New morphological and molecular data on the little-known pontellid Calanopia media Gurney, 1927 (Crustacea, Copepoda, Calanoida) from the Red Sea, with notes on its diel vertical distribution

Abstract As a part of the routine neritic zooplankton collection program in Obhur Creek (central Red Sea, Saudi Arabia), specimens of a pontellid calanoid copepod, Calanopia media Gurney, 1927, were observed and studied. Since the original description was rather brief and drawings limited, especially of mouthparts and legs, which were not illustrated and described, the species is here fully redescribed. Red Sea specimens showed considerable variation in the female genital compound somite, the right caudal ramus and leg 5, as well as in the presence of a medial knob ventrally on the male right prosomal corner. DNA sequences of mtCOI of different specimens did not show any significant differences and supported their identity as one species. Calanopia media exhibited clear diel vertical migration, with high densities of 106 and 150 ind. m-3 during sunset (6:00 pm; UTC+3) and midnight (12:00 am; UTC+3) collections, respectively. However, this species was not observed in other zooplankton collections from the surface to 20 m depth, at 6:00 am and 12:00 pm (UTC+3).


Introduction
The pontellid (Calanoida) fauna of the Red Sea contains a surprisingly low proportion of the Indo-Pacific fauna, from which it is apparently derived (El-Sherbiny and Ueda 2008;El-Sherbiny 2009) Dana, 1849, C. minor A. Scott, 1902, C. media Gurney, 1927, C. kideysi Ünal & Shmeleva, 2002, C. thompsoni A. Scott, 1909and C. tulina El-Sherbiny & Al-Aidaroos, 2017. The original description of C. media is rather brief, does not include the cephalic and thoracic limbs, and illustrations are confined to habitus drawings of the female and male in dorsal view, the female urosome, the male right antennule ancestral segments 19 and 20 and the male and female leg 5.
During the examination of plankton, samples collected from the Saudi Arabian waters of the central Red Sea, in Obhur Creek, Jeddah, specimens of what we provisionally called C. media were observed. These specimens differ in some respects from Gurney's (1927) original description. Here, we provide a full description of the species and an account of variability among the Obhur Creek specimens. Comparison with the type specimens held in the Natural History Museum, London (BMNH 1926.2.16.69-88) was carried out, and information on the vertical migration in the water column was provided. In addition, the mitochondrial COI gene of some species from the Red Sea was sequenced and compared with the sequences available in Gen-Bank (NCBI) to determine their affinity.

Material and methods
Zooplankton samples were collected from Obhur Creek (21°42'32.23"N, 39°5'41.56"E) using a 50 cm diameter plankton net of 150 µm mesh size, towed near the surface for 10 minutes at a speed of about 1-1.5 knots and vertically from 20m depth to the surface on 21 January 2016 at 7:00am, 12:00pm, 6:00pm and 12:00am (UTC+3) local time (sunrise at 7:04am and sunset at 6:05pm; UTC+3). A flowmeter (Hydrobios) was attached to the net mouth for estimating the volume of water filtered. Samples were fixed immediately in 95% alcohol. Subsequently, Calanopia media specimens were picked from zooplankton samples collected at midnight. For microscopic examination, specimens were dissected in lactic acid and were observed using brightfield and differential interference microscopy (Nikon DM 6000). Drawings and measurements were made with a camera lucida attached to the microscope and an ocular micrometer. Morphological terminology follows Huys and Boxshall (1991), except for maxillary and maxillipedal appendages, which follow Ferrari and Ivanenko (2008). For scanning electron microscopy, Calanopia specimens were washed three times in filtered seawater and clean distilled water, then dehydrated through a 30-100% ethanol series and dried with hexamethyldisilazane. The specimens were mounted on a stub, coated with gold palladium, and observed with a SEM FEI-QUANTA 250.
Mandible (Fig. 3A). Gnathobase with eight teeth on cutting edge, third and fourth ventralmost teeth bicuspidate; patches of dagger-like spinules arranged at base of third to sixth ventralmost teeth; mandibular palp basis with 4 setae; endopod 2-segmented, first and second segments carrying 3 and 6 setae, respectively; exopod 5-segmented, first to fourth segments each with one seta and fifth segment with 3 setae.
Legs 1-4 as in other members of the genus, with 3-segmented exopods and 2-segmented endopods as well as lateral spines with serrated hyaline margins (  Leg 5 (Fig. 4E) asymmetrical but with same number of spines and processes; coxa and intercoxal sclerite completely fused; right basis broader and slightly shorter than left basis, each with one posterior plumose seta; exopod 2-segmented; first exopodal segment of right leg shorter than that of left leg; with 1 lateral fused process and 1 strong spine distally; second exopodal segment of right leg slightly longer than that of left leg, extending into tapering process fused to its segment, with 2 lateral articulated spines (proximal one smaller).
Male. Prosome (Fig. 5A) 2.1 times as long as urosome; cephalosome and first pedigerous somite completely separated; fourth and fifth pedigerous somites completely fused (Figs 5A, 6B); rostrum as in female (Fig.5 B, C); posterior corners of prosome slightly asymmetrical (right one slightly longer than left), with a sharp triangular process directed posteriorly and with a distinct ventral knob or process on its right medial margin, which cannot be seen in dorsal view (Figs 5D, 6B). Urosome composed of 5 free somites, genital somite with genital aperture located ventrolaterally on posterior left side margin; second urosomite longer than other somites; anal somite shorter than preceding somite; caudal rami symmetrical, 2.2 times longer than wide, each ramus with 6 setae (II-VI) and seta VII small, inserted in ventrodistal medial margin.
Antenna, mouthparts and legs 1-4 as in female. Leg 5 (Figs 5G, 6D) uniramous, asymmetrical; coxae and intercoxal sclerite completely fused. Left leg basis carrying 1 plumose seta posteriorly near two-thirds of its length; exopod 2-segmented, first segment shorter than basis (0.46 times) with small laterodistal spine; second segment nearly 1.35 times as long as first one, bearing 3 articulated spines (2 stout apically and one small laterally), medial hirsute margin with one distal fused spine. Right leg 5 (Figs 5G, 6D), longer than left; basis slightly longer than coxa, carrying one posterior plumose seta; right exopod 2-segmented, first segment with small thumb-like process located at approximately one-third of segment length, with small seta near base of thumb, lateral margin concave with bilobed flap-like process; second exopodal segment approximately 0.7 as long as first exopod segment, curved at about mid-length and bluntly rounded distally, bearing 2 setae in depression (one proximal and one central) and 2 unequal outer setae at mid-length (proximal one longer than distal).

Variations.
On the ventral surface of the female genital compound somite of some specimens, a small fold in the cuticle may be found on the right or left side. Also, the degree of anteromedial expansion of the female right caudal ramus varies among specimens. The anteromedial expansion of the female right caudal ramus was present in most of the specimens collected from the study area (about 90% of the population), and sometimes the degree of this expansion varied greatly among specimens. In some specimens, the right caudal ramus had a concave or straight medial margin. Moreover, the ventral knob on the right side of the male prosome posterior corners varies in size.   Remarks. We compared our specimens with the paratypes deposited at the Natural History Museum, London (BMNH 1926.2.16.69-88), and concluded that our specimens are C. media. Both our specimens and the paratypes shared most of the diagnostic features of the species, such as: the shape of the fifth pediger, the presence of 2 ventral spinules on the right side of the female genital compound somite, and the structure of both female and male leg 5. However, the asymmetry of female leg 5 (right leg basis broader and slightly shorter than left, first exopodal segment of right leg shorter than that of left leg and second exopodal segment of right leg slightly longer than on left leg) and the presence of a ventral knob on the right side of the prosome were probably overlooked in the original description by Gurney (1927). Nevertheless, our specimens differ in the asymmetry of the caudal rami, of which the right ramus is broader and expanded anteromedially, and slightly shorter than the left one.
Distribution. Calanopia media was originally described from the Suez Gulf and the southern part of the Suez Canal (Gurney 1927) during the CAMBRIDGE Expedition. Subsequently, Pesta (1941) collected this species during the POLA Expedition in the southern Red Sea (15°26'12"N, 40°05'24"E). In 1956, Rose recorded this species from the Vietnamese waters. Later, it was recorded from the Levantine Basin by Berdugo (1968) and Lakkis (1984) and considered to be a Lessepsian migrant species. In the present study, C. media was found in considerable abundance (106 ind. m -3 ) in samples collected at sunset (6:00 pm; UTC+3), with the highest densities at midnight (150 ind. m -3 ). Copepodid stages were relatively low, constituting 9 and 5% of the population at sunset and midnight, respectively. It was completely absent from near the surface in morning and midday samples. The sex ratio (males/females) of C. media varied between 0.46 and 0.54 at 6:00 pm and 12:00 am (UTC+3), respectively. Molecular diversity. A 624-bp region of the mtCOI was obtained for four female individuals of C. media, which varied in the degree of anteromedial expansion of the female right caudal ramus in specimens collected from Obhur Creek, central Red Sea. Results showed that the four analyzed specimens have nearly identical mt-COI sequences, with a distance ranging between 0.013 and 0.016 based on the pairwise distance method and Kimura 2 parameter model. The intraspecific variation in the mtCOI sequences of the other Red Sea species, C. minor and C. thompsoni, were 0.000 and 0.002, respectively.
Moreover, in the current analysis, sequences were obtained for three other Calanopia species collected from the study area (C. elliptica, C. minor and C. thompsoni) and sequences of one species (C. thompsoni) were obtained from NCBI. The mtCOI sequences of Calanopia species (i.e., C. elliptica, C. media, C. minor and C. thompsoni) from the Red Sea differ between 21.3% and 29.4% (Table 1 and Fig. 7). A Neighbor Joining phylogenetic analysis using the Kimura 2 parameter model showed that C. media was clearly distinct from its congeneric species (Fig. 7). Concerning C. thompsoni, the only sequenced mtCOI in NCBI, it is clear that the average distance between Red Sea specimens and Indian Ocean specimens (KP068656-KP068659) was 0.035 (0.030-0.042), whereas for the China seas' specimens it was 0.201 (0.193-0.211).

Discussion
In his original description of C. media, Gurney (1927) provided drawings only for the whole body and leg 5 of both sexes, as well as of the female urosome and the geniculate part of the male right antennule (segments XX and XXI), and compared it with C. elliptica. His description was brief and focused mainly on the female genital compound somite and female leg 5. For the male, he only mentioned that the major differences between C. media and C. elliptica lay in a lack of serrations on the second exopodal segment of right leg 5. In his words, "Fifth legs closely resembling those of C. elliptica, but without the scalloped edge to the broad subterminal joint of the right leg". Re-examination of C. media from the study area allowed us to more accurately describe this species. The original characters reported by Gurney (1927) are given in brackets: 1) the female genital compound carries one or two spinules on the right side (2 spinules); 2) the female caudal rami are asymmetrical, the right one is expanding anteromedially (symmetrical); 3) the female leg 5 is slightly asymmetrical, with the left leg slightly shorter than the right due to shortness of both the basis and first exopodal segment (mentioned as symmetrical by Gurney, but from his drawings the first exopodal segment is longer on the right side and also the right second exopod segment is longer than the left one); 4) the male prosomal corners project posterolaterally into asymmetrical pointed processes, the right one of which is slightly longer than the left, with a distinct ventral knob on its medial margin, invisible in dorsal view (symmetrical); and 5) the second exopod segment of the male right leg 5 carries one seta medially, which is lacking in Gurney's description. Moreover, Pesta (1941), in his description from the southern Red Sea, mentioned the lack of spinules on the genital compound somite, while the female leg 5 exopod proportions differed slightly from Gurney's description (in Pesta's description, the second exopod segment is 1.1 times as long as the first exopod segment) and the second distal spine on the second exopod segment is shorter than in Gurney's description.
In addition to the variability noted in the caudal rami of C. media, variation has been reported in C. sewelli Jones & Park, 1967, collected from Marquesas, central Pacific, in which the right ramus was sometimes longer and with a concave medial margin. Moreover, asymmetry of the female caudal rami has been noted in C. asymmetrica Mulyadi & Ueda, 1996 collected from Indonesian waters, where the right ramus was much longer than the left one and expanded posteriorly. This asymmetry was found also in C. australica Bayly & Greenwood, 1966 collected from Moreton Bay, Australia, in which the left ramus was longer than the right. Asymmetry of the male prosomal points in C. media is similar to that in C. sarsi C.B. Wilson, 1950 collected from Fiji waters and C. tulina (El-Sherbiny and Al-Aidaroos 2017) described from the Red Sea.
Calanopia media is closely related to C. tulina from the central Red Sea, but they can be distinguished from each other by the characters listed in Table 2. The most distinctive characters of C. media are: 1) the presence of 2 ventral spinules on the right side of the female genital compound somite, 2) the extreme asymmetry of the caudal rami of the female, 3) the presence of 2 long articulated and 1 short fused terminal spines on the second exopodal segment of the male left leg 5, 4) the first exopodal segment of male right leg 5 with a small thumb located approximately at one-third of the segment length and with the semicircular processes on a flap on its lateral part, and 5) the second exopodal segment of the male right leg 5 shorter than the first exopodal segment, which is curved at mid-length with a blunt apex and 2 medial setae within a shallow medial depression.
In our study, C. media exhibited a clear diel vertical migration (DVM). Sunset ascend and sunrise descent were performed at very low light intensities. This pattern is known as the nocturnal or normal DVM (Forward 1988). Similar observations were made in previous studies in the southern part of the Suez Canal and the southern Red Sea by Gurney (1927) and Pesta (1941), respectively, who reported that C. media was found in high abundance in the coastal waters mainly in the night samples. This pattern was recorded also for C. americana F. Dahl, 1894 in the eastern Gulf of Mexico (Turner et al. 1979) and in the Brazilian waters (Pessoa et al. 2014). This DVM may With 3 articulated spines (2 stout and terminal, and one small and lateral) With 2 relatively long curved, terminal medially-serrated articulated spines and 1 lateral spine directed medially First exopodal segment of right leg 5 With small thumb-like process located at one-third of segment length, with bilobed flap-like process With very small rounded-tip thumb-like process located nearly mid-length, central medial part smooth, not bilobed Second exopodal segment of right leg 5 Curved at mid-length with 2 setae on concave surface, and with 2 unequal setae at mid-length on convex surface Curved at two-thirds length, with 2 setae on concave surface, and 2 unequal setae at mid-length on convex surface.
be performed to avoid UV radiation, light and predators, as well as for availability of food and favorable temperature (Bollens and Frost 1989;Lampert 1989;Andersen and Nival 1991;Pearre 2003;Cohen and Forward 2005). Generally, the light intensity in Obhur Creek drops steeply below 5 m depth, and very little light penetrates into waters below 15 m (Al-Aidaroos et al., unpublished data). Following the same trend, UVB radiation decreased by 74.15% at 5 m depth, while only 2% reached 15 m depth (Duarte et al., unpublished data). This suggests that C. media is avoiding high temperature, high light intensity and/or visual predation during the day and coming up to the surface layers for feeding during night-time. This species was observed to be abundant in night collections by light trap from a shallow coral reef in the central Red Sea (unpublished data). This might be attributed to its benthopelagic behavior, which is clearly visible in terms of the stout, large outer spines on its legs (1-4), as reported in the benthopelagic copepod genus Platycopia (Ohtsuka et al. 1998). Morphology can be considered as a fundamental method for copepod species identification. However, some pontellid species display considerable intraspecific morphological variations in the female genital compound somite, caudal rami and fifth leg (Jeong et al. 2014). Such differences might be sufficient to prove the presence of a new species, as it is the case for some centropagoid species (e.g. Sakaguchi and Ueda 2010;Soh et al. 2012). The mtCOI gene was proposed as a unique tool for copepod identification with 'barcoding' (Bradford et al. 2010;Blanco-Bercial et al. 2014), which can verify the species identity within morphologically varying pontellid specimens. Within Crustacea, the level of genetic variation between congeneric species is 17.16%, while the level of intraspecific variability is 0.46% (Costa et al. 2007). Moreover, variation between calanoid copepod species varied between 13-22%, 17.6-26.7% and 21-23% in previous studies by Bucklin et al. (1999), Eyun et al. (2007) and Soh et al. (2013), respectively. In this study, the intraspecific variation in the COI sequences from the Red Sea specimens of C. media, C. minor and C. thompsoni would confirm the hypothesis of Costa et al. (2007). The Calanopia species nucleotide sequences collected from GenBank indicate no genetic differentiation between the specimens of C. thompsoni from the Red Sea and the Indian Ocean. In contrast, the nucleotide data of C. thompsoni from the Red Sea revealed considerable variations with specimens collected from the China seas, indicating that the two populations are genetically different. This high level of deviation between both populations can by supported by the allopatric speciation hypothesis of Carpenter and Springer (2005), which states that in the Pleistocene the migration of marine organisms from the West Pacific Ocean to the Indian Ocean seems to be blocked by an ecological vicariant. Moreover, sequences included herein from the Red Sea are the first barcodes for these species, and it will be useful in future pontellid barcode studies.
During the last two decades, six species of pontellids have been originally described as new species or were first recorded from the Red Sea: Calanopia kideysi by Ünal and Shmeleva (2002), Labidocera boxshalli El-Sherbiny & Ueda, 2010 by El-Sherbiny and Ueda (2010)