Parasitol Res (2012) 111:819–826 DOI 10.1007/s00436-012-2904-z

ORIGINAL PAPER

Three novel myxobolid species of genera Henneguya and Myxobolus (Myxosporea: Bivalvulida) from marine fish in Japan Yang-Chun Li & Hiroshi Sato & Yoichi Kamata & Takahiro Ohnishi & Yoshiko Sugita-Konishi

Received: 28 January 2012 / Accepted: 19 March 2012 / Published online: 5 April 2012 # Springer-Verlag 2012

Abstract Myxosporean genera Henneguya and Myxobolus (Bivalvulida: Myxobolidae) are closely related in morphology and molecular phylogeny, speciose with approximately 1,000 nominal species. The majority of them are recorded from freshwater fish worldwide, and few are known from marine fish. In this study, three myxobolid spp. are described from marine fish around Japan. Two novel Henneguya spp., Henneguya ogawai sp. n. and Henneguya yokoyamai sp. n., are described from two black sea breams (Acanthopagrus schlegelii) fished in the Inland Sea (Setonaikai), Japan. Plasmodia of the former species were localized in the esophageal or intestinal wall, and those of the latter species were in the wall of the gall bladder and peritoneum. Spore development in plasmodia of these two species was synchronous. The spore body of H. ogawai sp. n. was 11.0 (8.9–12.2)μm in length, 6.9 (6.3–7.5)μm in width, 5.9 (5.2–6.6)μm in thickness, with a bifurcated caudal process of equal length, 10.0 (8.4–12.7)μm long; total spore length, 21.1 (19.2–23.4)μm. It contained two polar capsule, 4.3 (3.8–5.2)×1.9 (1.4–2.3)μm. The spore body of H. yokoyamai sp. n. was 11.0 (10.1–13.7)μm in length, 7.1 (6.6–7.5)μm in width, and 5.6 (4.5–6.4)μm in thickness, with a bifurcated caudal process of equal length, 14.1 (10.8– 17.0)μm long; total spore length, 25.0 (21.9–29.2)μm. It contained two polar capsules, 3.7 (3.1–4.2) × 2.0 (1.8– Y.-C. Li : H. Sato (*) Laboratory of Veterinary Parasitology, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan e-mail: [email protected] Y. Kamata : T. Ohnishi : Y. Sugita-Konishi Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

2.4)μm. A novel Myxobolus sp., Myxobolus machidai sp. n., is described from a spotted knifejaw (Oplegnathus punctatus) fished in the Sea of Japan, off Shimonoseki, Yamaguchi Prefecture, Japan. Plasmodia were embedded in the esophageal wall. Its round spore was small in size, 9.0 (8.1–9.4)μm in length, 7.8 (7.5–8.3)μm in width, and 5.5 (5.1–6.0)μm in thickness. It contained two polar capsules, 3.5 (3.2–3.8)×2.3 (2.2–2.5)μm. Spore development in a plasmodium was asynchronous. Nucleotide sequencing of the small subunit ribosomal RNA gene (SSU rDNA) of these two novel Henneguya spp. revealed a close phylogenetic relationship with the marine clade of Henneguya spp.; however, they were distinct in morphology and SSU rDNA sequence from any known species. M. machidai sp. n. was grouped with freshwater Henneguya spp. in a phylogenetic tree based on the SSU rDNA, distant from a known marine clade of Myxobolus spp. reported mainly from the Mediterranean Sea. This is the first record of Henneguya–Myxobolus spp. from natural marine water in Japan.

Introduction The small subunit ribosomal RNA gene (SSU rDNA) sequence of varied myxosporean species discloses the presence of the Henneguya–Myxobolus clade (Myxozoa: Myxosporea: Bivalvulida: Myxobolidae), where different species of both genera are placed in a complex (Fiala 2006; Liu et al. 2010). Morphologically, myxobolids of genera Henneguya Thélohan, 1892 and Myxobolus Bütschli, 1882 have basically similar myxosporean spores; however, only the former possesses a caudal process (Lom and Dyková 2006). The synopsis of the species of the genus Henneguya provided by Eiras in 2002 included 146 nominal species (Eiras 2002). Since publication of Eiras’ synopsis, to

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the best of our knowledge, more than 33 species have been newly recorded and added to this genus (e.g., Dyková et al. 2011; Barassa et al. 2012). However, in 2005, Lom and Dyková (2006) counted 204 nominal species for this genus. Similarly, the genus Myxobolus has been reported to contain at least 744 nominal species (Eiras et al. 2005) or 792 nominal species (Lom and Dyková 2006), with, to the best of our knowledge, additional records of approximately 40 new species since the publication of the synopsis by Eiras et al. in 2005 (e.g., Kaur and Singh 2011; U-taynapum et al. 2011). Thus, myxobolids of these two genera, Myxobolus and Henneguya, represent the first and second largest groups in the Myxozoa, respectively, with more than 2,200 species in total (Lom and Dyková 2006). The majority of them are described from freshwater fish worldwide. During our continuous survey of myxosporean plasmodia in daily consumed marine fish in Japan (Matsukane et al. 2010, 2011), we detected plasmodia due to Henneguya in the alimentary tract and gall bladder of two black sea breams (Acanthopagrus schlegelii), a common fish in daily markets in Japan and a popular fish for consumption by anglers. Furthermore, we detected plasmodia due to Myxobolus in the esophageal wall of a spotted knifejaw (Oplegnathus punctatus), a common fish in Japanese-style restaurants and also popular with anglers. The present study reports the morphological and genetic characterization of these marine Henneguya and Myxobolus spp. and compares them with their congeners.

Materials and methods Two black sea breams, approximately 30 cm in body length, were fished in Mitajiri Bay, Hofu City, Yamaguchi Prefecture, Japan (N34° 03′, E131° 35′) on 11 and 15 July 2011. This bay faces Japan’s Inland Sea (Setonaikai). A spotted knifejaw was fished in the Sea of Japan, off Shimonoseki, Yamaguchi Prefecture, Japan, and prepared in a Japanese restaurant near Yamaguchi University for serving as sashimi on 5 October 2011. The whole body of the black sea breams and a portion of the spotted knifejaw, i.e., the remainder after the majority of the trunk muscles had been removed, were examined by the naked eye or using a stereomicroscope in the laboratory. Fresh spores of Henneguya spp. were observed using a microscope equipped with differential interference contrast imaging, photographed at a magnification of ×400, and transformed into photographs with Adobe® Photoshop® ver. 11.0 (Adobe Systems, San Jose, CA, USA). For the Myxobolus sp., fresh spores were observed and photographed with an oil immersion objective (×1,000) using the agar-embedding technique (Lom 1969). Photographs

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were then printed at a high magnification. Measurements were conducted on multiple printed photographs according to the guidelines of Lom and Arthur (1989). Parasite DNA was extracted from plasmodia isolated from different organs using an Illustra™ tissue and cells genomicPrep Mini Spin Kit (GE Healthcare UK, Buckinghamshire, UK) according to the instructions of the manufacturer. PCR amplification of two overlapping fragments of SSU rDNA was performed in a 20-μl volume containing a DNA polymerase, Blend Taq-Plus (TOYOBO, Dojima Hama, Osaka, Japan), and two combinations of universal eukaryotic primers: (1) NSF4/18 (5′-CTGGTTGATCCTGCCAGT-3′) and NSR1438/20 (5′-GGGCATCACAGACCTGTTAT-3′), and (2) NSF573/19 (5′-CGCGGTAATTCCAGCTCCA-3′) and SSU18R (5′-TGATCCTTCYGCAGGTTCAC-3′). The PCR cycling protocol was 3 min at 94 °C, then 40 cycles of 45 s at 94 °C, 1 min at 64 °C, and 1 min at 72 °C, followed by a final extension at 72 °C for 7 min. Alternatively, a combination of universal eukaryotic primers, Eurib1 (5′ACCTGGTTGATCCTGCCAG-3′) and reverse Eurib2 (5′CTTCCGCTGGTTCACCTACGG-3′), was used to amplify almost the whole length of the SSU rDNA at once (Kopečná et al. 2006). The PCR cycling protocol was 2 min at 95 °C, then 35 cycles of 1 min at 95 °C, 1 min at 48 °C, and 90 s at 72 °C, followed by a final extension at 72 °C for 7 min. The PCR products were purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany), and sequenced directly using the primers described above for PCR amplification and three additional primers for sequencing: NSF1179/18 (5′-AATTTGACTCAACACGGG-3′), NSF1624/20 (5′-TTTGTACACACCGCCCGTCG-3′), and NSR581/18 (5′-TCTCAGGCTCCCTCTCCGG-3′). For phylogenetic analysis, the newly obtained SSU rDNA sequences of the myxobolid spp. in the present study (DDBJ/EMBL/GenBank accession nos. AB693050– AB693054) and Henneguya/Myxobolus-related sequences retrieved from the DDBJ/EMBL/GenBank databases were aligned using the Clustal W multiple alignment program (Thompson et al. 1994), with subsequent manual adjustment. The accession numbers of the sequences analyzed in this study are given in the figure showing the phylogenetic tree. Regions judged to be poorly aligned and characters with a gap in any sequences were excluded from subsequent analyses (Smythe et al. 2006); 1,161 characters, of which 554 were variable, remained for subsequent analysis for SSU rDNA. Maximum likelihood (ML) analysis was performed with the program PhyML (Guindon and Gascuel 2003; Dereeper et al. 2008) provided on the “phylogeny.fr” website (http://www.phylogeny.fr/). The probability of inferred branch was assessed by the approximate likelihood ratio test, an alternative to the nonparametric bootstrap estimation of branch support (Anisimova and Gascuel 2006).

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Results Occurrence of plasmodia A few white cysts (e.g., 0.78×0.58 and 0.66×0.66 mm) were found in the esophageal wall of the black sea bream obtained on 11 July. Similarly, a few white cysts (e.g., 0.78×0.32 and 0.38×0.20 mm) were detected in the intestinal wall of the fish obtained on 15 July. In addition, several minute white nodules were observed in the wall of the gall bladder in the latter fish. Their size, expressed as range with mean and standard deviation in parentheses, was 0.22–0.71 (0.42±0.19)×0.17–0.57 (0.28±0.17)mm (n05). Four white plasmodia were embedded in the esophageal wall of the spotted knifejaw, measuring 0.16–0.30 (average, 0.22)× 0.13–0.28 (0.19)mm. The spores obtained from the black sea breams were elliptical with two polar capsules and a bifurcated caudal process, typical features of the genus Henneguya. The length of the caudal process was different between spores in plasmodia collected from the alimentary tract (esophagus and intestine) and the gall bladder or peritoneal wall (Fig. 1). As detailed later, the SSU rDNA sequences of spores collected from different organs were different, suggesting that two different Henneguya spp. parasitized the black sea bream. The spores collected from plasmodia in the esophageal wall of the spotted knifejaw were round with two polar capsules and no caudal process, typical features of the genus Myxobolus (Fig. 2). From the results of morphological and genetic analyses, these three species obtained from marine fish are undescribed myxobolid species. Description Henneguya ogawai sp. n. (Myxosporea: Bivalvulida) Spores elliptical with a bifurcated caudal process (tails). Morphometrics (in micrometers) based on the measurements of 30 spores from the intestinal wall of a black sea bream obtained on 15 July 2011. Spore body length, 8.9– 12.2 (11.0±0.8); spore width, 6.3–7.5 (6.9±0.4); spore body thickness, 5.2–6.6 (5.9±0.4); length of two tails, equal, 8.4– 12.7 (10.0±1.2); total spore length, 19.2–23.4 (21.1±1.3); polar capsule length, 3.8–5.2 (4.3 ± 0.4); polar capsule width, 1.4–2.3 (1.9 ±0.2); and dimensions of two polar capsules, slightly different. Coils of polar filament were not visible. Spore development in a plasmodium, synchronous (Figs. 1a–h and 3a, b). Taxonomic summary Type host: Black sea bream (A. schlegelii). Site in host: Alimentary tract wall (lamina propria/ connective tissue of muscular layers).

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Type locality: Inland Sea (Setonaikai), off Hofu City, Yamaguchi Prefecture, Japan. Specimen deposited: Hapantotype NSMT-Pr332; Parahapantotype NSMT-Rr331, National Science Museum, Tokyo, Japan. Etymology: The species is named after Dr. Kazuo Ogawa, professor emeritus of Tokyo University, in recognition of his significant contribution to the knowledge of fish parasitology, including myxozoans. Prevalence: Unknown. Remarks The morphology of this myxosporean species is consistent with the definition of the genus Henneguya, such as elliptical spores with a suture separating biconvex valves and a caudal process (Lom and Dyková 2006). Most of the Henneguya spp. have been recorded from the gills, skin, and kidneys of freshwater fish (Eiras 2002). Several Henneguya spp. have been recorded from the gastrointestinal wall of freshwater and brackish water fish as follows: Henneguya branchialis Ashmawy et al., 1989 from Clarias lazera in Egypt; Henneguya ghaffari Ali, 1999 from Lates niloticus in Egypt; Henneguya gigas Chen and Hsieh, 1960 from Channa argus in China; Henneguya hainanensis Chen and Ma 1998 from Misgurnus anguillicaudatus in China; Henneguya ocellata Iversen and Yokel, 1963 from Sciaenops ocellatus in the USA; Henneguya ovaliformis Ma, Wang and Cai, 1986 from Channa striatus in China; Henneguya preintestinalis Ozaki and Isizaki, 1941 from Tridentiger obscurus in Japan; Henneguya rhinogobii Li and Nie, 1973 from Rhinogobius giurinus in China; Henneguya sinensis Chen and Hsieh, 1960 from Channa argus in China; Henneguya tangschensis Wu, 1997 from C. argus in China; Henneguya tenuis Vaney and Conte, 1901 from Gymnocephalus cernuus in France; and Henneguya zikawiensis Sikama, 1938 from Carassius auratus auratus in Japan (Eiras 2002; Reed et al. 2007). All these species have distinct dimensions regarding the spore body and/or the length of the caudal process from those of H. ogawai sp. n. A limited number of Henneguya spp. from marine fish has been recorded to date, including Henneguya lateolabracis Yokoyama et al. 2003 from aquacultured Lateolabrax sp. in Japan, Henneguya pagri Yokoyama et al. 2005 from aquacultured Pagrus major in Japan, and Henneguya akule Work et al. 2008 from Selar crumenophthalmus in Hawaii. Most of these marine Henneguya spp. have been recorded from the gills, bulbus arteriosus, and kidneys (Kpatcha et al. 1997; Eiras 2002; Yokoyama et al. 2003, 2005; Reed et al. 2007; Work et al. 2008). In addition, H. ogawai sp. n. can be morphologically differentiated from all of

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Fig. 1 Phase contrast micrographs of fresh spore preparations of two new Henneguya spp. from the alimentary tract wall of black sea breams. a–d H. ogawai sp. n. from a plasmodium localized in the esophageal wall; e–h H. ogawai sp. n. from a plasmodium localized in the intestinal wall; and i–l H. yokoyamai sp. n. from a plasmodium localized in the gall bladder wall. a, b, e, f, i, j, frontal views; c, d, g, h, k, l, lateral views

these marine Henneguya spp., except for Henneguya ouakamensis Kpatcha et al. 1997, on the basis of its shorter caudal process. There are, however, critical differences in the

dimensions of the polar capsules and the relative sizes of the polar capsules in a spore between H. ogawai sp. n. and H. ouakamensis, in addition to different hosts and sites concerning plasmodia development. Description

Fig. 2 Phase contrast micrographs of fresh spore preparations of M. machidai sp. n. from the esophageal wall of a spotted knifejaw. a frontal view; b, c lateral views

Henneguya yokoyamai sp. n. (Myxosporea: Bivalvulida) Spores elliptical with a bifurcated caudal process. Morphometrics (in micrometer) based on the measurements of 35 spores from the gall bladder wall of a black sea bream obtained on 15 July 2011. Spore body length, 10.1–13.7 (11.0±0.8); spore width, 6.6–7.5 (7.1±0.4); spore body thickness, 4.5–6.4 (5.6±0.4); length of two tails, equal, 10.8–17.0 (14.1±1.6); total spore length, 21.9–29.2 (25.0± 1.7); polar capsule length, 3.1–4.2 (3.7±0.4); polar capsule width, 1.8–2.4 (2.0 ±0.2); and dimensions of two polar capsules, slightly different. Coils of polar filament were not visible. Spore development in a plasmodium, synchronous (Figs. 1i–l and 3c, d).

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Fig. 3 Stylized drawings of H. ogawai sp. n. (a and b), H. yokoyamai sp. n. (c and d), and M. machidai sp. n. (e and f) in frontal and lateral views

Taxonomic summary Type host: Black sea bream (A. schlegelii). Site in host: Gall bladder wall (lamina propria/connective tissue of muscular layers). Type locality: Inland Sea (Setonaikai), off Hofu City, Yamaguchi Prefecture, Japan. Specimen deposited: Hapantotype NSMT-Pr334; Parahapantotype NSMT-Rr333, National Science Museum, Tokyo, Japan. Etymology: The species is named after Dr. Hiroshi Yokoyama, Tokyo University, in recognition of his significant contribution to the knowledge of myxozoans and their biology. Prevalence: Unknown. Remarks The morphology of this myxobolid species is almost identical to the previously described H. ogawai sp. n. except for the length of the bifurcated caudal process. Differentiation of the present species from other described Henneguya spp. is possible, as discussed above. Several Henneguya spp. have been recorded from the gall bladder of freshwater fish: Henneguya limatula Meglitsch, 1937 from Ictalurus punctatus and Ictalurus furcatus in the USA; Henneguya ntemensis Fomena and Bouix 1996 from Brienomyrus brachyistius in Cameroon; Henneguya kawangtungensis Chen, 1998 from Mylopharyngodon piceus in China; and Henneguya arapaima Feijó et al. 2008 from Arapaima gigas in Brazil (Eiras 2002; Feijó et al. 2008). All of these species have more elongated spore bodies and longer caudal processes than H. yokoyamai sp. n., except for H. ntemensis, which has a spore body comparable in size to H. yokoyamai sp. n. but a shorter caudal process (length, 3.2–9.6 μm; average 5.1 μm) (Fomena and Bouix 1996; Eiras 2002; Feijó et al. 2008). Furthermore, the phylogenetic analysis based on the SSU rDNA (detailed later)

supports the concept that H. yokoyamai sp. n. is distinct from any other Henneguya spp. characterized genetically as well as H. ogawai sp. n. Description Myxobolus machidai sp. n. (Myxosporea: Bivalvulida) Spores round, without a mucous envelope nor an intercapsular appendix. Morphometrics (in micrometer) based on the measurements of five spores from the esophageal wall of a spotted knifejaw obtained on 5 October 2011. Spore length, 8.1–9.4 (9.0±0.6); spore width, 7.5–8.3 (7.8±0.4); spore thickness, 5.1–6.0 (5.5±0.3); polar capsule length, 3.2–3.8 (3.5±0.2); polar capsule width, 2.2–2.5 (2.3±0.1); and dimensions of two polar capsules, identical. Coils of polar filament were not visible. Spore development in a plasmodium, asynchronous (Figs. 2 and 3e and f). Taxonomic summary Type host: Spotted knifejaw (O. punctatus). Site in host: Esophageal wall (lamina propria/connective tissue of muscular layers). Type locality: Sea of Japan, off Shimonoseki, Yamaguchi Prefecture, Japan. Specimen deposited: Hapantotype NSMT-Pr335, National Science Museum, Tokyo, Japan. Etymology: The species is named after Dr. Masaaki Machida, director emeritus of Meguro Parasitological Museum, Tokyo, in recognition of his significant contribution to the knowledge of fish parasitology. Prevalence: Unknown. Remarks The morphology of this myxobolid species is almost identical to Henneguya spp. except for the lack of a

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bifurcated caudal process. Spores of Myxobolus spp. are diminutive and resemble one another in morphology, making species identification difficult based on morphology alone. Along with morphology, the host preference as well as organ or tissue tropism are important keys in the classical species differentiation of myxozoans. In this regard, there is no record of Myxobolus sp. from marine fish of the family Oplegnathidae in a synopsis of the species published in 2005 (Eiras et al. 2005), and to the best of our knowledge, there have been no reports since 2005. Although 45 Myxobolus spp. have been recorded from the alimentary tract wall (Eiras et al. 2005), all of them, except for Myxobolus chiungchowensis Chen, 1998 from the intestine of Mugil cephalus off China, have been detected in freshwater fish. As the myxosporean spores and polar capsules of M. chiungchowensis have larger dimensions than Myxobolus machidai sp. n. (Chen and Ma 1998), these two species are differentiated by morphometrics.

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Phylogenetic analysis By direct sequencing, 2,038 and 2,029-bp sequences of the SSU rDNA region of H. ogawai sp. n. and H. yokoyamai sp. n. were obtained, respectively, and deposited in the DDBJ/ EMBL/GenBank databases (accession nos. AB693050– AB693053). Similarly, a 2,020-bp sequence of the SSU rDNA region of M. machidai sp. n. was obtained and deposited (DDBJ/EMBL/GenBank accession no. AB693054). The SSU rDNA sequences of H. ogawai sp. n. obtained from the esophageal wall of the black sea bream fished on 11 July and from the intestinal wall of the black sea bream fished on 15 July were absolutely identical. At the same time, the SSU rDNA sequences of H. yokoyamai sp. n. obtained from the wall of the peritoneum and gall bladder of the black sea bream fished on 15 July were completely identical. No deposited sequences of Henneguya spp. or other myxobolid species were identical to the newly obtained SSU rDNA sequences of the present study.

Fig. 4 ML phylogenetic tree based on the SSU rDNA sequences. On the right side of the figure, water type of the host fish (M marine and brackish water, F freshwater), location in the host, host name and country, and DDBJ/EMBL/GenBank accession no. are shown for each species

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A ML phylogenetic tree constructed on the basis of the SSU rDNA revealed that H. ogawai sp. n. and H. yokoyamai sp. n. formed a clade with Henneguya spp. of marine fish, particularly those forming plasmodia in the bulbus arteriosus, such as Henneguya pagri, Henneguya lateolabracis, and Henneguya akule (Yokoyama et al. 2003, 2005; Work et al. 2008), and H. tunisiensis localized in gills (Bahri et al. 2010) (Fig. 4). The newly obtained SSU rDNA sequences of H. ogawai sp. n. and H. yokoyamai sp. n. were placed in the closest position on the phylogenetic tree, but their SSU rDNA sequence similarity was 95.4 %. On the contrary, M. machidai sp. n. localized in a clade of freshwater Henneguya spp., taking a distant position from a clade of Myxobolus spp. of marine fish (Kent et al. 2001; Bahri et al. 2003).

the traditional morphology-based taxonomy and a new taxonomy reflecting the molecular phylogeny. Despite the speciose state of the Henneguya–Myxobolus myxobolids, the scarcity of reported species in some confined clades continues to hamper the implementation of a new taxonomic systematics. In this regard, the addition of two Henneguya spp. and one Myxobolus sp. reported in the present study from marine fish to the Henneguya–Myxobolus clade (Fiala 2006; Liu et al. 2010) may help us to speculate the molecular phylogeny and taxonomic relationship of freshwater and marine myxobolids.

Discussion

References

Only a single species, Myxobolus acanthopagri Lom and Dyková 1994, from the speciose Henneguya–Myxobolus clade (Fiala 2006) has been recorded from fish of the genus Acanthopagrus worldwide (Lom and Dyková 1994) and none from other marine fish of the family Oplegnathidae. M. acanthopagri was recorded from subepithelial tissue and muscularis of the intestine of A. australis fished in Coffs Harbour, NSW, Australia. Unfortunately, its SSU rDNA sequence is not currently available in the DDBJ/EMBL/GenBank databases. Thus, its phylogenetic relationship with the two new Henneguya spp. reported in the present study remains to be clarified. Myxozoans infecting the same target organs, rather than those taking the same host species or groups or the same spore morphology, appear to cluster in the phylogenetic tree (Andree et al. 1999; Eszterbauer 2004; Holzer et al. 2004; Fiala 2006). Furthermore, it is currently hypothesized that the bifurcated caudal process of the genus “Henneguya” arose on separate occasions during the evolution of ancient myxobolids in the world water (Kent et al. 2001; Fiala 2006; Lom and Dyková 2006), and so new myxobolid systematics awaits proposal (Lom and Dyková 2006; Bartosová et al. 2009; Picon-Camacho et al. 2009; Liu et al. 2010). Liu et al. (2010) observed a single species, M. turpisrotundus Zhang, 2009, showing both Myxobolus-type and Henneguya-type spores (albeit the latter type occupied about 10 %), and indicated the inadequacy of the use of the caudal process as the key character to separate these two genera, as having been addressed by Kent et al. (2001). Thus, genetic analyses, exclusively dependent on the SSU rDNA sequence at present, are being increasingly applied to taxonomic study; however, the stock of data is currently insufficient. In light of such conditions, we should inevitably follow the classical taxonomic methods with genetic characterization of the identified species for future resolution of the conflict between

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Acknowledgments This study was supported in part by a Grant-inAid (H23-shokuhin-ippan-007) from the Ministry of Health, Labour and Welfare of Japan.

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