Phytoflagellate diversity in Roskilde Fjord (Denmark), including the description of Pyramimonas octopora sp. nov. (Pyramimonadales, Chlorophyta)

Figures & data

Figs 1, 2. Seasonal cycle of environmental parameters in Roskilde Fjord during the study period.

Fig. 1. Abiotic variables: temperature and salinity.

Fig. 2. Nutrient concentrations: dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorus (DIP). Large full dots represent samples in which serial dilution cultures (SDC) were established.

Figs 3–10. Seasonal variation in phytoplankton community composition in Roskilde Fjord.

Fig. 3. Chlorophyll a concentration. Large dots represent samples in which serial dilution cultures (SDC) were established.

Fig. 4. Biomass estimated from the proportion of accessory pigments using the Bayesian Compositional estimator (BCE). Phytoplankton group abbreviations: Chloro =  chlorophytes, Crypto = cryptophytes, Cyanop = cyanophytes, Dino = autotrophic dinoflagellates, Eugl = euglenophytes, Pelago = pelagophytes, Prasino = prasinophytes, and Prymne = prymnesiophytes.

Figs 5, 6. Main groups enumerated by SDC and MPN as carbon biomass and cell abundances. Phytoplankton group abbreviations: Crypto = cryptophytes, Diat = diatoms, Dino = autotrophic dinoflagellates, Eugl = euglenophytes, Hapto = haptophytes, HNF = miscellaneous heterotrophic nanoflagellates, NID = non-identified organisms, Pico_euk = eukaryotes <2 µm, and Prasino = prasinophytes.

Fig. 5. Estimates based on carbon biomass.

Fig. 6. Estimates based on cell abundances.

Figs 7, 8. Cryptophyte composition at genus level as carbon biomass and cell abundances.

Fig. 7. Estimates based on carbon biomass.

Fig. 8. Estimates based on cell abundances.

Figs 9, 10. Prasinophyte composition at genus level as carbon biomass and cell abundances.

Fig. 9. Estimates based on carbon biomass.

Fig. 10. Estimates based on cell abundances.

Figs 11–27. Examples of identified organisms in Roskilde Fjord. All micrographs are LM, except Fig. 20, which is SEM. Scale bars = 5 µm.

Figs 11, 12. Eutreptiella sp.

Figs 13, 14. Diacronema ennorea, palmeloid stage (Fig. 13) and single cell (Fig. 14).

Fig. 15. Nephroselmis pyriformis.

Fig. 16. Mantoniella squamata.

Fig. 17. Teleaulax amphioxeia.

Fig. 18. Chrysochromulina simplex.

Figs 19, 20. Teleaulax acuta, LM (Fig. 19) and SEM (Fig. 20), showing the pattern on cell surface and the deep furrow.

Fig. 21. Chroomonas vectensis.

Fig. 22. Rhodomonas salina.

Fig. 23. Hemiselmis cf. cryptochromatica.

Figs 24, 25. Hemiselmis sp., isolated in October (Fig. 24) and July 2016 (Fig. 25).

Figs 26, 27. Hemiselmis virescens.

Table 1. Flagellates identified to species level, the month in which they were isolated, strain code, techniques used for identification, and GenBank accession number

Species	Month	Strain	Identification method	Accession number
Cryptophyceae
Chroomonas vectensis	Jan/17	Cvec_01	LM, DNA	SSU (MW451606)
Hemiselmis cf. cryptochromatica	Jun/16	Hcry_06	LM, DNA, SEM	LSU (MW451669) SSU (MW451605)
H. virescens	Mar/16	Hvir_03	LM, DNA, SEM	LSU (MW451668) SSU (MW451604)
Hemiselmis sp.	Jul/16	Hemi_07	LM	-
Hemiselmis sp.	Oct/16	Hemi_10	LM, SEM	-
Hemiselmis sp.	Nov/16	Hemi_11	LM	-
Rhodomonas salina	Jun/16	Rsal_06	LM, DNA	LSU (MW451670) SSU (MW451607)
Teleaulax amphioxeia	Mar/16	Tamp_03	LM, DNA	SSU (MW451609)
T. amphioxeia	Apr/16	Tamp_04	LM, DNA	SSU (MW451610)
T. acuta	Oct/16	Tacu_10	LM, DNA, SEM	SSU (MW451608)
Coccolithophyceae
Chrysochromulina simplex	Nov/16	Csim_11	LM, TEM, DNA	SSU (MW451615)
Pavlovophyceae
Diacronema ennorea	May/16	Denn_05	LM, DNA	SSU (MW451614)
Mamiellophyceae
Mantoniella squamata	Mar/16	Msqu_03	LM, TEM, DNA	SSU (MW451611)
M. squamata	Apr/16	Msqu_04	LM, TEM, DNA	SSU (MW451612)
M. squamata	May/16	Msqu_05	LM, TEM, DNA	SSU (MW451613)
Ostreococcus sp.	Aug/16	OSTR_08	DNA	SSU (MW451616)
Ostreococcus cf. tauri	Dec/16	OSTR_12	DNA	SSU (MW451617)
Nephroselmidophyceae
Nephroselmis pyriformis	May/16	Npyr_05	LM, TEM	-
Pyramimonadophyceae
Pyramimonas grossii	May/16	Pgro_05	LM, TEM	-
P. grossii	Aug/16	Pgro_05	LM, TEM	-
P. octopora	Jun/16	Poct_06	LM, TEM, DNA	SSU (MW451603)

Figs 28–31. Pyramimonas octopora sp. nov., LM. Scale bars = 10 µm.

Figs 28, 29, 31. Cells with different morphologies in lateral view showing eyespots (E) close to the anterior part of the cell and pyrenoid (P) displaced towards the cell side.

Fig. 30. Apical view showing the location of the eyespots, displaced towards the cell anterior end.

Figs 32–42. Pyramimonas octopora sp. nov., TEM. Figures 32–34, 36–41 are from thin sections; Figs 35, 42 are from whole mount preparations stained with uranyl acetate. All scale bars = 200 nm, except for Figs 32, 34 and 36 in which the scale bars = 1 µm.

Fig. 32. Transverse section through the flagellar pit, with four flagella (1–4), scale reservoir (S), nucleus (N) and eyespots (E) near the cell corner.

Fig. 33. Section through the flagella showing hair scales (HS), limuloid scales (LS) and under-layer of pentagonal scales (PS).

Fig. 34. Longitudinal section showing nucleus (N), bi-layered eyespot (E) and scale reservoir (S) close to the apical part of the cell.

Fig. 35. Body scales: box scales (BS), crown scales (CS) and limuloid scales (LS). Note detail on the ornamentation of the box scales, with eight perforation on the base.

Fig. 36. longitudinal section showing scale reservoir (S), nucleus (N) and pyrenoid (Py) penetrated by thylakoids.

Fig. 37. Detail of a longitudinal section showing the square sysnistosome (Sy) and the four flagella (1–4).

Fig. 38. Details of the underlayer body scale.

Fig. 39. Details of the crown scale base.

Fig. 40. Detail showing the footprint scales (indicated by arrows) between the box scales.

Fig. 41. Cell surface detail showing the underlayer body scales (US), box scales (BS) and crown scales (CS) at the surface.

Fig. 42. Limuloid (LS) and box scales (BS).

Figs 43–45. Diagrammatic representation of the most characteristic scales of Pyramimonas octopora sp. nov. Drawings by Nikolaj Toftemark Nielsen.

Fig. 43. Box scale with solid wall.

Fig. 44. Crown scale.

Fig. 45. Limuloid flagellar scale.

Fig. 46. Phylogenetic analysis of Pyramimonas spp based on sequences of SSU rDNA. Bootstrap values supporting the branches are given at each node as estimated by different methods (Neighbour Joining NJ/Maximum Parsimony MP/Maximum Likelihood ML).