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PUBLISHED: 11 APRIL 2016 | ARTICLE NUMBER: 16048 | DOI: 10.1038/NMICROBIOL.2016.48

A new view of the tree of life
Laura A. Hug1†, Brett J. Baker2, Karthik Anantharaman1, Christopher T. Brown3, Alexander J. Probst1,
Cindy J. Castelle1, Cristina N. Butterfield1, Alex W. Hernsdorf3, Yuki Amano4, Kotaro Ise4,
Yohey Suzuki5, Natasha Dudek6, David A. Relman7,8, Kari M. Finstad9, Ronald Amundson9,
Brian C. Thomas1 and Jillian F. Banfield1,9*

The tree of life is one of the most important organizing prin- Contributing to this expansion in genome numbers are single cell
ciples in biology1. Gene surveys suggest the existence of an genomics13 and metagenomics studies. Metagenomics is a shotgun
enormous number of branches2, but even an approximation of sequencing-based method in which DNA isolated directly from the
the full scale of the tree has remained elusive. Recent depic- environment is sequenced, and the reconstructed genome fragments
tions of the tree of life have focused either on the nature of are assigned to draft genomes14. New bioinformatics methods yield
deep evolutionary relationships3–5 or on the known, well-classi- complete and near-complete genome sequences, without a reliance
fied diversity of life with an emphasis on eukaryotes6. These on cultivation or reference genomes7,15. These genome- (rather than
approaches overlook the dramatic change in our understanding gene) based approaches provide information about metabolic poten-
of life’s diversity resulting from genomic sampling of previously tial and a variety of phylogenetically informative sequences that can
unexamined environments. New methods to generate genome be used to classify organisms16. Here, we have constructed a tree
sequences illuminate the identity of organisms and their meta- of life by making use of genomes from public databases and 1,011
bolic capacities, placing them in community and ecosystem con- newly reconstructed genomes that we recovered from a variety of
texts7,8. Here, we use new genomic data from over 1,000 environments (see Methods).
uncultivated and little known organisms, together with pub- To render this tree of life, we aligned and concatenated a set of 16
lished sequences, to infer a dramatically expanded version of ribosomal protein sequences from each organism. This approach
the tree of life, with Bacteria, Archaea and Eukarya included. yields a higher-resolution tree than is obtained from a single gene,
The depiction is both a global overview and a snapshot of the such as the widely used 16S rRNA gene16. The use of ribosomal pro-
diversity within each major lineage. The results reveal the dom- teins avoids artefacts that would arise from phylogenies constructed
inance of bacterial diversification and underline the importance using genes with unrelated functions and subject to different evol-
of organisms lacking isolated representatives, with substantial utionary processes. Another important advantage of the chosen
evolution concentrated in a major radiation of such organisms. ribosomal proteins is that they tend to be syntenic and co-located
This tree highlights major lineages currently underrepresented in a small genomic region in Bacteria and Archaea, reducing
in biogeochemical models and identifies radiations that are binning errors that could substantially perturb the geometry of
probably important for future evolutionary analyses. the tree. Included in this tree is one representative per genus for
Early approaches to describe the tree of life distinguished organisms all genera for which high-quality draft and complete genomes
based on their physical characteristics and metabolic features. exist (3,083 organisms in total).
Molecular methods dramatically broadened the diversity that could Despite the methodological challenges, we have included repre-
be included in the tree because they circumvented the need for direct sentatives of all three domains of life. Our primary focus relates to
observation and experimentation by relying on sequenced genes as the status of Bacteria and Archaea, as these organisms have been
markers for lineages. Gene surveys, typically using the small subunit most difficult to profile using macroscopic approaches, and substan-
ribosomal RNA (SSU rRNA) gene, provided a remarkable and novel tial progress has been made recently with acquisition of new genome
view of the biological world1,9,10, but questions about the structure sequences7,8,13. The placement of Eukarya relative to Bacteria and
and extent of diversity remain. Organisms from novel lineages have Archaea is controversial1,4,5,17,18. Eukaryotes are believed to be evol-
eluded surveys, because many are invisible to these methods due to utionary chimaeras that arose via endosymbiotic fusion, probably
sequence divergence relative to the primers commonly used for gene involving bacterial and archaeal cells19. Here, we do not attempt
amplification7,11. Furthermore, unusual sequences, including those to confidently resolve the placement of the Eukarya. We position
with unexpected insertions, may be discarded as artefacts7. them using sequences of a subset of their nuclear-encoded riboso-
Whole genome reconstruction was first accomplished in 1995 mal proteins, an approach that classifies them based on the inheri-
(ref. 12), with a near-exponential increase in the number of draft tance of their information systems as opposed to lipid or other
genomes reported each subsequent year. There are 30,437 genomes cellular structures5.
from all three domains of life—Bacteria, Archaea and Eukarya— Figure 1 presents a new view of the tree of life. This is one of a
which are currently available in the Joint Genome Institute’s relatively small number of three-domain trees constructed from
Integrated Microbial Genomes database (accessed 24 September 2015). molecular information so far, and the first comprehensive tree to

Department of Earth and Planetary Science, UC Berkeley, Berkeley, California 94720, USA. 2 Department of Marine Science, University of Texas Austin,
Port Aransas, Texas 78373, USA. 3 Department of Plant and Microbial Biology, UC Berkeley, Berkeley, California 94720, USA. 4 Sector of Decommissioning
and Radioactive Wastes Management, Japan Atomic Energy Agency, Ibaraki 319-1184, Japan. 5 Graduate School of Science, The University of Tokyo,
Tokyo 113-8654, Japan. 6 Department of Ecology and Evolutionary Biology, UC Santa Cruz, Santa Cruz, California 95064, USA. 7 Departments of Medicine
and of Microbiology and Immunology, Stanford University, Stanford, California 94305, USA. 8 Veterans Affairs Palo Alto Health Care System, Palo Alto,
California 94304, USA. 9 Department of Environmental Science, Policy, and Management, UC Berkeley, Berkeley, California 94720, USA. †Present address:
Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. *e-mail:


© 2016 Macmillan Publishers Limited. All rights reserved

2 NATURE MICROBIOLOGY | VOL 1 | MAY 2016 | www.4 Gammaproteobacteria Micrarchaeota Diapherotrites Eukaryotes Nanohaloarchaeota Aenigmarchaeota Loki. The tree includes 92 named bacterial phyla. respectively. and are still in the process of definition at lower taxonomic levels. but not otherwise delineated due to the low resolution of these lineages. Latescibacteria Synergistetes Giovannonibacteria BRC1 Fusobacteria Wolfebacteria Melainabacteria Jorgensenbacteria Marinimicrobia RBX1 Bacteroidetes Ignavibacteria WOR1 Chlorobi Caldithrix Azambacteria PVC Parcubacteria superphylum Yanofskybacteria Planctomycetes Moranbacteria Elusimicrobia Chlamydiae. DPANN Korarch. encompassing the total diversity represented by sequenced genomes. in italics. Crenarch. The names Tenericutes and Thermodesulfobacteria are bracketed to indicate that these lineages branch within the Firmicutes and the Deltaproteobacteria. Lineages lacking an isolated representative are highlighted with non-italicized names and red dots. Magasanikbacteria Verrucomicrobia Uhrbacteria Falkowbacteria Candidate Omnitrophica Phyla Radiation SM2F11 Aminicentantes Rokubacteria NC10 Acidobacteria Peregrinibacteria Tectomicrobia.nature. Lentisphaerae. see Methods. Pacearchaeota Bathyarc. Parvarchaeota Thor.LETTERS NATURE MICROBIOLOGY DOI: 10. Eukaryotic supergroups are noted. The complete ribosomal protein tree is available in rectangular format with full bootstrap values as Supplementary Fig. Opisthokonta Altiarchaeales Halobacteria Z7ME43 Methanopyri Methanococci TACK Archaea Excavata Hadesarchaea Thermococci Thaumarchaeota Archaeplastida Methanobacteria Thermoplasmata Chromalveolata Archaeoglobi Methanomicrobia Amoebozoa Figure 1 | A current view of the tree of life. Nanoarchaeota YNPFFA Woesearchaeota Aigarch. For details on taxon sampling and tree inference. with well-characterized lineage © 2016 Macmillan Publishers Limited. Major lineages are assigned arbitrary colours and named. Acidithiobacillia Betaproteobacteria Major lineages with isolated representative: italics Major lineage lacking isolated representative: 0. All rights reserved . 1 and in Newick format in Supplementary Dataset 2.2016. Modulibacteria Gracilibacteria BD1-5.1038/NMICROBIOL.48 (Tenericutes) Bacteria Nomurabacteria Actinobacteria Armatimonadetes Kaiserbacteria Zixibacteria Atribacteria Adlerbacteria Cloacimonetes Aquificae Chloroflexi Campbellbacteria Fibrobacteres Calescamantes Gemmatimonadetes Caldiserica Firmicutes WOR-3 Dictyoglomi TA06 Thermotogae Cyanobacteria Poribacteria Deinococcus-Therm. The CPR phyla are assigned a single colour as they are composed entirely of organisms without isolated representatives. GN02 Nitrospinae Absconditabacteria SR1 Nitrospirae Saccharibacteria Dadabacteria Berkelbacteria Deltaprotebacteria (Thermodesulfobacteria) Chrysiogenetes Deferribacteres Hydrogenedentes NKB19 Spirochaetes Woesebacteria Shapirobacteria Wirthbacteria Amesbacteria TM6 Collierbacteria Epsilonproteobacteria Pacebacteria Beckwithbacteria Dojkabacteria WS6 Roizmanbacteria Gottesmanbacteria CPR1 Levybacteria CPR3 Daviesbacteria Microgenomates Katanobacteria Curtissbacteria Alphaproteobacteria WWE3 Zetaproteo. 26 archaeal phyla and all five of the Eukaryotic supergroups.

1038/NMICROBIOL. Methanobacteria. NC10. Acidobacteria Chrysiogenetes. Bacteroidetes Cloacamonetes Fibrobacteres Uncultured bacteria Elusimicrobia Uncultured bacteria (CP RIF1) O ph Omnitrophica Omnitrophica p Planctomycetes y Ch C Chlamydiae a yd Lentisphaerae p Verrucomicrobia Verrucomicrobia Modulibacteria. The threshold for groups (coloured wedges) was an average branch length of <0.. Jorgensenbacteria.48 LETTERS 0.. Kaiserbacteria Parcubacteria Yanofskybacteria P b i Parcubacteria SM2F11 te a Parcubacteria Parcubacteria Kuenenbacteria.. Tectomicrobia. Dadabacteria. 3 © 2016 Macmillan Publishers Limited. Wolfebacteria. Thermodesulfobacteria. Zetaproteobacteria. D Deferribacteres f b Alphaproteobacteria. Notably. Cand. Nanoarchaeota Archaea Woesearchaeota Altiarchaeales Z7ME43 E43 Methanopyri.NATURE MICROBIOLOGY DOI: 10.. 1 in which each major lineage represents the same amount of evolutionary distance. b t i M Melainabacteria l i b t i WOR-1 RBX-1 Firmicutes. Falkowbacteria. p . some well-accepted phyla become single groups and others are split into multiple distinct groups. Parcubacteria Nomurabacteria. Nitrospirae.nature.2016.. Rokubacteria.. Aq ifi . Collierbacteria Mi Microgenomates Sh i b i Shapirobacteria Mi Microgenomates t W Woesebacteria b Microgenomates Amesbacteria Mi Microgenomates g t LLevybacteria yb t i Microgenomates Gottesmanbacteria Microgenomatest R i b t i Roizmanbacteria Microgenomates Roizmanbacteria KAZAN CPR2. Thermoplasmata Uncultured Thermoplasmata p Thermoplasmata Opisthokonta. g Methanomicrobia. NATURE MICROBIOLOGY | VOL 1 | MAY 2016 | www. Armatimonadetes. Archaeplastida Chromalveolata.. Ignavibacteria. and do not propose the resulting groups to have special taxonomic status. Caldithrix. The complete ribosomal protein tree is available in rectangular format with full bootstrap values as Supplementary Fig. Chloroflexi. Halobacteria Aciduliprofundum. Poribacteria Latescibacteria WS3 Gemmatimonadetes. Methanococci. Thermococci Archaeoglobi. b M Marinimicrobia. Actinobacteria Fusobacteria. 1 and in Newick format in Supplementary Dataset 2. Bootstrap support values are indicated by circles on nodes—black for support of 85% and above. Tenericutes. Magasanikbacteria Parcubacteria Parcubacteria Parcubacteria Figure 2 | A reformatted view of the tree in Fig. Giovannonibacteria. The massive scale of diversity in the CPR and the large fraction of major lineages that lack isolated representatives (red dots) are apparent from this analysis. Nitrospinae. Deltaprot. Uhrbacteria. Chlorobi. All rights reserved . Pacebacteria. WOR-3. Calescamantes C EM19 Caldiserica. Betaproteobacteria. Amoebozoa Eukaryotes Thorarchaeota Th h t Lokiarchaeota Korarchaeota Crenarchaeota Crenarchaeota YNPFFA FFA Aigarchaeota. Caldiarchaeum subterraneum Thaumarchaeota Thaumarchaeota C Cyanobacteria. Campbellbacteria.65 substitutions per site. p . Adlerbacteria. We undertook this analysis to provide perspective on the structure of the tree. Gammaproteobacteria TM6 Epsilonproteobacteria Deltaproteobacteria b H Hydrogenedentes d d t N NKB19 Spirochaetes S Spirochaetes i h t Dojkabacteria WS6 CPR3 Katanobacteria WWE3 Candidate Phyla Radiation Katanobacteria WWE3 Katanobacteria WWE3 g Microgenomates Microgenomates g Curtissbacteria Microgenomates Daviesbacteria Microgenomates Beckwithbacteria. Dictyoglomi Atribacteria ((OP9)) BRC1. Nanoarchaeota Woesearchaeota. Hadesarchaea. Saccharibacteria TM7 Berkelbacteria Berkelbacteria Berkelbacteria Berkelbacteria CPR Uncultured unclassified bacteria Peregrinibacteria Peregrinibacteria Gracilibacteria BD1-5 / GNO2 Abs Absc Absconditabacteria SR1 Parcubacteria Parcubacteria Moranbacteria g Parcubacteria Azambacteria. Synergistetes y g Bacteria Uncultured bacteria (CP RIF32) Deinococcus-Thermus Thermotogae Aquificae.2 Diapherotrites Nanohaloarchaeota Bootstrap ≥ 85% Unclassified archaea Unclassified archaea 85% > Bootstrap ≥ 50% Pacearchaeota Woesearchaeota. TA06 Zixibacteria. grey for support from 50 to 84%. Excavata.

with the Korarchaeota.25. What emerges from analysis of this tree is the depth of Interestingly. The organisms were present in samples collected from a and single-cell genomics methods to date. 1. 34).011 newly sampled teristics that hint at an early metabolic platform for life. 2). the CPR. a group that includes protists.24 (ref. with moderate. Finally. 1 recapitulates expected organism groupings at genomics and single-cell genomics methods detect members of most taxonomic levels and is largely congruent with the tree calcu. see Methods). If inherited. We highlight all major lineages with genomic represen. but the branch. The lower apparent phylo- to-strong support for Phyla (>75% in most cases). (Supplementary Fig. L6.berkeley. as than the other Domains. in part which has the three-domain topology proposed by Woese and co.13. Thus far. All rights reserved . Genomes were additionally required to have consistent nucleotide composition and coverage across scaffolds. a group of parasitic fungi. the Archaea relative to Bacteria to sampling bias because meta- The tree in Fig. which appears to subdivide the domain. grassland meadow soil in northern California. From IMG-M.840 computational hours on the CIPRES supercomputer. genome.1. Taxa were removed if their available sequence data represented less than 50% of the expected alignment columns (90% of taxa had more than 80% of the in Fig. so that the main independently using MUSCLE v. Genomes were only included if they were estimated to be have been shown) to be symbionts7. 2 is the (PROTGAMMALG in the RAxML model section). Consistent with this view. The support values for taxonomic groups seawater27. both domains equally well. The concatenated ribosomal protein alignment was constructed as described vation by these organisms once more complex organisms appeared.48 be published since the development of genome-resolved meta.LETTERS NATURE MICROBIOLOGY DOI: 10. initial tree reconstructions lineages without isolated representatives (red dots in Fig. a deep subsurface research site in Japan. as implemented on many currently accepted phyla and larger values collapsed accepted the CIPRES web server36. fungi. Most workers in 19901 (Supplementary Fig. Genomes were reconstructed from metagenomes restricted metabolic capacities7. SSU rRNA gene sequences and more strongly resolves the deeper jgi. a previously developed data set of eukaryotic genome information30. 1 for full bootstrap support values).20). S3. the analysis highlights the large fraction of diversity tree and the three-domain tree are competing hypotheses for the that is currently only accessible via cultivation-independent origin of Eukarya5. driven by thermophilic. illustrating the progress that has been and animals. L3. S17 and S19) were aligned the tree are defined using evolutionary © 2016 Macmillan Publishers Limited. phylogenies. A striking feature of this tree is the large number of major thus removing strain. genomes were sampled such that a single representative for each defined genus was selected. the phylum is not monophyletic (for example. forming a final length to the leaf taxa. However. hydrothermal vents28. >70% complete based on presence/absence of a suite of 51 single copy genes for plete citric acid cycles and respiratory chains and most have limited Bacteria and 38 single copy genes for Archaea.2016. S8. Subsequently. but with groups defined on the basis of average branch expected alignment columns). further analyses to resolve these and other genome-resolved approaches.31 (ref. highlighted in purple in Fig. 1.doe. As proposed microbial lineages. organisms. symbiotic lifestyles. L18. The Microsporidia are known to of these lineages are clustered together into discrete regions of the contribute long branch attraction artefacts confounding placement of the Eukarya33. For phyla and genetic code23. This contributed marker gene information for 1. We chose an average branch length that alignment comprising 3. The two-domain Eocyte importantly. specifically within the made in the last two decades following the first published TACK superphylum21 and sibling to the Lokiarchaeota22.011 organisms from lineages for which genomes were from hundreds of genomes from genome-resolved metagenomics not previously available. A maximum best recapitulated the current taxonomy (smaller values fragmented likelihood tree was constructed using RAxML v. every member of the phylum was sequences for non-extremophile Archaea. 2). L24. 3. but not in the SSU rRNA tree (Supplementary tation. A total of enormous extent of evolution that has occurred within the CPR. this placement is not evident in the SSU rRNA tree.8.8. L14. branches within the Archaea. 2). where the major lineages of L4. Of particular note is the Candidate Phyla Radiation (CPR)7. The 16 alignments were concatenated. radiations. previously16. (Supplementary Fig. 1). In brief. Many identified aberrant long-branch attraction effects placing the Microsporidia. published separately. in combination Supplementary Fig. may allow clarification of these compositional biases. 1). 35). The lower support for deep branch place. tree.and species-level overlaps. all cells lack com. reconstructed genomes from current metagenome projects (see Supplementary mesophilic and halophilic lifestyles as well as by a primitive Table 1 for NCBI accession numbers). based on their ing order of the deepest branches cannot be confidently resolved comparatively recent evolution. due to the CPR.nature. 1). and two dolphin mouths. deep relationships will be strengthened with the availability of genomes for a greater diversity of organisms. L16.596 amino-acid positions. It remains determined using the ggkbase binning software (ggkbase. shallow aquifer system. The diversity within the CPR could be a result of the early emergence with 100 sampled to generate proportional support values. We do not attribute the smaller scope of previously reported2. plants genomic sampling of life. Regardless of branching order. these radiations were sampled to an approximate lineages8. Ribosomal proteins have been shown to contain Important advantages of the ribosomal protein tree compared with the SSU rRNA gene Methods A data set comprehensively covering the three domains of life was generated using tree are that it includes organisms with incomplete or unavailable publicly available genomes from the Joint Genome Institute’s IMG-M database (img.083 genomes and 2. such as the DPANN initially included. the Eukarya.31. The full tree inference of this group and/or a consequence of rapid evolution related to required 3. the terrestrial subsurface15 and are strong at the Species through Class levels (>85%).1038/NMICROBIOL. whose genomes were reconstructed for metabolic analyses to be then adoption of symbiotic lifestyles may have been a later inno. a CO2-rich geyser tively small genomes and most have somewhat (if not highly) system. This depiction of the tree captures the current recently.24. 2). because Fig. L5. Evident in Fig. the 16 ribosomal protein data sets (ribosomal proteins L2. Continued expansion of the number of genome candidate phyla lacking full taxonomic definition. all members have rela. evolutionary history that is contained within the Bacteria. a salt crust in the Atacama Desert.32 and newly sitional biases across the three domains. most of which are phylum-level branches (see Fig. human-associated microbiomes29).26. Alignments were trimmed to groups become apparent without bias arising from historical remove ambiguously aligned C and N termini as well as columns composed of more naming conventions. protein tree (Fig. S10. This depiction uses the same inferred tree as than 95% gaps. and to show unclear whether these reduced metabolisms are a consequence of consistent placement across both SSU rRNA and concatenated ribosomal protein superphylum-wide loss of capacities or if these are inherited charac. Figure 2 presents another perspective. 4 NATURE MICROBIOLOGY | VOL 1 | MAY 2016 | www. The tree of life as we know it has dramatically expanded due to ments is a consequence of our prioritization of taxon sampling new genomic sampling of previously enigmatic or unknown over number of genes used for tree construction. genetic diversity of Eukarya is fully expected. 8. The CPR is early-emerging on the ribosomal genomics. with other lineages that lack isolated representatives (red dots in we separately identify the Classes of the Proteobacteria.13. the Domain Bacteria includes more major lineages of organisms Deltaproteobacteria branch away from the other Proteobacteria. genus level of divergence based on comparison with taxonomically described phyla. Many are inferred (and some as described previously7. clearly comprises the majority of life’s current diversity. under the LG plus gamma model of evolution phyla into very few lineages. as or no ability to synthesize nucleotides and amino acids. L22. Based on information available This study includes 1. 156 bootstrap replicates were conducted under the rapid bootstrapping algorithm. previously published genomes derived from metagenomic data sets7. Archaea are lated using traditional SSU rRNA gene sequence information less prominent and less diverse in many ecosystems (for example. and were subsequently removed from the analysis. and with the number of bootstraps automatically determined (MRE-based bootstopping criterion).

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Seitz for sequence submission assistance. generated data sets and conducted reproduce the material.B. L. All authors read and approved the Acknowledgements final manuscript. N.A. available online at www. R.H.F. L.LETTERS NATURE MICROBIOLOGY DOI: 10.W. Correspondence and requests for materials should AC02-05CH11231.B. The images or other third party material in for generating the metagenome sequence via the Community Science Program. defined the research objective. Merigan Endowment at Stanford University. and J.J. In addition. Carlin and E. and J. Jensen (US Navy Marine Mammal Program) for dolphin This work is licensed under a Creative Commons Attribution 4.W. and by the Thomas C. Y.A. be addressed to J. Eisen for comments.. this article are included in the article's Creative Commons license.I. K. and the DOE Joint Genome Institute International License.B. conducted data analysis..C. B. funding was provided by the The authors declare no competing financial interests.C. Venn-Watson. DOE-SC10010566..N. Kantor.F. A.. L. et al. unless indicated otherwise in the credit line.48 phylogenetic tree inferences.B.P.A.0/ 6 NATURE MICROBIOLOGY | VOL 1 | MAY 2016 | www. K. D. 12-PLANET12R-0025 and NSF DEB grant and J.H.F.H.0 samples.. K. Supplementary information is available online.A. licenses/by/4.nature.. C.1038/NMICROBIOL. users will need to obtain permission from the license holder to L. if the material is not included under the Author contributions Creative Commons license.F. This research was largely supported by the Lawrence Berkeley National Laboratory (LBNL) Genomes to Watershed Scientific Focus Area funded by the US Department of Energy (DOE). N00014-07-1-0287.H. B.M. B. C..B.. Reprints and permissions information is DE-AC02-05CH11231.A.J. Office of Biological and Environmental Research (OBER) under contract no. S. K. contributed to metagenome binning and genome analysis. e00708–13 (2013). Small genomes and sparse metabolisms of sediment.B.B. Ministry of Economy. Trade and Industry of Japan. and Competing interests Joan M.2016. K. N00014-10-1-0233 and N00014-11-1-0918. DOE OBER grant no. L.A. 1406956. C. Additional support was provided by LBNL EFRC award no.F.A.F.. wrote the manuscript with input from all authors.. and J. Additional information Office of Science.nature. associated bacteria from four candidate phyla. The authors thank J.A. R.S... MBio 4.. Y. Office of Naval Research grants nos.J. A.B.T. To view a copy of this license. DE..H.