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Sequences from the Stramenopile class Eustigmatophyceae are rarely reported in metabarcoding studies, and when they have been reported, there are very few haplotypes. We hypothesized that the paucity of eustigmatophyte species detected in these studies may be a result of the metabarcoding techniques used, which have primarily employed universal ribosomal RNA gene regions. In this study, we examined environmental DNA samples from 22 sites in southwestern Virginia, some of which had previously been studied using ribosomal RNA analysis. We used metabarcoding techniques targeting the plastidrbcL gene with new primers designed to produce a 370 bp amplicon from all lineages of the Eustigmatophyceae in a reference collection. The amplicons were then analyzed with DADA2 to produce amplicon sequence variants (ASVs). Our results revealed 184rbcL haplotypes that can be tentatively assigned to the Eustigmatophyceae from these sites, representing much higher diversity than has been detected by ribosomal DNA‐based studies. The techniques employed can be used for future studies of population structure, ecology, distribution, and diversity of this class. With these techniques, it should be possible to make realistic estimates of the species‐level diversity of the Eustigmatophyceae on local, regional, and perhaps global scales.

 

Filamentous representatives of Cladophorales (Chlorophyta) are major contributors to algal biomass of littoral communities. In the present study, community analysis of a reported bloom in the Ogeechee River in Georgia provided an opportunity to combine morphological and genetic analyses with ecological information related to an understudied nuisance alga. A polyphasic approach of incorporating genotypic and phenotypic methods led to the identification of the algal community as Pithophora roettleri (Roth) Wittrock. Morphological analysis showed a monospecific community based on the average length and diameter of the heterosporous intercalary and terminal akinetes, along with the diameter of the principal filaments. Single-gene and concatenated-gene phylogenetic analyses of the LSU (28S rRNA) and SSU (18S rRNA) markers further confirmed this species identification. In this study, we conducted a morphological treatment of P. roettleri, produced 17 novel gene sequences, and produced a new, schematic diagram illustrating the four steps of the asexual reproduction of an intercalary akinete. Morphological characteristics, like the position and shape of akinetes documented here, and the availability of genetic sequences can improve identification and further ecological understanding of filamentous green algae. Flowing mats of P. roettleri, like those observed in this study, can lower light availability for other biota and structurally alter the habitat. 

Mitochondria carry the remnant of an ancestral bacterial chromosome and express those genes with a system separate and distinct from the nucleus. Mitochondrial genes are transcribed as poly-cistronic primary transcripts which are post-transcriptionally processed to create individual translationally competent mRNAs. Algae post-transcriptional processing has only been explored in Chlamydomonas reinhardtii (Class: Chlorophyceae) and the mature mRNAs are different than higher plants, having no 5′ UnTranslated Regions (UTRs), much shorter and more variable 3′ UTRs and polycytidylated mature mRNAs. In this study, we analyzed transcript termini using circular RT-PCR and PacBio Iso-Seq to survey the 3′ and 5′ UTRs and termini for two green algae, Pediastrum duplex (Class: Chlorophyceae) and Chara vulgaris (Class: Charophyceae). This enabled the comparison of processing in the chlorophyte and charophyte clades of green algae to determine if the differences in mitochondrial mRNA processing pre-date the invasion of land by embryophytes. We report that the 5′ mRNA termini and non-template 3′ termini additions in P. duplex resemble those of C. reinhardtii, suggesting a conservation of mRNA processing among the chlorophyceae. We also report that C. vulgaris mRNA UTRs are much longer than chlorophytic examples, lack polycytidylation, and are polyadenylated similar to embryophytes. This demonstrates that some mitochondrial mRNA processing events diverged with the split between chlorophytic and streptophytic algae. 

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. 

Species within the green algal order Cladophorales have an unconventional plastome structure where individual coding regions or small numbers of genes occur as linear single-stranded DNAs folded into hairpin structures. Another group of photosynthetic organisms with an equivalently reduced chloroplast genome are the peridinin dinoflagellates of the Alveolata eukaryotic lineage whose plastomes are mini-circles carrying one or a few genes required for photosynthesis. One unusual aspect of the Alveolata is the polyuridylylation of mRNA 30 ends among peridinin dinoflagellates and the chromerid algae. This study was conducted to understand if an unconventional highly reduced plastome structure co-occurs with unconventional RNA processing. To address this, the 50 and 30 mRNA termini of the known chloroplast genes of Pithophora roettleri (order Cladophorales) were analyzed for evidence of post-transcriptional processing. Circular Reverse Transcriptase PCR (cRT-PCR) followed by deep sequencing of the amplicons was used to analyze 50 and 30 mRNA termini. Evidence of several processing events were collected, most notably the 30 termini of six of the eight genes were polyuridylylated, which has not been reported for any lineage outside of the Alveolata. Other processing events include poly(A) and heteropolymeric 30 additions, 50 primary transcript start sites, as well as the presence of circularized RNAs. Five other species representing other green algal lineages were also tested and poly(U) additions appear to be limited to the order Cladophorales. These results demonstrate that chloroplast mRNA polyuridylylation is not the sole provenance of photosynthetic alveolates and may have convergently evolved in two distinct photosynthetic lineages. 

Species within the green algal order Cladophorales have an unconventional plastome structure where individual coding regions or small numbers of genes occur as linear single-stranded DNAs folded into hairpin structures. Another group of photosynthetic organisms with an equivalently reduced chloroplast genome are the peridinin dinoflagellates of the Alveolata eukaryotic lineage whose plastomes are mini-circles carrying one or a few genes required for photosynthesis. One unusual aspect of the Alveolata is the polyuridylylation of mRNA 30 ends among peridinin dinoflagellates and the chromerid algae. This study was conducted to understand if an unconventional highly reduced plastome structure co-occurs with unconventional RNA processing. To address this, the 50 and 30 mRNA termini of the known chloroplast genes of Pithophora roettleri (order Cladophorales) were analyzed for evidence of post-transcriptional processing. Circular Reverse Transcriptase PCR (cRT-PCR) followed by deep sequencing of the amplicons was used to analyze 50 and 30 mRNA termini. Evidence of several processing events were collected, most notably the 30 termini of six of the eight genes were polyuridylylated, which has not been reported for any lineage outside of the Alveolata. Other processing events include poly(A) and heteropolymeric 30 additions, 50 primary transcript start sites, as well as the presence of circularized RNAs. Five other species representing other green algal lineages were also tested and poly(U) additions appear to be limited to the order Cladophorales. These results demonstrate that chloroplast mRNA polyuridylylation is not the sole provenance of photosynthetic alveolates and may have convergently evolved in two distinct photosynthetic lineages. 

The relatively recent focus on the widespread occurrence and abundance of circular RNAs (circRNA) in the human cell nucleus has sparked an intensive interest in their existence and possible roles in cell gene expression and physiology. The presence of circRNAs in mammalian mitochondria, however, has been under-explored. Mitochondrial mRNAs differ from those produced from nuclear genes because they lack introns and are transcribed as poly-cistronic transcripts that are endonucleolytically cleaved, leaving transcripts with very small 5′ and 3′ UTRs. Circular RNAs have been identified in the semi-autonomous organelles of single-celled organisms and plants but their purpose has not been clearly demonstrated. The goal of our project was to test the hypothesis, processed mRNAs are circularized in vertebrate mitochondria as a necessary RNA processing step prior to translation. Mitochondrial mRNAs were isolated from the human cell line HEK293 and evidence of circularization sought by treating RNA with RNAse-R and then amplifying putative 3′-5′ junction sites. Sequence results demonstrated the occurrence of mRNA circularization within each coding region of the mitochondrial genome. However, in most cases the circRNAs carried coding regions that had been truncated, suggesting they were not translatable. Quantification of the circularized versions of the mRNAs revealed they comprise a small portion (~10%) of the total mRNA. These findings demonstrate that mRNA circularization occurs in mammalian mitochondria but it does not appear to play a role in making translatable mRNAs.