Laura A. Katz

Estimating the timing of early eukaryotic diversification with multigene molecular clocks

Although macroscopic plants, animals, and fungi are the most familiar eukaryotes, the bulk of eukaryotic diversity is microbial. Elucidating the timing of diversification among the more than 70 lineages is key to understanding the evolution of eukaryotes. Here, we use taxon-rich multigene data combined with diverse fossils and a relaxed molecular clock framework to estimate the timing of the last common ancestor of extant eukaryotes and the divergence of major clades. Overall, these analyses suggest that the last common ancestor lived between 1866 and 1679 Ma, consistent with the earliest microfossils interpreted with confidence as eukaryotic. During this interval, the Earths surface differed markedly from today; for example, the oceans were incompletely ventilated, with ferruginous and, after about 1800 Ma, sulfidic water masses commonly lying beneath moderately oxygenated surface waters. Our time estimates also indicate that the major clades of eukaryotes diverged before 1000 Ma, with most or all probably diverging before 1200 Ma. Fossils, however, suggest that diversity within major extant clades expanded later, beginning about 800 Ma, when the oceans began their transition to a more modern chemical state. In combination, paleontological and molecular approaches indicate that long stems preceded diversification in the major eukaryotic lineages.

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing.

Current sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the worlds oceans.

Synthesis of phylogeny and taxonomy into a comprehensive tree of life

Significance Scientists have used gene sequences and morphological data to construct tens of thousands of evolutionary trees that describe the evolutionary history of animals, plants, and microbes. This study is the first, to our knowledge, to apply an efficient and automated process for assembling published trees into a complete tree of life. This tree and the underlying data are available to browse and download from the Internet, facilitating subsequent analyses that require evolutionary trees. The tree can be easily updated with newly published data. Our analysis of coverage not only reveals gaps in sampling and naming biodiversity but also further demonstrates that most published phylogenies are not available in digital formats that can be summarized into a tree of life. Reconstructing the phylogenetic relationships that unite all lineages (the tree of life) is a grand challenge. The paucity of homologous character data across disparately related lineages currently renders direct phylogenetic inference untenable. To reconstruct a comprehensive tree of life, we therefore synthesized published phylogenies, together with taxonomic classifications for taxa never incorporated into a phylogeny. We present a draft tree containing 2.3 million tips—the Open Tree of Life. Realization of this tree required the assembly of two additional community resources: (i) a comprehensive global reference taxonomy and (ii) a database of published phylogenetic trees mapped to this taxonomy. Our open source framework facilitates community comment and contribution, enabling the tree to be continuously updated when new phylogenetic and taxonomic data become digitally available. Although data coverage and phylogenetic conflict across the Open Tree of Life illuminate gaps in both the underlying data available for phylogenetic reconstruction and the publication of trees as digital objects, the tree provides a compelling starting point for community contribution. This comprehensive tree will fuel fundamental research on the nature of biological diversity, ultimately providing up-to-date phylogenies for downstream applications in comparative biology, ecology, conservation biology, climate change, agriculture, and genomics.

Evaluating support for the current classification of eukaryotic diversity.

Perspectives on the classification of eukaryotic diversity have changed rapidly in recent years, as the four eukaryotic groups within the five-kingdom classification—plants, animals, fungi, and protists—have been transformed through numerous permutations into the current system of six “supergroups.” The intent of the supergroup classification system is to unite microbial and macroscopic eukaryotes based on phylogenetic inference. This supergroup approach is increasing in popularity in the literature and is appearing in introductory biology textbooks. We evaluate the stability and support for the current six-supergroup classification of eukaryotes based on molecular genealogies. We assess three aspects of each supergroup: (1) the stability of its taxonomy, (2) the support for monophyly (single evolutionary origin) in molecular analyses targeting a supergroup, and (3) the support for monophyly when a supergroup is included as an out-group in phylogenetic studies targeting other taxa. Our analysis demonstrates that supergroup taxonomies are unstable and that support for groups varies tremendously, indicating that the current classification scheme of eukaryotes is likely premature. We highlight several trends contributing to the instability and discuss the requirements for establishing robust clades within the eukaryotic tree of life.

Diversity and geographic distribution of ciliates (Protista: Ciliophora)

About 4,500 free-living ciliate morphospecies have been described, applying an average synonymy rate of 20%. We estimate that 83–89% of the ciliate diversity is still undescribed, using the following probabilities: detailed habitat studies suggest that the described number of morphospecies must be doubled: 4,500 → 9,000; this figure has to be increased by about 50% due to species with similar interphase morphology but different resting cysts: 9,000 → 13,500; the genetic and molecular data suggest that this value must be doubled or trebled: 13,500 → 27,000 to 40,000 free-living, biological ciliate species. The knowledge on geographic distribution of ciliates heavily depends on flagship species and statistical analyses because reliable faunistic studies are rare and molecular data are still in its infancy. We present a list of 52 ciliate flagship species as a testable hypothesis, i.e., the hypothesis of restricted distribution of certain ciliate species must be refused when a considerable number of them is found in all or most biogeographic regions. Flagship species and statistical analyses consistently show Gondwanan and Laurasian ciliate communities, suggesting that the split of Pangaea deeply influenced ciliate distribution and rare species play a key role in geographic differentiation. However, there is also substantial evidence for continental, regional, and local endemism of free-living ciliates. The molecular studies usually show a high level of genetic diversity underlying ciliate morphospecies, suggesting that morphologic and molecular evolution may be decoupled in many ciliate species. Molecular studies on ciliate biogeography are at variance, possibly because most are still focusing on single molecular markers. In sum, the data indicate that ciliate biogeography is similar to that of plants and animals, but with an increased proportion of cosmopolites, favouring the moderate endemicity model.

Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase

PCR-mediated recombination can greatly impact estimates of diversity, both in environmental studies and in analyses of gene family evolution. Here we measure chimera (PCR-mediated recombinant) formation by analyzing a mixture of eight partial actin sequences isolated from the amoeba Arcella hemisphaerica amplified under a variety of conditions that mimic standard laboratory situations. We further compare a new-generation proofreading processivity-enhanced polymerase to both a standard proofreading enzyme and previously published results. Proofreading polymerases are preferred over other polymerases in instances where evolutionary inferences must be made. Our analyses reveal that reducing the initial template concentration is as critical as reducing the number of cycles for decreasing chimera formation and improving accuracy. Furthermore, assessing the efficiency of recovery of original haplotypes demonstrates that multiple PCR reactions are required to capture the actual genetic diversity of a sample. Finally, the experiments confirm that processivity-enhanced polymerases enable a substantial decrease in PCR-mediated recombination through reducing starting template concentration, without compromising the robustness of PCR reactions.

Insights into the diversity of choreotrich and oligotrich ciliates (Class: Spirotrichea) based on genealogical analyses of multiple loci.

To examine relationships among spirotrich ciliates using multi-locus sequence analyses and to provide preliminary insights into molecular diversity within species, we sequenced the small subunit rDNA (SSU rDNA), 5.8S rDNA, alpha-tubulin and the internally transcribed spacer regions (ITS1 and ITS2) of the rDNA genes from seven choreotrich (Class: Spirotrichea) and three oligotrich (Class: Spirotrichea) taxa. Genealogies constructed from SSU rDNA and ITS sequences are concordant and broadly support current classifications based on morphology. The one exception is the freshwater oligotrich Halteria grandinella, which, as has been previously noted, falls outside of the clade containing the other oligotrichs. In contrast, analyses of alpha-tubulin sequences are discordant with traditional taxonomy and rDNA genealogies. These analyses also indicate that considerably more genetic variation exists among choreotrich and oligotrich genera than among stichotrich genera. To explore the level of genetic variation among individuals in temporally isolated populations, we collected additional samples of a subset of planktonic choreotrichs and oligotrichs and characterized polymorphisms in ITS1, ITS2 and 5.8S rDNA. Analyses of these data indicate that, at least for some ciliate lineages, DNA polymorphisms vary temporally, and that genetic heterogeneity underlies some very similar morphological types.

Comprehensive Phylogenetic Reconstruction of Amoebozoa Based on Concatenated Analyses of SSU-rDNA and Actin Genes

Evolutionary relationships within Amoebozoa have been the subject of controversy for two reasons: 1) paucity of morphological characters in traditional surveys and 2) haphazard taxonomic sampling in modern molecular reconstructions. These along with other factors have prevented the erection of a definitive system that resolves confidently both higher and lower-level relationships. Additionally, the recent recognition that many protosteloid amoebae are in fact scattered throughout the Amoebozoa suggests that phylogenetic reconstructions have been excluding an extensive and integral group of organisms. Here we provide a comprehensive phylogenetic reconstruction based on 139 taxa using molecular information from both SSU-rDNA and actin genes. We provide molecular data for 13 of those taxa, 12 of which had not been previously characterized. We explored the dataset extensively by generating 18 alternative reconstructions that assess the effect of missing data, long-branched taxa, unstable taxa, fast evolving sites and inclusion of environmental sequences. We compared reconstructions with each other as well as against previously published phylogenies. Our analyses show that many of the morphologically established lower-level relationships (defined here as relationships roughly equivalent to Order level or below) are congruent with molecular data. However, the data are insufficient to corroborate or reject the large majority of proposed higher-level relationships (above the Order-level), with the exception of Tubulinea, Archamoebae and Myxogastrea, which are consistently recovered. Moreover, contrary to previous expectations, the inclusion of available environmental sequences does not significantly improve the Amoebozoa reconstruction. This is probably because key amoebozoan taxa are not easily amplified by environmental sequencing methodology due to high rates of molecular evolution and regular occurrence of large indels and introns. Finally, in an effort to facilitate future sampling of key amoebozoan taxa, we provide a novel methodology for genome amplification and cDNA extraction from single or a few cells, a method that is culture-independent and allows both photodocumentation and extraction of multiple genes from natural samples.

Origin and Diversification of Eukaryotes

The bulk of the diversity of eukaryotic life is microbial. Although the larger eukaryotes-namely plants, animals, and fungi-dominate our visual landscapes, microbial lineages compose the greater part of both genetic diversity and biomass, and contain many evolutionary innovations. Our understanding of the origin and diversification of eukaryotes has improved substantially with analyses of molecular data from diverse lineages. These data have provided insight into the nature of the genome of the last eukaryotic common ancestor (LECA). Yet, the origin of key eukaryotic features, namely the nucleus and cytoskeleton, remains poorly understood. In contrast, the past decades have seen considerable refinement in hypotheses on the major branching events in the evolution of eukaryotic diversity. New insights have also emerged, including evidence for the acquisition of mitochondria at the time of the origin of eukaryotes and data supporting the dynamic nature of genomes in LECA.

The chastity of amoebae: re-evaluating evidence for sex in amoeboid organisms

Amoebae are generally assumed to be asexual. We argue that this view is a relict of early classification schemes that lumped all amoebae together inside the ‘lower’ protozoa, separated from the ‘higher’ plants, animals and fungi. This artificial classification allowed microbial eukaryotes, including amoebae, to be dismissed as primitive, and implied that the biological rules and theories developed for macro-organisms need not apply to microbes. Eukaryotic diversity is made up of 70+ lineages, most of which are microbial. Plants, animals and fungi are nested among these microbial lineages. Thus, theories on the prevalence and maintenance of sex developed for macro-organisms should in fact apply to microbial eukaryotes, though the theories may need to be refined and generalized (e.g. to account for the variation in sexual strategies and prevalence of facultative sex in natural populations of many microbial eukaryotes). We use a revised phylogenetic framework to assess evidence for sex in several amoeboid lineages that are traditionally considered asexual, and we interpret this evidence in light of theories on the evolution of sex developed for macro-organisms. We emphasize that the limited data available for many lineages coupled with natural variation in microbial life cycles overestimate the extent of asexuality. Mapping sexuality onto the eukaryotic tree of life demonstrates that the majority of amoeboid lineages are, contrary to popular belief, anciently sexual, and that most asexual groups have probably arisen recently and independently. Additionally, several unusual genomic traits are prevalent in amoeboid lineages, including cyclic polyploidy, which may serve as alternative mechanisms to minimize the deleterious effects of asexuality.