Tropical regions contain many more species than do temperate and polar regions; however, explanations as to why remain unclear; for example, see [1, 2]. The Amazon Basin is particularly species rich and harbors arguably the world’s greatest terrestrial [3, 4] and freshwater [5] biodiversity. Numerous potential processes generating this diversity within the Amazon basin have been proposed; for example, see [6–8]. Testing of processes that have generated Amazonian biodiversity depends on solid taxonomy, since species are the operational units in all studies of biodiversity; for example, see [9]. The taxonomy of many Amazonian groups is still poorly known [3, 5] and at least in fishes a number of instances of haplotype sharing between closely related species have been reported; for example, see [10–14]. This makes it difficult to apply the phylogenetic species concept in species discovery and diagnosis. The delimitation of species of the genus Symphysodon has also been problematic in part due to sharing of mitochondrial DNA haplotypes between species [15–17] and a complete lack of resolution with nuclear DNA haplotypes [15, 17]