Taxonomy and Molecular Phylogeny of the genus Nannopus (Copepoda: Harpacticoida: Nannopodidae) in Korean Waters

Abstract

The present study aimed to identify and describe Nannopus species with integrative approach (morphological and molecular taxonomy). For this purpose, a non-destructive DNA extraction method was adopted, consequently, morphological and molecular data were obtained from same specimens. The four genes: mitochondrial cytochrome c oxidase subunit I (COI) from 190 specimens, cytochrome b (CYTB) from 53 specimens, 18SrDNA from 25 specimens, and 28SrDNA from 43 specimens were amplified to investigate species boundaries. Several species delimitation methods were employed, uncorrected p-distances for COI, CYTB, 18SrDNA, and 28SrDNA, Automatic Barcode Gap Discovery – ABGD, and Bayesian implemented of Poisson tree processes – bPTP for COI and CYTB. Further, maximum likelihood and Bayesian phylogeny for COI, CYTB, 18SrDNA, and 28SrDNA were also constructed as a single genes and combined dataset. The species delimitation and phylogenetic analyses were all suggested the presence of three cryptic and six morphologically distinct species. These all species are sympatrically coexisted with wide distribution across the mudflats of Yellow Sea to South Sea of South Korea. After confirming genetically, six species were morphologically described. Within the group of species having seven elements in P3–P4 exp-3, Nannopus ganghwaensis shared naked caudal seta III with N. flexibilis Lilljeborg, 1902, N. didelphis Fiers and Kotwicki 2013, and N. hirsutus Fiers and Kotwicki 2013. However, distinguished from three species based on combination of characters, dorsal surface of cephalothorax without integumental window, lateral margins of urosomites are not densely hirsutus, and caudal seta IV slightly inflated and without globular expansion near insertion site. Within the group of species having seven elements in P3–P4 exp-3, Nannopus minutus and N. dimorphicus shared pinnate caudal seta III. However, they both differed in number of integumental windows (three in N. minutus, but only one in N. dimorphicus), caudal seta V (normal type, and anterior part wide in N. minutus, but bulbous, and anterior part heavily inflated in N. dimorphicus). Nannopus serratus and N. unisegmentatus Shen and Tai, 1964 shared two setae on P2 enp-2 but differed in ornamentation and position of setae on mandibular palp, and shape of inner seta on P3 enp-2. Nannopus parvus and N. bulbiseta shared two bulbiform pinnate setae on antennule second segment but they both displayed unique characters, caudal ramus trapezoidal in N. parvus and sub-cylindrical in N. bulbiseta. Several sexual dimorphisms were also discussed for future taxonomical purpose. A global key to species is provided based on 15 valid species including six newly described species. Overall, the present study shed light on cryptic complexity in N. ganghwaensis clade, which is endemic in intertidal mudflats of South Korea. They displayed conflicting divergence pattern among four genes. They also had conflicting topologies between BI and ML among single gene phylogenies. The topology was also conflicted between single gene phylogenies and combined phylogeny. The combined phylogeny was most robust and congruent with morphology, suggested with two major clades. Further, the validity of reinstating Ilyophilus Lilljeborg, 1902 was discussed and suggested to include all the congeners in Nannopus until the type species (N. palustris Brady, 1880) is re-described. Also, the validity of I. canui sensu was pinpointed. Moreover, this study represents additional example of integrative taxonomy, linking DNA methods and classical taxonomy based on morphology. Hence, it lays foundation for further taxonomical studies and biogeography of this taxon.