Pit Plugs Research Articles

Evidence of spermatia in Hildenbrandia (Rhodophyta, Hildenbrandiales)

ABSTRACT Specimens of Hildenbrandia bearing conceptacles that contained small, bead-shaped reproductive cells, only 3–4 µm wide, features diagnostic of Hildenbrandia sanjuanensis, were collected from the high intertidal zone at the type locality of the species. Examination of conceptacle contents by transmission electron microscopy was used to detail the differentiation of the catenate reproductive cells, and it revealed that these cells were joined by pit connections. Tetraspores are the only known reproductive cells in the Hildenbrandiales, but the process of cleaving tetrasporangia to form tetraspores has never been found to include the deposition of pit plugs in any red alga. An alternative interpretation of the nature of the reproductive cells of H. sanjuanensis is that they are spermatia or carpospores. The features of small size, sparse chloroplasts with few thylakoids, and near absence of starch contents in the reproductive cells favours the view that these cells are spermatia. If this interpretation is correct, it represents the first evidence of spermatia in any member of the Hildenbrandiophycidae, and it indicates that the possibility of a sexual life history in members of this order should be re-examined.

Intercellular transport across pit-connections in the filamentous red alga Griffithsia monilis

Intercellular nutrient and signal transduction are essential to sustaining multicellular organisms and maximizing thebenefits of multicellularity. It has long been believed that red algal intercellular transport of macromolecules is preventedby the protein-rich pit plug within pit-connections, the only physical connection between cells. Fluorescein isothiocyanate-dextran and recombinant green fluorescence protein (rGFP) of various molecular sizes were injected into vegetativecells of Griffithsia monilis using a micromanipulator, and intercellular transport of the fluorescent probes was examined.Pit-connections were found to provide intercellular transport of tracers at rates comparable to plasmodesmata in otherorganisms. The time necessary for the transport to an adjacent cell was dependent on the molecular size and the directionof the transport. Fluorescent dextran of 3 kDa was transported to adjacent cells in 1–2 h after injection and migratedto all cells of the filament within 24 h, but fluorescent dextran of 10–20 kDa took 24 h to transfer to neighboring cells. Themigration occurred faster towards adjacent reproductive cells and to apical cells than basally. Fluorescent tracers above40 kDa and rGFP was not transported to neighboring cells, but accumulated near the pit plug. Our results suggest thatpit-connections are conduit for macromolecules between neighboring cells and that these size-specific conduits allowintercellular communication between the vegetative cells of red algae.

Formation of secondary pit connections by conjunctor cells in a coralline red alga

ABSTRACT For more than eighty years, the accepted view of the origin of secondary pit connections in coralline red algae has been that a narrow channel of direct fusion forms between adjacent intercalary meristematic cells and a pit plug is subsequently deposited within the fusion channel; no cell or nuclear division is involved. Such a mechanism contrasts with the prevailing pattern of secondary pit connection formation among red algae in which a highly unequal, intercalary cell division produces a short-lived diminutive cell, the conjunctor cell, that fuses with an adjacent cell; the pit connection produced in the formation of the conjunctor cell is then shared by the two surviving cells. Study of Lithophyllum impressum by light and transmission electron microscopy revealed that asymmetrical lateral divisions of meristematic cells, as well as cells throughout the perithallus, produce tiny nucleated conjunctor cells. Thus, these findings indicate that secondary pit connection formation in this coralline alga does not occur by a direct mechanism nor are these connections generated only in the layer of intercalary meristematic cells as long believed.

Ultrastructure of epithallial cells and trichocyte initials in Amphiroa (Corallinales, Rhodophyta), and possible systematic value of trichocyte development in corallines

ABSTRACT To better understand the dynamics of the coralline algal thallus surface, scanning and transmission electron microscopy were used to examine the structure and development of epithallial cells and trichocyte initials in three species of Amphiroa. Epithallial cells possessed several distinctive organic wall layers. A densely fibrillar, moderately electron-dense layer completely surrounded each epithallial cell. Inward of this layer on the distal cell face, irregular deposits of wall material projected into the cell lumen. Outward on the distal cell surface, an electron-dense layer tapered from a central conical projection. This cone of distal wall material originated before the epithallial cell that ultimately bore it, having been deposited by an initial cell to sever its connection with a senescing epithallial cell in advance of epithallial cell replacement. This wall feature provided an indication of epithallial cell regeneration. Trichocyte initials were present beneath the layer of epithallial cells. The calcified sidewall of the epithallial cell overlaying the trichocyte chamber contained a channel that terminated in a pore through which the shaft of a trichocyte could pass. Lateral to the trichocyte initial, uncalcified walls still bearing pit plugs once shared by the initial cell and multiple generations of lost and regenerated trichocytes were compressed against the side of the trichocyte chamber. A single section of one trichocyte chamber showed 17 remnant pit plugs, indicating a high frequency of trichocyte turnover. Comparisons drawn from the published literature suggest that trichocyte development has the potential to serve as a source of systematic characters for coralline algae.

Rhodachlya westii sp. nov. (Rhodachlyales, Rhodophyta), a new species from Brazil, revealed by an integrative taxonomic approach

ABSTRACT A small red alga was isolated into culture from rhodoliths collected on the Piauí coast, northeastern Brazil. Molecular data from the plastid-encoded rbcL gene, combined with morphological and ultrastructural evidence, demonstrated that it belongs to the genus Rhodachlya and supports the proposal of a new species, R. westii. The new species was characterised by basal and erect uniseriate filaments, cells with parietal H-shaped or lobed plastids with one or two prominent pyrenoids, absence of unicellular hairs, and with monosporangia sessile or on branched stalks. Ultrastructure revealed pit plugs with two cap layers without a cap membrane, typical of Rhodachlyales. In our rbcL phylogeny, R. westii sp. nov. was recovered in a well-supported clade containing R. hawaiiana and R. madagascarensis (generitype) with interspecific divergence ranging from 5% to 10%, respectively. Characters previously used for species-level taxonomy (e.g. cell dimensions, number of pyrenoids and presence or absence of colourless hairs) are often plastic and overlap. We conclude that Rhodachlya species can be accurately identified only based on DNA sequence data and their geographic distribution. Rhodachlya westii becomes the third species of the genus, representing the first record in the Atlantic Ocean. Our data suggest that the genus is probably widely distributed. In view of the scarcity of reliable morphological features available for comparison, molecular-assisted alpha taxonomy is needed for uncovering the long-overlooked diversity in this red algal group.

Three-dimensional preservation of cellular and subcellular structures suggests 1.6 billion-year-old crown-group red algae.

The ~1.6 Ga Tirohan Dolomite of the Lower Vindhyan in central India contains phosphatized stromatolitic microbialites. We report from there uniquely well-preserved fossils interpreted as probable crown-group rhodophytes (red algae). The filamentous form Rafatazmia chitrakootensis n. gen, n. sp. has uniserial rows of large cells and grows through diffusely distributed septation. Each cell has a centrally suspended, conspicuous rhomboidal disk interpreted as a pyrenoid. The septa between the cells have central structures that may represent pit connections and pit plugs. Another filamentous form, Denaricion mendax n. gen., n. sp., has coin-like cells reminiscent of those in large sulfur-oxidizing bacteria but much more recalcitrant than the liquid-vacuole-filled cells of the latter. There are also resemblances with oscillatoriacean cyanobacteria, although cell volumes in the latter are much smaller. The wider affinities of Denaricion are uncertain. Ramathallus lobatus n. gen., n. sp. is a lobate sessile alga with pseudoparenchymatous thallus, “cell fountains,” and apical growth, suggesting florideophycean affinity. If these inferences are correct, Rafatazmia and Ramathallus represent crown-group multicellular rhodophytes, antedating the oldest previously accepted red alga in the fossil record by about 400 million years.

Win some, lose some: genome evolution in red algae.

What's special about red algae? You might say that they have distinct pigments, the phycobiliproteins; unusual plastids with unstacked thylakoids; a different storage saccharide, floridean starch; or that some species have a unique triphasic life history and a special connection between cells, pit plugs. To me, what is most fascinating is what they don't have. There are no flagella and no centrioles. What is the result of not having flagella? One may think that this means at no stage in the life cycle can the organism swim. Red algae are, thus, aquatic organisms that sexually reproduce, but with extremely limited possibilities with respect to chemotaxic responses toward gametes of the other sex, complicating the fusion of gametes. For example, in red algae the male gametes are released and, by random movement of waves and currents, find a female gamete on a female plant; remember that the ocean is large with small areas where seaweeds are found and there are many red algal species, further complicating reproductive success. A lack of motility also means that unicellular planktonic red algae have a decreased capability to position themselves in the light environment. It is generally believed that the ancestral eukaryote had a flagellum (Koumandou et al. 2013), and therefore it must have been lost in red algae. Therefore, the flagella along with the capacity for directed movement, except limited (<1 mm) amoebic movement (Pickett-Heaps et al. 2001), have disappeared. This is likely to be a large handicap for red algae, suggesting that the evolutionary force that triggered the loss of flagella was very strong. So what happened, when and why? The answers to these questions have for a long time been a mystery, but recent increased knowledge on genes and genomes of red algae is starting to shed some light on this subject and on the general evolution of red algal genes and genomes. The article by Qiu et al. (2015) in this issue provides new, important insights into the evolution of red algal genomes, including the loss of genes, the loss of function, and the subsequent gain of genes through horizontal gene transfer. [...]

Characterization of a novel freshwater gigartinalean red alga from Belize, with a description of Sterrocladia belizeana sp. nov. (Rhodophyta)

Sherwood A.R., Necchi O. Jr., Carlile A.L., Laughinghouse H.D. IV, Fredericq S., and Sheath R.G. 2012. Characterization of a novel freshwater gigartinalean red alga from Belize, with a description of Sterrocladia belizeana sp. nov. (Rhodophyta). Phycologia 51: 627–635. DOI: 10.2216/12-012.1Freshwater representatives of the red algal order Gigartinales are extremely rare, with the monotypic genus Sterrocladia being the only truly freshwater example. This alga was described in 1850 from material originating from French Guiana, and although its affinities at the ordinal and familial levels have been debated based on morphological and anatomical characteristics, its placement has not been confirmed with the assistance of molecular analyses. Here we report several collections of a freshwater red alga from Belize that share many of the characteristics of the single described species, Sterrocladia amnica, but also display some important differences. Based on microscopic analysis, our collections were distinguished from S. amnica based on thickness of the cortical layer, cell size, possession of a loose versus entire medulla and absence of reproductive nemathecia. Transmission electron microscopy confirmed a pit plug anatomy consistent with placement of the new collections in the order Gigartinales, and a new method for resin infiltration into thick cortical tissue was described. Although recent material of S. amnica was unavailable, molecular phylogenetic analyses of the rbcL and SSU rRNA gene for our new samples indicated that it was indeed a member of the Gigartinales, and that it was positioned basal to the Gigartinaceae and Phyllophoraceae clades, likely representing a novel family. The COI-5P barcode sequence showed no close matches, but the vast majority of the closest hits were to representatives of the Phyllophoraceae. Here we describe a second species within the genus Sterrocladia to accommodate our Belizean collections, Sterrocladia belizeana sp. nov., and discuss the phylogenetic affinities of this unique genus of freshwater gigartinalean red algae. We also propose a lectotype of Sterrocladia amnica based on examination of syntype materials.

A Second Species ofRhodachlya(Rhodachlyales, Rhodophyta) from Hawaii, with a Description ofR.hawaiianasp. nov.

Rhodachlya hawaiiana Kurihara, J.A. West, Conklin et A.R. Sherwood sp. nov. is described based on cultured material from the island of Hawaii as the second member of the genus Rhodachlya. The pit plug ultrastructure of this alga is characterized by the presence of inner and outer cap layers but lacking a cap membrane between the layers, which is a diagnostic feature of the order Rhodachlyales and the genus Rhodachlya. Morphologically, R. hawaiiana is distinctive from R. madagascarensis in vegetative cell dimensions, frequency of colorless hair cell formation, and spore germination pattern. Rhodachlya hawaiiana and R. madagascarensis are also distinguishable by their nuclear SSU rRNA gene sequence divergence (p-distance = 0.0064), which is comparable to other species-level divergences among members of the Nemaliophycidae.Rhodachlya hawaiiana Kurihara, J.A. West, Conklin et Sherwood sp. nov., décrite à partir de materiel cultivé provenant de l'île d'Hawaii, est la seconde espèce appartenant au genre Rhodachlya. L'ultrastructure des synapses de cette algue montre la présence d'une couche interne et d'une couche externe recouvrant le bouchon synaptique, sans membrane de revêtement entre les couches, ce qui est une des caractéristiques de l'ordre des Rhodachlyales. Morphologiquement R. hawaiiana diffère de R. madagascarensis par la taille de ses cellules végétatives, par la fréquence des cellules formant des poils hyalins et par le mode de germination des spores. Ils diffèrent aussi par la séquence de leur SSU rRNA avec une divergence (p-distance=0.0064) comparable à d'autres divergences entre espèces parmi les Nemaliophycidae.

Observations of Pihiella liagoraciphila (Pihiellales, Rhodophyta)

Pueschel C.M. and Huisman J.M. 2010. Observations of Pihiella liagoraciphila (Pihiellales, Rhodophyta). Phycologia 49: 42–47. DOI: 10.2216/08-92.1The structure of the diminutive endo/epiphytic red alga Pihiella liagoraciphila was examined by light and transmission electron microscopy. The structure of the pit plugs consisted of a plug core with cap membranes but no cap layers, thus supporting the independence of this species from the Ahnfeltiales, to which it is most closely related in a molecular phylogeny but whose members have pit plugs without cap membranes. Hair cells were common and prominent and were characterized by exceptionally large pit plugs and an unusual membranous lattice adjacent to the pit plugs. The presence of large, degenerating pit plugs on the flanks of the cells subtending hair cells indicates that the hair cells can regenerate by division of the subtending cell, which acts as an initial. Sporangia also appear to regenerate from their stalk cells. These findings have implications for understanding the incompletely known life history of P. liagoraciphila.

Rhodachlya madagascarensis gen. et sp. nov.: a distinct acrochaetioid represents a new order and family (Rhodachlyales ord. nov., Rhodachlyaceae fam. nov.) of the Florideophyceae (Rhodophyta)

J.A. West, J.L. Scott, K.A. West, U. Karsten, S.L. Clayden and G.W. Saunders. 2008. Rhodachlya madagascarensis gen. et sp. nov.: a distinct acrochaetioid represents a new order and family (Rhodachlyales ord. nov., Rhodachlyaceae fam. nov.) of the Florideophyceae (Rhodophyta). Phycologia 47: 203–212. DOI: 10.2216/07-72.1A filamentous ‘acrochaetioid’ red algal epiphyte on Posidonia from Madagascar was isolated into culture. It reproduces solely by monosporangia and the spherical spores have gliding motility that differs from amoeboid spore motility seen with time-lapse video microscopy in most Acrochaetiales and Colaconematales. Electron microscopy reveals that a peripheral encircling thylakoid is absent in the chloroplast, Golgi bodies are associated with mitochondria, and pit plugs have platelike outer and inner caps and lack a cap membrane. Low-molecular-weight carbohydrates are floridoside and trehalose. Molecular evidence (small subunit, large subunit and EF2 sequences) resolved this taxon as a completely novel lineage of the Nemaliophycidae, and it is here described as Rhodachlya madagascarensis J.A. West et al., gen. et sp. nov. In light of the unusual combination of ultrastructural features and the lack of affinity to any of the currently recognized families and orders in molecular analyses, this alga forms the basis of a new family, Rhodachlyaceae G.W. Saunders et al., fam. nov., and order, Rhodachlyales G.W. Saunders et al., ord. nov., of the subclass Nemaliophycidae.

Reexamination of the pit plugs and the characteristic membranous structures in Porphyra yezoensis (Bangiales, Rhodophyta)

C. Ueki, C. Nagasato, T. Motomura and N. Saga. 2008. Reexamination of the pit plugs and the characteristic membranous structures in Porphyra yezoensis (Bangiales, Rhodophyta). Phycologia 47: 5–11. DOI: 10.2216/07–12.1Pit plug formation during conchosporogenesis in Porphyra yezoensis Ueda was studied with electron microscopy using freeze substitution. Just after nuclear division, cytokinesis started with furrowing of the septum. During furrowing, small vesicles were produced by projections of the plasma membrane in the space between the plasma membrane and the cell wall material and occasionally formed long chains. Afterwards, the plug core was gradually formed by endoplasmic reticulum, becoming vertical to the septum. The plasma membrane expanded like a loop along the pit plug from both sides of the furrowing septum. Lomasomes were located near the plasma membrane, beside the pit plug. With maturation of conchosporangia, the number of lomasomes increased near the pit plug. P. yezoensis has a characteristic expanding process of the plasma membrane along the pit plug, which is unique in the red algae.

Formation of primary pit connection during conchocelis phase ofPorphyra yezoensis (Bangiophyceae, Rhodophyta)

The formation of pit connection during conchocelis phase ofPorphyra yezoensis Ueda was observed and examined with transmission electron microscope (TEM) and epifluorence microscope. It is indicated that the pit connection was formed in late stage of conchocelis phase and the early stages of conchosporangial cell development, and disappeared in bispore stage. The pit connection contained a thin membrane layer at outer pit plug. Stained with 4′, 6′-diamidino-2-phenylidole dihydrochloride hydrate (DAPI), transferring of DNA or RNA between adjacent cells were observed in late stage of conchocelis development, it was deduced that pit connection might serve as a channel for signal transduction and genetic substance transportation in conchocelis phase.

Cellular and subcellular localization of kainic acid in the marine red alga Digenea simplex

Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as "granule bodies", which were found only in the pericentral cells of the thallus.

Fine structural study of the red seaweed Gymnogongrus torulosus (Phyllophoraceae, Rhodophyta)

The present study analyzed several characters of the red seaweed Gymnogongrus torulosus, such as cellular structure of the thallus, cuticle, pit plug and cell wall ultrastructure, and morphology of some organelles like plastids, Golgi bodies and mitochondria. Also, anomalous chloroplasts with thylakoid disorganization were found in medullary cells. The significance of this thylakoid disposition is still unclear. This is one of the first studies focused on the fine structure of a red alga recorded in Argentina.

A PROPOSAL FOR A NEW RED ALGAL ORDER, THE THOREALES1

Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well‐supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate‐like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate‐like.

Ultrastructure of trichocyte (hair cell) complexes in Jania adhaerens (Corallinales, Rhodophyta)

Trichocyte complexes of Jania adhaerens (Corallinales, Rhodophyta) were studied by scanning and transmission electron microscopy. The trichocyte complex is composed of trichocytes (hair cells), their associated initial cells, and a previously unknown ephemeral cell, here termed the crown cell. The crown cell, which is part of the epithallial layer, is the original apical cell of each filament forming a trichocyte complex. The trichocyte initial divides to produce both a trichocyte devoid of chloroplasts and a subtending cell that serves as the initial cell for the next trichocyte generation (secondary trichocyte initial). A hair growing from the apical end of the trichocyte passes through a collar of distinctive wall material deposited originally around the apical end of the primary trichocyte initial and the base of the crown cell. The first hair passing through this channel destroys the crown cell before emerging from the thallus surface. The cytoplasm of hair-bearing trichocytes is particularly rich in Golgi vesicles. After the hair is lost and as the trichocyte base degenerates, the secondary trichocyte initial enlarges, filling the chamber that formerly housed both the initial and the trichocyte. The pit plug and crosswall that had been shared by the initial cell and the senescent trichocyte are pushed aside and compressed by the enlarging initial. These wall structures persist between the initial cell and the surrounding calcium carbonate wall. The enlarged secondary initial cell either becomes quiescent, persisting in the undivided state and being equivalent to a megacell or heterocyst, or it divides, producing a new trichocyte and initial. Remnants of more than 20 generations of walls and pit plugs were observed within the chambers of some trichocyte complexes. These wall remnants potentially provide a record of every cycle of regeneration of each trichocyte produced over the life of the alga.

Heydrichia homalopasta sp. nov. (Sporolithaceae, Rhodophyta) from Australia

Heydrichia homalopasta sp. nov. is described from the east coast of Australia. The new species lacks the prominent pit plug caps found in the two South African species Heydrichia woelkerlingii Townsend, Keats et Chamberlain and Heydrichia groeneri Keats et Chamberlain. In habit, H. homalopasta grows as a thin crust (to 1 mm thick), similar to H. groeneri, but differing from the massive crusts (to 15 mm thick) found in H. woelkerlingii. The tetrasporangial nemathecia of H. homalopasta are single sporangial complexes whereas those of the South African species are composed of numerous sporangial complexes.

FITC-Phalloidin Staining of F-Actin in Aglaothamnion oosumiense and Griffithsia japonica (Rhodophyta)

Fluorescein isothiocyanate (FITC) conjugated phalloidin was used to label F-actin in Aglaothamnion oosumiense Itono, and Griffithsia japonica Okamura. Labelling was achieved without treatment by wall-digesting enzymes. Formaldehyde fixation followed by Triton X-100 wash gave good preservation of F-actin and cell components. Extensive arrays of F-actin were observed in the peripheral cytoplasm and nuclear area of the two species. Actin was strongly associated with the pit plugs in both species. Quantitative analysis using confocal microscopy showed that the amount of F-actin increased during cell division in Aglaothamnion oosumiense. Actin also appeared to play an important role in cytokinesis and karyokinesis during tetrasporogenesis.

A PROPOSAL FOR A NEW RED ALGAL ORDER, THE THOREALES

The freshwater red algal genera Nemalionopsis and Thorea are currently classified in the family Thoreaceae of the Batrachospermales. However, these two genera differ from other members of the order in having multiaxial thalli. The phylogenetic relationships of the genera were investigated using a combination of DNA sequence analysis (rbcL and 18S rRNA genes) and transmission electron microscopy. In both gene trees, Nemalionopsis and Thorea form well supported clades (87–100% bootstrap values) separate from other members of the Batrachospermales as well as taxa from the Acrochaetiales, Palmariales, and Nemaliales. In addition, analysis of the secondary structure of the 18S rRNA gene in Nemalionopsis and Thorea reveals an additional helix, which is not present in any of the other taxa within the Rhodophyta. Pit plugs of the Thoreaceae also differ from those of other members of the Batrachospermales in not having an outer cap layer that is not universally domed. Based on these findings, it is proposed that Nemalionopsis and Thorea represent a distinct taxonomic group that should be recognized as a new order, the Thoreales.