Biogeography and Phylogeny of Aigarchaeota, A Novel Phylum of Archaea
Gisele Braga Goertz, McNair Scholar, Biological Sciences Major
Dr. Brian P. Hedlund, Faculty Mentor, School of Life Sciences
ABSTRACT
‘Aigarchaeota’ is a candidate phylum of Archaea known only by 16S rRNA gene fragments from cultiva-
tion-independent microbial surveys and a single composite genome from Candidatus ‘Caldiarchaeum subterraneum’, an inhabitant of a subterranean gold mine in Japan. Gene sequences associated with ‘Aigarchaeota’ have been found in a variety of geothermal habits, however a comprehensive analysis of the phylogeny
and distribution of ‘Aigarchaeota’ has not yet been done. Public databases were mined for 16S rRNA gene
sequences related to known ‘Aigarchaeota’ and a combination of approaches were used to rigorously define
the phylogenetic boundaries of the phylum, investigate its distribution, and design potential ‘Aigarchaeota’specific primers. Approximately 300 16S rRNA genes and gene fragments affiliated with ‘Aigarchaeota’ were
identified, phylogenic analyses suggested that ‘Aigarchaeota’ belonged to at least three family- to order-level
groups and at least 13 genus-level groups, and supported the proposed relationship between ‘Aigarchaeota’,
Thaumarchaeota, Crenarchaeota, and Korarchaeota in the so-called ‘TACK’ superphylum. A global distribution was suggested with genus-level group habitat differentiation. Some groups were predominantly terrestrial (1A, 2B, 3, 4 & 5) while other groups were mostly found in Marine habitats (6-8). Fifteen strong ‘Aigarchaeota’-specific primer candidates for the polymerase chain reaction (PCR) amplification of 16S rRNA
genes were designed for further analyses.
• ‘Aigarchaeota’ gene sequences mined from GenBank and out-group sequences were aligned in mothur
using a 50,000 character SILVA reference alignment capable of aligning sequences from all three domains of
life.
• Alignments were manually curated in BioEdit version 7.1.3.0, trimmed, then saved as Phylogenetic Inference Package (PHYLIP) version 4-compatible file.
• A distance matrix was computed using “Dnadist”, then used in the “Neighbor” program, which clusters
lineages and constructs a tree.
• Sequences were subsequently evaluated at the species (>97%), genus (>95%), family (>90%), order
(>85%), and phylum (>80%) level.
PRIMER EVALUATION
• ‘Aigarchaeota’-specific qRT-PCR primers were designed by three different programs the SP-Designer,
ARB, and AlleleID.
• Primers were classified as forward or reverse and submitted to the Ribosomal Database Project (RDP)
Probe Match tool to eliminate primers that were non- ‘Aigarchaeota’ specific.
• Primers that did match well with ‘Aigarchaeota’ were reviewed in RDP to determine which genus-level
groups were associated with each of the individual primers.
• Primers were analyzed using OligoCalc to ensure appropriate GC content (~50%), melting temperature
(60°C), and absence of hairpin formation and potential self-annealing sites.
RESULTS
CONCLUSION
This study revealed, for the first time, the phylogenetic diversity and distribution of the novel phylum ‘Ai-
garchaeota’. Currently available 16S rRNA gene sequences from global studies suggest that the phylum consists of at least 13 genus-level groups, and that ‘Aigarchaeota’ is situated firmly in the ‘TACK’ superphylum,
but exists as a separate, unique phylum that does not possess a phylum-level relationship with the Miscellaneous Cren Group (see Figure 1). Genus-level Group 2A was detected in both terrestrial and marine environments. In contrast, Groups 1A, 2B, 3, 4 & 5 and Groups 6-8 are predominant in terrestrial and marine habitats
respectively. Groups 1B and 1C were only found in terrestrial habitats, while Group 1D was only found in marine habitats. Potential primers designed in this study range from genus-level group specific to a combination
of groups allowing for the detection of all potential ‘Aigarchaeota’ defined in this study. It’s important to note
that there were fewer numbers of marine studies (17) than terrestrial studies (54), thus making it difficult to
make solid inferences.
Figure 1. Neighbor-Joining Tree showing 16S rRNA gene phylogenetic relationship between ‘Aigarchaeota’
Groups 1A – 8 and archaeal outgroups, with unsupported nodes identified with boxes. Scale bar represents one
substitution per 100 nucleotides.
LEGEND (°C)
<15
15-50
50-80
>80
Unpublished/not available
Number of Gene Sequences Reported
1-5
6-10
11-25
METHODOLOGY
PHYLOGENETIC TREE CONSTRUCTION
• The 16S rRNA gene sequence of Ca. ‘C. subterraneum’ was submitted to BLAST, and the accession numbers of all gene sequences with greater than 80% identity were recorded.
1A
1B
1C
1D
1E
2A
2B
3
4
5
6
7
8
Figure 3. Global distribution of ‘Aigarchaeota’ sequences by genus-level groups.
INTRODUCTION
Despite our knowledge of the diversity of life on earth there are major lineages of microbial life that have
never been studied that are referred to as “biological dark matter” or “microbial dark matter” (Marcy et al.,
2007; Rinke, et al., 2013). One of these so-called “microbial dark matter” groups was originally described
as pSL4 and related gene sequences were later grouped under the name Hot Water Crenarchaeotic Group 1
(HWCG 1) (Barns et al., 1996; Nunoura et al., 2005). A metagenomics study of a microbial mat community
led to the construction of a complete composite genome for a member of the pSL4/HWCG 1 lineage proposed as Candidatus ‘Caldiarchaeum subterraneum’ and further analysis of the genome led to the proposition of the phylum ‘Aigarchaeota’ (Nunoura et al., 2005; Nunoura et al., 2011).
Genomic analysis of Ca. subterraneum revealed unique eukaryote-type features which led to the proposal
of a superphylum level relationship called the ‘TACK’ superphylum (Guy and Ettema, 2011). The cultivationindependent studies suggest a world-wide distribution of ‘Aigarchaeota’ and dominance of 16S rRNA gene
clone libraries from habitats such as a deep sea hydrothermal vent in Okinawa Trough at 35-60 °C and Great
Boiling Spring at 79-87°C ( Nunoura et al., 2010; Cole et al., 2012). Cole at al 2012 reported that ‘Aigarchaeota’ 16S rRNA gene sequences had the highest relative abundance at Site A (~54%), with a general trend
of decreasing relative abundance down to ~5% at 62°C (Site E), suggesting ‘Aigarchaeota’ niche differentiation along a thermal gradient.
The purpose of this study was to gather 16S rRNA gene sequences potentially belonging to ‘Aigarchaeota’,
and to use those sequences to address several goals:
(i) Rigorously define the phylum
(ii) Gain insight into the phylogenetic and potential taxonomic structure of the phylum
(iii) Assess the distribution of ‘Aigarchaeota’ as a whole, along with individual groups of ‘Aigarchaeota’ in
nature
(iv) Design ‘Aigarchaeota’- specific 16S rRNA gene primers that combined will target genus-level groups
GROUPS
26-50
51-75
Figure 2. ‘Aigarchaeota’ sequences organized to represent global sample location, sampling temperature, number of
sequences per location, and associated genus-level groups.
REFERENCES
Barns SM, Delwiche CF, Palmer JD, Pace NR (1996) Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences. Proc. Natl. Acad. Sci. USA 93:9188–9193
Cole JK, Peacock JP, Dodsworth JA, Williams AJ, Thompson DB, Dong H, Wu G, Hedlund BP (2012) Sediment microbial communities in Great Boiling Spring are controlled by temperature and distinct from water communities. ISME J. 7: 718–729.
Guy L & Ettema TJG (2011). The archaeal ‘TACK’ superphylum and the origin of eukaryotes.
Trends Microbiol. 19: 580–587.
Marcy Y, Ouverney C, Bik EM, Lösekann T, Ivanova N, Martin HG, Szeto E, Platt D, Hugenholtz P, Relman DA, Quake SR (2007)
Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth.
Proceedings of the National Academy of Science 104: 11889-11894. Print.
Nunoura T, Hirayama H, Takami H, Oida H, Nishi S, Shimamura S, Suzuki Y, Inagaki F, Takai K, Nealson KH, Horikoshi K (2005)
Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of
genome fragments. Environ. Microbiol. 7:1967–1984.
Nunoura T, Oida H, Nakaseama M, Kosaka A, Ohkubo SB, Kikuchi T et al. (2010). Archaeal diversity and distribution along thermal
and geochemical gradients in hydrothermal sediments at the Yonaguni Knoll IV Hydrothermal Field in the Southern Okinawa Trough.
Appl Environ Microbiol. 76: 1198–1211
Nunoura T, Takaki Y, Kakuta J, Nishi S, Sugahara J, Kazama H, Chee GJ, Hattori M, Kanai A, Atomi H, Takai K, Takami H (2011) Insights into the evolution of archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group. Nucleic
Acids Research 39: 3204-3223.
Rinke C, Schwientek P, Sczyrba A, Ivanova N, Anderson IJ, Cheng JF, Darling A, Malfatti S, Swan BK, Gies EA, Dodsworth JA, Hedlund
BP, Tsiamis G, Sievert SM, Liu WT, Eisen JA, Hallam SJ, Kyrpides NC, Stepanauskas R, Rubin EM, Hugenholtz P, Woyke T, (2013
Insights into the phylogeny and coding potential of microbial dark matter. Nature 00: 1-7