Croaking Science

Croaking Science: Venomous Amphibians

December 21, 2020 by editor

Venomous animals are able to inject their toxins into another organism while poisons are ingested, inhaled and absorbed. The ability to deliver venom into another animal has distinct evolutionary advantages such as in defence, prey capture and even sexual selection.

Amphibians secrete a wide variety of compounds from their skin glands. Generally, mucous glands help provide a moist coating on their skin to facilitate cutaneous respiration while granular glands secrete substances that amphibians use as a chemical defence against predators (eg. toxic and noxious compounds) and microorganisms (eg. antimicrobial peptides). This is sometimes displayed with bright, aposematic colouration (Duellman and Trueb 1996).

Many different toxic secretions have been found in amphibian skin which act in numerous ways to disrupt the physiology of potential enemies (Daly et al. 2005, Savitzky et al. 2012). For example, newts in the genera Taricha and Notophthalmus synthesise tetrodotoxins in high concentrations and are co-evolving in an evolutionary arms race with Thamnophis garter snakes where toxicity is a selective pressure (Brodie et al. 2005, Mailho-Fontana et al. 2019, Hague et al. 2020). Fire salamanders secrete samandarin alkaloids through their paratoid glands which can cause convulsions, hypertension and respiratory paralysis in potential predators. Frogs can sequester an array of toxins such as the potent neurotoxin Zetekitoxin in the Panamanian golden frog and the batrachotoxins in highly toxic Phyllobates poison frogs (Duellman and Trueb 1996). The bright yellow Australian corroboree frogs of the genus Pseudophryne synthesise their own pseudophrynamine toxins as well as sequestering pumiliotoxins from their environment to deter predators (Smith et al. 2002). Fossorial caecilians are also known to produce defensive toxins with poison glands being discovered on the tails of Siphonops annulatus ringed caecilians as a possible defence against predators as they burrow into the soil (Jared et al. 2019).

Venom, however, is rare in amphibians with only a few species possessing a system to deliver their toxins into another organism.

The Iberian ribbed newt (Pleurodeles waltii) is a fascinating salamandrid from Spain and Portugal with an incredible defence behaviour. They have the ability to use their ribs to protrude through the skin to envenomate and ‘sting’ a predator. When distressed these newts can flatten themselves or arch their backs in an antipredator posture. They will then rotate their ribs 65° forwards which increases the angle of the spine to allow its ribs to penetrate through the skin wall and project as ‘spines’. This allows them to coat their ribs with a viscous, milky substance from their skin tubercles and inject it into the mouth of the predator making them unpalatable and allowing them to escape (Heiss et al. 2010). This defence mechanism doesn’t cause any permanent damage as antimicrobial peptides are able to prevent infection in the lacerated skin and their tissue is able to regenerate remarkably quickly. The tip of the ribs are also covered with a periosteum layer which is also thought to prevent microbial infection when the ribs puncture the skin.

The Echinotriton genus of crocodile newts are a sister group to the sharp ribbed newt and are also able to use their ribs to pierce their body wall when attacked by predators (Brodie et al. 1984).

Brazil is home to two tree frogs which have incredible cranial morphologies and venomous defensive mechanisms. The Greening’s frog Corythomantis greeningi live in the semi-arid caatinga ecosystem of Eastern Brazil and conceal themselves in tree hollows and rock crevices to stay moist and evade predators. Bruno’s casque headed frog Aparasphenodon brunoi is another endemic Brazilian hylid with a fascinating skull morphology. They inhabit lowland tropical forests and shrubland and like C. greeningi, will hide in water-filled tree or bamboo hollows and bromeliad phytotelmata.

Both of these peculiar frogs have flattened, casqued heads with their skin co-ossified with underlying bones. They use their heads to aid in a behaviour known as phragmosis (Jared et al. 2006, Blotto et al. 2020). Phragmosis occurs when an animal enters a hole and blocks the entrance with their head to defend themselves from predators. In the lab, these frogs will exhibit this behaviour by entering test tubes backwards and blocking themselves off with their casqued heads. This phragmotic behaviour along with their venomous spines means these frogs have never been observed being predated in the wild. It is also thought that cranial ossification and phragmosis also indirectly reduces water loss and prevents desiccation by creating a humid microclimate within their tree holes (Jared et al. 2006).

These frogs have bony spines, ridges and protrusions on their skulls in areas with high concentrations of granular and mucous glands which secrete a potent venom. Their mobile heads allow the frogs to deliver the venom into animals via head-butting and jabbing with their spines which are coated with the toxic secretions as the spines pierce their venom glands. This provides a highly effective chemical defensive mechanism as the toxin coupled with the wound caused by the head spines ensures would-be predators have a bad time when they attempt to ingest these frogs (Jared et al. 2015). Their cutaneous secretions include alkaloids and steroids which can induce oedema and intense pain in predators (Mendes et al. 2016). The venom contains both proteolytic and fibrinolytic agents as well as hyaluronidase which aids the toxins in diffusing around their enemies’ bodies (Jared et al. 2015).

The venom of Greening’s frog is thought to be twice as lethal as fer-de-lance snakes of the genus Bothrops while Bruno’s casque headed frog secretes a venom 25x as toxic as these notorious neotropical vipers with an LD₅₀ of 94.8µg in mice. A single gram of A. brunoi venom could kill 300,000 mice or 80 humans (Jared et al. 2015)! However, A. brunoi has smaller spines and granular glands than C. greening and so may not be able to inject as much venom when defending against a predator.

Top: Bruno’s casque headed frog (Aparasphenodon brunoi) L: Renato Augusto Martins R: Carlos Jared

Bottom: Greening’s frogs (Corythomantis greeningi) Carlos Jared

There are many other frogs with complex cranial morphology including immense variation in skull shape and hyperossification which often relate to their interesting and diverse ecologies (Paluh et al. 2020). With an array of other anurans having mineralised and spiny skulls, it is possible that there are a few more venomous frogs which are waiting to be studied. Contenders include the fascinating shovelhead tree frog Triprion, the crowned tree frog Anotheca spinosa and Polypedates ranwellai (Jared et al. 2015).

In a recent paper by Mailho-Fontana et al. 2020, a new set of specialised dental glands were discovered in Brazilian ringed caecilians (Siphonops annulatus) that may produce venomous enzymes – but further research is needed to confirm this. These enzymes were demonstrated to have gelatinolytic, caseinolytic and fibrinogenolytic properties. This incredible discovery may allow researchers to rethink the evolution of venom in vertebrates (since it could have evolved independently in both amphibians and reptiles) and inspire new studies about caecilian toxinology.

Through histological and biochemical analysis of saliva samples, researchers found A2 phospholipase enzymes which could mean that some fossorial caecilians inject venomous saliva via these dental glands into their earthworm prey in order to incapacitate and digest them. These enzymes are found in many other venomous creatures such as scorpions, snakes and insects. It is also worth noting that many venoms have originated as saliva such as in komodo dragons, shrews, bats and slow lorises making the prospect of venomous gymnophiones very exciting!

Other caecilians including the basal genus Rhinatrema showed similar dental glands to the ringed caecilians which could suggest that caecilians evolved to inject oral venom early on in their evolution (Jared et al. 2020).

Written by Xavier Mahele

References

Blotto, B.L., Lyra, M.L., Cardoso, M.C., Trefaut Rodrigues, M., R. Dias, I., Marciano‐Jr, E., Dal Vechio, F., Orrico, V.G., Brandão, R.A., Lopes de Assis, C., Lantyer‐Silva, A.S., Rutherford, M.G., Gagliardi‐Urrutia, G., Solé, M., Baldo, D., Nunes, I., Cajade, R., Torres, A., Grant, T., Jungfer, K.‐H., da Silva, H.R., Haddad, C.F. and Faivovich, J. (2020) The phylogeny of the Casque‐headed Treefrogs (Hylidae: Hylinae: Lophyohylini). Cladistics

Brodie, E.D., Feldman, C.R., Hanifin, C.T. et al. (2005) Parallel Arms Races between Garter Snakes and Newts Involving Tetrodotoxin as the Phenotypic Interface of Coevolution. Journal of Chemical Ecology 31, 343–356.

Brodie, E., Nussbaum, R., & Marianne DiGiovanni. (1984) Antipredator Adaptations of Asian Salamanders (Salamandridae). Herpetologica, 40(1), 56-68.

Daly, J. W., Spande, T. F. & Garraffo, H. M. (2005) Alkaloids from Amphibian Skin: A Tabulation of Over Eight-Hundred Compounds. Journal of Natural Products 68, 1556–1575

Duellman, W. E. & Trueb, L. (1996) Biology of Amphibians. McGraw-Hill.

Hague, M.T.J., Stokes, A.N., Feldman, C.R., Brodie, E.D., Jr. and Brodie, E.D., III. (2020) The geographic mosaic of arms race coevolution is closely matched to prey population structure. Evolution Letters 4: 317-332.

Heiss, E., Natchev, N., Salaberger, D., Gumpenberger, M., Rabanser, A. and Weisgram, J. (2010), Hurt yourself to hurt your enemy: new insights on the function of the bizarre antipredator mechanism in the salamandrid Pleurodeles waltl. Journal of Zoology 280: 156-162.

Jared, C., Antoniazzi, M.M., Navas, C.A., Katchburian, E., Freymüller, E., Tambourgi, D.V. and Rodrigues, M.T. (2005) Head co‐ossification, phragmosis and defence in the casque‐headed tree frog Corythomantis greeningi. Journal of Zoology 265: 1-8.

Jared C, Mailho-Fontana PL, Antoniazzi MM, Mendes VA, Barbaro KC, Rodrigues MT, Brodie ED (2015) Venomous Frogs Use Heads as Weapons. Current Biology Volume 25, Issue 16,

Jared, C., Mailho-Fontana, P.L., Marques-Porto, R. et al. (2018) Skin gland concentrations adapted to different evolutionary pressures in the head and posterior regions of the caecilian Siphonops annulatus. Scientific Reports 8, 3576.

Mailho-Fontana, P.L., Jared, C., Antoniazzi, M.M. et al. (2019) Variations in tetrodotoxin levels in populations of Taricha granulosa are expressed in the morphology of their cutaneous glands. Scientific Reports 9, 18490.

Mailho-Fontana, P. L. et al. (2020) Morphological Evidence for an Oral Venom System in Caecilian Amphibians. iScience 23, 101234.

Mendes VA, Barbaro KC, Sciani JM, Vassão RC, Pimenta DC, Jared C, Antoniazzi MM. (2016) The cutaneous secretion of the casque-headed tree frog Corythomantis greeningi: Biochemical characterization and some biological effects. Toxicon Volume 122.

Paluh D, Stanley EL, Blackburn DC. (2020) Evolution of hyperossification expands skull diversity in frogs. Proceedings of the National Academy of Sciences 117 (15) 8554-8562.

Savitzky, A.H., Mori, A., Hutchinson, D.A. et al. (2012) Sequestered defensive toxins in tetrapod vertebrates: principles, patterns, and prospects for future studies. Chemoecology 22, 141–158.

Smith, B. P. et al. (2002) Evidence for Biosynthesis of Pseudophrynamine Alkaloids by an Australian Myobatrachid Frog (Pseudophryne) and for Sequestration of Dietary Pumiliotoxins. Journal of Natural Products 65, 439–447.l

Croaking Science: How many amphibian species are there, how do we know, and how many are threatened with extinction?

November 29, 2020 by admin

I’m sure most Froglife supporters and Croaking Science readers will be aware that the world’s amphibian species are in crisis. However, you may not know just how bad the crisis is, and how we know about it. This Croaking Science article aims to show how strong the evidence is, how it is gathered, and how difficult it is to keep up to date.

Herpetologists began to become aware in the 1990s that, although all kinds of wildlife were in trouble all over the world, amphibians were a special case. Simon Stuart and colleagues put numbers to this feeling in 2004. Their Global Amphibian Assessment estimated that 32.5% of amphibian species were globally threatened (i.e. they fitted into the IUCN Red List categories Critically Endangered, Endangered or Vulnerable: see later for definitions) compared to 12% of birds and 23% of mammals. Further, 22.5% of amphibians fell into the Data Deficient category i.e. too little was known about their status to make an assessment, a substantially higher proportion than for birds and mammals whose status tends to be better known. This meant that only about 44% of amphibian species could be classed as of Least Concern. Stuart and colleagues discussed possible reasons for amphibians being in worse shape than the other mainly terrestrial vertebrate groups (at that time, too little was known about the status of reptiles to make a similar estimate for them). Although anthropogenic habitat loss and change were the underlying main causes for all wildlife declines, amphibian populations seemed to be declining even in good quality habitat, for what Stuart et al. called ‘enigmatic’ reasons. Within a few years, it was clear that the main cause of the ‘enigma’ was the spread of the fungal disease chytridiomycosis against which many amphibian species had no or only very limited resistance. It is interesting in a time of a global pandemic affecting the human population to reflect on the causes and effects of a novel disease affecting wildlife.

Stuart et al.’s assessment was based on the then total number of described amphibian species, 5743. However, James Hanken (1999) had earlier noted the irony that, at a time when amphibian species were in severe decline, the number of known species was rapidly increasing. This trend has continued. The Amphibian Species of the World website (Frost, 2020) currently (October, 2020) lists 8226 species, a 43% increase on the number assessed by Stuart et al. in 2004. Over the last 10 years, the number of described species of amphibians (mostly anurans: frogs and toads) has increased on average by 150 per year.

How has this happened? The biggest discoveries of new species are in the tropics where amphibian diversity is highest, and where, until recently, resources and expertise for amphibian research were very limited. Amphibians are mostly small, mainly active at night, and often quite localised, all of which create difficulties in cataloguing biodiversity. It also turns out that external appearances can conceal underlying differences, so that it is only recently, with the availability of DNA sequencing, that herpetologists can work out that some species, previously thought to have extensive ranges, should really be split into several distinct species. Over my time studying the frogs of Trinidad and Tobago, new species descriptions of this kind have occurred in several cases. For example, the stream frogs of northern Venezuela, Trinidad and Tobago (see Croaking Science September 2020 for an account of colour changes in these frogs) were earlier thought to belong to one species: now they are three, Mannophryne trinitatis, M. olmonae and M. venezuelensis. Will this process of finding new amphibian species come to an end? Presumably it will, but currently there is no sign of the new discovery trend slowing down.

How do we establish the conservation status of a species? The task of keeping tabs on amphibians falls to the IUCN Species Survival Commission’s Amphibian Specialist Group (ASG). Their aim is to ‘provide the scientific foundation to inform effective amphibian conservation around the world’. A key part of their work is the compilation and revision of the Amphibian Red List i.e. an assessment of all species relative to their status in the wild. The main Red List categories (IUCN, 2001), with their definitions, and the current proportions of amphibian species in each category (from a total of 6932 species) are shown below:

Category, definition, percentage of amphibian species

Critically endangered (CR): extremely high risk of extinction in the wild, 8.9%

Endangered (EN): very high risk…, 14.4%

Vulnerable (VU): high risk…., 9.8%

Near threatened (NT): close to qualifying as threatened, 5.6%

Least concern (LC): widespread and abundant, 41.5%

Data deficient (DD): inadequate information available to make an assessment, 19.3%

There is a final category of ‘not evaluated’: these are mainly newly identified species for which information on their population status is often very limited. Where new species have been named from the splitting of a widespread species, this causes a particular problem for the Red Listing process. For example, the widespread small tree frog Dendropsophus minutus is an abundant neotropical LC species; the population in Trinidad is now classed as a Trinidad endemic, D. goughi, so its status now needs assessed separately.

Clearly, a species’ status can change with time, so the Red List needs regular updating. In addition, as noted above, the identification of new species means there is a constant need for new assessments. At present, the ASG is nearing completion of a major re-assessment, due for release in December 2020: it will be fascinating to find how amphibians as a whole have fared since the last major revision in 2010.

What sort of evidence goes into determining a species conservation status? The Red Listing process is as objective as possible and relies on input from experts around the world. Key questions are: have populations changed, and if so, by how much? What is the geographic range of the species, and how much of this range does it occupy? How large or small is the population? These can be quite difficult questions to answer with any degree of certainty. Consider the UK, with its considerable resources and abundant wildlife experts. We only have a small number of amphibian species. How good is our knowledge of their population sizes and trends? Even our presence/absence distribution maps are very variable in quality, and knowledge of populations is very patchy, even for the species that are of conservation concern, like natterjack toads and great crested newts.

Now consider Venezuela: a large country in political and economic turmoil with only a small number of dedicated herpetologists, but over 300 species of amphibians. The task of Red Listing here is immense and likely to be much based on educated guesswork. I have had some involvement over the last year in re-assessing the conservation status of Trinidad and Tobago’s much smaller number (35) of species, including three previously classed as threatened, and which share their ranges with Venezuela. The process has involved several herpetologists knowledgeable about the amphibians of the three territories pooling their information to complete the IUCN’s detailed evaluation forms, and coming to a judgement. The three species are:

Phytotriades auratus, the golden tree frog (see Figure). Formerly CR on the basis of occurrence only on two separate Trinidad mountain peaks, where it lives among the leaves and in the water tanks of huge arboreal bromeliads. Recent discoveries of populations in northern Venezuela and on another Trinidad mountain have led us to revise the assessment to EN.
Hyalinobatrachium orientale, the eastern glass frog (see Croaking Science, July 2020). Found along forest streams in northern Venezuela and in north east Tobago, but not Trinidad. Previously assessed as VU. Because of its limited range in Tobago and threats from deforestation in Venezuela, we retain VU as its Red List status.
Flectonotus fitzgeraldi, the dwarf marsupial tree frog (see Croaking Science October 2020). Found on forest and forest edge vegetation that holds pools of water, such as bromeliads and Heliconia, in northern Venezuela, Trinidad and Tobago. The previous assessment was EN, but when we examined the evidence, it became clear that this was a case where the previous assessors had made a judgment based on very limited data. Fortunately, we had been involved in extensive surveys of the presence/absence of this species in Trinidad and Tobago, as well as population estimates at a few locations. Added to data on the species’ range in Venezuela, we have been able to publish a paper (Smith et al. in press) and to assess this species as LC.

In these three cases, we have two reductions in the estimated threats. In both cases, these are not linked to active conservation effort, but rather to better data on the numbers and distribution of species. Much of the hard work has been done, not by experts, but by students and amateur naturalists taking part in actions like Bioblitzes. Mobilising citizens in this way has been important in the UK for taxa likes birds and butterflies. It needs to be done all over the world for more taxa.

References

Frost, D.R. (2020). Amphibian Species of the World, version 6.0. Available online.

Hanken, J. (1999). Why are there so many new amphibian species when amphibians are declining? Trends in Ecology and Evolution 14, 7-8.

IUCN (2001). IUCN Red List Categories and Criteria, version 3.1. Available online.

Smith, J. et al. (in press). The distribution and conservation status of the dwarf marsupial frog (Flectonotus fitzgeraldi; Anura, Hemiphractidae) in Trinidad, Tobago and Venezuela. Amphibian and Reptile Conservation.

Stuart, S. et al. (2004). Status and trends of amphibian declines and extinctions worldwide. Science 306, 1783-1786.

Croaking Science: Amphibians in the ‘Anthropause’

October 27, 2020 by admin

On damp nights in the spring in temperate regions, amphibians emerge from their winter hideouts to migrate to their breeding pools. Nothing will stop their drive to reach water to spawn and so they brave a barrage of cars and artificial barriers along their route which often results in them getting squashed.

Amphibians have complex, biphasic life histories which makes their conservation challenging. Most species require high quality aquatic and terrestrial habitats which must both be connected by a transition habitat during migrations (Baldwin et al. 2006).

As urbanisation expands around the world, many aspects of wildlife physiology, movement patterns, population genetics and overall health are affected due to anthropogenic stressors such as habitat loss and artificial light pollution. The unprecedented expansion in transport infrastructure in the past century has had massive impacts on amphibian populations around the world (Beebee 2013, Glista et al. 2008). Roads fragment habitats and cause high mortality as well as being a significant source of chemical contaminants (Petrovan and Schmidt 2016, White et al. 2017, Bird et al. 2019).

Roads which bisect migratory routes severely restrict the dispersal of amphibians between their breeding areas and terrestrial habitat. This can have catastrophic impacts on gene flow and metapopulation dynamics potentially leading to population declines, genetic isolation and even extinction (Fahrig and Rytwinski 2009, Marsh and Trenham 2001). Therefore, mitigating road-kill and habitat fragmentation is a conservation priority (Puky 2005, Beebee 2013, Cushman 2005). Genetic bottlenecks as a result of fragmentation reduce the viability of populations and can leave amphibians more vulnerable to other synergistic environmental stressors such as climate change, pollution and emerging wildlife diseases (Cushman 2005, Laurence and Useche 2009). Urban fragmentation is known to decrease species diversity and abundance and coupled with wider habitat degradation such as edge effects and loss of breeding habitats, can make cities particularly challenging for amphibians (Bickford et al. 2011, Scheffers and Paszkowski 2012). At some field sites, mass mortality on roads can cause complete extirpation of anurans and salamanders within years (Cooke 2011, Hels and Buchwald 2000). With more and more cars on the roads, it is increasingly difficult for amphibians to avoid them.

Temperate amphibians such as the common toad have experienced significant and continuous declines. In the UK it is estimated that 20 tonnes of common toads (Bufo bufo) are killed annually as they migrate across roads to reach their breeding ponds, with up to 13,000 annual mortalities being recorded even at manned amphibian crossings. This has contributed to a 68% decline in their population size in the past 50 years at a rate of 2.26% per year. The severity of the population reduction means that these toads are no longer ‘common’ toads and could soon qualify as vulnerable on the IUCN Red List (Petrovan and Schmidt 2016).

The COVID-19 pandemic has caused immense human tragedy as it has spread around the world straining healthcare services and paralysing economic systems. Quarantine restrictions imposed by governments in order to slow the spread of the virus, have caused extraordinary disruption to our globalised society but have created rare opportunities for some wildlife species (Zellmer et al. 2020). As bustling metropolises slowed down they were occupied by usually elusive fauna; wild boars were seen roaming Barcelona’s quiet neighbourhoods, flamingos flocked to New Delhi’s lakes, river otters and puma stalked silent streets in Chile and sea turtles thrived on empty Brazilian beaches. A recent paper by Rutz et al. (2020) coined the term ‘anthropause’ for this rapid and unprecedented “global slowing down of modern human activities, notably travel.” It has resulted in profound short term environmental benefits such as improved air and water quality as well as reduced wildlife disturbance.

Research conducted during the Anthropause has highlighted both known and less known impacts that we are having on the natural world and is providing a unique opportunity to study wildlife populations during a time of reduced human presence all around the world (Forti et al. 2020, Zellmer et al. 2020, Bates et al. 2020). Monitoring the ways wildlife has responded to covid lockdowns in urban areas allows researchers to quantify the impacts human mobility is having on wildlife in the Anthropocene (the term given to our geological age, dominated by human activities) and potentially find strategies to mitigate biodiversity loss (Rutz et al. 2020, Stokstad 2020). This research provides invaluable insight into the mechanisms of human-wildlife interactions that will inform conservation for decades to come.

Lockdowns in spring and summer of 2020 meant fewer vehicles on the road which has significantly decreased road mortality for many species including amphibians. A “roadkill reprieve” this year may have made amphibian migratory journeys much safer according to Goldfarb (2020). A study by Grilo et al. (2020) calculated that 194 million birds and 29 million mammals are killed on European roads each year under normal circumstances while in Brazil, road mortality has superseded hunting to become the “leading cause of direct human caused mortality in terrestrial vertebrates” (Carvalho et al. 2014). This year they may not suffer the same fate. Simply driving less could have been “the biggest conservation action” taken by humanity over the past 50 years according to Fraser Shilling of the Road Ecology Centre at UC Davis and could have potentially saved the lives of 500 million vertebrates (Nguyen et al. 2020).

In March and April as traffic flows dipped by 73% across the US, many creatures received temporary respite from road collisions, with Californian mountain lion fatalities down 58% and a 48% decrease in roadkill deaths in Maine. Although much of the data focuses on large mammals, the same is true for smaller, less mobile animals such as amphibians, snakes and turtles (Katz 2020).

At amphibian crossings this spring in Maine, wood frogs, salamanders and newts were twice as likely to survive than in previous years, which is great news for these sensitive creatures that are very vulnerable to habitat fragmentation. Four amphibians successfully crossed the road for every one squashed this year, half as many as in previous years (Goldfarb 2020, Keim 2020).

Wood frog (Photo Credit: Brian Gratwicke)

A study by Manenti et al. (2020) examined the effects of the COVID-19 lockdown and reduced human disturbance on wildlife in Italy and describes both positive and negative effects on biodiversity conservation. As well as allowing wildlife to explore new habitats and extend their activity periods, species richness and reproductive success increased while roadkill diminished in temporarily quieter areas. Some negative ecological effects included reduced enforcement of regulations, and postponed conservation action such as invasive species management.

A decrease in road traffic led to significantly improved survival of migrating amphibians on Italian roads this spring at eight toad crossings. Data suggests that there was an 80-90% decrease in nocturnal road traffic at these study sites during the anthropause. Four hundred and eight common toads (Bufo bufo) and 16 agile frogs (Rana dalmatina) were recorded as roadkill in 2019, and this dropped to only 38 toads and zero agile frog mortalities in 2020. The median mortality of road killed amphibians at each site decreased from 53 individuals to only one in 2020 (with some sites recording no mortalities) indicating that an increased number of adults made it to their breeding pools this spring giving populations a much needed boost. Manenti et al. (2020) also surveyed transects in Liguria, Northern Italy to quantify the effects of reduced traffic on common wall lizards (Podarcis muralis) and Western green lizards (Lacerta bilineata) and found a similar 10-fold decrease in road mortality.

Many of the conservation benefits the Anthropause created are ephemeral, as traffic returned to baseline levels once economic activity restarted. Although breeding booms this spring may bring some respite to struggling populations, alone, they will not be able to counteract the extreme loss of genetic diversity as a result of population bottlenecks in the past.

The information collected over the Anthropause highlights the need to improve the landscape to make it safer for all wildlife and particularly amphibians (Merrow 2007). If we can alter our transport networks to counter the effects of large-scale urban development and increased transport infrastructure we could go some way to mitigating the catastrophic impact of habitat fragmentation. The observed benefits that limiting road traffic can provide for migrating amphibians could inform more decisions about temporary, focused road closures around the world in the future to replicate this effect and to better conserve endangered amphibian and reptile species (Manteni et al. 2020).

Building fauna tunnels and bridges across dangerous roads to link habitat fragments allows animals to cross safely (Dodd et al. 2004, Gonzalez-Gallina 2018, Vartan 2017). Road mortality decreases by up to 85%-95% with animals responding surprisingly quickly to the change as ecosystems are reconnected (Vartan 2019). Wildlife tunnel projects can mitigate the effects of fragmentation and have been shown to allow amphibians to move successfully between sites bisected by roads. Tunnels and drift fences reduce road mortality, promote habitat connectivity, and allow amphibians to colonise new ponds leading to population increases and expansion into reconnected habitat (Jarvis et al. 2019). These movement corridors also allow roads to be permeable to a host of other wildlife species like reptiles and small mammals (Purky 2005, Jarvis et al. 2019).

The incredible work done by volunteers at amphibian crossings who help migrating amphibians make it across roads (Petrovan and Schmidt 2016) could also contribute to sustaining population increases this spring from quieter road traffic.

COVID-19 has also highlighted how explicitly intertwined human and wildlife health are and the catastrophic impacts of the overexploitation of nature. Emerging wildlife diseases such as chytrid fungi and ranaviruses threaten amphibians around the world and their spread has been catalysed by anthropogenic trade in our globalised world (O’Hanlon et al. 2019). A slowdown in global trade and human flows as a result of COVID-19 could also have gone a way to mitigating the spread of the pathogens that imperil amphibians (Forti et al. 2020). Although this benefit may only be short-lived as economies restart, there is hope that better regulation of the wildlife trade, in order to decrease the chances that future novel zoonotic diseases spill over from wildlife reservoirs in the future, could benefit amphibians too. Since the wildlife trade is responsible for the translocation of many amphibian pathogens (Kolby et al. 2014, O’Hanlon et al. 2018) better regulation and enforcement of the trade in live animals could help stem the spread of emerging amphibian diseases and invasive species (Forti et al. 2020).

The Anthropause provides us with an excellent opportunity to reimagine our relationship with nature and our socio-economic systems so that biodiversity can flourish and acts as a reminder that biodiversity is essential for maintaining human health and wellbeing too.

The disruption to the economic status quo that the pandemic has created gives us a pivotal opportunity to consider a greener, more sustainable future where people and wildlife can thrive. In order to effectively stem biodiversity loss in the Anthropocene, we must use this pivotal moment to plan a green recovery which incorporates wildlife conservation into all economic rebound projects. In the UK this means not viewing the protection of great crested newts as a delay to construction (Howard 2020) but valuing them as integral parts of a plan to balance our economic and environmental ambitions. It is also a perfect opportunity to invest in road mitigation measures such as wildlife tunnels and fauna bridges to reduce the detrimental impact they can have on wildlife populations (Merrow 2007), with the added benefit of being excellent economic multipliers.

November 2020 Croaking Science article by Xavier Mahele

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Croaking Science: Red Alert! The alarming state of marsupial frogs

September 22, 2020 by admin

Amphibians have evolved a fascinating variety of ways to produce and look after their young. Parental care occurs in many families, with either the mother or the father, or occasionally both, spending time and effort to give their young a better chance in life. Recent Croaking Science articles have described the glass frogs (July), where fathers guard incubating eggs on leaves above streams; Phyllomedusa tree frogs (August) where the parents enclose the eggs in folded leaves above water; Mannophryne stream frogs (September), where fathers guard their eggs on land, then transport the hatched tadpoles on their backs to safe water. This month, we look at the marsupial frogs, where mothers carry their developing eggs on their backs. As well as considering their reproductive arrangements, we look at their conservation status: overall, an alarming picture.

The Hemiphractidae are a family of New World frogs, composed of 118 species in six genera. They are distributed across Central and South America, as well as some Caribbean islands. They are very small arboreal frogs, with a primarily nocturnal lifestyle. The main distinguishing feature of the family is the storage and development of eggs on the backs of females, either externally or within a pouch (which has earned some the nickname of marsupial frogs). They also have unusually large embryonic external gills compared to other frogs.

Males of several species have been seen to actively place the eggs on the backs of females during amplexus, during which time they are assumed to contribute their half of the required gametes. They have mostly been seen to breed during wet seasons in tropical areas, as most frogs do. In northern parts of South America there are two wet seasons, a long one lasting from April until August and a shorter one lasting from November until January. Hemiphractid frogs have been seen carrying eggs during both of these, suggesting that they do not possess a strict reproductive timetable but carry out opportunistic breeding when conditions suit.

You can see a range of specialisations between hemiphractid genera, none more apparent than the egg-carrying strategy (to pouch or not to pouch). The genera that utilise enclosed dorsal pouches include Gastrotheca, Flectonotus and Fritziana. Cryptobatrachus, Stefania and Hemiphractus don’t have pouches, instead using mucosal secretions to keep the eggs in place.

Within the pouchless genera, Stefania stand out due to the development of young. In this genus there is no free-swimming tadpole stage, resulting in the hatching of froglets directly from the eggs carried on the female’s back. Female Stefania individuals have been found carrying up to 25 eggs on their back! With a gestation period of around 3 months, those kids are one heck of a burden. Within the pouched or “marsupial” species, the two belonging to the genus Flectonotus stand out due to their mysterious nature, with little research being conducted on either. It is known, however, that hatching occurs within the pouch and that individuals are then deposited into small bodies of water, such as in the ‘tanks’ enclosed by bromeliad leaves; they are at the stage of being well developed tadpoles, which soon metamorphose, without feeding. It is not clear why Flectonotus retains this requirement for a brief aquatic phase, rather than progressing to the froglet stage in the pouch.

Conservation status

Analysis of IUCN’s Red List status for the family Hemiphractidae shows 32% of species in the four threatened categories (critically endangered to near threatened), similar to the figure for amphibians as a whole. However, only 25% of species are in the least concern category, which provides a much more alarming picture. This is because 24% of species are listed as data deficient, and 20% more have yet to be assessed. This is the reality behind some of the global figures for the state of nature. In some parts of the world, especially the tropics, the state of our knowledge of many groups and individual species is very poor. Because of the continuing loss and alteration of natural habitats around the world, the expectation is that species whose status is poorly known are unlikely to be doing well. However, this is not always the case, as we show below for one species of hemiphractid frog.

The dwarf marsupial frog (Flectonotus fitgeraldi) was brought into the spotlight by a recent piece of collaborative research between researchers in Scotland, Trinidad, the USA, Portugal and Venezuela. This study aimed to clarify the conservation status of the species, by establishing an accurate picture of their distribution. Currently, IUCN lists the species as Endangered, but this status appeared to be based on no published original research. Finding these frogs is easier said than done, since they are sized between 2 and 3cm (roughly the size of a 2 pence coin). They have been reported to exist across the islands of Trinidad and Tobago, as well as northern parts of Venezuela. However, there was uncertainty as to whether these three populations fit within a single species, since there had been several cases previously when close examination of widely distributed amphibian species revealed the different populations to be divergent enough to merit description as separate species.

A frog sitting on a wooden surface

Description automatically generated — **Pictured:** *F. fitzgeraldi* male perched on a leaf, caught mid-croak by Paul Hoskisson

F. fitzgeraldi are most reliably found by identification of the plant species they use as homes and tadpole deposition sites. These are commonly bromeliads, heliconias and aroids. The larval young of F. fitzgeraldi are deposited in phytotelmata, small pools of water contained within plant structures. The hatching tadpoles are at a late non-feeding stage, having been nourished by yolk content of the egg. In the days following deposition in a bromeliad tank, tadpoles develop limbs and emerge from the water as froglets. Metamorphosis is normally complete within 5 days, during which the tail is reabsorbed and froglets graduate to become fully-fledged frogs.

In order to review the Endangered classification of F. fitzgeraldi, the study made use of molecular techniques as well as surveys over the course of several years. Genomic DNA samples were compared for sequence similarity to determine whether the populations in Trinidad, Tobago and Venezuela all belonged to the same species. Surveys were initially conducted to determine the hours of peak activity in F. fitzgeraldi and found that they exclusively call between 6pm and 8pm, roughly an hour either side of sunset. Based on this information, presence/absence surveys were carried out using the call to determine presence (seeing the frogs is difficult, and moving vegetation to attempt to see them disturbs the frogs) at locations across Trinidad, Tobago and Venezuela. These took the form of transects through forested areas, either on foot or in vehicles, while listening for the raspy calls of F. fitzgeraldi, which sound like running your finger along a comb. This presence/absence data was used to build up a map of their distribution.

**Pictured:** *F. fitzgeraldi* perched on a leaf

Results from the study showed the three populations belong to the same species, with no taxonomic changes needed. It was also shown that F. fitzgeraldi was distributed more widely across Trinidad, Tobago and Venezuela than previously thought. This, combined with the lack of evidence showing deforestation on these two islands, was sufficient to demonstrate that ‘Endangered’ is too extreme a label for the species. However, deforestation in Venezuela, mainly for cattle farming, was found to threaten the habitat quality of F. fitzgeraldi, so the study concluded that a categorisation of the species as ‘Vulnerable’ was appropriate.

**Pictured:** Female *F. fitzgeraldi*: left- showing slit on the back, entrance to the brood pouch; right- ‘pregnant’ female. Each bump is a developing embryo in the brood pouch.

Although F. fitzgeraldi is sheltered from mainland deforestation by the island refuges provided by Trinidad and Tobago, many other hemiphractid frogs do not share the same luxury. This means that habitat loss caused by deforestation poses a threat to species whose distribution is restricted to northern areas of the South American continent, such as Venezuela and Guyana. For this reason, species of the hemiphractid family must be monitored closely in these areas over the following decade.

The work on F. fitzgeraldi that we summarise here shows that detailed fieldwork can sometimes reveal that the status of a species is better than previously thought. It also shows the value of student expeditions, which can deploy significant numbers of enthusiastic young people to carry out detailed field research which would be prohibitively expensive otherwise.

Acknowledgements

We thank Paul Hoskisson and Joanna Smith for permission to use their photographs.

Further reading

Our account of the conservation status of F. fitzgeraldi is based on a research paper currently under review.

Cameron Boyle and Roger Downie
University of Glasgow

Croaking Science: More than meets the eye- cryptic frogs are more colourful than you think

August 28, 2020 by admin

Mannophryne is a genus of 20 frog species found in the forests of Venezuela and Trinidad and Tobago. They belong to the family Aromobatidae, the sister family to the well-known Dendrobatidae, or poison dart frogs. Unlike poison dart frogs, that are brightly coloured to advertise their toxicity, aromobatids use cryptic colouration to blend into their surroundings as they do not have toxic compounds in their skins. In addition, they have a very effective escape response and can identify and reach cover quickly. This has led to one of their common names of rocket frogs, as well as being known as stream frogs, because of their normal habitat of stream margins.

In Trinidad and Tobago, we studied the endemic Mannophryne trinitatis (Trinidad) and M. olmonae (Tobago). Previous assessments of both species for IUCN indicated that they are threatened with extinction on account of habitat loss or degradation. An additional worry was the finding that both species were infected with chytrid fungus, the cause of numerous amphibian population declines across the world, although Mannophryne seemed not to suffer from the infection. We conducted population surveys of M. trinitatis in the Northern Range of Trinidad and found them to be numerous, no longer infected with chytrid, and with no sign of significant habitat loss. In Tobago, M. olmonae are very restricted in their distribution and are found at much lower numbers along streams in the northeast of the island. However, they also showed a lack of chytrid infection and no serious loss of habitat. We contributed to a review of IUCN’s frog assessments and recommend that M. trinitatis now be classed as Least Concern, and M. olmonae as Vulnerable.

Mannophryne are small mottled brown frogs, only a few centimetres in length, which are active during the day close to small mountain streams in primary and secondary forests. They feed on small arthropods such as beetles, flies and spiders present on the abundant rotting fruit. During the rainy season (June to December), calling males can be heard morning and afternoon trying to attract a mate with their quick “weep, weep, weep” whistling calls. If you follow the calls and look carefully, they can be seen perched on rocks, roots, plants or at the mouths of small caves but will jump quickly away to safety if approached. Meanwhile, females hold a territory, which they defend from other females. When a female has chosen a mate, she follows the male into his cave and lays up to 14 eggs on the damp leaf litter. The males provide parental care, defending and cleaning the eggs until they hatch. This takes around 21 days and then each tadpole flips onto its father’s back and he begins the journey to look for a safe pool of water for his tadpoles to develop in. When a pool free from predatory fish or crustaceans and with a good supply of food is located, the father deposits some of the tadpoles and then searches for another. Tadpoles from a single clutch may be deposited in several bodies of water and will take around 2 months to develop to the stage of metamorphosis.

*Figure 1: Calling male (left) and non-calling male (right) M. trinitatis.*

One of the most interesting and recently studied aspects of this genus is their colouration. Sexual dichromatism is the difference in colouration between the males and females of a species. This is often used in communication as a visual signal to warn off competitors or highlight quality to potential mates. Sexual dichromatism can be split into two categories. Dynamic dichromatism only occurs in males and involves the development of temporary colouration, often as part of a courtship display, whilst ontogenetic dichromatism can occur in males or females and involves the development of a permanent colour difference as they mature. Mannophryne are a very rare example among frogs in showing both, where males turn a jet-black colour when they call, and females develop a yellow patch on their throats as they mature.

This is noteworthy, because both forms of dichromatism are energetically expensive, stressing their importance. As male Mannophryne are active and calling during the day, their colour change seems counter- productive as it makes them more conspicuous. Additionally, it appears that open areas are a preferred calling site, and in turn attract a mate, further increasing their predation risk. Therefore, both signals (visual and audio) must be essential in attracting a mate. The role of the yellow throat in females is likely a signal of quality. We found that throat colour is individually variable in the extent of the colour patch and in its hue, ranging from pale yellow to bright orange. Larger and heavier females have more orange throats. Animals often use bright or bold colours to signal their quality to others and to avoid physical confrontation. In M. trinitatis, females can be see raising their heads to show their pulsing throat patch when confronted by another female. This signal shows that the defending female has the additional energy to dedicate to the “expensive” orange colouration and often provides enough incentive to dissuade the challenger from further conflict. Yellow and orange colours are based on carotenoid pigments, and there is evidence from many kinds of animals that carotenoids signal health and quality, as they are related to immune system functions. As a test of this idea that colour differences in the females are a sign of quality, we tested their escape responses in a semi-natural experiment: frogs with the brightest yellow throats showed more effective escape responses than the others.

*Figure 2: Female (yellow throat) and male (grey throat) M. trinitatis*

An extra facet to throat colour use by Mannophryne females may be signalling their quality to males. As males take such exhaustive care of their clutch through to deposition of tadpoles, it is unlikely that they could manage more than one at a time. Therefore, it is important that they choose a high-quality female. Mate selection on both sides may be a more significant and understudied aspect to sexual selection in animals where males take all or the majority of parental care responsibilities.

Another fascinating piece of the puzzle is the timing of these colour changes. We observed the colour change on a male M. trinitatis, with the whole process taking around 20 minutes. The male was located by erratic calling and found to still be in its mottled brown colouration. As the calling became more consistent and frequent, the colouration changed to become darker until its change was complete. This is interesting because colour change in frogs is generally quite slow, mediated by hormones, and it will be interesting to discover if there is a special mechanism in Mannophryne. We found through rearing juveniles for several months after metamorphosis, that the yellow colouration develops very gradually in females, well before they reach sexual maturity.

Even though they do not have the bright colours or toxicity of their dart frog cousins, these little frogs are a lot more interesting and special than you might think at first glance.

Further Reading

Downie, J. R., Livingstone, S. R., & Cormack, J. R. (2001). Selection of tadpole deposition sites by male Trinidadian stream frogs, Mannophryne trinitatis (Dendrobatidae): an example of anti-predator behaviour. Herpetological Journal 11, 91-100.

Downie, J. R., Robinson, E., Linklater‐McLennan, R. J., Somerville, E., & Kamenos, N. (2005). Are there costs to extended larval transport in the Trinidadian stream frog, Mannophryne trinitatis (Dendrobatidae)?. Journal of Natural History 39, 2023-2034.

Greener, M. S., Hutton, E., Pollock, C. J., Wilson, A., Lam, C. Y., Nokhbatolfoghahai, M., … & Downie, J. R. (2020). Sexual dichromatism in the neotropical genus Mannophryne (Anura: Aromobatidae). PloS ONE 15(7), e0223080.

Greener, M. S., Shepherd, R., Hoskisson, P. A., Asmath, H., & Downie, J. R. (2017). How many Trinidad stream frogs (Mannophryne trinitatis) are there, and should they be regarded as vulnerable to extinction?. Herpetological Journal 27, 5-11.

Jowers, M. J., & Downie, J. R. (2005). Tadpole deposition behaviour in male stream frogs Mannophryne trinitatis (Anura: Dendrobatidae). Journal of Natural History 39, 3013-3027.

Lehtinen, R. M., Mannette, R. P., Naranjit, K. T., & Roach, A. C. (2007). Ecological observations on the critically endangered Tobago endemic frog Mannophryne olmonae. Applied Herpetology 4, 377-386.

Manzanilla, J., Jowers, M. J., La Marca, E., & García-París, M. (2007). Taxonomic reassessment of Mannophryne trinitatis (Anura: Dendrobatidae) with a description of a new species from Venezuela. Herpetological Journal 17, 31-42.

Contributing authors : Mark S. Greener and Roger Downie

Croaking Science: Egg protection is a sealed deal for Trinidad leaf frogs

July 29, 2020 by admin

Classification and Biogeography

Figure1: Distribution of *Phyllomedusa trinitatis*. (IUCN)

The Trinidadian leaf frog (Phyllomedusa trinitatis) is a neotropical tree frog belonging in the Family Phyllomedusidae. The family currently includes over 60 species in eight genera, notably Agalychnis (including the poster species, the red-eyed tree frog) and Phyllomedusa (16 species). Phyllomedusa are commonly known as leaf frogs (because of their bright green colour) or monkey frogs (because of the way they perch and grasp twigs with their long fingers and toes). Members of the family are found throughout the tropical forests of Central and South America. P. trinitatis are distributed across the forests along the northern coast of Venezuela and throughout the island of Trinidad. This distribution suggests that the species has changed very little since Trinidad split from the South American mainland. Tobago, on the other hand, was created through volcanic activity, and is not home to any P. trinitatis individuals.

General Ecology

P. trinitatis are sized between 50 and 90 cm, with females larger (roughly 1.5x) than males. The species shows a mostly nocturnal activity pattern, which allows escape from unfavourable hot and dry conditions present during the dry season in tropical regions. They have been seen to feed on insects present in tropical forests, with a particular penchant for grasshoppers. However, the general ecology of the species is relatively understudied, with not much more known. We made a start by using radio-trackers and thread bobbin tracking to follow where the frogs go after leaving the breeding site. Females moved more than males, with some climbing high into the trees, and others staying at lower levels. The majority of studies on the species focus on breeding ecology. All Phyllomedusa species lay egg clutches on leaves, usually above standing water, and cover the eggs by folding the leaves around them. In contrast, the egg clutches of Agalychnis are laid on leaves, but not covered.

Figure 2: Male *P. trinitatis* individual gracefully moving between perches at breeding site.

Courtship and Mate Selection

During the wet season in Trinidad, typically starting with rains in early June and ending in November, individuals begin to gather around suitable bodies of still water at sunset. Higher densities of frogs are seen on nights showing higher levels of humidity, mostly due to rains from the preceding day. Sunset occurs at roughly 6.30pm in Trinidad, with little variation throughout the year due to the island’s location close to the Equator. Male individuals are the first to descend from the trees, taking up positions within the vegetation surrounding a pond.

From these perches, males begin emitting advertisement calls to entice females and demonstrate their superiority over other males present. Calls of a lower frequency have been associated with a greater body condition. The calls of P. trinitatis individuals are relatively quiet in comparison to other anuran species present in the forests of Trinidad, and can be drowned out by the raucous sounds of other species. An interesting and unexplored question is whether the calls of other species interfere with Phyllomedusa mating behaviour. Some frogs are explosive breeders, with large numbers gathering at a breeding site over a day or two, mating, then dispersing. Phyllomedusa does not show this pattern. Variable numbers of individuals appear at breeding sites over a period of weeks, even months. Some males appear to adopt a strategy of attending the breeding site frequently, others only occasionally. There may be a trade-off here: since the attendance of fertile females is unpredictable, frequent attendance increases the chance of mating opportunities. On the other hand, frequent attendance may be costly, through reduced opportunities to feed and increased chances of predation. Studying site attendance patterns requires the ability to recognise individuals. We found that Phyllomedusa have individually variable ventral patterns of ‘islands’ and spots, which allow the construction of a photo-catalogue of all the individuals in an area. Fortunately, these are quite docile frogs which are not fazed by being picked up to photograph their belly patterns!

Figure 3: Trinidadian leaf frogs are known to be placid around humans, but are not fans of paparazzi. This frog demonstrates the ventral ‘island’ marking which is variable enough to allow the identification of individuals.

Fertile females arrive after hearing males calling and select a mate from the males on display. The females are weighed down with a clutch of eggs ready to lay. They are commonly outnumbered, which can produce a high level of competition between males, resulting in conflict behaviours including scramble competition where several males crawl over each other and wrestle to achieve amplexus with a female.

Figure 4: Scramble competition events in *P. trinitatis*. (Left) Female sandwiched between two competing males. (Right) Amplexing pair hassled by two rival males during nest building.

Nest-building Process

Once amplexus is achieved a period of inactivity takes place, presumably for the mating pair to gain familiarity and for the female to judge the suitability of the male. After courtship is completed a mating pair will move to find an appropriate leaf on which to lay the clutch and construct a nest. The leaf selected is either overhanging still water or in close proximity to it, and broad enough to support the weight of an egg clutch (although in locations lacking broad leaves, they are able to construct nests from a number of narrower leaves, even grass stems). The pair remains in amplexus while constructing the leaf nest, using their back legs to hold the edges of the leaf together in a narrow cone shape while the female lays the egg mass which is covered in adhesive jelly to secure the nest structure. Nest construction can last anywhere between 30 and 90 minutes, after which the male departs, leaving the female to hold the leaf in place while the adhesive compounds set to cement the nest structure.

Figure 5: Breeding pair after completing nest construction. Male (rear) has left female (front) to hold the nest in place while it sets.

The egg mass within also contains eggless capsules which provide hydration for the developing eggs (since the sealed nest does not allow the entry of rainwater). Hatching tends to take place around 8 days after nest construction, with tadpoles falling out of the bottom end of nest into the water below where they develop until metamorphosis. The development time between tadpole and froglet is roughly 3 weeks in P. trinitatis, during which juveniles grow to over five times their original size. Upon exiting the pond froglets ascend into the surrounding vegetation where the adolescent tail is fully reabsorbed, metamorphosis is completed, and life as an adult frog begins.

Figure 6: *P. trinitatis* juvenile, reaching end of metamorphosis, climbing from pond to finish development.

Further reading

Downie, J.R. et al. (2013). Nest structure, incubation and hatching in the Trinidadian leaf-frog Phyllomedusa trinitatis. Phyllomedusa 11, 13-32.

Gourevitch, E.H.Z. and Downie, J.R. (2018). Evaluation of tree frog tracking methods using the Trinidad monkey frog Phyllomedusa trinitatis. Phyllomedusa17, 233-246.

Smith, J.M. et al. (2019). Stable individual variation in ventral spotting patterns in Phyllomedusa trinitatis and other Phyllomedusa species: a minimally invasive method for recognising individuals. Phyllomedusa 18, 13-26.

IUCN (International Union for Conservation of Nature), Conservation International & NatureServe. 2008. Phyllomedusa trinitatis. The IUCN Red List of Threatened Species. Version 2020-2 (https://www.iucnredlist.org/species/55867/11382870; accessed on 8/7/2020)

Authors Cameron Boyle and Roger Downie, University of Glasgow

Croaking Science

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