Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2017, 64: 035 doi: 10.14411/fp.2017.035 http://folia.paru.cas.cz Research Article Description and phylogenetic position of a new species of Rhabdias Stiles et Hassall, 1905 (Nematoda: Rhabdiasidae) from the banded rubber frog, Phrynomantis bifasciatus (Smith) (Amphibia: Microhylidae), in South Africa Yuriy Kuzmin1, Ali Halajian2, Sareh Tavakol2, Wilmien J. Luus-Powell2 and Vasyl V. Tkach3 1 Department of Parasitology, I. I. Schmalhausen Institute of Zoology, Kyiv, Ukraine; 2 Department of Biodiversity (Zoology), University of Limpopo, Sovenga, South Africa; 3 Department of Biology, University of North Dakota, Grand Forks, ND, USA Abstract: The lung-dwelling nematode Rhabdias engelbrechti[- ƽRhabdias Stiles et Hassall, 1905 occurring in the Afrotropical !""""\"""""$ ""%&'M+)&>'M>?)J$"&M?M'>JRhabdias engelbrechti is the tenth species of the genus found in Afrotropical anurans. Our molecular phylogenetic analysis based on the complete sequences of the ITS region and partial sequences of large subunit (28S) gene of the nuclear ribosomal RNA demonstrates that the new species is more closely related to the Eurasian species Rhabdias bufonis (Schrank, 1788) than to two other species from sub-Saharan Africa represented in the tree. In addition, partial sequences of the mitochondrial protein coding cox1 and ribosomal 12S genes of the new """$"QRhabdias. Keywords: Rhabdias engelbrechti sp. n., amphibians, Limpopo Province, morphology, molecular phylogeny, rDNA, cox1 Rhabdias Stiles et Hassall, 1905 is the largest genus of throleptidae, Bufonidae and Microhylidae (Chabaud et al. the Rhabdiasidae Railliet, 1915 comprising about 80 nom- 1961, Baker 1982, 1987, Kuzmin 2001, Junker et al. 2010, inal species (Kuzmin and Tkach 2017) of lung-dwelling Kuzmin et al. 2013, Tkach et al. 2014a). parasites of amphibians and reptiles. Species of the genus In the present paper we describe a new species W""%""- of Rhabdias collected from the banded rubber frog, arctica. Currently, 23 species of the genus have been re- Phrynomantis bifasciatus (Smith) (Microhylidae), in Lim- ported from the Afrotropical realm, ten of them as parasites popo Province, South Africa. Morphological and molecu- in amphibians. Of these, Rhabdias bdellophis Baylis, 1929 "ƽ&cytochrome oxidase 1 (cox1) is the only parasite of caecilians (Gymnophiona) (Baylis and 12S mitochondrial genes) of the new species from 1929), while the remaining nine species parasitise hosts of previously described African species of Rhabdias parasitic the order Anura. in anurans is provided along with an analysis of its phy- Three species, R. blommersiae Kuzmin, Junker, du logenetic position based on sequences of the complete ITS Preez et Bain, 2013, R. madagascariensis Chabaud, Bry- region and partial 28S gene of nuclear ribosomal DNA. goo et Petter, 1961 and R. vencesi Junker, Lhermitte-Valla- Y[\>\] MATERIALS AND METHODS remaining species, R. africanus Kuzmin, 2001, R. collaris Eight banded rubber frogs were found dead in January 2013 Baker, 1987, R. ohlerae Junker, Lhermitte-Vallarino et on the farm De Loskop (23°30'S; 29°18'E) in the vicinity of Bain, 2010, R. picardiae Junker, Lhermitte-Vallarino et Polokwane, Limpopo Province, South Africa (permit number Bain, 2010, R. sylvestris (Baker, 1982) and R. tanyai Jun- 001-CPM403-00012 from the Limpopo Department of Econom- ker, Lhermitte-Vallarino et Bain, 2010 occur on mainland ic Development, Environment & Tourism). Collected frogs were sub-Saharan Africa, parasitising hosts of the families Ar- transferred on ice to the Laboratory of Parasitology, University of Address for correspondence: V.V. Tkach, Department of Biology, University of North Dakota, 10 Cornell Street, Grand Forks, ND 58202, USA. Phone: 1-701-777-4675; E-mail: [email protected] Zoobank number for article: urn:lsid:zoobank.org:pub:2D3F9905-F70E-4B46-9479-3E1066A3F7DA 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.2017.035 Kuzmin et al.: A new species of Rhabdias from South Africa of R. engelbrechti and R. africanus and previously published 4.7–7.9 g (average 6.4 g) and a snout to vent length of 4.0–4.7 cm (Tkach et al. 2006, 2014a,b) sequences of 16 additional species of (average 4.5 cm). All frogs had cestodes in their intestine, seven RhabdiasƽPneumonema "[" tiliquae Johnston, 1916 was chosen as outgroup based on the re- "- sults of the phylogenetic analysis of the Rhabdiasidae by Tkach der a light microscope, the nematodes were cleared in lactophe- "&[\>JYW- " & Q" "" " "$" sented in Tables 2 and 3. and lactic acid). Cleared specimens were studied on temporary The new sequences and sequences obtained from GenBank "jx]>Q were initially aligned using MEGA6 software (Tamura et al. ""$- [\>MJ " ""$ [ ]' """$en face. }"Y$"$ Drawings were made from series of photomicrographs. Meas- $""$ urements were taken with the use of the digital imaging system. out using Bayesian inference as implemented in the MrBayes pro- Measurements of 20 specimens from three frogs are given in gram, version 2.01 (Huelsenbeck et al. 2001) with the following Table 1. All measurements are in micrometres unless otherwise nucleotide substitution parameters: lset nst=6, rates=invgamma, indicated. ncat=4, shape=estimate, inferrates=yes and basefreq=empirical, Genomic DNA of one individual of the new species as well that correspond to a general time reversible (GTR) model includ- as a specimen of another species from sub-Saharan Africa, ing estimates of the proportion of invariant sites (I) and gamma R. africanus (collected from Sclerophrys gutturalis [Power, (G) distributed among-site rate variation. 1927] and kindly provided by Odile Bain and Kerstin Junker) The substitution model was selected using Jmodeltest ver. W"W&>+++J}- 0.1.1 (Posada 2008) software. Posterior probabilities were ap- ments of the nuclear DNA region spanning the 3' end of the 18S M\\\\\\ ""W" nuclear rRNA gene, ITS region (ITS1+5.8S+ITS2) and 5' end of ""$"\ the 28S &"">MMJ" $QR\\\\- by PCR on an Eppendorf Master Gradient thermal cycler (Haup- erations. The resulting phylogenetic trees were visualised using pauge, NY, USA), using forward primer ritf (5'-GCG GCT TAA FigTree ver. 1.4 software (Rambaut 2012). TTT GAC TCA ACA CGG-3'), and reverse primer 1500R (5'- Newly obtained mitochondrial sequences from the new spe- GCT ATC CTG AGG GAA ACT TCG-3'). PCR reactions were cies R. engelbrechti and R. africanus were compared with com- performed using New England Biolabs® (Ipswich, MA, USA) patible sequences of species of Rhabdias published by Junker et OneTaq® Quick-Load®]]R al. (2010). In the publication by Junker at al. (2010) the GenBank instructions. Annealing temperature in these PCR reactions was accession numbers for sequences of R. tanyai were provided in- M![QW™ (Valen- correctly and duplicated those of R. vencesi. In the present work cia, CA, USA) columns and sequenced directly on an ABI Prism we use corrected GenBank numbers for sequences of R. tanyai. 3100™ (Carlsbad, CA, USA) automated capillary sequencer using Pairwise nucleotide comparison data and genetic identity matri- ABI BigDye™$R" ces for cox1 and 12S sequences were generated using MEGA6 DNA product was sequenced in both directions using the two PCR software. primers and, additionally, internal primers ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3'), 300R (5'-CAA CTT TCC CTC ACG RESULTS GTA CTT G-3'), 300F (5'-CAA GTA CCG TGA GGG AAA GTT G-3') and ECD2 (5'-CTT GGT CCG TGT TTC AAG ACG GG- Rhabdias engelbrechti sp. n. Figs. 1, 2 3'). Considering that most of the Afrotropical species of Rhabdias are represented in GenBank by partial sequences of mitochondri- ZooBank number for species: al cox1 and ribosomal 12S genes (Junker et al. 2010), we have se- urn:lsid:zoobank.org:act:9636FB19-DFC7-4872-88C4-EF910925AC78 quenced these genes from both the new species and R. africanus. Cox1 amplicons were obtained using primers LCO1490 Description (based on the holotype and 4 paratypes, all (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 gravid hermaphrodites; measurements of the holotype are (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') published by followed by the ranges for the type series in parentheses. Casiraghi et al. (2004). Partial 12S gene amplicons were obtained Measurements of all studied specimens are given in Ta- using primers 12SF (5'-GTTCCAGAATAATCGGCT A-3') and ble 1). Body length 5.06 mm (4.70–5.49 mm). Body 362 12SR (5'-ATTGACGGATG(AG)TTTGTACC-3') published by (306–362) wide at mid-body, gradually tapering anterior- Casiraghi et al. (2004). An annealing temperature of 45 °C was ly and posteriorly (Fig. 2A). Body width 156 (127–156) used in both cases. PCR primers were used as sequencing primers at junction of oesophagus and intestine, 145 (119–145) at for both mitochondrial genes. anus. Anterior end rounded, posterior end tapered. Body Contiguous sequences were assembled and edited using Se- ""$\- quencher™ ver. 4.1.1 (GeneCodes Corp., Ann Arbor, MI, USA) or thirds of body, thin at mid-region. Spherical cuticular and deposited in GenBank under accession numbers MG428406, \&""J$ MG428408, MG428410 (R. engelbrechti sp. n.) and MG428407, more or less distinctly separated from cuticle of remainder MG428409, MG428411 (R. africanus). The phylogenetic anal- of body (Figs. 1A,C, 2B).