Paracaesicola nanshaensis n. gen., n. sp. (Monogenea, Microcotylidae) a gill parasite of Paracaesio sordida (Teleostei, Lutjanidae) from the South China Sea.

Paracaesicola n. gen., is erected herein to accommodate a new microcotylid species, Paracaesicola nanshaensis n. sp., collected from the Yongshu Reef, South China Sea. This species is the first monogenean to be recorded from the gills of Paracaesio sordida. The new species is characterized by the following features: (i) haptor short, with clamps arranged in two equal bilateral rows; (ii) testes numerous, arranged in two roughly alternating longitudinal rows, extending into the haptor; (iii) genital atrium armed with 16 robust spines, which are vertically arranged on top of the sausage shaped muscular male copulatory organ; and (iv) single vagina, bottle-shaped, with a distinctly bulbous vaginal atrium. The terminals of the reproductive system discriminate Paracaesicola n. gen. from all other genera in the Microcotylidae. Molecular phylogenetic analyses, based on partial 28S rDNA, places Paracaesicola nanshaensis n. sp. within the microcotylid clade, but its sequence differs from all known available microcotylid sequences.

Coral reefs are well known for their remarkable biodiversity. Many monogenean species have been described from the coral reefs of the Hawaiian Islands, the Great Barrier Reef, and New Caledonia [17,18,35,38]. The Nansha Islands of the South China Sea are a typical coral reef ecosystem, from which numerous coral reel fishes have been recorded [25]. However, the diversity of monogeneans in this region is poorly known. During an ongoing investigation of the monogenean fauna from the South China Sea, specimens of an undescribed species of Microcotylidae were collected from the Yongshu Reef. The morphological features of this new species are described herein, and a new genus is proposed to accommodate this new species.

Specimen collection and morphological analysis
Fishes were caught by line angling off the Yongshu Reef in the South China Sea from March to May, 2016. Parasites were collected and treated as described by Zhang et al. [41]. Twelve parasitic specimens were preserved in 70% ethanol for subsequent staining with acetic carmine, four were mounted in Berlese fluid for the morphological study of the hard parts, and two were digested with proteinase K (20 lg/lL) for 30 min at room temperature for the analysis of genital spines. The remaining specimens were fixed in 95% ethanol for DNA extraction. Illustrations were drawn with the help of an Olympus LB (Olympus Corporation, Japan), scanned, and processed with Photoshop CS4.0 (Adobe, USA). Specimens were measured using Olympus DP22 software. All measurement ranges are given in micrometres, followed by the mean and the number of specimens studied (n) in parentheses. The method for measurement of genital atrium spines is shown in Figure 1.

Sequences of monogeneans
Total genomic DNA was extracted using the TIANamp Marine Animals DNA Kit (Tiangen, China), following the manufacturer's instructions. We amplified the C1-D2 fragment of the 28S ribosome RNA subunit with PCR using previously published primer pairs (C1F: 5 0 -ACCCGCTGAATTTAAG-CAT -3 0 and D2R: 5 0 -TGGTCCGTGTTTCAAGAC -3 0 ) [11]. Each 25 lL PCR contained 12.5 lL Master Mix (2x), 7.5 lL ddH 2 O, 1 lL of each primer, and 3 lL genomic DNA. The PCR cycling conditions were initial denaturation at 94°C for 5 min; followed by 35 cycles of 94°C for 1 min, 56°C for 45 s, and 72°C for 1 min; and a final elongation at 72°C for 10 min. PCR products were confirmed by 1% agarose gel electrophoresis, and sequenced by the Sangon Biotech Company (Guangzhou, China). The sequences obtained were analyzed using DNAMAN 7.0 and Sequencher 5.0 (Gene Codes Corp.) software, compared to the GenBank database content with BLAST, and deposited in GenBank under accession number MH700264, with a final length of 881 bp.

Trees of monogeneans and distances
A tree was constructed from our new sequence of Paracaesicola nanshaensis n. sp. and 28S sequences of microcotylids available in GenBank; a sequence of Polystomoides asiaticus Rohde, 1965 was used as the outgroup. The dataset included 29 nucleotide sequences (Table 1). There were 855 positions in the dataset (including gaps). After estimating the best model with MEGA7 [22], the tree was inferred using the maximum likelihood (ML) method based on the general time reversible model with gamma distribution (GTR + G) in MEGA7 [31], with 1000 replications. The neighbour-joining (NJ) method was also used for comparison in MEGA7. Distances between sequences (Kimura-2 parameter distances) were computed from the same dataset with MEGA7 [19].
For trees, the neighbour-joining and maximum likelihood methods led to slightly different topologies. Here we present only the ML tree with Bootstrap support values at nodes (Fig. 2). The topology of the ML showed representative species belonging to different families (Microcotylidae, Heteraxinidae, Gotocotylidae, Protomicrocotylidae, Allodiscotylidae), while forming a monophyletic clade in their own family. Within the microcotylid clade, the new species P. nanshaensis clustered with Cynoscionicola branquialis AF382050 and Diplostamenides sciaenae FJ432589 to form a separate monophyletic subclade (68% bootstrap support in ML, 76% in NJ). However, members of Microcotylinae do not form an exclusive clade in their own subfamily. The molecular phylogenetic results supported P. nanshaensis n. sp. as a new taxon.      Etymology: The species is named after the locality, the Nansha Islands in the South China Sea.

Differential diagnosis
Based on the revision of Mamaev [29], we here assign the new species to the Microcotylinae by the following morphological features: a symmetrical haptor with microcotylid-type clamps, armed genital atrium, intestinal caeca unfused posteriorly, absence of terminal lappet and anchors, a pretesticular ovary in the form of a question mark, numerous testes, a single middorsal vagina. The new genus is erected herein primarily on the basis of the morphological features of terminal structures of the reproductive system: the sausage-shaped muscular MCO, the armed genital atrium, and the bottle-shaped vagina with distinctly bulbous vaginal atrium.
As suggested by Yoon et al. [39], genera within Microcoty- Microcotylinae, Paracaesicola n. gen. most closely resembles Atriostella Unnithan, 1971 as indicated by the armed genital atrium and unarmed MCO [39]. However, it can be distinguished from it by the haptor, the MCO, and genital spines [34]. The haptor of species of Atriostella spp. is slender taillike, slightly less than half total body length, the genital atrium is armed with usually long spines of dissimilar lengths, and the cirrus is roughly spheroidal with lobes. Moreover, the vagina of Atriostella spp. is unarmed. All these features are highly dissimilar to those in the new species described.

Discussion
Within Microcotylidae, the armatures of male and female terminalia are complex and diverse. Paracaesicola nanshaensis n. g. n. sp. is characterised by its sausage-shaped muscular male copulatory organ (MCO) and bottle-shaped vagina. Except for species in Microcotyloides Fujii, 1940 and Polynemicola Unnithan, 1971, the MCOs of most microcotylid species are conical in shape, bulbous, or not differentiated [9,29]. In addition, the unique arrangement of the genital spines, MCO, and vagina in P. nanshaensis n. sp. (closely clustered in a line, such that the vagina is located immediately posterior to the MCO, and the MCO is also immediately posterior to the genital spines) and the disposition of genital atrium spines (tightly gathered and standing vertically on top of the MCO) are not seen in the other microcotylid species (Fig. 4B). Moreover, in P. nanshaensis, testes extend well into the haptor. In all other microcotylid species with a separate haptor, there is little to no overlap between the haptor and the testicular field [23,36]. Morphological characters are, however, often affected by sampling and fixation conditions, as has been observed in studies of other species of Platyhelminthes [1,2]. Here, the samples placed directly in alcohol exhibited a strong contraction of the body, causing the testes to transform from a normal follicular shape into a long transverse strip. Machkewskyi et al. [26] identified seven measurements in microcotylids as independent of body length: pharynx length, genital atrium length, vitello-vaginal duct length, number of testes, number of clamps, length of the clamps, and width of clamp. The size of the haptor is determined by the number and the size of clamps. In P. nanshaensis, the haptor bears relatively few clamps (55-65). As a consequence, the haptor of P. nanshaensis is short and presents as an inverted equilateral triangle. Some microcotylid species have approximately the same or fewer clamps, as compared to P. nanshaensis (e.g., some species in Metamicrocotyla Yamaguti, 1953 [2,12,21,36]. However, the number of clamps in microcotylids varies greatly, even within the same genus. The erection of this new genus was further supported by our molecular phylogeny, which placed P. nanshaensis in the microcotylid clade, but with a high genetic difference from all other ingroup microcotylids. Our phylogenetic topology was congruent with previous phylogenetic studies that recovered Omanicotyle heterospina grouping with Bivagina pagrosomi, and further forming a clade with Microcotyle spp. [16,32,39]. This clade has a relatively close relationship with species in the subfamily Atriasterinae (Sparicotyle Mamaev, 1984, Pagellicotyle Mamaev, 1984 and Atrispinum Euzet & Maillard, 1974) [15]. However, Paracaesicola nanshaensis do not share a recent common ancestor with other members of Microcotylinae. Additionally, other members of the subfamily Microcotylinae do not form an exclusive clade. Diplostamenides Unnithan, 1971 and Caballeraxine Lebedev, 1972 (Microcotylinae), for example, group with Cynoscionicola Price, 1962 (Anchoromicrocotylinae Bravo-Hollis, 1981) and Metamicrocotyla (Metamicrocotylinae Yamaguti, 1963), respectively. This result suggests that the Microcotylinae maybe require division into smaller subfamilies. The phylogeny of the Microcotylidae is not well resolved, because the bootstrap values in some branches are low. Owing to the paucity of molecular sequences for species in the Microcotylidae, it is difficult to accurately determine phylogenetic relationships within its families. Therefore, sequence data for additional taxa are required.