Evol Biol (2009) 36:256–266 DOI 10.1007/s11692-008-9047-2

ESSAY

Modelling the Transport of Nutrients in Early Animals

N. J. Beaumont

Received: 20 June 2008 / Accepted: 6 November 2008 / Published online: 10 January 2009 Ó Springer Science+Business Media, LLC 2008

Abstract The single-celled ancestors of multi-cellular Introduction animals (metazoans) did not need to transport nutrients between cells, but this ability is vital for modern animals. The origins of multi-cellularity are obscure. It is unclear How could intercellular nutrient transport have begun? how early multi-cellular animals evolved, and why it And how did this influence the early evolution of animals? happened at that particular time. Suggestions or hypotheses In this hypothesis, I suggest that nutrients could have about the origins can be tested using the main lines of passed directly between the cytoplasm of conjoined cells in available evidence about the earliest animals: the fossil early compacted cell-balls, along the plane of the closed record and the molecular and developmental data from epithelium. This would have limited early animals to the modern species. size and form of modern embryos. The mechanisms that The Precambrian fossil record provides evidence of the indirectly transport nutrients between discrete cells, via the earliest known multi-cellular forms. Multi-cellular organ- extracellular fluid within the body-space, are modelled to isms have been identified from rocks up to 600 million years have evolved sequentially; so comparison of nutrient old. A diverse group of large fossils, the Ediacaran Fauna, transport processes could provide evidence of any early have been found at sites around the World in rocks from divergences of phyla. When the last of the indirect inter- between approximately 580 million and 550 million years cellular transport processes for essential nutrients had been ago. It has been suggested that these frondose organisms developed, the extracellular fluid within the body-space were ancestral to some modern animals (Glaessner and would have contained all necessary nutrients. Then the Wade 1966). However, an alternative hypothesis has been epithelium could have greatly expanded, and cells lived suggested: that they were an independent ‘‘experiment’’ in and divided within the body-space. This development of multi-cellularity (Seilacher 1984). At this stage, it is unclear nutrient transport processes would have enabled animals to whether these organisms were ancestral to modern phyla. greatly increase in size and complexity. Microfossils have been found in southern China from approximately 580 million years ago (Xiao et al. 1998; Keywords Metazoan Evolution Cambrian Chen et al. 2000). These minute fossils are less than a Proterozoic Á Á Á millimetre across but are exquisitely preserved. They have been interpreted as embryonic forms; but the adult forms do not seem to be present—so it is unclear how they should be incorporated into an account of early multi-cellularity. A putative bilaterian from this area has also been reported. Vernanimalcula (Chen et al. 2004a) has been interpreted as N. J. Beaumont (&) having a mouth, gut and anus in a hollow form of less than Department of Medicine, Royal Free & University College a millimetre; however this interpretation is controversial Medical School, Royal Free Campus, Rowland Hill Street, (Bengtson and Budd 2004; Chen et al. 2004b). London NW3 2PF, UK e-mail: [email protected]; Animals from extant phyla definitely start to appear in [email protected] the fossil record from around 530 million years ago. The

123 Evol Biol (2009) 36:256–266 257 apparently sudden appearance of a broad range of complex in the fossil record. Nevertheless, it is clear that multi- animals from virtually all recognised phyla has been called cellular creatures arose from simpler organisms, as surely the Cambrian ‘‘explosion’’. Taking the fossil record on its as the adult develops from the fertilized egg by way of the own, it suggests that animals with bodyplans similar to embryo, so it is intriguing to consider the reasons and modern animals appeared suddenly, with no obviously requirements for such changes. simpler precursors. There is continuing debate over whe- The aim here is to understand the advent of multi-cel- ther this is an artefact of the fossil record or an accurate, if lularity by considering how nutrients move between the rather puzzling, record of events. cells of modern animals, and then considering how this The comparison of homologous molecular sequences distribution could have begun. I argue that these processes from different modern phyla, calibrated with reference to would not have developed in unicellular organisms, but the fossil record, has been used to create ‘‘molecular that they are necessary for triploblastic metazoans. I pres- clocks’’. These data have been used to extrapolate back to ent a model of early nutrient transport in animals, suggest the metazoan radiation, to estimate the time when the stem how this may have evolved into modern forms and con- groups that led to modern phyla diverged. In contrast to the sider some of the implications this had for evolution. impression formed from the fossil record, many of the studies based upon molecular data have suggested that The Transport of Nutrients in Animals modern phyla diverged a long time before the Cambrian period (Wray et al. 1996; Pisani et al. 2004). These studies The cells of multi-cellular organisms must obtain sufficient suggest that the ancestral stem groups of modern phyla had nutrients from either the environment or from within the diverged for a considerable length of Precambrian time organism. Nutrients pass between cells in triploblastic without leaving traces in the fossil record. This is surpris- animals by a variety of methods. These processes can be ing because the stem groups of modern phyla were divided into direct and indirect transport, depending upon probably multi-cellular. However, there may have been a whether the molecules that are being transported pass number of gene, and even genome, duplications around the across cell membranes and through another compartment time of the metazoan radiation, and rates of genetic change when moving between cells. In direct transport, nutrients could have varied between phyla. These factors may have pass between cells via cytoplasmic bridges (such as gap jeopardized the assumption of uniform rates of variation junctions). These link the cytoplasm of conjoined cells and upon which this approach is based (Peterson et al. 2004). allow small molecules to non-specifically equilibrate It is difficult to understand the beginning of multi-cel- between them without having to cross extracellular space lular animals because it is unclear how the metazoan or the plasma membrane (Fig. 1a). This can happen in cell- radiation, when the variety of modern phyla first arose— types such as epithelia or syncitia. which has been investigated by analyzing molecular However, there are some cell types, such as muscle, sequences, was related to the Cambrian ‘‘explosion’’ seen bone and blood cells, which are not conjoined with other

AB

Fig. 1 Nutrients can pass between cells by direct or indirect Nutrients are released into the enclosed body-space by the cells that nutrient transport. Simplified representation in which only two take them up from the gut. They are then available for other cells nutrients (blue and red) are depicted in cross-sections of hypothetical (such as the token isolated internal cell depicted). Note that a separate gastrula-like forms. a Direct nutrient transport: Both nutrients are indirect nutrient transport process is required for each nutrient. taken up into the organism and pass from cell to cell by cytoplasmic Nutrients are only depicted in the top half of the figure links across the closed epithelium. b Indirect nutrient transport:

123 258 Evol Biol (2009) 36:256–266 cells and take up all the nutrients they require from the Therefore, metazoan nutrient uptake processes could have extracellular fluid. This requires that they have all the derived from those used by unicellular organisms. necessary transport mechanisms to take up each nutrient as needed across the plasma membrane, and also that all Nutrients Could have Moved Between Cells Within essential nutrients are present in the extracellular fluid. Early Animals by Direct Transport This in turn requires that the cells that take up the nutrients from food (for example, the enterocytes that line the Some cells in early multi-cellular organisms would have mammalian gut) also release these nutrients into the needed to receive nutrients from other cells when their extracellular space within the organism so that they may be ability to take up nutrients from the environment became taken up by other cells. This mode of intercellular nutrient limited because their access to food was hampered by their transport is here designated indirect, to denote that the position in the organism. This movement of nutrients could nutrients pass between cells by crossing the plasma mem- have happened by direct transport. Cytoplasmic bridges brane, moving across the extracellular space within the would have established a physical link between cells, organism and then crossing the membrane of another cell which would have coupled the pools of solutes in them (Fig. 1b). without requiring the development of specialized transport The two processes that move nutrients between the cells processes for each nutrient. This ability to share all nutri- of metazoans, direct and indirect transport, are similar ents between cells by a single mechanism would have concepts to the symplastic and apoplastic nutrient transport enabled organisms to evolve in which some cells received that has been described in plants and fungi. The two pro- their nutrients from other cells rather than the environment. cesses differ in the mechanisms which transfer the nutrients from cell to cell. Direct transport uses a single physical Could Early Animals have Used Only the Indirect nexus between the cytoplasm of conjoined cells to enable Transport of Nutrients Between Cells? the movement of small molecules between cells; whilst indirect transport requires that specific nutrient transport If this were the case, then early metazoans would have processes have been developed for each nutrient. Direct needed indirect transport mechanisms for all the carbon transport is simpler than indirect nutrient transport, but and nitrogen sources, cations, anions, fatty acids, vitamins unlike plants and fungi, most nutrients are indirectly and trace elements that are essential nutrients. It would not transported via the circulation in metazoans. This required have been viable to have only some of these mechanisms, the development or recruitment of specific transporter or to have developed them one after another. For this proteins to convey each nutrient across the plasma suggestion to have worked, these indirect nutrient transport membrane. mechanisms would all have had to have started either Having distinguished the two processes by which before the first multi-cellular organisms or at the same nutrients can move between cells, the methods by which time. nutrients could have been transported between cells in Would it have been possible for all the indirect nutrient early multi-cellular organisms can be considered. We can transport processes to have been selected before the for- then attempt to reconstruct how modern transport processes mation of multi-cellular organisms? The uptake of arose from that beginning. nutrients from the environment would not have been problematic for either free-living cells or those at the sur- The Uptake of Nutrients by Early Animals face of a multi-cellular form, as described earlier. However, it is in the interest of single cells to retain their Food would have had to have been digested by early ani- nutrients, so there was no reason for free-living cells to mals. This could either have been performed by secreting evolve mechanisms to release all their nutrients, as if in digestive enzymes to break down complex food items anticipation of them clustering together. Isolated cells with extracellularly, or by phagocytosing food items and then processes for both the uptake and release of specific digesting them intracellularly. Both means of feeding nutrients would have performed a futile cycle, a micro- would have required that nutrients from the digested food scopic Torment of the Danaids. In modern metazoans, the were transported across the membrane into the cytoplasm. cells that take nutrients up from the lumen of the gut These would have been active processes, similar to those release them into the body-space, which is isolated from for taking nutrients up into discrete cells. These would the environment by the closed epithelial layer, so that only have been mechanisms that needed metabolic energy to other cells of the organism can take them up. It is not take the substrate across the membrane into the cytoplasm, apparent why precursors of metazoans, presumably free- against the concentration-gradient. These processes would living unicellular organisms, would have benefited from not have been reversible under physiological conditions. developing mechanisms to release nutrients, and then have

123 Evol Biol (2009) 36:256–266 259 held them in reserve, ready for when cells aggregated to closed epithelial (or syncytial) sheets, a tissue that is shared form multi-cellular creatures. by all eumetazoans. All the cells of these hollow cell-balls Could unicellular organisms have aggregated and would have been coupled by cytoplasmic connections, so simultaneously created all the necessary indirect transport that the direct transport of nutrients between all cells could processes to form viable organisms? This would have have occurred. The epithelial sheet also enclosed an required that there was a simultaneous creation of an epi- internal body-space that could have been the basis for the thelial layer to enclose the body-space, along with all the evolution of indirect nutrient transport processes. processes for transporting essential nutrients out of the cells that took them up from the environment, creating extracellular fluid containing all of these nutrients, and the A Model of the Evolution of Intercellular Nutrient transport processes for taking them up into the cells iso- Transport in Early Animals lated from the environment. This would have required an unfeasible set of coincidences (Cereijido et al. 2004) so the A model of the evolution of nutrient transport in early ani- spontaneous creation of multi-cellular organisms which mals will be developed upon the basis that isolated cells first relied only upon indirect nutrient transport processes is a formed clusters, which led to groups of cells capable of direct poor model for the earliest metazoans. nutrient transport. This development later made possible the evolution of organisms capable of indirect nutrient transport. Possible Forms of the Earliest Multi-Cellular Animals Modern animals make extensive use of the indirect transport of nutrients via the circulation, unlike other multi-cellular The earliest multi-cellular organisms, in which some cells organisms such as plants and fungi. It should be possible to rely upon others for their nutrients, must be based upon propose a model of early animals based upon direct nutrient direct nutrient transport. So the earliest metazoans would transport, compatible with the subsequent evolution of forms have used the direct transport of nutrients between cells. capable of indirect nutrient transport. What possible forms could those earliest multi-cellular Simple clonal clusters of cells may have independently animals have been? arisen a number of times, and selective pressures made A clue may be an attribute that is common to all met- them evolve into a variety of forms. In Precambrian seas, azoans, which suggests that their common ancestor also perhaps selected for through predation by organisms shared this feature. Haeckel (1874) noted that the early capable of engulfing whole cells, some eukaryotic cells stages of embryonic development are similar for all phyla; remained attached after division, which created small they develop from a cluster of cells into a hollow ball of bunches of cells. If ecological interactions favoured cells, a blastula, which then invaginates to form a gastrula. increasingly larger forms, then cell-balls could have He proposed that the earliest multi-cellular animals were increased in size by increasing the number of cells, and similar to these modern embryonic forms. The hypothesis them becoming attached to each other sufficiently tightly to proposed here recapitulates and then develops upon this form closed epithelial sheets (Fig. 2). This would have part of his theory: Developmental pathways were estab- required cell–cell interactions, such as the formation of lished which created viable multi-cellular forms of life, and tight junctions, through specific proteins that may have they became the basis for further developments including been possessed by the common ancestor of metazoans. The indirect nutrient transport processes. evolution of the closed epithelium would have isolated the An advantage of modelling early metazoans upon interior of the cell-ball from the environment, creating a modern embryonic stages is that these forms comprise of new space, in a process similar to the development of an

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Fig. 2 A model of the evolution of direct nutrient transport in the have created an internal space within the cell-ball, and led to the earliest animals. A single cell (a) could have remained attached to its polarisation of the cells as different transport proteins were trafficked sister cell after division (b). This process could have been repeated so to the interior membrane (red) (e) the internal body-space could have that a cluster of clonal cells formed (c). The cells of the cluster could been expanded (f) become attached by links that conjoined their cytoplasm (d) this could

123 260 Evol Biol (2009) 36:256–266 epithelium by the compaction of the modern embryo. This difference in orientation would have meant that the proteins would have formed hollow balls of cells that could have involved in importing nutrients from vesicles into the cyto- expanded by increasing the volume of the extracellular plasm could not simply have been redeployed to transport space within the cell-ball (Fig. 2). nutrients out of the cell into the extracellular space. Even if The hollow cell-ball, similar in form to a modern blas- the earliest multi-cellular creatures did feed by phagocytosis, tula, would have comprised of clonal cells, so it would this does not explain how indirect intercellular nutrient have been spherically symmetrical. The cells were all on transport processes arose. the surface of the sphere and could have performed similar What would have been the maximum size of hollow roles. If such forms were in open water then they would cell-balls reliant upon the direct transport of nutrients? have been exposed to similar food-sources over their sur- When a cell-ball was taking up nutrients from a limited face, so transporting nutrients from cell to cell would not area, such as the point of contact with a food source, or an have been vital (Fig. 3a). invagination of the surface, then the size would have been However, the spherical symmetry would effectively limited by the very nature of the direct transport of nutri- have been broken if food was available only on one side of ents between cells. As nutrients diffused around the plane the sphere. For example, if the cell-ball took up nutrients of the conjoined cells, away from the point of uptake, they asymmetrically, i.e. from a point of contact with a micro- passed through the cytoplasm of all the other cells, which bial mat, either by secreting digestive enzymes and then would have consumed them. Therefore, the concentration taking up simple nutrients, or by phagocytosis, then it of nutrients would have decreased as they passed around would have been necessary to transport nutrients from cells the organism (Fig. 3b). So the longest length of conjoined in contact with the source of nourishment to others cells that could have been supported by direct nutrient (Fig. 3b). Once the nutrients had been taken up by cells at transport, which would have been a constraint on the one point of the cell-ball they could have passed to adja- possible size of the organism, would have been determined cent cells (and then on to others away from the source of by the longest concentration-gradient that could have nourishment) diffusing through intercellular cytoplasmic supported all the cells. That is the highest concentration bridges within the epithelium. that the cells which took up the nutrients could have It has been suggested that the last common ancestor of withstood, and the lowest concentration that the cells fur- metazoans was phagotrophic, taking food particles up by thest down the gradient could have tolerated. phagocytosis. Nutrients would have to have been transported The concentration-gradients would not have been the from the phagocytic vacuole into the cytoplasm: so could same for all nutrients. Nutrients were not used at the same these transport processes have been the basis for the devel- rate. The concentration of the nutrient used most quickly opment of indirect intercellular nutrient transport processes? would have decreased most along the conjoined cells. The The transport of nutrients out of vesicles, across the mem- combination of a different gradient for each nutrient and brane into the cytoplasm is in the opposite direction from the different tolerances for the concentration ranges of these first step of indirect intercellular nutrient transport, in which nutrients would have meant that only one nutrient would nutrients travel from the cytoplasm of the absorptive cell, have set the upper limit of the length of epithelium that could across the membrane into the extracellular space. This have been maintained by the direct transport of nutrients

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Fig. 3 Indirect nutrient transport processes could have arisen depicted in red is steepest. c Later organism in which an indirect separately in early metazoans which relied upon the direct mechanism for the transport of the red nutrient has been developed. transport of nutrients. Simplified representation in which only three Now the concentration-gradient of the blue nutrient cannot be nutrients (blue, red and yellow) are depicted in early blastula-like increased, so it limits the size of the organism. d Further development forms (a) no nutrient gradients were formed in pelagic form. b In a in which an indirect transport process has developed for the blue benthic form in contact with a microbial mat, nutrient-gradients were nutrient. The interior of this organism contains both the red and blue established. All three nutrients pass between the conjoined cells by nutrients. The size of the organism is constrained by the concentra- direct transport. Note that the concentration-gradient for the nutrient tion-gradient of the yellow nutrient along the epithelium

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(Fig. 3b). When an indirect transport process was estab- limited to the size at which the cells at the extremes of lished for this nutrient, it would have passed from those cells nutrient concentration-gradients could have lived. The that took it up into the enclosed body-space for any cell of the diameter of the cell-ball, or invaginated cell-ball, would organism to take up. This would have lifted this constraint on have got larger incrementally with the acquisition of the size of the organism and, providing sufficient of this indirect nutrient transport processes. However, early met- nutrient was available, the cell-ball could have increased in azoans that were dependent, at least in part, upon direct size until the concentration-gradient of the next nutrient nutrient transport would have remained tiny—even smaller became the limiting factor (Fig. 3c). If there were still eco- than this . logical pressures to increase in size, then there would have In contrast to this model, it has been suggested that cal- been an advantage to developing an indirect transport pro- cisponges and silicisponges are paraphyletic, and that cess for this nutrient too (Fig. 3d). Similar selective sponges with calcareous skeletons are closer to eumetazo- pressures would have favoured the development of indirect ans. From this, it has been inferred that a water-canal system nutrient transport processes for further essential nutrients. and choanocytes are shared-primitive characteristics of A hypothesis has been outlined in which the size of the metazoans (Halanych 2004; Eernisse and Peterson 2004). organism could have changed after the development of an This would be consistent with choanoflagelates being the individual indirect nutrient transport process. As the indi- sister-group of metazoans, and their having homologous cell rect intercellular nutrient transport processes sequentially adhesion and cell communication processes. This presents a evolved, there would have been slight increases in the size very different model of the earliest metazoans as filter- of the organisms. feeders, similar in form to modern poriferans. Through these developments, the interior of these However, a recent study has challenged the long-held organisms came to contain some nutrients, but these would view that poriferans are closest to the basal form of met- have been insufficient to support a permanent population of azoans by suggesting that ctenophores were the earliest cells independent from the surrounding epithelial sheet. diverging metazoans (Dunn et al. 2008). This suggests that However, it may have been possible for cells to have vis- a diploblastic stem group split from other animals first, and ited the interior of the organism, or lived there and taken may not have shared in subsequent innovations in indirect the rest of their nutrients directly from other cells. nutrient transport. If this happened, then some of the During this series of acquisitions of indirect transport indirect nutrient transport processes of diploblasts would processes, hollow cell-balls invaginated at the point in con- not be expected to be synapomorphic with those of tri- tact with the food, which created a specialized internal ploblasts. The relatively large size, but simple form, of surface where nutrients would have been taken up into the some modern diploblasts suggests that they are not animal. So the evolution of eumetazoans is echoed by the dependent solely upon direct nutrient transport from a development of the blastula into the gastrula, as Haeckel single site of uptake. Characterisation of diploblastic suggested. Both of these forms allowed the direct transport of nutrient transport processes should help discover when they nutrients along the plane of the closed epithelial layer to all diverged from other animals. cells, whilst favouring the serial acquisition of indirect The order in which phyla diverged may be unclear at nutrient transport processes across the enclosed body-space. present but, as noted earlier, extant metazoans pass through This infolding of the cell-ball distinguished cells that spe- an embryonic phase as a hollow ball of cells. For example, cialized in digesting food and taking up nutrients, from those calcareous sponge embryos develop through a phase as a at the surface and the reproductive cells. Those indented balls hollow ball of cells which invaginates, although this may of cells were organisms with anterior–posterior axes, so cell not be true gastrulation (Larroux et al. 2006). This suggests differentiation and simple body-plans started with these that they shared an ancestor with eumetazoans which had a stages. similar form comprising of connected cells surrounding an enclosed body-space. Perhaps the body-space was sec- ondarily lost in adult poriferans whilst the cell-cell contact Corollaries and Questions that Develop was maintained after the divergence of these groups from from this Hypothesis eumetazoans. The closed epithelium around an enclosed body-space, The Earliest Metazoans had to be Small and Simple proposed in this model of the earliest metazoans, would Because they Relied upon the Direct Transport seem a better candidate for the form of the early metazoans of Nutrients than the water-canal system of poriferans. The need to directly transport nutrients between the cells surrounding a According to this model of the evolution of metazoans, the body-space was the reason that early animals were limited size of early multi-cellular animals would have been to a very small size.

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Upon the Acquisition of All Indirect Nutrient Transport acquisition of all indirect nutrient transport processes was Processes independent from the metazoan radiation. If this were the case, then we could expect some fossil evidence of the There would have been a fundamental change when a line ‘‘long fuse’’ of gradual divergence and development prior of organisms, having developed all the indirect intercellu- to the Cambrian ‘‘explosion’’. This model is consistent with lar nutrient transport processes apart from one, evolved an a relatively early divergence of phyla, which is consistent indirect transport process for this last essential nutrient. with a number of studies based upon molecular data (Blair When this final mechanism evolved, the extracellular fluid and Hedges 2005). in the interior of these organisms would have contained all Scenario 2: A number of lines independently acquired their the nutrients that cells required. This would have freed final indirect nutrient transport processes relatively those early metazoans from dependence upon the direct early. This would not be consistent with the last common transport of nutrients. Their size was no longer constrained ancestor of bilaterians having been as advanced as a small, by the length of epithelium that could be sustained by the roundish flatworm. It would have been much smaller and concentration-gradients established during the direct simpler in form, still reliant, at least in part, upon the direct transport of nutrients. At this point, providing sufficient transport of nutrients. The separate acquisition of the final food was available, the closed epithelium could have indirect nutrient transport process by a number of lines expanded greatly. Furthermore, cells could have lived and would have allowed triploblastic bodyplans to have divided enclosed within the body-space, relying upon evolved a number of times, and this could help explain the indirect nutrient transport, without needing to be linked by metazoan radiation. This timing would be consistent with cytoplasmic coupling for the direct transport of nutrients. the many studies based upon molecular data and would So the acquisition of this final indirect transport process for suggest a long fossil record of incremental development the last essential trace nutrient would have suddenly and the radiation of metazoans through the Ediacaran enabled this line to have developed dramatically larger Period. This would need a separate explanation for why animals and triploblastic body-plans. This development early animals remained so small and well hidden from the could have happened only once, or it could have occurred fossil record for so long. in a number of lines. Scenario 3: A single line acquired the final indirect nutrient transport process relatively late. This timing would be consistent with the fossil record—providing the Ediacaran When, and in How Many Separate Lines, Did Animals Fauna are not direct ancestors of modern phyla. This would Evolve the Last of the Indirect Transport Processes leave microfossils as possible ancestors of modern phyla for Essential Nutrients? from the Ediacaran period, and they may have been restricted to this size by their need to directly transport The serial acquisition of indirect nutrient transport pro- some nutrients between conjoined cells. This timing would cesses, which has been suggested here, would have challenge many studies based upon molecular data because happened towards the end of the Proterozoic Era. The it suggests that phyla diverged very late, just before the acquisition of the final indirect nutrient transport process, Cambrian ‘‘explosion’’. However, this scenario could which enabled triploblastic bodyplans and much greater explain why animals remained small for such a long time size to be developed, could have happened across a range prior to the Cambrian period, but it would require a sepa- of times: from the relatively early (before the Ediacaran rate account for how and why phyla diverged so period) to the relatively late (close to the start of the extraordinarily rapidly, and created such distinct Cambrian period). It could have happened only once, or bodyplans. separately in a number of lines. These scenarios would have left different evidence, so it should be possible to Scenario 4: A number of lines independently acquired the distinguish between them. final indirect nutrient transport processes relatively late. As stated above, a late acquisition of the final indirect Scenario 1: A single line acquired the final indirect nutrient transport process would seem to fit with an interpretation of transport process relatively early. This description would the fossil record, but might be contradicted by some studies be consistent with this line developing to form a relatively based upon molecular data. There again, the matter is complex last common ancestor of metazoans, such as the unresolved and other studies suggest a later divergence of ‘‘small, roundish flatworm’’ hypothesized by Gerhardt and phyla (Aris-Brosou and Yang 2003). Kirschner (1997). This would have left a considerable amount of time for different bodyplans to have arisen, but If a number of lines completed the set of indirect does not suggest any reason why this happened. So the nutrient transport processes then they could have gone on

123 Evol Biol (2009) 36:256–266 263 to form triploblastic bodyplans independently, and these the highest concentration that could be withstood by the could have been the stem groups of individual phyla. If this cells at the site of uptake, the lowest concentration that were the case, then we would predict a later metazoan could be tolerated by the antipodal cells and the rates of radiation would be found by studies based upon molecular metabolism and diffusion. data. We could also expect that the last indirect nutrient Indirect nutrient transport processes for individual transport processes acquired would not be homologous for essential nutrients would have been established sequen- those lines that had diverged, so these transport functions tially. The processes for indirectly transporting the first for trace nutrients could now be performed by proteins nutrients, probably glucose, amino acids and abundant from different families. ions, were likely to have been established when modern phyla would have shared a common ancestor, so the mechanisms in modern phyla should be similar. That is, Summary of Testable Predictions from this Hypothesis homologous proteins will perform the same transport About Nutrient Transport in Early Metazoans function for these substrates for all animal phyla. If further indirect transport mechanisms for the same nutrients If some cells remained conjoined as a response to a evolved later, then proteins from different families could hypothesized predator that could engulf individual cells, be found to transport the same nutrient in species from then such a selective pressure would also have had effects within the same phylum now. on other organisms. There may have been defensive It is likely that the indirect transport of trace nutrients changes that are apparent in the fossils of other groups at would have been established later. There may still have the same time as the earliest animal evolution. This may be been a common metazoan ancestor at the time at which any part of the explanation for the sudden appearance of the particular indirect transport process evolved, in which case acanthomorphic acritarchs. we would expect modern phyla to use homologous pro- The earliest multi-cellular organisms, in which some cesses. However, if any lines had already diverged then cells rely upon others for their nutrients, must be based they would have developed transport processes for sub- upon direct nutrient transport, rather than indirect nutrient sequent essential nutrients separately; therefore, the transport. This will apply to all multi-cellular organisms in transport mechanisms would not be homologous and could which some cells rely upon others for their nutrients. now be different (i.e. performed by members of different This hypothesis suggests that animals shared a multi- protein families) in animals stemming from those lines. cellular ancestor that directly transported nutrients between This could be tested empirically by establishing which cells. If this were the case then the linked cytoplasm of proteins indirectly transport essential nutrients such as early cells would be a synapomorphy for animals. These vitamins and trace elements, for all available phyla. links may have been in the form of a syncitium, gap junctions or cytoplasmic bridges. The forms of cytoplasmic links within embryos from different phyla may provide Experimental Methods for Studying the Indirect useful data for testing this idea. Nutrient Transport Processes of Different Phyla This hypothesis predicts that there was a long period when animals were small, relatively simple, hollow cell- Indirect nutrient transporter proteins can be studied by a balls (or indented cell-balls), prior to the sudden develop- variety of methods, such as heterologous expression in a ment of triploblastic forms. These later forms would have suitable system. The cDNA of individual genes can be quickly become much larger, and this development would cloned and the encoded mRNA synthesized by in vitro have coincided with a rapid increase in complexity after transcription. The mRNA can be micro-injected into large the acquisition of the last indirect nutrient transport pro- cells that have the mechanisms necessary for the synthesis cesses. This would have left a dramatic mark upon the of proteins, such as unfertilized oocytes from Xenopus la- fossil record. This is consistent with metazoan life being evis. The activity of the putative transporter proteins limited to the minute hollow forms in the Ediacaran Period, expressed by the oocytes can be assessed using radio- before the small, shelly fossils of the early Cambrian labelled nutrients in standard uptake or release assays. Period, prior to the advent of modern phyla by the mid- The intercellular transport of common nutrients is best Cambrian. understood in vertebrates. For example, the uptake of It should be possible to discover the order in which glucose by active transport from the lumen of the gut, or indirect nutrient transport processes would have been the nephron, is performed by members of the SGLT family established. The limit on the size of a cell-ball that had to of proteins; whilst the passive transport of glucose across directly transport a particular nutrient around a closed the basolateral membrane of the enterocyte, and other cells, epithelium would have been determined by a few factors: is performed by members of the GLUT family of solute

123 264 Evol Biol (2009) 36:256–266 transporters (Scheepers et al. 2004; Manolescu et al. selected for individually; without relying upon the extre- 2007). This did not have to be the case because in some mely unlikely simultaneous origin of all necessary indirect plants and bacteria members of the GLUT family translo- nutrient transport processes. cate solutes by active transport. But it is inferred from this This hypothesis emphasizes the influence of nutrient hypothesis that indirect transport processes for some sub- transport upon early metazoan evolution, but does not strates (it remains to be established which) were developed consider other factors. This model is simplified and does early, before any stem groups had diverged, so the indirect not consider fluctuations in nutrient availability or the transport of those nutrients will be performed by homo- possibility of other cell-types, besides epithelial, in early logues of those proteins in all animals today. animals. Other intrinsic factors such as the development of The uptake and intercellular transport of less abundant novel cell-types, senses and signalling molecules would essential nutrients are much less well understood. As the also have to be taken into account for a more complete proteins that transport these nutrients are identified in description of the early evolution of animals. Extrinsic vertebrates, homologues of these genes from other phyla factors such as changes in the climate, or the composition can be tested. However, it may be necessary to screen a of the sea and atmosphere, would have influenced the pace wider range of putative transporter proteins to identify and timing of the advent of metazoans. Interactions with those responsible for the indirect transport of trace nutri- other organisms would also have been important. For ents in other phyla. example, the first line of animals which established all the indirect transport mechanisms would have begun the first macroscopic fauna. Any subsequent lines that established Discussion their last remaining nutrient transport processes afterwards would have had to grow up in the environment and ecology Fundamental differences between the nutrient transport created and dominated by that first line. The first line processes of unicellular and multi-cellular organisms have would have been the dominant early fauna, and the fossil been described. The importance of the processes that dis- record from the Cambrian suggests that this could have tribute nutrients between the cells of animals has been been the stem group that lead to arthropods (Xian-Guang emphasized. This created a puzzle about how unicellular et al. 2003). organisms could have evolved into the first multi-cellular Gould (1989) suggested that if we could ‘‘replay the animals, which had some cells that could not get their tape’’ of the early evolution of animals then we could nutrients from the environment; a development that discover the contingent events in this natural history. Those required fundamental changes in their nutrient transport events which were not necessarily so might be different in processes. subsequent replays. A similar argument might be applied to A model of the intercellular movement of nutrients in the acquisition of indirect nutrient transport processes, and the earliest animals has been developed. A distinction has the metazoan radiation. If some stem-groups had diverged been drawn between the direct and indirect transport of before those simple forms developed the last of their nutrients between animal cells. It has been inferred that indirect nutrient transport mechanisms, then lines might, in small, relatively simple multi-cellular organisms could effect, have ‘‘replayed’’ the development from early simple have evolved which relied upon a direct nutrient transport multi-cellular animals into triploblastic forms. This would mechanism. This hypothesis, and here it overlaps the the- have given a number of chances for simple multi-cellular ory of Haeckel (1874), proposes early animal forms which organisms to have evolved into triploblastic animals inde- are still echoed in the development of modern embryos. pendently, which might account for the wide range of The similarities between early animal embryos, and the body-plans across phyla. early formation of closed epithelia, suggest that similar Contingency might be seen in the acquisition of the last structures were possessed by a common ancestor shared by of the indirect nutrient transport processes. In mammals, animals. I propose that early multi-cellular organisms were some less abundant nutrients are indirectly transported by limited to their small size and simple forms by the need to proteins of families that had initially been associated with transport nutrients directly between conjoined cells, rather the transport of other substrates. For example, iodide is than these forms being determined by Haeckel’s biogenetic transported by a member of the SGLT Na?/glucose co- law. transporter family (Dai et al. 1996), whilst biotin is trans- The model was then developed to explain how the body- ported by another member of this family (Prasad et al. space enclosed by the epithelium favoured the sequential 1996). This suggests that there has been adaptability of the development of indirect nutrient transport processes, whilst substrate-specificity of some transporter proteins. Genes enabling the direct transport of nutrients. This explains how encoding transporter proteins have been duplicated in indirect nutrient transport processes could have been animal genomes, and this presented opportunities for some

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