Fig 1.
Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium pendula isolated from the polychaete Scolelepis squamata.
A-C. DIC micrographs showing a spindle-shaped trophozoite in different positions of the pendular movement. The mucron at the anterior end is rounded (arrowhead) and visually separated from the rest of the cell with a slight restriction. The axial canal (ac) is conspicuous at the anterior end, but ran along the entire cell. The oval nucleus (n) is situated in the middle of the cell. D-E. Two gamonts (G1, G2) in caudal syzygy. The mucrons (arrowhead) are visible. The double arrow marks the junction between the two gamonts. Gamonts are changing from elongated to stumpy. F. Young gametocyst, the junction (double arrow) is still visible. G. Gametocysts in different focal planes, packed with round oocysts. H-K. SEM micrographs of trophozoites showing the epicytic longitudinal folds (arrow), the mucron (arrowhead) free of folds and some transverse striations in the inner curvature of the cell. Scale bars: Fig 1A-E, 30 μm; Fig 1F, 50 μm; Fig 1G, 70 μm; Fig 1H-I, 5μm; Fig J-K, 2μm.
Table 1.
Morphological comparison of Selenidium species presented in this study and other species that belong to the same clades in the phylogenetic tree (data from this study highlighted in bold).
Fig 2.
Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium hollandei isolated from the polychaete Sabellaria alveolata.
A-B. DIC micrographs of elongated and flattened trophozoites. The anterior end (mucron area, arrowhead) is narrower than the posterior end of the cell. C-D. Contracted trophozoites during peristaltic movement. The ovoid nucleus is visible (n). Broad epicytic folds (arrow) inscribe the surface of the entire cell. E-F. DIC and SEM micrographs of two gamonts (G1, G2) in lateral syzygy with overlapping posterior ends (double arrow). G-I. Trophozoites in different stages of movement. When contracted or bended, transverse striations (double arrow) form on the surface. The mucron (arrowhead) at the anterior end is free of folds. The broad longitudinal epicytic folds (arrows) cover most of the body. The lateral view shows dorso-ventrally extremely flattened trophozoites. J. Longitudinal epicytic folds (arrow) with visible pores (arrowheads) in the grooves between the folds. Scale bars: Fig 2A, C-D, G-H, 20 μm; Fig 2B, E-F, 30 μm; Fig 2I, 25 μm; Fig 2J, 5 μm.
Fig 3.
Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium sabellariae isolated from the polychaete Sabellaria alveolata.
A- C. DIC micrographs of trophozoites in different focal planes and different stages of movement. The mucron (arrowhead) is pointed to rounded and the ovoid nucleus (n) is situated in the middle of the cell. D. Two gamonts (G1, G2) in lateral syzygy with their orientation in opposite directions. The anterior ends are marked with arrowheads. Both gamonts are widest in the area of the junction (double arrow). E. Higher magnification SEM of the surface ultrastructure. There are numerous micropores (arrowheads) along the grooves of the main folds (arrow), whereas the grooves of the secondary folds (double arrow) do not display any micropores. F. Trophozoite, showing a rounded mucron (arrowhead) and longitudinal epicytic folds (arrow) with secondary folds expanding over parts of the cell’s length. G. Anterior end showing the rounded mucron (arrowhead) that is free of folds, but has a basal cluster of transverse striations (double arrowhead). Shortly after the mucron the main folds (arrow) start splitting (double arrowhead) into secondary folds. H. Posterior end of the trophozoite, showing the merging secondary folds (arrow), before they reach the posterior end. Scale bars: Fig 3A-D, 30 μm; Fig 3F, 20 μm; Fig 3E, G-H, 2 μm.
Fig 4.
Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium fallax isolated from the polychaete Cirriformia tentaculata.
A-C. DIC micrographs of trophozoites in different stages of movement. Trophozoits can be elongated to curled. The mucron (arrowhead) is rounded and sometimes quite flattened at the tip. The ellipsoid nucleus (n) is situated in the anterior part of the cell. D. SEM micrograph of a trophozoite, showing a flattened mucron (arrowhead) and longitudinal epicytic folds (arrows) with secondary folds expanding over parts of the cell’s length. The posterior end is very pointed. E. Anterior end showing the flat-topped mucron (arrowhead) that is free of folds, but has a nipple-like structure (arrow) in the middle. The upper rim appears to be a bit bulgy (double arrowhead). The region directly after the bulge is inscribed by short, narrow, superficial folds. The broader main folds start splitting (asterisks) into secondary folds shortly after. F. Posterior end of the trophozoite showing the tip free of folds, but with some indentations. G. Higher magnification SEM of the surface ultrastructure. The grooves between the secondary folds (asterisks) are often narrower than the grooves between the main folds (arrows). Scale bars: Fig 4A-B, 25 μm; Fig 4C, 15 μm; Fig 4D, 10 μm; Fig 4E-G, 1 μm.
Fig 5.
Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium sabellae isolated from the polychaete Sabella pavonina.
A. DIC micrographs of trophozoites in different focal planes. The mucron (arrowhead) is cone-shaped. The ellipsoid nucleus (n) is situated in the middle of the cell. Longitudinal epicytic folds (arrow) are visible along the cell. The posterior part ends in a dorso-ventrally flattened, blunt end. B. PC micrographs showing the plasticity of the cell. The nucleus (n) keeps its position in the middle of the trophozoite. C. DIC micrograph of an attached trophozoite with visible epicytic folds (arrow). D. High magnification SEM of the anterior end showing the cone-shaped mucron (arrowhead) that is free of folds. The longitudinal epicytic folds (arrows) start to broaden after their emergence. E. Trophozoite, showing the cone-shaped mucron (arrowhead) and longitudinal epicytic folds (arrows) that fan out right after the mucron. The posterior end is broadly rounded or blunt. F. Posterior end of trophozoite in lateral view, showing that the very tip is free of folds. The cell is dorso-ventrally flattened. There are micropores (arrowheads) visible in between the folds. G. Surface ultrastructure towards the posterior end. The epicytic folds (arrow) enlarge towards the posterior end, and gradually terminate. There are numerous micropores (arrowheads) along the grooves between the folds. Scale bars: Fig 5A, C, 20 μm; Fig 5B, 30 μm; Fig 5E, 10 μm; Fig 5F, D, 3 μm; Fig 5G, 1 μm.
Fig 6.
Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium spiralis sp. n. isolated from the polychaete Amphitritides gracilis.
A. PC micrograph showing the general spindle-like shape of the trophozoites with spirally arranged epicytic folds (double arrowhead) that appear to overlap each other in a crisscross pattern. The ovoid nucleus (n) with its round nucleolus (nu) is situated in the posterior half of the cell. The mucron (arrowhead) is set apart from the rest of the cell through a slight indentation. B. DIC micrographs of the same cell as in A in different focal planes. The main epicytic folds (double arrowhead) and the secondary epicytic folds (asterisks) are spirally arranged along the longitudinal axis of the cell. C. SEM micrograph showing the surface ultrastructure of the trophozoite. The mucron (arrowhead) is free of folds. The main epicytic folds (double arrowheads) start right after the mucron and split very early into secondary folds. The grooves (asterisks) between the secondary folds appear quite deep from the anterior end and become gradually shallower towards the posterior end at which point they disappear completely. The cell is adorned with transverse striations (arrows) along its whole length apart from the anterior and posterior tip. D. The mucron (arrowhead) is free of folds. The very prominent transverse striations (arrow) start immediately posterior to the mucron, similar to the main epicytic folds (double arrowhead) and the split (asterisks) into secondary folds soon after. E. The transverse striations (arrows) continue to almost the tip of the posterior end, but are less prominent compared to the anterior end. One slight indentation (asterisk) of a secondary fold is visible on one of the main epicytic folds (double arrowhead). F. High magnification SEM of the transverse striations (arrow) and the grooves (asterisk) between the folds. Scale bars: Fig 6A-B, 25 μm; Fig 6C, 10 μm; Fig 6D-F, 2 μm.
Fig 7.
Differential interference contrast (DIC) light micrographs showing the general morphology of the gregarine Selenidium antevariabilis sp. n. isolated from the polychaete Amphitritides gracilis.
A-C. DIC micrographs of spindle-shaped trophozoites with plastic anterior (arrowhead) and rounded posterior ends. The arrowhead marks the mucron area, which can be rounded or flattened, but often showing finger-like protrusions (double arrowhead). The ovoid nucleus (n) is situated in the middle of the cell or slightly shifted to the anterior end. Broad longitudinal epicytic folds (asterisks) run along the cells anterior-posterior axis with a helical turn, which appears like a criss-cross pattern on the surface (arrow). Scale bars: Fig 7A-C, 30 μm.
Fig 8.
Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium opheliae sp. n. isolated from the polychaete Ophelia roscoffensis.
A-C. DIC micrographs of different trophozoite cells showing the general elongated cell shape that is slightly reminiscent of a pea pod. The mucron (arrowhead) anterior end is slightly elongated and ends in a rounded tip, while the posterior end (arrow) is wider and either blunt or heart-shaped. The ovoid nucleus (n) is situated in the middle of the cell or shifted slightly to the posterior end. D. Two gamonts (G1, G2) in syzygy. This species forms a caudo-lateral syzygy with the two posterior ends overlapping (arrow). Both gamonts have a visible nucleus (n) in the middle. E. SEM micrograph showing the general morphology and ultrastructure of the trophozoite, with an elongated mucron (arrowhead) and a heart- shaped (arrow) posterior end. The surface seemed to be crinkled in places (double arrow). F. Some surface areas showed few transverse striations (asterisks) but in no obvious pattern. G. Anterior end with elongated mucron (arrowhead). H. Posterior end with visible crinkles (double arrow) on the surface. Scale bars: Fig 3A–3D, 30 μm; Fig 3E, 20 μm; Fig 3F–3H, 2 μm.
Fig 9.
Phylogenetic relationships of apicomplexans.
A. Bayesian tree of apicomplexans inferred using the GTR + C40 model of substitution in Phylobayes on an alignment of 171 small subunit (SSU) rDNA sequences and 1488 unambiguously aligned sites. Thickened lines at branches denote Bayesian posterior probabilities and bootstrap support (see legend in the center of the figure for details). The eight sequences derived from this study are highlighted in bold. A1 to A5 show the positions of the five ‘archigregarine’clades. B. Schematized Maximum likelihood phylogeny analysis of the same dataset inferred under the GTR+G model as implemented in RAxML 8.2. The outgroup composition and topology is the same as in Fig 9A and is simplified due to space limitations. C. Bayesian topology of the dataset S with exhaustive sampling of Selenidiidae, also enriched for environmental sequences inferred in Phylobayes under the GTR + C40 model of substitution. The branching support follows parameters of tree A.