Characterization of the Complete Mitochondrial Genome of Uvitellina Sp., Representative of the Family Cyclocoelidae and Phylogenetic Implications
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Parasitology Research (2019) 118:2203–2211 https://doi.org/10.1007/s00436-019-06358-y GENETICS, EVOLUTION, AND PHYLOGENY - ORIGINAL PAPER Characterization of the complete mitochondrial genome of Uvitellina sp., representative of the family Cyclocoelidae and phylogenetic implications Suleman1 & Mian Sayed Khan2 & Petr Heneberg3 & Cheng-Yan Zhou4 & Nehaz Muhammad1 & Xing-Quan Zhu1,4 & Jun Ma1 Received: 2 February 2019 /Accepted: 16 May 2019 /Published online: 1 June 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Mitochondrial (mt) DNA has been useful in revealing the phylogenetic relationship of eukaryotic organisms including flatworms. Therefore, the use of mitogenomic data for the comparative and phylogenetic purposes is needed for those families of digenetic trematodes for which the mitogenomic data are still missing. Molecular data with sufficiently rich informative characters that can better resolve species identification, discrimination, and membership in different genera is also required for members of some morphologically difficult families of trematodes bearing few autapomorphic characters among its members. Here, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) and the complete mt genome of the trematode Uvitellina sp. (Cyclocoelidae: Haematotrephinae) was determined and annotated. The mt genome of this avian trematode is 14,217 bp in length, containing 36 genes plus a single non-coding region. The ITS rDNA sequences were used for the pairwise sequence comparison of Uvitellina sp. with European cyclocoelid species, and the mitochondrial 12 protein-coding genes (PCGs) and two ribosomal RNA genes were used to evaluate the position of the family within selected trematodes. The ITS rDNA analysis of Uvitellina sp. showed less nucleotide differences with Hyptiasmus oculeus (16.77%) than with other European cyclocoelids (18.63–23.58%). The Bayesian inference (BI) analysis using the 12 mt PCGs and two rRNA genes supported the placement of the family Cyclocoelidae within the superfamily Echinostomatoidea (Plagiorchiida: Echinostmata). The availability of the mt genome sequences of Uvitellina sp. provides a novel resource of molecular markers for phylogenetic studies of Cyclocoelidae and other trematodes. Keywords Cyclocoelidae . Uvitellina sp. Nuclear ribosomal DNA . Mitochondrial genome . Phylogenetic implications Section Editor: Neil Bruce Chilton Electronic supplementary material The online version of this article Introduction (https://doi.org/10.1007/s00436-019-06358-y) contains supplementary material, which is available to authorized users. The family Cyclocoelidae Stossich, 1902, contains medium- sized to large trematodes, which parasitize the airways, air * Jun Ma sacs, nasal and infraorbital sinuses, hypothalamus, liver and [email protected] body cavity of their avian hosts that are feeding on molluscs 1 State Key Laboratory of Veterinary Etiological Biology, Key (Kanev et al. 2002;DronenandBlend2015). For the life cycle Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou of these avian trematodes, freshwater pulmonate snails or xe- Veterinary Research Institute, Chinese Academy of Agricultural rothermic snails serve as both first and second intermediate ’ Sciences, Lanzhou, Gansu Province 730046, People s Republic of hosts (Poulin and Cribb 2002; Dronen and Blend 2015). The China tailless cercariae do not emerge from the snails; instead, they 2 Department of Zoology, University of Swabi, Khyber encyst either in rediae or in the tissue of the snail, develop to Pakhtunkhwa, Pakistan metacercariae and await ingestion by the avian host (Poulin 3 Third Faculty of Medicine, Charles University, 100 and Cribb 2002; Dronen and Blend 2015). Thus, birds feeding 00 Prague, Czech Republic on molluscs, including waders, get infected upon ingestion of 4 Jiangsu Co-innovation Center for the Prevention and Control of an infected snail (Kanev et al. 2002; Shutler et al. 2016). Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu The classification of Cyclocoelidae underwent many chang- Province 225009, People’s Republic of China es over time with the proposal of additional subfamilies, genera 2204 Parasitol Res (2019) 118:2203–2211 and species. Dronen and Blend (2015) published an extensive composition and conducted a phylogenetic analysis with other monograph with keys to the six subfamilies, 22 genera and 128 selected trematodes using the amino acid sequences of 12 species. The subfamily Haematotrephinae was characterized by mitochondrial protein-coding genes (PCGs). having the ovary ranging from pretesticular in position to op- posite the anterior testis forming a triangle with the testes (Dronen and Blend 2015). Within Haematotrephinae, the genus Materials and methods Uvitellina Witenberg, 1923, was distinguished from other four genera by having a distinctly pretesticular ovary forming a tri- Sampling, identification and DNA extraction angle with the testes, postpharyngeal genital pore and posteri- orly confluent vitelline fields (Dronen et al. 2017). Dronen and Three cyclocoelid specimens were collected from the air sacs Blend (2015) assigned nine species to Uvitellina i.e. Uvitellina (lungs) of their definitive host, the red-wattled Lapwing pseudocotylea Witenberg, 1923, Uvitellina macroisophaga (Vanellus indicus) (Boddaert, 1783) (Charadriiformes: Hannum and Wilson, 1934, Uvitellina vanelli Rudolphi, Charadriidae) in Swabi, Khyber Pakhtunkhwa (KP), Pakistan. 1819, Uvitellina titiri Chatterji, 1958, Uvitellina simile The live worms were killed and relaxed in hot water and stored Stossich, 1902, Uvitellina kaniharensis Gupta, 1958, in 80% ethanol (Lutz et al. 2017). For light microscopy, a spec- Uvitellina adelphus Johnston, 1917, Uvitellina iraquensis imen was stained, dehydrated and mounted permanently ac- Dronen, Ali and Al-Amura, 2013, and Uvitellina himantopi cording to the recommended protocol (Lutz et al. 2017)and Dronen and Tkach, 2013. identified to the genus level following the existing identification All the hitherto proposed classification systems of keys and descriptions of cyclocoelids (Kanev et al. 2002; Cyclocoelidae are primarily based on morphological charac- Dronen 2007;DronenandBlend2015;Gupta1958;Siddiqi ters, but the family has been recognized as a Bmorphologically and Jairajpuri 1962). For molecular work, the total genomic difficult group^ (Dronen et al. 2017), Bunsettled taxonomic DNA was extracted from 80% ethanol-preserved specimens group^ (Dronen and Blend 2015;Sitkoetal.2017)and according to a published protocol (Gasser et al. 2007). Bcontroversial family^ (Kalani et al. 2015). Among the main reasons which lead to misidentification are the presence of ITS rDNA sequencing and comparison with European their low numbers in definitive hosts, low chance of regaining cyclocoelids specimens studied previously, morphological similarity be- tween genera, for example, Cyclocoelum, Harrahium and The nuclear ribosomal DNA region spanning the 3′ end of the Hyptiasmus, and higher similarities in their developmental 18S rDNA gene, the complete ITS (ITS1, 5.8S, ITS2) and the cycles (Dronen and Blend 2015; Sitko et al. 2006, 2017). 5′ end of the 28S rDNA gene was amplified by PCR using two Thus, molecular studies could benefit more for the family sets of primers: BD1 (forward; 5′-GTCGTAACAAGGTT Cyclocoelidae than probably any other family of flukes TCCGTA-3′) and BD2 (reverse; 5′-ATGCTTAAATTCAG (Dronen et al. 2017). Surprisingly, few studies have been done CGGGT-3′) (Morgan and Blair 1995) and NC13 (ITS2)/F on the molecular phylogenetics, positioning the (5′-ATCGATGAAGAACGCAGC-3′) (Jacobs et al. 1997) Cyclocoelidae within the higher taxa based on nuclear ribo- and Dd28SR1 (5′-ACAAACAACCCGACTCCAAG-3′) somal DNA (rDNA) (Littlewood and Bray 2001; Olson et al. (Otranto et al. 2007). The positive amplicons were purified 2003; Libert et al. 2012; Tkach et al. 2016). Sitko et al. (2017) and subjected to Sanger sequencing by GENEWIZ sequenc- performed the phylogenetic analyses of central European ing company (Beijing, China). While the complete ITS rDNA cyclocoelids based on two nuclear (ITS2, 18S rDNA) and sequences were not available for all cyclocoelids, the nuclear two mitochondrial (cox1, nad1) DNA loci. However, for the ribosomal DNA region spanning partial 5.8S, the complete genus Uvitellina (Haematotrephinae), there are no sequences ITS2 and the partial 28S rDNA has been sequenced for available, neither from nuclear nor from mitochondrial DNA. European cyclocoelids (Sitko et al. 2017). Therefore, this nu- The mitochondrial DNA (mtDNA) is an effective and rich clear rDNA region was used for comparative purposes with source of genetic markers (Smith 2016; Locke et al. 2018)for other European cyclocoelids. The nucleotide differences in molecular identification and phylogenetics of many organ- percent (including gaps within alignment) across the rDNA isms. However, mt genomes of only a limited number of region (mentioned above) were estimated using pairwise trematodes have been sequenced. The mitogenomic data is alignment in ClustalX 1.83 (Thompson et al. 1997)and still missing for some morphologically difficult groups of BioEdit 7.0.9.0 (Hall 1999). trematodes including Cyclocoelidae. Here, we determined the internal transcribed spacer region of nuclear ribosomal Long-PCR and mt genome sequencing DNA (ITS1-5.8S-ITS2 rDNA) and the complete mt genome of Uvitellina sp. as the representative