Early Metazoan Research Articles

Expansion microscopy in Placozoa: improving resolution and preservation of fragile samples during marine expedition

Placozoa are small disc-shaped animals representing one of the early branching metazoan lineages with only a dozen cell types, fast effector reactions, and complex behaviors. The simplest organization and small cell sizes limit standard microscopy applications. Here, we implement a new methodology and protocol for expansion microscopy, improving both the resolution and preservation of fragile placozoans and kin. As a result, the proposed approaches can be applicable to a diversity of microscopic animals and their larvae with broad applicability for both laboratory and cross-disciplinary field research during long-term marine expeditions.

Low oxygen but dynamic marine redox conditions permitted the Cambrian Radiation.

Whether metazoan diversification during the Cambrian Radiation was driven by increased marine oxygenation remains highly debated. Repeated global oceanic oxygenation events have been inferred during this interval, but the degree of shallow marine oxygenation and its relationship to biodiversification and clade appearance remain uncertain. To resolve this, we interrogate an interval from ~527 to 519 Ma, encompassing multiple proposed global oceanic oxygenation events. We integrate the spatial and temporal distribution of shallow water, in situ reef metazoans, and trilobites, with high-resolution multi-proxy redox data through the highly biodiverse Siberian Platform. We document primarily dysoxic water column conditions, suggesting that early Cambrian metazoans, including motile skeletal benthos, had low oxygen demands. We further document oxygenation events coincident with positive carbon isotope excursions that led to modestly elevated oxygen levels. These events correspond to regional increases in species richness and habitat expansion of mainly endemic species, offering a potentially globally applicable model for biodiversification during the Cambrian Radiation.

Tolerance of Placozoa for temperate climates: Evidence for known and new placozoan clades in the southern waters of Australia.

Placozoans are small multicellular sea-dwelling animals that are typically found in shallow, warm ocean waters and have been reported in various marine environments worldwide. Their unique morphology makes them a powerful new model organism to study the evolutionary cell biology in early metazoans. Yet, knowledge on their biodiversity and ecological distribution is severely limited. Here, we report the isolation of placozoans in the temperate waters of Victoria, Australia, their most southern location known to date. Using light, electron, and confocal microscopy, we describe their morphology and behaviour. In addition to the known haplotypes H2 and H17, we have identified a new haplotype, here designated as H20, which defines a new placozoan clade. This study provides novel insights into the distribution, ecological niche separation and genetic diversity of placozoans, and reports the first morphological and ultrastructural characterisation of placozoan clades isolated from the southern waters of Victoria, Australia.

A transphyletic study of metazoan β-catenin protein complexes

Beta-catenin is essential for diverse biological processes, such as body axis determination and cell differentiation, during metazoan embryonic development. Beta-catenin is thought to exert such functions through complexes formed with various proteins. Although β-catenin complex proteins have been identified in several bilaterians, little is known about the structural and functional properties of β-catenin complexes in early metazoan evolution. In the present study, we performed a comparative analysis of β-catenin sequences in nonbilaterian lineages that diverged early in metazoan evolution. We also carried out transphyletic function experiments with β-catenin from nonbilaterian metazoans using developing Xenopus embryos, including secondary axis induction in embryos and proteomic analysis of β-catenin protein complexes. Comparative functional analysis of nonbilaterian β-catenins demonstrated sequence characteristics important for β-catenin functions, and the deep origin and evolutionary conservation of the cadherin–catenin complex. Proteins that co-immunoprecipitated with β-catenin included several proteins conserved among metazoans. These data provide new insights into the conserved repertoire of β-catenin complexes.

Transport of ‘Nama’‐type biota in sediment gravity and combined flows: Implications for terminal Ediacaran palaeoecology

ABSTRACTThe lower Nama Group in southern Namibia contains trace fossils and soft‐bodied and biomineralized macro‐organisms from the terminal Ediacaran Period (ca 550 to 539 Ma), offering insights into early metazoan evolution. Interpretation of the fossilized Nama Group organisms as being preserved in, or very close to, the environments in which they originally lived has yielded insights into organism feeding habits, reproduction and life histories. Sedimentological evidence presented here reveals that typical ‘Nama‐type’ Ediacaran macro‐fossils (Cloudina, Ernietta, Pteridinium and Rangea) in the Dabis and Zaris formations of the Witputz Sub‐basin seldom preserve organisms in life position in their original palaeoenvironments. Both soft‐bodied and biomineralizing organisms were transported in sediment gravity flows (debris flows, turbidity flows and transitional debris flow‐turbidity flow ‘hybrid’ event beds) or combined flow (hummocky cross‐strata) to their terminal environment of deposition in shoreface and offshore shelf settings. Transport has placed studied beds and their associated macro‐organisms in depositional settings detached from the original life habitat, with macro‐organisms sourced from shallower‐water, up‐slope environments. Integrated sedimentological and palaeontological data indicate that the Nama Group may not provide a high‐fidelity record of original Nama ecosystems. Individual macro‐organisms are clasts within beds, and can be horizontal, imbricated or chaotic in orientation. Transport can blend different communities at various scales (bed, outcrop and basin), complicating interpretations of life habitats, species interactions and taxon‐specific ecology, such as feeding behaviour and life position of organisms. Recognition of organism transport also impacts datasets used for comparing global Ediacaran fossil assemblages, with implications for tracking spatial and temporal patterns in early animal evolution.

Evolution and palaeoenvironmental migration of a Cambrian undermat miner

Evolution and palaeoenvironmental migration of a Cambrian undermat miner

Coordinated cellular behavior regulated by epinephrine neurotransmitters in the nerveless placozoa

Understanding how cells communicated before the evolution of nervous systems in early metazoans is key to unraveling the origins of multicellular life. We focused on Trichoplax adhaerens, one of the earliest multicellular animals, to explore this question. Through screening a small compound library targeting G protein-coupled receptors (GPCRs), we found that Trichoplax exhibits distinctive rotational movements when exposed to epinephrine. Further studies suggested that, akin to those in humans, this basal organism also utilizes adrenergic signals to regulate its negative taxis behavior, with the downstream signaling pathway being more straightforward and efficient. Mechanistically, the binding of ligands activates downstream calcium signaling, subsequently modulating ciliary redox signals. This process ultimately regulates the beating direction of cilia, governing the coordinated movement of the organism. Our findings not only highlight the enduring presence of adrenergic signaling in stress responses during evolution but also underscore the importance of early metazoan expansion of GPCR families. This amplification empowers us with the ability to sense external cues and modulate cellular communication effectively.

Multiple Non-Destructive Approaches to Analysis of the Early Silurian Chain Coral Halysites from South China.

Cnidarians are among the most important diploblastic organisms, elucidating many of the early stages of Metazoan evolution. However, Cnidarian fossils from Cambrian deposits have been rarely documented, mainly due to difficulties in identifying early Cnidarian representatives. Halysites, a tabulate coral from Silurian reef systems, serves as a crucial taxon for interpreting Cambrian cnidarians. Traditionally, the biological characteristics of Halysites have been analyzed using methods limited by pretreatment requirements (destructive testing) and the chamber size capacity of relevant analytical instruments. These constraints often lead to irreversible information loss and inadequate data extraction. This means that, to date, there has been no high-resolution three-dimensional mineralization analysis of Halysites. This study aims to introduce novel, non-destructive techniques to analyze the internal structure and chemical composition of Halysites. Furthermore, it seeks to elucidate the relationship between coral organisms and biomineralization in reef settings and to compare Silurian Tabulata with putative Cambrian cnidarians. Techniques such as micro-X-ray fluorescence spectrometry (micro-XRF), micro-X-ray computed tomography (micro-CT), and scanning electron microscopy (SEM) were employed in this research. With the help of high-resolution micro-CT scanning, we identify the growth pattern of Halysites, showing both lateral and vertical development. The lateral multiple-branching growth pattern of Halysites corals is first established herein. The flaggy corallite at the initial stage of branching is also observed. The micro-XRF mapping results reveal the occurrence of septa spines for Halysites, a trait previously thought rare or absent. Additionally, the ratio of coral volume to the surrounding rock was assessed, revealing that Halysites reefs were relatively sparse (volume ratio = ~30%). The cavities between Halysites likely provided more space for other organisms (e.g., rugose corals and bryozoans) when compared to other coral reef types. Additionally, we provide a comparative analysis of post-Cambrian colonial calcareous skeletons, offering insights into the structural features and growth patterns of early skeletal metazoans across the Ediacaran-Cambrian boundary.

Sea level controls on Ediacaran-Cambrian animal radiations.

The drivers of Ediacaran-Cambrian metazoan radiations remain unclear, as does the fidelity of the record. We use a global age framework [580-510 million years (Ma) ago] to estimate changes in marine sedimentary rock volume and area, reconstructed biodiversity (mean genus richness), and sampling intensity, integrated with carbonate carbon isotopes (δ13Ccarb) and global redox data [carbonate Uranium isotopes (δ238Ucarb)]. Sampling intensity correlates with overall mean reconstructed biodiversity >535 Ma ago, while second-order (~10-80 Ma) global transgressive-regressive cycles controlled the distribution of different marine sedimentary rocks. The temporal distribution of the Avalon assemblage is partly controlled by the temporally and spatially limited record of deep-marine siliciclastic rocks. Each successive rise of metazoan morphogroups that define the Avalon, White Sea, and Cambrian assemblages appears to coincide with global shallow marine oxygenation events at δ13Ccarb maxima, which precede major sea level transgressions. While the record of biodiversity is biased, early metazoan radiations and oxygenation events are linked to major sea level cycles.

Shallow-water redox evolution from the Ediacaran to the early Cambrian: Linkages to the early animal innovations

Shallow-water redox evolution from the Ediacaran to the early Cambrian: Linkages to the early animal innovations

Cyanobacterial and fungi-like microbial fossils from the earliest Cambrian phosphorite of South China

Cyanobacterial and fungi-like microbial fossils from the earliest Cambrian phosphorite of South China

Evolution and extinction in a supercontinental world: did the breakup of Rodinia provide metazoans with evolutionary salvation?

A time discrepancy of at least 200–300 million years exists between the generally accepted onset of metazoan evolution as currently evidenced from fossils compared with that from studies at the molecular level. That temporal disparity coincides with the existence and subsequent breakup of the Rodinia Supercontinent when the earliest evolutionary crucibles were isolated intracratonic basins rather than the globally connected shallow seaways of subsequent eras. However, the discovery of fossil evidence to complement the extrapolations from molecular data has been, and continues to be, hampered, by both the geographical limitations of these isolated intracratonic basins and the fact that any such discovery would conflict with the global geological mindset that macrofossils of such age do not exist. Yet recently identified within the intracratonic Amadeus Basin of central Australia is a suite of macroscopic metazoan fossils that dates to ca 850–840 Ma, exemplifying an early attempt at animal evolution within the restrictions of the Rodinia Supercontinent. Such early metazoan evolution within such isolated crucibles, however, was extremely vulnerable to variations in climatic conditions, which threatened total extinction because the isolation of these basins limited metazoan distribution by denying them the possibility of migrating to safer havens. The Amadeus Basin evidence suggests that early metazoan evolution perhaps comprised cycles of about 10 million years when evolution flourished followed by climate change induced extinction and a subsequent evolutionary void of between 50 and 100 million years. Such cycles possibly characterised metazoan evolution until the Rodinia Supercontinent broke-up, after which connections between smaller oceans, shallow seas and narrow waterways provided metazoan life passageway to sanctuary during times of environmental stress. As the Amadeus Basin was just one of a number of isolated Rodinian-aged intracratonic basins, it is likely that similar fossils of the earliest metazoans are preserved elsewhere awaiting discovery.

Morphology shapes community dynamics in early animal ecosystems

The driving forces behind the evolution of early metazoans are not well understood, but key insights into their ecology and evolution can be gained through ecological analyses of the in situ, sessile communities of the Avalon assemblage in the Ediacaran (~565 million years ago). Community structure in the Avalon is thought to be underpinned by epifaunal tiering and ecological succession, which we investigate in this study in 18 Avalon communities. Here we found that Avalon communities form four distinctive Community Types irrespective of succession processes, which are instead based on the dominance of morphologically distinct taxa, and that tiering is prevalent in three of these Community Types. Our results are consistent with emergent neutrality, whereby ecologically specialized morphologies evolve as a consequence of neutral (stochastic or reproductive) processes within niches, leading to generalization within the frond-dominated Community Type. Our results provide an ecological signature of the first origination and subsequent loss of disparate morphologies, probably as a consequence of community restructuring in response to ecological innovation. This restructuring led to the survival of non-tiered frondose generalists over tiered specialists, even into the youngest Ediacaran assemblages. Such frondose body plans also survive beyond the Ediacaran–Cambrian transition, perhaps due to the greater resilience afforded to them by their alternative ecological strategies.

Adaptations to changing substrates in diploblastic dinomischids from the early Cambrian

The significant changes in seafloor conditions during the Ediacaran-Cambrian transition presumably bear on the autecology of the benthic animals living in this time interval. However, little evidence shows morphological modifications and life strategy transitions in early animals in response to this substrate revolution. Dinomischidae is a family of diploblastic-grade with a sessile, suspension-feeding lifestyle, which includes Xianguangia, Daihua and Dinomischus. Here, we redescribe Calathites spinalis, a poorly known taxon from the early Cambrian (Epoch 2, Age 3, ∼518 Ma), as a new member of this family. Our new material reveals that the body of C. spinalis consists of a stalked calyx and 18 tentacle-sheath complexes, which is a typical trait of dinomischids. Our phylogenetic analyses recover Calathites as a sister group of Xianguangia and Daihua and support dinomischids being a monophyletic group. Members of dinomischids possess unique attachment structures that vary in shape and size, presenting a unique example of morphological adaptations of early sessile animals during the Cambrian substrate revolution. Comparison with early metazoan fossils reveals that dinomischids and other early basal metazoans lagged behind sessile bilaterians in terms of adaptations to substrate changes; the former tends to persist with attachment strategies adapted to Proterozoic-style seafloor conditions that were relatively firm.

‘Conga lines’ of Ediacaran fronds: insights into the reproductive biology of early metazoans

Late Ediacaran strata from Newfoundland, Canada (~574–560 Ma) document near-census palaeocommunities of some of the earliest metazoans. Such preservation enables reproductive strategies to be inferred from the spatial distribution of populations of fossilized benthic organisms, previously revealing the existence of both propagule and stoloniferous reproductive modes among Ediacaran frondose taxa. Here, we describe ‘conga lines’: linear arrangements of more than three closely spaced fossil specimens. We calculate probabilistic models of point maps of 13 fossil-bearing bedding surfaces and show that four surfaces contain conga lines that are not the result of chance alignments. We then test whether these features could result from passive pelagic propagules settling in the lee of an existing frond, using computational fluid dynamics and discrete phase modelling. Under Ediacaran palaeoenvironmental conditions, preferential leeside settlement at the spatial scale of the conga lines is unlikely. We therefore conclude that these features are novel and do not reflect previously described reproductive strategies employed by Ediacaran organisms, suggesting the use of mixed reproductive strategies in the earliest animals. Such strategies enabled Ediacaran frondose taxa to act as reproductive generalists and may be an important facet of early metazoan evolution.

Conserved Genes in Highly Regenerative Metazoans Are Associated with Planarian Regeneration.

Metazoan species depict a wide spectrum of regeneration ability which calls into question the evolutionary origins of the underlying processes. Since species with high regeneration ability are widely distributed throughout metazoans, there is a possibility that the metazoan ancestor had an underlying common molecular mechanism. Early metazoans like sponges possess high regenerative ability, but, due to the large differences they have with Cnidaria and Bilateria regarding symmetry and neuronal systems, it can be inferred that this regenerative ability is different. We hypothesized that the last common ancestor of Cnidaria and Bilateria possessed remarkable regenerative ability which was lost during evolution. We separated Cnidaria and Bilateria into three classes possessing whole-body regenerating, high regenerative ability, and low regenerative ability. Using a multiway BLAST and gene phylogeny approach, we identified genes conserved in whole-body regenerating species and lost in low regenerative ability species and labeled them Cnidaria and Bilaterian regeneration genes. Through transcription factor analysis, we identified that Cnidaria and Bilaterian regeneration genes were associated with an overabundance of homeodomain regulatory elements. RNA interference of Cnidaria and Bilaterian regeneration genes resulted in loss of regeneration phenotype for HRJDa, HRJDb, DUF21, DISP3, and ARMR genes. We observed that DUF21 knockdown was highly lethal in the early stages of regeneration indicating a potential role in wound response. Also, HRJDa, HRJDb, DISP3, and ARMR knockdown showed loss of regeneration phenotype after second amputation. The results strongly correlate with their respective RNA-seq profiles. We propose that Cnidaria and Bilaterian regeneration genes play a major role in regeneration across highly regenerative Cnidaria and Bilateria.

How studies in developmental epithelial-mesenchymal transition and mesenchymal-epithelial transition inspired new research paradigms in biomedicine.

Epithelial-mesenchymal transition (EMT) and its reverse mechanism, mesenchymal-epithelial transition (MET), are evolutionarily conserved mechanisms initially identified in studies of early metazoan development. EMT may even have been established in choanoflagellates, the closest unicellular relative of Metazoa. These crucial morphological transitions operate during body plan formation and subsequently in organogenesis. These findings have prompted an increasing number of investigators in biomedicine to assess the importance of such mechanisms that drive epithelial cell plasticity in multiple diseases associated with congenital disabilities and fibrosis, and, most importantly, in the progression of carcinoma. EMT and MET also play crucial roles in regenerative medicine, notably by contributing epigenetic changes in somatic cells to initiate reprogramming into stem cells and their subsequent differentiation into distinct lineages.

Neuromuscular organization of the benthic ctenophore, Vallicula multiformis

Ctenophora is the earliest metazoan taxon with neurons and muscles. Recent studies have described genetic, physiological, and cellular characteristics of the neural and muscular systems of this phylogenically important lineage. However, despite the ecological diversity of ctenophore niches, including both pelagic and benthic forms, studies have focused predominantly on pelagic species. In the present study, we describe the neural and muscular architectures of the benthic ctenophore, Vallicula multiformis (Order Platyctenida), employing immunohistochemical analysis using antibodies against amidated neuropeptides with the C-terminal sequences VWYa, NPWa, FGLa, or WTGa to compare it to pelagic species. In V. multiformis, which lacks the characteristic comb rows seen in pelagic ctenophores, neural structures that develop beneath the comb were not detected, whereas the subepithelial and tentacle neural networks showed considerable similarity to those of pelagic species. Despite significant differences in morphology and lifestyle, muscle organization in V. multiformis closely resembles that of pelagic species. Detailed analysis of neurons that express these peptides unveiled a neural architecture composed of various neural subtypes. This included widely distributed subepithelial neural networks (SNNs) and neurosecretory cells located primarily in the peripheral region. The consistent distribution patterns of the VWYa-positive SNN and tentacle nerves between V. multiformis and the pelagic species, Bolinopsis mikado, suggest evolutionarily conserved function of these neurons in the Ctenophora. In contrast, NPWa-positive neurons, which extend neurites connecting the apical organ and comb rows in B. mikado, showed a neurite-less neurosecretory cell morphology in this flattened, sessile species. Evaluation of characteristics and variations in neural and muscular architectures shared by benthic and pelagic ctenophore species may yield valuable insights for unraveling the biology of this rapidly evolving yet enigmatic metazoan lineage. These findings also provide important insight into neural control modalities in early metazoan evolution.

Illustrated Neuroanatomy of Ctenophores: Immunohistochemistry.

Ctenophores or comb jellies are representatives of an enigmatic lineage of early branching metazoans with complex tissue and organ organization. Their biology and even microanatomy are not well known for most of these fragile pelagic and deep-water species. Here, we present immunohistochemical protocols successfully tested on more than a dozen ctenophores. This chapter also illustrates neural organization in several reference species of the phylum (Pleurobrachia bachei, P. pileus, Mnemiopsis leidyi, Bolinopsis microptera, Beroe ovata, and B. abyssicola) as well as numerous ciliated structures in different functional systems. The applications of these protocols illuminate a very complex diversification of cell types comparable to many bilaterian lineages.

Authigenic clay mineral constraints on spatiotemporal evolution of restricted, evaporitic conditions during deposition of the Ediacaran Doushantuo Formation

The carbonate-rich shelf facies of the Ediacaran Doushantuo Formation (South China) have produced a broad array of microfossil and isotope proxy records which underpin much of our understanding of environmental change and biospheric evolution during this key time period. Recent reports of locally abundant authigenic, Mg-rich saponite clay in the Yangtze Gorges Area hint at potentially widespread evaporitic conditions in a lagoonal setting. Despite the implications for interpretation of proxy records and the environmental setting of early metazoans, the spatiotemporal extent of restricted, evaporitic conditions across the Yangtze Block remains largely unexplored. Here we use mineralogical and petrographic techniques to document the spatial and stratigraphic distribution of authigenic clay minerals across seven sites, representing a transect from shallow proximal shelf to deep basinal settings. Our results demonstrate the widespread and persistent occurrence of authigenic saponite in proximal shelf settings, whereas talc is identified at more distal shelf sites, consistent with Mg-clay authigenesis in an evaporitic lagoon with spatially variable detrital aluminosilicate input. Slope and basinal sites contain no Mg-clays and are instead characterized by abundant authigenic illite, consistent with an open marine setting. Stratigraphically, illite was found in the basal cap carbonate and uppermost Doushantuo Formation, with Mg-clays only present in the interval in-between, suggesting gradual development of an extensive carbonate rimmed lagoon over the Yangtze shelf that restricted seawater exchange, followed by stronger marine influence due to erosion of the carbonate rim and subsequent marine transgression. We conclude that restricted, evaporitic conditions were much more expansive than previously assumed, potentially providing favorable conditions for the Doushantuo Biota. Further, the widespread presence of authigenic clays across the shelf supports the case for enhanced reverse weathering before the advent of biosilicification.