Although collar cells are conserved across animals and their closest relatives, the choanoflagellates, little is known about their ancestry, their subcellular architecture, or how they differentiate. The choanoflagellate Salpingoeca rosetta expresses genes necessary for animal development and can alternate between unicellular and multicellular states, making it a powerful model for investigating the origin of animal multicellularity and mechanisms underlying cell differentiation. To compare the subcellular architecture of solitary collar cells in S . rosetta with that of multicellular ‘rosette’ colonies and collar cells in sponges, we reconstructed entire cells in 3D through transmission electron microscopy on serial ultrathin sections. Structural analysis of our 3D reconstructions revealed important differences between single and colonial choanoflagellate cells, with colonial cells exhibiting a more amoeboid morphology consistent with higher levels of macropinocytotic activity. Comparison of multiple reconstructed rosette colonies highlighted the variable nature of cell sizes, cell–cell contact networks, and colony arrangement. Importantly, we uncovered the presence of elongated cells in some rosette colonies that likely represent a distinct and differentiated cell type, pointing toward spatial cell differentiation. Intercellular bridges within choanoflagellate colonies displayed a variety of morphologies and connected some but not all neighbouring cells. Reconstruction of sponge choanocytes revealed ultrastructural commonalities but also differences in major organelle composition in comparison to choanoflagellates. Together, our comparative reconstructions uncover the architecture of cell differentiation in choanoflagellates and sponge choanocytes and constitute an important step in reconstructing the cell biology of the last common ancestor of animals.

12 13 *Correspondence pawel.burkhardt@uib.no 14 15 16 17 18 19 20 21 22 23 24 the nucleus and mitochondria than choanoflagellates and more of their volume to 50 food vacuoles. Together, our comparative reconstructions uncover the architecture 51 of cell differentiation in choanoflagellates and sponge choanocytes and constitute an 52 important step in reconstructing the cell biology of the last common ancestor of the 53 animal kingdom. 54 55 56 57 RESULTS AND DISCUSSION 58 Three-dimensional cellular architecture of choanoflagellates 59Collar cells were likely one of the first animal cell types [1,8,9] and persist in most 60 animal phyla ( Figure 1A). Therefore, characterising the microanatomy of 61 choanoflagellates and sponge choanocytes has important implications for the origin 62 and evolution of animal cell types. To fully characterise and reconstruct both single 63 and colonial S. rosetta cells, we used high-pressure freezing and 3D serial ultrathin 64 TEM sectioning (3D ssTEM), in addition to fluorescent microscopy. Three randomly 65 selected single cells and three randomly selected colonial cells from a single colony 66 were chosen for the reconstruction of entire choanoflagellate cells and subcellular 67 structures (Figures 1 and S1-2, Videos S1-6). Both single and colonial S. rosetta 68 cells exhibited a prominent, central nucleus enveloped by a mitochondrial reticulum 69 and basal food vacuoles -as well as intracellular glycogen reserves -consistent with 70 the coarse choanoflagellate cellular architecture reported in previous studies [10,11] 71 (reviewed in [7,12]) (Figures 1 and S1-2, Videos S1-6). However, with the increased 72 resolution of electron microscopy we detected three morphologically distinct 73 populations of intracellular vesicles with distinct subcellular localizations ( Figure 1G 74 and S1): 1) Large vesicles (extremely electron-lucent, 226 ± 53 nm in diameter), 2) 75Golgi-associated vesicles (electron-dense inclusions, 50 ± 10 nm in diameter), and 763) Apical vesicles (electron-lucent, 103 ± 21 nm in diameter). Extracellular vesicles 77 were also observed associated with two of the single cells (electron-lucent, 173 ± 36 78 nm in diameter) and appeared to bud from the microvillar membrane ( Fig S1L). 79Choanoflagellate cells subjected to fluorescent labelling were congruent with 3D 80 ssTEM reconstructions in terms of organelle localization ( Figure 1B-C), providing 81 evidence that the 3D models presented herein are biologically representative. 82 Ultrastructural commonalities and differences between single and colonial 83 choanoflagellate cells 84Our 3D ssTEM reconstructions allowed for detailed volumetric and numerical 85 comparisons among single and colonial S. rosetta cells (Figures 2 and S2, Table S1 86 and S2). Overall, the general deposition of organelles was unchanged in both cell 87 types (Figures 2A, B and S2A-C). In addition, single and colonial cells devote a 88 similar proportion of cell volume to most of their major organelles (nucleus: single 89 cells 12.92 ± 0.58%...

Key to the ecological prominence of fungi is their distinctive cell biology, our understanding of which has been principally based on dikaryan hyphal and yeast forms. The early-diverging Chytridiomycota (chytrids) are ecologically important and a significant component of fungal diversity, yet their cell biology remains poorly understood. Unlike dikaryan hyphae, chytrids typically attach to substrates and feed osmotrophically via anucleate rhizoids. The evolution of fungal hyphae appears to have occurred from rhizoid-bearing lineages and it has been hypothesized that a rhizoid-like structure was the precursor to multicellular hyphae. Here, we show in a unicellular chytrid, Rhizoclosmatium globosum , that rhizoid development exhibits striking similarities with dikaryan hyphae and is adaptive to resource availability. Rhizoid morphogenesis exhibits analogous patterns to hyphal growth and is controlled by β-glucan-dependent cell wall synthesis and actin polymerization. Chytrid rhizoids growing from individual cells also demonstrate adaptive morphological plasticity in response to resource availability, developing a searching phenotype when carbon starved and spatial differentiation when interacting with particulate organic matter. We demonstrate that the adaptive cell biology and associated developmental plasticity considered characteristic of hyphal fungi are shared more widely across the Kingdom Fungi and therefore could be conserved from their most recent common ancestor.