Phylotranscriptomics points to multiple independent origins of multicellularity and cellular differentiation in the volvocine algae

The volvocine algae, which include the single-celled species Chlamydomonas reinhardtii and the colonial species Volvox carteri, serve as a model in which to study the evolution of multicellularity and cellular differentiation. Studies reconstructing the history of this group have by and large relied on datasets of one to a few genes for phylogenetic inference and ancestral character state reconstruction. As a result, volvocine phylogenies lack concordance depending on the number and/or type of genes (i.e., chloroplast vs nuclear) chosen for phylogenetic inference. While multiple studies suggest that multicellularity evolved only once in the volvocine algae, that each of its three colonial families is monophyletic, and that there have been at least three independent origins of cellular differentiation in the group, other studies call into question one or more of these conclusions. An accurate assessment of the evolutionary history of the volvocine algae requires inference of a more robust phylogeny. We performed RNA sequencing (RNA-seq) on 55 strains representing 47 volvocine algal species and obtained similar data from curated databases on 13 additional strains. We then compiled a dataset consisting of transcripts for 40 single-copy, protein-coding, nuclear genes and subjected the predicted amino acid sequences of these genes to maximum likelihood, Bayesian inference, and coalescent-based analyses. These analyses show that multicellularity independently evolved at least twice in the volvocine algae and that the colonial family Goniaceae is not monophyletic. Our data further indicate that cellular differentiation arose independently at least four, and possibly as many as six times, within the volvocine algae. Altogether, our results demonstrate that multicellularity and cellular differentiation are evolutionarily labile in the volvocine algae, affirming the importance of this group as a model system for the study of major transitions in the history of life.M.S

Evolution of Individuality: A Case Study in the Volvocine Green Algae

While numerous criteria have been proposed in definitions of biological individuality, it is not clear whether these criteria reflect the evolutionary processes that underlie transitions in individuality. We consider the evolution of individuality during the transition from unicellular to multicellular life. We assume that “individuality” (however it is defined) has changed in the volvocine green algae lineage during the transition from single cells, to simple multicellular colonies with four to one hundred cells, to more complex multicellular organisms with thousands of differentiated cells. We map traits associated with the various proposed individuality criteria onto volvocine algae species thought to be similar to ancestral forms arising during this transition in individuality. We find that the fulfillment of some criteria, such as genetic homogeneity and genetic uniqueness, do not change across species, while traits underpinning other aspects of individuality, including degrees of integration, group-level fitness and adaptation, and group indivisibility, change dramatically. We observe that different kinds of individuals likely exist at different levels of organization (cell and group) in the same species of algae. Future research should focus on the causes and consequences of variation in individuality

緑藻ボルボックス系列を用いた多細胞化初期段階の進化生物学的研究

学位の種別: 課程博士審査委員会委員 : （主査）東京大学准教授 入江 直樹, 東京大学教授 寺島 一郎, 東京大学教授 塚谷 裕一, 東京大学准教授 野﨑 久義, 国立遺伝学研究所教授 宮城島 進也University of Tokyo(東京大学

Stable nuclear transformation of Gonium pectorale

Lerche K, Hallmann A. Stable nuclear transformation of Gonium pectorale. BMC Biotechnology. 2009;9(1): 64.BACKGROUND: Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri. RESULTS: Stable nuclear transformation of G. pectorale was achieved using a heterologous dominant antibiotic resistance gene, the aminoglycoside 3'-phosphotransferase VIII gene (aphVIII) of Streptomyces rimosus, as a selectable marker. Heterologous 3'- and 5'-untranslated flanking sequences, including promoters, were from Chlamydomonas reinhardtii or from Volvox carteri. After particle gun bombardment of wild type Gonium cells with plasmid-coated gold particles, transformants were recovered. The transformants were able to grow in the presence of the antibiotic paromomycin and produced a detectable level of the AphVIII protein. The plasmids integrated into the genome, and stable integration was verified after propagation for over 1400 colony generations. Co-transformants were recovered with a frequency of ~30-50% when cells were co-bombarded with aphVIII-based selectable marker plasmids along with unselectable plasmids containing heterologous genes. The transcription of the co-transformed, unselectable genes was confirmed. After heterologous expression of the luciferase gene from the marine copepod Gaussia princeps, which was previously engineered to match the codon usage in C. reinhardtii, Gonium transformants show luciferase activity through light emission in bioluminescence assays. CONCLUSION: Flanking sequences that include promoters from C. reinhardtii and from V. carteri work in G. pectorale and allow the functional expression of heterologous genes, such as the selectable marker gene aphVIII of S. rimosus or the co-transformed, codon-optimized G. princeps luciferase gene, which turned out to be a suitable reporter gene in Gonium. The availability of a method for transformation of Gonium makes genetic engineering of this species possible and allows for detailed studies in molecular evolution using the unicellular Chlamydomonas, the 16-celled Gonium, and the multicellular Volvox

Dominantne vrste mrežnog fitoplanktona u Visovačkom jezeru, NP Krka

Species composition of the net-phytoplankton assemblage and seasonality of the dominant species were investigated in the travertine monomictic stratified riverine Lake Visovac in relation to the environmental variables. The one year investigation was based on monthly sampling from April 1995 to March 1996 at the two deepest vertical profiles, with a maximum depth of 25 m. Diatoms, especially Asterionella formosa Hass. dominated net-phytoplankton assemblages except in late summer and autumn when dinoflagellate species Ceratium hirundinella (O. F. Müller) Bergh became the dominate form. The effects of the short term dynamics of environmental factors on the algal growth in the barrage lenitic area of the calcareous river stressed several variables as important factors controlling annual net-phytoplankton distribution. Temperature was the variable that most affected species abundance, changes in dominance and species composition (Primer 5, BIO-ENV; http://www.primer-e.com/). CCA analysis performed on abundance dataset and environmental variables confirm the importance of temperature but also highlighted total phosphorus, conductivity and silica.Tijekom jednogodišnjeg razdoblja od travnja 1995. do ožujka 1996. istraživana je, u jednomjesečnim intervalima, vertikalna i sezonska distribucija mrežnog fitoplanktona u lenitičkom području Visovačkog jezera. Naglasak tijekom istraživanja bio je na sastav vrsta te povezanost dominantnih vrsta i okolišnih čimbenika. Najzastupljenija vrsta tijekom kasnog proljeća je dijatomeja Asterionella formosa Hass, dok ljeti i u jesen dominaciju preuzima dinoflagelat Ceratium hirundinella (O. F. Müller) Bergh. Od mjerenih ekoloških parametara, temperatura vode najznačajnije je utjecala na promjene u sastavu vrsta, njihovu zastupljenost i dominaciju (Primer 5, BIO-ENV). CCA analiza dobivena na osnovi zastupljenosti fitoplanktonskih stanica i praćenih ekoloških čimbenika potvrdila je znatan utjecaj temperature vode, kao i ukupnog fosfora, provodljivosti i silicija

Origins and stepwise expansion of R2R3-MYB transcription factors for the terrestrial adaptation of plants

The R2R3-MYB transcription factors play critical roles in various processes in embryophytes (land plants). Here, we identified genes encoding R2R3-MYB proteins from rhodophytes, glaucophytes, Chromista, chlorophytes, charophytes, and embryophytes. We classified the R2R3-MYB genes into three subgroups (I, II, and III) based on their evolutionary history and gene structure. The subgroup I is the most ancient group that includes members from all plant lineages. The subgroup II was formed before the divergence of charophytes and embryophytes. The subgroup III genes form a monophyletic group and only comprise members from land plants with conserved exon–intron structure. Each subgroup was further divided into multiple clades. The subgroup I can be divided into I-A, I-B, I-C, and I-D. The I-A, I-B, and I-C are the most basal clades that have originated before the divergence of Archaeplastida. The I-D with the II and III subgroups form a monophyletic group, containing only green plants. The II and III subgroups form another monophyletic group with Streptophyta only. Once on land, the subgroup III genes have experienced two rounds of major expansions. The first round occurred before the origin of land plants, and the second round occurred after the divergence of land plants. Due to significant gene expansion, the subgroup III genes have become the predominant group of R2R3-MYBs in land plants. The highly unbalanced pattern of birth and death evolution of R2R3-MYB genes indicates their important roles in the successful adaptation and massive radiation of land plants to occupy a multitude of terrestrial environments

Molecular Systematics of the Green Algal Order Trentepohliales (Chlorophyta).

Molecular, karyological, and nuclear genome quantification data from representatives of the order Trentepohliales (Chlorophyta) were used to infer evolutionary relationships with other green algal classes and orders. Phylogenetic analyses of the nuclear-encoded small subunit ribosomal DNA (18S rDNA) sequences from taxa representing all of the major lineages of green algae consistently indicated that the subaerial Trentepohliales are closely related to Ulvophycean marine green algae, particularly to the siphonous and hemisiphonous orders. The phylogenetic distribution of continuous and discontinuous types of mitochondrial large subunit ribosomal RNAs (mtLSU rRNA) in green algae has been shown to be consistent with phylogenetic relationships previously suggested by both ultrastructural data from the flagellar apparatus and nuclear rRNA sequence analysis. Our studies indicated the presence of a continuous mtLSU rRNA in Cephaleuros parasiticus ; continuous mtLSU rRNA have been reported in all the investigated zoosporic green algae with a counterclockwise orientation of the flagellar apparatus and their autosporic descendants; a result that is consistent with an ulvophycean affinity. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA content in eight species representing three genera of the subaerial green algal order Trentepohliales (Chlorophyta). Comparisons of mean fluorescence intensity (If) values of algal nuclear genomes to those of chicken erythrocytes (RBC) resulted in an estimate of 1. 1--4.1 pg for the algae. DNA levels in Cephaleuros parasiticus Karsten for 2 C nuclei in gametophytic phase closely approximate 50% of the 4 C values in the sporophytic phase, confirming previous observations based on culture and ultrastructural studies on the presumptive sexual life cycle in this genus. Genome quantification data for eight Trentepohlialean taxa suggest a doubling sequence for nuclear DNA contents. This phenomenon might reflect the presence of a polyploid series in the order Trentepohliales. Amplification and sequencing of the chloroplast-encoded large subunit ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) gene was used to assess its reliability as a phylogenetic marker of the order Trentepohliales and the green algal classes. Our present results suggest using the rbcL gene sequences is more useful within green algal classes than for the analysis of phylogenetic analysis among major groups of algae

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva’s rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance “green tides.” Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage

Unraveling The Evolution And Diversity Of Giant Plastid Genomes In Chlamydomonadalean Green Algae

Organelle genomes are known to have large sizes and substantial non-coding content, despite conserved coding regions and low substitution rates. Notably, volvocine green algae exhibit significant variation in plastid genome size, with some species harboring ptDNA ten times larger than the average. To explain this variability, my thesis explores two hypotheses. The first proposes that genetic divergence accumulates due to weak negative selection and genetic drift, resulting in similar evolution rates for coding and non-coding regions. The second suggests high evolution rates in non-coding sequences are due to error-prone repair mechanisms. Analyzing new plastid genomes from volvocine green algae, I found a potential for high silent-site substitution rates in intergenic regions. My analysis shows that these hypotheses can be applied to plastid genomes of close relatives to advance our understanding of the mechanisms of sequence evolution specific to non-coding DNA accumulation within the volvocine green algae

Chlamydomonas reinhardtii, an Algal Model in the Nitrogen Cycle

Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3−) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalga Chlamydomonas reinhardtii as an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms