772 J. Parasitol., 93(4), 2007, pp. 772–780  American Society of Parasitologists 2007 THREE NEW SPECIES OF LIGOPHORUS (MONOGENEA: DACTYLOGYRIDAE) ON THE GILLS OF MUGIL CEPHALUS (TELEOSTEI: MUGILIDAE) FROM THE JAPAN SEA* Nataliya Yu. Rubtsova, Juan A. Balbuena†, and Volodimir L. Sarabeev Department of Biology, Zaporizhzhya National University, 66 Zhukovskogo Street, 69600 Zhaporizhzhya, Ukraine. e-mail: volodimir.sarabeev@uv.es ABSTRACT: Comparative morphology and multivariate morphometric analysis of monogeneans collected on flathead mullets Mugil cephalus from 2 Russian localities of the Japan Sea revealed the presence of 3 new species of Ligophorus, namely, L. domnichi n. sp., L. pacificus n. sp., and L. cheleus n. sp., which are described herein. So far, only 1 species of dactylogyrid monogenean identified as Ligophorus chabaudi was known on flathead mullets in this sea, but after comparison with the present material, we propose that this form actually represents L. domnichi n. sp. Results support previous zoogeographical evidence, suggesting that flathead mullets from different seas harbor different species complexes of Ligophorus. One interesting finding is that the 3 new species have a U-shaped ovary, whereas ovate ovaries have been reported in previous descriptions of species of the genus, e.g., L. vanbenedenii, L. parvicirrus, L. imitans, and L. chongmingensis. The U-shaped ovary was revealed only when the worms were observed in lateral view. The additional examination of L. vanbenedenii, L. parvicirrus, L. imitans, and L. pilengas specimens from our collections also revealed a U-shaped ovary in these forms as well. Further studies should establish whether or not this character is shared by all members of the genus. Data on the monogenean parasites of mullets (Mugilidae) from the Japan Sea are very scarce. To our knowledge, only 4 species have been recorded or described: Gyrodactylus mugili Zhukov, 1970; Ligophorus kaohsianghsieni (Gussev, 1962); L. pilengas Sarabeev and Balbuena, 2004, and L. chabaudi Euzet and Suriano, 1977 (Gussev, 1955; Zhukov, 1970; Gussev, 1985; Sarabeev and Balbuena, 2004, and our unpublished data). The first 3 species occur on the so-iuy mullet Mugil soiuy Basi- lewsky, 1855 and have also been reported in the Azov and the Black Seas after the introduction of this host in this area (Dmi- trieva, 1996; Miroshnichenko and Maltsev, 1998; Sarabeev and Balbuena, 2004). The fourth species has been reported on the flathead mullet Mugil cephalus Linnaeus, 1758. It was first re- corded as Ancyrocephalus vanbenedenii (Parona and Perugia, 1890) but, after the erection of Ligophorus by Euzet and Sur- iano (1977), these authors transferred it to L. chabaudi Euzet and Suriano, 1977. However, in the light of recent zoogeograph- ic evidence suggesting that flathead mullets from different geo- graphic areas harbor different species of Ligophorus (Sarabeev et al., 2005), and because L. chabaudi is commonly found on flathead mullets from the Mediterranean (Rubtsova et al., 2006), the occurrence of this species in the Japan Sea was in need of scrutiny. During a parasitological survey of flathead mullets in 2 Russian localities of the Japan Sea, several dactylogyrid monogeneans were collected, and a detailed morphological and morphometric study revealed that they could be ascribed to 3 new species of Ligophorus, which are described herein. In ad- dition, after comparison with the description of specimens orig- inally identified as A. vanbenedenii from the Japan Sea, we propose that this form actually conforms to 1 of these new species. MATERIALS AND METHODS Adult flathead mullets were collected in 2 Russian localities of the Japan Sea, i.e., Kiyevka Bay and Delta of the Razdol’naya River (Table Received 3 March 2006; revised 5 June 2006; accepted 5 June 2006. * The term ‘‘Japan Sea’’ is used in conformity with the current usage set by the International Hydrographic Organization, and does not nec- essarily reflect the authors’ stance over the international dispute about the naming of this sea. † Marine Zoology Unit, Cavanilles Institute of Biodiversity and Evo- lutionary Biology, University of Valencia, P.O. Box 22085, 46071 Valencia, Spain. I). Fish were examined microscopically for monogeneans within the day of capture, or after thawing if frozen. This study is based on 89 spec- imens belonging to 3 new species of Ligophorus (Table I). Specimens were mounted in glycerin jelly, following Gussev (1983). Specimens were stained in iron acetocarmine, passed through a series of increasing ethanol concentrations (from 70 to 100%), cleared in dimethyl phthal- ate, and mounted in Canada balsam to study details of their internal anatomy. We measured 35 characters, 25 of them belonging to sclerotized structures. Nineteen sclerotized characters were measured as defined in Sarabeev and Balbuena (2004) and Rubtsova et al. (2006), whereas the remainder (ventral and dorsal anchors shaft lengths; distal part length and width, shaft length of upper lobe, and lower lobe length of acces- sory piece of male copulatory complex) were determined as shown on Figure 1. These 6 additional characters were chosen because they seemed useful for species discrimination after a preliminary study of the present specimens. Dimensions of soft internal organs and body represent their largest measurement. The following abbreviations for the characters are used throughout the text: BL, body length; BW, body width; VAA, ventral anchor total length; VAB, ventral anchor main part length; VAC, ventral anchor outer root length; VAD, ventral anchor inner root length; VAE, ventral anchor point length; VAF, ventral anchor shaft length; DAA, dorsal anchor total length; DAB, dorsal anchor main part length; DAC, dorsal anchor outer root length; DAD, dorsal anchor inner root length; DAE, dorsal anchor point length; DAF, dorsal anchor shaft length; UTL, uncinulus total length; USHL, uncinulus shaft length; USL, uncinulus sickle length; VBL, ventral bar length; VBAP, distance between membranous anterior processes (protuberances) of ventral bar; DBL, dorsal bar length; PAPL, penis accessory piece total length; PA- PUL, penis accessory piece distal portion of upper lobe length; PAPUW, penis accessory piece distal portion of upper lobe width; PAPSHL, pe- nis accessory piece upper lobe shaft length; PAPLL, penis accessory piece lower lobe length; PL, total length of penis; VL, vagina length. Measurements are given in micrometers as mean  standard devia- tion with range in brackets, and number of specimens measured for each character in parentheses. Measurements and drawings were made with the use of a Leica DMLB2 microscope and a Nikon Optiphot-2 microscope with interference contrast (magnification 1010 for the body and 10100 [under immersion oil] for sclerotized structures and internal organs). Photographs of sclerotized structures of the haptor and male copulative apparatus were made with a Leica DMR microscope with interference contrast and a Leica DC300 camera. Because variation ranges of all metric characters of the 3 new species overlapped, a principal-components analysis (PCA) based on the overall covariation matrix was used to visualize and evaluate the morphometric differentiation between the species. Because correlation between most of the morphometric characters of Ligophorus spp. have been observed (Mariniello et al., 2004), PCA seems a suitable tool to circumvent the problem of multicollinearity of metric traits (Ter Braak, 1995) and their discrimination. The PCA was performed on all but 1 (VL), log-trans- formed metric characters of 79 specimens for which data for all vari-