Expansion of the cytochrome C oxidase subunit I database and description of four new lobose testate amoebae species (Amoebozoa; Arcellinida).

Arcellinida is ascending in importance in protistology, but description of their diversity still presents multiple challenges. Furthermore, applicable tools for surveillance of these organisms are still in developing stages. Importantly, a good database that sets a correspondence between molecular barcodes and species morphology is lacking. Cytochrome oxidase (COI) has been suggested as the most relevant marker for species discrimination in Arcellinida. However, some major groups of Arcellinida are still lacking a COI sequence. Here we expand the database of COI marker sequences for Arcellinids, using single-cell PCR, transcriptomics, and database scavenging. In the present work, we added 24 new Arcellinida COI sequences to the database, covering all unsampled infra- and suborders. Additionally, we added six new SSUrRNA sequences and described four new species using morphological, morphometrical, and molecular evidence: Heleopera steppica, Centropyxis blatta, Arcella uspiensis, and Cylindrifflugia periurbana. This new database will provide a new starting point to address new research questions from shell evolution, biogeography, and systematics of arcellinids.

When ecological transitions are not so infrequent: independent colonizations of athalassohaline water bodies by Arcellidae (Arcellinida; Amoebozoa), with descriptions of four new species.

The salinity and humidity barriers divide biodiversity and strongly influence the distribution of organisms. Crossing them opens the possibility for organisms to colonize new niches and diversify, but requires profound physiological adaptations and is supposed to happen rarely in evolutionary history. We tested the relative importance of each ecological barrier by building the phylogeny, based on mitochondrial cytochrome oxidase gene (COI) sequences, of a group of microorganisms common in freshwater and soils, the Arcellidae (Arcellinida; Amoebozoa). We explored the biodiversity of this family in the sediments of athalassohaline water bodies (i.e. of fluctuating salinity that have non-marine origins). We found three new aquatic species, which represent, to the best of our knowledge, the first reports of Arcellinida in these salt-impacted ecosystems, plus a fourth terrestrial one in bryophytes. Culturing experiments performed on Arcella euryhalina sp. nov. showed similar growth curves in pure freshwater and under 20 g/L salinity, and long-term survival at 50 g/L, displaying a halotolerant biology. Phylogenetic analyses showed that all three new athalassohaline species represent independent transition events through the salinity barrier by freshwater ancestor, in contrast to the terrestrial species, which are monophyletic and represent a unique ecological transition from freshwater to soil environments.

A needle in a haystack: A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea).

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost-effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies.

Deconstructing Difflugia: The tangled evolution of lobose testate amoebae shells (Amoebozoa: Arcellinida) illustrates the importance of convergent evolution in protist phylogeny.

Protists, the micro-eukaryotes that are neither plants, animals nor fungi build up the greatest part of eukaryotic diversity on Earth. Yet, their evolutionary histories and patterns are still mostly ignored, and their complexity overlooked. Protists are often assumed to keep stable morphologies for long periods of time (morphological stasis). In this work, we test this paradigm taking Arcellinida testate amoebae as a model. We build a taxon-rich phylogeny based on two mitochondrial (COI and NADH) and one nuclear (SSU) gene, and reconstruct morphological evolution among clades. In addition, we prove the existence of mitochondrial mRNA editing for the COI gene. The trees show a lack of conservatism of shell outlines within the main clades, as well as a widespread occurrence of morphological convergences between far-related taxa. Our results refute, therefore, a widespread morphological stasis, which may be an artefact resulting from low taxon coverage. As a corollary, we also revise the groups systematics, notably by emending the large and highly polyphyletic genus Difflugia. These results lead, amongst others, to the erection of a new infraorder Cylindrothecina, as well as two new genera Cylindrifflugia and Golemanskia.

Cyphoderia ampulla (Cyphoderiidae: Rhizaria), a tale of freshwater sailors: The causes and consequences of ecological transitions through the salinity barrier in a family of benthic protists.

The salinity barrier that separates marine and freshwater biomes is probably the most important division in biodiversity on Earth. Those organisms that successfully performed this transition had access to new ecosystems while undergoing changes in selective pressure, which often led to major shifts in diversification rates. While these transitions have been extensively investigated in animals, the tempo, mode, and outcome of crossing the salinity barrier have been scarcely studied in other eukaryotes. Here, we reconstructed the evolutionary history of the species complex Cyphoderia ampulla (Euglyphida: Cercozoa: Rhizaria) based on DNA sequences from the nuclear SSU rRNA gene and the mitochondrial cytochrome oxidase subunit I gene, obtained from publicly available environmental DNA data (GeneBank, EukBank) and isolated organisms. A tree calibrated with euglyphid fossils showed that four independent transitions towards freshwater systems occurred from the mid-Miocene onwards, coincident with important fluctuations in sea level. Ancestral trait reconstructions indicated that the whole family Cyphoderiidae had a marine origin and suggest that ancestors of the freshwater forms were euryhaline and lived in environments with fluctuating salinity. Diversification rates did not show any obvious increase concomitant with ecological transitions, but morphometric analyses indicated that species increased in size and homogenized their morphology after colonizing the new environments. This suggests adaptation to changes in selective pressure exerted by life in freshwater sediments.

Arcellinida testate amoebae as climate miner's canaries in Southern Spain.

Southern Spain is currently under threat of desertification as a consequence of global climate change, which pressures on fragile ecosystems such as caves. The organisms living in these extremely stable environments are particularly sensitive and prone to extinction, therefore they can be used as bioindicators for climate change. Cyanobacterial mats form peculiar and vulnerable micro-ecosystems at the entrance of caves and house a diversity of protists. Amongst them, Arcellinida testate amoebae have been traditionally used as bioindicators for environmental quality, notably because their narrow ecological tolerance and their key ecological position as top predators of the microbial foodwebs. We report here two new species of Arcellinida found in the cyanobacterial mats of cave Hundidero, in Sierra de Grazalema, Malaga province, whose traits suggest a narrow tolerance for changes in humidity. We provide a formal description for Difflugia alhadiqa sp. nov. and Heleopera baetica sp. nov. based on morphometrics and 18S rRNA gene data, and propose using the presence of these species to indicate the good health of the cyanobacterial mats, like miner's canaries for local climate.

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.