Monogenea, Gyrodactylidae) on Gobiid fishes: Combined DNA and Morphological Analyses
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Systematic Parasitology 59: 103–120, 2004. 103 © 2004 Kluwer Academic Publishers. Printed in the Netherlands. Four new species of Gyrodactylus von Nordmann, 1832 (Monogenea, Gyrodactylidae) on gobiid fishes: combined DNA and morphological analyses Tine Huyse1,2,∗, Göran Malmberg3 & Filip A.M. Volckaert1 1Katholieke Universiteit Leuven, Laboratory of Aquatic Ecology, Ch. de B´eriotstraat 32, B-3000 Leuven, Belgium 2Parasitic Worms Division, Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK 3Department of Zoology, Stockholm University, S-10691 Stockholm, Sweden Accepted for publication 19th March, 2004 Abstract Four Gyrodactylus species parasitising four closely related gobiid species in European coastal waters were studied and compared with G. arcuatus Bychowsky sensu Bychowsky & Poljansky (1953) from Gasterosteus aculatus. These were G. gondae n. sp. from Pomatoschistus minutus and P. lozanoi, G. flavescensis n. sp. from Gobiusculus flavescens, G. arcuatoides n. sp. from P. minutus and G. branchialis n. sp. from P. microps. Combined molecular and morphological analyses, as well as morphometric and statistical methods, were used. The ssrRNA V4 region and the complete ITS rDNA region were sequenced. Genetically the four new species are clearly distinct from G. arcuatus. From a morphological point of view, the haptoral hard parts of G. gondae n. sp., G. flavescensis n. sp. and G. arcuatoides n. sp. are related to those of G. arcuatus, while these parts of G. branchialis n. sp. are different, but related to those of G. quadratidigitus Longshaw, Pursglove & Shinn, 2003. For the latter two species, a new species group is formed. The V4 and ITS sequence analyses, however, indicate a close relationship between G. branchialis and the three G. arcuatus-like species. Introduction In a previous study by Geets et al. (1999), Gyro- dactylus arcuatus-like specimens were found on the Species of Gyrodactylus von Nordmann, 1832 are gills of three goby species, Pomatoschistus minutus common ectoparasites on fish in Eurasian fresh and (Pallas), P. lozanoi (de Buen) and P. pictus (Malm), salt waters. According to Cable et al. (1999), progen- from the Voordelta Area (SW Netherlands). These esis and viviparity have led to a low number of useful closely related sympatric goby species are the most taxonomic characteristics. A morphological identifica- abundant benthic fish species in the North Sea (Fonds, tion method has been developed by Malmberg (1970), 1973; Miller, 1986). Gyrodactylus specimens were mainly based on the hard parts of the haptor, with collected in different seasons, from the three goby the marginal hook features being crucial for discrim- hosts and from Gasterosteus aculeatus L. (Geets et al. ination of very closely related species. Cunningham 1999). A morphometric multivariate analysis on 17 et al. (1995) introduced molecular markers, namely hook characters demonstrated both seasonal and host- the rDNA region with the V4 region and the internal related variation. The authors could not establish, transcribed spacers as a new tool for species identifica- however, whether these host-related forms belonged tion. These molecular tools, combined with morpholo- to the same species or represented three distinct spe- gical analysis, have been applied by many researchers cies. In this paper, on the basis of combined molecular, (Cunningham et al., 2001; Huyse & Volckaert, 2002; morphological and statistical analyses, the two Gyro- Zietara & Lumme, 2003). dactylus arcuatus-like species in Geets et al. (1999), ∗ G. sp. 1 from P. minutus and P. lozanoi and G. sp. 2 TH takes responsibility for the molecular, morphometric and statistical parts and GM for the morphological aspects of this work. from P. pictus are described together with another un- 104 Table 1. Gyrodactylus species analysed: G. arcuatus, G. gondae n. sp., G. flavescensis n. sp., G. arcuatoides n. sp. and G. branchialis n. sp. Gyrodactylus Host spp. Locality Date, water temperature, N P* A* Site nS salinity ◦ G. arcuatus Gasterosteus Edesö, Sweden 11/06/01, 10 C, 4 3/4 12 F/G 1 ◦ ◦ arcuatus 59 22 N, 18 27 E 5.0 ppm ◦ G. gondae Pomatoschistus Texel, The Netherlands 15/06/99, 12 C 11 91 330 G/F 2 ◦ ◦ G. gondae minutus 53 N, 4 48 E 31.0 ppm ◦ P. minutus Texel, The Netherlands 25/11/00, 12 C 23 61% 372 G/F 3 ◦ ◦ G. gondae P. lozanoi 53 N, 4 48 E 31.0 ppm 7 5/7 40 G/F 2 ◦ P. lozanoi North Sea, Belgium 26/10/99, 12 C 31 19% 14 2 ◦ ◦ 51 35 N, 2 18 E 35.0 ppm ◦ G. gondae P. minutus Trondheim, Norway 13/06/00, 12 C32/314F2 ◦ ◦ 63 32 N, 10 26 E 32.0 ppm ◦ G. gondae P. minutus Bergen, Norway 21/05/01, 9-10 C 10 60% 18 F 3 ◦ ◦ 60 16 N, 5 10 E 33.0 ppm ◦ G. flavescensis Gobiusculus Bergen, Norway 22/06/00, 9–10 C66/670F/G3 ◦ ◦ flavescens 60 16 N, 5 10 E 33.0 ppm ◦ G. flavescensis G. flavescens Bergen, Norway 21/05/01, 8–9 C 12 83% 78 F/G 1 ◦ ◦ 60 16 N, 5 10 E 33.0 ppm ◦ G. flavescensis G. flavescens Trondheim, Norway 13/06/00, 12 C32/38F/G3 ◦ ◦ 63 32 N, 10 26 E 32.0 ppm ◦ G. arcuatoides P. pictus Bergen, Norway 22/06/00, 9–10 C 16 81% 98 G/F 3 ◦ ◦ 60 16 N, 5 10 E 33.0 ppm ◦ G. arcuatoides P. pictus Bergen, Norway 21/05/01, 8–9 C 10 70% 29 G/F 2 ◦ ◦ 60 16 N, 5 10 E 33.0 ppm ◦ G. branchialis P. microps Spuikom Ostend, Belgium 18/08/99, 16–18 C* 25 84% 248 G 4 ◦ ◦ 51 14 N, 2 57 E 31.1 ppm ◦ G. branchialis P. microps Ambleteuse, France 23/09/99, 15 C 15 47% 75 G 2 ◦ ◦ 50 N, 1 36 E 16–30 ppm ◦ P. microps Texel, The Netherlands 15/06/99, 12 C 8 50% 9 G 2 ◦ ◦ 53 N, 4 48 E 31.0 ppm ◦ P. microps Yerseke, The Netherlands 06/11/99, 16.7 C 8 88% 53 G 1 ◦ ◦ 51 30 N, 4 4 E 30.1 ppm ◦ G. rarus Spinachia Trondheim, Norway 13/06/00, 12 C32/38G3 ◦ ◦ spinacha 63 32 N, 10 26 E 32.0 ppm N, number of host specimens collected. P, prevalence; A, abundance. Sites: F, fins; G, gills; G/F, >50% on gills, but also found on fins; F/G, >50% on fins, but also found on gills. nS, number of sequences. ∗ ∗ P , prevalence, calculated as the ratio of the number of infected fish specimens to the total number of fish (if N ≥ 10) examined; A , abundance, calculated as the total number of Gyrodactylus specimens per fish population. described G. arcuatus-like species from Gobiusculus Materials and methods flavescens. Gyrodactylus sp. from P. microps in Geets et al. (1999) is described as a G. quadratidigitus-like Hosts and parasites (see Longshaw, Pursglove & Shinn, 2003) species. The material for the present study was collected along the Eastern Atlantic seaboard, from Trondheim, Nor- way (the northernmost sampling point) to Ambleteuse, France (the southernmost sampling point). Data re- garding host, locality, date of collection, water tem- 105 perature, salinity, number of host specimens, pre- Ecology, Catholic University Leuven, Belgium and valence, abundance, site on host and number of the the Department of Zoology, Stockholm University, included Gyrodactylus specimens are listed below and Sweden. In Belgium measurements of ventral bars and in Table 1. Fish were transported alive in local water anchors were made using a Zeiss HBO50 microscope to the laboratory and killed by pithing prior to in- (×100 oil immersion objective, with ×10 micrometer vestigation. Using a stereomicroscope, Gyrodactylus eyepieces). Digital images were analysed with the specimens were individually removed from the fish by program SigmaScan Pro 5. In Stockholm the micro- means of preparation needles. Whenever possible, the scopical analyses were carried out using oil immersion same specimen was used for both morphological and (×90 objective), phase contrast and drawing prism molecular analysis. If so, the haptor was separated equipment (Malmberg, 1970) connected to a Leica from the body and fixed between slide and coverslip DC 300 digital camera and archiving system. Images in ammonium picro-glycerine according to Malmberg of the haptoral hard parts of all specimens and of (1970), and the body was transferred into 5 µlof large embryos in the uterus, when present, were stored milli-Q water and stored at −20 ◦C for further DNA and printed (LazerPrint system; Reality Imaging Sys- analysis. The excretory bladders were studied in some tem, Munich, Germany) for further analysis. The live specimens of all four new species. results were compared to drawings made by means of a drawing tube (Malmberg, 1970). Measurements of Molecular analysis marginal hook sickles were performed by means of image analysis (Leica Q500IW with a Hamamatsu 3 Amplification and sequencing of the complete ITS re- CCD camera, C5810), the sickle area by detection and gion, including the 5.8S gene and the V4 region of the other measurements by interactive measuring on the ssrRNA, have been performed previously (Huyse a computer screen. Drawings of the holotype and the et al., 2003; Accession Nos AY338432- AY338434). paratype specimens of the Gyrodactylus species in the Sequences were aligned with the Clustal X multiple senior author’s collection were compared to drawings sequence alignment program (version 1.81, Thompson of G. arcuatus specimens in the ‘Malmberg collec- et al., 1997); regions with an ambiguous alignment tion’. In total, 10 G. arcuatus specimens (Swedish were excluded from further analyses. To infer phylo- Museum of Natural History, SMNH, Acc. No. 48440) genetic relationships, maximum parsimony (MP), were studied: 10 digitally recorded, three drawn by maximum likelihood (ML) and distance-based meth- means of a drawing apparatus and 12 marginal hooks ods were applied using PAUP* v.