VENUS 65 (1-2): 51-70, 2006

Review

Fertilization in : Morphological Clues to Phylogeny*

John Buckland-Nicks Biology Department, St Francis Xavier University, St Ninian’s Street, Antigonish, Nova Scotia, B2G 2W5, Canada; [email protected]

Abstract: The last common ancestor of , the largest order of chitons, evolved unique mechanisms of fertilizing elaborate eggs with dart-like sperm that are typical of the group. In contrast Leptochiton asellus and other Lepidopleurida [sensu Sirenko 1993], have retained the plesiomorphic condition with smooth-hulled eggs and sperm with prominent acrosomes. The mechanism of fertilization in L. asellus is expected to be similar to scaphopods and some other mollusks, because of basic similarities in sperm and egg design. By this mechanism, the acrosome reaction releases enzymes that digest a large hole in the jelly layer and vitelline layer. An acrosomal process polymerizes and extends the inner acrosomal membrane down to fuse with an egg microvillus. A fertilization cone is raised up through the vitelline layer and engulfs the sperm including the nucleus, centrioles, mitochondria and part of the flagellum. The transition to elaborate egg hulls and reduced acrosomes, which characterize all Chitonida, may be evident as intermediate stages among certain lepidopleurids, such as Deshayesiella curvata and Hanleya hanleyi, which have eggs with smooth hulls but sperm with smaller acrosomes on short nuclear filaments. Furthermore, dentatus is of special interest, as it has retained a similar egg to D. curvata but its sperm is derived, like all other chitonids. Fertilization follows the chitonid pattern, in which a tiny acrosome digests a hole in the vitelline layer that permits only the injection of chromatin into the egg. The fertilization cone remains below the vitelline layer and so does not engulf the body of the sperm. Thus sperm organelles seem to be abandoned on the egg surface in a bag of sperm membrane. This suggests that centrioles, as well as mitochondria, of Chitonida are maternally inherited. New characters developed for Callochiton dentatus, Deshayesiella curvata and Hanleya hanleyi, have enabled a revision of previous phylogenies. The revised analysis suggests that Callochitonidae is the sister taxon to all of Chitonida and is not part of the order Lepidopleurida [sensu Sirenko, 1993]). Furthermore, Lepidopleurida appear to be a paraphyletic grouping. The Chitonida are still clearly divisible into the two suborders, Chitonina and Acanthochitonina.

Keywords: Polyplacophora, evolution, sperm, egg, fertilization

Introduction

The study of fertilization among chitons has revealed key differences in the mechanism of sperm entry which are taxonomically, as well as morphologically, important (Buckland-Nicks, 1993, 1995; Buckland-Nicks & Eernisse, 1992; Buckland-Nicks & Hodgson, 2000; Buckland- Nicks et al., 1988a, 1988b, 1990; Pashchenko & Drozdov, 1998). Fertilization was misinterpreted for many years because it was thought that an acrosome was lacking (see review by Buckland-

*Invited paper to the special number of Venus for the 2nd International Symposium, Tsukuba 52 J. Buckland-Nicks

Nicks, 1995). Alternative mechanisms of sperm entry were proposed to account for fertilization without an acrosome, including direct piercing of the hull or penetration via a micropyle (Sakker, 1984; Al-Hajj, 1987). As it turns out, an acrosome is present in all chiton examined to date. It shows a gradual reduction in the group from the anticipated plesiomorphic condition of a large, typical molluscan acrosome cone found in lepidopleurids, such as Leptochiton asellus (Hodgson et al., 1988), through forms of intermediate size in Deshayesiella curvata (Pashchenko & Drozdov, 1998), to the highly reduced acrosome vesicles that characterize all Chitonida (see review by Buckland-Nicks, 1995). Changes in the size of the acrosome correlate with changes in the form of the nucleus. L. asellus has a bullet-shaped nucleus with a large acrosome cone, whereas D. curvata has a dart-shaped nucleus including a short nuclear filament capped by a smaller acrosome cone (Pashchenko & Drozdov, 1998). Finally, sperm of all Chitonida have a dart-shaped nucleus with a long nuclear filament that terminates in a tiny acrosome vesicle. Thus, besides acrosome reduction from a cone to a vesicle, other key sperm features that have changed during the evolution of chitons, include; the disappearance of the subacrosomal granule and hence the acrosomal process (perforatorium) formed from it, and the appearance of the needle- like nuclear filament and the basal plate, separating acrosome from nucleus. All of these features correlate with changes in the mechanism of fertilization. In the past decade the identification of many new species of Lepidopleurida [sensu Sirenko 1993] (Sirenko, 2000; Saito, 1994-1998; Strack, 1992), has paved the way for a re-evaluation of chiton phylogeny based on key morphological characters, such as; the structure of soft body parts and shell valves (Sirenko, 1993; 1997), the structure of gametes and their interaction at fertilization (Buckland-Nicks 1995; Pashchenko & Drozdov 1998), as well as molecular structure (Okusu et al., 2003). Studies of Deshayesiella sp., Hanleya sp. and Callochiton sp. have revealed eggs or sperm with apparent intermediate conditions, which could provide an understanding of how Chitonida evolved their unique versions of gametes from ancestral forms. If true it would mean that both the order Lepidopleurida [sensu Sirenko 1993] and the suborder Lepidopleurina [sensu van Belle 1983] are paraphyletic. On the other hand if lepidopleurids are monophyletic, then these apparent intermediate states would be homoplasies. This paper reviews recent work done on gamete structure and fertilization in all chitons and brings to light new evidence from species that have evolved mixtures of plesiomorphic and derived traits, that may help reveal a more accurate phylogeny of chitons.

Materials and Methods

Specimens The species and their sources used in this study, were as follows: Stenoplax conspicua (Pilsbry, 1892) was collected off the coast of southern California (33˚5´ N, 117˚30´ W) in 1993; Hanleya hanleyi W. Bean in Thorpe, 1844 was collected by Ross Mayhew from scallop shells dredged off Lurcher Bank, Nova Scotia (43˚15´ N, 65˚30´ W) in 1994; Leptochiton asellus (Linnaeus, 1767) was collected off the east coast of Scotland at Rubh’ a’ Bhaid Bheithe near Loch Linnhe (56˚41´ N, 5˚13´ W) by Mike Sturrock in 2001; Deshayesiella curvata Carpenter in Pilsbry 1892, Leptochiton rugatus (Pilsbry, 1892), and Tonicella undocaerulea Sirenko, 1973 were collected from Vostok Bay (42˚53N´, 132˚44´ E), by Boris Sirenko in 1997; Chiton tulipa Quoy and Gaimard, 1835 and Callochiton dentatus (Spengler, 1797) (syn.: C. castaneus Wood 1815), were collected from East London, South Africa in 1999 (33˚03´ S, 28˚03´ E) [Note: Buckland- Nicks & Hodgson (2000), and also Okusu et al. (2003), referred to the same species by a more recent name: Callochiton castaneus Wood 1815]; Chiton nigrovirescens De Blainville, 1825 was collected from the west coast of the Cape Peninsula, South Africa (34˚16´ S, 18˚40´ E) in 1999; Lepidochitona dentiens (Gould, 1846), Lepidochitona fernaldi Eernisse, 1986, and Fertilization in Chitons: Morphological Clues to Phylogeny 53 muscosa (Gould, 1846) were collected off San Juan Island, USA (48˚28´ N, 122˚54´ W) in 1990. Acanthochitona viridis Pease 1872, was collected off the west coast of Oahu, Hawaii (21˚18´ N, 158˚09´ W) in 1987.

Electron Microscopy Fixation for T.E.M. and SEM followed several methods detailed in Buckland-Nicks (1993) and Buckland-Nicks & Hodgson (2000). Variation in fixation quality occurred in this study, because some specimens had to be stored in glutaraldehyde for longer periods (up to 4 weeks), as they were shipped to Canada from other countries. Various useful techniques have been devised over the years in this laboratory to examine sperm penetration of chiton eggs with electron microscopy, these include: 1. Isolating females in petri dishes containing filtered seawater at room temperature, or adding some sperm to the same dish often induces spawning. Once females begin spawning eggs, they can be washed and removed to a fresh container to provide unfertilized eggs. Fertilizing chiton eggs stripped from ovaries is rarely successful, as egg morphology is usually immature. 2. Slicing eggs in half with a Kesei microknife (Tokyo, Japan) to expose and visualize penetrating sperm (Figs. 1, 2). 3. Using 000 gauge, stainless steel, insect pins (Fine Science Tools, USA) to roll dried, fixed eggs on double sided carbon (or other) sticky tape to remove cupules or spines and reveal fertilizing sperm for SEM studies (Fig. 3). 4. Using millipore-filtered fixatives and dehydrating solutions throughout processing of SEM specimens, reduces particulate contaminants and should be coupled with the complete removal of each solution at each exchange. Microporous specimen capsules (Cedar Lane Ltd) are key to processing SEM samples through critical point drying.

Results and Discussion

Lepidopleurida [sensu Sirenko 1993] Sperm Structure: Hodgson et al. (1988) showed that sperm of Leptochiton asellus have a very large acrosome cone (2.4 µm long), enclosing some diffuse subacrosomal material, separated from a bullet-shaped nucleus (4.1 µm long) by an indistinct basal plate. A similar sperm type, but with variation in lengths of components, is seen in L. rugatus (Figs. 4, 5), as well as L. assimilis (Pashchenko & Drozdov, 1998) and has been classified as Type 4 chiton sperm (Buckland-Nicks, 1995). The proximal and distal centrioles of this sperm become connected at right angles to each other but each centriole is still discernible as a separate entity, unlike those of Chitonida. The space around the centrioles usually contains glycogen granules (Fig. 8). Five or six spherical mitochondria form a symmetrical ring around the centrioles, to complete the mid-piece. The distal centriole, which gives rise to the flagellum, is connected to the annulus and plasma membrane by a centriolar satellite complex, as is usual in aquasperm (Buckland-Nicks, 1995; Paschenko & Drozdov, 1998). The Leptochiton sp. sperm type is currently considered plesiomorphic to chitons, because it resembles sperm of some other molluscs and annelids but not any Chitonida. An apparent intermediate condition was discovered in Deshayesiella curvata (Pashchenko & Drozodov, 1998). In this species a short nuclear filament (1.9 µm long), terminates in an acrosome cone (1 µm long), smaller than that of L. asellus, but complete with subacrosomal granular material and a basal plate (Fig. 6). The nuclear filament exits eccentrically from the main body of the nucleus (Fig. 7). These features apply also to the sperm of Hanleya hanleyi (Hanleyidae) (Figs. 9, 10). The nuclear filament in this sperm is 2 µm long and the acrosome is 1 µm long. In these and other respects the sperm of H. hanleyi is very similar to that of D. curvata, suggesting a close phylogenetic relationship. This would be classified as sperm Type 5, in keeping with the format started by Buckland-Nicks (1995). Egg Structure: Species of the genus Leptochiton produce eggs with smooth jelly-like hulls, 54 J. Buckland-Nicks comprising a thick (14-30 µm) outer layer lacking pores, which encloses a thin vitelline layer (about 1 µm thick) that overlies microvilli projecting into it from the egg membrane (Hodgson et al., 1988; Pashchenko & Drozdov, 1998). This egg type is considered plesiomorphic to chitons, as it is similar to that of other molluscs including some scaphopods (Dufresne-Dubé et al., 1983) and bivalves (Longo et al., 1993), but is quite different from the egg hulls that develop in all other Chitonida. It is also different from the egg of Deshayesiella curvata, which has a smooth jelly- like hull (up to 80 µm thick) but is perforated with large pores (8-10 µm in diameter), which are spaced 30 µm apart and lead directly to the vitelline layer (Pashchenko & Drozdov, 1998). The jelly layer of all smooth eggs studied thus far, breaks down much more rapidly than the thinner hulls of chitonid eggs, and it must be fixed rapidly after exposure to sperm in order to preserve it at all. In L. asellus, (Fig. 14), as well as D. curvata, the jelly layer disappears completely after exposure to sperm for ten minutes. This is because the jelly layer is dissolved rapidly by the release of acrosomal enzymes from the many sperm. The smooth egg of Callochiton dentatus behaves similarly, in that the jelly layer is clearly visible in eggs dissected from the ovary (Figs. 12, 13) but this layer breaks down rapidly in the presence of many sperm (Buckland-Nicks & Hodgson, 2000). It will be important to examine the eggs of Hanleyidae to see if they are more like Leptochiton sp., smooth and continuous, or like those of Deshayesiella sp. and Callochiton sp., smooth but perforated by pores. Fertilization: Fertilization has not been studied in detail in any lepidopleurid. However, sperm of Leptochiton asellus have been observed to elongate in the region of the acrosome during exposure to eggs (Fig. 15), which suggests the formation of an acrosomal process. Furthermore, the basic features of egg and sperm described for L. asellus and Deshayesiella curvata above, are similar to those described for scaphopods (Dufresne-Dubé et al., 1983), some bivalves (Longo, 1987; 1993); and sea urchins (Epel, 1978; Longo, 1987), and can be expected to interact in a similar way. A hypothetical model of fertilization in Leptochiton sp. is shown in Diagram 1. The key points to notice in this mechanism are: 1. The acrosome reaction releases enzymes that digest a large hole in the jelly layer and vitelline membrane. 2. This is followed immediately by formation of an acrosomal process (perforatorium) through the polymerization of actin in the subacrosomal granule. 3. The acrosomal process delivers the inner acrosomal membrane to an egg microvillus, resulting in fusion. 4. Other microvilli now fuse with the incoming sperm and a large fertilization cone is formed that breaches the vitelline layer and engulfs the entire sperm, except most of the flagellum (refer to Figures 29-33 in Dufresne-Dubé et al., 1983). 5. A cortical granule reaction seals the vitelline layer which forms the fertilization membrane and provides a block to polyspermy (see review by Gilbert, 2003). It is likely that D. curvata and H. hanleyi that retain the same acrosome structure as L. asellus, would fertilize in a similar way.

Chitonida ̶ Chitonina Sperm Structure: The sperm of Chitonina, like all Chitonida, are dart-shaped with a needle- like extension to the nucleus that terminates in a tiny acrosomal vesicle (see review by Buckland- Nicks, 1995). In species such as apiculata the acrosome contains a single granule of uniform density (Figs. 16, 17). The mid-piece usually comprises four to seven mitochondria distributed asymmetrically around the centrioles. The centrioles are fused to form a dense basal body, which is eccentric to the main axis of the sperm (Fig. 17). The portion of the basal body that corresponds to the distal centriole, is harnessed to the annulus by the centriolar satellite complex, and gives rise to the flagellum posteriorly (Fig. 17). The plasma membrane adjacent to the annulus is expanded posteriorly to form a collar around the base of the flagellum (Fig. 17), which may be large in some species. This collar of membrane is usually reinforced by dense granular material making it thicker in the region of the annulus (Fig. 17). This describes sperm Type 2 according to Buckland-Nicks (1995), as illustrated in Diagram 2. Fertilization in Chitons: Morphological Clues to Phylogeny 55

Diagram 1. Hypothetical mechanism of fertilization in Lepidopleurida. A. Mature sperm approaches egg with intact acrosome cone (AC), bullet-shaped nucleus (N), spherical mitochondria (M), proximal centriole (PC) and distal centriole (DC), giving rise to a central flagellum (F) [shown in part]. B. The sperm contacts the jelly layer (JL) and the acrosome reaction occurs, releasing digestive enzymes that create a hole in the jelly layer. This is accompanied by the polymerization of actin forming the acrosomal process (AP) and the rolling back of fused acrosome and plasma membranes (FM). C. A hole is then digested in the vitelline layer (VL) and the acrosomal process delivers the inner acrosomal membrane into contact with an egg microvillus and they fuse. More microvilli extend up through the breached vitelline layer and fuse with the sperm, creating a fertilization cone (FC). D. The fertilization cone engulfs the entire sperm, including nucleus, a portion of the flagellum, centrioles and mitochondria. The jelly layer begins to disintegrate (DJL). E. The sperm passes into the egg cortex, leaving just a portion of the flagellum behind (FP). Cortical granules (CG) are released by exocytosis, which transforms the vitelline layer into the fertilization membrane. This is later raised above the surface of the egg and blocks polyspermy. Not exactly to scale.

Egg Structure: Eggs of Chitonina typically have hulls elaborated into long spines with narrow bases (5-30 µm) (Sirenko, 1993). Most spines emanate from hexagonal bases, which interlock closely. Spine form is a good indicator of phylogenetic relationship in Chitonina (Eernisse, 1984; Sirenko, 1993). Within spines may vary from having petalloid tips in Chiton tulipa (Figs. 18, 19), to having hooks, as in C. nigrovirescens (Fig. 20). Among , spines usually have bifurcating tips, like Stenoplax conspicua (Figs. 21, 22) and Lepidozona mertensii (Sirenko, 1993). Brooding forms typically have reduced spines, which favours storage of more eggs in the confined space of the pallial grooves (Eernisse, 1984; Buckland-Nicks & Eernisse, 1992). Long spines have been found to dramatically slow sinking rates by six fold and thus keep 56 J. Buckland-Nicks

Diagram 2. Fertilization in Chitonina. A. Mature sperm arrives at hull between two spines (Sp), with intact acrosome vesicle (A), dart-shaped nucleus (N) with nuclear filament (NF), basal body (BB), collar (C), posterior spherical mitochondria (M) and flagellum (shown in part). B. After contact with the rough layer (RL) of the hull, the acrosome releases digestive enzymes that dissolve an area around the nuclear filament (arrow), as well as a pore through the hull’s fibrous layer (FL) and the vitelline layer (VL). The nuclear filament delivers the inner acrosomal membrane to an egg microvillus and they fuse creating a tube into the egg. C. Chromatin (Ch) is injected as a long thread into the egg cortex and a fertilization cone (FC) is formed beneath the vitelline layer. The vitelline layer is not breached and remains as a barrier to sperm organelles, which appear to be abandoned on the surface of the egg in a bag of membrane (arrowhead). Cortical granules (CG) are released by exocytosis into the perivitelline space.

spawned eggs suspended for longer in the water column (Buckland-Nicks, 1993). C. nigrovirescens is an exception, as it has eggs with long spines but it broods them to the crawl- away stage (Branch & Branch, 1981; Schwabe, 2000). When eggs touch against each other in the pallial grooves, the hooked spines interlock, thus binding them together in clusters, which keep them in the pallial grooves. However, females were observed also releasing individual eggs into petri dishes, which suggests that they have the ability to free-spawn. In this way females may be able to take advantage of the benefits of dispersal for some offspring, while brooding others to crawl-away juveniles. The extent to which this happens in nature needs to be more fully investigated. Fertilization in Chitons: Morphological Clues to Phylogeny 57

Diagram 3. Fertilization in Acanthochitonina. A. Mature sperm swim inside an open cupule with intact acrosome (A), dart-shaped nucleus (N) with long nuclear filament (NF), oblong lateral mitochondria (LM), as well as spherical posterior mitochondria (PM), basal body (BB) and a distinct fibrous complex (FC), posterior to the collar but adjacent to the flagellum (F). B. After contact with the base of the hull (H) the acrosome reaction releases digestive enzymes that create a small pore in the hull and vitelline layer (VL), through which the nuclear filament delivers the inner acrosomal membrane to an egg microvillus and they fuse. Following fusion, the chromatin (Ch) is injected into the egg. C. A fertilization cone has formed below the vitelline layer in the perivitelline space, leaving sperm organelles abandoned on the surface in a bag of membrane (arrowhead), as in Diagram 2. Cortical granules (CG) are released into the perivitelline space and later the fertilization membrane is raised. (see Buckland-Nicks et al., 1988)

The hull of most Chitonina, though much thinner than in lepidopleurids, is more resilient, because it does not deteriorate as quickly. It comprises a thin outer dense layer (0.1 µm thick), of mostly small tightly packed spheres overlying a lighter fibrous matrix, which is more robust within the spines than between them (Fig. 24). Total thickness of the hull between spines is about 1µm. Underneath the hull is a thin vitelline layer (0.2 µm thick), which overlies the egg membrane. During oogenesis, in some Chitonina, such as Stenoplax fallax, long microvilli project up between the hexagonal plates and can be seen between the spines (Eernisse personal communication). Retraction of these microvilli at maturity may create micropores between the spines that provide 58 J. Buckland-Nicks

Diagram 4. Fertilization in Callochitonidae. A. Mature sperm enters large pore (P) in jelly layer with an intact acrosome vesicle (A), dart-shaped nucleus (N) with long nuclear filament (NF). Oblong mitochondria (M) surround a basal body (BB) that gives rise to the flagellum (F). B. Acrosomal enzymes digest a pathway through the rest of the jelly layer as well as the vitelline layer (VL), creating a small pore, through which the nuclear filament delivers the inner acrosomal membrane to an egg membrane cup (MC). Following fusion a fertilization cone (FC) is raised beneath the vitelline layer and chromatin (Ch) is injected into the egg . The vitelline layer prevents sperm organelles from entering the egg and they remain abandoned on the surface in a bag of membrane (arrowhead). Cortical granules (CG) undergo exocytosis releasing their contents into the perivitelline space, which later causes the fertilization membrane to be raised (see Buckland-Nicks and Hodgson, 2000). more direct pathways for sperm to reach the egg membrane beneath the vitelline layer. Fertilization: Sperm penetration of the egg in most Chitonina occurs between spines, usually at the junctions of their hexagonal bases (Figs. 19, 22). When a sperm comes close to the hull, the acrosome reaction occurs and the dense outer layer of the hull is dissolved in an area four or five times the diameter of the nuclear filament (Figs. 23, 25, 26). The nuclear filament then penetrates the hull and the remaining acrosomal contents digest a minute hole in the vitelline layer, through which the nuclear filament is inserted. The final steps of fertilization are similar to those described later for C. dentatus and illustrated in Diagram 2. Fertilization in Chitons: Morphological Clues to Phylogeny 59

Diagram 5. Diagram 6.

Diagram 5. Phylogenetic tree redrawn from Okusu et al., (2003, fig. 7A) based on molecular data (but, for simplicity, omitting taxa that were not examined in the present study). Epimenia used as outgroup in both Diagram 5 and 6. Note Callochiton is placed with Leptochiton within Lepidopleurida (sensu Sirenko 1993, 1997). The position of Lepidozona was found to be inconsistent with some trees placing it within Chitonina but others (fig. 7B) near Mopalia within Acanthochitonina. Diagram 6. Updated analysis of Buckland-Nicks (1995) based on characters of sperm, eggs and general morphology. Modified to include new data on the genera Deshayesiella and Hanleya. (Species used in this study include: Epimenia australis (outgroup); Leptochiton asellus, Deshayesiella curvata, Hanleya hanleyi, Callochtion dentatus, , Lepidozona mertensii, Stenoplax conspicua, Chiton tulipa, Ischnochiton albus, Lepidochitona dentiens, Acanthochitona viridis, Tonicella lineata, and Mopalia muscosa). Key points to note are: 1). The separation of Chitonina (Chaetopleura to Ischnochiton) from Acanthochitonina (Lepidochitona to Mopalia). 2). The placement of Callochiton as sister group to all Chitonida (Chitonina + Acanthochitonina). 3). The clade Deshayesiella + Hanleya and the paraphyly of Leptochiton. Other trees (not shown) showed considerable paraphyly within Acanthochitonina. The future inclusion of accurate molecular sequences in this data matrix will further improve the resolution of these taxa (see Eernisse et al., in prep.; Okusu et al., in prep.).

Chitonida ̶ Acanthochitonina Sperm structure: Sperm Type 1, which is typical of Acanthochitonina (Diagram 3) have a distinctive fibrous complex located next to the flagellar axoneme, posterior to the mid-piece (Fig. 34). This is quite different from the thickening of the collar posterior to the annulus in Chitonina. The synapomorphy of the fibrous complex has been confirmed in the following families: Acanthochitonidae, Cryptochitonidae, Lepidochitonidae, , and Tonicellidae, but other Acanthochitonina have not been examined yet. There often are lateral mitochondria (Fig. 34), whereas in Chitonina mitochondria are usually all posterior to the nucleus. Compared to Chitonina, the acrosome of Acanthochitonina appears more complex in some species, such as Acanthochitona viridis (Fig. 33), containing two granules rather than the one observed in Chaetopleura apiculata (Fig. 16) and Callochiton dentatus (Fig. 11). It has not been possible yet to consistently reveal the ultrastructure of acrosomes, as fixation is often inadequate. Improved fixation techniques, such as freeze-substitution (Steinbrecht & Muller, 1987) need to be tried in order to elucidate any differences more clearly. Egg structure: Eggs of Acanthochitonina have hulls elaborated into wide based cupules, 50-90 µm in diameter, as seen in Tonicella undocaerulea [Tonicellidae] (Figs. 27, 28) and Lepidochitona dentiens [Lepidochitonidae] (Fig. 32). Cupules may be open, as in Tonicellidae and Mopaliidae (Fig. 27), or closed, as in Acanthochitonidae and most Lepidochitonidae (Fig. 32). An exception 60 J. Buckland-Nicks is Lepidochitona cinerea which has been shown to have open cupules at maturity (Eernisse 1984), not closed ones (Durfort et al., 1982; Okusu et al., 2003). The reason for this discrepancy, perhaps, is that chiton eggs with open cupules (such as Tonicella lineata), may appear closed when immature (see Buckland-Nicks et al., 1988). Retraction of egg microvilli during oogenesis has been shown to alter the integrity of hull and vitelline layer by creating pathways of least resistance, which sperm locate both in open cupule and closed cupule species (Buckland-Nicks et al., 1988; Buckland-Nicks & Eernisse, 1993). However, micropores are not necessarily created by this retraction of microvilli. For example, species with open cupules have not been observed to develop micropores inside cupules (Fig. 30), whereas some species with closed cupules do develop micropores (Buckland-Nicks & Eernisse, 1993). These observations were supported by Pashchenko & Drozdov (1998), who studied various other species in these groups. Exactly how the structure of the hull or acrosome affects fertilization is not completely understood. Fertilization: In open cupule species, sperm are attracted inside cupules to penetrate the hull (Figs. 29, 30). When eggs of Tonicella sp. or Mopalia muscosa were exposed to sperm concentrates, very few sperm were found between the cupules (Figs. 29, 30), yet microvilli were located in all regions (Fig. 28). This suggests that sperm chemoattractants, which have been demonstrated in chitons (Miller, 1977), are released from inside the egg cupules. Eggs with closed cupules, including Lepidochitona dentiens, restrict sperm to the intercupule area (Fig. 31), which is riddled with micropores (Buckland-Nicks, 1993). Sperm locating one of these micropores makes contact with a single egg microvillus and fusion occurs. Events following this are similar to Callochiton dentatus. These events are summarized in Diagram 3.

Callochitonidae Callochitonidae are unusual in the sense that they have retained plesiomorphic smooth eggs with pores like those of Deshayesiella curvata, yet their sperm are derived, like those of Chitonida. Current classifications disagree as to where to place this unique family (Sirenko, 1993, 1997; Buckland-Nicks, 1995; Okusu et al., 2003). This is discussed in detail in the final section entitled ‘On the Phylogeny of Chitons’. Sperm Structure: Sperm of Callochiton dentatus (syn.: C. castaneus Wood 1815) have an eccentric, needle-like extension to the oval nucleus, which is capped by a tiny acrosomal vesicle and is separated from it by a basal plate (Fig. 11; Buckland-Nicks & Hodgson, 2000), like all other Chitonida. During spermiogenesis, the centrioles fuse to form a dense basal body, in which individual centrioles cannot be distinguished easily, as occurs in all other Chitonida. This contrasts with Lepidopleuridae, in which centrioles are connected but remain distinct. In C. dentatus the flagellum and basal body are slightly eccentric and are surrounded by three spherical and two elongate mitochondria (refer to fig. 16 in Buckland-Nicks & Hodgson, 2000), whereas in C. septemvalvis all five mitochondria are spherical and are arranged symmetrically (Russel-Pinto et al., 1984). Thus, the sperm of C. dentatus is unlike lepidopleurid sperm, because all components are modified in ways that correspond with the pattern in Chitonida, with the exception of greater symmetry in the mid-piece than in most other species of this order. This sperm Type 3 is illustrated in Diagram 4. Egg Structure: Callochiton dentatus eggs are surrounded by a 20 µm thick jelly layer permeated by numerous large pores about 2 µm in diameter and spaced about 9 µm apart. Below the surface the pores enlarge to about 8 µm at their widest point (Figs. 12, 13), and extend down to the vitelline layer. Within each pore, fine fibres criss-cross and overlap in the middle (Fig. 12, Diagram 4). Each pore coincides with a cup in the egg membrane, below the vitelline layer, which has few microvilli (Buckland-Nicks & Hodgson, 2000). The description of the egg of Deshayesiella curvata (Pashchenko & Drozdov, 1998) is quite similar to the egg of C. dentatus, although the jelly layer of D. curvata eggs is four times thicker (80 µm) and pores are Fertilization in Chitons: Morphological Clues to Phylogeny 61 correspondingly further apart (30 µm). It remains to be seen if these eggs are truly homologous. Fertilization: When sperm meets egg in Callochiton dentatus, the sperm quickly locates a pore in the jelly layer and swims down to the surface of the egg, where release of acrosomal enzymes digests a hole in the vitelline layer (Figs. 36, 37). After the nuclear filament penetrates the vitelline layer, the inner acrosomal membrane fuses with the egg membrane at the base of a cup, probably with one of the sparse microvilli there (Buckland-Nicks & Hodgson, 2000). A narrow tube connecting sperm and egg is formed, which permits the injection of chromatin as a long thread, into the egg cortex (Fig. 37). More microvilli then fuse with the nucleus beneath the vitelline layer and a fertilization cone is formed in the perivitelline space (Figs. 35, 36). The release of cortical granules, as in other invertebrates, transforms the vitelline layer into the fertilization membrane and raises it, preventing polyspermy (Buckland-Nicks & Hodgson, 2000, and Fig. 35). The fertilization cone does not breach the vitelline layer, as it does in scaphopods (Dufresne-Dubé et al., 1983), some bivalves (Longo, 1987, 1993) and sea urchins (Epel, 1978; Longo, 1987). Rather, the sperm organelles, such as mitochondria and centrioles, are prevented from entering the egg cortex and appear abandoned in a bag of membrane on the egg surface (Fig. 37). This stage has been observed numerous times in this and other species but we cannot yet be certain, without confirmation using fluorescent markers, that sperm organelles never enter the egg. Another important point is that, since the centrioles are bound together as a basal body, which is in turn bound to the annulus on the plasma membrane in all Chitonida (Figs. 17, 34), these organelles probably do not function independently. In sea urchins, for example, the sperm proximal centriole contributes both to movement of pronuclei in the zygote, as well as to the formation of the first mitotic spindle (see review by Gilbert, 2003). These are functions that we expect for lepidopleurids, however, because each centriole is still distinct and can be expected to enter the egg at fertilization, along with the sperm mitochondria. It should be possible in the future, through the use of organelle specific probes, to determine precisely the fate of paternal centrioles and mitochondria in zygotes of both lepidopleurids and chitonids. Fertilization in Callochiton dentatus is summarized in Diagram 4.

On the Phylogeny of Chitons Diagram 5 redrawn from Okusu et al. (2003), but for simplicity omitting taxa that were not examined in this study, shows a consensus tree based on all their molecular data, which seems to be consistent with the analysis of Sirenko (1993) and Buckland-Nicks (1995), in that it identifies two suborders of Chitonida; Acanthochitonina and Chitonina. However, Okusu et al., found weak support for Acanthochitonina in their molecular analysis as there appear to be two other clades within this taxon, which would suggest that it is paraphyletic. Current analyses based on morphology alone, updated for this review (Diagram 6), still support keeping these two suborders intact. Members of Acanthochitonina [sensu Sirenko, 1993] are readily identifiable by several synapomorphic characters (see Eernisse et al., 2006 and Table 1). For example, Acanthochitonina all have wide based egg cupules and abanal gills (Eernisse, 1984; Sirenko, 1993) and their sperm have a fibrous complex on one side of the flagellum posterior to the mid-piece (Buckland-Nicks, 1995). None of these features occur in any Chitonina, to my knowledge. The thickening of the collar membrane posterior to the annulus in Chitonina is likely not homologous to the fibrous complex in the flagellum of Acanthochitonina, as may have been implied previously (Buckland- Nicks, 1995). In the same molecular analysis by Okusu et al. (2003), Lepidozona mertensii was an anomaly, being grouped in one tree near Stenoplax alata within Chitonina and in another tree with Mopalia muscosa within Acanthochitonina, partly because sperm characters were unavailable to resolve this dichotomy but also because the molecular data may not have been quite right for this group. Both sperm and egg structure of L. mertensii. have been examined in this laboratory (egg structure 62 J. Buckland-Nicks

Fig. 1. SEM of egg of Mopalia muscosa, sectioned with a Kesei microknife, showing sperm entry inside cupules. Nuclear filament of one sperm can be seen penetrating into egg cortex (arrowhead). Scale bar = 6 µm. Fig. 2. Close-up of sectioned egg of M. muscosa showing several sperm penetrating egg hull (arrows). One sperm reveals the injection of chromatin into the egg cortex (arrowhead). Note, as often happens, sperm nuclear filament bends before it locates a pathway through the vitelline layer. Scale bar = 2 µm. Fig. 3. Egg of M. muscosa with cupules removed. Note that majority of sperm are penetrating hull inside cupule. Few sperm between cupules are successful. Scale bar = 10 µm. Fig. 4. Sperm of Leptochiton rugatus showing elongate acrosome cone (A) on top of bullet-shaped nucleus (N). Scale bar = 1 µm. Fig. 5. Portion of acrosome cone of L. rugatus containing loose subacrosomal material (arrowhead). Scale bar = 0.2 µm. Fig. 6. Acrosome cone (A) of Deshayesiella curvata sperm is separated from nuclear filament (NF) by sub-acrosomal plate (arrowhead). Scale bar = 0.2 µm. Fig. 7. SEM of D. curvata sperm, showing acrosome (A) atop nuclear filament (NF). Note nuclear filament emerges eccentrically from body of nucleus. Spherical mitochondria (M) are visible posteriorly. Scale bar = 1 µm. Fertilization in Chitons: Morphological Clues to Phylogeny 63

Fig. 8. Sperm of Leptochiton asellus. Note: Nucleus (N), mitochondria (M), proximal (PC) and distal centriole (DC), which gives rise to flagellum. Scale bar = 1 µm. Fig. 9. Sperm of Hanleya hanleyi, showing how nuclear filament (NF) emerges eccentrically to rest of nucleus and terminates in the acrosome (A). Scale bar = 0.6 µm. Fig. 10. Portion of sperm of H. hanleyi, showing distinct subacrosomal plate (arrowhead) between acrosome cone (A) and nuclear filament (NF). Scale bar = 0.2 µm. Fig. 11. Portion of sperm of Callochiton dentatus showing subacrosomal plate (arrowhead), separating acrosome (A) from nuclear filament (NF). Scale bar = 0.1 µm. Fig. 12. 1 µm section of immature egg of C. dentatus stained with Richardson’s stain. Note intact jelly coat with pores (arrow) enclosed by layer of follicle cells (FC). Scale bar = 36 µm. Fig. 13. Portion of immature egg of C. dentatus showing pores in jelly coat (arrowheads) that give sperm access to vitelline layer (VL). Jelly coat is enclosed by a layer of follicle cells (FC). Egg membrane forms cups (MC) beneath each pore in the jelly coat. Scale bar = 10 µm. Fig. 14. SEM of egg of L. asellus after 10 mins exposure to sperm concentrate. Note absence of jelly layer; sperm are lying almost directly on vitelline layer. Scale bar = 50 µm. Fig. 15. Sperm of L. asellus interacting with egg surface. Unreacted sperm at top has short nuclear filament and acrosome, whereas sperm below has elongated filament (arrowhead), indicating acrosome reaction has occurred. Scale bar = 30 µm. 64 J. Buckland-Nicks

Fig. 16. Tip of sperm of Chaetopleura apiculata showing acrosome with single granule, separated from nuclear filament (NF) by basal plate (arrowhead). Scale bar = 0.2 µm. Fig. 17. Sperm of C. apiculata, showing bullet-shaped nucleus and start of nuclear filament (NF). Adjacent is the tip of another sperm showing the acrosome (A). Visible posterior to the nucleus is the basal body (BB) comprising fused proximal and distal centrioles. One spoke of the centriolar satellite complex (CS) can be seen connecting the distal centriole to the annulus (An), which is labelled on the opposite side. Mitochondria (M) are housed in a posterior swelling of the plasma membrane which is filled with glycogen granules (G). Extending from the annulus and visible on both sides of the collar (C) is a distinct thickening of the plasma membrane (arrowhead). Scale bar = 0.4 µm. Fig. 18. SEM of egg of Chiton tulipa showing elongate spines with petalloid tips. Scale bar = 100 µm. Fig. 19. Close-up of fertilized egg of C. tulipa showing several sperm penetrating hull (arrowhead) between petalloid-tipped spines. Scale bar = 10 µm. Fig. 20. Portion of egg of Chiton nigrovirescens, showing hooked spines. When two eggs touch, their hooks interlock and prevent them from separating. This occurs in the pallial grooves and is a novel adaptation to brooding. Females can also release some eggs individually to the plankton and perhaps benefit from the advantages of dispersal. Scale bar = 10 µm. Fertilization in Chitons: Morphological Clues to Phylogeny 65

Fig. 21. SEM of egg of Stenopolax conspicua, showing elongate spines with bifurcating tips. Scale bar = 50 µm. Fig. 22. Close-up of fertilized egg of S. conspicua showing several sperm penetrating hull between spines (arrowheads). Egg is characterized by spines with bifurcating tips (arrows). Scale bar = 10 µm. Fig. 23. Sperm penetrating hull of S. conspicua showing where rough surface layer has been dissolved making it smooth (arrows). Scale bar = 0.5 µm. Fig. 24. Section of egg of C. tulipa showing rough surface layer comprising small dense spheres (arrowhead). Inside the spines and above the vitelline layer (VL) is a region of loose fibres (large arrow). After sperm-egg fusion begins, cortical granules (CG) are released from beneath the egg membrane into the perivitelline space (PVS). Scale bar = 1 µm. Fig. 25. Light micrograph of fertilizing sperm (Sp) on egg of C. tulipa, showing that rough surface layer (RL) has been dissolved around penetrating sperm filament (arrowhead). Scale bar = 5 µm. Fig. 26. Surface of egg of S. conspicua showing region of rough surface layer (RL) of hull that has been dissolved (arrowhead) by fertilizing sperm (Sp). Scale bar = 1 µm. 66 J. Buckland-Nicks

Fig. 27. SEM of egg of Tonicella undocaerulea showing open cupule type. Scale bar = 50 µm. Fig. 28. SEM of egg of T. undocaerulea with some cupules removed by rolling on sticky tape. Revealed are the patterns of microvilli beneath the hull and vitelline layer, which can be seen to correspond to open channels inside cupules (arrowheads), as well as regions between cupules (arrow). Scale bar = 44 µm. Fig. 29. Close-up of egg of T. undocaerulea showing a fertilizing sperm inside hull cupule (arrowhead). Sperm lying between cupules did not penetrate the hull. Scale bar = 5 µm. Fig. 30. Four sperm penetrating the hull inside a cupule of the egg of Mopalia muscosa. Micropores in the hull are not evident; rather each sperm digests its own pathway into the hull. Scale bar = 5 µm. Fig. 31. Fertilized egg of Lepidochitona dentiens, showing sperm penetrating hull in narrow region between closed cupules (arrowheads). Scale bar = 10 µm. Fig. 32. Egg of L. dentiens showing closed cupule type. Scale bar = 50 µm. Fertilization in Chitons: Morphological Clues to Phylogeny 67

Fig. 33. Section of entire nuclear filament of sperm of Acanthochitona viridis, showing acrosome at tip (arrowhead). Scale bar = 0.3 µm. Inset: Acrosome (A) separated from nuclear filament by basal plate (arrowhead). Scale bar = 0.1 µm. Fig. 34. Sperm of A. viridis, showing bullet-shaped portion of nucleus (N), lateral and basal mitochondria (M), basal body (BB), and fibrous complex (FC) on one side of flagellum. Scale bar = 4 µm. Fig. 35. Light micrograph of sperm penetrating egg of Callochiton dentatus. Jelly layer has been dissolved. A successful sperm (arrowhead) has penetrated the vitelline layer (VL) and fused with the egg membrane, which has raised a fertilization cone (FC) around it. Scale bar = 6 µm. Fig. 36. SEM of C. dentatus egg in similar region as shown in Fig. 35. A successful sperm has caused a fertilization cone (FC) to be raised beneath vitelline layer (VL). Only a small pore is made in the vitelline layer to permit the nuclear filament through (arrowhead). This prevents, at least at this stage, the engulfment of the entire sperm. Scale bar = 1 µm. Fig. 37. TEM C. dentatus egg in similar region to Figs. 35 and 36, showing successful sperm which has made a small pore (P) in the vitelline layer and has injected a long strand of chromatin into the egg cortex (small arrows). What remains is a bag of membrane on the surface of the egg that contains the mitochondria (M), centrioles and flagellum. Scale bar = 1 µm. 68 J. Buckland-Nicks also examined by Eernisse, 1984) and were found to be very close to that of Stenoplax conspicua (Buckland-Nicks unpublished observations), suggesting the former option, that Lepidozona sp. is within Chitonina. Callochitonidae has been placed within the Chitonida in most previous classifications (see review by Okusu et al., 2003) and basal within Chitonida by Buckland-Nicks (1995). More recently Buckland-Nicks & Hodgson (2000) suggested that it: “may warrant separation as a sister taxon to the suborders Chitonina and Acanthochitonina” (i.e. Chitonida), based on plesiomorphic egg morphology and derived sperm morphology. Okusu et al. (2003) found strong support from their molecular analysis for placing Callochitonidae as a sister taxon to Lepidopleuridae, outside the Chitonida (Diagram 5). The present morphological analysis (Diagram 6) is clearly in disagreement with this, placing Callochitonidae as sister taxon to Chitonida, because several features of Callochiton sperm are synapomorphies for all Chitonida (including: elongate nuclear filament; acrosome reduced to a small vesicle; absence of subacrosomal material; centrioles fused to form a single basal body and slightly acentric; mitochondria not all spherical or symmetrical. See figures 15, 16 in Buckland-Nicks & Hodgson, 2000). This analysis is supported by recent preliminary results from 16S rDNA (D. J. Eernisse, personal communication) which show that Callochiton dentatus groups closer to other members of Chitonida, and not with any Lepidopleuridae. Okusu et al. (2003) did not examine the 16S rDNA gene in Callochiton sp., although it was used for other genera in their analysis. The fact that the eggs of C. dentatus are more like those of Deshayesiella curvata, may mean that C. sp. retained some features of the plesiomorphic egg design but is a descendant of a chitonid ancestor that had already evolved a new mechanism of sperm entry; the one that now characterizes all Chitonida. The current analysis (Diagram 6) shows Leptochiton to be paraphyletic to a clade including Deshayesiella and Hanleya. However, this is mainly due to coding of sperm and egg features as intermediate, which may not be accurate. Eernisse’s 16s rDNA analysis finds that there is strong support for a monophyletic Lepidopleuridae (currently including, Leptochiton, Lepidopleurus, Deshayesiella, and Oldroydia). The order Lepidopleurida [sensu Sirenko 1993] or the suborder Lepidopleurina [sensu van Belle 1983] quite likely are paraphyletic taxa, which could be resolved by molecular analysis of other lepidopleurids not yet examined such as, Nierstraszella, Ferreiraella and Hanleya.

Acknowledgements

Thanks are due to the following colleagues who collected ripe species of chitons for me, or helped identify them: Boris Sirenko, Russia (for Deshayesiella curvata, Leptochiton rugatus and Tonicella undocaerulea); Ross Mayhew, Canada (for Hanleya hanleyi); Mike Sturrock, Scotland (for Leptochiton asellus); and Alan Hodgson, South Africa (for Callochiton dentatus and Chiton tulipa). Thanks also to D.J. Eernisse for helpful criticisms with phylogeny. This research was supported by a Hugh Kelly Fellowship, Rhodes University, South Africa and by an NSERC of Canada Discovery Grant to JB-N.

References

Al-Hajj, H. A. 1987. Ultrastructural study of spermiogenesis in the chiton Acanthopleura haddoni from the Jordan Gulf of Aqaba (Red Sea). International Journal of Invertebrate Reproduction and Development 12: 295-306. Branch, G. M. & Branch, M. 1981. The Living Shores of Southern Africa. 272 pp. Struik, Cape Town. Buckland-Nicks, J. 1993. Hull cupules of chiton eggs: parachute structures and sperm focusing devices? Biological Bulletin, Marine Biological Laboratory Woods Hole 184: 269-276. Buckland-Nicks, J. 1995. Ultrastructure of sperm and sperm-egg interaction in Aculifera: implications for molluscan phylogeny. In: Jamieson, B. G. M., Ausio, J. & Justine, J.-L. (eds.), Advances in Spermatozoal Phylogeny and , pp. 129-153. Mémoirs du Muséum National d’Histoire Fertilization in Chitons: Morphological Clues to Phylogeny 69

Naturelle, Paris 166: 129-153. Buckland-Nicks, J., Koss, R. & Chia, F.-S. 1988a. The elusive acrosome of chiton sperm. International Journal of Invertebrate Reproduction and Development 13: 193-198. Buckland-Nicks, J., Koss, R. & Chia, F.-S. 1988b. Fertilization in a chiton: acrosome-mediated sperm-egg fusion. Gamete Research 21: 199-212. Buckland-Nicks, J., Koss, R. & F.-S. Chia 1990. Spermiogenesis in Polyplacophora, with special reference to acrosome formation. Zoomorphology 109: 179-188. Buckland-Nicks, J. & Eernisse, D. J. 1992. Ultrastructure of mature sperm and eggs of the brooding hermaphroditic chiton, Lepidochitona fernaldi Eernisse 1986, with special reference to the mechanism of fertilization. Journal of Experimental Zoology 265: 567-574. Buckland-Nicks, J. & Hodgson, A. N. 2000. Fertilization in Callochiton castaneus (). Biological Bulletin 199: 59-67. Dufresne-Dubé, L., Picheral, B. & Guerrier, P. 1983. An ultrastructural analysis of Dentalium vulgare (Mollusca: Scaphopoda) gametes with special reference to early events at fertilization. Journal of Ultrastructure Research 83: 242-257. Durfort, M., Bargalló, B., Bozzo, M. G., Fontarnau, R. & López-Camps, J. 1982. Relationship between follicular cells and oocytes of Trachydermon cinereus Thiele (Mollusca, Polyplacophora). Malacologia 22: 211-217. Eernisse, D. J. 1984. Lepidochitona Gray, 1821 (Mollusca: Polyplacophora), from the Pacific coast of the United States: systematics and reproduction. Ph.D. Thesis, University of California at Santa Cruz. Eernisse, D. J., Buckland-Nicks, J. & Hodgson, A. N. 2006. Phylogeny of chitons based on morphology. Venus 65: 169-170. (Abstract) Eernisse, D. J. & Reynolds, P. D. 1994. Polyplacophora. In: F. W. Harrison (ed.), Microscopic Anatomy of Invertebrates, vol. 5 Mollusca 1. pp. 55-110. Wiley-Liss, New York. Epel, D. 1978. Mechanisms of activation of sperm and egg during fertilization of sea urchin gametes. Current Topics in Developmental Biology 12: 185-246. Gilbert, S. F. 2006. Developmental Biology. 8th edition. 817 pp. Sinauer Assoc., New York. Hodgson, A. N., Baxter, J. M., Sturrock, M.G. & Bernard, R. T. F. 1988. Comparative spermatology of 11 species of Polyplacophora (Mollusca) from the suborders Lepidopleurina, Chitonina and Acanthochitonina. Proceedings of the Royal Society of London, Ser. B 235: 161-177. Longo, F. J. 1987. Fertilization. 183 pp. Chapman and Hall, New York. Longo, F. J., Mathews, L. & Hedgecock, D. 1993. Morphogenesis of maternal and paternal genomes in fertilized oyster eggs (Crassostrea gigas): Effects of cytochalasin B at different periods during meiotic maturation. Biological Bulletin 185: 197-214. Miller, R. L. 1977. Chemotactic behavior of the sperm of chitons (Mollusca: Polyplacophora). Journal of Experimental Zoology 202: 203-212 Okusu, A., Schwabe, E., Eernisse, D. J. & Giribet, G. 2003. Towards a phylogeny of chitons (Mollusca, Polyplacophora) based on combined analysis of five molecular loci. Organisms, Diversity & Evolution 3: 281-302. Pashchenko, S. V. & Drozdov, A. L. 1998. Morphology of gametes in five species of far-eastern chitons. Invertebrate Reproduction & Development 33: 47-56. Russell-Pinto, F., Azevedo C. & Oliveira, E. 1984. Comparative ultrastructural studies of spermiogenesis and spermatozoa in some species of Polyplacophora (Mollusca). Invertebrate Reproduction & Development 7: 267-277. Saito, H. 1994. The shallow water chiton fauna of Eastern Hokkaido, Japan. Memoirs of the National Science Museum, Tokyo 27: 93-104. Saito, H. 1995. The chiton fauna of Onagawa bay, Northeastern Honshu, Japan. Memoirs of the National Science Museum, Tokyo 28: 99-112 Saito, H. 1996. Seven new species of the genus Parachiton (Polyplacophora: Leptochitonidae) from the Northwest Pacific. Venus (Japanese Journal of Malacology) 55: 161-187. Saito, H. 1997. Deep-sea chiton fauna of Suruga Bay (Mollusca: Polyplacophora) with descriptions of six new species. National Science Museum Monographs (12): 31-58. Saito, H. 1998. The chitons (Mollusca: Polyplacophora) of the Goto Islands, West Coast of Kyushu, Japan. Memoirs of the National Science Museum, Tokyo 31: 145-163. Sakker, E. R. 1984. Sperm morphology, spermatogenesis and spermiogenesis of three species of chitons (Mollusca, Polyplacophora). Zoomorphology 104: 111-121. Schwabe, E. 2000. Chiton nigrovirescens de Blainville 1825 (Mollusca: Polyplacophora: Chitonidae) ̶ eine gut bekannte brutpflegende Art von Süd-Afrika. Club Conchylia Informationen 32: 63-67. Sirenko, B. I. 1993. Revision of the system of the order Chitonida (Mollusca: Polyplacophora) on the 70 J. Buckland-Nicks

basis of correlation between the type of gills arrangement and the shape of the chorion processes. Ruthenica 3: 93-117. Sirenko, B. I. 1997. The importance of the development of articulamentum for taxonomy of chitons (Mollusca, Polyplacophora). Ruthenica 7: 1-24. Sirenko, B. I. 2000. A new and unusual species of Parachiton (Mollusca: Polyplacophora) from South Africa. African Zoology 35: 93-98. Steinbrecht, R. A. & Muller, M. 1987. Freeze-substitution and freeze-drying. In: Steinbrecht, R.A & Zierold, K. (eds.), Cryotechniques in Biological Electron Microscopy, pp. 149-172. Springer Verlag, New York. Strack, H. L. 1992. The Polyplacophora of the Red Sea. Journal of the Malacological Society of Australia 14: 1-40. Van Belle, R. A. 1983. The systematic classification of the chitons (Mollusca: Polyplacophora). Informations de la Société Belge de Malacologie 11: 1-164.

(Received March 30, 2004 / Accepted October 1, 2004)

多板類の受精：系統関係を解く形態学的手がかり

J. バックランド‐ニックス

要 約

多板綱の最大の目であるクサズリガイ目 Chitonida [sensu Sirenko, 1993] の共通祖先は，特異な形態の 卵膜を発達させた卵と頭部が糸状に伸長した精子による独特の受精メカニズムを発達させ，この目の 特徴となっている。一方，Leptochiton asellus とそのほかのサメハダヒザラガイ目 Lepidopleurida [sensu Sirenko, 1993] では，祖先形態である平滑な卵膜を持つ卵と突起した先体を持つ精子が遺存している。L. asellus の受精メカニズムは，精子と卵のデザインの基本的な類似性から，堀足綱やその他の軟体動物に 類似したものと思われる。その受精過程は，まず先体反応により酵素が放出されてゼリー層と卵黄膜に 大きな穴があけられる。その後，先体反応のプロセスにより先体の内膜が重合して伸長し，卵の微絨毛 と融合する。さらに，受精丘が卵黄膜より盛り上がり，精子が，その核・中心体・ミトコンドリアおよ び鞭毛の一部も含めて吸収される。 サメハダヒザラガイ目から，発達した卵膜を持つ卵と縮小した先体の精子を持つクサズリガイ目へと 移行するにあたって，その中間段階としての特徴をもつカギヅメヒザラガイ Deshayesiella curvata とハン レイヒザラガイ Hanleya hanleyi は，平滑な卵膜の卵と短い核質の先端部の上に小さな先体を備えた精子 を持っている。さらに，ハチノスヒザラガイ属の 1 種，Callochiton dentatus では興味深いことに，カギヅ メヒザラガイと同様の卵を遺していながら精子はクサズリガイ目の種と同様の特殊化をしている。その 受精過程はクサズリガイ目のパターンに沿っており，微小な先体によって卵黄膜に小さな穴があけられ 染色質だけが卵内に注入される。受精丘が卵黄膜下で形成されるが，精子の本体が吸収されることはな い。精子の細胞小器官は精子の膜に包まれたまま卵表面に遺されているものと思われる。このことから， クサズリガイ目では中心体もミトコンドリアと同様に母系的に伝えられていることが示唆される。 上記のハチノスヒザラガイ属の 1 種，カギヅメヒザラガイ，ハンレイヒザラガイに新たに見られ るこれらの形質から，これまで考えられてきた系統関係を以下のように見直すことができる。ハリ ハダヒザラガイ科 Callochitonidae はクサズリガイ目全体からの姉妹群とされ，サメハダヒザラガイ目 Lepidopleurida [sensu Sirenko 1993] には含まれない。さらに，サメハダヒザラガイ目は側系統群となり， クサズリガイ目はより明瞭にクサズリガイ亜目 Chitonina とケハダヒザラガイ亜目 Acanthochitonina の 2 つの亜目に区分することができる。