Two new cestode species of Tetragonocephalum Shipley & Hornell, 1905 (Lecanicephalidea, Tetragonocephalidae) from Himantura randalli Last, Manjaji-Matsumoto & Moore (Myliobatiformes, Dasyatidae) from the Gulf of Oman

Abstract The original description of the genus Tetragonocephalum was published more than one hundred years ago but its taxonomic status was clarified only recently. To date, approximately 30 nominal species of this genus have been described, mostly from the northern Indian Ocean, but nearly half of them are invalid and only 14 species are recognized as valid. In the present study two new species of Tetragonocephalum are described from the spiral intestine of Himantura randalli from off Jod, on the northern coast of the Gulf of Oman. Tetragonocephalum sabae sp. n. is distinguishable from the valid species of Tetragonocephalum based on number of proglottids (43−53), number of testes (42−50), and size of scolex (401−453×328−455), acetabula (87−109×72−116), mature proglottids (802−1,333×226−336), cirrus sac (92−160×103−154), and eggs (16−19×11−13). Tetragonocephalum salarii sp. n. can be distinguished from Tetragonocephalum sabae sp. n. and all other valid species of Tetragonocephalum based on number of proglottids (77−86). Furthermore, it differs from its congeners based on a combination of some characteristics, including the number of mature (3−7) and gravid (18−20) proglottids, the number of testes (30−38), and the size of acetabula (84−111×80−96), mature proglottids (497−833×334−403), gravid proglottids (1,036−1,482×440−575), testes (20−34×31−50), ovary (123−215×210−278), and eggs (24−45×13−21).

The only study on Tetragonocephalum from the Gulf of Oman was conducted by Golestaninasab et al. (2014) who showed that the genus Tetragonocephalum can act as a heavy metal bioindicator in the marine environment.
The present article is the first taxonomic study of the genus Tetragonocephalum from the Gulf of Oman. We describe two new species of this genus collected from the spiral intestine of the Arabian banded whipray, Himantura randalli Last, Manjaji-Matsumoto & Moore, 2012.

Materials and methods
A total of 36 specimens of H. randalli was collected from northern waters of the Gulf of Oman, 29 individuals in May 2011 and seven in October 2012. They were caught by local fishermen off Jod, Zarabad, Iran (25°26'58"N, 59°30'29"E). Each specimen was given a unique collection number for author reference. All host individuals were photographed and morphometric and morphological characteristics were recorded to facilitate species identification. Species identity was confirmed using Naylor et al. (2012), Last et al. (2012), and Henderson et al. (2015).
Host specimens were dissected along the mid-ventral line; spiral intestines were removed and opened by a longitudinal incision. Subsequently, spiral intestines were fixed in 10% seawater buffered formalin, shaken vigorously, and held for approximately seven days. The samples were then transferred to the Zoology Laboratory, School of Biology, University of Tehran for detailed examination of parasitic infection. Spiral intestines and intestinal contents were examined under a stereomicroscope. Tapeworms were carefully removed from the spiral intestine and washed in distilled water for about one hour before being preserved in 70% ethanol. Parasite specimens were prepared as whole mounts for light microscopy observation according to Koch et al. (2012).
Whole mounts were studied using a Leica DM500 light microscope. Images of Tetragonocephalum specimens were taken using a Leica ICC50 HD color digital camera mounted on the Leica DM500 light microscope (Buffalo Grove, Illinois, United States) and measurements were taken using the image analysis software Leica Application Suite (LAS EZ v.3.0.0) (Leica 2013). Measurements were analyzed in IBM® SPSS® Statistics Package v.22 (IBMCorp. 2013). All measurements of the reproductive organs were taken from mature proglottids. Measurements are given in micrometers (µm) unless otherwise indicated; they are given as the range followed in parentheses by the mean, number of worms examined, and the total number of measurements if more than one measurement was taken per worm. Illustrations were prepared with Adobe® Illustrator® CC (Adobe Incorp. 2013) based on the drafts sketched under a Reichert Biovar microscope with the aid of a drawing tube.
Some scoleces were prepared for ultrastructural studies using SEM following the protocol of Jensen (2005). The specimens were sputter coated with approximately 10 nm of gold/palladium, and examined with a field emission scanning election microscope (HIT4160102, Hitachi, Tokyo, Japan) at the School of Electrical and Computer Engineering (ECE), University of Tehran. Microthrix terminology follows Chervy (2009).
Type and voucher specimens are deposited at The Zoological Museum, University of Tehran, Tehran, Iran (ZUTC), and The Natural History Museum, London, England (BMNH).

Description (Figures 1 and 3a−d).
Based on three whole mounts of gravid specimens and two scoleces prepared for SEM and their vouchers (partially measured).
Etymology. This species is named in honor of the first author's wife, Saba Saadati Safa, for her unconditional support and patience over the last five years.
Remarks One scolex for SEM, ZUTC Platy. 1549s (one stub) and its whole-mounted voucher, ZUTC Platy. 1549v (one slide), from the same host individual as the holotype.
Description (Figures 2 and 3e−h). Based on three whole mounts of gravid specimens and one scolex prepared for SEM and its voucher (partially measured).
Etymology. This species is named in honor of Mr. Naser Salari in gratitude for his assistance with the collection of host specimens.
Remarks. Tetragonocephalum salarii sp. n. possesses the characteristics of the genus and can be distinguished from T. sabae sp. n. and all 14 valid congeners based on the following characteristics. Tetragonocephalum salarii sp. n. differs from T. sabae sp. n. in the total number of proglottids (

Discussion
The genus Tetragonocephalum possesses the following characteristics: unique bisaccate uterus, acraspedote strobila, testes distributed anterior to the cirrus sac, ovary C-shaped in cross-section, conspicuously enlarged genital atrium and pore (Ivanov and Campbell 2000, Jensen 2005, Jensen et al. 2016. The specimens described here as two new species of Tetragonocephalum from H. randalli from the Gulf of Oman are consistent with the current concept of the genus. The genus Tetragonocephalum has a very controversial taxonomic history (Jensen 2005). After being erected (Shipley and Hornell 1905), between 1906 and 2000, the taxonomic status of this genus remained uncertain, accepted as valid by some authors, but only as a synonym of Tylocephalum Linton, 1890 by others (Ivanov andCampbell 2000, Jensen 2005). The validity of Tetragonocephalum was confirmed by Ivanov and Campbell (2000) in a revision of this genus and Tylocephalum. Further on, Jensen (2005) revised the order Lecanicephalidea, and Jensen et al. (2016) carried out a comprehensive molecular phylogenetic study of the order Lecanicephalidea. In the most recent revision of Tetragonocephalum, Jensen (2005) considered this genus to consist of 14 valid species.
The fourteen valid and the current two new species of Tetragonocephalum differ from each other based on the morphometric characteristics, including the number of testes, mature, and gravid proglottids and the size of the scolex, acetabula, testes, cirrus sac, ovary, eggs, mature and gravid proglottids.
The two new species were collected from different host individuals. One out of twenty-nine host specimens collected in May 2011 was parasitized by T. sabae sp. n. (n = 5) and one out of seven in October 2012 by T. salarii sp. n. (n = 4). These cestodes appear to be rare in the waters near Jod, Iran, Gulf of Oman. The occurrence of these species does not appear to be by chance, as collections were made over two years and at two different seasons. Furthermore, these host records do not appear to be aberrant or due to accidental infection because fish of the genus Himantura are known hosts for Tetragonocephalum spp. (Jensen 2005, Jensen et al. 2016, and many members of the Lecanicephalidea tend to have a high degree of host-specificity. This is often recorded for congeneric hosts; for example, Tylocephalum in the genus Rhinoptera Cuvier (see Ivanov and Campbell 2000) or Hexacanalis Perrenoud, 1931 in the genus Gymnura van Hasselt (Cielocha and Jensen 2011).
In order to compare the new and valid species, the original descriptions of valid ones were used. However, there are some limitations and uncertainties which undermine comprehensive and detailed comparisons. Some original descriptions are incomplete and lacking important morphometric data. For example, there are no appropriate measurements and/or drawings of the internal organs in the original descriptions of T. trygonis and T. uarnak Hornell 1905, 1906); Pintner (1928) introduced T. simile as a new species without formal description and provided five drawings. Another problem in comparisons between different congeneric species of Tetragonocephalum is unreliable data provided in some original descriptions. Considering the descriptions of previous species, it seems that some measurements might be incorrect; for example, the maximum width of T. trygonis (30 at scolex [probably 300?]) (Shipley and Hornell 1905). As a result, such measurements should be treated with caution. Furthermore, it appears that some features that are common to different species of the genus Tetragonocephalum were described differently in various species, such as absence of cephalic peduncle, spatial pattern of testes, shape of ovary, and armature of cirrus. Also, there are some features, which have not been taken into account so far and might be important in species identification, such as the attachment pattern of apical organ to scolex proper modification and strobila to scolex proper, the degrees of overlap between fields of testes and anterolateral vitelline follicles, and the histological structure of testes.
In conclusion, the problematic taxonomic status of some previously described species, due to inappropriate and/or incomplete descriptions, and type materials which are either unspecified or missing, make it necessary to designate neotypes and redescribe all previously described species from the type hosts and localities, except for T. passeyi which is thoroughly described by Jensen (2005).