Forty Years of Inferential Methods in the Journals of the Society for Molecular Biology and Evolution.

We are launching a series to celebrate the 40th anniversary of the first issue of Molecular Biology and Evolution. In 2024, we will publish virtual issues containing selected papers published in the Society for Molecular Biology and Evolution journals, Molecular Biology and Evolution and Genome Biology and Evolution. Each virtual issue will be accompanied by a perspective that highlights the historic and contemporary contributions of our journals to a specific topic in molecular evolution. This perspective, the first in the series, presents an account of the broad array of methods that have been published in the Society for Molecular Biology and Evolution journals, including methods to infer phylogenies, to test hypotheses in a phylogenetic framework, and to infer population genetic processes. We also mention many of the software implementations that make methods tractable for empiricists. In short, the Society for Molecular Biology and Evolution community has much to celebrate after four decades of publishing high-quality science including numerous important inferential methods.

Old genes in new places: A taxon-rich analysis of interdomain lateral gene transfer events.

Vertical inheritance is foundational to Darwinian evolution, but fails to explain major innovations such as the rapid spread of antibiotic resistance among bacteria and the origin of photosynthesis in eukaryotes. While lateral gene transfer (LGT) is recognized as an evolutionary force in prokaryotes, the role of LGT in eukaryotic evolution is less clear. With the exception of the transfer of genes from organelles to the nucleus, a process termed endosymbiotic gene transfer (EGT), the extent of interdomain transfer from prokaryotes to eukaryotes is highly debated. A common critique of studies of interdomain LGT is the reliance on the topology of single-gene trees that attempt to estimate more than one billion years of evolution. We take a more conservative approach by identifying cases in which a single clade of eukaryotes is found in an otherwise prokaryotic gene tree (i.e. exclusive presence). Starting with a taxon-rich dataset of over 13,600 gene families and passing data through several rounds of curation, we identify and categorize the function of 306 interdomain LGT events into diverse eukaryotes, including 189 putative EGTs, 52 LGTs into Opisthokonta (i.e. animals, fungi and their microbial relatives), and 42 LGTs nearly exclusive to anaerobic eukaryotes. To assess differential gene loss as an explanation for exclusive presence, we compare branch lengths within each LGT tree to a set of vertically-inherited genes subsampled to mimic gene loss (i.e. with the same taxonomic sampling) and consistently find shorter relative distance between eukaryotes and prokaryotes in LGT trees, a pattern inconsistent with gene loss. Our methods provide a framework for future studies of interdomain LGT and move the field closer to an understanding of how best to model the evolutionary history of eukaryotes.

Foraminifera as a model of the extensive variability in genome dynamics among eukaryotes.

Knowledge of eukaryotic life cycles and associated genome dynamics stems largely from research on animals, plants, and a small number of "model" (i.e., easily cultivable) lineages. This skewed sampling results in an underappreciation of the variability among the many microeukaryotic lineages, which represent the bulk of eukaryotic biodiversity. The range of complex nuclear transformations that exists within lineages of microbial eukaryotes challenges the textbook understanding of genome and nuclear cycles. Here, we look in-depth at Foraminifera, an ancient (∼600 million-year-old) lineage widely studied as proxies in paleoceanography and environmental biomonitoring. We demonstrate that Foraminifera challenge the "rules" of life cycles developed largely from studies of plants and animals. To this end, we synthesize data on foraminiferal life cycles, focusing on extensive endoreplication within individuals (i.e., single cells), the unusual nuclear process called Zerfall, and the separation of germline and somatic function into distinct nuclei (i.e., heterokaryosis). These processes highlight complexities within lineages and expand our understanding of the dynamics of eukaryotic genomes.

Taxon-rich transcriptomics supports higher-level phylogeny and major evolutionary trends in Foraminifera.

Foraminifera, classified in the supergroup Rhizaria, are a common and highly diverse group of mainly marine protists. Despite their evolutionary and ecological importance, only limited genomic data (one partial genome and nine transcriptomic datasets) have been published for this group. Foraminiferal molecular phylogeny is largely based on 18S rRNA gene sequence analysis. However, due to highly variable evolutionary rates of substitution in ribosomal genes plus the existence of intragenomic variation at this locus, the relationships between and within foraminiferal classes remain uncertain. We analyze transcriptomic data from 28 species, adding 19 new species to the previously published dataset, including members of the strongly under-represented class Monothalamea. A phylogenomic reconstruction of Rhizaria, rooted with alveolates and stramenopiles, based on 199 genes and 68 species supports the monophyly of Foraminifera and their sister relationship to Polycystinea. The phylogenomic tree of Foraminifera is very similar to the 18S rRNA tree, with the paraphyletic single-chambered monothalamids giving rise to the multi-chambered Tubothalamea and Globothalamea. Within the Monothalamea, our analyses confirm the monophyly of the giant, deep-sea xenophyophores that branch within clade C and indicate the basal position of monothalamous clades D and E. The multi-chambered Globothalamea are monophyletic and comprise the paraphyletic Textulariida and monophyletic Rotaliida. Our phylogenomic analyses support major evolutionary trends of Foraminifera revealed by ribosomal phylogenies and reinforce their current higher-level classification.

Macronuclear development in ciliates, with a focus on nuclear architecture.

Ciliates are defined by the presence of dimorphic nuclei as they have both a somatic macronucleus and germline micronucleus within each individual cell. The size and structure of both germline micronuclei and somatic macronuclei vary tremendously among ciliates. Except just after conjugation (i.e. the nuclear exchange in their life cycle), the germline micronucleus is transcriptionally inactive and contains canonical chromosomes that will be inherited between generations. In contrast, the transcriptionally active macronucleus contains chromosomes that vary in size in different classes of ciliates, with some lineages having extensively fragmented gene-sized somatic chromosomes while others contain longer multigene chromosomes. Here, we describe the variation in somatic macronuclear architecture in lineages sampled across the ciliate tree of life, specifically focusing on lineages with extensively fragmented chromosomes (e.g. the classes Phyllopharyngea and Spirotrichea). Further, we synthesize information from the literature on the development of ciliate macronuclei, focusing on changes in nuclear architecture throughout life cycles. These data highlight the tremendous diversity among ciliate nuclear cycles, extend our understanding of patterns of genome evolution, and provide insight into different germline and somatic nuclear features (e.g. nuclear structure and development) among eukaryotes.

Epigenetic influences of mobile genetic elements on ciliate genome architecture and evolution.

Mobile genetic elements (MGEs) are transient genetic material that can move either within a single organism's genome or between individuals or species. While historically considered "junk" DNA (i.e., deleterious or at best neutral), more recent studies reveal the potential adaptive advantages MGEs provide in lineages across the tree of life. Ciliates, a group of single-celled microbial eukaryotes characterized by nuclear dimorphism, exemplify how epigenetic influences from MGEs shape genome architecture and patterns of molecular evolution. Ciliate nuclear dimorphism may have evolved as a response to transposon invasion and ciliates have since co-opted transposons to carry out programmed DNA deletion. Another example of the effect of MGEs is in providing mechanisms for lateral gene transfer (LGT) from bacteria, which introduces genetic diversity and, in several cases, may drive ecological specialization in ciliates. As a third example, the integration of viral DNA, likely through transduction, provides new genetic materials and can change the way host cells defend themselves against other viral pathogens. We argue that the acquisition of MGEs through non-Mendelian patterns of inheritance, coupled with their effects on ciliate genome architecture and persistence throughout evolutionary history, exemplify how the transmission of mobile elements should be considered a mechanism of transgenerational epigenetic inheritance.

Newly designed foraminifera primers identify habitat-specific lineages through metabarcoding analyses.

Foraminifera include diverse shell-building lineages found in a wide array of aquatic habitats from the deep-sea to intertidal zones to brackish and freshwater ecosystems. Recent estimates of morphological and molecular foraminifera diversity have increased the knowledge of foraminiferal diversity, which is critical as these lineages are used as bioindicators of past and present environmental perturbation. However, a comparative analysis of foraminiferal biodiversity between their major habitats (freshwater, brackish, intertidal, and marine) is underexplored, particularly using molecular tools. Here, we present a metabarcoding survey of foraminiferal diversity across different ecosystems using newly designed foraminifera-specific primers that target the hypervariable regions of the foraminifera SSU-rRNA gene (~250-300 bp long). We tested these primer sets on four foraminifera species and then across several environments: the intertidal zone, coastal ecosystems, and freshwater vernal pools. We retrieved 655 operational taxonomic units (OTUs); the majority of which are undetermined taxa that have no closely matching sequences in the reference database. Furthermore, we identified 163 OTUs with distinct habitat preferences. Most of the observed OTUs belonged to lineages of single-chambered foraminifera, including poorly explored freshwater foraminifera which encompass a clade of Reticulomyxa-like forms. Our pilot study provides the community with an additional set of newly designed and taxon-specific primers to elucidate foraminiferal diversity across different habitats.

Opinion: Genetic Conflict With Mobile Elements Drives Eukaryotic Genome Evolution, and Perhaps Also Eukaryogenesis.

Through analyses of diverse microeukaryotes, we have previously argued that eukaryotic genomes are dynamic systems that rely on epigenetic mechanisms to distinguish germline (i.e., DNA to be inherited) from soma (i.e., DNA that undergoes polyploidization, genome rearrangement, etc.), even in the context of a single nucleus. Here, we extend these arguments by including two well-documented observations: (1) eukaryotic genomes interact frequently with mobile genetic elements (MGEs) like viruses and transposable elements (TEs), creating genetic conflict, and (2) epigenetic mechanisms regulate MGEs. Synthesis of these ideas leads to the hypothesis that genetic conflict with MGEs contributed to the evolution of a dynamic eukaryotic genome in the last eukaryotic common ancestor (LECA), and may have contributed to eukaryogenesis (i.e., may have been a driver in the evolution of FECA, the first eukaryotic common ancestor). Sex (i.e., meiosis) may have evolved within the context of the development of germline-soma distinctions in LECA, as this process resets the germline genome by regulating/eliminating somatic (i.e., polyploid, rearranged) genetic material. Our synthesis of these ideas expands on hypotheses of the origin of eukaryotes by integrating the roles of MGEs and epigenetics.

High Diversity of Testate Amoebae (Amoebozoa, Arcellinida) Detected by HTS Analyses in a New England Fen using Newly Designed Taxon-specific Primers.

Testate (shell-building) amoebae, such as the Arcellinida (Amoebozoa), are useful bioindicators for climate change. Though past work has relied on morphological analyses to characterize Arcellinida diversity, genetic analyses revealed the presence of multiple cryptic species underlying morphospecies. Here, we design and deploy Arcellinida-specific primers for the SSU-rDNA gene to assess the community composition on the molecular level in a pilot study of two samplings from a New England fen: (1) 36-cm horizontal transects and vertical cores; and (2) 26-m horizontal transects fractioned into four size classes (2-10, 10-35, 35-100, and 100-300 µm). Analyses of these data show the following: (1) a considerable genetic diversity within Arcellinida, much of which comes from morphospecies lacking sequences on GenBank; (2) communities characterized by DNA (i.e. active + quiescent) are distinct from those characterized by RNA (i.e. active, indicator of biomass); (3) active communities on the surface tend to be more similar to one another than to core communities, despite considerable heterogeneity; and (4) analyses of communities fractioned by size find some lineages (OTUs) that are abundant in disjunct size categories, suggesting the possibility of life-history stages. Together, these data demonstrate the potential of these primers to elucidate the diversity of Arcellinida communities in diverse habitats.

De novo Sequencing, Assembly, and Annotation of the Transcriptome for the Free-Living Testate Amoeba Arcella intermedia.

Arcella, a diverse understudied genus of testate amoebae is a member of Tubulinea in Amoebozoa group. Transcriptomes are a powerful tool for characterization of these organisms as they are an efficient way of characterizing the protein-coding potential of the genome. In this work, we employed both single-cell and clonal populations transcriptomics to create a reference transcriptome for Arcella. We compared our results with annotations of Dictyostelium discoideum, a model Amoebozoan. We assembled a pool of 38 Arcella intermedia transcriptomes, which after filtering are composed of a total of 14,712 translated proteins. There are GO categories enriched in Arcella including mainly intracellular signal transduction pathways; we also used KEGG to annotate 11,546 contigs, which also have similar distribution to Dictyostelium. A large portion of data is still impossible to assign to a gene family, probably due to a combination of lineage-specific genes, incomplete sequences in the transcriptome and rapidly evolved genes. Some absences in pathways could also be related to low expression of these genes. We provide a reference database for Arcella, and we highlight the emergence of the need for further gene discovery in Arcella.

Microbial Diversity in the Eukaryotic SAR Clade: Illuminating the Darkness Between Morphology and Molecular Data.

Despite their diversity and ecological importance, many areas of the SAR-Stramenopila, Alveolata, and Rhizaria-clade are poorly understood as the majority (90%) of SAR species lack molecular data and only 5% of species are from well-sampled families. Here, we review and summarize the state of knowledge about the three major clades of SAR, describing the diversity within each clade and identifying synapomorphies when possible. We also assess the "dark area" of SAR: the morphologically described species that are missing molecular data. The majority of molecular data for SAR lineages are characterized from marine samples and vertebrate hosts, highlighting the need for additional research effort in areas such as freshwater and terrestrial habitats and "non-vertebrate" hosts. We also describe the paucity of data on the biogeography of SAR species, and point to opportunities to illuminate diversity in this major eukaryotic clade. See also the video abstract here: https://youtu.be/_VUXqaX19Rw.

The concept of the hologenome, an epigenetic phenomenon, challenges aspects of the modern evolutionary synthesis.

John Tyler Bonner's call to re-evaluate evolutionary theory in light of major transitions in life on Earth (e.g., from the first origins of microbial life to the evolution of sex, and the origins of multicellularity) resonate with recent discoveries on epigenetics and the concept of the hologenome. Current studies of genome evolution often mistakenly focus only on the inheritance of DNA between parent and offspring. These are in line with the widely accepted Neo-Darwinian framework that pairs Mendelian genetics with an emphasis on natural selection as explanations for the evolution of biodiversity on Earth. Increasing evidence for widespread symbioses complicates this narrative, as is seen in Scott Gilbert's discussion of the concept of the holobiont in this series: Organisms across the tree of life coexist with substantial influence on one another through endosymbiosis, symbioses, and host-associated microbiomes. The holobiont theory, coupled with observations from molecular studies, also requires us to understand genomes in a new way-by considering the interactions underlain by the genome of a host plus its associated microbes, a conglomerate entity referred to as the hologenome. We argue that the complex patterns of inheritance of these genomes coupled with the influence of symbionts on host gene expression make the concept of the hologenome an epigenetic phenomenon. We further argue that the aspects of the hologenome challenge of the modern evolutionary synthesis, which requires updating to remain consistent with Darwin's intent of providing natural laws that underlie the evolution of life on Earth.

John Tyler Bonner: Remembering a scientific pioneer.

Throughout his life, John Tyler Bonner contributed to major transformations in the fields of developmental and evolutionary biology. He pondered the evolution of complexity and the significance of randomness in evolution, and was instrumental in the formation of evolutionary developmental biology. His contributions were vast, ranging from highly technical scientific articles to numerous books written for a broad audience. This historical vignette gathers reflections by several prominent researchers on the greatness of John Bonner and the implications of his work.

Ubiquity or not ubiquity: That is the question.

The nature of population structure in eukaryotic microbes has been the subject of intense debate, but until recently the tools to test these hypotheses were either problematic (e.g., allozymes that cannot detect all genetic changes) or beyond financial and technological limits of most laboratories (e.g., high throughput sequencing). In a recent issue of Molecular Ecology, Craig et al. (2019) use a genomic approach to investigate the population structure of a model alga, the chlorophyte Chlamydomonas reinhardtii (Figure 1). Using high throughput sequencing, read mapping, and variant calling, they detected strong signals of differentiation at a continental scale, while local patterns of admixture were complex. Population genomic techniques such as these have not been used extensively in studies of microbial eukaryotes and the fields of conservation genetics and evolution stand to benefit vastly from the adoption of these techniques to studies of diverse protist lineages.

Nuclear Features of the Heterotrich Ciliate Blepharisma americanum: Genomic Amplification, Life Cycle, and Nuclear Inclusion.

Blepharisma americanum, a member of the understudied ciliate class Heterotrichea, has a moniliform somatic macronucleus that resembles beads on a string. Blepharisma americanum is distinguishable by its pink coloration derived from the autofluorescent pigment blepharismin and tends to have a single somatic macronucleus with 3-6 nodes and multiple germline micronuclei. We used fluorescence confocal microscopy to explore the DNA content and amplification between the somatic and germline nuclei of B. americanum through its life cycle. We estimate that the DNA content of the macronucleus and micronucleus are 43 ± 8 Gbp and 83 ± 16 Mbp respectively. This correlates with an approximate DNA content difference of 500-fold from micronucleus to macronucleus and a macronuclear ploidy of ~1,100 N as compared to the presumably diploid micronucleus. We also investigate a previously reported macronuclear inclusion, which is present sporadically across all life cycle stages; this inclusion looks as if it contains blepharismin based on its fluorescent properties, but its function remains unknown. We also provide additional detail to our understanding of life cycles changes in B. americanum by analyses of fluorescent images. Overall, the data analyzed here contribute to our understanding of the diversity of nuclear architecture in ciliates by providing details on the highly polyploid somatic macronucleus of B. americanum.

Synthesis of phylogeny and taxonomy into a comprehensive 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.

Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error.

Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: Halteria grandinella, Blepharisma americanum and Strombidium stylifer We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of Halteria grandinella is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size.

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.

Unexpected biodiversity of ciliates in marine samples from below the photic zone.

Marine microbial eukaryotes play critical roles in planktonic food webs and have been described as most diverse in the photic zone where productivity is high. We used high-throughput sequencing (HTS) to analyse the spatial distribution of planktonic ciliate diversity from shallow waters (<30 m depth) to beyond the continental shelf (>800 m depth) along a 163 km transect off the coast of New England, USA. We focus on ciliates in the subclasses Oligotrichia and Choreotrichia (class Spirotrichea), as these taxa are major components of marine food webs. We did not observe the decrease of diversity below the photic zone expected based on productivity and previous analyses. Instead, we saw an increase of diversity with depth. We also observed that the ciliate communities assessed by HTS cluster by depth layer and degree of water column stratification, suggesting that community assembly is driven by environmental factors. Across our samples, abundant OTUs tend to match previously characterized morphospecies while rare OTUs are more often undescribed, consistent with the idea that species in the rare biosphere remain to be characterized by microscopy. Finally, samples taken below the photic zone also reveal the prevalence of two uncharacterized (i.e. lacking sequenced morphospecies) clades - clusters X1 and X2 - that are enriched within the nano-sized fraction (2-10 µm) and are defined by deletions within the region of the SSU-rDNA analysed here. Together, these data reinforce that we still have much to learn about microbial diversity in marine ecosystems, especially in deep-waters that may be a reservoir for rare species and uncharacterized taxa.

Phylogenomics of 'Discosea': A new molecular phylogenetic perspective on Amoebozoa with flat body forms.

The majority of amoeboid lineages with flattened body forms are placed under a taxonomic hypothetical class 'Discosea' sensu Smirnov et al. (2011), which encompasses some of the most diverse morphs within Amoebozoa. However, its taxonomy and phylogeny is poorly understood. This is partly due to lack of support in studies that are based on limited gene sampling. In this study we use a phylogenomic approach including newly-generated RNA-Seq data and comprehensive taxon sampling to resolve the phylogeny of 'Discosea'. Our analysis included representatives from all orders of 'Discosea' and up to 550 genes, the largest gene sampling in Amoebozoa to date. We conducted extensive analyses to assess the robustness of our resulting phylogenies to effects of missing data and outgroup choice using probabilistic methods. All of our analyses, which explore the impact of varying amounts of missing data, consistently recover well-resolved and supported groups of Amoebozoa. Our results neither support the monophyly nor dichotomy of 'Discosea' as defined by Smirnov et al. (2011). Rather, we recover a robust well-resolved clade referred to as Eudiscosea encompassing the majority of discosean orders (seven of the nine studied here), while the Dactylopodida, Thecamoebida and Himatismenida, previously included in 'Discosea,' are non-monophyletic. We also recover novel relationships within the Eudiscosea that are largely congruent with morphology. Our analyses enabled us to place some incertae sedis lineages and previously unstable lineages such as Vermistella, Mayorella, Gocevia, and Stereomyxa. We recommend some phylogeny-based taxonomic amendments highlighting the new findings of this study and discuss the evolution of the group based on our current understanding.