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Species(Annelida:Clitellata: from Lake JapaneseJapaneseSociety Society ofSystematicZoologyof Systematic Zoology Species Diversity, 2008, 13, 221-230 A New Phreodrilid Species (Annelida: Clitellata: Phreodrilidae) from Lake Biwa, Japan Patrick Martiniand Akifumi Ohtaka2 i Freshwater Biology, Rayal Beigian institute of IVcttural Sciences, Rue Vtiutier 29, B-1000 Brussels, Belgriunz E-maig:Patrich.Martin@naturalsciences,be 2Department ofIVicitural Sciences, haculty ofEducation, Hirosaki Uhiver:sdy, 1 Bunltyo-cho, Hirosaki, 036-8560 Jic]pan E-mail: [email protected] (Received 24 January 2008; Accepted 15 October 2008) using A new species of Phreodrilidae is described flrom Lake Biwa, Japan, classical morphology and a 658bp long fragment of the mitochondrial cy- "DNA tochrome c oxidase subunit I (COI) gene as a potenttal barcode". Asta- cQpsidrilus ru,uteki sp. nov, is unusual in possessing dorsal bundles each rep- resented by only a single seta, instead of the usual hair and support setae. Among other phreodrilids, this feature has been reported only in IVesodrilus isochaeta Pinder and Brinkhurst, 1997 and insulodrilus butdus Pinder and Brinkhurst, 1997, both described firom Australia. The new species pessesses This is paired spermathecal setae and peeuliar voluminous, pendant penes, the first record of the family in Japan, a circumstance that contradiets the assumed Gondwanan origin of the Phreodrilidae. Because any hypothesis of Asia) a former zoogeographical connection between Japan (or other parts of and Australia is unsupported by any other biogeographical data, it is proba- ble that A. ryuteki was introduced to Japan from the Southern Hemisphere. Conversely, the tubificid Embolocqphalus yanzaguchii (Brinkhurst, 1971) ap- pears to have been introduced into Australia from Japan. Key Words: Clitellata, oligochaetes, Phreodrilidae, AstacQpsidrigus, new species, Lake Biwa. Introduction excluding To date, there are 51 species in the family Phreodrilidae, Tttsmani- aedritus tasmaniaensis Goddard, 1909, a taxon too poorly described to be assigned to any of the current genera. Two subfamilies are distinguished, the Phreodrilinae including Phreodrilus and Antarctodrilus, and the Phreodriloidinae with Phreo- driloides, AstacQpsielritus, insulodrilus, AJesodritus, and Schizodrilus (Brinkhurst 1991; Pinder and Brinkhurst 1997). The global distribution ef the family indicates a Gondwanan origin (Martin et al, 2008), The majority of species occur in the South- Hemisphere: ern Hemisphere, although a few have been recorded in the Northern Morocco (Giani et al. 1995), Sri Lanka (Stephenson 1913), the Arabian Peninsula (Martinez-Ansemil et al. 2002), and Ireland (Gunn et al. 2003). In this context, the find- present new species found in Lake Biwa, Japan, is an especially significant ing, since it faIIs well outside of the biogeographical range predicted by the hypoth- NII-Electronic Library Service JapaneseJapaneseSociety Society ofSystematicof Systematic Zoology 222 P. Martin and A. Ohtaka esis of a Gondwanan origin, For this reason, we have decided to publish a descrip- tion of this new species even though only one specimen (one fragment) is available for description, in spite of many unsuccessful attempts to find additional material at the type locality, This decision is additionally justMed by the fact that the new species has quite distinct morphological features that unambiguously characterize it vis-a-vis its congeners. Material and Methods The material was collected in Lake Biwa in January, 2001, by means of a dredge during a survey of the aquatic oligochaete fauna (Ohtaka and Nishino 1995, 1999, 2006; Nishino et al. 1999). The collected material was sieved through a net (250,um mesh size) anct preserved in absolute ethanol fbr subsequent DNA analy- sis. Morphological study The anterior part of the specimen (head and sexual segments) was used fbr classical morphological work. Prior to dissection, this part was removed from ethanol and fixed overnight in the laboratory in 70,Xu neutralised fbrmalin. It was cut along the midsagittal plane and the intestine was partly removed. The frag- ments were stained in alcoholic carmine, dehydrated, cleared in xylene, mounted in Canada balsam, and examined under a Reichert compound microscope with di/e ferential interference contrast. Drawings were made by means of a camera lueida. The specimen has been deposited as the holotype in the Hokkaide University Mu- seum, Sapporo, Japan, with the acronym ZIHU, representing the former Zoological Institute, Hokkaido University. In the description, segments are designated by Roman numerals and septa are indicated by the Arabic numbers of the adjacent segments (e.g., septum 415 lies between segments IV and V). DNA extraction, PCR amplification, and sequencing DNA was extracted from the posterior part of the specimen using QIAgen DNA Mini Kit according to the manufacturer's protocol for animal tissue. A 658bp re- gion of the COI gene was amplified using 10ul DNA extract, The primers HC02198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') and LCO1490 (5'-GGTCAACAAAT CATAAAGATATTGG-3r) (Folmer et al. 1994) were used in quantities ef 2.5"1, with 2.5"1 dNTP,, 2.5"I buffer, O,26ul Taq DNA polymerase (5Upt1-i), and 4.74"1 steril- ized water. The samples were subjected to 30 cycles of 60s at 940C, 60s at 510C, and 90 s at 72eC, with an additional initial step of 5 min denaturation at 94eC, and a final step of 5min extension at 720C. The PCR product was purified using the GEXTM PCR DNA and Gel Band Purincation Kit (Amersham Biosciences) according to the manufacturer's protocol. The sample was prepared for sequencing reaction with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). The sequenc- mg reactlon mixture included 3ul DNA, 2 ready reaction mix, × se- purified ,ul 1 pt15 quencing buffer, 2 pt1 of 2"M primer, and 2 "1 sterilized water. Sequencing-PCR was carried out in both directions using the primers Iisted above, with 25 cycles of 10s at 960C, 5s at 50eC, and 4min at 600C, with an initial step of 60s denaturation at 96oC. Automatic sequencing was perfbrmed on an ABI310 Genetic Analyser (Ap- NII-Electronic Library Service JapaneseJapaneseSociety Society ofSystematicof Systematic Zoology New phreodrilid species from Lake Biwa 223 plied Biosystems), The sequence was deposited in EMBL under accession number FM163436, Taxonomy AstacQpsidrilus r vuteki sp. nov. (Fig, 1) mature specimen, Holotype. ZIHU-3261, slide Ol.036.02, sexually incomplete (first 21 segments), Type Iocality. Northern basin off Hayasaki (35023'N, 136007'E), Lake Biwa, Japan, 29 January 2001, in the profundal zone of the lake at 90 m depth (see NishinQ et al. 1999). "ryuteki", "dragon Etymology. A literally a fiute", is a Japanese transverse flute used in gagaku, the Shinto traditional orchestra associated with the imperial court and ceremonies. The sound of the ryuteki is said to represent the dragons that ascend into the skies between the heavenly lights and the people of the earth, The name, a noun in apposition, is used allegorically, and is meant to represent a dragon (Astacopsidrilus rly"teki sp, nov.) calling to its closest Gondwanan relatives (the Australasian Phreodrilidae) across the Pacific Ocean. Deseription. Length 3,1mm, 21 segments (incomplete specimen with length to male pore 1.55mm). Width at XII (clitellum) O.35mm. Prostomium rounded. Clitel- lum extending over XII-XIII (Fig. IC). Ventral setae from II, two per bundle, 96-126 um long, about 6"m thiek, becom- ing slightly longer from II to following segments, sigmoid with nodulus on upper third of shaft, bifid with reduced upper teeth (Fig. IA, vs), Ventral setae absent in XII. Two modified genital, spermathecal setae per bundle in XIII; one of these elon- inconspicuous gate, 129"m long, 3.8um thick, and straight with bent ental tip, nodulus slightly distal to middle of shaft, and hollow distal tip, seta prqecting from enlarged, glandular setal sac (Fig. IB, ss); other seta shorter, 60um long, 2.3um thick, and differentiated into a simple rod mostly contained within the sac lengest (Fig. IA, sps). Large, discrete glandular cells associated with ental end of spermathecal setae and musculature (Fig. IB, gc). G!andular setal sacs opening into spermathecal vestibulae near spermathecal pores. Dorsal setae from III, one seta (exceptionally twoabne a replacement seta?) representing each bundle, similar in shape to ventral setae but slightly smaller, 70-108,um long, 6"m thick (Fig. IA, ds). Neither hair setae nor support setae pres- ent. roughly in Male and sperrnathecal pores paired (Fig. IB, mp, spp), opening of XII, same plane slightly medial of ventral seta] line; male pores on posterior part spermathecal pores on anterier margin of XIII. Eversible dorsal pad of pharynx well developed in II to IV, oesophagus in V-IX, suddenly widening te intestine in X. One pair of testes in XI. Male ducts paired. Prestate glands absent, Sperm funnels small, located in ventral part of 11!12 (Fig. IB, sfi. Vasa deferentia thin, about 10um wide and roughly ef same width along whole length, anterio-ventrally coiled in part of XII, extending dorsally to join atria near union with penes (Fig. IB, vd, u). Atria tubular, convoluted, confined to NII-Electronic Library Service JapaneseJapaneseSociety Society ofSystematicZoologyof Systematic Zoology 224 P. Martin and A. Ohtaka co raQen vnco>acoaaco s ¢ y-E8aEacagts anQut F-.--=---=--L E gg 8 NN .JV ( Fig. 1, Astacopsidritus ro,uteki sp, nov., holotype (ZIHU-3261), A, Somatic and spermathecal setae; B, right lateral view of genitalia
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