First reports of Grania (Clitellata: Enchytraeidae) from Africa and South America: molecular phylogeny and descriptions of nine new species

In this article, our knowledge of the geographic distribution of Grania species is expanded by describing seven new species, G rania bekkouchei sp. nov. , G rania brasiliensis sp. nov. , G rania capensis sp. nov. , G rania chilensis sp. nov. , G rania cryptica sp. nov. , G rania hinojosai sp. nov. , and G rania simonae sp. nov. , from poorly investigated regions of the Southern Hemisphere, plus two new species, G rania carolinensis sp. nov. and G rania unitheca sp. nov. , from off the east coast of the USA. An immature achaetous specimen that we call Grania cf. levis was also included. The newly generated data were combined with a previously published data set in order to update the hypothesis of phylogenetic relationships among Grania species. All new species except G . cryptica sp. nov. are supported by both morphological and molecular data. In addition, we find that G . chilensis sp. nov. is structured in at least four distinct populations along the Chilean coast. The species described from South Africa form a monophyletic clade where two are morphologically indistinguishable but diverging in both mitochondrial and nuclear ribosomal genes, and thus we describe them as different species, G . bekkouchei sp. nov. and G . cryptica sp. nov. Among the North Carolinian species, G . unitheca sp. nov. is indicated as a close relative of Grania monospermatheca Erséus & Lasserre, 1976, and G . carolinensis sp. nov. is indicated as a close relative of G. cf. levis. The updated phylogeny is strongly concordant with geographical species distributions, thus supporting a low level of dispersal within this genus, as has previously been hypothesized. © 2015 The Linnean Society of London     

Genetic variation and phylogeny of Scandinavian species of Grania (Annelida: Clitellata: Enchytraeidae), with the discovery of a cryptic species

Individuals of five nominal species of Grania (Annelida: Clitellata: Enchytraeidae) were collected from locations in Sweden, Norway and France, for studies on the intraspecific variation at the Cytochrome Oxidase I (COI) locus of mitochondrial DNA and internal transcribed spacer (ITS) region of nuclear DNA. It was found that the previously described morphospecies in general contain low variation compared to the between‐species variation in both loci. In one instance, however, an individual morphologically indistinguishable from G. ovitheca was found to be deviant and instead cluster with G. postclitellochaeta both by COI and ITS. We describe this individual as a new species: G. occulta sp.n. Furthermore, phylogenetic analyses were conducted, showing a close relationship between G. variochaeta, G. occulta, G. ovitheca and G. postclitellochaeta, as well as between G. pusilla and G. maricola. Using the results from the phylogenetic analyses, we discuss the evolution of morphological characters in Scandinavian species of Grania.     

Cryptic diversity among the achaetous Marionina (Annelida,Clitellata, Enchytraeidae)

This study investigates the diversity and taxonomy of a mainly marine group of species lacking chaetae currently assigned to the genus Marionina. This achaetous group includes four nominal species: M. achaeta (Hagen, 1954 Hagen, G. 1954. Michaelsena achaeta nov. sp., ein neuer mariner Oligochaet aus der Kieler Bucht. Faunistische Mitteilungen aus Norddeutschland, 1: 12–13.  [Google Scholar]), M. achaeta sensu Lasserre, 1964 Lasserre, P. 1964. Notes sur quelques oligochètes Enchytraeidae présents dans les plages du Bassin d’Arcachon. Procés-Verbaux des Séances de la Société Linnéenne de Bordeaux, 101: 87–91.  [Google Scholar], M. nevisensis Righi & Kanner, 1979 Righi, G. and Kanner, E. 1979. Marine Oligochaeta (Tubificidae and Enchytraeidae) from the Caribbean Sea. Studies of the Fauna of Curaçao and other Caribbean Islands, 58: 44–68.  [Google Scholar] and M. arenaria Healy, 1979 Healy, B. 1979a. Marine fauna of County Wexford. 1 – Littoral and brackishwater Oligochaeta. The Irish Naturalists' Journal, 19: 418–422.  [Google Scholar]. As Lasserre's (1964 Lasserre, P. 1964. Notes sur quelques oligochètes Enchytraeidae présents dans les plages du Bassin d’Arcachon. Procés-Verbaux des Séances de la Société Linnéenne de Bordeaux, 101: 87–91.  [Google Scholar]) M. achaeta appears to be morphologically different from its (then) senior homonym M. achaeta (Hagen, 1954 Hagen, G. 1954. Michaelsena achaeta nov. sp., ein neuer mariner Oligochaet aus der Kieler Bucht. Faunistische Mitteilungen aus Norddeutschland, 1: 12–13.  [Google Scholar]), the replacement name M. nothachaeta nom. nov. is proposed for it. We studied the genetic and morphological diversity of achaetous specimens of Marionina collected in Florida, the Great Barrier Reef, New Caledonia, Sweden, England and the Bahamas. The collection localities are almost all supralittoral and often brackish-water habitats. Parts of the mitochondrial genes 12S, 16S, COI and the nuclear genes 18S, 28S and ITS were analysed to assess the genetic variation and phylogeny of the achaetous Marionina species. The molecular data reveal one monophyletic group of 11 separately evolving lineages, and between these lineages, K2P distances in the barcoding gene COI vary between 5.4 and 25.0%. On a morphological basis, the lineages could be assigned to seven different groups (morphotypes), of which only two could be identified as described nominal taxa: M. nevisensis s. lat. (several lineages) and M. nothachaeta. Since the former taxon appears to be a complex of cryptic species around the world and the original type material no longer exists, a neotype from the Caribbean was designated for M. nevisensis s. str. The remaining achaetous lineages represent five morphologically distinct species that are left unnamed, awaiting finer morphological scrutiny and detailed comparisons with new collections of M. achaeta and M. arenaria. Summing up, the group of achaetous Marionina now seems to contain up to 13 different species, seven of which are yet to be formally described and named.     

Comparative study of the cuticle in some aquatic oligochaetes (Annelida: Clitellata)

An examination of the cuticle of six aquatic oligochaete species using transmission electron microscopy revealed a larger morphological variation than previously known. Three freshwater species, Aulodrilus pluriseta, Spirosperma ferox (both Tubificidae), and Pristina breviseta (Naididae), and three marine species, Clitellio arenarius, Heterochaeta costata (both Tubificidae), and Paranais litoralis (Naididae), were investigated. The arrangement of the collagen fibers in the cuticle differs among the studied species. Only S. ferox shows an "orthogonal grid," i.e., layers of parallel fibers perpendicular to each other, as earlier described for lumbricids and enchytraeids. Clitellio arenarius and H. costata have fibers arranged in layers, while A. pluriseta and P. litoralis have irregularly distributed fibers. Pristina breviseta lacks cuticular fibers. The matrix surrounding the collagen fibers (when present) continues outside the fiber layer, making up a thin epicuticle, which has a unique banding in each of the studied species. The external surface of the epicuticle is covered with epicuticular projections. Their number, shape, and attachment to the epicuticle vary among the studied species. Furthermore, a distinctive internal substructure of the projections was observed in H. costata, A. pluriseta, S. ferox, and P. breviseta. Microvilli, extensions from the epidermal cells, penetrate the cuticle and terminate at its outer surface. In three species microvilli were observed to pinch off the epicuticular projections. The size, number, and shape of the latter vary; no typical microvilli were observed in S. ferox.     

Specific criteria in Grania (Oligochaeta,Enchytraeidae)

The structure of the penial bulb and male efferent duct system of Grania species may be used in addition to setal pattern and spermathecal shape to distinguish species. Six penial bulb types are distinguished: (1) a simple, small, glandular bulb surrounding the male pore; (2) a small, glandular bulb, with a large, associated, dorso-medial gland mass; (3) a small glandular bulb, medial to the male pore, with an elongate male bursa (the aglandular sac), the vas deferens exitting directly into the invaginated male pore; (4) a glandular bulb with an aglandular sac and a small, cuticular stylet embedded in the bulb, extending from the ectal end of the vas deferens; (5) a glandular bulb and an aglandular sac with a long stylet extending from the vas deferens, through the bulb into the sac; and (6) glandular bulb reduced or absent, with or without an aglandular sac; with a long stylet and other prominent modifications, usually muscular, of the vas deferens. The details of the male duct structure were consistent within specimens grouped on the basis of setal distribution and shape and detailed spermathecal structure. Diverse male duct patterns are found within the polytypic species G. macrochaeta and G. postclitellochaeta. The positions of the spermathecal and male pores in their respective segments are distinctive for some species.     

Ultrastructure of the rotifer integument: peculiarities of Sinantherina socialis (Monogononta: Gnesiotrocha)

The rotifer integument is a well‐described syncytium that contains an apical intracytoplasmic lamina (ICL) that functions for both skeletal support and muscle insertion. To date, there is limited information on the structure of the integument in species of Gnesiotrocha, a diverse subclade of Monogononta that consists of solitary, colonial, sessile, and planktonic species. In this study, we examined the ultrastructure of the integument in the colonial rotifer Sinantherina socialis to determine how it corresponds to that of other monogononts. The integument of S. socialis was broadly similar to that of other rotifers, consisting of a thickened glycocalyx, multilaminate ICL, and syncytial epidermis. However, it was different in several regards. The ICL consisted of three distinct layers from apical to basal: layer 1 consisted of at least two electron‐dense laminae; layer 2 was a thickened matrix of amorphous, electron‐dense material or was fibrous; and layer 3 was an electron‐dense lamina of varying thickness that covered the underlying syncytium. Significantly, layers 1 and 2 formed a ridge‐and‐groove like system of finger‐like projections across the trunk surface that has not been observed in other rotifers. A voluminous syncytial cytoplasm (up to 3 μm thick) was present beneath the ICL and was mostly electron lucent and with few organelles. Bundles of potential microtubules were scattered throughout the syncytium. We hypothesize that the voluminous cytoplasm with microtubules serves as skeletal support for the rotifer's sessile lifestyle, while the external ridges may function as a texture‐based deterrent to predators, or serves to trap secretions from the species' defensive glands. Basally, the epidermis was highly folded and bordered by a thin basal lamina that separated the plasmalemma from the blastocoel. Membrane‐bound vesicles were present throughout the integument's cytoplasm and are hypothesized to function in the secretion of extracellular matrix and in the maintenance of the ICL.     

A new freshwater oligochaete species (Clitellata: Enchytraeidae) from Livingston Island,Antarctica

The new enchytraeid species Lumbricillus healyae sp. n. is described from freshwater streams, with well-oxygenated and poorly mineralised waters, situated in Byers Peninsula (Livingston Island, South Shetland Islands, Antarctica). Lumbricillus healyae sp. n. is morphologically close to L. antarcticus and L. incisus, and it is mainly distinguished by the structure of the spermatheca with a short, distinct ectal duct, the oval penial bulb (greatest diameter in the transverse body axis) associated with strong dorso-ventral muscular fibres, and a protrusible pseudopenis. A second undetermined Lumbricillus species is described from a small stream. Study specimens are not fully mature; however, the highly irregular form and size of the testis-sac lobes and the absence of a penial bulb encapsulated under a muscular layer are remarkable. It is probably related to a small group of Lumbricillus species reported from the Antarctic maritime region (L. colpites, L. griseus and L. aestum), characterised by the structure of the male duct, which ends in a simple pore surrounded by glands.

Comparative structure of the epidermis in polychaetes (Annelida)

The polychaete epidermis generally consists of a single layer of supportive cells, gland cells and sensory cells. Except for the latter, this paper reviews the recent literature on the annelid epidermis, focussing on the mentioned cell types and the cuticle. The annelid epidermis is compared to that of Sipuncula, Echiura and Myzostomida. Supportive cells predominate in the polychaete epidermis. They show a high structural diversity even within single specimens. Ciliated cells are usually multiciliary and only two cases of monociliary epidermis cells are known. Unambigous epithilio-muscle cells are only described in feeding palps of a Magelona species. Secretory cells release a large number of gland products and some of them are essential for tube secretion. Rather pecularities of the cells and its arrangement within glands than the ultrastructure of the secretions is useful for phylogenetic considerations. One of the main components of the cuticle is collagen. Recent studies indicate that annelid cuticular collagen differs in several aspects from collagen of the connective tissue and might be of interest for systematics.     

Comparative body wall musculature and muscle fibre ultrastructure in branchiobdellidans (Annelida: Clitellata), and their phylogenetic significance

The body wall muscles in five species of branchiobdellidans are all arranged in the oligochaete pattern and the muscle fibres are obliquely striated. The structure of the circular muscle fibres do vary to some degree. The longitudinal muscle fibres in Ankyrodrilus legaeus, Branchiobdella kozarovi, and Xironogiton instabilis all are round circomyarian and thus double-obliquely striated. These species represent three of the four genera composing the family Branchiobdellidae. Although Bdellodrilus illuminatus and Cambarincola fallax, from the families Bdellodrilidae and Cambarincolidae, respectively, also possess a few round circomyarian fibres, most are polyplatymyarian comparable to single-obliquely striated fibres. A similar division of branchiobdellidan families is obtained based on the number of anterior nephridial pores. The muscular structure in the branchiobdellidans shows both similarities and differences with the leeches and the lumbriculid oligochaetes. One phylogenetic explanation for this is that the branchiobdellidans separated from the common clitellate ancestor before the oligochaetes and leeches became recognizable taxa.     

Phylogenetics of some Enchytraeidae (Annelida: Oligochaeta): A preliminary investigation of relationships to the Haplotaxidae

In phylogenetic considerations of the Oligochaeta and the family Enchytraeidae, Propappus has been considered the ancestral or even the archetypal enchytraeid genus. Three nominal species are presently included in Propappus Michaelsen, 1905, P. volki Michaelsen, 1916, P. arhyncotus Sokolskaja, 1972, and the type species of the genus, P. glandulosus Michaelsen, 1905. Examination of type material of P. glandulosus and P. volki, as well as other material of the latter, showed that these species have a single pair of testes in segment XI and a single pair of ovaries in segment XIII. According to current classifications of the Oligochaeta, these species of Propappus cannot be retained in the Enchytraeidae. A phylogenetic analysis including two species of Propappus and 29 species of Haplotaxidae suggest a sister group relationship between Propappus and some Enchytraeidae and Haplotaxidae.     

Body wall of juvenile and adult Gastromermis boophthorae (Nematoda: Mermithidae): Ultrastructure and nutritional role

During the life cycle of the mermithid nematode Gastromermis boophthorae the main components of the body wall, namely the cuticle, hypodermis and somatic musculature, undergo radical changes in structure and relative proportions. In the non-feeding, free-living stages the cuticle is a well-defined, multilayered structure which lies upon a generally thin hypodermis; the somatic musculature has the extensive and highly organised appearance typical of motile nematodes. In parasitic juveniles the body wall has a completely different structure which is adapted for the suggested purpose of nutrient uptake from the host insect's haemocoelomic fluid; the cuticle is characteristically thin and rather diffuse, the underlying hypodermis becomes very extensive and metabolically active, and there is a corresponding reduction in the proportion of somatic musculature. The sub-cuticular surface of the enlarged hypodermis has a microvillous configuration and has associated alkaline phosphatase activity.     

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.     

Species separation and identification in the Enchytraeidae (Oligochaeta, Annelida): combining morphology with general protein data

In the Enchytraeidae, species separation and identification is often problematic due to high morphological similarity of closely related species and considerable intraspecific variability of crucial characteristics. Immature specimens are almost undeterminable. To meet these difficulties, this paper recommends the consulting of general protein patterns as exhibited by non-specific silver-staining after isoelectric focusing. A method is presented which allows the successful inclusion of protein data in taxonomic studies and field surveys. Two examples from an investigation on Fridericia field populations show that, with the help of general protein pattern analysis, a clear taxonomic decision on the identity of morphologically aberrant forms and of juvenile specimens as well can be achieved with comparatively little expenditure of time. A combined use of morphological and protein data for taxonomic purposes is suggested.     

Bioaccumulation of lindane and hexachlorobenzene by the oligochaetes Enchytraeus luxuriosus and Enchytraeus albidus (Enchytraeidae,Oligochaeta, Annelida)

The uptake of chemicals in soil organisms, especially earthworms, has been studied many times. However, in Europe no internationally accepted standardised test guideline for the assessment of bioaccumulation in the soil ecosystem exists. Therefore, the German Federal Environmental Agency recently funded a project in which a standardisable test method for measuring bioaccumulation of chemicals using earthworms and enchytraeids is being developed. In this contribution, initial results with the new method are presented, using two model chemicals (the insecticide lindane and the fungicide hexachlorobenzene). Two enchytraeid species (Enchytraeus luxuriosus and Enchytraeus albidus) were selected as test organisms due to their easy handling and their important ecological role in the soil compartment. Artificial soil and a natural standard soil were used as test substrates. Test concentrations were based on previous results of acute and reproduction toxicity tests performed with the same species. Uptake as well as the elimination of the test substances were examined under standardised conditions in a closed test system. The first results show that both chemicals were accumulated considerably by both enchytraeid species. The bioaccumulation factors (BAFs) of lindane and hexachlorobenzene found for enchytraeids are significantly higher compared to those for lumbricid earthworms. Evaluation of the preliminary data suggests that the smaller species E. luxuriosus accumulated the two chemicals to a greater extent than E. albidus. In most cases, both chemicals were eliminated completely. The use of this new test method appears suitable for the ecotoxicological risk assessment of bioaccumulative chemicals.     

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     

The occurrence of species of semi-aquatic Enchytraeidae (Oligochaeta) in Ireland

The Enchytraeidae are essentially terrestrial oligochaetes but many species have marked aquatic tendencies. Over two thirds of recorded Irish species were found in soils which were submerged or frequently flooded and 35% showed a distinct preference for these conditions. Relatively few species were living in soils subject to drought. Red blood was present in 28 species, all but one from soils with more than 55% water. Cognettia sphagnetorum and C. glandulosa developed red blood in very wet conditions. In a survey of Irish wetlands, samples were taken from bog, heath, marsh, fen, margins of lakes and rivers, and saltmarsh. The influence of various environmental parameters was determined using ordination techniques. Magnesium and pH were found to be the most important factors. A high level of magnesium distinguished coastal sites and pH 5.2 separated two clusters representing acid peat and marsh-fen-aquatic sites. Groups of indicator species characterized each of the three clusters. The ecological distribution of the indicator species is described, and their usefulness in classifying enchytraeid communities is discussed.