Xarifiid Copepods (Copepoda: Cyclopoida: Xarifiidae) Parasitic in the Coral Psammocora columna Dana, 1846 from Taiwan

Simple Summary The coral reef is the crucial habitat for numerous marine creatures, and scleractinian corals are building blocks of this community. Therefore, the health condition of scleractinian corals is an essential factor for the sustainability of coral reef ecosystems. Recent studies indicate that global warming, seawater acidification as well as coral diseases are the main threats to scleractinian corals. In addition, coral endoparasites may impact the health of scleractinian corals by consuming coral tissues and potentially transferring disease pathogens. However, we have limited knowledge about the distributions of coral endoparasites across scleractinian corals. It may lead to the assessment bias on the health condition of the coral reef community due to no consideration on the impact of the interaction between corals and their endoparasites. Here, we performed an elaborate survey on a widely distributed scleractinian coral species, Psammocora columna, and discovered two new parasitic copepod species, Xarifia yanliaoensis and Xarifia magnifica. We also summarized a classification key of morphological characteristics for the identification of Xarifia copepods in Psammocora corals. The findings of this study present new records of copepod-coral relationships in the Indo-West Pacific for the biological resource database. Furthermore, these are the footstone knowledge for further studies on coral reef conservation. Abstract A comprehensive knowledge of relationships between coral and coral-associated organisms is essential for the conservation studies of the coral reef community, yet the biodiversity database of coral-inhabiting copepods remains incomplete. Here we surveyed in a widely distributed scleractinian coral, Psammocora columna Dana, 1846, and newly discovered two endoparasitic copepod species, Xarifia yanliaoensis sp. nov. and Xarifia magnifica sp. nov. These two new species are described based on specimens collected in Taiwan, and they share several common morphological characters of Xarifia copepods, i.e., region dorsal to fifth legs having three posteriorly directed processes unequally. However, X. yanliaoensis sp. nov. is distinguishable from other species by the morphology of the endopods of legs, antenna, maxilla, and maxilliped (in both genders). The morphological characters of X. magnifica sp. nov. are the endopods of legs, leg 5, and maxilliped in the male. Including the two new species described in the present work, the genus Xarifia Humes, 1960 belongs to the cyclopoid family Xarifiidae Humes, 1960 currently consists of 94 species, and eight of them live in association with the Psammocora coral. A comparison table and a key to the species of Xarifia from Psammocora corals are given herein.


Introduction
Parasitic copepods use a wide range of scleractinian corals as hosts [1]. Up to date, a total of 363 copepod species representing 99 genera, 19 families and three orders (Cyclopoda, Siphonostomatoida, and Harpactioida) have been recognized as parasites in 148 shallow-water stony corals [2]. The total included 288 cyclopoids, 68 siphonostomatoids, and seven harpacticoids. These coral-associated copepods exhibit a marked diversity in morphology in accordance with their respective ecological niches. Among the coralassociated copepods, Xarifiidae Humes, 1960 [3] is a family of endoparasitic copepods living in the gastrovascular cavities of coral polyps and more than 90 valid species have been discovered in Indo-West Pacific coral reefs [2,4,5]. It has been evident that their virulence may be related to their life history strategies, Symbiodinium density, surface area of host coral colonies, and concentration of nitrate and chlorophyll-a in the surrounding seawater. Therefore, the potential of using these parasites as bioindicators for predicting the future physiological performance of host corals in response to environmental change can be developed by tracking their abundance and species composition [2].
According to the previous studies carried out from 1967 to 2010 [4,[6][7][8][9][10], 15 species of parasitic copepods have been discovered from a widely distributed scleractinian genus, Psammocora Dana, 1846 [11] throughout the Indo-West Pacific, including three highly modified cyclopoids: Xarifia diminuta Humes and Ho, 1967 [6], Xarifia formosa Humes, 1985 [4], Xarifia imitans Humes, 1985 [4]. Recently, Cheng and Lin reported three new species of Xarifia, Xarifia conrepta Cheng and Lin 2021 [12], Xarifia gracilis Cheng and Lin 2021 [12], and Xarifia lata Cheng and Lin 2021 [12], parasitic in Psammocora digitata Milne Edwards and Haime 1851 [13] from Taiwan. Thus, among 92 Xarifia species recorded up to date, six of them have been discovered in Psammocora corals. All six species can be found in P. digitata, while X. diminuta also occurred in Psammocora contigua [14]. (Table 1). Obviously, the current knowledge about coral-associated copepods of the widespread Psammocora corals remains limited. There are ten valid species of Psammocora around the world [15], but only two species of Psammocora corals have been examined for coralassociated copepods. Psammocora columna Dana, 1846 [11] is a widely distributed coral and has not been examined for coral-associated copepods yet. Herein, we perform a survey in two colonies of P. columna collected from a shallow-water reef on the north coast of Taiwan (Figure 1), and describe two new species of xarifiid copepods. Combining our findings with previous records, 20 species of parasitic copepods including eight species of Xarifia have been found in Psammocora corals.

Specimen Collection
Several fragments of two colonies of Psammocora columna Dana, 1846 [11] were sampled by scuba diving on coral reefs in northern Taiwan (Figures 1 and 2). Coral fragments were placed in plastic bags underwater and transported to the laboratory. We adopted the standard methods from previous studies to collect copepod specimens [4,6,12]. Briefly, coral samples in sea water were placed in a 500 mL beaker and enough 95% ethyl alcohol was added to make an approximately 5% solution, which was left to sit for at least 4-6 h. Then, coral fragments were removed and the liquid with any sediment was poured through a fine net (approximately 100 µm mesh size). The copepods then were acquired from the sediment using forceps and preserved in 70% ethanol. Individuals were later cleared in 85% lactic acid for 1-2 h, then dissected on a wooden slide under a dissecting microscope [16]. The appendages were examined under a compound microscope using magnifications of up to 1000×. All drawings were made with the aid of a drawing tube.

Specimen Collection
Several fragments of two colonies of Psammocora columna Dana, 1846 [11] were sampled by scuba diving on coral reefs in northern Taiwan (Figures 1 and 2). Coral fragments were placed in plastic bags underwater and transported to the laboratory. We adopted the standard methods from previous studies to collect copepod specimens [4,6,12]. Briefly, coral samples in sea water were placed in a 500 mL beaker and enough 95% ethyl alcohol was added to make an approximately 5% solution, which was left to sit for at least 4-6 h. Then, coral fragments were removed and the liquid with any sediment was poured through a fine net (approximately 100 µm mesh size). The copepods then were acquired from the sediment using forceps and preserved in 70% ethanol. Individuals were later cleared in 85% lactic acid for 1-2 h, then dissected on a wooden slide under a dissecting microscope [16]. The appendages were examined under a compound microscope using magnifications of up to 1000×. All drawings were made with the aid of a drawing tube.  Humes, 1960. Genus Xarifia Humes, 1960. Xarifia yanliaoensis sp. nov. (Figures 3-5).
Spermatophore not observed. Etymology: The specific name magnifica, Latin adjective for "big" or "majestic" refers to the relatively strong element on the third segment of maxilliped.

Discussion
Based on the findings from this study, the genus Xarifia currently consists of 94 species and eight of them live in association with the Psammocora coral. The armature of endopods of legs 1-4 is the main diagnostic feature of Xarifia. The terminal segment of endopods of legs 1-4 of X. yanliaoensis sp. nov. is a unique character, armed with 1, 2, 0, 1 setae, respectively. After comparison with another valid species of Xarifia, we recognized that only Xarifia simplex Humes, 1985 [4] parasitic in the Scapophyllia cylindrica (=Merulina cylindrica (Milne Edwards and Haime, 1849) [17]) present the same armature of endopods of legs 1-4. However, the body surface of X. simplex covered with scattered small hairs (setules), while the body surface of X. yanliaoensis sp. nov. is smooth. The X. yanliaoensis sp. nov. differs from X. simplex by (1) the armature formula of the maxilla: three setae in X. yanliaoensis sp. nov. but two in X. simplex [4] (p. 561: Figure 49l); (2) the armature of terminal segment of antenna: I + 2 in X. yanliaoensis sp. nov., while I + 1 in X. simplex [4] (p. 561: Figure 49h); (3) the size of lobe or protuberance on the second segment of maxilliped: the protuberance in X. yanliaoensis sp. nov. is obviously bigger than that in X. simplex [4] (p. 562: Figure 50a,b) and its length is similar to the second segment of maxilliped. In addition, the maxilliped of male between these two species also showed some differences: with trifurcate tip and triangular process on the concave edge of the fourth segment in X. simplex [4] (p. 562: Figure 50j), but with bifurcate tip and 3 serrations on concave margin in X. yanliaoensis sp. nov. The other new species described herein, X. magnifica sp. nov., is similar to Xarifia anopla Humes and Dojiri, 1982 [18], Xarifia brevicauda Humes and Ho, 1968 [19], Xarifia filata Humes, 1985 [4], Xarifia hadra Humes and Dojiri, 1983 [20], Xarifia scutipes Humes and Dojiri, 1983 [20], Xarifia longa Cheng, Ho and Dai, 2007 [21], Xarifia capillata Cheng, Ho and Dai, 2011 [22], and Xarifia parva Cheng, Ho and Dai, 2011 [22] in the armature formula of the terminal endopodal segments of legs 1-4 (2, 2, 0, 0). Xarifia anopla, X. filata, X. hadra, X. longa, and X. parva can be excluded first because of their endopods of legs 1-4 exhibit only 1 segment. Xarifia capillata can be distinguished from X. magnifica sp. nov. by the region dorsal to fifth legs with a single central process which is tipped with tuft of setules [22] (p. 228: Figure 1A-C). Xarifia scutipes may be distinguished easily from X. magnifica sp. nov. in the shield-like leg 5 in the female [20] (p. 282: Figure 18a-c) and the claw of the maxilliped in the male with a large hyaline excrescence on the concave margin [20] (p. 285: Figure 21d,e). Xarifia brevicauda differs from X. magnifica sp. nov. by its abbreviated genital somite and abdomen [19] (p. 448: Figures 114-116), and by the serrated excrescence on the claw of the male maxilliped [19] (p. 449: Figure 130).
Eight species of Xarifia have been known using Psammocora corals as hosts (Table 1). We selected certain external features of Xarifia that are useful for the determination of species. Eleven features are shown in Table 2 and a useful key is provided as follows: However, this assessment could be feasible only if the interactions and ecological impacts between parasitic copepods and host corals are well recognized. In the present study, we have improved the understanding of species diversity of coral-associated copepods in the world. As accumulating knowledge of parasitic copepod and host coral relationships, we expect to reduce the stress of harms and diseases to host corals by applying biological control of parasitic copepods in the future.

Conclusions
To fulfil distribution information of xarifiid copepods in reef-building corals and enrich the completeness of the biological resource database of the Indo-West Pacific area, we studied Xarifia copepods in Taiwan for decades. So far, 22 species of Xarifia have been described as endoparasites of corals in Taiwanese waters. Although P. columna is a widely distributed coral, there is no available information on the parasite fauna of this coral species. In the present study, we examined several fragments of two colonies of this coral species for parasitic copepods and described two new species of Xarifia. Totally, 20 species of parasitic copepods including eight species of Xarifia are now known to be associated with Psammocora corals. Furthermore, we summarized a classification key of eight Xarifia species in Psammocora corals, which would be a handy index for a broader of coral reef researchers to identify copepod species by themselves. The discovery of so many species of parasitic copepods from Psammocora corals is unusual. Further studies to investigate whether the distinct morphological traits of these parasitic copepods might increase the fitness and allow them to adapt to their micro-niches inside the corals are required. Uncovering the phylogenetic relationships and interactions between these parasitic copepods and corals may enrich the fundamental knowledge to conservation biologists for assessments of health conditions of coral reef community, as well as the co-evolution between host-parasite relationships.  Institutional Review Board Statement: The ethical approval is not necessary to the research. This study addressed the taxonomy of two new species of parasitic copepods. The research project followed the regulations of experimental animals management and did not carry any potential harm and conflict to the study participants.