Comparative mitochondrial genomics in Nematoda reveal astonishing variation in compositional biases and substitution rates indicative of multi-level selection.

BACKGROUND: Nematodes are the most abundant and diverse metazoans on Earth, and are known to significantly affect ecosystem functioning. A better understanding of their biology and ecology, including potential adaptations to diverse habitats and lifestyles, is key to understanding their response to global change scenarios. Mitochondrial genomes offer high species level characterization, low cost of sequencing, and an ease of data handling that can provide insights into nematode evolutionary pressures. RESULTS: Generally, nematode mitochondrial genomes exhibited similar structural characteristics (e.g., gene size and GC content), but displayed remarkable variability around these general patterns. Compositional strand biases showed strong codon position specific G skews and relationships with nematode life traits (especially parasitic feeding habits) equal to or greater than with predicted phylogeny. On average, nematode mitochondrial genomes showed low non-synonymous substitution rates, but also high clade specific deviations from these means. Despite the presence of significant mutational saturation, non-synonymous (dN) and synonymous (dS) substitution rates could still be significantly explained by feeding habit and/or habitat. Low ratios of dN:dS rates, particularly associated with the parasitic lifestyles, suggested the presence of strong purifying selection. CONCLUSIONS: Nematode mitochondrial genomes demonstrated a capacity to accumulate diversity in composition, structure, and content while still maintaining functional genes. Moreover, they demonstrated a capacity for rapid evolutionary change pointing to a potential interaction between multi-level selection pressures and rapid evolution. In conclusion, this study helps establish a background for our understanding of the potential evolutionary pressures shaping nematode mitochondrial genomes, while outlining likely routes of future inquiry.

18S-NemaBase: Curated 18S rRNA Database of Nematode Sequences.

Nematodes are the most abundant and diverse animals on the planet but lack representation in biodiversity research. This presents a problem for studying nematode diversity, particularly when molecular tools (i.e., barcoding and metabarcoding) rely on well-populated and curated reference databases, which are absent for nematodes. To improve molecular identification and the assessment of nematode diversity, we created and curated an 18S rRNA database specific to nematodes (18S-NemaBase) using sequences sourced from the most recent publicly available 18S rRNA SILVA v138 database. As part of the curation process, taxonomic strings were standardized to contain a fixed number of taxonomic ranks relevant to nematology and updated for the most recent accepted nematode classifications. In addition, apparent erroneous sequences were removed. To test the efficacy and accuracy of 18S-NemaBase, we compared it to an older but also curated SILVA v111 and the newest SILVA v138 by assigning taxonomies and analyzing the diversity of a nematode dataset from the Western Nebraska Sandhills. We showed that 18S-NemaBase provided more accurate taxonomic assignments and diversity assessments than either version of SILVA, with a much easier workflow and no need for manual corrections. Additionally, observed diversity further improved when 18S-NemaBase was supplemented with reference sequences from nematodes present in the study site. Although the 18S-NemaBase is a step in the right direction, a concerted effort to increase the number of high-quality, accessible, full-length nematode reference sequences is more important now than ever.

Nematode mitochondrial metagenomics: A new tool for biodiversity analysis.

DNA barcoding approaches have greatly increased our understanding of biodiversity on the planet, and metabarcoding is widely used for classifying members of the phylum Nematoda. However, loci typically utilized in metabarcoding studies are often unable to resolve closely related species or are unable to recover all taxa present in a sample due to inadequate PCR primer binding. Mitochondrial metagenomics (mtMG) is an alternative approach utilizing shotgun sequencing of total DNA to recover the mitochondrial genomes of all species present in samples. However, this approach requires a comprehensive reference database for identification and currently available mitochondrial sequences for nematodes are highly dominated by sequences from the order Rhabditida, and excludes many clades entirely. Here, we analysed the efficacy of mtMG for the recovery of nematode taxa and the generation of mitochondrial genomes. We first developed a curated reference database of nematode mitochondrial sequences and expanded it with 40 newly sequenced taxa. We then tested the mito-metagenomics approach using a series of nematode mock communities consisting of morphologically identified nematode species representing various feeding traits, life stages, and phylogenetic relationships. We were able to identify all but two species through the de novo assembly of COX1 genes. We were also able to recover additional mitochondrial protein coding genes (PCGs) for 23 of the 24 detected species including a full array of 12 PCGs from five of the species. We conclude that mtMG offers a potential for the effective recovery of nematode biodiversity but remains limited by the breadth of the reference database.

Host identity is the dominant factor in the assembly of nematode and tardigrade gut microbiomes in Antarctic Dry Valley streams.

Recent work examining nematode and tardigrade gut microbiomes has identified species-specific relationships between host and gut community composition. However, only a handful of species from either phylum have been examined. How microbiomes differ among species and what factors contribute to their assembly remains unexplored. Cyanobacterial mats within Antarctic Dry Valley streams host a simple and tractable natural ecosystem of identifiable microinvertebrates to address these questions. We sampled 2 types of coexisting mats (i.e., black and orange) across four spatially isolated streams, hand-picked single individuals of two nematode species (i.e., Eudorylaimus antarcticus and Plectus murrayi) and tardigrades, to examine their gut microbiomes using 16S and 18S rRNA metabarcoding. All gut microbiomes (bacterial and eukaryotic) were significantly less diverse than the mats they were isolated from. In contrast to mats, microinvertebrates' guts were depleted of Cyanobacteria and differentially enriched in taxa of Bacteroidetes, Proteobacteria, and Fungi. Among factors investigated, gut microbiome composition was most influenced by host identity while environmental factors (e.g., mats and streams) were less important. The importance of host identity in predicting gut microbiome composition suggests functional value to the host, similar to other organisms with strong host selected microbiomes.

Crossing Treeline: Bacterioplankton Communities of Alpine and Subalpine Rocky Mountain Lakes.

From the aboveground vegetation to the belowground microbes, terrestrial communities differ between the highly divergent alpine (above treeline) and subalpine (below treeline) ecosystems. Yet, much less is known about the partitioning of microbial communities between alpine and subalpine lakes. Our goal was to determine whether the composition of bacterioplankton communities of high-elevation mountain lakes differed across treeline, identify key players in driving the community composition, and identify potential environmental factors that may be driving differences. To do so, we compared bacterial community composition (using 16S rDNA sequencing) of alpine and subalpine lakes in the Southern Rocky Mountain ecoregion at two time points: once in the early summer and once in the late summer. In the early summer (July), shortly after peak runoff, bacterial communities of alpine lakes were distinct from subalpine lakes. Interestingly, by the end of the summer (approximately 5 weeks after the first visit in August), bacterial communities of alpine and subalpine lakes were no longer distinct. Several bacterial amplicon sequence variants (ASVs) were also identified as key players by significantly contributing to the community dissimilarity. The community divergence across treeline found in the early summer was correlated with several environmental factors, including dissolved organic carbon (DOC), pH, chlorophyll-a (chl-a), and total dissolved nitrogen (TDN). In this paper, we offer several potential scenarios driven by both biotic and abiotic factors that could lead to the observed patterns. While the mechanisms for these patterns are yet to be determined, the community dissimilarity in the early summer correlates with the timing of increased hydrologic connections with the terrestrial environment. Springtime snowmelt brings the flushing of mountain watersheds that connects terrestrial and aquatic ecosystems. This connectivity declines precipitously throughout the summer after snowmelt is complete. Regional climate change is predicted to bring alterations to precipitation and snowpack, which can modify the flushing of solutes, nutrients, and terrestrial microbes into lakes. Future preservation of the unique alpine lake ecosystem is dependent on a better understanding of ecosystem partitioning across treeline and careful consideration of terrestrial-aquatic connections in mountain watersheds.

Gullies and Moraines Are Islands of Biodiversity in an Arid, Mountain Landscape, Asgard Range, Antarctica.

Cold, dry, and nutrient-poor, the McMurdo Dry Valleys of Antarctica are among the most extreme terrestrial environments on Earth. Numerous studies have described microbial communities of low elevation soils and streams below glaciers, while less is known about microbial communities in higher elevation soils above glaciers. We characterized microbial life in four landscape features (habitats) of a mountain in Taylor Valley. These habitats varied significantly in soil moisture and include moist soils of a (1) lateral glacial moraine, (2) gully that terminates at the moraine, and very dry soils on (3) a southeastern slope and (4) dry sites near the gully. Using rRNA gene PCR amplicon sequencing of Bacteria and Archaea (16S SSU) and eukaryotes (18S SSU), we found that all habitat types harbored significantly different bacterial and eukaryotic communities and that these differences were most apparent when comparing habitats that had macroscopically visible soil crusts (gully and moraine) to habitats with no visible crusts (near gully and slope). These differences were driven by a relative predominance of Actinobacteria and a Colpodella sp. in non-crust habitats, and by phototrophic bacteria and eukaryotes (e.g., a moss) and predators (e.g., tardigrades) in habitats with biological soil crusts (gully and moraine). The gully and moraine also had significantly higher 16S and 18S ESV richness than the other two habitat types. We further found that many of the phototrophic bacteria and eukaryotes of the gully and moraine share high sequence identity with phototrophs from moist and wet areas elsewhere in the Dry Valleys and other cold desert ecosystems. These include a Moss (Bryum sp.), several algae (e.g., a Chlorococcum sp.) and cyanobacteria (e.g., Nostoc and Phormidium spp.). Overall, the results reported here broaden the diversity of habitat types that have been studied in the Dry Valleys of Antarctica and suggest future avenues of research to more definitively understand the biogeography and factors controlling microbial diversity in this unique ecosystem.

Microbial Species-Area Relationships in Antarctic Cryoconite Holes Depend on Productivity.

The island species-area relationship (ISAR) is a positive association between the number of species and the area of an isolated, island-like habitat. ISARs are ubiquitous across domains of life, yet the processes generating ISARs remain poorly understood, particularly for microbes. Larger and more productive islands are hypothesized to have more species because they support larger populations of each species and thus reduce the probability of stochastic extinctions in small population sizes. Here, we disentangled the effects of "island" size and productivity on the ISAR of Antarctic cryoconite holes. We compared the species richness of bacteria and microbial eukaryotes on two glaciers that differ in their productivity across varying hole sizes. We found that cryoconite holes on the more productive Canada Glacier gained more species with increasing hole area than holes on the less productive Taylor Glacier. Within each glacier, neither productivity nor community evenness explained additional variation in the ISAR. Our results are, therefore, consistent with productivity shaping microbial ISARs at broad scales. More comparisons of microbial ISARs across environments with limited confounding factors, such as cryoconite holes, and experimental manipulations within these systems will further contribute to our understanding of the processes shaping microbial biogeography.

Comparison of Microbial Communities in the Sediments and Water Columns of Frozen Cryoconite Holes in the McMurdo Dry Valleys, Antarctica.

Although cryoconite holes, sediment-filled melt holes on glacier surfaces, appear small and homogenous, their microbial inhabitants may be spatially partitioned. This partitioning could be particularly important for maintaining biodiversity in holes that remain isolated for many years, such as in Antarctica. We hypothesized that cryoconite holes with greater species richness and biomass should exhibit greater partitioning between the sediments and water, promoting greater biodiversity through spatial niche partitioning. We tested this hypothesis by sampling frozen cryoconite holes along a gradient of biomass and biodiversity in the Taylor Valley, Antarctica, where ice-lidded cryoconite holes are a ubiquitous feature of glaciers. We extracted DNA and chlorophyll a from the sediments and water of these samples to describe biodiversity and quantify proxies for biomass. Contrary to our expectation, we found that cryoconite holes with greater richness and biomass showed less partitioning of phylotypes by the sediments versus the water, perhaps indicating that the probability of sediment microbes being mixed into the water is higher from richer sediments. Another explanation may be that organisms from the water were compressed by freezing down to the sediment layer, leaving primarily relic DNA of dead cells to be detected higher in the frozen water. Further evidence of this explanation is that the dominant sequences unique to water closely matched organisms that do not live in cryoconite holes or the Dry Valleys (e.g., vertebrates); so this cryptic biodiversity could represent unknown microbial animals or DNA from atmospheric deposition of dead biomass in the otherwise low-biomass water. Although we cannot rule out spatial niche partitioning occurring at finer scales or in melted cryoconite holes, we found no evidence of partitioning between the sediments and water in frozen holes. Future work should include more sampling of cryoconite holes at a finer spatial scale, and characterizing the communities of the sediments and water when cryoconite holes are melted and active.

Diversity patterns of microbial eukaryotes mirror those of bacteria in Antarctic cryoconite holes.

Ice-lidded cryoconite holes on glaciers in the Taylor Valley, Antarctica, provide a unique system of natural mesocosms for studying community structure and assembly. We used high-throughput DNA sequencing to characterize both microbial eukaryotic communities and bacterial communities within cryoconite holes across three glaciers to study similarities in their spatial patterns. We expected that the alpha (phylogenetic diversity) and beta (pairwise community dissimilarity) diversity patterns of eukaryotes in cryoconite holes would be related to those of bacteria, and that they would be related to the biogeochemical gradient within the Taylor Valley. We found that eukaryotic alpha and beta diversity were strongly related to those of bacteria across scales ranging from 140 m to 41 km apart. Alpha diversity of both was significantly related to position in the valley and surface area of the cryoconite hole, with pH also significantly correlated with the eukaryotic diversity. Beta diversity for both bacteria and eukaryotes was significantly related to position in the valley, with bacterial beta diversity also related to nitrate. These results are consistent with transport of sediments onto glaciers occurring primarily at local scales relative to the size of the valley, thus creating feedbacks in local chemistry and diversity.

Spatial autocorrelation of microbial communities atop a debris-covered glacier is evidence of a supraglacial chronosequence.

Although microbial communities from many glacial environments have been analyzed, microbes living in the debris atop debris-covered glaciers represent an understudied frontier in the cryosphere. The few previous molecular studies of microbes in supraglacial debris have either had limited phylogenetic resolution, limited spatial resolution (e.g. only one sample site on the glacier) or both. Here, we present the microbiome of a debris-covered glacier across all three domains of life, using a spatially-explicit sampling scheme to characterize the Middle Fork Toklat Glacier's microbiome from its terminus to sites high on the glacier. Our results show that microbial communities differ across the supraglacial transect, but surprisingly these communities are strongly spatially autocorrelated, suggesting the presence of a supraglacial chronosequence. This pattern is dominated by phototrophic microbes (both bacteria and eukaryotes) which are less abundant near the terminus and more abundant higher on the glacier. We use these data to refute the hypothesis that the inhabitants of the glacier are randomly deposited atmospheric microbes, and to provide evidence that succession from a predominantly photosynthetic to a more heterotrophic community is occurring on the glacier.

A Naganishia in high places: functioning populations or dormant cells from the atmosphere?

Here, we review the current state of knowledge concerning high-elevation members of the extremophilic Cryptococcus albidus clade (now classified as the genus Naganishia). These fungi dominate eukaryotic microbial communities across the highest elevation, soil-like material (tephra) on volcanoes such as Llullaillaco, Socompa, and Saírecabur in the Atacama region of Chile, Argentina, and Bolivia. Recent studies indicate that Naganishia species are among the most resistant organisms to UV radiation, and a strain of N. friedmannii from Volcán Llullaillaco is the first organism that is known to grow during the extreme, diurnal freeze-thaw cycles that occur on a continuous basis at elevations above 6000 m.a.s.l. in the Atacama region. These and other extremophilic traits discussed in this review may serve a dual purpose of allowing Naganishia species to survive long-distance transport through the atmosphere and to survive the extreme conditions found at high elevations. Current evidence indicates that there are frequent dispersal events between high-elevation volcanoes of Atacama region and the Dry Valleys of Antarctica via "Rossby Wave" merging of the polar and sub-tropical jet streams. This dispersal hypothesis needs further verification, as does the hypothesis that Naganishia species are flexible "opportunitrophs" that can grow during rare periods of water (from melting snow) and nutrient availability (from Aeolian inputs) in one of the most extreme terrestrial habitats on Earth.