Five new coexisting species of copepod crustaceans of the genus Spaniomolgus (Poecilostomatoida: Rhynchomolgidae), symbionts of the stony coral Stylophorapistillata (Scleractinia)

Abstract Spaniomolgus is a symbiotic genus of copepods of the poecilostomatoid family Rhynchomolgidae and is known to be associated with shallow-water reef-building hermatypic corals. Three species of this genus were previously found only in washings of Acropora and Stylophora in northern Madagascar. Four coral morphotypes of Stylophorapistillata (Pocilloporidae) were collected by SCUBA at 1 to 28 m depth in five sites in the Saudi Arabian Red Sea in 2013. Copepods found on these colonies were studied using light, confocal and scanning electron microscopy. Five new, and one known, species of the genus Spaniomolgus were discovered in washings and inside the galls of the hermatypic coral S.pistillata. The description of these new species (Spaniomolgusglobussp. n., S.stylophorussp. n., S.dentatussp. n., S.maculatussp. n., and S.acutussp. n.) and a key for the identification of all of its congeners is provided herein.


Introduction
Rhynchomolgidae Humes and Stock, 1973 is one of the largest families of poecilostomatoid copepods comprising over 250 species living in association with various marine invertebrates (Ho and Kim 2001;Boxshall and Halsey 2004). There are 44 genera in the family Rhynchomolgidae with the genus Doridicola Leydig, 1853 being the largest in the family and comprising 52 species (Ho andIvanenko 2013, Walter andBoxshall 2018). Thirty-eight genera of the family include only up to six species. One of these small genera, Spaniomolgus Humes & Stock, 1973, consists of three species: the type species S. compositus (Humes & Frost, 1964), S. geminus (Humes & Ho, 1968) and S. crassus (Humes & Ho, 1968), all previously attributed to the genus Lichomolgus Thorell, 1859. Spaniomolgus are found in association with scleractinians of the genera Acropora Oken, 1815, Seriatopora Lamarck, 1816, and Stylophora Schweigger, 1820 from Madagascar (Humes and Ho 1968, Humes and Stock 1972, 1973. There have been no records of Spaniomolgus since the revision of the lichomolgoid complex Stock 1972, 1973) and until the discovery of an unidentified species of Spaniomolgus living in modified polyps (galls) of Stylophora pistillata Esper, 1797 in the Red Sea (Ivanenko et al. 2014, Shelyakin et al. 2018. Branching corals of Stylophora pistillata are widely distributed around the Indo-Pacific and are phenotypically plastic, i.e., morphological variation across different habitats, depths, and geographic regions can be observed. The latest study based on seven DNA loci demonstrated that Stylophora corals from the Red Sea belong to a single molecular clade, and that morphospecies of Stylophora pistillata, S. danae Milne Edwards & Haime, 1850, S. subseriata (Ehrenberg, 1834), and S. kuehlmanni Scheer & Pillai, 1983 from the Red Sea are now considered as synonyms of S. pistillata (Arrigoni et al. 2016).
This paper describes five new species of Spaniomolgus living in symbiosis with four morphotypes of Stylophora pistillata from the Red Sea. Comments on the relationships with other congeners are given, and a key to the species of the genus Spaniomolgus is presented.

Materials and methods
The sampling was undertaken in accordance with the policies and procedures of the King Abdullah University of Science and Technology (KAUST). Permissions for KAUST to undertake the research were obtained from the appropriate governmental agencies of the Kingdom of Saudi Arabia.
Four colonies of Stylophora pistillata from the Thuwal reefs in the central Red Sea and one colony from the reef close to Al Lith in the southern Red Sea were sampled (distance between the sampling locations is about 280 km) (Fig. 1, Table 1). The map was created using Python scripts (Jones et al. 2001), labels were included using the software Adobe Photoshop CS4 (Adobe Systems, San Jose, CA, USA). The coral colonies were collected using a hammer and chisel, and encased in sealed plastic bags while snorkeling and SCUBA diving at depths ranging from 1 to 28 m. The coral samples were rinsed on board as follows: 96% ethanol was added to each sample until the overall solution reached a concentration 10% to relax the animals attached to the coral. After 15 minutes, the samples were shaken, and the water with the detached symbionts was filtered through a 100 μm sieve. Copepods were sorted under a Carl Zeiss™ Stemi 2000-C stereomicroscope. Coral colonies were also examined for copepods in modified corallites and galls. Galls were dissected, and copepods were extracted from inhabited polyps using entomological needles and preserved in 96% ethanol.
In the lab, copepods were dissected in lactic acid and then stained with Chlorazol black E (Sigma C-1144) for contrast enhancement (Ivanenko and Defaye 2004). Specimens were then examined as temporary mounts in lactophenol and later sealed with Entellan as permanent mounts. The coral hosts (Fig. 2) were bleached in sodium hypochlorite for 48 h, rinsed with fresh water, dried and photographed. The copepods were kept in 2 mL vials in 96% ethanol with a small drop of glycerol.
For confocal microscopy, exoskeletons were individually transferred to distilled water and then stained with Fuchsin (Ivanenko et al. 2012;Corgosinho et al. 2018).  Table 1). The copepods were inspected using an inverted Nikon A1 confocal laser scanning microscope (CLSM, Nikon Corporation, Tokyo, Japan) at Lomonosov Moscow State University, using a 40× oil immersion objective and lasers with wavelengths of 532 and 640 nm. The laser power was set to 60%. The amplitude offset and detector gain were manually adjusted. CLSM image stacks were obtained throughout the whole animal, and the scanning software was adjusted to perform the optimal number of scans. Image size was set for 2000×2000 dpi and the reconstruction of the external anatomy was obtained by maximum projection. The final images were adjusted for contrast and brightness using the software Adobe Photoshop CS4. All figures were prepared using a Leica DM5500B differential interference microscope equipped with a camera lucida. The armature formula of swimming legs 1-4 follows Sewell (1949), spines are indicated by Roman numerals and setae by Arabic numerals. Mean body length (MBL) of copepods was measured from the anterior margin of the rostrum to the posterior margin of the caudal rami.
For scanning electron microscopy (SEM), copepods were dehydrated through increasing ethanol concentrations, critical point dried, mounted on aluminium stubs, coated with gold, and examined in a CamScan SEM (CamScan Electron Optics Ltd, London, UK) at the Faculty of Biology of Lomonosov Moscow State University. The bleached fragments of corals were mounted on metal stands using glue, coated with a conductive gold film and examined with the same SEM.
Type specimens of copepods are deposited in the collection of the Zoological Museum of Lomonosov Moscow State University (ZMMU). The coral hosts are deposited in the collection of King Abdullah University of Science and Technology (KAUST).

Results
Five new and one described species of the genus Spaniomolgus were found in washings and inside of polyps of four morphotypes of the hermatypic coral Stylophora pistillata collected from five sites (  (Humes & Frost, 1964), by original designation. (Humes & Ho, 1968), S. crassus (Humes & Ho, 1968), S. globus sp. n., S. stylophorus sp. n., S. dentatus sp. n., S. maculatus sp. n., S. acutus sp. n. Etymology. The specific Latin epithet globus, globe, refers to the body shape in life when the urosome forms an s-shaped flexure.
Urosome s-shaped when alive, with the genital double-somite drawn forward under the metasome and the postgenital somites in line with the prosome (Fig. 3a); 5-segmented, comprising fifth pedigerous somite, genital double-somite, and three free abdominal somites (Fig. 3b). In dorsal view, only the postgenital somites are visible. Leg 5-bearing somite bell-shaped, slightly wider than long.

Antenna
Mandible (Fig. 3g). Basal region with a rounded hyaline expansion and a distal row of small teeth on inner margin, and a fringe of setules on the outer margin. Terminal lash long, denticulated.
Maxillule ( Fig. 3e) a single segment with a small seta and three hyaline prolongations (seemingly not articulated), one of them ornamented with setules.
Legs 1-4 ( Fig. 4a-d) with 3-segmented rami except for 2-segmented leg 4 endopod. Inner coxal seta long and plumose in legs 1-3, short and naked in leg 4. Outer basal seta short and naked in all legs. Endopod of leg 4 reaching beyond middle of third exopodal segment; with two terminal spines unequal in length, outer 32 μm long, inner 55 μm long, the latter spines with hyaline. Outer spines on leg 4 exopod with smooth lamellae. Armature formula as follows:
Sixth leg (Fig. 3b) represented by two very small articulated spines near attachment of eggs sacs.
Male unknown.
Etymology. The specific name from the Latin dentatus, refers to the denticulated margin of the maxillipedal claw.
Description. Adult female. Body cyclopiform, with oval cephalothorax and cylindrical urosome (Fig. 5a). Body length 750 μm and maximum width 390 μm. Prosome comprising cephalothorax and three free pedigerous somites. Second and third pedigerous somites with slightly rectangular epimeral areas. Fourth pedigerous somite smaller than preceding ones, its epimeral areas much less expanded.
Antenna (Fig. 5c) 3-segmented; first segment 53 μm long with small terminal hyaline seta; second segment 68 μm long with seta medially; third segment 60 μm long with three hyaline setae medially and two apical hyaline setae, small recurved terminal claw 24 μm long. Second and third segments measured along inner margin subequal in length.
Leg 4 ( Fig. 5e) with 3-segmented exopod and 2-segmented endopod. Inner coxal seta and outer basal seta naked. Endopod reaching beyond middle of third exopodal segment; second segment with two apical spines unequal in length, outer 30 μm long, inner 50 μm long, the latter spines with hyaline and weakly serrated margins. Outer spines of exopod with barbed lamellae.
Fifth leg (Fig. 5b) with protopod incorporated into somite; outer basal seta not observed. Free segment long, slender and recurved, 4.2 times as long as wide, bearing two apical setae unequal in length, inner most about twice as long as outer one.
Sixth leg (arrowed in Fig. 5b) represented by two very small articulated projections near attachment of eggs sacs.
Male unknown.  Description. Adult female. Body cyclopiform; oval cephalothorax slightly pointed on top and cylindrical urosome (Fig. 6a). Mean body length 710 μm (with range of 700 -720 μm) and mean maximum width 315 μm (with range of 270 -360 μm), based on two specimens. Prosome comprising cephalothorax and three free pedigerous somites. Second pedigerous somite with epimeral area slightly angular and third pedigerous somite with epimeral area rounded. Fourth pedigerous somite smaller than preceding ones, almost invisible in dorsal view.
Antenna (Fig. 6c) 3-segmented; first segment 45 μm long with small hyaline apical seta; second segment 87 μm long with one hyaline seta medially; third segment 55 μm long with two hyaline setae medially, and one apical hyaline seta, with small recurved terminal claw 22 μm long. Length ratio of second to third segments (measured along inner margin) 1.7:1.
Maxilliped (Fig. 6d) 3-segmented; first segment unarmed; second segment robust, with two naked inner setae; third segment claw-like, with two setae medially equal in length; apex with pore.
Leg 4 (Fig. 6e) with 3-segmented exopod and 2-segmented endopod. Inner coxal seta short and naked, outer basal seta short and plumose. Endopod reaching beyond middle of third exopodal segment; with two distal spines unequal in length, outer 30 μm long, inner 50 μm long, the latter spines with hyaline and weakly serrated margins. Outer spines of exopod with smooth lamellae.
Fifth leg (Fig. 6b) with protopod incorporated into somite; outer basal smooth seta short. Free segment long, slender and recurved, 7.6 times as long as wide, bearing two apical setae unequal in length, inner most about twice as long as outer one.
Male unknown.
Etymology. The specific Latin epithet acutus, pointed, refers to the pointed epimeral areas of the second and third pedigerous somites.
Antenna (Fig. c) 3-segmented; first segment 48μm long with small terminal hyaline seta; second segment 60 μm long, with similar seta medially; third segment 76 μm long, with two hyaline setae medially, and two apical hyaline setae, with small recurved terminal claw 20 μm long. Length ratio of second to third segments (measured along inner margin) 1:1.2.
Leg 4 (Fig. 7e) with 3-segmented exopod and 2-segmented endopod. Inner coxal seta and outer basal seta short and naked. Endopod reaching tip of third exopodal segment, with two apical spines unequal in length, outer 39 μm long, inner 52 μm long, the latter spines with hyaline and smooth margins. Outer spines on leg 4 exopod with smooth lamellae.
Fifth leg (Fig. 7b) with protopod incorporated into somite; outer basal seta smooth. Free segment long, slender and recurved, 9.3 times as long as wide, bearing two apical setae unequal in length, inner most 3.6 times the length of outer one.
Sixth leg (Fig. 7f ) represented by two very small articulated projections near attachment of eggs sacs.
Male unknown. Etymology. The specific epithet stylophorus refers to the host name Stylophora. Description. Adult female. Body cyclopiform, with oval cephalothorax and cylindrical urosome (Figs 8a, 9b). Mean body length 1.15 mm (with range of 1.1 -1.2 mm) and mean maximum width 365 μm (with range of 320 -410 μm), based on two specimens. Somite bearing leg 1 completely separated from cephalosome. Epimeral areas of metasomal somites slightly angular. Fourth pedigerous somite smaller than preceding ones, its epimeral areas not visible in dorsal view.
Antenna (Fig. 8c) 3-segmented; first segment 80μm long with small terminal hyaline seta; second segment 115 μm long with a seta medially; third segment 78 μm long with three hyaline setae medially, and two apical hyaline setae, with small recurved terminal claw 30 μm long. Length ratio of second to third segments (measured along inner margin) 1.5:1. Maxilliped (Fig. 8d) 3-segmented; first segment unarmed; second segment robust, with two naked inner setae; third segment claw-like, with two setae medially equal in length; apex with pore.
Leg 4 (Fig. 8e) with 3-segmented exopod and 2-segmented endopod. Inner coxal seta and outer basal seta short and naked. Endopod reaching beyond middle of third exopodal segment, with two apical spines unequal in length, outer 38 μm and inner 70 μm, the latter spines with hyaline and serrated margins. Outer spines of exopod with smooth lamellae.
Leg 5 (Fig. 8b) with protopod incorporated into somite; outer basal seta naked. Free segment long, slender and recurved, 5.0 times as long as wide, bearing two apical setae unequal in length, inner most more than twice the length of outer one.

Taxonomy
Designation of the genus Spaniomolgus Humes & Stock, 1973 was based on three previously known species of Lichomolgus copepods associated with scleractinian corals: the type species S. compositus, S. geminus, and S. crassus from northern Madagascar Frost 1964, Humes andHo 1968). The finding of five new species and S. crassus in the Red Sea is the first record since 1968. Although Spaniomolgus is a rather homogenous genus, there are differences among its eight species. The body has a broadened and thickened prosome in S. crassus and S. globus, but it is moderately widened, and the epimeral areas of the second and third pedigerous somites are slightly rectangular or angular in S. stylophorus, S. geminus, S. compositus, S. dentatus, S. maculatus, and S. acutus. Another key character to separate the species of Spaniomolgus is the body organization. For example, the first pedigerous somite is clearly set off from the cephalosome in S. crassus and S. stylophorus, incompletely separated from the cephalosome by an indistinct furrow in S. geminus, S. compositus, and S. globus, and completely fused to the cephalosome in S. dentatus, S. maculatus, and S. acutus.
The antennules are very similar in all eight species, with the only difference being the presence of an extra seta in the sixth segment in S. globus, S. stylophorus, S. dentatus, S. maculatus, and S. acutus.
The maxillules of S. globus, S. stylophorus, S. dentatus, S. maculatus, and S. acutus are represented by a single segment bearing a small seta and three hyaline prolongations without evident articulation. However, according to Humes and Frost (1964) and Humes and Ho (1968), the maxillule shows four hyaline prolongations without articulation in S. geminus, S. compositus, and S. crassus. The condition of the maxillulary projections of the latter three species needs to be reassessed because the articulation of one of these elements was probably overlooked.
As for the maxilliped, small interspecific differences in the third claw-like segment were detected. The margin of the claw has three very small subterminal spinules in S. geminus, S. compositus, and S. crassus, but it is smooth and with an apical pore in S. stylophorus and S. maculatus. The distal half of the claw's margin is denticulated in S. globus and S. dentatus; but with as single subapical tooth in S. acutus.
The armature of the legs is the same for the eight species; only the ornamentation of the fourth leg varies among the species. The exopodal spines have barbed lamellae in S. geminus, S. compositus, S. dentatus, S. maculatus, and S. acutus, but they are smooth in S. crassus, S. globus, and S. stylophorus. With respect to the terminal spines of the second endopodal segment, they are hyaline and smooth in S. acutus and S. crassus, but serrated in S. stylophorus, S. dentatus, S. maculatus, S. compositus, and S. geminus. In S. globus the outer terminal spine is serrated and the inner one is smooth.
The genital double-somite, generally rather narrow, can be present in three different shapes. In S. crassus, S. compositus, and S. geminus it is wider in its anterior third than in its posterior two-thirds; it is longer than wide with almost parallel margins in S. dentatus, S. maculatus and S. acutus, and completely square and bell-shaped in S. globus and S. stylophorus (wider in its posterior part).
The fifth leg in all species shows a long, slender and recurved segment of exopod with two apical setae. The length:width ratio of the free segment varies among the species, it is 10.5 times as long as wide in S. geminus, 9.3 times in S. acutus, 7.9 times in S. compositus, 7.6 times in S. maculatus, 6.7 times in S. globus, 6.3 times in S. crassus, 5.0 times in S. stylophorus, and 4.2 times in S. dentatus. Noteworthy, the outer basal seta of is minute in S. globus and has not been observed in S. dentatus.
The length:width ratio of the caudal rami, characteristically elongated in all the species, is also variable. The caudal rami are 9.1 times as long as wide in S. geminus, 5.0 times in S. compositus and S. maculatus, between 4.0 and 4.5 times in S. globus, S. stylophorus and S. dentatus, 3.7 times in S. acutus, and 2.8 times in S. crassus. The eight species present six terminal setae that are characteristically short and naked, except for S. acutus in which the dorsal seta has not been observed.

Hosts
Spaniomolgus compositus found by Humes and Frost (1964) in washings of Stylophora subseriata, and Spaniomolgus crassus and S. geminus reported by Humes and Ho (1968) from washings of Stylophora mordax (Dana, 1846) should be now considered as cooccurring symbionts of one coral host, Stylophora pistillata. We assume that the coral indicated by Humes and Frost (1964) as Seriatopora subseriata is actually Stylophora subseriata (Ehrenberg, 1834) as the name Seriatopora subseriata is not valid. Thus, all eight species of Spaniomolgus reported in the present paper are now considered as associates of a single host species, Stylophora pistillata.

Ecological comments
The scleractinian coral Stylophora is considered to be one of the main Indo-Pacific reef-framework builders and is one of the dominant species in shallow-water reef environments exposed to strong wave action (Veron 2000). Stylophora pistillata hosts a great variety of copepods, including highly transformed xarifiids, which live in the gastrovascular cavities of the polyps. These symbiotic copepods were first noticed by Dr. Sebastian A. Gerlach during the Xarifa Expedition to the Red Sea and the Maldives Archipelago in 1957-1958(Humes 1985a. Since then, copepods of three different orders have been found in association with this scleractinian coral: one species of Harpacticoida, Alteuthellopsis corallina Humes, 1981 (Peltidiidae, ectosymbiotic), three species of Siphonostomatoida, Asteropontius corallophilus Stock, 1966, A. magnisetiger Kim, 2010, Gascardama longisiphonata Kim, 2010, and seven species of Poecilostomatoida (Stock 1966, Humes 1981, Kim 2010. Among these poecilostomatoid copepods, five endosymbiotic species belong to the family Xarifiidae, Xarifia decorata Humes & Ho, 1968, X. dissona Humes, 1985, X. lissa Humes & Ho, 1968, X. obesa Humes & Ho, 1968, and X. lissa Humes & Ho, 1968, and three ectosymbiotic species belong to the family Rhynchomolgidae, S. crassus, S. compositus, and S. geminus (Humes and Frost 1964, Humes and Ho 1968, Humes 1985b. Though coral-associated copepods have been studied for a considerable period of time, there remains a scarcity of data on their biology and ecology (Humes 1994, Ho 2001, Cheng et al. 2016. Relationships between copepods and their hosts remain poorly studied due to the microscopic size of these crustaceans making in situ observations difficult. There are only few studies that include information about the interactions between copepods and corals (e.g. Ivanenko et al. 2014, Shelyakin et al. 2018. Recent experiments by Cheng and Dai (2009) showed the ability of xarifiid copepods to get inside of the polyp of S. pistillata and to stay there as a symbiont. These copepods can make a polyp open its mouth either by releasing specific chemicals which induce feeding behaviour or act as muscle relaxants. However, it is still unclear which mechanism is actually utilized. It is also unknown if other coral species may be infected in a similar manner. Gall-inducing copepods are another example of coral hosts being affected by copepods. These copepods appear to attach to the soft tissues of the coral, and by disturbing it with their swimming legs, elicit the defence mechanism of a coral to grow a calcareous barrier (Dojiri 1988, Ivanenko et al. 2014. The multifocal purple spots syndrome of sea fans, which was thought to be caused by a fungous pathogen, appears to be induced by endoparasitic copepods sitting in the tissue outgrowths (Ivanenko et al. 2017).
It is often unclear whether copepods should be classified as parasites, because of the absence of rigorous experimental documentation. If we want to study copepod-coral relationships, it is crucial to know which copepod species are involved in symbiosis and what is their effect on the host. Therefore, it is important to provide detailed descriptions as well as identification keys for all copepod species associated with corals, so species composition and abundance of copepod communities can be tracked and used as a bioindicator for environmental changes and coral health (Ho 2001, Zeppilli et al. 2015, 2018. Moreover, most of the symbiotic copepods depend entirely on the well-being of their hosts, and with the loss of corals during the recent bleaching events, many species of copepods associated with these corals could disappear, some even before being described. For instance, reefs close to Al Lith in the central Red Sea, where some of our samples were collected, were severely affected by the major bleaching event of 2015-2016 (Monroe et al. 2018, Osman et al. 2018). Most of the colonies of S. pistillata at the Al Lith reefs and about 20% of colonies at the Thuwal reefs were bleached and died (Monroe et al. 2018, Osman et al. 2018, personal observations of V.N. Ivanenko and S.V. Mudrova in May 2017). Therefore, abundance and diversity of copepods could have also been strongly affected, and some of the species collected from the reefs near Al Lith may already be gone from this region.