18S rDNA phylogeny of Clitellata (Annelida)

The phylogeny of Clitellata was analysed using 18S rDNA sequences of a selection of species representing Hirudinida, Acanthobdellida, Branchiobdellida and 10 oligochaetous families. Eleven new 18S sequences of Capilloventridae (one), Haplotaxidae (one), Propappidae (one), Enchytraeidae (two), Lumbricidae (one), Almidae (one), Megascolecidae (two), Lumbriculidae (one), and Phreodrilidae (one) are reported and aligned together with corresponding sequences of 28 previously studied clitellate taxa. Twelve polychaete species were used as an outgroup. The analysis supports an earlier hypothesis based on morphological features that Capilloventridae represents a basal clade of Clitellata; in the 18S tree it shows a sister-group relationship to all other clitellates. The remaining clitellate taxa form a basal dichotomy, one clade containing Tubificidae (including the former 'Naididae'), Phreodrilidae, Haplotaxidae, and Propappidae, the other clade with two subgroups: (1) Lumbriculidae together with all leech-like taxa (Acanthobdellida, Branchiobdellida and Hirudinida), and (2) Enchytraeidae together with a monophyletic group of all earthworms included in the study (Lumbricidae, Almidae and Megascolecidae). These earthworms are members of the taxon Crassiclitellata, the monophyly of which is thus supported by the data. The tree also shows support for the hypothesis that the first clitellates were aquatic. The position of the single species representing Haplotaxidae is not as basal as could have been expected from earlier morphology-based conclusions about the ancestral status of this family. However, if Haplotaxidae is indeed a paraphyletic assemblage of relict taxa, a higher number of representatives will be needed to resolve its exact relationships with the other clitellates.     

Phylogeny of oligochaetous Clitellata

Clitellata, with more than one third of all annelid species described, is briefly introduced, and an overview of the hypotheses of phylogenetic relationships among the groups traditionally referred to as oligochaetes is given. The presentation is placed in a historical context and describes the trend to move from intuitive, narrative approaches to more formal analyses of character patterns. Monophyly of the earthworms (the megadriles, or Metagynophora sensu Jamieson), or at least a major part of them (Crassiclitellata sensu Jamieson), and paraphyly of the ‘microdrile’ largely aquatic, groups are supported by both morphological and molecular data. Further, DNA sequences as well as spermatozoal ultrastructure corroborate that all leech-like taxa (Hirudinida, Acanthobdellida and Branchiobdellida) constitute a clade derived within ‘Oligochaeta’, closely related to the family Lumbriculidae. Molecular systematic studies also support relationships already identified on the basis of morphological data, e.g., the position of Naididae within Tubificidae, the position of Phreodrilidae close to, but outside, the same family, and the putative sistergroup relationship between the newly discovered Capilloventridae and the rest of Clitellata. A recent study using 18S rDNA suggests that Enchytraeidae is closely related to Metagynophora, and that these two taxa, which contain all terrestrial oligochaetous clitellates, form a clade derived from aquatic ‘microdriles’ This refutes a recent hypothesis proposing that the ancestor of Clitellata was terrestrial. To a great extent, however, the basal resolution of the oligochaetous clitellates remains unclear.     

The phylogenetic position of the Aeolosomatidae and Parergodrilidae, two enigmatic oligochaete-like taxa of the 'Polychaeta', based on molecular data from 18S rDNA sequences

The Aeolosomatidae and the Parergodrilidae are meiofaunal Annelida showing different combinations of clitellate‐like and non‐clitellate character states. Their phylogenetic positions and their systematic status within the Annelida are still in debate. Here we attempt to infer their systematic position using 18S rDNA sequences of the aeolosomatid Aeolosoma sp. and the parergodrilid Stygocapitella subterranea and several other meiofaunal taxa such as the Dinophilidae, Polygordiidae and Saccocirridae. The data matrix was complemented by sequences from several annelid, arthropod and molluscan species. After evaluation of the phylogenetic signal the data set was analysed with maximum‐parsimony, distance and maximum‐likelihood algorithms. Sequences from selected arthropods or molluscs were chosen for outgroup comparison. The resolution of the resulting phylogenies is discussed in comparison to previous studies. The results do not unequivocally support a sister‐group relationship of Aeolosoma sp. and the Clitellata. Instead, depending on the algorithms applied, Aeolosoma clusters in various clades within the polychaetes, for instance, together with eunicidan species, the Dinophilidae, Harmothoë impar or Nereis limbata. The position of Aeolosoma sp. thus cannot be resolved on the basis of the data available. S. subterranea always falls close to a cluster comprising Scoloplos armiger, Questa paucibranchiata and Magelona mirabilis, all of which were resolved as not closely related to both Aeolosoma sp. and the Clitellata. Therefore, convergent evolution of clitellate‐like characters in S. subterranea and hence in the Parergodrilidae is suggested by our phylogenetic analysis. Moreover, the Clitellata form a monophyletic clade within the paraphyletic polychaetes.     

Microorganization of ovaries and oogenesis of Haplotaxis sp. (Clitellata: Haplotaxidae)

Ovaries of Haplotaxis sp. were studied in active and nonactive states, that is, in a sexually mature specimen and in specimens outside of the reproductive period. Two pairs of ovaries were found in segments XI and XII. Especially in the nonactive state, they were in close contact with copulatory glands. Each ovary was composed of germ cells interconnected with syncytial cysts, which were enveloped by a layer of somatic cells. Within cysts each germ cell had one ring canal connecting it to the common anuclear cytoplasmic mass called a cytophore. During oogenesis clustering germ cells differentiated into nurse cells and oocytes; thus, the oogenesis was recognized as meroistic. Vitellogenic oocytes were detached from the ovaries and continued yolk absorption within the body cavity. Because recent studies have shown the variety of ovaries and germ line cyst organization in clitellates and suggest their evolutionary conservatism at the family or subfamily level, the data presented here can be valid in understanding the phylogenetic relationships among Clitellata. In this context, ovaries found in Haplotaxis sp. resembled those of the “Tubifex” type. “Tubifex” ovaries are characteristic for numerous microdrile taxa (tubificines, limnodriloidines, propappids, lumbriculids, and leech‐like branchiobdellids) and can be regarded as the primary character for these Clitellata in which germ‐line cysts are formed during early oogenesis. As the family Haplotaxidae is currently considered to be paraphyletic and the species studied here belongs to Haplotaxidae sensu stricto, our results support the close relationship of Haplotaxidae sensu stricto to the clade consisting of Lumbriculidae, Branchiobdellida, and Hirudinida, in which lumbriculids are sister to the latter two.     

On the origin of the Hirudinea and the demise of the Oligochaeta

The phylogenetic relationships of the Clitellata were investigated with a data set of published and new complete 18S rRNA gene sequences of 51 species representing 41 families. Sequences were aligned on the basis of a secondary structure model and analysed with maximum parsimony and maximum likelihood. In contrast to the latter method, parsimony did not recover the monophyly of Clitellata. However, a close scrutiny of the data suggested a spurious attraction between some polychaetes and clitellates. As a rule, molecular trees are closely aligned with morphology-based phylogenies. Acanthobdellida and Euhirudinea were reconciled in their traditional Hirudinea clade and were included in the Oligochaeta with the Branchiobdellida via the Lumbriculidae as a possible link between the two assemblages. While the 18S gene yielded a meaningful historical signal for determining relationships within clitellates, the exact position of Hirudinea and Branchiobdellida within oligochaetes remained unresolved. The lack of phylogenetic signal is interpreted as evidence for a rapid radiation of these taxa. The placement of Clitellata within the Polychaeta remained unresolved. The biological reality of polytomies within annelids is suggested and supports the hypothesis of an extremely ancient radiation of polychaetes and emergence of clitellates.     

Validating Livanow: Molecular Data Agree That Leeches, Branchiobdellidans, and Acanthobdella peledina Form a Monophyletic Group of Oligochaetes

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.     

A phylogenetic analysis of Tubificinae and Limnodriloidinae (Annelida, Clitellata, Tubificidae) using sperm and somatic characters

Marotta, R., Ferraguti, M. & Erséus, C. (2003). A phylogenetic analysis of Tubificinae and Limnodriloidinae (Annelida, Clitellata, Tubificidae) using sperm and somatic characters. — Zoologica Scripta , 32 , 255–278.
The spermatozoa and the sperm aggregates of 13 species belonging to four genera ( Smithsonidrilus, Limnodriloides, Thalassodrilides, Doliodrilus ) in the tubificid subfamily Limnodriloidinae (Annelida, Oligochaeta) were studied and compared with the spermatozoal patterns already described in the subfamily Tubificinae. Two characters considered exclusive for the Tubificinae were found in the more spermatologically variable Limnodriloidinae: the production of two kinds of spermatozoa, eusperm and parasperm, and the presence, in the spermathecae, of sperm aggregates formed by a combination of the two sperm types. A parsimony analysis was performed on the spermatozoal data of the species examined and compared with that based on the somatic characters of the same species: a critical revision of the already codified eusperm characters was carried out and the ultrastructure of parasperm was used as a new subset of spermatozoal characters. In a total evidence approach, a further parsimony analysis was run using a matrix combining both sets of characters. This analysis suggested that the double sperm line and the sperm aggregates composed of both eusperm and parasperm may well be homologous in tubificines and limnodriloidines. It thus supported the previous notion that Tubificinae and Limnodriloidinae are closely related and indicated that these subfamilies may be sister taxa.     

Molecular phylogenetic study of the systematic position of Baikalian oligochaetes in Clitellata

Phylogenetic relationships within the class Clitellata were examined using partial nucleotide sequences of the nuclear 18S ribosomal RNA gene. In the analysis, the already determined sequences for the individual species representing the class members Hirudinea, Acanthobdellida, Branchiobdellida, and Oligochaeta were included. Furthermore, newly determined sequences of the thirteen representatives of the family Lumbriculidae, including 12 Baikalian endemic species, were analyzed. The hypothesis on the close relatedness of these four groups of Clitellata was supported. Leeches, branchiobdellids, and lumbriculids form three independent parallel branches of evolution. These results were consistent with the hypothesis on the role of the family Lumbriculidae as a connecting link, or the transition form between the parasitic and free-living groups of Clitellata. At the same time, these data refute the suggestion that Lumbriculidae could be the ancestral lineage of other Oligochaeta. Moreover, polymorphic group of Baikalian lumbriculids clustered independently from the other representatives of the family, pointing to the uniqueness of the Baikalian fauna of oligochaetes, which was formed within relatively closed system of this ancient lake.     

Phylogenetic analysis of Tubificidae (Annelida, Clitellata) based on 18S rDNA sequences

Tubificids are aquatic clitellate worms, but recent analyses of morphological characters suggested that this family, as currently recognized, is paraphyletic. Sequences of the 18S rDNA gene of 40 protostome worm species (including 13 representatives of the Tubificidae) and 2 mollusc species were cladistically analyzed to test the monophyly of the Tubificidae and that of some of its constituent subfamilies. Under all alignments tested, the same general phylogenetic pattern emerged. The data support the idea that the Naididae, another clitellate taxon, is associated with some "rhyacodriline" groups within the Tubificidae. The data also corroborate the idea that the Tubificinae and the Limnodriloidinae are monophyletic but indicate that the Rhyacodrilinae and the Phallodrilinae are not. Bathydrilus does not appear to be closely related to other "phallodriline" genera.     

Validating Livanow: molecular data agree that leeches, Branchiobdellidans, and Acanthobdella peledina form a monophyletic group of oligochaetes.

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.     

Rapidly evolving lineages impede the resolution of phylogenetic relationships among Clitellata (Annelida)

The phylogenetic relationships of the Clitellata were investigated using a data set with published and new complete or partial 18S rRNA and mtCOI gene sequences of 13 and 49 taxa representing 8 and 14 families, respectively. Three different alignments were considered for 18S, and the possible influence of departures from rate constancy among sites was evaluated by analyses using a Gamma model of rate heterogeneity. Maximum-likelihood estimates of the shape parameter alpha of the Gamma distribution were very low, whatever the alignment or the gene considered, suggesting that phylogenetic reconstructions taking into account the rate heterogeneity among sites are likely to be the most reliable. Analyzed separately, the two genes did not resolve the relationships among the Clitellata, but the consensus tree was congruent with the morphology-based relationships. Our data suggest the inclusion of the Euhirudinea, Acanthobdellida, and Branchiobdellida in the Oligochaeta and suggest the Lumbriculidae as the link between both assemblages. Although separate analyses of both genes, as well as different alignments for the 18S rRNA sequences, yielded conflicting results concerning the phylogenetic position of leeches and leech-like worms vis-à-vis the Oligochaeta, subsequent analyses using the Gamma model greatly reduced the observed inconsistencies. Our analyses show that among the Clitellata, the leeches and the leech-like and gutless worms represent significantly faster evolving lineages. It is suggested that the observed higher mutation rates may be explained by the fact that these lineages contain almost exclusively commensal and/or parasitic taxa.     

Molecular phylogeny of leeches: Congruence of nuclear and mitochondrial rDNA data sets and the origin of bloodsucking

Complete 18S rDNA sequences and sequences of domain III of mitochondrial 12S rDNA were obtained to assess phylogenetic relationships among major suprageneric taxa of leeches and the possibly closely related clitellate taxa Branchiobdellida and Acanthobdellida. The monophyly of the families Erpobdellidae, Piscicolidae, and Glossiphoniidae, the suborders Erpobdelliformes and Hirudiniformes, and the order Arhynchobdellida have been confirmed by parsimony and maximum likelihood phylogenetic analysis of separate and combined data sets. Both the nuclear 18S rDNA sequences and the mitochondrial 12S rDNA sequences were consistent in not supporting a monophyletic order Rhynchobdellida, represented by the families Piscicolidae and Glossiphoniidae. A topology with the Piscicolidae as the first branch in the leech tree followed by the Glossiphoniidae received the highest support in terms of taxonomic, character, and outgroup congruence. According to this topology, the putative apomorphies of the Rhynchobdellidae (e.g. the proboscis) can be parsimoniously explained as plesiomorphies already present in the ancestral leech. This common ancestor was probably a bloodsucking leech with a proboscis rather than an unspecialized ectocommensal, as suggested by previous hypotheses. During the course of leech evolution, a reduction of the proboscis could have taken place in predatory arhynchobdellid ancestors to enable swallowing of larger prey. A second gain of sanguivory by the jawed Hirudiniformes could have been facilitated by pre-adaptations to ectoparasitic blood feeding. The 18S rDNA analysis further indicates a close relationship between the clitellate groups Branchiobdellida and Acanthobdellida, although this relationship is not strongly supported.     

Molecular phylogenetic study of the systematic position of Baikalian oligochaetes in the system Clitellata

Phylogenetic relationships within the class Clitellata were examined using nucleotide sequences of nuclear 18S ribosomal RNA gene fragments. In the analysis, the already determined sequences for the individual species representing the class members Hirudinea, Acanthobdellida, Branchiobdellida, and Oligochaeta were included. Furthermore, newly determined sequences of the thirteen representatives of the family Lumbriculidae. including 12 Baikalian endemic species, were analyzed. The hypothesis on the close relatedness of these four groups of Clitellata was supported. Leeches, branchiobdellidans, and lumbriculides form three independent parallel branches of evolution. These results were consistent with the hypothesis on the role of the family Lumbriculidae as a connecting link, or the transition form between the parasitic and free-living groups of Clitellata. At the same time, these data refute the suggestion that Lumbriculidae could be the ancestral lineage of other Oligochaeta. Moreover, polymorphic group of Baikalian lumbriculides clustered independently from the other representatives of the family, pointing to the uniqueness of the Baikalian fauna of oligochaetes, which was formed within relatively closed system of this ancient lake.     

Ovary organization and oogenesis in two species of Lumbriculida (Annelida,Clitellata)

The aim of the present study is to describe the organization of the ovary and mode of oogenesis at the ultrastructural level in two representatives of Lumbriculida – Lumbriculus variegatus and Stylodrilus heringianus. In both species studied, the ovaries are small and conically shaped structures that are attached to the intersegmental septum via a thin ligament. The ovaries are composed of germline cysts formed by germ cells interconnected by stable cytoplasmic bridges. As a rule, the cyst center is occupied by a poorly developed anuclear cytoplasmic mass, termed a cytophore, whereas the germ cells are located at the periphery of the cyst. Germline cysts are enveloped by somatic cells. The ovaries of the species studied are polarized, i.e., along the long axis of the ovary there is an evident gradient of germ cell development. The data obtained suggest ovary meroism, i.e., two categories of germ cells were found: oocytes, which continue meiosis, gather nutrients, grow and protrude into the body cavity, and nurse cells, which do not grow and are supposed to supply oocytes with cell organelles and macromolecules via the cytophore. The ovary structure and mode of oogenesis in the species studied were compared with those of other clitellate annelids. As a rule, in all clitellates studied to date, the ovaries are composed of germline cysts equipped with a cytophore and associated with somatic cells; however, the ovary morphology differs between taxa regarding several quantitative and qualitative features. The ovary organization and mode of oogenesis in L. variegatus and S. heringianus strongly resemble those found in Tubificinae and Branchiobdellida studied to date. Our results also support a sister-group relationship between Lumbriculida and a clade comprising ectoparasitic clitellates (i.e., Branchiobdellida, Acanthobdellida and Hirudinida) with Branchiobdellida as a plesiomorphic sister group to Acanthobdellida and Hirudinida.     

The sister group relationship of Aeolosomatidae and Potamodrilidae (Annelida: “Polychaeta”) — a molecular phylogenetic approach based on 18S rDNA and cytochrome oxidase I

Aeolosomatidae and Potamodrilidae are small meiofauna annelids of apparently simple organization and uncertain phylogenetic position. Potamodrilidae was regarded either as a subtaxon of Aeolosomatidae, united with them as Aphanoneura, or entirely unrelated to Aeolosomatidae. Moreover, the groups have been placed in various positions within Annelida: as sister group of Clitellata, as a highly derived clitellate taxon, or excluded from Clitellata and not closely related to them due to great morphological differences. Although molecular studies give strong support for the exclusion of these two taxa from Clitellata their questionable sister group relationship to each other has not been addressed specifically. In the present study sequences of the nuclear 18S rDNA and the mitochondrial Cytochrome Oxidase I gene were used for addressing this question. In addition to the available nuclear 18S rDNA sequences, partial sequences of Cytochrome Oxidase I of Rheomorpha neiswestnovae (Lastochkin, 1935) and Potamodrilus fluviatilis Lastochkin, 1935 along with other polychaete taxa were determined. Combined analyses of these two genes were conducted using Maximum Parsimony and Bayesian analysis. A sister group relationship of Aeolosomatidae and Potamodrilidae is significantly supported in all. As in previous studies a relationship to Clitellata is not supported but the phylogenetic position of both Aeolosomatidae and Clitellata within the polychaetes remains enigmatic.     

Molecular phylogeny of North American Branchiobdellida (Annelida: Clitellata)

Branchiobdellidans, or crayfish worms, are ectosymbiotic clitellate annelids associated primarily with freshwater crayfishes. The main objectives of our study were to infer a molecular phylogeny for the North American Branchiobdellida, examine its congruence with morphology-based hypotheses of relationships at the subfamily and genus level, and use our dataset to assess consistency of GenBank-archived branchiobdellidan sequences. We used nucleotide sequence data from two mtDNA genes (COI and 16S rDNA) and three nuclear genes (28S rDNA, 18S rDNA, and ITS1) to estimate phylogenetic relationships among 47 described and one undescribed species of Branchiobdellida. We recovered a monophyletic branchiobdellidan clade with generally short branch lengths, suggesting that a large portion of the taxon has likely undergone a recent and rapid radiation in North America. Results from our phylogenetic analyses indicate that current taxonomic groupings are largely unsupported by the molecular data. All four subfamilies are either paraphyletic or polyphyletic, and only three of seven sampled non-monotypic genera were monophyletic. We found a high rate (49%) of inconsistency in GenBank-archived sequences, over 70% of which can be attributed to field- or laboratory-based error.     

A test of monophyly of the gutless Phallodrilinae (Oligochaeta, Tubificidae) and the use of a 573-bp region of the mitochondrial cytochrome oxidase I gene in analysis of annelid phylogeny

A 573-bp region of the mitochondrial gene cytochrome c oxidase subunit I (COI) of two species of Inanidrilus Erséus and four species of Olavius Erséus (Phallodrilinae, Tubificidae) is used in a parsimony analysis together with a selection of 35 other annelids (including members of Polychaeta, Pogonophora, Aphanoneura, and the clitellate taxa Tubificidae, Enchytraeidae, Naididae, Lumbriculidae, Haplotaxidae, Lumbricidae, Criodrilidae, Branchiobdellida and Hirudinea), and with two molluscs as outgroups. The data support the monophyly of the Olavius and Inanidrilus group, with a monophyletic Inanidrilus . However, parsimony jackknife analyses show that most of the other groups are unsupported by the data set, thus revealing a large amount of homoplasy in the selected gene region. Practically no information is given of within/between family relationships except for a few, closely related species. This suggests that the analysed COI region is not useful, when used alone, for inferring higher level relationships among the annelids.     

DNA‐based phylogeny of the marine genus Heterodrilus (Annelida,Clitellata, Naididae)

Heterodrilus is a group of marine Naididae, common worldwide in subtropical and tropical areas, and unique among the oligochaetes by their tridentate chaetae. The phylogenetic relationships within the group are assessed from the nuclear 18S rDNA gene, and the mitochondrial cytochrome c oxidase subunit I (COI) and 16S rDNA genes. Sequence data were obtained from 16 Heterodrilus species and 13 out‐group taxa; 48 sequences are new for this study. The data were analysed by Bayesian inference. Monophyly of the genus is corroborated by the resulting tree, with Heterodrilus ersei (a taxon representing a small group of species with aberrant male genitalia) proposed to be outside all other sampled species. Although earlier regarded as a member of the subfamily Rhyacodrilinae, both molecular and morphological data seem to support that Heterodrilus is closely related to Phallodrilinae. However, the results are not conclusive as to whether the genus is the sister group of, or a group nested inside, or separate from this latter subfamily. The studied sample of species suggests at least two major clades in Heterodrilus with different geographical distributions, in one of the clades, most species are from the Indo‐West Pacific Ocean, while in the other, the majority are from the Western Atlantic Ocean. Morphological characters traditionally used in Heterodrilus taxonomy are optimized on the phylogenetic tree, revealing a high degree of homoplasy.     

Evolution of habitat preference in Clitellata (Annelida)

Clitellata (earthworms, leeches, and allies) is a clade of segmented annelid worms that comprise more than 5000 species found worldwide in many aquatic and terrestrial habitats. According to current views, the first clitellates were either aquatic (marine or freshwater) or terrestrial. To address this question further, we assessed the phylogenetic relationships among clitellates using parsimony, maximum likelihood and Bayesian analyses of 175 annelid 18S ribosomal DNA sequences. We then defined two ecological characters (Habitat and Aquatic‐environment preferences) and mapped those characters on the trees from the three analyses, using parsimony character‐state reconstruction (i.e. Fitch optimization). We accommodated phylogenetic uncertainty in the character mapping by reconstructing character evolution on all the trees resulting from parsimony and maximum likelihood bootstrap analyses and, in the Bayesian inference, on the trees sampled using the Markov chain Monte Carlo algorithm. Our analyses revealed that an ‘aquatic’ ancestral state for clitellates is a robust result. By using alterations of coding characters and constrained analyses, we also demonstrated that the hypothesis for a terrestrial origin of clitellates is not supported. Our analyses also suggest that the most recent ancestor of clitellates originated from a freshwater environment. However, we stress the importance of adding sequences of some rare marine taxa to more rigorously assess the freshwater origin of Clitellata. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 447–464.