Spermiogenesis in Aplacophora (Mollusca) provides insight into evolution of Bilateria

Spermiogenesis in Aplacophora (Mollusca) provides insight into evolution of Bilateria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Spermiogenesis in Aplacophora (Mollusca) provides insight into evolution of Bilateria John Buckland-Nicks, Alan N. Hodgson This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4902173/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Dec, 2024 Read the published version in Zoomorphology → Version 1 posted 16 You are reading this latest preprint version Abstract Sperm structure among species in each Class of Aplacophora is highly conserved but between the two Classes is radically different. This together with numerous morphological differences between the two groups, suggests a long separation and a likely paraphyletic relationship, which has been suggested in the past but is not supported by molecular analyses. All Caudofoveata examined have unique externally-fertilizing ect-aquasperm found nowhere else in the Animal Kingdom. Solenogastres fertilize internally with introsperm like those described for Epimenia australis but differ in details of length and number of specific components, providing insights to relationships among them. Furthermore, the solenogaster introsperm shares at least nine characters with introsperm of the bilaterian lineage Nemertodermatida, but shares none of these characters with the sperm of Caudofoveata, Polyplacophora (chitons), or Xenoturbellida and few with Acoela. However, a recent re-analysis of molecular data points to the re-organization of Xenacoelomorpha due to Long Branch Attraction and its separation into a basal Nemertodermatida plus a “Xenacoela” clade related to Ambulacraria. If the shared sperm characters of Solenogastres and Nemertodermatida are plesiomorphies not homoplasies, this would provide support for phylogenies that place Solenogastres basal to Mollusca. If true, then basal Bilateria would have a direct link with Protostomia. Recent discoveries of minute worm-like bilaterian body and trace fossils in Ediacaran deposits suggest that earliest bilaterians were meiofaunal and preceded the great Cambrian explosion by millions of years. chaetoderms solenogasters nemertodermatids phylogeny sperm structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Aplacophora are a group of marine, benthic, worm-like molluscs, covered in aragonitic spicules that were proposed by some morphologists to be the sister group to Polyplacophora forming the Aculifera (Scheltema 1993 ; Scheltema et al. 1994 ); a conclusion since supported by molecular studies (Vinther et al. 2012 ; Kocot et al. 2019 ; Mikkelsen et al. 2019 ; Han et al. 2024 ). Aculifera is currently considered to be basal among extant Mollusca (Kocot et al. 2019 ; Mikkelsen et al. 2019 ). Aplacophora are divided into two distinct Classes, the Caudofoveata (= Chaetodermomorpha) and the Solenogastres (= Neomeniomorpha) that have major differences in structure, habits, and reproductive biology (Todt et al. 2008 ; Scheltema et al. 2008 ). This has led some authors to consider them to be paraphyletic (Salvini-Plawe1978; Salvini-Plawen and Steiner 1996 ; Haszprunar 2000 ; Vinther et al. 2012 ), but more recent molecular analyses have found them to be monophyletic (Kocot et al. 2019 ; Mikkelsen et al. 2019 ). Caudofoveata or ‘chaetoderms’ are infaunal and lack a foot, feeding on organisms in bottom sediments with a reduced radula (Scheltema et al. 1994 ; Todt et al. 2008 ). The body is divided into three segments, the anterium, trunk and posterium and the gut comprises a stomach, midgut and intestine. They have a pair of ctenidia in the posterior pallial cavity and the blood contains hemocyanin. Caudofoveata are dioecious, fertilizing externally with “ect-aquasperm” (for sperm terminology see Jamieson 1987 ). Sperm structure, before details were known, was thought to be similar to Polyplacophora (the chitons) (Franzén 1955 ; Hadfield 1979 ). However, this proved to be wrong, when a detailed study of one species, Chaetoderma argenteum Heath, 1911 (Buckland-Nicks and Chia 1989 ), revealed a series of unique features, as yet found nowhere else in the Animal Kingdom. Solenogastres are more uniformly cylindrical and crawl on a ciliated pedal groove that is considered to be a modified foot (Scheltema et al. 1994 ). They feed largely on Cnidaria (Scheltema et al. 1994 ; 2008 ), have an undivided gut and they lack ctenidia. They have an atrial sense organ which is absent in Caudofoveata. The blood contains a red pigment that may be a derivative of hemoglobin but they do not contain hemocyanin. Also, unlike Caudofoveata, Solenogastres are monoecious, copulate and fertilize internally with “introsperm” (Buckland-Nicks and Scheltema 1995 ). The sperm of one species of Solenogastres, Epimenia australis (Thiele, 1897), has been described in detail and exhibits a suite of characters shared with introsperm of several other animal groups, including the basal bilaterian taxon Nemertodermatida (Xenacoelomorpha) (Buckland-Nicks 1995 ; Buckland-Nicks and Scheltema 1995 ; Buckland-Nicks et al. 2019 ). These remarkably similar characters are considered by most to be homoplasies (Bieler et al. 2014 ), as they predict a different ancestry than that indicated by most recent molecular analyses (Cannon et al. 2016 ; Meyer-Wachsmuth and Jondelius 2016 ; Ruiz-Trillo and Paps 2016 ; Kocot et al. 2019 ; Mulhair et al. 2022 ; Yap-Chiongco et al. 2024 ). However, Redmond ( 2023 ) found that Acoelomorpha flatworm monophyly is a severe long branch-attraction artefact obscuring a clade of Acoela and Xenoturbellida, that he called “Xenacoela”, which is related to Ambulacraria. The link between Xenoturbellida (but not Acoela) and Ambulacraria was predicted by a re-analysis of sperm morphology of Xenoturbella bocki Westblad, 1949, which was shown to be very similar to hemichordate sperm (Buckland-Nicks et al. 2019 ). The separation of Acoela and Nemertodermatida also would agree with Wallberg et al. ( 2007 ) who, based on detailed analyses of 18S and 28S RNA, dismissed the taxon Acoelomorpha, finding that “Acoela and Nemertodermatida are separate clades”. Moreover, sperm structure of Nemertodermatida suggests that they are closer to the Urbilaterian with their typical 9 + 2 flagellum and linear arrangement of acrosome, nucleus and mitochondria which is not found in any Acoela (Tyler and Rieger 1975 ; Petrov et al. 2004 ; Buckland-Nicks et al. 2019 ). Sperm structure of Caudofoveata and Solenogastres is known in detail from only two species (Buckland-Nicks and Chia 1989 ; Buckland-Nicks and Scheltema, 1995 ) that do not represent the diversity of the families in these taxa. Nevertheless, the results from these studies suggest that there are vast differences in sperm structure between the two Classes indicating a long period of separation between them. The purpose of this paper is to compare and contrast spermiogenesis and sperm structure of representative species of several different families in both Classes and examine the phylogenetic implications of these findings in the light of recent morphological and molecular analyses of Aculifera, Xenacoelomorpha and related taxa. Materials and Methods Specimens of Caudofoveata from Family Chaetodermatidae: Chaetoderma canadense Nierstrasz, 1902 and C. argenteum Heath, 1911 were collected off the east and west coasts of Canada respectively in 1987 and 1992. Chaetoderma nitidulum Lovén, 1844; Falcidens crossotus Salvini-Plawen 1968; and from Family Limifossoridae: Scutopus ventrolineatus Salvini-Plawen, 1968, were collected off Blomsterdalen, Norway in 2007. Specimens of Solenogastres from Family Dondersiidae: Stylomenia sulcodoryata Handl and Salvini Plawen 2001; from Family Gymnomeniidae: Gymnomenia pellucida Odhner, 1920 and Wirenia argentea Odhner 1920; from Family Simrothiellidae: Simrothiella margaritacea (Koren and Danielssen, 1877); from Family Proneomeniidae: Dorymenia sarsii (Koren and Danielssen, 1877); from Family Simrothiellidae: a species of Helicoradomenia sp., Scheltema and Kuzirian with some digital micrographs was donated by Christiane Todt in 2007. Specimens of the gastropod Nerita polita Linnaeus, 1758 (Gastropoda) were collected near Kihei, Maui in 1987. for the purpose of comparing aspects of spermiogenesis with Solenogastres. The best fixation of tissues for transmission electron microscopy (TEM) was achieved by using a primary fixative of 2.5% glutaraldehyde in 0.45µm filtered seawater (FSW) and 0.2M sodium cacodylate buffer (pH 7.4) kept on ice for 2hr and allowed to warm to ambient temperature overnight. Tissues were washed in FSW / sodium cacodylate buffer and transferred to 1% osmium tetroxide in the same buffer on ice for 1hr. Tissues were dehydrated in an ethanol series, exchanged through propylene oxide and infiltrated then embedded in Epon/Spurrs mixed 1:1. Sections for light microscopy were cut at 1µm and stained with 1% toluidine blue (made to pH 9 with sodium bicarbonate). For transmission electron microscopy, serial thin sections with silver/gold interference colour were cut with a diamond knife (Diatome, Switzerland), picked up on nickel slot grids (EM Sciences), and gently placed on formvar windows in a rack (EM Sciences). Dried grids were picked up with fine forceps and stained with uranyl acetate (1hr) and lead citrate (10 min) before being examined in a Philips 410 TEM operated at 80kV and photographed with a Kodak L12C digital camera at 1200dpi. For scanning electron microscopy (SEM) of Chaetoderma argenteum sperm, “egg water” was made by mixing ripe eggs dissected from an ovary with 0.45µm filtered sea water and after 10 mins filtering the water again. Fresh sperm was dissected from testes and pipetted either into glass vials containing just 0.45µm filtered sea water, or into other vials containing “egg water”. Sperm from the two treatments were kept separate and pipetted into fine pore Flo-Thru vials (Pelco) and placed in glutaraldehyde fixative, as above. After 2hr vials were washed in several changes of 0.45µm filtered sea water before post-fixing for 1hr in 1% osmium tetroxide in 0.2M sodium cacodylate buffer. The vials were washed repeatedly in distilled water and then dehydrated in an ethanol series to 3 changes of 100%. Vials were then exchanged through amyl acetate and critical-point dried. Vials containing dried sperm were upended onto aluminium stubs coated with double-sided sticky carbon tabs. The stubs were sputter-coated with gold in an SPI-Module Sputter Coater and examined in a JEOL JSM 5300 SEM and photographed with Quartz PCI imaging at 1200dpi. Results The testes of all species studied produced only fertilizing spermatozoa (euspermatozoa). Caudofoveata The five species of Caudofoveata examined here have fundamentally similar sperm that are a type of “ect-aquasperm” (Fig. 1 A-E), which fertilize externally in sea water. Each sperm consists of a small pear-shaped head that contains a nucleus capped by an apical horn, apical dense tube (terminology after Buckland-Nicks and Chia 1989 ) and acrosome, a mid-piece of five spherical mitochondria that surround the orthogonally arranged proximal and distal centrioles, and a flagellum (Fig. 1 A) (Table 1 for some dimensions). When Chaetoderma argenteum sperm were immersed in egg water, the apical dense tube extended visibly about 1.5 times its original length (Fig. 1 B). The sperm of all species investigated are formed by the same basic process that was described in detail for Chaetoderma argenteum (Chaetodermatidae) by Buckland-Nicks and Chia ( 1989 ). Therefore, a single description only follows with a focus on Scutopus ventrolineatus because spermiogenesis has not been described in any species of this Family (Limifossoridae). Very early spermatids are typical of those of other molluscs (e.g. see Hodgson and Bernard 1988 ; Hodgson et al. 1988 ; Eckelbarger and Davis 1996 ) and therefore we have not illustrated these. However, they contain a spheroidal nucleus with heterochromatin, and the cytoplasm contains numerous small mitochondria, some rough endoplasmic reticulum, a Golgi body and a pair of centrioles. Early in spermiogenesis, the Golgi body secretes vesicles next to the plasma membrane (Fig. 2 A) which fuse to form the proacrosome. As spermiogenesis progresses, the proacrosome then migrates along the plasma membrane to what will be the anterior of the spermatozoon (Fig. 2 B). At the same time the numerous smaller mitochondria accumulate at the presumptive posterior of the developing sperm (not illustrated but see Fig. 3 A,B for diagrammatic illustration) and fuse to form five larger, spherical mitochondria surrounding the centrioles at the base of the nucleus (Fig. 2 C,D,E). In addition, the annulus attaches the distal centriole to the plasma membrane, via the centriolar satellites, and produces the distal flagellum (Figs. 2 E, 3 C). The Golgi body moves anteriorly to a depression on one side of the apex of the elongating nucleus where it begins to secrete vesicles that contribute to structures called the apical horn, the apical dense tube, as well as a large vacuole (Figs. 2 B, 3 C). The proximal centriole, which is positioned at the apex of the distal centriole nearest to the nucleus, develops a rootlet that attaches it to a slight depression in the nucleus (the nuclear fossa) that contains some electron-dense material (Figs. 2 E, 3 C). The proximal centriole develops the proximal flagellum, which passes anteriorly beside the mitochondria and nucleus and later extends beyond its tip (Fig. 2 C,D,H, 3 C). Occasional droplets thought to be lipid were observed in the cytoplasm of some spermatids (Fig. 2 F). In mid to late spermatids the chromatin in the spherical nucleus undergoes condensation becoming granular in appearance (Figs. 2 B,C,D, 3 C), then more homogeneously electron-dense (Fig. 2 E,F,H,I, 3 D). At the same time the nucleus elongates and begins to attain its final pear shape (Fig. 2 I, 3 D), the apical horn surrounds the pointed apex of the nucleus and extends into the developing apical dense tube (Fig. 2 F, 3 C,D). The apical dense tube elongates adjacent to the proximal flagellum (Fig. 2 H, 3 C) and extends over the large vacuole (Fig. 2 F,G,I, 3 C). The elongating apical dense tube eventually contacts the acrosome, the contents of which are not differentiated, on the plasma membrane and becomes fixed in position (Fig. 2 I, 3 C). Very late spermatids have also lost the apical flagellum by a mechanism we were unable to determine. Finally, the residual cytoplasm containing the Golgi body and large vacuole of each spermatid is released (not illustrated), freeing mature sperm as individuals. Details of these events are summarized diagrammatically in Fig. 3 (A-D). Table 1 Lengths (µm) of sperm components of different species of Caudofoveata and Solenogastres. ADT = apical dense tube; AH = apical horn; Mitoch = mitochondrial CAUDOFOVEATA Location Acrosome Nucleus Length Mid-Piece Mitoch Sheath Annulus Adjunct ADT AH Scutopus ventrolineatus Norway 0.5 2.5 X X X 2.4 1.5 Falcidens crossotus Norway 0.5 2.8 X X X 3.3 1.4 Chaetoderma argenteum Canada West Coast 0.5 2.0 X X X 3.0 1.7 Chaetoderma canadense Canada East Coast 0.5 1.6 X X X 2.5 1.5 Chaetoderma nitidulum Norway 0.5 1.5 X X X 2.5 1.3 SOLENOGASTRES Acrosome Nucleus Length Mid Piece Mitoch. Sheath Annulus Adjunct #Annulus Adjuncts Simrothiella margaritacea Norway 1.0 3.5 9.0 X X 2 Helicoradomenia sp. East Pacific Hydro. Vent 1.0 4.0 9.5 2.5 7.0 2 Dorymenia sarsii Norway 1.1 5.0 9.0 7.5 3.5 2 Epimenia australis (B-N & Scheltema 1995) Papua New. Guinea 1.3 4.5 9.0 8.0 1.0 2 Gymnomenia pellucida Norway 1.8 24.0 16.0 14.0 2.9 2 Wirenia argentea Norway 1.2 8.0 9.0 7.3 2.5 2 Stylomenia sulcodoryata Norway 1.7 7.0 13.0 11.3 1.7 4 Solenogastres Sperm structure All six species from five families of Solenogastres, have elongate introsperm that are similar in structure to that described in some detail for Epimenia australis by Buckland-Nicks ( 1995 ) and Buckland-Nicks and Scheltema ( 1995 ). The sperm head consists of an anteriorly positioned narrow conical acrosome, invaginated posteriorly atop a rod-shaped nucleus, containing highly condensed chromatin (Figs. 4 , 5 B and inset) and separated from it by a subacrosomal plate (Fig. 5 A). Posteriorly, the nucleus has a small invagination, the nuclear fossa, that houses the basal body (fused distal + proximal centrioles) (Figs. 4 , 5 B and inset). The distal centriole of this structure produces a long flagellum with a 9 + 2 arrangement of microtubules (Figs. 5 C,D,F), that tapers to singlet microtubules in the end-piece and ends in a terminal bulb (Fig. 5 G and inset). The mid-piece region consists of mitochondria that are arranged as a sheath around the axoneme (Figs. 4 , 5 B inset,C,E). A spiral ridge is formed between the mitochondria and axoneme (Figs. 6 C,D,F). Where the mid-piece meets the glycogen-piece there is an invaginated junction housing the annulus and anterior to it, the annulus adjuncts (Figs. 4 , 5 D,E) of which there are four in S. sulcodoryata and two in the other species (Table 1 ). The annulus adjuncts may extend anteriorly for several microns. Extending posteriorly from the annulus in the glycogen-piece for about 0.5 microns, are a series of coarse fibres overlying the axoneme (Figs. 5 E,F). Transverse sections of the acrosome, mid-piece, glycogen piece and end-piece are shown in Figs. 5 A,C,D,F,G and these correspond with specific sections drawn in the detailed summary diagram Fig. 8 D. Although the basic structure of the sperm is similar among all species studied, differences between species occur in the mid-piece structure, the number and length of the ‘annulus adjuncts’, and the length of various components such as the acrosome, nucleus, and mid-piece (Table 1 ). Spermiogenesis All stages of spermiogenesis were observed in the gonads of Stylomenia sulcodoryata and Gymnomenia pellucida , whereas only late stages were present in Helicoradomenia sp., Simrothiella margaritacea , Dorymenia sarsii and Wirenia argentea . However, the structure of these late spermatids indicates that the sperm of all species form in a very similar way and therefore a single description only follows. Very early spermatids are typical of those of the majority of Mollusca and are not illustrated. They have a spherical nucleus with heterochromatin and a cytoplasm containing numerous small mitochondria, a Golgi body, some rough endoplasmic reticulum, and a pair of centrioles. Early in spermiogenesis the distal centriole attaches to the plasma membrane via the annulus and then migrates to the base of the nucleus dragging the plasma membrane with it, thus creating a flagellar canal (Fig. 6 A). Spherical mitochondria aggregate at the base of the nucleus and fuse to form larger spherical mitochondria (Fig. 6 A). At the early to mid- spermatid stage when the nucleus is still spherical, the Golgi body secretes vesicles that fuse to form the proacrosome that migrates to the apex of the nucleus (Fig. 6 A,B), where it undergoes further elongation and is separated from the anterior nucleus by electron-dense material (Fig. 6 C). The Golgi body then separates and migrates posteriorly (Fig. 6 B), coming to lie near the mitochondria at the base of the nucleus. In this position, the Golgi body secretes numerous vesicles that coalesce to form a ‘spiral ridge’ of dense material around the naked axoneme (Fig. 6 B,C,D,F) from the distal centriole to the annulus at the mid-piece and glycogen-piece junction; a distance of about 9–16 microns in the mature sperm of the different species (Table 1 ). The mitochondria become oval-shaped when seen in mid-longitudinal section (Fig. 6 C,) and begin to elongate, extending posteriorly to wrap around the spiral ridge and axoneme (Figs. 6 F; 7 C), eventually forming the mitochondrial sheath of the mid-piece (Figs. 6 E, 7 B). At the same time the nucleus undergoes elongation (Figs. 6 C,E; 7 C) as the chromatin condenses (granular then fibrillar) with the posterior nuclear fossa deepening to house the basal body (= fused centrioles) (Figs. 7 B,C). The annulus breaks away from the distal centriole and migrates posteriorly to the invaginated junction between the mid-piece and glycogen-piece, eliminating the flagellar canal as it does so (Fig. 7 D). As the components of the late spermatids elongate and differentiate 6 distinct regions (acrosome, nucleus, mid-piece, annulus adjuncts, glycogen-piece and end-piece) are created (Figs. 5 A-F; 8D1-7) and residual cytoplasm (Fig. 6 D) is eliminated. At maturity the acrosome has become a narrow cone that is invaginated posteriorly and contains a diffuse subacrosomal granule within the subacrosomal space (Fig. 7 A). It is also separated from the tip of the dense rod-shaped nucleus by a subacrosomal plate (Fig. 7 A). The base of the nucleus is invaginated into the nuclear fossa that houses the “basal body” (Fig. 7 B) formed from the fused centrioles. Throughout spermiogenesis spermatids in a cohort are linked by cytoplasmic bridges (Fig. 6 B,D) so that they are at the same stage of development (e.g. Figures 5 B inset, 6E, 7C). The detailed events of spermiogenesis are summarized diagrammatically in Fig. 8 (A-D) Discussion Phylogenetic relationships among Aplacophora in relation to sperm structure and morphology Although most morphological (Scheltema 1993 ; 1996 ; Todt 2013) and molecular analyses (Kocot et al. 2011 ; Smith et al. 2011 ; Vinther et al. 2012 ; Kocot et al. 2019 ; Kocot et al. 2020 ) place Caudofoveata as sister taxon to Solenogastres, there are nevertheless significant morphological differences between the two groups of Aplacophora. The Caudofoveata lack a foot, have a body and gut divided into three parts, have a pair of ctenidia, hemocyanin in the blood, and are dioecious. By contrast, Solenogastres crawl on a foot, have an undivided cylindrical body, lack ctenidia and hemocyanin, and are monoecious. The results of this study and two previous studies (Buckland-Nicks and Chia 1989 ; Buckland-Nicks and Scheltema 1995 ) also show that the sperm of Caudofoveata and Solenogastres described to date are completely different. Whereas the Caudofoveata have a type of “ect-aquasperm” (terminology after Jamieson, 1987 ), the sperm of the Solenogastres are of the introsperm type. These dramatic differences in sperm structure between these sister taxa, as well as their many morphological differences, suggests a long evolutionary separation from each other and their common ancestor and begs the question first posed by Salvini-Plawen and colleagues: are these groups paraphyletic? (Salvini-Plawen 1978 ; Salvini-Plawen and Steiner 1996 ). Currently, three families are recognized within the Caudofoveata: Chaetodermatidae, Limifossoridae and Prochaetodermatidae. Sperm structure has now been described from species belonging to the first two. Although few morphological characters are shared between the Chaetodermatidae and Limifossoridae (Mikkelson et al. 2018) the structure of their sperm is remarkably conserved and is not found anywhere else in the Animal Kingdom. Because these species produce ect-aquasperm, it indicates that fertilization is external with gametes being released into the sea water. However, as far as we are aware, spawning has never been observed in any caudofoveate in the field. Recent molecular studies indicate that the Prochaetodermatidae are monophyletic and sister to the two other families (Mikkelson et al. 2019; Kocot et al. 2019 ). Although there is only one study on the reproductive biology of a prochaetodermatid (Scheltema, 1985 ), we suggest that they will also have ect-aquasperm. Studies on sperm structure from members of this family are now required to test this hypothesis. On sperm structure and fertilization in Caudofoveata The spermatozoa of Caudofoveata have two unique structures: the apical horn and an apical dense tube in the sperm head (Buckland-Nicks and Chia, 1989 and current study). Whilst spermiogenesis is basically similar to that described for other molluscs, there are unique features of spermiogenesis. The first, is the presence of a large apical vacuole in early to mid-spermatids that is closely associated with the developing apical dense tube and has been hypothesized to help guide its formation (Buckland-Nicks and Chia, 1989 ). The second is the formation of a flagellum from the proximal centriole that exits anteriorly adjacent to the developing apical dense tube possibly aiding in its development. The flagellum from the proximal centriole is subsequently eliminated leaving a uniflagellate mature sperm (Buckland-Nicks and Chia, 1989 and this study). Whilst the mechanism by which the proximal flagellum is eliminated is not known, it could occur by deflagellation (Quarmby 2004 ) or microtubule disassembly, as commonly occurs during cell division (Bloodgood 1994; Hu et al. 2015 ). Whilst the function of the apical dense tube of Caudofoveata during fertilization, is unknown, in Chaetoderma argenteum it elongates to 1.5x its normal length when exposed to egg water (Buckland-Nicks, 1995 ; also see Fig. 1 B), suggesting that it may be a unique mechanism for bridging the egg envelopes similar to the actin-based ‘perforatorium’ of other molluscs that is activated by the acrosome reaction (see review by Tilney, 2012 ). How fertilization could work: The simple acrosomal vesicle could operate like that of Chitonida and dissolve a pore in the egg envelopes through which the extending apical dense tube would project, delivering the inner acrosomal membrane into contact with the egg membrane. Following membrane fusion, the chromatin would be injected through the apical dense tube into the egg cortex. To this end, we have noted that the tip of the nucleus is pointed, where it inserts into the apical horn. Although speculative, this scenario would work based on known details of fertilization in other animals, including chitons (Buckland-Nicks 1995 ; 2006 ; 2008 ; Colwin and Colwin 1967 ; Summers et al.1975; Buckland-Nicks and Hodgson 2000 ; and see review by Tilney, 2012 ). It should be made clear that the reduced acrosome and needle-tipped nucleus of Chitonida are secondarily derived characters, since basal members of chitons, the Leptochitonidae have a typical acrosome, nucleus structure and fertilization mechanism (Buckland-Nicks 2008 ). On sperm structure in Solenogastres Including the current study, spermiogenesis and sperm structure in Solenogastres has been described from 7 species representing six of the 24 families (as stated by Kocot et al. 2019 ; Yap-Chiongco et al. 2024 ). All species have introsperm confirming internal fertilization in this class of hermaphrodite Aplacophora and spermiogenesis in all species is similar to that described by Buckland-Nicks and Scheltema ( 1995 ) for Epimenia australis . Thus, spermiogenesis described here, is highly conserved across the phylogenetic tree of Solenogastres as stated by Yap-Chiongco et al. ( 2024 ). The phylogeny of Solenogastres has undergone major re-organization in recent years with Order Cavibelonia being found to be paraphyletic (Kocot et al 2019 ; Yap-Chiongo et al. 2024). From the standpoint of sperm structure alone, Helicoradomenia , Simrothiella and Dorymenia all group in the former Order Cavibelonia (Kocot et al. 2019 ), as they have the shortest acrosomes and nuclei and longest annulus adjuncts. Epimenia , formerly included in Order Cavibelonia, is intermediate in acrosome and nucleus length and has the shortest annulus adjuncts and therefore fits better with others from Order Pholidoskepia, Gymnomenia , Stylomenia and Wirenia , which have the longest acrosomes and nuclei and shorter annulus adjuncts. There has been debate as to whether the earliest solenogasters were small and meiofaunal or more macrofaunal like their ‘predicted’ chiton ancestors (Kocot et al. 2019 ; Yap-Chiongco et al. 2024 ). Molecular analyses differ on this point. Maximum Likelihood analysis places the small bodied Meiomeniidae as basal, suggesting a meiofaunal ancestry. Whereas, Bayesian Likelihood Inference places the larger bodied Amphimeniidae as basal, more in keeping with a chiton ancestry (Yap-Chiongco et al. 2024 ). Analysis of genome size by these authors shows that solenogasters tend to have much smaller genomes (e.g.243Mbp for Neomenia ) than their sister group the Caudofoveata (e.g.2.45Gbp (= 2450 Mp) in Chaetoderma ) or the Polyplacophora (chitons) (2.84Gbp in Cryptochiton ). As Yap-Chiongco et al. ( 2024 ) said in some groups “genome size may co-evolve with life-history”, so in this case rather than supporting a larger bodied chiton ancestry, this could mean that small body size (smaller genome) is ancestral. Supporting this is our analysis of sperm structure, which shows that all solenogasters studied have complex introsperm, very similar to those of the basal bilaterian Nemertodermatida; whereas both Caudofoveata and chitons (Buckland-Nicks 2008 ) have derived and simpler ect-aquasperm. On sperm structure as a source of characters for phylogenetic inference Wilson ( 1925 ) stated that: “Sperms of different species exhibit remarkable differences in size, form and structure……that it is hardly an exaggeration to say that most species of animals might be identified by the morphology of their sperm alone”. Indeed, sperm structure has been used in many studies to infer phylogenetic relationships (review by Bartolomaeus et al. 2024 ) and in some to create detailed character analyses that improved existing phylogenetic trees (Buckland-Nicks 1995 ). However, there has always been the worry that functional constraints result in convergent evolution of similar sperm traits. Some recent studies tested this notion and found that functional constraints do not overpower historical signals in sperm of frogs and nemerteans (Cherneyshev 2021 ; Valchi et al. 2023 ; Bartolomaeus et al. 2024 ), which suggests that sperm ultrastructure may be used “as a valuable source of characters for phylogenetic inferences” (Bartolomaeus et al. 2024 ). Recently, Fitzpatrick et al. ( 2022 ) have opened a sperm character database, which should help enormously with future phylogenetic analyses of animals, in particular the early bilaterian groups. One problem with establishing an outgroup for sperm derived characters is that early Metazoa, such as sponges and Cnidaria, fertilize with ect-aquasperm but earliest bilaterians, perhaps similar to Nemertodermatida, may have already developed the complex introsperm. Thus, where ect-aquasperm are developed secondarily, in more recent taxa, there is the danger that a phylogenetic analysis using sperm characters will place them more basal than they should be, unless this is taken into consideration. The sperm structure and spermiogenesis of Solenogastres shares nine key characters (listed below) with sperm of the Order Nemertodermatida (Phylum Xenacoelomorpha). These features are not found in either the Acoela or the Xenoturbellida. Importantly, Redmond ( 2023 ) recently dismissed Acoelomorpha within Xenacoelomorpha, as an artefact of long branch attraction (LBA), which is in agreement with Wallberg et al. ( 2007 ). This discovery suggests that Nemertodermatida are likely basal among current Bilateria, a view long held by several other scientists (Tyler and Rieger 1975 , 1977 ; Buckland-Nicks and Scheltema 1995 ; Hejnol and Martindale 2008 ; Buckland-Nicks et al. 2019 ) and warrants further discussion below. On sperm structure and the origin of Bilateria: implications of the dismissal of Acoelomorpha Wallberg et al. ( 2007 ) stated (based on their detailed study of 18S and 28S RNA sequences): “Acoela and Nemertodermatida are separate bilaterian clades” and they dismissed the clade Acoelomorpha. However, the Phylum Xenacoelomorpha, which connects Xenoturbellida with Acoelomorpha, has been widely accepted as an important link to our bilaterian ancestry and is supported by numerous molecular analyses (Bourlat et al. 2003 ; 2006 ; Achatz et al. 2013 ; Cannon et al. 2016 ; Rouse et al. 2016 ; Philippe et al. 2019 ; Kapli and Telford 2020 ; Mulhair et al. 2022 ; Schiffer et al. 2022 , to name a few). The recent paper by Redmond ( 2023 ) once again sheds considerable doubt on Acoelomorpha instead supporting the conclusion that they are a paraphyletic group (Wallberg et al. 2007 ). Redmond ( 2023 ) found that LBA, as well as Compositional Bias and other phenomena have been corrupting the accuracy of molecular analyses and are responsible for the artefactual linking of Nemertodermatida with Acoela. Instead, he found that Xenoturbellida grouped with Ambulacraria (Redmond 2023 ), which supports morphological evidence from the re-analysis of sperm structure of Xenoturbella bocki by Buckland-Nicks et al. ( 2019 ), showing close similarity to the sperm of the hemichordate Schizocardium (Franzén 2001 ). The link between Xenoturbellida and Ambulacraria was found in several previous molecular analyses (Bourlat et al. 2003 , 2006 ; Philippe et al. 2011 ; Telford 2008 ; Nakano 2015 ; Mulhair et al. 2022 ; Schiffer et al. 2022 ), but all of these included Acoelomorpha within Xenacoelomorpha. Redmond ( 2023 ) still places Xenacoelomorpha within Deuterostomia. One problem with this is that although the molecular machinery for excretory system ultrafiltration is present in Xenacoelomorpha, they lack the Osr gene that is present in both Protostomia and Deuterostomia and which enables the production of discrete excretory organs like nephridia and nephrons (Abalde et al 2023 ), which are absent in all Xenacoelomorpha. Furthermore, most morphological and phylogenomic analyses support a sister relationship between Xenacoelomorpha and the rest of Bilateria (Nephrozoa hypothesis) (Jondelius et al 2019 ). Interestingly, Redmond ( 2023 ) found that Acoela formed a separate clade with Xenoturbellida, which he called the “Xenacoela”. This fits with larval development which is direct in both cases (Nakano et al. 2013 ) but not with sperm structure (Buckland-Nicks et al. 2019 ). The sperm of Nemertodermatida has a more plesiomorphic structure than Acoela, because of the arrangement of its component acrosome, nucleus, mid-piece, and typical single 9 + 2 flagellum (Tyler and Rieger 1975 , 1977 ; Petrov et al. 2004 ; Buckland-Nicks et al. 2019 ), which suggests that Nemertodermatida are basal within Xenacoelomorpha. This is supported by analyses of their development as Nemertodermatida exhibit a suite of characters that are considered plesiomorphic to Acoela, including: a more regulative cleavage pattern, superficial nerve net in the epithelium, epithelial digestive endoderm and lack of simple eyes (Hejnol and Martindale 2008 ; Achatz et al. 2013 ). Furthermore, Xenoturbella possesses a much larger gene complement of the Hox, Wnt, bHLH and GPCR families than acoels (Hejnol and Pang 2016 ), suggesting that acoels are more basal, which is supported by sperm structure (Buckland-Nicks et al. 2019 ). It is perhaps significant that Nemertodermatida has evolved nine specific sperm characters, that do not appear in the ect-aquasperm of any Radiata but are all found in the Solenogastres. 1. Small conical acrosome atop nucleus, 2. Rod-like condensed nucleus, in which condensation of chromatin progresses further into fibres before becoming uniformly dense, 3. Proximal and distal centrioles fuse to form a basal body which is housed in the nuclear fossa. 4. The annulus breaks away from the distal centriole and moves posteriorly everting the flagellar canal, 5. After producing the acrosome, the Golgi body migrates posteriorly to below the mitochondria and secretes the complex ‘spiral ridge’ around the axoneme, 6. The mitochondria fuse, elongate and partially or completely enclose the axoneme to form a mitochondrial sheath, 7. Annulus adjuncts extend anteriorly from the region of the annulus. 8. Coarse fibres overlying the outer microtubule doublets, project posteriorly from the annulus region into the tail. 9. The tail region contains glycogen granules as an energy reserve. Not all nemertodermatids share all nine characters but all solenogasters examined, do, suggesting that one or other character may have been lost in a particular nemertodermatid species or lineage but was retained in their descendants. Until now, these characters have been considered to be the result of convergent evolution (Bieler et al. 2014 ) but if Nemertodermatida are basal bilaterians they could be symplesiomorphies. Some of these characters are found in other basal molluscs with internal fertilization (but none with external fertilization), including the Neritimorpha (Gastropoda) in which the Golgi body choreographs a closely similar series of complex movements and secretions as in Solenogastres, to deposit first, the conical acrosome, and then the spiral ridge around the axoneme (Fig. 7 E) (see also: Buckland-Nicks and Chia 1986 ). If these sperm characters are homologous (i.e. symplesiomorphic) as we suggest, it again raises the question of whether there is a deep phylogenetic link between Nemertodermatida and Solenogastres as well as Protostomia in general (Buckland-Nicks and Scheltema 1995 ). Based on sperm structure alone, it reiterates arguments that Solenogastres are basal to Mollusca and that the caudofoveates and chitons evolved later, possibly re-developing ect-aquasperm by “progenetic spermiogenesis” (Buckland-Nicks 1995 ; Buckland-Nicks and Scheltema 1995 ); a concept that more recently has led to the discovery of a modern equivalent in molluscs (Winik et al. 2009 ). The Nemertodermatida and Solenogastres are largely microscopic, meiofaunal taxa, living in marine sediments. Interest in the meiofauna has increased markedly in the last twenty years (Giere and Schratzberger 2023 ; Worsaae et al. 2023 ) with trace fossils of earliest worm-like bilaterians ( Helminthoidichnites ) dating to before the Cambrian being found more often (Buatois and Mangano 2016; Parry et al. 2017 ; Gehling and Droser 2018 ). However, recently, Evans et al. ( 2020 ) discovered the earliest known worm-like bilaterian, Ikaria warioota , in trace fossils, as well as body fossils, that date back to the early Ediacaran in Southern Australia and persist through the Cambrian boundary. Based on the above arguments and our analysis of sperm structure, we support the conclusion that the Urbilaterian was a small, meiofaunal, flatworm-like organism with sexual reproduction by internal fertilization followed by direct development (Tyler and Rieger 1975 ; Buckland-Nicks and Scheltema 1995 ; Hejnol and Martindale 2008 ; Buckland-Nicks et al. 2019 ). Declarations Competing Interests: The authors have no competing interests to declare that are relevant to the content of this article. Funding: Supported by Natural Sciences and Engineering Research Council of Canada, grant 46205 to J.B-N. Author Contribution Both J.B-N. and A.N.H. contributed to the study conception and design. Material preparation, data collection and analysis were performed by [J.B-N]. The first draft of the manuscript was written by [J.B-N.] and both J.B-N. and A.N.H. commented on previous versions of the manuscript, read and approved the final manuscript. Acknowledgements Special thanks to Christiane Todt who supplied and identified almost all of the solenogasters and some of the chaetoderms from Norway. Amelie Scheltema (deceased) helped collect and identify specimens of C. canadense and C. argenteum . Thanks also to Moira Galway, Chair of the Biology Department at St. Francis Xavier University for use of TEM facilities. References Abalde S, Tellgren-Roth C, Heintz J, Vinnere Pettersson O, Jondelius U (2023). The draft genome of the microscopic Nemertoderma westbladi sheds light on the evolution of Acoelomorpha genomes. Front Genet, 14:1244493 Achatz JG, Chiodin M, Salvenmoser W, Tyler S, Martinez M (2013). The Acoela: on their kind and kinships, especially with nemertodermatids and xenoturbellids ( Bilateria incertae sedis). 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Proc R Soc Lond B 279:1259-1268 Wallberg A, Curini‐Galletti M, Ahmadzadeh A, Jondelius U (2007) Dismissal of Acoelomorpha: Acoela and Nemertodermatida are separate early bilaterian clades. Zool Scripta 36(5):509-523 Wilson EB (1925) The cell in development and heredity. Macmillan Winik BC, Catalan M, Gamarra-Luques C, Castro-Vazquez A (2009) Ectaquasperm-like parasperm in an internally fertilizing gastropod. Invert Biol 128:223-231 Worsaae,K, Vinther J, Sorensen MV (2023) Evolution of Bilateria from a meiofauna perspective – miniaturization in the focus. New Horizons in Meiobenthos. Springer Yap-Chiongco MK, Bergmeier FS, Roberts NG, Jörger KM, Kocot KM (2024) Phylogenomic reconstruction of Solenogastres (Mollusca, Aplacophora) informs hypotheses on body size evolution. Molec Phylogenet Evol, 194:108029 Additional Declarations No competing interests reported. Supplementary Files FrontCoverIllustration.pdf A. Scanning electron microscope image of the mature sperm of showing distal flagellum (f), mitochondrion, (m). nucleus (n), apical horn (ah), apical dense tube (adt), and acrosome (a). B. Scanning electron microscope image of the mature sperm of treated with egg water, showing dramatic elongation of apical dense tube (arrow). C,D,E. Diagrammatic representations of longitudinal sections through the spermatozoa of three species of Caudofoveata. acrosome (a); apical dense tube (adt); apical horn (ah); glycogen (gly); proximal centriole (pc); distal centriole (dc) attached to annulus by spokes; m, mitochondrion; nucleus (n) with lacunae; flagellum (f). Scale bars: A,B 1.0 µm; C,D,E 0.8µm (approx.). 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4902173","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":352255434,"identity":"79a53f9a-380f-4269-8e8d-6d3da15ec815","order_by":0,"name":"John Buckland-Nicks","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACxhlA3FDAwMAP5HwgQYsBA4NkAzOITQyQgGoxOECsFubZPYYfZxjY5RmfP3+wmYfBTp6ww+acMZbcYJBcbHYjmbFxBkOyYQNBLTNyzBgfGDAnbrvBzP7gA8MBRmK11Cdu7j/M2JDAcMCeOC0bDA4nbmBIZmwA2pJIWMucY8WSMwyOJ864kWzYOMMgOZmgFsPZzRs/9lRUJ/b3H3zYzFNhZ0tYC6oKA0LqgYBwNIyCUTAKRsEoAAD7Gz76OflLUQAAAABJRU5ErkJggg==","orcid":"","institution":"St Francis Xavier University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Buckland-Nicks","suffix":""},{"id":352255435,"identity":"4972fb87-fd61-4039-b505-ffc172a94c24","order_by":1,"name":"Alan N. Hodgson","email":"","orcid":"","institution":"Rhodes University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alan","middleName":"N.","lastName":"Hodgson","suffix":""}],"badges":[],"createdAt":"2024-08-12 17:10:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4902173/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4902173/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00435-024-00688-x","type":"published","date":"2024-12-05T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64301869,"identity":"32f8339b-ad58-4ad8-bd70-57c746f54170","added_by":"auto","created_at":"2024-09-11 11:54:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1152992,"visible":true,"origin":"","legend":"\u003cp\u003eA. Scanning electron microscope image of the mature sperm of \u003cem\u003eChaetoderma argenteum\u003c/em\u003eshowing distal flagellum (f), mitochondrion, (m). nucleus (n), apical horn (ah), apical dense tube (adt), and acrosome (a). B. Scanning electron microscope image of the mature sperm of \u003cem\u003eC. argenteum\u003c/em\u003e treated with egg water, showing dramatic elongation of apical dense tube (arrow). C,D,E. Diagrammatic representations of longitudinal sections through the spermatozoa of three species of Caudofoveata. acrosome (a); apical dense tube (adt); apical horn (ah); glycogen (gly); proximal centriole (pc); distal centriole (dc) attached to annulus by spokes; m, mitochondrion; nucleus (n) with lacunae; flagellum (f). Scale bars: A,B 1.0 µm; C,D,E 0.8µm (approx.).\u003c/p\u003e","description":"","filename":"AplacFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/dce3101fcc17e4860af7f15f.png"},{"id":64301873,"identity":"0cea2c90-066e-44f5-9575-713f1def0b36","added_by":"auto","created_at":"2024-09-11 11:54:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3164269,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscope images of some stages of spermiogenesis in \u003cem\u003eScutopus ventrolineatus\u003c/em\u003e (A,B,C,E,F,I) and \u003cem\u003eChaetoderma canadense \u003c/em\u003e(D,G,H).\u003c/p\u003e\n\u003cp\u003eA. Golgi vesicles (v) collecting near the plasma membrane and proacrosome (pa) in an early spermatid. Scale bar 0.2mm.\u003c/p\u003e\n\u003cp\u003eB. Mid-spermatids. In one spermatid the proacrosome (pa) is migrating along the plasma membrane close to the nucleus (n). In the second spermatid the Golgi body (G) has taken up a position adjacent to the developing apex of the nucleus (n) where it contributes vesicles (arrows) to the apical horn (ah) and a vacuole (va). Note the granular appearance of the condensing chromatin within the nucleus. Scale bar 0.3mm.\u003c/p\u003e\n\u003cp\u003eC. Oblique section through a mid-spermatid in which the centrioles (c) have produced both a proximal (pf) and distal (df) flagellum that extend anteriorly either side of the nucleus (n) and between the mitochondria (m). Scale bar 0.4mm.\u003c/p\u003e\n\u003cp\u003eD. Oblique section through a mid-spermatid showing proximal flagellum (pf) extending anteriorly beyond the nucleus (n). Centrioles (c) and mitochondria (m). Scale bar 0.8mm.\u003c/p\u003e\n\u003cp\u003eE. Longitudinal section through a mid-spermatid showing mitochondria (m) at base of nucleus (n) surrounding the orthogonally arranged proximal (pc) and distal (dc) centrioles. Note that the distal centriole, which is anchored to the annulus (an) by spokes, is producing the flagellum (f). The proximal centriole is located within the posterior nuclear fossa and anchored by electron-dense material (arrow). Scale bar 0.3mm.\u003c/p\u003e\n\u003cp\u003eF. Oblique section through the anterior region of a mid-spermatid showing the apex of the nucleus (n) and apical horn (ah) as well as the apical dense tube (adt) that is curving around the vacuole (va). Note the position of the proacrosome (pa) and the presence of a lipid droplet (L). Scale bar 0.4mm.\u003c/p\u003e\n\u003cp\u003eG. Higher magnification of an apical dense tube terminating in the acrosome (a), and its association with the vacuole (va). Scale bar 0.3mm.\u003c/p\u003e\n\u003cp\u003eH. Association of the apical dense tube (adt) and proximal flagellum (pf) in a late spermatid. Note position of apical horn (ah). Scale bar 0.8mm.\u003c/p\u003e\n\u003cp\u003eI. Longitudinal section through a late spermatid in which the chromatin of the nucleus (n) is almost fully condensed. The apical dense tube (adt) has extended as far as the acrosome (a) and is still lying over the vacuole (va). Only a small portion of the apical horn (ah) is visible in the section. Spherical mitochondria (m). Scale bar 0.8mm.\u003c/p\u003e","description":"","filename":"AplacFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/0954cbf34f5b7128f26e669a.png"},{"id":64301871,"identity":"8fce230b-de60-4b7f-bd95-f479c961069d","added_by":"auto","created_at":"2024-09-11 11:54:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1108718,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic representation of spermiogenesis in Caudofoveata.\u003c/p\u003e\n\u003cp\u003eA. In the early spermatid small mitochondria (m) are distributed throughout the cytoplasm along with some rough endoplasmic reticulum. The Golgi body (G) is producing vesicles that fuse to form the proacrosome (pa) on the plasma membrane. The distal centriole, with proximal centriole above it, has attached to the plasma membrane by the annulus (arrow). Chromatin in the nucleus (n) has begun condensation and at its base the nuclear membrane has thickened and depressed to form the nuclear fossa preparation for receiving the proximal centriole.\u003c/p\u003e\n\u003cp\u003eB. Mitochondria are migrating posteriorly and fusing to form larger spherical mitochondria around the centrioles (dc and pc) at the base of the nucleus (n). The distal centriole (dc) is producing the distal flagellum (df) which creates a temporary flagellar canal posterior to the annulus. The proacrosome (pa) is migrating anteriorly and the Golgi body is secreting vesicles that will form the large vacuole (va) and other structures.\u003c/p\u003e\n\u003cp\u003eC. The nucleus (n) has condensed, elongated and developed a pointed apex where it enters the apical horn (ah). Spherical mitochondria (m) surround the centrioles that are producing the distal (df) and proximal (pf) flagella that extend anteriorly. The Golgi body has taken up a position at the apex of the nucleus where it is completing the large vacuole (va) as well as producing other vesicles that contribute to forming the apical horn (ah) and apical dense tube (adt). The latter has contacted the acrosome (a).\u003c/p\u003e\n\u003cp\u003eD. Mature sperm with condensed chromatin containing some lacunae in the nucleus (n). The residual cytoplasm containing Golgi body and proximal flagellum has been eliminated. The distal flagellum now projects posteriorly between the mitochondria (m) and through a small flagellar collar (fc) containing glycogen granules, terminates in the tapered end-piece (ep). The apical horn (ah) extends into the elongate apical dense tube (adt) which terminates at the acrosome (a). Scale bar 0.6µm (approx.).\u003c/p\u003e","description":"","filename":"AplacFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/402c096335dc9d1dbbf16950.png"},{"id":64301872,"identity":"3ebcb5f1-9e13-461a-9cfd-1bd2eb301814","added_by":"auto","created_at":"2024-09-11 11:54:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1296649,"visible":true,"origin":"","legend":"\u003cp\u003eTEM of sperm of \u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLongitudinal sections of mature sperm of \u003cem\u003eS.\u003c/em\u003e \u003cem\u003esulcodoryata \u003c/em\u003ewith fully formed acrosomes (a) and elongate electron-dense nuclei (n) each with nuclear fossa, housing a basal body (bb); showing mid-piece (mp) formed by fused elongated mitochondria enclosing the axoneme. The annulus (an) is located at the junction between mid-piece and glycogen-piece (gp) and annulus adjuncts (aa) extend anteriorly from there. Scale bar 0.5mm\u003c/p\u003e","description":"","filename":"AplacFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/4d5d8fbebfb402affab902b3.png"},{"id":64301870,"identity":"ca749fe2-02d6-49a2-9ecf-74303124b772","added_by":"auto","created_at":"2024-09-11 11:54:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2419651,"visible":true,"origin":"","legend":"\u003cp\u003eTEMs of spermatozoa of Solenogastres.\u003c/p\u003e\n\u003cp\u003eA. Transverse section of mature sperm acrosomes of \u003cem\u003eStylomenia\u003c/em\u003e \u003cem\u003esulcodoryata\u003c/em\u003e with subacrosomal plate (sp) and subacrosomal granule (sg). Scale bar 0.2mm\u003c/p\u003e\n\u003cp\u003eB. Longitudinal section of mature sperm of \u003cem\u003eDorymenia sarsii \u003c/em\u003eshowing acrosome (a), electron-dense nucleus (n) and basal body in nuclear fossa (nf). Elsewhere a TS of the mid-piece shows the annulus adjuncts (aa) outside the mitochondrial sheath. Scale bar 0.3mm. \u003cstrong\u003eInset:\u003c/strong\u003eLS mature sperm of \u003cem\u003eHelicoradomenia \u003c/em\u003esp. showing acrosome (a), electron-dense nucleus (n) with nuclear fossa housing the basal body (bb) and the mitochondrial sheath (ms) of the mid-piece. Scale bar 0.7mm\u003c/p\u003e\n\u003cp\u003eC. Transverse section of mature sperm of\u003cem\u003e Dorymenia sarsii\u003c/em\u003e, showing sections of nucleus (n) one with the nuclear fossa (nf) and basal body; sections of mid-piece, showing mitochondria (m) and annulus adjuncts (aa); glycogen-piece (gp) and end-piece (ep) containing singlet microtubules. Scale bar 0.4mm\u003c/p\u003e\n\u003cp\u003eD.\u003cstrong\u003e \u003c/strong\u003eTransverse section of mature sperm of \u003cem\u003eStylomenia\u003c/em\u003e \u003cem\u003esulcodoryata \u003c/em\u003e, showing nucleus (n) and mid-piece at the level of the four annulus adjuncts (aa) [level 4 in Figure 8]. Scale bar 0.3mm\u003c/p\u003e\n\u003cp\u003eE.\u003cstrong\u003e \u003c/strong\u003eLongitudinal section of mature sperm of \u003cem\u003eStylomenia\u003c/em\u003e \u003cem\u003esulcodoryata\u003c/em\u003e at invaginated junction between mid-piece and glycogen-piece (arrow), containing the annulus (an) with coarse fibers (cf) extending posteriorly into the glycogen piece. The annulus adjuncts (aa) extend anteriorly around the axoneme (ax) into the mid-piece. Scale bar 0.4mm\u003c/p\u003e\n\u003cp\u003eF.\u003cstrong\u003e \u003c/strong\u003eTransverse section of glycogen-piece of \u003cem\u003eStylomenia\u003c/em\u003e \u003cem\u003esulcodoryata\u003c/em\u003e showing coarse fibers (cf) [at level 5 in Figure 8]; and end-piece (ep) of flagellum [posterior to level 7 in Figure 8]. Scale bar 0.2mm\u003c/p\u003e\n\u003cp\u003eG.\u003cstrong\u003e \u003c/strong\u003eLongitudinal section of tip of end-piece (ep) of \u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e tapering to the terminal bulb (tb), where doublet microtubules have reduced to zero. Scale bar 0.6mm. \u003cstrong\u003eInset:\u003c/strong\u003eTS of tapered end-piece of tail where axonemal doublet microtubules are reduced to singlets [posterior to level 7 in Figure 8]. Scale bar 0.1mm\u003c/p\u003e","description":"","filename":"AplacFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/9b32a8302b52246fd62d1ba5.png"},{"id":64301877,"identity":"dcce764a-f683-4a44-8291-9cfa8391e823","added_by":"auto","created_at":"2024-09-11 11:54:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3433868,"visible":true,"origin":"","legend":"\u003cp\u003eTEMs of spermiogenesis in Solenogastres.\u003c/p\u003e\n\u003cp\u003eA.\u003cstrong\u003e \u003c/strong\u003eRound spermatids of \u003cem\u003eGymnomenia pellucida\u003c/em\u003e, showing Golgi bodies (G) forming pro-acrosomes (pa). Note: mitochondria (m), proximal centriole (pc) in nuclear fossa (nf) and distal centriole (dc) forming flagellum and flagellar canal (fc). Scale bar 0.8mm\u003c/p\u003e\n\u003cp\u003eB. Round spermatid of \u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e in which the Golgi body (G) has moved posteriorly beside nucleus (n) and is secreting vesicles that coalesce to form the spiral ridge (sr) around the axoneme. Also note the cytoplasmic bridge (*) joining two of the spermatids. Scale bar 0.8mm\u003c/p\u003e\n\u003cp\u003eC. Mid-spermatid of \u003cem\u003eGymnomenia pellucida\u003c/em\u003e with elongate acrosome (a) and condensing nucleus (n) with nuclear fossa (nf) containing remnants of proximal centriole (pc). Note: oval mitochondria (m), distal centriole (dc), and axoneme (ax) with spiral ridge (sr). Scale bar 0.8mm\u003c/p\u003e\n\u003cp\u003eD. Mid-spermatid of \u003cem\u003eStylomenia sulcodoryata \u003c/em\u003eshowing elongate acrosome (a) atop nucleus (n) with condensed, fibrous chromatin. Golgi body (G) positioned posteriorly to the mitochondria (m) is releasing vesicles that coalesce to form the spiral ridge (sr) around the axoneme (ax). Note the cytoplasmic bridge (*) still linking two spermatids and elsewhere residual cytoplasm (rc) collecting beside the mid-piece. Scale bar 0.6mm\u003c/p\u003e\n\u003cp\u003eE. Late spermatids of \u003cem\u003eGymnomenia pellucida\u003c/em\u003e with chromatin forming fibres in the elongate nuclei (n) and mitochondria (m) spiralling around the axoneme to form the mid-piece. Scale bar 1.0mm\u003c/p\u003e\n\u003cp\u003eF. Spermatid of \u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e in which large oval mitochondria (m) below the nucleus (n) are wrapping around the completed spiral ridge (sr) on the axoneme (ax). Scale bar 0.6mm\u003c/p\u003e","description":"","filename":"AplacFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/bd7dd3dd2d399fa30317e6e3.png"},{"id":64301875,"identity":"1ae0b8f4-1500-4635-ab66-c5cb84de7484","added_by":"auto","created_at":"2024-09-11 11:54:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3104918,"visible":true,"origin":"","legend":"\u003cp\u003eTEMs of spermiogenesis in Solenoagastres and the gastropod \u003cem\u003eNerita polita\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA. Longitudinal section of mature sperm of \u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e showing conical acrosome (a) with subacrosomal granule (sg) and subacrosomal plate (sp) separating it from the electron-dense nucleus (n). Scale bar 0.2mm\u003c/p\u003e\n\u003cp\u003eB. Longitudinal section of late spermatids of \u003cem\u003eStylomenia sulcodoryata \u003c/em\u003eshowing electron-dense nucleus with nuclear fossa housing the basal body (bb). Axoneme with spiral ridge (sr) extends posteriorly inside the mitochondrial sheath (ms) of mid-piece. Scale bar 0.6mm\u003c/p\u003e\n\u003cp\u003eC Longitudinal section of late spermatids of \u003cem\u003eHelicoradomenia \u003c/em\u003esp., showing sections of electron-dense nucleus (n) with nuclear fossa (nf) housing the basal body. Mitochondria (m) are wrapping around the axoneme in mid-piece and long annulus adjuncts (aa) extend anteriorly from above the annulus (an) at mid-piece junction.\u003cem\u003e \u003c/em\u003eScale bar 0.8mm. I\u003cstrong\u003enset:\u003c/strong\u003e Transverse section of late spermatid of \u003cem\u003eHelicoradomenia\u003c/em\u003e sp. showing electron-dense nucleus in one spermatid and in an adjacent spermatid, mitochondria in mid-piece (m) [at levels 2\u0026amp;3 in Figure 8]. Scale bar 0.2mm\u003c/p\u003e\n\u003cp\u003eD. Longitudinal section of sperm of \u003cem\u003eGymnomenia pellucida\u003c/em\u003e at invaginated junctions (arrows) between mid-piece and glycogen-piece, showing annulus (an). Annulus adjuncts (aa) extend anteriorly. Scale bar 0.4mm\u003c/p\u003e\n\u003cp\u003eE. TEM close-up of mid-spermatid of the gastropod \u003cem\u003eNerita polita\u003c/em\u003e showing details of spiral ridge formation by the Golgi body (G) secreting vesicles towards individual ridges as they are deposited around the flagellum. Note: In this species, the annulus (arrow) does not break away from the distal centriole (dc) at this time, maintaining its attachment to the plasma membrane. The spiral ridge, therefore, binds the flagellar canal (fc) permanently around the axoneme. Scale bar 0.4mm\u003c/p\u003e","description":"","filename":"AplacFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/907e86d229b17ec9c99c1bb7.png"},{"id":64302535,"identity":"a56df58a-0e61-42d1-b040-3204c6bd67c0","added_by":"auto","created_at":"2024-09-11 12:02:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2591867,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic representation of details of spermiogenesis in \u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA. Round spermatid showing spherical nucleus (n) with uncondensed chromatin, many small mitochondria (m); distal centriole (dc) is attached to the plasma membrane by the annulus (an) and is producing the flagellum (f). The Golgi body (G) is secreting vesicles that coalesce to form the pro-acrosome (pa) attached to the plasma membrane.\u003c/p\u003e\n\u003cp\u003eB.\u003cstrong\u003e \u003c/strong\u003eThe pro-acrosome (pa) has migrated to the apex of the nucleus (n), that has begun condensation of chromatin. The proximal centriole (pc) has connected to the nuclear fossa at the base of the nucleus (n). The annulus (an) has separated from the distal centriole and migrated posteriorly with the elongating flagellum, everting the flagellar canal as it does so. Meanwhile the Golgi body (G) has moved to a position posterior to the fusing mitochondria (m) where it is secreting vesicles that coalesce to form the spiral ridge (sr) around the axoneme\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC. The acrosome (a) continues to elongate and invaginate posteriorly condensation of nuclear chromatin has progressed to fibre formation. The Golgi body (G) has completed the spiral ridge (sr) and moved anteriorly to where the residual cytoplasm will collect. Large oval mitochondria (m) are visible at the base of the nucleus. The nuclear fossa (nf) has deepened further to house the fused centrioles that become the basal body.\u003c/p\u003e\n\u003cp\u003eD. Mature sperm with elongate conical acrosome containing a diffuse sub-acrosomal granule (sg) and separated from the nucleus by a sub-acrosomal plate (sp).\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eTS 1: \u003c/em\u003eAcrosome. \u003cem\u003eTS 2:\u003c/em\u003e Nucleus. \u003cem\u003eTS 3:\u003c/em\u003e Mitochondrial sheath. \u003cem\u003eLS 2-3\u003c/em\u003e shows nuclear fossa (nf) housing basal body, mitochondrial sheath (ms) and start of spiral ridge (sr) around axoneme\u003cstrong\u003e. \u003c/strong\u003e\u003cem\u003eTS 4\u003c/em\u003e: Annulus Adjuncts (aa). \u003cem\u003eTS 5:\u003c/em\u003e Coarse fibres (cf) outside the axoneme. \u003cem\u003eLS 4-5\u003c/em\u003e shows invaginated junction (arrow) housing the annulus between mid-piece and glycogen-piece. Annulus adjuncts (aa) extend anteriorly \u003cem\u003eTS 6:\u003c/em\u003e Glycogen-piece. \u003cem\u003eTS 7:\u003c/em\u003e End-piece.\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eLS 6-7: \u003c/em\u003eshows tapering of flagellum which ends in terminal bulb (tb). Asterisks (*) show cytoplasmic bridges connecting adjacent spermatids in a cohort. Scale bar 0.6mm (approx.)\u003c/p\u003e","description":"","filename":"AplacFig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/e120c2ce42da5b8d14c57689.png"},{"id":70964675,"identity":"7685474e-e177-445c-a927-16b942843cb0","added_by":"auto","created_at":"2024-12-09 16:14:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25905588,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/6e6c1c9e-1c59-4a6e-9c60-27d229a39e97.pdf"},{"id":64302536,"identity":"713784ab-90da-4f11-a3d1-eea5e8049132","added_by":"auto","created_at":"2024-09-11 12:02:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":730012,"visible":true,"origin":"","legend":"\u003cp\u003eA. Scanning electron microscope image of the mature sperm of showing distal flagellum (f), mitochondrion, (m). nucleus (n), apical horn (ah), apical dense tube (adt), and acrosome (a). B. Scanning electron microscope image of the mature sperm of treated with egg water, showing dramatic elongation of apical dense tube (arrow). C,D,E. Diagrammatic representations of longitudinal sections through the spermatozoa of three species of Caudofoveata. acrosome (a); apical dense tube (adt); apical horn (ah); glycogen (gly); proximal centriole (pc); distal centriole (dc) attached to annulus by spokes; m, mitochondrion; nucleus (n) with lacunae; flagellum (f). Scale bars: A,B 1.0 µm; C,D,E 0.8µm (approx.).\u003c/p\u003e","description":"","filename":"FrontCoverIllustration.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4902173/v1/9dc625d47f47d1067888a786.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spermiogenesis in Aplacophora (Mollusca) provides insight into evolution of Bilateria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAplacophora are a group of marine, benthic, worm-like molluscs, covered in aragonitic spicules that were proposed by some morphologists to be the sister group to Polyplacophora forming the Aculifera (Scheltema \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Scheltema et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1994\u003c/span\u003e); a conclusion since supported by molecular studies (Vinther et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mikkelsen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Aculifera is currently considered to be basal among extant Mollusca (Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mikkelsen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Aplacophora are divided into two distinct Classes, the Caudofoveata (=\u0026thinsp;Chaetodermomorpha) and the Solenogastres (=\u0026thinsp;Neomeniomorpha) that have major differences in structure, habits, and reproductive biology (Todt et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Scheltema et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This has led some authors to consider them to be paraphyletic (Salvini-Plawe1978; Salvini-Plawen and Steiner \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Haszprunar \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Vinther et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), but more recent molecular analyses have found them to be monophyletic (Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mikkelsen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCaudofoveata or \u0026lsquo;chaetoderms\u0026rsquo; are infaunal and lack a foot, feeding on organisms in bottom sediments with a reduced radula (Scheltema et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Todt et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The body is divided into three segments, the anterium, trunk and posterium and the gut comprises a stomach, midgut and intestine. They have a pair of ctenidia in the posterior pallial cavity and the blood contains hemocyanin. Caudofoveata are dioecious, fertilizing externally with \u0026ldquo;ect-aquasperm\u0026rdquo; (for sperm terminology see Jamieson \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Sperm structure, before details were known, was thought to be similar to Polyplacophora (the chitons) (Franz\u0026eacute;n \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1955\u003c/span\u003e; Hadfield \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). However, this proved to be wrong, when a detailed study of one species, \u003cem\u003eChaetoderma argenteum\u003c/em\u003e Heath, 1911 (Buckland-Nicks and Chia \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), revealed a series of unique features, as yet found nowhere else in the Animal Kingdom.\u003c/p\u003e \u003cp\u003eSolenogastres are more uniformly cylindrical and crawl on a ciliated pedal groove that is considered to be a modified foot (Scheltema et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). They feed largely on Cnidaria (Scheltema et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), have an undivided gut and they lack ctenidia. They have an atrial sense organ which is absent in Caudofoveata. The blood contains a red pigment that may be a derivative of hemoglobin but they do not contain hemocyanin. Also, unlike Caudofoveata, Solenogastres are monoecious, copulate and fertilize internally with \u0026ldquo;introsperm\u0026rdquo; (Buckland-Nicks and Scheltema \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The sperm of one species of Solenogastres, \u003cem\u003eEpimenia australis\u003c/em\u003e (Thiele, 1897), has been described in detail and exhibits a suite of characters shared with introsperm of several other animal groups, including the basal bilaterian taxon Nemertodermatida (Xenacoelomorpha) (Buckland-Nicks \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Buckland-Nicks and Scheltema \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Buckland-Nicks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These remarkably similar characters are considered by most to be homoplasies (Bieler et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), as they predict a different ancestry than that indicated by most recent molecular analyses (Cannon et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Meyer-Wachsmuth and Jondelius \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ruiz-Trillo and Paps \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mulhair et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yap-Chiongco et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, Redmond (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that Acoelomorpha flatworm monophyly is a severe long branch-attraction artefact obscuring a clade of Acoela and Xenoturbellida, that he called \u0026ldquo;Xenacoela\u0026rdquo;, which is related to Ambulacraria. The link between Xenoturbellida (but not Acoela) and Ambulacraria was predicted by a re-analysis of sperm morphology of \u003cem\u003eXenoturbella bocki\u003c/em\u003e Westblad, 1949, which was shown to be very similar to hemichordate sperm (Buckland-Nicks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The separation of Acoela and Nemertodermatida also would agree with Wallberg et al. (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) who, based on detailed analyses of 18S and 28S RNA, dismissed the taxon Acoelomorpha, finding that \u0026ldquo;Acoela and Nemertodermatida are separate clades\u0026rdquo;. Moreover, sperm structure of Nemertodermatida suggests that they are closer to the Urbilaterian with their typical 9\u0026thinsp;+\u0026thinsp;2 flagellum and linear arrangement of acrosome, nucleus and mitochondria which is not found in any Acoela (Tyler and Rieger \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Petrov et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Buckland-Nicks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSperm structure of Caudofoveata and Solenogastres is known in detail from only two species (Buckland-Nicks and Chia \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Buckland-Nicks and Scheltema, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) that do not represent the diversity of the families in these taxa. Nevertheless, the results from these studies suggest that there are vast differences in sperm structure between the two Classes indicating a long period of separation between them. The purpose of this paper is to compare and contrast spermiogenesis and sperm structure of representative species of several different families in both Classes and examine the phylogenetic implications of these findings in the light of recent morphological and molecular analyses of Aculifera, Xenacoelomorpha and related taxa.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eSpecimens of Caudofoveata from Family Chaetodermatidae: \u003cem\u003eChaetoderma canadense\u003c/em\u003e Nierstrasz, 1902 and \u003cem\u003eC. argenteum\u003c/em\u003e Heath, 1911 were collected off the east and west coasts of Canada respectively in 1987 and 1992. \u003cem\u003eChaetoderma nitidulum\u003c/em\u003e Lov\u0026eacute;n, 1844; \u003cem\u003eFalcidens crossotus\u003c/em\u003e Salvini-Plawen 1968; and from Family Limifossoridae: \u003cem\u003eScutopus ventrolineatus\u003c/em\u003e Salvini-Plawen, 1968, were collected off Blomsterdalen, Norway in 2007.\u003c/p\u003e \u003cp\u003eSpecimens of Solenogastres from Family Dondersiidae: \u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e Handl and Salvini Plawen 2001; from Family Gymnomeniidae: \u003cem\u003eGymnomenia pellucida\u003c/em\u003e Odhner, 1920 and \u003cem\u003eWirenia argentea\u003c/em\u003e Odhner 1920; from Family Simrothiellidae: \u003cem\u003eSimrothiella margaritacea\u003c/em\u003e (Koren and Danielssen, 1877); from Family Proneomeniidae: \u003cem\u003eDorymenia sarsii\u003c/em\u003e (Koren and Danielssen, 1877); from Family Simrothiellidae: a species of \u003cem\u003eHelicoradomenia\u003c/em\u003e sp., Scheltema and Kuzirian with some digital micrographs was donated by Christiane Todt in 2007.\u003c/p\u003e \u003cp\u003eSpecimens of the gastropod \u003cem\u003eNerita polita\u003c/em\u003e Linnaeus, 1758 (Gastropoda) were collected near Kihei, Maui in 1987. for the purpose of comparing aspects of spermiogenesis with Solenogastres.\u003c/p\u003e \u003cp\u003eThe best fixation of tissues for transmission electron microscopy (TEM) was achieved by using a primary fixative of 2.5% glutaraldehyde in 0.45\u0026micro;m filtered seawater (FSW) and 0.2M sodium cacodylate buffer (pH 7.4) kept on ice for 2hr and allowed to warm to ambient temperature overnight. Tissues were washed in FSW / sodium cacodylate buffer and transferred to 1% osmium tetroxide in the same buffer on ice for 1hr. Tissues were dehydrated in an ethanol series, exchanged through propylene oxide and infiltrated then embedded in Epon/Spurrs mixed 1:1. Sections for light microscopy were cut at 1\u0026micro;m and stained with 1% toluidine blue (made to pH 9 with sodium bicarbonate). For transmission electron microscopy, serial thin sections with silver/gold interference colour were cut with a diamond knife (Diatome, Switzerland), picked up on nickel slot grids (EM Sciences), and gently placed on formvar windows in a rack (EM Sciences). Dried grids were picked up with fine forceps and stained with uranyl acetate (1hr) and lead citrate (10 min) before being examined in a Philips 410 TEM operated at 80kV and photographed with a Kodak L12C digital camera at 1200dpi.\u003c/p\u003e \u003cp\u003eFor scanning electron microscopy (SEM) of \u003cem\u003eChaetoderma argenteum\u003c/em\u003e sperm, \u0026ldquo;egg water\u0026rdquo; was made by mixing ripe eggs dissected from an ovary with 0.45\u0026micro;m filtered sea water and after 10 mins filtering the water again. Fresh sperm was dissected from testes and pipetted either into glass vials containing just 0.45\u0026micro;m filtered sea water, or into other vials containing \u0026ldquo;egg water\u0026rdquo;. Sperm from the two treatments were kept separate and pipetted into fine pore Flo-Thru vials (Pelco) and placed in glutaraldehyde fixative, as above. After 2hr vials were washed in several changes of 0.45\u0026micro;m filtered sea water before post-fixing for 1hr in 1% osmium tetroxide in 0.2M sodium cacodylate buffer. The vials were washed repeatedly in distilled water and then dehydrated in an ethanol series to 3 changes of 100%. Vials were then exchanged through amyl acetate and critical-point dried. Vials containing dried sperm were upended onto aluminium stubs coated with double-sided sticky carbon tabs. The stubs were sputter-coated with gold in an SPI-Module Sputter Coater and examined in a JEOL JSM 5300 SEM and photographed with Quartz PCI imaging at 1200dpi.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe testes of all species studied produced only fertilizing spermatozoa (euspermatozoa).\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCaudofoveata\u003c/h2\u003e \u003cp\u003eThe five species of Caudofoveata examined here have fundamentally similar sperm that are a type of \u0026ldquo;ect-aquasperm\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E), which fertilize externally in sea water. Each sperm consists of a small pear-shaped head that contains a nucleus capped by an apical horn, apical dense tube (terminology after Buckland-Nicks and Chia \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) and acrosome, a mid-piece of five spherical mitochondria that surround the orthogonally arranged proximal and distal centrioles, and a flagellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for some dimensions). When \u003cem\u003eChaetoderma argenteum\u003c/em\u003e sperm were immersed in egg water, the apical dense tube extended visibly about 1.5 times its original length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe sperm of all species investigated are formed by the same basic process that was described in detail for \u003cem\u003eChaetoderma argenteum\u003c/em\u003e (Chaetodermatidae) by Buckland-Nicks and Chia (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Therefore, a single description only follows with a focus on \u003cem\u003eScutopus ventrolineatus\u003c/em\u003e because spermiogenesis has not been described in any species of this Family (Limifossoridae).\u003c/p\u003e \u003cp\u003eVery early spermatids are typical of those of other molluscs (e.g. see Hodgson and Bernard \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Hodgson et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Eckelbarger and Davis \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and therefore we have not illustrated these. However, they contain a spheroidal nucleus with heterochromatin, and the cytoplasm contains numerous small mitochondria, some rough endoplasmic reticulum, a Golgi body and a pair of centrioles. Early in spermiogenesis, the Golgi body secretes vesicles next to the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) which fuse to form the proacrosome. As spermiogenesis progresses, the proacrosome then migrates along the plasma membrane to what will be the anterior of the spermatozoon (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). At the same time the numerous smaller mitochondria accumulate at the presumptive posterior of the developing sperm (not illustrated but see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B for diagrammatic illustration) and fuse to form five larger, spherical mitochondria surrounding the centrioles at the base of the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D,E). In addition, the annulus attaches the distal centriole to the plasma membrane, via the centriolar satellites, and produces the distal flagellum (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The Golgi body moves anteriorly to a depression on one side of the apex of the elongating nucleus where it begins to secrete vesicles that contribute to structures called the apical horn, the apical dense tube, as well as a large vacuole (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The proximal centriole, which is positioned at the apex of the distal centriole nearest to the nucleus, develops a rootlet that attaches it to a slight depression in the nucleus (the nuclear fossa) that contains some electron-dense material (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The proximal centriole develops the proximal flagellum, which passes anteriorly beside the mitochondria and nucleus and later extends beyond its tip (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D,H, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Occasional droplets thought to be lipid were observed in the cytoplasm of some spermatids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn mid to late spermatids the chromatin in the spherical nucleus undergoes condensation becoming granular in appearance (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB,C,D, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), then more homogeneously electron-dense (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE,F,H,I, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). At the same time the nucleus elongates and begins to attain its final pear shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), the apical horn surrounds the pointed apex of the nucleus and extends into the developing apical dense tube (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D). The apical dense tube elongates adjacent to the proximal flagellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and extends over the large vacuole (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF,G,I, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The elongating apical dense tube eventually contacts the acrosome, the contents of which are not differentiated, on the plasma membrane and becomes fixed in position (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Very late spermatids have also lost the apical flagellum by a mechanism we were unable to determine. Finally, the residual cytoplasm containing the Golgi body and large vacuole of each spermatid is released (not illustrated), freeing mature sperm as individuals. Details of these events are summarized diagrammatically in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (A-D).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLengths (\u0026micro;m) of sperm components of different species of Caudofoveata and Solenogastres. ADT\u0026thinsp;=\u0026thinsp;apical dense tube; AH\u0026thinsp;=\u0026thinsp;apical horn; Mitoch\u0026thinsp;=\u0026thinsp;mitochondrial\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCAUDOFOVEATA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcrosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNucleus Length\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMid-Piece\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMitoch Sheath\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnnulus Adjunct\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eADT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScutopus ventrolineatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFalcidens crossotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eChaetoderma argenteum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCanada West Coast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eChaetoderma canadense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCanada East Coast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eChaetoderma nitidulum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSOLENOGASTRES\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcrosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNucleus Length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMid Piece\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMitoch. Sheath\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnnulus Adjunct\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e#Annulus Adjuncts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSimrothiella margaritacea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHelicoradomenia\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEast Pacific Hydro. Vent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDorymenia sarsii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEpimenia australis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(B-N \u0026amp; Scheltema 1995)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePapua New. Guinea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGymnomenia pellucida\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWirenia argentea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSolenogastres\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eSperm structure\u003c/h2\u003e \u003cp\u003eAll six species from five families of Solenogastres, have elongate introsperm that are similar in structure to that described in some detail for \u003cem\u003eEpimenia australis\u003c/em\u003e by Buckland-Nicks (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and Buckland-Nicks and Scheltema (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The sperm head consists of an anteriorly positioned narrow conical acrosome, invaginated posteriorly atop a rod-shaped nucleus, containing highly condensed chromatin (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and inset) and separated from it by a subacrosomal plate (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Posteriorly, the nucleus has a small invagination, the nuclear fossa, that houses the basal body (fused distal\u0026thinsp;+\u0026thinsp;proximal centrioles) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and inset). The distal centriole of this structure produces a long flagellum with a 9\u0026thinsp;+\u0026thinsp;2 arrangement of microtubules (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D,F), that tapers to singlet microtubules in the end-piece and ends in a terminal bulb (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and inset). The mid-piece region consists of mitochondria that are arranged as a sheath around the axoneme (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB inset,C,E). A spiral ridge is formed between the mitochondria and axoneme (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC,D,F). Where the mid-piece meets the glycogen-piece there is an invaginated junction housing the annulus and anterior to it, the annulus adjuncts (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD,E) of which there are four in \u003cem\u003eS. sulcodoryata\u003c/em\u003e and two in the other species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The annulus adjuncts may extend anteriorly for several microns. Extending posteriorly from the annulus in the glycogen-piece for about 0.5 microns, are a series of coarse fibres overlying the axoneme (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE,F). Transverse sections of the acrosome, mid-piece, glycogen piece and end-piece are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,C,D,F,G and these correspond with specific sections drawn in the detailed summary diagram Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eD. Although the basic structure of the sperm is similar among all species studied, differences between species occur in the mid-piece structure, the number and length of the \u0026lsquo;annulus adjuncts\u0026rsquo;, and the length of various components such as the acrosome, nucleus, and mid-piece (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSpermiogenesis\u003c/h2\u003e \u003cp\u003eAll stages of spermiogenesis were observed in the gonads of \u003cem\u003eStylomenia sulcodoryata\u003c/em\u003e and \u003cem\u003eGymnomenia pellucida\u003c/em\u003e, whereas only late stages were present in \u003cem\u003eHelicoradomenia\u003c/em\u003e sp., \u003cem\u003eSimrothiella margaritacea\u003c/em\u003e, \u003cem\u003eDorymenia sarsii\u003c/em\u003e and \u003cem\u003eWirenia argentea\u003c/em\u003e. However, the structure of these late spermatids indicates that the sperm of all species form in a very similar way and therefore a single description only follows.\u003c/p\u003e \u003cp\u003eVery early spermatids are typical of those of the majority of Mollusca and are not illustrated. They have a spherical nucleus with heterochromatin and a cytoplasm containing numerous small mitochondria, a Golgi body, some rough endoplasmic reticulum, and a pair of centrioles. Early in spermiogenesis the distal centriole attaches to the plasma membrane via the annulus and then migrates to the base of the nucleus dragging the plasma membrane with it, thus creating a flagellar canal (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Spherical mitochondria aggregate at the base of the nucleus and fuse to form larger spherical mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). At the early to mid- spermatid stage when the nucleus is still spherical, the Golgi body secretes vesicles that fuse to form the proacrosome that migrates to the apex of the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,B), where it undergoes further elongation and is separated from the anterior nucleus by electron-dense material (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The Golgi body then separates and migrates posteriorly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), coming to lie near the mitochondria at the base of the nucleus. In this position, the Golgi body secretes numerous vesicles that coalesce to form a \u0026lsquo;spiral ridge\u0026rsquo; of dense material around the naked axoneme (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,C,D,F) from the distal centriole to the annulus at the mid-piece and glycogen-piece junction; a distance of about 9\u0026ndash;16 microns in the mature sperm of the different species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The mitochondria become oval-shaped when seen in mid-longitudinal section (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC,) and begin to elongate, extending posteriorly to wrap around the spiral ridge and axoneme (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF; \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), eventually forming the mitochondrial sheath of the mid-piece (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). At the same time the nucleus undergoes elongation (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC,E; \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) as the chromatin condenses (granular then fibrillar) with the posterior nuclear fossa deepening to house the basal body (=\u0026thinsp;fused centrioles) (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB,C). The annulus breaks away from the distal centriole and migrates posteriorly to the invaginated junction between the mid-piece and glycogen-piece, eliminating the flagellar canal as it does so (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). As the components of the late spermatids elongate and differentiate 6 distinct regions (acrosome, nucleus, mid-piece, annulus adjuncts, glycogen-piece and end-piece) are created (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F; 8D1-7) and residual cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) is eliminated. At maturity the acrosome has become a narrow cone that is invaginated posteriorly and contains a diffuse subacrosomal granule within the subacrosomal space (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). It is also separated from the tip of the dense rod-shaped nucleus by a subacrosomal plate (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The base of the nucleus is invaginated into the nuclear fossa that houses the \u0026ldquo;basal body\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) formed from the fused centrioles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThroughout spermiogenesis spermatids in a cohort are linked by cytoplasmic bridges (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,D) so that they are at the same stage of development (e.g. Figures\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB inset, 6E, 7C). The detailed events of spermiogenesis are summarized diagrammatically in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e (A-D)\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic relationships among Aplacophora in relation to sperm structure and morphology\u003c/h2\u003e \u003cp\u003eAlthough most morphological (Scheltema \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Todt 2013) and molecular analyses (Kocot et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Smith et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Vinther et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kocot et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) place Caudofoveata as sister taxon to Solenogastres, there are nevertheless significant morphological differences between the two groups of Aplacophora. The Caudofoveata lack a foot, have a body and gut divided into three parts, have a pair of ctenidia, hemocyanin in the blood, and are dioecious. By contrast, Solenogastres crawl on a foot, have an undivided cylindrical body, lack ctenidia and hemocyanin, and are monoecious. The results of this study and two previous studies (Buckland-Nicks and Chia \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Buckland-Nicks and Scheltema \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) also show that the sperm of Caudofoveata and Solenogastres described to date are completely different. Whereas the Caudofoveata have a type of \u0026ldquo;ect-aquasperm\u0026rdquo; (terminology after Jamieson, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), the sperm of the Solenogastres are of the introsperm type. These dramatic differences in sperm structure between these sister taxa, as well as their many morphological differences, suggests a long evolutionary separation from each other and their common ancestor and begs the question first posed by Salvini-Plawen and colleagues: are these groups paraphyletic? (Salvini-Plawen \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Salvini-Plawen and Steiner \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrently, three families are recognized within the Caudofoveata: Chaetodermatidae, Limifossoridae and Prochaetodermatidae. Sperm structure has now been described from species belonging to the first two. Although few morphological characters are shared between the Chaetodermatidae and Limifossoridae (Mikkelson et al. 2018) the structure of their sperm is remarkably conserved and is not found anywhere else in the Animal Kingdom. Because these species produce ect-aquasperm, it indicates that fertilization is external with gametes being released into the sea water. However, as far as we are aware, spawning has never been observed in any caudofoveate in the field. Recent molecular studies indicate that the Prochaetodermatidae are monophyletic and sister to the two other families (Mikkelson et al. 2019; Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although there is only one study on the reproductive biology of a prochaetodermatid (Scheltema, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), we suggest that they will also have ect-aquasperm. Studies on sperm structure from members of this family are now required to test this hypothesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eOn sperm structure and fertilization in Caudofoveata\u003c/h2\u003e \u003cp\u003eThe spermatozoa of Caudofoveata have two unique structures: the apical horn and an apical dense tube in the sperm head (Buckland-Nicks and Chia, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1989\u003c/span\u003e and current study). Whilst spermiogenesis is basically similar to that described for other molluscs, there are unique features of spermiogenesis. The first, is the presence of a large apical vacuole in early to mid-spermatids that is closely associated with the developing apical dense tube and has been hypothesized to help guide its formation (Buckland-Nicks and Chia, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). The second is the formation of a flagellum from the proximal centriole that exits anteriorly adjacent to the developing apical dense tube possibly aiding in its development. The flagellum from the proximal centriole is subsequently eliminated leaving a uniflagellate mature sperm (Buckland-Nicks and Chia, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1989\u003c/span\u003e and this study). Whilst the mechanism by which the proximal flagellum is eliminated is not known, it could occur by deflagellation (Quarmby \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) or microtubule disassembly, as commonly occurs during cell division (Bloodgood 1994; Hu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhilst the function of the apical dense tube of Caudofoveata during fertilization, is unknown, in \u003cem\u003eChaetoderma argenteum\u003c/em\u003e it elongates to 1.5x its normal length when exposed to egg water (Buckland-Nicks, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; also see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), suggesting that it may be a unique mechanism for bridging the egg envelopes similar to the actin-based \u0026lsquo;perforatorium\u0026rsquo; of other molluscs that is activated by the acrosome reaction (see review by Tilney, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHow fertilization could work: The simple acrosomal vesicle could operate like that of Chitonida and dissolve a pore in the egg envelopes through which the extending apical dense tube would project, delivering the inner acrosomal membrane into contact with the egg membrane. Following membrane fusion, the chromatin would be injected through the apical dense tube into the egg cortex. To this end, we have noted that the tip of the nucleus is pointed, where it inserts into the apical horn. Although speculative, this scenario would work based on known details of fertilization in other animals, including chitons (Buckland-Nicks \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Colwin and Colwin \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Summers et al.1975; Buckland-Nicks and Hodgson \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; and see review by Tilney, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). It should be made clear that the reduced acrosome and needle-tipped nucleus of Chitonida are secondarily derived characters, since basal members of chitons, the Leptochitonidae have a typical acrosome, nucleus structure and fertilization mechanism (Buckland-Nicks \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOn sperm structure in Solenogastres\u003c/h2\u003e \u003cp\u003eIncluding the current study, spermiogenesis and sperm structure in Solenogastres has been described from 7 species representing six of the 24 families (as stated by Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yap-Chiongco et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). All species have introsperm confirming internal fertilization in this class of hermaphrodite Aplacophora and spermiogenesis in all species is similar to that described by Buckland-Nicks and Scheltema (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) for \u003cem\u003eEpimenia australis\u003c/em\u003e. Thus, spermiogenesis described here, is highly conserved across the phylogenetic tree of Solenogastres as stated by Yap-Chiongco et al. (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe phylogeny of Solenogastres has undergone major re-organization in recent years with Order Cavibelonia being found to be paraphyletic (Kocot et al \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yap-Chiongo et al. 2024). From the standpoint of sperm structure alone, \u003cem\u003eHelicoradomenia\u003c/em\u003e, \u003cem\u003eSimrothiella\u003c/em\u003e and \u003cem\u003eDorymenia\u003c/em\u003e all group in the former Order Cavibelonia (Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), as they have the shortest acrosomes and nuclei and longest annulus adjuncts. \u003cem\u003eEpimenia\u003c/em\u003e, formerly included in Order Cavibelonia, is intermediate in acrosome and nucleus length and has the shortest annulus adjuncts and therefore fits better with others from Order Pholidoskepia, \u003cem\u003eGymnomenia\u003c/em\u003e, \u003cem\u003eStylomenia\u003c/em\u003e and \u003cem\u003eWirenia\u003c/em\u003e, which have the longest acrosomes and nuclei and shorter annulus adjuncts.\u003c/p\u003e \u003cp\u003eThere has been debate as to whether the earliest solenogasters were small and meiofaunal or more macrofaunal like their \u0026lsquo;predicted\u0026rsquo; chiton ancestors (Kocot et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yap-Chiongco et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Molecular analyses differ on this point. Maximum Likelihood analysis places the small bodied Meiomeniidae as basal, suggesting a meiofaunal ancestry. Whereas, Bayesian Likelihood Inference places the larger bodied Amphimeniidae as basal, more in keeping with a chiton ancestry (Yap-Chiongco et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Analysis of genome size by these authors shows that solenogasters tend to have much smaller genomes (e.g.243Mbp for \u003cem\u003eNeomenia\u003c/em\u003e) than their sister group the Caudofoveata (e.g.2.45Gbp (=\u0026thinsp;2450 Mp) in \u003cem\u003eChaetoderma\u003c/em\u003e) or the Polyplacophora (chitons) (2.84Gbp in \u003cem\u003eCryptochiton\u003c/em\u003e). As Yap-Chiongco et al. (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) said in some groups \u0026ldquo;genome size may co-evolve with life-history\u0026rdquo;, so in this case rather than supporting a larger bodied chiton ancestry, this could mean that small body size (smaller genome) is ancestral. Supporting this is our analysis of sperm structure, which shows that all solenogasters studied have complex introsperm, very similar to those of the basal bilaterian Nemertodermatida; whereas both Caudofoveata and chitons (Buckland-Nicks \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) have derived and simpler ect-aquasperm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOn sperm structure as a source of characters for phylogenetic inference\u003c/h2\u003e \u003cp\u003eWilson (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1925\u003c/span\u003e) stated that: \u0026ldquo;Sperms of different species exhibit remarkable differences in size, form and structure\u0026hellip;\u0026hellip;that it is hardly an exaggeration to say that most species of animals might be identified by the morphology of their sperm alone\u0026rdquo;. Indeed, sperm structure has been used in many studies to infer phylogenetic relationships (review by Bartolomaeus et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and in some to create detailed character analyses that improved existing phylogenetic trees (Buckland-Nicks \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). However, there has always been the worry that functional constraints result in convergent evolution of similar sperm traits. Some recent studies tested this notion and found that functional constraints do not overpower historical signals in sperm of frogs and nemerteans (Cherneyshev \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Valchi et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bartolomaeus et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which suggests that sperm ultrastructure may be used \u0026ldquo;as a valuable source of characters for phylogenetic inferences\u0026rdquo; (Bartolomaeus et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Recently, Fitzpatrick et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have opened a sperm character database, which should help enormously with future phylogenetic analyses of animals, in particular the early bilaterian groups. One problem with establishing an outgroup for sperm derived characters is that early Metazoa, such as sponges and Cnidaria, fertilize with ect-aquasperm but earliest bilaterians, perhaps similar to Nemertodermatida, may have already developed the complex introsperm. Thus, where ect-aquasperm are developed secondarily, in more recent taxa, there is the danger that a phylogenetic analysis using sperm characters will place them more basal than they should be, unless this is taken into consideration.\u003c/p\u003e \u003cp\u003eThe sperm structure and spermiogenesis of Solenogastres shares nine key characters (listed below) with sperm of the Order Nemertodermatida (Phylum Xenacoelomorpha). These features are not found in either the Acoela or the Xenoturbellida. Importantly, Redmond (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) recently dismissed Acoelomorpha within Xenacoelomorpha, as an artefact of long branch attraction (LBA), which is in agreement with Wallberg et al. (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This discovery suggests that Nemertodermatida are likely basal among current Bilateria, a view long held by several other scientists (Tyler and Rieger \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1975\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Buckland-Nicks and Scheltema \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Hejnol and Martindale \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Buckland-Nicks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and warrants further discussion below.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOn sperm structure and the origin of Bilateria: implications of the dismissal of\u003c/em\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eAcoelomorpha\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWallberg et al. (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) stated (based on their detailed study of 18S and 28S RNA sequences): \u0026ldquo;Acoela and Nemertodermatida are separate bilaterian clades\u0026rdquo; and they dismissed the clade Acoelomorpha. However, the Phylum Xenacoelomorpha, which connects Xenoturbellida with Acoelomorpha, has been widely accepted as an important link to our bilaterian ancestry and is supported by numerous molecular analyses (Bourlat et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Achatz et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Cannon et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rouse et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Philippe et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kapli and Telford \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mulhair et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Schiffer et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, to name a few). The recent paper by Redmond (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) once again sheds considerable doubt on Acoelomorpha instead supporting the conclusion that they are a paraphyletic group (Wallberg et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Redmond (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that LBA, as well as Compositional Bias and other phenomena have been corrupting the accuracy of molecular analyses and are responsible for the artefactual linking of Nemertodermatida with Acoela. Instead, he found that Xenoturbellida grouped with Ambulacraria (Redmond \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which supports morphological evidence from the re-analysis of sperm structure of \u003cem\u003eXenoturbella bocki\u003c/em\u003e by Buckland-Nicks et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), showing close similarity to the sperm of the hemichordate \u003cem\u003eSchizocardium\u003c/em\u003e (Franz\u0026eacute;n \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The link between Xenoturbellida and Ambulacraria was found in several previous molecular analyses (Bourlat et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Philippe et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Telford \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Nakano \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mulhair et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Schiffer et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but all of these included Acoelomorpha within Xenacoelomorpha.\u003c/p\u003e \u003cp\u003eRedmond (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) still places Xenacoelomorpha within Deuterostomia. One problem with this is that although the molecular machinery for excretory system ultrafiltration is present in Xenacoelomorpha, they lack the \u003cem\u003eOsr\u003c/em\u003e gene that is present in both Protostomia and Deuterostomia and which enables the production of discrete excretory organs like nephridia and nephrons (Abalde et al \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which are absent in all Xenacoelomorpha. Furthermore, most morphological and phylogenomic analyses support a sister relationship between Xenacoelomorpha and the rest of Bilateria (Nephrozoa hypothesis) (Jondelius et al \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, Redmond (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that Acoela formed a separate clade with Xenoturbellida, which he called the \u0026ldquo;Xenacoela\u0026rdquo;. This fits with larval development which is direct in both cases (Nakano et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) but not with sperm structure (Buckland-Nicks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The sperm of Nemertodermatida has a more plesiomorphic structure than Acoela, because of the arrangement of its component acrosome, nucleus, mid-piece, and typical single 9\u0026thinsp;+\u0026thinsp;2 flagellum (Tyler and Rieger \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1975\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Petrov et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Buckland-Nicks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which suggests that Nemertodermatida are basal within Xenacoelomorpha. This is supported by analyses of their development as Nemertodermatida exhibit a suite of characters that are considered plesiomorphic to Acoela, including: a more regulative cleavage pattern, superficial nerve net in the epithelium, epithelial digestive endoderm and lack of simple eyes (Hejnol and Martindale \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Achatz et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Furthermore, \u003cem\u003eXenoturbella\u003c/em\u003e possesses a much larger gene complement of the Hox, Wnt, bHLH and GPCR families than acoels (Hejnol and Pang \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), suggesting that acoels are more basal, which is supported by sperm structure (Buckland-Nicks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is perhaps significant that Nemertodermatida has evolved nine specific sperm characters, that do not appear in the ect-aquasperm of any Radiata but are all found in the Solenogastres. 1. Small conical acrosome atop nucleus, 2. Rod-like condensed nucleus, in which condensation of chromatin progresses further into fibres before becoming uniformly dense, 3. Proximal and distal centrioles fuse to form a basal body which is housed in the nuclear fossa. 4. The annulus breaks away from the distal centriole and moves posteriorly everting the flagellar canal, 5. After producing the acrosome, the Golgi body migrates posteriorly to below the mitochondria and secretes the complex \u0026lsquo;spiral ridge\u0026rsquo; around the axoneme, 6. The mitochondria fuse, elongate and partially or completely enclose the axoneme to form a mitochondrial sheath, 7. Annulus adjuncts extend anteriorly from the region of the annulus. 8. Coarse fibres overlying the outer microtubule doublets, project posteriorly from the annulus region into the tail. 9. The tail region contains glycogen granules as an energy reserve. Not all nemertodermatids share all nine characters but all solenogasters examined, do, suggesting that one or other character may have been lost in a particular nemertodermatid species or lineage but was retained in their descendants. Until now, these characters have been considered to be the result of convergent evolution (Bieler et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) but if Nemertodermatida are basal bilaterians they could be symplesiomorphies.\u003c/p\u003e \u003cp\u003eSome of these characters are found in other basal molluscs with internal fertilization (but none with external fertilization), including the Neritimorpha (Gastropoda) in which the Golgi body choreographs a closely similar series of complex movements and secretions as in Solenogastres, to deposit first, the conical acrosome, and then the spiral ridge around the axoneme (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) (see also: Buckland-Nicks and Chia \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). If these sperm characters are homologous (i.e. symplesiomorphic) as we suggest, it again raises the question of whether there is a deep phylogenetic link between Nemertodermatida and Solenogastres as well as Protostomia in general (Buckland-Nicks and Scheltema \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on sperm structure alone, it reiterates arguments that Solenogastres are basal to Mollusca and that the caudofoveates and chitons evolved later, possibly re-developing ect-aquasperm by \u0026ldquo;progenetic spermiogenesis\u0026rdquo; (Buckland-Nicks \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Buckland-Nicks and Scheltema \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e); a concept that more recently has led to the discovery of a modern equivalent in molluscs (Winik et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Nemertodermatida and Solenogastres are largely microscopic, meiofaunal taxa, living in marine sediments. Interest in the meiofauna has increased markedly in the last twenty years (Giere and Schratzberger \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Worsaae et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) with trace fossils of earliest worm-like bilaterians (\u003cem\u003eHelminthoidichnites\u003c/em\u003e) dating to before the Cambrian being found more often (Buatois and Mangano 2016; Parry et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gehling and Droser \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, recently, Evans et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) discovered the earliest known worm-like bilaterian, \u003cem\u003eIkaria warioota\u003c/em\u003e, in trace fossils, as well as body fossils, that date back to the early Ediacaran in Southern Australia and persist through the Cambrian boundary.\u003c/p\u003e \u003cp\u003eBased on the above arguments and our analysis of sperm structure, we support the conclusion that the Urbilaterian was a small, meiofaunal, flatworm-like organism with sexual reproduction by internal fertilization followed by direct development (Tyler and Rieger \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Buckland-Nicks and Scheltema \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Hejnol and Martindale \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Buckland-Nicks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eSupported by Natural Sciences and Engineering Research Council of Canada, grant 46205 to J.B-N.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBoth J.B-N. and A.N.H. contributed to the study conception and design. Material preparation, data collection and analysis were performed by [J.B-N]. The first draft of the manuscript was written by [J.B-N.] and both J.B-N. and A.N.H. commented on previous versions of the manuscript, read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eSpecial thanks to Christiane Todt who supplied and identified almost all of the solenogasters and some of the chaetoderms from Norway. Amelie Scheltema (deceased) helped collect and identify specimens of \u003cem\u003eC. canadense\u003c/em\u003e and \u003cem\u003eC. argenteum\u003c/em\u003e. Thanks also to Moira Galway, Chair of the Biology Department at St. Francis Xavier University for use of TEM facilities.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbalde S, Tellgren-Roth C, Heintz J, Vinnere Pettersson O, Jondelius U (2023). 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Springer\u003c/li\u003e\n\u003cli\u003eYap-Chiongco MK, Bergmeier FS, Roberts NG, J\u0026ouml;rger KM, Kocot KM (2024) Phylogenomic reconstruction of Solenogastres (Mollusca, Aplacophora) informs hypotheses on body size evolution. Molec Phylogenet Evol, 194:108029\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"zoomorphology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"zomo","sideBox":"Learn more about [Zoomorphology](http://link.springer.com/journal/435)","snPcode":"435","submissionUrl":"https://submission.nature.com/new-submission/435/3","title":"Zoomorphology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chaetoderms, solenogasters, nemertodermatids, phylogeny, sperm structure","lastPublishedDoi":"10.21203/rs.3.rs-4902173/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4902173/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSperm structure among species in each Class of Aplacophora is highly conserved but between the two Classes is radically different. This together with numerous morphological differences between the two groups, suggests a long separation and a likely paraphyletic relationship, which has been suggested in the past but is not supported by molecular analyses. All Caudofoveata examined have unique externally-fertilizing ect-aquasperm found nowhere else in the Animal Kingdom. Solenogastres fertilize internally with introsperm like those described for \u003cem\u003eEpimenia australis\u003c/em\u003e but differ in details of length and number of specific components, providing insights to relationships among them. Furthermore, the solenogaster introsperm shares at least nine characters with introsperm of the bilaterian lineage Nemertodermatida, but shares none of these characters with the sperm of Caudofoveata, Polyplacophora (chitons), or Xenoturbellida and few with Acoela. However, a recent re-analysis of molecular data points to the re-organization of Xenacoelomorpha due to Long Branch Attraction and its separation into a basal Nemertodermatida plus a \u0026ldquo;Xenacoela\u0026rdquo; clade related to Ambulacraria. If the shared sperm characters of Solenogastres and Nemertodermatida are plesiomorphies not homoplasies, this would provide support for phylogenies that place Solenogastres basal to Mollusca. If true, then basal Bilateria would have a direct link with Protostomia. Recent discoveries of minute worm-like bilaterian body and trace fossils in Ediacaran deposits suggest that earliest bilaterians were meiofaunal and preceded the great Cambrian explosion by millions of years.\u003c/p\u003e","manuscriptTitle":"Spermiogenesis in Aplacophora (Mollusca) provides insight into evolution of Bilateria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-11 11:54:22","doi":"10.21203/rs.3.rs-4902173/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-27T20:20:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-24T23:53:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-23T18:49:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-12T13:21:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-10T23:37:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144987904991833195853656582706314689615","date":"2024-09-02T08:08:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258128774638314689673517923848575261988","date":"2024-08-29T08:15:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319151179386120227258616598126375652122","date":"2024-08-28T13:48:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279435966440916369045055398612103871243","date":"2024-08-26T12:28:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-26T07:40:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273331022495913321892898342191434581993","date":"2024-08-25T23:07:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278505242130357318518470439375261804118","date":"2024-08-24T05:44:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-23T19:03:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-14T07:08:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-14T07:06:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Zoomorphology","date":"2024-08-12T17:09:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"zoomorphology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"zomo","sideBox":"Learn more about [Zoomorphology](http://link.springer.com/journal/435)","snPcode":"435","submissionUrl":"https://submission.nature.com/new-submission/435/3","title":"Zoomorphology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a95aa0de-41a3-4dc7-a29e-90e4aaf8ab1d","owner":[],"postedDate":"September 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T15:59:55+00:00","versionOfRecord":{"articleIdentity":"rs-4902173","link":"https://doi.org/10.1007/s00435-024-00688-x","journal":{"identity":"zoomorphology","isVorOnly":false,"title":"Zoomorphology"},"publishedOn":"2024-12-05 15:57:05","publishedOnDateReadable":"December 5th, 2024"},"versionCreatedAt":"2024-09-11 11:54:22","video":"","vorDoi":"10.1007/s00435-024-00688-x","vorDoiUrl":"https://doi.org/10.1007/s00435-024-00688-x","workflowStages":[]},"version":"v1","identity":"rs-4902173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4902173","identity":"rs-4902173","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}