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Compassionate approaches for the conservation and protection of fire salamanders

In: Israel Journal of Ecology and Evolution
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Leon Blaustein Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Ori Segev Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Valentina Rovelli Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Shirli Bar-David Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel
Mitrani Department of Desert Ecology, Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Sede Boqer Campus, Sede Boker 84990 Israel

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Lior Blank Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel
Department of Plant Pathology and Weed Research, ARO, Volcani Center, Bet-Dagan 50250, Israel

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Antonina Polevikov Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Nadav Pezaro Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Tamar Krugman Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Simona Showstack Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Avi Koplovich Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Lital Ozeri Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel

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Alan R. Templeton Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel
Department of Biology and Division of Statistical Genomics, Washington University, St. Louis, MO 63130-4899 USA

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The Near Eastern fire salamander, Salamandra infraimmaculata, is considered an endangered species in Israel and is near-threatened regionally. For 25 years, our laboratory has sought ethical sampling methods to protect individuals and populations of Salamandra. To “mark” individuals for estimating dispersal and population size, we use non-invasive individual-specific markings from photographs of larvae and adults. We demonstrated through mesocosm experiments (which are less mortality-driven than in nature) that exotic Gambusia affinis have extreme negative mortality effects on Salamandra larvae. From a compassionate conservation aspect, G. affinis should not be killed and placed in habitats where amphibians are not in danger and mosquitoes can be controlled. We identified breeding-site characteristics demonstrating that permanent breeding sites support larger adult populations than temporary breeding sites. For population genetics studies, we take minimal sized tail tips from adults (which have no adverse effects) for microsatellite data. For gene expression studies, rather than sacrifice entire bodies, we demonstrated that by taking only small larval tail tips, we could follow gene expression. We additionally demonstrated that tail tip removal does not affect survival, time to or size at metamorphosis. We documented high road kill rates at a specific breeding site. To prevent potential disease spread, we sterilize boots and sampling gear. We use results for implementing or recommending conservation of individuals and populations – e.g., identifying: movement corridors for breeding site dispersal; roadkill hotspots for under-road tunnels; suitable habitat for pool construction for more effective conservation; utilizing population genetics for recommending management units; information on demography and genetic diversity to identify hotspots for conservation; removal of Gambusia for amphibian protection.

Abstract

The Near Eastern fire salamander, Salamandra infraimmaculata, is considered an endangered species in Israel and is near-threatened regionally. For 25 years, our laboratory has sought ethical sampling methods to protect individuals and populations of Salamandra. To “mark” individuals for estimating dispersal and population size, we use non-invasive individual-specific markings from photographs of larvae and adults. We demonstrated through mesocosm experiments (which are less mortality-driven than in nature) that exotic Gambusia affinis have extreme negative mortality effects on Salamandra larvae. From a compassionate conservation aspect, G. affinis should not be killed and placed in habitats where amphibians are not in danger and mosquitoes can be controlled. We identified breeding-site characteristics demonstrating that permanent breeding sites support larger adult populations than temporary breeding sites. For population genetics studies, we take minimal sized tail tips from adults (which have no adverse effects) for microsatellite data. For gene expression studies, rather than sacrifice entire bodies, we demonstrated that by taking only small larval tail tips, we could follow gene expression. We additionally demonstrated that tail tip removal does not affect survival, time to or size at metamorphosis. We documented high road kill rates at a specific breeding site. To prevent potential disease spread, we sterilize boots and sampling gear. We use results for implementing or recommending conservation of individuals and populations – e.g., identifying: movement corridors for breeding site dispersal; roadkill hotspots for under-road tunnels; suitable habitat for pool construction for more effective conservation; utilizing population genetics for recommending management units; information on demography and genetic diversity to identify hotspots for conservation; removal of Gambusia for amphibian protection.

Introduction

Compassionate conservation is a discipline that not only acts to prevent extinctions of populations but also protects individuals from unnecessary harm and death (Ben-Ami et al., 2014; Ramp and Beckoff, 2015, Wallach et al., 2015). The Near Eastern fire salamander, Salamandra infraimmaculata, is considered an endangered species in Israel (Dolev and Perevolotsky, 2004) and near-threatened regionally ( IUCN Red List, 2017). For 2.5 decades, our laboratory has sought ethical sampling methods to protect individuals and populations of S. infraimmaculata while performing research to further our knowledge and understanding of their life history, ecology, population genetics and ecological genomics, which in turn, can be used to help preserve individuals and populations.

Natural History of Salamandra infraimmaculata

The Near Eastern fire salamander, S. infraimmaculata, exists in Iran, Iraq, Syria, Turkey, Lebanon, and Israel (IUCN Red List, 2017). In Israel, S. infraimmaculata occurs in the northern, mountainous and more rainy regions that are at least 190 m above sea level (asl) (Blank and Blaustein, 2012, 2014). In Syria also, they are also not found below 200 m asl (Bogaerts et al., 2013). Salamandra infraimmaculata retreat into deep crevices and underground caves when rains stop, where they remain relatively dormant throughout the hot and dry season (approximately early May through mid-October). Adults resume activity and emerge from estivation sites once rains begin in October or November. Gravid females emerge from their over-summering sites, carrying a full complement of larvae (as many as 193 larvae has been deposited by a single clutch from a single mother although S. infraimmaculata females from Tel Dan are smaller and deposit far fewer larvae [Degani, 1996]). Adults are typically active on rainy nights and females larviposit in a variety of water bodies including temporary lentic ponds, permanent lentic ponds, temporary ponds in ephemeral wadis and in permanent streams (Degani, 1996; Blank and Blaustein, 2012). Females have been shown to adjust the number of larvae they deposit in a particular water body depending on the water volume (Segev et al., 2011) or preferentially select structurally complex habitats when older cohorts of conspecific individuals are already present, presumably to avoid cannibalism (Sadeh et al., 2009). Larvae are key predators that have a large impact on community structure, including negative effects on many predators and herbivores that lead to trophic cascades (Blaustein et al., 1996, Eitam et al., 2005, Segev and Blaustein, 2007). They are also predators on mosquito larvae and egg rafts (Blaustein et al., 2014). Some crustacean species also avoid hatching in response to chemical cues of Salamandra larvae, apparently to avoid being consumed (Blaustein, 1997; Spencer and Blaustein, 2001). The larvae are also cannibalistic (Degani, 1993; Markman et al., 2009; Sadeh, 2012). Once larvae metamorphose and exit the water body, it takes about 3–5 years to become reproductively mature (Warburg, 1994) and they may live to be 25 years or more (Warburg, 1994). Here, we report on our research activities, taking compassionate conservation into account.

Estimating S. infraimmaculata population size and dispersal by mark-recapture

In assessing population size and dispersal using capture-mark-recapture methods, many species are tracked by cutting off different toes for assessing individual identification. In many cases, it is not known whether cut-off toes can affect mobility, survival or dispersal of particular species (Perry et al., 2011). Toe-clipping is a common technique that has been widely used in studies of mammals, reptiles, birds and amphibians but both the efficacy and humane nature of the technique are subject to compassionate criticism (Clark, 1972; May, 2004; McCarthy and Paris, 2004). Moreover, the technique may not be effective, especially for long-term studies, if toes are lost naturally or for animals that have the ability to regenerate their appendages (Ferner, 1979). In the case of estimating dispersal and population size of the fire salamander, we (Bar-David et al., 2007; Segev et al., 2010; Goedbloed et al., 2017. I. Sinai et al., unpublished results) and others (Warburg, 1994) have used photography for specific dorsal spot patterns in S. infraimmaculata adults to identify specific individuals (Fig. 1). Our mark-recapture studies have demonstrated population size at specific breeding sites (Segev et al., 2010; I. Sinai et al., unpublished results). We have found that temporary breeding sites have far fewer adults in the population than permanent breeding sites (Segev et al., 2010; I. Sinai et al., unpublished results). Moreover, mark-recapture studies based on dorsal spot patterns have demonstrated dispersal among breeding sites (Bar-David et al., 2007). Based on these findings, we recommend increasing the traditional conservation unit for salamanders – from focusing on the protection of a breeding site to the protection of several breeding sites and their surroundings to maintain landscape connectivity among them (Kershenbaum et al., 2014).

Figure 1.
Figure 1.

Identification of the same individual adult of Salamandra infraimmaculata collected nearly three years apart (3 February 2010 and 8 December 2012) based on identical dorsal spot patterns.

Citation: Israel Journal of Ecology and Evolution 63, 3-4 (2017) ; 10.1163/22244662-06303001

Photo credit: ORI SEGEV.

We have also used individual-specific non-invasive photographs of tail fins in Salamandra larvae. Using individual-specific tail fins (Eitam and Blaustein, 2002; Fig. 2), we were able to study priority effects in cohorts of Salamandra larvae (Eitam et al., 2005) and also down-stream dispersal of S. infraimmaculata larvae in ephemeral wadis (Segev and Blaustein, 2014). Using the tailfin identification, we were able to document that some Salamandra larvae did not remain in their rock pools, but dispersed downstream or were forced downstream to another rock pool. Also by using non-invasive photographs of adults for mark-recapture, we have been able to assess how different breeding habitats support different population sizes. Our research group has also recently been testing the use of Visible Implant Elastomer (VIE) tags as a marking technique for identifying Salamandra larvae in both field and laboratory experiments. The VIE tags consist of a silicon-based liquid polymer that is injected subdermally (under anesthesia) and cures rapidly into a biocompatible and pliable solid (Northwest Marine Technology, Inc.). The multi-color tagging options of the VIE provides minimally invasive and harmless means to mark large numbers of larvae when pattern recognition methods are unavailable (Low, 2003; Grant, 2008; Dodd, 2010). Larvae marked using VIE in our lab have also shown no indication of suffering adverse effects in either survival or behavioural changes (Segev et al., 2015; Pezaro et al., unpublished results).

Figure 2.
Figure 2.

Identification of four different individual larvae of Salamandra infraimmaculata based on tailfin spot patterns. All larvae have different tailfin spot patterns.

Citation: Israel Journal of Ecology and Evolution 63, 3-4 (2017) ; 10.1163/22244662-06303001

Photo credit: ORI SEGEV.

Is assessing impacts of introduced predators of Salamandra larvae compassionate conservation?

In 2003, we found that Salamandra larvae in ponds in the presence of high densities of the mosquitofish, Gambusia affinis (which were apparently introduced for mosquito control), had chewed-off tail fins and other appendages while most Salamandra larvae in ponds without Gambusia had normal intact tail fins and other appendages although Salamandra may bite off other individuals’ tails due to cannibalism (Degani 1993; Sadeh et al., 2009; Sadeh 2012). We found that at the ponds of Kaukab Springs (Lower Galilee Mountains), with high densities of Gambusia, no salamander larvae survived to metamorphosis. We submitted this work to the Israel Journal of Zoology as a likely warning that Gambusia had strong deleterious effects on Salamandra larvae. However, rather than accept this fairly convincing short communication, reviewers felt that stronger inference was needed – i.e., that an experiment should be done, manipulating the presence or absence of Gambusia, and assessing Salamandra larvae. Though we were strongly convinced that Gambusia was causing strong deleterious effects on fire salamander larvae based on field surveys, and, an experimental manipulation would result in some Salamandra larval mortality, we decided to conduct an outdoor mesocosm experiment to demonstrate the deleterious effects due to reviewer insistence. The small numbers of salamander larvae used in mesocosm experiments generally do not result in as much mortality as is found in natural pools. We obtained newborn salamander larvae from four gravid females from a site where the total adult population was estimated to be >500 adults and we used and estimated <0.3 percent of the larvae born at that breeding site. The experiment consisted of presence or absence of Gambusia crossed with presence or absence of artificial vegetation (total: 16 mesocosms). The artificial vegetation did not increase refuge for Salamandra larvae but instead, increased the number of Gambusia. Gambusia had very strong negative effects on the salamander larvae. Vegetation did not act as a refuge against Gambusia and Salamandra is exceptionally unlikely to be able to coexist with Gambusia. Given the two controls without Gambusia (i.e. no vegetation and vegetation), much higher survival occurred than the larvae that were subjected to Gambusia in mesocosms.

Though the initial reviewers of Israel Journal of Zoology would not recommend acceptance of this work based on correlations, a new biological conservation journal also refused to consider the manuscript containing both correlational evidence and experimental evidence because mortality was caused to individuals. This work encouraged us to come up with our own formula for compassionate conservation: if an experiment sacrifices fewer individuals that would have occurred in nature anyway, and the information accrued from the experiment (that lead to mortality of some individuals) leads to better conservation management and fewer mortalities in the future, then we would say that demonstrating the deleterious effects of mosquitofish with a small number of Salamandra larvae was “compassionate conservation”. The paper was then accepted by the journal, Animal Conservation (Segev et al., 2009). As a consequence of the surveys and experimental work, the Israel Nature and Parks Authority removed all Gambusia from the Salamandra breeding sites that saved many thousands of Salamandra larvae each year. Given the high density of Gambusia, mortality to the salamander larvae are virtually 100 percent. Since there are about 250 females at Kaukab springs, and minimally, each one larviposits at least 100 larvae. Thus, minimally at Kaukab, ~25,000 larvae are saved from Gambusia each year by removing Gambusia. In removing the Gambusia from the permanent ponds of Kaukab, many of the Gambusia were killed but some Gambusia were also placed in environments where Salamandra did not exist for mosquito control. Gambusia are harmful predators as they also prey upon many other amphibian larvae (e.g., Cabrera-Guzman et al., 2017; Pollard et al., 2017). Salamandra anyway have very strong effects on mosquito larvae where they prey upon egg rafts, mosquito larvae and pupae (Blaustein et al., 2014). The results of this experiment resulted in far fewer mortalities than would have been caused in natural breeding sites by Gambusia. This is akin to medical experiments where some individuals are given placebo medication or even sham surgery while others are given the real medication or real surgery (McCullough, 2017; Tambone et al., 2017). Though a number of patients taking the placebo die from lack of medication, the experiment, giving strong inference, allowed for much higher human survival in the future. For all our laboratory and outdoor mesocosm experiments, Salamandra adults, juveniles and surviving larvae are all returned to their breeding site during the breeding season. To date, not a single gravid Salamandra or juvenile died in the lab though a very small fraction of larvae do die in the lab. From a compassionate conservation aspect, we believe that Gambusia should not be killed and rescued from amphibian-breeding sites, and placed in non-natural habitats where Gambusia can control mosquitoes.

Assessing phenotypic plasticity and local adaptation in fire salamander metamorphosis

S. infraimmaculata larvae possess phenotypic plasticity and local adaptation in metamorphosing more quickly in response to pond desiccation (Sadeh et al., 2011; Polevikov, 2017). However, temporary pond desiccation is a common form of mortality in salamanders and other amphibians when larvae have not reached a critical minimum size to metamorphose (e.g., Warburg, 1992; Blaustein et al., 2001). Approximately 20 years ago, one of the authors of this paper, Leon Blaustein, came across hundreds of fire salamander larvae that were within a small desiccating pool and it was clear that within 1–2 days, with no forecast of rains, these hundreds of larvae would be dead – either due to desiccation or because certain birds such as crows can easily prey upon them when in shallow water (Showstack, 2016). Blaustein called the Israel Nature and Parks Authority (INPA) to request permission to remove these larvae, place them in artificial pools and then return them once it rained again filling the natural pool with rain water. The representative of the INPA refused to allow this stating “let nature take its course”. Was this decision a dispassionate act or an important evolutionary move? Rescuing the larvae would have been going against natural selection; larvae will of course die if they have not reached a minimum critical size to metamorphose, but natural selection acts on these populations for allowing these larvae to develop phenotypic plasticity and local adaptation (Polivekov 2017). In a recent experiment to assess phenotypic plasticity and local adaptation among populations from temporary and permanent breeding sites, when allowing water to desiccate, we also placed wet towels at the bottom of the experimental tubs once the depth was approximately zero; if salamander larvae have reached the minimum critical size to metamorphose, we know from many observations that larvae hide under moist rocks and can accelerate metamorphosis rather than dying. Thus, adding the wet towels were both mimicking nature and a compassionate natural act (Polevikov, 2017). Only 3 of 216 larvae died as a result of these wet towels (Polevikov, 2017).

Compassionate and effective methods for measuring Salamandra body morphometrics as a function of habitat type

Aquatic animals demonstrate phenotypic plasticity in body shape in response to different habitat types, such as lentic versus lotic habitats (Gaston and Lauer, 2015), or in response to risk of predation (Hossie et al., 2010). Body dimensions were traditionally measured by using a simple ruler, while holding the animal and stretching the measured organ or body. Each measurement was taken separately which prolongs the process. For aquatic animals (e.g. fish and amphibian larvae) measurements taken out of the water can potentially increase stress for the measured animal. Traditional modern morphometrics compares lengths, as well as the ratios and angles between them but it ignores the shape as a whole and is susceptible to biases when size measurements overlap, run in similar directions, or when several measurements radiate from a single point which adds dependency among measurements (Zelditch et al., 2004).

Geometric morphometrics (GMM) uses homologous features of the organism to explore morphological shape changes (Bookstein, 1982). In this sense, shape is stripped off of non-pure shape properties like location, scale, and rotation (Kendall, 1977). It preserves the shape through all the steps of analysis, and thus the relationship between measurements (Adams et al., 2004).

The use of digital cameras and water-filled transparent photo chambers makes data collection and digitization cheaper, efficient, and less stressful for the measured animal. In parallel, developed software for the analysis of shapes became common, from stand-alone programs like tpsDig (Gerth and Maia, 2017), PAST, SHAPE, MORPHOJ, to R packages like Geomorph, Morpho, Shapes, and Momocs.

Three years ago, our lab started using GMM, to identify and determine the degree of phenotypic plasticity in fire salamander larvae from different habitat types, i.e. lotic versus lentic breeding sites. We exposed laboratory-born larvae from different habitat types to flowing and stagnant condition in a fully factorial mesocosm manipulation experiment. We use the R package Geomorph (Adams and Otárola-Castillo, 2013), to analyze the changes in larval body shape.

Minimally invasive methods for studying population and landscape genetics

Studying population genetics and landscape genetics of fire salamanders are important for conservation concerns. We have been especially concerned about testing for genetic diversity along the core and periphery of the species’ range (Blank et al., 2013; I. Sinai et al., unpublished results). To do this, we have taken very small tips off the end of Salamandra adult tails. The clipped tail tip is ~0.3–0.4 cm while the total size of Salamandra adults generally range from 28–32 cm in total length. Subsequent studies have demonstrated that the Salamandra adults do well, even after these small tail tips have been removed (O. Segev et al., unpublished results; I. Sinai et al., unpublished results). To assess population genetics and landscape genetics, we conducted microsatellite studies, using up to 15 loci. Our studies have shown that the isolated peripheral Mount Carmel has considerably lower allelic richness than the Galilee populations and various levels of genetic similarity across many populations (Blank et al., 2013; I. Sinai et al., unpublished results). Based on these results, we advocated that conservation plans for this species should account for these patterns. Moreover, the microsatellite data also reveal population structuring and limited dispersal in a very heterogeneous landscape. We have also considered landscape features to gene flow by assessing least cost path, random walk and isolation by resistance models (Kershenbaum et al., 2014). These models have helped us understand where dispersal corridors exist.

Minimally invasive methods for studying genome-wide analysis of gene expression

Studying genome-wide analysis of gene expression in salamanders can give a better handle on phenotypic plasticity, local adaptation and development (Czypionka et al., 2015, Polevikov, 2017). When we were going to begin such a study, we were advised that gene expression needed to be studied using RNA extracted from the entire body of fire salamanders. We decided to test whether simply clipping the larval tailfin was enough for assessing gene expression – i.e., yield gene expression patterns that are representative of the whole organism. We first showed (Segev et al., 2015) that taking a partial tail-fin clip was not harmful to the fire salamander larvae; cannibalistic fire salamander larvae often bite off a section of the tail fin. We further demonstrated that the tailfin grows back and survival to, size at and time to metamorphosis is unaffected (Segev et al., 2015). Several full bodies of Salamandra salamandra in Germany had previously been sacrificed for transcriptomic analysis. We compared the full body transcriptomes of S. salamandra to larval tailfins in response to temperatures and found that 51% of the expressed genes were common for both the larval tailfin (Czypionka et al., 2015). This study demonstrated that we could study gene expression without killing larvae and without using whole body transcriptomes.

Protecting Salamandra infraimmaculata adults from road kills

Our studies of fire salamander adults have demonstrated that adults are quite prone to vehicles on roads that are near breeding sites, and there are many road kills by vehicles. In particular, I. Sinai and T. Oron (unpublished results) have shown that ~150 adults over four seasons were found killed by vehicles along ~2 km of road near Dishon Stream. The high mortalities, which is against compassionate conservation ethics, call for underground tunnels below roads with drift fences to funnel the adults through the tunnel (D’Amico et al., 2015; Bain et al., 2017).

Preventing disease spread among breeding sites in fire salamanders

When studying wild populations, researchers should always be aware of the impact of their activities on the health of individuals and populations. In the last decades, infectious diseases have been recognized as main threats to wildlife and amphibians in particular have faced a severe decline due to the spread of a disease called chytridiomycosis (Kilpatrick et al., 2010). Chytridiomycosis is caused by Batrachochytrium dendrobatidis (Bd), a chytrid fungus belonging to the Chytridiomycota Division, Order Chytridiales (Kilpatrick et al., 2010). Although it is still not clear if this pathogen was first introduced by humans or if it was already present when its virulent activity started, it seems that its spreading has been fostered by human movements (Kilpatrick et al., 2010). In recent years, a new pathogen called Batrachochytrium salamandrivorans appeared in Europe, and its outbreak is severely threatening fire salamander populations in many countries (Stegen et al., 2017; Gray et al., 2017). As far as Israel is concerned, up until now, the amphibian chytrid fungus, Batrachochytrium dendrobatidis was detected on Hula painted frogs (Latonia nigriventer) around the Hula wetlands, but there was no chytridiomycosis in L. nigriventer, probably because the skin mucosome contains antimicrobial peptides (Perl et al., 2017). Until now, no such infectious disease as has been reported in S. infraimmaculata in Israel which occur only several tens of km from the Hula wetlands, but the implementation of a biosecurity protocol is a priority, as best practice in conservation (Gray et al., 2017). In fact, Salamandra populations in Israel are usually small and relatively isolated, and the emergence and spreading of infectious diseases might be catastrophic, possibly leading to the local extinction of genetically unique populations. For this reason, to avoid contamination between individuals belonging to different populations, when using salamanders for our experiments, we disinfect all the equipment with Virkon S (DuPont), a broad-spectrum disinfectant. Moreover, prevention of contamination in the field should be mandatory not only for herpetologists, but for all scientists and naturalists working in the field. In this regard, the situation in Israel is complicated by the fact that the country is very small and that many of the sites where Salamandra breed are close to roads, making them easily accessible to the public. Therefore, even if we are doing our best to prevent contamination among different populations, human activities in general can potentially represent a main threat for S. infraimmaculata in Israel. The Israel Nature and Parks Authority has recommended that we do not return Salamandra used in experiments, which has validity, but thus far, we have returned all individuals to their breeding sites for 25 years and there have not been any infections.

Conclusions

When dealing with an endangered species, biological conservation research is often needed to provide the information necessary to design and implement a recovery plan that hopefully will increase the numbers of individuals and ensure the viability of the species. Table 1 summarizes our compassionate conservation methods. Such research should always be done in a manner that minimizes the suffering and death of individuals, but another ethical consideration must be the cost of not doing the research. For example, our research on the impact of Gambusia upon S. infraimmaculata larval survival did cause the death of some larvae (however, not nearly as much mortality as is observed in nature), but it provided knowledge that was convincing to agencies that altered their policies in a manner that greatly increased the survival of many more larvae and helped in the sustainability of this endangered species. This short-term individual versus long-term population trade-off is a unique ethical dilemma in nature conservation research on endangered species. From a compassionate conservation standpoint, Gambusia should also be saved and placed in non-amphibian breeding sites. Answers to this dilemma are not always easy to obtain, but this is a recurrent issue in many endangered species’ survival programs and should not be ignored. We hope that research in our group will serve as an example that the ethical principles of compassionate conservation can be upheld while employing rigorous scientific methods and a striving for the development of evidence-based conservation strategies.

T000001

We thank Dror Ben-Ami for motivating Palestinians, Jordanians and Israelis by organizing a compassionate conservation conference in Israel. Author Leon Blaustein was highly influenced in a compassionate conservation setting by four people: his father, Isak Blaustein, of blessed memory, who refused to kill anything, Iris Toren from Sydney, Australia, who has campaigned relentlessly for animal rights and ethics, Ofir Elbaum, a young man who recently was killed in an automobile accident who also campaigned for animal ethics and Israel-Arab peaceful coexistence, and Arian Wallach who showed amazing compassion for her graduate studies on deer. We also thank Olga Rybak for her exceptionally compassionate treatment of fire salamanders – adults and larvae – when they were in our laboratory. We are grateful to Iftach Sinai and Talya Oron for sharing some unpublished compassionate conservation general results to include in this paper. Funding for this study was provided by the Deustche-Israel Projects (DIP) (BL 1271/1-1) awarded to Leon Blaustein and Alan R. Templeton and to Arne Nolte and Sebastian Steinfartz (STE 1130/8-1).

References

  • Adams D.C. ,Otárola-Castillo E . (2013). geomorph: an r package for the collection and analysis of geometric morphometric shape data. Methods in Ecology and Evolution. 4:393399.

    • Search Google Scholar
    • Export Citation
  • Adams D.C. , Rohlf F.J. , and Slice D.E . (2004). Geometric morphometrics: Ten years of progress following the ‘revolution.’ Italian Journal of Zoology. 71:516.

    • Search Google Scholar
    • Export Citation
  • Bain T.K. , Cook D.G. , and Girman D.J. (2017). Evaluating the effects of abiotic and biotic factors on the movement through wildlife crossing tunnels during migration of the California tiger salamander, Ambystoma californiense . Herpetological Conservation and Biology 12:192201.

    • Search Google Scholar
    • Export Citation
  • Bar-David S. , Segev O. , Peleg N. , Hill N. , Templeton A.R. , Schultz C.B. , and Blaustein L. (2007). Long distance movements by fire salamanders (Salamandra salamandra infraimmaculata) and implications for habitat fragmentation. Israel Journal of Ecology and Evolution 53:179196.

    • Search Google Scholar
    • Export Citation
  • Ben-Ami D. , Boom K. , Boronyak L. , Townend C. , Ramp D. , Croft D.B. , Bekoff M. (2014). The welfare ethics of the commercial killing of free-ranging kangaroos: An evaluation of the benefits and costs of the industry. Animal Welfare 23: 110.

    • Search Google Scholar
    • Export Citation
  • Blank L. , and Blaustein L. (2012). Using ecological niche modeling to predict the distributions of two endangered amphibian species in aquatic breeding sites. Hydrobiologia 693:157167.

    • Search Google Scholar
    • Export Citation
  • Blank L. and Blaustein L. (2014). A multi-scale analysis of breeding site characteristics of the endangered fire salamander (Salamandra infraimmaculata) at its extreme southern range limit. Hydrobiologia 726:229244.

    • Search Google Scholar
    • Export Citation
  • Blank L. , Sinai I. , Bar-David S. , Peleg N. , Segev O. , Sadeh A. , Kopelman M.N. , Templeton A.R. , Merilä J. , and Blaustein L. (2013) Genetic population structure of the endangered fire salamander (Salamandra infraimmaculata) at the southernmost extreme of its distribution. Animal Conservation 16:412421.

    • Search Google Scholar
    • Export Citation
  • Blaustein L. (1997). Nonconsumptive effects of larval Salamandra on its crustacean prey: can eggs detect predators? Oecologia 110:212217.

    • Search Google Scholar
    • Export Citation
  • Blaustein A.R. , Wildy E.L , Belden L.K. , and Hatch A. (2001). Influence of abiotic and biotic factors on amphibians in ephemeral ponds with special reference to log-toed salamanders (Ambystoma macrodactylum). Israel Journal of Zoology 47:333345.

    • Search Google Scholar
    • Export Citation
  • Blaustein J. , Sadeh A. , Blaustein L. (2014). Influence of fire salamander larvae on among-pool distribution of mosquito egg rafts: Oviposition habitat selection or egg raft predation? Hydrobiologia 723:157165.

    • Search Google Scholar
    • Export Citation
  • Blaustein L. , Friedman J. , and Fahima T. (1996). Larval Salamandra drive temporary pool community dynamics: evidence from an artificial pool experiment. Oikos 76:392402.

    • Search Google Scholar
    • Export Citation
  • Bogaerts S. , Sparreboom M. , Pasmans F. , Almasri A. , Beukema W. , Shehab A. , and Amr Z.S. (2013). Distribution, ecology and conservation of Ommatotriton vittatus and Salamandra infraimmaculata in Syria. Salamandra 49:8796.

    • Search Google Scholar
    • Export Citation
  • Bookstein FL . 1982. Foundation of morphometrics. Annual Review of Ecology and Systematics 13:451470.

  • Cabrera-Guzman E. , Diaz-Paniagua C. , and Gomez-Mestre I. 2017. Competitive and predatory interactions between invasive mosquitofish and native larval newts. Biological Invasions 19:14491460.

    • Search Google Scholar
    • Export Citation
  • Clarke R.D. (1972). The effect of toe clipping on survival in Fowler’s toad (Bufo woodhousei fowleri). Copeia, 1972 (1), 182185.

  • Czypionka T. , Krugman T. , Altmüller J. , Blaustein L , Steinfartz S. , Templeton A.R. , and Nolte A.W. 2015. Ecological transcriptomics – a nonlethal sampling approach for endangered fire salamanders. Methods in Ecology and Evolution 6:14171425.

    • Search Google Scholar
    • Export Citation
  • D’Amico M. , Roman J. , de los Reyes L. , and Revilla E. (2015). Vertebrate road-kill patterns in Mediterranean habitats: Who, when and where. Biological Conservation 191:234242.

    • Search Google Scholar
    • Export Citation
  • Degani G. (1993) Cannibalism among Salamandra salamandra larvae. Israel Journal of Zoology 39:125129.

  • Degani G. (1996) Salamandra salamandra at the southern limit of its distribution. Laser Pages Publishing, Jerusalem, Israel.

  • Dodd C.K. (Ed.). (2010). Amphibian ecology and conservation: a handbook of techniques. Oxford University Press.

  • Dolev A. , and Perevolotsky A. eds. (2004). The red book: Vertebrates in Israel. Israel Nature and Parks Authority and Society for Protection of Nature, Israel Press, Jerusalem.

    • Search Google Scholar
    • Export Citation
  • Eitam A. , and Blaustein L. (2002). Non-invasive individual identification of larval Salamandra using tail-fin spot patterns . Amphibia-Reptilia 23:215219.

    • Search Google Scholar
    • Export Citation
  • Eitam A. , Blaustein L. , and Mangel M. (2005). Density and intercohort priority effects on larval Salamandra salamandra in temporary pools. Oecologia 146:3642.

    • Search Google Scholar
    • Export Citation
  • Ferner J.W. (1979). Review of marking techniques for amphibians and reptiles. Herpetological Circular 35. Society for the Study of Amphibians and Reptiles, Atlanta, USA.

    • Search Google Scholar
    • Export Citation
  • Gaston K.A. , and Lauer T.E . 2015. Morphometric variation in bluegill Lepomis macrochirus and green sunfish Lepomis cyanellus in lentic and lotic systems. Journal of Fish Biology 86: 317332.

    • Search Google Scholar
    • Export Citation
  • Gerth C.J. , and Maia A . (2017). Shape analysis of the jaws between two innow species over ontogeny. Journal of Morphology 278:14121420.

    • Search Google Scholar
    • Export Citation
  • Goedbloed J.D. , Segev O. , Küpfer E. , Pietzsch N. , Matthe M. , and Steinfartz S. (2017). Evaluation of a new Amphident module and sources of automated photo identification errors using data from Salamandra infraimmaculata. Salamandra 53:314318.

    • Search Google Scholar
    • Export Citation
  • Grant E.H.C. (2008). Visual implant elastomer mark retention through metamorphosis in amphibian larvae. Journal of Wildlife Management 72:12471252.

    • Search Google Scholar
    • Export Citation
  • Gray M.J. , Duffus A.L.J. , Haman K.H. , Harris R.N. , Allender M.C. , Thompson T.A. , Christman M.R. , Sacerdote-Velat A. , Sprague L.A. , Williams J.M. , and Miller D.L. (2017). Pathogen surveillance in Herpetofaunal populations: Guidance on Study Design, Sample Collection, Biosecurity, and Intervention strategies. Herpetological Review 48:334351.

    • Search Google Scholar
    • Export Citation
  • Hossie T.J. , Ferland-Raymond B. , Burness G. , and Murray D.L. (2010). Morphological and behavioral responses of frog tadpoles to perceived predation risk: A possible role for corticosterone mediation? Ecoscience 17:100108.

    • Search Google Scholar
    • Export Citation
  • IUCNRed List. 2017: http://www.iucnredlist.org/details/59466/0

  • Kendall D.G . (1977). The Diffusion of Shape. Advances in Applied Probability. 9:428.

  • Kershenbaum A. , Blank L. , Sinai I. , Merila J. , Blaustein L. and Templeton A.R. (2014). Landscape influences on dispersal behaviour: A theoretical model and empirical test using the fire salamander, Salamandra infraimmaculata . Oecologia 175:509520.

    • Search Google Scholar
    • Export Citation
  • Kilpatrick A.M. , Briggs C.J. , and Daszak P. (2010). The ecology and impact of chytridiomycosis: an emerging disease of amphibians. Trends in Ecology & Evolution 25:109118.

    • Search Google Scholar
    • Export Citation
  • Lowe W.H. (2003). Linking dispersal to local population dynamics: a case study using a headwater salamander system. Ecology 84:21452154.

    • Search Google Scholar
    • Export Citation
  • Markman S. , Hill N. , Todrank J , Heth G. and Blaustein L. (2009). Differential aggressiveness covaries with genetic similarity in fire salamander larvae. Behavioral Ecology and Sociobiology 63:11491155.

    • Search Google Scholar
    • Export Citation
  • May R.M. (2004). Ecology: ethics and amphibians. Nature 431(7007):403403.

  • McCarthy M.A. , and Parris K.M. (2004). Clarifying the effect of toe clipping on frogs with Bayesian statistics. Journal of Applied Ecology 41:780786.

    • Search Google Scholar
    • Export Citation
  • McCullough L.B. (2017). Philosophical Provocation: The Lifeblood of Clinical Ethics. Journal of Medicine and Philosophy 42(1):16. doi: 10.1093/jmp/jhw034.

    • Search Google Scholar
    • Export Citation
  • Perl R.G.B. , Gafny S. , Malka Y. , Renan S. , Woodhams, D.C, Rollins-Smith L. , Pask J.D. , Bletz M.C. , Geffen E. , and Vences M. (2017). Natural history and conservation of the rediscovered Hula painted frog, Latonia nigriventer. Contributions to Zoology 86:1137.

    • Search Google Scholar
    • Export Citation
  • Perry G. , Wallace M.C. , Perry D. , Curzer H. , and Muhlberger P. (2011). Toe Clipping of Amphibians and Reptiles: Science, Ethics, and the Law. Journal of Herpetology 45:547555.

    • Search Google Scholar
    • Export Citation
  • Pollard C.J. , Stockwell M.P. , Bower D.S. , Garnham J.I. , Pickett E.J. , Darcovich K ., O’meara J ., Clulow J ., and Mahony M.J . 2017. Removal of an exotic fish influences amphibian breeding site selection. Journal of Wildlife Management 81:720727.

    • Search Google Scholar
    • Export Citation
  • Polevikov A. (2017). Local adaptation, phenotypic plasticity and gene expression of Salamandra infraimmaculata larvae in permanent vs. ephemeral breeding habitats. MSc thesis, University of Haifa.

    • Search Google Scholar
    • Export Citation
  • Ramp D. , and Bekoff M. (2015). Compassion as a practical and evolved ethic for conservation. Bioscience 65:323327.

  • Sadeh A. (2012). Kin-selective cannibalism and compensatory performance in larval salamander cohorts inhabiting temporary ponds. Evolutionary Ecology Research 14: 113123.

    • Search Google Scholar
    • Export Citation
  • Sadeh A. , Mangel M. , and Blaustein L. (2009). Context-dependent reproductive habitat selection: the interactive roles of structural complexity and cannibalistic conspecifics. Ecology Letters 12:11581164.

    • Search Google Scholar
    • Export Citation
  • Sadeh A. Mangel M. , Polevikov A. , and Blaustein L. (2015). Intercohort size structure dynamics of fire salamander larvae in ephemeral habitats: a mesocosm experiment. Oecologia 179:425433.

    • Search Google Scholar
    • Export Citation
  • Segev O. , and Blaustein L. (2007). Priority effects of the early breeding fire salamander on the late breeding banded newt. Hydrobiologia 583:275283.

    • Search Google Scholar
    • Export Citation
  • Segev O. , and Blaustein L. (2014). Influence of water velocity and predation risk on fire salamander (Salamandra infraimmaculata) larval drift between temporary pools within ephemeral streams. Freshwater Science 33:950957.

    • Search Google Scholar
    • Export Citation
  • Segev O. , Mangel M. , and Blaustein L. (2009). Deleterious effects of mosquitofish (Gambusia affinis) on fire salamander (Salamandra infraimmaculata) larvae. Animal Conservation 12:2937.

    • Search Google Scholar
    • Export Citation
  • Segev O. , Hill N. Templeton A.R. and Blaustein L. (2010). Population size, structure and phenology of an endangered salamander, Salamandra infraimmaculata, at temporary and permanent breeding sites. Journal for Nature Conservation 18:189195.

    • Search Google Scholar
    • Export Citation
  • Segev O. , Mangel M. , Wolf N. , Sadeh A. , Kershenbaum A. , and Blaustein L. (2011). Spatio-temporal reproductive strategies in the fire salamander: a model and empirical test. Behavioral Ecology 22:670678.

    • Search Google Scholar
    • Export Citation
  • Segev O. Polevikov A. , Blank L. , Goedbloed D , Küpfer. E. Gershberg, A., Koplovich, A., and Blaustein, L. (2015). Effects of tail clipping on larval performance and tail regeneration rates in the Near Eastern Fire Salamander, Salamandra infraimmaculata. PLoSONE10(6):e0128077. doi:10.1371/journal. pone.0128077.

    • Search Google Scholar
    • Export Citation
  • Showstack S. (2016). Influence of various biotic and abiotic factors on predation and coloration of Salamandra infraimmaculata larvae. MSc thesis. University of Haifa.

    • Search Google Scholar
    • Export Citation
  • Spencer M. , and Blaustein L. (2001). Hatching responses of temporary pool invertebrates in response to environmental signals. Israel Journal of Zoology 47:397418.

    • Search Google Scholar
    • Export Citation
  • Stegen G. , Pasmans F. , Schmidt B.R. , Rouffaer L.O. , Van Praet S. , Schaub M. , … & Haesebrouck F. (2017). Drivers of salamander extirpation mediated by Batrachochytrium salamandrivorans. Nature 544(7650):353356.

    • Search Google Scholar
    • Export Citation
  • Tambone V. , Sacchini D. , Spagnolo A.G. , Menga R. , Ricci G. , Valenti R. , Vitali M.A ., and Ciccozzi M. (2017). A Proposed Road Map for the Ethical Evaluation of Sham (Placebo) Surgery. Annals of Surgery 265(4):658661. doi: 10.1097/sla.0000000000002007.

    • Search Google Scholar
    • Export Citation
  • Wallach A.D. , Bekoof M. , Nelson M.P. , and Ramp D. (2015). Promoting predators and compassionate conservation. Conservation Biology 49:14811484.

    • Search Google Scholar
    • Export Citation
  • Warburg M.R. (1992). Breeding patterns in a fringe population of fire salamanders, Salamandra salamandra. Herpetological Journal 2:5458.

    • Search Google Scholar
    • Export Citation
  • Warburg M.R. (1994). Population ecology, breeding activity, longevity, and reproductive strategies of Salamandra salamandra during an 18-year long study of an isolated population on Mt. Carmel, Israel. Mertensiella 4:399421.

    • Search Google Scholar
    • Export Citation
  • Zelditch M.L ., Swiderski D.L ., and Sheets D.S. (2004) Geometric morphometrics for biologists. A primer. Academic Press, Elsevier, Inc.

    • Search Google Scholar
    • Export Citation