ORIGINAL ARTICLE Wataru Makino • Natsumi Maruoka • Megumi Nakagawa • Noriko Takamura DNA barcoding of freshwater zooplankton in Lake Kasumigaura, Japan Received: 22 November 2016 / Accepted: 13 April 2017 Ó The Ecological Society of Japan 2017 Abstract Although DNA barcoding is a promising tool for the identification of organisms, it requires the development of a specific reference sequence library for sample application. In the present study we developed a Lake Kasumigaura, Japan, zooplankton DNA barcode library to increase the sensitivity of future zooplankton monitoring for detecting lake ecosystem condition changes. Specifically, the mitochondrial cytochrome c oxidase subunit I (mtCOI) haplotype, i.e., the primary DNA barcode, was examined for each zooplankton taxon. In crustaceans, 37 mtCOI haplotypes were ob- tained from 99 individuals, representing four and 15 morpho-species of Copepoda and Cladocera, respec- tively. Comparing these sequences with those in Gen- Bank shows that the lake harbors putative non- indigenous species, such as Daphnia ambigua. In rotifers, 132 mtCOI haplotypes were obtained from 302 indi- viduals, representing 11 genera and one unclassified taxon. The automatic barcode gap discovery (ABGD) algorithm separated these haplotypes into 43 species. Brachionus cf. calyciflorus was divided into five ABGD species, and different ABGD species tended to occur in different seasons. Seasonal ABGD-species succession was also observed within Polyarthra spp. and Synchaeta spp. These seasonal successions were not detected by inspections of external morphology alone. Accepting up to 7% sequence divergence within the same species, mtCOI reference sequences were available in GenBank for three, 13, and 17 species in Copepoda, Cladocera, and Rotifera, respectively. The present results, therefore, reveal the serious shortage of mtCOI reference se- quences for rotifers, and underscore the urgency of developing rotifer mtCOI barcode libraries on a global scale. Keywords Biodiversity Æ DNA barcoding Æ Freshwater zooplankton Æ Lake Kasumigaura Æ Zooplankton monitoring programs Introduction Microscopic crustaceans (cladocerans and copepods) and rotifers are the major zooplankton components in terms of biomass in lakes and ponds (e.g., Pace 1986; Jeppesen et al. 2001). These organisms play an impor- tant ecological role by linking primary producers to planktivorous fish. Because the strength of predation upon zooplankton is reflected in their community structure (see Lampert and Sommer 1997), examining zooplankton community structure is critical to under- standing ecosystem conditions in lakes and ponds. Subsequently, zooplankton monitoring programs have been established in many parts of the world, including Lake Kasumigaura (Japan), for which the monthly re- cord of in situ zooplankton abundance since 1980 has been published (as a data paper, Takamura et al. 2017). Workers in the various zooplankton monitoring programs (non-taxonomists in many cases) must avoid misidentification, which could result in future confusion, such as misreads of the chronological changes in species richness and/or geographic range. Nonetheless, there are two practical problems impeding the morphological identification of freshwater zooplankton. First, although the morphological identification of freshwater zoo- Electronic supplementary material The online version of this article (doi:10.1007/s11284-017-1458-z) contains supplementary material, which is available to authorized users. W. Makino (&) Graduate School of Life Sciences, Tohoku University, 6-3 Aramaki aza aoba, Sendai, Miyagi 980-8578, Japan E-mail: makinowataru@m.tohoku.ac.jp N. Maruoka Æ W. Makino Department of Biology, Tohoku University, 6-3 Aramaki aza aoba, Sendai, Miyagi 980-8578, Japan M. Nakagawa Æ N. Takamura Center for Environmental Biology and Ecosystem Studies, Na- tional Institute for Environmental Studies, 16-2 Onogawa, Tsu- kuba, Ibaraki 305-8506, Japan Ecol Res DOI 10.1007/s11284-017-1458-z