Figures 2–4 in More than meets the eye: regional specialisation and microbial cover of the blade of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)

Figures 2–4: Microbial colonisation of blade margins from a wild plant of Porphyra UMbilicalis (Figures 2 and 3) and a cultured descendent (Figure 4) of a wild plant brought into laboratory culture and maintained without antibiotic treatment. Bacteria occur in patches along portions (Figure 2, arrows) but not all (Figure 3, arrows mark neutral spores) of the same wild blade, with much greater density of filamentous bacteria in Figure 2. Cultured blades (Figure 4) have a dense cover of bacteria, including filamentous cyanobacteria (arrows); contemporaneous light microscopic observations found at least three distinct cyanobacteria on these cultured blades, including a probable Calothrix. Wild P. UMbilicalis (n = 6 plants) were collected along a 30-m transect (~5 m apart) in the high intertidal zone at Schoodic Point, Acadia National Park (44.33380000, −68.05805556; 30 December 2016, permit ACAD-2017-SCI-0006). For each plant, one piece of reproductive margin (0.5–1 cm2) and the holdfast were removed with sterile techniques, transported on ice, fixed (4°C) in 5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.0) containing 0.2 M sucrose, post-fixed in 1% OsO4 in 0.07 M sodium cacodylate, dehydrated in an ethanol series, critical point dried, and sputter-coated with gold-palladium to give a final coating that was 27 nm thick; see Royer 2017 for complete details. All six plants were observed, and representative images are presented. The cultured specimens (Figure 4) were maintained through successive generations by standard techniques (Royer et al. 2018) after collection of the parent on 5 May 2015 at Lubec, Maine.