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1 Department of Biology, University of Milan, 20133 Milan, Italy
2 Department of Marine Biology, University of Vienna, A-1090 Vienna, Austria
To whom correspondence should be addressed. E-mail: roberto.marotta{at}unimi.it
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Abstract |
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Ideas about the phylogenetic affinities of Vestimentifera, and their close relatives, the Frenulata, have shifted remarkably since their discovery in 1969 (Webb, 1969; see Rouse, 2001). In recent years vestimentiferans have been considered as a derived group nested inside the polychaetes, within Siboglinidae (Rouse and Fauchald, 1997; McHugh, 1997, Halanych et al., 2001), which also includes the frenulate and moniliferan Pogonophora. The phylogenetic relationships within this group are still undecided, although much evidence supports the hypothesis that vestimentiferans are a monophyletic group (Kojima et al., 1993; Rouse, 2001; Schulze, 2002).
The reproductive biology of vestimentiferans continues to be the subject of considerable debate. Although apparent spawning events have been observed in Riftia pachyptila (Van Dover, 1994) and Lamellibrachia luymesi (Hilário et al., 2005), direct sperm transfer and internal fertilization seem to be the main means of fertilization among vestimentiferans, as suggested by the observation, in five species, of spermatozoa close to the eggs in diverticula of the oviduct (Hilário et al., 2005). These authors suggest (p. 21) that "the cloud of presumed gametes observed in these ‘spawning events’ could consist of zygotes, embryos, or even larvae if fertilization is internal."
In all vestimentiferan species investigated up to now (Van der Land and Nørrevang, 1977; Gardiner and Jones, 1985; reviews in Gardiner and Jones, 1993; Rouse, 1999), the spermatozoa are of the "modified type" (sensu Franzén, 1956; ent-aquasperm of Rouse and Jamieson, 1987).
Despite the contribution that the comparative study of sperm morphology has made to the understanding of fertilization biology and to the solution of phylogenetic problems in several metazoan groups (Jamieson, 1987), we have data on spermiogenesis from only 5 of the 25 known vestimentiferan species (Schulze, 2002): Riftia pachyptila (Gardiner and Jones, 1985; Jones and Gardiner, 1985) and Lamellibrachia luymesi (Van der Land and Nørrevang, 1977), Lamellibrachia barhami (Southward, 1991), Paraescarpia echinospica (Southward et al., 2002), and Ridgeia piscesae (Southward and Coates, 1989) (for the most recent review, see Rouse, 1999). Among the remaining siboglinids, sperm ultrastructure has been described only in the frenulate Siboglinum ekmani (Franzén, 1973) and confirmed in Siboglinum fiordicum by Southward (1993).
The spermatozoa of Siboglinidae share a similar ground plan: they are filiform cells formed, in sequence, by a helical acrosome, an elongated coiled nucleus surrounded by a variable number of helical mitochondria, and by a long and simple flagellum (Rouse, 1999). In spite of this uniformity in sperm architecture, frenulates and vestimentiferans differ in the way they transfer spermatozoa to the females. Internal fertilization, via characteristic spermatophores, appears to be the norm in frenulates (Ivanov, 1963; Bakke, 1990). In contrast, in vestimentiferans, with the exceptions of Ridgeia piscesae (Southward and Coates, 1989) and Tevnia jerichonana (Southward, 1999), spermatozoa are released as bundles (Van der Land and Nørrevang, 1977; Webb, 1977; Gardiner and Jones, 1985; Macdonald et al., 2002; Hilário et al., 2005), which are capable of swimming and eventually disintegrate in seawater (Cary et al., 1989). In contrast, sperm masses have been recorded in vivo adhering to various parts of the body of female Ridgeia piscesae (Macdonald et al., 2002). Masses of spermatozoa no longer grouped in bundles were in fact found in the spermathecae of Riftia pachyptila, Tevnia jerichonana, Lamellibrachia luymesi, and Seepiophila jonesi (Hilário et al., 2005).
The present study provides the first detailed description of the ultrastructure of mature spermatozoa and sperm bundles of Lamellibrachia luymesi and a comparison with that of Riftia pachyptila. Our aim is, first, to resolve the fine structure of both sperm cells and aggregates, using different microscopical techniques—transmission electron microscopy (TEM), scanning electron microscopy (SEM), and immunofluorescence—and, second, to provide data on sperm ultrastructure for phylogenetic studies within this taxon.
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Materials and Methods |
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Microscopical techniques
For transmission electron microscopy (TEM), the specimens were washed in 0.1 M cacodylate buffer (pH = 7.4), postfixed in 1% osmium tetroxide in the same buffer for 2 h, washed in distilled water, stained en block overnight in 2% aqueous uranyl acetate, dehydrated in a graded ethanol series, and embedded in EPON resin. Sections were cut with a Reichert Ultracut E microtome and observed with a JEOL 100SX electron microscope.
For scanning electron microscopy (SEM), the sperm cells, once transferred onto coverslips coated by poly-l-lysine, were washed in 0.1 M cacodylate buffer (pH = 7.4). Adhesion of spermatozoa to the poly-l-lysine coat permitted us to wash and handle the coverslips without loss of material. Graded dehydration with ethanol was followed by replacement with hexamethyldisilazane (Melone and Ferraguti, 1994). The coverslips processed in this way were glued with silver paint to SEM stubs, coated with gold, and observed with a Leo 1430 scanning electron microscope.
For immunocytochemical staining, the specimens, once transferred onto slides coated with poly-l-lysine, were incubated in a 1% solution of sodium borohydride in phosphate-buffered saline (PBS; 110 mM, pH 7.4) for 5 min to eliminate the fixative-induced autofluorescence. Then the slides were washed in PBS for 1 h, incubated with a solution of 0.25% Triton X-100 and 0.1% Tween in PBS for 30 min, followed by an incubation with 4% bovine serum albumin (in PBS) for 20 min and an incubation with the mouse monoclonal antibodies anti 
-tubulin (Sigma, 1:200 in PBS) at 4°C overnight. Afterward, the slides were rinsed in PBS, incubated with anti-mouse antibodies conjugated to FITC (Sigma; 1:50 in PBS) for 3 h, washed in PBS, and finally incubated with a mixture of DAPI (4',6-diamidino-2-phenylindole, Sigma, 1:500 in PBS) and phalloidin conjugated to TRITC (Sigma; 1:200 in PBS) for 30 min. The coverslip was mounted with DABCO (Aldrich). The fluorescence images were acquired with a Leica TCS-NT confocal microscope.
Phylogenetic analysis
Thirteen spermatozoal characters, all treated as unordered, were considered for phylogenetic analysis (see Fig. 4B). The ingroup comprised four siboglinid species representing one genus of frenulate and three genera of vestimentiferans, as shown in Table 1. To root the tree, the ent-aquasperm of the sabellid Fabriciola liguronis (Rouse, 1993) and the ect-aquasperm of the sabellarid Phragmatopoma lapidosa (Eckelbarger, 1984) were chosen. Phylogenetic analyses were performed using PAUP (Phylogenetic Analysis Using Parsimony), version 4.0b10 for 32-bit Microsoft Windows (Swofford, 2002). The exact branch-and-bound search algorithm was selected, using default settings. AutoDecay (Eriksson, 2001), together with PAUP (settings as above), was used to calculate Bremer supports (Bremer, 1988).
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Acrosome.
The acrosome is formed by a thread-like acrosome vesicle helically coiled around an elongated, cylindrical, subacrosomal chamber that is devoid of any structured subacrosomal material (Fig. 2C, H, Q). The acrosome of R. pachyptila, up to 6 µm long, has a diameter of about 0.5 µm in its central region, narrowing to about 0.2 µm at both ends (pitch 1.6 µm, 4 gyres). Its major axis forms an angle with respect to the nuclear major axis (Fig. 1H). The basal boundary of each acrosomal vesicle gyre has ribbon-like thickenings (Fig. 2L). The acrosome of L. luymesi forms a smaller angle with the nucleus; is about 4 µm long; and has a basal diameter of about 0.5 µm, narrowing to 0.3 µm at the apex (Fig. 1C). Anteriorly, the pitch of the acrosome vesicle is 0.6 µm, increasing to 1 µm posteriorly, with as many as six gyres. Ribbon-like thickenings are present in the central region of each gyre (Fig. 2G). Immunofluorescence staining of sperm aggregates in R. pachyptila reveals that the unstructured subacrosomal material is actin-negative (Fig. 3B, C).
Nucleus and mitochondria.
The nucleus is about 26 µm long in both species and tapers from its concave base to the apex. It consists of two portions: a posterior main portion, more than 23 µm long, filled with completely condensed chromatin and surrounded by a single mitochondrial helix (Fig. 2B, U); and an apical, shorter one (about 3 µm long) (Fig. 2A, T) that is completely devoid of chromatin, as shown by immunocytochemistry (Fig. 3A, D), and shows a thickening of the nuclear envelope with a fibrillar appearance (Fig. 2A, inset). The nuclear envelope starts to thicken gradually, beginning from the apex of the region with chromatin of the nucleus. The two portions of the nucleus have a different and complex shape: the main portion may be visualized as a cylinder in which one of the two parallel helical grooves is excavated (Fig. 2M). One of the two parallel helical grooves hosts two mitochondria, arranged in sequence and superimposed for a short distance (Fig. 2F, S). The other groove has only amorphous cytoplasm. The short apical portion is corkscrew-shaped because of thickenings of the nuclear envelope, and it terminates apically in an electron-dense button, connecting the nucleus with the acrosome (Fig. 2D, R). The pitch of the nuclear helix increases towards the apex, and in parallel, the width of each gyre narrows.
Centriolar region and flagellum.
The axoneme, with a simple 9 x 2 + 2 pattern, originates from an elongated basal body in which no microtubule is visible. A prominent anchoring apparatus surrounds the basal body and a short tract of the axoneme (Fig. 2B, U). This apparatus (Fig. 2E, N) is formed by two systems of pericentriolar processes, originating respectively from the apical and the middle portion of the basal body and connected by an electron-dense subplasmalemmal necklace of small particles, each with a diameter of about 18 nm. The pericentriolar system originating from the middle portion of the basal body branches out in a second order of arms that diverge towards the plasma membrane and end in an electron-dense subplasmalemmal structure, the annulus sensu Baccetti and Afzelius (1976).
The plasma membrane is separated from the axoneme around its basal portion, then gradually becomes adherent to it for the rest of its length (Fig. 2J, O, P): in this portion with cytoplasm of the flagellum, our immunofluorescence staining reveals the presence of actin (Fig. 3B, C). The flagellum terminates in a long endpiece showing a progressive reduction of the axonemal doublets (Fig. 2K).
Sperm bundles of Riftia pachyptila and Lamellibrachia luymesi
The sperm bundles of both R. pachyptila and L. luymesi show the same fine-structural organization; they differ mainly in the number of spermatozoa, with an average of 350 and 60, respectively. In both species the spermatozoa are grouped in characteristic bud-like sperm bundles (Fig. 1B, G). Each bundle is formed by two main regions: (a) an apical portion formed by the helical heads (Fig. 1C, H), the centriolar regions, and the portions with cytoplasm of the flagella (Fig. 1E, J), tightly packed together to form a calyx-like structure; and (b) an elongated main portion formed by tightly packed flagella to form a stalk-like structure. In addition, the sperm bundles of R. pachyptila also have a terminal region, about 12.5 µm long, formed by the endpieces of the flagella, which are tightly packed to form a peduncle-like structure (Fig. 1I). Single transversal rows of microfilaments encircle the apical portion of the sperm bundles, connecting the apical nuclear portion and the centriolar regions of each spermatozoon (Fig. 1H, J); a web of filaments surrounds the peduncle portion of the sperm bundles of R. pachyptila (Fig. 1I).
Parsimony analysis
The parsimony analysis of the 13 selected spermatozoal characters resulted in three most parsimonious trees (MPTs), with 19 steps and a consistency index (CI) of 1. In all the MPTs obtained, the two outgroups, Fabriciola liguronis and Phragmatopoma lapidosa, group by themselves at the base of the tree. The examined ingroup species form a strongly supported monophyletic Siboglinidae (Bremer support value = 5). The frenulate Siboglinum ekmani is the sister group of a well-supported vestimentiferan clade (Bremer support value = 3), comprising Lamellibrachia luymesi, Ridgeia piscesae, and Riftia pachyptila. A basal polytomy does not allow any resolution among the vestimentiferan taxa considered. The phylogram of one of the MPTs obtained with the same topology as the strict consensus tree is shown in Figure 4A.
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Discussion |
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In R. pachyptila and L. luymesi—as well as in Ridgeia piscesae, the other vestimentiferan in which sperm ultrastructure is partially known (Southward and Coates, 1989)—the acrosome is formed by a thread-like vesicle helically coiled around an elongated subacrosomal chamber. In a study of the spermatozoa in spawned sperm masses of Ridgeia piscesae, Southward and Coates (1989) showed that the acrosome slides down over the nucleus to enclose its apical portion after the emission of the sperm, because the acrosome does enclose the apical portion of the nucleus at this stage. Gardiner and Jones (1993; p. 438) commented that "it appears reasonable to suggest a similar realignment of the acrosome occurs in the final maturation of spermatozoa in Riftia pachyptila." We have not observed this process of maturation in R. pachyptila and L. luymesi. In the sperm bundles of both species, the acrosome is situated at the top of the nuclear apex.
A helical nucleus showing an apical region with a lower electron density is another common feature of all vestimentiferans (Rouse, 1999). Gardiner and Jones (1985), studying the spermiogenesis in R. pachyptila, concluded that the hollow tube following the electron-dense portion of the nucleus was in fact part of the nucleus, even if it showed a superficial similarity to the helical acrosome tube that characterizes clitellate spermatozoa (Ferraguti, 1999). In this region, the nuclear envelope is formed by a thick layer of microfilaments surrounding a clear space in which chromatin may be absent (Gardiner and Jones, 1985). Our immunofluorescence staining on mature sperm and sperm aggregates in both R. pachyptila and L. luymesi reveals that, in fact, DNA is present only in the main basal region of the nucleus, whereas the apical portion is DNA-free, thus confirming the hypothesis of Gardiner and Jones (1985). These authors also suggested an active role for the filaments that form the nuclear wall during fertilization. If composed of actin, their elongation after the acrosomal reaction could, for example, facilitate the entry of the nucleus into the egg, in analogy with the subacrosomal material of other species. However, our immunofluorescent stainings did not reveal actin in this area; thus the function of the nuclear filaments remains obscure.
Functional aspects of the spermatozoa in vestimentiferans
The functional relationships between various sperm structures and aspects of reproductive mechanisms in polychaetes have been the subject of considerable speculation (Rouse and McHugh, 1994). A modified sperm morphology has been correlated with some form of indirect sperm transfer (Franzén, 1956), in particular to increase the penetration capacity through viscous media, to allow optimal packaging of spermatozoa in spermatophores or spermathecae (Westheide, 1984; Rouse, 1992), and to support the sperm propagation in the female tracts (Afzelius, 1971).
The vestimentiferan spermatozoa share the following features:
Elongate acrosome.
The length of the acrosome among vestimentiferans ranges from around 4 µm in L. luymesi to 8 µm in Ridgeia piscesae. The acrosomal length may be attributed to features of the eggs, since a positive correlation between acrosome length and thickness of the vitelline envelope has been demonstrated in other groups (Franzén, 1983; Jamieson et al., 1983).
Elongate nucleus.
The extreme nuclear elongation (26 µm) in Riftia pachyptila and L. luymesi parallels that found in other vestimentiferans such as Ridgeia piscesae (30–40 µm; Southward and Coates, 1989), Lamellibrachia columna (30 µm; Southward, 1991), Paraescarpia echinospica (27 µm; Southward et al., 2002) and is exceeded among polychaetes studied to date only by the sperm nucleus of Micromaldane sp. (average nucleus length 25 µm; Rouse, 1992) and Streblospio benedicti (average nucleus length 47.7 µm; Rice, 1981). Sperm elongation among polychaetes appears to be strongly correlated with copulation, storage in spermathecae or spermatophores, and large egg size (Jamieson and Rouse, 1989; Franzén, 1983). The presence of small, yolky eggs, with a diameter of about 100 µm in vestimentiferans, suggests that sperm elongation is due to sperm storage in spermathecae rather than to egg size.
Absence of a true midpiece.
The absence of a true midpiece is very rare among polychaetes. A midpiece is absent in the ent-aquasperm (sensu Jamieson and Rouse, 1989) of the terebellids Ramex californiensis and Nicolea zostericola (Rouse and McHugh, 1994), and of the capitellid Micromaldane sp. (Rouse, 1992). In these species the mitochondria occupy longitudinal grooves along the posterior region of the nucleus, as is also the case in the introsperm of Nerilla antennata (Franzén and Sensenbaugh, 1984). The absence of a true midpiece evolved convergently in different metazoan phyla such as gastrotrichs (Marotta et al., 2005) or chordates (Fukomoto, 1981). The absence of a midpiece in conjunction with the presence of mitochondria wound around the nucleus has been considered to be an adaptation for locomotion, allowing the sperm to swim more freely (Fukomoto, 1981).
Flagellum.
The length of the flagellum, like that of the acrosome, varies significantly among vestimentiferan spermatozoa, ranging from about 70 µm in L. luymesi to around 110 µm in Riftia pachyptila. In species with internal fertilization, the increase in length of the flagellum is considered to be an adaptation to increase the propulsive activity of the spermatozoa along the female genital ducts. The different length of the flagella in the two species could be correlated to differences in the length of their oviducts.
Although the fertilization biology among vestimentiferans is still unclear, their spermatozoa have been tentatively classified as ent-aquasperm sensu Jamieson and Rouse (1989) (Rouse, 1999). Indeed internal fertilization has been inferred from the presence of seminal receptacles at the posterior end of the oviduct in several vestimentiferan species (Hilário et al., 2005); and, on the basis of observations of spawning in the field (Van Dover, 1994), it seems likely that sperm bundles are released by males into the water column, from which they are collected by the females or find their way into the female gonopores (Macdonald et al., 2002).
Morphology of sperm aggregates in vestimentiferans
Vestimentiferans, unlike frenulates, do not have spermatophores. The difference in the number of mature spermatozoa present in the sperm bundles of both Riftia pachyptila and Lamellibrachia luymesi may be related to the size of their spermathecae, which range from a diameter of about 600 µm in L. luymesi to more than 1300 µm in Riftia pachyptila (Hilário et al., 2005). The sperm aggregates of Ridgeia piscesae are spermatozeugmata embedded in a sticky matrix, and they differ strongly from those in both Riftia pachyptila and L. luymesi (Southward and Coates, 1989). The filamentous structures observed in Riftia pachyptila sperm bundles may play some role in their active transfer from males to females. It has been hypothesized that in frenulate pogonophorans the spermatophoral filaments play an important role in active transfer, flotation, and adhesion of released spermatophores (Flügel, 1977).
Sperm morphologies and biology of fertilization in deep-sea polychaetes
Polychaetes living at hydrothermal vents and methane seeps have heterogeneous reproductive methods (Van Dover, 2000) accompanied by different sperm types. The ampharetid hydrothermal vent polychaete Amphisamytha galapagensis (McHugh and Tunnicliffe, 1994) and the hesionid Hesiocaeca methanicola from the cold seeps of the Gulf of Mexico (Eckelbarger et al., 2001) are broadcast spawners, and both have ect-aquasperms. The orbinid Methanoaricia endrobranchiata, although co-occurring in the same habitat as H. methanicola, has modified spermatozoa with long nuclei and a greatly elongated and curved acrosome; they are tentatively classified as ent-aquasperm (Eckelbarger and Young, 2002). The spermatozoa of the alvinellids Paralvinella pandorae and P. grasslei from hydrothermal vents are highly autapomorphic: those of P. grasslei have been tentatively classified as introsperm, since pseudocopulation and internal fertilization has been hypothesized for this species (Zal et al., 1994). In contrast, the unique spermatozoa of P. pandorae, tentatively classified as ent-aquasperm, are probably transferred to the females in bundles, and fertilization most likely takes place in the female’s tube or in a glutinous mass outside the tube (McHugh, 1995).
It has been suggested that the reproductive strategies of deep-sea hydrothermal vent organisms have evolved in relation to the highly varying hydrodynamic, chemical, and thermic conditions (Zal et al., 1994). For example, indirect sperm transfer and modified sperm morphology could have evolved to limit the exposure of gametes to high sulfide levels (Eckelbarger and Young, 2002). The variability of sperm types found among deep-sea polychaetes may suggest that sperm morphology and fertilization biology are more correlated with phylogenetic history than with the selective pressures imposed by this extreme habitat (McHugh, 1995). The similarity of sperm ultrastructure and fertilization biology between the deep-sea vestimentiferans and their close relatives, the frenulates, some of which occur in shallow water, seems to support this hypothesis.
Phylogenetic analysis
Although our study included only a very small fraction of the biodiversity in Siboglinidae, our analysis reveals that sperm ultrastructure can contribute to the understanding of the phylogeny of the group. The results of this phylogenetic analysis are congruent with those coming from general morphology and molecules. Sperm ultrastructure supports the monophyly of Siboglinidae, as indicated by morphological (Rouse, 2001) and molecular data (Winnepenninckx et al., 1995; Black et al., 1997; McHugh, 1997). As shown in Figure 4A, the presence of a helical acrosome (#1) at the top of an elongated nucleus (more than 20 µm long, #5), of mitochondria helically coiled around the main nuclear portion (#7), and of a complex anchoring apparatus (#10) are autapomorphies for the siboglinid spermatozoon.
The nested position of vestimentiferans inside siboglinids (Black et al., 1997) and the monophyly of vestimentiferans based on morphology (Schulze, 2002) and molecules (Black et al., 1997; Halanych et al., 2001) are paralleled by our sperm-based phylogenetic analysis. Sperm autapomorphies for vestimentiferans are a thread-like acrosome vesicle (#2), a complex helical nucleus (#4), the absence of chromatin at the nuclear apex (#6), two mitochondria (#8), a single centriole (#9), and spermatozoa aggregated in sperm bundles (#12) (see Fig. 1A).
The homogeneity in sperm ultrastructure among vestimentiferans, as confirmed by the high support for the vestimentiferan clade, matches with the identity of amino acid sequences in the cytochrome oxidase c subunit I of several vestimentiferan species (Kojima et al., 1997) and supports the idea that extant vestimentiferans constitute a recent evolutionary radiation (Black et al., 1997).
The proposed close relationship between the recently discovered siboglinid genus Osedax and vestimentiferans, based on molecular data (Rouse et al., 2004), seems to be supported by sperm ultrastructure. Indeed, the single micrograph of the longitudinal section of the spermatozoon of Osedax rubiplumus (Rouse et al., 2004) shows a vestimentiferan-like nucleus, formed by two parallel helical grooves hosting mitochondria.
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Acknowledgments |
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. J. Mol. Evol. 37: 66–70.[ISI][Medline]
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