Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2018, 65: 014 doi: 10.14411/fp.2018.014 http://folia.paru.cas.cz Research Article Two new species of Stillabothrium (Cestoda: Rhinebothriidea) from stingrays of the genus Fontitrygon from Senegal Elsie A. Dedrick1, Florian B. Reyda1, Elise K. Iwanyckyj1, Timothy R. Ruhnke2 1 Biology Department & Biological Field Station, State University of New York, College at Oneonta, Ravine Parkway Oneonta, NY, USA; 2 Department of Biology, West Virginia State University, Institute, WV, USA Abstract: Morphological and molecular analyses of cestode specimens collected during survey work of batoid elasmobranchs and their parasites in Senegal revealed two new species of the rhinebothriidean cestode genus Stillabothrium Healy et Reyda 2016. Stillabothrium allisonae Dedrick et Reyda sp. n. and Stillabothrium charlotteae Iwanyckyj, Dedrick et Reyda sp. n. are both de- scribed from Fontitrygon margaritella (Compagno et Roberts) and Fontitrygon margarita (Günther). Both new cestode species overlap in geographic distribution, host use and proglottid morphology, but are distinguished from each other, and from the other seven described species of Stillabothrium, on the basis of their pattern of bothridial loculi. Phylogenetic analyses based on sequence data for 1,084 bp from the D1–D3 region of 28S rDNA that included multiple specimens of both new species and eight other spe- cies of Stillabothrium corroborated the morphologically-determined species boundaries. The phylogenetic analyses indicate that S. allisonae sp. n. and S. charlotteae sp. n. are sister species, a noteworthy pattern given that the two species of the stingray genus Fontitrygon they both parasitise, F. margaritella and F. margarita, are also sister species. Although species of Stillabothrium vary widely in their patterns of facial loculi, the variation does not appear to correlate with phylogeny. Most species of Stillabothrium parasitise myliobatiform elasmobranch genera of the Dasyatidae Jordan. This study brings the number of described species of Stil- labothrium to nine, three of which occur in the eastern Atlantic, two of which occur off the northern coast of Australia, and four of which are from coastal Borneo. Keywords: tapeworms, taxonomy, 28S rDNA, phylogeny, survey, sister species, biodiversity, species boundaries Our understanding of the species diversity of many single species. Except for Sungaicestus, each of these gen- orders of cestodes has expanded substantially in recent era is potentially more diverse; in both studies (Reyda et al. years owing to a global effort by an international team of 2016, Caira et al. 2017) the authors referred to specimens scientists to survey, inventory and describe new species. of additional undescribed species of the new genera. This progress was recently summarised in a volume ed- This paper focuses on two previously undescribed spe- ited by Caira and Jensen (2017) in which taxonomic and cies of Stillabothrium that were obtained during field work systematic information on each of the 19 cestode orders on the coast of Senegal in 2003, 2004 and 2005, and re- is provided by the experts of the respective orders. As part ferred to in the study by Healy et al. (2009) as Rhinebothri- of this effort, the generic and species diversity the cestode inae New genus 3 sp. n. 1 and Rhinebothriinae New genus order Rhinebothriidea Healy, Caira, Jensen, Webster et 3 sp. n. 2 (or simply New genus 3 sp. n. 1 and New genus 3 Littlewood, 2009 has been substantially expanded since its sp. n. 2). The two new species described in this study over- establishment in 2009 (Healy et al. 2009) to accommodate lap in geographic distribution and in host use. Both species newly discovered taxa with unique scolex morphologies. were found in the spiral intestines of Fontitrygon margar- Four of the rhinebothriidean genera originally referred ita (Günther) and Fontitrygon margaritella (Compagno et to by Healy et al. (2009) as potentially new, based on both Roberts) and there were several instances of concurrent in- morphological and molecular data, were subsequently de- fections. In this study, as in the one by Reyda et al. (2016), scribed in two studies (Reyda et al. 2016, Caira et al. 2017). morphological data were complemented by sequence data These genera are Stillabothrium Healy et Reyda, 2016, for the D1–D3 region of the 28S rDNA gene. Both sources with seven species, Barbeaucestus Caira, Healy, Marques of data played a key role in delineating the boundaries of et Jensen, 2017 with four species, Divaricobothrium Cai- the new species in the context of a phylogeny of species of ra, Healy, Marques et Jensen, 2017 with two species, and Stillabothrium. Sungaicestus Caira, Healy, Marques et Jensen, 2017 with a Address for correspondence: F.B. Reyda, Biology Department & Biological Field Station, State University of New York, College at Oneon- ta, Ravine Parkway Oneonta, NY, 13820-4015, USA. Phone: +1-607.436.3719; Fax: +1-607.436.3646; E-mail: [email protected] Zoobank number for article: urn:lsid:zoobank.org:pub:7FD2D9BE-83F5-4051-B13A-6893349BD3DF This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.14411/fp.2018.014 Dedrick et al.: Two new cestode species from stingrays MATERIALS AND METHODS Czech Republic; LRP, Lawrence R. Penner Parasitology Collec- The cestodes described here came from two individuals of tion, Department of Ecology & Evolutionary Biology, University Fontitrygon margarita (Collection Code and Numbers SE-232 of Connecticut, Storrs, Connecticut, U.S.A.; MNHN, Muséum and SE-241) and eight individuals of Fontitrygon margaritella National d’Histoire Naturelle, Paris, France; USNM, United (Collection Code and Numbers SE-125, SE-228, SE-229, SE- States National Museum, Smithsonian Institution, Washington, 233, SE-262, SE-279, SE-306 and SE-308). The stingray speci- D.C. U.S.A. Nomenclatural acts in this manuscript are registered mens were collected from the coast of Senegal during field work at Zoobank.org. that occurred in 2003, 2004 and 2005. Stingrays were collected in The molecular analyses included newly sequenced specimens conjunction with local fishermen. Each host was identified in the and sequences from GenBank that were generated for previous field, assigned a Collection Code and unique Collection Number, studies (Healy et al. 2009, Reyda et al. 2016). All newly se- and photographed, and relevant information (e.g. sex, size) was quenced specimens originally fixed in 95% were initially cut and recorded. A tissue sample was also collected for subsequent DNA either the scolex and/or terminal proglottid was removed and pre- analysis. Additional data for each host specimen can be accessed pared as whole mounts as described above, and deposited in the at the Global Cestode Database (Caira et al. 2012) at www. elas- LRP as hologenophores, The remaining portion of each specimen mobranchs.tapewormdb.uconn.edu by entering its assigned was subjected to the molecular protocols mentioned below. Collection Code and Collection Number (e.g. SE-232). Elasmo- Table 1 provides taxon name, host, collection locality, mu- branch classification follows Naylor et al. (2012a); elasmobranch seum accession number for hologenophores (sensu Pleijel et al. taxonomy follows Last et al. (2016). Field identifications of host 2008) and GenBank accession numbers for each of the specimens specimens were verified using NADH2 sequence data (see Nay- included in the molecular analyses. Sequence data were generated lor et al. 2012b). for two or more individuals of both new species of Stillabothrium In the case of each host specimen, the spiral intestine was and included in the molecular analysis, given that the goal was to removed and opened with a longitudinal incision. A subsample test species boundaries of the new species within the context of a of worms was removed, washed in seawater and sorted into two phylogenetic hypothesis of members of the genus Stillabothrium. subsets. The first subset was fixed in 10% seawater-buffered -for Sequence data obtained from GenBank for the analysis consisted malin and subsequently stored in 70% ethanol; the other subset of a single sequence for each of the seven described species of was fixed in 95% ethanol. Spiral intestines were fixed in 10% sea- Stillabothrium as well as for a specimen of an undescribed spe- water-buffered formalin and additional worms were removed un- cies, referred to as Stillabothrium sp. n. 4 by Reyda et al. (2016). der a dissecting microscope upon return to the laboratory. Worms Protocols for DNA extraction, PCR amplification, DNA sequenc- prepared as whole mounts were hydrated in a graded series of eth- ing, sequence analysis and sequence alignment are as given in anols, stained in Delafield’s hematoxylin, destained in 70% acid Reyda et al. (2016). ethanol, neutralised in 70% basic ethanol, dehydrated in a graded Phylogenetic analysis was conducted on sequences of a total ethanol series, cleared in methyl salicylate, and mounted on glass of 19 specimens of 12 cestode species (Table 1). Anthocephalum slides in Canada balsam. Worms examined with SEM (scanning michaeli Ruhnke et Seaman, 2009 and Escherbothrium sp. were electron microscopy) were cut in half and the strobila of each used as outgroup species. Bayesian inference was conducted us- was prepared as a whole mount as described above to serve as a ing MrBayes version 3.2 (Ronquist and Huelsenbeck 2003) with voucher, and the scolex was examined with SEM. Scoleces were the following settings: lset nst = 6 rates = invgamma ngammacat hydrated in a graded ethanol series, placed in 1% osmium tetroxide = 4; ngen = 5,000,000; samplefreq = 1,000. Fifty percent of the overnight, dehydrated in a graded ethanol series, transferred to samples were discarded on burnin. Bootstrap analysis was also hexamethyldisilazane for 15 min in an exhaust hood and allowed conducted using PAUP* verion 5.4.0b (Swofford 2000).