The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

The association of immobility and camouflage is widespread as a defensive mechanism in prey from varied taxa. However, many experiments assessing the reaction of prey to predator cues are conducted under artificial laboratory conditions. In a previous experiment we observed the tadpoles of Ololygon machadoi (Hylidae) to respond to predator visual and/or chemical cues by choosing backgrounds that improve their disruptive properties, but detected no associated reduction of movement. Here we experimentally demonstrate this response in the species' natural habitat, on backgrounds where the tadpoles are likely to achieve their best camouflage. We also tested whether previous experiences could influence both background choice and immobility in O. machadoi tadpoles. these novel experimental results suggest that a defensive behavior-i.e., reduction of movement-in these tadpoles is more strongly expressed under the natural conditions where they evolved, compared to laboratory conditions where prey and predator were brought into closer contact. Besides, previous experiences are likely to play an important role in expressed defensive responses. Prey species are under constant selection to escape predation. They can develop predator specific responses, but some responses seem to be widespread, such as spatial avoidance of predators and reduction of activity 1. In this context prey species that count on camouflage to reduce the probability of being detected by predators tend to remain motionless, aiding to the protective properties of their colouration 2,3. Reduction of movement is likely to reduce detection probability and can emerge as a spontaneous response when the detected threat is at a distance (background threat), but an approaching (immediate) threat may otherwise elicit an escape response 4. Previous experience can also determine the reaction of prey to specific predators 5 , however there is evidence that evolutionary pressures may have led prey species to develop different defensive mechanisms and express each one in appropriate contexts determined by the assessed threat 4. Both innate and learned defensive behaviours can be expressed even in very early life stages 6,7. Tadpoles, the larvae of biphasic anuran amphibians, are frequently employed in experiments conducted to study the reaction of prey to predator cues 8 , due to their broad range of predators and defensive mechanisms 1. It has been shown that many species of tadpoles reduce movement in the presence of predators 1,9. However, these studies are usually conducted under laboratory or mesocosm conditions, sometimes within reduced spaces in artificial standardized containers where predator cues are manipulated, other factors kept equal, to record tadpole reaction to particular cues 1,10. Under such circumstances, both visual and chemical stimuli have been