24-isopropyl cholestane is an organic molecule produced by specific sponges, protists^[1] and marine algae.^[2] The identification of this molecule at high abundances in Neoproterozoic rocks has been interpreted to reflect the presence of multicellular life prior to the rapid diversification and radiation of life during the Cambrian explosion.^[3][4] In this transitional period at the start of the Phanerozoic, single-celled organisms evolved to produce many of the evolutionary lineages present on Earth today.^[5] Interpreting 24-isopropyl cholestane in ancient rocks as indicating the presence of sponges before this rapid diversification event alters the traditional understanding of the evolution of multicellular life and the coupling of biology to changes in end-Neoproterozoic climate. However, there are several arguments against causally linking 24-isopropyl cholestane to sponges based on considerations of marine algae and the potential alteration of organic molecules over geologic time.^[6] In particular the discovery of 24-isopropyl cholestane in rhizarian protists implies that this biomarker cannot be used on its own to trace sponges.^[1] Interpreting the presence of 24-isopropyl cholestane in the context of changingglobal biogeochemical cycles at the Proterozoic-Phanerozoic transition remains an area of active research.

While Love et al. (2009) argues for the presence of sponges in rocks below the Marinoan cap carbonate at ~635 Ma (millions of years ago),^[3] Antcliffe (2013) estimates the age of the biomarker-bearing rock to be between 645 Ma and ~580 Ma.^[6] Most recently, Gold et al. (2016) writes that the age of rocks containing 24-isoproylcholestane have an age between ~650 Ma and 540 Ma.^[4] In all cases, estimates agree that the age of the rocks containing 24-isoproylcholestane pre-date the Cambrian explosion at ~541 Ma.

The presence of sponges before ~540 Ma has profound implications for the evolution of multicellular life and the coupling of the biosphere to Neoproterozoic climate. Climate change before the Cambrian explosion and the subsequent diversification of life are intricately intertwined with understanding the causes of Snowball Earth episodes,^[11] the deposition of Banded Iron Formations,^[12] and the second step in the rise of atmospheric oxygen.^[13] In particular, the presence of sponges raises questions of the minimum dissolved O₂ content of the oceans in the late Neoproterozoic and the transition from a euxinic Canfield ocean to the modern oxygenated deep-ocean. However, sponges appear to require very little O₂ to survive, so their presence in the Precambrian may not provide strong constraints on Proterozoic O₂ levels.^[14]

There are several lines of logic against interpreting 24-isopropyl cholestane as a biomarker for demosponge:^[6]

1. Much of the argument for Precambrian sponges is grounded in the observation that pelagophyte algae produce organic matter with a low ratio of 24-isopropyl cholestane to 24-n-propyl cholestane, but that this ratio is high in ancient rocks. However, the observed change in the sterane ratio could also be explained if algae changed the relative abundances in which they produce steranes over the past 600 million years. In a similar line of argument, it is possible that another extinct organism from which the algae descended produced organic matter with a higher ratio of 24-isopropyl cholestane to 24-n-propyl cholestane. As argued above, recent evidence has suggested that the algae's synthesis pathway only arose during the Phanerozoic,^[4] which tempers this argument. More generally, these concerns address the issue of insufficient specificity in molecular fossils, which plagues many biomarker studies.
2. 24-isopropyl cholestane can be formed through sedimentary diagenesis of other organic molecules, so the high ratio of 24-isopropyl cholestane to 24-n-propyl cholestane could simply reflect the post-depositional transformation of organic matter. Some research has addressed this concern by showing a lack of alteration in other organic molecules,^[3] such as hopanes, but subsequent analysis has questioned whether the molecules with minimal alteration could have been contamination from modern petroleum-derived oil.^[6]
3. There may have been a group of bacteria living symbiotically with the sponges that also produced 24-isopropyl cholestane.^[15] If these bacteria produced the biomarker throughout geologic time, its presence would not be strictly indicative of demosponge. However, as with the marine algae, analysis of the ratio of 24-isopropyl cholestane to 24-n-propyl cholestane may clarify the source of the compounds.
4. It is strange to find sponge biomarkers before the Cambrian explosion without accompanying sponge fossils (although there is tentative evidence for sponge-like structures in the latest-Neoproterozoic^[10]). Moreover, once fossils of sponges do appear during the Paleozoic, the ratio of 24-isopropyl cholestane to 24-n-propylcholestane returns to its background value. This is surprising because we might expect the ratio to stay elevated or even to increase as metazoa diversified and sponges proliferated.