t i l Ophiotaenia tessellata sp. n. (Eucestoda: Proteocephalinae) from Natrix tessellata (Laurenti, 1768) (Serpentes: Colubridae) in Egypt

Ophiotaenia tessellata sp. n. (Proteocephalidea: Proteocephalinae) is described from the intestine of the dice water snake Natrix tessellata (Laurenti, 1768) (Serpentes: Colubridae) collected from El Faiyoum Governorate, Egypt. Standard methods of collection of the snakes and examination of the cestode tapeworms for taxonomic studies were used. Ophiotaenia tessellata sp. n. was identified and being separable from Ophiotaenia species found in African snakes as well as those from colubrid snakes based on many morphological characteristics. Analysis of a dataset based on 473 bp of its 18S rRNA gene regions was carried out to determine the phylogenetic position of the new species among other proteocephalideans. Ophiotaenia tessellata sp. n. shows a close relationship to O. lapata Rambeloson, Ranaivoson and de Chambrier, 2012 parasite of the endemic snake Madagascarophis colubrinus from Madagascar; both infect African colubrid snakes.

Most species of Ophiotaenia are strictly host-specific (oioxenous sensu Euzet and Combes, 1980), infecting only one species of definitive host (see de Chambrier et al., 2006;Ammann and de Chambrier, 2008;Rambeloson et al., 2012;Scholz et al., 2013) and limited in their distribution to individual continents and/or zoogeographical regions (Freze, 1965). Molecular data have revealed this genus O n l i n e

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as polyphyletic and as many as 10 distinct lineages of Ophiotaenia were found (see de Chambrier et al., 2015). In the present paper, a new species of Ophiotaenia is described from the dice water snake Natrix tessellata (Laurenti, 1768) (Serpentes: Colubridae) in Egypt. New morphological data as revealed by SEM and TEM are provided. To determine the position of the present worm within order Proteocephalidea, we aligned its 18S rRNA gene with a representative selection of proteocephalidean tapeworms.

MATERIALS AND METHODS
A total of 10 dice water snake Natrix tessellata (Laurenti, 1768) were collected from the irrigation canals and the surrounding terrestrial habitat from El Faiyoum Governorate, Egypt, (29°19'38.3" N 30°51'03.4"E) by professional hunters during summer 2015, average temperature 34/21°C (day/night). The snakes were transported alive to the laboratory, where they were euthanized and dissected. Intestinal tapeworms were collected, washed in physiological saline, fixed in 4% hot neutral formalin solution and subsequently stored in 70% ethanol, then stained with hematoxylin and eosin, dehydrated in an ethanol series, cleared with eugenol and mounted in Canada balsam as permanent preparations. Pieces of the strobila were embedded in paraffin wax, longitudinally and transversely sectioned at 12-15μm and stained with hematoxylin and eosin. Eggs were studied in distilled water and illustrated.
In preparation for electron microscopy studies, some tapeworms were transferred to 4% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated through a graded ethanol series; critically point dried. The scolex with the proliferative zone and parts of the strobila were coated with gold and examined using a JEOL scanning electron microscope (JSM-5500LV) at an accelerating voltage of 20kV. Parts of mature tapeworm proglottides were embedded in epoxy resin and a series of ultrathin sections were cut (for further spermatogenesis study) with Leica EM UC6 ultramicrotome, post-stained with uranyl acetate and lead citrate lead citrate. Next, they were examined using a transmission electron microscope at 80kV. Microthrix terminology follows that described by Chervy (2009).
All measurements are given in micrometres unless otherwise indicated. Abbreviations used in the description are as follows: x, mean; n, number of measurements; CS, relative size of the cirrus sac expressed as percentage of its length to the width of the proglottis; GP, genital pore position expressed as percentage of its position to the proglottis length; OV, percent of ovary width to width of the proglottis; ROV, relative size of the ovary, defined as the proportion of its size to the size of the proglottis (see . Paratype of the whole cestode worm has been deposited in the Institute of Parasitology, České Budějovice, Czech Republic (IPCAS) with a collection number C-825.
Gradient temperature PCR runs were conducted in the range of 50 °C to 60 °C to determine the optimal annealing temperature in the final volume of 50μl. The reaction mixture contained 25μl of GoTaq® Green Master Mix (Promega, Cat #M712c, Madison, USA), 5 μl of genomic DNA, 3 μl of each forward and reverse primers (30 pmole), and an amount of nuclease free water corresponding to a final volume of 50μl. The cycling conditions were as follows: 1 cycle at 95°C for 1 min followed by 40 cycles at 94°C for 30 sec, 50-65°C for 45 sec, and 68°C for 60 sec. A final extension was carried out at 68°C for 5 min followed by cooling to 4°C. The amplified DNA regions were analysed with electrophoresis in 1.5% agarose gel in TAE buffer. The most effective annealing temperature was found to be 61.5°C for 18S primers. The separated DNA bands of the expected size were cut from the gel and purified using the QiAquick gel extraction kit (Qiagen, Cat # 28706, Ambion, Courtabeuf, France). The fragments were then cloned into the pGEM®-T Easy vector (Promega, Cat # A1360, Madison, USA) following the manufacturer's instructions. The ligation mixture was used to transform Escherichia coli JM 109 competent cells according to Sambrook et al. (1989), and the positive clones were screened in selective LB/IPTG/X-gal/Ampicillin/agar plates. The plasmids of the mostly white colonies were extracted using the PureYield™ Plasmid Miniprepsystem (Promega, Cat# 1222, Madison, USA), and the presence of the correct insert was judged by PCR using T7 and SP6 universal primers at an annealing temperature of 55°C as described previously.

Sequence alignment and phylogenetic analysis
Sequencing was performed in both directions using either the T7 or SP6 primers obtained from Macrogen Inc. (http://www.macrogen.com/en/main/index.php).

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The sequences were analysed in both directions and assembled using the Seqman PROGRAM (Seqman, version 5.07;DNASTAR, Inc., Madison, WI, USA, 2003). A BLAST search (ver.2.2.30; http://blast.ncbi.nlm.nih. gov/Blast.cgi) was performed to search for similarities between the obtained sequence and previously deposited sequences in the GenBank database. Data derived from the 18S sequence were aligned using CLUSTAL-X multiple sequence alignment according to Thompson et al. (1997) and compared with previously recorded data from GenBank. The alignments were manually corrected using the alignment editor in BioEdit 4.8.9 according to Hall (1999). A phylogenetic tree was constructed using maximum parsimony (neighbour-interchange [CNI] level 3, random addition trees 100). To evaluate the robustness of tree topologies, a bootstrap analysis was performed based on 1000 replicates using MEGA 4.0 according to Tamura et al. (2007). A total of 473 bp of the 18S rRNA gene regions of the examined tapeworm parasite were deposited in GenBank with accession number KJ917783.1. A list of species with hosts, collection sites, associated GenBank accession numbers and percent identity is provided in Table IV, however, few taxonomical changes have taken place: Ophiotaenia gallardi is now Austalophiotaenia gallardi (Johnston, 1911) n. comb. (see de Chambrier et al., 2018).
Testes medullary, in one layer arranged in two lateral fields, not overlapping the cirrus-sac, vagina or vas deferens and occupying 83-92% (x= 88%, n= 10) of the total length Ovary medullary, bilobed, follicular with wide lateral wings of irregular shape, 340-500 wide (x= 427, n= 13) and up to 90 in length; OV 61-76% (x= 68%, n= 13) and ROV 4.1%. Mehlis' gland 55-90 (x= 72, n= 17) in diameter, representing 11-17% (x= 13%, n= 6) of proglottis width (Figs. 1B, C, 2A). Vaginal canal 12-25 (x= 17, n= 12) wide, anterior or posterior to cirrus sac, with terminal part near genital atrium surrounded by intensely staining cells and a circular vaginal sphincter 39-67 (x= 51, n= 9) in diameter that exhibits a ratio of 3:1 to the vaginal canal width (Fig.  1D). Vagina anterior (40%) or posterior (60%) (n= 56) to cirrus sac. Vitelline follicles round to oval, medullary occupying 70-88% (x= 80%, n= 9) of the proglottis length, interrupted at level of cirrus-sac, and extending at a distance of 40-85 (x= 59, n= 39) from the lateral margin of the proglottis and parallel to it. They reach the posterior end of the ovary (Figs. 1B-D, 2A, B). is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 23 nucleotide sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 453 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.
Uterus medullary showing type 1 of development according to de Chambrier et al. (2004b). In immature proglottides, uterine stem is a medial straight longitudinal tube of intensely staining cells. Lumen of uterus appearing in first mature proglottides. Uterine diverticula formed before first eggs appear in uterine stem. In gravid proglottides, uterus occupying up to 47% of proglottis width and reach up to 97% of proglottis length, with 18-30 (x= 23, n= 22) lateral uterine diverticula on each side. Uterus opening ventrally by 4-5 uterine pores in gravid proglottides (Figs. 1B, C, 2B, 3K).
Voucher specimens: Paratype of the whole cestode worm has been deposited in the Institute of Parasitology, České Budějovice, Czech Republic (IPCAS) with a collection number C-825. Etymology: The specific name is derived from the host specific name.

Differential diagnosis
The new species is placed in the genus Ophiotaenia La Rue, 1911 (Subfamily: Proteocephalinae) on the basis of the medullary position of the reproductive organs and vitellaria, the unarmed scolex with four simple uniloculate suckers of normal type and the distribution of testes in two lateral fields (La Rue, 1911;Freze, 1965;Schmidt, 1986;Rego, 1994).
The new species is separable from Ophiotaenia species found in African snakes in the followings: This species is marked by (1) a transverse slit-like structure in the anterior margin of each sucker which could be recognized O n l i n e

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I.S. Gamil and D. Fouad as the opening of gland cells located posteromedially to the suckers, (2) the tumuli formed by the apocrinetype secretion of gland cells which are abundant in the proliferative zone as also observed in the whole strobila, and (3) the digital projections on the embryophore. Ophiotaenia tessellata sp. n. can be distinguished from O. europaea Odening, 1963, both infecting Natrix tessellata, in the possession of a vaginal sphincter, smaller body width (1 versus 2.3 mm), fewer testes (65-135 versus 115-344), smaller ratio of ovary width to proglottis width (61-76% versus 86%) and lower relative size of the ovary (4.1% versus 12.7%) (see , in addition to different type of uterus formation (type 1 versus type 2, see de Chambrier et al., 2004b) (Table I).
Although O. tessellata sp. n. resembles O. georgievi de Chambrier et al., 2010 and O. lapata Rambeloson et al., 2012 infecting African colubrid snakes in the number of testes, the relative size of the cirrus sac, percent of ovary width to proglottis width and the presence of vaginal sphincter, it differs from both species in the scolex width and by the number of embryophore layers around the oncosphere, and from O. georgievi by the presence of digitiform projections on the external surface of the embryophore and from O. lapata by the absence of apical organ and fewer uterine diverticula (18-30 versus 41-68) ( Tables I, III). The new species differs from O. congolensis Southwell and Lake, 1939 in the higher egg diameter (29-33 versus 15) and from O. crotaphopeltis Sandground, 1928, O. nybelini Hilmy, 1936 and sanbernardinensis Rudin, 1917 by a higher scolex width. It differs also from O. dubinini Freze and Scharpilo, 1965 and O. viperis (Beddard, 1913) in the position of the genital pore as well as by a lower percent of ovary width to proglottis width. O. tessellata sp. n. can be distinguished from O. faranciae (MacCallum, 1921), O. gilberti O. joanae (de Chambrier andPaulino, 1997) by the absence of apical organ and by the scolex width. It differs from O. flava Rudin, 1917 andO. hyalina Rudin, 1917 Rudin, 1917 andO. zschokkei Rudin, 1917 infecting the Egyptian cobra Naja haje in Africa; O. tessellata sp. n. differs from both species by the lower body width, lower number and dimension of testes and fewer uterine diverticula.

Phylogenetic analysis
In the present study, the maximum likelihood method was used to construct the phylogenetic tree and representatives of protocephalidea, tetraphyllidea, phyllobothriidea and cyclophyllidea, with strongly supported independent clades. Our phylogenetic analysis, incorporating new and existing data investigated the placement of the examined proteocephalid species within Proteocephalidea.
Pairwise comparison of the isolated genomic sequence from Ophiotaenia tessellata sp. n. with a variety of species and genotypes disclosed unique genetic sequences. Comparison of this novel genetic sequence with others retrieved from GenBank demonstrated a high degree of similarity with range of 91%-99% for 18S rRNA. The results showed that the sequence exhibited the highest percent identity (99%) with Ophiotaenia lapata Rambeloson et al., 2012 (Table IV). Molecular phylogenetic analysis based on 18S rRNA gene sequence of Ophiotaenia tessellata sp. n. shows that it is closely related to O. lapata parasite of the endemic snake Madagascarophis colubrinus from Madagascar and grouped with Australophiotaenia gallardi (Johnston, 1911) n. comb. and an assembly of four Ophiotaenia species infecting snakes (Fig. 4) with a total of three Ophiotaenia species infecting colubrid snakes, in addition, the highest BLAST scores with the lowest divergence values were recorded for O. lapata.
In the present study, although, the present worm records a large total body length in comparison with other Ophiotaenia species found in colubrid snakes and O n l i n e

Present study
Abbreviations: a, absent; gp, gravid proglottis; mp, mature proglottis; on, oncosphere diameter; p, present; CS, relative size of the cirrus-sac expressed as percentage of its length to the width of the proglottis; GP, genital pore position expressed as percentage of its position to the proglottis length; OV, percent of ovary width to width of the proglottis; ROV, relative size of the ovary defined as the proportion of its size to the size of the proglottis (see Table II in Ammann and de Chambrier, 2008 and Table I

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Ophiotaenia tessellata sp. n. (Eucestoda: Proteocephalinae) from Natrix tessellata represents the largest one in all species of Ophiotaenia infecting African colubrid snakes (Table I), its width is somewhat small in relation to its length. O. tessellata sp. n. lacks an apical organ. Similarly, most species of Ophiotaenia infecting colubrid snakes (Table I) as well as those infecting African snakes (see Rambeloson et al., 2012) lack the apical organ. On the contrary, the apical organ was recorded in proteocephalidean tapeworms infecting amphibians, fishes, lizards and snakes (Table III). Numerous acicular filitriches and few gladiate spinitriches were observed covering O. tessellata sp. n. Arredondo et al. (2013) recorded that the gladiate spinitriches is the most frequent microthrix type on the surface of the scolex and strobila in proteocephalidean cestodes and may be either alone or interspersed with filitriches. Filitriches are thought to contribute to the amplification of the absorptive surface of the tegument while the spinitriches in fixation (see Scholz et al., 1999;Žd'árská et al., 2004). The scolex of O. tessellata sp. n. is characterized by the presence of a concentration of cells with granular contents, which may represent gland cells, situated posteromedially to the suckers, with secretions that may participate in the attachment of the scolex between the villi of the host intestine, as previously suggested (see Befus and Freeman, 1973), and recorded (see Gamil, 2012) and explaining the observation of the transverse slit-like structure of the suckers. Cells with granular contents differently located in the scolex and proliferative zone or evidently defined as glands (sometimes as eccrine glands) with ducts transporting their secretory products to the tegument surface have been recorded in the proteocephalideans Nomimoscolex suspectus, Proteocephalus exiguus, P. macrocephalus, P. percae, P. soniae, P. torulosus, Silurotaenia siluri (de Chambrier and Vaucher, 1994;Scholz et al., 1998Scholz et al., , 1999Žd'árská and Nebesářová, 1999;Zehnder et al., 2000;Žd'árská et al., 2004); all these proteocephalideans are fish parasites, in addition to Austalophiotaenia mjobergi (Nybelin, 1917) and Ophiotaenia azevedoi  infecting snakes (de Chambrier et al., , 2018  Abbreviations: a, absent; om, oncospheral membrane; p, present; CS, relative size of the cirrus-sac expressed as percentage of its length to the width of the proglottis; GP, genital pore position expressed as percentage of its position to the proglottis length; OV, percent of ovary width to width of the proglottis; ROV, relative size of the ovary defined as the proportion of its size to the size of the proglottis (see Table I in ; // number of lateral uterine diverticula on each side; † ratio of the length of lateral bands of vitelline follicles to proglottis length and clarified as aporal (ap) and poral (po); ¤ taken from figures in Freze (1965).

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I.S. Gamil and D. Fouad     Abbreviations: a, absent; ao, species with apical organ; ap, aporal; Int., Intermediate type of uterus formation according to de Chambrier et al. (2017a);M, mammal;om, oncospheral membrane;p, present;po, poral;*, according to de Chambrier et al. (2004b); **, in pregravid and/or gravid proglottides; ‡, diameter of the external layer of embryophore, the diameter of the hyaline outer envelope is written in parentheses; ^, on the external surface of the embryophore; §, new combination; •, the uterine aperture is of Crepidobothrium type (see de Chambrier, 1989b) defined as a single longitudinal pore occupying the whole length of the gravid proglottis with narrow internal uterine pores; †, illustrated but not described in text; African species are in bold. The hosts from Litoria aurea to Apalone spinifera are amphibians, and from Clarias cf. anguillaris to Phractocephalus hemioliopterus are fishes, and from Norops trachyderma to Tupinambis teguixin are lizards, and from Pseudechis porphyriacus to Tropidophis cf. taczanowskyi are snakes. with the assumed attachment function. Proteolytic, adhesive and protective functions for cestode scolex gland secretions have also been suggested (see McCullough and Fairweather, 1989). Notably, O. euzeti  infecting the snake Bothrops jararaca is the only species of Ophiotaenia sharing the present worm the posteromedially situation of gland cells to the suckers (although there are no detectable pores to the surface) (de O n l i n e F i r s t A r t i c l e . Such situation and distribution of gland cells was never recorded in any proteocephalidean species infecting colubrid snakes generally and represents one of the three types of gland cells described in proteocephalidean scoleces (see de Chambrier et al., 2017a). Another character for O. tessellata sp. n. is the tumuli (mound-like structures) which are more abundant on the proliferative zone than the whole strobila; tumuli burst and form large pores which seem to be that of gland cells that discharge their secretions by apocrine-like mechanism. Such structures were firstly observed in O. tessellata sp. n. and require further studies for their nature and function. A similar structure has been observed earlier by Threadgold (1965) who had proposed that the bulbous evaginations of the proteocephalidean P. pollanicoli tegument exhibited either excretory or secretory activity; some evaginations swell, burst, and release their contents or are separated as spherical bodies. Tumuli have been also observed on the monticellideans Spatulifer cf. maringaensis Pavanelli and Rego, 1989 and Synbranchiella mabelae Arredondo et al., 2017, and supposed to be formed by the secretory products of unicellular glands (Arredondo and Gil de Pertierra, 2008;Arredondo et al., 2017); Note that all are fish parasites. Nebesářová (1997, 1999) had observed in the apocrine gland type the destruction of secretory projections accompanied with the discharge of the secretion which form a part of the secretory materials. It should also be mentioned that the eccrinelike mechanism of releasing secretory materials is the only type of releasing recorded in proteocephalideans to date (which is elaborated upon earlier in this section).

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In Proteocephalidea, secondary canals branch from ventral osmoregulatory canals and extend mainly in proglottides; they are rarely observed in the scolex and proliferative zone parts (Žd'árská and Nebesářová, 2006). In the present study, secondary osmoregulatory canals are observed in the scolex, proliferative zone and the strobila; such canals open outside by fine pores irregularly scattered all over the worm's body. Interestingly, such excretory pores have been recorded previously in O. nankingensis Hsü, 1935 andO. sanbernardinensis Rudin, 1917, both infecting colubrid snakes, as well as O. adiposa Rudin, 1917 infecting the African snake Bitis arietans (see Freze, 1965) in addition to Ophiotaenia species infecting the Brazilian snake Bothrops jararaca (de Chambrier et al., 1991.
The relative ovarian size in Ophiotaenia sp. n. is 4.1%; such percent is used recently as a novel and useful diagnostic character for proteocephalidean tapeworms by Ammann and de Chambrier (2008) and  who found that the relative ovarian size in species of Ophiotaenia from reptiles from all parts of the world except Europe ranges between 1.5% and 6.7%. The relative ovarian size of O. tessellata spp. n. (4.1%) falls into the range reported for Ophiotaenia spp. It is to be noted that the relative ovarian size in Ophiotaenia species infecting colubrid snakes from all parts of the world ranges from 2.1% in O. faranciae to 9.8% in O. dubinini (Table  I), and that in Ophiotaenia infecting African snakes from 2.1% to 6.4% (see . In O. tessellata sp. n., type 1 of uterus development is recorded and has 18-30 uterine diverticula on each side; Table III shows the presence of type 1, type 2 in addition to the intermediate type of uterus formation (recorded by de Chambrier et al., 2017) in some proteocephalidean tapeworms infecting amphibians, fishes and snakes. In fact, there is a divergent uterine diverticula-range observed in Ophiotaenia species infecting snakes (Tables  I, II) and (Table I in Rambeloson et al., 2012) (Table II). It is worthwhile noting that no precise number of uterine pores is observed, and this number ranges from one to more in proteocephalidean tapeworms from different hosts (Table III), in addition to the specified uterine aperture typical of Crepidobothrium species (see Beddard, 1913;de Chambrier, 1988de Chambrier, , 1989ade Chambrier, , b, 1990de Chambrier et al., 1991).
Although the oncosphere diameter of O. tessellata sp. n. is more or less similar in most species of Ophiotaenia infecting colubrid snakes and the Egyptian cobra (Tables  I, II), it is easy to detect that O. tessellata sp. n. exhibits a greater range of hyaline outer envelope compared to many proteocephalidean tapeworms infecting snakes (Table III). O. tessellata sp. n. is with a 2-layered embryophore sharing O. congolensis, O. crotaphopeltis and O. nybelini (see Southwell and Lake, 1939;Freze, 1965) infecting African colubrid snakes, but it is interesting to highlight that the 3-layered embryophore (provided with an additional thick supplementary layer) has been also recorded in O. georgievi and O. lapata , Rambeloson et al., 2012, both are also infecting African colubrid snakes. The variation in the number of embryophore layers is clearly observable in proteocephalidean tapeworms infecting amphibians, lizards and snakes (Table III) and considered by Rambeloson et al. (2012) as a good discriminant character.
Ophiotaenia tessellata sp. n. shows an evident oncospheral membrane, observed by TEM, surrounding O n l i n e F i r s t A r t i c l e the oncosphere. Although this membrane is one of the four envelopes that are classically described to surround the proteocephalidean eggs (see de Chambrier, 2006), it is poorly recorded or illustrated (Table III) and defined as a thin supplementary layer between the embryophore and oncosphere of Sandonella sandoni eggs (see de Chambrier et al., 2008) which must be differed from the thick layer forming the 3-layered embryophore. The small digitiform projections (outgrowths) on the external surface of the embryophore in O. tessellata sp. n. have been recorded in only two species of Ophiotaenia infecting colubrid snakes: O. lapata and O. nattereri (as fine hooklets or processes, see La Rue, 1914), as well as in few proteocephalideans infecting amphibians, lizards, snakes and the only species infecting a mammal (Table III). The cluster form of the egg recorded in the majority of Australian proteocephalideans from varanids and snakes (see de Chambrier, 2006;Jones and de Chambrier, 2016;de Chambrier et al., 2018) and the only species infecting mammal (Cañeda-Guzmán et al., 2001) was not observed from any species of Ophiotaenia from colubrid snakes.
Ophiotaenia is a species-rich genus, and in particular, phylogenetic analyses indicate that the genus is polyphyletic and include assemblages of distantly related taxa with similar morphology, apparently as a result of convergent evolution (de Chambrier et al., 2017b). The most comprehensive molecular phylogenetic analysis, based on 28S rDNA revealed the grouping of Ophiotaenia spp. in three main clades by de Chambrier et al. (2015) who added that all these Ophiotaenia species do not differ significantly in their morphology except for that the species of Ophiotaenia of clade K possess type 1 uterus whereas those in the other two clades (N and O) have type 2. A major interest has been in determining that Ophiotaenia tessellata sp. n. seems to belong to the clade K, whereas O. europaea parasitizing Natrix maura in Europe belongs to the clade O  and previously it did not group with any other species from its genus (Zehnder and Mariaux, 1999). In the present study, a close phylogenetic relationship between O. tessellata sp. n. and O. lapata (both are found in African colubrid snakes) was observed. The 18S rRNA regions yielded congruent results for the taxonomic position of O. tessellata sp. n.; it has a unique genetic sequence embedded in the genus Ophiotaenia and exhibits a close relationship with O. lapata as a putative sister taxon.

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Ophiotaenia tessellata sp. n. (Eucestoda: Proteocephalinae) from Natrix tessellata