A new species of Pilarella (Copepoda, Calanoida, Arietellidae) from the hyperbenthic layer of Japan, with a molecular phylogenetic analysis of some representative genera of the Arietellidae

Abstract A new species of the continental shelf hyperbenthic genus Pilarella is described, the first from the Indo-Pacific. This is the second species of Pilarella known, and the first description of a male in the genus. The new species is easily distinguished from other species of Pilarella (P. longicornis) based on: (1) short caudal rami, approximately 1.5 times longer than wide; (2) 2 setae on the mandibular endopod; (3) 6 setae on the maxillular coxal epipodite; and (4) in the female, a short left antennule reaching the posterior border of the genital double-somite. The new diagnosis of Pilarella differs from Metacalanus in the separation of ancestral segments IX–XII and XIV–XV of the antennule, and the presence of 5–6 setae on the maxillular praecoxal arthrite. Pilarella is also separated from Metacalanalis based on the absence of a seta on the third ancestral segment of the antennary exopod, the symmetry of legs 1–3, the presence of a medial basal seta on the female leg 5, and 2 lateral exopodal spines on the female leg 5. A molecular phylogenetic analysis of some representative genera of the family Arietellidae, including the present new species, recovers two arietellid clades (Metacalanus- and Arietellus-clades) as in previous studies. Dichotomous keys for the genera of Arietellidae and the species of Pilarella are included.

Recently, molecular markers such as ribosomal and mitochondrial DNA sequences have been used as tools for the exploration of phylogenetic relationships among various calanoid taxa. However, only data on one arietellid species (Paraugaptilus buchani) is available in the DDBJ/EMBL/GenBank databases, and no molecular phylogenetic analysis has been conducted yet among arietellid genera.
This study describes a new species of Pilarella collected off the Amami Oshima Island, Kagoshima Prefecture, Japan. This is the second species of Pilarella and the first record of this genus in the Indo-Pacific. The male of Pilarella is described for the first time. Moreover, we explore the phylogenetic relationships among seven arietellid species based on 28S ribosomal DNA. Furthermore, new dichotomous keys to arietellid genera and species of Pilarella are provided.

Sample collection and morphological observations
The arietellid copepods used in this study were collected off the Nansei Islands in southwest Japan in May 2018 and 2019 by the training and research vessel "Toyoshio-Maru" of Hiroshima University. Sampling dates, localities, and collection gears (cf. Ohtsuka et al. 1992) are shown in Table 1. Specimens were fixed in 99.5% ethanol.
Specimens of the new species of Pilarella were dissected under a stereomicroscope (SZX7, Olympus), and their appendages and urosome cleared in lactophenol. Illustrations were drawn under a biological microscope (BX53, Olympus) with a drawing tube.

DNA extraction, PCR, and sequencing
Total DNA of samples, except of Metacalanus sp., was extracted from the whole body using DNeasy Blood & Tissue kits (Qiagen, Venlo, Netherlands). Total DNA extraction from a small sample of Metacalanus sp. was performed using the whole body according to the method described by Suyama (2011). DNA was quantified using a NanoDrop 2000 instrument (Thermo-Fisher, Waltham, MA, USA) and then adjusted to 1 ng μL -1 with sterilized water for PCR amplification. The 28S nuclear ribosomal DNA region (28S) was amplified using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) with Taq PCR Master Mix Kit (Qiagen) and primers; 28S-F1a (5ʹ-GCG GAG GAA AAG AAA CTA AC-3ʹ) and 28S-R1a (5ʹ-GCA TAG TTT CAC CAT CTT TCG GG-3ʹ) (Blanco-Bercial et al. 2011). Thermocycling conditions for 28S were 94 °C for 7 min; 35-40 cycles at 94 °C for 45 s, 50 °C for 1 min, and 72 °C for 1 min; and a final extension at 72 °C for 7 min. Amplification results were verified using 2% (w/v) agarose electrophoresis. Excess primers and dNTPs were removed with ExoSAP-IT (Thermo-Fisher, Scientific, Waltham, MA, USA), and the sequencing was performed commercially (Macrogen Japan, Kyoto, Japan).
Seven sequences for 28S (Accession numbers: LC510290-LC510295, LC516702) obtained in this study were deposited in the DNA Data Bank of Japan (DDBJ) and GenBank. Paraheterorhabdus compactus (Sars, 1900) (Accession number: HM997026) was used as the outgroup taxon. Sequence alignment was performed using CLUSTAL W (Thompson et al. 1994) in MEGA 7.026 (Tamura et al. 2011), and final alignments of 649 bp were used for the phylogenetic analysis. Phylogenetic relationships among the partial 28S sequences were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. ML analysis was performed using software RAxML 8 (Stamatakis 2006, Stamatakis et al. 2008) under the GTR+Γ model, and the data set was run with 10,000 bootstrap replicates. BI analysis was performed using the soft-  (Ronquist et al. 2012) under the GTR+Γ model. Two parallel analyses of Metropolis-Coupled Markov Chain Monte Carlo (MC 3 ) were conducted for 1,000,000 generations, and topologies were sampled every 100 generations. The convergence of MCMC was checked with the value of the average standard deviation of split frequencies (ASDSF) in MrBayes and trace plots in the software Tracer 1.7.1 (Rambaut et al. 2018). The first 2500 trees (25% of all trees) were discarded as burn-in, and the consensus was estimated by summarizing the remainder 7500 trees. The phylogenetic trees of ML and BI analyses were visualized with Figtree 1.4.4 (Rambaut 2009).
Remarks. The diagnosis of Metacalanus from Ohtsuka et al. (1994) differs from Pilarella in the fusion of ancestral segments IX-XII and XIV-XV on the antennule, and the display of 0-2 setae on the maxillular praecoxal arthrite (vs. 5-6 setae in Pilarella). The diagnosis of Metacalanalis from Ohtsuka et al. (2005) differs also from Pilarella in the presence of 1 seta on the third ancestral segment of the antennary exopod, the asymmetry of legs 1-3, the absence of medial basal seta on female leg 5, and the presence of 3 lateral exopodal spines on the female leg 5 (vs. 2 in Pilarella).
Etymology. The specific name of the new species is derived from the Latin adjective compactus meaning "stocky" to denote the habitus of the present new species.
Remarks. The present new species falls within the diagnosis of the monotypic Pilarella (Alvarez 1985) except for the following features: (1) left antennule not reaching the caudal rami; (2) short caudal rami; (3) 6 spines on the maxillular praecoxal arthrite (5 in the previous diagnosis); and (4) 6 setae on the maxillular coxal epipodite (5 in the previous diagnosis). For the differences from other genera, see generic remarks in the present study.
Phylogenetic analysis. The Maximum Likelihood tree based on 16 species of the superfamily Arietelloidea is shown in Figure 5. Both ML and BI trees showed two clades in Arietellidae. In clade I, Pilarella compacta sp. nov. and Metacalanus sp. were placed in the same clade (BP = 69%; PP = 0.948). Hyperbionyx athesphatos (HM997029) (belonging to the Hyperbionychidae) and clade I formed a cluster with a low bootstrap value (BP = 54%; PP = 0.868). In clade II, four arietellid genera (Paraugaptilus, Paramisophria, Sarsarietellus, and Arietellus) were grouped in the same cluster with high bootstrap value (ML = 81%; PP = 0.991), and Arietellus was placed in the same clade as Sarsarietellus with a high bootstrap value (BP = 87%; PP = 0.997). The sequence of Paraugaptilus buchani (LC510293) in this study showed a lower identity of 96% to P. buchani (HM997028) in the DDBJ/EMBL/GenBank databases, which differed by five gaps and 19 single nucleotide polymorphisms (SNP).

Key to genera of the family Arietellidae
The homology of the maxilla and maxilliped by Ivanenko (2001, 2008) is shown in parenthesis. Female genital double-somite with single copulatory pore; third ancestral segment on exopod of female leg 5 with at most 3 elements and 1 process .

Discussion
This study is the first report of a male Pilarella. The male antennule of Pilarella compacta sp. nov. has long, tape-like aesthetascs on the basal part. Similar antennules were found in a male Metacalanus acutioperculum and Metacalanus adriaticus (Ohtsuka 1984;Kršinić and Boxshall 2021). The segmentation and setation of the male antennule are similar to those of Crassarietellus and more segments are retained than in the diagnosis of Metacalanus (Ohtsuka et al. 1994). The male leg 5 has the same segmentation as in Metacalanus aurivilli (Chen and Zhang 1965), and corresponds to the Metacalanus-clade in the reduction of endopod (Ohtsuka et al. 1994). These features of the male Pilarella suggest its close relation to Crassarietellus and Metacalanus, which belong to the Metacalanus-clade in Ohtsuka et al. (1994). As regards the molecular analysis, clades I (Metacalanus, Paramisophria, and Pilarella) and II (Arietellus, Paraugaptilus, and Sarsarietellus) in Figure 5 correspond to two lineages, the Metacalanus-and Arietellus-clades, which were discussed by Ohtsuka et al. (1994Ohtsuka et al. ( , 2005 and Soh et al. (2013). Although Ohtsuka et al. (1994Ohtsuka et al. ( , 2005 considered Paramisophria to be in the Metacalanus-clade, Soh et al. (2013) regarded the position of Paramisophria as equivocal based on their cladistic analysis. In the present study, Paramisophria was included in clade II (Arietellus-clade), although with low bootstrap value and posterior probability (BP = 49%, PP = 0.678).
Hyperbionix, belonging to the Hyperbionychidae, was clustered in clade I and included in the lineage of the Arietellidae (Fig. 5). However, estimating the phylogenetic position of the Hyperbionychidae is difficult because the cluster of Hyperbionychidae + clade I was supported by a low bootstrap value (BP = 54%). Blanco-Bercial et al. (2011) indicated the close relationship between Paraugaptilus buchani (Arietellidae) and Hyperbionyx athesphatos (Hyperbionychidae) in their molecular phylogeny. Ohtsuka et al. (1993) described many autapomorphic characteristics in the Hyperbionychidae, and Soh (1998) indicated that the Hyperbionychidae has some plesiomorphic characteristics that are not observed in the Arietellidae. These studies suggested that the Hyperbionychidae is closely related to the Arietellidae but is not derived from the Arietellidae.
In clade II (Arietellus-clade), a comparison of the 28S sequences of Paraugaptilus buchani between the present (material from the western North Pacific) and a previous study (material from the western North Atlantic; Blanco-Bercial et al. 2011) showed a low concordance rate (96%), differing by five gaps and 19 SNPs. However, no differences in morphological characteristics were observed between the Paraugaptilus buchani material collected from the western North Pacific in the present study and the original description of the species based on Atlantic material (Deevey 1973). As a possible explanation of this discordance, we consider it as due to intra-specific variation. Differences in genetic structure among populations of the same species have been reported in many other calanoids (Bucklin et al. 2003). In our case, it may be caused by the significant geographic distance separating the Pacific and the Atlantic locations where the material was collected.
The present study suggests a genetically closer relationship of Arietellus to Sarsarietellus based on high bootstrap value and posterior probability (BP = 87%, PP = 0.997) rather than to Paraugaptilus, as previous studies based on morphological features pointed out (Ohtsuka et al. 1994(Ohtsuka et al. , 2005Soh et al. 2013). Furthermore, Arietellus and Sarsarietellus also share a synapomorphic fused right and left copulatory pores (Ohtsuka et al. 1994;Ohtsuka et al. 2004). These two closely related genera, Arietellus and Sarsarietellus, are distributed across the pelagic realm and the deep-sea hyperbenthic layers, respectively (Ohtsuka et al. 1994). Ohtsuka et al. (2005) suggested that, based on morphology, the two pelagic genera (Arietellus and Paraugaptilus) could have diverged from a hyperbenthic ancestor. However, the present study shows that Arietellus (pelagic genus) is closer to Sarsarietellus (hyperbenthic genus) rather than to Paraugaptilus, and that Arietellus and Paraugaptilus could have colonized the pelagic realm independently, or that Sarsarietellus secondarily turned back to the deepsea hyperbenthic layers. In conclusion, the molecular phylogenetic relationships of the Arietellidae supported the Metacalanus-and Arietellus-clades as depicted by Ohtsuka et al. (1994Ohtsuka et al. ( , 2005 and Soh et al. (2013), and provide new information about other possible colonization routes followed by members of the Arietellus-clade.