Further confirmation of the presence of DNA in the pyrenoid core of the siphonous green algal genus Caulerpa (Caulerpales, Ulvophyceae, Chlorophyta)

We re-examined the distribution of chloroplast DNA (ct-DNA) in the pyrenoid core of Caulerpa okamurae Weber van Bosse and C. lentillifera J. Agardh by fluorescence microscopy after staining the squashes and Technovit sections with DNA fluorochromes such as 4′6-diamidino-2-phenylmdole (DAPI), ethidium bromide, Hoechst 33258 and chromomycin A3. All fluorochromes stained specifically the pyrenoid core on the squashes and Technovit sections. In addition, we present new data on the localization of ct-DNA in the pyrenoid core of two other species of the genus Caulerpa: C. cactoides (Turner) Agardh and C. geminata Harvey.     

A FORENSIC AND PHYLOGENETIC SURVEY OF CAULERPA SPECIES (CAULERPALES,CHLOROPHYTA) FROM THE FLORIDA COAST,LOCAL AQUARIUM SHOPS,AND E‐COMMERCE: ESTABLISHING A PROACTIVE BASELINE FOR EARLY DETECTION1

Baseline genotypes were established for 256 individuals of Caulerpa collected from 27 field locations in Florida (including the Keys), the Bahamas, US Virgin Islands, and Honduras, nearly doubling the number of available GenBank sequences. On the basis of sequences from the nuclear rDNA‐ITS 1+2 and the chloroplast tufA regions, the phylogeny of Caulerpa was reassessed and the presence of invasive strains was determined. Surveys in central Florida and southern California of >100 saltwater aquarium shops and 90 internet sites revealed that >50% sold Caulerpa. Of the 14 Caulerpa species encountered, Caulerpa racemosa was the most common, followed by Caulerpa sertularioides, Caulerpa prolifera, Caulerpa mexicana, and Caulerpa serrulata. None of the >180 field‐collected individuals (representing 13 species) was the invasive strain of Caulerpa taxifolia or C. racemosa. With one exception (a sample of C. racemosa from a shop in southern California belonged to the invasive Clade III strain), no invasive strains were found in saltwater aquarium stores in Florida or on any of the internet sites. Although these results are encouraging, we recommend a ban on the sale of all Caulerpa species (including “live rock”) because: morphological identification of Caulerpa species is unreliable (>12% misidentification rate) and invasive strains can only be identified by their aligned DNA sequences, and because the potential capacity for invasive behavior in other Caulerpa species is far from clear. The addition of the Florida region to the genetic data base for Caulerpa provides a valuable proactive resource for invasion biologists as well as researchers interested in the evolution and speciation of Caulerpa.     

OBSERVATIONS ON THE FINE STRUCTURE OF OEDOGONIUM. V. EVIDENCE FOR THE DE NOVO FORMATION OF PYRENOIDS IN ZOOSPORES OF OE. CARDIACUM123

Ultrastructural studies of the chloroplasts of zoospores and developing zoospores of Oedogonium carcliacum have disclosed the occurrence of numerous incipient pyrenoids. A single developing zoospore may possess several score of these structures which appear to arise de novo in the chloroplast stroma and seem to lack any direct association with mature pyrenoids which are also present in the cells. The incipient pyrenoids lack the associated starch grains and the membrane-limited channels characteristic of mature pyrenoids, but they are readily recognized in the chloroplasts since they demonstrate a greater granularity and electron density than the surrounding chloroplast stroma. The granularity and electron density of the incipient pyrenoids match the ultra-structural appearance of the matrix of mature pyrenoids. The smallest of the incipient pyrenoids examined from serial sections had a maximum diameter of less than 0.3 μ. This may be compared with the size of mature pyrenoids, many with a maximum diameter of over 5.0 μ. In all the zoospores and developing zoospores examined, only one mature pyrenoid was observed in an apparent stage of division.     

EVOLUTIONARY ORIGIN OF GLOEOMONAS (VOLVOCALES,CHLOROPHYCEAE), BASED ON ULTRASTRUCTURE OF CHLOROPLASTS AND MOLECULAR PHYLOGENY1

Gloeomonas is a peculiar unicellular volvocalean genus because it lacks pyrenoids in the chloroplasts under the light microscope and has two flagellar bases that are remote from each other. However, ultrastructural features of chloroplasts are very limited, and no molecular phylogenetic analyses have been carried out in Gloeomonas. In this study, we observed ultrastructural features of chloroplasts of three species of Gloeomonas and Chloromonas rubrifilum (Korshikov ex Pascher) Pröschold, B. Marin, U. Schlösser et Melkonian SAG 3.85, and phylogenetic analyses were carried out based on the combined data set from 18S rRNA, ATP synthase beta‐subunit, and P700 chl a–apoprotein A2 gene sequences to deduce the natural phylogenetic positions of the genus Gloeomonas. The present EM demonstrated that the chloroplasts of the three Gloeomonas species and C. rubrifilum SAG 3.85 did not have typical pyrenoids with associated starch grains, but they possessed pyrenoid matrices that protruded interiorly within the stroma regions of the chloroplast. The pyrenoid matrices were large and broad in C. rubrifilum, whereas those of the three Gloeomonas species were recognized in only the small protruded regions of the chloroplast lobes. The present multigene phylogenetic analyses resolved that the three species of Gloeomonas belong to the Chloromonas lineage or Chloromonadinia of the Volvocales, and Chloromonas insignis (Anakhin) Gerloff et H. Ettl NIES‐447 and C. rubrifilum SAG 3.85, both of which have pyrenoids without associated starch grains, were positioned basally to the clade composed of the three species of Gloeomonas. Therefore, Gloeomonas might have evolved from such a Chloromonas species through reduction in pyrenoid matrix size within the chloroplast and by separating their two flagellar bases.     

A PRELIMINARY COMPARISON OF RESTRICTION FRAGMENT PATTERNS IN THE GENUS CAULERPA (CHLOROPHYTA) AND THE UNIQUE STRUCTURE OF THE CHLOROPLAST GENOME OF CAULERPA SERTULARIOIDES1

Comparisons of chloroplast DNA restriction fragments in four species of Caulerpa revealed that patterns between the species were different, with few and possibly no homologous bands. Two forms of Caulerpa sertularioides also revealed different patterns, and it is possible that the forms are separate species. The chloroplast genome in Caulerpa sertularioides f sertularioides (S. G. Gmelin) Howe is 131.4 kb in size and lacks large repeat units. The discovery of another green-algal chloroplast genome that lacks an inverted repeat indicates that this feature is either not ancestral to the Chlorophyceae or has been lost several times. Several gene clusters commonly found in chloroplast DNAs were found to occur in Caulerpa chloroplast DNA, for example, psbD/C, atpF/H, and psaA/B. The 16S and 23s rRNA, which are typically adjacent, contained in an inverted repeat, and cotranscribed, are over 40 kb apart. Genes rps12 and tufA, members of the str operon in eubacteria, are over 50 kb in distance from each other in Caulerpa. The gene order in Caulerpa is unlike any other chloroplast genome characterized to date.     

Changes in structure of isolated chloroplasts ofCodium fragile andCaulerpa filiformis in response to osmotic shock and detergent treatment

Summary The ultrastructure of chloroplasts from two genera of coenocytic green algae,Codium andCaulerpa, were examined after suspension in hypotonic solution and in detergent at various concentrations. The capacity of the suspensions to carry out CO₂-dependent and ferricyanide-dependent O₂ evolution was measured under the same conditions of osmotic strength and detergent concentration.The chloroplasts in the preparations were in the form of cytoplasts and gave rates of O₂ evolution comparable with those expected from undamaged chloroplasts. Suspension in hypotonic solution depressed the rate of CO₂-dependent O₂ evolution in both species, but this was partially restored in theCodium chloroplasts when these were re-suspended in iso-osmotic solutions. Major structural changes were observed only after suspension in buffer when theCodium chloroplasts lost their outer envelope, most of their stroma, and the thylakoids became swollen.Caulerpa chloroplasts were more variable in their response and, even when suspended in buffer only, the proportion of the plastids which had lost all of their stroma and thylakoid swelling was never as common as inCodium chloroplasts. However, once suspended in hyper-osmotic medium below 700 mosmolar,Caulerpa chloroplasts could not regain their capacity for CO₂-dependent O₂ evolution.Detergent treatment removed the cytoplast membrane but not the cytoplasmic material adhering to the chloroplast envelope. High concentrations of detergent were needed to cause loss of the chloroplast envelope, loss of stromal contents and unstacking of the thylakoids.Caulerpa chloroplasts were less sensitive to detergent than those ofCodium. There was no indication that specific structures such as the thylakoid organizing body were resistant to detergent action. The results show that exposure to hypotonic solutions and to detergent results in less damage to these chloroplasts than it would to those of higher plants. It is proposed that the basis of this unusual resistance is not due to the properties of the chloroplast membranes but to the presence of material which coats the organelles during isolation. This material is likely to be identical with the sulphated xylo-mannogalactan isolated from the vacuole contents of these algae and which has the visco-elastic properties essential to allow the organelles to resist disruption by osmotic forces and disintegration by detergents.     

MOLECULAR PHYLOGENY OF THE GENUS CAULERPA (CAULERPALES,CHLOROPHYTA) INFERRED FROM CHLOROPLAST tufA GENE1

The genus Caulerpa consists of about 75 species of tropical to subtropical siphonous green algae. To better understand the evolutionary history of the genus, a molecular phylogeny was inferred from chloroplast tufA sequences of 23 taxa. A sequence of Caulerpella ambigua was included as a potential outgroup. Results reveal that the latter taxon is, indeed, sister to all ingroup sequences. Caulerpa itself consists of a series of relatively ancient and species‐poor lineages and a relatively modern and rapidly diversifying clade, containing most of the diversity. The molecular phylogeny conflicts with the intrageneric sectional classification based on morphological characters and an evolutionary scheme based on chloroplast ultrastructure. High bootstrap values support monophyly of C. mexicana, C. sertularioides, C. taxifolia, C. webbiana, and C. prolifera, whereas most other Caulerpa species show para‐ or polyphyly.     

ANALYSIS OF A PLASTID MULTIGENE DATA SET AND THE PHYLOGENETIC POSITION OF THE MARINE MACROALGA CAULERPA FILIFORMIS (CHLOROPHYTA)1

Molecular phylogenetic relationships within the Chlorophyta have relied heavily on rRNA data. These data have revolutionized our insight in green algal evolution, yet some class relationships have never been well resolved. A commonly used class within the Chlorophyta is the Ulvophyceae, although there is not much support for its monophyly. The relationships among the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae are also contentious. In recent years, chloroplast genome data have shown their utility in resolving relationships between the main green algal clades, but such studies have never included marine macroalgae. We provide partial chloroplast genome data (～30,000 bp, 23 genes) of the ulvophycean macroalga Caulerpa filiformis (Suhr) K. Herig. We show gene order conservation for some gene combinations and rearrangements in other regions compared to closely related taxa. Our data also revealed a pseudogene (ycf62) in Caulerpa species. Our phylogenetic results, based on analyses of a 23‐gene alignment, suggest that neither Ulvophyceae nor Trebouxiophyceae are monophyletic, with Caulerpa being more closely related to the trebouxiophyte Chlorella than to Oltmannsiellopsis and Pseudendoclonium.     

Re-examination of ultrastructures of the stellate chloroplast organization in brown algae: Structure and development of pyrenoids

Some taxa of brown algae have a so‐called ‘stellate’ chloroplast arrangement composed of multiple chloroplasts arranged in a stellate configuration, or else a single chloroplast with radiating lobes. The fine structures of chloroplasts and pyrenoids have been studied, but the details of their membrane configurations as well as pyrenoid ontogeny have not been well understood. The ultrastructure of the single stellate chloroplast in Splachnidium rugosum and Scytothamnus australis were re‐examined in the present study, as well as the stellate arrangement of chloroplasts in Asteronema ferruginea and Asterocladon interjectum, using freeze‐substitution fixation. It was confirmed that the chloroplast envelope invaginated into the pyrenoid in Splachnidium rugosum, Scytothamnus australis and Asteronema ferruginea, but chloroplast endoplasmic reticulum (CER) remained on the surface of the chloroplast. The space between the invaginated chloroplast envelope and CER was filled with electron‐dense material. In Asteronema ferruginea, CER surrounding each pyrenoid was closely appressed to the neighboring CER over the pyrenoids, so that the chloroplasts formed a stellate configuration; however, in the apical cells chloroplasts formed two or more loose groups, or were completely dispersed. The pyrenoids of Asterocladon interjectum did not have any invagination of the chloroplast envelope, but a unique membranous sac surrounded the pyrenoid complex and occasionally other organelles (e.g. mitochondria). Immunolocalization of β‐1,3‐glucans showed that the membranous sac in Asterocladon interjectum did not contain photosynthetic products such as chrysolaminaran. Observations in the dividing cells of Splachnidium rugosum and Scytothamnus australis indicated that the pyrenoid in the center of the chloroplast enlarged and divided into two before or during chloroplast division.     

NEW ULTRASTRUCTURAL ASPECTS OF PYRENOIDS OF THE LICHEN PHOTOBIONT TREBOUXZA (MICROTHAMNIALES,CHLOROPHYTA)1

The pyrenoid structure of Trebouxia, a photobiont of two lichen species, Umbilicaria cinereorufescens (Schaer.) Frey and Parmelia sulcata Taylor, was investigated. In both lichen species, the pyrenoid of the photobiont exhibited straight, unbranched, long or short tubules. In the first lichen species, multiple pyrenoids were observed occasionally, while in the second one, homogeneous masses, called protein bodies, appeared between the thylakoids. These protein bodies were previously observed in some other species of the family Umbilicariaceae. Serial sections from single pyrenoids showed that tubules of the Impressa-type pyrenoid were closely associated with pyrenoglobuli. The three-dimensional reconstruction of a complete chloroplast of a P. sulcata algal cell showed that the protein bodies were spatially separate structures. Immunolocalization techniques to detect the presence of ribulose-bisphosphate carboxylase (Rubisco) in the chloroplast showed that this enzyme was present primarily in the pyrenoid matrix. When protein bodies were present in the chloroplast, Rubisco appeared to be localized in these structures. The presence of pyrenoid satellites and protein bodies with reactivity to anti-Rubisco may be related to the nutritional conditions of the thalli.     

LIGHT AND ELECTRON MICROSCOPY OF PYRENOIDS AND SPECIES DELIMITATION IN VOLVULINA (CHLOROPHYTA,VOLVOCACEAE)1

The appearances of pyrenoids in the vegetative cells of Volvulina steinii Playfair and V. pringsheimii Starr were observed in detail by light and electron microscopy in relation to the culture age to clarify the taxonomic relationship between the two species. In V. pringsheimii, the pyrenoids were always present in the bottom of the cupshaped chloroplasts and their gross morphology did not vary in relation to the culture age, while those of V. steinii appeared de novo and developed as the culture aged. In 24-h cultures of V. steinii, pyrenoids were not observed in the chloroplasts. In 48-h cultures, a pyrenoid matrix developed apparently de novo in the brim of the cupshaped chloroplast. Subsequently, starch grains appeared around the pyrenoid matrix in 72-h cultures. The volume of the matrix and the associated starch grains increased and tubular channels entered into the pyrenoid matrix in 96-h cultures. In addition, the pyrenoid in the parental chloroplast of V. pringsheimii divided and was distributed to each daughter cell during cell divisions in daughter colony formation, while the parental pyrenoid of V. steinii did not divide and went to one of the daughter cells. Therefore, these two species can be clearly distinguished by the differences in the position of pyrenoids in the cupshaped chloroplasts and stability of pyrenoid appearance in relation to the culture age, as well as in the fate of parental pyrenoids during daughter colony formation.     

Electron Microscopic Observations on the Symbiosis of Paramecium bursaria and its Intracellular Algae*

SYNOPSIS. Observations were made on the fine structure of Paramecium bursaria and its intracellular Chlorella symbionts. Emphasis was placed on the structure of the algae and structural aspects of the relationship between the organisms. The algae are surrounded by a prominent cell wall and contain a cup-shaped chloroplast which lies just beneath the plasma membrane. Within the cavity formed by the chloroplast are a large nucleus, a mitochondrion, one or more dictyosomes, and numerous ribosomes. The chloroplast itself is made up of a series of lamellar stacks each containing 2–6 or more thylakoids with a granular stroma and starch grains intercalated between the stacks. The thylakoid stacks of mature algae are frequently more compact than those of recently divided algae. A large pyrenoid is located within the base of the chloroplast. It is made up of a granular or fibrillar matrix surrounded by a shell of starch. The matrix is bisected by a stack of 2 thylakoids. Prior to the division of the chloroplast the pyrenoid regresses; pyrenoids subsequently form in the daughter chloroplasts thru condensation of the matrix material and the reappearance of a starch shell. This shell appears to be formed by the hollowing-out of starch grains already present in the chloroplast stroma. Accordingly, in this case, starch moves from the stroma to the pyrenoid. The algae are located thruout the peripheral cytoplasm of the Paramecium. Each alga is located in an individual vacuole except immediately following division of the algae when the daughter cells are temporarily located in the vacuole which harbored the parental cell. Shortly thereafter the vacuole membrane invaginates, thereby isolating the daughter algae into individual vacuoles. Degenerating symbiotic algae are seen; because these are frequently found in vacuoles with bacteria, they are presumed to be undergoing digestion. Due to the conditions of culture these algae could have been either of intracellular or extracellular origin.     

PYRENOID FORMATION ASSOCIATED WITH THE CELL CYCLE IN THE BROWN ALGA,SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

Vegetative cells of the brown alga Scytosiphon lomentaria (Lyngbye) Link characteristically have only one chloroplast with a prominent protruding pyrenoid, whereas zygotes have both paternal and maternal chloroplasts. In zygotes, before cell and chloroplast division, each chloroplast has an old and a new pyrenoid. In this study, we raised a polyclonal antibody to RUBISCO and examined the distribution of RUBISCO by immunofluorescence microscopy, focusing on new pyrenoid formation in vegetative cells of gametophytes and zygotes in Scytosiphon. In interphase, only one old pyrenoid was positively indicated by anti‐RUBISCO antibody in vegetative cells of gametophytes. From mid‐S phase, small fluorescence aggregates reflecting RUBISCO localization started to appear at stroma positions other than adjacent to the old protruding pyrenoid. The fluorescent spots eventually coalesced into a protrusion into the adjacent cytoplasm. We also used inhibitors to clarify the relationship between the cell cycle and new pyrenoid formation, using zygotes after fertilization. When DNA replication was blocked by aphidicolin, new pyrenoid formation was also inhibited. Washing out aphidicolin permitted new pyrenoid formation with the progression of the cell cycle. When mitosis was prolonged by nocodazole, which disrupted the spindle microtubules, the fluorescent masses indicating RUBISCO localization continued to increase when compared with pyrenoid formation in untreated zygotes. During treatment with chloramphenicol, mitosis and cytokinesis were completed. However, there was no occurrence of new RUBISCO localization within the chloroplast stroma beyond the old pyrenoid. From these observations, it seems clear that new pyrenoid formation in the brown alga Scytosiphon depends on the cell cycle.     

ULTRASTRUCTURE OF THE CONCHOCELIS STAGE OF THE MARINE RED ALGA PORPHYRA LEUCOSTICTA1

The ultrastructure of the Conchocelis or filamentous stage of Porphyra leucosticta was investigated. Each cell contains 1 or 2 parietal, stellate chloroplasts with a single pyrenoid in each chloroplast. The centrally located nucleus is irregularly shaped and contains 1–2 nucleoli. The cytoplasm has typical floridean starch grains and nonmernbrane-bound lipid bodies. The cell wall is divided into an outer and an inner wall. Many lomasomes are associated with the cell membrane. Pit connections are found between cells, and their taxonomic significance is discussed.     

CYTOLOGY AND ULTRASTRUCTURE OF CHLORARACHNION REPTANS (CHLORARACHNIOPHYTA DIVISIO NOVA,CHLORARACHNIOPHYCEAE CLASSIS NOVA)1

The green amoeboid cells of Chlorarachnion reptans Geitler are completely naked and each contains a central nucleus, several bilobed chloroplasts each with a central projecting pyrenoid enveloped by a capping vesicle, several Golgi bodies, mitochondria with tubular cristae, extensive rough ER, and a distinct layer of peripheral vesicles. Complex extrusome-like organelles occur rarely in both the amoeboid and flagellate stages. The only organelles entering the reticulopodia are mitochondria, but microtubules are also present. The chloroplasts contain chlorophylls a and b, but histochemical tests suggest that the carbohydrate storage product probably is not a starch. The chloroplast lamellae are composed of one to three thylakoids or form deep stacks. A girdle lamella and interlamellar partitions are absent. Each chloroplast is bounded by either four separate membranes, a pair of membranes with vesicular profiles between them, or three membranes; all three arrangements may occur in the same chloroplast. A periplastidal compartment occurs near the base of the pyrenoid where there are always four surrounding membranes. The compartment has a relatively dense matrix and contains ribosome-like particles and small dense spheres; it extends over and into a deep invagination in the pyrenoid where its contents are enclosed in a double-membraned envelope which is penetrated by wide pores. The zoospores are ovoid and each bears a single laterally inserted flagellum which appears to be wrapped helically around the cell body during swimming. The flagellum lies in a groove in the cell surface and bears fine lateral hairs. Neither a second flagellum or vestige of one, nor an eyespot, is present. A single microtubular root and a larger homogeneous root run from the flagellar base parallel to the emerging flagellum, between the nuclear envelope and the plasmalemma. In the simple flagellar transition region, fine filaments connect adjacent axonemal doublets. A detailed comparison of C. reptans with all other algal taxa results in the conclusion that it must be segregated in the new class Chlorarachniophyceae, the only class in the new division Chlorarachniophyta. The possibility that C. reptans evolved from a symbiosis between a colorless amoeboid cell and a chlorophyll b- containing eukaryote is considered, but the possible affinities of the symbiont remain enigmatic. The implications of the unique chloroplast structure of C. reptans for current hypotheses concerning the origin of chloroplasts are discussed.     

CACHONINA ILLDEFINA SP. NOV. (DINOPHYCEAE): CHLOROPLAST TUBULES AND DEGENERATION OF THE PYRENOID1

A new species of the dinoflagellate genus Cachonina, C. illdefina sp. nov., was isolated from a red tide off El Capitan State Park, Santa Barbara County, California, in October 1973. The organism is light yellowgreen in color with deeply incised girdle and sulcal grooves. Electron microscopy of the organism, revealed a typical dinokaryotic nucleus. The chloroplasts of the organism are connected, and often contain microtubule-like elements, 25 nm diam. The pyrenoids are characterized as excluding chloroplast thylakoids and ribosomes, although containing an amorphous matrix and numerous tubular invaginations from the cytoplasm. The pyrenoids become detached from the chloroplasts and degenerate into small vesicles. C. illdefina is not bioluminescent.     

LIGHT MICROSCOPY AND ELECTRON MICROSCOPY OF NEPHROSELMIS SPINOSA SP. NOV. (PRASINOPHYCEAE,CHLOROPHYTA)1

Nephroselmis spinosa Suda sp. nov. is described based on LM and EM observations. Two strains of N. spinosa (S222 and SD959‐3) were isolated from sand samples collected from the northwest coast of western Australia. The cells were remarkably right–left flattened and appeared ellipse or bean‐shaped when viewed from their right or left side. A single, parietal, crescent chloroplast was pale green to yellowish green and contained one conspicuous eyespot in its anterior ventral edge near the base of the short flagellum. A pyrenoid with three starch plates was located at the dorsal of the chloroplast. The cells divided by transverse binary cell division, as is common in other species of this genus. This alga possessed four types of body scales, and three scale types were similar to N. olivacea Stein and N. astigmatica Inouye & Pienaar. However, the fourth and outermost scale type was distinctive because although it was a spiny stellate scale with nine spines, one of them extended about 1 μm and was slightly curved with a hook at the end. This scale morphology, an important taxonomic characteristic, has never been described for the genus Nephroselmis. The cell's morphology, pyrenoid structure, hair scales, and cell size were distinctive from previously described Nephroselmis species, and its unique scale characteristic led me to name this newly proposed species “spinosa,” meaning spiny.     

Chloroplast structure and starch grain production as phylogenetic indicators in the lower Rhodophyceae

The position of starch grain production, the shape of the starch grains and the depth to which the pyrenoid is embedded in the chloroplast are used as indicators of evolution in the lower Rhodophyceae. A cell with cytoplasmic allantoid starch grains encasing the pyrenoid and no thylakoids between the chloroplast envelope and the pyrenoid is considered to be evolutionarily primitive. A cell with oval starch grains not associated with the pyrenoid and with a pyrenoid deeply embedded in the chloroplast is thought to be evolutionarily advanced. A polyphyletic origin of the Porphyridiales is discussed.