CHORUSING PATTERNS OF A DIVERSE ANURAN COMMUNITY, WITH AN EMPHASIS ON SOUTHERN CRAWFISH FROGS (LITHOBATES AREOLATUS AREOLATUS)

Wildlife surveys have a critical role in conservation efforts and the collection of life history data. For anuran amphibians these surveys often focus on calling males. In order to further our understanding of anuran ecology, we used automated recording systems to monitor the calling activities of the anuran communities at two beaver-formed lakes and one cattle pond in southeastern Oklahoma. We documented 14 anuran species between 5 February and 28 April 2012. Temperature had a significant effect on the calling patterns of Eastern Narrow-mouthed Toads (Gastrophryne carolinensis), Green Treefrogs (Hyla cinerea), Gray Treefrogs (Hyla versicolor), Southern Crawfish Frogs (Lithobates areolatus areolatus), and Cajun Chorus Frogs (Pseudacris fouquettei). Temperature did not have a significant effect on the calling patterns of Dwarf American Toads (Anaxyrus americanus charlesmithi), American Bullfrogs (Lithobates catesbeianus), or Green Frogs (Lithobates clamitans). There was not a significant relationship between rainfall and calling for L. a. areolatus. The presence of several of these species, including L. a. areolatus and Hurter’s Spadefoots (Scaphiopus hurterii) was unusual because these anurans typically breed in ephemeral, fishless pools, but the beaver lakes are permanent and sustain populations of carnivorous fishes

Life finds a way: the recovery of frog populations from a chytridiomycosis outbreak

Emerging infectious diseases are a serious threat to wildlife, but not all populations or species have the same response to outbreaks. In some cases, diseases shift from being epizootic to enzootic, allowing populations to recover, but both the causes of recoveries and the long-term consequences of disease outbreaks remain poorly understood. My PhD aimed to further our knowledge of these important topics by using a frog assemblage in the Australian Wet Tropics as a model system for understanding recoveries from disease outbreaks. This region was impacted by an outbreak of the fungal disease chytridiomycosis (caused by the pathogen Batrachochytrium dendrobatidis [Bd]) in the late 1980s and early 1990s, during which high elevation populations of several frog species declined or disappeared, while low elevation populations remained stable. Following the outbreak, some species recovered at upland sites, but the patterns of both declines and recoveries vary among species. Litoria dayi disappeared from upland sites and has never recovered. Litoria nannotis disappeared from upland sites and has largely recovered. Litoria serrata declined at upland sites and has recovered, and Litoria wilcoxii did not decline substantially at any elevation. These different histories with the disease presented a great opportunity for studying the factors that allowed some species to recover, while apparently precluding recovery in others, and my thesis examined both population genetics and microbiomes of frogs in this system. My primary goals were to examine the long-term consequences of the outbreak (e.g., fragmentation, inbreeding, loss of diversity) and test several hypotheses for the differences in the history of declines and recoveries among species (e.g., differences in dispersal abilities, a lack of adaptive potential due to lost diversity, differences in microbiomes). I used single nucleotide polymorphisms to examine connectivity patterns, test for a loss of diversity, and test for Bd-driven selection. I examined low elevation populations of L. nannotis, L. serrata, and L. dayi that survived the outbreak, and compared them to recovered upland populations of L. nannotis and L. serrata. I sampled L. dayi at three national parks and L. nannotis and L. serrata at two national parks. All three species showed high levels of connectivity within a given park, and there was no structuring along streams, suggesting that all three species have good dispersal abilities. No inbreeding was present in any species, and all species showed high genetic diversity levels north of Paluma Range National Park. At Paluma, however, both L. nannotis and L. serrata had reduced genetic diversity, and diversity levels followed a west–east pattern, with higher diversity on the western half of the park (L. dayi does not occur at Paluma). These diversity patterns matched habitat patterns, with higher diversity in wetter areas with larger sections of rainforest, suggesting that the size and quality of refuge habitat may play an important role in the retention of genetic diversity during a disease outbreak. I did not find consistent evidence of selection in L. nannotis, but there was consistency among outlier testes for L. dayi. These tests could not conclusively demonstrate that L. dayi was undergoing diseaseinduced selection, but they were suggestive. Prior to analysing the microbiomes of the frog species, it was necessary to test or develop several microbiome methodologies. First, microbiome data often need to be normalized prior to analysis, and many methods are available, but several of the most popular methods use variance standardizing techniques that can distort ecological data. Therefore, I compared six methods (rarefaction, proportions, upper quartile, CSS, edgeRTMM, and DESeq-VS) using both a published data set and simulations. My results showed that upper quartile, CSS, edgeR-TMM, and DESeq-VS failed to fully standardize reads, and inflated minor differences among rare micro-organisms while suppressing large differences among common micro-organisms, thus distorting community comparisons. In contrast, using proportions or rarefaction produced accurate results, with proportions outperforming rarefaction. Another common issue with microbiome studies is the ubiquitous presence of bacterial contamination. This problem has been widely documented, but no method of accurately removing contaminate reads exists. Therefore, I developed an algorithm for identifying and removing contaminate reads, wrote an R package (microDecon) to implement it, and tested it using two large simulations, a published data set, and a sequencing experiment. All tests showed that microDecon was highly accurate and improved the results in 98.1% of cases. Having tested and developed these methods, I was able to apply them to the microbiomes of frog populations. Multiple laboratory studies have documented beneficial effects of bacteria for amphibian hosts during Bd infections, and several field studies have suggested that microbiomes may play important roles in infection dynamics. Nearly all of this research has focused on bacteria, while the fungal microbiomes of amphibians remain largely unexplored. Therefore, I examined both the fungal and bacterial microbiomes of L. dayi, L. nannotis, L. serrata, and L. wilcoxii to make one of the first comparisons of bacteria and fungi in frog populations and test the hypothesis that differences in microbiomes could explain the differences in patterns of declines and recoveries in the Wet Tropics frog assemblage. I also used qPCR to examine Bd infection prevalence and intensity. Bacterial microbiomes generally had higher operational taxonomic unit (OTU) richness but lower evenness than the fungal microbiomes. Bacterial microbiomes also tended to be less variable within groups of samples (e.g., frog species), resulting in stronger clustering in ordination plots. Nevertheless, fungal and bacterial Bray-Curtis dissimilarities were positively correlated within frog species (i.e., two individuals with similar fungal microbiomes tended to also have similar bacterial microbiomes). Fungal and bacterial richness were also correlated. This is a somewhat novel result that suggests that either one microbiome is driving the other, or both are being affected similarly by environmental variables. Results for associations with Bd were mixed. I did not find associations between Bd and beta-diversity for fungi or bacteria. Also, the relative abundance of bacteria that are inhibitory to Bd (based on previous culturing studies) did not follow the expected patterns of association with Bd. Litoria dayi had the highest relative abundance of inhibitory bacteria despite having never recovered from the outbreak, while L. wilcoxii (which never declined) had a low relative abundance of inhibitory bacteria. Additionally, for L. dayi and L. wilcoxii there were significant positive associations between the relative abundance of inhibitory bacteria and Bd infection intensity. In contrast, OTU richness showed negative associations with Bd infection intensity for both fungi and bacteria. Additionally, for both fungi and bacteria, L. dayi had the lowest OTU richness of any frog species. These results are consistent with a protective effect of OTU richness and suggest that a lack of richness in L. dayi has played a role in its inability to recover from the outbreak. In summary, I found that having large areas of high-quality lowland habitat is likely important for allowing populations to retain genetic diversity during an outbreak, and they should be a focus of conservation efforts. Additionally, neither differences in genetic diversity nor differences in dispersal abilities could explain why L. dayi has been unable to recover from population declines. There was some evidence that L. dayi is in the process of adapting, but this was not conclusive. The microbiome data did not show significant associations between Bd and either total community composition or the relative abundance of inhibitory bacteria, but there were associations with the OTU richness of both fungal and bacterial microbiomes, suggesting that richness may be an important factor in infection dynamics

CHORUSING PATTERNS OF A DIVERSE ANURAN COMMUNITY, WITH AN EMPHASIS ON SOUTHERN CRAWFISH FROGS (LITHOBATES AREOLATUS AREOLATUS)

Wildlife surveys have a critical role in conservation efforts and the collection of life history data. For anuran amphibians these surveys often focus on calling males. In order to further our understanding of anuran ecology, we used automated recording systems to monitor the calling activities of the anuran communities at two beaver-formed lakes and one cattle pond in southeastern Oklahoma. We documented 14 anuran species between 5 February and 28 April 2012. Temperature had a significant effect on the calling patterns of Eastern Narrow-mouthed Toads (Gastrophryne carolinensis), Green Treefrogs (Hyla cinerea), Gray Treefrogs (Hyla versicolor), Southern Crawfish Frogs (Lithobates areolatus areolatus), and Cajun Chorus Frogs (Pseudacris fouquettei). Temperature did not have a significant effect on the calling patterns of Dwarf American Toads (Anaxyrus americanus charlesmithi), American Bullfrogs (Lithobates catesbeianus), or Green Frogs (Lithobates clamitans). There was not a significant relationship between rainfall and calling for L. a. areolatus. The presence of several of these species, including L. a. areolatus and Hurter’s Spadefoots (Scaphiopus hurterii) was unusual because these anurans typically breed in ephemeral, fishless pools, but the beaver lakes are permanent and sustain populations of carnivorous fishes

Seasonal, environmental and anthropogenic influences on nocturnal basking in turtles and crocodiles from North-Eastern Australia

Many ectotherms bask in the sun as a behavioural mechanism to increase body temperature and facilitate metabolism, digestion or gamete production, among other functions. Such behaviours are common during the day, but some nocturnal species are also known to thermoregulate at night, in the absence of solar radiation, through shifts in body posture or microhabitat selection. Additionally, recent work has documented nocturnal basking in freshwater turtles in tropical Australia, though the purpose of the behaviour remains unknown. Here, we have built upon that work to test: 1. seasonal differences, 2. the influence of environmental factors and 3. the influence of anthropogenic development (e.g. river-front houses) on nocturnal basking behaviour. We visually surveyed transects repeatedly at night on the Ross River, Townsville, QLD, Australia from March to November 2020 and documented nocturnal basking in both freshwater turtles (Emydura macquarii krefftii) and freshwater crocodiles (Crocodylus johnstoni). For both taxa, we found significantly more nocturnal basking activity during the hotter months. Likewise, water surface temperature significantly influenced nocturnal basking in both taxa, especially when water temperatures were both high and warmer than air temperatures. We propose that nocturnal basking provides a mechanism for thermoregulatory cooling when water temperatures are high (e.g. 30°C) and above-preferred temperatures. After accounting for availability in basking habitat, both turtles and crocodiles basked more frequently on the undeveloped side of the river, suggesting avoidance of human activity or disturbance. This study is the first to document nocturnal basking activity temporally throughout the year as well as the first to identify the influences of environmental factors. Nocturnal thermoregulation has been documented in many reptiles, however, thermoregulatory cooling in tropical systems is less well-known

Where are the turtles? Looking for Western Chicken Turtles, Deirochelys reticularia miaria, in Mississippi

Turtles are among the world’s most threatened vertebrates, with roughly two-thirds of species listed as threatened or endangered (Buhlmann et al., 2009). Factors such as habitat loss, overharvesting, poaching, disease, and climate change are driving these declines (Stanford et al., 2020), and it is imperative that we carefully monitor turtle populations to determine the extent of declines and prioritize conservation actions for threatened species. Western Chicken Turtles (Deirochelys reticularia miaria Schwartz, 1956) are among the turtles that appear to be experiencing rapid declines. This subspecies is distinct from the other subspecies in morphology (Schwartz, 1956), diet (McKnight et al., 2015c), nesting season (McKnight et al., 2015a, 2018; Carr and Tolson, 2018), and activity season (McKnight et al., 2015a; Bowers 2020). Genetic comparisons are currently limited, but available data also suggest a deep phylogenetic split between the Eastern Chicken Turtle, D. r. reticularia, (Latreille, 1801) and D. r. miaria Schwart, 1956 (Walker and Avise 1998; Hilzinger 2009). Historically, D. r. miaria occurs in Missouri, Oklahoma, Arkansas, Louisiana, Texas, and a small portion of western Mississippi, and the ranges of D. r. miaria and D. r. reticularia are primarily divided by the Mississippi River, which likely acts as a strong barrier to interbreeding

Learned avoidance of trap locations in freshwater turtles

Context. Understanding the effects that learned responses to being captured have on subsequent recapture rates and associated abundance estimates is important for developing accurate descriptions of populations and communities. Although variation in the willingness of individual turtles to be trapped is commonly mentioned in the literature, few studies have experimentally tested learned trap avoidance (or fondness) in turtles. Aims. To determine whether turtles learn to avoid traps, whether repositioning traps will lead to increased capture rates, whether this effect varies among species, and whether such relocations yield more accurate depictions of community structure. Methods. We studied a community of turtles in a small lake in southeastern Kansas that included populations of red-eared slider turtles (Trachemys scripta elegans) and common musk turtles (Sternotherus odoratus). We trapped the lake for 35 consecutive days by using two concurrently deployed groups of traps. One group remained stationary for the duration of the study, whereas traps comprising the other group were moved to new locations on Day 14 and returned to their original locations on Day 28, thus dividing the trapping season into three periods. Key results. For both species, capture rates declined over time. However, traps in the moved group captured more T. s. elegans than did those in the stationary group during the second period and more S. odoratus during the third period. Traps in the moved group also had higher recapture rates in the second period. Population abundance estimates based on captures from the moved group, the stationary group, and the pool of all captures were similar for T. s. elegans, but for S. odoratus the stationary group of traps produced an abundance estimate much lower than those generated from the moved group and the pool of all captures. Conclusions. Both species exhibited learned avoidance of trap locations, but relocating traps had distinct effects on different species, and the accuracy of the observed community structure was improved by relocating traps. Implications. The movement patterns and catchability of individuals of different species within a community must be taken into consideration when developing trapping protocols. Even high-intensity trapping over a long period may not generate an accurate sample of the community if different species use the spatial environment in substantially different ways and learn to avoid trap locations

Habitat Preferences of Breeding Amphibians in Eastern Spain

The aim of this research was to determine the reproductive habitat preferences of several species of amphibians in eastern Spain. We recorded amphibia presence/absence and measured biotic and abiotic variables at 67 ponds in a 43.5 km2 area representing a wide variety of aquatic ecosystems, such as temporary and semipermanent cisterns, drinking troughs, and natural and artificial ponds, all of various sizes and depths. We used this information to predict occupancy using Generalized Linear Models. We built models for the Iberian Ribbed Newt (Pleurodeles waltl), Iberian Green Frog (Pelophylax perezi), Common Midwife Toad (Alytes obstetricans), Natterjack Toad (Epidalea calamita), and Mediterranean Parsley Frog (Pelodytes hespericus). We also found Common Toad (Bufo spinosus) and Spadefoot Toad (Pelobates cultripes) but did not build models for them. The variables that explained occupancy were species specific, with depth and, especially, the type of substratum playing key roles in most of them. Type of substratum reflected pond age and was represented by hard substratum (associated with new artificial ponds and structures), hard substratum covered by soft sediment (associated with old artificial ponds and structures), and soft substratum (associated with old natural ponds). The differences among the species in occupancy models indicate that species-specific management actions may be necessary to preserve the amphibian community in the long term

Too cold is better than too hot: Preferred temperatures and basking behaviour in a tropical freshwater turtle

Thermoregulation is critical to the survival of animals. Tropical environments can be particularly thermally challenging as they reach very high, even lethal, temperatures. The thermoregulatory responses of tropical freshwater turtles to these challenges are poorly known. One common thermoregulatory behaviour is diurnal basking, which, for many species, facilitates heat gain. Recently, however, a north-eastern Australian population of Krefft's river turtles (Emydura macquarii krefftii) has been observed basking nocturnally, possibly to allow cooling. To test this, we determined the thermal preference (central 50% of temperatures selected) of E. m. krefftii in an aquatic thermal gradient in the laboratory. We then conducted a manipulative experiment to test the effects of water temperatures, both lower and higher than preferred temperature, on diurnal and nocturnal basking. The preferred temperature range fell between 25.3°C (±SD: 1.5) and 27.6°C (±1.4) during the day, and 25.3°C (±2.4) and 26.8°C (±2.5) at night. Based on this, we exposed turtles to three 24 h water temperature treatments (‘cool’ [23°C], ‘preferred’ [26°C] and ‘warm’ [29°C]) while air temperature remained constant at 26°C. Turtles basked more frequently and for longer periods during both the day and night when water temperatures were above their preferred range (the ‘warm’ treatment). This population frequently encounters aquatic temperatures above the preferred thermal range, and our results support the hypothesis that nocturnal basking is a mechanism for escaping unfavourably warm water. Targeted field studies would be a valuable next step in understanding the seasonal scope of this behaviour in a natural environment

Microbiome diversity and composition varies across body areas in a freshwater turtle

There is increasing recognition that microbiomes are important for host health and ecology, and understanding host microbiomes is important for planning appropriate conservation strategies. However, microbiome data are lacking for many taxa, including turtles. To further our understanding of the interactions between aquatic microbiomes and their hosts, we used next generation sequencing technology to examine the microbiomes of the Krefft's river turtle (Emydura macquarii krefftii). We examined the microbiomes of the buccal (oral) cavity, skin on the head, parts of the shell with macroalgae and parts of the shell without macroalgae. Bacteria in the phyla Proteobacteria and Bacteroidetes were the most common in most samples (particularly buccal samples), but Cyanobacteria, Deinococcus-thermus and Chloroflexi were also common (particularly in external microbiomes). We found significant differences in community composition among each body area, as well as significant differences among individuals. The buccal cavity had lower bacterial richness and evenness than any of the external microbiomes, and it had many amplicon sequence variants (ASVs) with a low relative abundance compared to other body areas. Nevertheless, the buccal cavity also had the most unique ASVs. Parts of the shell with and without algae also had different microbiomes, with particularly obvious differences in the relative abundances of the families Methylomonaceae, Saprospiraceae and Nostocaceae. This study provides novel, baseline information about the external microbiomes of turtles and is a first step in understanding their ecological roles

Ranaviruses in captive and wild Australian lizards

Ranaviral infections have been associated with mass mortality events in captive and wild amphibian, fish, and reptile populations globally. In Australia, two distinct types of ranaviruses have been isolated: epizootic haematopoietic necrosis virus in fish and a Frog virus 3-like ranavirus in amphibians. Experimental studies and serum surveys have demonstrated that several Australian native fish, amphibian, and reptile species are susceptible to infection and supported the theory that ranavirus is naturally circulating in Australian herpetofauna. However, ranaviral infections have not been detected in captive or wild lizards in Australia. Oral-cloacal swabs were collected from 42 wild lizards from northern Queensland and 83 captive lizards from private collections held across three states/territories. Samples were tested for ranaviral DNA using a quantitative PCR assay. This assay detected ranaviral DNA in 30/83 (36.1%) captive and 33/42 (78.6%) wild lizard samples. This is the first time molecular evidence of ranavirus has been reported in Australian lizards