Two new species of the genus Caligus (Crustacea, Copepoda, Siphonostomatoida) from the Sea of Japan, with a note on the establishment of a new species group

Abstract Two new species of Caligus are described from the Japanese coast of the Sea of Japan. Caligus chinglonglinisp. nov. is based on a male specimen found in a plankton sample, whereas C. kajiisp. nov. was collected from the body surface of the host flathead Platycephalus sp. These two new species can be assigned to a distinct species group, the pseudorhombi group newly named and defined by the morphology of the genital complex in both sexes, and by the structure and armature of legs 2 and 4. The species group so far accommodates 19 species including these two new species. The morphology, host specificity and zoogeography of the species group are discussed herein and keys to species groups of Caligus and to species of the C. pseudorhombi species group are provided.


Introduction
Members of the genus Caligus Müller, 1785 are known as sea lice and several species are known to cause serious economic losses in marine fish farming facilities (Ho and Lin 2004b;Johnson et al. 2004, Costello 2009Shinn et al. 2015a, b). The life cycle of sea lice and their host specificity have been the subject of intensive studies designed to develop methods of controlling these pests. The life cycles of caligid copepods have been shown to be more diverse than expected. The general pattern is for a species to utilize a single host for all post-copepodid stages, after infection of the host fish (Ho and Lin 2004b;Dojiri and Ho 2013). A less common type of life cycle is found in the so-called "planktonic caligids" such as Caligus undulatus Shen & Li, 1959, C. ogawai Venmathi Maran, Ohtsuka & Shang, 2012and C. ilhoikimi Suárez-Morales & Gasca, 2016, which exhibit a dual mode of life with adults found both on the host fish and free in the water column (Shen and Li 1959;Ho and Lin 2004a;Venmathi Maran and Ohtsuka 2008;Venmathi Maran et al. 2012a, b, 2016Suárez-Morales et al. 2012a, b; Suárez-Morales and Gasca 2016; Kim et al. 2019). A third life cycle pattern is found in species which conduct host switching after the final molt of the chalimus phase and require both an intermediate and a final host (Hayward et al. 2009;Ohtsuka et al. 2018). The third type is rare and has so far been recorded only in three species infecting farmed fish (Hayward et al. 2008, Ohtsuka et al. 2018, although Cressey and Cressey (1980) suspected that the adults of Caligus biseriodentatus Shen, 1957 occurred on different host species from the immature stages.
An undescribed species of Caligus was found in a plankton sample collected at Ashibe Port, Iki Island, Nagasaki Prefecture, Japan on May 24, 2014. Another undescribed species was found infecting the flathead Platycephalus sp. caught off Shimonoseki City, Yamaguchi Prefecture, Japan in the Sea of Japan in 2016. Since these two undescribed species belong to a distinct species group of Caligus, they are described together in the present paper, together with remarks on taxonomy, host specificity and distribution of members of the species group.

Materials and methods
A single male specimen of Caligus was found in a plankton sample collected by towing a small plankton net around an underwater fishing light (KU-5MB, Koto Electric Co., Ltd.) at Ashibe Port, Iki Island, Nagasaki Prefecture, Japan (33°48.54'N, 129°45.231'E) during the night-time of May 24, 2014. This becomes the holotype of a new species. A second undescribed species was found infecting the body surface of the flathead Platycephalus sp. (total length 58 cm) caught by fishing off Shimonoseki City, Yamaguchi Prefecture, Japan (34°00.686'N, 130°53.756'E) in the morning of August 24, 2016. The copepods were fixed in 70% ethanol immediately after capture. After immersing the copepod specimens in lactophenol, these were examined using Humes and Gooding's (1964) slides on a differential interference microscope (BX-53, Olympus Co., Ltd.) equipped with a drawing tube. Body lengths were measured from the frontal margin of the cephalothorax to the posterior margin of the caudal ramus excluding the caudal setae. Protists epibiontic on the undescribed caligid collected from Iki Island were photographed with a digital camera (DP21, Olympus Co., Ltd.) attached to the microscope. Terminology essentially follows Ho and Lin (2004b).

Taxonomy
Armature and elements of legs 1-4 as in Table 1. Leg 1 ( Fig. 2A) with massive protopod bearing 1 inner and 1 outer small plumose setae plus bifid setule on outer margin; intercoxal sclerite slender, unornamented; endopod reduced to club-shaped process located near base of exopod; exopod 2-segmented, first segment with row of fine setules along inner margin and 1 naked seta at outer distal corner, second segment with 3 large plumose setae along inner (posterior) margin and 4 elements terminally, middle two of which each bearing accessory process. Leg 2 ( Fig. 2B-D) with intercoxal sclerite ornamented with trapezoidal marginal membrane along posterior margin; coxa with large plumose seta at posterior corner and minute setule on anterior surface; basis ornamented with marginal membrane on both inner and outer edges, bearing 1 minute seta on knob at outer distal corner ( Fig. 2C) plus setule near midpoint of inner margin; endopod 3-segmented, outer margins of second and third segments with dense patches of minute setules; exopod 3-segmented, first segment with long outer spine directed obliquely across surface of second segment, second segment with relatively short outer spine, third segment with small outer knob (arrowed in Fig. 2D), 1 reduced outer spine, 1 short terminal spine and 5 plumose setae increasing in size from apical to innermost. Leg 3 ( Fig. 2E-G) apron (protopod) without surface processes, bearing well developed inner seta and 1 minute outer seta, plus 2 relatively long setules along posterior margin; outer basal margin of apron undulating; endopod 2-segmented, proximal segment small, with 1 long plumose seta; velum developed, hirsute along free posterior margin; second segment expanded along outer margin (Fig. 2F); exopod 3-segmented ( Fig. 2G), proximal segment small, with slightly curved outer spine not reaching distal border of next segment, middle segment with 1 inner plumose and 1 outer naked seta, third segment with 3 spiniform setae increasing in size distally plus 4 inner setae.
Leg 4 (Figs 2H, I, 3A) with protopod bearing low outer prominence at mid-length and minute plumose seta at outer distal corner; exopod distinctly 2-segmented, first exopodal segment with long outer spine almost fused basally to segment and reaching more than half distance to origin of proximalmost outer spine on compound second segment; second segment with 1 terminal and 2 slender spines on distal margin plus lateral spine, plus 2 bifurcate processes terminally; each process complex, with 1 or 3 minute prominences basally (Fig. 2I).

Female. Unknown.
Remarks. The new species is most closely related to C. acanthopagri Ho, Lin & Chen, 1994, C. dieuzeidei sensu Shiino (1954b, and C. latigenitalis Shiino, 1954 in general appearance and in the structure of the appendages and sternal furca. As Izawa and Choi (2000) and Ho and Lin (2004b) suggested, the minor but most distinct dif- ference can be found in the structure of the pectens of the second exopod segment of leg 4 among these three species. Those of C. chinglonglini sp. nov., C. dieuzeidei sensu Shiino (1954b) (see Discussion), and C. latigenitalis are sharply indented, whereas that of C. acanthopagri is composed of a hyaline membrane. The former three species can be distinguished by the number and shape of dentate processes (divided into 3 or 5 prominences but not hand-like in C. chinglonglini; 3 or 4 and hand-like in C. dieuzeidei sensu Shiino; 4 or 5 and hand-like in C. latigenitalis). In addition, the shape and numbers of processes of the maxillipedal myxal area differ among the males of these three species. In C. chinglonglini sp. nov. and C. latigenitalis, there are 3 processes arrayed along the myxal margin, but the middle process is furnished with serrated tips in the former but is rounded in the latter. In C. dieuzeidei sensu Shiino (1954b), there are only two processes, one quadrangular and the other low triangular, present along the margin.
Although the present new species is described on the basis of a single male, no other species belonging to the newly proposed pseudorhombi species group (see Discussion) has so far been recorded from Japanese waters except for C. latigenitalis (Nagasawa et al. 2010) in which only females were originally described by Shiino (1954a) and subsequently both sexes were redescribed in detail by Izawa and Choi (2000). These two species are distinguishable as mentioned above. In addition, C. bifurcus Shen, 1958, assigned to the same species group was described from Chinese waters based only on two females, but the non-sexually dimorphic characteristics such as sternal furca and legs differ distinctly from those of C. chinglonglini sp. nov. Therefore, the establishment of the present new species is justified.
The new species is the fourth species of Caligus found exclusively from plankton samples in Japan (see Venmathi Maran et al. 2016, table 2).
Peritrich ciliates were attached along the posterior margin of both maxillipeds ( Fig.  3B, C) and on the ventral side of the cephalothorax. Epibiont suctorian and peritrich ciliates have already been recorded from species of Caligus and Lepeophtheirus von Nordmann, 1832 (Stone and Bruno 1989;Gresty and Warren 1993;Fernandez-Leborans et al. 2005). In L. salmonis (Krøyer, 1837) collected from Hokkaido, Japan, the peritrich Epistylis sp. attached mainly to the antennae and legs 2 and 3. This is the first record of the occurrence of epibiont peritrich ciliates on "pelagic caligids" (Ho and Lin 2004a;Venmathi Maran and Ohtsuka 2008;Venmathi Maran et al. 2012a, b, 2016. Etymology. The new species is named in honor of the late Dr Ching-long Lin who made a great contribution to the taxonomy of parasitic copepods together with Prof. Ju-shey Ho.  Description. Female. Body length of holotype 6.16 mm, 4.86-6.16 mm in holotype and female paratypes (mean ± standard deviation = 5.49 ± 0.32 mm, N = 38). Dorsal cephalothoracic shield subcircular, almost as long as wide (Fig. 4A). Lunules (Fig. 4A) relatively small. Pediger 4 almost completely fused to genital complex. Genital complex (Fig. 4A) subquadrate, about 1.14 times longer than wide, produced posteroventrally into pair of rounded processes between which paired copulatory pores located (Fig. 4B). Spermatophores (Fig. 4B) attached to copulatory pores via fine tubules; spermatophore proper globular, ca. 0.12 mm in diameter. Paired egg strings of holotype female containing 22 and 24 eggs. Abdomen (Fig. 4A, B) 1-segmented, about as long as wide. Caudal ramus (Fig. 4A, B) furnished with rows of setules along inner and outer margins; seta II minute, located near base of seta III on subterminal ventral surface, seta III subterminal, setae IV-VI terminal, well developed, seta VII minute, located at inner distal corner.
Leg 2 (Fig. 5D, E) with intercoxal sclerite bearing semi-circular marginal membrane along posterior margin; coxa with large plumose seta at posterior corner and setule on anterior surface; basis ornamented with marginal membrane along both inner and outer edges and long setule near midpoint of inner margin; armed with minute seta at distal outer corner; rami 3-segmented; first endopod segment with notch bearing tuft of setules, second segment furnished with row of setules along outer margin, third segment with tuft of setules near base of proximal outer seta; first exopod segment with anterior marginal membrane reflexed dorsally over segment surface and long, stout outer spine directed obliquely across surface of second segment; second segment with smaller outer spine than in first segment; third segment armed with 1 reduced spine (arrowed in Fig. 5E) and 1 moderate-size outer spine. Leg 3 (Fig. 5F-H) apron (protopod) with no distinct ornamentation on surface, armed with 1 long inner seta and 1 small outer seta terminally; endopod 2-segmented, proximal segment reduced, velum developed, decorated entirely with row of setules along free posterior margin; distal segment with outer margin expanded and hirsute Fig. 5G; exopod 3-segmented, first segment with strong, slightly curved, inwarddirected outer spine not reaching distal margin of second segment, second segment with expanded, hirsute outer margin and 1 minute outer seta (arrowed in Fig. 5H), third segment with 3 small spines plus 4 inner setae.
Leg 4 (Fig. 5I, J) protopod slightly shorter than exopod, bearing minute plumose seta at outer distal corner; exopod 2-segmented, with pecten at base of each exopodal spine; first exopodal segment bearing spinulose spine reaching nearly half of distance to proximalmost outer spine on second segment; innermost terminal spine more than 3 times longer than middle spine. Leg 5 (Fig. 4B) represented by small outer knob bearing protopodal seta and inner (exopodal) knob bearing 2 setae.
Remarks. The female of the new species most closely resembles Caligus bifurcus, C. musaicus Cavaleiro, Santos & Ho, 2010, C. pectinatus Shiino, 1965, C. pseudorhombi Boxshall, 2018, C. pterois Kurian, 1949 andC. xystercus Cressey, 1991. All these species share a 2-segmented exopod on leg 4 armed with 4 spines on the distal exopodal segment, the female genital complex is nearly as long as wide and about twice as long as the abdomen, and the abdomen is about as long as wide. However, the present new species is distinguished from these species by the combination of the following characteristics: (1) the genital complex is as long as wide (cf. wider than long in C. bifurcus, C. musaicus and C. pterois; slightly longer than wide in C. xystercus); (2) the genital complex is about 2.1 times longer than the abdomen (cf. 1.2 times longer in C. bifurcus; 2.2 in C. musaicus; 2.1 in C. pseudorhombi; 1.6 in C. pterois; 3.6 in C. xystercus); (3) the corpus of the maxilliped lacks processes (cf. ridge-like process present in C. pseudorhombi and C. pterois); (4) the tines of the sternal furca taper distally (cf. uniform in width and with a truncate tip in C. pectinatus); (5) the terminal exopod segment of leg 1 is furnished with 3 large spines terminally (2 in C. pseudorhombi); and (6) the maxillipedal subchela is more than half the length of the corpus (cf. much shorter in C. musaicus).
In males, the new species is most similar to C. musaicus, C. nuenonnae Andrews, Bott, Battaglene &Nowak, 2009, C. pterois, andC. priacanthi Pillai, 1962. These five species share the following characteristics: (1) the genital complex is laterally expanded and produced into 2 posterolateral protuberances representing leg 5, armed with 1 (outer lobe) and 2 setae (inner lobe); (2) the abdomen is completely or incompletely 2-segmented and shorter than the genital complex; (3) the maxillipedal corpus is well developed and carries anteriorly-produced processes on the myxal surface. However, the new species is easily distinguishable from these congeners by the combination of the following features: (1) the maxillipedal corpus has a rounded process on the posterior surface (absent in the other species); (2) the sternal furca has pointed tines which are widely separated (tines that are close at base and with rounded tips in C. priacanthi; rounded in C. nuenonnae); (3) the mxyal surface of the maxilliped carries 3 large, rounded processes along the margin, (cf. the processes are different in shape and number in the other species); (4) the posterior dentiform process of the maxillule lacks a surface ornamentation of minute prominences (present in C. nuenonnae).
Etymology. The new species is named in honor of the late, supremely talented carcinologist Tomonari Kaji who passed away in May 2019.

Discussion
Five named species groups were recognized within the genus Caligus by Boxshall (2018) but as part of the justification for establishing C. pseudorhombi Boxshall, 2018 as a new species, he informally recognized an additional distinct species group. This unnamed species group was diagnosed on the basis of female morphology. The following features are shared by species within the group: (1) the exopod of leg 4 is 2-segmented and the compound distal segment carries 4 spines; (2) the genital complex of the female is as long as wide, without posterolateral lobes, and about twice as long as the abdomen; and (3) the abdomen is about as long as wide (see Boxshall 2018). An additional characteristic which we identify here relates to leg 2: the proximal spine on the outer margin of the third exopodal segment is markedly reduced and the adjacent distal spine is also relatively small in almost all members of the species group for which information on leg 2 is available. The males of the species group are defined for the first time as follows: (1) leg 4 is as in the female; (2) the genital complex is subquadrate with legs 5 and 6 located close together at the posterolateral corner; (3) the abdomen is 1-or, typically, 2-segmented; and (4) the myxal surface of the maxilliped has 1 to 3 pointed or rounded processes (except for C. longirostris Hewitt, 1964). We recognize that the following 19 species can be included in this species group: C. acanthopagri (♀♂ known), C. bifurcus (♀), C. buechlerae Hewitt, 1964 (♀♂), C. chinglonglini sp. nov. (♂), C. dieuzeidei Brian, 1932 (♀♂), C. hobsoni Cressey, 1969, (♀♂), C. kajii sp. nov. (♀♂), C. latigenitalis (♀♂), C. ligatus (♀♂), C. longirostris (♀♂), C. musaicus (♀♂), C. nuenonnae (♀♂), C. olsoni Pearse, 1953 (♀♂), C. pectinatus (♀), C. pseudorhombi (♀♂), C. priacanthi (♀♂), C. pterois (♀♂), C. similis Ho, Kim & Nagasawa, 2005 (♀♂), and C. xystercus (♀). Unfortunately for C. olsoni, no information is available on leg 2. This species group is newly named as the pseudorhombi group, partly because it was first pointed out when C. pseudorhombi was originally described by Boxshall (2018), and partly because both sexes of the species were described in detail by Boxshall (2018).
Both sexes of C. dieuzeidei were described by Brian (1932) based on material collected in the Mediterranean, but this species has not been found since the original description. This species has been recorded by Shiino (1954bShiino ( , 1959Shiino ( , 1960. According to Izawa and Choi's (2000) direct observations of Shiino's material: his "C. dieuzeidei" from Acanthopagrus schlegeli (Shiino 1954b) was identical with Caligus latigenitalis, as already pointed out by Lin et al. (1994), and his material of "C. dieuzeidei" from Siganus fuscescens (Houttuyn, 1782) (Shiino 1959) was Caligus oviceps Shiino, 1952 (Izawa andChoi 2000). Shiino's (1960) specimens of "C. dieuzeidei" collected from two elasmobranchs were identified as an unknown congener, although the evidence was not presented. Excluding Shiino's (1960) C. dieuzeidei, a total of 19 species, including the two new species described herein can be assigned to this species group. Although C. dieuzeidei Brian, 1932 and C. latigenitalis were not listed as members of the species group by Boxshall (2018), it is clear that leg 4 and the genital complex and abdomen of the female could fall within the diagnosis (see Brian 1932, Shiino 1954a, b, Izawa and Choi 2000. Unfortunately, leg 2 was neither figured nor mentioned in the text by Brian (1932) in his original description of C. dieuzeidei, so the configuration of the spines on leg 2 exopod cannot be confirmed.
The hosts and geographical distributions of the members of the newly recognised species group are summarized in Table 3. The host fish for the species group vary widely and include both pelagic and benthic taxa. The host specificity seems to be relatively low in C. acanthopagri, C. hobsoni, C. ligatus, and C. xystercus, but may be higher in other species. Four members of the species group most frequently utilize the family Sparidae as hosts: C. acanthopagri, C. dieuzeidei, C. latigenitalis and C. xystercus. Two species are associated with each of the following host families: Atherinidae (C. ligatus, C. olsoni), Aulostomidae (C. ligatus, C. xystercus), Pomacanthidae (C. hobsoni, C. xystercus), and Priacanthidae (C. priacanthi, C. xystercus). This is the first record of the occurrence of a species (C. chinglonglini sp. nov.) belonging to the species group in plankton samples. According to Venmathi Maran et al. (2016), 11 species of Caligus were found exclusively from plankton samples. Of these pelagic caligids, only C. adunctus is assigned to a species group, the C. macarovi group, while the remaining species have not as yet been classified into the five groups defined by Boxshall (2018).
Males of 16 species belonging to the C. pseudorhombi group are known, including the present two new species. With the exception of C. buechlerae, these males can be divided into two sub-groups on the basis of the morphology of the genital complex: in one sub-group, both of the anterior and posterior knobs representing leg 5 are produced posteriorly, and leg 6 (genital operculum) is armed with 1 (or rarely 2) small setae terminally; whereas in the other sub-group only the posterior (exopodal) knob is distally produced, and leg 6 has 2 (or rarely 3) setae terminally. The first sub-group consists of C. kajii sp. nov., C. musaicus, C. nuenonnae, C. pseudorhombi, C. priacanthi, and C. pterois. The second sub-group comprises C. acanthopagri, C. chinglonglini sp. nov., C. dieuzeidei, C. hobsoni, C. latigenitalis, C. ligatus, C. longirostris, C. olsoni and C. similis. Members of the first sub-group are widely distributed in the Indo-Pacific and the Atlantic, whereas the second sub-group is restricted to the Pacific (Kurian 1949;Pearse 1953;Shen 1958, Heegaard 1962Hewitt 1964;Lewis 1967;Shiino 1965;Cressey 1969;Pillai 1985;Izawa and Choi 2000;Ho and Lin 2004b;Ho et al. 2005;Andrews et al. 2009;Cavaleiro et al. 2010;Boxshall 2018; present study) (see Table 3).