Only a few major research programs are currently targeting COI barcoding of amphibians and reptiles (including chelonians and crocodiles), two major groups of tetrapods. Amphibian and reptile species are typically old, strongly divergent, and contain deep conspecifi c lineages which might lead to problems in species assignment with incomplete reference databases. As far as known, there is no single pair of COI primers that will guarantee a suffi cient rate of success across all amphibian and reptile taxa, or within major subclades of amphibians and reptiles, which means that the PCR amplifi cation strategy needs to be adjusted depending on the specifi c research question. In general, many more amphibian and reptile taxa have been sequenced for 16S rDNA, which for some purposes may be a suitable complementary marker, at least until a more comprehensive COI reference database becomes available. DNA barcoding has successfully been used to identify amphibian larval stages (tadpoles) in species-rich tropical assemblages. Tissue sampling, DNA extraction, and amplifi cation of COI is straightforward in amphibians and reptiles. Single primer pairs are likely to have a failure rate between 5 and 50\% if taxa of a wide taxonomic range are targeted; in such cases the use of primer cocktails or subsequent hierarchical usage of different primer pairs is necessary. If the target group is taxonomically limited, many studies have followed a strategy of designing specifi c primers which then allow an easy and reliable amplifi cation of all samples.
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Understanding patterns of species diversity can only be achieved by long-term research and the integration of taxonomical, ecological and behavioral data. Here we studied the diversity and ecology of ants of Iguazu National Park (INP), Argentina, using six sampling techniques. DNA barcodes were used to uncover cryptic diversity, test species/morphospecies delimitation accuracy, and link unidentified male and queen specimens with their worker caste. Results: INP houses 195 described ant species and an additional 49 morphospecies. Leaf litter sampling and pitfall traps were the most efficient sampling methods, while surface baiting revealed the prevalence of large predatory species at different times of the day. Comparing baiting to other sampling methods provided information on species co-existence and the presence of possible dominance hierarchies among ant species. We obtained the DNA barcodes of 312 specimens from 124 species (51% of the ants of INP). Our analyses evidenced a clear barcode gap in all species but two, with an average distance to the nearest neighbor of 15.75%, almost eight times larger than the mean distance to the furthest conspecific (2.07%). Eighty-three percent of the sequence clusters obtained with different clustering algorithms (ABGD, RESL, TCS) matched the reference species or morphospecies, while 10% highlighted possible cryptic diversity. In terms of efficacy, this barcode library allowed a correct identification in more than 94% of the species/morphospecies, and to assign a species name to 69% of the unidentified males and queens. Significance: This study evidences that DNA barcodes are a valuable tool for identifying the ants of the Atlantic Forest, a global diversity hotspot. Furthermore, our project provides a framework for understanding the ecology and the taxonomic diversity of the ants of this region, including the identification of currently undescribed reproductive castes and the discovery of possible cryptic species.
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RBINS Staff Publications 2017
