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Genetic variation and phylogeny of the cosmopolitan marine genus Tubificoides(Annelida: Clitellata: Naididae: Tubificinae)

Sebastian Kvist a,b,⇑, Indra Neil Sarkar c, Christer Erséus aaDepartment of Zoology, University of Gothenburg, Box 463, SE-405 30 Göteborg, SwedenbRichard Gilder Graduate School, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024, USAcCenter 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

a r t i c l e i n f o

Article history:Received 15 February 2010Revised 16 August 2010Accepted 17 August 2010Available online 27 August 2010

Keywords:Genetic variationHaplotype diversitySpecies delimitationGeographic distributionPhylogenyCombined analysisDNA barcodingTubificoidesTubificinaeNaididae

a b s t r a c t

Prior attempts to resolve the phylogenetic relationships of the cosmopolitan, marine clitellate genusTubificoides, using only morphology, resulted in unresolved trees. In this study, three mitochondrialand three nuclear loci (5912 aligned sites) were analyzed, representing 14 morphologically separatespecies. Genetic distances within and between these forms on the basis of the mitochondrial genes(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(i.e., phylogenetic species) were represented, including three new, cryptic species closely related to T.pseudogaster, T. amplivasatus and T. insularis, respectively. Special emphasis was put on the DNA barcod-ing gene (COI), which was subject to haplotype diversity analysis and, for four species, diagnostic posi-tion (as determined by the Characteristic Attribute Organization System [CAOS]) screening. Typically,the intralineage variation was 1–2 orders of magnitude smaller than the interlineage divergence, mak-ing COI useful for identification of species within Tubificoides. The genetic data corroborate that many ofthe morphospecies are coherent but widely distributed metapopulations. Monophyly of the genus issupported and the evolutionary history of parts of the genus is revealed by phylogenetic analysis ofthe combined data set. A northern hemisphere origin of the genus is suggested, and most of the widelydistributed species are members of one particular clade. Two morphological characters previouslyemphasized in Tubificoides taxonomy (hair chaetae and cuticular papillation) were optimized on thephylogenetic tree, revealing considerable homoplasy, belying the utility of these features as phyloge-netic markers.

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1. Introduction

Naididae (including the former Tubificidae; see ICZN, 2007;Erséus et al., 2008) is a large group of aquatic clitellates, compris-ing about 1000 described species (Erséus, 2005). Currently dividedinto eight subfamilies (Tubificinae, Rhyacodrilinae, Phallodrilinae,Naidinae, Telmatodrilinae, Limnodriloidinae, Pristininae and Opist-ocystinae [Erséus et al., 2008, 2010]), representatives of the familyare found in marine as well as freshwater habitats. Tubificoides Las-tockin, 1937 is a species-rich genus of Tubificinae consisting of 57nominal species (unpublished compilation) including two recentlydescribed taxa (Kvist et al., 2008). Some species of Tubificoidesshow a worldwide distribution while others appear endemic to

smaller areas. Most species are euhaline, others are tolerant to fluc-tuations in salinity or prefer oligohaline conditions, and many ofthem are favored by organic pollution and sulfide-rich sediments(e.g. Giere et al., 1988; Dubilier et al., 1995; Lerberg et al., 2000).By and large, the genus can be found in most coastal or deep-seasediments at least in the northern hemisphere (e.g. Cook, 1969;Erséus, 1975; Brinkhurst and Baker, 1979; Brinkhurst, 1985; Kvistet al., 2008), some species being vastly abundant (e.g. Erséus andDiaz, 1989; Harrel, 2004). However, all taxa examined in thisstudy, except T. amplivasatus (Erséus, 1975), appear restricted toshallow waters.

The early taxonomyof current Tubificoides specieswas confusing.Disagreements concerning the generic status of some species,mainly based on chaetal features, resulted in these being transferredbetween other genera; e.g. Tubifex Lamarck, 1816, Clitellio Savigny,1820 and Limnodrilus Claparède, 1862 (Dahl, 1960; Brinkhurst,1962, 1965; see Brinkhurst and Baker, 1979; Baker, 1980). These

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⇑ Corresponding author at: Richard Gilder Graduate School, American Museum ofNatural History, Central Park West at 79th Street, New York, NY 10024, USA.

E-mail address: [email protected] (S. Kvist).

Molecular Phylogenetics and Evolution 57 (2010) 687–702

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species were later included in (and several new species were as-signed to) Peloscolex Leidy, 1851 (e.g., Hrabě, 1966; Cook, 1969), agenus primarily defined by its characteristic cuticular papillation.However, Holmquist (1978, 1979) regarded Peloscolex as an artificialassemblage and re-established Tubificoides, placing all marine spe-cies of Peloscolex within it; except for P. benedii (d’Udekem, 1855),whichwas transferred to EdukemiusHolmquist, 1978. These speciesshow great resemblance in the morphology of the genitalia, whichled Brinkhurst and Baker (1979) to transfer 13 additional taxa(including E. benedii) to Tubificoides. Members of Tubificoides sensulato are recognized by the principal morphology of their male geni-talia; the vas deferens always enters the atrium subapically, moreor less opposite to where the stalk of the prostate gland enters thesame structure. The different species are then largely delimited byunique combinations of details in the arrangement and distributionof chaetae, the presence or absence of cuticular papillae and theshape and size of thepenis sheaths. These features give amosaic pat-tern to any morphological character matrix of Tubificoides and for-mal cladistic analyses of such data sets have so far failed toproduce any resolved phylogenetic trees (Erséus, unpublished).

The reported wide range of some Tubificoides species (e.g. Baker,1984; Brinkhurst, 1986; Helgason and Erséus, 1987) is suggestiveof their significant impact on benthic coastal ecosystems in thenorthern hemisphere. Despite this, little is known about the evolu-tionary relationships and the population genetic structure of thevarious species. In a previous study, Erséus and Kvist (2007) exam-ined the variation in the cytochrome c oxidase subunit I gene (COI)of four Scandinavian species of Tubificoides to evaluate its applica-bility in DNA barcoding (see Hebert et al., 2003a,b, 2004; Hajiba-baei et al., 2007). The interspecific divergence was found to betwo orders of magnitude greater than the intraspecific variationfor this gene. However, this factor (the ‘‘barcoding gap”) may havebeen inflated by the limited size of the geographic area sampled(Meyer and Paulay, 2005).

The purpose of the present study was to assess the genetic varia-tion in a larger sample of Tubificoides, including nuclear as well asmitochondrial data, and re-examine COI using both distance andcharacter based methods. In particular, we evaluated the haplotypedistribution among conspecific, widely separated populations. Wealso conducted a phylogenetic analysis to estimate the general evo-lutionary history of the genus and,more specifically, to establish thenumber of separately evolving lineages within some nominal taxawith suspected cryptic speciation. Finally, we used the evolutionarytree to assess the phylogenetic signal of some of themost commonlyused morphological characters in the taxonomy of Tubificoides.

2. Material and methods

2.1. Genes and taxa studied

Six loci were used for the combined phylogenetic analysis: par-tial COI mtDNA, partial 16S and 12S ribosomal mtDNA, partial nu-clear 18S and 28S rDNA and the nuclear ITS region (with theInternal Transcribed Spacers 1 and 2 flanking the 5.8S ribosomalgene). Fourteen morphologically separated Tubificoides specieswere investigated and eight other tubificine naidids were used asoutgroup taxa. The trees were rooted with Limnodriloides anxius(Limnodriloidinae). A complete list of specimens, sampling sites,and gene and voucher accession numbers can be found in Table 1.All vouchers are deposited in the Swedish Museum of Natural His-tory (SMNH), Stockholm.

2.2. Collection of new material

Our material was assembled from 14 countries, representingboth sides of the Atlantic Ocean, the West Pacific Ocean (one spec-

imen) and the Caspian Sea (one specimen) (Fig. 1). In most cases,the specimens were collected by Kvist and/or Erséus, otherwiseby colleagues noted at the bottom of Table 1. Intertidal and subtid-al samples were sieved by elutriation using a mesh size of 125–300 lm, and the specimens were in toto preserved in 80% ethanol.Subsequently, the worms were cut in two parts, and the posteriorpart was transferred to 95% ethanol to be used for DNA extraction,while the anterior part was stained in alcoholic paracarmine, dehy-drated in an ethanol–xylene series, and mounted whole in Canadabalsam to serve as a voucher.

2.3. DNA extraction, amplification and purification

Total genomic DNA was extracted using DNeasy Blood and Tis-sue Kits! (Qiagen Ltd.) according to the manufacturer’s protocol.The six loci were amplified using PuReTaq Ready-To-Go™ PCRbeads (GE Healthcare) and 1 ll of each primer (Table 2). For thePCR-reactions, the DNA extract was used in quantities of 2–4 lland 19–21 ll of sterilized water was added to each amplification,giving a total sample size of 25 ll. Either a PTC-100! (MJ ResearchInc.) or an Eppendorf Mastercycler" was used. The thermal profilewas gene dependent and varied as follows: an initial step of 5 mindenaturation at 95 #C (for all samples) followed by 30 (for 18S), 35(for COI, 16S, 28S and ITS) or 43 (for 12S) cycles of denaturation at95 #C (30 s for 16S, 18S and ITS; 40 s for COI, 28S and 12S), anneal-ing at 45 #C (for COI, 16S [35 cycles] and 12S [43 cycles]), 50 #C (forITS [35 cycles]), 52 #C (for 28S [35 cycles]) or 54 #C (for 18S [30 cy-cles]). The cycles were run for 30 s for 16S, 18S and ITS, 40 s for28S, and 45 s for COI and 12S. Extension was performed at 72 #C(60 s for COI, 16S, 12S and 28S; 90 s for 18S and ITS) and a finalextension step at 72 #C (8 min) was performed for all samples.

PCR products were purified using the E.Z.N.A™ Cycle-Pure kit(Omega Bio-Tek Inc.) following the manufacturer’s protocol. There-after, sequencing was carried out in both directions and, for thispurpose, additional primers were used for ITS and 18S. COI se-quences of specimens CE539, 541, 551, 552, 1064, 1269, 1274and 1724–1762 were obtained from Erséus and Kvist (2007). Forthe new specimens, PCR products were sent to MacroGen Co.Ltd., South Korea for sequencing.

2.4. Sequence assembly and alignment

Forward and reverse sequences were assembled using SeqManII (DNAStar" Inc.). Both ends were trimmed in EditSeq (DNAStar"

Inc.) to delete primer sequences and manual editing was per-formed on obvious misreadings. Moreover, the Tubificoides COI se-quences EF675192–675228, used by Erséus and Kvist (2007), wererevisited and several of these were extended in both ends; thesewere previously cut by the authors based on the rather low, yet va-lid, base-calling scores. To ensure the absence of stop codons in theCOI sequence, these were translated using six frame translation onthe Baylor College of Medicine Search Launcher website ().The consensus sequences were aligned using the web version ofMUSCLE ver. 3.7 (Edgar, 2004) on the European BioinformaticsInstitute (EBI) server applying default settings and for ITS, whichproved hard to correctly align, minor changes were further doneby eye (TreeBASE study S10593; matrix M5892).

2.5. Data analyses

Four data sets were used: (i) 187 specimens for the COI varia-tion analysis, (ii) 62 specimens for the genetic variation analysisof the 16S and ITS loci, and also for the phylogenetic analysis ofthe ITS locus, (iii) 34 specimens of Tubificoides and eight outgrouptaxa for the phylogenetic analysis of each of the remaining five loci

688 S. Kvist et al. /Molecular Phylogenetics and Evolution 57 (2010) 687–702

Author's personal copyTable 1List of included specimens, collection localities and GenBank accession numbers. Country codes used in the site column, SE, Sweden; DK, Denmark; NL, The Netherlands; US, United States of America; ES, Spain; UK, United Kingdom; HK,Hong Kong; IR, Iran; FR, France; ET, Estonia; BS, Bahamas; CA, Canada. Accession numbers shown in bold face were included in the extended 16S and ITS data sets used for the genetic variation analyses. Sequences of CE196 Heterochaetacostata were derived from two different specimens from the same locality. Most specimens are collected by first and/or third author, and exceptions from this are noted as footnotes.

Species andauthority

Site (depth) Source Voucher# GenBank accession #

COI 12S 16S 18S 28S ITS

T. benedii I (d’Udekem, 1855) SE, Bohuslän, Koster area, Ursholmen (3 m) CE539-1 SMNH85171 HM460078 – – – – –T. benedii I SE, Bohuslän, Koster area, Ursholmen (3 m) CE539-2 SMNH85172 HM460079 – – – – –T. benedii I SE, Bohuslän, Koster area, Persgrunden (17 m) CE1269-2 SMNH85169 HM460080 – – – – –T. benedii I SE, Bohuslän, Koster area, Persgrunden (17 m) CE1269-3 SMNH85170 HM460081 – – – – –T. benedii I SE, Bohuslän, Koster area, Persgrunden (44–52 m) CE1638 SMNH108894 HM460082 – – – – –T. benedii I DK, The Sound, Elsinore, Ellekilde have, near shore (6 m) CE1744 SMNH85158 HM460083 – – – – –T. benedii I DK, The Sound, Elsinore, Ellekilde have (10 m) CE1746 SMNH85160 HM460084 HM459894 HM459929 HM459994 HM460034 HM460273T. benedii I DK, The Sound, S of Elsinore harbour (16 m) CE1747 SMNH85161 HM460085 – – – – –T. benedii I SE, Bohuslän, Koster area, Saltö, mudflat (0.5 m) CE1754 SMNH85162 HM460086 – – – – –T. benedii I SE, Bohuslän, Koster area, Saltö, mudflat (0.5 m) CE1755 SMNH85163 HM460087 – – – – –T. benedii I SE, Bohuslän, Koster area, Saltö, mudflat (0.5 m) CE1756 SMNH85164 HM460088 – – – – –T. benedii I SE, Bohuslän, Koster area, Saltö, mudflat (0.5 m) CE1758 SMNH85165 HM460089 – – – – –T. benedii I SE, Bohuslän, Koster area, Saltö, mudflat (0.5 m) CE1759 SMNH85166 HM460090 – – – – –T. benedii I SE, Bohuslän, Koster area, Saltö, mudflat (0.5 m) CE1760 SMNH85167 HM460091 – – – – –T. benedii I SE, Bohuslän, off Lysekil, Bonden (26–31 m) CE1778 SMNH108895 HM460092 – – – – –T. benedii I SE, Bohuslän, off Lysekil, Bonden (26–31 m) CE1779 SMNH108896 HM460093 – – – – –T. benedii I SE, Bohuslän, off Lysekil, Bonden (26–31 m) CE1781 SMNH108897 HM460094 HM459895 HM459930 HM459995 HM460035 HM460274T. benedii I SE, Bohuslän, off Lysekil, Bonden (26–31 m) CE1782 SMNH108898 HM460095 – – – – –T. benedii I SE, Bohuslän, Koster area, Ramsö, at Ullvillarna (20–30 m) CE2080 SMNH108899 HM460096 – HM459931 – – HM460275T. benedii I SE, Bohuslän, Koster area, Ramsö, at Ullvillarna (20–30 m) CE2081 SMNH108900 HM460097 – HM459932 – – HM460276T. benedii I SE, Bohuslän, Koster area, Ramsö, at Ullvillarna (20–30 m) CE2083 SMNH108901 HM460098 – – – – –T. benedii I SE, Västergötland, Gothenburg, Saltholmen (0.7 m) CE2541 SMNH108902 HM460099 – – – – –T. benedii I SE, Västergötland, Gothenburg, Saltholmen (0.7 m) CE2543 SMNH108903 HM460100 – – – – –T. benedii I SE, Västergötland, Gothenburg, Saltholmen (1.5 m) CE2587 SMNH108904 HM460101 – HM459933 – – HM460277T. benedii I SE, Västergötland, Gothenburg, Saltholmen (1.5 m) CE2588 SMNH108905 HM460102 – – – – –T. benedii I SE, Västergötland, Gothenburg, Saltholmen (1.5 m) CE2589 SMNH108906 HM460103 – – – – –T. benedii Ia FR, Atlantic coast, Concarneau (lower intertidal) CE2955 SMNH108907 HM460104 – – – – –T. benedii Ia FR, Atlantic coast, Concarneau (lower intertidal) CE2956 SMNH108908 HM460105 – – – – –T. benedii Ia FR, Atlantic coast, Roscoff, W of harbour (lower intertidal) CE2957 SMNH108909 HM460106 – – – – –T. benedii I SE, Halland, Kungsbacka, Gottskär (intertidal) CE3102 SMNH108910 HM460107 – – – – –T. benedii I SE, Halland, Kungsbacka, Gottskär (intertidal) CE3109 SMNH108911 HM460108 – – – – –T. benedii I SE, Bohuslän, Koster area, W of Ursholmen (38 m) CE3220 SMNH108912 HM460109 – HM459934 – – HM460278T. benedii I SE, Bohuslän, Koster area, W of Ursholmen (38 m) CE3221 SMNH108913 HM460110 – – – – –T. benedii Ib NL, Zeeland, Walcheren (8 m) CE5320 SMNH108914 HM460111 – HM459935 – – HM460279T. benedii Ic NO, Bergen, near Biological Station, Espegrend (5–10 m) CE5430 SMNH108915 HM460112 – – – – –T. benedii Ic NO, Bergen, near Biological Station, Espegrend (5–10 m) CE5431 SMNH108916 HM460113 – – – – –T. benedii Ic NO, Bergen, near Biological Station, Espegrend (10–15 m) CE5433 SMNH108917 HM460114 HM459896 HM459936 HM459996 HM460036 HM460280T. benedii I CA, New Brunswick, Bocabec, Passamaquoddy Bay (0.5 m) CE7282 SMNH108918 HM460115 – – – – HM460281T. benedii I CA, NB, Blacks Harbour, Passamaquoddy Bay (0.5 m) CE7283 SMNH108919 HM460116 – – – – –T. benedii IId UK, Plymouth, Millbrook Lake, Palmer Point (intertidal) CE2691 SMNH108920 HM460117 – HM459937 – – HM460282T. benedii IId UK, Plymouth, Millbrook Lake, Palmer Point (intertidal) CE2692 SMNH108921 HM460118 – HM459938 – – HM460283T. benedii IId UK, Plymouth, Millbrook Lake, Palmer Point (intertidal) CE2693 SMNH108922 HM460119 – – – – –T. benedii II UK, Wales, S of Newport, St. Brides Wentloog (5 m) CE3390 SMNH108923 HM460120 HM459897 HM459939 HM459997 HM460037 HM460284T. benedii II UK, Wales, S of Newport, St. Brides Wentloog (5 m) CE3392 SMNH108924 HM460121 – – – – –T. benedii II UK, Wales, S of Newport, St. Brides Wentloog (5 m) CE3394 SMNH108925 HM460122 – – – – –T. benedii II UK, Wales, S of Newport, Peterstone Wentloog (2–3 m) CE3397 SMNH108926 HM460123 – HM459940 – – HM460285T. benedii II UK, Wales, S of Newport, Peterstone Wentloog (2–3 m) CE3398 SMNH108927 HM460124 – – – – –T. benedii II UK, Wales, S of Newport, Peterstone Wentloog (2–3 m) CE3399 SMNH108928 HM460125 – – – – –T. benedii II UK, Wales, S of Newport, Peterstone Wentloog (2 m) CE3401 SMNH108929 HM460126 – – – – –T. benedii II UK, Wales, S of Newport, Peterstone Wentloog (2 m) CE3402 SMNH108930 HM460127 HM459898 HM459941 HM459998 HM460038 HM460286T. benedii II UK, Wales, S of Newport, Peterstone Wentloog (2 m) CE3403 SMNH108931 HM460128 – – – – –

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Author's personal copyTable 1 (continued)




T. benedii II UK, Wales, S of Newport, Peterstone Wentloog (2 m) CE3404 SMNH108932 HM460129 – – – – –T. benedii II UK, Wales, S of Newport, Peterstone Wentloog (1 m) CE3405 SMNH108933 HM460130 – – – – –T. benedii IIb NL, Zeeland, Middelburg, Walcheren, canal (8 m) CE5319 SMNH108934 HM460131 – HM459942 – – HM460287T. benedii II CA, New Brunsw., St. Andrews, Passamaquoddy Bay (0.5 m) CE7284 SMNH108935 HM460132 – – – – HM460288T. benedii II CA, New Brunsw., St. Andrews, Passamaquoddy Bay (0.5 m) CE7285 SMNH108936 HM460133 – – – – –T. diazi Brinkhurst and Baker, 1979a FR, Atlantic coast, Pouldohan Bay (intertidal) CE2964 SMNH108937 HM460134 – – – – –T. diazi UK, Wales, Swansea, Swansea Bay, Oxwich (3 m) CE3409 SMNH108938 HM460135 HM459899 HM459943 HM459999 HM460039 HM460289T. diazi UK, Wales, Swansea, Swansea Bay, Oxwich (3 m) CE3410 SMNH108939 HM460136 – HM459944 – – HM460290T. diazi UK, Wales, Swansea, Swansea Bay, Oxwich (3 m) CE3411 SMNH108940 HM460137 HM459900 HM459945 HM460000 HM460040 HM460291T. diazi UK, Wales, Swansea, Swansea Bay, Oxwich (3 m) CE3482 SMNH108941 HM460138 – – – – –T. diazi UK, Wales, Swansea, Swansea Bay, Mumbles Pier (3 m) CE3484 SMNH108942 HM460139 – – – – –T. amplivasatus I (Erséus, 1975) SE, Bohuslän, Koster area, Koster fjord trench (250 m) CE541–1 SMNH85181 HM460140 – – – – –T. amplivasatus I SE, Bohuslän, Koster area, Koster fjord trench (250 m) CE541–2 SMNH85182 HM460141 – – – – –T. amplivasatus I SE, Bohuslän, Koster area, Koster fjord trench (250 m) CE541–3 SMNH85183 HM460142 – – – – –T. amplivasatus I SE, Bohuslän, Koster area, Koster fjord trench (250 m) CE541–4 SMNH85184 HM460143 – HM459946 – – HM460292T. amplivasatus I SE, Bohuslän, Koster area, inner archipelago (

Author's personal copyT. heterochaetusg NL, Zuid-Holland, Dirksland, Havelose Weg CE2264 SMNH108967 HM460183 – – – – –T. heterochaetush NL, Zuid-Holland, Dirksland, Wittebrug Goededreede CE2266 SMNH108968 HM460184 HM459903 HM459959 HM460008 HM460048 HM460305T. heterochaetus US, Virginia, Middlesex Co., La Grange Creek (1–2 m) CE2446 SMNH108969 HM460185 – – – – –T. heterochaetus US, Virginia, Middlesex Co., La Grange Creek (1–2 m) CE2447 SMNH108970 HM460186 HM459904 HM459960 HM460009 HM460049 HM460306T. heterochaetus US, Virginia, Middlesex Co., Grooms Landing (0.5 m) CE2468 SMNH108971 HM460187 – – – – –T. heterochaetus US, Virginia, Middlesex Co., Grooms Landing (0.5 m) CE2469 SMNH108972 HM460188 – – – – –T. heterochaetus US, Virginia, Middlesex Co., Grooms Landing (0.5 m) CE2470 SMNH108973 HM460189 – – – – –T. heterochaetus US, Virginia, Middlesex Co., La Grange Creek (1–2 m) CE2496 SMNH108974 HM460190 – HM459961 – – –T. kozloffi Baker, 1983e SE, Halland, Fladen (15–20 m) CE1064 SMNH95193 HM460191 – – – – –T. kozloffi DK, The Sound, Elsinore, Ellekilde have (10 m) CE1272 SMNH108975 HM460192 – – – – –T. kozloffi SE, Bohuslän, Koster area, W of Nord-Koster (16–18 m) CE2062 SMNH108976 HM460193 HM459905 HM459962 HM460010 HM460050 HM460307T. kozloffi SE, Bohuslän, Koster area, W of Nord-Koster (16–18 m) CE2134 SMNH108977 HM460194 HM459906 HM459963 HM460011 HM460051 HM460308T. kozloffi SE, Bohuslän, Koster area, W of Nord-Koster (16–18 m) CE2135 SMNH108978 HM460195 – – – – –T. kozloffi SE, Bohuslän, Koster area, W of Nord-Koster (16–18 m) CE2136 SMNH108979 HM460196 – – – – –T. kozloffi SE, Bohuslän, Koster area, W of Ursholmen (38 m) CE3210 SMNH108980 HM460197 – – – – –T. kozloffi SE, Bohuslän, Koster area, W of Ursholmen (38 m) CE3211 SMNH108981 HM460198 – – – – –T. kozloffi SE, Bohuslän, Koster area, W of Ursholmen (38 m) CE3212 SMNH108982 HM460199 – HM459964 – – HM460309T. kozloffi SE, Bohuslän, Koster area, W of Ursholmen (38 m) CE3213 SMNH108983 HM460200 – – – – –T. kozloffi SE, Bohuslän, Koster area, S of Sydkoster, Skåreskär (15 m) CE3250 SMNH108984 HM460201 – – – – –T. pseudogaster I (Dahl, 1960) SE, Bohuslän, Koster area, Tjärnö, at marine lab (0.5 m) CE199–3 SMNH108985 HM460202 – HM459965 – – –T. pseudogaster I SE, Bohuslän, Koster area, Tjärnö, at marine lab (intertidal) CE2077 SMNH108986 HM460203 HM459907 HM459966 HM460012 HM460052 HM460310T. pseudogaster I SE, Halland, Kungsbacka, Hanhalsholme (1 m) CE3107 SMNH108987 HM460204 – – – – –T. pseudogaster I SE, Bohuslän, Koster area, between Saltö and Tjärnö (1 m) CE3205 SMNH108988 HM460205 HM459908 HM459967 HM460013 HM460053 HM460311T. pseudogaster II SE, Bohuslän, Koster area, Tjärnö, at marine lab (0.5 m) CE199–2 SMNH108989 HM460206 HM459909 HM459968 HM460014 HM460054 HM460312T. pseudogaster II SE, Bohuslän, Koster area, between Saltö and Tjärnö (1 m) CE1753 SMNH108990 HM460207 HM459910 HM459969 HM460015 HM460055 HM460313T. pseudogaster II SE, Västergötland, Gothenburg, Saltholmen (1.5 m) CE2578 SMNH108991 HM460208 – – – – –T. pseudogaster II SE, Västergötland, Gothenburg, Saltholmen (1.5 m) CE2580 SMNH108992 HM460209 – – – – –T. pseudogaster II SE, Västergötland, Gothenburg, Saltholmen (1.5 m) CE2581 SMNH108993 HM460210 – – – – –T. pseudogaster II SE, Bohuslän, Koster area, between Saltö and Tjärnö (1 m) CE3206 SMNH108994 HM460211 – – – – –T. pseudogaster II UK, Wales, N of Caerphilly, Rhymney River (3–4 m) CE3355 SMNH108995 HM460212 – – – – –T. pseudogaster II UK, Wales, S of Newport, Peterstone Wentloog (1 m) CE3406 SMNH108996 HM460213 – – – – –T. pseudogaster IIc NO, Bergen, at Biological Station, Espegrend (intertidal) CE5442 SMNH108997 HM460214 – – – – –T. pseudogaster IIc NO, Bergen, at Biological Station, Espegrend (intertidal) CE5443 SMNH108998 HM460215 – – – – –T. wasselli Brinkhurst and Baker, 1979 US, Virginia, York Co., York Bridge Harbour (2 m) CE2408 SMNH108999 HM460216 HM459911 HM459970 HM460016 HM460056 HM460314T. wasselli US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2491 SMNH109000 HM460217 HM459912 HM459971 HM460017 HM460057 HM460315T. parapectinatus Brinkhurst, 1985 US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2473 SMNH109001 HM460218 – HM459972 – – HM460316T. parapectinatus US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2476 SMNH109002 HM460219 – – – – –T. parapectinatus US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2486 SMNH109003 HM460220 – – – – –T. parapectinatus US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2489 SMNH109004 HM460221 HM459913 HM459973 HM460018 HM460058 HM460317T. parapectinatus US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2490 SMNH109005 HM460222 HM459914 HM459974 HM460019 HM460059 HM460318T. parapectinatusb NL, Zeeland, Middelburg, Walcheren, canal (8 m) CE5321 SMNH109006 HM460223 – – – – –T. parapectinatusb NL, Zeeland, Middelburg, Walcheren, canal (8 m) CE5322 SMNH109007 HM460224 – – – – –T. parapectinatusb NL, Zeeland, Middelburg, Walcheren, canal (8 m) CE5323 SMNH109008 HM460225 HM459915 HM459975 HM460020 HM460060 HM460319T. brownaei Brinkhurst and Baker, 1979 US, Virginia, Gloucester Co., York River, Gloucester Pt (2 m) CE2376 SMNH109009 HM460226 – HM459976 – – HM460320T. brownaei US, Virginia, Gloucester Co., York River, Gloucester Pt (2 m) CE2377 SMNH109010 HM460227 – – – – –T. brownae US, Virginia, Gloucester Co., York River, Clay Bank (8 m) CE2428 SMNH109011 HM460228 – – – – –T. brownae US, Virginia, Gloucester Co., York River, Clay Bank (8 m) CE2429 SMNH109012 HM460229 – – – – –T. brownae US, Virginia, Gloucester Co., York River, Clay Bank (8 m) CE2430 SMNH109013 HM460230 HM459916 HM459977 HM460021 HM460061 HM460321T. brownae US, Virginia, Gloucester Co., York River, Clay Bank (8 m) CE2438 SMNH109014 HM460231 – – – – –T. brownae US, Virginia, Gloucester Co., York River, Clay Bank (8 m) CE2439 SMNH109015 HM460232 – – – – –T. brownae US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2474 SMNH109016 HM460233 – – – – –T. brownae US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2482 SMNH109017 HM460234 – – – – –T. brownae US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2484 SMNH109018 HM460235 – – – – –T. brownae US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2485 SMNH109019 HM460236 – – – – –T. brownae US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2487 SMNH109020 HM460237 – – – – –T. brownae US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2488 SMNH109021 HM460238 – – – – –T. brownae US, Delaware, Sussex Co., Lewes, Marine Lab (4 m) CE2475 SMNH109022 HM460239 – – – – –T. brownae UK, Wales, S of Newport, St. Brides Wentloog (5 m) CE3387 SMNH109023 HM460240 HM459917 HM459978 HM460022 HM460062 HM460322T. brownae UK, Wales, S of Newport, St. Brides Wentloog (5 m) CE3389 SMNH109024 HM460241 – – – – –

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Author's personal copyTable 1 (continued)




T. brownae US, New York, New York, Staten Island,Rossville (0.5 m)

CE5460 SMNH109025 HM460242 – – – – –

T. imajimaij Brinkhurst, 1985 HK, Conic Island, submarine cave (19 m) CE536 SMNH109026 HM460243 HM459918 HM459979 HM460023 HM460063 HM460323T. insularis I (Stephenson, 1922) UK, Wales, Swansea, Swansea Bay, Oxwich (3 m) CE3415 SMNH109027 HM460244 – – – – –T. insularis I UK, Wales, Swansea, Swansea Bay, Mumbles Pier (3 m) CE3416 SMNH109028 HM460245 – – – – –T. insularis I UK, Wales, Swansea, Swansea Bay,

Mumbles Pier (3 m)CE3417 SMNH109029 HM460246 HM459919 HM459980 HM460024 HM460064 HM460324

T. insularis I UK, Wales, Swansea, Swansea Bay,Mumbles Pier (3 m)

CE3418 SMNH109030 HM460247 HM459920 HM459981 HM460025 HM460065 HM460325

T. insularis I UK, Wales, Swansea, Swansea Bay,Mumbles Pier (3 m)

CE3424 SMNH109031 HM460248 – HM459982 – – HM460326

T. insularis IIk UK, Scotland, Cambpeltown, Campbeltown Loch (18 m) CE4438 SMNH109032 HM460249 – HM459983 HM460026 HM460066 HM460327T. insularis IIk UK, Scotland, Campbeltown, Campbeltown Loch (18 m) CE4439 SMNH109033 HM460250 – HM459984 HM460027 HM460067 HM460328T. parviductus Helgason and

Erséus, 1987BS, Great Exuma, Lee Stocking Island, Lobster Pond (0.5 m) CE71 SMNH109034 HM460251 HM459921 HM459985 HM460028 HM460068 HM460329

T. fraseril Brinkhurst, 1986 IR, Caspian Sea, Noor coast (15–30 m; salinity 13 ppt) CE1273 SMNH109035 HM460252 – – – – –T. fraseri US, Virginia, Gloucester Co., Glouc. Pt., Sarah’s Cr. (3 m) CE2396 SMNH109036 HM460253 – HM459986 – – HM460330T. fraseri US, Virginia, Gloucester Co., Glouc. Pt., Heywood Cr. (2 m) CE2412 SMNH109037 HM460254 HM459922 HM459987 HM460029 HM460069 HM460331T. fraseri US, Virginia, Gloucester Co., Glouc. Pt., Heywood Cr. (2 m) CE2413 SMNH109038 HM460255 – – – – –T. fraseri US, Virginia, Gloucester Co., Glouc. Pt., Heywood Cr. (2 m) CE2414 SMNH109039 HM460256 – – – – –T. fraseri US, Virginia, Gloucester Co., Glouc. Pt.,

Heywood Cr. (2 m)CE2422 SMNH109040 HM460257 – – – – –

T. fraseri US, Virginia, Middlesex Co.,La Grange Creek (1–2 m)

CE2448 SMNH109041 HM460258 – – – – –

T. fraseri US, Virginia, Middlesex Co., La Grange Creek (1–2 m) CE2450 SMNH109042 HM460259 – – – – –T. fraseri US, Virginia, Middlesex Co., Urbanna Creek harbour (2 m) CE2458 SMNH109043 HM460260 – – – – –T. fraseri US, Virginia, Middlesex Co., Urbanna Creek harbour (2 m) CE2459 SMNH109044 HM460261 – HM459988 – – HM460332T. fraseri US, Virginia, Middlesex Co., Urbanna Creek harbour (2 m) CE2460 SMNH109045 HM460262 – HM459989 – – HM460333T. fraseri US, Virginia, Middlesex Co., Urbanna Creek harbour (2 m) CE2495 SMNH109046 HM460263 – – – – –T. fraseri UK, Wales, N of Caerphilly, Rhymney River (3–4 m) CE3354 SMNH109047 HM460264 HM459923 HM459990 HM460030 HM460070 HM460334

Outgroup taxaClitellio arenarius (Müller, 1776) SE, Bohuslän, Koster area, Tjärnö Marine lab (0.3 m) CE112 – HM460265 HM459924 AY885615 AF411863 HM460071 –Heterochaeta costata (Claparède, 1863) SE, Bohuslän, Koster area, near Tjärnö (>10 m) CE196 – HM460266 HM459925 AY340460 AY340432 AY340397 –Tubifex ignotus (Stolc, 1886) SE, Västergötland, Vårgårda, Fly,

Lången Lake (littoral)CE211 – HM460267 DQ459921 AY885610 AF411879 HM460072 –

Aulodrilus pluriseta (Piguet, 1906) EE, lab culture kept by Tarmo Timm CE281 – HM460268 HM459926 HM459991 HM460031 HM460073 –Ilyodrilus templetoni (Southern, 1909) EE, lab culture kept by Tarmo Timm CE282 – HM460269 HM459927 HM459992 HM460032 HM460074 –Psammoryctides barbatus (Grube, 1861) EE, lab culture kept by Tarmo Timm CE289 – HM460270 HM459928 HM459993 HM460033 HM460075 –Limnodrilus hoffmeisteri Claparède, 1862 EE, lab culture kept by Tarmo Timm CE290 – HM460271 DQ459923 AY885613 AF469007 HM460076 –Limnodriloides anxius Erséus, 1990 BS, Great Exuma, Lee Stocking Island (0.5 m) CE131 – HM460272 DQ459919 AY885621 AF411866 HM460077 –

Individuals collected by:a Pierre De Wit (2007).b Ton van Haaren (2008).c Pierre De Wit (2008).d Colin Kilvington (2007).e Pierre De Wit (2005).f Judith Fuchs (2007).g Rienk Geene (2006).h Tjeerd du Bois (2006).i Alice Brylawski (2007).j Hong Zhou (2002).k Andrew Mackie (2008).l Mershad Taheri (2005).

m Individuals identified as T. heterochaetus by Erséus and Kvist (2007).

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and the mitochondrial and the nuclear loci separately, and (iv) 18specimens of Tubificoides and eight outgroup taxa for the phyloge-netic analysis of the six loci combined. Failure to recover 12S for T.insularis II, T. amplivasatus I and II, and ITS for the outgroup taxa re-sulted in these being represented by the five remaining loci,respectively.

Nucleotide diversities ± standard errors (S.E.) of COI, 16S andITS were calculated in MEGA ver. 4 (Tamura et al., 2007) usingthe Kimura 2-parameter (K2P) model of base substitution

(Kimura, 1980) and with the following settings: uniform ratesamong sites and complete deletion of gaps. Haplotypediversities ± S.E. were calculated in DnaSP ver. 4.90.1 (Rozaset al., 2003) using the Jukes and Cantor model of nucleotidesubstitution (Jukes and Cantor, 1969) and the sliding windowoption (window length 100, step size 25). DnaSP does nothandle ambiguous nucleotides (e.g. N, M or Y) and, therefore,33 such positions were changed to gaps in the extended ITS dataset.

Fig. 1. Map of the collection localities for the specimens of Tubificoides. Fourteen countries and three continents are represented among the 187 samples used in the presentstudy.

Table 2List of primers used in the amplification and sequencing PCR-reactions.

Gene Primer Sequence Reference

ITS ITS4 50-TCCTCCGCTTATTGATATGC-30 White et al. (1990)ITS5 50-GGAAGTAAAAGTCGTAACAAGG-30 White et al. (1990)5.8MussR 50-GATGTCGATGTTCAATGTGTCCTGC-30 Källersjö et al. (2005)5.8MussF 50-CGCAGCCAGCTGCGTGAATTAATGT-30 Källersjö et al. (2005)

18S TimA 50-AMCTGGTTGATCCTGCCAG-30 Tim Littlewood (pers. comm. in Norén and Jondelius, 1999)TimB 50-TGATCCATCTGCAGGTTCACCT-30 Tim Littlewood (pers. comm. in Norén and Jondelius, 1999)1100R 50-GATCGTCTTCGAACCTCTG-30 Tim Littlewood (pers. comm. in Norén and Jondelius, 1999)660F 50-GATCTCGGGTCCAGGCT-30 Erséus et al. (2002)1806R 50-CCTTGTTACGACTTTTACTTCCTC-3 Michael Norén (pers comm. in Hovmöller et al., 2002)18S4FB 50-CCAGCAGCCGCGGTAATTCCAG-30 Norén and Jondelius (1999)18S4FBK 50-CTGGAATTACCGCGGCTGCTGG-30 Norén and Jondelius (1999)18S5F 50-GCGAAAGCATTTGCCAAGAA-30 Marta Riutort (pers comm. in Norén and Jondelius, 1999)18S7FK 50-GCATCACAGACCTGTTATTGC-30 Marta Riutort (pers. comm. in Norén and Jondelius, 1999)

28S 28SC1́ 50-ACCCGCTGAATTTAAGCAT-30 Dayrat et al. (2001)28SC2 50-TGAACTCTCTCTTCAAAGTTCTTTTC-30 Dayrat et al. (2001)

12S 12SE1 50-AAAACATGGATTAGATACCCRYCTAT-30 Jamieson et al. (2002)12SH 50-ACCTACTTTGTTACGACTTATCT-3 Jamieson et al. (2002)

COI LCO1490 50-GGTCAACAAATCATAAAGATATTGG-30 Folmer et al. (1994)HCO2198 50-TAAACTTCAGGGTGACCAAAAAATCA-30 Folmer et al. (1994)COI-E 50-TACTTCTGGGTGTCCGAAGAATCA-3 Bely and Wray (2004)

16S 16SAR-L 50-CGCCTGTTTATCAAAAACAT-30 Palumbi (1996)16SBRH 50-CCGGTCTGAACTCAGATCACGT-30 Palumbi (1996)

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Because morphological discrepancies exist for most of our sam-pled species but not for the groups with suspected cryptic forms,the Characteristic Attribute Organization System (CAOS; Sarkar

et al., 2002a,b) was used to find diagnostic positions (molecularsynapomorphies or Characteristic Attributes [CA’s]) in a phyloge-netic-free context (sensu Lowenstein et al., 2009) for these groups.

Table 3Gene and input data for the combined analysis.

Locus and codon position Total number of characters Number of informative characters Model Priors

COI 1st position 219 83 GTR + G Lset: nst = 6, rates = gammaPrset: statefreqpr = dirichlet (1, 1, 1, 1)

COI 2nd position 219 17 SYM + I + G Lset: nst = 6, rates = invgammaPrset: statefreqpr = fixed (equal)

COI 3rd position 220 185 GTR + I Lset: nst = 6, rates = propinvPrset: statefreqpr = dirichlet (1, 1, 1, 1)

16S 507 200 GTR + I + G Lset: nst = 6, rates = invgammaPrset: statefreqpr = dirichlet (1, 1, 1, 1)


18S 1777 56 K80 + I + G Lset: nst = 2, rates = invgammaPrset: statefreqpr = fixed (equal)


ITS 2224 579 GTR + I + G Lset: nst = 6, rates = invgammaPrset: statefreqpr = dirichlet (1, 1, 1, 1)

Total 5912 1331

Fig. 2. Midpoint-rooted Neighbor-Joining (NJ) trees of the COI locus. (a) Tree derived from the NJ analysis of the complete 187 taxa data set with each of T. fraseri, T. brownae,T. heterochaetus and T. swirencowi (discussed in detail in Section 4.3) highlighted. The tree shows a distinct separation of 18 different COI lineages within Tubificoides. (b–e)show NJ trees of the isolated alignments of T. heterochaetus, T. swirencowi, T. brownae and T. fraseri, respectively.



For the present study, single positions (‘‘simple” CA’s) were identi-fied, which were further categorized as ‘‘pure” (occurring across allmembers of a given group) or ‘‘private” (occurring exclusivelywithin a given group but not across all members).

PAUP* 4.0b10 (Swofford, 2002) was used to construct a Neigh-bor-Joining (NJ) tree of the COI locus using the K2P correction mod-el with gaps treated as missing data and using midpoint-rooting.Using PAUP*, stability of the branches (in all but the NJ tree) wasestimated by bootstrap re-sampling under the maximum parsi-mony (MP) criterion with 1000 bootstrap replicates each consist-ing of 10 random addition sequence replicates and employingTBR branch swapping.

MrModeltest ver. 2.2 (Nylander, 2004) was used to find themost likely model of evolution under a Bayesian framework (seeJohnson and Omland, 2004), with the data partitioned for eachgene and also for each codon position in COI. The partitions weretested separately according to the Akaike Information Criterion(Akaike, 1974; see Table 3) and the posterior probabilities wereestimated through Bayesian analysis (BA) using MrBayes3 (Ron-quist and Huelsenbeck, 2003). Two simultaneous runs, each withone cold and three hot Markov chains, were performed for 20 mil-lion generations (except for the extended nuclear analysis, whichended prematurely at 16 million generations due to temporalrestrictions of the server) on the CBSU computer cluster at the Cor-nell Theory Center, New York. Trees were sampled every 1000thgeneration and the first 2000 trees were discarded as ‘‘burn-in”.This yielded 14,001–18,001 trees to be used for the estimation ofposterior probabilities. Using the cumulative function, the webversion of AWTY (Nylander et al., 2008) was used to verify thatpost burn-in generations had reached stationarity in all data sets.

3. Results

3.1. The correct identity of previously deposited GenBank sequences

Specimens CE1732–1740 (see Table 1) were previously identi-fied as T. heterochaetus by Erséus and Kvist (2007), but re-examina-tion of the morphological vouchers showed that they are T.swirencowi. Thus, five previously published COI sequences (Gen-Bank Nos. EF675222–675226) should be attributed to the latterspecies.

Moreover, a worm erroneously identified as T. pseudogaster waspreviously used for sequencing of 18S (AF411873; Erséus et al.,2002), 16S (AY885609; Sjölin et al., 2005), and 12S (DQ459922;Envall et al., 2006; Erséus et al., 2010). During the present study,we found that this worm instead is identical to Heterochaeta costa-ta Claparède, 1863. This is further treated in Section 4.4.

3.2. New geographical records

Tubificoides swirencowi, previously known from the Black andMediterranean Seas (Jaroschenko, 1948; Casellato, 1999; Casellatoand Salmaso, 2000) is here formally reported for the first time fromDenmark and Sweden. As noted above, this replaces the erroneousrecord of T. heterochaetus (from Denmark) by Erséus and Kvist(2007). Furthermore, T. fraseri, previously known from Australia,New Zealand and several sites on the Atlantic and Pacific coastsof the US (Brinkhurst, 1986; Erséus, 1989), is reported for the firsttime from Wales and the Caspian Sea. Finally, T. parapectinatus,previously known from the Pacific coast of North America (Brink-hurst, 1985) is reported for the first time from the Netherlandsand from the US Atlantic coast.

3.3. Genetic variation, haplotype diversity and CA’s in COI

In preliminary diagnoses, specimens of T. benedii, T. amplivasa-tus, T. pseudogaster and T. insularis all showed morphological con-formity with the original descriptions of these taxa, but COI soonsuggested that each of them is separated into two different lin-eages. The suffices I and II are hereafter used to distinguish be-tween the different types within these morphospecies and theterms ‘‘intralineage” and ‘‘interlineage” variation are used to avoidconfusion between the terms ‘‘lineages” and ‘‘species”. Thus, a totalof 18 different mitochondrial lineages of Tubificoides were investi-gated (Table 1).

The mean intralineage COI variation in the total data set was0.33% ± 0.11 (T. fraseri showing a notably high value[0.920% ± 0.210]; see Supplementary Table 1), and the mean valueof the interlineage divergence was 24.31% ± 2.19 (T. benedii I vs. IIshowing a lower value than other comparisons [4.19% ± 0.74];see Supplementary Table 2). Low values were also noted for T. ins-ularis I vs. II (9.04% ± 1.20), T. amplivasatus I vs. II (12.74% ± 1.50)and T. imajimai vs. T. parapectinatus (13.60% ± 1.55). The mean dis-tance between lineages I and II of the morphospecies T. pseudogas-ter was 22.07% ± 2.01. These five pairings are further discussed inSection 4.1.

Fig. 2 shows the midpoint-rooted NJ tree of the COI locus, inwhich 18 distinct lineages can be identified; the four highlightedand enlarged portions of the tree are important for the discussionon geographical distribution (Section 4.3). In the COI haplotype fre-quency analysis, T. fraseri showed the largest number of differenthaplotypes (10) despite being represented by only 13 specimens(Supplementary Table 1). The highest diversity (1.00 ± 0.50) wasfound in T. wasselli but this is only based on two specimens; theclosest value to this was 0.95% ± 0.02, present in T. fraseri.

At the COI locus, CA’s were recovered for the four taxa with sus-pected cryptic forms. The CA’s are listed in their entirety in Table 4.When separating T. benedii I and II, 24 pure and 15 private attri-butes were found; for T. amplivasatus I and II, 71 pure and 7 privateattributes were found; for T. pseudogaster I and II, 124 pure and 3private attributes were found; and for T. insularis I and II, 55 pureand no private attributes were found.

3.4. Genetic variation in 16S

The ratio between interspecific and intraspecific variation in16S largely reflects that of COI, but 16S showed consistently lowervalues than COI. The mean intralineage variation was 0.15% ± 0.12(notably, the highest variation occurring in T. fraseri [0.58% ± 0.30])and the mean interlineage divergence was 18.11% ± 2.57, with thecomparisons T. benedii I vs. II, T. insularis I vs. II, T. amplivasatus I vs.II and T. imajimai vs. T. parapectinatus showing low values (Supple-mentary Tables 3 and 4).

3.5. Genetic variation in ITS

The ITS region failed to separate T. benedii I and II (see Fig. 3).Therefore, these were lumped into one lineage in both the geneticvariation and haplotype diversity analyses of this region (Supple-mentary Table 5). The mean intralineage variation of the ITS regionwas 0.28% ± 0.16 and the mean interlineage divergence was9.36% ± 1.72, with low values noted for T. imajimai vs. T. parapectin-atus (0.94% ± 0.51), T. pseudogaster I vs. II (1.26% ± 0.63) and T. bene-dii vs. T. kozloffi (1.28% ± 0.61). T. imajimai and T. parviductus(represented by single specimens) were separated from other spe-cies by a mean of 8.33% ± 1.70 and 8.70% ± 1.58, respectively (Sup-plementary Table 6).



Table 4Characteristic Attributes (CA’s) in the COI locus of the four different morphospecies as retrieved by CAOS. ‘‘Position” indicates in which nucleotide position at the locus the difference is situated. Nucleotides in parentheses indicate theminority private attributes, i.e., attributes present in some representatives of one lineage and not in the other; all other attributes in the table are pure.

Position 003 009 019 047 082 088 100 127 142 181 184 187 200 202 205 208 214 220 223 242 265 278 343 370 379 382 405T. benedii I C T C G(C) G A(T) T T T T(C) T C(T) G A A(G) G A C G C G(A) A C T T(A) T CT. benedii II A T(C) T G A T C C C T T(C) C A T A A G T A A G C T C A A T

Position 415 477 508 521 542 556 576 586 618 625 630 636T. benedii I A(G) G G T T A T T G C(G) T GT. benedii II G A A(G) T(C) C A(G) T(A) C A C A A

Position 001 010 019 021 049 067 085 109 112 133 142 157 158 160 178 181 187 197 206 214 220 235 247 251 253 259 268T. amplivasatus I A T T G G(A) A A G C T T C T A C C T C T G A T T C G T AT. amplivasatus II G C C A G C G A A C C T C G T G C T C A G C C T A C G

Position 271 274 292 293 295 298 307 313 315 328 331 350 373 407 418 428 436 454 460 478 484 487 505 508 514 526 538T. amplivasatus I T T(C) A C(T) T A C A A T G T G T T T T C C G A T C G(A) G(A) C AT. amplivasatus II C A C C C G A G G C A G A C C C C T T A C C T A A T T

Position 547 553 565 571 574 583 586 592 593 595 598 599 610 616 619 622 625 628 631 637 643 646 649 658T. amplivasatus I C T G C C G C C A(C) C C A(C) T T T T A A G T C T A TT. amplivasatus II T A A T T A T A A T T A C C C A C G A C A C G C

Position 005 010 011 014 016 022 023 030 031 037 043 046 049 055 059 061 076 079 088 094 118 121 122 124 130 133 139T. pseudogaster I T C C C C A A G G G A A T C C G A C C C C A A T A C AT. pseudogaster II C T T T A A(T) G C C A T C A T T A G A A T T C C A C T T

Position 142 143 145 158 160 163 172 181 184 187 197 202 205 208 211 212 214 220 223 226 232 238 241 250 256 263 271T. pseudogaster I C T G T G A C G A T T G C G G C G C A T A C T C G C CT. pseudogaster II T C A C C G A T T C C C A A A T A T T C T A A T A T

Position 277 284 286 295 298 307 319 325 343 346 347 350 358 359 361 379 373 376 379 385 388 391 397 400 415 421 424T. pseudogaster I T C C A C G T A G C C T T C T A A T A T A C T C T C AT. pseudogaster II C T A C A A C C A T GT C T A C C A C C G A C T G T T T

Position 436 439 457 460 469 474 478 484 496 502 505 508 512 513 515 517 523 526 532 538 539 541 547 550 556 562 565T. pseudogaster I C T A T C G A(T) A T G C C A G G(A) T C T A T C C C T T G AT. pseudogaster II T C G C A A T C C A T A T C G C T C T A T A A C A C G

Position 568 574 577 580 581 583 586 589 595 596 598 604 613 631 637 640 643 646 655T. pseudogaster I T C G A C G A T C T A C C T T C C C TT. pseudogaster II C T A G T A C C T C T T T G C T A T C

Position 005 010 016 031 034 049 064 076 100 109 112 122 127 148 190 214 217 229 241 250 265 274 283 328 334 340 373T. insularis I T C T A T C T G G G C T A G G A T A A T T T C T T T AT. insularis II C T C G A T C A A A A C T A A G C G G C C A T C C C G

Position 403 409 428 439 451 456 474 481 484 493 496 502 508 520 538 562 571 598 601 607 613 616 619 622 628 631 652T. insularis I C G C C C A T A T A C C A T C T T C C T C C C T A G TT. insularis II T A T T T G A G C G T T G C T C C A T A T T T A T A C

Position 653T. insularis I CT. insularis II T

696S.K

vistet

al./Molecular

Phylogeneticsand

Evolution57

(2010)687–702


3.6. Phylogenetic analyses

Parsimony and Bayesian analyses were accomplished for thecombined data set, the mitochondrial loci, the nuclear loci andeach of the six loci, separately. The tree from the combined dataset is shown in Fig. 4 and the mitochondrial and nuclear treesare shown in Supplementary Figs. 1 and 2, respectively (some sup-port values present in the single gene trees are presented in Sup-plementary Table 7). To test if the resulting topology of thecombined tree was compromised by the absent ITS data for theoutgroups, this region was also excluded from the ingroup taxaand the analysis was re-run. The resulting topology showed amonophyletic Tubificoides, and the locus was therefore includedin the combined analysis.

The combined data set included 5912 characters; 1331 of whichwere parsimony informative (Table 3). The BA tree (Fig. 4) is lar-gely congruent with, but more resolved than, the MP tree (notshown). Thirteen of its nodes, relevant to the phylogeny of Tubifi-coides, show posterior probabilities (pp) > 0.95, but only nine ofthese received bootstrap support (bs) > 90% in the MP analysis(see Fig. 4). Monophyly of Tubificoides is strongly supported (pp1.00, bs 94%), and Ilyodrilus templetoni is suggested as this genus’sister taxon (pp 1.00, bs < 50%).

Tubificoides shows a basal dichotomy, with a clade of T. kozloffiand T. benedii (supported by pp 0.96) being sister to all remainingtaxa (with pp 1.00). The latter group consists of T. pseudogaster(I + II, supported by pp 1.00, bs 96%), and a large assemblage (sup-ported by pp 1.00) with four clades that are unresolved from eachother. The first two of these clades are T. parviductus and T. swirenc-owi, respectively. The third (supported by pp 1.00, bs 99%) containsT. amplivasatus I + II and T. insularis I + II, both morphospecies withmaximum support. The fourth clade (pp 1.00) is a group of sevenspecies forming two primary subclades, one (with only pp 0.93)comprising T. heterochaetus and T. fraseri, the other (pp 1.00) con-taining T. parapectinatus + T. imajimai (pp 1.00, bs 100%), and T.diazi + T. brownae + T. wasselli (pp 0.94); among the last three spe-cies, T. brownae and T. wasselli are sister taxa (pp 1.00, bs 96%).

The extended mitochondrial and nuclear data sets, with 34Tubificoides taxa each, consisted of 1577 and 4549 characters,respectively. The mitochondrial data set, under both optimality cri-teria, supports the monophyly of Tubificoides with pp 1.00 and bs82% (Supplementary Fig. 1), but for the remaining interrelation-ships, the MP tree was somewhat unresolved. In total, 13 nodesrelevant to Tubificoides (excluding the terminal nodes showingthe alliance of the two individuals of the same species, which allshowed maximum support) received ppP 0.95 and 12 of these

Fig. 3. Bayesian tree of the ITS region (average ln L of !14437.61 and an average harmonic mean of the two runs of !14501.56). The tree shows 17 distinct lineages ofTubificoides and a failure to resolve T. benedii I from II. However, for T. amplivasatus, T. pseudogaster and T. insularis the tree shows a distinct separation of the different types (Iand II). Posterior probabilities (>0.5) are shown above, and bootstrap support values (>50%) below the branches.



also exist with high support (save for one node showing pp 0.59,bs < 50%) in the combined tree. However, only six nodes showbsP 90% and these are all present with high support in the com-bined data set tree. Using the nuclear data set, both the MP andBA trees nested Ilyodrilus templetoni unresolved within Tubificoides(Supplementary Fig. 2). Nine nodes relevant to the phylogeny ofTubificoides (excluding the terminal nodes as noted above, whichagain received maximum support) received pp > 0.95 and all ofthese exist with high support in the combined tree. Only six nodesshow bs > 90% and these also overlap with highly supported nodesin the combined tree.

4. Discussion

4.1. Species delimitation and cryptic speciation

De Queiroz (2007), when reviewing the concepts of species andspecies delimitation, suggested a unifying concept of species beingdefined as ‘‘separately evolving metapopulation lineages”. Further,he argued that the actual delimitation of such lineages as species isbrought through the practical process of establishing lines of evi-dence for independent evolution (e.g. morphological differences,genetic divergence, evidence of ceased gene flow, reproductive iso-lation). That is, the more evidence supporting that two populationshave separate histories, the stronger the case for them being differ-ent species. Each of the morphospecies T. benedii, T. pseudogaster, T.amplivasatus and T. insularis were found to contain two distinctmitochondrial lineages and the question is whether or not theselineages are separate species.

All the studied mitochondrial loci separate T. benedii into twodifferent lineages (Fig. 2a; Supplementary Fig. 1). These were bothrepresented in the same localities in the Netherlands and Canadabut only one of the lineages was found in other localities (T. benediiI: Sweden, Norway, Denmark and France; T. benedii II: Wales andEngland; see Fig. 1). Thus, both lineages are distributed acrossthe Atlantic Ocean. There is no evidence for ceased gene flow be-tween or within populations and we therefore conclude that themitochondrial divergence is due to a former separation of mito-chondrial lineages and we suggest that T. benedii be regarded asa single species.

The sister group relationship of the two lineages of T. pseudog-aster is surprising considering their large COI distance (Supplemen-tary Table 2). In fact, the comparisons between T. pseudogaster IIand, respectively, T. benedii I, T. benedii II and T. kozloffi all showsmaller distance than that between and T. pseudogaster I and II.The nuclear genes (especially ITS) also resolve T. pseudogaster line-age I from lineage II. Specimens of the two lineages are almostidentical morphologically but a putative difference may be thenoted rich mucus formation on the body surface of T. pseudogasterII, not seen in lineage I (personal observation). As each lineageexhibits (reciprocal) monophyly in the combined tree it thus seemsappropriate to regard them as separate species.

Tubificoides amplivasatus I and II are morphologically indistin-guishable, and yet genetically distinct in both their mitochondrialand nuclear genomes (Supplementary Tables 2 and 6; Supplemen-tary Figs. 1 and 2). Specimens of T. amplivasatus I were all collectedin the northeastern Atlantic Ocean (the area of the type locality forthe species; Erséus, 1975) and the three specimens of T. amplivas-atus II were collected on the Atlantic coast of southern Spain. We

Fig. 4. Bayesian tree derived from the combined analysis of the six loci. The two BA runs returned trees with an average ln L of !34869.98 and an average harmonic mean of!34945.10. Posterior probabilities (>0.5) are shown above, and bootstrap support values (>50%) below the branches. Following each taxon is an abbreviation of the collectioncountry and, when present, a ‘‘leaf” indicates cuticular papillation and a ‘‘needle” indicates hair chaetae. The image of the worm is taken from Erséus and Bonomi (1987).



thus conclude that speciation has occurred and a formal descrip-tion of ‘‘T. amplivasatus II” as a new (cryptic) taxon is currently inpreparation (Kvist and Erséus). Interestingly, the two species pairsT. amplivasatus I and II, and T. imajimai and T. parapectinatus showsimilar values of genetic divergence in both the mitochondrial andnuclear loci suggesting that the species split occurred at approxi-mately the same time for both pairs.

The COI divergence between T. insularis I and II is intermediatebetween that of T. benedii I and II, and T. amplivasatus I and II, and inthe few specimens available (five of T. insularis I, two of T. insularisII) there was only one COI haplotype in each lineage. The ITS dis-tance between T. insularis I and II, however, is comparable to theintralineage variation within T. wasselli as well as the interlineagedivergence between T. pseudogaster I and II. The T. insularis speci-mens were collected over a narrow range (T. insularis I: Wales, T.insularis II: Scotland) and are morphologically inseparable yet thetrees based on nuclear data suggest that they are evolving sepa-rately. For the time being, T. insularis I and II may be regarded astwo putative cryptic species, but a formal taxonomic revision isnot warranted until the population genetics of this complex hasbeen studied in greater detail, using additional specimens not onlyfrom the Northeast Atlantic; the morphospecies T. insularis has alsobeen recorded from New Brunswick and Maine in the NorthwestAtlantic (Brinkhurst, 1985).

The need for additional data also applies to T. wasselli, the twospecimens of which differed significantly in both COI and ITS. Itsintraspecific COI variation was about twice the size of the secondhighest value (present in T. fraseri), and there would be evidencefor cryptic speciation also in T. wasselli should the rather high ITSdivergence show a consistent pattern of separation in a larger sam-ple of this taxon.

Most of the other nominal species examined in this study aredistinct on the basis of both mitochondrial and nuclear genetic dis-tances (Fig. 2a; Supplementary Tables 1–6), but also in terms ofphylogeny (Figs. 3 and 4; Supplementary Figs. 1–2). In additionto this, there are clear morphological discrepancies between theseother lineages. In summary, we thus conclude that our material ofTubificoides represents 17 separately evolving metapopulations(i.e., species), three of which are cryptic forms within T. pseudogas-ter, T. amplivasatus, and T. insularis, respectively.

4.2. DNA barcoding and genetic variation

In line with the results presented by Erséus and Kvist (2007),our new data show that the interlineage divergence of COI is 1–2orders of magnitude larger than the intralineage variation of thesame gene (Supplementary Tables 1–2) also in this taxonomicallyand geographically wider representation of Tubificoides. Only afew interlineage comparisons show mean divergence values below20% and, conversely, only the mean distance within T. wasselli (twospecimens only) is above 1%. In the two putative species within T.insularis, as well as in T. pseudogaster I, COI has shown no variationthus far. These results indicate that, regardless of the increasedgeographical area sampled and the inclusion of more taxa, the bar-coding gap is still large enough to effectively separate species fromcongeners (see Hebert et al., 2003a) in Tubificoides. These COI val-ues parallel prior studies of oligochaetous clitellates. For example,Huang et al. (2007) reported an interspecific divergence of P15%as opposed to an intraspecific variation of 16% between, and


senting a specific clone. It is then noteworthy that the single wormfrom the Caspian Sea (CE1273) belongs to the same clone as twoworms from Virginia, USA (CE2422, CE2460), whereas the remain-ing nine individuals from Virginia represent seven other clones andthe single specimen from the UK (Cardiff) represents yet anotherclone. This high diversity suggests that the Virginia population ofT. fraseri either is the result of multiple introductions, or belongsto the source population of this taxon.

Great COI diversity was also found in T. brownae (17 specimens,eight haplotypes; Fig. 2d) and T. heterochaetus (12 specimens, se-ven haplotypes; Fig. 2b). The former is often a dominant contribu-tor to biomass of benthic invertebrates (Diaz, 1984), and it hasbeen shown to inhabit degraded (Lerberg et al., 2000) as well asunpolluted sediments (Nichols and Thompson, 1985). Thus, itsgeographic expansion is probably not confined to any particularoxygen level or nutritional preference and it is probably capableof rapid reproduction, regardless of mode. Our sample of T. brow-nae contains two European specimens (CE3387, CE3389), and a to-tal of 15 worms from Virginia, Delaware and New York, all off theEast coast of USA (Fig 2d; Table 1). CE3387 is close to CE2475, anaberrant US haplotype from Delaware, while CE3389 is nestedamong the other American haplotypes, and the sample as a wholemay be part of one large, genetically diverse, amphi-Atlanticpopulation.

Tubificoides heterochaetus has also been found dominant inmany types of environments, particularly in oligohaline tidalcreeks and estuaries (Diaz, 1989). For example, de Vos (1936)had not found this species while collecting in Zuiderzee, TheNetherlands in 1920–21, but when sampling the same brack-ish-water area in 1927–32, he found that T. heterochaetus hadbecome the most dominant oligochaete at many sampling sta-tions. He therefore concluded that it was introduced to Zuider-zee after 1921 (as cited by Wolff, 2005). Interestingly, thisspecies was first described and named, from the Southern BalticSea, by Michaelsen (1926) at about the same time. The rapid in-crease in abundance of T. heterochaetus in Zuidersee suggeststhat this species is not native to the Dutch coast. Our six speci-mens from a single site in Suid-Holland (CE2260-2264, CE2266)represent four different haplotypes (Fig. 2b), suggesting multipleintroductions. At the other side of the Ocean, at two sites in Vir-ginia, the six other specimens of T. heterochaetus represent fiveseparate haplotypes, two of which are identical to those of Dutchspecimens. Thus, this species exhibits a rather high diversity inboth areas investigated.

Tubificoides swirencowi, first described from the Black Sea (Jar-oschenko, 1948), and later reported from the Mediterranean Sea(Casellato, 1999), now appears to be established also in the Katte-gat–Skagerrak region of Denmark and Sweden (Erséus and Kvist,2007; present paper). This taxon shows rather high haplotypediversity (Supplementary Table 1), but this diversity only corre-sponds to five nucleotide substitutions, evenly distributed amongthe individuals, indicating that the Scandinavian population is a re-cent introduction, possibly from the Mediterranean area. Furthersupporting this notion is the fact that the ITS region shows no var-iation in our (limited) sample. In the COI NJ tree presented by Ersé-us and Kvist (2007), the five specimens of T. swirencowi (thenlabeled T. heterochaetus; see Section 3.1.) also display five differenthaplotypes. However, the haplotype difference between CE1732and CE1736 is solely a length inequality; no actual nucleotide sub-stitutions separated the two specimens. These sequences were ex-tended to the same length in the present study and no furtherdifferences were found between them (Fig. 2c).

The overall tendency in these examples of the widely distrib-uted species of Tubificoides is that there is great mitochondrial var-iation throughout their ranges, but that there has been, and maystill be, considerable gene flow between some of the various pop-

ulations. No sample of any reasonable size from one area bears anysigns of bottlenecked diversity.

There may be a few records of recent introductions in our dataset; one is the finding of T. heterochaetus in The Netherlands (seeabove), another the finding of an otherwise ‘‘American” haplotypeof T. fraseri in the Caspian Sea. However, as both these species areknown also from several other parts of the world it is still difficultto establish their native areas. Moreover, as will be discussed be-low, there is not much help from their phylogeny either. Withthe emerging patterns of so many different species being discov-ered across the expected boundaries of marine biogeography, thereseems to be overwhelming evidence that these taxa are ubiquitous,opportunistic worms that have become common in several coastalsediments. In some sense of the word they are thus ‘‘invasive”, butas they appear to be so well established, it is still difficult to under-stand if they adversely have affected, or will affect, the habitatsthey invade economically, environmentally or ecologically.

4.4. Phylogeny

Based on a comparative study of the arrangement of chaetae,and the morphology and orientation of the male genital organs,Erséus (1984) discussed the possibility of Tubificoides being re-cently diverged from the otherwise mainly freshwater-based gen-era comprising the subfamily Tubificinae. Moreover, using DNAdata, this subfamily has been shown to be closely related to Limno-driloidinae, an exclusively marine taxon (Sjölin et al., 2005; Envallet al., 2006; Erséus et al., 2010). Therefore, the phylogenetic analy-ses of the combined data set of the present study included seventubificine and one limnodriloidine species as outgroups. The anal-ysis (Fig. 4) supports Tubificoides as a monophyletic group in Tub-ificinae (pp 1.00, bs 94%), and that Ilyodrilus templetoni is its sistertaxon (pp 1.00, bs 93%). In some regards, I. templetoni is morpho-logically similar to Tubificoides, e.g., both taxa have cuticularizedpenis sheaths and the placement of the prostate gland is subapicalon the atrium (for I. templetoni, see Southern, 1909, Plate VIII,Fig. 6E–F). However, in I. templetoni the vas deferens enters the at-rium apically whereas in Tubificoides this structure enters the at-rium subapically in a position more or less opposite to theprostate gland; this latter vas-atrium arrangement is likely to bean autapomorphy for Tubificoides.

Further, Ilyodrilus Eisen, 1879 is a freshwater genus as is Psam-moryctides Hrabě, 1964, which in our Bayesian analysis (Fig. 4) issister to Ilyodrilus + Tubificoides (with pp 0.97), whereas all mem-bers of Tubificoides live in brackish or fully marine waters. Thisphylogeny thus supports a secondary invasion of the sea by Tubif-icoides (as suggested by Erséus, 1984) unrelated to the origins ofother marine groups within the Naididae. It should be noted, how-ever, that this placement of Psammoryctides is not well-supportedin the bootstrap analysis and because Bayesian approaches havebeen shown to be more prone to incorrectly supporting false phy-logenetic relationships than a bootstrap approach (Douady et al.,2003), this node should be viewed with appropriate caution.

Some of the previous molecular assessments, including mem-bers of Tubificoides, failed to recover the monophyly of the genus(Erséus et al., 2002, 2010; Envall et al., 2006) but these studieswere based on fewer loci and included a misidentified specimen(‘‘T. pseudogaster”), now known to be H. costata (see Section 3.1.).

The basal topology of the Tubficoides phylogeny involves a para-phyletic assemblage of taxa (T. benedii, T. kozloffi, T. pseudogaster Iand T. pseudogaster II), which share a more northern distributionrange than the remaining species (Fig. 4). This suggests that thecommon ancestor of Tubificoides (i.e., of the species sampled here)evolved in the colder part of the northern hemisphere. The remain-ing part of the tree contains mostly temperate and boreal species,but also a few known to extend into tropical waters (T. parviductus



in the Caribbean, T. imajimai in Hong Kong and Florida, T. wasselli inFlorida). This large group contains a particular clade (T. heterocha-etus through T. wasselli in Fig. 4), in which all species show a distri-bution involving more than one continent or ocean. Among theother species in this group, T. swirencowi, T. amplivasatus I and IIhave yet to be found outside Europe and T. swirencowi has probablybeen introduced to Scandinavia in recent times (see above). Themorphospecies T. insularis, however, has also been recorded fromthe North American coast of the Atlantic Ocean. At present, it is dif-ficult to see any biogeographical pattern reflected in the topologyof this large group, but it can be noted that all its species, exceptT. amplivasatus I and II, and T. swirencowi, have at some point beenrecorded from the Northwest Atlantic. Moreover, considering thatin addition to the taxa included in the present study, about 25other species of Tubificodes have been described from this area(from Canada in the north to Central America in the south [ourcompilation]), the Northwest Atlantic seems to be a hotspot forspecies diversity of Tubificoides. Therefore, it is not unreasonableto suggest that at least the group containing the seven species, T.heterochaetus through T. wasselli (in Fig. 4), may have originatedand diversified in this region. However, additional taxa from theNorth American continental shelves and the Caribbean are neededto test this hypothesis.

4.5. Evolution of morphological characters

As mentioned above, penis sheath morphology, presence/ab-sence of hair chaetae and cuticular papillation, as well as thearrangement of the bifid chaetae, general body shape and sizeare characters that are commonly used in diagnoses of Tubificoidesspecies. The molecular phylogeny presented here allows for evalu-ation of the stableness of these characters as phylogenetic markers,as the morphological traits can be optimized on the tree. Here wefocus on two conspicuous characters: hair chaetae and cuticularpapillation and these were optimized under a parsimonyframework.

Judging from the topology of the tree (Fig. 4), there seems to bea significant amount of homoplasy in the occurrence of hair chae-tae, as the species with hairs (T. kozloffi, T. amplivasatus I and II, T.swirencowi, T. imajimai, T. insularis and T. parapectinatus) are partlyscattered across the tree. In light of the phylogeny, the hair chaetaefeature seems to have been lost or gained on at least four indepen-dent occasions in Tubificoides (see Fig. 4).

Cuticular papillation is not unique for Tubificoides but is presentin several other tubificine naidids (e.g. Spirosperma, Quistadrilus), aswell as representatives of other subfamilies including Phallodrili-nae (Duridrilus) and Limnodriloidinae (Tectidrilus). The species thatshow cuticular papillation (T. wasselli, T. benedii, T. heterochaetusand T. insularis) are disunited in our tree (Fig. 4), suggesting thatthis character is also homoplasious in Tubificoides; cuticular papil-lation appears to have been gained independently on at least fourdifferent occasions in the phylogeny of Tubificoides.

However, the supporting bootstrap values for many of thenodes discussed above are very low (


Erséus, C., 1975. Peloscolex amplivasatus sp.n. and Macroseta rarisetis gen. et sp.n.(Oligochaeta, Tubificidae) from the West coast of Norway. Sarsia 58, 1–8.

Erséus, C., 1984. Cladistic analysis of the subfamilies within the Tubificidae(Oligochaeta). Zool. Scr. 19, 57–63.

Erséus, C., 1985. Annelida of Saudi Arabia. Marine Tubificidae (Oligochaeta) of theArabian Gulf coast of Saudi Arabia. Fauna of Saudi Arabia 6, 130–154.

Erséus, C., 1989. Two new species of the marine genus Limnodriloides and a record ofTubificoides fraseri Brinkhurst (Oligochaeta: Tubificidae) from New Zealand. NZJ. Mar. Freshwat. Res. 23, 557–561.

Erséus, C., 2005. Phylogeny of oligochaetous Clitellata. Hydrobiologia 535 (536),357–372.

Erséus, C., Bonomi, G., 1987. A new species of Tubificoides (Oligochaeta, Tubificidae)from the Adriatic Sea. Ital. J. Zool. 54, 165–168.

Erséus, C., Diaz, R.J., 1989. Population dynamics of Tubificoides amplivasatus(Oligochaeta, Tubificidae) in the Oresund, Denmark. Hydrobiologia 180, 161–176.

Erséus, C., Envall, I., Marchese, M., Gustavsson, L., 2010. The systematic position ofOpistocystidae (Annelida, Clitellata) revealed by DNA data. Mol. Phylogenet.Evol. 54, 309–313.

Erséus, C., Källersjö, M., Ekman, M., Hovmöller, R., 2002. 18S rDNA phylogeny of theTubificidae (Clitellata) and its constituent taxa: dismissal of the Naididae. Mol.Phylogenet. Evol. 22, 414–422.

Erséus, C., Kvist, S., 2007. COI variation in Scandinavian marine species ofTubificoides (Annelida: Clitellata: Tubificidae). J. Mar. Biol. Assoc. UK 87,1121–1126.

Erséus, C., Wetzel, M., Gustavsson, L., 2008. ICZN rules – a farewell to Tubificidae(Annelida, Clitellata). Zootaxa 1744, 66–68.

Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers foramplification of mitochondrial cytochrome c oxidase subunit I from diversemetazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299.

Frankham, R., 2005. Resolving the genetic paradox in invasive species. Heredity 94,385.

Giere, O., Rhode, B., Dubilier, N., 1988. Structural peculiarities of the body wall ofTubificoides benedii (Oligochaeta) and possible relations to its life in sulphidicsediments. Zoomorphology 108, 29–39.

Gustafsson, D.R., Price, D.A., Erséus, C., 2009. Genetic variation in the popular labworm Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae) revealscryptic speciation. Mol. Phylogenet. Evol. 51, 182–189.

Hajibabaei, M., Singer, G.A.C., Hebert, P.D.N., Hickey, D.A., 2007. DNA barcoding:how it complements taxonomy, molecular phylogenetics and populationgenetics. Trends Genet. 23, 167–172.

Halanych, K.M., Janosik, A.M., 2006. A review of molecular markers used for Annelidphylogenetics. Integr. Comp. Biol. 46, 533–543.

Harrel, R.C., 2004. Systematic and ecological notes on Tubificoides heterochaetus(Oligochaeta: Tubificidae) from the Neches River estuary, Texas. Texas J. Sci. 56,263–267.

Hebert, P.D.N., Cywinska, A., Ball, S.L., deWaard, J.R., 2003a. Biological identificationsthrough DNA barcodes. Proc. R. Soc. Lond. B. 270, 313–321.

Hebert, P.D.N., Ratnasingham, S., deWaard, J.R., 2003b. Barcoding animal life:cytochrome c oxidase subunit 1 divergences among closely related species.Proc. R. Soc. Lond. B 270, 96–99.

Hebert, P.D.N., Stoeckle, M.Y., Zemlak, S.T., Francis, C.M., 2004. Identification of birdsthrough DNA barcodes. PLoS Biol. 2, 1657–1663.

Helgason, G.V., Erséus, C., 1987. Three new species of Tubificoides (Oligochaeta,Tubificidae) from the North-west Atlantic and notes on geographic variation inthe circumpolar T. Kozloffi. Sarsia 72, 159–169.

Holmquist, C., 1978. Revision of the genus Peloscolex (Oligochaeta, Tubificidae). 1.Morphological and anatomical scrutiny; with discussion on the generic level.Zool. Scr. 7, 187–209.

Holmquist, C., 1979. Revision of the genus Peloscolex (Oligochaeta, Tubificidae). 2.Scrutiny of the species. Zool. Scr 8, 37–60.

Hovmöller, R., Pape, T., Källersjö, M., 2002. The Palaeoptera problem: basalpterygote phylogeny inferred from 18S and 28S rDNA sequences. Cladistics18, 313–323.

Hrabě, S., 1966. New or insufficiently known species of the family Tubificidae. SpisyPrirodoved Fak. Univ. J.E. Purkyne Brne. 470, 57–76.

Huang, J., Xu, Q., Sun, Z.J., Tang, G.L., Su, Z.Y., 2007. Identifying earthworms throughDNA barcodes. Pedobiologia 51, 301–309.

International Commission of Zoological Nomenclature [ICZN], 2007. Opinion 2167(Case 3305). Naididae Ehrenberg, 1828 (Annelida, Clitellata): precedence overTubificidae Vejdovsky, 1876 maintained. Bull. Zool. Nomen. 64, 71–72.

Jamieson, B.G.M., Tillier, S., Tillier, A., Justine, J.-L., Ling, E., James, S., McDonald, K.,Hugall, A.F., 2002. Phylogeny of the Megascolecidae and Crassiclitellata(Annelida, Oligochaeta): combined versus partitioned analysis using nuclear(28S) and mitochondrial (12S, 16S) rDNA. Zoosystema 24, 707–734.

Jaroschenko, M.F., 1948. Oligochaetes of the Dnieper-Bug Liman. Nauc Zap. Modlav.Nauc. Issl. Bazy Acad. Nauk. SSSR 1, 57–68.

Johnson, J.B., Omland, K.S., 2004. Model selection in ecology and evolution. TrendsEcol. Evol. 19, 101–108.

Jukes, T.H., Cantor, C.R., 1969. Evolution of protein molecules. In: Munro, H.N. (Ed.),Mammalian Protein Metabolism. Academic Press, New York, pp. 21–123.

Källersjö, M., von Proschwitz, T., Lundberg, S., Eldenäs, P., Erséus, C., 2005.Evaluation of ITS rDNA as a complement to mitochondrial gene sequences forphylogenetic studies in freshwater mussels: an example using Unionidae fromnorth-western Europe. Zool. Scr. 34, 415–424.

Kimura, M., 1980. A simple method for estimating evolutionary rates of basesubstitutions through comparative studies of nucleotide sequences. J. Mol. Evol.16, 111–120.

Kvist, S., Dreyer, J., Erséus, C., 2008. Two new species of Tubificoides (Annelida:Clitellata: Naididae) from the Blake Ridge methane seep in the North-westAtlantic Ocean. Proc. Biol. Soc. Wash. 121, 531–540.

Lerberg, S.B., Holland, F.A., Sanger, D.M., 2000. Responses of tidal creekmacrobenthic communities to the effects of watershed development.Estuaries 23, 838–853.

Lowenstein, J.H., Amato, G., Kolokotronis, S.-O., 2009. The real maccoyii: identifyingtuna sushi with DNA barcodes–contrasting characteristic attributes and geneticdistances. PLoS ONE 4, e7866.

Meyer, C.P., Paulay, G., 2005. DNA barcoding: error rates based on comprehensivesampling. PLoS Biol. 3, 2229–2238.

Michaelsen, W., 1926. Oligochäten aus dem Ryck bei Greifswald und vonbenachbarten Meeresgebieten. Mitt. Hamb. Zool. Mus. Inst. 42, 21–29.

Milligan, M.R., 1991. Two new species of Tubificoides (Oligochaeta, Tubificidae) andnew records of T. Brownae and T. imajimai from the Gulf of Mexico andCaribbean, with a redescription of T. bakeri. Zool. Scr. 20, 339–345.

Nichols, F.H., Thompson, J.K., 1985. Persistance of an introduced mudflatcommunity in South San Fransisco Bay, California. Mar. Ecol. Prog. Ser. 24,83–97.

Norén, M., Jondelius, U., 1999. Phylogeny of the Prolecithophora (Platyhelminthes)inferred from 18S rDNA sequences. Cladistics 15, 103–112.

Nylander, J.A.A., 2004. MrModeltest V2. Program distributed by the author,Evolutionary Biology Centre. Uppsala University.

Nylander, J.A.A., Wilgenbusch, J.C., Warren, D.L., Swofford, D.L., 2008. AWTY (are wethere yet?): a system for graphical exploration of MCMC convergence inBayesian phylogenetics. Bioinformatics 24, 581–583.

Palumbi, S.R., 1996. Nucleic acids II: the polymerase chain reaction. In: Hillis, D.M.,Mortiz, C., Mable, B.K. (Eds.), Molecular systematics, 2nd ed. Sinauer AssociatesSunderland, MA, pp. 205–247.

Popescu-Marinescu, V., Botea, F., Brezeanu, G., 1966. Untersuchungen über dieOligochaeten im rümanischen Sektor des Donaubassins. Arch. Hydrobiol. Suppl.30, 161–179.

Rach, J., DeSalle, R., Sarkar, I.N., Schierwater, B., Hadrys, H., 2008. Character-basedDNA barcoding allows discrimination of genera, species and populations inOdonata. Proc. R. Soc. B. 275, 237–247.

Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inferenceunder mixed models. Bioinformatics 19, 1572–1574.

Rozas, J., Sánchez-DelBarrio, J.C., Messeguer, X., Rozas, R., 2003. DnaSP, DNApolymorphism analyses by the coalescent and other methods. Bioinformatics19, 2496–2497.

Sarkar, I.N., Planet, P.J., Bael, T.E., Stanley, S.E., Siddall, M., DeSalle, R., Figurski, D.H.,2002a. Characteristic attributes in cancer microarrays. J. Biomed. Inform. 35,111–122.

Sarkar, I.N., Thornton, J.W., Planet, P.J., Figurski, D.H., Schierwater, B., DeSalle, R.,2002b. An automated phylogenetic key for classifying homeoboxes. Mol.Phylogenet. Evol. 24, 388–399.

Sjölin, E., Erséus, C., Källersjö, M., 2005. Phylogeny of Tubificidae (Annelida,Clitellata) based on mitochondrial and nuclear sequence data. Mol.Phylogenet. Evol. 35, 431–441.

Southern, R., 1909. Contributions towards a monograph of the British and IrishOligochaeta. Proc. Roy. Irish Acad. 27, 119–182.

Swofford, D., 2002. PAUP*: phylogenetic analysis using parsimony (*and othermethods). Version 4.0b. Computer software and manual: sinauer associates,Sunderland, MA.

Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionarygenetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599.

Thompson, B., Lowe, S., Kellogg, M. 2000. Macrobenthic assemblages of the SanFrancisco bay-delta, and their responses to abiotic factors. Results of the BenthicPilot Study 1994–1997 Part 1, pp. 1–41.

Voua Otomo, P., Jansen van Vuuren, B., Reinecke, S.A., 2009. Usefulness of DNAbarcoding in ecotoxicological investigations: resolving taxonomic uncertaintiesusing Eisenia Malm 1877 as an example. Bull. Environ. Contam. Toxicol. 82,261–264.

White, T.M., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing offungal ribosomal RNA for phylogenetics. In: Innis, M.A., Gelfrand, D.H., Sninsky,J.J., White, T.J. (Eds.), PCR Protocols. Academic Press, San Diego, CA, pp. 315–322.

Wolff, W.J., 2005. Non-indigenous marine and estuarine species in the Netherlands.Zool. Meded. (Leiden) 79, 1–116.


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