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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Molecular Phylogenetics and Evolution 57 (2010) 687–702 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Genetic variation and phylogeny of the cosmopolitan marine genus Tubificoides (Annelida: Clitellata: Naididae: Tubificinae) a,b, c a Sebastian Kvist ⇑, Indra Neil Sarkar , Christer Erséus a Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Göteborg, Sweden b Richard Gilder Graduate School, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024, USA c Center for Clinical and Translational Science, Department of Microbiology & Molecular Genetics and Department of Computer Science, University of Vermont, 89 Beaumont Avenue, Given Courtyard N309, Burlington VT 05405, USA article info a b s t r a c t Article history: Prior attempts to resolve the phylogenetic relationships of the cosmopolitan, marine clitellate genus Received 15 February 2010 Tubificoides, using only morphology, resulted in unresolved trees. In this study, three mitochondrial Revised 16 August 2010 and three nuclear loci (5912 aligned sites) were analyzed, representing 14 morphologically separate Accepted 17 August 2010 species. Genetic distances within and between these forms on the basis of the mitochondrial genes Available online 27 August 2010 (COI, 16S and 12S) revealed that 18 distinct mitochondrial lineages were represented in the data set. After analyzing also nuclear data (28S, 18S and ITS) we conclude that 17 separately evolving lineages Keywords: (i.e., phylogenetic species) were represented, including three new, cryptic species closely related to T. Genetic variation pseudogaster, T. amplivasatus and T. insularis, respectively. Special emphasis was put on the DNA barcod- Haplotype diversity Species delimitation ing gene (COI), which was subject to haplotype diversity analysis and, for four species, diagnostic posi- Geographic distribution tion (as determined by the Characteristic Attribute Organization System [CAOS]) screening. Typically, Phylogeny the intralineage variation was 1–2 orders of magnitude smaller than the interlineage divergence, mak- Combined analysis ing COI useful for identification of species within Tubificoides. The genetic data corroborate that many of DNA barcoding the morphospecies are coherent but widely distributed metapopulations. Monophyly of the genus is Tubificoides supported and the evolutionary history of parts of the genus is revealed by phylogenetic analysis of Tubificinae the combined data set. A northern hemisphere origin of the genus is suggested, and most of the widely Naididae distributed species are members of one particular clade. Two morphological characters previously emphasized in Tubificoides taxonomy (hair chaetae and cuticular papillation) were optimized on the phylogenetic tree, revealing considerable homoplasy, belying the utility of these features as phyloge- netic markers. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction smaller areas. Most species are euhaline, others are tolerant to fluc- tuations in salinity or prefer oligohaline conditions, and many of Naididae (including the former Tubificidae; see ICZN, 2007; them are favored by organic pollution and sulfide-rich sediments Erséus et al., 2008) is a large group of aquatic clitellates, compris- (e.g. Giere et al., 1988; Dubilier et al., 1995; Lerberg et al., 2000). ing about 1000 described species (Erséus, 2005). Currently divided By and large, the genus can be found in most coastal or deep-sea into eight subfamilies (Tubificinae, Rhyacodrilinae, Phallodrilinae, sediments at least in the northern hemisphere (e.g. Cook, 1969; Naidinae, Telmatodrilinae, Limnodriloidinae, Pristininae and Opist- Erséus, 1975; Brinkhurst and Baker, 1979; Brinkhurst, 1985; Kvist ocystinae [Erséus et al., 2008, 2010]), representatives of the family et al., 2008), some species being vastly abundant (e.g. Erséus and are found in marine as well as freshwater habitats. Tubificoides Las- Diaz, 1989; Harrel, 2004). However, all taxa examined in this tockin, 1937 is a species-rich genus of Tubificinae consisting of 57 study, except T. amplivasatus (Erséus, 1975), appear restricted to nominal species (unpublished compilation) including two recently shallow waters. described taxa (Kvist et al., 2008). Some species of Tubificoides The early taxonomy of current Tubificoides species was confusing. show a worldwide distribution while others appear endemic to Disagreements concerning the generic status of some species, mainly based on chaetal features, resulted in these being transferred Corresponding author at: Richard Gilder Graduate School, American Museum of between other genera; e.g. Tubifex Lamarck, 1816, Clitellio Savigny, ⇑ Natural History, Central Park West at 79th Street, New York, NY 10024, USA. 1820 and Limnodrilus Claparède, 1862 (Dahl, 1960; Brinkhurst, E-mail address: [email protected] (S. Kvist). 1962, 1965; see Brinkhurst and Baker, 1979; Baker, 1980). These 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.08.018 Author's personal copy 688 S. Kvist et al. / Molecular Phylogenetics and Evolution 57 (2010) 687–702 species were later included in (and several new species were as- imen) and the Caspian Sea (one specimen) (Fig. 1). In most cases, signed to) Peloscolex Leidy, 1851 (e.g., Hrabeˇ, 1966; Cook, 1969), a the specimens were collected by Kvist and/or Erséus, otherwise genus primarily defined by its characteristic cuticular papillation. by colleagues noted at the bottom of Table 1. Intertidal and subtid- However, Holmquist (1978, 1979) regarded Peloscolex as an artificial al samples were sieved by elutriation using a mesh size of 125– assemblage and re-established Tubificoides, placing all marine spe- 300 lm, and the specimens were in toto preserved in 80% ethanol. cies of Peloscolex within it; except for P. benedii (d’Udekem, 1855), Subsequently, the worms were cut in two parts, and the posterior which was transferred to Edukemius Holmquist, 1978. These species part was transferred to 95% ethanol to be used for DNA extraction, show great resemblance in the morphology of the genitalia, which while the anterior part was stained in alcoholic paracarmine, dehy- led Brinkhurst and Baker (1979) to transfer 13 additional taxa drated in an ethanol–xylene series, and mounted whole in Canada (including E. benedii) to Tubificoides. Members of Tubificoides sensu balsam to serve as a voucher. lato are recognized by the principal morphology of their male geni- talia; the vas deferens always enters the atrium subapically, more 2.3. DNA extraction, amplification and purification or less opposite to where the stalk of the prostate gland enters the same structure. The different species are then largely delimited by Total genomic DNA was extracted using DNeasy Blood and Tis- unique combinations of details in the arrangement and distribution sue KitsÓ (Qiagen Ltd.) according to the manufacturer’s protocol. of chaetae, the presence or absence of cuticular papillae and the The six loci were amplified using PuReTaq Ready-To-Go™ PCR shape and size of the penis sheaths. These features give a mosaic pat- beads (GE Healthcare) and 1 ll of each primer (Table 2). For the tern to any morphological character matrix of Tubificoides and for- PCR-reactions, the DNA extract was used in quantities of 2–4 ll mal cladistic analyses of such data sets have so far failed to and 19–21 ll of sterilized water was added to each amplification, produce any resolved phylogenetic trees (Erséus, unpublished). giving a total sample size of 25 ll. Either a PTC-100Ó (MJ Research The reported wide range of some Tubificoides species (e.g. Baker, Inc.) or an Eppendorf MastercyclerÒ was used. The thermal profile 1984; Brinkhurst, 1986; Helgason and Erséus, 1987) is suggestive was gene dependent and varied as follows: an initial step of 5 min of their significant impact on benthic coastal ecosystems in the denaturation at 95 °C (for all samples) followed by 30 (for 18S), 35 northern hemisphere. Despite this, little is known about the evolu- (for COI, 16S, 28S and ITS) or 43 (for 12S) cycles of denaturation at tionary relationships and the population genetic structure of the 95 °C (30 s for 16S, 18S and ITS; 40 s for COI, 28S and 12S), anneal- various species. In a previous study, Erséus and Kvist (2007) exam- ing at 45 °C (for COI, 16S [35 cycles] and 12S [43 cycles]), 50 °C (for ined the variation in the cytochrome c oxidase subunit I gene (COI) ITS [35 cycles]), 52 °C (for 28S [35 cycles]) or 54 °C (for 18S [30 cy- of four Scandinavian species of Tubificoides to evaluate its applica- cles]). The cycles were run for 30 s for 16S, 18S and ITS, 40 s for bility in DNA barcoding (see Hebert et al., 2003a,b, 2004; Hajiba- 28S, and 45 s for COI