Population genetic analysis of the recently rediscovered Hula painted frog (Latonia nigriventer) reveals high genetic diversity and low inbreeding.

After its recent rediscovery, the Hula painted frog (Latonia nigriventer) has remained one of the world's rarest and least understood amphibian species. Together with its apparently low dispersal capability and highly disturbed niche, the low abundance of this living fossil calls for urgent conservation measures. We used 18 newly developed microsatellite loci and four different models to calculate the effective population size (Ne) of a total of 125 Hula painted frog individuals sampled at a single location. We compare the Ne estimates to the estimates of potentially reproducing adults in this population (Nad) determined through a capture-recapture study on 118 adult Hula painted frogs captured at the same site. Surprisingly, our data suggests that, despite Nad estimates of only ~234-244 and Ne estimates of ~16.6-35.8, the species appears to maintain a very high genetic diversity (HO = 0.771) and low inbreeding coefficient (FIS = -0.018). This puzzling outcome could perhaps be explained by the hypotheses of either genetic rescue from one or more unknown Hula painted frog populations nearby or by recent admixture of genetically divergent subpopulations. Independent of which scenario is correct, the original locations of these populations still remain to be determined.

Living quarters of a living fossil-Uncovering the current distribution pattern of the rediscovered Hula painted frog (Latonia nigriventer) using environmental DNA.

One of the greatest challenges of effective conservation measures is the correct identification of sites where rare and elusive organisms reside. The recently rediscovered Hula painted frog (Latonia nigriventer) has not been seen for many decades and was therefore categorized extinct. Since its rediscovery in 2011, individuals from the critically endangered species have been found, with great effort, only in four restricted sites. We applied the environmental DNA (eDNA) approach to search for new populations of the Hula painted frog in suitable aquatic habitats. We further used the eDNA data to classify the landscape factors associated with the species distribution and to predict its suitable habitats. We sampled 52 aquatic sites in the Hula Valley during the spring of 2015 and 2016 and amplified the samples with a species-specific qPCR assay. DNA of the Hula painted frog was detected in 22 of the sites, all of which clustered within three main areas. A boosting classification model showed that soil type, vegetation cover and the current and former habitats are all key predictors of the frog's current distribution. Intriguingly, the habitat suitability models reveal a high affinity of the species to its long-lost habitat of the historical wetlands. Our findings encourage a series of informed searches for new populations of this threatened frog and provide guidance for future conservation management programmes. In the era of global conservation crisis of amphibians, developing the eDNA approach, a reliable detection method for many critically endangered and elusive amphibians, is of particular importance.

The rediscovered Hula painted frog is a living fossil.

Amphibian declines are seen as an indicator of the onset of a sixth mass extinction of life on earth. Because of a combination of factors such as habitat destruction, emerging pathogens and pollutants, over 156 amphibian species have not been seen for several decades, and 34 of these were listed as extinct by 2004. Here we report the rediscovery of the Hula painted frog, the first amphibian to have been declared extinct. We provide evidence that not only has this species survived undetected in its type locality for almost 60 years but also that it is a surviving member of an otherwise extinct genus of alytid frogs, Latonia, known only as fossils from Oligocene to Pleistocene in Europe. The survival of this living fossil is a striking example of resilience to severe habitat degradation during the past century by an amphibian.