Currently, freshwater zooplankton sampling and identification methodologies have remained virtually unchanged since they were first established in the beginning of the XX century. One major contributing factor to this slow progress is the limited success of modern genetic methodologies, such as DNA barcoding, in several of the main groups. This study demonstrates improved protocols which enable the rapid assessment of most animal taxa inhabiting any freshwater system by combining the use of light traps, careful fixation at low temperatures using ethanol, and zooplankton‐specific primers. We DNA‐barcoded 2,136 specimens from a diverse array of taxonomic assemblages (rotifers, mollusks, mites, crustaceans, insects, and fishes) from several Canadian and Mexican lakes with an average sequence success rate of 85.3%. In total, 325 Barcode Index Numbers (BINs) were detected with only three BINs (two cladocerans and one copepod) shared between Canada and Mexico, suggesting a much narrower distribution range of freshwater zooplankton than previously thought. This study is the first to broadly explore the metazoan biodiversity of freshwater systems with DNA barcodes to construct a reference library that represents the first step for future programs which aim to monitor ecosystem health, track invasive species, or improve knowledge of the ecology and distribution of freshwater zooplankton.

Our work shows the efficacy of DNA barcoding for recognizing the early stages of freshwater fish. We collected 3195 larvae and juveniles. Of them, we identified 43 different morphotypes. After DNA barcodes of 350 specimens, we ascertained 7 orders, 12 families, 19 genera, 20 species, and 20 Barcode Index Numbers, corresponding to putative species. For the first time, we reported the presence of the brackish species, Gobiosoma yucatanum in Lake Bacalar. Specimens of the genus Atherinella sp. and Anchoa sp. are possibly new species. Using both methods, morphology, and DNA barcodes, we identified 95% of the total larvae collected (2953 to species, and 78 to genus), and all of them were native. From them, the order Gobiiformes represented 87%. The most abundant species were Lophogobius cyprinoides and Dormitator maculatus, followed by Gobiosoma yucatanum and Ctenobius fasciatus. The Muyil and Chuyanché lagoons have the highest number of species. We present for the first time a short description of Cyprinodon artifrons and Floridichthys polyommus. This information conforms an indispensable baseline for ecological monitoring, to evaluate impacts, and developing management and conservation plans of biodiversity, principally in areas under human pressure such as Sian Ka’an, and Lake Bacalar, where tourism is high and growing in disorder.