Species of Pseudorhabdosynochus (Monogenea, Diplectanidae) from Groupers (Mycteroperca spp., Epinephelidae) in the Mediterranean and Eastern Atlantic Ocean, with Special Reference to the ‘Beverleyburtonae Group’ and Description of Two New Species

Pseudorhabdosynochus Yamaguti, 1958 is a species-rich diplectanid genus, mainly restricted to the gills of groupers (Epinephelidae) and especially abundant in warm seas. Species from the Mediterranean are not fully documented. Two new and two previously known species from the gills of Mycteroperca spp. (M. costae, M. rubra, and M. marginata) in the Mediterranean and Eastern Atlantic Ocean are described here from new material and slides kept in collections. Identifications of newly collected fish were ascertained by barcoding of cytochrome c oxidase subunit I (COI) sequences. Pseudorhabdosynochus beverleyburtonae (Oliver, 1984) Kritsky & Beverley-Burton, 1986 and P. sosia Neifar & Euzet 2007 are redescribed from type-specimens and new specimens collected off Tunisia and Libya from M. marginata and M. costae, respectively. Pseudorhabdosynochus oliveri n. sp., from M. marginata (type-host) off the Mediterranean coast of France (type-locality), is described from specimens found among voucher specimens of P. beverleyburtonae deposited by Guy Oliver in the collection of the Muséum National d’Histoire Naturelle, Paris. Pseudorhabdosynochus oliveri is distinguished by the shape of its sclerotised vagina; it was not found in the other localities investigated. Pseudorhabdosynochus hayet n. sp. is described from M. rubra (type host) off Senegal (type-locality) and Tunisia. Pseudorhabdosynochus hayet is morphologically similar to P. sosia (type-host: M. costae) but was distinguished by differences in measurements of the vagina and male copulatory organ, different host, and divergent COI sequences. The four species (P. beverleyburtonae, P. sosia, P. oliveri, and P. hayet) share common characteristics such as squamodiscs with 2 innermost circular rows of rodlets and a similar general structure of the sclerotised vagina; we propose to group them into a ‘beverleyburtonae group’ within Pseudorhabdosynochus.


Introduction
Pseudorhabdosynochus Yamaguti, 1958 is a species-rich diplectanid genus [1][2][3][4]; its members are mainly restricted to the gills of groupers (Epinephelidae), with a few exceptions, and, since most groupers inhabit warm seas, they are especially numerous in tropical seas [5]. Species from the Mediterranean are not fully documented, although eleven nominal species have been listed [6,7].
Pseudorhabdosynochus beverleyburtonae (Oliver, 1984) Kritsky & Beverley-Burton, 1986 is a gill parasite of the dusky grouper Mycteroperca marginata in the Mediterranean Sea and both sides of the Atlantic [2,[8][9][10][11]. We found that three species of Pseudorhabdosynochus from groupers assigned to Mycteroperca Gill in the Mediterranean Sea and Eastern Atlantic Ocean were morphologically very close to P. beverleyburtonae. Among these species, one, namely P. sosia Neifar & Euzet, 2007, was already known, and two are new and are described in this paper. Based on the sclerotised vagina, the primary character for species diagnosis within Pseudorhabdosynochus, we propose the 'beverleyburtonae group' to accommodate them. Monogenean COI sequences were used to complement the morphological analysis of parasites. Fish COI sequences were used to confirm the morphological identification of hosts [12,13].

Fish sampling and identification
Fish were purchased at the fish markets in Sfax and Tunis, Tunisia and in Tripoli, Libya. These were previously caught by fishermen in the nearby coastal waters of the Mediterranean Sea. In all cases, the fish were dead when available for parasitological studies. No permits were required for the described study. Fish were identified morphologically according to keys [14] and books [15], and these identifications were challenged by analyses of COI sequences of individual fish (Table 1). Fish nomenclature follows [16] and [17].

COI sequence of fish host
We used the QIAamp DNA Mini Kit (Qiagen), as per the manufacturer's instructions, to perform DNA extraction. The 5 0 region of the cytochrome oxidase I (COI) mitochondrial gene was amplified with the primers FishF1 (5 0 -TCAACCAACCACAAAGACATTGGCAC-3 0 ) and FishR1 (5 0 -TAGACTTCTGGGTGGCCAAAGAATCA-3 0 ) [12]. PCR reactions were performed in 20 μl, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl 2 , 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94°C, followed by 40 cycles at 94°C for 30 sec, 48°C for 40 sec, and 72°C for 50 sec, with a final extension at 72°C for 7 min. PCR products were purified and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems). We used CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers KX255747 -KX255751 (Table 1). Species identification was confirmed with the BOLD identification engine [18].
Details about molecular identification of host fish are provided briefly in the description of the monogenean species.

Monogenean morphology
Diplectanids collected from fish gills were prepared by three methods: a) mounted in ammonium picrate-glycerine [19] (designated as 'p'); b) mounted in Berlese fluid (designated as 'b'); and c) dehydrated in an ethanol series, stained with carmine and permanently mounted in Canada balsam (designated as 'c') [20]. Specimens were drawn using an Olympus BH2 microscope equipped with drawing apparatus and DIC optics. The terminology for the sclerotised parts, i.e. the male quadriloculate organ and the vagina follows Justine (2007) [1]. Measurements, in micrometres, were taken with the help of a custom-made transparent rule and are expressed as the mean followed in parentheses by the range, the standard deviation when n30, and (n) the number of observations; measurements were taken as in Fig 1 in Chaabane et al. (2015) [6]. The measurements of the right-hand haptoral hard-parts and left-hand equivalents were pooled. The measurements of the holotype are separated and indicated by 'h'. Drawings were scanned and redrawn on a computer using Adobe Illustrator. The museum abbreviation used is as follows: MNHN, Muséum National d'Histoire Naturelle, Paris.

COI sequence of monogeneans
We used a QIAmp DNA Micro Kit (Qiagen) to extract DNA from whole monogenean specimens. The specific primers COI-ASmit1 (forward 5 0 -TTTTTTGGGCATCCTGAGGTTTAT-3 0 ) and COI-ASmit2 (reverse 5 0 -TAAAGAAAGAACATAATGAAAATG-3 0 ) were used to amplify a fragment of 424 bp of the COI gene [21]. The PCR reaction was performed in 20 μl, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl 2 , 0.25 mM dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). Thermocycles consisted of an initial denaturation step at 94°C for 2 min, followed by 37 cycles of denaturation at 94°C for 30 sec, annealing at 48°C for 40 sec, and extension at 72°C for 50 sec. The final extension was conducted at 72°C for 5 min. Sequences were edited with CodonCode Aligner software (Codon-Code Corporation, Dedham, MA, USA), compared to the GenBank database content with BLAST, and deposited in GenBank under accession numbers KX255741 -KX255746 (Table 1).
Pairwise nucleotide distances were assessed using the Kimura 2-parameter (K2P) model [22] in MEGA 7. The phylogenetic tree was constructed using the Neighbour Joining (NJ) method based on the Kimura 2-parameter (K2P) model in MEGA 7 [23]; all codon positions were used.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:CB13D383-7994-4BCE-BB8F-A46E874E26D3. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Results
Pseudorhabdosynochus beverleyburtonae (Oliver, 1984 Molecular identification of fish via DNA barcoding: The COI sequence of our specimen (KX255749) ( Table 1) was identical to three sequences (KU739519-521) previously obtained from the same fish species in the same locality (Tunisia) [24]. We conclude that our specimen belongs to M. marginata.
Comments. Pseudorhabdosynochus beverleyburtonae was originally described by Oliver as Cycloplectanum beverleyburtonae, from M. marginata (as Epinephelus guaza) in the Western Mediterranean Sea off France [10]. Kritsky & Beverley-Burton (1986) regarded Cycloplectanum Oliver, 1968 a junior synonym of Pseudorhabdosynochus Yamaguti, 1958 and placed several species, including C. beverleyburtonae, in the latter genus [28]. Most subsequent authors have accepted Pseudorhabdosynochus as the valid genus; however, Oliver continued to designate the species as C. beverleyburtonae [25].
Pseudorhabdosynochus beverleyburtonae parasitises M. marginata, which is a trans-Atlantic grouper fish. The original description does not include any measurements [10]. Measurements in specimens from various origins show differences ( Table 2) but these should be attributed to various methods of fixation and degree of flattening, as is usual in diplectanids [20]. The morphology of the sclerotised vagina is similar in all specimens, including our newly examined specimens from Tunisia. We conclude, as previous authors [2,11], that the populations assigned to M. marginata from the Mediterranean Sea and both sides of the Atlantic Ocean are conspecific. Tunisia is a new geographical record for the species, and is the first record on the southern shore of the Mediterranean Sea.
Differential diagnosis. Pseudorhabdosynochus beverleyburtonae is close to P. oliveri n. sp., P. sosia and P. hayet n. sp. in terms of general morphology of body, presence of two circular innermost rows of rodlets in squamodiscs and general morphology of the sclerotised vagina. It is separated from P. oliveri, which also occurs on M. marginata, by the shape of its primary canal (narrow in P. beverleyburtonae vs. wide in P. oliveri), and from P. sosia and P. hayet by the shape of its primary chamber, wider and more sclerotised in the two latter species.
Vitelline follicles lateral, coextensive with intestinal caeca and contiguous posterior to testis. Ovary on right side, looping dorsoventrally around right intestinal caecum.
Sclerotised vagina comprises sclerotised trumpet, primary canal, primary chamber, secondary canal and secondary chamber. Trumpet in anterior part; primary canal with thick wall, curved in its anterior part, enlarged in its posterior part with wide lumen; connection between primary canal and primary chamber posterior; primary chamber elongate, roughly parallel to primary canal but reversely oriented, its wall the same thickness as primary canal; secondary canal short, its lumen thin; secondary chamber located just posterior to trumpet, roughly spherical, its wall thick, smaller than primary chamber (Figs 3C and 4A-4C). Total length of sclerotised vagina c 63 (59-65, n = 4). Diameter of primary canal h 11, c 14 (11-16, n = 4).
Comments. This new species was found among voucher specimens labelled as Cycloplectanum beverleyburtonae by Guy Oliver (now P. beverleyburtonae). These specimens were deposited by Guy Oliver in the MNHN collections after his retirement and were not mentioned  [30] contained drawings of specimens from both species; in the case of the P. beverleyburtonae/P. oliveri couple, we did not find any indication of P. oliveri being included in the original description of P. beverleyburtonae.
Although P. oliveri and P. beverleyburtonae apparently co-occurred on dusky groupers from off France examined by Oliver, we did not find the former in our fish specimen from Tunisia.
Differential diagnosis. Pseudorhabdosynochus oliveri is close to P. beverleyburtonae, P. sosia and P. hayet n. sp. in terms of general morphology of body, presence of two circular innermost rows of rodlets in squamodiscs and general morphology of the sclerotised vagina. It is separated from the three others by the development of its primary canal, which is wide, vs. thin, and the small primary chamber, vs. large in the other three species.
Molecular identification of fish via DNA barcoding: Four specimens from Tunisia were sequenced (Table 1), including three new and one already sequenced as KT805240 [31]. All sequences had 99-100% similarity with sequences already identified as M. costae in GenBank, confirming the morphological identification of the host.
Site of infection: Gills Type-locality: Off Sfax, Tunisia.
Sclerotised vagina comprises sclerotised trumpet, primary canal, primary chamber, secondary canal and secondary chamber. Trumpet anterior, ring-shaped; primary canal with regular diameter and medium-thick wall, curved or S-shaped; connection between primary canal and primary chamber posterior; primary chamber ovoid or pear-shaped, roughly parallel to primary canal but reversely oriented, its wall thick; secondary canal well visible, its lumen thin, its wall less sclerotised than chambers; secondary chamber spherical, its wall thick, smaller than primary chamber; secondary chamber located just posterior to trumpet (Fig 5E-5H). Sclerotised vagina length b 44±4.3 (35-58, n = 30), external diameter of primary chamber b 15 (11-17, n = 28).  Differential diagnosis. By the morphology of its sclerotised vagina, P. sosia is close to P. beverleyburtonae, P. oliveri n. sp. and P. hayet n. sp. Indeed, Neifar & Euzet named the species for its resemblance with P. beverleyburtonae [3]. Pseudorhabdosynochus sosia and P. beverleyburtonae have similar vaginal lengths but can be differentiated, as indicated by Neifar & Euzet, 2007, by the external diameter of the primary chamber (larger in P. sosia) and its shape (pearshaped in P. sosia vs elongated in P. beverleyburtonae), and by the shape and length of the primary canal (longer and more coiled in P. beverleyburtonae). Pseudorhabdosynochus sosia is readily distinguished from P. oliveri by the shape of its primary canal (thin vs. wide in the latter).
The morphology of the vagina is very similar in P. sosia and P. hayet, but P. sosia differs by smaller male copulatory organ (see details under P. hayet, and   Prevalence: In Tunisia, 1/1, in Libya, 0/1. Molecular characterisation: A COI sequence was obtained for a specimen from off Tunis, Tunisia (Table 1).
Etymology: The specific name, hayet, is from Arabic ( ‫ﺡ‬ ‫ﻱ‬ ‫ﺍ‬ ‫ﺓ‬ ). The species is named after the mother of the first author. Name in apposition, invariable.
Description (Figs 6 and 7).  intercaecal, pre-testicular, encircles right caecum. Vitelline follicles present from pre-pharynx level to haptoral peduncle. Sclerotised vagina comprises sclerotised trumpet, primary canal, primary chamber, secondary canal and secondary chamber. Trumpet anterior, ring-shaped; primary canal with regular diameter and medium-thick wall, curved or S-shaped; connection between primary canal and primary chamber posterior; primary chamber pear-shaped or elongate, roughly parallel to primary canal but reversely oriented, its wall thick; secondary canal well visible, its lumen narrow; secondary chamber spherical, smaller than primary chamber; wall of secondary chamber thick; secondary chamber located just posterior to trumpet (Figs 6C and 7A-7D Differential diagnosis. Pseudorhabdosynochus hayet n. sp. can be distinguished from P. oliveri n. sp. by the shape of its primary canal (thin vs. wide in the latter), and from P. beverleyburtonae by the shape of its primary chamber (wide vs. thin in the latter). The morphology of the sclerotised vagina and other organs is very similar to that of P. sosia. The two species can, however, be distinguished by larger dimensions of the vagina in P. hayet. It is well known that measurements of sclerotised parts change with degree of flattening, i.e. the method for preparing slides [20]. Measurements between the two species are different when compared between specimens prepared with the same methods ( Table 2): picrate (43 in P. hayet vs 35-36 in P. sosia) and Berlese (61 vs 44). The male copulatory organ is longer in P. hayet: picrate (89 vs 81-84 in P. sosia), and Berlese (150 vs 92). In addition, the hosts are different (M. rubra for P. hayet vs M. costae for P. sosia) ( Table 1). The COI sequences are divergent (Table 4). The 'beverleyburtonae group' The four species assigned to Pseudorhabdosynochus described and/or redescribed herein, namely P. beverleyburtonae and P. oliveri n. sp. from M. marginata, P. sosia from M. costae, and P. hayet n. sp. from M. rubra, exhibit great similarity in the morphology of the sclerotised vagina, the main character on which Pseudorhabdosynochus species are distinguished [3,4,6,32,33]. Fig 8 shows a comparison of the sclerotised vagina in all four species; colours were used to show the various parts of the vaginae and the homologies between different species. In addition, all these four species are characterised by a similar structure of the squamodiscs, with two innermost rows as closed circles; this character was used as a primary characteristic to erect Cycloplectanum Oliver, 1968, which was later considered a junior synonym of Pseudorhabdosynochus [9] [28]. Squamodiscs with two innermost rows as closed circles are found in many Pseudorhabdosynochus species and even in species now placed in other genera such as Echinoplectanum Justine & Euzet, 2006 and thus cannot be used for defining a genus [34].
We propose, for these four species, a group which is referred to here as the 'beverleyburtonae group'. The four species are parasitic on groupers of the genus Mycteroperca in the Mediterranean Sea and eastern Atlantic (with P. beverleyburtonae also found in the western Atlantic).
Three species of Pseudorhabdosynochus from groupers in the Mediterranean Sea share vaginal characters with species of the beverleyburtonae group but do not belong to it. These species are: -Pseudorhabdosynochus regius Chaabane, Neifar & Justine, 2015, from M. rubra [6]. This species possesses the same general vaginal structure but it has a straight primary canal (vs curved or S-shaped in species of beverleyburtonae group), a less differentiated trumpet, and the primary and secondary chambers are grouped in a heavily sclerotised structure.
-Pseudorhabdosynochus dolicocolpos Neifar & Euzet, 2007, from M. costae [3]. This species has a well-defined trumpet but the primary canal is extremely long and thin, the primary chamber is very small and smaller than the secondary chamber.
-Pseudorhabdosynochus sinediscus Neifar & Euzet, 2007, from M. costae [3]. This species has a vaginal structure superficially resembling species of the beverleyburtonae group but there is no secondary chamber. In addition, it is distinguished by the lack of squamodiscs. , and, as an outgroup, Lamellodiscus ignoratus Palombi, 1943, a member of the Diplectanidae family to which Pseudorhabdosynochus belongs. In the tree obtained by the NJ method (Fig 9), the two species P. hayet and P. sosia were sister-groups. The genetic distance between species of Pseudorhabdosynochus varied from 13 to 26%, and the distance between P. hayet and P. sosia was 13-14% (Table 4).

Discussion
Gills of groupers (Epinephelidae) harbour numerous diplectanid monogeneans assigned to Pseudorhabdosynochus [1-5, 20, 29, 35-38]. This genus currently contains more than 80 valid species [39] which are generally specialists. Based on the great similarity in the shape of the sclerotised vagina, the primary character for Pseudorhabdosynochus species diagnosis, the 'beverleyburtonae group' is proposed herein to include two new and two previously described species on Mycteroperca spp. in the Mediterranean and Eastern Atlantic Ocean. Several groups of Pseudorhabdosynochus species have already been suggested such as those on Epinephelus spp. in the southern Pacific, namely the 'P. cupatus group' [35] [38,42]. Kritsky et al. 2015 also proposed three groups of Pseudorhabdosynochus species on Epinephelus spp., Mycteroperca spp., and Hyporthodus spp., respectively, in the western Atlantic Ocean [2]. These groups do not have formal systematic value but are useful for distinguishing species among the numerous valid Pseudorhabdosynochus species.
In this paper, we used mainly the morphology of the sclerotised vaginae to differentiate the diplectanid species. However, we added molecular information on two species for which the differentiation was mainly based on differences of measurements, but not on differences of morphology, namely P. sosia and the new species P. hayet. Using multiple and complementary sources of data, i.e. 'integrative taxonomy' [43], is important for better species characterisation and delimitation. In the case of P. sosia and P. hayet, the fact that the hosts are different is additional information advocating different parasite species, but is certainly not sufficient because it has been demonstrated that, in some cases, the same Pseudorhabdosynochus species can parasitize different host fish [44]; in that particular case, the genetic differences between specimens of P. cyanopodus Sigura & Justine, 2008 from two hosts were very low (0-1.5%). The genetic distance between P. sosia and P. hayet was 13-14%, thus lower than the differences observed between other Pseudorhabdosynochus species in our dataset (19-26%), but much higher than the differences between specimens of P. cyanopodus found on two different hosts [44]. The literature on monogenean COI sequences and their interspecific distances is scarce. Vanhove et al. (2013) have emphasized the difficulties encountered in using COI for the differentiation of species in various groups of flatworms, including monogeneans; however, they cite a case in which two valid diplectanid species were divergent by only 3.2% [45]. In other flatworms (geoplanid triclads), such as Microplana spp., intraspecific variation was up to 4%, and interspecific variation was 19% [46], and a difference of 4.8% between Platydemus manokwari haplotypes was considered intraspecific [47]. New species are hypotheses that should be tested [48]. We conclude that the 13-14% difference of COI sequences between P. sosia and P. hayet is indicative of two different species, in addition to morphological differences and different hosts, and that P. hayet should be erected as a new species. A list of 10 grouper-diplectanid species in the Mediterranean Sea has been provided (see Table 1 in [6]), and another species, P. sulamericanus, was recently added [6,7]. The two new species of Pseudorhabdosynochus described here bring the total of species known in the Mediterranean to thirteen; however, the biodiversity of diplectanids on Mediterranean groupers is not yet fully documented.