A new species of Clausidium Kossmann , 1874 ( Crustacea , Copepoda , Cyclopoida , Clausidiidae ) associated with ghost shrimps from Iran

A new species of the Clausidium Kossmann, 1874 copepods is described, illustrated and provided by Confocal Laser Scanning Microscope photo on the basis of specimens from the gill chamber of Callianidea typa H. Milne Edwards, 1837 from the Iranian coast of the Persian Gulf. Clausidium persiaensis sp. nov is unique in the possession of a fi ne spine on endopod-1 of the antennae, large blade-like setae with an acute tip on P1, and the shape of P5 and the anal somite. Th e new species extends the group distribution into the northwest Indian Ocean and represents the fi rst records of the genus in Iran. key words CLSM, Persian Gulf, Indian Ocean, taxonomy, Callianidea typa.

A new species of Clausidium Nauplius, 25: 2017018 burrowing shrimps recorded from the littoral zone of Iran have associated copepods from the genus Clausidium Kossmann, 1874 (Clausidiidae, Cyclopoida).Although these clausidiid copepods are relatively rarely recorded because of the cryptic lifestyle of their hosts, a total of 11 species of Clausidium have been described so far (Kihara and Rocha, 2013).This genus has seven species recorded from the Atlantic Ocean and two species from the Pacific Ocean.The remaining two species, Clausidium travancorense Pillai, 1959 and Clausidium chelatum Pillai, 1959 have been found in the Indian Ocean (Pillai, 1959).In the present study, we investigate Clausidium copepods of Iranian coastal waters of the Persian Gulf, and describe a new species associated with the ghost shrimp Callianidea typa H. Milne Edwards, 1837.This new species extends the group distribution to the northwest Indian Ocean and represents the first record of the genus in Iran.

Material and Methods
Sampling was carried out at three localities along the Iranian coast of the Persian Gulf and Qeshm Island (Fig. 1).Clausidium persiaensis sp.nov.was collected from the gill chamber of Callianidea typa H. Milne Edwards, 1837 in three different sampling sites in the Persian Gulf and Qeshm Island.At the sampling site, the copepods were relaxed with drops of Menthol 1.5% added to the sea water and separated from the host by filtration through a 63 µm mesh size net.The collected specimens were transferred to 75% ethanol.Whole specimens were temporarily mounted on slides with glycerin, and adhesive plastic discs were used to support the coverslip (Kihara and Rocha 2013).Specimens were dissected under a Leica MZ12 stereomicroscope (Leica, Wetzlar, Germany).Dissected parts were mounted on slides using glycerin as mounting medium, and preparations were sealed with transparent nail varnish.The material was studied with a Leica DMR differential interference contrast microscope (Leica, Wetzlar, Germany) equipped with a drawing tube.The descriptive terminology follows Huys et al. (1996).Abbreviations used in the text are: ae, aesthetasc; P1-P5, legs 1-5; exp and enp, exopod and endopod, respectively; exp (enp)-1 (-2, -3), proximal (middle, distal) segments of a ramus.
The body length was measured from the anterior apex of the cephalothorax to the posterior margin of the caudal rami, excluding caudal setae.The type material is deposited in the collection of the Zoological Museum, University of Tehran (ZUTC).

Confocal laser scanning microscopy (CLSM)
For confocal laser scanning microscopy (CLSM), selected material was stained with 1:1 solution of Congo Red and Acid Fuchsin overnight.Whole specimens and dissected parts were mounted on slides with glycerin following the procedure described by Michels and Büntzow (2010).The material was scanned using a Leica TCS SP5 (Leica, Wetzlar, Germany) equipped with a Leica DM5000 B upright microscope (Leica, Wetzlar, Germany) and 3 visible-light lasers (DPSS 10 mW 561 nm; HeNe 10 mW 633 nm; Ar 100 mW 458 nm, 476 nm, 488 nm and 514 nm), combined with the software Leica Application Suite Advanced Fluorescence (LAS AF 2.2.1) (Leica, Wetzlar, Germany).To obtain a three-dimensional representation from selected body parts, the data produced during the CLSM scanning was processed with the free software Drishti (http://anusf.anu.edu.au/Vizlab/drishti/).       , 12C) biramous, both rami 3-segmented, and modified for prehension.Coxa and basis fused forming protopod with naked seta on outer corner near exopod insertion; large blade-like element on inner corner, with rounded projection along the outer margin, concentrically lines and acute apex.Exp-1 with outer seta.Exp-2 with 1 small seta.Exp-3 with 3 outer setae (proximal and distal ones reduced), 2 apical naked setae and 2 inner pinnate setae.
Enp-1 with 1 stout curved process with adhesive areas along the distal margin (marked with square in Fig. 4A).Enp-2 with a long seta (marked with asterisk in Fig. 4A).Enp-3 elongated, irregular segment
P2-P4 (Fig. 10B) with larger coxae and not so elongated basis when compared with female.Armature formula of P2-P4 as follows (Roman numerals representing spines, Arabic numerals representing setae): P5 (Fig. 7A) elongated, 7 times longer than wide and almost stright.Exopodal segment elongated with 2 serrate spines and naked seta along outer margin and 1 pinnate spine apically, spine I inserted near spine II, on the distal third of the segment.P6 (Fig. 7A) with 1 pinnate seta.
Etymology.The species name persiaensis is derived from the Latin meaning of or belonging to Persia referring to the provenance of the material.

discussion
There are a large number of different classification schemes proposed for the copepods in molecular, morphological or integrated approaches (Ho, 1990;1994;Huys and Boxshall, 1991).The phylogenetic relationships of the copepods are very controversial as Huys and Boxshall (1991)  Poecilostomatoida as a valid order.Boxshall and Hasley (2004) proposed 9 orders for copepods and Cyclopoida treated as an order that comprises all those families previously attributed to both the Cyclopoida and the Poecilostomatoida.It should be noted that Huys et al. (2012), by the use of molecular evidence, confirmed that the Poecilostomatoida order was not a monophyletic group, but rather it splits from the Cyclopoida.Hence this supports the decision to include all these in one order.
In the present study, we accept the concept discussed by Boxshall and Hasley (2004) and assign our new species to Cyclopoida.The generic characters of Clausidium are not completely clear as many species descriptions lack male specimens, and so depend heavily on female morphology.On the other hand, many species of Clausidium described many years ago are not well illustrated and nor well described (see Pearse, 1947;Wilson, 1932;Kensley, 1974).Following Boxshall and Halsey's (2004) key, the present new species is assigned to Clausidium by the small sucker on endopods of legs 1 to 4. It seems that the mentioned character is the most reliable homology for recognition and to use in a phylogenetic study.Furthermore, the following characters can be used as supplementary ones for defining Clausidium: body oval and flattened, prosome comprising cephalothorax and 3 free pedigerous somites, urosome comprising 5 or 6 segments, antennule 7-segmented, antenna 4-segmented, legs 1 to 4 biramous, first pair highly modified, fifth leg uniramous and 2-segmented.
Clausidium persiaensis sp.nov. shares with C. travancorense the armature formula of legs 2 to 4, but can be easily distinguished from its congener by the unique characteristics observed in the female's antenna with 2 strong pectinate spines on endopod 2, as well as in the armature and swimming legs with elongated basis.Another differential feature observed in males is the maxilliped with distinct projections.The most obvious differentiation between the two species is in the shape of the body, P1 and P5.In a dorsal view of C. persiaensis sp.nov., the shape of the females body is suboval and the anterior margin is narrower than the posterior one, and the third pedigerous somites are 2.6 as wide as long.In contrast, C. travancorense is more flattened, slightly narrowing posteriorly, the posterior margin tapers to a rounded apex, and the third pedigerous somite is 1.2 as wide as long.The P1 in C. persiaensis sp.nov.with a blade like process acute tip (versus blunt tip) and Exp2 of P1 with 1 setae (versus 2 setae) is also diagnostic.The shape and armature of the P5 are different between the two species.The free exopodal segment of C. persiaensis sp.nov. is wide, with a curved proximal part in the general outline, rather than being elongate and slender in C. travancorense.The P5 in C. persiaensis sp.nov.also has 3 serrate spines along the outer margin and 1 serrate spine apically (versus 3 setae along outer margin and 1 seta apically).
Clausidium persiaensis sp.nov.further shares with Clausidium rodriguesi Kihara and Rocha, 2013 the armature of P2 -P5, and maxilla and mandible, but is distinguished from the later by the free exopodal segment of P5, which is elongated about 2 times longer than wide and with 4 serrate spines (versus  3 times longer than wide with 3 serrate spines and 1 naked seta).The basis of the syncoxa has a large serrate process, bearing 3 setae (1 pinnate, 1 serrate and 1 naked) and 1 pinnate spine (versus 2 pinnate, 1 naked setae and 1 pinnate spine).The basis of the maxilliped has 1 naked and 1 pinnate seta (versus 1 pinnate seta and 1 spine), and the endopod is 2-segmented; first segment armed with 1 naked seta; second segment A new species of Clausidium Nauplius, 25: 2017018 bearing 1 naked lateral seta, 3 stout distal spines and 1 naked inner seta (versus endopod being 2-segmented; first segment unarmed; second segment bearing 2 naked lateral setae, 3 pinnate distal setae and stout distal spine).
The present species agrees with the female description of Clausidium maximus Hwang, Lee & Kim, 2016 in some aspects, which are: the armature formula of legs 1 to 4, maxilla, mandible and caudal ram.Clausidium persiaensis sp.nov. is easily defined from that species by the following characters: P5 in the later species having 4 serrate spine (versus 4 naked setae), segment 2 of the antennal endopod with 2 pectinate spines and 2 setae (versus 4 setae); segment 3 with 6 apical setae (versus 7 setae).Also, there are some differences between the male specimens of the above species that includes: free exopodal segment of P5 in Clausidium persiaensis elongated with armed with 3 serrate spines with 1 naked seta (versus 5 setae).
Early researchers studying Clausidium assumed some species were symbiotic and associated of ghost shrimps (Corsetti and Strasser 2003;Kihara and Rocha 2013), although some species were thought to be parasitic (Kossmann, 1874;Wilson, 1937;Pearse, 1947;Humes, 1949;Pillai, 1959).This complex interaction is a potentially important aspect of ghost shrimp biology.Because of the influential role that ghost shrimps play in aquatic systems, Clausidium copepods may have indirect effects on local communities and ecosystem processes via their direct effects on ghost shrimps.

Final
plates were composed and adjusted for contrast and brightness using the software Adobe Photoshop CS4 (Adobe Systems, San José, U.S.A.).