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Hybrid zones act as natural laboratories where divergent genomes interact, providing powerful systems for examining the evolutionary processes underlying biological diversity. In this study, we characterized patterns of genomic and phenotypic variation resulting from hybridization between divergent intraspecific lineages of the Neotropical red-eyed treefrog (Agalychnis callidryas). We found genetic evidence of a newly discovered contact zone and phenotypic novelty in leg colour-a trait suspected to play a role in mediating assortative mating in this species. Analysis of hybrid ancestry revealed an abundance of later-generation Fn individuals, suggesting persistence of hybrids in the contact zone. Hybrids are predominantly of southern ancestry but are phenotypically more similar to northern populations. Genome-wide association mapping revealed QTL with measurable effects on leg-colour variation, but further work is required to dissect the architecture of this trait and establish causal links. Further, genomic cline analyses indicated substantial variation in patterns of introgression across the genome. Directional introgression of loci associated with different aspects of leg colour are inherited from each parental lineage, creating a distinct hybrid colour pattern. We show that hybridization can generate new phenotypes, revealing the evolutionary processes that potentially underlie patterns of phenotypic diversity in this iconic polytypic frog. Our study is consistent with a role of hybridization and sexual selection in lineage diversification, evolutionary processes that have been implicated in accelerating divergence in the most phenotypically diverse species.

Geographic variation in courtship behavior can affect reproductive success of divergent phenotypes via mate choice. Over time, this can lead to reproductive isolation and ultimately to speciation. The Neotropical red-eyed treefrog (Agalychnis callidryas) exhibits high levels of phenotypic variation among populations in Costa Rica and Panama, including differences in color pattern, body size, and skin peptides. To test the extent of behavioral premating isolation among differentiated populations, we quantified male advertisement calls from six sites and female responses to male stimuli (acoustic and visual signals) from four sites. Our results show that both male advertisement calls and female behavior vary among populations: Discriminant function analyses can predict the population of origin for 99.3% ± 0.7 of males based on male call (dominant frequency and bandwidth) and 76.1% ± 6.6 of females based on female response behavior (frequency and duration of visual displays). Further, female mate choice trials (n = 69) showed that population divergence in male signals is coupled with female preference for local male stimuli. Combined, these results suggest that evolved differences among populations in male call properties and female response signals could have consequences for reproductive isolation. Finally, population variation in male and female behavior was not well explained by geographic or genetic distance, indicating a role for localized selection and/or drift. The interplay between male courtship and female responses may facilitate the evolution of local variants in courtship style, thus accelerating premating isolation via assortative mating.

Premating reproductive isolation (RI) may reduce gene flow across populations that have differentiated in traits important for mate choice. Examining RI across genetic and phenotypic clines can inform the fundamental evolutionary processes that underlie population and lineage differentiation. We conducted female mate-choice studies across an intraspecific red-eyed treefrog cline in Costa Rica and Panama with 2 specific aims: (1) to characterize RI across the cline and examine the relationship between premating RI and genetic and phenotypic distance and (2) to evaluate our results within a broader evolutionary and taxonomic perspective through examination of other RI studies. We found that female red-eyed treefrogs prefer local males relative to non-local males, indicating that some premating RI has evolved in this system, but that preference strength is not associated with phenotypic or geographic distance. Our analysis of 65 other studies revealed no clear pattern between the strength of RI and geographic distribution (allopatry, parapatry, cline) or phenotypic distance, but revealed extreme variation and overlap in levels of intra- and interspecific levels of RI. This work contributes to a growing body of literature that examines intraspecific RI across a cline to understand the selective processes that shape evolutionary patterns at the earliest stages of divergence.

The Central American red-eyed treefrog (Agalychnis callidryas) is a nocturnal/crepuscular frog that exhibits high population-level phenotypic variation in a suite of traits generally important in anuran communication. Females of this species demonstrate a preference for local over non-local males, although the contribution of different communication modes to mate choice remains untested. We performed eight sets of experiments at La Selva Biological Station (Costa Rica) to test the relative roles of male advertisement call and flank-stripe pattern on female mate choice. For each trial, we used pre-recorded calls for acoustic signals and/or 3D-printed, hand-painted models for visual stimulus. Females selected conspecifics over heterospecifics when presented with either acoustic or visual stimuli only, indicating that both evolve as species-specific signals used for mate discrimination. Bayesian modeling showed that females chose local males over non-locals based on unimodal; responses to multimodal signals varied. Female mate choice in this species is likely nuanced and subject to the constraints of the local environment in which animals are signaling. Our data show that at least two traits that vary among populations are important for mate selection. Evolution shapes animal communication in diverse ways to accommodate complicated signaling contexts, including environmental noise, the presence of eavesdroppers, and changing landscapes. Among species that use complex signals or signals in multiple modalities, understanding the role of signaling traits in reproductive behavior and their concomitant shifts between populations can shed light on lineage divergence, speciation, and the forces shaping communication. We used choice tests with Agalychnis callidryas to determine which traits lead to local-male mate preference. We found that both call and stripe are each sufficient to allow this nocturnal frog to choose mates in absence of the other stimulus. Multimodal signals were marginally better than call—but not color—alone. This may be a result of statistical noise or preliminary evidence of asymmetric use of traits across a complex landscape. These results suggest that call and color may encode both redundant information and multiple messages to receivers in this species: either is sufficient to elicit mate choice (redundant), but together, they enhance local mate preference (multiple messages). Further tests on how other communication modalities inform behavior will add to our understanding of complex signaling and lineage divergence among differentiated populations in this species.

Finite element error estimates are derived for the incompressible Stokes equations with variable viscosity. The ratio of the supremum and the infimum of the viscosity appears in the error bounds. Numerical studies show that this ratio can be observed sometimes. However, often the numerical results show a weaker dependency on the viscosity.

Background The non-lethal collection of sperm from live males is an important component for multiple captive-breeding techniques, including assisted reproductive technology (ART) protocols, sperm cryopreservation and in vitro fertilization. However, in amphibians, the type and amount of hormone necessary to induce spermiation can be highly variable, even among closely related species. We are unaware of any studies that have examined the spermiation response to exogenous hormones across highly differentiated populations within a species. Methods We examined variation in sperm viability and production in response to the hormone LHRH among four divergent populations of the Red-eyed Treefrog (Agalychnis callidryas). We hypothesized that these highly differentiated populations would show variability in sperm count and viability in response to two dosages, 2 μg/g and 4 μg/g, of the hormone LHRH. We collected spermic urine 3 h post injection (PI). We then examined variation in spermiation at 3, 7, 12, and 24 h PI of LHRH for two allopatric populations that previously showed evidence of premating behavioral isolation. Results One population of Red-eyed Treefrog exhibited reduced sperm viability but not count in response to the hormone LHRH compared to all other populations. In addition, we found peak viability at 3 h PI for the allopatric population comparison. There was no difference in sperm production within or between populations at 3, 7, 12, or 24 h PI. For both studies, intrapopulation variation was high. Conclusion ART often focuses on threatened species with small, isolated populations, which could evolve localized differences due to the evolutionary process of drift and selection. The high variation in response and the population-level differences in sperm viability we observed demonstrate that practitioners of ART should consider the possibility of divergent responses to hormones which may affect study design and animal receptivity to ART protocols. Electronic supplementary material The online version of this article (doi:10.1186/s12958-016-0216-3) contains supplementary material, which is available to authorized users.

Many organisms depend on acoustic communication for myriad functions, and have evolved behaviours to minimize effects of naturally occurring acoustic interference. However, as habitats are subject to increased alteration, anthropogenic noise becomes unavoidable, and how animals overcome such interference is not well understood. In most ecosystems, only a subset of frog species is associated with disturbed habitats; the ability of these species to overcome exogenous noise suggests that habitat associations may be related to species' response to noise. We tested the hypothesis that frogs associated with largely undisturbed forest habitat would be less likely to increase call output in response to exogenous noise than would those associated with disturbed or open habitat. While this relationship was not significant, we found a slight trend supporting the hypothesis. We then asked whether anthropogenic noise affects chorus tenure at individual- or at chorus-levels. Male frogs exposed to anthropogenic noise decreased both the number of days present at the chorus and the nightly chorus duration relative to controls. Because females generally join choruses late at night to breed, the effects of noise shown here are likely to substantially decrease frog reproductive success; thus, the acoustic environment may play an important role in shaping population dynamics and in amphibian declines.

In Belize, Central America, many farms surrounding the Protected Areas of the Maya Mountains rely heavily on the application of agrochemicals. The purpose of this study was to test whether orographic drift of glyphosate and organophosphates into the nearby Maya Mountain Protected Areas occurred by collecting phytotelmic water from seven sites over 3 years. Regardless of location within the Maya Mountain Protected Areas, glyphosate was present; organophosphates were more common at ridge sites. Although glyphosate concentrations were low, due to the number of threatened species and the human use of stream water outside the Maya Mountain Protected Areas, better understanding of these effects is warranted.

Anthropogenic change such as road construction and subsequent traffic noise in pristine habitats has been shown to be detrimental to a range of vertebrate taxa. The effect of anthropogenic noise on anuran communication is not well known, and has only recently been a topic of investigation. We tested the effect of anthropogenic noise on the calling behaviour of the Amazonian treefrog Dendropsophus triangulum . We performed four experiments. We first presented frogs with the noise of a pre-recorded motorcycle engine, and recorded the call rates of focal frogs for 5 min, then for another 5 min during which they were presented with a broadcast of noise, and again for 5 min post-stimulus. We repeated this experiment using music as the stimulus. We then tested the effect of intermittent engine noise, recording baseline call rate and response to six cycles of intermittent noise. In all experiments, call rates nearly doubled during playbacks. Finally, we compared frogs' responses to anthropogenic noise to responses to chorus noise broadcast at the same intensity; call rates did not differ between treatments. These results demonstrate a clear effect of exogenous noise on male D. triangulum .

The laminar scales of the fern Elaphoglossum paleaceum were studied to determine if they act as a morphological mechanism to protect leaves from excess light. We hypothesized that if scales serve this purpose, then individual plants growing in high light would have greater laminar scale density than those in low light. For our first experiment, plants from high light roadsides were collected and subjected to artificial scale removal and then exposed to super-saturating pulse of light for 30 min. We found that leaves with their scales removed exhibited significantly greater photoinhibition than leaves with scales intact. Leaves with intact scales recovered fully after twelve hours whereas those with scales removed remained photoinhibited. Scale density on the adaxial side of leaves was positively correlated with light intensity. The data from this study indicate that fern laminar scales help reduce photoinhibition and potentially function as a morphological defense against photodamage.

Urbanization brings the introduction of roads and anthropogenic noise. Noise has negative health outcomes across vertebrates. White's treefrogs (Litoria caerulea) exposed to ecologically relevant levels of traffic noise for one week had elevated circulating corticosterone levels and decreased sperm count and sperm viability relative to controls., Urbanization is a major driver of ecological change and comes with a suite of habitat modifications, including alterations to the local temperature, precipitation, light and noise regimes. Although many recent studies have investigated the behavioural and ecological ramifications of urbanization, physiological work in this area has lagged. We tested the hypothesis that anthropogenic noise is a stressor for amphibians and that chronic exposure to such noise leads to reproductive suppression. In the laboratory, we exposed male White's treefrogs, Litoria caerulea, to conspecific chorus noise either alone or coupled with pre-recorded traffic noise nightly for 1 week. Frogs presented with anthropogenic noise had significantly higher circulating concentrations of corticosterone and significantly decreased sperm count and sperm viability than did control frogs. These results suggest that in addition to having behavioural and ecological effects, anthropogenic change might alter physiology and Darwinian fitness. Future work should integrate disparate fields such as behaviour, ecology and physiology to elucidate fully organisms’ responses to habitat change.

Rationale: Psychological (psych) distress is a putative risk factor for coronary artery disease (CAD) and may affect compliance with lifestyle & treatment. We sought to determine the impact of psych distress in cardiac rehab (CR) patients (pts) on cardiovascular event rates (CVE) including PCI/CABG, coronary angiogram, acute coronary syndrome (ACS). Methods: Psych profile of 440 CAD pts was assessed on entry to CR using Symptom Check List-90 (SCL-90). Questionnaire screened for depression (dep), anxiety (anx), hostility, and a global severity index (GSI). Mean SCL-90 T-score for each parameter in the adult population is 50 with a standard deviation of 10. SCL-90 score ≥60 = 84th percentile, SCL≥63 = 90th percentile, SCL score ≥70 is the 98th percentile. Complete data on repeat admissions and subsequent non-elective and elective CV procedures performed in the one-year following enrollment to CR was available on 410 pts. Results: Of the 440 pts 28.4% were female, 75.7% married and 89% Caucasian; 25.5% were hypertensive, 20.5% diabetic and 47% had a BMI>30. Mean age was 61.5+10.7. Clinical indication for CR: 56.4% PCI/CABG, 8.4% stable angina (SA) and 35.2% ACS. There was a high prevalence of dep, anx, and overall psych distress as measured by the GSI (Table1.) SA pts had significantly higher prevalence of anx (p=0.018) and hostility (p=0.009) compared to PCI/CABG and ACS. There was no difference in SCL-90 scores by gender, age, education, body mass index (BMI) or diabetes. 89 CVE required hospital admission; 62 (15.1%) pts once and 27(6.5%) two or more times. Reasons for the 89 readmissions included myocardial infarction (7.9%), SA (11.2%), unstable angina (50.6%) and other non-cardiac (30.3%). 62 procedures were performed, which included 6(10%) CABG, 21(34%) PCI, 20(32%) heart catheterizations, and 15(24%) other procedures. Forty percent of procedures performed were elective. Odds of readmission at one year was 3.14 greater in pts with dep score ≥70 (p=0.003) and 2.95 times greater in pts with Anx score ≥70 (p=0.037). Pts with dep score ≥70 were 3.4 times more likely to get a non-elective procedure (p Conclusions: Psych distress in CR pts has a significant impact on outcomes. Consideration should be given to routine psych profiling at entry to CR and appropriate treatment for these pts.

The amphibian pathogen Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines around the globe. Although it has been found in most countries in Central America, its presence has never been assessed in Belize. We set out to determine the range, prevalence, and diversity of Bd using quantitative PCR (qPCR) and sequencing of a portion of the 5.8 s and ITS1-2 regions. Swabs were collected from 524 amphibians of at least 26 species in the protected areas of the Maya Mountains of Belize. We sequenced a subset of 72 samples that had tested positive for Bd by qPCR at least once; 30 samples were verified as Bd. Eight unique Bd haplotypes were identified in the Maya Mountains, five of which were previously undescribed. We identified unique ecological niches for the two most broadly distributed haplotypes. Combined with data showing differing virulence shown in different strains in other studies, the 5.8 s - ITS1-2 region diversity found in this study suggests that there may be substantial differences among populations or haplotypes. Future work should focus on whether specific haplotypes for other genomic regions and possibly pathogenicity can be associated with haplotypes at this locus, as well as the integration of molecular tools with other ecological tools to elucidate the ecology and pathogenicity of Bd.

Long-term elevation of glucocorticoid hormones associated with chronic stress may be detrimental. Chronic treatment of White's treefrogs with exogenous corticosterone reduced eosinophil numbers but did not alter sperm count or sperm viability. We suggest that corticosterone inhibits immune but not reproductive function in this species in response to chronic stress., Amphibian populations are declining globally. The potential contribution of glucocorticoid hormones to these declines has received little attention, but chronic elevation of glucocorticoids has been linked to a suite of negative outcomes across vertebrate taxa. Recently, chronic environmental stress has been associated with precipitous declines in sperm count and sperm viability in White’s treefrogs (Litoria caerulea), but the mechanism remains unknown. In order to determine whether corticosterone is responsible for suppressing reproductive and immune function in this species, we elevated circulating concentrations of corticosterone in 10 male captive-bred frogs via transdermal application for 7 days. We compared sperm count, sperm viability, splenic cell count and circulating leucocyte counts in corticosterone-treated frogs with those in untreated control frogs. Chronic application of exogenous corticosterone led to supraphysiological circulating concentrations of corticosterone, but had no effect on sperm count or viability. However, corticosterone-treated frogs demonstrated a significant decrease in circulating eosinophils, which are immune cells implicated in fighting a variety of pathogens, including extracellular parasites. These findings suggest that although chronic elevation of circulating corticosterone is not necessarily associated with reproductive suppression in this species, it may cause immunosuppression. Thus, chronic glucocorticoid elevations in amphibians might enhance susceptibility to infection with pathogens and parasites, and their potential contributions to global population declines warrant further study.