New species of Pavlovophyceae (Haptophyta) and revision of the genera Exanthemachrysis, Rebecca and Pavlova Author Véron, Benoît Laboratoire de Biologie des Organismes et Écosystèmes Aquatiques (BOREA) Université de Caen-Normandie, MNHN, SU, UA, CNRS, IRD, 14000 Caen, France. & Normandie Université, UNICAEN, UMR 8067 BOREA, MNHN, SU, CNRS, IRD- 207, UA, 14000 Caen, France. benoit.veron@unicaen.fr Author Rougier, Etienne Laboratoire de Biologie des Organismes et Écosystèmes Aquatiques (BOREA) Université de Caen-Normandie, MNHN, SU, UA, CNRS, IRD, 14000 Caen, France. & Normandie Université, UNICAEN, UMR 8067 BOREA, MNHN, SU, CNRS, IRD- 207, UA, 14000 Caen, France. etienne.rougier@gmail.com Author Taylor, Anthony Laboratoire de Biologie des Organismes et Écosystèmes Aquatiques (BOREA) Université de Caen-Normandie, MNHN, SU, UA, CNRS, IRD, 14000 Caen, France. & Normandie Université, UNICAEN, UMR 8067 BOREA, MNHN, SU, CNRS, IRD- 207, UA, 14000 Caen, France. & Institute of Marine Sciences, University of Portsmouth, Portsmouth, Hampshire, UK. anthony.taylor2590@gmail.com Author Goux, Didier Normandie Université, UNICAEN, CMAbio 3 (Centre de Microscopie Appliquée à la Biologie), SF EMerode, 14000 Caen, France. & Normandie Université, ENSICAEN, UNICAEN, CNRS, UMR 6508 CRISMAT, 14000 Caen, France. didier.goux@unicaen.fr text European Journal of Taxonomy 2023 2023-03-08 861 21 47 journal article 58894 10.5852/ejt.2023.861.2063 98f15b31-8fcd-4ea4-9621-6bff596e64fe 2118-9773 7710258 Genus Pavlova Butcher emend. Véron Figs 10–13 The unidentified strains of Pavlova (AC248 and AC250) were chosen for study because none had ever been examined within the sub-clade 3.2 ( Bendif et al. 2011 ); both strains were found to have the same cytomorphological characteristics as the genus Pavlova . Description of strains AC248 and AC250 Non-motile cells are occasionally present in cultures, clustered to a few in a loose mucilage ( Fig. 11A ) and showing a reduced flagellar appendage. Motile cells are slightly ovoid (5.9 µm ± 0.5 × 5.2 µm ± 0.4, n = 49), free swimming and highly metabolic ( Fig. 10 ). Emergence of the appendages is from a narrow, shallow sub-apical pit ( Fig. 12A ). Except at its base ( Fig. 12E ), the AF (9.5 µm ± 3.8, n = 12) is coated with several layers of regularly spaced ( Fig. 12D ) flat and ovoid KS (≈ 47 × 34 nm , n = 4) with a slight median constriction and with fine non-tubular hairs ( Fig. 12C ). The smooth and short PF (1.9 µm ± 0.5, n = 6) is tapered distally ( Fig. 12B ). The bipartite H (1.1 µm ± 0.2, n = 7) consists of a proximal part of constant diameter and a distal part of equal length and smaller diameter. The single cup-shaped parietal C ( Fig. 11B ) contains bundles of thylakoids grouped in stacks three to five ( Fig. 11C ). One end of the C, near the pit and F bases, contains a conspicuous orange E ( Fig. 10A–B ) consisting of a cluster of osmiophilic globules located along its inner surface ( Fig. 11B, D ). In the centre of the C, opposite the F base, is a PY forming an ovoid bulge at the cell surface ( Figs 10C , 11B , 12A ). In transverse section, this protruding PY has the unusual aspect of a thick, wide utricle ( Fig. 13A–B, D ) curving in on itself ( Fig. 13B–C ) and entirely surrounded by the C-membrane bordered by the periplastic ER. Details of the pyrenoid of Pavlova spp. This particular form of PY, present in strains AC248, AC250 and also AC33 ( Fig. 13D ), had previously been observed in various species of Pavlova (i.e., P. pinguis , P. gyrans and P. granifera ) but had not been retained as a marker of the genus. It turns out that with all strains of Pavlova for which we now have sections, this PY is a very distinctive feature of the genus. Fig. 10. Pavlova sp. AC248, motile cells, LM images. Side views of curved oblong ( A ), ovoid ( B ) and spherical ( C ) free-swimming cells with conspicuous S-shaped AF emerging apically. Greenish brown parietal C (black arrows) with an E and a posterior bulging PY. Vacuolar crystals of barium sulphate (white arrows). Scale bars = 10 µm. Indeed, at the time of the revision of the species P. pinguis , Green (1980) observed this pyrenoid very clearly in posterior position which he described as “large and conspicuous, frequently being pushed into a bulge at the posterior end of the cell.”. He also noted that this PY is “...frequently penetrated by a tubular invagination containing cytoplasmic material...”. In fact, his illustrations (see his figs 8, 44, 45) clearly show the curved shape of this PY in P. pinguis , as does fig. 7F of Bendif et al. (2011) . In their revision of P. gyrans , Green & Manton (1970) noted the central position of the PY within the C as well as its prominent bulging shape but did not examine thin-sections in TEM allowing them to see its curved shape. In their revision of the genus Pavlova they retained the fact that the C is bilobed with a prominent PY. Bendif et al. (2011) also showed this recurving PY in P. gyrans (see their fig. 6G) but retained only the bulge it forms on the cell. For P. granifera , Green (1973) showed the same shape and organisation of the PY (see his fig. 4) with an extension this time towards the interior of the cell (see his fig. 35), a situation we also observed only in the case of Pavlova AC 250 ( Fig. 13C ). Green (1973) did not retain the singular shape of this PY but reports in his revision of the P. granifera , that the PY is “discretely bulging towards the interior of the cell” ( Green 1980 ). Bendif et al. (2011) also observe this pyrenoid in P. granifera but with less detail. Fig. 11. Pavlova sp. AC248, chloroplast details, TEM images (Fix 1). A . Benthic colony of non-motile cells surrounded by a single, loose, non-layered M (white stars); single cup-shaped parietal C. B . Longitudinal section of a flagellate cell with parallel arrangement of TH in the C. Posterior PY protruding from the centre of the C, opposite a F, with an E at the tip of the C on its inner face. C . Detail of parallel TH lamellae forming stacks of 4 or 5 ending at the beginning of the PY. D . Detail of F insertion area showing proximity of the intrachloroplastic E to the long flagellar root (white arrows). Scale bars: A–B = 2 µm; C–D = 200 nm. The singular shape of this PY that we describe as campylotropous is indeed a distinctive feature of the species of the genus Pavlova since in Exanthemachrysis (the other genus of Pavlovophyceae with a bulging pyrenoid) the two species now described do not show such a recurving shape, but a simple sphero-ovoid PY (see for E. gayraliae : Gayral & Fresnel 1979 : figs 11–12, 21–22 and Bendif et al. 2011 : fig. 4E; for E. fresneliae sp. nov. : Fig. 3 ). When Butcher (1952) erected the genus Pavlova , he noted the presence of “leucosin bodies” in the posterior part of the cells and his drawings (see Butcher 1952 : pl. II, figs 35–37) clearly show what is now known to be this type of PY. He did not retain this character as distinctive of the genus, nor did Green (1967) , as cited above, when describing P. pinguis and his subsequent revision of the genus Pavlova . Bendif et al. (2011) introduced a more detailed description of the single C in their revised description by stating that it had a “posterior bulging pyrenoid and conspicuous eyespot on the inner surface near the flagellar pit”. Fig. 12. Pavlova sp. AC248, flagellar apparatus details of motile cells. A . SEM image of apical view of a swimming cell with a complete F apparatus emerging almost in the centre of a narrow, shallow pit: tomentose S-shaped AF, end-tapered PF and bipartite H inserted between both. Bulging PY on the opposite side. B–E . Negative staining images. B . Details of the base of the F apparatus showing the AF, PF with a tapered end, and H consisting of a proximal part of constant diameter and a distal part of subequal length and smaller diameter. C . Distal rounded part of the AF (with a blistering of the membrane) showing covering of long non-tubular hairs and regular arrangement of KS. D . Central part of the AF showing flat KS with a slight median constriction (black arrows) arranged in several more or less regular layers. E . Proximal part of the AF with multilayered KS, extending after a bare area in the immediate vicinity of the cell. Scale bars: A–B = 1 µm; C = 0.5 µm; D = 50 nm; E = 200 nm. Fig. 13. Three Pavlova strains, details of the PY. A–E . TEM images. A . AC250, whole cell with a longitudinally sectioned C showing the E composed of osmiophilic clustered globules on its inner side and on the opposite side, the transversely sectioned campylotropous PY forming a slight hump on the cell surface and showing the narrow median space. (Fix 1). B . AC248, protruding campylotropous PY, longitudinally sectioned, completely surrounded by the C-membrane, bordered by periplastic ER on the cell surface and showing a narrow medial stromal space. (Fix 1). C . AC250, campylotropous PY, longitudinally sectioned emerging from the C towards the interior of the cell. (Fix 1). D . AC33, section of a palmelloid benthic cell surrounded by M with a cross sectioned campylotropous PY, in the middle of single cup-shaped parietal C, forming an ovoid bulge at the cell surface. (Original image, courtesy of C. Billard). E . AC248, detail of a cross section of the campylotropous pyrenoid attached to the plasma membrane (white arrows) showing the surrounding chloroplastic envelope lined on the inside by the periplastic ER (black arrows). (Fix 1). F . SEM image of AC248, postero-lateral view of a swimming cell with a complete F apparatus. PY bulging opposite the flagella with a tongue-like part projecting outwards from the cell. Scale bars: A = 1 µm; B = 20 nm; C = 0.5 µm; E = 200 nm; F = 2 µm. Taxonomic outcome: a revised description of Pavlova The particular and very characteristic shape of the pyrenoid in all species of Pavlova makes it a very distinctive feature that leads us to a revision of the genus description.