Molecular phylogeny of the Lecudinoidea (Apicomplexa): A major group of marine gregarines with diverse shapes, movements and hosts

Abstract

Gregarine apicomplexans are ubiquitous endosymbionts of invertebrate hosts. Despite their ecological and evolutionary importance, inferences about the phylogenetic relationships of major gregarine groups, such as the Lecudinidae and Urosporidae, have been hindered by vague taxonomic definitions and limited molecular and morphological data. In this study, we investigated five gregarine species collected from four families of polychaete hosts (Nereididae, Oenonidae, Hesionidae, and Phyllodocidae) using light microscopy (LM) and scanning electron microscopy (SEM). We also generated small subunit ribosomal DNA sequences from these species and conducted molecular phylogenetic analyses to elucidate the evolutionary relationships within the Lecudinoidea. Our results include new molecular and morphological data for two previously described species (Lecudina cf. platynereidis and Lecudina cf. arabellae), the discovery of a new species of Lecudina (L. oxydromus n. sp.), and the discovery of two novel species, namely Amplectina cordis n. gen. et. n. sp. and Sphinctocystis inclina n. sp. These two species exhibited unique shapes and movements, resembling those of urosporids but with a phylogenetic affinity to lecudinids, blurring the border between lecudinids and urosporids. Our study emphasizes the need for further investigations into this highly diverse group, which has achieved great success across multiple animal phyla with diverse shapes and movements.

Keywords: Lecudina; Gregarinasina; Urosporidae; molecular phylogeny; scanning electron microscopy.

FIGURE 1

Differential interference contrast light micrographs (LM) and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of a trophozoite of Lecudina cf. platynereidis. (A) LM showing an elongated, bottle‐like shaped trophozoite. The anterior end exhibits a ball‐ or dome‐like shape. The oval nucleus is located near the middle of the cell slightly toward the posterior end (scale bar: 20 μm). (B) SEM showing dense longitudinal folds on the trophozoite. An apical papilla is visible at the tip of the epimerite (scale bar: 20 μm). (C) The posterior end of a trophozoite displaying dense epicytic folds with undulations, shows slight spiral patterns that stop near the end (scale bar: 10 μm). ap, Apical papilla; ep, Epimerite; n, Nucleus; nu, Nucleolous.

FIGURE 2

Differential interference contrast light micrographs (LM) and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of trophozoites of Lecudina cf. arabellae. (A) LM images of two different trophozoites observed on an inverted microscope showing slightly different shapes. Both cells exhibit a blunt anterior end and a sharp posterior end. A rounded nucleus is located between the middle and anterior end of the cell. The cell on the left shows a more crescent shape, while the one on the right shows a straight shape and bending movement at the anterior part, which was commonly observed in many cells (scale bar: 100 μm). (B) LM images of the anterior parts of two trophozoites. A slight indentation is observed after an epimerite (indicated with an arrowhead) or where the bending movement occurs (indicated with a double arrowhead). Some cells harbored two nucleoli. Some transverse striations were visible (scale bar: 50 μm). (C) SEM of a trophozoite showing a crescent shape, blunt end, and a sharp posterior end (scale bar: 50 μm) (D) SEM showing the anterior part of a cell showing an apical papilla (scale bar: 10 μm). (E) High magnification SEM showing longitudinal epicytic folds with a density of 3–4 folds/μm (scale bar: 4 μm). ap, Apical papilla; ep, Epimerite; n, Nucleus; nu, Nucleolus; pe, Posterior end.

FIGURE 3

Differential interference contrast light micrographs (LM) and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of trophozoites of Lecudina oxydromus. (A) The trophozoite exhibits a crescent shape and brown color due to dense amylopectin granules. The anterior end lacks granules. An oval nucleus is situated near the anterior end (scale bar: 10 μm). (B) SEM image showing a crescent‐shaped trophozoite (scale bar: 10 μm). (C) High magnification SEM showing dense longitudinal folds with a density of 4–5 folds per μm (scale bar: 2 μm).

FIGURE 4

Differential interference contrast light micrographs (LM) and scanning electron micrographs (SEM) showing the general morphology, movement, and surface ultrastructure of trophozoites and gamonts of Amplectina cordis n. gen. et n.sp. (A) sequential series of LMs of gamonts in syzygy observed with an inverted microscope, showing peristaltic movement of cells (scale bar: 20 μm). (B) LM image of gamonts in syzygy observed with a compound microscope. Nuclei in each gamont are visible (scale bar: 20 μm). (C and D) Two different single trophozoites also demonstrating peristaltic movement (scale bar: 10 μm). (E) SEM images of two gamonts in sygyzy showing longitudinal folds and the syzygy junction (indicated with arrow heads) (scale bar: 10 μm). (F) SEM image of a single trophozoite showing longitudinal folds (scale bar: 10 μm). (G) High magnification SEM showing syzygy junction (indicated with arrow heads) and longitudinal folds with a density of 2–3 folds per μm (scale bar: 1 μm). (H) High magnification SEM showing longitudinal folds 3–4‐folds per μm (scale bar: 3 μm).

FIGURE 5

Differential interference contrast light micrographs (LM) and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of trophozoites and gamonts of Sphinctocystis inclina n. sp. (A) LM of two trophozoites observed on an inverted microscope. Both cells exhibit bending movement (scale bar: 100 μm). (B) LM of a trophozoite attached to host tissue showing annular constrictions. An oval shaped nucleus is located between the middle and the anterior end of the cell (scale bar: 50 μm). (C) SEM of a trophozoite showing annular constrictions (scale bar: 50 μm). The anterior end is smooth without noticeable apical papilla (scale bar: 50 μm). (D) SEM of a trophozoite showing its flexibility. Numerous annular constrictions are visible due to bending (scale bar: 25 μm). (E) High magnification SEM showing a dense array of undulating epicytic folds (scale bar: 2 μm).

FIGURE 6

A phylogenetic tree of apicomplexans inferred from 88 SSU rDNA sequences and 1239 sites using IQ‐tree. Only branches with strong support (BS > 90 on IQ‐tree, PP > 0.95 in MrBayes) are shown with gray circles. Although some family‐level and genus‐level groups received strong support, the relationships among them are not clear. All the sequences reported in this study (highlighted in blue font) belong to the Lecudinoidea (highlighted with a blue bar). Lecudinidae (green box) and Urosporidae (light blue box) are shown. Genera that were once placed within the Lecudinidae are highlighted with pink asterisks.

FIGURE 7

A phylogenetic tree of the Lecudinoidea inferred from 53 SSU rDNA sequences and 1377 sites using IQ‐tree. Both bootstrap support (BS) from IQtree and posterior probabilities (PP) from MrBayes analyses are displayed for internal branches. Two major families of Lecudinoidea, “Lecudinidae” and “Urosporidae”, are shown (highlighted with colored boxes). Well‐supported clades containing the type species of Lankesteria and Lecudina are highlighted with dashed boxes. Host information, as well as some important characteristics of the trophozoites (cell surface, infection sites, movement, and syzygy), are shown next to the tree. LF, Longitudinal folds; I, intestinal; G, Gliding; ?, Unknown. ‘observed’ under the syzygy category indicates that syzygy was observed but the exact orientation was not determined.