Prorocentrum bisaeptum Hoppenrath, Gottschling & Tillmann, 2024

Tillmann, U., Gottschling, M., Sunesen, I., Wietkamp, S., Dzhembekova, N., Rodriguez Hernández, F., Tardivo Kubis, J., Sar, E., Kaufmann, M. & Hoppenrath, M., 2024, Morphological and molecular characterization of Prorocentrum bidens (formerly known as P. compressum) and description of the closely related Prorocentrum bisaeptum sp. nov. (Prorocentrales, Dinophyceae), Phycologia 63 (5), pp. 431-452 : 438-441

Prorocentrum bisaeptum Hoppenrath, Gottschling & Tillmann , sp. nov.

Figs 47–87

DESCRIPTION: Symmetrical prorocentroid cells, broadly to narrowly elliptic to ovate, 34.8–42.9 µm long, 28.6–33.5 µm deep and 15.0–21.6 µm wide. Smooth thecal surface with pores of two size classes; more densely packed towards the plate margins; posterior central pore cluster; with a narrow central area devoid of pores. Periflagellar area with 10 platelets (1, 2, 3, 4, 5, 6a, 6b, 7, 8a, 8b) and anterior projections appearing as two opposed wings. Two pyrenoids visible on one lateral side in anterior and posterior position. Encapsulated stage (two hyaline envelopes, one nesting in the other) containing up to four cells with actively beating flagella.

HOLOTYPE: SEM-stub (designated here: CEDiT2023H165) of the clonal strain Madeira deposited at Senckenberg am Meer, German Centre for Marine Biodiversity Research, Centre of Excellence for Dinophyte Taxonomy, Germany. All SEM images from this strain ( Figs 67–87 ) were taken from this stub.

ISOTYPE: Lugol-fixed sample of the clonal strain Madeira (CEDiT 2024I 181) deposited at Senckenberg am Meer, German Centre for Marine Biodiversity Research, Centre of Excellence for Dinophyte Taxonomy, Germany .

DNA SEQUENCE OF HOLOTYPE STRAIN: GenBank accession number: PP873957 (ITS + LSU rRNA).

ETYMOLOGY: Latin: bi = double; saeptum = enclosure, envelope; referring to the two nested, hyaline envelops enclosing the cells in the encapsulated stage.

TYPE- LOCALITY: North-East Atlantic Ocean , south off Funchal, Madeira, Portugal (32°36.36’ N; 16°53.54’ W).

HABITAT: Open subtropical surface water. The species is part of the plankton community.

PHYCOBANK REGISTRATION: http://phycobank.org/104624

MORPHOLOGICAL DESCRIPTION

Cells of P. bisaeptum were symmetrical and broadly to narrowly elliptic to ovate (narrowing slightly in the posterior cell half) in lateral view ( Fig. 47–53 ). Cell size ranged from 34.8 to 42.9 µm in length and 28.6 to 33.5 µm in depth ( Table 2 ) with a length/depth ratio of c. 1.28. Cells were laterally compressed and lens-shaped in ventral or dorsal view. Cell width ranged from 15.0 to 21.6 µm with a mean length/width ratio of c. 2.33. Cells had both a longitudinal and a wavy transverse flagellum arising from the apical periflagellar area ( Fig. 47 ). In LM, the surface was smooth, and no ornamentation of the theca was visible except for scattered thecal pores ( Fig. 50 ). The apical area was slightly flattened and terminated with characteristic anterior projections appearing as two opposed wings and additional central structure(s) ( Fig. 47, 48 ). A large, round and hyaline area, the pusule, was visible in the anterior part of the cell ( Fig. 47 ). Cells had two retiform chloroplasts ( Fig. 51, 52 ). Two round pyrenoids per chloroplast in the anterior and posterior half of the cell were visible in LM of cells in lateral view ( Fig. 49, 51, 52 ). A large U-shaped nucleus ( Fig. 48, 54 ) with thick, dinokaryotic chromosomes was located in the posterior half of the cell ( Fig. 47, 48, 54 ).

Fig. 23. Right lateral view.

Fig. 24. Left lateral view.

Fig. 25. Ventral right-lateral view.

Fig. 26. Ventral left-lateral view.

Fig. 27. Apical right-lateral view.

Fig. 28. Apical dorsal view.

Fig. 29. Dorsal view.

Fig. 30. Apical view.

Fig. 31. Antapical lateral view.

In culture, cells occurred in two different stages (Suppl. video V2). The cells were either freely motile ( Fig. 47 ) with a typical helical swimming path or in an encapsulated stage, they were enclosed with actively beating flagella in two hyaline envelopes, one nesting in the other. Each envelope was bounded by a thin, barely visible surface layer that was most distinct under phase contrast. The smaller inner envelope contained one to four cells (rarely three; Fig. 55–61 ). Single cells surrounded by only a single, narrow envelope were rarely seen ( Fig. 55 ). Encapsulated cells, which were sometimes seen to undergo cell division by desmoschisis along the sagittal suture ( Fig. 62–66 ), exhibited reduced motility (Suppl. video V2). The flagella of these cells continued to beat, at times rotating the cells within the envelope (Suppl. video V2). The spherical outer envelope was much larger, and of different sizes (Suppl. video V2). Cells were occasionally observed to abandon their envelopes (Suppl. video V2).

The general appearance of the cell ( Fig. 67–76 ) was confirmed by SEM revealing ultrastructural details of thecal plates and the microarchitecture of the periflagellar area. The surface of both thecal plates was smooth (rarely with faint depressions, Figs 76 , 77 ). The intercalary bands were of varying width and striated transversally ( Figs 73–76 , 79 ). Thecal pores were abundant on both thecal plates, more densely packed towards the plate margins, and a narrow, central area was (nearly) devoid of pores ( Fig. 67–76 ). A posterior pore cluster in the central marginal area was recognizable ( Figs 70, 71 , 81 ). Two types of pores were distinguished. Large pores with an outer round opening, a counter-sunk short cavity and at its bottom an inner round opening of smaller diameter ( Fig. 77, 78 ). In addition, there were small pores with a round opening ( Fig. 77, 78 ). Some pore tubes of small pores with oblique outlet ports were visible on the plate surface ( Fig. 77, 78 ) and in some cases, these ports were V-shaped. Internal views of thick plates revealed inner pore openings of two size classes ( Fig. 80, 81 ).

The periflagellar area ( Fig. 82–87 ), which was 6.0– 7.5 µm deep and 3.0–4.0 µm wide (n = 13), was located in a small excavation mainly of the right thecal plate ( Figs 67, 68, 73 , 87 ). It was composed of 10 platelets (pattern: 1, 2, 3, 4, 5, 6a, 6b, 7, 8a, 8b) surrounding the fp and an ap ( Fig. 82–87 ). A number of platelets had characteristic structures. Wings were the most conspicuous extensions on platelets 1 and 4 ( Fig. 83, 85, 87 ) and sometimes platelet 8b ( Fig. 83–85 ). Platelet lists were present on the narrow platelets 2 and 6a ( Fig. 82–85 ), sometimes reaching the size of wings ( Fig. 87 ). Platelets 3, 5 and 6b had a protrusion at their fp margin ( Fig. 82–84 ). Platelet 7 could have a platelet list bordering the ap ( Fig. 82, 85 ). Platelet 6 was consistently split into two parts (6a and 6b), and platelet 8 was also split into two parts (8a and 8b, Fig. 85, 86 ), although platelet 8a was often covered by the wing of platelet 8b ( Fig. 83, 84 ). The fp was irregular in shape, generally longer than wide, and surrounded by platelets 3, 5, 6b, and 8b ( Fig. 82, 83, 86 ). The ap was elliptic and smaller than the fp, and was surrounded by platelets 7 and 8a ( Fig. 83, 85, 86 ).

Molecular phylogenetics

The SSU + ITS + LSU alignment was 1800 + 739 + 3490 bp long and composed of 305 + 537 + 840 parsimonyinformative sites (28%, mean of 20.51 per terminal taxon) and 2654 distinct RAxML alignment patterns. The ML tree (–ln = 56,602.26), was highly similar to the Bayesian tree, with many nodes having high if not maximal support ( Fig. 88 ). With respect to Dinophysales and Gymnodiniales , the studied ingroup was monophyletic (82LBS, 1.00BPP) and segregated into four supported monophyletic lineages, namely Adenoides Balech (100LBS, 1.00BPP), Plagiodinium M.A.Faust & Balech (100LBS, 1.00BPP) and the clades PRO1, including the type species of Prorocentrum P. micans (93LBS, 1.00BPP), and PRO2 (93LBS, 1.00BPP).

The PRO1 clade consisted of six well-supported lineages, two of which comprised benthic species such as Prorocentrum tsawwassenense Hoppenrath & B.S.Leander and P. emarginatum Fukuyo. Three sublineages constituted the P. cordatum (Ostenfeld) J.D.Dodge species group, the P. micans species group and the Prorocentrum triestinum J. Schiller species group. The P. bidens species group comprised P. bidens (78LBS, 0.99BPP) and P. bisaeptum (single accession) and showed low sequence divergence. ITS1 sequences of P. bidens differed from those of P. bisaeptum in four positions and ITS2 sequences in five positions and LSU sequences in four positions. No compensatory base-pair changes (CBCs) could be detected.

Figures

Fig. 47–54. Prorocentrum bisaeptum, strain from Madeira, LM of living and formalin-fixed cells. Scale bars = 10 µm. Fig. 47. Cell in left lateral view. Note the anterior projections (arrowheads), the large pusule (p), the posterior nucleus (n) and the longitudinal flagellum. Fig. 48, 49. The same cell in lateral view of two different focal planes showing the anterior projections (arrowheads) and the nucleus (n) in 48. Note the two pyrenoids (py) in 49. Fig. 50. Cell in surface focus showing the thecal plate with pores. Fig. 51, 52. The same cell showing chloroplast details; (51) differential interference contrast, (52) epifluorescence showing the autofluorescence of the chloroplast. Note the two pyrenoids (py). Fig. 53, 54. The same fixed cell with DAPI-stained nucleus (n) visible (54).

Fig. 55–66. Prorocentrum bisaeptum, strain from Madeira, LM of living cells enclosed in two nested, hyaline envelopes. Fig. 55. A cell at high magnification. Note the inner narrow envelope (arrows), the anterior projections (arrowheads) and the elongated elliptic cell outline. Scale bar = 10 µm. Fig. 56, 57. Two cells enclosed in the inner envelopes (white arrows), which are nesting in the outer, spherical envelope (black arrows). Figure 56. Bright field optics. Figure 57. Differential Interference Contrast microscopy. Scale bars = 10 µm (57), 25 µm (56). Fig. 58, 59. Three cells in the envelopes. Figure 58. Three individual cells in one envelope. Figure 59. One individual cell and a pair in a late division stage. Scale bars = 25 µm. Fig. 60. Four cells in the envelopes. Scale bar = 25 µm. Fig. 61. One cells in the inner of the two envelopes. Scale bar = 25 µm. Fig. 62–66. Different stages of cell division within the envelopes. Scale bars = 25 µm.

Fig. 67–76. Prorocentrum bisaeptum, strain from Madeira, SEM of different cells. Scale bars = 10 µm. Fig. 67,68. Right lateral view. Fig. 69. Left lateral view. Note the narrow central area devoid of pores in 67–69. Fig. 70, 71. Separated thecal plates. Note the posterior pore cluster in 70. Fig. 72. Internal view of the (likely) left lateral plate still connected with the periflagellar area. Note the distinct growth bands, also of the tiny platelets, and the internal openings of the thecal pores. Fig. 73. Ventral right-lateral view. Fig. 74. Dorsal left-lateral view. Fig. 75. Dorsal view. Fig. 76. Dorsal right-lateral view. Note the faint depressions in the plate center.

Fig. 77–81. Prorocentrum bisaeptum, strain from Madeira, SEM of the surface ornamentation and of thecal pores. Scale bars = 5 µm. Fig. 77, 78. External views of the thecal plate surface. Note the smooth surface, the presence of larger (white arrows) and smaller pores (black arrows). Small pores can have an oblique outlet port visible on the plate surface. In some cases, these ports had a V-shape. Fig. 79. Striated thecal plate growth bands next to the sagittal suture (white arrow). Fig. 80, 81. Internal views of thecal plates showing the internal structure of thecal pores in the thick thecal plates. Note that larger and smaller internal openings are distinguishable and the posterior pore cluster in 81.

Fig. 82–87. Prorocentrum bisaeptum, strain from Madeira, SEM of details of the periflagellar area. Numbers label the periflagellar platelets, fp = flagellar pore; ap = accessory pore. Scale bars = 1 µm. Fig. 82. Apical view of a separated periflagellar area. Fig. 83–85. Apical views. Fig. 86. Internal view of a separated periflagellar area. Note the clearly visible suture splitting platelet 8 into two parts, platelets 8a and 8b. Fig. 87. Right-lateral apical view.

Fig. 88. Molecular phylogenetics of prorocentralean dinophytes (with the outgroup taxa Dinophysales and Gymnodiniales cropped), including all accessions assignable to P. bidens and P. bisaeptum. ML tree (–ln = 56,602.26), as inferred from an rRNA nucleotide alignment (1682 parsimony-informative sites) with strain number information. Accessions corresponding to type or at least reference material are indicated by bold type; freshwater accessions are shown in grey. Numbers on branches are ML bootstrap (above) and Bayesian support values (below). Asterisks indicate maximal support values; values <50% and <0.90 are not shown. Clades are indicated (abbreviations: ADE, Adenoides; Bid, P. bidens species group; cor, P. cordatum species group; ema, P. emarginatum species group; mic, P. micans species group; PLA, Plagiodinium; PRO, Prorocentrum; tri, P. triestinum species group; tsa, P. tsawwassenense species group).