Cold climate adaptation is a plausible cause for evolution of multicellular sporulation in Dictyostelia

Unicellular protozoa that encyst individually upon starvation evolved at least eight times into organisms that instead form multicellular fruiting bodies with spores. The Dictyostelia are the largest and most complex group of such organisms. They can be subdivided into 4 major groups, with many species in groups 1–3 having additionally retained encystment. To understand fitness differences between spores and cysts, we measured long-term survival of spores and cysts under climate-mimicking conditions, investigated spore and cyst ultrastructure, and related fitness characteristics to species ecology. We found that spores and cysts survived 22 °C equally well, but that spores survived wet and dry frost better than cysts, with group 4 spores being most resilient. Spore walls consist of three layers and those of cysts of maximally two, while spores were also more compacted than cysts, with group 4 spores being the most compacted. Group 4 species were frequently isolated from arctic and alpine zones, which was rarely the case for group 1–3 species. We inferred a fossil-calibrated phylogeny of Dictyostelia, which showed that its two major branches diverged 0.52 billion years ago, following several global glaciations. Our results suggest that Dictyostelium multicellular sporulation was a likely adaptation to a cold climate.

Species are phylogenetically ordered within their taxon group. We use the older given species names in this work instead of the novel classification (4 th column) based on the SSU rDNA phylogeny 1,2 , which did not resolve several genera and may need to be revised in the near future. Species are colour-coded to reflect the major taxon group of which they are part. Species in black text belong to minor intermediate groupings.

Spore and cyst ultrastructure
To understand why spores survive long term storage better than cysts and why group 4 spores are more frost resistant than others, we examined ultrastructural differences between spores and cysts. Spore ultrastructure was investigated in at least six species from each of the major taxon groups and one species each for the minor groups, while cyst ultrastructure was studied for those species that readily formed cysts. Spores across taxon groups have many ultrastructural features in common. Spore cytoplasm is highly condensed with ribosomes and organelles packed closely together. This condensation obscures resolution of finer ultrastructural detail and features like endoplasmic reticulum and Golgi cisternae can generally not be distinguished. Membrane-bound vesicles with or without inclusions are common, and electron-transparent granules without membranes are present, mostly at the poles in groups 1-3. Dictyostelium nuclei have no identifiable single nucleosome, but can harbour distinct patches or more dispersed darker heterochromatin. The spore wall typically consists of two narrow dense layers that flank a broader more transparent layer. In D. discoideum, the latter was shown to consist mostly of cellulose fibrils, while the dense layers are more (glyco)protein-rich 3 . The spore surface can vary from smooth to undulating or sharply ridged.
Spores from all investigated species per group are shown in figures S3-S6, while cysts are shown in figure  S7. Apart from D. deminutivum with a two-layered wall, group 1 spores have the typical three-layered spore wall and are further characterized by their tightly clustered polar granules embedded in smooth dark material. They usually show many grouped vesicles and round to oval mitochondria with visible cristae (Fig. S3). Characteristic for most group 2 spores are the loosely grouped polar granules, which can be surrounded by a double layer of ribosomes, likely rough endoplasmic reticulum (RER), but not by smooth dense material as in groups 1, 3 and 4. RER flanked granules are however also present in spores of the other groups and in cysts. Mitochondria are generally smooth-surfaced with well-defined cristae. Spores of many group 2 species also have a distinctive opaque sub-membrane cortex and many vesicles (Fig. S4). Group 3 spores are characterized by dense cytoplasm and relatively thin walls. Spore granules are clustered at the poles and are mostly surrounded by denser smooth material. Mitochondria are often broadly or more finely lobed (Fig. S5). Group 4 spores stand out by having spore granules that are not distinctly polar, but dispersed throughout the cell. Most mitochondria are finely lobed (crenate), with almost invisible cristae and, unique for this group, they are fringed with ribosomes ( Fig. S6).
Cysts contain semi-transparent granules that are similar to the spore polar granules and many vesicles. However, their wall architectures are more variable (Fig. S7). D. deminutivum cysts in group 1 have a narrow inner transparent layer and even thinner outer slightly denser layer. Group 2 cysts have a one or two-layered wall, the latter with the inner being more opaque and the outer layer loosely fibrous. D. polycephalum, an outgroup species to both groups 3 and 4 has a relatively wide wall that is less structured than the spore wall. See the legends to figures S3-S6 for more detailed descriptions of spores and cysts for each species and table S2 for species-specific features. The D. deminutivum spores are very small with a thin 2-layered wall consisting of a thin dense inner and loosely fibrous outer layer. There are both large vesicles, stretched out along the length of the spore and smaller grouped vesicles. There is generally only one spore granule per pole. The overview or inset images are shown at the same magnification. Bar in overview: 1 µm; bar in inset: 0.1 µm. P: plasma membrane, L1, L2, L3: inner, middle and outer layers of spore wall. Figure S4. Group 2 spores P. pallidum spores have a fairly wide three-layered walls and a distinctive dark cortex (C) below the plasma membrane (P). Spore granules are loosely grouped at the poles. There are many grouped and dispersed vesicles and mitochrondria show distinctive cristae. P. pseudocandidum, P. filamentosum, P. equisetoides and P. arachnoideum spores are similar to those of P. pallidum, except that the cytoplasm of P. arachnoideum spores shows typical empty patches. D. oculare spores are small with a three-layered wall that sometimes forms acute angles at the poles. Mitochondria show distinct cristae and some inclusions. There are many small vesicles, mostly grouped and present throughout cell. Most spores also have some larger highly indented vesicles with dense granular content, resembling the contents of K. aerogenes food bacteria (B?). One or two large granules are present at the poles, giving the impression of eyes, to which this species owes its name.
The plasma membrane and underlying cortex of A. leptosomum and A. subglobosum spores are intensely scallopped. Spore granules are dispersed throughout the cells. The elliptical spores of A. ellipticum have a relatively narrow wall and display spore granules of variable size. Mitochondria show distinctive cristae. D. polycarpum spores have a thick spore wall and loosely grouped polar granules. The plasma membrane and underlying cortex is in places discontinuous with the underlying cytoplasm, which may be due to shrinkage during fixation. Bar in overview = 1 µm; bar in inset: 0.1 µm.
- Figure S5. Group 3 spores. D. coeruleo-stipes spores are long and narrow. The spore wall is relatively thin, but the middle layer is rather fibrous with some dense spots. Spore granules are grouped together at or just below poles, mostly embedded in darker smooth material. Mitochondria are often broadly lobed. The polar granule content of the thin-walled The content of the D. purpureum spores is very condensed. The ultrastructure of the organelles is therefore poorly defined, but similar to that of D. mucoroides and the other group 4 species. P. violaceum is a member of a small sister group to group 4. Its spore granules are more specifically localized at the poles and are sometimes embedded in darker smooth material. Mitochondria have open crista and are less frequently fringed by ribosomes than those of group 4 spores. The outer spore surface shows sharp peaks or ridges. Bar in overview: 1 µm; bar in inset: 0.1 µm. Figure S7. Cyst ultrastructure D. polycephalum cysts show a fairly dense inner wall and a loosely fibrous outer wall of varying thickness. There is a somewhat denser top layer, which is however much less defined as the spore outer layer and therefore not considered as a third layer here. Mitochondria are round with distinct cristae and some inclusions. Granules are dispersed through the cell. Several hemispherical dark bodies are present underneath the plasma membrane and bacteria-like inclusions (B?), similar to those seen in D. polycephalum spores, are present in most cysts. The P. pallidum cyst walls consist of an inner layer of intermediate density and an outer loosely fibrous layer. The cyst content is less condensed than that of the spore. Mitochondria with well-defined cristae, sometimes with inclusions, are localized throughout the cell. Granules and vesicles of varying size are present throughout the cell. A narrow discontinuous cortex is visible below the plasma membrane. The ultrastructure of P. pallidum cysts has been reported previously 5 and is almost identical to that described here. P. pseudocandidum cysts show a single layered highly fibrous cell wall and opaque inner membranes. The P. filamentosum cysts show similar hemispherical opaque patches below the plasma membrane as D. polycephalum cysts. The wall appears to be only single layered and is rather transparent. Similar to those of P. pallidum, A. leptosomum cysts walls show a narrow dark inner and wider more transparent outer layer. However, a cortex is not obvious. Mitochondria are round to oval with well-defined cristae and both granules and vesicles are present throughout the cytoplasm. The P. equisetoides cytoplasm contains typical "empty" patches, devoid of ribosomes. D. deminutivum cysts are, like the spores, very small with highly condensed contents. The cyst wall is insubstantial and consists of an inner translucent layer and narrow denser outer layer. Single and grouped vesicles are present and granules are dispersed throughout the cell, often enclosed in smooth darker material. Bar in overview: 1 µm; bar in inset: 0.1 µm.

Quantification of ultrastructural features
To aid objective evaluation of differences between spores or cysts of individual species, we measured or enumerated ultrastructural features from 10 spores or cysts for each species. These features are the width of each layer of the spore and cyst wall, the location, grouping, number and diameter of granules per section and their fractional contribution to the cell cross-section area, the grouping, number and fractional cross-section area of membrane-bound vesicles per section, the distribution of heterochromatin and the morphology of mitochondria in each cell. Features that were only found in one or a few species are listed in Table S2.

Figure S8. Spore and cyst walls.
The width of spore and cyst wall layers were measured at 20 positions across the periphery of TEM sections of 10 different cells and averaged per cell. These values were then averaged per species and displayed with standard error of the mean (SE) values (n=10) in the left panels. All per cell values were also averaged over entire taxon groups and shown with SE in the right panels. Because D. deminutivum with its very small spores and narrow walls is a distinct outlier, the values for group 1 were also calculated without those for D. deminutivum (Grp1-Ddemin.). For complete wall width significant differences between groups (P<0.05) were tested using Analysis Of Variance (ANOVA) on ranks. P-values of the comparisons are shown in the colour coded matrix. All data and statistical analyses are listed in supplemental spreadsheet Data2_Walls.xlxs.
Within groups there is considerable variation in the width of the spore wall, which is largely due to variation in the central cellulosic layer. Measurements of the gray scale of the wall layers from EM images show that the middle layer is always the least electron opaque of the three layers. For all cysts, except D. deminutivum, the inner layer is the most opaque. Note that due to variation in section thickness and counter-staining, the gray scale values only provide a relative estimate of electron opacity. Across taxon groups, group 3 walls are significantly narrower than those of groups 2, 4 and 1.

Figure S9. Organelles -Granules
Spore and cyst granules are defined as circular semi-transparent organelles that are not enclosed in membranes and usually reside at the poles of spores of groups 1 and 3. They are considered to be lipid droplets although this was never proven by (histo)chemical analysis. We determined the number (A) and average diameter (B) of granules per section over sections of 10 cells for each species as well as the total section area without the wall (Fig. S12A) and calculated the total (C) and relative area (E) occupied by granules per section. In addition we recorded the location (E) and distribution (F) of granules, the latter only when more than two granules were present. The bar graphs (A-C) represent means and SE of quantitative features for individual species on the left panels and for taxon groups on the right panels. Qualitative features (E,F) are presented as stacked bars, where the length of each stack represents the fraction of cells that display the feature.
Cysts contained significantly more granules than spores (A), which were relatively large (B). However, due due to cysts being larger than spores, their proportional contribution to the section area was about the same (D). Spore granules were relative large in groups 1 and 2 and were significantly smaller in group 4 (B). Their proportional contribution to the cell area was also significantly smaller in group 4 (D). In both cysts and group 4 spores, granules were mostly distributed throughout the cell (E), while in spores from groups 1-3, granules were predominantly located at the poles. Group 2 granules tended to be more loosely grouped than those in groups 1 and 3 (E). Note that the low number of granules visible per section makes assessment of their grouping in TEM images less reliable than from whole spore observations under phase contrast 6 . All data, calculations and statistical analyses are listed in supplemental spreadsheet Data3_Organelles.xlxs.

Figure S10. Organelles -Vesicles
Vesicles here comprise all membrane bounded organelles that are not mitochondria or the nucleus. Their numbers were counted per section (A) and their total area was determined by circling the circumference of individual or of tightly packed clusters of vesicles onscreen in ImageJ (C). Individual vesicle area was assessed by dividing total vesicle area per section by vesicle number (B). Relative vesicle areas (D) and the location (E) and distribution (F) of vesicles were determined as described for granules. Partially due to the large variation in vesicle abundance and size between sections and between species, none of the measured parameters showed significant differences between spores of different taxon groups. The relatively large cysts contained significantly larger but not more numerous vesicles than spores, but the overall contribution of vesicles to their cross section area was about the same.

Figure S11. Organelles -Mitochondria
The outlines of the relatively opaque mitochondria could be difficult to distinguish in the equally opaque spore cytoplasm. We therefore did not attempt to measure their area, but counted their numbers per cell (A) and the fraction of mitochondria per cell that displayed indented (crenate) versus smooth outlines (B), closed versus open cristae (C) and presence versus absence of a border of ribosomes (D). These features appeared to show group-specific differences upon initial examination of EM images. We additionally recorded the distribution of mitochondria over the cell (E).
Mitochondria are most numerous in cysts followed by group 4 and groups 3, 2 and 1 in sequence, which may be related to cell size also decreasing in roughly that order (Fig. S12). Mitochondria with an indented surface are significantly most abundant in groups 3 and 4 than in the other groups or in cysts, while mitochondria with closed cristae and a ribosome fringe are particularly prominent in group 4. The location of mitochondria does not differ significantly between groups.

Figure S12. Cell and nuclear features
Cross-section areas of spore and cysts were measured to determine relative contributions of granules and vesicles to cells and show that cysts are significantly larger than spores (A). Both cell types take equally long to differentiate upon starvation, so the spores either go through another round of division or the cysts retain more water than spores. As reported previously group 1 species have relatively small spores 6 . We also measured some nucleus associated features such as the percentage of the circumference of the nucleus that is fringed by ribosomes (D), the distribution pattern of heterochromatin (C) and when present in distinct foci, the number of foci (B). These features show large variation between species and no group-specific trends, except that in cysts heterochromatin was either present in one or two foci, or not visible in the section.  Table S3. Correlation matrix.

Correlations between spore/cyst fitness and morphology
Matrix of correlation coefficients (R) between spore/cyst survival under different conditions and ultrastructural features of spores and cysts as reported in figures S8 to S12 and measured across 27 species. The colour coding intensity reflects the value of R, with positive and negative correlations shown in red and blue respectively. Values surrounded by black borders were statistically significant with P<0.05. Values were estimated using both Pearson product moment and Spearman rank order correlation using datasets comprising both spores and cysts and spores only. The complete data tables and correlation matrices with P-values are shown in Supplemental Data 4_Correlations.xlsx. The width of spore walls and the presence of mitochondria with closed cristae and/or fringed by ribosomes is positively correlated with spore survival in wet or dry frost, while the relative area occupied by granules and vesicles is negatively correlated. Figure S13. Global distribution of Dictyostelia and optimal culture temperatures Data on geographical location, distribution and optimal culture temperature were retrieved from original species diagnosis and further studies on species ecology (see supplemental Data5_Ecology.xlxs), and are concisely plotted onto phylogenetically ordered Dictyostelium species 6 .  (Table S4) were used to infer divergence times at interior nodes of the tree using the MCMCTree software in PAML v. 4.9 7 (See Materials and Methods). The nodes whose estimates were constrained by fossil calibrations are labelled with italic numbers, and the specific minimum and maximum dates that were used are shown in the subset legend (maximums are only shown when available and dates are scaled in 100 mya). The mean estimate of the divergence times in hundred millions of years ago are shown at the nodes, with 95% Highest Posterior Density (HPD) intervals outlined by blue bars. The divergence between the two major branches of Dictyostelia is estimated to have occurred at 520 million years ago.