Although amphibians as a group are relatively well-studied, data deficiency still remains an issue that has to be overcome before conservation efforts can be effectively developed. Prior to its supposed extinction, the available information on the Hula painted frog was merely descriptive and only based on three adult specimens and two tadpoles. Without any details on its habitat requirements, activity patterns and reproductive biology, this secretive species went unnoticed for decades, despite numerous attempts to rediscover it in its former, but highly modified habitat. Even though habitat degradation remains a prime concern for most species, recently emerged pathogens have led to an increased awareness of disease-related threats. In this multifaceted threat scenario, basic biological information on the target species is still fundamental for developing risk assessments and conservation strategies, but is not sufficient in itself anymore.
In our study, we have provided detailed morphological descriptions of both adults and tadpoles and have compiled first findings on the behaviour, activity patterns and vocalisation of L. nigriventer as well as initial information on its habitat. Together with our preliminary results on the presence of Bd on the Hula painted frog, our findings are important means to direct future research and conservation management of this critically endangered amphibian.
Our surveys in the Hula Valley and adjacent regions of northern Israel confirmed that L. nigriventer is a localised species that occurs within and in close vicinity to the Hula Nature Reserve. This wetland site became Israel’s first nature reserve in 1964 (Israel Nature and Parks Authority 2015) and is a closely monitored haven for a rich vertebrate fauna (Table S1) that – with exception of a fenced circular path – is not accessible to the public. By contrast, the site at Yesod HaMa’ala is subject to heavy anthropogenic influences, e.g. extensive rubbish dumping, petrol spills, near agricultural pesticide applications as well as occasionally free-roaming horses. The newly discovered large population at this heavily disturbed site not only demonstrates the adaptability of this rare frog to different environmental conditions, but is furthermore likely to be crucial for its overall survival. In addition, the confirmation of recent reproductive events at that site as well as the fact that adult individuals can reach snout-vent lengths of up to almost 130 mm while juveniles and tadpoles are comparatively tiny, illuminate aspects of the species’ life history that will guide future conservation planning.
Latonia nigriventer is shy in its aquatic habitat, rarely seen active during the day, and apparently not emitting loud vocalisations. Given these elusive habits, additional undetected populations might exist in the Hula Valley. Nevertheless, it is most likely that L. nigriventer will be restricted to particular kinds of habitats that allow the large adults to hide in the aquatic habitat (e.g., in deep layers of aquatic vegetation and mud) as well as in the terrestrial environment. It is yet unclear whether our observation of less adult individuals staying in the water from September through January is associated with the rather sparse vegetation along and within the water during the colder months or simply a reflection of our sampling effort. Based on our knowledge on Discoglossus species we initially directed our sampling effort mainly on the terrestrial habitat. We therefore cannot rule out the possibility of L. nigriventer being present in its aquatic habitat year-round.
We acknowledge that our radio-tracking attempt only yielded preliminary insights and implanted or digested devices will be necessary to confirm the results. However, our capture-recapture data also reflect a reduced movement. This apparently low dispersal capability of the Hula painted frog and its extended presence in the breeding waters are a major difference in comparison to species of its sister genus Discoglossus. These smaller-sized frogs (SVL 36–80 mm) breed in a large variety of shallow and often ephemeral water bodies following episodes of rain and an increase of temperatures (Vences and Grossenbacher, 2012). While Discoglossus males might stay close to or in water bodies during some weeks for reproduction, females usually show aquatic habits only for reproduction. In contrast, the large-sized Hula painted frog was so far without exception found in the proximity of and mostly in permanent water bodies.
These seemingly more aquatic habits of L. nigriventer convey risks as well as opportunities for conservation. On one hand, the year-round dependence on specific aquatic habitats might leave the individuals particularly vulnerable to local environmental changes, especially during the hot and dry summer months. On the other hand, if indeed small, deep, densely vegetated, muddy and permanent ditches represent an optimal habitat for this species, it should be easy to engineer exactly this kind of habitat in different parts of the Hula Valley where drainages and canals are omnipresent.
Like the closely related Discoglossus species, we found L. nigriventer to apparently breed opportunistically whenever conditions are advantageous. However, the role of rainfall for triggering egg deposition in this rare frog that mostly dwells in permanent water bodies remains unclear. The comparatively few tadpoles and juveniles found (Fig. 12) suggest that reproduction and recruitment in Latonia is comparatively low, and populations might survive less favourable periods with low reproductive success due to the longevity of the adults. Although the large size of this frog suggests a rather long lifespan, this hypothesis requires further testing. In contrast, the rather aquatic habits and reproduction in permanent water bodies might reduce the dependence of Latonia on rainfall compared to species of its sister genus Discoglossus.
Even though a large number of Hula painted frogs were found in the newly discovered site near Yesod HaMa’ala (131 out of 137 individuals), the conditions at this site might not be optimal. We observed a variety of detrimental factors of anthropogenic origin that might affect larval stages and productivity of adults. One of those factors is the sheer quantity of plastic and metal waste in the ditch, the degradation by-products of which have already been found to affect larval development and reproduction in amphibians (Oehlmann et al., 2009; Severtsova and Gutierrez, 2013). Another potentially detrimental factor is the nearby pesticide application. Studies that addressed the effect of pesticides on amphibians have shown that exposures even to low concentrations of the most commonly applied pesticide atrazine result in the feminisation of male frogs and cause the retardation of larval development and growth (Hayes et al., 2002, 2006; Boone and James, 2003; Carr et al., 2003). So far, however, the impact of both rubbish deposition and pesticide application at that new site on the development and reproduction of Latonia remains purely speculative.
Apart from anthropogenic influences, the high percentage of injury found in adults and the overall low numbers of small individuals might indicate a high predatory pressure on the early life stages of L. nigriventer. Besides White-throated kingfishers (Halcyon smyrnensis (Linneaeus, 1758)) that are known to prey upon adult L. nigriventer (Biton et al., 2013), juveniles might fall prey to a plethora of widespread and abundant animals that are known or suspected to prey on amphibians and present at the locality, such as Western mosquitofish (Gambusia affinis (Baird & Girard, 1853); Goodsell and Kats, 1999; Komak and Crossland, 2000; Segev et al., 2009), freshwater crustaceans (Gherardi and Micheli, 1989; Capolongo and Cilia, 1987), dragonfly nymphs (Stav et al., 2007), Caspian turtles (Mauremys caspica (Gmelin, 1774); Sidis and Gasith, 1985), lycosid spiders and carabid beetles (McCormick and Polis, 1982; Toledo, 2005), or larger frog individuals comprising conspecifics (Mendelssohn & Steinitz, 1943) and P. bedriagae (own observations).
Our data show that the fungal pathogen, Bd, is present on L. nigriventer (27%) as well as on P. bedriagae in the Hula Valley. During our surveys in northern Israel, we did not observe mass mortality events in any amphibian species, nor did we notice skin lesions in any individual. This finding is not unexpected given Bd has not wreaked havoc in amphibian populations in much of the Old World, with the exception of Australia. Also for Europe only a limited number of incidences with Bd-induced mortalities have been documented so far (Bosch et al., 2001; Bosch and Martínez-Solano, 2006; Bosch and Rincón, 2008). All in all, many species appear to coexist with Bd at low infection intensities (Spitzen-van der Sluijs et al., 2014). Albeit our study has relatively low sample size and we conducted Bd screening only during one season, we can hypothesise that the low infection levels observed in our study could be associated both with environment and host-associated factors. In temperature experiments with Bd isolates, Piotrowski et al. (2004) showed that Bd optimally grows at temperatures between 10–25 °C, and suggest that lower or higher temperatures may not induce epidemics. The high water temperatures (> 25 °C) prevailing in the investigated ditches during the summer months, could thus be one factor explaining the low prevalence and intensity of Bd. Our observations that Latonia individuals do not seem to form dense aggregations, but keep some distance to each other might also have affected the ability of Bd to spread from individual to individual.
The presence of antimicrobial peptides in L. nigriventer could also, in part, explain the observed low Bd prevalence and intensity, although we acknowledge that two screened individuals are not an adequate sample size for broad conclusions to be made. However, our results provide a baseline for all future studies that endeavour to analyse the innate immunity of L. nigriventer. Furthermore, the cutaneous bacterial community could play a role in mitigating chytrid infection in these frogs; as a perspective for future studies it is worth mentioning that seven of the most abundant bacterial OTUs found on the skin of L. nigriventer belong to species or genera known to potentially exhibit antifungal activity (Harris et al., 2006; Woodhams et al., 2007; Lauer et al,. 2008; Becker et al., 2015). The infection dynamics of Bd are likely a complex interaction of external environmental parameters, host behaviour, and host immunity factors that may differ across life stages. However, continued monitoring of Bd infection rates as well as an identification of the Bd strain present in northern Israel remain important. Furthermore, minimising the anthropogenic impact at the localities and preventing the introduction of new amphibian pathogens by carefully disinfecting utilised equipment before and after usage at the respective sites are imperative.
Our findings have direct relevance for the ongoing restoration project in the Hula Valley which offers enormous opportunities for improving and extending this rare species’ suitable habitat. In close collaboration with local stakeholders and the Israel Nature and Parks Authority we will design a prospective monitoring system for this species and continue to raise local awareness in order to improve conservation at known and potential other locations. Environmental DNA studies to detect further populations of L. nigriventer are currently underway. Even though none of the Latonia individuals discovered within the Hula Nature Reserve was found in an aquatic habitat, first eDNA results indicate that individuals are at least crossing one of the water bodies within the north-eastern part of the reserve (Renan et al., 2016).
As our first tadpole-rearing attempt proved fruitful, a further step will be to set up a captive breeding programme. Such a programme could tide this unique frog over the most critical early tadpole and juvenile stages enabling translocation of individuals to restored and newly created habitats within its presumed former range, which would help to increase the population size and promote the survival of the Hula painted frog.