Population demographics for amphibian larvae are rarely estimated due to marking technique limita... more Population demographics for amphibian larvae are rarely estimated due to marking technique limitations on small body size, morphological change (metamorphosis), and the associated habitat changes (aquatic to terrestrial environments). A technique that may meet some of these limitations is visible implant elastomer (VIE) tagging. In this study, we report on the efficacy of VIE tagging a tree frog (Hylidae) at the tadpole stage for cohort identification across metamorphosis to the adult stage, in a field environment. During our preliminary captive trial, post-metamorphosis tag retention was 100% over three months, with no adverse effects observed on survival, growth or time to metamorphosis. During our field study tag retention in recaptured Litoria aurea was 95% for tadpoles and 88% across metamorphosis. By 200 days post-tagging, retention declined to 75% in the adult stage and stabilised around 50% by 300 days. Post metamorphosis the retention rate was less reliable and dependent upon sex and life-stage. Females showed the highest retention rate (max. 62%, 760 days post tagging), followed by juveniles (max. 45%, 400 days post tagging) and males (max. 20%, 760 days post tagging). We conclude that VIE tagging is a viable method for studying cohort larval movements and population demographics of amphibians up to a 50 day postmetamorphosis stage.
The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose)... more The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose) as diluents, in cryoprotective additives containing 15% (v/v) DMSO or glycerol as cryoprotectants, were investigated on the recovery of sperm motility after cryopreservation of cane toad (Bufo marinus) spermatoazoa at low (approximately 5 degrees C/min(-1)) and high cooling rates (approximately 35 degrees C/min(-1)). The results show that: 1. recovery of percentage motility was higher with slow cooling than with high cooling rates (37.0 +/- 2.5%, 15.3 +/- 1.6%, P<0.001, respectively), 2. disaccharides were more effective than monosaccharides in protecting spermatozoa with slow cooling (43.9 +/- 1.2%, 26.8 +/- 2.5%, P<0.02, respectively), 3. glycerol was more effective than DMSO with fast cooling (18.3 +/- 2.2%, 12.6 +/- 2.3%, P<0.02, respectively), 4. trehalose with glycerol was the most effective cryoprotective additive with fast cooling (31.0 +/- 3.2%, P<0.05), and 5. overall the recovery of degree (vigour) of motility (range, 1.9 - 3.2) was more resilient to cryopreservation than recovery of percentage motility (range, 8.9 - 51.5 %). Comparison of post-thaw percentage and vigour of sperm motility up to 24 minutes after activation showed disaccharides supported greater duration sperm motility than monosaccharides This result and the recovery of spermatozoa immediately after freeze-thaw, show the main effect of saccharides are as cryoprotectants and not as exogenous energy substrates.
The short-term storage (at 0 degrees C) and cryopreservation of spermatozoa may be useful for pro... more The short-term storage (at 0 degrees C) and cryopreservation of spermatozoa may be useful for providing gametes for fertilisations performed in programmes for the conservation and management of endangered amphibians. The current study was undertaken to examine the applicability of amphibian spermatozoa storage protocols developed with the cane toad (Bufo marinus) to a wider range of amphibian species, with a view to ultimately using these protocols for endangered species. In Australia, at least 29 species of recently extinct or endangered frogs are from the families the Myobatrachidae and the Hylidae. This study investigated the applicability of short-term storage and cryopreservation protocols developed for cane toad (Bufo marinus) spermatozoa to those of hylid and myobatrachid species. Storage of spermatozoa in intact testes or in suspensions for six days at 0 degrees C showed spermatozoa maintained higher motility in suspensions than those in testes, and hylid spermatozoa maintained greater motility than myobatrachid spermatozoa. However, the protocols for optimal storage at 0 degrees C varied with testis size when spermatozoa were stored in whole testes. Spermatozoa from 13 frog species representing both families were cryopreserved using sucrose as diluent with Me(2)SO or glycerol as cryoprotectants. After cryopreservation hylid spermatozoa showed a greater recovery than myobatrachid spermatozoa and Me(2)SO provided higher recovery than glycerol. The freeze-thaw recovery of spermatozoa was independent of testes weight of the species studied. These results show spermatozoa from the Hylidae and Myobatrachidae may be stored both in the short-term (at 0 degrees C) and long-term by cryopreservation using protocols established for B. marinus.
Reintroduction programs are used widely in conservation to reduce a species' risk of extincti... more Reintroduction programs are used widely in conservation to reduce a species' risk of extinction and amphibians are considered suitable candidates for such programs because of their behavioural simplicity and high reproductive output. The Green and Golden Bell Frog Litoria aurea is an endangered species that has been reintroduced into several areas within its natural range, but the outcome of these programs remain unknown. This paper presents the results from the first release of the bell frog in the Hunter Region of New South Wales. This reintroduction released 850 tadpoles into a closed system of three ponds and rehabilitated habitat. Tadpole survival was high but following metamorphosis a decline in numbers began that continued for 13 months and resulted in the disappearance of all released bell frogs. The cause of this decline was investigated and eventually attributed to infection by the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis. These results emphasize the importance of including regula...
bioRxiv (Cold Spring Harbor Laboratory), Jul 16, 2023
There are strong potential benefits of incorporating assisted reproductive technologies (ARTs) in... more There are strong potential benefits of incorporating assisted reproductive technologies (ARTs) into conservation programs for the management of threatened amphibians as the global amphibian decline continues. As sperm cryopreservation and other ARTs advance in common species, focus on non-lethal sperm collection methods for threatened amphibians is imperative. We aimed to realise this goal by testing various doses of exogenous hormones for non-lethal induction of spermiation in a threatened frog (Litoria aurea) and develop cold storage and cryopreservation protocols following the recovery of urinic sperm. Our major findings include: (1) that sperm release could be induced in high concentrations with 20 IU/g bodyweight of human chorionic gonadotrophin (hCG); (2) high levels (>50%) of live, motile. CC-BY-NC-ND 4.
Zenodo (CERN European Organization for Nuclear Research), Apr 23, 2014
Global climate change poses major threats to biodiversity (Thomas et al., 2004). A potential weak... more Global climate change poses major threats to biodiversity (Thomas et al., 2004). A potential weakness of previous studies that project biodiversity impacts of climate change is their reliance on correlative bioclimatic habitat models that do not consider species life history. Important advances that overcome this limitation are: coupled species modelling approaches that integrate stochastic population models with bioclimatic habitat models (Keith et al., 2008); and mechanistic habitat models based on physiological budgets of energy and water (Kearney et al., 2010). These approaches combine complementary strengths of the component models (mechanistic realism, broad generality).
Climate change directly affects the suitability of habitats for species, but also indirectly alte... more Climate change directly affects the suitability of habitats for species, but also indirectly alters natural disturbances such as fire, which can negatively impact species' persistence. Developing accurate predictions of climate change impacts requires estimates of the interactive effects of climate and disturbance regimes at both population and landscape scales. Here we couple a habitat suitability model with a population viability model to examine the interactive effects of climate change and altered fire regimes on a fireresponsive frog species across its geographic range in southeastern Australia. By 2100, we predict expected minimum abundances (EMA) to decline by 66% (under GFDL-CM2 A1FI climate projections) or 87% (CSIRO Mk3.5 A1FI) in the absence of fire. Increased frequency of low-intensity fires reduced EMA by less than 5%, whereas increased frequency of highintensity fires reduced EMA by up to 40% compared with the no-fire scenario. While shifts in fire regimes are predicted to impact metapopulation viability, these indirect effects of fire are far less severe than the direct impact of climate change on habitat suitability. Exploring the interactive impacts of climate change and altered disturbance regimes can help managers prioritize threats across space and time.
Mixophyes are large ground-dwelling myobatrachid frogs from eastern Australia and New Guinea. Sev... more Mixophyes are large ground-dwelling myobatrachid frogs from eastern Australia and New Guinea. Several of the species found in mid-eastern and southeastern 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.
In brief Sperm cryopreservation has been recognised as a tool for preventing loss of genetic dive... more 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. 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 &gt;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 amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is an infectious disease respo... more The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is an infectious disease responsible for the worldwide decline of amphibian species. To mitigate these declines, it is necessary to identify the various vectors by which the fungus can be transmitted between individuals and populations. The objective of this study was to determine whether adult female mosquitoes can carry and transfer Bd fungal cells. Mosquitoes were exposed to net soaked in a live Bd zoospore suspension to determine whether they are able to externally acquire the fungus. Another group was placed into containers with a sterile and Bd-inoculated agar plate to determine whether mosquitoes could transfer Bd between these surfaces. Bd DNA was found to be present on mosquito legs exposed to inoculated netting and agar plates suggesting that Bd can be transmitted by the mosquito over short distances. This is the first study to demonstrate that an insect host may be a mechanical vector of Bd and suggests that we should begin to consider the role of mosquitoes in the dissemination and control of the fungus.
Amphibians have declined due to multiple impacts including invasive fish and the disease chytridi... more 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.
The status of many amphibian populations remains unclear due to undetected declines driven by dis... more The status of many amphibian populations remains unclear due to undetected declines driven by disease and difficulties in obtaining accurate population estimates. Here, we used genome complexity reduction-based sequencing technology to study the poorly understood Littlejohn’s treefrog, Litoria littlejohni across its fragmented distribution in eastern Australia. We detected five identifiable genetic clusters, with moderate to strong genetic isolation. At a regional scale, population isolation was likely driven by population crashes, resulting in small populations impacted by founder effects. Moderate genetic isolation was detected among populations on the Woronora Plateau despite short distances between population clusters. Evidence of recent declines was apparent in three populations that had very small effective population size, reduced genetic diversity and high inbreeding values. The rates of inbreeding detected in these populations combined with their small size leave these popu...
The status of many amphibian populations remains unclear due to undetected declines driven by dis... more The status of many amphibian populations remains unclear due to undetected declines driven by disease and difficulties in obtaining accurate population estimates. Here, we used genetic data (Single Nucleotide Polymorphisms) to investigate Australia’s poorly understood Littlejohn’s treefrog, Litoria littlejohni across its fragmented distribution. We detected five identifiable genetic clusters, with moderate to strong genetic isolation. At a regional scale, population isolation likely of population crashes, resulting in small populations with evidence of founder effects. Moderate genetic isolation was detected among populations on the Woronora Plateau despite short distances between population clusters. Evidence of recent declines was apparent in three populations that had very small effective population size, reduced genetic diversity and high inbreeding values. The rates of inbreeding detected in these populations combined with their small size leave these populations at elevated ri...
Biodiversity is in global decline during the Anthropocene. Declines have been caused by multiple ... more 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 r...
AimAfter environmental disasters, species with large population losses may need urgent protection... more AimAfter environmental disasters, species with large population losses may need urgent protection to prevent extinction and support recovery. Following the 2019–2020 Australian megafires, we estimated population losses and recovery in fire‐affected fauna, to inform conservation status assessments and management.LocationTemperate and subtropical Australia.Time period2019–2030 and beyond.Major taxaAustralian terrestrial and freshwater vertebrates; one invertebrate group.MethodsFrom > 1,050 fire‐affected taxa, we selected 173 whose distributions substantially overlapped the fire extent. We estimated the proportion of each taxon’s distribution affected by fires, using fire severity and aquatic impact mapping, and new distribution mapping. Using expert elicitation informed by evidence of responses to previous wildfires, we estimated local population responses to fires of varying severity. We combined the spatial and elicitation data to estimate overall population loss and recovery tra...
Population demographics for amphibian larvae are rarely estimated due to marking technique limita... more Population demographics for amphibian larvae are rarely estimated due to marking technique limitations on small body size, morphological change (metamorphosis), and the associated habitat changes (aquatic to terrestrial environments). A technique that may meet some of these limitations is visible implant elastomer (VIE) tagging. In this study, we report on the efficacy of VIE tagging a tree frog (Hylidae) at the tadpole stage for cohort identification across metamorphosis to the adult stage, in a field environment. During our preliminary captive trial, post-metamorphosis tag retention was 100% over three months, with no adverse effects observed on survival, growth or time to metamorphosis. During our field study tag retention in recaptured Litoria aurea was 95% for tadpoles and 88% across metamorphosis. By 200 days post-tagging, retention declined to 75% in the adult stage and stabilised around 50% by 300 days. Post metamorphosis the retention rate was less reliable and dependent upon sex and life-stage. Females showed the highest retention rate (max. 62%, 760 days post tagging), followed by juveniles (max. 45%, 400 days post tagging) and males (max. 20%, 760 days post tagging). We conclude that VIE tagging is a viable method for studying cohort larval movements and population demographics of amphibians up to a 50 day postmetamorphosis stage.
The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose)... more The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose) as diluents, in cryoprotective additives containing 15% (v/v) DMSO or glycerol as cryoprotectants, were investigated on the recovery of sperm motility after cryopreservation of cane toad (Bufo marinus) spermatoazoa at low (approximately 5 degrees C/min(-1)) and high cooling rates (approximately 35 degrees C/min(-1)). The results show that: 1. recovery of percentage motility was higher with slow cooling than with high cooling rates (37.0 +/- 2.5%, 15.3 +/- 1.6%, P<0.001, respectively), 2. disaccharides were more effective than monosaccharides in protecting spermatozoa with slow cooling (43.9 +/- 1.2%, 26.8 +/- 2.5%, P<0.02, respectively), 3. glycerol was more effective than DMSO with fast cooling (18.3 +/- 2.2%, 12.6 +/- 2.3%, P<0.02, respectively), 4. trehalose with glycerol was the most effective cryoprotective additive with fast cooling (31.0 +/- 3.2%, P<0.05), and 5. overall the recovery of degree (vigour) of motility (range, 1.9 - 3.2) was more resilient to cryopreservation than recovery of percentage motility (range, 8.9 - 51.5 %). Comparison of post-thaw percentage and vigour of sperm motility up to 24 minutes after activation showed disaccharides supported greater duration sperm motility than monosaccharides This result and the recovery of spermatozoa immediately after freeze-thaw, show the main effect of saccharides are as cryoprotectants and not as exogenous energy substrates.
The short-term storage (at 0 degrees C) and cryopreservation of spermatozoa may be useful for pro... more The short-term storage (at 0 degrees C) and cryopreservation of spermatozoa may be useful for providing gametes for fertilisations performed in programmes for the conservation and management of endangered amphibians. The current study was undertaken to examine the applicability of amphibian spermatozoa storage protocols developed with the cane toad (Bufo marinus) to a wider range of amphibian species, with a view to ultimately using these protocols for endangered species. In Australia, at least 29 species of recently extinct or endangered frogs are from the families the Myobatrachidae and the Hylidae. This study investigated the applicability of short-term storage and cryopreservation protocols developed for cane toad (Bufo marinus) spermatozoa to those of hylid and myobatrachid species. Storage of spermatozoa in intact testes or in suspensions for six days at 0 degrees C showed spermatozoa maintained higher motility in suspensions than those in testes, and hylid spermatozoa maintained greater motility than myobatrachid spermatozoa. However, the protocols for optimal storage at 0 degrees C varied with testis size when spermatozoa were stored in whole testes. Spermatozoa from 13 frog species representing both families were cryopreserved using sucrose as diluent with Me(2)SO or glycerol as cryoprotectants. After cryopreservation hylid spermatozoa showed a greater recovery than myobatrachid spermatozoa and Me(2)SO provided higher recovery than glycerol. The freeze-thaw recovery of spermatozoa was independent of testes weight of the species studied. These results show spermatozoa from the Hylidae and Myobatrachidae may be stored both in the short-term (at 0 degrees C) and long-term by cryopreservation using protocols established for B. marinus.
Reintroduction programs are used widely in conservation to reduce a species' risk of extincti... more Reintroduction programs are used widely in conservation to reduce a species' risk of extinction and amphibians are considered suitable candidates for such programs because of their behavioural simplicity and high reproductive output. The Green and Golden Bell Frog Litoria aurea is an endangered species that has been reintroduced into several areas within its natural range, but the outcome of these programs remain unknown. This paper presents the results from the first release of the bell frog in the Hunter Region of New South Wales. This reintroduction released 850 tadpoles into a closed system of three ponds and rehabilitated habitat. Tadpole survival was high but following metamorphosis a decline in numbers began that continued for 13 months and resulted in the disappearance of all released bell frogs. The cause of this decline was investigated and eventually attributed to infection by the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis. These results emphasize the importance of including regula...
bioRxiv (Cold Spring Harbor Laboratory), Jul 16, 2023
There are strong potential benefits of incorporating assisted reproductive technologies (ARTs) in... more There are strong potential benefits of incorporating assisted reproductive technologies (ARTs) into conservation programs for the management of threatened amphibians as the global amphibian decline continues. As sperm cryopreservation and other ARTs advance in common species, focus on non-lethal sperm collection methods for threatened amphibians is imperative. We aimed to realise this goal by testing various doses of exogenous hormones for non-lethal induction of spermiation in a threatened frog (Litoria aurea) and develop cold storage and cryopreservation protocols following the recovery of urinic sperm. Our major findings include: (1) that sperm release could be induced in high concentrations with 20 IU/g bodyweight of human chorionic gonadotrophin (hCG); (2) high levels (>50%) of live, motile. CC-BY-NC-ND 4.
Zenodo (CERN European Organization for Nuclear Research), Apr 23, 2014
Global climate change poses major threats to biodiversity (Thomas et al., 2004). A potential weak... more Global climate change poses major threats to biodiversity (Thomas et al., 2004). A potential weakness of previous studies that project biodiversity impacts of climate change is their reliance on correlative bioclimatic habitat models that do not consider species life history. Important advances that overcome this limitation are: coupled species modelling approaches that integrate stochastic population models with bioclimatic habitat models (Keith et al., 2008); and mechanistic habitat models based on physiological budgets of energy and water (Kearney et al., 2010). These approaches combine complementary strengths of the component models (mechanistic realism, broad generality).
Climate change directly affects the suitability of habitats for species, but also indirectly alte... more Climate change directly affects the suitability of habitats for species, but also indirectly alters natural disturbances such as fire, which can negatively impact species' persistence. Developing accurate predictions of climate change impacts requires estimates of the interactive effects of climate and disturbance regimes at both population and landscape scales. Here we couple a habitat suitability model with a population viability model to examine the interactive effects of climate change and altered fire regimes on a fireresponsive frog species across its geographic range in southeastern Australia. By 2100, we predict expected minimum abundances (EMA) to decline by 66% (under GFDL-CM2 A1FI climate projections) or 87% (CSIRO Mk3.5 A1FI) in the absence of fire. Increased frequency of low-intensity fires reduced EMA by less than 5%, whereas increased frequency of highintensity fires reduced EMA by up to 40% compared with the no-fire scenario. While shifts in fire regimes are predicted to impact metapopulation viability, these indirect effects of fire are far less severe than the direct impact of climate change on habitat suitability. Exploring the interactive impacts of climate change and altered disturbance regimes can help managers prioritize threats across space and time.
Mixophyes are large ground-dwelling myobatrachid frogs from eastern Australia and New Guinea. Sev... more Mixophyes are large ground-dwelling myobatrachid frogs from eastern Australia and New Guinea. Several of the species found in mid-eastern and southeastern 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.
In brief Sperm cryopreservation has been recognised as a tool for preventing loss of genetic dive... more 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. 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 &gt;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 amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is an infectious disease respo... more The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is an infectious disease responsible for the worldwide decline of amphibian species. To mitigate these declines, it is necessary to identify the various vectors by which the fungus can be transmitted between individuals and populations. The objective of this study was to determine whether adult female mosquitoes can carry and transfer Bd fungal cells. Mosquitoes were exposed to net soaked in a live Bd zoospore suspension to determine whether they are able to externally acquire the fungus. Another group was placed into containers with a sterile and Bd-inoculated agar plate to determine whether mosquitoes could transfer Bd between these surfaces. Bd DNA was found to be present on mosquito legs exposed to inoculated netting and agar plates suggesting that Bd can be transmitted by the mosquito over short distances. This is the first study to demonstrate that an insect host may be a mechanical vector of Bd and suggests that we should begin to consider the role of mosquitoes in the dissemination and control of the fungus.
Amphibians have declined due to multiple impacts including invasive fish and the disease chytridi... more 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.
The status of many amphibian populations remains unclear due to undetected declines driven by dis... more The status of many amphibian populations remains unclear due to undetected declines driven by disease and difficulties in obtaining accurate population estimates. Here, we used genome complexity reduction-based sequencing technology to study the poorly understood Littlejohn’s treefrog, Litoria littlejohni across its fragmented distribution in eastern Australia. We detected five identifiable genetic clusters, with moderate to strong genetic isolation. At a regional scale, population isolation was likely driven by population crashes, resulting in small populations impacted by founder effects. Moderate genetic isolation was detected among populations on the Woronora Plateau despite short distances between population clusters. Evidence of recent declines was apparent in three populations that had very small effective population size, reduced genetic diversity and high inbreeding values. The rates of inbreeding detected in these populations combined with their small size leave these popu...
The status of many amphibian populations remains unclear due to undetected declines driven by dis... more The status of many amphibian populations remains unclear due to undetected declines driven by disease and difficulties in obtaining accurate population estimates. Here, we used genetic data (Single Nucleotide Polymorphisms) to investigate Australia’s poorly understood Littlejohn’s treefrog, Litoria littlejohni across its fragmented distribution. We detected five identifiable genetic clusters, with moderate to strong genetic isolation. At a regional scale, population isolation likely of population crashes, resulting in small populations with evidence of founder effects. Moderate genetic isolation was detected among populations on the Woronora Plateau despite short distances between population clusters. Evidence of recent declines was apparent in three populations that had very small effective population size, reduced genetic diversity and high inbreeding values. The rates of inbreeding detected in these populations combined with their small size leave these populations at elevated ri...
Biodiversity is in global decline during the Anthropocene. Declines have been caused by multiple ... more 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 r...
AimAfter environmental disasters, species with large population losses may need urgent protection... more AimAfter environmental disasters, species with large population losses may need urgent protection to prevent extinction and support recovery. Following the 2019–2020 Australian megafires, we estimated population losses and recovery in fire‐affected fauna, to inform conservation status assessments and management.LocationTemperate and subtropical Australia.Time period2019–2030 and beyond.Major taxaAustralian terrestrial and freshwater vertebrates; one invertebrate group.MethodsFrom > 1,050 fire‐affected taxa, we selected 173 whose distributions substantially overlapped the fire extent. We estimated the proportion of each taxon’s distribution affected by fires, using fire severity and aquatic impact mapping, and new distribution mapping. Using expert elicitation informed by evidence of responses to previous wildfires, we estimated local population responses to fires of varying severity. We combined the spatial and elicitation data to estimate overall population loss and recovery tra...
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Papers by Michael Mahony