Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

Systematic assessment of the brown tree frog (Anura: Pelodryadidae: Litoria ewingii) reveals two endemic species in South Australia.

The brown tree frog (Litoria ewingii) is a relatively widespread, commonly encountered pelodryadid frog from south-eastern Australia, known for its characteristic whistling call. The distribution of Litoria ewingii spans over more than 350,000 km2, encompassing a range of moist temperate habitats, and is fragmented by well-known biogeographic barriers. A preliminary analysis of mitochondrial DNA sequences revealed evidence for deep phylogenetic structure between some of these fragmented populations. In this study, we sought to re-evaluate the systematics and taxonomy of Litoria ewingii sensu lato by analysing variation in nuclear and mitochondrial DNA, adult morphology and male advertisement calls throughout the species range. Our analyses reveal two additional, deeply divergent and allopatric lineages in South Australia. We herein re-describe Litoria ewingii from Tasmania, southern New South Wales, Victoria and south-eastern South Australia, resurrect the name Litoria calliscelis for a species occurring in the Mount Lofty Ranges and Fleurieu Peninsula in South Australia, and describe a new species, Litoria sibilus sp. nov., endemic to Kangaroo Island.

Refrigerated storage and cryopreservation of hormonally induced sperm in the threatened frog, Litoria aurea.

As sperm cryopreservation and other assisted reproductive technologies (ARTs) advance in common amphibian species, focus on applying non-lethal sperm collection methods to the conservation and genetic management of threatened species is imperative. The goal of this study was to examine the application of logistically practical ART protocols in a threatened frog (Litoria aurea). First, we tested the efficacy of various concentrations of human chorionic gonadotropin (hCG) (20, 40 IU/g bodyweight) and Gonadotropin releasing hormone antagonist (0.25 µg/g and 0.5 µg/g body weight GnRH-a) on the induction of spermatozoa. Using the samples obtained from the previous trials, we tested the effect of cold storage and cryopreservation protocols on long-term refrigerated storage and post-thaw sperm recovery. Our major findings include: (1) high quality sperm were induced with 20 and 40 IU/g bodyweight of (hCG); (2) proportions of live, motile sperm post-thaw, were recovered at higher levels than previously reported for L. aurea (>50%) when preserved with 15% v/v DMSO and 1% w/v sucrose; and (3) spermic urine stored at 5 °C retained motility for up to 14 days. Our findings demonstrate that the protocols developed in this study allowed for successful induction and recovery of high-quality spermatozoa from a threatened Australian anuran, L. aurea, providing a prime example of how ARTs can contribute to the conservation of rare and threatened species.

Cell culture and karyotypic description of Pseudophrynecoriacea (Keferstein, 1868) (Amphibia, Anura) from the New South Wales Central Coast.

The karyotype of the IUCN least concern red-backed toadlet Pseudophryne (P.) coriacea (Keferstein, 1868) from the New South Wales Central Coast is described following tissue culture of toe clipping macerates and conventional DAPI staining. The diploid number is 2n = 24. The karyotype is represented by six large and five small chromosomal pairs and one very small chromosomal pair. The very small chromosome 12 is 12% the size of chromosome 1. One of the large chromosomes is subtelocentric, two of the large chromosomes are submetacentric and the remaining chromosomes are metacentric. The putative nucleolus organiser region (NOR) is observed on chromosome 4. The diploid number and location of the putative NOR correlates to that of the previously published IUCN critically endangered P.corroboree (Moore 1953) and unpublished descriptions of the P.coriacea karyotype. This is the first described cell culture of a species from the genus Pseudophryne Fitzinger, 1843, first published analysis of the P.coriacea karyotype and the first published analysis of centromeric allocation of this genus. Globally there exists a large inventory of tissue samples in cryobanks that are not associated with known recovery mechanisms such as basic cell culture techniques. Detailed cytogenetic analyses of these cryobanked samples are therefore not possible. This work therefore enables: (i) a comparison of the P.coriacea karyotype with that of the critically endangered P.corroboree and (ii) a benchmark for repeat and future cytogenetic and genomic analyses of cryostored samples of this genus.

An invasive pathogen drives directional niche contractions in amphibians.

Global change is causing an unprecedented restructuring of ecosystems, with the spread of invasive species being a key driver. While population declines of native species due to invasives are well documented, much less is known about whether new biotic interactions reshape niches of native species. Here we quantify geographic range and realized-niche contractions in Australian frog species following the introduction of amphibian chytrid fungus Batrachochytrium dendrobatidis, a pathogen responsible for catastrophic amphibian declines worldwide. We show that chytrid-impacted species experienced proportionately greater contractions in niche breadth than geographic distribution following chytrid emergence. Furthermore, niche contractions were directional, with contemporary distributions of chytrid-impacted species characterized by higher temperatures, lower diurnal temperature range, higher precipitation and lower elevations. Areas with these conditions may enable host persistence with chytrid through lower pathogenicity of the fungus and/or greater demographic resilience. Nevertheless, contraction to a narrower subset of environmental conditions could increase host vulnerability to other threatening processes and should be considered in assessments of extinction risk and during conservation planning. More broadly, our results emphasize that biotic interactions can strongly shape species realized niches and that large-scale niche contractions due to new species interactions-particularly emerging pathogens-could be widespread.

A new species of barred frog, Mixophyes (Anura: Myobatrachidae) from south-eastern Australia identified by molecular genetic analyses.

Mixophyes are large ground-dwelling myobatrachid frogs from eastern Australia and New Guinea. Several of the species found in mid-eastern and south-eastern Australia are listed as threatened, due largely to declines presumably caused by the amphibian disease chytridiomycosis. Given the wide distribution of several of these species and that their distributions cross well-known biogeographic boundaries that often correspond to deep genetic breaks or species boundaries among closely related vertebrates, we undertook a molecular genetic assessment of population structure across the range of each species to determine the presence of undescribed species. Of the four species of Mixophyes subject to molecular population genetic analyses, one, the Stuttering Frog (Mixophyes balbus), showed a level of diversity consistent with the presence of two species. Morphometric, meristic and bioacoustic analyses corroborate these distinctions, and a new species is described for the populations south of the Macleay River valley in mid-eastern New South Wales to east Gippsland in Victoria. Applying the IUCN Red List threat criteria the new species meets the conservation status assessment criteria for Endangered 2B1a,b because its extent of occupancy and area of occupancy are below the threshold value and it has declined and disappeared from the southern two thirds of its distribution over the past 30 years.

Complex Organisms Must Deal with Complex Threats: How Does Amphibian Conservation Deal with Biphasic Life Cycles?

The unprecedented rate of global amphibian decline is attributed to The Anthropocene, with human actions triggering the Sixth Mass Extinction Event. Amphibians have suffered some of the most extreme declines, and their lack of response to conservation actions may reflect challenges faced by taxa that exhibit biphasic life histories. There is an urgent need to ensure that conservation measures are cost-effective and yield positive outcomes. Many conservation actions have failed to meet their intended goals of bolstering populations to ensure the persistence of species into the future. We suggest that past conservation efforts have not considered how different threats influence multiple life stages of amphibians, potentially leading to suboptimal outcomes for their conservation. Our review highlights the multitude of threats amphibians face at each life stage and the conservation actions used to mitigate these threats. We also draw attention to the paucity of studies that have employed multiple actions across more than one life stage. Conservation programs for biphasic amphibians, and the research that guides them, lack a multi-pronged approach to deal with multiple threats across the lifecycle. Conservation management programs must recognise the changing threat landscape for biphasic amphibians to reduce their notoriety as the most threatened vertebrate taxa globally.

Molecular systematic analysis demonstrates that the threatened southern bell frog, Litoria raniformis (Anura: Pelodryadidae) of eastern Australia, comprises two sub-species.

In south-eastern Australia, the pelodryadid Litoria aurea Group (sensu Tyler & Davies 1978) comprises three species: Litoria aurea (Lesson, 1829), Litoria raniformis (Keferstein, 1867), and Litoria castanea (Steindachner, 1867). All three species have been subject to declines over recent decades and taxonomic uncertainty persists among populations on the tablelands in New South Wales. We address the systematics of the Group by analysing mitochondrial and nuclear DNA sequences to assess divergence in the Litoria raniformis from across its current range in New South Wales (NSW), Victoria, South Australia (SA) and Tasmania. We also included samples of Litoria castanea from a recently rediscovered population in the southern tablelands of NSW. Our phylogenetic and population genetic analyses show that Litoria raniformis comprises northern and southern lineages, showing deep mitochondrial DNA sequence divergence (7% net average sequence divergence) and can be diagnosed by fixed allelic differences at more than 4,000 SNP loci. Samples of the northern lineage were collected from the Murray-Darling Basin while those of the southern lineage were collected from south-eastern South Australia, southern and south-eastern Victoria and Tasmania. Analysis of the morphology and bioacoustics did not unequivocally delineate the two lineages. The presence of a hybrid backcross individual in western Victoria at the northern margin of the southern lineage, leads us to assign sub-species status to the two lineages, L. r. raniformis for the northern lineage and L. r. major for the southern lineage. Our data do not unequivocally resolve the taxonomic status of L. castanea which will require molecular genetic analyses of museum vouchers from those parts of the range where L. castanea and L. raniformis are no longer extant. Our data also suggest that human mediated movement of frogs may have occurred over the past 50 years. Our genotyping of vouchers collected in the 1970s from the Mount Lofty Ranges in South Australia detected mitochondrial haplotypes of both sub-species and SNP analysis showed that a single Tasmanian specimen was a backcross with L. r. raniformis ancestry. Movement of L. r. raniformis into Tasmania and both sub-species into the Mount Lofty Ranges are not likely due to passive movements of animals through agricultural commerce, but due to the attractiveness of the species as pets and subsequent escapes or releases, potentially of the larval life stage.

Paradigm shift in frog sperm cryopreservation: reduced role for non-penetrating cryoprotectants.

In brief: Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs. Abstract: Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.

The effect of salt dosing for chytrid mitigation on tadpoles of a threatened frog, Litoria aurea.

The novel fungal pathogen Batrachochytrium dendrobatidis (chytrid) is one of the greatest threats to amphibians worldwide. Small increases in water salinity (up to ca. 4 ppt) have been shown to limit chytrid transmission between frogs, potentially providing a way to create environmental refugia to reduce its impact at a landscape scale. However, the effect of increasing water salinity on tadpoles, a life stage confined to water, is highly variable. Increased water salinity can lead to reduced size and altered growth patterns in some species, with flow-on effects to vital rates such as survival and reproduction. It is thus important to assess potential trade-offs caused by increasing salinity as a tool to mitigate chytrid in susceptible frogs. We conducted laboratory experiments to examine the effects of salinity on the survival and development of tadpoles of a threatened frog (Litoria aurea), previously demonstrated as a suitable candidate for trialling landscape manipulations to mitigate chytrid. We exposed tadpoles to salinity ranging from 1 to 6 ppt and measured survival, time to metamorphosis, body mass and locomotor performance of post-metamorphic frogs as a measure of fitness. Survival and time to metamorphosis did not differ between salinity treatments or controls reared in rainwater. Body mass was positively associated with increasing salinity in the first 14 days. Juvenile frogs from three salinity treatments also showed the same or better locomotor performance compared to rainwater controls, confirming that environmental salinity may influence life history traits in the larval stage, potentially as a hormetic response. Our research suggests that salt concentrations in the range previously shown to improve survival of frogs in the presence of chytrid are unlikely to impact larval development of our candidate threatened species. Our study lends support to the idea of manipulating salinity to create environmental refugia from chytrid for at least some salt-tolerant species.

A new species of Philoria (Anura: Limnodynastidae) from the uplands of the Gondwana Rainforests World Heritage Area of eastern Australia.

The six species of mountain frogs (Philoria: Limnodynastidae: Anura) are endemic to south-eastern Australia. Five species occur in headwater systems in mountainous north-eastern New South Wales (NSW) and south-eastern Queensland (Qld), centred on the Gondwana Rainforests of Australia World Heritage Area. A previous molecular genetic analysis identified divergent genetic lineages in the central and western McPherson Ranges region of Qld and NSW, but sampling was inadequate to test the species status of these lineages. With more comprehensive geographic sampling and examination of the nuclear genome using SNP analysis, we show that an undescribed species, P. knowlesi sp. nov., occurs in the central and western McPherson Ranges (Levers Plateau and Mount Barney complex). The new species is not phylogenetically closely related to P. loveridgei in the nuclear data but is related to one of two divergent lineages within P. loveridgei in the mtDNA data. We postulate that the discordance between the nuclear and mtDNA outcomes is due to ancient introgression of the mtDNA genome from P. loveridgei into the new species. Male advertisement calls and multivariate morphological analyses do not reliably distinguish P. knowlesi sp. nov. from any of the Philoria species in northeast NSW and southeast Qld. The genetic comparisons also enable us to define further the distributions of P. loveridgei and P. kundagungan. Samples from the Lamington Plateau, Springbrook Plateau, Wollumbin (Mt Warning National Park), and the Nightcap Range, are all P. loveridgei, and its distribution is now defined as the eastern McPherson Ranges and Tweed caldera. Philoria kundagungan is distributed from the Mistake Mountains in south-eastern Qld to the Tooloom Scrub on the Koreelah Range, southwest of Woodenbong, in NSW, with two subpopulations identified by SNP analysis. We therefore assessed the IUCN threat category of P. loveridgei and P. kundagungan and undertook new assessments for each of its two subpopulations and for the new taxon P. knowlesi sp. nov., using IUCN Red List criteria. Philoria loveridgei, P. kundagungan (entire range and northern subpopulation separately) and P. knowlesi sp. nov. each meet criteria for Endangered (EN B2(a)(b)[i, iii]). The southern subpopulation of P. kundagungan, in the Koreelah Range, meets criteria for Critically Endangered (CE B2(a)(b)[i, iii]). These taxa are all highly threatened due to the small number of known locations, the restricted nature of their breeding habitat, and direct and indirect threats from climate change, and the potential impact of the amphibian disease chytridiomycosis. Feral pigs are an emerging threat, with significant impacts now observed in Philoria breeding habitat in the Mistake Mountains.

Factors influencing persistence of a threatened amphibian in restored wetlands despite severe population decline during climate change driven weather extremes.

Biodiversity is in global decline during the Anthropocene. Declines have been caused by multiple factors, such as habitat removal, invasive species, and disease, which are often targets for conservation management. However, conservation interventions are under threat from climate change induced weather extremes. Weather extremes are becoming more frequent and devastating and an example of this was the 2019/2020 Australian drought and mega-fires. We provide a case study the impacts of these extreme weather events had on a population of the threatened frog Litoria aurea that occurs in a constructed habitat which was designed to reduce the impact of introduced fish and chytrid-induced disease. We aimed to determine what factors influenced persistence so that the design of wetlands can be further optimised to future-proof threatened amphibians. We achieved this with 4 years (2016-2020) of intensive capture-recapture surveys during austral spring and summer across nine wetlands (n = 94 repeat surveys). As hypothesized, drought caused a sharp reduction in population size, but persistence was achieved. The most parsimonious predictor of survival was an interaction between maximum air temperature and rainfall, indicating that weather extremes likely caused the decline. Survival was positively correlated with wetland vegetation coverage, positing this is an important feature to target to enhance resilience in wetland restoration programs. Additionally, the benefits obtained from measures to reduce chytrid prevalence were not compromised during drought, as there was a positive correlation between salinity and survival. We emphasize that many species may not be able to persist under worse extreme weather scenarios. Despite the potential for habitat augmentation to buffer effects of extreme weather, global action on climate change is needed to reduce extinction risk. Supplementary Information: The online version contains supplementary material available at 10.1007/s10531-022-02387-9.

Seasonal variation in the prevalence of a fungal pathogen and unexpected clearance from infection in a susceptible frog species.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is a primary driver for amphibian population declines and extinctions worldwide. For highly susceptible species, such as the green and golden bell frog Litoria aurea, large numbers of Bd-related mortalities are thought to occur during the colder season (winter), when low temperatures favour the growth of the pathogen. However, extant L. aurea populations are persisting with Bd. We measured Bd prevalence and infection levels of wild L. aurea using capture-mark-recapture and radio-tracking methods. Using this information, we sought to determine host and environmental correlates of Bd prevalence and infection load. Mean ± SE infection load was higher in frogs sampled in autumn (431.5 ± 310.4 genomic equivalents; GE) and winter (1147.5 ± 735.8 GE), compared to spring (21.8 ± 19.3 GE) and summer (0.9 ± 0.8 GE). Furthermore, prevalence of Bd infection in L. aurea was highest in winter (43.6%; 95% CI 33.1-54.7%) and lowest in summer (11.2%; 95% CI 6.8-17.9%). Both prevalence and infection load decreased with increasing temperature. Seven frogs cleared their fungal infection during the coolest months when Bd prevalence was highest; however, these clearances were not permanent, as 5 frogs became infected again. Understanding the factors that allow amphibians to clear their Bd infections when temperatures are optimal for Bd growth presents the potential for manipulating such factors and provides an important step in future research.

Taxonomic revision of south-eastern Australian giant burrowing frogs (Anura: Limnodynastidae: Heleioporus Gray).

The rarely encountered giant burrowing frog, Heleioporus australiacus, is distributed widely in a variety of sclerophyll forest habitats east of the Great Dividing Range in south-eastern Australia. Analyses of variation in nucleotide sequences of the mitochondrial ND4 gene and thousands of nuclear gene SNPs revealed the presence of two deeply divergent lineages. Multivariate morphological comparisons show the two lineages differ in body proportions with > 91% of individuals being correctly classified in DFA. The two lineages differ in the number and size of spots on the lateral surfaces and the degree by which the cloaca is surrounded by colour patches. The mating calls are significantly different in number of pulses in the note. The presence of a F2 hybrid in the area where the distribution of the two taxa come into closest proximity leads us to assign subspecies status to the lineages, as we have not been able to assess the extent of potential genetic introgression. In our sampling, the F2 hybrid sample sits within an otherwise unsampled gap of ~90km between the distributions of the two lineages. The nominate northern sub-species is restricted to the Sydney Basin bioregion, while the newly recognised southern subspecies occurs from south of the Kangaroo Valley in the mid-southern coast of New South Wales to near Walhalla in central Gippsland in Victoria. The habitat of the two subspecies is remarkably similar. Adults spend large portions of their lives on the forest floor where they forage and burrow in a variety of vegetation communities. The southern subspecies occurs most commonly in dry sclerophyll forests with an open understory in the south and in open forest and heath communities with a dense understory in the north of its distribution. The northern subspecies is also found in dry open forests and heaths in association with eroded sandstone landscapes in the Sydney Basin bioregion. Males of both taxa call from both constructed burrows and open positions on small streams, differing from the five Western Australian species of Heleioporus where males call only from constructed burrows. Using the IUCN Red List process, we found that the extent of occupancy and area of occupancy along with evidence of decline for both subspecies are consistent with the criteria for Endangered (A2(c)B2(a)(b)).

A new hip-pocket frog from mid-eastern Australia (Anura: Myobatrachidae: Assa).

The hip-pocket frog (Assa darlingtoni), a small terrestrial myobatrachid frog found in mid-eastern Australia, has a highly derived, unusual, reproductive mode involving a unique form of male parental care. Males have subcutaneous pouches that open near the hip, and the developing tadpoles are carried in these pouches to post metamorphosis. It is found on several isolated mountain ranges in closed forest habitats, associated with high rainfall and temperate or sub-tropical climates. We established genetic relationships among specimens sampled across the range using phylogenetic analyses of thousands of single nucleotide polymorphisms (SNPs) from the nuclear genome and mitochondrial ND2 gene nucleotide sequences. These analyses uncovered two lineages that are genetically distinct in both nDNA and mtDNA analyses and that have low levels of divergence in male advertisement calls and are morphologically cryptic. Our data support separate species status for each lineage, based on the molecular genetic data. The first, which we name as a new species, Assa wollumbin sp. nov., is restricted to a single mountain, Wollumbin (= Mount Warning), the eroded cone of an ancient shield volcanothe Tweed Volcano. The second, the nominal species A. darlingtoni, has a wider distribution in five geographically disjunct subpopulations along 430 km of the Great Dividing Range in south-eastern Queensland and north-eastern New South Wales. The distributions of the two species closely approach within 15 km of each other on the central plug and rim of the caldera of the Tweed Volcano. Assa wollumbin sp. nov. meets the conservation criteria for Critically Endangered [A3(e), B2(a,b)]. When all subpopulations of A. darlingtoni are combined the conservation assessment is Endangered [A3(e), B2(a,b)]. Because of the fragmented nature of the distribution of A. darlingtoni, combined with the genetic evidence of concordant sub-structuring, we also conducted a conservation assessment on the five subpopulations. Two were assessed as Critically Endangered (DAguilar Range and Conondale/Blackall Ranges), and the remainder as Endangered (Dorrigo Plateau, McPherson Ranges, and Gibraltar Ranges/Washpool).

Genetic evidence for polyandry in the threatened green and golden bell frog.

Identifying which species exhibit polyandry may lead to further insights into evolutionary biology and social behaviour. However, confirming polyandry can be difficult. High-resolution genetics provides a useful means to gain evidence. Although the threatened Pelodryadid frog, the green and golden bell frog Litoria aurea, has been subject to numerous ecological studies, there is uncertainty surrounding its reproductive ecology. Polyandry has not been formally identified in L. aurea or any species within the Pelodryadidae family. We aimed to identify if there was genetic evidence of polyandry in a population occurring in a wetland complex on Kooragang Island, New South Wales. To accomplish this, we collected genetic samples of tadpoles within the same size cohort about 20-30 days after explosive breeding events. Genotypes of 14 females, nine males and 70 tadpoles were analysed with COLONY (1988 single nucleotide polymorphisms after filtering) to identify parentage, full-siblings and half-siblings. We found support for the hypothesis that L. aurea is polyandrous. Based on previous observations of multi-male matings and the narrow time periods that breeding occurred in, it is likely this species exhibits simultaneous polyandry. We discuss these results in regards to behavioural adaptive processes and avenues for further research.

Large area used by squirrel gliders in an urban area, uncovered using GPS telemetry.

The squirrel glider (Petaurus norfolcensis) is a threatened, gliding marsupial that persists in fragmented landscapes despite its restricted capacity to cross large gaps. As measures to maintain and/or restore suitable habitat depend on knowledge about the species' ecological requirements, we investigated the area used by squirrel gliders in an urban area near Newcastle, Australia. Using GPS telemetry data and the autocorrelated kernel density estimator, we estimated area used to average 10.8 ha and varied from 4.6 to 15 ha, which is equal to or greater than found in previous studies that spanned longer time periods. This has implications when identifying the minimum patch size necessary for ensuring the long-term conservation of a squirrel glider population.

Rapid population increase of the threatened Australian amphibian Litoria aurea in response to wetlands constructed as a refuge from chytrid-induced disease and introduced fish.

Amphibians have declined due to multiple impacts including invasive fish and the disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Wetland restoration can be used to increase amphibian populations. However the design of created wetlands must account for threats such as Bd and introduced fish. There have been no attempts on a landscape level to manage these threats with habitat design. Here we monitored the green and golden bell frog (Litoria aurea) in 2.6 ha of constructed wetlands designed to enhance breeding and increase survival through passive mitigation of Bd and exotic fish. We compared the fecundity, adult population sizes, introduced fish occupancy, Bd prevalence and survival rates of frogs in created wetlands (CW) to three control sites to determine if and why the habitat design was successful. Monitoring involved weekly capture-recapture during the austral spring and summer for three L. aurea breeding seasons. We hypothesised that (1) if the CWs were successful in passively limiting fish colonisation, a larger number of breeding events would be detected compared to control sites which are known to be widely colonised by introduced fish. (2) If the wetlands were successful in passively mitigating Bd, then we would observe an equal or greater survival rate and equal to or lower Bd prevalence compared to control wetlands. We observed a 3.3-fold increase in adult population size in CW from season 1 to 2, and the population increased further in season 3.We found strong support for hypothesis (1) and weak support for (2). Based on these results, we conclude that this design was beneficial shortly after their formation primarily due to fish exclusion, but further study is required to determine if these benefits extend long-term. Future amphibian restoration studies are needed to improve the design of wetlands to enhance suppression of Bd.

How to make use of unlabeled observations in species distribution modeling using point process models.

Species distribution modeling, which allows users to predict the spatial distribution of species with the use of environmental covariates, has become increasingly popular, with many software platforms providing tools to fit such models. However, the species observations used can have varying levels of quality and can have incomplete information, such as uncertain or unknown species identity.In this paper, we develop two algorithms to classify observations with unknown species identities which simultaneously predict several species distributions using spatial point processes. Through simulations, we compare the performance of these algorithms using 7 different initializations to the performance of models fitted using only the observations with known species identity.We show that performance varies with differences in correlation among species distributions, species abundance, and the proportion of observations with unknown species identities. Additionally, some of the methods developed here outperformed the models that did not use the misspecified data. We applied the best-performing methods to a dataset of three frog species (Mixophyes).These models represent a helpful and promising tool for opportunistic surveys where misidentification is possible or for the distribution of species newly separated in their taxonomy.