Pandarus Leach, 1816 (Copepoda: Siphonostomatoida: Pandaridae) species collected from elasmobranchs off South Africa with the description of Pandarus echinifer n. sp.

Eight species of Pandarus Leach, 1816 collected from hosts caught off South Africa are reported. These species include P. bicolor Leach, 1816, P. niger Kirtisinghe, 1950 and P. carcharhini Ho, 1963 belonging to the “bicolor” group and P. cranchii Leach, 1819, P. satyrus Dana, 1849, P. smithii Rathbun, 1886 and P. sinuatus Say, 1818 belonging to the “cranchii” group. Notes on previous and new distinguishing features are provided with illustrations, specifically the relative lengths of the dorsal plates and caudal rami as well as the structure of the distomedial spine on the second segment of leg 1 exopod. Additionally, illustrated re-descriptions are provided for P. satyrus and P. sinuatus. Furthermore, a new species Pandarus echinifer n. sp., also belonging to the “cranchii” group, collected from the snaggletooth shark Hemipristis elongata (Klunzinger) is described. This species is most similar to P. sinuatus but can be distinguished from it by the heavily spinulated distomedial spine on the last segment of the first leg exopod. Molecular analysis of the cytochrome oxidase I partial gene is used to calculate sequence divergences amongst different individuals and species. According to the results (as well as based on morphological characters) P. rhincodonicus Norman, Newbound & Knott, 2000 is a synonym of P. cranchii. New hosts and geographic localities from South Africa (and Ningaloo Park, Western Australia) are reported.

According to Cressey (1967), the adult females can be divided into two groups based on their dorsal morphology, i.e., the "bicolor" group with the dorsal plates of thoracic somite two that extends only up to the posterior edge of the plate of somite three (including P. bicolor, P. niger, P. carcharini) and the "cranchii" group with the dorsal plates of the thoracic somite two extending well beyond the posterior edge of that of somite three (including P. sinuatus, P. cranchii, P. satyrus, P. zygaenae, P. smithii, P. floridanus, P. katoi and P. rhincodonicus).The three species in the "bicolor" group can be distinguished from each other mainly by the lengths of the caudal rami with those of P. bicolor barely visible in dorsal view, those of P. carcharhini just extending beyond the abdominal plate and those of P. niger extending well beyond the abdominal plate (Kirtisinghe 1950;Ho 1963;Cressey 1967).In the "cranchii" group, P. satyrus, P. cranchii and P. rhincodonicus are morphologically very similar (Cressey 1967;Norman et al. 2000) as are P. floridanus and P. sinuatus; and P. katoi and P. zygaenae (Cressey 1967) while P. smithii can be distinguished from all the other species in the "cranchii" group by the presence of basal medial expansions on the caudal rami (Izawa 2010).However, Pandarus species undergo considerable morphological changes during their ontogeny (Kabata 1979) as well as varying signs of pigmentation (Ho 1963;Cressey 1967;Kabata 1979) and therefore using their dorsal features for species identification may result in misidentifications.Additionally, the setation of the limbs also exhibits variability (Hewitt 1967) with some species having the same spine and setal formulas (Cressey 1967).
In the present study, species of Pandarus were collected from elasmobranchs caught from both the Atlantic and Indian oceans off South Africa.Thus, the paper reports on eight Pandarus species collected with additional notes on their characteristic features and redescribes and illustrates the adult females of P. satyrus and P. sinuatus, including new hosts and/ or geographical records of some collected species.Additionally, a new species, P. echinifer n. sp., is described.Sequences divergences of 13 individuals (10 downloaded from Genbank and three newly generated) using the cytochrome oxidase I partial gene were used to estimate inter-and intraspecific divergences within and amongst Pandarus species.

Sampling and morphological observation
Copepod specimens were collected from elasmobranch species caught mostly in the nets of the Kwa-Zulu-Natal Sharks Board (KZNSB) and some species caught as by-catch during hake assessment demersal cruises off the west coast of South Africa on board the Department of Agriculture, Forestry and Fisheries (DAFF) research vessel R/V (Africana) during 2008 as well as from fish caught by commercial fishermen off Gansbaai (West coast).Additionally, specimens were collected from white sharks during the Ocearch Project in South Africa as well as from a snaggletooth shark from uShaka Marine World, Durban.Furthermore, specimens were collected from a stranded whale shark off the west coast while collected specimens from whale sharks swimming in Ningaloo Marine Park (Western Australia) were also obtained.The fish hosts were mostly identified by scientists at KZNSB and researchers on board the vessels.Collected specimens were fixed and preserved in 70% ethanol.Selected specimens were cleared and stained in lactic acid with a small amount of dissolved lignin pink.These specimens were dissected and studied under both stereo-and light microscopes using the wooden slide technique (Humes & Gooding 1964).Selected specimens were prepared for scanning electron microscopy (SEM) by dehydrating them through a series of ethanol (70, 80, 90, 100, 100% for about 30 min each) followed by immersion in hexamethyldisilazane for about an hour.Excess hexamethyldisilizane, not evaporated, were removed and the specimens were allowed to dry completely before being sputter-coated with gold-palladium and carbon.Host names were verified using Froese & Pauly (2024).Morphological nomenclature mostly follows Cressey (1967) and Kabata (1979).Voucher specimens and the type-material were deposited in the Iziko South African Museum, Cape Town, South Africa.

DNA extraction and sequencing
Genomic DNA was extracted from specimens of Pandarus collected from the stranded whale shark in South Africa as well as from specimens received from Ningaloo Marine Park (Western Australia) using the QIAGEN® QIAamp® DNA micro kit (Whitehead Scientific (Pty) Ltd) according to the manufacturer's instructions.Polymerase Chain Reaction (PCR) was used to amplify a fragment of the mitochondrial (mtDNA) COI (Cytochrome Oxidase I) gene using primers LCO 1490 (forward) and HCO 2198 (reverse) (Folmer et al. 1994).The 20 µl PCR reaction mixture consisted of 10 µl Master mix (Ampliqon Taq DNA Polymerase Master Mix RED) containing HotStarTaq Plus DNA polymerase, 1 µl of each primer, 5-8 µl (depending on the DNA concentration) of DNA and ddH 2 O to top up the volume.The cycling conditions consisted of an initial denaturation at 95 °C (4 min); followed by 30 cycles of 94 °C (1 min) denaturation, 43-45 °C (2 min) annealing and 72 °C (3 min) extension, with a final extension of 72 °C (10 min) in the MiniOpticon real-time PCR system.Purification of PCR products and sequencing was done by Inqaba Biotechnical Industries (Pty) Ltd.The resulting chromatograms of the sequences were checked for nucleotide ambiguities, and the forward and reverse sequences assembled and edited using CLC main workbench 7.9.1 (QIAGEN).Generated sequences were aligned with sequences downloaded from Genbank (Accession numbers: HG942363, FJ447387-FJ447391, KJ551843 and OL457303-OL457305) using Clustal X 2.0.12 (Thompson et al. 1997).The aligned dataset was imported into MacClade 4.0 (Maddison & Maddison 2001) and translated into amino acids to verify the alignment.Uncorrected pairwise sequence divergences were estimated using MEGA 7 (Kumar et al. 2016).The generated sequences were submitted to Genbank (Accession numbers: PP434798-PP434800).Kirtisinghe, 1950;d, Pandarus carcharhini Ho, 1963.thoracic plates and the appearance of the caudal rami.The dorsal plates of the second thoracic somite extend only to the posterior margin of those of the third thoracic somite (Fig. 1c) while the caudal rami are long and reach well beyond the posterior margin of the dorsal abdominal plate (Fig. 1c) (caudal rami about 1 1 / 3 times as long as the dorsal abdominal plate (Rangnekar & Rangnekar 1972) (see Plate 1 in Rangnekar & Rangnekar (1972)).
Material examined: 1♀ from single host specimen.
Pandarus carcharhini females are distinguished from all other species by the same combination of features as P. bicolor and P. niger, namely that the dorsal plates of the second thoracic somite extend only to the posterior margin of those of the third thoracic somite (Fig. 1d) while the caudal rami are longer than those in P. bicolor reaching beyond the posterior margin of the dorsal abdominal plate (Ho 1963) (although slightly shorter than abdominal plate in current specimen), but shorter than those of P. niger (see Fig. 13 in Ho (1963) Pandarus cranchii females have dorsal plates of the second thoracic somite that extend well beyond the posterior margin of those of the third thoracic somite (Figs.2a, b), sometimes even beyond the posterior margin of the plate of the fourth thoracic somite (Fig. 2a).The caudal rami extend mostly beyond the posterior margin of the dorsal abdominal plate (Fig. 2a) or at least up to the posterior margin (see Fig. 2b and Fig. 1A in Izawa (2010)).Additionally, the second segment of leg 1 exopod is armed with a distomedial spinulated process (Figs.2c, d) (see Fig. 2A in Izawa (2010)).Pandarus satyrus Dana, 1849 Hosts: Carcharodon carcharias (Linnaeus); Isurus oxyrinchus Rafinesque (Lamniformes: Lamnidae); Carcharias taurus Rafinesque (Lamniformes: Carchariidae) from east coast (SA), and Rhincodon typus Smith (Orectolobiformes: Rhincodontidae) from west coast (SA) and Ningaloo Marine Park (Western Austalia).
Locality: Indian and Atlantic Oceans, South Africa and Ningaloo Marine Park (Western Australia).
Re-description (Figs. 3, 4) Adult female [based on 10 specimens].Cephalothorax dorsally with several small denticles medially on posterior margin (Fig. 3a).Dorsal thoracic plates of second somite about twice as long as that of third somite reaching more than half length of dorsal plate of fourth somite (sometimes almost to posterior margin of dorsal plate of fourth somite (Fig. 3b)).Genital complex with paired posterior protrusions each armed with a small spine (Figs.3c, d).Dorsal abdominal plate fan-shaped with small spinule on lateral margin (Fig. 3c).Caudal rami (Fig. 3e) lateral to dorsal abdominal plate, extending beyond middle of dorsal abdominal plate (Fig. 3a) (sometimes beyond posterior margin of dorsal abdominal plate (Fig. 3b)), sharply pointed distally with 2 processes and pinnate seta dorsally and 1 spine and small pinnate seta ventrally.

Remarks
Females of P. satyrus and P. cranchii are morphologically very similar and can apparently be distinguished by the lengths of the caudal rami relative to the length of the dorsal abdominal plate (Cressey 1967;Lewis 1966), while the leg armature is mostly the same for the two species (Cressey 1967).However, even though the plates of the second thoracic somite of P. cranchii mostly extends to or beyond the posterior margin of the plate of the fourth thoracic somite some individuals of P. satyrus have equally long plates of the second thoracic somite (current observations, see Fig. 3b).Furthermore, the caudal rami of P. satyrus mostly do not extend beyond the posterior margin of the dorsal abdominal plate, as often seen in P. cranchii (with exceptions, see Fig. 12D in Shiino (1954)), but some individuals also have equally long caudal rami as seen in P. cranchii (current observations, see Fig. 3b).Due to the variation in the armature of the legs present in members of Pandaridae (Hewitt 1967) reports of different numbers of armature elements are not unusual for P. satyrus females (e.g.Shiino 1954;Ho 1963;Lewis 1966;Cressey 1967;Hewitt 1967).However, a clear difference between P. satyrus and P. cranchii females is the structure of the distomedial spine on the last exopodal segment of leg 1 with that of P. cranchii being a spinulated process (see Figs. 2c, d and Fig. 2A in Izawa (2010), Fig. 13B in Shiino (1954), Fig. 12c in Lewis (1966) and Fig. 145 in Hewitt (1967)) while that of P. satyrus is a smooth, bifid process (see Fig. Locality: Indian and Atlantic Oceans, South Africa.
Material examined: Several ♀♀ from all host specimens.expansions (often shorter than posterior margin of abdomen (see Figs. 5b, 6a).The second segment of leg 1 exopod is armed with a distomedial smooth process with a slightly extended tip (Fig. 6b, c, d) (sometimes causing a slightly bifid appearance (Figs.6c, d Locality: Indian Ocean, South Africa.Material examined: Several ♀♀ from all host specimens. Voucher material: 10 adult ♀♀ (SAMC-A096833) collected from C. taurus.7a).Dorsal thoracic plates of second somite slightly longer than that of third somite (sometimes appearing almost equal in length), reaching only about a third length of dorsal plate of fourth somite (Fig. 7a) (some specimens reaching about half length of dorsal plate of fourth somite (Fig. 7b)).Genital complex with paired posterior protrusions (Fig. 7a).Dorsal abdominal plate mostly circular with thin anterior extension joining with genital complex (Fig. 7a).Caudal rami (Figs.7a, 8a) lateral to dorsal abdominal plate, extending slightly beyond middle of dorsal abdominal plate (Fig. 7a), slender, sharply pointed distally with 2 processes dorsally and 1 spine and small pinnate seta ventrally (Fig. 7c), slight basal medial expansions (Figs.7a, 8a).

Remarks
Females of P. sinuatus are part of the "cranchii group" with the dorsal plates of thoracic somite two extending "well beyond" the posterior margin of the plate of somite three (Cressey 1967) even though the plates of somite 2 only extend slightly beyond the plate of somite three.Furthermore, P. sinuatus is related to P. floridanus but are distinguishable by the lengths of the plates of thoracic somite 2, with those of P. floridanus extending beyond the middle of the plate of somite 4 whereas those of P. sinuatus are not extending to the middle of the plate of somite 4, but only to the anterior third (Cressey 1967).Additionally, the dorsal abdominal plate is about as long as the exposed caudal rami (Cressey 1967).However, current observations highlight the fact that the plates of thoracic somites two and three are often almost the same length (see Fig. 7a) while these plates may sometimes extend further than just the anterior third of the plate of somite four (see Fig. 7b).Additionally, the length of the caudal rami of the studied specimens were mostly shorter than the length of the dorsal abdominal plate (see Fig. 7a) rather than the same length (according to Wilson (1907)).The presence of slight basal medial expansions on the caudal rami of P. sinuatus individuals (see Figs. 7a,8a) as well as the variation in the sizes and shapes of the dorsal thoracic plates resulted in difficulty distinguishing between females of P. sinuatus and P. smithii.However, the structure of the distomedial spine on the last exopodal segment of leg 1 can be used to distinguish the females of the two species, with that of P. sinuatus appearing much more bulbous (Figs.8d-f) and not slightly bifid (Figs.6b-d Etymology: The specific name echinifer (sea-urchin bearer) refers to the structure of the distomedial spine on the last exopodal segment of leg 1 that reminds of the spines on a sea-urchin.Description (Figs. 10,11,12) Adult female [Based on two specimens, one without abdominal plate].Female typical Pandarus morphology.Body length from anterior margin to posterior margin of abdominal plate/abdomen 6.25-6.5 mm (mean 6.4), cephalothorax length 2.5-2.8mm (mean 2.65), cephalothorax width 3.75-3.85mm (mean 3.8), abdominal plate length and width 1.25 mm.Cephalothorax dorsally with uneven posteromedial margin with several sharp denticles (about 5 on either side of middle) (Fig. 10a).Dorsal thoracic plates of second somite slightly longer than that of third somite, reaching only about a third length of dorsal plate of fourth somite (Fig. 10a).

Remarks
Females of P. echinifer n. sp. are also part of the "cranchii group" with the dorsal plates of thoracic somite two extending "well beyond" the posterior margin of the plate of somite three (Cressey 1967), although they are only slightly longer than the plate of somite three.Additionally, the plates of thoracic somite two extends only to the anterior third of the plate of somite four while the dorsal abdominal plate is longer than the exposed caudal rami.These features are very similar to those P. sinuatus.Additionally, the presence of slight basal medial expansions on the caudal rami of P. echinifer n. sp.individuals similar to those in P. sinuatus also cause difficulty distinguishing among females of P. echinifer n. sp., P. sinuatus and P. smithii.The structure of the distomedial spine on the last exopodal segment of leg 1 can be used to distinguish among the three species with similar dorsal morphology i.e.P. echinifer n. sp., P. sinuatus and P. smithii with that of P. echinifer n. sp.heavily spinulated (Figs. 11b,12a,b), that of P. sinuatus appearing more bulbous 9a) and that of P. smithii a bulbous process with a slightly extended tip, giving it a slightly bifid appearance (Figs.6b-d).The spinules covering the tip of the distomedial spine on the last exopodal segment of leg 1 of P. echinifer n. sp. is reminiscent of that of P. cranchii (see Fig. 2c, d) but with more prominent spinules (see Fig. 12b) while the relative lengths of the dorsal thoracic plates and the lengths of the caudal rami vastly differ between P. echinifer n. sp. and P. cranchii.Furthermore P. cranchii does not have slight basal medial expansions on the caudal rami and thus the two species are easily distinguished.
Significant non-synonymous substitutions (where more than one sequence share the substitution) in the dataset occurred due to three transitions and one transversion.A first codon position transition is shared by all the P. cranchii sequences (FJ447387, PP434799, PP434800, KJ551843) and P. rhincodonicus (HG942363) from GTC/A to ATT at position 94 resulting in a change in the coded amino acid from Valine to Isoleucine.Pandarus satyrus (PP434798, FJ447391) and P. sinuatus (FJ447389, FJ447390) share a change in the coded amino acid from Valine to Isoleucine due to a first codon position transition from GTT/C to ATT at position 328.A transversion at the first codon position 346 from TCT/C to GCT/C resulted in a change in amino acid from Serine to Alanine in P. sinuatus (FJ447389, FJ447390) and P. smithii (FJ447388).Lastly a first codon position transition at position 502 from GTT/A to ATC resulted in a change from the amino acid Valine to Isoleusine in P. sinuatus (FJ447389, FJ447390).

Remarks
The morphological description of P. rhincodonicus mentioned distinguishing features such as the dorsal plates of thoracic somite two extending "almost to the limit of the fused plates of" thoracic somite four while the plates of thoracic somite two of P. cranchii are "considerably shorter" than those of somite four (Norman et al. 2000).However, due to the considerable variation amongst individuals this is not always the case (see Fig. 2a) and additionally, P. satyrus also have plates of thoracic somite two almost as long as those of somite four (see Fig. 3b, Fig. 1 in Cressey (1967)).Additionally, "the posterior margin of the cephalon is armed with four tubercules instead of the serrated margin" (Norman et al. 2000) is also seen in other species e.g.P. cranchii (see Izawa 2010), P. satyrus (see Fig. 3a), P. sinuatus (see Fig. 7a) and P. echinifer n. sp.(see Fig. 10a) while that of P. smithii seems more serrated (see Fig. 5a, Fig. 7a in Izawa ( 2010))."The shape of the caudal rami differ as they lack the large inner lobe characteristic of P. cranchii" (Norman et al. 2000) is invalid since P. cranchii do not have caudal rami with basal medial expansions (see Figs. A, D in Izawa (2010)).Thus, no valid distinguishing morphological characteristics are found for P. rhincodonicus while the description included features applicable to both P. cranchii and P. satyrus (see Fig. 1A in Norman et al. (2000)).It is therefore suspected that P. rhincodonicus is a synonym of P. cranchii.Based on evidence from the molecular analysis which included the sequence of P. rhincodonicus, downloaded from Genbank (and others collected from R. typus at Ningaloo Park and morphologically identified as P. cranchii) indicated that the maximum sequence divergence amongst these sequences is 3% (Table 1) similar to expected intraspecific variation and thus P. rhincodonicus is a synonym of P. cranchii.

Discussion
Of the current 14 valid species of Pandarus (Walter & Boxshall 2024), P. rouxii, P. brevicaudis and P. ambiguus should be considered as species inquirenda.Additionally, P. rhincodonicus is a synonym of P. cranchii and P. echinifer n. sp. is newly described.Thus, the number of valid Pandarus species currently is 11.
The identification of Pandarus species is compromised due to different Pandarus species that share many characteristics e.g. the relative lengths of the dorsal thoracic plates as well as varying signs of pigmentation amongst individuals (Ho 1963;Cressey 1967;Kabata 1979) and the same spine and setal formulas (Cressey 1967), while different individuals of the same species exhibit variation in the setation of the limbs (Hewitt 1967).Even though the division into the "bicolor" and "cranchii" groups can still be used as a starting point, researchers should be careful as in some individuals this is not absolutely clear (see Fig. 7a) as is the presence of the basal medial expansions to distinguish P. smithii (cf. Figs. 5b,6a,8a,10b).Additionally, different species are symbionts of several host species (see current study, Shiino (1954), Lewis (1966), Cressey (1967), Alvarez & Winfield (2001) and Izawa (2010)).
From the current study, it seems like the detailed structure of the distomedial spine on the last segment of the exopod of leg 1 may be a distinguishing feature amongst similar Pandarus species (cf.Figs. 2c,d,4f,9a,11b,12a,b).Even though the distomedial spine is quite small, differences can be observed using light microscopy (see Figs. 2d,6d,8f,12a,b).This character may be specifically useful if used in combination with other less distinguishing features such as the relative lengths of the dorsal plates and the caudal rami.
Voucher material: Six adult ♀♀ (SAMC-A096831) collected from C. carcharias; four adult ♀♀ (SAMC-A096832) collected from I. oxyrinchus.Pandarus smithii females have dorsal plates of the second thoracic somite that extend beyond the posterior margin of those of the third thoracic somite, almost reaching middle of plate on fourth thoracic somite (Fig. 5a, see Fig. 34 in Cressey (1967) and Fig. 7A in Izawa (2010)).The caudal rami are mostly shorter than the dorsal abdominal plate or extend almost to the posterior margin of the dorsal abdominal plate (Fig. 5a, see Fig. 34 in Cressey (1967) and Fig. 7A in Izawa (2010)) bearing basal medial

Fig
Fig. 8 Pandarus sinuatus Say, 1818.a, scanning electron micrograph of abdomen and caudal rami, ventral view; b, scanning electron micrograph of maxillule; c, scanning electron micrograph of maxilliped; d, scanning electron micrograph of bulbous distomedial process of leg 1 exopod distal segment; e, scanning electron micrograph of distomedial bulbous process of leg 1 exopod distal segment (different view); f, light microscope photo of leg 1 exopod with distomedial bulbous process of distal segment and distal segment of endopod.