Environmental DNA 18s rRNA amplicon sequencing Raw sequence reads

Community ecology is an important tool to inform conservation managers about threatened and endangered species, but it has largely been ignored in reintroduction planning. Characterizing communities at potential reintroduction release sites may help ensure that biotic environments are favorable for translocations. Eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) are fully aquatic salamanders that are declining and undergoing conservation measures throughout much of their range. In the state of Indiana, eastern hellbenders are currently endangered and restricted to a single river system. As such, there is an effort to reintroduce hellbenders to other rivers in the historic range. We implemented a hierarchical approach to evaluate the abiotic and biotic environment across potential release sites. We first used known habitat preferences and expert evaluation to characterize the physical habitat at five candidate reintroduction streams and the source river (the Blue River). We then implemented environmental DNA (eDNA) methodology to characterize the eukaryote diversity at multiple sites in each river. One potential reintroduction river (Indian Creek) showed very similar physical habitat and eukaryotic community structure to the source river. We subsequently performed a fine-scale analysis of crayfish abundance and macroinvertebrate community structure at potential release sites within Indian Creek. We chose four potential reintroduction sites based on high habitat ranking and researcher accessibility and compared them to four sites in the source river known to support hellbender populations. The abundance and size of crayfish, the main prey base of hellbenders, did not differ between the rivers. Overall macroinvertebrate richness was higher in Indian Creek, but we detected no difference in Shannon diversity indices or overall community composition between the two rivers. None of the potential reintroduction sites stood out as clearly superior to the others suggesting that multiple sites within Indian Creek might be appropriate for Eastern Hellbender reintroductions. This study provides a methodological framework for an ecosystem-centric approach to the selection of reintroduction sites for aquatic species.