Fossilized spermatozoa preserved in a 50-Myr-old annelid cocoon from Antarctica.

The origin and evolution of clitellate annelids--earthworms, leeches and their relatives--is poorly understood, partly because body fossils of these delicate organisms are exceedingly rare. The distinctive egg cases (cocoons) of Clitellata, however, are relatively common in the fossil record, although their potential for phylogenetic studies has remained largely unexplored. Here, we report the remarkable discovery of fossilized spermatozoa preserved within the secreted wall layers of a 50-Myr-old clitellate cocoon from Antarctica, representing the oldest fossil animal sperm yet known. Sperm characters are highly informative for the classification of extant Annelida. The Antarctic fossil spermatozoa have several features that point to affinities with the peculiar, leech-like 'crayfish worms' (Branchiobdellida). We anticipate that systematic surveys of cocoon fossils coupled with advances in non-destructive analytical methods may open a new window into the evolution of minute, soft-bodied life forms that are otherwise only rarely observed in the fossil record.

Alike but different: the evolution of the Tubifex tubifex species complex (Annelida, Clitellata) through polyploidization.

BACKGROUND: Tubifex tubifex is a widespread annelid characterized by considerable variability in its taxonomic characteristics and by a mixed reproductive strategy, with both parthenogenesis and biparental reproduction. In a molecular phylogenetic analysis, we detected substantial genetic variability among sympatric Tubifex spp. from the Lambro River (Milano, Italy), which we suggested comprise several cryptic species. To gain insights into the evolutionary events that generated this differentiation, we performed a cytogenetic analysis in parallel with a molecular assay. Approximately 80 cocoons of T. tubifex and T. blanchardi were collected and dissected. For each cocoon, we sequenced a fragment of the 16S rRNA from half of the sibling embryos and karyotyped the other half. To generate a robust phylogeny enabling the reconstruction of the evolutionary processes shaping the diversity of these sympatric lineages, we complemented our original 16S rRNA gene sequences with additional COI sequences. RESULTS: The chromosome number distribution was consistent with the presence of at least six sympatric euploid chromosome complements (one diploid, one triploid, three tetraploids and one hexaploid), as confirmed by a FISH assay performed with an homologous 18S rDNA probe. All the worms with 2n = 50 chromosomes belonged to an already identified sibling species of T. tubifex, T. blanchardi. The six euploid sets were coherently arranged in the phylogeny, with each lineage grouping specimens with the same chromosome complement. CONCLUSIONS: These results are compatible with the hypothesis that multiple polyploidization events, possibly enhanced by parthenogenesis, may have driven the evolution of the T. tubifex species complex.

The world in a river? A preliminary analysis of the 16S rDNA variability of Tubifex species (Clitellata: Tubificidae) from the Lambro River.

Tubifex tubifex Müller, 1774 is a cosmopolitan freshwater tubificid widely used as a model in ecotoxicology, population dynamics and developmental biology. It is traditionally recognized as a polytypic species and in Lambro River (Milano, Northern Italy) it occurs in two of the three recognized forms, named "tubifex" and "blanchardi", alternatively considered as ecological forms or distinct species. To investigate the genetic differentiation of the populations occurring in the Lambro River we sequenced a fragment of the 16S rDNA mitochondrial gene. T. blanchardi, characterized by a low genetic diversity, was genetically segregated from the other sympatric T. tubifex. The ancestral state reconstruction was used to define the morphological traits that support its distinctness. On the contrary, the other T. tubifex from the Lambro community, although morphologically indistinguishable, revealed an astonishing degree of genetic variability, both between and within the three identified clades that proved to be genetically isolated. Using samples from the mixed Lambro River community and from other countries around the world we present an overview of the species complex' 16S rDNA variability. Our results show that the genetic variability did not sensibly increase widening the data set, suggesting that the Lambro River populations meet the species' worldwide genetic variability.

The spermatozoon of a 'living fossil': Tubiluchus troglodytes (Priapulida).

The spermatozoon of Tubiluchus troglodytes, the first priapulid formally described from the Mediterranean Sea has a head composed of an acrosome and a nucleus. The acrosome is divided in two branches coiled around the nucleus. The nucleus is basally columnar, but apically generates two rods helically coiled one around the other. The midpiece is formed by an axoneme with 27 accessory microtubules, surrounded by three mitochondria. An annulus separates the midpiece from the tail that contains a 9 + 2 axoneme surrounded by nine accessory microtubules. The spermatozoon of T. troglodytes is similar to that of the other two species known from the genus, and completely different from the 'primitive' one of the other priapulids. Since Tubiluchus is considered the most basal of the extant priapulids, and the only genus with an internal fertilization, it may be that in priapulids the external fertilization is a derived character.

Spermatozoa and sperm aggregates in the vestimentiferan Lamellibrachia luymesi compared with those of Riftia pachyptila (Polychaeta: Siboglinidae: Vestimentifera).

The spermatozoa and the sperm bundles of the vestimentiferans Riftia pachyptila and Lamellibrachia luymesi (Annelida: Siboglinidae) were studied using several microscopical techniques (transmission and scanning electron microscopy, and confocal microscopy) and compared with some other annelid sperm. The spermatozoa and sperm bundles of both species show a similar structure, but they differ in the dimensions of the components of individual cells and in the number of spermatozoa forming each sperm bundle. The spermatozoa of R. pachyptila and L. luymesi are filiform cells composed, in sequence, by an acrosome in the form of a thread-like helical vesicle, an elongated coiled nucleus surrounded by two helical mitochondria, and a long flagellum. In the spermatozoa of both species, the apical portion of the nucleus is completely devoid of chromatin and is delimited by a thickened nuclear envelope with a fibrillar appearance. Both species have sperm bundles that resemble buds, having a calyx-like portion formed by the helical heads, and a stalk-like portion formed by the tightly packed flagella. A parsimony analysis based on spermatozoal characters showed monophyly of the Siboglinidae and the Vestimentifera. We propose a new set of autapomorphies characterizing vestimentiferan spermatozoa. Our analysis suggests that spermatozoal characters are useful to the understanding of the phylogeny of the group.

The occurrence of strongylid nematodes in the epididymides of wood mice.

The recent discovery of a larval nematode in the epididymides of free-living wood mice (Apodemus sylvaticus) suggests a sexual transmission of these parasites. They have been placed within the bursate nematodes (order Strongylida) through 18S rDNA analysis, suggesting that they are undetermined metastrongyloid nematodes. The possibility that these parasites are transmitted sexually opens an intriguing field of research because sexually transmitted metazoan parasites are known to occur mainly in invertebrates, whereas in vertebrates sexually transmitted parasites are usually microparasites such as viruses, bacteria and protozoa.

Mixed reproductive strategy in Tubifex tubifex (Oligochaeta, Tubificidae)?

Despite the wide use of the tubificid oligochaete Tubifex tubifex in eco-toxicological studies, the reproductive strategy adopted by the species, that can reasonably be assumed to be the key to its ecological success, is still not well investigated. This study reports on breeding experiments analysed by allozyme markers (Pgi, Pgm, and Idh), accompanied by a study of the sperm production in the species, with the aim to: (1) clarify the type of uniparental reproduction adopted by the species, (2) test the hypothesis that uniparental reproduction is reversible, (3) investigate the occurrence of biparental reproduction in laboratory cultures. Studies of parent-offspring comparison at polymorphic allozyme loci showed parthenogenetic reproduction and maintenance of asexuality in the parthenogenetic individuals. A cross-breeding test performed with couples made up of randomly assorted individuals, whose genotype combinations were suitable for discriminating between sexual and asexual reproduction, failed to show biparental reproduction in laboratory cultures: T. tubifex always reproduced parthenogenetically. Unexpectedly, spermiogenetic analysis indicated that both kinds of sperm produced by the species (eusperm and parasperm) were differentiated in individuals raised either in cohort cultures or in isolation (first, second, and third parthenogenetic generations), with a similar pattern of sperm production correlated to the sexual stage. Interestingly, there was no avoidance of mating in any of the collective cultures analysed. Concomitance between parthenogenetic reproduction and a "normal" male functionality, which is typical of a sexually reproducing species, could be justified by a mixed reproductive strategy or a pseudogamy process occurring in T. tubifex. However, several aspects of the reproductive behaviour of the species deserve further investigation.

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.

PARSIMONY ANALYSIS OF THE PHYLOGENY OF SOME OLIGOCHAETA (ANNELIDA) USING SPERMATOZOAL ULTRASTRUCTURE.

Abstract- Quantitative and qualitative ultrastructural data from the spermatozoa of 11 oligochaete species, representing all orders (Tubificida, Lumbriculida, and Haplotaxida, yielded a single most parsimonious tree, using the Wagner-tree (PAUP) method of Swofford (1984). The Haplotaxida (Haplotaxis through Megascolecidae) form a discrete monophyletic group, with inclusion, however, of the reproductively exceptional tubificidan Phreodrilus. Bythonomus, representing the Lumbriculidae, the phylogenetic position of which has been so controversial, forms the plesiomorphic sister group of the Haplotaxida, a position which is supported from other evidence. The Tubificida, represented by two tubificids (Rhizodrilus and Limnodriloides) and by the enchytraeid Lumbricillus, are plesiomorphic relative to the lumbriculid+haplotaxid assemblage and lie at the base of the tree, but all three appear mutually paraphyletic. Monophyly of the Tubificida cannot, however, be considered conclusively refuted from the small sample used. Lumbricillus appears to have the most plesiomorphic sperm in the investigated oligochaetes. The Megascolecidae (Amynthas and Fletcherodrilus) form the highest affinity and most apomor-phic group. The implications of relative apomorphy of the Lumbriculida (Bythonomus) are profound. Branchiobdellids and leeches are generally regarded as sharing a common ancestry with lumbriculids and would, because of the revised position of the latter, cladistically constitute part of the Oligochaeta sensu lato.

Two sperm types in the spermatozeugmata of Tubifex tubifex (annelida, oligochaeta).

The spermatozeugmata (sperm bundles lacking a distinct wall) from the spermathecae of Tubifex tubifex are composed of two different zones: an internal axial cylinder containing conventional spermatozoa and an external cortex composed of modified spermatozoa, tightly packed together. The conventional spermatozoa conform to the classical clitellate scheme: very long and thin with a complex acrosome, a filiform nucleus, small mitochondria, and a flagellum with Y links and ß glycogen granules as accessory structures. The modified spermatozoa show "empty" acrosomes, degenerating nuclei, and tails which contain γ glycogen granules. The tails are helically wound around the spermatozeugma and are connected to each other by junctional complexes. The tips of the cortical tails are free and move with a metachronal wave. The presence of two sperm types in tubificids is discussed and a protective function for the modified cortical spermatozoa is proposed.