Nearly all land plants post‐transcriptionally modify specific nucleotides within RNAs, a process known as RNA editing. This adaptation allows the correction of deleterious mutations within the asexually reproducing and presumably non‐recombinant chloroplast and mitochondrial genomes. There are no reports of RNA editing in any of the green algae so this phenomenon is presumed to have originated in embryophytes either after the invasion of land or in the now extinct algal ancestor of all land plants. This was challenged when a recent in silico screen for RNA edit sites based on genomic sequence homology predicted edit sites in the green alga Chara vulgaris, a multicellular alga found within the Streptophyta clade and one of the closest extant algal relatives of land plants. In this study, the organelle transcriptomes of C. vulgaris and Chlamydomonas reinhardtii were deep sequenced for a comprehensive assessment of RNA editing. Initial analyses based solely on sequence comparisons suggested potential edit sites in both species, but subsequent high‐resolution melt analysis, RNase H‐dependent PCR (rhPCR), and Sanger sequencing of DNA and complementary DNAs (cDNAs) from each of the putative edit sites revealed them to be either single‐nucleotide polymorphisms (SNPs) or spurious deep sequencing results. The lack of RNA editing in these two lineages is consistent with the current hypothesis that RNA editing evolved after embryophytes split from its ancestral algal lineage.

Caves are often assumed to be static environments separated from weather changes experienced on the surface. The high humidity and stability of these subterranean environments make them attractive to many different organisms including microbes such as bacteria and protists. Cave waters generally originate from the surface, may be filtered by overlying soils, can accumulate in interstitial epikarst zones underground, and emerge in caves as streams, pools and droplets on speleothems. Water movement is the primary architect of karst caves, and depending on the hydrologic connectivity between surface and subsurface, is the most likely medium for the introduction of microbes to caves. Recently published metabarcoding surveys of karst cave soils and speleothems have suggested that the vast majority of bacteria residing in these habitats do not occur on the surface, calling into question the role of microbial transport by surface waters. The purpose of this study was to use metabarcoding to monitor the aquatic prokaryotic microbiome of a cave for 1 year, conduct longitudinal analyses of the cave’s aquatic bacterioplankton, and compare it to nearby surface water. Water samples were collected from two locations inside Panel Cave in Natural Tunnel State Park in Duffield, VA and two locations outside of the cave. Of the two cave locations, one was fed by groundwater and drip water and the other by infiltrating surface water. A total of 1,854 distinct prokaryotic ASVs were detected from cave samples and 245 (13.1%) were not found in surface samples. PCo analysis demonstrated a marginal delineation between two cave sample sites and between cave and surface microbiomes suggesting the aquatic bacterioplankton in a karst cave is much more similar to surface microbes than reported from speleothems and soils. Most surprisingly, there was a cave microbe population and diversity bloom in the fall months whereas biodiversity remained relatively steady on the surface. The cave microbiome was more similar to the surface before the bloom than during and afterwards. This event demonstrates that large influxes of bacteria and particulate organic matter can enter the cave from either the surface or interstitial zones and the divergence of the cave microbiome from the surface demonstrates movement of microbes from the epikarst zones into the cave.

Summary: The purpose of this study was to survey the eukaryotic microbiome of two karst caves in the Valley and Ridge physiographic region of the Appalachian Mountains. Caves are known to harbour eukaryotic microbes but their very low densities and small cell size make them difficult to collect and identify. Microeukaryotes were surveyed using two methodologies, filtering water and submerging glass microscope slides mounted in periphytometers in cave pools. The periphyton sampling yielded 13.5 times more unique amplicon sequence variants (ASVs) than filtered water. The most abundant protist supergroup was Alveolata with large proportions of the ASVs belonging to dinoflagellate, ciliate and apicomplexan clades. The next most abundant were Rhizarians followed by Stramenopiles (diatoms and chrysophytes) and Ameobozoans. Very few of the ASVs, 1.5%, matched curated protist sequences with greater than 99% identity and only 2.5% could be identified from surface plankton samples collected in the same region. The overall composition of the eukaryotic microbiome appears to be a combination of bacterial grazers and parasitic species that could possibly survive underground as well as cells, cysts and spores probably transported from the surface.

• Premise of the study: Focused ion beam–scanning electron microscopy (FIB‐SEM) combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron‐level resolution. We have applied FIB‐SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution.• Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type.• Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue.• Discussion: The application of FIB‐SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB‐SEM for organelle distribution and cell architecture studies.