A new species in the genus Acartia Dana, 1846 (Crustacea, Copepoda, Calanoida, Acartiidae) from the South Pacific coastal waters of Nadi Bay, Fiji

Abstract A new species in the genus Acartia, Acartia nadiensissp. nov., is described from Fijian coastal waters. This species belongs to the subgenus Odontacartia based on the following morphological features: presence of a rostral filaments, a pointed process on the last prosomite, a serrated terminal spine on female P5, and the absence of a protrusion on the basis of the male right P5. This new species can be differentiated from its congeners by the combination of the absence of a spine on the first segment of the antennules, the short outer seta of female P5, and a medial spine on the exp-2 of the left male P5. Phylogenetic analyses using mitochondrial COI partial sequences show that the new species is distinct from its congeners.


Sample collection and identification
Specimens were collected from Nadi Bay, Fiji, using a 100 μm mesh plankton net having a 30 cm diameter mouth, and then preserved in 99% ethanol. Specimens were dissected in lactic acid, and mounted on slides with lactophenol. Preparations were sealed with transparent nail varnish. All drawings were prepared using a drawing tube attached to an Olympus BX51 differential interference contrast microscope. For scanning electron microscope (SEM) preparation, specimens were dehydrated in a series of graded ethanol solutions, then placed in isoamyl acetate, critical point dried, mounted on stubs, coated in platinum, and observed under a Hitachi S4700 field-emission electron microscope at Eulji University, Seoul, Korea. Descriptive terminology was adopted from Huys and Boxshall (1991).

DNA extraction and amplification
For DNA extraction, ethanol was removed from fixed specimens (99% EtOH) by washing with distilled water, and DNA was extracted using a tissue DNA purification kit (COSMO GENETECH, Co. Ltd, Korea). DNA was extracted from individual specimens. mtCOI DNA was amplified in 20 μl reaction volumes containing extracted tissue DNA and primers LCO-1490 (5'-GGT CAA CAA ATC ATA AAG ATA AAG ATA TTG G-3') and HCO-2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') (Folmer et al. 1994). PCR conditions comprised initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 46 °C for 2 min, and extension at 72 °C for 3 min. This was followed by a final extension step at 72 °C for 10 min. PCR products were evaluated by electrophoresing amplification products on 1% agarose gel containing ethidium bromide. Purification of amplified products was performed using a PCR purification kit (COSMO GENETECH Co. Ltd, Korea), and both strands were sequenced using an ABI 3730XL sequencer (COSMO GENETECH Co. Ltd, Korea).
Antennule incompletely 18-segmented ( Fig. 2A, B), fourth to seventh segments partly fused on dorsal surface; ninth to eleventh segment each with one row of setules, twelfth segment with three rows of setules, thirteenth and seventeenth segment each with one row of setules; segmentation and setation patterns as follows: (1) Antenna ( Fig. 2C): coxa with seta; basis and first endopodal segment fused to form elongated allobasis bearing eight setae medially and one seta terminally along inner marin, and spinular row on distal area; second endopodal segment elongated, with seven setae, rows of spinules on lateral margin; third exopododal segment short, with seven setae. Exopod 4-segmented; setation formula 1, 2, 2, 3.

Molecular analysis
A 581 bp partial region of mtCOI was sequenced from five species: A. nadiensis sp. nov., A. erythraea, A. japonica, A. ohtsukai, and A. omorii. Sequences of two species (A. pacifica and A. spinicauda) were obtained from NCBI and also included in the analysis. All species belong to the subgenus Odontacartia except A. omorii, which belongs to the subgenus Acartiura and was used as the outgroup. The mtCOI sequences of A. nadiensis differed in a 24.1% from A. japonica, and in up to 29.0% from A. pacifica (Table 2). Neighbor joining and minimum evolution phylogenetic analyses using the Tamura-Nei model showed that A. nadiensis was clearly distinct from its congeneric species (Fig. 9).

Discussion
The new species, Acartia nadiensis sp. nov., clearly belongs to the subgenus Odontacartia. This subgenus displays the following diagnostic characters compared to the other five subgenera of Acartia: presence of rostral filaments, symmetrical pointed process on the posterior corner of the last prosomite, minutely serrated terminal spine on female P5, and absence of protrusion on the basis of male right P5 (Steuer 1915;Ueda and Bucklin 2006;Soh 2010). The new species can be distinguished from other Odontacartia species by several distinctive characters (Table 3). First, Odontacartia species, including the new  (Acartiura) omorii as outgroup.

Female
Body length Long seta Long seta Long seta Spine species, can be divided into two groups based on the presence of a spine on the first segment of antennules (Steuer 1923;Srinui et al. 2019). Species with this spine include A. amboinensis, A. australis, A. bispinosa, A. erythraea, A. japonica, and A. lilljeborgi. Species lacking spine include A. bowmani, A. centura, A. mertoni, A. ohtsukai, A. pacifica, and A. spinicauda. Acartia nadiensis sp. nov. also lacks a spine on the first segment of antennules. Second, the outer seta of the female P5 of A. nadiensis sp. nov. is much shorter than the terminal spine, and the length ratio of the outer seta/terminal spine is 0.4. Most species of Odontacartia have an outer seta that is longer than the terminal spine in female P5. There are two species (A. bowmani and A. japonica) that have a short terminal seta on female P5, and the length ratio of outer seta/terminal seta are 0.9 and 0.7, respectively. Third, the male P5 of A. nadiensis is clearly distinguishable from the rest of species based on its length and the type of medial process on the exp-2 of the left leg.
Furthermore, the new species shows other minor differences compared to the other 13 Odontacartia species, such as the number of dorsal spines on the urosomite, the length/ width ratio of the female P5 basis, and the length/width ratio of caudal rami.
To supplement the morphological evidences, we conducted molecular phylogenetic analyses using partial mtCOI sequences of six Odontacartia species, including the new species. The mtCOI gene is widely used to identify sibling species due to its higher evolutionary rate than 16s and 18s rDNA (Knowlton and Weight 1998;Hebert et al. 2003;Schindel and Miller 2005;Karanovic et al. 2018). In previous studies of calanoid copepods, mtCOI sequence divergence between species have been shown to range from 13.0-22.0% (Bucklin et al. 1999), 17.6-26.7% (Eyun et al. 2007, and 21.0-23.0% (Soh et al. 2013). The mtCOI partial sequence of A. nadiensis sp. nov. differed by 24.1-29.0% from the sequences of congeneric species, which is greater than the range of interspecific differences reported in previous studies.
The length ratio of the outer seta/terminal spine of the female P5 is the most diagnostic morphological feature in Odontacartia species. However, this character is also used to determine the subgenus Euacartia (Soh et al. 2013). This confusion between subgenus systems has been documented previously (Madhupratap and Haridas 1994). Barthélémy (1999) compared female genital structure of 25 species of Acartiidae using light and scanning electron microscopy and concluded that there is no support for the current subdivision of Acartia into subgenera. Although the new species A. nadiensis belongs to the subgenus Odontacartia based on the current identification system, the validity subgeneric taxa, as proposed by Steuer (1915Steuer ( , 1923, within Acartia should be reevaluated.