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2001 Annual Science Report

Marine Biological Laboratory Reporting  |  JUL 2000 – JUN 2001

Executive Summary

Executive Summary — MBL (dm)

The evolution and survival of Earth’s biota would not be possible without single-cell organisms. For nearly 90% of this planet’s history, microbes were the only kind of life. These organisms dominate every corner of our biosphere, and they are likely to be the only life-forms that might be encountered in other parts of our solar system if not the entire cosmos. There is only sparse information about the true diversity of microorganisms and how they orchestrate and drive key bio-geochemical cycles that shape our ever-changing planet. We are only now beginning to appreciate the tremendous diversity of microorganisms, which dwarfs animal/plant conventions. A more complete understanding of microbial diversity and evolution, descriptions of ecosystem-wide patterns of gene expression, and detailed analyses of biogeochemistry would provide a new foundation for interpreting paleontological and geological studies that describe Earth’s early history. In addition to contributing towards the design of life-detection experiments, information about microbial evolution and their limits of habitability provides guidance for seeking extraterrestrial life.

The MBL Astrobiology team employs molecular techniques to explore how relatively simple organisms and their genomes (compared to those of metazoans) evolved into more complex forms. We are united by questions that parallel the evolutionary biology goals in the Astrobiology Roadmap: Where did we come from? How did we get here? and Where are we going? We seek to understand how multi-member gene families and entire genomes have evolved, how evolution of genotype is related to changes phenotype, how processes other than simple mutation influence evolution of life on earth e.g. endosymbiosis, how microbial lineages have adapted to extreme environments and how eukaryotes originated and evolved in to complex, multicellular forms. Our investigations span the levels of individual genes, genomes, cells, populations, communities and entire ecosystems.

An important goal for projects that investigate evolution of biological complexity is to develop an understanding of protist diversity and a robust phylogeny for all eukaryotes. To address these questions we have taken advantage of the remarkable conservation of both small subunit (SSU) and large subunit (LSU) ribosomal RNA sequences to infer eukaryotic phylogenies. This work addresses two important questions. 1) What is the identity of early diverging eukaryotic lineages? and 2) What is the identify of protist groups that were ancestral to the metazoans? The rRNA analyses are complemented by studies of other genes including alpha-tubulins, beta-tubulins and fumerases. One of our guiding hypotheses is based upon SSU rRNA trees, which show the earliest diverging eukaryotes lack mitochondria and peroxisomes. In nearly all examples, early diverging eukaryotic lineages in rRNA trees are represented by organisms of medical relevance.

Since it is unlikely that clinicians would have discovered the origins of eukaryotes, an important goal of the MBL astrobiology program is to search for novel eukaryhote diversity in rarely studied environments, some of which may resemble conditions that could have existed millions/billions of years ago on other solar system bodies. We use molecular techniques to explore protist diversity in extreme environments including sedimentary cores from the Guaymas hydrothermal vent environment, the iron-rich, acidic environment of the Rio Tinto in southwestern Spain, and in marine and freshwater anoxic sediments. Rio Tinto is of particular interest because in many ways it may represent an analogous environment to early martian conditions when liquid water might have been present. In support of these investigations and as part of a very significant expansion of our outreach program, we have also developed a new WEB site titled micro*scope ( The site provides an image-rich means to access and identify microbial diversity. The following are highlights from these investigations.

â?¢ Analysis of rRNAs show that the absence of organelles occurs in both deep branching and more recently diverged eukaryotic lineages. Such losses have occurred independently several times.

â?¢ Based upon comparisons of both rRNAs and tubulins, the excavate taxa including diplomonads and other species that possess similar ultrastructure features are not a monophyletic assemblage.

â?¢ Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin genes place the excavate taxon Carpediemonas as a close relative of diplomonads (and with SSUrRNA, retortamonads), to the exclusion of all other eukaryotes. This novel relationship is recovered across phylogenetic methods and various taxon-deletion experiments and under different SSUrRNA alignments.

â?¢ Phylogenetic analaysis of combined data sets from both small subunit and large subunit ribosomal RNA sequences show that Bilateria are not more closely related to the Ctenophora than to the Cnidaria. The Bilateria most likely are the sister group to the Silicea.

â?¢ The molecular survey of eukaryotic microbial diversity in a hydrothermal vent environment shows that these anoxic sediments and the overlying seawater harbor a mixture of genetically diverse protists. Many sequence isolates represent novel protists, including early branching eukaryotic lineages or extended diversity within described taxa.

â?¢ At least two mechanisms, with overlapping consequences, account for the eukaryotic community structure of this environment; adaptation to warm anoxic environments, and wide distribution of aerophilic protists in marine environments, some of which may migrate into and survive in the sediment, while others e.g. phototrophs, are simply deposited by sedimentary processes.

â?¢ In contrast to most environments, eukaryotes micro organisms rather than prokaryotes dominate parts of Rio Tinto.

â?¢ Microscope, a comprehensive WEB site for microbial diversity, was released for public use in May, 2001.

â?¢ Our investigations of eukaryotic microbial diversity in the hydrothermal sediments of the Guaymas Basin (Gulf of California, Mexico) are complemented by 13C isotopic analysis of archaeal and bacterial lipids studies and molecular diversity surveys based upon comparisons of 16S rRNAs. This allows us to correlate community composition with modes of nutrition. A major lineage of uncultured euryarchaeota (ANME-1 including ANME-1b), and uncultured members (ANME-2) of the Methanosarcinales, the acetoclastic and methyl-disproportionating methanogens dominate this ecosystem. There are also associated bacterial populations that are predominantly made up of gamma, delta and epsilon-Proteobacteria, green non-sulfur bacteria, and the uncultured candidate subdivision OP11.

â?¢ We have identified a methanotrophic microbial consortium at a hydrothermal vent site that utilizes geothermal and biogenic methane.

â?¢ In contract to oxygen-respiring, sulfide-oxidizing hydrothermal vent microbes that are commonly the basis of the hydrothermal vent food chain, the archaeal/bacterial consortium in Guaymas thrives without oxygen and requires only sulfate as oxidant.

The projects on phootosymbioses and studies of genetic changes to phenotypic are progressing. Both of these efforts required field trips to obtain samples for analysis. Gast has isolated several cDNA clones that may be important in formation of photosymbiotic algal relationships but more experiments are required before reporting definitive results. Cummings’ study of opsin evolution has made substantial progress in both the analytical areas and data acquisition. Sequences are in progress or have been completed for 35 opsin cDNA clones and RNA samples have been prepared for more than 60 species. In anticipation of a large data base of opsin clones, Cummings has made progess in the implementation of computationally demanding methods of sequence analysis for linking phenotypic change with genotypic evolution. He has completed the largest and most comprehensive study of spectral tuning in vertebrate color vision. Furthermore this work has demonstrated the successful application of novel analytical techniques related to those that have been applied to space shuttle landing to genotype-phenotype relationships. Highlights include:

â?¢ Few of the hundreds of variable amino acid positions in vertebrate opsin have an influence of wavelength of light absorbed

â?¢ Several positions appear to be equivalent, or indistinguishable because they covary, with regard to their influence on spectral tuning

â?¢ Most of the variance in spectral tuning can be explained by 2 or three amino acid changes

Riley’s evolution of protein group has been very active over the course of the past year with the publication of six manuscripts. They have been exploring the link between genotype and phenotype evolution through studies of related gene families in E. coli. Through these analyses the Riley group has identified a potential role of gene fusion in evolution which may be extremely important in the evolution of genomic complexity. Gene fusions may be responsible for achieving efficiency in localizing related reactions in physical proximit, not in equiplibrium with the aqueous phase of the cell. Highlights include:

â?¢ The function of 81% of the gene in E. coli have been identified (one year ago the functions of 1/3 of the E. coli coding regions were unknown)

â?¢ Multimodular genes are multifunctional and define physiologically related activities.

Finally, we have provided a report on progress of the Ecogenomics Focus Group (not just the MBL-related activities). Beyond our contributions to the Ecogenomics focus group organization activities and meetings, the MBL group has restricted its efforts to constructing libraries for proxy cyanobacterial cultures and making strategic decisions about equipment and methodology that will be employed for analyzing gene expression patterns throughout the hypersaline mat cores. In the very near future, samples returned from the field site will provide a second proxy culture for a sulfate reducing species. Because of the delay in reaching the field site, this project is just getting underway. Highlights for the Ecogenomics Focus Group include:

â?¢ The NAI Ecogenomics Focus Group has initiated a comprehensive sampling of biogeochemical processes and microbial diversity in the hypersaline mats of the Guerrero Negro.

â?¢ The Keck Foundation awarded funds to the MBL for the construction of an advanced laboratory of ecological and evolutionary genomics.