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.     

New alluroidids (Annelida,Clitellata) from Guyana

A total of 30 microbialites at two sites in Lake Clifton, Yalgorup National Park, Western Australia, were sampled by coring to quantify the associated fauna with these organo-sedimentary structures. Twenty five species of aquatic fauna were recorded from the cores, comprising 20 species of metazoan, predominantly Crustacea (including Melita kauerti (Amphipoda), Exosphaeroma cf. serventii (Isopoda); and Cyprideis australiensis(Ostracoda); Polychaeta (Capitella cf. capitata); nematodes; and five species of Foraminifera (Protista). Multivariate analysis of the five numerically most abundant taxa (amphipods, isopods, ostracods, polychaetes, nematodes) separated microbialites by season and submergence. Numbers of all taxa, particularly polychaetes and amphipods, were much higher in spring than in autumn, and in permanently-inundated than in seasonally-inundated microbialites. The exception was higher numbers of juvenile polychaetes in seasonally-inundated microbialites at the northern site in spring. This study showed that modern thrombolitic microbialites can co-exist with a diverse invertebrate fauna and serves as a baseline for future studies of interactions between microbialites and fauna.     

Ovaries of Tubificinae (Clitellata, Naididae) resemble ovary cords found in Hirudinea (Clitellata)

The ultrastructure of the ovaries and oogenesis was studied in three species of three genera of Tubificinae. The paired ovaries are small, conically shaped structures, connected to the intersegmental septum between segments X and XI by their narrow end. The ovaries are composed of syncytial cysts of germ cells interconnected by stable cytoplasmic bridges (ring canals) and surrounded by follicular cells. The architecture of the germ-line cysts is exactly the same as in all clitellate annelids studied to date, i.e. each cell in a cyst has only one ring canal connecting it to the central, anuclear cytoplasmic mass, the cytophore. The ovaries found in all of the species studied seem to be meroistic, i.e. the ultimate fate of germ cells within a cyst is different, and the majority of cells withdraw from meiosis and become nurse cells; the rest continue meiosis, gather macromolecules, cell organelles and storage material, and become oocytes. The ovaries are polarized; their narrow end contains mitotically dividing oogonia and germ cells entering the meiosis prophase; whereas within the middle and basal parts, nurse cells, a prominent cytophore and growing oocytes occur. During late previtellogenesis/early vitellogenesis, the oocytes detach from the cytophore and float in the coelom; they are usually enveloped by the peritoneal epithelium and associated with blood vessels. Generally, the organization of ovaries in all of the Tubificinae species studied resembles the polarized ovary cords found within the ovisacs of some Euhirudinea. The organization of ovaries and the course of oogenesis between the genera studied and other clitellate annelids are compared. Finally, it is suggested that germ-line cysts formation and the meroistic mode of oogenesis may be a primary character for all Clitellata.     

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.     

Mass production of basal bodies in paraspermiogenesis of Tubificinae (Annelida,Oligochaeta)

The oligochaete annelids, belonging to the subfamily Tubificinae, produce two types of spermatozoa: eusperm (the fertilising ones) and parasperm, protecting and carrying the eusperm. The pathway for the production of the two types is common until the onset of meiosis, but then a regular meiosis produces eusperm, whereas parasperm is generated through a peculiar mechanism of nuclear fragmentation giving rise to an irregular, but very high, number of paraspermatozoa. Since every parasperm has its own flagellum, this entails the necessity of producing a very high number of basal bodies. In the present paper, we describe how basal bodies are generated through a mechanism similar to that producing the basal bodies in ciliated epithelia, but never observed up to now during the genesis of a uniflagellated cell. Basal bodies form in close proximity to a precursor structure called deuterosome, which originates de novo in the cytoplasm from fibrogranular material. The various stages of centriologenesis are positive to anti-centrin antibodies and, observed by electron microscopy, correspond closely to the ones described for ciliated epithelia. However, once formed, the basal bodies migrate to their final position and produce the parasperm flagellum.     

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.     

Ultrastructure of the nephrostome in Enchytraeus albidus (Annelida, Clitellata)

 The nephrostome of Enchytraeus albidus exhibits a longitudinal slit-like opening on the dorsal side of a bulbous organ which is mainly composed of three cells, one flame cell situated centrally and endowed with a ciliary flame, and two cells lying superficially, called the mantle cell and the accessory mantle cell. The mantle cell occupies the ventral side of the organ extending on both sides up to the opening to constitute its immediate border on the frontal and lateral sides. Here it forms a kind of crest which is heavily subdivided into many protrusions and infoldings endowed with long cilia which exclusively spread into the coelomic cavity. The accessory mantle cell borders the narrow posterior slit of the opening, forming the roof of the initial canal which is devoid of cilia. From its anterior region a projection arises extending above the opening. The flame cell forms a groove from which the ciliary flame arises which extends into the canal. At its posterior region it replaces the accessory mantle cell displacing it onto the dorsal surface of the organ. It is argued that the mantle cell and the accessory mantle cell have presumbly originated from coelothelial cells. Thus the metanephridium may be a composite organ developing from a nephridioblastic and a coeloblastic source. A discussion of results in other annelid species indicates that metanephridia in the Annelida may have evolved more than once. Accepted: 13 October 1997     

Yamaguchia toyensis n. sp., n. gen. (Annelida, Clitellata, Lumbriculidae) from profundal Lake habitat in Japan

Yamaguchia toyensis n. sp., n. gen. is described from an oligotrophic caldera lake, Lake Toya, Hokkaido, Japan. Although the taxonomic affinities are unknown, the genus differs from all other Lumbriculidae in having the combination of testes and atria in X, a single, prosoporous male funnel per atrium, and spermathecae in XI. Unlike other Japanese lakes that have thus far been surveyed, Lake Toya supports abundant populations of lumbriculids in the profundal benthos.     

The double sperm line in Isochaetides (Annelida,Clitellata, Tubificidae)

Sperm morphology and spermatogenesis were examined in the oligochaete annelid Isochaetides arenarius, a species belonging to the subfamily Tubificinae inhabiting the sediments of Lake Baikal. As all tubificines, Isochaetides produces two types of spermatozoa, named eusperm and parasperm. The eusperm are the fertilizing male gametes and consist, in sequence, of an acrosome, a nucleus, a mitochondrial mid-piece, and a tail. The parasperm have the same general architecture, but differ in cytological details: the acrosome is shorter, devoid of a perforatorium, and the acrosome vesicle has a different, simpler, shape. The nucleus is much shorter and rectilinear (the eusperm nucleus is twisted). The mid-piece mitochondria are less numerous but their overall volume is larger. The flagellum has a plasma membrane largely separated from the axoneme, and is devoid of glycogen granules. After mating, the two sperm types gather in the spermathecae to form spermatozeugmata; in these structures the parasperm form an external sheath involving the centrally located eusperm and their tails are connected by conspicuous septate junctions. Parasperm nuclei are produced through a process of fragmentation of the 'spermatocytes', whereas the flagellar basal bodies are produced by a process similar to that giving rise to basal bodies in ciliated epithelia.     

Genetic and chaetal variation in Nais worms (Annelida,Clitellata, Naididae)

The genus Nais is a group of oligochaetous clitellates, common in eutrophic freshwater habitats. About 30 species are described. Species identification is based primarily on chaetal characters, which are often subtle, inconsistent, and even overlapping between nominal species. We investigated the correlation between genetic variation and chaetal morphology in this genus. Eighty‐one individuals from Europe, North America, and China were included in the study. Seventy‐five of these were preserved as vouchers. They were scrutinized with regard to chaetal morphology, and ten different morphotypes were identified. Three molecular markers, two mitochondrial (the COI gene and 16S rDNA) and one nuclear (the ITS region), were used to establish the genetic lineages in the material. Genetic variation was found to be largely congruent with chaetal character patterns. However, at least nine separately evolving lineages (all supported by mitochondrial as well as nuclear data) correspond to at most six nominal species. Four morphotypes/lineages are recognized as Nais barbata, Nais christinae, Nais elinguis, and Nais stolci, respectively, whereas five, or possibly more, lineages represent a morphological continuum covering the variation of the Nais communis/variabilis complex. Thus, cryptic speciation is revealed. Our results indicate that a taxonomic revision of the genus will be needed in the future.     

Morphological reinvestigation and phylogenetic relationship of Acanthobdella peledina (Annelida,Clitellata)

Summary In recently collected specimens of Acanthobdella peledina the nervous system, the genital organs and the coelomic organisation were reinvestigated after complete serial sections. These anatomical results are schematically represented. In addition, the integument, the chaetae and the peripheral muscle layer were investigated by electron microscopy. In general, the results confirm Livanow's classic monograph (1906), with the exception of a few details. The body apparently possesses neither a prostomium nor an achaetous buccal region (peristomium). The number of 29 true segments is concluded from the number of segmental ganglia. The five anteriormost segments, each with four pairs of hookshaped chaetae arranged around the mouth opening, are considered to be functionally equivalent to an anterior sucker. The ultrastructure of the integument and the chaetae generally conforms to the typical annelidan pattern. The muscle cells are of the typical hirudinean type. The outer male genital pore is positioned in segment 10; the female organs open in segment 11 directly behind the septum between segments 10 and 11. The main emphasis is laid on the evaluation of the position of the taxon within the Clitellata, including a discussion of the Branchiobdellida, and the cladograms presented show the Acanthobdellida to be the sister group of the Euhirudinea. Characters shared by the Branchiobdellida and Hirudinea (including A. peledina) are considered to be convergently evolved.     

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.     

Redescription of Piguetiella denticulata (Annelida, Clitellata, Naididae) from Japan and the Russian Far East, with a description of the genital organs in the species

A naidid oligochaete, Piguetiella denticulata Liang and Xie (Invertebrates of Wuling Mountains area, Southwestern China, pp 383–394, 1997), is redescribed based on new material from Japan and the Russian Far East, and its generic position is verified by an examination of the reproductive organs. P. denticulata closely resembles P. michiganensis in lacking eyes and dorsal hair chaetae, but it is distinguished from the latter by having intermediate teeth on both its dorsal and ventral crotchets. In Japan, P. denticulata is widely distributed and often abundant in mountain streams with sandy and gravelly bottoms.     

Observations on three species of branchiobdellid (Annelida: Clitellata) worms from eastern Asia

Three jars from the Hamburg Museum contained specimens of Branchiobdella minuta, presumably from the original Pierantoni (1912) material, and Cirrodrilus cirratus and C. uchidai from Yamaguchi's (1932a) collection. The head of a syntype of B. minuta was described and B. cheni becomes its junior subjective synonym. One specimen each of C. cirratus and C. uchidai was sectioned and described, particularly the male genitalia. The diet of all three branchiobdellids consists of microorganisms. In Cirrodrilus sp. basic protein granules were demonstrated in the gland cells of the intestine and its chloragogen cells. The peristomial and lateral epidermal gland cell secretions were characterized histochemically.     

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.     

Molecular evidence for the non-monophyletic status of Naidinae (Annelida, Clitellata, Tubificidae)

Naidinae (former Naididae) is a group of small aquatic clitellate annelids, common worldwide. In this study, we evaluated the phylogenetic status of Naidinae, and examined the phylogenetic relationships within the group. Sequence data from two mitochondrial genes (12S rDNA and 16S rDNA), and one nuclear gene (18S rDNA), were used. Sequences were obtained from 27 naidine species, 24 species from the other tubificid subfamilies, and five outgroup taxa. New sequences (in all 108) as well as GenBank data were used. The data were analysed by parsimony and Bayesian inference. The tree topologies emanating from the different analyses are congruent to a great extent. Naidinae is not found to be monophyletic. The naidine genus Pristina appears to be a derived group within a clade consisting of several genera (Ainudrilus, Epirodrilus, Monopylephorus, and Rhyacodrilus) from another tubificid subfamily, Rhyacodrilinae. These results demonstrate the need for a taxonomic revision: either Ainudrilus, Epirodrilus, Monopylephorus, and Rhyacodrilus should be included within Naidinae, or Pristina should be excluded from this subfamily. Monophyly of four out of six naidine genera represented by more than one species is supported: Chaetogaster, Dero, Paranais, and Pristina, respectively.     

Ultrastructural investigation of coelomocytes in representatives of Naidinae and Rhyacodrilinae (Annelida,Clitellata, Tubificidae)

Various types of free‐floating cells are found in the coelomic fluid of representatives of several annelid groups. The ultrastructure of these “coelomocytes,” however, has been studied to a limited degree. In this study, we used a transmission electron microscope to investigate the coelomocytes in specimens of five species of Naidinae and three species of Rhyacodrilinae (all oligochaetous clitellates within the family Tubificidae). These were compared with each other and with previously described coelomocytes of representatives of other oligochaete taxa. Only one distinguishable coelomocyte type was found in the studied specimens: a round to oblong cell without pseudopodia or other appendages, primarily containing membrane‐bound granules of varying electron density, a prominent network of rough endoplasmic reticulum, and free ribosomes. This type differs to a great extent from most of the previously described coelomocytes, but shows similarities to certain types found in members of Enchytraeidae and Megascolecidae. Although we noticed some variation, we did not find any ultrastructural characters in these cells obviously useful for phylogenetic studies within Tubificidae. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Phylogeny of Tubificidae (Annelida, Clitellata) based on mitochondrial and nuclear sequence data

The tubificid clitellates are a common component in the freshwater bottom fauna and are also the most abundant oligochaete group in marine habitats. There are over 800 described species classified in six subfamilies; Tubificinae, Limnodriloidinae, Rhyacodrilinae, Telmatodrilinae, Phallodrilinae, and Naidinae. In this study we examine the phylogenetic relationships in Tubificidae using a combination of mitochondrial 16S rDNA and nuclear 18S rDNA sequence data. Sequences were obtained from five outgroup and 56 ingroup taxa, including five of the six subfamilies of Tubificidae. The data were analysed by maximum parsimony and Bayesian inference. The resulting tree topologies are virtually without conflict. Several associations traditionally recognized within the family Tubificidae are supported, in the Bayesian analysis including a sister group relationship between Tubificinae and Limnodriloidinae. The results also indicate that Rhyacodrilinae is polyphyletic--some of its members (Heterodrilus spp.) fall into a clade with Phallodrilinae, all other groups with Naidinae. Naidinae is also polyphyletic with two rhyacodriline genera, Monopylephorus and Ainudrilus, nested within. Most of the tubificid genera included in the study are supported as monophyletic; however, Tubifex and Limnodriloides are refuted, and Tubificoides is unresolved from other tubificine taxa.