The most widely held, but rarely tested, hypothesis for the origin of animals is that they evolved from a unicellular ancestor with an apical cilium surrounded by a microvillar collar that structurally resembled modern sponge choanocytes and choanoflagellates 1-4. Here we test this traditional view of animal origins by comparing the transcriptomes, fates and behaviours of the three primary sponge cell types-choanocytes, pluripotent mesenchymal archeocytes and epithelial pinacocytes-with choanoflagellates and other unicellular holozoans. Unexpectedly, we find the transcriptome of sponge choanocytes is the least similar to the transcriptomes of choanoflagellates and is significantly enriched in genes unique to either animals or sponges alone. In contrast, pluripotent archeocytes up-regulate genes controlling cell proliferation and gene expression, as in other metazoan stem cells and in the proliferating stages of two unicellular holozoans, including a colonial choanoflagellate. Choanocytes in the sponge Amphimedon queenslandica exist in a transient metastable state and readily transdifferentiate into archeocytes, which can differentiate into a range of other cell types. These sponge cell type conversions are similar to the temporal cell state changes that occur in unicellular holozoans 5. Together, these analyses offer no support for the homology of sponge choanocytes and choanoflagellates, nor for the view that the first multicellular animals were simple balls of cells with limited capacity to differentiate. Instead, our results are consistent with the first animal cell being able to transition between multiple states in a manner similar to modern transdifferentiating and stem cells. References 1 Cavalier-Smith, T. Origin of animal multicellularity: precursors, causes, consequences-the choanoflagellate/sponge transition, neurogenesis and the Cambrian explosion.

The Porifera (sponges) is one of the earliest phyletic lineages to branch off the metazoan tree. Although the body-plan of sponges is among the simplest in the animal kingdom and sponges lack nervous systems that communicate environmental signals to other cells, their larvae have sensory systems that generate coordinated responses to environmental cues. In eumetazoans (Cnidaria and Bilateria), the nervous systems of larvae often regulate metamorphosis through Ca(2+)-dependent signal transduction. In sponges, neither the identity of the receptor system that detects an inductive environmental cue (hereafter "metamorphic cues") nor the signaling system that mediates settlement and metamorphosis are known. Using a combination of behavioral assays and surgical manipulations, we show here that specialized epithelial cells-referred to as flask cells-enriched in the anterior third of the Amphimedon queenslandica larva are most likely to be the sensory cells that detect the metamorphic cues. Surgical removal of the region enriched in flask cells in a larva inhibits the initiation of metamorphosis. The flask cell has an apical sensory apparatus with a cilium surrounded by an apical F-actin-rich protrusion, and numerous vesicles, hallmarks of eumetazoan sensory-neurosecretory cells. We demonstrate that these flask cells respond to metamorphic cues by elevating intracellular Ca(2+) levels, and that this elevation is necessary for the initiation of metamorphosis. Taken together, these analyses suggest that sponge larvae have sensory-secretory epithelial cells capable of converting exogenous cues into internal signals via Ca(2+)-mediated signaling, which is necessary for the initiation of metamorphosis. Similarities in the morphology, physiology, and function of the sensory flask cells in sponge larvae with the sensory/neurosecretory cells in eumetazoan larvae suggest this sensory system predates the divergence of Porifera and Eumetazoa.

Main 46The last common ancestor of all living animals appears to have possessed epithelial and 47 mesenchymal cell types that could transdifferentiate over an ontogenetic life cycle 48 ( Fig.1a) 1,4 . This capacity to develop and differentiate required a regulatory capacity to 49 control spatial and temporal gene expression, and included a diversified set of signalling 50 pathways, transcription factors, enhancers, promoters and non-coding RNAs (Fig. 1a) [5][6][7][8][9] . 51Recent analyses of the genomes and life cycles of unicellular holozoan relatives of 52 animals have revealed that the regulatory repertoire present in multicellular animals 53 largely evolved first in a unicellular ancestor ( Fig. 1a) 2,5,6 . These insights contrast with a 54 widely-held view that all animals evolved from a stem organism that was a simple ball 55 of ciliated cells 1,3,4 . Implicit in this traditional perspective is that (i) regulatory systems 56 necessary for cell differentiation evolved after the divergence of metazoan and 57 choanoflagellates lineages, and (ii) morphological features shared between 58 choanoflagellate and choanocytes are homologous and were present in the original 59 animal cell. While the former is not supported by recent data -unicellular holozoans 60 can change cell states by environmentally-induced temporal shifts in gene expression 61 ( Fig. 1a) 5,6,10-12 -the latter is contingent upon the still controversial aspect of whether 62 extant choanocytes and choanoflagellates accurately reflect the ancestral animal cell 63 type. 64To test this, we first compared cell type-specific transcriptomes 13 from the sponge 65Amphimedon queenslandica with each other, and with transcriptomes expressed during 66 the life cycles of closely-related unicellular holozoans, the choanoflagellate Salpingoeca 67 rosetta, the filasterean Capsaspora owczarzaki and the ichthyosporean Creolimax 68 fragrantissima (Fig. 1a) 10-12 . We chose three sponge somatic cell types hypothesised to 69 be homologous to cells present in the last common ancestor of contemporary Extended Data Figure 2: Percentage of KEGG cellular processes and 663 environmental information processing (i.e. cell signalling) genes present in each 664 cell type, corresponding to the number of components making up each KEGG 665 category identified. 666 a, Cellular processes genes. b, Environmental information processing (i.e. cell 667 signalling) genes. 668 669 Extended Data Figure 3: Evolutionary age of genes expressed in Amphimedon 670 queenslandica choanocytes, archeocytes and pinacocytes using different 671 expression thresholds. 672 *

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