Evaluation of ITS2-28S as a molecular marker for identification of calanoid copepods in the subtropical western North Pacific

Abstract

We evaluated the internal transcribed spacer 2 and large subunit ribosomal DNA (ITS2-28S) region as a molecular marker for identifying calanoid copepods in the subtropical western North Pacific. The ITS2-28S sequence was successfully amplified in 232/244 individuals, a much higher rate of success than for cytochrome oxidase I amplification (77/244 individuals). A total of 194 sequences of ITS2-28S were obtained using a single primer pair. A high degree of genetic variability in ITS2-28S was observed in Lucicutia flavicornis (n = 13), Nannocalanus minor (n = 9), Pleuromamma abdominalis (n = 9) and Spinocalanus spinosus (n = 2), each showing several sequence types based on phylogenetic analysis of the ITS2-28S. A difference in the body size that corresponded with two sequence types of P. abdominalis was observed. The other species showed consistently lower genetic variability within species (0–0.001) than between species (≥0.005) using ITS2-28S of 810–968 bp aligned in each family. Although further morphological and genetic analysis of a larger sample size are necessary, our results suggest that ITS2-28S is a nuclear marker that does not need to be cloned and is easily amplified and sufficiently variable as a possible marker for DNA barcoding or to identify genetic groups of calanoid copepods.