Are sponges the most ancient multicellular animals on Earth?

Sponges (Porifera) are one of the oldest currently living groups of animals and possibly the oldest animals belonging to Metazoa. High polymorphism caused by the totipotency of all sponge cells, primitive structure: absence of nervous and, probably, muscular tissue; absence of light-sensitive organs and cells (except for cross-shaped cells at some stages of embryogenesis); weak tissue specialization, for example, many functions of connective tissue in more complex animals are performed by only a few types of mesoglea cells – all this allows us to judge the primitiveness of this group. But sponges also possess a well-developed collagen matrix, contractile ability (oscular and total), complex molecular receptor complexes on the cell surface (tyrosine kinases, integrins), elements of a sensory system (metabotropic glutamate receptors, crystallins), and an immune system (immunoglobulin-like molecules, Rh system, cysteine utility cells), which classifies sponges as true Metazoa [6].

The problem of the "rooting" of the entire group of multicellular animals is debated, and the branching order from the common branch of multicellular sponges (Porifera), ctenophores (Ctenophora), placozoans (Placozoa), cnidarians (Cnidaria), and bilaterians (Bilateria) is not reliably known, except that cnidarians and bilaterians are the closest relatives. Other groups are independent and ancient relative to all multicellular animals.

Fig. 1 The branching order of existing animal groups is rather unclear (hatched areas indicate groups with an uncertain phylogenetic position) (Adamska, 2015).

The main problem is that clarifying the phylogenetic relationships of early Metazoa and the history of multicellularity formation as a whole is complicated by the antiquity of such evolutionary events. Regarding sponges, even here, alpha-taxonomy is not fully known, nor are the phylogenetic relationships within the group. Therefore, even if we accept the hypothesis that sponges are at the origin of multicellularity and give rise to other groups, a separate class, such as Calcarea, could also claim this role (as shown by scientists from the Oceanology Center in Marseille). Their data also suggest that the class Hexactinellida in sponges differs significantly not only morphologically, physiologically, but also phylogenetically from demosponges and calcareous sponges [2].

Studies in molecular phylogenetics provide different insights into the position of sponges on the Metazoa tree, due to the selectivity of the compared groups. There is also a strong dependence of the tree topology on the chosen outgroup, which will root the tree, as well as on statistical models of DNA site substitutions (essentially, this determines the rate of mutation accumulation and is reflected in the divergence strength of the tree and the length of its branches).

It has been shown that if a more distant outgroup to multicellular animals is chosen, for example, fungi (as part of the monophyletic group Opisthokonta), it is possible to confirm the alternative hypothesis that Placozoa are at the root of multicellularity, while choosing a more closely related group, choanoflagellates (Choanoflagellata), yields a well-supported bootstrap topology that confirms the hypothesis of the origin of all multicellular animals from a sponge ancestor [9]. Such a choice may be more appropriate, as choanoflagellates are considered a group closely related to multicellular animals not only due to common morphological features (e.g., they are often compared to sponge choanocytes [4]) but also as confirmed by phylogenetic methods [3,7].

Fig. 2: A whole-genome study shows the proximity of the choanoflagellate M. Brevicollis to multicellular organisms compared to fungi. (King, 2008)

Overall, a number of molecular phylogenetic studies indicate that sponges are at the base of Metazoa, using various methods for finding optimal topologies (e.g., maximum parsimony, neighbor-joining) as well as different nucleotide substitution models [2,9].

Fig. 3: A phylogenetic tree indicating the basal position of sponges (H, D, Ca) relative to other multicellular animals: cnidarians, placozoans, ctenophores (Cn, PI, Ct). It also shows that calcareous sponges (Ca) form a single clade with them with a high level of support (the article even suggests their classification as a separate phylum giving rise to other multicellular animals). The assumption of the close relationship between choanoflagellates (C) and sponges and other multicellular animals is also confirmed. (Borchiellini, 2001)

Fig. 4: A phylogenetic tree constructed with choanoflagellates as the outgroup, using complex nucleotide substitution models (A – CAT, B – CAT-GTR), with high support for sponges (Demospongiae, Hexactinellida, Calcarea, Homoscleromorpha) occupying a basal position. It is worth noting that here, branching sponges, along with another group of homoscleromorphs, are also distinguished as a separate "advanced" clade. (Pisani, 2015)

Regarding comparative physiology: back in the late 1990s, scientists from the University of Erfurt studied some receptor molecules, collagen, immune system proteins of sponges, and their DNA sequences, using amino acid substitution models, which also suggested sponges as the most ancient representatives of multicellular animals. Based on the analysis, the divergence of sponges and choanoflagellates was dated, and these estimates coincide with other methods of determining the appearance of sponges (early Cambrian). Interestingly, it is suggested that the possibility of multicellularity, cell differentiation, and the subsequent Cambrian explosion as a whole became possible after the initiation of an evolutionarily significant mechanism of alternative splicing (exon shuffling during transcription) 1000 million years ago. Comparison of sequences encoding protein kinase C in this work indicates Calcarea as the closest relative of Eumetazoa and separates Hexactinellida from other sponges [6]. Here, one can also point to the "competition" between hypotheses about the primacy of ctenophores and sponges, and sponges win here, as they have an even more primitive contractile system (we only know about the possible involvement of endopinacocytes in this [8]) than ctenophores. Comparison of synapses and neurotransmitters of ctenophores with those of bilaterians, however, also reveals significant differences, but convergent evolution of the nervous system may play a role here [10].

Fig. 5: Comparative analysis of proteins (collagen, integrin, galectin, myosin), signaling molecules (tyrosine kinases, serine-tyrosine kinases, G-protein), transcription factors (homeodomain, MADS-box), immune receptors (heat shock proteins, proteasomes, CRCR-SCR, Ph-like proteins), cell lineages and ancestral cells for sensory functions, also indicates the basal position of sponges with a temporal dating of divergence from choanoflagellates (about 600 million years ago). The analysis does not present data on ctenophores or placozoans. Also, on the timeline, stages of the emergence of mechanisms that significantly increase the variability of complex proteins (formation of domains, modules, alternative splicing) are depicted. (Muller, 1998)

What if we assume the existence of a non-sponge ancestor that had a simpler structure, yet had a relatively differentiated multicellular body? This assumption is indeed plausible, but it raises a number of problems caused by the need for paleontological data or the current inability to model such an ancestor. Thus, this question can be left open. What if such an ancestor was more complex?

Recent studies published by a team of Australian biologists, conducted at the International Centre for Marine Molecular Biology at the University of Bergen, in the field of comparing transcription factors of embryogenesis in three classes of sponges, suggest that the relatively primitive body structure of sponges may be related to gene loss as a result of evolution from a more complex ancestor that gave rise to sponges and cnidarians. Such a hypothetical common ancestor would have had a richer set of genes in the compared gene families (in this case, T-box genes, Sox genes, ANTP class genes, Six genes, Pax genes, GATA, and Smad genes), which are only partially preserved for each class of sponges. Phylogenetic analysis also showed that all genes from these families exhibit similarity to the closest group – cnidarians. The authors also note that such gene loss has occurred repeatedly during evolution in different taxonomic groups (e.g., in the same cnidarians (Hydra) or even in vertebrates (Tunicata)) [1]. A significant point in favor of the hypothesis is that the studied genes sometimes play a key role in sponge embryogenesis (e.g., during the excurvation of calcareous sponges, the Brachyury gene is actively expressed, which, interestingly, is also involved in the formation of the axial skeleton in chordates), so events like gene loss from these families or duplication indeed affect the course of evolution and are informative for the proposed hypothesis. At the same time, this may only indicate that many genes have simply changed or been modified in the genome over the course of evolution, or even that the gene's function has changed, and this is not a consequence of gene loss from a more complex ancestor. In any case, without paleontological data, this remains only a hypothesis.

What about paleontological data? A recent study in 2016 by scientists from the Massachusetts Institute of Technology not only confirms the hypothesis of sponges as the most ancient multicellular animals but also pushes their "antiquity" back to the Precambrian period (about 640 million years ago). The scarcity of traditional sponge fossils can be compensated by a large number of "molecular" fossils (mineralized deposits). In this study, these were deposits of the sterol 24-isopropylcholestane, for which the gene responsible for lipid synthesis – SMT gene – was identified. Comparative genomics methods were then used, which showed two possible lipid donors: algae and sponges, with molecular clock methods (dating evolutionary events) indicating that it appeared earlier in sponges, specifically 640 million years ago [5].

Fig. 6: A – alternative hypotheses of SMT gene evolution, where the asterisk denotes the moment of gene duplication (formation of the SMT gene responsible for the synthesis of 24-isopropylcholestane), and the blue circle denotes the origin of siliceous sponges. B – dating the origin of the SMT gene for sponges (Demosponge Duplication) and algae (Algae Duplication). The diagram shows that the time difference between these events is up to 300 million years. (Golda, 2016)

Therefore, it is most likely that sponges are indeed the most ancient multicellular animals on Earth, but this raises many additional questions. For example, are they the first multicellular organisms, or simply a more successful group that has survived to this day? If so, what was this sponge like, given the incredible diversity of sponges we have, even including syncytial forms (glass sponges), and perhaps there are even grounds to classify them as separate phyla. The study of their physiology within the framework of evolution is also of fundamental importance, as, for example, their contractile abilities could play a significant role in understanding how the nervous system developed in its initial stages. The question also remains open whether sponges were the ancestors of cnidarians and bilaterians, or simply the closest sister group that has survived to this day. I believe that for this, we still need to collect molecular, paleontological, and physiological data from all groups of early multicellular animals for a long time to be able to model a hypothetical ancestor that first acquired many specialized cells.

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