Phylogenomics and Morphological Reconstruction of Arcellinida Testate Amoebae Highlight Diversity of Microbial Eukaryotes in the Neoproterozoic

Life was microbial for the majority of Earth's history, but as very few microbial lineages leave a fossil record, the Precambrian evolution of life remains shrouded in mystery. Shelled (testate) amoebae stand out as an exception with rich documented diversity in the Neoproterozoic as vase-shaped microfossils (VSMs). While there is general consensus that most of these can be attributed to the Arcellinida lineage in Amoebozoa, it is still unclear whether they can be used as key fossils for interpretation of early eukaryotic evolution. Here, we present a well-resolved phylogenomic reconstruction based on 250 genes, obtained using single-cell transcriptomic techniques from a representative selection of 19 Arcellinid testate amoeba taxa. The robust phylogenetic framework enables deeper interpretations of evolution in this lineage and demanded an updated classification of the group. Additionally, we performed reconstruction of ancestral morphologies, yielding hypothetical ancestors remarkably similar to existing Neoproterozoic VSMs. We demonstrate that major lineages of testate amoebae were already diversified before the Sturtian glaciation (720 mya), supporting the hypothesis that massive eukaryotic diversification took place in the early Neoproterozoic and congruent with the interpretation that VSM are arcellinid testate amoebae.Arcellinid testate amoebae are some of the earliest eukaryotic microbes. Lahr et al. show that these amoebae have seven major deep phylogenetic lineages, probably diverged as early as 730 mya. Diversification of these microbes in the mid-Neoproterozoic was probably happening alongside the gradual oxygenation of oceans

Agglutinated tests in post-Sturtian cap carbonates of Namibia and Mongolia

Paleomagnetic data suggest that the early Cryogenian (Sturtian) glaciation extended to sea level at low latitude. The impact of this dramatic environmental change on biota, and the composition of ecosystems in the immediate aftermath of the Sturtian glaciation remain virtually unknown. Here we report the discovery of abundant agglutinated tests in organic-rich carbonates directly overlying Sturtian glacial deposits from two different paleocontinents: the Rasthof Formation of the Congo craton in northern Namibia and the Tsagaan Oloom Formation of the Dzabkhan terrane in Mongolia. The most abundant tests preserve morphological and compositional characters consistent with those found in at least two different families of modern lobose testate amoebae (Amoebozoa), a group of heterotrophic microbial eukaryotes. The presence of spatially and compositionally variable clay minerals, quartz and microcline on the test walls is a signature of widespread biological agglutination. The post-glacial fossil assemblages differ from the most common pre-Sturtian vaseshaped fossil testate amoebae, perhaps as a result of different preservational mechanisms or of the appearance of new forms after the glaciation. The apparent local abundance of eukaryotic body fossils in the post-Sturtian carbonates suggests that the Cryogenian limestones and dolostones may host a currently unexplored fossil record of modern eukaryotes.Earth and Planetary Science

Molecular investigation of Phryganella acropodia Hertwig et Lesser, 1874 (Arcellinida, Amoebozoa)

Phryganella acropodia Hertwig and Lesser, 1874, is one of the most common and abundant testate amoeba species. It represents the type species of the genus Phryganella Penard, 1902, which in turn is the type genus for the suborder Phryganellina (Arcellinida) Bovee, 1985, but despite its taxonomic importance it was not yet analyzed with molecular methods. We established two cultures of putative Phryganella acropodia, designed Phryganellina-specific primers, amplified SSU rDNA data and subjected these sequences to phylogenetic analyses. Morphological and genetic differences were found between both strains. With SSU rDNA phylogenetic analyses we confirm that Phryganella acropodia branches with Phryganella paradoxa Penard, 1902 and Cryptodifflugia Penard, 1890 in the Phryganellina. We thus give further evidence that pseudopodia morphology in the Arcellinida is a character of high taxonomic value, as suggested by Bovee and Jung when erecting the suborder Phryganellina. Moreover, we provide evidence for cryptic diversity and for the first time confirm the existence of a naked life stage in Arcellinida by molecular means

Phylogenetic divergence within the Arcellinida (Amoebozoa) is congruent with test size and metabolism type

Arcellinida (lobose testate amoebae) are abundant and diverse in many ecosystems, especially in moist to aquatic environments. Molecular phylogeny has shown that overall test morphology (e.g., spherical or elongate) is generally conserved in Arcellinida lineages, but the taxonomic value of other traits (e.g., size, ornamentation, mixotrophy/heterotrophy metabolism type) has not been systematically evaluated. Morphological and physiological traits that correspond to genetic differences likely represent adaptive traits of ecological significance. We combined high-resolution phylogenetics (NAD9-NAD7 genes) and advanced morphometrics to assess the phylogenetic signal of morphological traits of a group of elongate Difflugia species (Arcellinida). The phylogenetic analyses revealed two clades which could be reliably separated by test size and the presence/absence of mixotrophy. Differences in test size may reflect trophic level, with smaller organisms occupying lower trophic levels. In addition to having larger tests, elongate mixotrophic Difflugia are characterised by wide, flat bases and an inflation of the lower two thirds of their test. These morphological traits may provide additional volume for endosymbionts and/or increased surface area to aid light transmission. Our results showcase greater diversity within the elongate Difflugia and highlight morphological traits of ecological and evolutionary significance

Population and molecular responses to warming in Netzelia tuberspinifera – An endemic and sensitive protist from East Asia

With the effects of global warming becoming ever more obvious, biodiversity conservation is facing severe challenges. Currently, a deeper understanding the mechanisms of the effects of warming on sensitive species has become an important topic in aquatic biodiversity and ecological management. Our study first overcame the “challenge” for a sensitive indicator species (Netzelia tuberspinifera, an endemic testate amoeba species in East Asia) of culturing under laboratory conditions, and then explored its molecular response mechanisms to warming using transcriptomic analysis. Our data indicate that temperature mainly drove the geographical and seasonal variation of N. tuberspinifera populations. Transcriptomic results indicate that when the temperature is 25 °C, it triggers molecular processes related with cell division, test formation and general biomass increase. However, once the temperature exceeds 40 °C, N. tuberspinifera is unable to survive. Following from these results, the distribution of N. tuberspinifera might expand towards higher altitude or latitude regions under global warming. For the first time, our study showed direct evidence for sensitive protozoa species that presents a very narrow adaptation mechanism to local climate. Our work provides fundamental data for regional biodiversity conservation and scientific reference in subtropical and tropical waterbodies

Quadrulella texcalense sp. nov. from a Mexican desert: An unexpected new environment for hyalospheniid testate amoebae

Quadrulella (Amoebozoa, Arcellinida, Hyalospheniidae) is a genus of testate amoebae with unmistakable morphology, which secretes characteristic square plates to reinforce the test. They are mainly known from fens and freshwater habitats and have never been documented in deserts. We describe a new species, Quadrulella texcalense, from biological soil crusts in the intertropical desert of Tehuacán (state of Puebla, Mexico). Quadrulella texcalense occurred only at altitudes between 2140 and 2221 m.a.s.l., together with the bryophyte genera Pseudocrossidium, Weissia, Bryum, Didymodon, Neohyophyla and Aloina. The soil was extremely dry (moisture of 1.97–2.6%), which contrasts sharply with previous reports for the Quadrulella genus. Single cell mitochondrial cytochrome oxidase I (COI) barcoding of thirteen isolated cells showed an important morphological variability despite having all the same COI barcode sequence. Quadrulella texcalense was placed in a tree containing other Hyalsopheniidae, including a newly barcoded South African species, Q. elegans. Q. texcalense unambiguously branched within genus Quadrulella in a compact clade but with a long branch, suggesting accelerated evolution due to a transition towards a new environment and/or under-sampling

Diverse vase-shaped microfossils within a Cryogenian glacial setting in the Urucum Formation (Brazil)

International audienceVase-shaped microfossils (VSMs) attributed to testate amoebae occur globally in diverse assemblages in Tonian rocks. These microfossils have thus been considered a potential biostratigraphic tool, especially for the interval between 789 and 729 Ma. Here we report a diverse and well-preserved in situ VSM assemblage, including several taxa previously considered as Tonian, within glacially influenced deposits for which sedimentological data support a Cryogenian age. However, the more robust recent multi-proxy correlation proposed by Freitas et al. (2021) indicates a Marinoan age for the studied succession. Detrital zircon data provide a maximum depositional weighted mean age of 749 ± 3 Ma for the VSM-bearing, organic-rich, fine-grained deposits within the Marinoan sequence in the Urucum Formation. Nine taxa are described from the fine-grained deposits in the upper Urucum Formation, Jacadigo Group, Brazil: Cycliocyrillium simplex, Bonniea dacruchares, Bonniea pytinaia, Bombycion micron, Limeta lageniformis, Palaeoarcella athanata, Trigonocyrillium horodyskii, Pakupaku kabin and cf. Taruma rata. The discovery of well-preserved in situ VSMs attributable to specific Tonian taxa within a Cryogenian succession challenges previous thinking that these organisms disappeared from marine ecosystems at the end of the Tonian

Testate Amoeba Functional Traits and Their Use in Paleoecology

This review provides a synthesis of current knowledge on the morphological and functional traits of testate amoebae, a polyphyletic group of protists commonly used as proxies of past hydrological changes in paleoecological investigations from peatland, lake sediment and soil archives. A trait-based approach to understanding testate amoebae ecology and paleoecology has gained in popularity in recent years, with research showing that morphological characteristics provide complementary information to the commonly used environmental inferences based on testate amoeba (morpho-)species data. We provide a broad overview of testate amoeba morphological and functional traits and trait-environment relationships in the context of ecology, evolution, genetics, biogeography, and paleoecology. As examples we report upon previous ecological and paleoecological studies that used trait-based approaches, and describe key testate amoebae traits that can be used to improve the interpretation of environmental studies. We also highlight knowledge gaps and speculate on potential future directions for the application of trait-based approaches in testate amoeba research