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Four new species of Acanthobothrium van Beneden, 1849 (Cestoda: Onchoproteocephalidea) from the spotted skate, Raja straeleni Poll, off the Western Cape, South Africa

The examination of eight spotted skates, Raja straeleni Poll, resulted in the discovery of four new species of Acanthoboth­ rium van Beneden, 1849, namely A. microhabentes sp. n., A. microtenuis sp. n., A. crassus sp. n., and A. dolichocollum sp. n., located off the Western Cape of South Africa. With a total of over 200 valid species of Acanthobothrium recognised worldwide, the use of an integrative approach becomes imperative in the interest of simplifying interspecific comparisons between congeners. In accordance with this, the four new species were incorporated into the category classification system established by Ghoshroy and Caira in 2001, where they were identified as category 2 species, which, at present, includes 47 recognised species of Acanthobothrium. Nevertheless, each of the four new species exhibits postovarian testes, a most intriguing and highly unusual feature among Acanthobothrium, instantly differentiating them from most congeners. This feature has been reported in 12 congeners, which have previously been considered to be restricted to waters of the Indo-Pacific Ocean. Not only do the four new congeners represent the first species of Acanthobothrium reported from southern Africa, but they also represent the first reported species with postovarian testes from the southern Atlantic Ocean. Regarding the legitimacy of the four new species, only two other category 2 species are reported to exhibit this feature, namely A. popi Fyler, Caira et Jensen, 2009, and A. bobconniorum Fyler et Caira, 2010, to which the four congeners were compared to. Acanthobothri­ um microhabentes sp. n. is the smallest of the congeners and differs from A. popi and A. bobconniorum by having fewer testes and postovarian testes, a shorter body, fewer proglottids, a shorter scolex, and longer cephalic peduncle. Acanthobothrium microtenuis sp. n. differs from A. popi and A. bobconniorum by having fewer testes and postovarian testes, a shorter scolex, longer cephalic peduncle, and the possession of columnar spinitriches on the anterior region of the terminal proglottid. Acanthobothrium crassus sp.n. differs from A. popi and A. bobconniorum by having fewer postovarian testes, a narrower cirrus-sac, larger vitelline follicles, and a longer cephalic peduncle. Acanthobothrium dolichocollum sp. n. is the longest of the four new species and differs from A. popi and A. bobconniorum by having fewer postovarian testes, more postporal testes, a larger body, more proglottids, larger testes and vitelline follicles, and an exceptionally long cephalic peduncle. Apart from differences in overall size, the four new species differ in a combination of measurements for the scolex, vitelline follicles, muscular pad and cephalic peduncle, and the number of proglottids and testes. The four species were recovered from a previously unexplored host and locality, expanding the host associations and geographical distribution of the genus.

al. 2017), and have been reported in four of the seven orders of sharks. Species parasitising sharks typically occur in carcharhiniform, heterodontiform and squaliform sharks .
Although having a cosmopolitan distribution, the majority of records of onchoproteocephalideans from elasmobranchs were derived from species of Acanthobothrium van Beneden, 1849 in tropical and subtropical waters . Acanthobothrium is the species-richest genus of all elasmobranch-infecting cestode genera (Maleki et al. 2015, Caira et al. 2018). The genus is characterised by a scolex with four bothridia, each containing one pair of bi-pronged hooks.
Despite the large number of valid species of the genus, it is estimated that an additional 800, if not more, species of Acanthobothrium are yet to be discovered worldwide . One region that potentially hosts many undescribed species of Acanthobothrium is the extremely biodiverse, biogeographical realm of Temperate southern Africa (sensu Spalding et al. 2007). The diversity, biogeography and biology of elasmobranchs off southern Africa have been well documented, with 204 elasmobranch species reported from this region (Ebert and van Hees 2015). However, only 18 of these species have been examined for cestode infections (Schaeffner and Smit 2019).
As part of a larger study on the biodiversity of marine fish parasites of southern Africa, specimens of Acantho bothrium were collected from Raja straeleni Poll (Rajidae) off the Western Cape of South Africa. This study provides information on the taxonomic status of species belonging to the genus Acanthobothrium, based on light and scanning electron microscopy, leading to the description of four species new to science.

MATERIALS AND METHODS
Eight specimens of Raja straeleni, seven males and one female, were collected by longline. One specimen was collected during October 2018 from Vermont (-34.4927, 19.4136), whereas the other seven specimens were collected in February 2019 from Walker Bay, Hermanus (-34.5283, 19.4102), Hawston (-34.4419, 19.3697), and De Kelders (-34.5925, 19.2925). Figure 1 indicates the sampling localities from which each species was obtained. Ethical approval for this project was received from the North-West University Animal Care, Health and Safety, Research Ethics Committee (NWU-AnimCareREC) with ethics number NWU-00065-19-A5. Permits for the collection and possession of batoid specimens for the purpose of research were issued by the South African Department of Agriculture, Forestry and Fisheries (permit nos. RES2019/45 and RES2019/105 issued to BCS; RES2019/58 and RES2019/61 issued to the South African Shark Conservancy).
Skates were sacrificed by means of an adjunctive procedure of trauma to the neurocranium, followed immediately by pithing of the brain. The body cavity was opened with a mid-ventral incision to remove the spiral intestine. Spiral intestines and their contents were fixed in hot, 4% formalin for morphological observations, and ultimately transferred to 70% ethanol. Specimens of Acanthobothrium were hand-picked from the spiral intestines and contents. They were hydrated in a graded ethanol series and stained in Delafield's haematoxylin, dehydrated in a graded ethanol series, cleared in clove oil, and permanently mounted on microscope slides using Canada balsam. Images of various internal organs and characteristic body structures used for measurements were acquired using a Nikon Y-TV55 video camera mounted on a Nikon ECLIPSE Ni microscope (Nikon, Tokyo, Japan). Image analysis software Image Pro Express (Nikon, Japan) was used to obtain all necessary measurements for descriptive analyses.
All measurements were taken according to specifications given by Ghoshroy and Caira (2001). Measurements are given both in text descriptions as well as in tabular form. Text descriptions include the range of measurements taken, whereas Table 1 presents metrical data as the mean, followed by standard deviation and number of specimens examined. Measurements are all presented in micrometres, with the exception of the total length, which is presented in millimetres. Drawings were made by the use of a drawing attachment tube.
Two to three specimens of each species were used for scanning electron microscopy (SEM). Each specimen selected for SEM observation was cleaned by lightly brushing the surface of the scolex. Cleaned specimens were then dehydrated in a graded ethanol series and either placed in hexamethyldisilisane (HMDS) overnight, or dried by critical point drying. Specimens were mounted onto carbon tape, placed on aluminium stubs and sputter-coated with either 20 to 30 nm of gold/palladium (SPI-module, SPI Supplies, Pennsylvania, USA), or first with carbon (Emscope TB500, Quorum Technologies, Puslinch, Ontario, USA), followed by 20 to 30 nm gold/palladium (Eiko IB2 ion coater, Eiko, Japan).
Specimens were examined with a Phenom Pro Desktop scanning electron microscope (ThermoScientific, Waltham, Massachusetts, USA) and a FEI Nova NanoSEM 450 scanning electron microscope (FEI, Hillsboro, Oregon, USA). Images were taken of similar regions of the strobila and scolex, aiding in microthrix comparisons among species. Images of microtriches of immature proglottids were taken directly posterior to the cephalic peduncle. The presence of additional microthrix morphologies on the terminal proglottid was noted in which case images are also provided.
The specific regions of micrographs taken are indicated on SEM plates for each of the respective species. Species allocation followed the category classification system developed by Ghoshroy and Caira (2001) to facilitate the differentiation among congeners. Following this classification system, species were characterised and assessed based on four morphological features: total length (< or > 15 mm), number of proglottids (< or > 50 proglottids), number of testes (< or > 80), and symmetry or asymmetry of aporal and poral ovarian lobes. Allocating each species to a specific category consequently simplifies differentiating between congeners. Accordingly, the four new species were incorporated into this system and compared particularly to the described species assigned to the same category as each of the four new species. Since members of different categories differ from one another in either one or more of the four morphological features constituent of this classification system (Fyler and Caira 2006), this subsequently warrants the elimination of congeners designated to different categories.
Specimens have been deposited in the helminthological collection of the National Museum, Bloemfontein, South Africa (NMB), the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (IPCAS), and the Natural History Museum, Geneva, Switzerland (MHNG-PLAT). Specimens used for SEM were retained in the parasite collection of the Water Research Group, North-West University, South Africa.

RESULTS
Four morphologically distinct species of Acanthobothri um were collected from subsets of eight specimens of Raja straeleni measuring 64.9-74.2 cm in length, disc widths of 45.8-53.4 cm, and a weight of 1,820-6,630 g ( Table  2). Of the eight skates, two were parasitised by all four morphotypes, two host specimens by three morphotypes, two by two morphotypes and two skates with a single morphotype. All four morphotypes were present in skates from Vermont, Walker Bay and De Kelders, but only a single morphotype was found in the skate collected at Hawston. The only female caught during the present study was parasitised by only one of the four morphotypes (Table 2).
Testes conspicuous in mature proglottids, irregularly oval in dorsoventral view, 32-55 long by 23-35 wide, arranged in two regular columns, one layer deep, 21-31  in total number, 4-7 in postporal field, 1-2 in postovarian field. Cirrus-sac ovoid, bent posteriorly, 57-80 long by 32-47 wide, containing armed cirrus; cirrus greatly expanded at base. Vagina narrow, relatively thin-walled and straight proximally, extending from ootype along medial line of proglottid to posterior margin of cirrus-sac, then laterally to common genital atrium. Ovary takes up approximately half proglottid length, stopping short of posterior margin of proglottid, H-shaped in dorsoventral view, lobulated. Ovary asymmetrical, poral lobe 243-444 in length, aporal lobe 210-525 in length, both lobes stopping before cirrus-sac; width of ovary at level of ovarian isthmus 54-76; ovarian isthmus located slightly posterior to mid-level of poral lobe of ovary. Mehlis' gland posterior to ovarian isthmus. Vitellarium follicular, in form of two lateral bands each consisting of two columns of relatively large follicles; bands extending from posterior margin of anterior-most testes to near posterior margin of ovary, interrupted by vagina and cirrus-sac; vitelline follicles 6-13 long by 10-24 wide. Vitelline follicle length relative to testis length 0.1-0.2 : 1. Uterus thin-walled, extending from ovarian isthmus to near anterior margin of proglottid. Eggs not observed. T y p e m a t e r i a l : Holotype deposited at NMB (P-604), paratypes in NMB (P-605-P-606), IPCAS (C-848) and MHNG (PLAT-137324, 137334). E t y m o l o g y : The species name "microhabentes" is derived from Latin ("micro", small; "habentes", stumpy) and relates to the small size of the specimens.
Remarks. Acanthobothrium microhabentes sp. n. is identified as a category 2 species according to specifications set by Ghoshroy and Caira (2001). At present, 47 species of Acanthobothrium recognised as category 2 species were reported worldwide (Maleki et al. 2013, Yang et al. 2016, Zaragoza-Tapia et al. 2019. Additionally, Franzese and Ivanov (2020) recognised two more species belonging to both category 1 and 2 as a result of intraspecific variability observed in ovarian symmetry. For the purpose of a more reliable comparison, the latter is considered as a category 2 species in this study. Acanthobothrium microhabentes fea-tures the presence of testes located posterior to the ovarian isthmus, therefore clearly distinguishing it from 45 of the recognised category 2 species, as well as the additional two species recognised by Franzese and Ivanov (2020).
Vagina narrow, relatively thin-walled and coiled proximally, greatly expanded distally, extending from ootype along medial line of proglottid to anterior margin of cirrus-sac, then laterally along anterior margin of cirrus-sac to common genital atrium. Ovary takes up approximately half of proglottid length, stopping short of posterior margin of proglottid, H-shaped in dorsoventral view, lobulated. Ovary asymmetrical, poral lobe 340-523 in length, aporal lobe 350-610 in length, both lobes extending past cirrus-sac; width of ovary at ovarian isthmus 95-116; ovarian isthmus located slightly posterior to mid-level of poral lobe of ovary. Mehlis' gland posterior to ovarian isthmus.
Vitellarium follicular, in form of two lateral bands, each consisting of two columns of relatively large follicles; bands extending from posterior margin of anterior-most testes to near posterior margin of ovary, interrupted by vagina and cirrus-sac; vitelline follicles 11-21 long by 20-34 wide. Vitelline follicle length relative to testes length 0.2-0.3 : 1. Uterus thin-walled, sacciform, extending from ovarian isthmus to near anterior margin of proglottid. Eggs not observed. Latin ("micro", small; "tenuis", slender) and relates to the small and slender body shape of specimens.
Vagina narrow, relatively thin-walled and coiled proximally, greatly expanded distally, extending from ootype along medial line of proglottid to anterior margin of cirrus-sac, then laterally to common genital atrium. Ovary takes up approximately half of proglottid, stopping short of posterior margin of the proglottid, H-shaped in dorsoventral view, lobulated. Ovary asymmetrical, poral lobe 440-652 in length, aporal lobe 464-711 in length; both lobes extending shortly past cirrus-sac; width of ovary at ovarian isthmus 98-115; ovarian isthmus located slightly posterior to midpoint of poral lobe of ovary. Mehlis' gland posterior to ovarian isthmus.
Vitellarium follicular, in form of two lateral bands, each consisting of two columns of relatively large follicles; bands extending from posterior margin of anterior-most testes to near posterior margin of ovary, interrupted by vagina and cirrus-sac; vitelline follicles 12-19 long by 32-38 wide. Vitelline follicle length relative to testes length 0.2-0.3 : 1. Uterus thin-walled, sacciform, extending from ovarian isthmus to near anterior margin of proglottid. Eggs not observed. Remarks. Acanthobothrium crassus sp. n. following the classification criteria set by Ghoshroy and Caira (2001), is identified as a category 2 species. By including both A. microhabentes sp. n. and A. microtenuis sp. n., A. crassus is among the 50th category 2 species reported to date. Acanthobothrium crassus differs from all but four of its congeners in the possession of postovarian testes. Acan thobothrium crassus is distinguished from A. bobconnio rum, A. microhabentes, A. microtenuis, and A. popi by a combination of morphological features. One such feature is the presence of craspedote proglottids in A. crassus, whereas its congeners possess acraspedote proglottids.
Vagina narrow, relatively thin-walled and extensively coiled proximally, thick-walled and greatly expanded distally, extending from ootype along medial line of proglottid to anterior margin of cirrus-sac, then laterally along anterior margin of cirrus-sac to common genital atrium. Ovary takes up almost half of length of proglottid, stopping short of posterior margin of proglottid, H-shaped in dorsoventral view, lobulated. Ovary asymmetrical, poral lobe 538-692 in length, aporal lobe 565-689 in length, both lobes extending past cirrus-sac; width of ovary at ovarian isthmus 108-120; ovarian isthmus located slightly posterior to mid-level of poral lobe of ovary. Mehlis' gland posterior to ovarian isthmus.
Vitellarium follicular, in form of two lateral bands, each consisting of two columns of relatively large follicles; bands extending from posterior margin of anterior-most testis to near posterior margin of ovary, interrupted by vagina and cirrus-sac; vitelline follicles 17-23 long by 34-41 wide. Vitelline follicle length relative to testes length 0.3-0.4 : 1. Uterus thin-walled, sacciform, extending from ovarian isthmus to near anterior margin of proglottid. Eggs not observed.  -137342, 137343). E t y m o l o g y : The species name "dolichocollum" is derived from Latin ("dolicho", elongate; "collum", neck) and relates to the unique morphological characteristic of the species i.e., its elongate cephalic peduncle.

DISCUSSION
Sampling efforts focussed on parasitological observations of elasmobranchs off South Africa have been extremely sparse Hadfield 2015, Schaeffner andSmit 2019). This particularly applies to the spotted skate, Raja straeleni, a relatively common species along the western and southern coastlines of southern Africa that has only once been examined for cestode infections (see Caira et al. 2014). Caira et al. (2014) discovered a new diphylidean cestode, a species that has not been encountered in the present study. On a global scale, a total of 18 species of Acanthobothrium have thus far been reported from 12 of the 23 valid species of Raja Linnaeus (Global Cestode Database 2020), clearly expressing the fact that host species are not limited to the infection of merely a single species of Acanthobothrium. Not only has this been previously reported in multiple studies , Fyler and Caira 2010, Maleki et al. 2015, but this observation was also reflected in the present study, where four new species have been described from a single host species.
The present study marks the first to report species of Acanthobothrium from South Africa. With the addition of these four species, a total of 207 valid species of Acan thobothrium are currently recognised worldwide , Franzese and Ivanov 2018, Maleki et al. 2018, Rodríguez-Ibarra et al. 2018, Zaragoza-Tapia et al. 2019, making the taxonomic classification of new species, and intrageneric comparisons between congeners within this genus taxing (Ghoshroy and Caira 2001). Hence, in the interest of simplifying this matter, Ghoshroy and Caira (2001) have developed a category classification system on the basis of four morphological characteristics. The South African species all measure less than 15 mm in total length, with fewer than 50 proglottids, fewer than 80 testes, and an asymmetric ovary, designating all of them as category 2 species.
Comparisons between the four species presented in the current study suggest the possibility that these congeners could be each other's closest relatives. This assumption, however, is not only grounded upon the fact that these species resembled each other in numerous morphological characteristics, but also due to the presence of four synhospitalic congeners parasitising the same host species (Caira and Tracy 2003, Caira et al. 2004, Fyler and Caira 2006. To ensure that the South African species are in fact new to science, interspecific comparisons were made between all category 2 species recognised worldwide.
With the addition of the South African species, the total number of species of Acanthobothrium allocated to category 2 increased to 51 species. With this still being quite a substantial number of species, the South African congeners are very easily distinguished from all except two of these congeners by the presence of postovarian testes, undoubtably being a very interesting, yet highly unusual feature among species of Acanthobothrium (see Fyler et al. 2009, Fyler and Caira 2010, Maleki et al. 2015. In point of fact, this feature has been previously documented in merely 6% (12 out of 203) of all species of Acanthobothrium known to infect elasmobranchs (Subhapradha 1955, Robinson 1959, Campbell and Beveridge 2002, Fyler and Caira 2006, Reyda and Caira 2006, Twohig et al. 2008, Fyler and Caira 2010, Maleki et al. 2015.
The South African congeners not only parasitise the same host species, but also share the rare feature of postovarian testes with 12 other species of Acanthobothrium. This makes the possibility of their relatedness even more evident. Given the fact that two other species with postovarian testes have also been documented in two other species of Raja (i.e. Zearaja nasuta [Müller et Henle], and Spiniraja whitleyi [Iredale]), the four new congeners might not only be grouped together as each other's closest relatives, but also with the 12 species of Acanthobothrium exhibiting the same unique feature, indorsing the notion that these species might comprise their own monophyletic clade (Fyler and Caira 2010). Moreover, as this is the first time that species of Acanthobothrium have been reported in R. straeleni, we can assume that this is the result of a single host invasion. Conversely, molecular analyses conducted by Fyler et al. (2009), including numerous synhospitalic congeners, concluded that these species do not represent closely related lineages. Fyler and Caira (2010) conducted a similar study, subsequently reaching the same conclusion.
Phylogenetic trees do not only serve insights on host associations but also on the geographical distribution of species. According to earlier studies (Goldstein 1967, Williams 1969, Ghoshroy and Caira 2001, Fyler 2009), congeners displaying the same geographical affinities are more likely to group together. Surprisingly, species of Acantho bothrium parasitising the same host species of different geographical localities do not group in the same clade (Fyler 2009).
If a phylogenetic tree would display the four new species as distinct or independent lineages it would indicate that the infection of R. straeleni does not resemble a single host invasion as previously suggested, but rather be the result of multiple disparate host invasions (Fyler 2009). Regardless of the host's specific nature, extensive host substitutions may have occurred throughout a much longer evolutionary time, accordingly proceeding unnoticed during shortlived investigations (Fyler 2009). The combined effect of different geographical affinities as well as host substitution possibly resulted in an extraordinary host specificity, as they have been documented from eight of 11 elasmobranch orders .
This is truly intriguing, considering the fact that the South African species are the first species to possess postovarian testes outside of the Indo-Pacific realm. Furthermore, the host from which the species were obtained, R. straeleni, has only been documented to populate the Eastern Atlantic Ocean from the coastline of the western Sahara to southern Africa. Nevertheless, regardless of geographical affinities, the possibility of potential relatedness between species of Acanthobothrium possessing this rare feature still persists, supported by the fact that two of these species (i.e. A. popi and A. zimmeri) have already grouped together, during a molecular study performed by Fyler et al. (2009). Unfortunately, as of yet, the degree of relatedness among these unique species is still unknown, recognising the need for further investigation and a collection of additional specimens of R. straeleni from the same localities. A more conclusive molecular analysis including both the South African and Indo-Pacific species is furthermore fundamental in defining their phylogenetic relationships, informing us on whether these species do, in fact, form a monophyletic clade.
In addition, a molecular phylogenetic analysis on newly collected specimens will reveal their phylogenetic position and will highlight new perspectives on the evolutionary relationships between these cestodes and their elasmobranch hosts. With merely 9% (18 of 204) of elasmobranchs (Ebert and van Hees 2015, Schaeffner and Smit 2019) off southern Africa examined for cestode infections, it is also expected that the species diversity of cestodes infecting elasmobranchs in South African waters is much higher than currently reported (Schaeffner and Smit 2019) and future large-scale surveys on elasmobranch parasites will, without any doubt, lead to numerous new species discoveries. Caira and Jensen (2017) estimated that 5,126 species of cestodes infect approximately 1,269 elasmobranch species worldwide, representing an average of four cestode species per elasmobranch host. As a consequence, the diversity of cestodes in the waters of South Africa is predicted to be either equal to, or, more likely, greater than the diversity of the chondrichthyan hosts present in these waters (Schaeffner and Smit 2019). Nevertheless, the current extensive and rapid rate of loss in biodiversity, not just in this region, but globally, limits the time remaining for the discovery and description of the cestode diversity. However, since species of Acanthobothrium are known to parasitise multiple elasmobranch orders, along with the ability to switch hosts, parasites of this genus could possibly be able to avoid extinction. Nonetheless, when accounting other cestode orders and genera parasitising elasmobranchs (i.e. cestodes without the ability to switch hosts), the loss of parasite diversity would then likely be higher than that of their hosts (Dobson et al. 2008). This is alarming given this will likely result in the extinction of numerous cestode species before they have even been discovered.
New, sustainable conservation schemes should be enforced, including a combination of multidisciplinary approaches. Hereby, information can be gained from parasites, providing us with a better understanding of the host, such as its evolutionary and demographic history and migratory patterns (Gomez and Nichols 2013). Collections of additional hosts and localities, such as the ocean basins surrounding southern Africa, which have previously not been properly explored, are required in this regard. New studies should also be concurrent with previously applied methods (i.e. taxonomy, molecular systematics) and the incorporation and implementation of novel, multisource approaches (i.e. biogeography, ecology, conservation science, ecotoxicology). When we consider to use these parasites as indicator organisms for pollution and global change, we may yield a much better understanding on the evolutionary interrelationships of host-parasite systems present within our world's remarkable ecosystems, contributing to various scientific fields, and ultimately contributing to the conservation of declining populations of endangered species and positive environmental change.