Peer Review #1 of "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) (v0.1)"

1 Department of Invertebrate Zoology, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russian Federation 2 CNRS, UMR 7144, Laboratoire Adaptation et Diversité en Milieu Marin, Roscoff, France 3 CNRS, UMR 7144, Station Biologique de Roscoff, Sorbonne Universités, Université Pierre et Marie Curie Paris 6, Roscoff, France 4 Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic 5 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation 6 Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow, Russian Federation 7 CNRS 7245, Molécules de Communication et Adaptation Moléculaire (MCAM), Paris, France 8 Sorbonne Universités, Muséum National d’Histoire Naturelle (MNHN), UMR 7245, Paris, France

495 cytopharyngeal complex has been observed -only with the possibly exception of the earliest 496 developmental stage (Fig. 10, F1, G3). A large frontal vacuolar structure (usually flattened and 497 containing fibrillar matter) develops just beneath the cell junction region; however, the vacuole 498 can sometimes be completely replaced with a dense fibrillar zone  499 . No evidence of its involvement in the eugregarine feeding has 500 been observed. Finally, archigregarines retain their mucron (together with the apical complex) 501 well into the syzygy stage (Fig. 10, D)  . Although the fate of the 502 attachment apparatus in aseptate eugregarines is poorly studied, it is presumably the same that in 503 the septate eugregarines, whose gamonts lose their epimerite upon the detaching from the host 504 epithelium Devauchelle, 1968;505 Schrével et al., 2013). 506 The structure and cytoplasmic content of developed epimerites in septate gregarines vary 507 substantially (Devauchelle, 1968;Desportes, 1969;Ormierès & Daumal, 1970; 508 Valigurová & Koudela, 2005): numerous mitochondria, granules of 509 amylopectin, lipid drops and vacuoles, well developed arrays of ER, and numerous fibrillar 510 structures (microtubules and microfilaments) -especially in the basal region if it is shaped like a 511 neck or stalk (so-called "diamerite") as, e.g., in Epicavus araeoceri (Ormierès & Daumal, 1970), 512 can be present.
513 The attachment apparatus of A. sagittata displays the main features of a simple epimerite (Fig. 4) 514 lacking cytoplasmic organelles and inclusions (absence of mitochondria, ER, and lipid drops), 515 although, like in epimerites of septate gregarines, there are amylopectin granules and a large 516 frontal vacuole -although not flattened, but rather bulky. Detached gregarines (mature 517 gamonts?) have no epimerite, which has been apparently discarded, judging from the appearance 518 and behaviour of individuals that were artificially dislodged from the host epithelium (see 519 Results). Some other aseptate gregarines also possess complex attachment organelles that are 520 comparable to true epimerites of septate gregarines in shape, ultrastructure, and fate (absent in 521 mature gamots), e.g., Lecudina (Cygnicollum) lankesteri. Unlike A. sagittata, the epimerite of L.
529 Taking into account the homologies of the eugregarine attachment organelles, the term "mucron" 530 should be restricted to the attachment apparatus in archigregarines, which contains the apical 531 complex and performs myzocytosis. In eugregarines, both aseptate and septate, the term 532 "epimerite" appears to be more appropriate, and is in accordance with the definitions and 533 gregarine descriptions in the "classic" literature on gregarines (e.g., Watson . This 534 terminological correction will remove ambiguity in taxonomical diagnoses and emphasize that 535 the epimerite is a shared evolutionary innovation (synapomorphy) of eugregarines. More 536 representative data are required to distinguish different types of epimerites: for example, the 544 In SSU rDNA phylogenies published to date, gregarines (sensu class Gregarinomorpha Grassé, 545 1953) have been not monophyletic. The most probable reason for that is that the SSU rDNA 546 sequences of many gregarines are highly divergent, therefore the topologies of resulting 547 phylogenetic trees are sensitive to changes in alignment site selection and taxon sampling.
548 Additionally, the presence of many long branches among gregarines and other apicomplexans 549 may lead to long branch attraction (LBA) artefacts (Bergsten, 2005). Only after careful manual 550 editing of the alignment (see supplemental raw data) and the exclusion of single gregarine 551 sequences corresponding to three extremely long branches (Pyxinia crystalligera, Stenophora 552 robusta, and Trichotokara spp.), all of the other gregarines did form a monophyletic lineage, 553 albeit weakly supported (Fig. 7). Despite their weakly supported monophyly in the SSU rDNA 554 phylogenies, all gregarines display a distinct morphological synapomorphy: the gametocyst, 555 which is an encysted syzygy (Frolov, 1991). Among other apicomplexans, only adeleid 556 coccidians have syzygy, but without the subsequent encystment into a gametocyst. Unlike SSU 557 rDNA alone, both the LSU rDNA and ribosomal operon-based phylogenies support the 558 monophyly of gregarines, although with a significantly limited taxon sampling without 559 archigregarine and some eugregarine lineages (Fig. 9). Similarly to SSU rDNA phylogenies, 560 long branch attraction can affect these tree topologies, although its negative effects are expected 561 to be lower than in SSU rDNA-alone phylogenies because the relative evolution rates of the LSU 562 rDNA in apicomplexans are more even than those of the SSU rDNA (Simdyanov, Diakin & 563 Aleoshin, 2015). The tegument of mucron is trimembrane pellicle excepting small region in front of conoid, where a cytostome and duct of mucron vacuole is intermittently formed, IMC is absent and there is just a single plasma membrane.
The IMC of the pellicle terminates at the edge of the cell junction zone, so the tegument of the attachment organelle is represented only by a single plasma membrane.
The IMC of the pellicle terminates at the edge of the cell junction zone, so the tegument of the attachment organelle is represented only by a single plasma membrane.

Cell junction between host and parasite
Septate cell junction; no peculiar structures on the edge of the junction zone.
Two closely adjacent plasma membranes (of host and parasite) forming high electron density zone. A circular groove in the gregarine tegument (plasma membrane) runs along the edge of the region of the cell junction (where the IMC terminates) and pinches a small portion of the host cell.
Two closely adjacent plasma membranes (of host and parasite) forming high electron density zone. A circular groove in the gregarine tegument (plasma membrane) runs along the edge of the region of the cell junction (where the IMC terminates) and pinches a small portion of the host cell.