First record of Clausidium (Copepoda, Clausidiidae) from Brazil: a new species associated with ghost shrimps Neocallichirus grandimana (Gibbes, 1850) (Decapoda, Callianassidae)

Abstract A new clausidiid copepod was found living in galleries of ghost shrimps Neocallichirus grandimana (Gibbes, 1850) in Natal, Brazil. The new species resembles to Clausidium senegalense Humes, 1957 and Clausidium vancouverense (Haddon, 1912) in the armature of P2–P5 of the female, and shares with Clausidium senegalense similar segmentation and armature of the antenna and maxilla of the female. Nevertheless, it can be easily distinguished from its congeners by the unique characteristics observed in the antenna, maxilliped and first leg of males, as well as by the anal somite, maxillule and maxilliped of the females. This new species extends the group distribution to the Southwest Atlantic and represents the first record of the genus in Brazil. A key for the identification of the species based on females of Clausidium is provided.


introduction
Clausidiids characterized by the presence of sucking discs on endopods of legs 1 to 4, the genus Clausidium Kossmann, 1874 was established to accommodate C. apodiforme (Philippi, 1839). During the revision by Light and Hartman (1937), C. californiense Wilson, 1935 was considered a synonym of C. vancouverense, and at present this genus contains 10 species found in the Atlantic, Pacific and Indian oceans (Table 1). Wilson (1921) and Humes (1949) provided keys to species.
The representatives of Clausidium are typically external associates of marine decapods. They can be found inhabiting the burrows of ghost or mud shrimps of the families Callianassidae Dana, 1852 andUpogebiidae Borradaile, 1903 (Table 1).
Although Clausidium is reported in population studies of the ghost shrimp Callichirus seilacheri (Bott, 1955) from Chilean coast (Marin and George-Nascimento 1993;Hernáez and Wehrtmann 2007), these species remain unidentified. Clausidium searsi Wilson, 1937 and C. vancouverense (Haddon, 1912), collected along the Peruvian coast, are the only described species in South America.
A new clausidiid copepod, which can not be reconciled to any of the 10 species of Clausidium that have been described so far, was found living in galleries of ghost shrimps Neocallichirus grandimana (Gibbes, 1850) in the intertidal zone of a beach in Natal, state of Rio Grande do Norte (N.E. of Brazil). This is the first record of genus Clausidium in Brazil.

Methods
The copepods were recovered from water drawn from the burrows and collected from pleopods of the ghost shrimp Neocallichirus grandimana in the intertidal zone of a beach in Natal, state of Rio Grande do Norte, Brazil (5°45'S, 35°11'W).
Whole specimens were examined in temporary lactic acid mounts. Chips of cover slip were used to support the cover glass of the preparation. After examination, material was returned to and preserved in 70% ethanol. Dissections were made in glycerine and the dissected parts were placed on slides and sealed with Glyceel.
A Leitz Laborlux D ® phase-contrast microscope and a Zeiss Axioskop 2 Plus ® compound microscope equipped with differential interference contrast, digital camera Nikon Coolpix 995 ® and camera lucida were used to examine and prepare illustrations of the specimens.
Two females and two males were prepared for scanning electron microscopy (SEM). Specimens were dehydrated through a series of graded acetone; criticalpoint dried, mounted on stubs, sputter-coated with palladium and observed using a Philips XL 30 Field Emission Scanning Electron microscope (Philips, Eindhoven, Netherlands).
For confocal laser scanning microscopy (CLSM), a female was stained with Congo Red and mounted on slide following the procedure described by Michels and Büntzow  Series of stacks were obtained, collecting overlapping optical sections throughout the whole preparation; the optimal number of scans and the imaging settings according to the software, are given in Table 2. Final images were obtained by maximum projection, and CLSM illustrations were composed and adjusted for contrast and brightness using the software Adobe Photoshop CS4 (Adobe Systems, San José, U.S.A.).
The type material is deposited in the collection of the Museu de Zoologia, Universidade de São Paulo, São Paulo, Brazil.  -19, 32-38, 42, 45, 47, 48): Total length, excluding setae on caudal rami, 1.36-1.40mm (N=6). Body cyclopiform (Figs 1, 32-33), maximum width measured at posterior margin of cephalic shield. Prosome twice longer than urosome. First pedigerous somite fused with cephalosome. Body prosomites with minute integumental pits, sensilla and numerous pores distributed as illustrated in Fig. 1. Somites bearing P2-P3 subequal, both with latero-posterior margin sharply drawn out and posterior margin smooth. Somite bearing P4 trapezoid in form, longer than the two anterior somites combined, posterior margin with row of sensilla. Urosome (Figs 1-3, 32-35) 3-segmented, distinctly narrower than prosome. Urosome comprising fifth pedigerous somite, genital double-somite, and anal somite. Somite bearing P5 (Figs 1-2, 32-33) 1.4 times broader than long in dorsal view and with P5 arising ventrolaterally. Genital double-somite (Figs 1-3, 32-33) 1.3 times longer than broad, dorsal and ventro-lateral cuticular ridges marking plane of fusion between genital and first abdominal somite. Genital apertures (Fig. 2) located dorsolaterally on each side, near posterior margin of fifth pedigerous somite. Presence of pairs of pores near genital apertures and medial pore on dorsal view. Ventral surface with pores along medial region (Fig. 3). Egg sacs dorsolaterally located on each side, reaching posterior edge of anal somite and containing 13-15 eggs each. Anal somite (Figs 1-3, 32-33) well developed, formed by second to fourth abdominal somites fused in single somite; dorsal surface with well sclerotized leaf-like areas laterally displaced and intricate folders as illustrated in figures 2 and 34, clearly incised medially, posterior borders with pointed curved extensions on outer corners; almost quadrate in ventral view, with pointed posterior inner corners and fringed with membrane medially. Caudal ramus (Figs 2-5) about 3.5 times longer than wide, and armed with 6 setae. Seta I absent, setae II and III slender and naked; setae IV and V strongly developed and bipinnate, plumose on inner edge and spinulose on outer edge (seta V, 2.5 times longer than seta IV); seta VI the shortest; seta VII triarticulate and located at inner posterior corner, both naked. Caudal ramus with rounded lappet on posterior margin of ventral surface covering basal portion of setae III-V. Rostrum (Fig. 33) incorporated into cephalothorax, demarcated by sclerotized areas laterally; with pair of sensilla ventrally and pattern of pores as illustrated.
Maxilliped 39) well developed, strongly modified. Syncoxa with 2 pinnate setae. Basis with unequal denticulate projections and distal half of border curved and with irregular margin. Endopod 1-segmented; with strong serrate claw implanted near curved projection, and 1 small seta.
P1 (Figs 28,43) similar to female. Coxa and basis fused, with rows of stout spinules along proximal margin, row of long spinules near inner distal corner, long naked seta on outer edge and 2 pinnate setae on inner distal corner. Exp-1 and Exp-2 with 1 outer seta each, and row of spinules along outer margin. Exp-3 with row of denticles along outer margin, 3 outer setae (2 pinnate and 1 naked), 1 apical bipinnate seta and 2 inner bipinnate setae. Enp-1 with adhesive fringe along distal margin, stout curved process with adhesive fringe (Fig. 46) and long pinnate seta on inner distal corner. Enp-2 and Enp-3 as in female.
Etymology. The new species is named in honor of Prof. Dr. Sérgio de A. Rodrigues (Universidade de São Paulo) in recognition of his significant contributions to the taxonomy of Callianassidae and who kindly made available the studied material.

Discussion
Although the new species C. rodriguesi resembles C. senegalense and C. vancouverense in the armature of P2-P5 of the female, and shares with C. senegalense similar segmentation and armature of the female antenna and maxilla, it can be easily distinguished from its congeners by the unique characteristics observed in the males, -i. e., antenna with modified elements (enlarged seta on endopod-1 and spines on endopod-2 and endopod-3); maxilliped with distinct denticulate projections; and P1 coxobasis with 1 outer and 2 inner setae.
Other differential features refer to the morphology of the anal somite with sclerotized leaf-like areas and intricated folders dorso-laterally, posterior borders with pointed curved extensions on outer corners and clearly incised medially ; maxillule with apical outer lobe bearing 2 pinnate and 2 naked setae, as well as maxillular inner lobe bearing 2 pinnate and 1 naked setae; and female maxilliped with 1 pinnate seta and 1 pinnate spine on basis, and endopod-2 bearing 2 naked lateral setae and 4 pinnate apical elements (3 setae and 1 spine).
This new species, the first record of Clausidium in Brazil, not only extends the group distribution to the Southwest Atlantic but also enlarges the host list for the genus by adding N. grandimana (Gibbes, 1850).
A dichotomous key to the 11 valid species of Clausidium based exclusively on females is given below. Males differ from females by shape and size of the body, as well as by small differences in the segmentation and armature of antenna, setal formulae of legs, maxilliped strongly modified -adapted to grasp the female. In addition, many records of males are not well detailed. Thus, any identification of males must be verified against the best available description of the species.