Fig 1.
Schematic summary of the deep divergences in diatoms using auxospore type to define each of the branches.
Numbers below individual branches indicate references to molecular phylogenies recovering monophyly for these branches [14, 16–18, 24–26]. Auxospores and valves are schematically simplified and not intended to be attributed to any individual species. Asterisk (*) in Mediophyceae indicates exception for non-polar Thalassiosirales who lost perizonia secondarily; see explanation in text. Mediophyceae perizonium reprinted from [9] under a CC BY license, with permission of Koeltz Scientific Books, original copyright 1982.
Fig 2.
Cell sizes of the Paralia guyana parent and progeny individuals in the three auxosporulating clones.
Small filled circles indicate individual parental cells, unfilled circles represent progeny cells. Note three or four size-classes of the progeny cells (depending on clone). Top arrows point to the discontinuities between cell size-classes representing progeny cells of the consecutive rounds of auxosporulation. Note that some of the large cells in the clone West1C2 designated as parents (full circles) are vegetative cells derived from initial cells produced by previous round(s) of auxosporulation (by the smaller cell-size parents) and who became parents producing the next generation of still larger auxospores and initial cells.
Fig 3.
Auxospore enlargement as observed in live material and brightfield microscopy.
A—elongated cell represents auxospore development stage before it rounds up; B—auxospore with initially uneven lateral expansion; C—a small, almost spherical auxospore; D—near full size spherical auxospore filled up with chloroplasts; E—a nearly mature auxospore undergoing second partial plasmolysis, note small refractive structure present in parental theca, behind retracting initial cell protoplast; F—a mature auxospore with initial cell inside; G—a short filament of post auxospore cells with initial epi- and hypothecae incorporated into the viable end cells; H—expired auxospore, nearly clear of cell contents, showing an outline of initial epitheca (arrowheads); I-J—another deteriorating auxospore, nearly clear of cell contents at two different foci: I—focus on an initial epivalve, (arrowheads); J—focus on a large scale (arrowheads).
Fig 4.
Auxospore nuclei as observed in DAPI stained cells.
A—elongated cell stage of auxospore development, note cell nucleus divided and the smaller of the sister nuclei is undergoing pyknosis (arrowhead); B & C—the same cells shown in different illumination, another elongated cell with one functional and one pyknotic nucleus (two large arrows point to the domed valves of this cell), note a smaller cell below the young auxospore in C (small arrow points to the flat hypovalve of a smaller frustule (see also SEM figures below); D—nearly spherical, small auxospore with centrally located nucleus; E—larger, spherical auxospore with centrally located nucleus; F—nearly full size auxospore with centrally located nucleus; G—nearly mature auxospore undergoing first partial plasmolysis prior to depositing the initial epivalve, note position of the nucleus (arrowhead) immediately underneath the retracting protoplast surface (small arrows) and a dome-shaped auxospore wall above (large arrows); H—mature auxospore after second partial plasmolysis, note two compact pyknotic nuclei located peripherally (arrowheads) and one functional nucleus located deep in the cell (arrow); I—mature auxospore with initial frustule, note nucleus near cell center.
Fig 5.
Auxospores and initial cells as observed in SEM; A-I water rinsed, and J-L acid-cleaned specimens.
A—theca resulting from uneven division of the auxospore mother cell (compare to Fig 4C), note doughnut-shaped, lightly silicified rudimentary hypovalve (valve EDS spectrum shown in Fig 6A-A1-2); B—a young spherical auxospore with large scale (arrowheads) exposed (compare to Fig 3I and 3J); C—the same large scale showing detail of pitted ornamentation; D—partially collapsed, large auxospore probably captured during partial plasmolysis, note large diameter of parental theca demonstrating the second round of auxosporulation; E—a partially damaged auxospore with thick wall with siliceous elements (EDS spectrum shown in Fig 6B-E1-2), note that the small parent cell of this auxospore indicates first round of auxosporulation; F- spinose initial valve covered with thin and pliable remains of the auxospore wall; G—the same specimen as in F at greater magnification showing scaly incunabulae (arrowheads) in the auxospore wall; H—another initial frustule enshrouded in the auxospore wall demonstrating scaly incunabulae in the girdle region (arrowheads); I—individual incunabular scales (arrowheads) disassociated from the auxospore wall (EDS spectrum shown in Fig 6C-I1-2); J—complete initial frustule in girdle view, note epicingulum; K—external view of an initial valve; L—internal view of an initial valve.
Fig 6.
Spectra acquired from locations shown in Fig 5A, 5E and 5I, respectively for vestigial hypovalve, auxospore wall and incunabular scale (locations indicated by boxes). Note difference in vertical scale of the spectra in Fig 6B. Spectra from position 1 are taken from structure shown in Fig 5 and spectra from position 2 from filter substrate which shows no detectable silicon peak. Gold peaks were generated from the conductive coating.
Fig 7.
Acid cleaned initial valves observed in brightfield LM.
A & B—convex, relatively plain initial valves with single (A) and double (B) elevations, note band of quincunx pores at the valve margin; C through E—a strongly concentrically undulated valve at three different foci: C—focus on valve margin and striae; D—focus on peripheral part of the valve face, ribs and stout spines; E—focus near central elevation with smaller spines; F—inside view of initial valve with central elevation; G—outside view of a less convex initial valve, note ribbed margin.
Fig 8.
Post auxospore frustules and valves as observed in SEM.
Morphology is intermediate between initial and typical vegetative valves. A—a short chain (probably 3–4 cells), note strong cingulae and initial valves on the end cells; B—disarticulated post auxospore chain showing 6 valves with imperfect marginal linking spines and mantles, note varying degree of imperfection likely contributing to weakness of cell-linking, thus the need for strong cingulae; C—two post-auxospore valves with mismatching marginal linking spines; a sibling valve at top (arrowheads) also with atypically shallow mantle and malformed linking spines; D—external view of a post auxospore valve with concentric depression and underdeveloped, imperfectly ornamented mantle; E—domed post-auxospore valve with imperfect internal and external linking spines; F—relatively flat post-auxospore valve with concentric depression, shallow mantle and malformed linking spines; G—post-auxospore valve with pronounced radial striation, featureless flange and spiny protrusions; H—somewhat elliptical post-auxospore valve with network of peripheral thickenings; I—typical intercalary vegetative valve with incompletely silicified mantle, note typically narrow fenestra which in more strongly silicified valves are covered by siliceous outcroppings, obscuring fenestra in SEM images.
Fig 9.
Internal view of initial and post-auxospore valves as observed in SEM.
A & B—note absence of rimoportula; C & D—rimoportulae near the valve mantle edge aligned perpendicularly to the mantle rim (arrowheads); E & F—hammock-shaped rimoportulae (arrowheads) with internal openings variably oriented with respect to the mantle rim; G & H—low-profile rimoportulae in typical position for a vegetative valve (arrowheads), underneath the mantle rim and parallel to rim’s circumference (focus is on the distal rim). Note imperfect striation and external ornamentation of the mantle on some of the valves.
Fig 10.
Acid cleaned post auxospore valves in brightfield LM.
Morphology is intermediate between initial and typical vegetative valves. A—valve face with irregular rugose markings and narrow, striated rim (arrowhead); B—valve face with numerous spines dispersed about the valve face; C—face with irregular marginal and central radiate shallow ribs, not spines; D—slightly elliptical valve face without internal linking spines and irregularly thickened margin; E—slightly irregular valve with ill-formed marginal spines and puncta; F—valve with well-developed marginal region, but with irregular central ornamentation (arrowhead); valve below is a typical vegetative intercalary valve; G—valve with relatively well developed internal linking spines but underdeveloped marginal spines.