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Comparative ultrastructural study of the cuticle and spermatozoa in Propappus volki Michaelsen, 1916 (Annelida: Clitellata)
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Zoologischer Anzeiger 247 (2008) 123–132 www.elsevier.de/jcz
Comparative ultrastructural study of the cuticle and spermatozoa in Propappus volki Michaelsen, 1916 (Annelida: Clitellata) Lena M. Gustavssona,, Marco Ferragutib, Roberto Marottab a
Department of Invertebrate Zoology, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden Dipartimento di Biologia, Universita` degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
b
Received 3 May 2007; received in revised form 27 August 2007; accepted 7 September 2007 Corresponding editor: M. V. Sørensen
Abstract 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. r 2007 Elsevier GmbH. All rights reserved. Keywords: Propappidae; Enchytraeidae; Ultrastructure; Cuticle; Spermatozoa; Phylogeny.
1. Introduction In 1905, Michaelsen described a new oligochaete species, Propappus glandulosus, from Lake Baikal Corresponding author.
E-mail address: [email protected] (L.M. Gustavsson). 0044-5231/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2007.09.003
(Michaelsen 1905). A new genus was established for this species, which was placed in Enchytraeidae. Although the new species differed substantially in morphology from the other members of the family, Propappus and the remaining enchytraeids were believed to form a monophyletic group in which Propappus represented an early branch within the family. Later,
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Michaelsen (1916) described a second species, Propappus volki, from Elbe River, near Hamburg. It was distinguished from P. glandulosus by its prostomial proboscis and the sperm funnel morphology. A third species, Propappus arhyncotus, was described by Sokolskaya (1972) from the Kamchatka peninsula. The placement of Propappus within Enchytraeidae has been questioned first by Nielsen and Christensen (1959), and later by Coates (1986) who erected a new family, Propappidae, for the three species. Morphologically, the members of Propappus differ in several characters from other enchytraeids. Specimens of Propappus have: (1) sigmoid, nodulate, bifid chaetae, commonly three per bundle, (2) large epidermal glands posterior to each chaetal bundle, (3) spermathecal pores posterior to septum 3/4, (4) glandular part of vasa deferentia located posterior to septum 11/12 and, (5) one pair of ovaries in segment XIII with female funnels located on septum 13/14. Monophyly of the family has only been tested once, by Coates (1987) who included, in a morphology-based phylogenetic analysis, two of the three Propappus species (P. volki and P. glandulosus) together with several haplotaxids, one enchytraeid, and a few megadriles. Although the two representatives of Propappus appeared as sister taxa in the analysis, the monophyly of Propappidae was not strongly supported, merely being based on the ‘‘absence’’ of several characters rather than presence, except for the position and shape of the spermathecae. Propappus turned out to be closely related to Enchytraeidae and Metataxis (Haplotaxidae). One of the characters considered, the presence of an epidermal gland associated with each chaetal bundle with an opening through the body wall immediately posterior to the bundle, appeared as an apomorphy for Propappidae. The possible homology of this structure to a similar one present in, e.g. Haplotaxis, Achaeta, and in some megadriles has previously been proposed (Michaelsen 1923; Stephenson 1930; Nielsen and Christensen 1959; Coates 1986) but further studies are required to draw any conclusions. In recent years, P. volki has been included in two phylogenetic analyses based on DNA data. The first one (Nylander et al. 1999), with the purpose of testing the monophyly of the gutless genera Olavius and Inanidrilus, did not reveal any information about the phylogenetic position of Propappus; all taxa except the gutless group were too weakly supported. In the second paper, a phylogenetic analysis of Clitellata based on 18S rRNA gene (Erse´us and Ka¨llersjo¨ 2004), Propappus groups together with Phreodrilidae, Tubificidae, and Haplotaxidae. The enchytraeids form a monophyletic group with Crassiclitellata (sensu Jamieson 1988). This DNA-based phylogeny thus supports Coates’ (1986) removal of Propappidae from Enchytraeidae.
Apart from the characters commonly used in taxonomy (see Coates 1986, 1987; Timm 1994), there are almost no data available on the fine morphology of Propappus. With the aim of providing new additional characters that may clarify the phylogenetic position of the family, we describe the ultrastructure of the cuticle and spermatozoa of P. volki.
2. Material and methods Several individuals of P. volki were collected by C. Pavelesku in May 2004 in the Stream Valea Firii (Romania), at a height of 1100 m above sea level. Additional specimens were collected by the first author in May 2005 in River Bra¨knea( n, Blekinge province, (south-eastern part of Sweden). The exact location is about 4 km south of Bra¨kne-Hoby, 0–30 m downstream the bridge crossing the river. In this part of the river the current is moderate to strong with a varying depth from 0.2 to 1 m. P. volki was found downstream of boulders in limited areas where the sediment was sorted to a specific size. The kick-method (Frost et al. 1971) was used to get material into a large bag net with extremely small mesh size. Each sample was then sorted under a dissecting microscope and each individual was identified live, compressed in a drop of water under a cover-slip, using a light microscope. All specimens were fixed in PAFG (Ermak and Eakin 1976) for about a week and then rinsed overnight in cacodylate buffer. They were then postfixed in 1% osmium tetroxide dissolved in the same buffer for 1 h, washed in buffer, dehydrated in a graded ethanol series, and then embedded via propylene oxide in Epon 812. Ultrathin and semithin sections were obtained with a Leica Ultracut or a Reichert Ultracut E. Semithin sections were stained with 1% toluidine blue and studied using a light microscope. Ultrathin sections were collected on copper grids, stained with lead citrate and uranyl acetate after Daddow’s (1983) methodology, and observed with a Jeol 100 SX or a LEO 912 AB electron microscope at 80 kV. For epidermal and cuticular studies, cross-sections were taken from different regions of the worms: proboscis, clitellum and the posterior region. To study sperm ultrastructure, the spermatozoa were examined at the male funnels (i.e. the entrances to the deferent ducts).
3. Results The studied specimens of P. volki are 4.5–6.0 mm long and consist of 35–41 segments. They have bifid chaetae, normally three per bundle, but occasionally there are one or two more chaetae per bundle. In mature
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specimens the clitellum begins in the middle of segment XII, and covers segments XIII and XIV. The prostomium is furnished with a proboscis, about 80 mm long and with a diameter of 20 mm in fixed specimens. Slightly longer proboscises are observed in living specimens.
3.1. Cuticle morphology The body wall of P. volki consists of five layers. An outer, non-cellular cuticle overlies the monolayered epidermis (Fig. 1A). Beneath the epidermis there are two layers of muscles; an outer circular layer and an inner longitudinal layer. A thin peritoneal epithelium covers the inner muscle layer and separates it from the coelom. Epidermal sense organs (multiciliated cells with short cilia and sensory cells with one to three long cilia) are sparsely distributed in the clitellar and post-clitellar regions of the worm. The body wall in the protruding proboscis differs and consists of two layers only; the epidermis and cuticle (Fig. 1D). The epidermis in this region is dominated by multiciliated sensory cells covered by a relatively thick cuticle. No muscle layers or coelom were found beneath the epidermal layer, instead the internal space is filled with a loose tissue. The cuticle is composed of several structures; a fibre zone, an epicuticle, epicuticular projections, and microvilli (Fig. 1C). Minor differences in the morphology of these structures occur between different regions of the worm (proboscis, clitellum, and post-clitellar region). A brief summary of the differences is presented in Table 1. 3.1.1. Fibre zone The proximal part of the cuticle has fibres embedded in a homogeneous matrix (Figs. 1B and C). The fibres are arranged in layers of parallel fibres with the same orientation of the fibres in all layers. In a cross-section of the worm, six to eight layers can be seen. Each fibre is composed of thinner, intertwined fibrils. The morphology of the fibre zone differs between the studied regions of the worm, and only in the post-clitellar region are the fibres arranged in layers. The proboscis region lacks compound fibres, instead thinner and shorter fibrils run in all directions (Fig. 1E). In the clitellar region, compound fibres are oriented in the same direction, but they are not arranged into layers (Fig. 1F). 3.1.2. Epicuticle The epicuticle is relatively thick, about one fourth of the total thickness of the cuticle (Figs. 1E and F). It is a continuation of the matrix surrounding the fibres, but devoid of any fibres or fibrils. The matrix, however, consists of smaller filaments (or microfibrils) and granules. The outer surface of the epicuticle has no limiting membrane. Three layers of different electron
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density are distinguished depending on how closely the filaments are packed together (Fig. 1F). The innermost layer is about half the thickness of the epicuticle, with loosely arranged filaments in all directions. The two outer layers have more tightly packed filaments with alignments parallel to the surface. The outermost layer is thinnest. The epicuticle has the same appearance in all regions of the worm studied. 3.1.3. Epicuticular projections Projections cover the outer surface of the epicuticle. They are membrane-bound bodies with their major axis perpendicular to the surface, about two-three times longer than wide. The shape of the projections is weakly pyramidal with the base touching the epicuticle (Fig. 1F). Thin filaments radiate from the double membrane enclosing the epicuticular projection. The filaments pointing towards the epicuticle connect to the filaments present in it, and probably keep the epicuticular projections in position, while those pointing outwards probably stabilize the mucus layer covering the worm. Apart from an electron-dense, crescentshaped structure visible in the outer part of each epicuticular projection, the internal substance is homogeneous. The epicuticular projections in the proboscis are longer and narrower than in other regions of the worm (Fig. 1E, Table 1). Their density is, however, about the same which makes them look more sparsely distributed. No other morphological differences were found between epicuticular projections in different regions. 3.1.4. Microvilli Microvilli, processes from the epidermal cells, penetrate the cuticle and reach the free outer surface of the epicuticle (Figs. 1A, C and E). The diameter of the microvilli varies between 50 and 140 nm, measured at the same distance from the epidermis. Most microvilli, nevertheless, have the same diameter as the epicuticular projections, and they end amongst them. The wider microvilli continue far outside the epicuticular projections, but look otherwise identical. No microvilli pinching off epicuticular projections were observed. The cell matrix inside the microvilli is homogeneous, and looks similar to the substance present inside each epicuticular projection. The density of the microvilli varies; in some areas there are almost as many microvilli as there are epicuticular projections, while in other areas they are lacking. Sometimes microvilli are intertwined with the cilia arising from the sensory cells, especially in the proboscis region (Fig. 1D). 3.1.5. Knobs Knobs are extensions from the epidermal cells into the cuticle. They are short, hemisphere-shaped bulges that reach into the lower part of the fibre zone (Fig. 1E).
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Table 1.
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Characters of the cuticle
Character
Proboscis
Clitellar region
Postclitellar region
Cuticle: thickness (nm) Fibre zone: thickness (nm) Fibre zone: fibre diameter (nm) Fibre zone: organization Epicuticle: thickness (nm) Epicuticle: layers Epicuticular projections: length (nm) Epicuticular projections: diameter (nm) Epicuticular projections: number per mm Microvilli: diameter (nm) Microvilli: numbers per mm
660 330 No fibres – 110 3 135 40 12 66–88 Varies
440 200 14–40 No layers 110 3 125 55 11 70–139 Varies
570 300 11–33 6–8 Layers 130 3 100 55 12 54–130 Varies
Thin filaments radiate from the hemisphere into the matrix of the cuticle. An electron-dense area is visible in the outer part of each bulge. The knobs are sparsely distributed in all regions of the worm.
3.2. Sperm morphology The following description refers to the mature spermatozoa as observed at the sperm funnels. The spermatozoon of P. volki is a filiform cell, formed, in sequence, by an acrosome, an elongated nucleus, a long mitochondrial midpiece, and a flagellum. 3.2.1. Acrosome The acrosome is an elongated cylinder (average length 10.171.3 mm, N ¼ 6) tapering from its basis to the apex, and slightly bent to one side (Fig. 2C and K). It is formed by a short and thick acrosomal tube (average length 2.770.5 mm, N ¼ 8) proximally ending in an internal thickening (limen sensu Jamieson 1978) (Fig. 2A and C). A long, cup-shaped acrosome vesicle occupies almost the entire length of the acrosome, and is completely external to the tube (Fig. 2C, E, and F). A thin sheet of electron-dense material fills the space between the basal portion of the acrosome vesicle and the plasma membrane; the distal part of the acrosome
vesicle lies close to the cell membrane (Fig. 2C). An elongated acrosome rod (average length 8.1371.32 mm, N ¼ 6), protruding for nearly all its length from the distal margin of the acrosomal tube, ends apically inside the invagination of the acrosome vesicle, close to the vesicle tip (Fig. 2C). A thin and short secondary tube starts from the proximal margin of the acrosomal vesicle, and ends close to the proximal extremity of the acrosome rod, in correspondence of a wider, cylindrical body, about as long as wide (Fig. 2C and K), resembling the basal node (sensu Jamieson 1978) diagnostic of the sperm of megascolecid earthworms. A small empty space, the basal chamber, is present at the base of the acrosome (Fig. 2C and K). A thin, dome-shaped structure, the nuclear pad (sensu Jamieson 1982), is interposed between the apical portion of the nucleus and the acrosome (Fig. 2C and K). 3.2.2. Nucleus The nucleus is a long, filiform structure of fully condensed chromatin, apically corkscrew-shaped and basally nearly straight (Fig. 2A and B). It decreases in diameter from its concave basis (average diameter 0.3370.03 mm, N ¼ 10) to the convex apex (average diameter 0.170.01 mm, N ¼ 10).
Fig. 1. TEM-micrographs showing the morphology of the cuticle of Propappus volki. (A) Cross-section of the body wall of a postclitellar region showing longitudinal (lm) and circular muscle layers (cm). The monolayered epidermis (e) is covered with cuticle (c) with several microvilli reaching through from the epidermis. (B) Cross-section of the clitellar region. A portion of a gland cell (gc) with its granular inclusions (gi) is visible. Thin fibrils (fz) are aligned in the cuticle. Pyramidal epicuticular projections (ep) cover the surface of the epicuticle. (C) Cross-section of a post-clitellar region. Cuticular fibres (fz) are arranged in layers. The epicuticle is covered with weakly pyramidal projections (ep). (D) Longitudinal section of the proboscis with cuticle (c) visible on both sides. Sensory cells (sc) with cilia (ci) penetrating the cuticle occur in the epidermal layer. Several microvilli reach through the cuticle. No muscle layers or coelom are found in the proboscis. (E) Longitudinal section of the proboscis. Thin fibrils lay in all directions in the cuticle (c). One microvillus (mv) stretches through the cuticle from the epidermal cell (e). The outer surface of the cuticle is covered with long and narrow epicuticular projections (ep). Knobs (k) reach from the epidermis into the proximal part of the fibre zone. (F) Details of the clitellar cuticle showing the layered epicuticle (ec), the fiber zone (fz) and the internal structure of the epicuticular projections (ep). Scale bar is 1.5 mm in (A); 0.38 mm in (B); 0.48 mm in (C); 1.9 mm in (D); 0.24 mm in (E) and 0.15 mm in (F).
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3.2.3. Midpiece The midpiece is formed by five twisted, elongated mitochondria (average length 1.670.13 mm, N ¼ 6) (Fig. 2D and G) each with the shape of a cylindrical sector around a central axis filled with electron-dense material. 3.2.4. Flagellum The flagellum contains a simple 9+2 axoneme surrounded by the plasma membrane at regular distance (average distance 0.0470.006 mm, N ¼ 14) (Fig. 2I and J). The basal body area is deeply modified by the presence, in its interior, of a long basal cylinder (sensu Ferraguti 1984a) nearly reaching the midpiece (Fig. 2H and L. A prominent central sheath, sensu Ferraguti (1984b), is visible as a modification of the central axonemal apparatus (Fig. 2I and J). Traces of putative glycogen granules are visible in cross-section, external to the axonemal doublets. Each doublet is connected to the plasma membrane by faint, electron-dense links (Fig. 2I and J). The flagellum ends abruptly in a narrow endpiece, by progressive reduction of the axonemal microtubule number.
4. Discussion Information from morphology and molecular sequences are used in phylogenetic analyses separately or in combination. The morphology and DNA information is sometimes conflicting, as it is the case in the Propappidae. Based on morphological characters, Propappus has been positioned within Enchytraeidae (Michaelsen 1916), or as a sister group to certain members of Enchytraeidae and Haplotaxidae (Coates 1987). In a parsimony analysis among clitellates, based on the entire 18S rRNA gene, Propappus grouped far from enchytraeids, forming a monophyletic group together with Tubificidae, Phreodrilidae, and Haplotaxidae (Erse´us and Ka¨llersjo¨ 2004). In a recent analysis based on one mitochondrial and three nuclear genes, P. volki branches off as sister taxon to Haplotaxis, with
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an enchytraeid being the sister group to them, albeit with weak support (Rousset et al. 2007). The morphological information about Propappus is limited to the species descriptions and further morphological data can be useful to clarify its phylogenetic position. Sperm ultrastructure has been proved to be informative as a phylogenetic marker, at least in groups such as clitellates with uniform reproductive mechanisms (Jamieson 1981b; Jamieson et al. 1987; Ferraguti et al. 1999; Marotta et al. 2003). Less is known about the phylogenetic significance of characters related to the cuticle ultrastructure among clitellates. However, the collagenous cuticle of annelids, echiurids, and sipunculids is used as one argument for a common ancestry of these taxa (Ax 2000).
4.1. Cuticle The cuticular morphology in P. volki does not deviate from the basic pattern described in other clitellates (Richards 1978; Jamieson 1981a, 1992). It is composed of the same principle elements: collagenous fibres in a matrix, epicuticle, microvilli, and epicuticular projections. There are, however, features in the cuticle that differ among clitellate species, e.g., the organization of the collagenous fibres, the shape and density of the epicuticular projections, and the structure of the epicuticle. Parallel fibres are arranged into layers, each layer one fibre thick, with those of adjacent layers being approximately perpendicular to each other and at about 451 to the longitudinal axis of the worm. This has been referred to as an orthogonal grid and has been reported in all lumbricids studied (e.g. Ruska and Ruska 1961; Coggeshall 1966; Burke 1974; Richards 1974; Humphreys and Porter 1976), but only in a few enchytraeids (Djaczenko and Calenda Cimmino 1974; Richards 1977). Most of the studied enchytraeids have, compared to lumbricids, thinner fibres irregularly organized into layers but not in a criss-cross pattern (Hess and Menzel 1967; Goodman and Parrish 1971; Hess and Vena 1974). The organization of the fibre zone in the body of P. volki resembles the type without a grid,
Fig. 2. TEM-micrographs showing the morphology of the spermatozoa of Propappus volki. All photographs were taken at the ciliated funnels. (A) Apical portion of two spermatozoa showing the nuclei (n) and the acrosomes (a). (B) The transition region between the twisted and straight portions of the nucleus. (C) Longitudinal section of an acrosome. The dashed lines point to the corresponding cross-sections at the right. (D) Longitudinal section of the twisted mitochondrial midpiece. (E) Distal cross-sections of the acrosome (corresponding dashed line in C). (F) Proximal cross-sections of the acrosome (corresponding dashed line in C). (G) Cross-section of the midpiece. Five mitochondria are visible. (H) Cross-section of the basal portion of the flagellum, showing the basal cylinder (arrow; corresponding to the dashed line in L). (I) Cross-sections of the flagellum. Connections between the doublets and the plasma membrane are visible (arrow). Note the prominent central sheath (arrowhead) surrounding the central axonemal doublet. (J) Cross-sections of the flagellum. Glycogen granules are visible in some preparations (arrowheads). (K) Schematic drawing of the acrosome and the top of the apical nuclear portion. (L) Longitudinal section of the midpiece base and the beginning of the flagellum showing the basal cylinder (arrow) and the prominent central sheath (arrowhead). The dashed line indicates the level of the section shown in H, m, mitochondria. Scale bar is 0.2 mm in (A); 0.6 mm in (B); 0.15 mm in (C); 0.3 mm in (D); 0.15 mm in (E) and (F); 0.18 mm in (G–J); 0.15 mm in (L).
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as present in enchytraeids, more closely than the type described for lumbricids. Morphological deviations from a grid organization have also been reported in tubificids (including naidines). In Dero obtusa, for example, the fibres are thin and wavy and do not form a grid (Krall 1968), whereas in Pristina breviseta the fibres are lacking (Gustavsson 2001) (both species are representatives of Naidinae, Tubificidae). Other tubificids have the fibres either arranged in a grid, or irregularly distributed (Gustavsson and Erse´us 2000; Gustavsson 2001). The number of fibre layers and the depth of the cuticle are not proportional to the body size nor are they strictly correlated with other known factors (Storch 1988). Jamieson (1992) put forward that they may be correlated to locomotion or abrasion. Tube dwelling annelid species have been reported to have thin cuticles in parts of their bodies that are normally sheltered, but there are exceptions. In P. volki, the thickest cuticle occurs in the proboscis which might be interpreted as a defense against abrasion. However, the proboscis lacks fibres in the cuticle, having only small fibrils, which probably makes the proboscis more flexible but less resistant to abrasion. The collagen fibre pattern has instead been assumed to correlate to body size; large worms are supposed to have fibres arranged in a grid (Richards 1978; Westheide and Rieger 1978; Jamieson 1981a), whereas small ones are not, but there are also exceptions, especially in enchytraeids (Jamieson 1981a). In P. volki, the fibre pattern differs within a specimen, from fibres arranged in layers in the body to irregularly distributed fibrils in the proboscis. The proboscis has the smallest diameter, so the fibre arrangement might be dependent on size. A cuticle without latticed collagen fibres is also reported from polychaete larvae and hesionid polychaetes (Westheide and Rieger 1978). The epicuticular projections are universal in annelids but their shape and density vary between species. They are membrane-bound bodies densely filling the outer surface of the cuticle, and outnumbering the microvilli extending from the epidermal cells. The epicuticular projections have been shown to originate from the microvilli (Hess and Menzel 1967; Krall 1968; Humphreys and Porter 1976), but most often they are assumed to be formed from the microvilli having the same diameter and similar internal structure (Potswald 1971). In P. volki, both epicuticular projections and microvilli are found in all parts of the worm, but it could not be determined whether the microvilli are the source of the epicuticular projections. The function of the epicuticular projections has not been clarified, but Richards (1978) suggested that they trap and mechanically stabilize the mucus film around the worm, while Hess & Menzel (1967) suggested that they might function in secretion and absorbtion. In P. volki, the epicuticular projections have an overall oblong form but
the length and diameter vary, and they are longer and thinner in the proboscis region than in the rest of the body. The projections are everywhere surrounded by mucus. Ciliated sense organs in oligochaetes are scattered on the prostomium, peristomium, and pygidium, and arranged in transverse rows in the remaining segments. According to Yanez et al. (2006) there are two kinds of ciliated sense organs in freshwater oligochaetes: multiciliated organs with short cilia (o7.5 mm), which dominate on non-chaetal segments (also present in earthworms), and sense organs with one to three long cilia (length up to 19 mm, mostly 10–12 mm), which are more numerous on the chaetal segments. In P. volki, both kinds are found. The multiciliated sensory cells with relatively short cilia dominate the epidermal layer of the proboscis. These multiciliated cells are probably of tactile function, as previously suggested by Yanez et al. (2006), and Propappus probably uses the proboscis as a sensory organ.
4.2. Mature spermatozoon The spermatozoon of P. volki belongs to the modified sperm category sensu Franze´n (1956), being a filiform cell characterized by an elongated nucleus and a derived midpiece. It conforms to the spermatozoal pattern of oligochaetous clitellates in having an acrosome tube, mitochondria interposed between nucleus and flagellum, and a modification of the central apparatus of the axoneme (Ferraguti 2000). The similarity between the spermatozoa of the Swedish and Romanian specimens of P. volki shows that there is homogeneity in sperm morphology among different populations. This study follows Jamieson’s (1987) insight on the taxonomic purpose of comparative spermatology, according to which ‘‘yspermatozoa offer an ‘‘independent arbiter’’ capable in many cases of resolving contentious taxonomic and phylogenetic problems’’. Compared with all previously studied clitellate species, the spermatozoa of P. volki share many characters with those of Enchytraeidae, thus supporting a relationship between these two taxa. 4.2.1. Acrosome Both enchytraeids and P. volki have a short and straight acrosome tube. In both taxa the acrosome vesicle, deeply cup-like, lays external to the acrosome tube, and the acrosome rod reaches the apical portion of the vesicle. In contrast, the acrosome of P. volki is narrower than that of enchytraeids, with a more elongated acrosome vesicle extending for a longer portion outside the acrosome tube. Intriguingly, in the P. volki spermatozoon, the secondary tube is in contact with the basal portion of the acrosome rod in a wider
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cylindrical body. This structure resembles the distinctive basal node as described in spermatozoa of megascolecid earthworms, tentatively considered homologue to the node sheath present also in lumbricid spermatozoa (Jamieson 1978). Thus its presence inside the acrosome of P. volki may support a relationship between Propappus, together with enchytraeids, to earthworms, as indicated by a combined data phylogenetic analysis (Marotta et al. 2008, in press). 4.2.2. Nucleus In all previously studied enchytraeid species (Jamieson and Ferraguti, 2006), as well as two species of Fridericia (F. montaphonensis and F. discifera) and Mesenchytraeus pedatus (Marotta and Ferraguti, in litt. 2006), the nucleus of the spermatozoa is apically flanged or corkscrewshaped and, except in Enchytraeus, basally straight. The nucleus of P. volki spermatozoon, although thinner at the apex with respect to enchytraeids, corroborates this last pattern, as it is apically corkscrew-shaped and basally straight. 4.2.3. Midpiece As in enchytraeid spermatozoa (Ferraguti 2000), the midpiece in P. volki is elongated and twisted thus supporting the grouping of Propappus and enchytraeids. Note that, among clitellates, a twisted midpiece also characterizes the spermatozoa of Lumbriculidae, Branchiobdellidae (with some exceptions, i.e. Xironogiton victoriensis), Acanthobdella peledina, and Hirudinida. The presence of five mitochondria in the spermatozoon of P. volki, instead of the four mitochondria as present in the enchytraeid sperm model, seems less relevant, since the range of four to five mitochondria is considered to be the plesiomorphic condition among clitellates, and widespread among tubificids (Jamieson et al. 1987). 4.2.4. Flagellum Unlike enchytraeid spermatozoa, which show both a prominent central sheath and tetragonal fibres as modification of the central axonemal apparatus (Ferraguti 1984a), the spermatozoon of P. volki shows only the prominent central sheath arrangement. Among clitellate spermatozoa P. volki shares this axonemal modification, considered to be the plesiomorphic condition among clitellates (Ferraguti 1984b), together with Lumbriculidae, Acanthobdella and Hirudinida.
5. Conclusions Ultrastructural features in the cuticle and spermatozoa of Propappus volki indicate a phylogenetic position close to the enchytraeids among the clitellates. The
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grouping of P. volki with tubificids, haplotaxids, and phreodrilids, as proposed by a 18S rRNA parsimony analysis (Erse´us and Ka¨llersjo¨ 2004), is thus supported by neither sperm nor cuticle data. Instead, the sperm and cuticle characters corroborate the morphologybased traditional view with a close relationship to enchytraeids, in agreement with a phylogenetic analysis based on three nuclear and one mitochondrial gene (Rousset et al. 2007) and a bayesian analysis of 18S rRNA and morphology (Marotta et al. 2008, in press).
Acknowledgements We are grateful to Ylva Lilliemarck for technical assistance, and to Mikael Thollesson for collecting assistance.
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Krall, J.F., 1968. The cuticle and epidermal cells of Dero obtusa (family Naididae). J. Ultrastruct. Res. 25, 84–93. Marotta, R., Ferraguti, M., Erse´us, C., 2003. A phylogenetic analysis of Tubificinae and Limnodriloidinae (Annelida, Clitellata, Tubificidae) using sperm and somatic characters. Zool. Scr. 32, 255–278. Marotta, R., Ferraguti, M., Erse´us, C., Gustavsson, L., 2008. Combined data phylogenetics and character evolution of Clitellata (Annelida) using 18S rDNA and morphology. Zool. J. Linn. Soc., in press. Michaelsen, W., 1905. Die Oligochaeten des Baikal-Sees. Commissions-Verlag von R. Friedla¨nder & Sohn, Kiew und Berlin. Michaelsen, W., 1916. Ein eigentu¨mlicher neuer Enchytraeide der Gattung Propappus aus der Niederelbe. Verh. Naturwiss. Ver. Hamb. n.s 3, 51–55. Michaelsen, W., 1923. Die Oligochaeten der Wolga. Arbeiten der Biologischen Wolga-Station, Saratov 9, 1–11. Nielsen, C.O., Christensen, B., 1959. The Enchytraeidae, critical revision and taxonomy of European species. Natura Jutlandica 8/9, 1–160. Nylander, J.A.A., Erse´us, C., Ka¨llersjo¨, M., 1999. A test of monophyly of the gutless Phallodrilinae (Oligochaeta, Tubificidae) and the use of a 573-bp region of the mitochondrial cytochrome oxidase I gene in analysis of annelid phylogeny. Zool. Scr. 28, 305–313. Potswald, H.E., 1971. A fine structural analysis of the epidermis and cuticle of the oligochaete Aeolosoma bengalense Stephenson. J. Morphol. 135, 185–212. Richards, K.S., 1974. The ultrastructure of the cuticle of some British lumbricids (Annelida). J. Zool. Lond. 172, 303–316. Richards, K.S., 1977. Structure and function in the oligochaete epidermis (Annelida). In: Spearman, R.I.C. (Ed.), Comparative Biology of Skin. Academic Press, New York, pp. 171–193. Richards, K.S., 1978. Epidermis and cuticle. In: Mill, P.J. (Ed.), Physiology of Annelids. Academic Press, London, pp. 33–61. Rousset, V., Pleijel, F., Rouse, G.W., Erse´us, C., Siddall, M.E., 2007. A molecular phylogeny of annelids. Cladistics 23, 41–63. Ruska, C., Ruska, H., 1961. Die Cuticula der Epidermis des Regenwurms (Lumbricus terrestris L). Z. Zellforsch. 53, 759–764. Sokolskaya, N.L., 1972. New data on the fauna of aquatic Oligochaeta from Kamchatka. Sbornik trudov Zoologicheskogo Muzeya Moskovskogo Universiteta 12, 74–90. Stephenson, J., 1930. The Oligochaeta. The Clarendon Press, Oxford. Storch, V., 1988. Integument. In: Westheide, W., Hermans, C.O. (Eds.), The Ultrastructure of Polychaeta. Gustav Fischers Verlag, Stuttgart and New York, pp. 13–36. Timm, T., 1994. Propappidae and aquatic Enchytraeidae (Oligochaeta) from the farthest southeast of Russia. Hydrobiologia 278, 67–78. Westheide, W., Rieger, R.M., 1978. Cuticle ultrastructure of hesionid polychaetes (Annelida). Zoomorphologie 91, 1–18. Yanez, E., Cuadrado, S., Martinez-Ansemil, E., 2006. External sense organs in freshwater oligochaetes (Annelida, Clitellata) revealed by scanning electron microscopy. J. Morphol. 267, 198–207.
Zoologischer Anzeiger 247 (2008) 123–132 www.elsevier.de/jcz
Comparative ultrastructural study of the cuticle and spermatozoa in Propappus volki Michaelsen, 1916 (Annelida: Clitellata) Lena M. Gustavssona,, Marco Ferragutib, Roberto Marottab a
Department of Invertebrate Zoology, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden Dipartimento di Biologia, Universita` degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
b
Received 3 May 2007; received in revised form 27 August 2007; accepted 7 September 2007 Corresponding editor: M. V. Sørensen
Abstract 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. r 2007 Elsevier GmbH. All rights reserved. Keywords: Propappidae; Enchytraeidae; Ultrastructure; Cuticle; Spermatozoa; Phylogeny.
1. Introduction In 1905, Michaelsen described a new oligochaete species, Propappus glandulosus, from Lake Baikal Corresponding author.
E-mail address: [email protected] (L.M. Gustavsson). 0044-5231/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2007.09.003
(Michaelsen 1905). A new genus was established for this species, which was placed in Enchytraeidae. Although the new species differed substantially in morphology from the other members of the family, Propappus and the remaining enchytraeids were believed to form a monophyletic group in which Propappus represented an early branch within the family. Later,
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Michaelsen (1916) described a second species, Propappus volki, from Elbe River, near Hamburg. It was distinguished from P. glandulosus by its prostomial proboscis and the sperm funnel morphology. A third species, Propappus arhyncotus, was described by Sokolskaya (1972) from the Kamchatka peninsula. The placement of Propappus within Enchytraeidae has been questioned first by Nielsen and Christensen (1959), and later by Coates (1986) who erected a new family, Propappidae, for the three species. Morphologically, the members of Propappus differ in several characters from other enchytraeids. Specimens of Propappus have: (1) sigmoid, nodulate, bifid chaetae, commonly three per bundle, (2) large epidermal glands posterior to each chaetal bundle, (3) spermathecal pores posterior to septum 3/4, (4) glandular part of vasa deferentia located posterior to septum 11/12 and, (5) one pair of ovaries in segment XIII with female funnels located on septum 13/14. Monophyly of the family has only been tested once, by Coates (1987) who included, in a morphology-based phylogenetic analysis, two of the three Propappus species (P. volki and P. glandulosus) together with several haplotaxids, one enchytraeid, and a few megadriles. Although the two representatives of Propappus appeared as sister taxa in the analysis, the monophyly of Propappidae was not strongly supported, merely being based on the ‘‘absence’’ of several characters rather than presence, except for the position and shape of the spermathecae. Propappus turned out to be closely related to Enchytraeidae and Metataxis (Haplotaxidae). One of the characters considered, the presence of an epidermal gland associated with each chaetal bundle with an opening through the body wall immediately posterior to the bundle, appeared as an apomorphy for Propappidae. The possible homology of this structure to a similar one present in, e.g. Haplotaxis, Achaeta, and in some megadriles has previously been proposed (Michaelsen 1923; Stephenson 1930; Nielsen and Christensen 1959; Coates 1986) but further studies are required to draw any conclusions. In recent years, P. volki has been included in two phylogenetic analyses based on DNA data. The first one (Nylander et al. 1999), with the purpose of testing the monophyly of the gutless genera Olavius and Inanidrilus, did not reveal any information about the phylogenetic position of Propappus; all taxa except the gutless group were too weakly supported. In the second paper, a phylogenetic analysis of Clitellata based on 18S rRNA gene (Erse´us and Ka¨llersjo¨ 2004), Propappus groups together with Phreodrilidae, Tubificidae, and Haplotaxidae. The enchytraeids form a monophyletic group with Crassiclitellata (sensu Jamieson 1988). This DNA-based phylogeny thus supports Coates’ (1986) removal of Propappidae from Enchytraeidae.
Apart from the characters commonly used in taxonomy (see Coates 1986, 1987; Timm 1994), there are almost no data available on the fine morphology of Propappus. With the aim of providing new additional characters that may clarify the phylogenetic position of the family, we describe the ultrastructure of the cuticle and spermatozoa of P. volki.
2. Material and methods Several individuals of P. volki were collected by C. Pavelesku in May 2004 in the Stream Valea Firii (Romania), at a height of 1100 m above sea level. Additional specimens were collected by the first author in May 2005 in River Bra¨knea( n, Blekinge province, (south-eastern part of Sweden). The exact location is about 4 km south of Bra¨kne-Hoby, 0–30 m downstream the bridge crossing the river. In this part of the river the current is moderate to strong with a varying depth from 0.2 to 1 m. P. volki was found downstream of boulders in limited areas where the sediment was sorted to a specific size. The kick-method (Frost et al. 1971) was used to get material into a large bag net with extremely small mesh size. Each sample was then sorted under a dissecting microscope and each individual was identified live, compressed in a drop of water under a cover-slip, using a light microscope. All specimens were fixed in PAFG (Ermak and Eakin 1976) for about a week and then rinsed overnight in cacodylate buffer. They were then postfixed in 1% osmium tetroxide dissolved in the same buffer for 1 h, washed in buffer, dehydrated in a graded ethanol series, and then embedded via propylene oxide in Epon 812. Ultrathin and semithin sections were obtained with a Leica Ultracut or a Reichert Ultracut E. Semithin sections were stained with 1% toluidine blue and studied using a light microscope. Ultrathin sections were collected on copper grids, stained with lead citrate and uranyl acetate after Daddow’s (1983) methodology, and observed with a Jeol 100 SX or a LEO 912 AB electron microscope at 80 kV. For epidermal and cuticular studies, cross-sections were taken from different regions of the worms: proboscis, clitellum and the posterior region. To study sperm ultrastructure, the spermatozoa were examined at the male funnels (i.e. the entrances to the deferent ducts).
3. Results The studied specimens of P. volki are 4.5–6.0 mm long and consist of 35–41 segments. They have bifid chaetae, normally three per bundle, but occasionally there are one or two more chaetae per bundle. In mature
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specimens the clitellum begins in the middle of segment XII, and covers segments XIII and XIV. The prostomium is furnished with a proboscis, about 80 mm long and with a diameter of 20 mm in fixed specimens. Slightly longer proboscises are observed in living specimens.
3.1. Cuticle morphology The body wall of P. volki consists of five layers. An outer, non-cellular cuticle overlies the monolayered epidermis (Fig. 1A). Beneath the epidermis there are two layers of muscles; an outer circular layer and an inner longitudinal layer. A thin peritoneal epithelium covers the inner muscle layer and separates it from the coelom. Epidermal sense organs (multiciliated cells with short cilia and sensory cells with one to three long cilia) are sparsely distributed in the clitellar and post-clitellar regions of the worm. The body wall in the protruding proboscis differs and consists of two layers only; the epidermis and cuticle (Fig. 1D). The epidermis in this region is dominated by multiciliated sensory cells covered by a relatively thick cuticle. No muscle layers or coelom were found beneath the epidermal layer, instead the internal space is filled with a loose tissue. The cuticle is composed of several structures; a fibre zone, an epicuticle, epicuticular projections, and microvilli (Fig. 1C). Minor differences in the morphology of these structures occur between different regions of the worm (proboscis, clitellum, and post-clitellar region). A brief summary of the differences is presented in Table 1. 3.1.1. Fibre zone The proximal part of the cuticle has fibres embedded in a homogeneous matrix (Figs. 1B and C). The fibres are arranged in layers of parallel fibres with the same orientation of the fibres in all layers. In a cross-section of the worm, six to eight layers can be seen. Each fibre is composed of thinner, intertwined fibrils. The morphology of the fibre zone differs between the studied regions of the worm, and only in the post-clitellar region are the fibres arranged in layers. The proboscis region lacks compound fibres, instead thinner and shorter fibrils run in all directions (Fig. 1E). In the clitellar region, compound fibres are oriented in the same direction, but they are not arranged into layers (Fig. 1F). 3.1.2. Epicuticle The epicuticle is relatively thick, about one fourth of the total thickness of the cuticle (Figs. 1E and F). It is a continuation of the matrix surrounding the fibres, but devoid of any fibres or fibrils. The matrix, however, consists of smaller filaments (or microfibrils) and granules. The outer surface of the epicuticle has no limiting membrane. Three layers of different electron
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density are distinguished depending on how closely the filaments are packed together (Fig. 1F). The innermost layer is about half the thickness of the epicuticle, with loosely arranged filaments in all directions. The two outer layers have more tightly packed filaments with alignments parallel to the surface. The outermost layer is thinnest. The epicuticle has the same appearance in all regions of the worm studied. 3.1.3. Epicuticular projections Projections cover the outer surface of the epicuticle. They are membrane-bound bodies with their major axis perpendicular to the surface, about two-three times longer than wide. The shape of the projections is weakly pyramidal with the base touching the epicuticle (Fig. 1F). Thin filaments radiate from the double membrane enclosing the epicuticular projection. The filaments pointing towards the epicuticle connect to the filaments present in it, and probably keep the epicuticular projections in position, while those pointing outwards probably stabilize the mucus layer covering the worm. Apart from an electron-dense, crescentshaped structure visible in the outer part of each epicuticular projection, the internal substance is homogeneous. The epicuticular projections in the proboscis are longer and narrower than in other regions of the worm (Fig. 1E, Table 1). Their density is, however, about the same which makes them look more sparsely distributed. No other morphological differences were found between epicuticular projections in different regions. 3.1.4. Microvilli Microvilli, processes from the epidermal cells, penetrate the cuticle and reach the free outer surface of the epicuticle (Figs. 1A, C and E). The diameter of the microvilli varies between 50 and 140 nm, measured at the same distance from the epidermis. Most microvilli, nevertheless, have the same diameter as the epicuticular projections, and they end amongst them. The wider microvilli continue far outside the epicuticular projections, but look otherwise identical. No microvilli pinching off epicuticular projections were observed. The cell matrix inside the microvilli is homogeneous, and looks similar to the substance present inside each epicuticular projection. The density of the microvilli varies; in some areas there are almost as many microvilli as there are epicuticular projections, while in other areas they are lacking. Sometimes microvilli are intertwined with the cilia arising from the sensory cells, especially in the proboscis region (Fig. 1D). 3.1.5. Knobs Knobs are extensions from the epidermal cells into the cuticle. They are short, hemisphere-shaped bulges that reach into the lower part of the fibre zone (Fig. 1E).
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Table 1.
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Characters of the cuticle
Character
Proboscis
Clitellar region
Postclitellar region
Cuticle: thickness (nm) Fibre zone: thickness (nm) Fibre zone: fibre diameter (nm) Fibre zone: organization Epicuticle: thickness (nm) Epicuticle: layers Epicuticular projections: length (nm) Epicuticular projections: diameter (nm) Epicuticular projections: number per mm Microvilli: diameter (nm) Microvilli: numbers per mm
660 330 No fibres – 110 3 135 40 12 66–88 Varies
440 200 14–40 No layers 110 3 125 55 11 70–139 Varies
570 300 11–33 6–8 Layers 130 3 100 55 12 54–130 Varies
Thin filaments radiate from the hemisphere into the matrix of the cuticle. An electron-dense area is visible in the outer part of each bulge. The knobs are sparsely distributed in all regions of the worm.
3.2. Sperm morphology The following description refers to the mature spermatozoa as observed at the sperm funnels. The spermatozoon of P. volki is a filiform cell, formed, in sequence, by an acrosome, an elongated nucleus, a long mitochondrial midpiece, and a flagellum. 3.2.1. Acrosome The acrosome is an elongated cylinder (average length 10.171.3 mm, N ¼ 6) tapering from its basis to the apex, and slightly bent to one side (Fig. 2C and K). It is formed by a short and thick acrosomal tube (average length 2.770.5 mm, N ¼ 8) proximally ending in an internal thickening (limen sensu Jamieson 1978) (Fig. 2A and C). A long, cup-shaped acrosome vesicle occupies almost the entire length of the acrosome, and is completely external to the tube (Fig. 2C, E, and F). A thin sheet of electron-dense material fills the space between the basal portion of the acrosome vesicle and the plasma membrane; the distal part of the acrosome
vesicle lies close to the cell membrane (Fig. 2C). An elongated acrosome rod (average length 8.1371.32 mm, N ¼ 6), protruding for nearly all its length from the distal margin of the acrosomal tube, ends apically inside the invagination of the acrosome vesicle, close to the vesicle tip (Fig. 2C). A thin and short secondary tube starts from the proximal margin of the acrosomal vesicle, and ends close to the proximal extremity of the acrosome rod, in correspondence of a wider, cylindrical body, about as long as wide (Fig. 2C and K), resembling the basal node (sensu Jamieson 1978) diagnostic of the sperm of megascolecid earthworms. A small empty space, the basal chamber, is present at the base of the acrosome (Fig. 2C and K). A thin, dome-shaped structure, the nuclear pad (sensu Jamieson 1982), is interposed between the apical portion of the nucleus and the acrosome (Fig. 2C and K). 3.2.2. Nucleus The nucleus is a long, filiform structure of fully condensed chromatin, apically corkscrew-shaped and basally nearly straight (Fig. 2A and B). It decreases in diameter from its concave basis (average diameter 0.3370.03 mm, N ¼ 10) to the convex apex (average diameter 0.170.01 mm, N ¼ 10).
Fig. 1. TEM-micrographs showing the morphology of the cuticle of Propappus volki. (A) Cross-section of the body wall of a postclitellar region showing longitudinal (lm) and circular muscle layers (cm). The monolayered epidermis (e) is covered with cuticle (c) with several microvilli reaching through from the epidermis. (B) Cross-section of the clitellar region. A portion of a gland cell (gc) with its granular inclusions (gi) is visible. Thin fibrils (fz) are aligned in the cuticle. Pyramidal epicuticular projections (ep) cover the surface of the epicuticle. (C) Cross-section of a post-clitellar region. Cuticular fibres (fz) are arranged in layers. The epicuticle is covered with weakly pyramidal projections (ep). (D) Longitudinal section of the proboscis with cuticle (c) visible on both sides. Sensory cells (sc) with cilia (ci) penetrating the cuticle occur in the epidermal layer. Several microvilli reach through the cuticle. No muscle layers or coelom are found in the proboscis. (E) Longitudinal section of the proboscis. Thin fibrils lay in all directions in the cuticle (c). One microvillus (mv) stretches through the cuticle from the epidermal cell (e). The outer surface of the cuticle is covered with long and narrow epicuticular projections (ep). Knobs (k) reach from the epidermis into the proximal part of the fibre zone. (F) Details of the clitellar cuticle showing the layered epicuticle (ec), the fiber zone (fz) and the internal structure of the epicuticular projections (ep). Scale bar is 1.5 mm in (A); 0.38 mm in (B); 0.48 mm in (C); 1.9 mm in (D); 0.24 mm in (E) and 0.15 mm in (F).
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3.2.3. Midpiece The midpiece is formed by five twisted, elongated mitochondria (average length 1.670.13 mm, N ¼ 6) (Fig. 2D and G) each with the shape of a cylindrical sector around a central axis filled with electron-dense material. 3.2.4. Flagellum The flagellum contains a simple 9+2 axoneme surrounded by the plasma membrane at regular distance (average distance 0.0470.006 mm, N ¼ 14) (Fig. 2I and J). The basal body area is deeply modified by the presence, in its interior, of a long basal cylinder (sensu Ferraguti 1984a) nearly reaching the midpiece (Fig. 2H and L. A prominent central sheath, sensu Ferraguti (1984b), is visible as a modification of the central axonemal apparatus (Fig. 2I and J). Traces of putative glycogen granules are visible in cross-section, external to the axonemal doublets. Each doublet is connected to the plasma membrane by faint, electron-dense links (Fig. 2I and J). The flagellum ends abruptly in a narrow endpiece, by progressive reduction of the axonemal microtubule number.
4. Discussion Information from morphology and molecular sequences are used in phylogenetic analyses separately or in combination. The morphology and DNA information is sometimes conflicting, as it is the case in the Propappidae. Based on morphological characters, Propappus has been positioned within Enchytraeidae (Michaelsen 1916), or as a sister group to certain members of Enchytraeidae and Haplotaxidae (Coates 1987). In a parsimony analysis among clitellates, based on the entire 18S rRNA gene, Propappus grouped far from enchytraeids, forming a monophyletic group together with Tubificidae, Phreodrilidae, and Haplotaxidae (Erse´us and Ka¨llersjo¨ 2004). In a recent analysis based on one mitochondrial and three nuclear genes, P. volki branches off as sister taxon to Haplotaxis, with
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an enchytraeid being the sister group to them, albeit with weak support (Rousset et al. 2007). The morphological information about Propappus is limited to the species descriptions and further morphological data can be useful to clarify its phylogenetic position. Sperm ultrastructure has been proved to be informative as a phylogenetic marker, at least in groups such as clitellates with uniform reproductive mechanisms (Jamieson 1981b; Jamieson et al. 1987; Ferraguti et al. 1999; Marotta et al. 2003). Less is known about the phylogenetic significance of characters related to the cuticle ultrastructure among clitellates. However, the collagenous cuticle of annelids, echiurids, and sipunculids is used as one argument for a common ancestry of these taxa (Ax 2000).
4.1. Cuticle The cuticular morphology in P. volki does not deviate from the basic pattern described in other clitellates (Richards 1978; Jamieson 1981a, 1992). It is composed of the same principle elements: collagenous fibres in a matrix, epicuticle, microvilli, and epicuticular projections. There are, however, features in the cuticle that differ among clitellate species, e.g., the organization of the collagenous fibres, the shape and density of the epicuticular projections, and the structure of the epicuticle. Parallel fibres are arranged into layers, each layer one fibre thick, with those of adjacent layers being approximately perpendicular to each other and at about 451 to the longitudinal axis of the worm. This has been referred to as an orthogonal grid and has been reported in all lumbricids studied (e.g. Ruska and Ruska 1961; Coggeshall 1966; Burke 1974; Richards 1974; Humphreys and Porter 1976), but only in a few enchytraeids (Djaczenko and Calenda Cimmino 1974; Richards 1977). Most of the studied enchytraeids have, compared to lumbricids, thinner fibres irregularly organized into layers but not in a criss-cross pattern (Hess and Menzel 1967; Goodman and Parrish 1971; Hess and Vena 1974). The organization of the fibre zone in the body of P. volki resembles the type without a grid,
Fig. 2. TEM-micrographs showing the morphology of the spermatozoa of Propappus volki. All photographs were taken at the ciliated funnels. (A) Apical portion of two spermatozoa showing the nuclei (n) and the acrosomes (a). (B) The transition region between the twisted and straight portions of the nucleus. (C) Longitudinal section of an acrosome. The dashed lines point to the corresponding cross-sections at the right. (D) Longitudinal section of the twisted mitochondrial midpiece. (E) Distal cross-sections of the acrosome (corresponding dashed line in C). (F) Proximal cross-sections of the acrosome (corresponding dashed line in C). (G) Cross-section of the midpiece. Five mitochondria are visible. (H) Cross-section of the basal portion of the flagellum, showing the basal cylinder (arrow; corresponding to the dashed line in L). (I) Cross-sections of the flagellum. Connections between the doublets and the plasma membrane are visible (arrow). Note the prominent central sheath (arrowhead) surrounding the central axonemal doublet. (J) Cross-sections of the flagellum. Glycogen granules are visible in some preparations (arrowheads). (K) Schematic drawing of the acrosome and the top of the apical nuclear portion. (L) Longitudinal section of the midpiece base and the beginning of the flagellum showing the basal cylinder (arrow) and the prominent central sheath (arrowhead). The dashed line indicates the level of the section shown in H, m, mitochondria. Scale bar is 0.2 mm in (A); 0.6 mm in (B); 0.15 mm in (C); 0.3 mm in (D); 0.15 mm in (E) and (F); 0.18 mm in (G–J); 0.15 mm in (L).
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as present in enchytraeids, more closely than the type described for lumbricids. Morphological deviations from a grid organization have also been reported in tubificids (including naidines). In Dero obtusa, for example, the fibres are thin and wavy and do not form a grid (Krall 1968), whereas in Pristina breviseta the fibres are lacking (Gustavsson 2001) (both species are representatives of Naidinae, Tubificidae). Other tubificids have the fibres either arranged in a grid, or irregularly distributed (Gustavsson and Erse´us 2000; Gustavsson 2001). The number of fibre layers and the depth of the cuticle are not proportional to the body size nor are they strictly correlated with other known factors (Storch 1988). Jamieson (1992) put forward that they may be correlated to locomotion or abrasion. Tube dwelling annelid species have been reported to have thin cuticles in parts of their bodies that are normally sheltered, but there are exceptions. In P. volki, the thickest cuticle occurs in the proboscis which might be interpreted as a defense against abrasion. However, the proboscis lacks fibres in the cuticle, having only small fibrils, which probably makes the proboscis more flexible but less resistant to abrasion. The collagen fibre pattern has instead been assumed to correlate to body size; large worms are supposed to have fibres arranged in a grid (Richards 1978; Westheide and Rieger 1978; Jamieson 1981a), whereas small ones are not, but there are also exceptions, especially in enchytraeids (Jamieson 1981a). In P. volki, the fibre pattern differs within a specimen, from fibres arranged in layers in the body to irregularly distributed fibrils in the proboscis. The proboscis has the smallest diameter, so the fibre arrangement might be dependent on size. A cuticle without latticed collagen fibres is also reported from polychaete larvae and hesionid polychaetes (Westheide and Rieger 1978). The epicuticular projections are universal in annelids but their shape and density vary between species. They are membrane-bound bodies densely filling the outer surface of the cuticle, and outnumbering the microvilli extending from the epidermal cells. The epicuticular projections have been shown to originate from the microvilli (Hess and Menzel 1967; Krall 1968; Humphreys and Porter 1976), but most often they are assumed to be formed from the microvilli having the same diameter and similar internal structure (Potswald 1971). In P. volki, both epicuticular projections and microvilli are found in all parts of the worm, but it could not be determined whether the microvilli are the source of the epicuticular projections. The function of the epicuticular projections has not been clarified, but Richards (1978) suggested that they trap and mechanically stabilize the mucus film around the worm, while Hess & Menzel (1967) suggested that they might function in secretion and absorbtion. In P. volki, the epicuticular projections have an overall oblong form but
the length and diameter vary, and they are longer and thinner in the proboscis region than in the rest of the body. The projections are everywhere surrounded by mucus. Ciliated sense organs in oligochaetes are scattered on the prostomium, peristomium, and pygidium, and arranged in transverse rows in the remaining segments. According to Yanez et al. (2006) there are two kinds of ciliated sense organs in freshwater oligochaetes: multiciliated organs with short cilia (o7.5 mm), which dominate on non-chaetal segments (also present in earthworms), and sense organs with one to three long cilia (length up to 19 mm, mostly 10–12 mm), which are more numerous on the chaetal segments. In P. volki, both kinds are found. The multiciliated sensory cells with relatively short cilia dominate the epidermal layer of the proboscis. These multiciliated cells are probably of tactile function, as previously suggested by Yanez et al. (2006), and Propappus probably uses the proboscis as a sensory organ.
4.2. Mature spermatozoon The spermatozoon of P. volki belongs to the modified sperm category sensu Franze´n (1956), being a filiform cell characterized by an elongated nucleus and a derived midpiece. It conforms to the spermatozoal pattern of oligochaetous clitellates in having an acrosome tube, mitochondria interposed between nucleus and flagellum, and a modification of the central apparatus of the axoneme (Ferraguti 2000). The similarity between the spermatozoa of the Swedish and Romanian specimens of P. volki shows that there is homogeneity in sperm morphology among different populations. This study follows Jamieson’s (1987) insight on the taxonomic purpose of comparative spermatology, according to which ‘‘yspermatozoa offer an ‘‘independent arbiter’’ capable in many cases of resolving contentious taxonomic and phylogenetic problems’’. Compared with all previously studied clitellate species, the spermatozoa of P. volki share many characters with those of Enchytraeidae, thus supporting a relationship between these two taxa. 4.2.1. Acrosome Both enchytraeids and P. volki have a short and straight acrosome tube. In both taxa the acrosome vesicle, deeply cup-like, lays external to the acrosome tube, and the acrosome rod reaches the apical portion of the vesicle. In contrast, the acrosome of P. volki is narrower than that of enchytraeids, with a more elongated acrosome vesicle extending for a longer portion outside the acrosome tube. Intriguingly, in the P. volki spermatozoon, the secondary tube is in contact with the basal portion of the acrosome rod in a wider
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cylindrical body. This structure resembles the distinctive basal node as described in spermatozoa of megascolecid earthworms, tentatively considered homologue to the node sheath present also in lumbricid spermatozoa (Jamieson 1978). Thus its presence inside the acrosome of P. volki may support a relationship between Propappus, together with enchytraeids, to earthworms, as indicated by a combined data phylogenetic analysis (Marotta et al. 2008, in press). 4.2.2. Nucleus In all previously studied enchytraeid species (Jamieson and Ferraguti, 2006), as well as two species of Fridericia (F. montaphonensis and F. discifera) and Mesenchytraeus pedatus (Marotta and Ferraguti, in litt. 2006), the nucleus of the spermatozoa is apically flanged or corkscrewshaped and, except in Enchytraeus, basally straight. The nucleus of P. volki spermatozoon, although thinner at the apex with respect to enchytraeids, corroborates this last pattern, as it is apically corkscrew-shaped and basally straight. 4.2.3. Midpiece As in enchytraeid spermatozoa (Ferraguti 2000), the midpiece in P. volki is elongated and twisted thus supporting the grouping of Propappus and enchytraeids. Note that, among clitellates, a twisted midpiece also characterizes the spermatozoa of Lumbriculidae, Branchiobdellidae (with some exceptions, i.e. Xironogiton victoriensis), Acanthobdella peledina, and Hirudinida. The presence of five mitochondria in the spermatozoon of P. volki, instead of the four mitochondria as present in the enchytraeid sperm model, seems less relevant, since the range of four to five mitochondria is considered to be the plesiomorphic condition among clitellates, and widespread among tubificids (Jamieson et al. 1987). 4.2.4. Flagellum Unlike enchytraeid spermatozoa, which show both a prominent central sheath and tetragonal fibres as modification of the central axonemal apparatus (Ferraguti 1984a), the spermatozoon of P. volki shows only the prominent central sheath arrangement. Among clitellate spermatozoa P. volki shares this axonemal modification, considered to be the plesiomorphic condition among clitellates (Ferraguti 1984b), together with Lumbriculidae, Acanthobdella and Hirudinida.
5. Conclusions Ultrastructural features in the cuticle and spermatozoa of Propappus volki indicate a phylogenetic position close to the enchytraeids among the clitellates. The
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grouping of P. volki with tubificids, haplotaxids, and phreodrilids, as proposed by a 18S rRNA parsimony analysis (Erse´us and Ka¨llersjo¨ 2004), is thus supported by neither sperm nor cuticle data. Instead, the sperm and cuticle characters corroborate the morphologybased traditional view with a close relationship to enchytraeids, in agreement with a phylogenetic analysis based on three nuclear and one mitochondrial gene (Rousset et al. 2007) and a bayesian analysis of 18S rRNA and morphology (Marotta et al. 2008, in press).
Acknowledgements We are grateful to Ylva Lilliemarck for technical assistance, and to Mikael Thollesson for collecting assistance.
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