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Israel Journal of Ecology & Evolution, 2017 http://dx.doi.org/10.1163/22244662-06303001

Compassionate approaches for the conservation and protection of fire Leon Blausteina,*, Ori Segeva,**, Valentina Rovellia, Shirli Bar-Davida,b, Lior Blanka,c, Antonina Polevikova, Nadav Pezaroa, Tamar Krugmana, Simona Showstacka, Avi Koplovicha, Lital Ozeria and Alan R. Templetona,d aInstitute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838 Israel bMitrani Department of Desert Ecology, Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Sede Boqer Campus, Sede Boker 84990 Israel cDepartment of Plant Pathology and Weed Research, ARO, Volcani Center, Bet-Dagan 50250, Israel dDepartment of Biology and Division of Statistical Genomics, Washington University, St. Louis, MO 63130-4899 USA **Current Address: Faculty of Aerospace Engineering, Technion, Haifa, Israel

Abstract The Near Eastern fire , 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 where 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 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 protection.

Keywords ethics; compassionate conservation; Gambusia affinis; gene expression; Salamandra infraimmaculata

Introduction at least 190 m above sea level (asl) (Blank and Blaustein, Compassionate conservation is a discipline that not only 2012, 2014). In Syria also, they are also not found below acts to prevent extinctions of populations but also protects 200 m asl (Bogaerts et al., 2013). Salamandra infraim- individuals from unnecessary harm and death (Ben-Ami maculata retreat into deep crevices and underground caves et al., 2014; Ramp and Beckoff, 2015, Wallach et al., 2015). when rains stop, where they remain relatively dormant The Near Eastern , Salamandra infraim- throughout the hot and dry season (approximately early maculata, is considered an endangered species in Israel May through mid-October). Adults resume activity and (Dolev and Perevolotsky, 2004) and near-threatened re- emerge from estivation sites once rains begin in October gionally (IUCN Red List, 2017). For 2.5 decades, our or November. Gravid females emerge from their over- laboratory has sought ethical sampling methods to protect summering sites, carrying a full complement of larvae (as individuals and populations of S. infraimmaculata while many as 193 larvae has been deposited by a single clutch performing research to further our knowledge and under- from a single mother although S. infraimmaculata females standing of their life history, ecology, population genetics from Tel Dan are smaller and deposit far fewer larvae [De- and ecological genomics, which in turn, can be used to gani, 1996]). Adults are typically active on rainy nights and help preserve individuals and populations. females larviposit in a variety of water bodies including temporary lentic ponds, permanent lentic ponds, tempo- rary ponds in ephemeral wadis and in permanent streams Natural History of Salamandra infraimmaculata (Degani, 1996; Blank and Blaustein, 2012). Females have The Near Eastern fire salamander, S. infraimmaculata, ex- been shown to adjust the number of larvae they deposit in a ists in Iran, Iraq, Syria, Turkey, Lebanon, and Israel (IUCN particular water body depending on the water volume (Se- Red List, 2017). In Israel, S. infraimmaculata occurs in gev et al., 2011) or preferentially select structurally com- the northern, mountainous and more rainy regions that are plex habitats when older cohorts of conspecific individuals

*Corresponding author. E-mail: [email protected]

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44 L. Blaustein et al. are already present, presumably to avoid cannibalism (Sa- wadis (Segev and Blaustein, 2014). Using the tailfin iden- deh et al., 2009). Larvae are key predators that have a large tification, we were able to document that some Salaman- impact on community structure, including negative effects dra larvae did not remain in their rock pools, but dispersed on many predators and herbivores that lead to trophic cas- downstream or were forced downstream to another rock cades (Blaustein et al., 1996, Eitam et al., 2005, Segev and pool. Also by using non-invasive photographs of adults for Blaustein, 2007). They are also predators on mosquito lar- mark-recapture, we have been able to assess how different vae and egg rafts (Blaustein et al., 2014). Some crustacean breeding habitats support different population sizes. Our species also avoid hatching in response to chemical cues research group has also recently been testing the use of Vis- of Salamandra larvae, apparently to avoid being consumed ible Implant Elastomer (VIE) tags as a marking technique (Blaustein, 1997; Spencer and Blaustein, 2001). The larvae for identifying Salamandra larvae in both field and labora- are also cannibalistic (Degani, 1993; Markman et al., 2009; tory experiments. The VIE tags consist of a silicon-based Sadeh, 2012). Once larvae metamorphose and exit the wa- liquid polymer that is injected subdermally (under anesthe- ter body, it takes about 3–5 years to become reproductively sia) and cures rapidly into a biocompatible and pliable sol- mature (Warburg, 1994) and they may live to be 25 years id (Northwest Marine Technology, Inc.). The multi-color­ or more (Warburg, 1994). Here, we report on our research tagging options of the VIE provides minimally invasive activities, taking compassionate conservation into account. 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 Estimating S. infraimmaculata population size and our lab have also shown no indication of suffering adverse dispersal by mark-recapture ­effects in either survival or behaviouralchanges ­ (Segev In assessing population size and dispersal using capture- et al., 2015; Pezaro et al., unpublished results). mark-recapture methods, many species are tracked by cut- ting off different toes for assessing individual identification. In many cases, it is not known whether cut-off toes can Is assessing impacts of introduced predators of affect mobility, survival or dispersal of particular species Salamandra larvae compassionate conservation? (Perry et al., 2011). Toe-clipping is a common technique In 2003, we found that Salamandra larvae in ponds in the that has been widely used in studies of mammals, reptiles, presence of high densities of the mosquitofish,Gambusia birds and amphibians but both the efficacy and humane affinis (which were apparently introduced for mosquito nature of the technique are subject to compassionate criti- control), had chewed-off tail fins and other appendages cism (Clark, 1972; May, 2004; McCarthy and Paris, 2004). while most Salamandra larvae in ponds without Gambusia Moreover, the technique may not be effective, especially for had normal intact tail fins and other appendages although long-term studies, if toes are lost naturally or for Salamandra may bite off other individuals’ tails due to can- that have the ability to regenerate their appendages (Ferner, nibalism (Degani 1993; Sadeh et al., 2009; Sadeh 2012). We 1979). In the case of estimating dispersal and population found that at the ponds of Kaukab Springs (Lower Galilee size of the fire salamander, we (Bar-David et al., 2007; Mountains), with high densities of Gambusia, no salaman- Segev et al., 2010; Goedbloed et al., 2017. I. Sinai et al., der larvae survived to metamorphosis. We submitted this unpublished results) and others (Warburg, 1994) have used work to the Israel Journal of Zoology as a likely warning photography for specific dorsal spot ­patterns in S. infraim- that Gambusia had strong deleterious effects on Salamandra­ maculata adults to identify specific individuals Fig. ( 1). larvae. However, rather than accept this fairly convincing Our mark-recapture studies have demonstrated population short communication, reviewers felt that stronger infer- size at specific breeding sites (Segev et al., 2010; I. Sinai ence was needed – i.e., that an experiment should be done, et al., unpublished results). We have found that temporary manipulating the presence or absence of Gambusia, and breeding sites have far fewer adults in the population than assessing Salamandra larvae. Though we were strongly permanent breeding sites (Segev et al., 2010; I. Sinai et al., convinced that Gambusia was causing strong deleterious unpublished results). Moreover, mark-recapture studies effects on fire salamander larvae based on field surveys, based on dorsal spot patterns have demonstrated disper- and, an experimental manipulation would result in some sal among breeding sites (Bar-David et al., 2007). Based Salamandra larval mortality, we decided to conduct an out- on these findings, we recommend increasing the tradi- door mesocosm experiment to demonstrate the deleterious tional conservation unit for salamanders – from focusing effects due to reviewer insistence. The small numbers of on the protection of a breeding site to the protection of salamander larvae used in mesocosm experiments gener- several breeding sites and their surroundings to maintain ally do not result in as much mortality as is found in natural landscape connectivity among them (Kershenbaum et al., pools. We obtained newborn salamander larvae from four 2014). gravid females from a site where the total adult population We have also used individual-specific non-invasive was estimated to be >500 adults and we used and estimated photographs of tail fins in Salamandra larvae. Using <0.3 percent of the larvae born at that breeding site. The individual-specific tail finsEitam ( and Blaustein, 2002; experiment consisted of presence or absence of Gambusia Fig. 2), we were able to study priority effects in cohorts crossed with presence or absence of artificial vegetation of Salamandra larvae (Eitam et al., 2005) and also down- (total: 16 mesocosms). The artificial vegetation did not in- stream dispersal of S. infraimmaculata larvae in ephemeral crease refuge for Salamandra larvae but instead, increased

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Israel Journal of Ecology & Evolution 45

03.02.2010

08.12.2012

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. Photo credit: ORI SEGEV. the number of Gambusia. Gambusia had very strong ­management and fewer mortalities in the future, then we negative effects on the salamander larvae. Vegetation would say that demonstrating the deleterious effects of did not act as a refuge against Gambusia and Salaman- mosquitofish with a small number of Salamandra larvae dra is exceptionally unlikely to be able to coexist with was “compassionate conservation”. The paper was then ac- Gambusia. Given the two controls without Gambusia (i.e. cepted by the journal, Animal Conservation (Segev et al., no vegetation and vegetation), much higher survival oc- 2009). As a consequence of the surveys and experimental curred than the larvae that were subjected to Gambusia in work, the Israel Nature and Parks Authority removed all mesocosms. Gambusia from the Salamandra breeding sites that saved Though the initial reviewers of Israel Journal of Zool- many thousands of Salamandra larvae each year. Given ogy would not recommend acceptance of this work based the high density of Gambusia, mortality to the salaman- on correlations, a new biological conservation journal also der larvae are virtually 100 percent. Since there are about refused to consider the manuscript containing both cor- 250 females at Kaukab springs, and minimally, each one relational evidence and experimental evidence because larviposits at least 100 larvae. Thus, minimally at Kauk- ­mortality was caused to individuals. This work encouraged ab, ~25,000 larvae are saved from Gambusia each year us to come up with our own formula for compassionate by removing Gambusia. In removing the Gambusia from conservation: if an experiment sacrifices fewer individu- the permanent ponds of Kaukab, many of the Gambusia als that would have occurred in nature anyway, and the were killed but some Gambusia were also placed in en- information accrued from the experiment (that lead to vironments where Salamandra did not exist for mosquito mortality of some individuals) leads to better ­conservation control. Gambusia are harmful predators as they also prey

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46 L. Blaustein et al.

Figure 2. Identification of four different individual larvae of Salamandra infraimmaculata based on tailfin spot patterns. All larvae have dif- ferent tailfin spot patterns. Photo credit: ORI SEGEV. upon many other amphibian­ ­larvae (e.g., Cabrera-Guzman metamorphose (e.g., Warburg, 1992; Blaustein et al., 2001). et al., 2017; Pollard et al., 2017). Salamandra anyway have Approximately 20 years ago, one of the authors of this very strong effects on mosquito larvae where they prey paper, Leon Blaustein, came across hundreds of fire sala- upon egg rafts, mosquito larvae and pupae (Blaustein mander larvae that were within a small desiccating pool et al., 2014). The results of this experiment resulted in far and it was clear that within 1–2 days, with no forecast of fewer mortalities than would have been caused in natural rains, these hundreds of larvae would be dead – either due breeding sites by Gambusia. This is akin to medical experi- to desiccation or because certain birds such as crows can ments where some individuals are given placebo medica- easily prey upon them when in shallow water (Showstack, tion or even sham surgery while others are given the real 2016). Blaustein called the Israel Nature and Parks Author- medication or real surgery (McCullough, 2017; Tambone ity (INPA) to request permission to remove these larvae, et al., 2017). Though a number of patients taking the pla- place them in artificial pools and then return them once it cebo die from lack of medication, the experiment, giving rained again filling the natural pool with rain water. The strong ­inference, allowed for much higher human survival representative of the INPA refused to allow this stating “let in the future. For all our laboratory and outdoor mesocosm nature take its course”. Was this decision a dispassionate experiments, Salamandra adults, juveniles and surviving act or an important evolutionary move? Rescuing the lar- larvae are all returned to their breeding site during the vae would have been going against natural selection; lar- breeding season. To date, not a single gravid Salamandra vae will of course die if they have not reached a minimum or juvenile died in the lab though a very small fraction of critical size to metamorphose, but natural selection acts on larvae do die in the lab. From a compassionate conservation these populations for allowing these larvae to develop phe- aspect, we believe that Gambusia should not be killed and notypic plasticity and local (Polivekov 2017). rescued from amphibian-breeding sites, and placed in non- In a recent experiment to assess phenotypic plasticity and natural habitats where Gambusia can control mosquitoes. local adaptation among populations from temporary and permanent breeding sites, when allowing water to desic- cate, we also placed wet towels at the bottom of the ex- Assessing phenotypic plasticity and local adaptation in perimental tubs once the depth was approximately zero; if fire salamander metamorphosis salamander larvae have reached the minimum critical size S. infraimmaculata larvae possess phenotypic plasticity to metamorphose, we know from many observations that and local adaptation in metamorphosing more quickly in larvae hide under moist rocks and can accelerate metamor- response to pond desiccation (Sadeh et al., 2011; Polevikov, phosis rather than dying. Thus, adding the wet towels were 2017). However, temporary pond desiccation is a common both mimicking nature and a compassionate natural act form of mortality in salamanders and other amphibians (Polevikov, 2017). Only 3 of 216 larvae died as a result of when larvae have not reached a critical minimum size to these wet towels (Polevikov, 2017).

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Israel Journal of Ecology & Evolution 47

Compassionate and effective methods for measuring (O. Segev et al., unpublished results; I. Sinai et al., unpub- Salamandra body morphometrics as a function of lished results). To assess population genetics and landscape habitat type genetics, we conducted microsatellite studies, using up Aquatic animals demonstrate phenotypic plasticity in to 15 loci. Our studies have shown that the isolated body shape in response to different habitat types, such peripheral Mount Carmel has considerably lower allelic as lentic versus lotic habitats (Gaston and Lauer, 2015), richness than the Galilee populations and various levels of or in response to risk of (Hossie et al., 2010). genetic similarity across many populations (Blank et al., Body dimensions were traditionally measured by using a 2013; I. Sinai et al., unpublished results). Based on these re- simple ruler, while holding the animal and stretching the sults, we advocated that conservation plans for this ­species measured organ or body. Each measurement was taken should account for these patterns. Moreover, the micro- separately which prolongs the process. For aquatic animals satellite data also reveal population structuring and lim- (e.g. fish and amphibian larvae) measurements taken out of ited dispersal in a very heterogeneous landscape. We have the water can potentially increase stress for the measured also considered landscape features to gene flow by animal. Traditional modern morphometrics compares assessing least cost path, random walk and isolation by lengths, as well as the ratios and angles between them but resistance models (Kershenbaum et al., 2014). These mod- it ignores the shape as a whole and is susceptible to biases els have helped us understand where dispersal corridors when size measurements overlap, run in similar directions, exist. or when several measurements radiate from a single point which adds dependency among measurements (Zelditch Minimally invasive methods for studying genome-wide et al., 2004). analysis of gene expression Geometric morphometrics (GMM) uses homologous features of the organism to explore morphological shape Studying genome-wide analysis of gene expression in sal- changes (Bookstein, 1982). In this sense, shape is stripped amanders can give a better handle on phenotypic plastic- off of non-pure shape properties like location, scale, and ity, local adaptation and development (Czypionka et al., rotation (Kendall, 1977). It preserves the shape through 2015, Polevikov, 2017). When we were going to begin such all the steps of analysis, and thus the relationship between a study, we were advised that gene expression needed to be measurements (Adams et al., 2004). studied using RNA extracted from the entire body of fire The use of digital cameras and water-filled transpar- salamanders. We decided to test whether simply clipping ent photo chambers makes data collection and digitiza- the larval tailfin was enough for assessing gene expres- tion cheaper, efficient, and less stressful for the measured sion – i.e., yield gene expression patterns that are represen- animal. In parallel, developed software for the analysis tative of the whole organism. We first showed (Segev et al., of shapes became common, from stand-alone programs 2015) that taking a partial tail-fin clip was not harmful to the like tpsDig (Gerth and Maia, 2017), PAST, SHAPE, fire salamander larvae; cannibalistic fire salamander larvae ­MORPHOJ, to R packages like Geomorph, Morpho, often bite off a section of the tail fin. We further demonstrat- Shapes, and Momocs. ed that the tailfin grows back and survival to, size at and Three years ago, our lab started using GMM, to iden- time to metamorphosis is unaffected Segev( et al., 2015). tify and determine the degree of phenotypic plasticity in Several full bodies of Salamandra salamandra in Germany fire salamander larvae from different habitat types, i.e. lotic had previously been sacrificed for transcriptomic analysis. versus lentic breeding sites. We exposed laboratory-born We compared the full body transcriptomes of S. sala- larvae from different habitat types to flowing and stagnant mandra to larval tailfins in response to temperatures and condition in a fully factorial mesocosm manipulation ex- found that 51% of the expressed genes were common periment. We use the R package Geomorph (Adams and for both the larval tailfin Czypionka( et al., 2015). This Otárola-Castillo, 2013), to analyze the changes in larval study demonstrated that we could study gene expres- body shape. sion without killing larvae and without using whole body transcriptomes.

Minimally invasive methods for studying population and landscape genetics Protecting Salamandra infraimmaculata adults from Studying population genetics and landscape genetics of road kills fire salamanders are important for conservation concerns. Our studies of fire salamander adults have demonstrated We have been especially concerned about testing for ge- that adults are quite prone to vehicles on roads that are near netic diversity along the core and periphery of the species’ breeding sites, and there are many road kills by vehicles. In range (Blank et al., 2013; I. Sinai et al., unpublished re- particular, I. Sinai and T. Oron (unpublished results) have sults). To do this, we have taken very small tips off the end shown that ~150 adults over four seasons were found killed of Salamandra adult tails. The clipped tail tip is ~0.3–0.4 by vehicles along ~2 km of road near Dishon Stream. The cm while the total size of Salamandra adults generally high mortalities, which is against compassionate conser- range from 28–32 cm in total length. Subsequent stud- vation ethics, call for underground tunnels below roads ies have demonstrated that the Salamandra adults do with drift fences to funnel the adults through the tunnel well, even after these small tail tips have been removed (D’Amico et al., 2015; Bain et al., 2017).

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Preventing disease spread among breeding sites in fire fire salamander populations in many countries Stegen( salamanders et al., 2017; Gray et al., 2017). As far as Israel is concerned, When studying wild populations, researchers should al- up until now, the amphibian chytrid , Batrachochy- ways be aware of the impact of their activities on the health trium dendrobatidis was detected on Hula painted of individuals and populations. In the last decades, infec- (Latonia nigriventer) around the Hula wetlands, but there tious diseases have been recognized as main threats to was no chytridiomycosis in L. nigriventer, probably be- wildlife and amphibians in particular have faced a severe cause the skin mucosome contains antimicrobial peptides decline due to the spread of a disease called chytridiomy- (Perl et al., 2017). Until now, no such infectious disease as cosis (Kilpatrick et al., 2010). Chytridiomycosis is caused has been reported in S. infraimmaculata in Israel which by Batrachochytrium dendrobatidis (Bd), a chytrid fungus occur only several tens of km from the Hula wetlands, belonging to the Chytridiomycota Division, Order Chy- but the implementation of a biosecurity protocol is a tridiales (Kilpatrick et al., 2010). Although it is still not priority, as best practice in conservation (Gray et al., 2017). clear if this pathogen was first introduced by humans or In fact, Salamandra populations in Israel are usually small if it was already present when its virulent activity started, and relatively isolated, and the emergence and spread- it seems that its spreading has been fostered by human ing of infectious diseases might be catastrophic, possibly movements (Kilpatrick et al., 2010). In recent years, a new leading to the local extinction of genetically unique pathogen called Batrachochytrium salamandrivorans ap- populations. For this reason, to avoid contamination be- peared in Europe, and its outbreak is severely threatening tween individuals belonging to different populations, when

Table 1. Summary of compassionate conservation methods for the Near Eastern fire salamander,Salamandra infraimmaculata. In all cases, adult and larval Salamandra were returned to their breeding site when field or laboratory experiments were completed.

Objective Non-invasive (or compassionate References conservation) research methods

Estimating adult population size at Photographs of individual- specific Warburg (1994); Segev et al. (2010); breeding sites spot patterns (non-harmful) Goedbloed et al. (2017); Sinai et al. unpublished Estimating dispersal among breeding Photographs of individual adult specific-spot Bar-David et al. (2007) sites patterns (non-harmful) Following dispersal in ephemeral wadis Photographs of individual-specific spot Segev and Blaustein (2014) patterns on larval tailfins (non-harmful) Following inter-cohort interactions Photographs of individual-specific spot patterns Eitam et al. (2005) on tail fins (non-harmful photographs although, as in natural pools, there is some competition and very little cannibalism) Identifying individuals in mesocosms Photographs of individual-specific spot patterns Segev et al. (2015); Pezaro et al., on tail fins andVIE tags (non-harmful) unpublished results Assessing impacts of Gambusia on Experimental manipulation that sacrificed several Segev et al. (2009) Salamandra larvae tens of Salamandra larvae but subsequently saved many thousands after Gambusia removal from breeding sites. Gambusia affinis should also be saved and placed in non-amphibian breeding sites. Assessing pond drying on metamorphosis Using wet towels in mesocosms to allow Polevikov (2017) metamorphosis which mimics natural pools (only 3 of 216 larvae desiccated) Population and landscape genetics Determining genetic structure based on taking Blank et al. (2013); Kershenbaum Salamandra adult tail tips for microsatellite data et al. (2014); I. Sinai et al., unpub- (non-harmful) lished results Assessing phenotypic plasticity of Determining how body shape changes in response Segev et al., unpublished results Salamandra morphometrics to lentic versus lotic habitats and dissolved oxygen (photographs for morphometrics are non-harmful; no larvae died from lower dissolved oxygen) Studying gene expression Taking partial tail fins for gene expression rather Segev et al. (2015) than taking whole bodies for transcriptomics (non-harmful) Documentation of adult Salamandra Calls for underground road tunnels with drift I. Sinai and T. Oron, unpublished road-kills fences to concentrate adults through the tunnels results (should significantly reduce road kills) Preventing potential spread of pathogens Disinfecting equipment and boots with Virkon S Kilpatrick et al. (2010); Stegen of Salamandra (DuPont) (prevents infection) et al. (2017); Gray et al. (2017)

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Israel Journal of Ecology & Evolution 49 using salamanders for our experiments, we disinfect all Australia, who has campaigned relentlessly for animal the equipment with Virkon S (DuPont), a broad-spectrum rights and ethics, Ofir Elbaum, a young man who recently disinfectant. Moreover, prevention of contamination in the was killed in an automobile accident who also campaigned field should be mandatory not only for herpetologists, but for animal ethics and Israel-Arab peaceful coexistence, and for all scientists and naturalists working in the field. In this Arian Wallach who showed amazing compassion for her regard, the situation in Israel is complicated by the fact that graduate studies on deer. We also thank Olga Rybak for her the country is very small and that many of the sites where exceptionally compassionate treatment of fire salamanders Salamandra breed are close to roads, making them easily – adults and larvae – when they were in our laboratory. We accessible to the public. Therefore, even if we are doing are grateful to Iftach Sinai and Talya Oron for sharing some our best to prevent contamination among different popula- unpublished compassionate conservation general results tions, human activities in general can potentially represent to include in this paper. Funding for this study was pro- a main threat for S. infraimmaculata in Israel. The Israel vided by the Deustche-Israel Projects (DIP) (BL 1271/1-1) Nature and Parks Authority has recommended that we do awarded to Leon Blaustein and Alan R. Templeton and to not return Salamandra used in experiments, which has va- Arne Nolte and Sebastian Steinfartz (STE 1130/8-1). lidity, but thus far, we have returned all individuals to their breeding sites for 25 years and there have not been any infections. References Adams, D.C., and Otárola-Castillo, E. (2013). geomorph: an r package for the collection and analysis of geometric mor- Conclusions phometric shape data. Methods in Ecology and Evolution. 4:393–399. When dealing with an endangered species, biological con- Adams, D.C., Rohlf, F.J., and Slice, D.E. (2004). Geometric mor- servation research is often needed to provide the informa- phometrics: Ten years of progress following the ‘revolution.’ tion necessary to design and implement a recovery plan Italian Journal of Zoology. 71:5–16. that hopefully will increase the numbers of individuals Bain, T.K., Cook, D.G., and Girman, D.J. (2017). Evaluating the effects of abiotic and biotic factors on the movement through and ensure the viability of the species. Table 1 summarizes wildlife crossing tunnels during migration of the California our compassionate conservation methods. Such research tiger salamander, Ambystoma californiense. Herpetological should always be done in a manner that minimizes the suf- Conservation and Biology 12:192–201. fering and death of individuals, but another ethical con- Bar-David, S., Segev, O., Peleg, N., Hill, N., Templeton, A.R., sideration must be the cost of not doing the research. For Schultz, C.B., and Blaustein, L. (2007). Long distance move- ments by fire salamanders Salamandra( salamandra infraim- example, our research on the impact of Gambusia upon maculata) and implications for habitat fragmentation. Israel S. infraimmaculata larval survival did cause the death of Journal of Ecology and Evolution 53:179–196. some larvae (however, not nearly as much mortality as is Ben-Ami, D., Boom, K., Boronyak, L., Townend, C., Ramp, D., observed in nature), but it provided knowledge that was Croft, D.B., and Bekoff, M. (2014). The welfare ethics of the convincing to agencies that altered their policies in a man- commercial killing of free-ranging kangaroos: An evaluation of the benefits and costs of the industry.Animal Welfare 23: ner that greatly increased the survival of many more larvae 1–10. and helped in the sustainability of this endangered species. Blank, L., and Blaustein, L. (2012). Using ecological niche model- This short-term individual versus long-term population ing to predict the distributions of two endangered amphibian trade-off is a unique ethical dilemma in nature conserva- species in aquatic breeding sites. Hydrobiologia 693:157–167. tion research on endangered species. From a compassion- Blank, L. and Blaustein, L. (2014). A multi-scale analysis of breeding site characteristics of the endangered fire salaman- ate conservation standpoint, Gambusia should also be der (Salamandra infraimmaculata) at its extreme southern saved and placed in non-amphibian breeding sites. An- range limit. Hydrobiologia 726:229–244. swers to this dilemma are not always easy to obtain, but Blank, L., Sinai, I., Bar-David, S., Peleg, N., Segev, O., Sadeh, A., this is a recurrent issue in many endangered species’ sur- Kopelman, M.N., Templeton, A.R., Merilä, J., and Blaustein, vival programs and should not be ignored. We hope that L. (2013) Genetic population structure of the endangered fire salamander Salamandra( infraimmaculata) at the south- research in our group will serve as an example that the ernmost extreme of its distribution. Animal Conservation ethical principles of compassionate conservation can be 16:412–421. upheld while employing rigorous scientific methods and a Blaustein, L. (1997). Nonconsumptive effects of larvalSalaman - striving for the development of evidence-based conserva- dra on its crustacean prey: can eggs detect predators? Oeco- tion strategies. logia 110:212–217. 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 salaman- Acknowledgements ders (Ambystoma macrodactylum). Israel Journal of Zoology We thank Dror Ben-Ami for motivating Palestinians, Jor- 47:333–345. Blaustein, J., Sadeh, A., Blaustein, L. (2014). Influence of fire danians and Israelis by organizing a compassionate con- salamander larvae on among-pool distribution of mosquito servation conference in Israel. Author Leon Blaustein was egg rafts: Oviposition habitat selection or egg raft predation? highly influenced in a compassionate conservation setting Hydrobiologia 723:157–165. by four people: his father, Isak Blaustein, of blessed mem- Blaustein, L., Friedman, J., and Fahima, T. (1996). Larval Sala- ory, who refused to kill anything, Iris Toren from Sydney, mandra drive temporary pool community dynamics: evi- dence from an artificial pool experiment. Oikos 76:392–402.

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