Clitellate Species Research Articles

Investigating the Clitellata (Annelida) of Icelandic springs with alternative barcodes

DNA barcoding is an invaluable tool to identify clitellates, regardless of life stage or cryptic morphology. However, as COI (the standard barcode for animals) is relatively long (658 bp), sequencing it requires DNA of high quality. When DNA is fragmented due to degradation, alternative barcodes of shorter length present an option to obtain genetic material. We attempted to sequence 187 clitellates sampled from springs in Iceland. However, the material had been stored at room temperature for two years, and DNA of the worms had degraded, and only three COI sequences were produced (i.e., <2% success rate). Using two alternative barcodes of 16S (one ca. 320 bp, the other ca. 70 bp long) we increased the number of sequenced specimens to 51. Comparisons of the 16S sequences showed that even the short 70 bp fragment contained enough genetic variation to separate all clitellate species in the material. Combined with morphological examinations we recognized a total of 23 species, where at least 8 are new records for Iceland, some belonging to genera new for Iceland: Cernosvitoviella and Pristina. All the new taxa are included in an updated species list of Icelandic Clitellata. The material revealed some stygophilic species previously known to inhabit springs, but true stygobionts, which are restricted to groundwater habitats, were not found. Our study shows that short 16S fragments can be obtained from DNA too degraded to be used in traditional COI barcoding, and contain enough genetic variation to separate closely related clitellate species.

Climatically induced temperature instability of groundwater‐dependent habitats will suppress cold‐adapted Clitellata species

AbstractGroundwater‐dependent ecosystems are recognized as biodiversity hotspots, being, apart many negative human impacts, highly threatened also by ongoing climate warming. Clitellata (Annelida) are dominant invertebrates of permanent fauna in spring habitats, representing a heterogeneous group including both specialized cold‐stenothermic and ubiquitous eurythermic species. Therefore, they seem to be a good model group to compare the effects of local springwater temperature (recorded in situ by data loggers) and mesoclimate (i.e., local) air temperature. By the analysis of clitellate assemblages at 41 isolated Western Carpathian spring fens, we found that their species composition was significantly driven by mesoclimate air temperature and springwater temperature independently of other important environmental variables (i.e., water mineralization, oxygen content, and total organic carbon). The effect of various environment‐related and temperature‐related variables on the number of clitellate species was analyzed separately for two categories, that is, substrate dwellers (endobenthic species) and surface‐active (epibenthic) species. The decrease of the number of species with the increasing amount of inorganic particles <500 µm in substrate was observed in the substrate dwellers. Mesoclimate air temperature had no significant effect on the number of species of substrate dwellers. However, water temperature, specifically its daily fluctuation, turned out to have a strong effect. Only the sites with no or moderate fluctuation were inhabited by cold‐stenotherm spring specialists and cold‐water species. In contrast, no significant response to any temperature parameter was found for the number of surface‐active species, which was driven only by other environmental variables. Our results suggest that climatically induced increase in temperature fluctuation of spring waters can result in notable reduction of cold‐adapted clitellate species (mainly the family Lumbriculidae) at the expense of eurythermic species. Such a scenario predicts compositional changes leading to clitellate assemblages with a dominance of generalist and semi‐aquatic species.

Reproductive differences among species, and between individuals and cohorts, in the leech genus Helobdella (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae), with implications for reproductive resource allocation in hermaphrodites.

Leeches and oligochaetes comprise a monophyletic group of annelids, the Clitellata, whose reproduction is characterized by simultaneous hermaphroditism. While most clitellate species reproduce by cross-fertilization, self-fertilization has been described within the speciose genus Helobdella. Here we document the reproductive life histories and reproductive capacities for three other Helobdella species. Under laboratory conditions, both H. robusta and H. octatestisaca exhibit uniparental reproduction, apparently reflecting self-fertility, and suggesting that this trait is ancestral for the genus. However, the third species, H. austinensis, seems incapable of reproduction by self-fertilization, so we inferred its reproductive life history by analyzing reproduction in breeding cohorts. Comparing the reproductive parameters for H. robusta reproducing in isolation and in cohorts revealed that reproduction in cohorts is dramatically delayed with respect to that of isolated individuals, and that cohorts of leeches coordinate their cocoon deposition in a manner that is not predicted from the reproductive parameters of individuals reproducing in isolation. Finally, our comparisons of reproductive capacity for individuals versus cohorts for H. robusta, and between different sizes of cohorts for H. austinensis, reveal differences in resource allocation between male and female reproductive roles that are consistent with evolutionary theory.

Variation of Clitellata (Annelida) assemblages related to water saturation in groundwater-dependent wetlands

Aquatic invertebrates of groundwater-dependent wetlands can be sensitive to a drop in the groundwater table, which is considered one of the possible impacts of climate change. We investigated whether aquatic clitellate species are able to dwell in waterlogged substrates without surface water, in 27 spring fens studied across the Western Carpathians. A total of 78 clitellate species were recorded in pairs of samples from aquatic and semi-aquatic habitats. Semiaquatic habitats had 27 species in common with aquatic habitats, but algal and fungal feeders were less frequent and abundant, and predatory leeches and Haplotaxis gordioides completely lacking. Results indicate that, during extreme droughts, when aquatic habitats cease to exist, some aquatic clitellates can persist in the waterlogged substrate, while some aquatic specialists may face the risk of local extinction.

New specific primers for amplification of the Internal Transcribed Spacer region in Clitellata (Annelida).

Nuclear molecular evidence, for example, the rapidly evolving Internal Transcribed Spacer region (ITS), integrated with maternally inherited (mitochondrial) COI barcodes, has provided new insights into the diversity of clitellate annelids. PCR amplification and sequencing of ITS, however, are often hampered by poor specificity of primers used. Therefore, new clitellate‐specific primers for amplifying the whole ITS region (ITS: 29F/1084R) and a part of it (ITS2: 606F/1082R) were developed on the basis of a collection of previously published ITS sequences with flanking rDNA coding regions. The specificity of these and other ITS primers used for clitellates were then tested in silico by evaluating their mismatches with all assembled and annotated sequences (STD, version r127) from EMBL, and the new primers were also tested in vitro for a taxonomically broad sample of clitellate species (71 specimens representing 11 families). The in silico analyses showed that the newly designed primers have a better performance than the universal ones when amplifying clitellate ITS sequences. In vitro PCR and sequencing using the new primers were successful, in particular, for the 606F/1082R pair, which worked well for 65 of the 71 specimens. Thus, using this pair for amplifying the ITS2 will facilitate further molecular systematic investigation of various clitellates. The other pair (29F/1084R), will be a useful complement to existing ITS primers, when amplifying ITS as a whole.

When morphology and DNA are discordant: Integrated taxonomic studies on the Eisenia fetida/andrei complex from different parts of Iran (Annelida, Clitellata: Megadrili)

Recent studies have indicated the presence of high cryptic diversity in some clitellate species. Eisenia fetida/andrei is an earthworm species complex that is commonly used in ecotoxicological assays, where undetected differences in contaminant responses between cryptic lineages may lead to confusing or misleading results. Furthermore, because of the key role that earthworm species play in the soil ecosystem, a better understanding of cryptic diversity is necessary to investigate if divergent lineages play different roles within their environments. This can be a serious problem for the reliability of the standard ecotoxicological tests. Traditional earthworm (Annelida, Clitellata: Megadrili) classifications have been mainly built using morphological characters. However, due to the limited number of diagnostic external features, morphological differences alone sometimes do not allow correct identification, especially for sibling taxa which are morphologically similar and whose requirements, overall reproductive performances, and life cycles do not differ significantly. In this study we have demonstrated that the classical morphological distinction between E. fetida and E. andrei does not fit with their phylogenetic structure and we have confirmed that the Eisenia fetida/andrei complex consists of two cryptic sibling species. We have also evaluated the distribution of this species complex according to some scale ecological inferences.

The response of Clitellata (Annelida) to environmental gradients in spring fens

Patterns in aquatic Clitellata assemblage composition are known to be driven by several environmental gradients, with water chemistry and substratum characteristics being particularly important. In this study we explored 54 isolated spring fens across the eastern Czech Republic and Slovakia. These fens varied in calcium and magnesium concentrations, forming a sharp and well defined environmental gradient running from calcium-poor acidic fens to extremely calcium-rich tufa-forming fens. We found that the main changes in clitellate species composition were controlled by this gradient, and/or total organic carbon content, over a wide area, including fen sites differing in other environmental conditions and historical development. However, this pattern was weakened in sites with a high organic matter content, which represented a second driver of change in assemblage composition along with water temperature. Three main types of fens were determined using cluster analysis based on clitellate assemblage composition. However, only the first type, which included tufa-forming fens, was found to fit with the previously established spring fen types based on vegetation (i.e. extremely mineral-rich fens with a tufa, brown-moss mineral-rich fens, mineral-rich Sphagnum fens and mineral-poor Sphagnum fens). The second clitellate type included sites with low temperatures and occasional desiccation, while the third type was characterised by high temperatures and trophy. Using eight environmental predictors, we were able to significantly explain changes in the population abundances of all 12 common species (i.e. recorded at 15-plus sites). The results from individual species modelling also suggests that an increase in organic matter content can trigger compositional shifts towards assemblages of common eurytopic tubificid species. Thus, human-induced eutrophication and negative changes in spring fen hydrology, mainly drying up, can represent a serious threat for species-specific assemblages of aquatic clitellates, especially at alkaline sites due to their isolated and spatially limited nature.

Environmental impact of heated mining waters on clitellate (Annelida: Clitellata) assemblages

Mining is a relatively highly monitored industry. While chemical pollutants (toxic ions, radionuclides, etc.) have mostly been eliminated from mining waters, other types of environmental pollution (temperature regime alterations, high concentrations of various anions, etc.) can affect benthic invertebrates. In this study, we focused on the effect of mining water effluent on the diversity and density of aquatic Clitellata. Four sampling sites were selected. Three sites in a natural stream (the Nedvědicka River, Czech Republic), one upstream and two downstream from the mining effluent, and one site on the mining waters were sampled monthly during 2008–2009. Environmental variables were recorded in and samples were collected from two types of habitats — riffles and pools. The response of clitellate assemblages was evaluated using principal component analysis and generalised estimating equations. The results indicated that the mining effluent caused partial species exchange and had negative effects on clitellate taxa richness and abundance. These responses were specific to both the habitat (riffle/pool) and species sampled. In each of the different taxa studied, we observed one of four typical clitellate responses: (a) elimination of stenotherm species; (b) reduction of clitellate species followed by quick recovery; (c) neutral response; or (d) positive influence. We found that aquatic clitellates, which are considered to be eurytopic with broad ecological valences, are also sensitive to even slight environmental pollution.

Description of Pristina armata n. sp. (Clitellata: Naididae: Pristininae) from a carnivorous plant (Nepenthes sp.) in Borneo, Indonesia.

A new clitellate species of Pristininae (Naididae), Pristina armata n. sp., found in the pitcher of the carnivorous plant Nepenthes sp., is reported from East Kalimantan, Indonesia. P. armata n. sp. is a very small clitellate, less than 1 mm long in fixed state, and without proboscis on the prostomium. Signs of reproduction by paratomy were observed, but the generic placement remains preliminary because sexually mature individuals were not found. P. armata n. sp. is characterized by giant hook-like dorsal chaetae at IV. The description of P. armata n. sp. was based on six fixed specimens of different size and stage of development. Noteworthy is the habitat of P. armata n. sp. in Nepenthes pitchers, this being the first clitellate species described from such a habitat. P. armata n. sp. may be a member of the nepenthebionts' community, realizing its life cycle inside the digestive fluid of the Nepenthes pitcher, or it belongs to nepenthephiles, species that commonly occur in this habitat but do not specialize on it.

Species richness and composition patterns of clitellate (Annelida) assemblages in the treeless spring fens: the effect of water chemistry and substrate

Spring fens are isolated treeless wetlands of a high conservation value. Their environmental conditions are strongly related to their groundwater chemistry, which controls species distribution within various groups of organisms. Clitellates, a dominant group of non-insect aquatic fauna, however, have never been studied in these habitats. It is unclear from previous studies to what extent the distribution of aquatic non-insect taxa reflects water chemistry rather than the substrate structure. We studied 34 spring fens sampled in 17 isolated sites in the Western Carpathian Mountains to determine mainly the effects of water chemistry and substrate structure on variation in species richness and composition of clitellate assemblages as examples of the non-insect fauna. A total of 34 taxa were found, with 3–15 taxa collected per sample. Species richness was negatively correlated with water mineral concentration measured as water electric conductivity (r = −0.57, P < 0.001) and positively with TOC (r = 0.60, P < 0.001). Surprisingly, the lowest number of taxa was found in calcareous fens and richness increased towards Sphagnum-fens. There was a species turnover related to changes in mineral richness and substrate characters. The main change of species composition was promoted by changes in substrate structure. The second gradient of species composition was linked with the amount of nutrients, moisture, and dominance of sphagna, and was associated with an increase of eurytopic species in fens with high nutrient availability. It was difficult to separate the effects of water chemistry and substrate on clitellate species distributions owing to the fact that variation in tufa precipitation and vegetation was driven by water chemistry changes. This study presented the first quantitative data on fen clitellate assemblages, which appear to have an unusual pattern of species richness. In contrast to plants and molluscs, calcareous fens appeared to be a harsh environment for clitellate species. Only few specialized species, mainly Trichodrilus strandi, were able to establish viable populations. The significant effect of water chemistry on clitellate distribution patterns raises questions about the direct influence of water chemistry on non-insect aquatic taxa, which have previously been considered to be mostly determined by substrate characteristics.

Lineage analysis of micromere 4d, a super-phylotypic cell for Lophotrochozoa, in the leech Helobdella and the sludgeworm Tubifex

The super-phylum Lophotrochozoa contains the plurality of extant animal phyla and exhibits a corresponding diversity of adult body plans. Moreover, in contrast to Ecdysozoa and Deuterostomia, most lophotrochozoans exhibit a conserved pattern of stereotyped early divisions called spiral cleavage. In particular, bilateral mesoderm in most lophotrochozoan species arises from the progeny of micromere 4d, which is assumed to be homologous with a similar cell in the embryo of the ancestral lophotrochozoan, more than 650million years ago. Thus, distinguishing the conserved and diversified features of cell fates in the 4d lineage among modern spiralians is required to understand how lophotrochozoan diversity has evolved by changes in developmental processes. Here we analyze cell fates for the early progeny of the bilateral daughters (M teloblasts) of micromere 4d in the leech Helobdella sp. Austin, a clitellate annelid. We show that the first six progeny of the M teloblasts (em1–em6) contribute five different sets of progeny to non-segmental mesoderm, mainly in the head and in the lining of the digestive tract. The latter feature, associated with cells em1 and em2 in Helobdella, is seen with the M teloblast lineage in a second clitellate species, the sludgeworm Tubifex tubifex and, on the basis of previously published work, in the initial progeny of the M teloblast homologs in molluscan species, suggesting that it may be an ancestral feature of lophotrochozoan development.

Comparative ultrastructural study of the cuticle and spermatozoa in Propappus volki Michaelsen, 1916 (Annelida: Clitellata)

The ultrastructure of the cuticle and mature spermatozoa of the oligochaete Propappus volki Michaelsen, 1916 is described with the aim of providing additional data for clarifying the systematic position of the taxon. P. volki is a fresh-water species living in streams, and is easily recognized by its proboscis on the pre-segmental prostomium and, in mature specimens, by a clitellum covering the segments XII–XIV. The cuticle is composed of a proximal fibre zone and a distal layered epicuticle covered with membrane-bound epicuticular projections. The fibre zone consists of collagenous fibres in a matrix, arranged in either densely packed parallel layers with the fibres oriented in the same direction, or with more loosely distributed fibres, although with the same main orientation. The epicuticular projections are pyramidal with the base leaning on the outer surface of the epicuticle. The cuticle covering the proboscis differs in morphology from that of the rest of the worm; the fibre zone is composed of thin and short fibrils running in all directions, and the epicuticular projections are longer and more narrow than the projections in other regions of the worm. The spermatozoa are filiform cells formed, in sequence, by an acrosome, an elongated nucleus, a long midpiece, and a flagellum. The acrosomal tube is short and straight with a completely external acrosomal vesicle. Following the acrosome is a apically corkscrew-shaped and basally straight nucleus. The midpiece is twisted and formed by five mitochondria. The flagellum shows a prominent central sheath arrangement. A comparison with ultrastructurally described cuticles and spermatozoa from other clitellate species reveals most similarities with enchytraeids.

Evolutionary aspects of pattern formation during clitellate muscle development

As a taxon of the lophotrochozoans, annelids have re-entered scientific investigations focusing on plesiomorphic bilaterian features and the evolutionary changes therein. The view of a clitellate-like plesiomorphic muscle arrangement in annelids has been challenged by recent investigations of polychaete muscle organization. However, there are few investigations of muscle formation in clitellate species that address this problem. Direct comparison of potential homologous muscles between these annelid groups is thus hampered. Somatic muscle formation during embryogenesis of two clitellates-the oligochaete Limnodrilus sp. and the hirudinean Erpobdella octoculata-occurs by distinct processes in each species, even though they share a closed outer layer of circular and an inner layer of longitudinal muscles characteristic of clitellates. In E. octoculata, the first emerging longitudinal muscles are distributed irregularly on the body surface of the embryo whereas the circular muscles appear in an orderly repetitive pattern along the anterioposterior axis. Both primary muscle types consist of fiber-bundles that branch at both their ends. This way the circular muscle bundles divide into a fine muscle-grid. The primary longitudinal muscles are incorporated into a second type of longitudinal muscles, the latter starting to differentiate adjacent to the ventral nerve cord. Those secondary muscles emerge in a ventral to dorsal manner, enclosing the embryo of E. octoculata. In Limnodrilus sp., one dorsal and one ventral bilateral pair of primary longitudinal muscles are established initially, elongating toward posterior. Initial circular muscles are emerging in a segmental pattern. Both muscle layers are completed later in development by the addition of secondary longitudinal and circular muscles. Some features of embryonic longitudinal muscle patterns in Limnodrilus sp. are comparable to structures found in adult polychaete muscle systems. Our findings show that comparative studies of body-wall muscle formation during clitellate embryogenesis are a promising approach to gain further information on annelid muscle arrangements.

The existence of all three ParaHox genes in the clitellate annelid, Perionyx excavatus

A ParaHox gene cluster is composed of three genes (Gsx, Xlox, and Cdx). It has been proposed that all three ParaHox genes were present in the last common ancestor to the lophotrochozoan protostomes and the deuterostomes and that gene loss event has occurred in the ecdysozoan lineage. In this paper, we report the existence of all three ParaHox genes in Perionyx excavatus, a clitellate annelid. Although orthologs of each of the three ParaHox genes were previously discovered from other lopotrochozoan taxa, this study constitutes the first reported isolation of all three ParaHox genes in the same clitellate species.

Evolutionary diversification of specification mechanisms within the O/P equivalence group of the leech genusHelobdella

Developmental fates and cell lineage patterns are highly conserved in the teloblast lineages that give rise to the segmental ectoderm of clitellate annelids. But previous studies have shown that the pathways involved in specification of the ventrolateral O lineage and the dorsolateral P lineage differ to some degree in distantly related clitellate species such as the leeches Helobdella and Theromyzon, and the sludgeworm Tubifex. To examine this developmental variation at a lower taxonomic level, we have explored the specification pathways of the O and P lineages in the leech genus Helobdella. In leech, the O and P lineages arise from a developmental equivalence group of O/P teloblasts. In this study, we demonstrate that the cell-cell interactions involved in cell fate specification of the O/P equivalence group differ among three laboratory colonies of closely related species. In two populations, the Q lineage is necessary to specify the P fate in the dorsalmost O/P lineage, but in the third population the P fate can be specified by a redundant pathway involving the M lineage. We also observe interspecific variation in the role played by cell interactions within the O/P equivalence group, and in the apparent significance of extrinsic signals from the micromere cell lineages. Our data suggest that cell fate specification in the O/P equivalence group is a complex process that involves multiple cell-cell interactions, and that the developmental architecture of the O/P equivalence group has undergone evolutionary diversification in closely related species, despite maintaining a conserved morphology.

Early development of metanephridia in the caudal budding zone of a clitellate annelid, Dero digitata (Naidida): an electron‐microscopical study

AbstractUltrastructural analysis has revealed that metanephridia in Dero digitata arise from three nephroblast cells in the frontal epithelium of a septum suggesting its mesodermal origin. Each cell has a fixed developmental destination, one nephroblast cell produces the entire canal part and two cells give rise to the nephrostome. The nephroblast cell nearest to the body wall enlarges and proliferates a first set of canal cells, then one of the two proximally adjacent nephroblast cells differentiates into the envelope of the nephrostome generating the marginal cilia of the opening (mantle cell) and the second one transforms into the anteriormost cell of the funnel, producing a flame of cilia that beats into the canal lumen (flame cell). Thereafter, new canal cells appear, mainly by mitosis of the first cell, enlarging the body of the nephridium whose further differentiation was not analysed. Comparison with other clitellate species suggests a mantle cell (or some marginal cells) and a flame cell (or a central cell) to be special characters of the metanephridium in the stem species of the Clitellata and that, compared to many polychaete species, its early development assumes a special course by a precocious determination and arrangement of nephroblast cells, which, in both groups, probably originate from an identical mesodermal stem cell. Results further indicate that the nonclitellate Aeolosomatidae, by virtue of corresponding nephrostomata, are possibly closer related to the Clitellata.

Is Hrabeiella periglandulata (Annelida, "Polychaeta") the sister group of Clitellata? Evidence from an ultrastructural analysis of the dorsal pharynx in H. periglandulata and Enchytraeus minutus (Annelida, Clitellata)

The terrestrial polychaete species Hrabeiella periglandulata has many characters in common with Clitellata and with Parergodrilidae, the latter representing another taxon of "Polychaeta" comprising a terrestrial species. Absence of a typical clitellum, different structure and position of the genital organs, different ultrastructure of the spermatozoa and chaetae as well as the presence of nuchal organs, although internal and highly modified, precluded inclusion of H. periglandulata within Clitellata. Consequently, convergent evolution of the clitellate-like features in these groups due to similar selection pressure was generally assumed. Similarities between H. periglandulata and Clitellata are greater than between both of these and Parergodrilidae and include not only reductions and absences but comparatively complex structures as well, among them the dorsally positioned pharynx, so far only known for Clitellata. However, the possibility of homology and thus synapomorphy between Hrabeiella and Clitellata has never seriously been discussed, a view seemingly supported by molecular data. In the present study an ultrastructural investigation of the dorsal pharynx was undertaken in H. periglandulata and in the clitellate species Enchytraeus minutus. The structures in common include a dense ciliation of the dorsal epithelium with short and immobile cilia, a prominent rootlet system which is connected to the musculature, numerous openings of pharyngeal gland cells between the ciliated cells, a cuticular fold surrounding the cilia, an unciliated buccal cavity, and cell bodies of the pharyngeal glands situated outside the pharyngeal epithelium. This makes the general assumption of convergence at least questionable. Likelihood of homology of this type of pharynx is further supported by the corresponding structure of cerebral sense organs and the central nervous system. Thus, these characters may likewise and more parsimoniously be explained as synapomorphies, making a sister group relationship of H. periglandulata and Clitellata conceivable.

Ultrastructural investigations on the spermatozoon and its genesis in Aeolosoma litorale with considerations on the phylogenetic implications for the aeolosomatidae (annelida)

The spermatozoa of Aeolosoma litorale are threadlike cells with a slight swelling in the midregion. The acrosome consists of a simple elongate vesicle without any accessory structures. The nucleus is a long, slightly conical cylinder. A column-shaped body derived from the proximal centriole underlies the nucleus and is united with the distal centriole forming a conventional basal body. The centriolar complex is surrounded by a mitochondriril derivative arising from two ring-shaped mitochondria that fuse to constitute a short tube. Lysosome-like vesicles are assembled on one side of the middle piece. The axoneme displays the common 9+2 pattern of microtubules. During spermiogenesis the acrosome protrudes in the distal region of the late spermatid while the flagellar base is shifted toward the cytophoral side. The absence of synapomorphies with the spermatozoa of the clitellate species corroborates the view that the Aeolosomatidae are not closely related to these taxa.