation than that which was available in full sun. Only on rare occasions were chameleons observed in areas with a UV Index (UVI) greater than 3.0, and the median UVI for both species was only 0.3. There was no daily temporal pattern in UV exposure for C. nasutum, but C. brevicorne was found in areas with lower UV levels in the late afternoon when compared to late morning. Additionally, C. nasutum males showed higher UV exposure than females in late morning. Our results suggest that both C. brevicorne and C. nasutum can be classified as Ferguson Zone 1 species, and should be provided with a UV-B gradient in captivity that offers access to UV-B radiation as well as adequate shaded refuge.

Estimating Herd Immunity to Amphibian Chytridiomycosis in Madagascar Based on the Defensive Function of Amphibian Skin Bacteria.

For decades, Amphibians have been globally threatened by the still expanding infectious disease, chytridiomycosis. Madagascar is an amphibian biodiversity hotspot where Batrachochytrium dendrobatidis (Bd) has only recently been detected. While no Bd-associated population declines have been reported, the risk of declines is high when invasive virulent lineages become involved. Cutaneous bacteria contribute to host innate immunity by providing defense against pathogens for numerous animals, including amphibians. Little is known, however, about the cutaneous bacterial residents of Malagasy amphibians and the functional capacity they have against Bd. We cultured 3179 skin bacterial isolates from over 90 frog species across Madagascar, identified them via Sanger sequencing of approximately 700 bp of the 16S rRNA gene, and characterized their functional capacity against Bd. A subset of isolates was also tested against multiple Bd genotypes. In addition, we applied the concept of herd immunity to estimate Bd-associated risk for amphibian communities across Madagascar based on bacterial antifungal activity. We found that multiple bacterial isolates (39% of all isolates) cultured from the skin of Malagasy frogs were able to inhibit Bd. Mean inhibition was weakly correlated with bacterial phylogeny, and certain taxonomic groups appear to have a high proportion of inhibitory isolates, such as the Enterobacteriaceae, Pseudomonadaceae, and Xanthamonadaceae (84, 80, and 75% respectively). Functional capacity of bacteria against Bd varied among Bd genotypes; however, there were some bacteria that showed broad spectrum inhibition against all tested Bd genotypes, suggesting that these bacteria would be good candidates for probiotic therapies. We estimated Bd-associated risk for sampled amphibian communities based on the concept of herd immunity. Multiple amphibian communities, including those in the amphibian diversity hotspots, Andasibe and Ranomafana, were estimated to be below the 80% herd immunity threshold, suggesting they may be at higher risk to chytridiomycosis if a lethal Bd genotype emerges in Madagascar. While this predictive approach rests on multiple assumptions, and incorporates only one component of hosts' defense against Bd, their culturable cutaneous bacterial defense, it can serve as a foundation for continued research on Bd-associated risk for the endemic frogs of Madagascar.

Beautiful bright belly: A distinctive new microhylid frog (Amphibia: Stumpffia) from eastern Madagascar.

We describe a new red-bellied species of the microhylid frog genus Stumpffia from the Andasibe region (18°56' S, 48°25' E, ca. 900 m elevation) in central-eastern Madagascar. Stumpffia kibomena sp. nov. differs from all other described Stumpffia species in coloration, morphology, and by genetic differentiation in the mitochondrial 16S  rRNA gene (≥8.6% uncorrected p-distance to all other nominal species of the genus). It is furthermore distinguished from most other Stumpffia species by its advertisement calls. The new species is reliably known only from a few specimens collected in the Andasibe region and based on the limited knowledge we suggest its IUCN Red List classification as "Data Deficient".

Widespread presence of the pathogenic fungus Batrachochytrium dendrobatidis in wild amphibian communities in Madagascar.

Amphibian chytridiomycosis, an emerging infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), has been a significant driver of amphibian declines. While globally widespread, Bd had not yet been reported from within Madagascar. We document surveys conducted across the country between 2005 and 2014, showing Bd's first record in 2010. Subsequently, Bd was detected in multiple areas, with prevalence reaching up to 100%. Detection of Bd appears to be associated with mid to high elevation sites and to have a seasonal pattern, with greater detectability during the dry season. Lineage-based PCR was performed on a subset of samples. While some did not amplify with any lineage probe, when a positive signal was observed, samples were most similar to the Global Panzootic Lineage (BdGPL). These results may suggest that Bd arrived recently, but do not exclude the existence of a previously undetected endemic Bd genotype. Representatives of all native anuran families have tested Bd-positive, and exposure trials confirm infection by Bd is possible. Bd's presence could pose significant threats to Madagascar's unique "megadiverse" amphibians.

Multilevel statistical models and the analysis of experimental data.

Data sets from ecological experiments can be difficult to analyze, due to lack of independence of experimental units and complex variance structures. In addition, information of interest may lie in complicated contrasts among treatments, rather than direct output from statistical tests. Here, we present a statistical framework for analyzing data sets containing non-independent experimental units and differences in variance among treatments (heteroscedasticity) and apply this framework to experimental data on interspecific competition among three tadpole species. Our framework involves three steps: (1) use a multilevel regression model to calculate coefficients of treatment effects on response variables; (2) combine coefficients to quantify the strength of competition (the target information of our experiment); and (3) use parametric bootstrapping to calculate significance of competition strengths. We repeated this framework using three multilevel regression models to analyze data at the level of individual tadpoles, at the replicate level, and at the replicate level accounting for heteroscedasticity. Comparing results shows the need to correctly specify the statistical model, with the model that accurately accounts for heteroscedasticity leading to different conclusions from the other two models. This approach gives a single, comprehensive analysis of experimental data that can be used to extract informative biological parameters in a statistically rigorous way.