﻿A new species of Rhyncholagena Lang, 1944 (Copepoda, Harpacticoida, Miraciidae) from Palau

﻿Abstract A new species of Miraciidae Dana, 1846, Rhyncholagenacuspissp. nov., was described from Palau. Morphological descriptions and gene fragment sequence barcoding were performed on the 11th species of Rhyncholagena Lang, 1944 collected from sandy sediment samples in the subtidal zone of the Philippine Sea, Palau. Morphological characteristics were compared and an updated identification key was provided. A new species, Rhyncholagenacuspissp. nov., was found to be morphologically similar to Rhyncholagenalittoralis Por, 1967 and R.bermudensis Malt, 1990. This is the first record of the genus Rhyncholagena in Palau. The study provides basic data for future studies and highlights the need for continued exploration of marine biodiversity in Palau and other regions.


Introduction
Palau is renowned for its high biological diversity which is attributed to the influence of two currents: the North Equatorial Current and the North Equatorial Countercurrent passing through the area (Gopalakrishnan and Cornuelle 2019).Furthermore, coral reefs and marine biodiversity in Palau are well preserved (Friedlander et al. 2015).Several studies have been conducted on various marine organisms in Palau, such as coral reefs (Hamner et al. 2007), amphipods (Myers 2013(Myers , 2014)), pontoniine shrimps (Marin and Paulay 2010), laomediid mud shrimps (Alvarez et al. 2010) and benthic dinoflagellates (Horiguchi et al. 2011).Moreover, studies on copepods such as calanoid copepods (Ohtsuka et al. 2000), misophrioid copepods (Boxshall and Iliffe 1990), siphonostomatoid copepods (Ho 1980), pelagic copepods in marine lakes (Saitoh et al. 2011) and symbiotic cyclopoid copepods (Kim and Boxshall 2020) have also been reported.However, there has been relatively little research on the marine harpacticoid copepods in the area.
In October 2018 and January 2019, meiofauna samples were collected from the subtidal zone of Palau via SCUBA diving and the benthic copepods inhabiting the Palau coast were identified.In the present study, ZooKeys 1180: 181-199 (2023), DOI: 10.3897/zookeys.1180.109288Jisu Yeom & Wonchoel Lee: New Rhyncholagena from Palau we discovered a new species belonging to the genus Rhyncholagena in sandy sediments.
In this study, we discovered a new species of the genus Rhyncholagena for the first time in Palau.It is the 11 th member of Rhyncholagena and morphologically similar to R. littoralis Por, 1967 andR. bermudensis Malt, 1990.The most prominent feature of the new species is the development of a long lateral spinous process at the end of the anal somite.Herein, we provide taxonomic description of the new species and a revised identification key to the Rhyncholagena species.Additionally, we obtained the 18S ribosomal RNA (18S rRNA) and mitochondrial cytochrome oxidase I (mtCOI) sequences from the new species.

Material and methods
Sediment samples were obtained from two stations along the west coast of Palau (Fig. 1) at a depth of 15 m using SCUBA diving techniques during October 2018 and January 2019.The sandy sediments were then rinsed with fresh water and the supernatant was filtered through a 38 μm sieve before being fixed with 99% ethanol.Harpacticoid specimens were sorted under a dissecting microscope (Olympus SZX12) and stored in 99% ethanol at 4 °C.The identification of harpacticoids was carried out by following Huys et al. (1996) and Wells (2007) using a compound microscope (Olympus BX51) at 400-1000× magnification.All drawings (Figs 2-7) were prepared by using a drawing tube on an Olympus BX51 differential interference contrast microscope.
Scanning electron micrographs (Figs 8-10) were taken with the Hitachi S-3400N scanning electron microscope (SEM).Specimens were prepared for SEM by being transferred into pure isoamyl-acetate, then critical-point dried, mounted on stubs, coated in gold and observed under SEM on the in-lens detector at an accelerating voltage of 10.0 kV and 15.0 kV and working distance between 7.0 to 13.4 mm.Digital photographs were processed and combined into plates using Adobe Photoshop CS6.
To extract DNA templates, we used worm lysis buffer as described in Williams et al. (1992).We then amplified fragments from two genes, 18S rRNA and mtCOI genes, using polymerase chain reaction (PCR) premix (BIONEER Co. / Labopass, Korea) and 3 μl of genomic DNA as a template.For mtCOI, we used the primers, Cop-CO1-2189R (Bucklin et al. 2010) and LCO 1490 (Folmer et al. 1994), while for 18S rRNA, we used 18S-F1, 18S-F3, 18S-R7 and 18S-R9 (Yamaguchi and Endo 2003).We followed the protocols specified in the references for PCR amplification.Successful amplification was confirmed by 1% agarose gel electrophoresis.PCR products were sent to Macrogen (Seoul, Korea) for purification and DNA sequencing.DNA was sequenced on an ABI automatic capillary sequencer using the same sets of primers as those used for amplification.
The phylogenetic tree (Fig. 11) was constructed using the Maximum Likelihood method.The analysis of 18S rRNA involved 20 nucleotide sequences.Sequences of the Miraciidae and the outgroups were obtained from NCBI (Table 1).The obtained sequences were checked manually and aligned using the ClustalW algorithm (Thompson et al. 1994) in MEGA version 7.0 (Kumar et al. 2016).Maximum Likelihood analysis was performed using the Kimura 2-parameter model and Gamma distributed with Invariant sites (K2+G+I) (Kimura 1980;Nei and Kumar 2000), based on the model test results in MEGA.One thousand bootstrap replicates were performed to obtain a relative measure of node support for the resulting trees.2A, B, 9).Approximately 1.2 times as long as greatest width, armed with spinules on the lateral ventral surface and the inner dorsal surface.Each ramus armed with seven setae; seta I and seta II located the medial of the lateral margin, seta I bare, seta II short, seta III bare and located near seta I and seta II on the lateral margin; seta IV well developed, bipinnate, seta IV more than half as long as seta V, seta V as long as urosome; both terminal caudal setae with fractured plane; seta VI bare, located on inner distal corner; seta VII, bare, located on dorsal surface of caudal ramus.
Maxillule (Fig. 4C).Praecoxal arthrite bearing six elements distally with two pinnate spines, two bare setae laterally and two bare setae on surface; coxa with two bare setae; basis with six bare setae; endopod bearing four bare setae; exopod bearing two plumose setae.
Maxilla (Fig. 4D).Three endites of syncoxa with two, two and three setae, proximal to distal, respectively; allobasis transformed to thick pinnate claw bearing bare seta, two thin setae and short seta on surface; uniarticulated endopod with six bare setae.
Maxilliped (Fig. 4E).Subchelate; syncoxa with three pinnate setae and row of setules; basis with row of setules on surface, three times as long as broad, bearing two bare setae; endopod elongate, with strong claw and two bare setae.
In P1-P4, all rami three-segmented and coxa ornamented with several rows of spinules.Armature formula of the new species as follows:  P1 (Fig. 5A).Basis with outer ornamented seta, inner margin with setules and armed with bipinnate spine.Exp-1 inner margin bare, exp-1 and exp-2 with bipinnate outer spine and ornamented with spinules along outer margin, exp-2 inner margin with setules and bare inner seta; exp-3 with three outer spines and two geniculate setae distally.Enp-1 much longer than exopod, about 6.2 times longer than wide, with inner pinnate seta distally, which almost reaches the end of enp-3, inner margin ornamented with setules; enp-2 small and bare; enp-3 as long as enp-2, outer margin ornamented with spinules, bearing slender bare seta at inner distal edge, long seta and unipinnate claw-like spine apically.
P2 (Fig. 5B).Basis with setules distally and outer ornamented seta.Both exp-1 and exp-2 with inner plumose seta, inner margin ornamented with setules, bipinnate outer spine and outer margin ornamented with spinules; exp-3 with two plumose inner setae, long plumose seta at inner terminal, long spiniform seta with plumose inner side and pinnate outer side at outer terminal and three outer spines, proximal outer margin ornamented with spinules.Endopod as long as exopod, enp-1 with plumose inner seta; enp-2 with two plumose inner setae; enp-3 with pinnate inner seta, two setae distally and spine at outer distal corner, outer margin of endopod segments ornamented with spinules.
P3 (Fig. 6A).Basis with bare outer seta.Both exp-1 and exp-2 with inner seta and outer margin ornamented with spinules; exp-3 with two long inner se-  tae, long plumose seta and long pinnate seta at inner terminal and three outer pinnate spines, proximal outer margin ornamented with spinules.Endopod as long as exopod, both enp-1 and enp-2 with plumose inner seta; enp-3 with three plumose inner setae, two setae distally and short spine at outer distal corner; outer margin of endopod segments ornamented with spinules.
P4 (Fig. 6B).Basis with bare outer seta.Exp-1 with bare inner seta; exp-2 with plumose inner seta, inner margin ornamented with setules; exp-3 with two long setae and short seta on inner margin, long plumose seta and long seta with plumose inner side and pinnate outer side at distal and three outer spines, outer margin of exopod ornamented with spinules.Endopod shorter than exopod, both enp-1 and enp-2 with plumose inner seta; enp-3 with two plumose inner setae, two setae distally and spine at outer distal corner, outer margin of endopod segments ornamented with spinules.
P5 (Fig. 7A).Baseoendopod and exopod distinct, baseoendopod with long, slender and bare basal seta; endopodal lobe bearing three inner pinnate spines and two distal pinnate spines.Exopod elongated, 3.6 times longer than wide, with incision between the apical setae, with setules along inner margin and spinules along outer margins, bearing six bare setae.
Genital area as in Fig. 7B.P6 with two long setae and short pinnate spiniform seta.Genital double-somite with epicopulatory bulb.
P5 (Figs 7C, 10B).Baseoendopod and exp distinct; endopodal lobe ornamented with spinules at outer margin, armed with two spines.Exp with five se-tae in total, including bipinnate inner seta, long distal bare seta and three bare outer setae, outer margin ornamented with setules.
P6 (Figs 7C, 10B) represented by short inner seta and two long bare setae on outer distal corner of genital operculum.
Etymology.The scientific name is derived from the Latin cuspis (meaning a point), alluding to the spinous process on the anal somite.
Identification key Ma and Li (2018) suggested a key for Robertgurneya species, based on earlier keys provided by Lang (1948) and Wells (2007).The format of the existing iden-   tification key was maintained and minor modifications and additions to the new species were reflected in this study.The updated identification key below includes 11 species and subspecies of Rhyncholagena.All valid species of the genus are listed in Table 2 and their morphological characters are being compared.

Morphology
The new species can be placed in the genus Rhyncholagena, based on two characteristics: the incision between the apical setae of the P5 exp and the elongated rostrum.A notable trait that appears only in the new species within the genus is a spinous process on the anal somite.This character is suggested as autapomorphy of the new species.
Based on the discussion presented by Ma and Li (2018), we assigned the new species to group 1 as it lacks a seta in the P1 enp-2.However, according to the identification key provided in the same paper, the new species was identified as R. bermudensis belonging to group 2. The morphological differences between these species are as follows: (1) The long lateral spinous process at the end of the anal somite (absent in R. bermudensis), (2) The length of the rostrum (reaching the level of the third segment of A1 in R. bermudensis, but only the middle of the second segment in the new species), (3) The number of setae on the A2 exopod (two more in the new species), (4) P1 enp-2 seta (long in R. bermudensis).
Following the keys to harpacticoid species (Wells 2007), this copepod was identified as R. littoralis by the following two characteristics: A2 exp with two segments and setal formulae of swimming legs.However, they were distinguished by a combination of the following morphological characteristics: (1) The long lateral spinous process at the end of the anal somite (absent in R. littoralis), (2) The type of apical setae on the female P5 benp (plumose in the new species), (3) The length-width ratio of the female P5 exp (elongated in the new  4) The number of setae on the mandibular palp (more setae in the new species), (5) The segmentation of the exopod of the mandible (more segments in the new species), ( 6) The number of setae on basis and endopod of the maxillule (three more in the new species) and ( 7) The number of endites in the maxilla (four endites, one more in R. littoralis).
The new species tends to have more setae or segments in the mouthparts and more ornamentations than R. littoralis.It supports the possibility that the new species may be more ancestral than R. littoralis in terms of oligomerization.In addition, the factors of morphological differences in relation to the habitat can be considered.It has been reported to be distributed in gravel bottoms in shallow waters of the Red Sea (Por 1967) and the coral reef area of the Atlantic Ocean (Sarmento and Santos 2012).It can be inferred that, as the habitat changed from the subtidal zone with strong currents to shallow areas with weak currents, the general evolutionary trend of crustaceans may have occurred, such as the reduction of the setae and decorations of copepods and fusion of segments.It is assumed that the major factor in the change in the morphology of the mouthparts is the influence of changes in the feeding process.R. littoralis, which is known to inhabit areas with more gravel than the sandy environments where the new species inhabits, may filter less during its feeding process and, thus, it would have been less necessary to maintain abundant setae on its mouthparts in these environments.

Distribution
Considering the distribution records of the previously reported 10 species (Ma and Li 2018), this genus does not have any specificities for a specific regional distribution (Atlantic Ocean, Indian Ocean, Indo-Pacific Region, Red Sea and Mediterranean Sea), specific environmental distribution (mangrove, coral reefs and seagrass), depth (intertidal to subtidal zone, 0.5-700 m) and particle size of sediment (mud, sand and gravel).
Considering these distribution records, this genus has the potential to thrive in diverse regions and environments.The low specificity of region and habitat environment and high ecological flexibility suggest the possibility that this taxon can adapt well to environmental changes.Further research on this taxon through collection and excavation studies will not only allow us to find the forms of the genus that appear universally in various environments, but also provide evidence of evolutionary trends within taxa depending on the habitat.

Phylogeny
To infer the phylogenetic position of the genus Rhyncholagena within Miraciidae, a phylogenetic tree (Fig. 11) was constructed using the 18S rRNA sequences of Miraciidae species uploaded to the NCBI database.According to the phylogenetic tree, this genus is relatively close to genera, such as Amphiascoides Nicholls, 1941, Paramphiascella Lang, 1944and Robertgurneya Apostolov & Marinov, 1988, within the subfamily Diosaccinae.The low bootstrap value of this clade implies that there are still numerous gaps in our understanding of the systematics, as the genera included within the phylogenetic tree are of limited diversity.Nevertheless, Rhyncholagena is relatively distant from Diosaccus Boeck, 1873 and Amonardia Lang, 1944.This is consistent with the overall trend of the phylogenetic tree, which was created by Lang (1944), based on morphological characteristics.From a morphological standpoint, Diosaccus and Amonardia belong to a different clade from the other six genera in Diosaccinae included in the phylogenetic tree (Fig. 11).Their morphological differences include more inner setae in P3 exp-3 (three setae) and fewer setae in the P5 exp of males (four setae).
Further research is needed for detailed phylogenetic considerations; however, because the two genera, Diosaccus and Amonardia, were confirmed to be more closely related to subfamilies other than Diosaccinae, it is judged that the phylogenetic re-establishment of the family Miraciidae is necessary.
As a result of calculating the p-distance of the sequences obtained in this study and the mtCOI sequences of Miraciidae species uploaded to NCBI, it was confirmed that the sequences of the new species differed by more than 20% from those of the other species.Highlighting the novelty of this study, the genetic information of Rhyncholagena was registered in NCBI for the first time.

Table 1 .
GenBank numbers of 18S rRNA sequences used in phylogenetic analyses in this study.

Table 2 .
Morphological comparison of species within Rhyncholagena (*not exceeding half-length of exp: X / exceeding half-length of exp: O / slightly exceeding half-length of exp: ∆).