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. 2025 Mar 5;13(3):598.
doi: 10.3390/microorganisms13030598.

Global Archaeal Diversity Revealed Through Massive Data Integration: Uncovering Just Tip of Iceberg

Antonios Kioukis  1 , Antonio Pedro Camargo  2 , Pavlos Pavlidis  3 , Ioannis Iliopoulos  4 , Nikos C Kyrpides  2 , Ilias Lagkouvardos  1
Affiliations

Global Archaeal Diversity Revealed Through Massive Data Integration: Uncovering Just Tip of Iceberg

Antonios Kioukis et al. Microorganisms. .

Abstract

The domain of Archaea has gathered significant interest for its ecological and biotechnological potential and its role in helping us to understand the evolutionary history of Eukaryotes. In comparison to the bacterial domain, the number of adequately described members in Archaea is relatively low, with less than 1000 species described. It is not clear whether this is solely due to the cultivation difficulty of its members or, indeed, the domain is characterized by evolutionary constraints that keep the number of species relatively low. Based on molecular evidence that bypasses the difficulties of formal cultivation and characterization, several novel clades have been proposed, enabling insights into their metabolism and physiology. Given the extent of global sampling and sequencing efforts, it is now possible and meaningful to question the magnitude of global archaeal diversity based on molecular evidence. To do so, we extracted all sequences classified as Archaea from 500 thousand amplicon samples available in public repositories. After processing through our highly conservative pipeline, we named this comprehensive resource the 'Global Archaea Diversity' (GAD), which encompassed nearly 3 million molecular species clusters at 97% similarity, and organized it into over 500 thousand genera and nearly 100 thousand families. Saline environments have contributed the most to the novel taxa of this previously unseen diversity. The majority of those 16S rRNA gene sequence fragments were verified by matches in metagenomic datasets from IMG/M. These findings reveal a vast and previously overlooked diversity within the Archaea, offering insights into their ecological roles and evolutionary importance while establishing a foundation for the future study and characterization of this intriguing domain of life.

Keywords: Archaea; Asgardarchaeota; IMNGS; genetic diversity; microbial dark matter.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Depicted are 15.8 million Archaea sequences originating from 177K SRA samples preprocessed by the IMNGS. Every OTU sequence was searched against LTP and SILVA, with any match over 98% being replaced by the database sequence due to their higher quality. SINA was used for alignment and classification. After identification of the most represented region, Escherichia coli 16S rRNA gene was also aligned. The number of E. coli bases (n = 244) within the selected region was used as the lowest limit of required information that each Archaea sequence included in our dataset must contain, so the sequence is included in the next stage. A dereplication was performed on the extracted sub-sequences with the output acting as our final dataset.
Figure 2
Figure 2
The selected region almost perfectly mirrors the percentage dissimilarity of the full sequences (family level: 89%, genus level 93%, species level 97%). Thus, our region is representative of the diversity contained within the full 16S rRNA.
Figure 3
Figure 3
Krona plot quantifying the size of each order after TIC. Novel (created by TIC) and known molecular orders (provided by the SINA classifier) are included.
Figure 4
Figure 4
Schematic of the process for estimation of the low limit of archaeal diversity. A list of highly confident SOTUs was assembled by joining the selections of the most abundant SOTU per GOTU per sample for all samples.
Figure 5
Figure 5
Graphlan plot with the center depicting the taxonomic tree of the SOTUs after TIC that incorporates both novel (red) and known (white) clades up to the order level. The three inner rings quantify the number of (#) SOTUs, genera, and families within each order. Names of the orders containing more than 10 K SOTUs are also given on the left side. The fourth ring (Abundance) shows the environment of the original IMNGS sample, where the most abundant sequence contained within this order comes from. The outer ring (Prevalence) depicts the majority-rule-voted environment from all the sequences contained within the selected order.
Figure 6
Figure 6
Rarefaction curves indicating the cumulative archaeal diversity at broad ecological niches (gamma diversity).The X axis represents the number of (#) microbial profiles intergrated. The Y axis represents the number of (#) thousands SOTUs discovered up to the selected number of profiles. dsotu corresponds to the expected novel SOTUs by the integration of one additional sample of that niche past those already included in the study. Dashed line is the 1:1 relation rate (1 new SOTU per 1 additional sample). (a) Host (Table S2); (b) Plant; (c) Soil; (d) Freshwater; (e) Saline water.
Figure 7
Figure 7
A novelty score, which was based on the number of FOTUs, GOTUs, and sOTUs within the samples of each environment, was normalized by the number of environment samples present in our dataset. Saline water samples contain the highest levels of unexplored novelty across all taxonomic levels, while host-associated samples (Table S2) and plant samples are the most extensively studied.
Figure 8
Figure 8
SOTUs verification as a factor of matching to sequences in IMG/M at different similarity levels. Horizontal red lines correspond to similarity cutoffs used for assigning sequences to species (97%), genera (93%), and families (89%). Vertical blue lines correspond to how many SOTUs are verified at each level. Dashed black line indicates the total number of SOTUs.
Figure 9
Figure 9
(a) Environmental association for the Asgard Archaea SOTUs based on the origin of the IMNGS samples with soil being the most rich environment. (b) Distribution of the predicted SOTUs to the Asgard classes present in SILVA at the time of this analysis.
Figure 10
Figure 10
Asgard SOTUs with UNKCLASS placement on the phylogenetic tree. There are five clusters that remain unknown and should be studied more as they may represent novel Asgard classes. Cluster 5 contains 26 SOTUs (6 verified by different targets of IMG/M) from diverse environments and originating from 25 SRA samples.
See this image and copyright information in PMC

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