A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarine Ancora sagittata (Leuckart, 1860) Labbé, 1899 (Apicomplexa: Eugregarinida)

PeerJ. 2017 May 30;5:e3354. doi: 10.7717/peerj.3354. eCollection 2017.

Abstract

Background: Gregarines are a group of early branching Apicomplexa parasitizing invertebrate animals. Despite their wide distribution and relevance to the understanding the phylogenesis of apicomplexans, gregarines remain understudied: light microscopy data are insufficient for classification, and electron microscopy and molecular data are fragmentary and overlap only partially.

Methods: Scanning and transmission electron microscopy, PCR, DNA cloning and sequencing (Sanger and NGS), molecular phylogenetic analyses using ribosomal RNA genes (18S (SSU), 5.8S, and 28S (LSU) ribosomal DNAs (rDNAs)).

Results and discussion: We present the results of an ultrastructural and molecular phylogenetic study on the marine gregarine Ancora sagittata from the polychaete Capitella capitata followed by evolutionary and taxonomic synthesis of the morphological and molecular phylogenetic evidence on eugregarines. The ultrastructure of Ancora sagittata generally corresponds to that of other eugregarines, but reveals some differences in epicytic folds (crests) and attachment apparatus to gregarines in the family Lecudinidae, where Ancora sagittata has been classified. Molecular phylogenetic trees based on SSU (18S) rDNA reveal several robust clades (superfamilies) of eugregarines, including Ancoroidea superfam. nov., which comprises two families (Ancoridae fam. nov. and Polyplicariidae) and branches separately from the Lecudinidae; thus, all representatives of Ancoroidea are here officially removed from the Lecudinidae. Analysis of sequence data also points to possible cryptic species within Ancora sagittata and the inclusion of numerous environmental sequences from anoxic habitats within the Ancoroidea. LSU (28S) rDNA phylogenies, unlike the analysis of SSU rDNA alone, recover a well-supported monophyly of the gregarines involved (eugregarines), although this conclusion is currently limited by sparse taxon sampling and the presence of fast-evolving sequences in some species. Comparative morphological analyses of gregarine teguments and attachment organelles lead us to revise their terminology. The terms "longitudinal folds" and "mucron" are restricted to archigregarines, whereas the terms "epicystic crests" and "epimerite" are proposed to describe the candidate synapomorphies of eugregarines, which, consequently, are considered as a monophyletic group. Abolishing the suborders Aseptata and Septata, incorporating neogregarines into the Eugregarinida, and treating the major molecular phylogenetic lineages of eugregarines as superfamilies appear as the best way of reconciling recent morphological and molecular evidence. Accordingly, the diagnosis of the order Eugregarinida Léger, 1900 is updated.

Keywords: Apicomplexa; Environmental DNA sequences; Marine gregarines; Phylogeny; SSU and LSU rDNA; Taxonomy; Ultrastructure.

Grant support

This work was supported by the Russian Foundation for Basic Research (project No. 15-29-02601), ECO-NET project 2131QM (Égide, France), the European Project MaCuMBa (FP7-KBBE-2012-6-311975), the French governmental ANR Agency under ANR-10-LABX-0003 BCDiv, ANR-11-IDEX-0004-02, and ANR HAPAR 2014 défi 1, the Interdisciplinary Program of the MNHN (ATM-Emergence des clades, des biotes et des cultures), and the Czech Science Foundation, project No. GBP505/12/G112 (ECIP). The phylogenetic analyses of SSU rDNA presented in this study were supported by the Russian Science Foundation, project No. 14-50-00029. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.