Tigriopus iranicus sp. nov., a new species of Harpacticidae (Copepoda, Crustacea) from Iran, with a redescription of T. raki Bradford, 1967

Abstract The first representative of Tigriopus Norman, 1869 from the north-western Indian Ocean is described from rock pools on the Iranian coast. Tigriopus iranicussp. nov. is distinguishable from its congeners by i) the possession of two maxillary endites, each with two setae; ii) a two-segmented mandibular endopod; iii) P1enp-3 with one pinnate claw, a well-developed geniculate spine and a small seta; and iv) female P6 with two setae. Additionally, we present a complete redescription of Tigriopus raki Bradford, 1967 on the basis of paratype material and a key to the species of the genus.


Introduction
The genus Tigriopus was introduced by Norman (1869) based on a specimen of T. lilljeborgi Norman, 1869(currently accepted as T. fulvus (Fischer, 1860) with strong claws on the P1, from Shetland, on the coast of Scotland. Tigriopus brevicornis (Müller, 1776) (= Cyclops brevicornis Müller, 1776), from the intertidal zone of the Danish coast, is without any doubt one of the oldest described members of the genus. Lang (1948: 311) claimed that the first member of the genus to be described is that illustrated by Ström (1765: 590, pl. IX, figs 1-10) and later described as Cyclops brevicornis by Müller (1776). Lang's claim (1948) is based on Ström's (1765) illustrations and his explanation of the habitat of the specimen, which was found in rocky pools but not in the open sea. The type species of Tigriopus, Harpacticus fulvus Fischer, 1860, which was originally described from Madeira, was reallocated to Thalestris Claus, 1863 by Claus (1863). Morphological similarities between T. brevicornis and T. fulvus (e.g. five setae on the female P5 baseoendopod, the male P5 with four setae on the exopod and one seta on the baseoendopod) caused Lang (1948: 340) to synonymize the nominotypical T. fulvus, T. fulvus var. adriatica Douwe, 1913 from Rovinj (Croatia) (Douwe 1913), and T. fulvus var. algirica Monard, 1936 from Algeria (Monard 1936) with T. brevicornis; only T. brevicornis was thought to be present in Europe until 1960. Moreover, a variety of T. fulvus, northumbriensis, was recorded from Scotland by Mistakidis (1949). Upon the inspection of micromorphological characters, Bozic (1960) recognized two European species: the northern, T. brevicornis, and the Mediterranean, T. fulvus, and Carli and Fiori (1977) supported the separation of these two species based on few morphological differences. Some years later, Soyer et al. (1987) observed the presence of five groups of species based on their geographic distributions and on some morphological similarities. Recent genetic studies on the Mediterranean population of T. fulvus revealed the presence of a single species with a remarkable biogeography (Vecchioni et al. 2019).
Identification of Iranian species based on Wells (2007) and comparison with the description and illustrations of T. raki suggested that the Iranian specimens might be T. raki. However, the great distance between New Zealand and Iran and totally different ecological factors of these two areas made it doubtful the Iranian material was T. raki. Therefore, the paratype material of T. raki (NIWA [National Institute of Water and Atmospheric Research] 1610 P-33) was obtained and reinspected for a more precise identification of the Iranian specimens. Closer examination revealed important differences between T. raki and the Iranian material.
During an investigation on the intertidal copepod fauna of the Persian Gulf and the Oman Sea, a new member of the genus Tigriopus was discovered. Herein, we describe a new species of Tigriopus, T. iranicus sp. nov., which was found in rock pools in the Persian Gulf and the Oman Sea.
The description of some characters of T. raki were omitted in the original description, and a complementary redescription of the species is provided herein.

Material and methods
The studied material was collected from rock pools in the Persian Gulf and the Oman Sea during a short-term research project in 2016 on the harpacticoid fauna of Iran. Collected specimens were preserved in 96% ethanol for future investigation. One male and one female were stained in a 1:1 solution of Congo Red and Acid Fuchsin for 24 h (Michels and Büntzow 2010). These materials were scanned using a Leica TCS SP5 equipped with a Leica DM5000 B upright microscope and 3 visible-light lasers (DPSS 10 mW 561 nm; HeNe 10 mW 633 nm; Ar 100 mW 458, 476,488, and 514 nm), in combination with the LAS AF 2.2.1 software (Leica Application Suite Advanced Florescence). Confocal Laser Scanning Microscopy images were obtained applying 561-nm excitation wave-length with 80% acousto-optic tunable filter. The acquisition resolution was 2048 × 2048. Final images, gained by maximum projections, were composed and adjusted for contrast and brightness using Adobe Photoshop CS6.
Whole male and female specimens were used for the illustration of the dorsal and lateral views of the habitus. The material was then dissected using a Leica MZ12 stereomicroscope for a detailed description of mouth parts and appendages. Dissected appendages were mounted on permanent slides with glycerin as mounting medium and sealed with a mixture of honeybee wax and paraffin. Pencil drawings of dissected parts were prepared with a Leica DMR differential interference contrast microscope equipped with a drawing tube at a magnification of 1000×. Digital inking was done using Adobe Illustrator CS6. The type material was deposited in the collection of the Senckenberg Gesellschaft für Naturforschung (Frankfurt/ Main, Germany). The descriptive terminology follows Huys and Boxshall (1991) and Schminke (1976). Abbreviations used in the text: A1, antennule; A2, antenna; ae, aesthetasc; P1-P6, first to sixth swimming legs; enp, endopod; exp, exopod; enp-1, 2, 3, proximal, middle, distal segments of endopod; exp-1, 2, 3, proximal, middle, distal segments of exopod. Type locality. Rock tidal pool on the coast of Iran, Jask, Vanak, 25°32'5"N, 58°52'12"E.

Systematics
Differential diagnosis. With marked distinction between prosome and urosome. P1-bearing somite fused to cephalothorax. Female antennule nine-segmented; sevensegmented and chirocerate in male. Mandible with two naked setae on basis, palp with two-segmented endopod and exopod. Maxilla with two endites, each with two setae. P1 enp-3 with one claw, one spine, and one naked seta. P1 exp-3 with five welldeveloped claws. Endopodal lobe of female P5 with four pinnate setae. Female P6 with two setae. Inner seta of the male P2 enp-2 incorporated to the segment creating curved, pinnate process. Male P5 baseoendopods fused, forming a continuous plate, endopodal lobe without armature, exp with four elements.
Description. Female. Total body length 630 µm, measured from tip of rostrum to posterior margin of furcal rami. Prosome four-segmented (Figs 1A-C, 2A, B), consisting of cephalothorax and three free pedigerous somites. First pedigerous somite bearing P1 fused to cephalosome. Cephalothorax and pedigerous somites smooth, furnished with sensilla; hyaline frills smooth without spinules or denticles. Rostrum (Fig. 3A) well developed, as long as first segment of antennule, bell-shaped, with two pairs of sensilla near apical and lateral margins. Urosome (Figs 1A-C, 2A, B) five-segmented, comprising fifth pedigerous somite, genital double-somite, two free abdominal somites and telson. Genital double-somite completely fused dorsally and ventrally, subdivided laterally by internal chitinous rib. Urosome ornamented with row of spinules laterally. Genital double-somite and first free abdominal somite clothed with sensilla dorsally. First and second free abdominal somites with posterior row of spinules ventrally. Telson with spinular rows ventrally and laterally, with two sensilla on dorsal distal margin. Anal operculum semicircular, smooth ( Fig. 2A).
Antenna (Fig. 3C). Three-segmented, composed of coxa, allobasis, and one free endopodal segment. Coxa without ornamentation. Allobasis ornamented with row of spinules and long setules proximally, abexopodal seta bipinnate. Free endopodal segment ornamented with row of spinules close to insertion site of proximal lateral spine, and with frill and minute spinules on outer distal corner; with two pinnate spines on lateral margin, one pinnate spine, four geniculate and two tiny naked setae distally. Exopod three-segmented; first segment longest with two pinnate setae; second segment with one pinnate seta; last segment with one pinnate seta incorporated into segment, and one tiny bare seta with spinules at its base.
Labrum (Fig. 4A). Broad and well developed, triangular in lateral view, ornamented with setules along outer margin.
Mandible (Fig. 4B). Coxa with well-developed gnathobase. The latter with six blunt and two multicuspidate teeth and one pinnate seta. Basis rectangular with two smooth setae. Both exopod and endopod two-segmented. Enp-1 with three naked setae laterally, two of them fused basally; enp-2 with five naked setae on distal margin. Exp-1 with two smooth setae on inner margin and spinules on outer margin; exp-2 with three naked setae.  (Fig. 4C, D). Arthrite of praecoxa with one ventral pinnate spine, tiny apical spine, two pinnate and four naked apical spines, and two smooth surface setae, ornamented with spinules on posterior surface, and with few lateral spinules medially. Coxal endite (Fig. 4D) cylindrical, with three naked setae, with spinules subdistally. Basis with two lateral smooth setae, and one pinnate and three smooth distal setae. Endopod one-segmented, cylindrical, with three naked setae. Exopod one-segmented, with four naked setae. (Fig. 4E). Syncoxa bearing one praecoxal and one coxal endite, each armed with two setae, with two lobes (probably the remnant of endites) between syncoxal endites, with spinules on praecoxal endite, with some medial and some distal spinules on syncoxa. Allobasis prolonged into a strong pinnate claw, with one anterior naked seta and one posterior slender smooth, and one strong bipinnate seta. Endopod one-segmented, carrying two naked setae.

Maxilla
Maxilliped (Fig. 3D). Syncoxa with spinule patch on outer margin and with distal spinule row; with one naked inner distal seta. Basis with strong spinules on inner margin and one naked seta. Endopod drawn out into a developed pinnate claw carrying a process with two smooth setae.
Swimming legs P1-P4 (Figs 5A, B, 6A, B) biramous, each ramus three-segmented and well-ornamented with spinules, exopod longer than endopod. Setal formula as follows: 0-0 I-0 I-1; I-1; III, II,2 0-1; 0-1; I,2,1 Leg 3 0-0 1-0 I-1; I-1; III, II,2 0-1; 0-1; I,2,1 Leg 4 0-0 1-0 I-1; I-1; III, II,3 0-1; 0-0; I,2,1 P1 (Fig. 5A). Intercoxal sclerite rectangular, longer than wide and smooth. Praecoxa and coxa ornamented with spinular rows on distolateral anterior surface. Basis bearing one inner and one outer spinulose flagellate spine, with rows of spinules on anterior surface as shown. Endopod approximately as long as first exopodal segment; enp-1 longest, with one long inner seta inserted at two-thirds length of segment, ornamented with setules and spinules as depicted, with spinule row on inner and outer margins; enp-2 wider than long, unarmed, with few large outer spinules; enp-3 with one strong curved pinnate claw, one spine and one tiny smooth seta at base of the claw, with outer spinular row. Exp-1 longest, bearing one pinnate flagellate outer spine distally, with row of outer spinules; Exp-2 ornamented with setules and spinules as shown, with one outer pinnate flagellate spine at two-thirds length of segment, and one inner subapical pinnate seta; exp-3 shortest, armed with five clawlike spines. P2 (Fig. 5B). Intercoxal sclerite smooth, longer than wide and curved. Coxa triangular, with proximal and outer subdistal spinular rows, with distal frill on anterior surface, and with pore near inner margin. Basis with spinules at base of outer spine and at base of exopod, with inner distal frill and pore close to insertion site of exopod, with outer flagellate pinnate spine. Endopodal segments with strong outer spinules; enp-1 and -2 with one inner seta ornamented with setules proximally and with small spinules distally; enp-3 with one spinulose and two pinnate setae, and one blunt pinnate spine. Exopodal segments with strong outer spinules; exp-1 and exp-2 with anterior distal frill, with one outer naked spine and one inner pinnate seta; exp-3 with three naked outer spines, one outer distal element ornamented as shown and one inner distal pinnate seta, and two pinnate inner setae. P3 (Fig. 6A). Coxa with spinules on outer distal corner and frill on inner distal border. Basis with strong outer spinules, with smooth outer seta. Segments of both rami with spinules on outer margins. Enp-1 and enp-2 armed with one long inner seta ornamented as depicted; enp-1 with, enp-2 without inner distal frill; enp-3 with three pinnate setae and one unipinnate spine. Exp-1 and exp-2 with anterior frill distally, with one outer pinnate spine and one inner seta ornamented as figured; exp-3 with three outer pinnate spines, one outer and one inner distal element as shown, and two inner setae. P4 (Fig. 6B). Coxa with anterior frill distally and with outer spinules. Both exopodal and endopodal segments ornamented with outer spinules. Enp-1 with frill on anterior distal margin, bearing one inner pinnate seta; enp-2 unarmed; enp-3 with one pinnate spine and three pinnate setae. Exp-1 with distal frill and outer pore on anterior surface, with one pinnate outer spine and one pinnate inner seta; exp-2 with anterior distal frill, with pinnate outer spine and pinnate inner seta; exp-3 with three outer pinnate spines, one outer and one inner distal element, and three inner setae of which medial spinulose. P5 (Fig. 7B). Exopod and baseoendopod separate; ornamented with spinules as illustrated; baseoendopod bearing smooth outer basal seta; endopodal lobe with four pinnate setae, with one tube-pore near innermost seta and two pores on anterior surface; exopod rectangular, with five pinnate setae and one pore on anterior surface. P6 (Fig. 7A). One-segmented, bearing one naked and one pinnate seta. Genital field (Fig. 7A). Situated in the middle of the genital double-somite, with one median genital pore at the boundary between genital somite and first abdominal somite. Male ( Fig. 8A-C). Habitus as in female except for genital somite separated from first abdominal somite. Total body length 685 µm measured from tip of rostrum to posterior margin of furcal rami. Sexual dimorphism expressed in antennule, antenna (the latter as in female, but without abexopodal seta), P2, P5 and P6.
Etymology. The specific epithet iranicus refers to the country where the new species was found. It is in the nominative singular. Gender masculine.  Bradford, 1967 Material examined. One female (SMF 37261/1-13) and one male (SMF 37262/1-10) paratype (NIWA 1610 P-33) dissected and mounted on slides, and three females and two males preserved in ethanol (SMF 37263).
Redescription. Female. Total body length 560 µm, measured from tip of rostrum to posterior margin of furcal rami. Few sensilla scattered on body surface (Fig. 12A, B).
Furcal rami (Fig. 12C) slightly wider than long; with outer and distal spinulose rows. Lateral seta I smooth, implanted in middle of ramus. Seta II smooth, longer than seta I, located subdistally. Seta III smooth, displaced ventrally, subdistal. Setae IV and V pinnate, located on distal margin. Seta VI smooth inserted on distal, inner corner. Seta VII tri-articulated and smooth, dorsally located near distal inner margin.
Antenna (Fig. 13B). Consisting of coxa, allobasis and one free endopodal segment. Coxa without ornamentation. Allobasis longest, with proximal inner spinules, with  pinnate abexopodal seta. Exopod three-segmented; first segment longest with two pinnate setae; second segment shortest, with one pinnate seta; last segment bearing two pinnate setae. Free endopodal segment ornamented with long spinules and with outer distal frill, with two lateral and one subdistal inner spine, four apical pinnate setae, and one tiny smooth outer distal seta.
Mandible (Fig. 13C, D). Coxa with anterior row of spinules. Gnathobase well developed; with two rows of blunt teeth and a comb-like structure; with one long pinnate seta. Basis ornamented with spinules; with one naked seta. Both rami onesegmented. Endopod with two pinnate and one naked seta laterally; with five distal setae fused at their bases. Exopod with two lateral setae, and four apical naked elements fused basally to segment.
Maxillule (Fig. 14A). Arthrite of praecoxa well developed; ornamented with spinules; bearing one tiny and eight developed spines apically, with two surface setae. Coxal endite with spinular row on distal margin; with three spinulose setae. Basis ornamented with spinules on anterior and posterior surface; with three subdistal naked setae, and two distal elements of which one visibly stronger and spinulose. Endopod with three naked setae fused to segment. Exopod rectangular, armed with three naked setae.
Maxilliped (Fig. 14D). Syncoxa ornamented with hair-like outer spinules and with inner spinules; with one naked inner seta. Basis with medial spinules of variable lengths; with one spinulose seta. Endopod drawn out into strong claw; with one cylindrical process carrying one naked seta apically and one tiny naked seta basally.
P4 (16B). Praecoxa triangular and unornamented. Coxa and basis largely as in P3. Enp-1 with one large pinnate inner seta; enp-2 unarmed; enp-3 with three pinnate setae and one pinnate spine. Exp-1 and -2 each with one outer pinnate spine and one inner pinnate seta; exp-3 with three pinnate outer spines, one outer and inner distal element ornamented as shown, and three pinnate setae. P5 (Fig. 17B). Baseoendopod and exopod separated. Basal outer seta long and naked. Endopodal lobe ornamented with long spinules along inner and outer margin, with inner tube-pore and median pore on anterior surface, with four pinnate setae. Exopod rectangular, longer than endopodal lobe, furnished with spinules on anterior and posterior surface, with five pinnate setae. P6 (Fig. 17A). Situated on anterior part of genital-double somite, with two pinnate and one small naked seta.
Diagnostic key to the species of Tigriopus

Discussion
The new species, T. iranicus sp. nov., was allocated into Tigriopus on account of the combination of: 1) a nine-segmented antennule in the female; 2) a three-segmented antennary exopod (with setal formula 2.1.2); 3) a three-segmented P1 endopod; 4) male P2 enp-2 with outer apophysis; 5) P3 without sexual dimorphism, and 7) male P5 endopodal lobe reduced or absent. Tigriopus iranicus sp. nov. is the third species of the genus reported from the Indian Ocean. Tigriopus crozettensis and T. kerguelensis were reported by Soyer et al. (1987) from Crozet and Kerguelen islands, respectively, in the southern Indian Ocean. The new species is the first record of Tigriopus from the northern part of the Indian Ocean. The species was found in tide pools in the Persian Gulf and the Oman Sea. Rocky shores are restricted to three areas of the Iranian southern coast (Polgar 2017).
The great similarities between females of different species make subtle details necessary for separation of species (Wells 2007). The new species is defined by the following autapomorphies: i) the number of syncoxal endites of the maxilla is reduced from three to two (the genus Tigriopus has been diagnosed with three endites, the proximal one of which is bilobed; T. iranicus sp. nov. is unique within the Harpacticidae in having a reduced number of syncoxal endites of the maxilla); ii) the reduced number from three to two (as in other species of the genus) setae on the female P6.
In addition to differences with other congeners, T. iranicus sp. nov., displays a unique two-segmented mandibular endopod. Within the genus Tigriopus the mandibular endopod is one-segmented. The two-segmented condition in the new species indicates a plesiomorphic state in the genus. In comparison with other species, the P1 enp-3 armature of T. iranicus sp. nov. has a different armature. The P1 enp-3 has one developed pinnate claw, one strong geniculate spine, and one slender seta, which are undescribed and not shown in any other species of the genus.
The new species, T. brevicornis and T. minutus (see Bozic 1960: 195, fig. 9) share the incorporation of the inner element of the male P2 enp-2 into the segment to form a curved pinnate spinous process. Similarly, the male P5 of the new species, T. raki and T. igai share the complete absorption of the unarmed endopodal lobe and a tetrasetose exopod.
As most older descriptions of species are incomplete, it is difficult to make further comparisons. Nevertheless, based on available information, the new species is most closely related to T. igai and T. raki, and seems to be more closely related to the latter. In addition to the apomorphies of T. iranicus sp. nov., there are several significant differences that separate the new species from the other two species. The new species differs from T. igai in: i) the number of setae on the female P6; ii) the number of endopodal and exopodal segments of the mandible; iii) the number of setae on the exopod of the mandible; iv) the number of setae on the coxal endite of the maxillule; and v) the number of setae on the syncoxal endites of the maxilla. Tigriopus iranicus sp. nov. differs from T. raki in the presence of: i) two setae on the mandibular basis (one in T. raki); ii) six setae on the basis of the maxillule (five in T. raki); ii) four setae on the exopod of the maxillule (three in T. raki); iv) two setae on the proximal syncoxal endite of the maxilla (four in T. raki); and v) three setae on the allobasis of the maxilla (two in T. raki).
Tigriopus raki was originally described from splash-zone pools from Northland, New Zealand by Bradford (1967). Our redescription of T. raki upon careful examination of the paratype material allows the following amendments to Bradford's (1967) original description of the species: 1) Bradford (1967) omitted the description and illustration of the maxillule and the maxilla. These appendages have been fully described and illustrated here.
3) We observed that the male P2 enp-2 inner seta is not incorporated into the segment.
The number of species currently attributed to Tigriopus increases to 15. Following Soyer et al. (1987), T. iranicus sp. nov. belongs to a group of tropical species composed of T. brachydactylus, T. igai, T. minutus, and T. raki. A preliminary phylogenetic analysis (Nazari and Gómez unpubl.) supports such grouping.