Complex multicellularity in fungi: evolutionary convergence,single origin,or both?

Complex multicellularity represents the most advanced level of biological organization and it has evolved only a few times: in metazoans, green plants, brown and red algae and fungi. Compared to other lineages, the evolution of multicellularity in fungi follows different principles; both simple and complex multicellularity evolved via unique mechanisms not found in other lineages. Herein we review ecological, palaeontological, developmental and genomic aspects of complex multicellularity in fungi and discuss general principles of the evolution of complex multicellularity in light of its fungal manifestations. Fungi represent the only lineage in which complex multicellularity shows signatures of convergent evolution: it appears 8–11 times in distinct fungal lineages, which show a patchy phylogenetic distribution yet share some of the genetic mechanisms underlying complex multicellular development. To explain the patchy distribution of complex multicellularity across the fungal phylogeny we identify four key observations: the large number of apparently independent complex multicellular clades; the lack of documented phenotypic homology between these clades; the conservation of gene circuits regulating the onset of complex multicellular development; and the existence of clades in which the evolution of complex multicellularity is coupled with limited gene family diversification. We discuss how these patterns and known genetic aspects of fungal development can be reconciled with the genetic theory of convergent evolution to explain the pervasive occurrence of complex multicellularity across the fungal tree of life.     

Archaeocyaths: alternatively explained as consortia of siphonous algae and cyanobacteria-like microbes in shallow Cambrian seas

In order to test the systematic position of archaeocyaths, a group of Cambrian, exclusively marine fossils of unclear biological affinity, currently ascribed mostly to sponges (Porifera), we present an alternative explanation supporting the early idea of siphonous green algal affinity of archaeocyaths. The new arguments are based on a study of the morphology, microstructure, and microtaphonomy of early Cambrian (Tommotian and Atdabanian) archaeocyathan specimens from northeastern Siberia. Light microscopic and scanning electron microscopic (SEM) observations revealed that in many specimens the outer, often microlaminated, skeletal fabric grades continuously into the microfabric similar to that of microbial bush-like and dendritic microfossils such as Angulocellularia and Renalcis. The polyhedral to subglobular units characterizing the skeletal microfabric of the studied archaeocyaths and their microbial outgrowths are most similar to recent and fossil mineralized (calcified) colonies of benthic coccoidal cyanobacteria. This means that what had heretofore been interpreted as skeletons secreted by archaeocyathan organisms represent calcareous crusts precipitated by epibiontic cyanobacteria-like microbes, probably associated with a variety of other bacteria, colonizing surfaces and internal spaces of soft-bodied archaeocyathan organisms. Our study shows that a number of the morphological traits of archaeocyaths conserved by the mineralized microbial cover are similar to those of of modern siphonous green algae, like the marine genus Codium Stackhouse (Bryopsidales). Identifiable in well-preserved archaeocyaths are remains of such traits characteristic for Codium species as utricles, medullary threads, juvenile stages (primordia), and coalescence structures. The taxonomic and palaeoenvironmental implications of the recognition of archaeocyaths as possible consortia of siphonous green seaweeds (Chlorophyta) and cyanobacteria-like microbes are briefly discussed.     

Biomechanical properties and holdfast morphology of coenocytic algae (Halimedales, Chlorophyta) in Bocas del Toro, Panama

For attached marine organisms, specific biomechanical properties may result in detachment or in tissue loss, when sufficient tensile force is applied. Algae experience such forces through water movement, which may thus act to limit size, abundance, and species composition, of populations of algae.Coenocytic construction is uncommon in the algae, but it occurs relatively more frequently in green algae found in shallow subtidal sediments associated with coral reefs, e.g., at our study site of Isla Colon, Bocas del Toro, Panama. We studied the biomechanical properties of some tropical coenocytic algae (Udotea flabellum (Ellis et Solander) Lamouroux, Penicillus capitatus Lamarck, P. pyriformis A. and E.S. Gepp, and Halimeda gracilis Harvey) anchored in sediments. We compare our results with published data on other coenocytic algae, as well as with multicellular algae. Our results show that properties of sand-dwelling coenocytes, such as mean force to dislodge (4.9-12.7 N), mean force to break (6.6-22.1 N), and mean strength (1.0-7.0 MN m^− 2), are all within the range reported for temperate, multicellular, algae. In contrast, the coenocytes differed markedly from the temperate non-coenocytes in the consequences of applied tensile force: coenocytes were removed whole, while most temperate algae attached to rocks break within the thallus. Some multicellular algae can regrow from tissue left on the substratum; three of the four coenocytic species we examined had rhizoids connecting closely adjacent (0.1-0.15 m) individuals, and these rhizoids may serve to regrow a new individual. While our experiments indicated that sufficient tensile force results in dislodgment, calculations using the experimentally determined variables led us to conclude that water velocities sufficient to dislodge individuals are unlikely to occur. Since dislodgment is usually fatal for algae, the role of the holdfast is a critical one. All of the species we investigated had similar holdfast morphology, a mass of rhizoids which entrained sand, the entire unit forming a hemispherical to cylindrical mass. Despite the consistency in holdfast form, and the initial prediction that this was an optimal form for anchoring these algae, our data suggest that this is not the case.     

Cleavage, incomplete inversion, and cytoplasmic bridges in Gonium pectorale (Volvocales, Chlorophyta)

Multicellularity arose several times in evolution of eukaryotes. The volvocine algae have full range of colonial organization from unicellular to colonies, and thus these algae are well-known models for examining the evolution and mechanisms of multicellularity. Gonium pectorale is a multicellular species of Volvocales and is thought to be one of the first small colonial organisms among the volvocine algae. In these algae, a cytoplasmic bridge is one of the key traits that arose during the evolution of multicellularity. Here, we observed the inversion process and the cytoplasmic bridges in G. pectorale using time-lapse, fluorescence, and electron microscopy. The cytoplasmic bridges were located in the middle region of the cell in 2-, 4-, 8-, and 16-celled stages and in inversion stages. However, there were no cytoplasmic bridges in the mature adult stage. Cytoplasmic bridges and cortical microtubules in G. pectorale suggest that a mechanism of kinesin-microtubule machinery similar to that in other volvocine algae is responsible for inversion in this species.     

Morphological,ultrastructural, and genetic characterization of coalescence in the intertidal and shallow subtidal kelps Lessonia spicata and L. berteroana (Laminariales,Heterokontophyta)

Coalescing macroalgae may fuse with conspecifics, forming genetically heterogeneous entities known as chimera. This process has been shown in taxa from roughly half the red algal orders and in the Codium species, a green alga. Field observations indicate that common and dominant kelps along central Chile exhibit a fused holdfast. We evaluated whether such fusions are true coalescence processes in Lessonia spicata and Lessonia berteroana. To this end, we characterized the ultrastructural event involved in holdfast fusion in the laboratory. Additionally, coalescence in natural populations was quantified by measuring the frequency of individuals with genetically heterogenic stipes within the same holdfast. Results indicate that coalescence appears as a frequent process in laboratory, mostly restricted to intraspecific fusions. During fusion, the meristodermatic cells located in the contact area modify their morphology and reduce the number of plastids, mitochondria, and cell inclusions. The cell wall becomes much thinner and develops plasmodesmata, enhancing communication with equivalent cells of the other coalescencing individual. Stipe genotyping indicates that there is a widespread occurrence of chimerism in both species and genetic heterogeneity is increasing directly with the increasing number of stipes. The combination of results suggests that kelp frequently coalesce in the field, and the histological response observed approaches that of red algae. Since kelps are part of the dominant vegetation in low intertidal and shallow subtidal beds, the adaptive values of coalescence in these species should be evaluated. It is concluded that coalescence and chimerism are evolutionary convergent processes, occurring in all three major groups of seaweeds.     

Epidermal cell-patterning genes of the stem parasitic plant Cuscuta campestris are involved in the development of holdfasts

Cuscuta campestris, a stem parasitic plant, commences its parasitic behavior by forming a specialized disk-like adhesive structure called a holdfast, which facilitates tight adhesion to the stem surface of the host plant. The morphology of epidermal cells in the holdfast is similar to that of the leaf trichome and root hairs of dicotyledonous plants. However, the regulatory network underlying the development of the holdfast has not been elucidated to date. In this study, we assessed the roles of epidermal cell-patterning genes in the development of a holdfast. Epidermal cell-patterning genes of C. campestris, including CcWER, CcGL3, CcTTG1, CcGL2, and CcJKD, were expressed slightly before the initiation of the outgrowth of stem epidermal cells. CcJKD-silencing repressed CcJKD, CcWER, CcGL3, CcTTG1, CcGL2; therefore, CcJKD is an upstream regulator of other epidermal cell-patterning genes. Unlike other genes, CcCPC was not upregulated after attachment to the host, and was not repressed by CcJKD-silencing. Protein interaction assays demonstrated that CcJKD interacted with CcTTG1 and CcCPC. Furthermore, CcJKD-silencing repressed the outgrowth of holdfast epidermal cells. Therefore, C. campestris invokes epidermal cell-patterning genes for the outgrowth of holdfast epidermal cells, and their regulatory mechanism is different from those for leaf trichome or root hairs.     

microRNAs and the evolution of complex multicellularity: identification of a large,diverse complement of microRNAs in the brown alga Ectocarpus

There is currently convincing evidence that microRNAs have evolved independently in at least six different eukaryotic lineages: animals, land plants, chlorophyte green algae, demosponges, slime molds and brown algae. MicroRNAs from different lineages are not homologous but some structural features are strongly conserved across the eukaryotic tree allowing the application of stringent criteria to identify novel microRNA loci. A large set of 63 microRNA families was identified in the brown alga Ectocarpus based on mapping of RNA-seq data and nine microRNAs were confirmed by northern blotting. The Ectocarpus microRNAs are highly diverse at the sequence level with few multi-gene families, and do not tend to occur in clusters but exhibit some highly conserved structural features such as the presence of a uracil at the first residue. No homologues of Ectocarpus microRNAs were found in other stramenopile genomes indicating that they emerged late in stramenopile evolution and are perhaps specific to the brown algae. The large number of microRNA loci in Ectocarpus is consistent with the developmental complexity of many brown algal species and supports a proposed link between the emergence and expansion of microRNA regulatory systems and the evolution of complex multicellularity.     

Apical Growth in Algae

Cytological and physiological aspects of apical growth in algae are reviewed. Data on morphology and ultrastructural organization of siphonous thalli are presented. Special attention is paid to the organization of microtubules and microfilaments and the role of the cytoskeleton in morphogenesis. A comparative study of apical growth in algae, fungi, and higher plants is presented.     

A multi-locus time-calibrated phylogeny of the siphonous green algae

The siphonous green algae are an assemblage of seaweeds that consist of a single giant cell. They comprise two sister orders, the Bryopsidales and Dasycladales. We infer the phylogenetic relationships among the siphonous green algae based on a five-locus data matrix and analyze temporal aspects of their diversification using relaxed molecular clock methods calibrated with the fossil record. The multi-locus approach resolves much of the previous phylogenetic uncertainty, but the radiation of families belonging to the core Halimedineae remains unresolved. In the Bryopsidales, three main clades were inferred, two of which correspond to previously described suborders (Bryopsidineae and Halimedineae) and a third lineage that contains only the limestone-boring genus Ostreobium. Relaxed molecular clock models indicate a Neoproterozoic origin of the siphonous green algae and a Paleozoic diversification of the orders into their families. The inferred node ages are used to resolve conflicting hypotheses about species ages in the tropical marine alga Halimeda.     

Multicellularity arose several times in the evolution of eukaryotes (Response to DOI 10.1002/bies.201100187)

The cellular slime mold Dictyostelium has cell‐cell connections similar in structure, function, and underlying molecular mechanisms to animal epithelial cells. These similarities form the basis for the proposal that multicellularity is ancestral to the clade containing animals, fungi, and Amoebozoa (including Dictyostelium): Amorphea (formerly “unikonts”). This hypothesis is intriguing and if true could precipitate a paradigm shift. However, phylogenetic analyses of two key genes reveal patterns inconsistent with a single origin of multicellularity. A single origin in Amorphea would also require loss of multicellularity in each of the many unicellular lineages within this clade. Further, there are numerous other origins of multicellularity within eukaryotes, including three within Amorphea, that are not characterized by these structural and mechanistic similarities. Instead, convergent evolution resulting from similar selective pressures for forming multicellular structures with motile and differentiated cells is the most likely explanation for the observed similarities between animal and dictyostelid cell‐cell connections.     

ULTRASTRUCTURE OF THE GREEN ALGA DICHOTOMOSIPHON TUBEROSUS WITH SPECIAL REFERENCE TO THE OCCURRENCE OF STRIATED TUBULES IN THE CHLOROPLAST1

The ultrastructure of the siphonous green alga Dichotomosiphon tuberosus (A. Br.) Ernst is compared with that of other siphonous plants. There is a characteristic association between the Golgi bodies and endoplasmic reticulum, but. the mitochondria are not involved in the association as they are in Vaucheria and the phycomycete Saprolegnia. An unusual structure and arrangement of the chloroplasts is described as well as a previously unreported type of “striated tubule” which occurs in most if not all chloroplasts, and amyloplasts. The structure of these tubules is compared with that of other tubules recently found in green algae and higher plants. In addition, cytoplasmic microtubules arranged in the longitudinal direction of the siphon suggest a function in cytoplasmic streaming.     

RAP,the Sole Octotricopeptide Repeat Protein in Arabidopsis,Is Required for Chloroplast 16S rRNA Maturation

The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5′ region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.     

Light-dependent conformational change of neoxanthin in a siphonous green alga,Codium intricatum,revealed by Raman spectroscopy

Siphonous green algae, a type of deep-sea green algae, appear olive drab and utilize blue–green light for photosynthesis. A siphonous green alga, Codium (C.) intricatum, was isolated from Okinawa prefecture in Japan, and a clonal algal culture in filamentous form was established. The major light-harvesting antenna was analogous to the trimeric LHCII found in higher plants, but the C. intricatum complex contained an unusual carbonyl carotenoid siphonaxanthin. Culture conditions were optimized to achieve high siphonaxanthin content in intact lyophilized filamentous bodies. Interestingly, the carotenoid composition was different when cultured under high irradiance: all-trans neoxanthin was accumulated in addition to the normal 9′-cis form in whole cell extract. Resonance Raman spectra of intact filamentous bodies, cultured under high- and low-light conditions, confirmed the accumulation of all-trans neoxanthin under high irradiance conditions. A plausible function of the presence of all-trans neoxanthin will be discussed in relation to the regulation against high light stress.     

Electrophysiology of Turgor Regulation in Marine Siphonous Green Algae

We review electrophysiological measures of turgor regulation in some siphonous green algae, primarily the giant-celled marine algae, Valonia and Ventricaria, with particular comparison to the well studied charophyte algae Chara and Lamprothamnium. The siphonous green algae have a less negative plasma membrane potential, and are unlikely to have a proton-based chemiosmotic transport system, dominated by active electrogenic K⁺ uptake. We also make note of the unusual cellular structure of the siphonous green algae. Hypertonic stress, due to increased external osmotic pressure, is accompanied by positive-going potential difference (PD), increase in conductance, and slow turgor regulation. The relationship between these is not yet resolved, but may involve changes in K⁺ conductance (G _K) or active K⁺ transport at both membranes. Hypotonic turgor regulation, in response to decreased external osmotic pressure, is ∼3 times faster than hypertonic turgor regulation. It is accompanied by a negative-going PD, although conductance also increases. The conductance increase and the magnitude of the PD change are strongly correlated with the magnitude of hypotonic stress.     

Chemical and enzymatic fractionation of cell walls from Fucales: insights into the structure of the extracellular matrix of brown algae

Background and Aims

Brown algae are photosynthetic multicellular marine organisms evolutionarily distant from land plants, with a distinctive cell wall. They feature carbohydrates shared with plants (cellulose), animals (fucose-containing sulfated polysaccharides, FCSPs) or bacteria (alginates). How these components are organized into a three-dimensional extracellular matrix (ECM) still remains unclear. Recent molecular analysis of the corresponding biosynthetic routes points toward a complex evolutionary history that shaped the ECM structure in brown algae.

Methods

Exhaustive sequential extractions and composition analyses of cell wall material from various brown algae of the order Fucales were performed. Dedicated enzymatic degradations were used to release and identify cell wall partners. This approach was complemented by systematic chromatographic analysis to study polymer interlinks further. An additional structural assessment of the sulfated fucan extracted from Himanthalia elongata was made.

Key Results

The data indicate that FCSPs are tightly associated with proteins and cellulose within the walls. Alginates are associated with most phenolic compounds. The sulfated fucans from H. elongata were shown to have a regular α-(1→3) backbone structure, while an alternating α-(1→3), (1→4) structure has been described in some brown algae from the order Fucales.

Conclusions

The data provide a global snapshot of the cell wall architecture in brown algae, and contribute to the understanding of the structure–function relationships of the main cell wall components. Enzymatic cross-linking of alginates by phenols may regulate the strengthening of the wall, and sulfated polysaccharides may play a key role in the adaptation to osmotic stress. The emergence and evolution of ECM components is further discussed in relation to the evolution of multicellularity in brown algae.     

Niche expansion, body size, and survival in Galápagos marine iguanas

Foraging theory predicts that dietary niche breadth should expand as resource availability decreases. However, Galápagos marine iguanas often die during algae shortages (El Niños) although land plants abound where they rest and reproduce. On Seymour Norte island, a subpopulation of iguanas exhibited unique foraging behavior: they consistently included the succulent beach plant B. maritima in their diet. We investigated the consequences of land-plant feeding for body size and survival. Batis-eaters supplemented their algae diet both before and after intertidal zone foraging, and more Batis was eaten during tides unfavorable for intertidal zone foraging (dawn and dusk). Larger, energy-constrained iguanas fed more on land than did smaller animals. Compared to intertidal zone algae, Batis was 39% lower in caloric content (1.6 vs. 2.6 kcal g^–1 dry mass), 56% lower in protein (8.3 vs. 18.9% dry mass) and 57% lower in nitrogen (1.3 vs. 3.0% dry mass). In spite of its lower nutrient value, iguanas that supplemented their diet with this plant were able to attain nearly twice the body size of other iguanas on the island. Age estimates indicate that many Batis-eaters survived repeated El Niño episodes during which animals of their relative size-class experienced high mortality on other islands. The larger animals were, however, completely dependent upon this supplementary source of food to maintain condition, and all perished in the 1997–1998 El Niño when high tides inundated and killed Batis on Seymour Norte Island. We hypothesize that Batis feeding developed as a local foraging tradition, and that dietary conservatism and strong foraging site fidelity explain why the inclusion of land plants in the diet has been observed in only a single population. Ultimately, a unique algae-adapted hindgut morphology and physiology may limit a switch from marine to terrestrial diet.     

Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps

Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.