Light microscopic images of cultured strains of <i>A. maculatum</i> algae.

<p>The <i>Oophila</i> strains Hb_cul-rk (A−C) and BB_cul-B (D−F) belong to subclades I and III, respectively. Monotypic cultures displayed at least three different cell types, which include 1) free-swimming biflagellates (A, D), which correspond to zoospores or gametes, 2) cells enclosed within a mother cell wall (B, E), likely representing asexually dividing zoospores, and 3) larger non-motile zygotes (C, F). Scale bars: 10 µm (A−F).</p

Atomic-Layer Deposition into 2- versus 3‑Dimensionally Ordered Nanoporous Media: Pore Size or Connectivity?

Atomic-layer deposition (ALD) is now being recognized as a powerful, general tool for modifying the surfaces of nanomaterials in applications for many energy conversion devices. However, ALD involves slow processes particularly when it is subjected to nanoporous media with high-aspect ratios. Predicting the exact experimental conditions of the desired reactions for coating inside deep pores by ALD is not available because of the lack of complete understanding of diffusion in nanoporous media. Here, we report a comparative study of the ALD coating onto two distinctive templates having nanopores, i.e., 2- and 3-dimensionally ordered media (DOM), of similar porosity and pore dimension. Self-supporting, crack-free templates were carefully prepared in centimeters for both 2- and 3-DOM and thus avoid any possible sources of uncontrollable diffusion of precursor gas molecules through unwanted microvoids and cracks. Comparison of the ALD coating profiles across the thickness of both templates reveals a fundamentally distinct coating mechanism. While a uniform growth zone develops along the pores of the 2-DOM (i.e., 1-D diffusion path), a gradual decrease in the deposition is observed in those of the 3-DOM (i.e., 3-D diffusion path) as ALD pulse time increases. This observation suggests an essential role of the pore connectivity, rather than individual pore sizes, in the gas diffusion dynamics inside nanoporous media. The present model can universally predict the ALD behaviors in nanoporous media even with different types of pore connectivity

Maximum incidence of non-<i>Oophila</i> taxa in egg capsules, based on a>0.99 cumulative probability of detecting sequences and the actual number of sequences obtained (see equation 2 in Discussion).

<p>Maximum incidence of non-<i>Oophila</i> taxa in egg capsules, based on a>0.99 cumulative probability of detecting sequences and the actual number of sequences obtained (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108915#pone.0108915.e007" target="_blank">equation 2</a> in Discussion).</p

Map of the geographic range and collection sites for egg masses of four amphibian hosts.

<p>Species range maps are plotted on a map of North America (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108915#s2" target="_blank">Materials and Methods</a>). The dark green color represents a range overlap between <i>L. sylvatica</i> and <i>A. maculatum</i>, and the pink color represents a range overlap between <i>L. aurora</i> and <i>A. gracile</i>. Numbered locations correspond to higher detail panels below. The maps of collection sites for algae corresponding to egg masses from <i>A. maculatum</i> and L. sylvatica in Nova Scotia, Canada (1), <i>A. gracile</i> in California, USA (2), <i>L. aurora</i> and <i>A. gracile</i> in Vancouver Island, British Columbia, Canada, and <i>A. maculatum</i> in New Jersey and Tennessee of USA (4/5).</p

Maximum likelihood (ML) tree of algal 18S rDNA sequences from egg masses of four amphibian taxa from various North American localities.

<p>The data matrix included 1,653 characters and 180 sequences. Newly obtained sequences are bold-faced. ML and MP bootstrap values greater than 50% are shown at corresponding nodes. Subclades I−IV are collapsed into triangles for visual clarity; an un-collapsed version of the tree can be found as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108915#pone.0108915.s003" target="_blank">Figure S1</a>. Numbers in parentheses indicates the number of sequences obtained and analyzed for the corresponding sample. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108915#pone-0108915-t001" target="_blank">Table 1</a> for naming conventions and GenBank accession numbers.</p

Collection details of egg masses from which algae were sampled.

<p>Collection details of egg masses from which algae were sampled.</p

Percentage pairwise distances (uncorrected) of 18S rDNA among the <i>Oophila</i> subclades I−IV.

<p>Percentage pairwise distances (uncorrected) of 18S rDNA among the <i>Oophila</i> subclades I−IV.</p

Data_Sheet_1_Novel Diversity of Deeply Branching Holomycota and Unicellular Holozoans Revealed by Metabarcoding in Middle Paraná River, Argentina.ZIP

<p>Opisthokonta represents a major lineage of eukaryotes and includes fungi and metazoans, as well as other less known unicellular groups. The latter are paraphyletic assemblages that branch in between the former two groups, and thus are important for understanding the origin and early diversification of opisthokonts. The full range of their diversity, however, has not yet been explored from diverse ecological habitats. Freshwater environments are crucial sources for new diversity; they are considered even more heterogeneous than marine ecosystems. This heterogeneity implies more ecological niches where local eukaryotic communities are located. However, knowledge of the unicellular opisthokont diversity is scarce from freshwater environments. Here, we performed an 18S rDNA metabarcoding study in the Middle Paraná River, Argentina, to characterize the molecular diversity of microbial eukaryotes, in particular unicellular members of Opisthokonta. We identified a potential novel clade branching as a sister-group to Fungi. We also detected in our data that more than 60% operational taxonomic units classified as unicellular holozoans (animals and relatives) represent new taxa at the species level. Of the remaining, the majority was assigned to the newly described holozoan species, Syssomonas multiformis. Together, our results show that a large hidden diversity of unicellular members of opisthokonts still remain to be uncovered. We also found that the geographical and ecological distribution of several taxa considered exclusive to marine environments is wider than previously thought.</p

Table_1_Novel Diversity of Deeply Branching Holomycota and Unicellular Holozoans Revealed by Metabarcoding in Middle Paraná River, Argentina.XLSX

<p>Opisthokonta represents a major lineage of eukaryotes and includes fungi and metazoans, as well as other less known unicellular groups. The latter are paraphyletic assemblages that branch in between the former two groups, and thus are important for understanding the origin and early diversification of opisthokonts. The full range of their diversity, however, has not yet been explored from diverse ecological habitats. Freshwater environments are crucial sources for new diversity; they are considered even more heterogeneous than marine ecosystems. This heterogeneity implies more ecological niches where local eukaryotic communities are located. However, knowledge of the unicellular opisthokont diversity is scarce from freshwater environments. Here, we performed an 18S rDNA metabarcoding study in the Middle Paraná River, Argentina, to characterize the molecular diversity of microbial eukaryotes, in particular unicellular members of Opisthokonta. We identified a potential novel clade branching as a sister-group to Fungi. We also detected in our data that more than 60% operational taxonomic units classified as unicellular holozoans (animals and relatives) represent new taxa at the species level. Of the remaining, the majority was assigned to the newly described holozoan species, Syssomonas multiformis. Together, our results show that a large hidden diversity of unicellular members of opisthokonts still remain to be uncovered. We also found that the geographical and ecological distribution of several taxa considered exclusive to marine environments is wider than previously thought.</p

Data_Sheet_2_Novel Diversity of Deeply Branching Holomycota and Unicellular Holozoans Revealed by Metabarcoding in Middle Paraná River, Argentina.ZIP

<p>Opisthokonta represents a major lineage of eukaryotes and includes fungi and metazoans, as well as other less known unicellular groups. The latter are paraphyletic assemblages that branch in between the former two groups, and thus are important for understanding the origin and early diversification of opisthokonts. The full range of their diversity, however, has not yet been explored from diverse ecological habitats. Freshwater environments are crucial sources for new diversity; they are considered even more heterogeneous than marine ecosystems. This heterogeneity implies more ecological niches where local eukaryotic communities are located. However, knowledge of the unicellular opisthokont diversity is scarce from freshwater environments. Here, we performed an 18S rDNA metabarcoding study in the Middle Paraná River, Argentina, to characterize the molecular diversity of microbial eukaryotes, in particular unicellular members of Opisthokonta. We identified a potential novel clade branching as a sister-group to Fungi. We also detected in our data that more than 60% operational taxonomic units classified as unicellular holozoans (animals and relatives) represent new taxa at the species level. Of the remaining, the majority was assigned to the newly described holozoan species, Syssomonas multiformis. Together, our results show that a large hidden diversity of unicellular members of opisthokonts still remain to be uncovered. We also found that the geographical and ecological distribution of several taxa considered exclusive to marine environments is wider than previously thought.</p