Jean-Lou Justine

SPERMATOZOA AS PHYLOGENETIC CHARACTERS FOR THE EUCESTODA

Spermatozoon ultrastructure and spermiogenesis are significant characters for phylogenetic inference. Sperm ultrastructure is reviewed from the literature in 56 species of Eucestoda. Data are available for 11 of the 12 orders of Eucestoda (Lecanicephalidea excepted), but in some orders data are scarce and often limited to a single species. Spermiogenesis and sperm ultrastructure in the Eucestoda is compared to that of other parasitic Platyhelminthes, with emphasis on structures of phylogenetic interest. Not only the descriptions of sperm structure, but those of the process of spermiogenesis, are necessary to define characters. Synapomorphies based on sperm ultrastructure for the Eucestoda include the absence of a mitochondrion in mature sperm and the presence of a crested body. A proposed synapomorphy for the Cyclophyllidea + Tetrabothriidea is the twisting of the peripheral microtubules; the absence of intercentriolar body and the absence of striated roots in the spermatid may constitute additional synapomorphies for this assemblage. Absence of flagellar rotation during spermiogenesis is synapomorphic for the Cyclophyllidea, and absence of proximodistal fusion could be synapomorphic for a part of the Cyclophyllidea. Several other characters could be useful for understanding phylogeny within the Eucestoda. The polarity of these characters could in several cases be determined, but diagnoses for taxa or relationships based on synapomorphies cannot be specified unequivocally due to putative convergence. Such characters and their putative polarity include: (1) the number of axonemes in mature spermatozoon (plesiomorphic=2; apomorphic=1); (2) size and number of crested bodies (p=1; a=several); and (3) angle of twisted microtubules; shape of nucleus (p=compact cord; a=crescent and annulus). Additional apomorphic attributes include (1) presence of a periaxonemal sheath (a putative synapomorphy for the Cyclophyllidea + Tetrabothriidea if reversals are postulated in certain cyclophyllideans); (2) presence of proteinaceous transverse walls; (3) presence of dense granules; and (4) shape of apical cones and posterior structures. Studies of sperm structure in the poorly known orders and additional comparative studies in the Cyclophyllidea are expected to provide new information for elucidation of phylogenetic relationships.

Phylogeny of the orders of the Eucestoda (Cercomeromorphae) based on comparative morphology : Historical perspectives and a new working hypothesis

The phylogeny of the Eucestoda was evaluated based on a suite of 49 binary and multistate characters derived from comparative morphological and ontogenetic studies; attributes of adult and larval tapeworms were considered. A single most parsimonious tree (MPT) (consistency index = 0.872; retention index = 0.838; and homoplasy index = 0.527) was fully resolved and is specified by the following: (Gyrocotylidea, (Amphilinidea, ((Spathebothriidea, (Pseudophyllidea, ((Diphyllidea, (Trypanorhyncha, (Tetraphyllidea, (Lecanicephalidea, ((Nippotaeniidea, (Tetrabothriidea, Cyclophyllidea)), Proteocephalidea))))), Haplobothriidea))), Caryophyllidea))). Monophyly for the Eucestoda was firmly corroborated. Trees derived from the primary and bootstrap analyses were congruent, but low values, particularly for relationships among the tetrafossate tapeworms, indicated additional examination is warranted. The MPT was found to be the most efficient hypothesis for describing character evolution and in specifying relationships among the orders when compared to those concepts that had been developed for the tapeworms over the past century. Areas of congruence were shared among the current hypothesis and one or more of the prior hypotheses. Major conclusions include: (1) Caryophyllidea are basal and monozooy is ancestral; (2) difossate forms are primitive, and the Pseudophyllidea are the sister group of the strongly polyzoic tapeworms; (3) Nippotaeniidea are highly derived; (4) the higher tapeworms (Tetraphyllidea, Lecanicephalidea, Proteocephalidea, Nippotaeniidea, Tetrabothriidea, and Cyclophyllidea) are closely related or potentially coordinate groups: (5) Tetrabothriidea and the Cyclophyllidea are sister groups; and (6) Tetraphyllidea is paraphyletic, with the Onchobothriidae basal to the Phyllobothriidae. Character support for placement of the Tetrabothriidea continues to be contradictory, and this order may represent a key to understanding the phylogeny of the higher cestodes. The current study constitutes a complete historical review and poses a new and robust hypothesis for the phylogeny of the Eucestoda.

Cladistic study in the Monogenea (Platyhelminthes), based upon a parsimony analysis of spermiogenetic and spermatozoal ultrastructural characters

Abstract Justine J.-L. 1991. Cladistic study in the Monogenea (Platyhelminthes), based upon a parsimony analysis of spermiogenetic and spermatozoal ultrastructural characters. International Journal for Parasitology 21 : 821–838. Characters concerning spermiogenesis and spermatozoon ultrastructure in the Monogenea were analysed with the program PAUP. A synapomorphy for the entire Monogenea (Polyopisthocotylea + Monopisthocotylea) could not be defined on the basis of a spermatozoal character. In the polyopisthocotylean Monogenea, spermatozoon ultrastructure is relatively homogeneous and a parsimony analysis was not attempted. In the monopisthocotylean Monogenea, 18 unordered characters (17 binary, one multistate) were used. A parsimony analysis was done for 23 taxa (21 monopisthocotylean taxa, the Polyopisthocotylea were considered as one taxon, and the Digenea was used as the outgroup); among the Monopisthocotylea, nine taxa were redundant, generally because of incomplete data. A semi-strict consensus tree was obtained (consistency index 0.645). Monophyly was established on the basis of spermatozoal and spermiogenetic ultrastructural characters in the following groups: the Polyopisthocotylea (one synapomorphy), the Monopisthocotylea (three synapomorphies), the Capsalidae/Dionchidae (two synapomorphies), the Loimoidae/Monocotylidae (one synapomorphy), and a group (two synapomorphies) which contains all Monogenea with uniflagellate spermatozoa grouped in a polychotomy (Ancyrocephalidae, Calceostomatidae, Pseudodactylogyridae, Diplectanidae, Tetraonchidae and Amphibdellatidae). Autapomorphies were recognized for the Amphibdellatidae and the Calceostomatidae. Not only spermatozoal ultrastructure, but also ultrastructure of spermiogenesis, should be used for defining synapomorphies. The tree herein obtained was compared with the most recent classifications of the Monogenea, based on morphology (Lebedev, 1988, Angewandte Parasitologie 29 : 149–167; Malmberg, 1990, Systematic Parasitology 17 : 1–65); important similarities with Lebedevs classification were observed.

PHYLOGENETIC ANALYSIS OF THE MONOGENEA AND THEIR RELATIONSHIPS WITH DIGENEA AND EUCESTODA INFERRED FROM 28S RDNA SEQUENCES

Platyhelminth phylogeny is controversial. Phylogenetic analyses of the partial domain C1 and the full domains D1 and C2 (358 nucleotides) from the 28S ribosomal RNA gene for 21 species from the Monogenea, Digenea, Cestoda, and, as the outgroup, Tricladida reveal major departures from prevailing theory. The Digenea and not the Monogenea (Monopisthocotylea and Polyopisthocotylea) form the sister group of the cestodes; the Monopisthocotylea and Polyopisthocotylea are each monophyletic, but the Monogenea do not form a monophylum; the sister group of the Digenea + Cestoda is the Polyopisthocotylea; and Monopisthocotylea are the sister group of all other parasitic flatworms.

SPERMATOZOON ULTRASTRUCTURE AND PHYLOGENETIC RELATIONSHIPS IN THE MONOGENEANS (PLATYHELMINTHES)

Abstract Justine J.-L. , Lambert A. and Mattei X. 1985. Spermatozoon ultrastructure and phylogenetic relationships in the monogeneans (Platyhelminthes). International Journal for Parasitology 15: 601–608. New observations reported in this study together with bibliographical data allow comparisons of spermatozoon ultrastructure in 28 genera of monogeneans, belonging to 19 families. The authors propose to compare and classify monogenean spermatozoa using two simple ultrastructural characteristics: (a) the number of axonemes, 1 or 2, (b) the presence or absence of cortical microtubules. These traits make it possible to group monogenean spermatozoa in four patterns. Pattern 1 (2 axonemes plus microtubules) is characteristic of the polyopisthocotyleans (9 families). The three other patterns are found in the monopisthocotyleans. Pattern 2 (2 axonemes without microtubules) is found in the Capsalidae and Dionchidae, which seem closely related, and also in the Udonellidae, Gyrodactylidae and Euzetrema . Pattern 3 (1 axoneme plus 1 altered axoneme plus microtubules) is found in the Monocotylidae and Loimoidae. Pattern 4 (1 axoneme without microtubules) is found in the Amphibdellatidae, Ancyrocephalidae, Calceostomatidae and Diplectanidae. A phylogeny of the monogeneans is drawn from the data of comparative spermatology; this scheme coincides in many points with the phylogeny of Lambert (1980) which was based on the study of chaetotaxy and ciliated cells of the oncomiracidium.

Réinvestigation de l'ultrastructure du spermatozoïde d'Haematoloechus (Trematoda: Haematoloechidae)

en The threadlike spermatozoon of the frog lung fluke, Haematoloechus, shows two patterns of external ornamentation. The first type is anterior and consists of bristles on the membrane that coincide with certain peripheral microtubules. This already exists in the zone of differentiation (ZD) of the young spermatid. The ZD is pinched off from the cytoplasmic mass at the arching-membrane level. The centriolelike body and striated roots are depolymerized. The two centrioles, made up of triplets, and the bristles are retained in the anterior part of the spermatozoon. The second type is more posterior. At this level, the transverse sections of the spermatozoon are asymmetric; only the larger side has peripheral microtubules, arranged in a semicircle around the axoneme, and a thick external crenate layer on its membrane. After several micrometers, this zone ends in an open collar (collerette) enclosing the next region of the spermatozoon. Only the part anterior to the collar is motile; on spontaneously breaking off, this region is mobile, although the unbroken spermatozoon is not. After the collar there is a region without microtubules. Then comes the very long middle region with dorsally and ventrally placed microtubules; the nucleus is at the posterior end. This new spermatozoon pattern, with retained ZD and anterior centrioles, resembles what we described earlier in a didymozoid. It probably occurs often in Digenea and perhaps other Platyhelminthes as well.

Spermiogenesis and spermatozoon of Echinostoma caproni (Platyhelminthes, Digenea): transmission and scanning electron microscopy, and tubulin immunocytochemistry

Spermiogenesis and the spermatozoon of Echinostoma caproni (from experimentally infested laboratory mice) were investigated by several methods. Transmission electron microscopy shows that spermiogenesis consists of proximo-distal fusion of three processes followed by elongation of the spermatid. Scanning electron microscopy shows that the spermatozoon is a filiform cell, 235 microns in length, with a cylindrical anterior extremity and a broader posterior extremity. Epifluorescence microscopy, including immunocytochemistry of tubulin and labelling of nucleus with specific dyes, has provided valuable additional information. Migration of the nuclei from the common cytoplasmic mass of spermatids to the distal part of the elongating spermatids is visualized, and centrioles demonstrated in the proximal, anterior region, and the nucleus in the distal, posterior region of the spermatozoon. One axoneme has a distal extremity which in the mature spermatozoon extends 30 microns more distally than the other, with the result that the posterior part of the spermatozoon contains a single axoneme and nucleus. Immunocytochemistry experiments show that a region, 15 microns in length, not labelled by the anti-tubulin antibodies with certain fixation-permeabilization procedures, corresponds to a region which, by transmission electron microscopy, shows external ornamentation on the membrane. This region has a bilaterally asymmetric pattern (in TEM), forms angles or coils according to the fixation used, and marks the boundary between two distinct patterns of movement. Spermiogenesis and the spermatozoon in E. caproni correspond to the general pattern found in the digeneans, with the exception of this asymmetric region. It is emphasized that the use of various methods provides a better understanding of sperm structure than transmission electron microscopy alone, particularly in the case of long, filiform spermatozoa.

Phylogenetic relationships within the polyopisthocotylean monogeneans (Platyhelminthes) inferred from partial 28S rDNA sequences

Recent studies based on molecular data (18S rDNA and partial 28S rDNA) and morphology did not resolve a terminal polytomy within the Polyopisthocotylea. Here, we have used sequences from the full domain D2 of the 28S rDNA for 24 species (18 new sequences) with three phylogenetic methods, maximum parsimony, neighbour-joining and maximum likelihood, to infer the relationships among the Polyopisthocotylea. The analysis of the domain D2 of the 28S rDNA has been performed on two data sets. The first one, complete, included the Polystomatidae as the outgroup in order to infer general relationships, and the second one, reduced, excluded the Polystomatidae and the polyopisthocotylean parasites of chondrichthyans, but used the Mazocraeidae as the outgroup in order to resolve the relationships between the terminal groups. The topology found, sustained by high bootstrap and decay index value, is: (outgroup (Chimaericolidae (Mazocraeidae (Gastrocotylinea, other Polyopisthocotylea)))). The polyopisthocotylean parasites of chondrichthyans are the sister-group of the polyopisthocotylean parasites of teleosts. In the latter, the Mazocraeidae, essentially parasites of Clupeidae, have a basal position. The polytomy between Gastrocotylinea, Discocotylinea and Microcotylinea is partially resolved in this study for the first time: the Gastrocotylinea are the sister-group of an unresolved group including the Microcotylinea, Discocotylinea and Plectanocotylidae. Inclusion of the Plectanocotylidae in the suborder Mazocraeinea is rejected. Monophyly of the Microcotylinea and Plectanocotylidae is confirmed, but monophyly of the Discocotylinea is questioned by the exclusion of Diplozoon.

Etude ultrastructurale de la spermiogenèse et du spermatozoïde d'un Plathelminthe: Gonapodasmius (Trematoda:Didymozoidae)

The technique of serial sections has been employed to study spermiogenesis and spermatozoon of Gonapodasmius. The zone of differentiation (ZD) shows no centriole-like body and no striated root. Peripheral microtubules are of two types: laterally placed thick microtubules which are spaced and coincide with external bristles on the membrane; and dorsally and ventrally placed thin microtubules which are closely aligned. The two parallel flagella fuse with the median cytoplasmic process. The nucleus, mitochondrion, and thin microtubules migrate distally. The arching membranes, at the base of the ZD, mark the area where the mature spermatozoon is pinched off from the cytoplasmic mass. The spermatozoon consists of four regions. (a) The anterior region, made of the ZD, has peripheral microtubules with external bristles, viewed in toto as longitudinal ornamentations. Two centriolar derivatives, made up of nine singlets each, are continued as two 9+“1” flagella, classic in Platyhelminthes. (b) The intermediate region shows no peripheral microtubules. (c) The middle region shows nucleus, mitochondria, and ventral microtubules without ornamentations. (d) In the terminal region, the two flagella separate. The first two regions have never been described in previously studied Platyhelminthes spermatozoa.

Phylogeny of the monopisthocotylea and Polyopisthocotylea (Platyhelminthes) inferred from 28S rDNA sequences.

This study focuses on the phylogenetic relationships within the Polyopisthocotylea and Monopisthocotylea, two groups that are often grouped within the monogeneans, a group of disputed paraphyly. Phylogenetic analyses were conducted with multiple outgroups chosen according to two hypotheses, a paraphyletic Monogenea or a monophyletic Monogenea, and with three methods, namely maximum parsimony, neighbour joining and maximum likelihood. Sequences used were from the partial domain C1, full domain D1, and partial domain C2 (550 nucleotides, 209 unambiguously aligned sites) from the 28S ribosomal RNA gene for 16 species of monopisthocotyleans, 26 polyopisthocotyleans including six polystomatids, and other Platyhelminthes (61 species in total, 27 new sequences). Results were similar with outgroups corresponding to the two hypotheses. Within the Monopisthocotylea, relationships were: ¿[(Udonella, capsalids), monocotylids], (diplectanids, ancyrocephalids)¿; each of these families was found to be monophyletic and their monophyly was supported by high bootstrap values in neighbour joining and maximum parsimony. Within the Polyopisthocotylea, the polystomatids were the sister-group of all others. Among the latter, Hexabothrium, parasite of chondrichthyans, was the most basal, and the mazocraeids, mainly parasites of clupeomorph teleosts, were the sister-groups of all other studied polyopisthocotyleans, these, mainly parasites of euteleosts, being polytomous.