Spermiogenesis and spermatozoon ultrastructure in basal polyopisthocotylean monogeneans, Hexabothriidae and Chimaericolidae, and their significance for the phylogeny of the Monogenea

Sperm ultrastructure provides morphological characters useful for understanding phylogeny; no study was available for two basal branches of the Polyopisthocotylea, the Chimaericolidea and Diclybothriidea. We describe here spermiogenesis and sperm in Chimaericola leptogaster (Chimaericolidae) and Rajonchocotyle emarginata (Hexabothriidae), and sperm in Callorhynchocotyle callorhynchi (Hexabothriidae). Spermiogenesis in C. leptogaster and R. emarginata shows the usual pattern of most Polyopisthocotylea with typical zones of differentiation and proximo-distal fusion of the flagella. In all three species, the structure of the spermatozoon is biflagellate, with two incorporated trepaxonematan 9 + “1” axonemes and a posterior nucleus. However, unexpected structures were also seen. An alleged synapomorphy of the Polyopisthocotylea is the presence of a continuous row of longitudinal microtubules in the nuclear region. The sperm of C. leptogaster has a posterior part with a single axoneme, and the part with the nucleus is devoid of the continuous row of microtubules. The spermatozoon of R. emarginata has an anterior region with membrane ornamentation, and posterior lateral microtubules are absent. The spermatozoon of C. callorhynchi has transverse sections with only dorsal and ventral microtubules, and its posterior part shows flat sections containing a single axoneme and the nucleus. These findings have important implications for phylogeny and for the definition of synapomorphies in the Neodermata. We point out a series of discrepancies between actual data and interpretation of character states in the matrix of a phylogeny of the Monogenea. Our main conclusion is that the synapomorphy “lateral microtubules in the principal region of the spermatozoon” does not define the Polyopisthocotylea but is restricted to the Mazocraeidea.

In the Monogenea, the Monopisthocotylea and the Polyopisthocotylea have each been considered to bear respective sperm synapomorphies [34], but no spermatological character has been found to unite the two groups [40].
The Monopisthocotylea have revealed important variations of sperm structure, which led to the recognition of several synapomorphies [34,39,42] that were used in combination with other morphological characters to build phylogenies [12,14]. In contrast, the Polyopisthocotylea show a relatively uniform sperm morphology [34,39] with the significant exception of the Diplozoidae with an aberrant aflagellate spermatozoon [47]; the latter has been considered to be related to the exceptional biology of fertilization in diplozoids and especially the absence of sperm competition [39,47].
The Polyopisthocotylea include, in modern classifications [26], four orders: the Polystomatidea, Chimaericolidea, Diclybothriidea and Mazocraeidea. The spermatozoon and sometimes spermiogenesis ultrastructure are documented in several species of the Polystomatidae, in one species of Sphyranuridae (Polystomatidea), and in many families belonging to the Mazocraeidea (Table 1). However, no information was available for the Chimaericolidea and Diclybothriidea. Since the Chimaericolidea or these two orders were considered basal to the Mazocraeidea in both morphological [12] and molecular [32,75,78] analyses, missing data on sperm structure in these orders was a significant knowledge gap of sperm structure in Polyopisthocotylea, and even of the Neodermata as a whole.
In this paper we present, for the first time, observations on two species of the family Hexabothriidae (Diclybothriidea) and one of the family Chimaericolidae (Chimaericolidea), thus filling the gaps in our knowledge of sperm ultrastructure in the Monogenea. These observations complement previous studies on the tegument [88], attachment organs [84,85], reproductive organs [83,87], and digestive system [86] of the same species.

Material and Methods
For electron microscopy, adult specimens of three polyopisthocotylean monogeneans were recovered from the gills of naturally infected cartilaginous fishes: Chimaericola leptogaster (Leuckart, 1830) (Chimaericolidae) from the chimaera (rabbit fish) Chimaera monstrosa Linnaeus, 1758 (Holocephali), Rajonchocotyle emarginata (Olsson, 1876) (Hexabothriidae) from the thorny (starry) ray Amblyraja radiata (Donovan, 1808) (Elasmobranchii), and Callorhynchocotyle callorhynchi (Manter, 1955) (Hexabothriidae) from the chimaera (Cape elephant fish) Callorhynchus capensis Duméril, 1865 (Holocephali). The first two were collected in the Norwegian Sea off Tromsø, Norway, and the latter was from the Southeast Atlantic off St Helena Bay on the western coast of South Africa. Live specimens of all three monogenean species were fixed using 3 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 20 days at 5°C, rinsed four times for 20 min in the same buffer and post-fixed in 1 % osmium tetroxide for 1 h. For ultrathin studies, samples were then dehydrated in a graded series of ethanol and acetone, and embedded in a mixture of Araldite and Epon. Ultrathin sections (70-90 nm in thickness) were stained with uranyl acetate and lead citrate, and examined using a JEOL-JEM-1011 transmission electron microscope operating at 80 kV [86].
Observations with the electron microscope were performed by LGP; interpretation and choice of micrographs to be included in this study were done by JLJ.
All transverse sections of spermatozoa and spermatids in the figures, with a few exceptions when several sections are together in a micrograph, are orientated with the mitochondrion at the bottom, thus following the arbitrary convention of Sato, Oh & Sakoda [97] of mitochondrion as "ventral". Longitudinal sections of spermatids are orientated with the anterior part at the top, i.e., for the zone of differentiation, the arched membranes at the top, and with the free flagella and the median cytoplasmic process at the bottom [35,39]. Longitudinal schematic drawings of spermatozoa are oriented with the centrioles at the top and the nucleus at the bottom, since neodermatan spermatozoa are "inverted" in comparison with most phyla [1,39].

Results
Spermiogenesis in the chimaericolid Chimaericola leptogaster (Figures 1 and 2) Spermiogenesis involves the formation of a protuberance on each spermatid, termed zone of differentiation. The early zone of differentiation is visible as a small protuberance which is close to the extremity of the nucleus. An intercentriolar body is perpendicular to the cell membrane; the membrane shows peripheral microtubules ( Figure 1A). * The systematic placement of species has been updated when appropriate; the host fish is indicated when the monogenean species was not identified. ** "Polyopisthocotylean": The Polyopisthocotylean structure is defined as two axonemes and a continuous row of peripheral microtubules in the region which contains the nucleus; "Aberrant": special case of Diplozoidae; "Dubious": available micrographs do not show the Polyopisthocotylean structure; "no": available micrographs show that the structure is not polyopisthocotylean. The mature zone of differentiation has the typical structure found in most neodermatans (Polyopisthocotylea, Digenea and Cestoda). It is conical, with arching membranes at its base (proximal extremity) and bears, at its distal extremity, two lateral free flagella and a median cytoplasmic process. The zone of differentiation contains two centrioles flanking the intercentriolar body and a longitudinal striated root is associated with each centriole ( Figures 1B, 1C, 2). The mitochondrion and the nucleus pass through the zone of differentiation ( Figures 1B, 1C, 2) into the median cytoplasmic process ( Figure 1B). The two flagella fuse with the median cytoplasmic process; the late zone of differentiation contains the two centrioles, slightly slanted ( Figure 1E) and has arching membranes at its proximal extremity ( Figure 1D, 1E).

Spermatozoon of the chimaericolid Chimaericola leptogaster (Figures 3 and 4)
The mature spermatozoon is a very elongate and thin cell; the anteroposterior sequence of transverse sections was deduced from comparison of sections, based on the simple principle that the nucleus is posterior and the centrioles are anterior, as in all neodermatans [35,39]. The axonemes show a typical trepaxonematan 9 + "1" structure ( Figure 3E-W, transverse sections; Figure 3X, longitudinal section).
The general structure of the spermatozoon is biflagellate, with two axonemes incorporated into the sperm body, a longitudinal mitochondrion, a nucleus, and cortical longitudinal microtubules. The anterior region of the spermatozoon is pointed; it shows a few peripheral  A-C, anteriormost part of spermatozoon; peripheral microtubules and progressive appearance of the two centrioles. D, E, one fully formed axoneme and section of the centriole of the other axoneme. F-H, peripheral microtubules (full ring in G,H), two axonemes, and mitochondrion. I, two axonemes, peripheral row of microtubules reduced to a few units, and mitochondrion. J, one axoneme, distal extremity of other axoneme as scattered doublets and central core, a few remaining peripheral microtubules, and mitochondrion. K-M, one axoneme and mitochondrion, a few peripheral microtubules. N-P, one axoneme, a few peripheral microtubules, mitochondrion and section of the anterior thin part of the nucleus, which is wider as sections are more posterior. Q-S, one axoneme, a few peripheral microtubules, mitochondrion, and section of nucleus approximately as wide as axoneme. T-W, one axoneme, a few peripheral microtubules, mitochondrion, and wide section of nucleus. W is a very wide section with electron-transparent chromatin. X, typical trepaxonematan axoneme. Scale in W, valid for all figures: 500 nm. microtubules and the centriolar derivative of one of the axonemes ( Figure 3A-B). More posteriorly, sections show the second centriolar derivative ( Figure 3C) and the first axoneme, surrounded by a continuous row of peripheral microtubules ( Figure 3D-E). The next region, in an antero-posterior sequence, contains the two axonemes, the peripheral row of microtubules and a section of the mitochondrion ( Figure 3F-H). The following region has only a few peripheral microtubules ( Figure 3I-M); one of the axonemes finishes (as doublets, Figure 3J) and thus the rest of the cell contains a single axoneme. The anteriormost sections in this region show only the mitochondrion, which has a small diameter in cross sections, but more posterior sections show the first sections of the nucleus which are sections with very small diameter ( Figure 3N-P). The posterior region of the spermatozoon contains a single axoneme, a few peripheral microtubules, a small diameter section of the mitochondrion, and a section of the nucleus with a diameter increasing towards the posterior end ( Figure 3Q-W). The chromatin of the nucleus is compact and electron-dense in its thin, anterior part ( Figure 3N-Q) then partly compact and partly fibrous ( Figure 3R-V), and finally fibrous and electron-lucent in its posteriormost part ( Figure 3W). The distal posterior extremity of the spermatozoon contains only the nucleus and a single axoneme ( Figure 3W). While useful information about sperm structure should be sought only from sections of spermatozoa in the reproductive organs, we had the opportunity to observe sections in the caeca and in the genito-intestinal canal. Sections were generally indicative of cells in hypotonic media, with swollen cytoplasm with uniform contents and swollen mitochondria ( Figure 4A, B, D, E). Occasionally, a few sections which seemed normal ( Figure 4C, compare with Figure 3G) were found, and probably correspond to spermatozoa which were recently transferred to these organs.

Spermiogenesis in the hexabothriid Rajonchocotyle emarginata (Figures 5-7)
Spermiogenesis involves the formation of a zone of differentiation. The early zone of differentiation is visible as a small protuberance with subpellicular microtubules ( Figure 5A, D); at this stage, the nucleus is round and the mitochondria are gathered above the nucleus. Slightly more advanced zones of differentiation show the formation of the intercentriolar body and two centrioles, close to the cell membrane ( Figure 5C, D). The two centrioles give rise  Figure 6A). The next stage is a typical, conical, zone of differentiation, which contains peripheral microtubules, the elongating nucleus with lamellated chromatin, roundish mitochondria, and two centrioles each associated with a striated root and located on each side of the intercentriolar body. The distal extremity of the zone of differentiation bears three almost parallel processes, the two lateral free flagella, and the median cytoplasmic process ( Figure 6B-I). The mitochondria seem to be separate, as roundish discrete elements, in the zone of differentiation (Figure 6D, E, G-I) but are fused into a single ribbon in the median cytoplasmic process (Figure 7). The median cytoplasmic process contains the mitochondrial ribbon and two sets of peripheral microtubules, one ventral and one dorsal ( Figure 7B-G). Electron-dense zones of the membrane, known as "attachment zones" are visible on each side of the median cytoplasmic process (two on each side), where no peripheral microtubules are present ( Figure 7B). The free flagella show the typical trepaxonematan 9 + "1" structure ( Figure 7B-H) but their distal extremities lack the central core, thus producing distal sections with 9 + 0 structure ( Figure 7I). The free flagella fuse with the median cytoplasmic process on its lateral sides; one flagellum fuses before the other, thus producing sections with a single axoneme incorporated into the median cytoplasmic process ( Figure 7H). The advanced zone of differentiation is elongate and deeply embedded in a canal into the cytoplasm of the spermatid mass ( Spermatozoon of the hexabothriid Rajonchocotyle emarginata (Figures 9 and 10) The mature spermatozoon is a very elongate and thin cell (Figures 9 and 10), with, according to our reconstitution of the anteroposterior sequence of transverse sections, a wider posterior extremity containing the nucleus  ( Figure 10). The axonemes show a typical trepaxonematan 9 + "1" structure ( Figures 9 and 10, transverse sections; Figure 9H, longitudinal section). The general structure of the spermatozoon is biflagellate, with two axonemes incorporated into the sperm body, a longitudinal mitochondrion, a nucleus, and peripheral longitudinal microtubules. The anterior extremity of the spermatozoon is thinner than the rest; it contains a single axoneme and peripheral microtubules ( Figure 9A,B). The anterior region is identical to the elongate late zone of differentiation shown in Figure 8, with a circular section, an almost continuous circle of longitudinal parallel microtubules associated with characteristic external ornamentation on the membrane, two axonemes and a section of the mitochondrial ribbon ( Figure 9C-G); longitudinal sections show that the mitochondrion is a regular ribbon and that the external ornamentation looks like a continuous fuzzy layer ( Figure 9H). A section of the nucleus with very small diameter, reduced to the nuclear envelope without electron-dense chromatin, is seen in all sections except the anteriormost ones ( Figure 9C-Q). More posteriorly, the external ornamentation progressively disappears and the continuous row of peripheral microtubules is gradually replaced by two sets of microtubules without ornamentation, one dorsal and one ventral; attachment zones are visible, thus indicating that this region originates from the fusion of the free flagella with the median cytoplasmic process ( Figure 9I, 9L-Q). A part of this region has very few microtubules and sections are more oval than triangular in shape ( Figure 9O-Q). The posterior region of the spermatozoon contains the same elements (two axonemes, peripheral microtubules and a section of the ribbon-shaped mitochondrion) but the section of the nucleus represents an increasing portion of the section ( Figure 9R, 10A-D). At its wider part, the nucleus has additional membranes around the nuclear envelope and fibrous chromatin. The posterior part of the spermatozoon shows the same structure but only one axoneme is present ( Figure 10E-G). The posterior extremity contains a section of the nucleus with small diameter and only a few microtubules ( Figure 10H, I).   Spermatozoon of the hexabothriid Callorhynchocotyle callorhynchi (Figures 11 and 12) Spermiogenesis was not observed in this species. The mature spermatozoon is a very elongate and thin cell (Figures 11 and 12), with, according to our reconstitution of the anteroposterior sequence of transverse sections, a larger posterior extremity containing the nucleus and a particular region containing what looks like an undulating membrane. The axonemes show a typical trepaxonematan 9 + "1" structure ( Figures 11 and 12).
The general structure of the spermatozoon is biflagellate, with two axonemes incorporated into the sperm body, a longitudinal mitochondrion, a nucleus, and peripheral longitudinal microtubules. The anterior extremity of the spermatozoon is thin; it contains only a few microtubules ( Figure 11A-C). More posteriorly, the first axoneme appears ( Figure 11D, E) followed by the second axoneme ( Figure 11G). At the level of the first centriole and slightly posterior to it, two dense zones are visible within the cytoplasm, outside the microtubule row ( Figure 11C-F). The anterior region of the spermatozoon contains the two axonemes and numerous microtubules, organized at two rows of dorsal and ventral microtubules, each doubled by a more internal row of microtubules ( Figure 11H-K); no cortical microtubules are present on the sides. This region does not contain a section of the mitochondrion nor of the nucleus. Progressively, this region is transformed in a region with two sets of microtubules, one ventral and one dorsal, devoid of the internal row of microtubules; a section of the mitochondrion and a section of the nucleus appear ( Figure 11L-P); intermediary sections ( Figure 11L-P) show that the second, internal row of microtubules is progressively lost. The next region has only the "typical" elements of a mature neodermatan spermatozoon, i.e. two lateral axonemes, a mitochondrion, a nucleus, and two rows of peripheral microtubules, one on the ventral and one on the dorsal side ( Figure 11Q-T). More posteriorly, one of the axonemes disappears and sections contain only a single axoneme, a section of mitochondrion and nucleus, and dorsal and ventral peripheral microtubules; the nucleus has a wide section with fibrous chromatin (Figure 12 A-C). The next region is quite particular: the shape of transverse sections progressively flattens, with the axoneme on one side, the section of the nucleus on the other side, and a thin layer of cytoplasm containing only the two rows of peripheral microtubules, now close one to the other, between the axoneme and the nucleus ( Figure 12D-I).
Most posterior sections show the axoneme ending as singlets ( Figure 12K,L). The posterior extremity shows only a comma-shape section with peripheral microtubules ( Figure 12 M-O), then a small structure with only a few microtubules ( Figure 12P). Figure 15 is a schematic drawing of our interpretation of the structure of the mature spermatozoon of Callorhynchocotyle callorhynchi.
In most polyopisthocotylean monogeneans, the principal region of the spermatozoon (i.e. the region with the nucleus and mitochondrion) has two axonemes and a continuous row of peripheral microtubules. A single significant divergence from this pattern was found in the Diplozoidae [47,48]. Diplozoids are unique in the monogeneans (and even in the animal world) in that the two hermaphrodite members of a pair are united for life, with the genital ducts in permanent communication [15]. Sperm morphology is known to be driven by constraints of sperm function and competition [25]; it was hypothesized [39,47] that the aberrant sperm structure in diplozoids was the result of the absence of sperm competition, and thus more representative of a variation of fertilization biology than the mark of a distinctive diplozoid branch in the polyopisthocotylean tree. A parallel situation is also encountered in the schistosomes (Digenea) [31,36,39,45].
A special structure found in rare polyopisthocotyleans is an undulating membrane. A typical undulating membrane was described in Gotocotyla sp. [56]. This undulating membrane is composed of a lateral flange containing more than one hundred parallel microtubules, and observations of living spermatozoa showed that the undulating membrane was functional, i.e. showing active motility [56]. This was considered an autapomorphy of Gotocotyla [34]. Other structures superficially looking like this undulating membrane have been mentioned in various polyopisthocotyleans, generally in the form of a lateral flange with longitudinal peripheral microtubules. Examples, listed by Quilichini et al. [89] are Pricea multae, Discocotyle sagittata and Concinnocotyla australensis (references in Table 1); none of these are similar to what exists in Gotocotyla, and none have been shown to be functional. A single flange is found in P. multae and D. sagittata, but there are two flanges in C. australensis. We have no evidence that these various lateral flanges are homologous between themselves, and they should probably be considered as autapomorphies of the various taxa in  which they were found. The flat sections described here in the spermatozoon of Callorhynchocotyle callorhynchi are reminiscent of what is called, in urodele amphibians, an "undulating membrane" [3,23]. This structure is different from all other cases found in monogeneans since it includes the nucleus and a single axoneme, with a flattened part between them with longitudinal microtubules; we have no information whether this structure is functional or not, i.e. whether it has a special role in the movement of the spermatozoon. We consider it to be an autapomorphy of Callorhynchocotyle, and we point out that, given the current state of knowledge on hexabothriid sperm, it is not a synapomorphy of the Hexabothriidae since it was not found in Rajonchocotyle emarginata. We also point out that it does not resemble and is not homologous to the undulating membrane of Gotocotyla and the lateral flanges of certain other polyopisthocotyleans.  Various modifications of the nucleus shape, especially in cross sections, have been described. These include a crescent-shaped nucleus partially surrounding the axonemes, in Discocotyle sagittata, an annular nucleus completely surrounding the axonemes in Concinnocotyla australensis, and a special polygonally shaped nucleus in Atriaster spp. (references in Table 1). These variations should be considered autapomorphies of the taxa in which they were found. It is possible that variations of the structure of the anterior and posterior extremities of the spermatozoon provide additional structures useful for phylogenies [89], but this information is often lacking in published papers.

Significance of variations of sperm ultrastructure within the Neodermata and the Polyopisthocotylea
The Neodermata, which include all major groups of parasitic Platyhelminthes, i.e. the Digenea, Aspidogastrea, Eucestoda, Gyrocotylidea, Amphilinidea, Polyopisthocotylea, and Monopisthocotylea (the latter two often considered as forming the Monogenea) are characterised by spermatozoal synapomorphy, the proximo-distal fusion of axonemes during spermiogenesis [35]. Associated with this process is the presence of a characteristic structure termed the zone of differentiation [35,39]. The spermatozoon of the Neodermata has typically two axonemes and longitudinal peripheral microtubules [35,39].
Within the Neodermata, Justine (1991) [35] considered that there was a plesiomorphic pattern, with two axonemes and ventral and dorsal microtubules in the "principal region" of the spermatozoon, i.e. the region which contains the nucleus; it should be kept in mind that since the sperm of the Neodermata is "inverted", the region with the nucleus is posterior [35,39]. This plesiomorphic pattern is found in the Digenea and Cestoda. Two synapomorphies, defining major groups, were proposed: absence of the dorsal and ventral microtubules for the Monopisthocotylea, and presence of additional lateral microtubules for the Polyopisthocotylea. Figure 16 was drawn from Figure 5 in the 1991 paper by Justine [35].
For the Polyopisthocotylea, the synapomorphy "presence of lateral microtubules in the principal region of the spermatozoon" was defined by Justine in 1991 [34,35,38,39]. It was used in the two major attempts to develop a phylogeny of the monogeneans by Boeger & Kritsky in 1993 [12] and 2001 [14]; we will discuss mainly the more recent of these papers [14] because it includes the matrix. According to Boeger & Kritsky [14], this sperm synapomorphy was one of the six synapomorphies uniting the Polyopisthocotylea. It was used as character number 64 in their analysis as "lateral microtubules in the spermatozoon principal region of" and was character change 132 in their hypothesis [14]. Apomorphic state "present" was considered one of the six character changes that separates the Heteronchoinea (= Polyopisthocotylea) from the rest of the monogenes, considered as Polyonchoinea (= Monopisthocotylea); for equivalences between Polyopisthocotylea and Monopisthocotylea and between Heteronchoinea and Polyonchoinea see Table 1 in [75].
The present findings introduce an important change in the character matrix since this character was not found in the present study in the Chimaericolidae and Hexabothriidae, two of the most basal groups in the Polyopisthocotylea. Our knowledge of sperm structure in hexabothriids was limited to Erpocotyle catenulata; however, the published observation on this species by Tuzet & Ktari in 1971 [105] is a single micrograph, somewhat fuzzy, of spermatozoa which are clearly altered with open membranes; peripheral microtubules are present, but the presence of lateral microtubules in sections with a nucleus cannot be ascertained. We thus conclude that the character "lateral microtubules in principal region of spermatozoon" is not present in the hexabothriid species studied so far, which belong to three genera, Erpocotyle, Callorhynchocotyle and Rajonchocotyle.
Moreover, careful re-analysis of accounts of sperm structure in the Polystomatidae shows that this character was not general in this family. It can be found in Concinnocotyla australensis, but cannot be seen in any of the photographs of spermatozoa of Pseudodiplorchis americanus or Polystoma sp.; in Polystoma spratti, the  [14] and the actual information which was available at that time (see Table 1) and current information after present paper. microtubules at the level of the nucleus never form a complete row (references in Table 1). For the Sphyranuridae, our knowledge is limited to observations without illustrations [34]. Interpretation of the significance of presence and absence of the character in the Polystomatoinea would need additional observations; we provisionally consider that presence of the row of microtubules in certain polystome species has its origin in independent evolutionary events or convergences. It thus appears that the apomorphic character "presence of lateral microtubules in the principal region of the spermatozoon" is not a synapomorphy of the Polyopisthocotylea as a whole.

Discrepancies between actual data and the matrix of Boeger & Kritsky (2001)
A close examination of the character matrix in Boeger & Kritsky [14] shows that the character state was, in many cases, not coded in accordance with the data available at the time of publication of this paper (2001). The matrix includes 57 lines; for the Polyopisthocotylea, 33 families are listed, and the character state was coded as "1" (presence of lateral microtubules) in 32 of them À the only exception was the Montschadskyellidae, coded as "?", which was and is still correct. A comparison with Table 1 shows that the coding was correct in 15 families (i.e. character state "1" was actually visible in published papers), but that 13 families were erroneously coded "1" whereas the character state was unknown. Moreover, for two families, Pterinotrematidae and Axinidae, the character was coded as "1" whereas published information was 0. We interpret these discrepancies as "over-generalization", i.e. a character state common in a group was coded for all members of the group, while it was in fact present only for some members. In the matrix by Boeger & Kritsky [14], over-generalization concerns 15 polyopisthocotylean families amongst 33 ( Table 2). The inspiration for the over-generalization of spermatological characters can probably be found in the papers on sperm structure by Justine [34,35,39,46], who generally considered that sperm structure was homogeneous in the Polyopisthocotylea. Over-generalization and errors in the analysis of sperm characters probably calls for a re-examination of some other (non-spermatological) characters in the matrix of Boeger & Kritsky [14]. However, it should be outlined that small corrections in spermatological characters in the matrix would probably not change the resulting tree and the phylogeny proposed by Boeger & Kritsky [14].

Re-interpretation of the major synapomorphies of the Monogenea in light of this study
Hypotheses for the phylogeny of the Neodermata, the parasitic Platyhelminthes which include the Cestoda, Trematoda and Monogenea, have been based, successively or simultaneously, on four sets of characters over the years: (1) Traditional morphology; (2) Sperm ultrastructure; (3) Sequences of selected parts of the genome; (4) Complete mitochondrial genomes. We can expect that the next step will be the comparison of complete genomes; however, we believe that main morphological or spermatological characters are still to be considered even in the future.  [14]. The taxon defined by the character "presence of lateral microtubules in principal region of spermatozoon" is the Polyopisthocotylea as a whole. Right: Character changes as evidenced by present study. The taxon defined by the character "presence of lateral microtubules in principal region of spermatozoon" is restricted to the Mazocraeidea.
Major phylogenies based on morphology by Boeger & Kritsky (with the inclusion of several characters of spermatozoa) have concluded on monophyly of the Monogenea [12,14]. However, spermatological characters do not provide evidence for monophyly [35,39,40,42,43,46] and a reexamination of morphological characters found the monogenean non-monophyletic [22]. Molecular analyses based on 18S or 28S partial gene sequences [74,75,79] found the Monopisthocotylea and the Polyopisthocotylea each to be monophyletic, but the Monogenea were paraphyletic; an exception is the analysis by Lockyer et al. (2003) [69] in which the Monogenea were monophyletic. Complete mitochondrial genomes are known for about 40 species of Platyhelminthes [100], including a dozen monogeneans [4,30,64,[80][81][82][111][112][113][114]. Ye et al. (2014) [111] compared the mitogenomes of 10 species and found that Polyopisthocotylea and Monopisthocotylea had distinct gene arrangements. Therefore, molecular data currently available still point towards non-monophyly of the Monogenea. It is important to mention that all mitogenomes available for the Polyopisthocotylea are from a single superfamily, the Mazocraeidea, a situation which is reminiscent of the database on sperm ultrastructure before the present study.
In Figure 17, we try to reconcile the present information with a general phylogeny of the Polyopisthocotylea. We consider, finally, that the apomorphic character "presence of lateral microtubules in the principal region of the spermatozoon" is a synapomorphy of the Mazocraeidea. An investigation of the Diclybothriidae, the sister-group of the Hexabothriidae would be of interest; investigations in additional hexabothriids would be useful too, since our present knowledge is based only on three genera among the fifteen included in the family.
We believe that the present findings and interpretation are important not only for the polyopisthocotylean monogeneans, but for our understanding of phylogeny in the whole Neodermata. particular Dr Willy Hemmingsen, University of Tromsø (Norway), and the staff of the RV 'Johan Ruud' of the University for their help with the fishing and material collection, and Dr Cecile Reed, University of Cape Town (South Africa) for material collection. The authors are grateful to the staff of the Centre of Electron Microscopy, I.D. Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, Russia, for technical assistance. This research was performed in the framework of the state assignment of FASO Russia (theme No. 0122-2014-0007) and supported in part by RFBR (Project no. 15-04-02890-a to LGP). This paper was reviewed by three anonymous reviewers; the authors express their gratitude for the quality and detail of their comments.

Conflict of interest
The Editor-in-Chief of Parasite is one of the authors of this manuscript. COPE (Committee on Publication Ethics, http://publicationethics.org), to which Parasite adheres, advises special treatment in these cases. In this case, the peer-review process was handled by an Invited Editor, Jerôme Depaquit.