Inheritance pattern of chloroplast DNA is correlated with gamete types based on sex-specific arrangement of the cell fusion site in Caulerpa (Ulvophyceae, Chlorophyta)

Using field emission scanning electron microscopy (FE‐SEM) and fluorescence microscopy, the respective relationships between the arrangement of the gamete cell‐fusion site and the inheritance pattern of chloroplast DNA (cp‐DNA) were studied for Caulerpa brachypus Harvey, C. okamurae Weber‐van Bosse, C. racemosa (Forsskål) J. Agardh var. laete‐virens (Montagne) Weber‐van Bosse, and C. serrulata (Forsskål) J. Agardh var. serrulata f. lata (Weber‐van Bosse) Tseng. The eyespot of the biflagellate gamete was visualized using FE‐SEM. The female gamete, but not the male, has one eyespot on the cell body posterior. In most mating pairs, the female gamete is fused at the anterior left side of the eyespot and the male gamete at a cell surface that is perpendicular to the plane of the flagellar beat when both gametes are mixed. Then, the inheritance pattern of cp‐DNA was observed using fluorescence microscopy after staining with 4′6‐diamidino‐2‐phenylindole. Male and female gametes have one cell nucleus and one chloroplast each. Chloroplasts of the female gamete usually contain 1–11 spherical or rod‐shaped nucleoids. In contrast, nucleoids are not usually detected in the male gamete’s chloroplast. After mixing male and female gametes, the male gamete without nucleoids and female gametes with nucleoids are always associated at the lateral side and become planozygotes. Such a correlation between the arrangement of the cell fusion site and the inheritance pattern of cp‐DNA was found in another member of Caulerpales, Bryopsis maxima Okamura. These results suggest the possibility that the arrangement of the cell fusion site in the gamete is not determined randomly regardless of sex, but is rather correlated with specific mating types. The relation of these results to those for Chlamydomonas is discussed.     

First record of Caulerpa lentillifera J. Agardh (Bryopsidales,Chlorophyta) from China

ABSTRACT

An edible green algal species Caulerpa lentillifera J. Agardh is reported from China for the first time. The species was collected from the southwest of Hainan Island and morphologically identified to be C. lentillifera based on the grape-like branches arising from cylindrical stolons. Phylogenetic analysis using tufA and rbcL DNA sequences also confirmed the monophyly of C. lentillifera-microphysa clade.     

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.     

Larvicidal effects of some natural products on the Aedes aegypti mosquito,a vector of dengue fever and Zika virus

Natural products are considered a good choice in the biological control of mosquitoes because they are an effective way to eliminate larvae and prevent an increase in mosquito numbers, while simultaneously not polluting the environment or damaging health. This investigation was designed to study the potential toxicity of three species of algae, Caulerpa racemosa (Weber-van Bosse, 1909), Padina boryana (Thivy, 1966), and Turbinaria ornata (Turner J. Agardh, 1848), on the larvae of Aedes aegypti mosquito, the vector of dengue and Zika viruses. Among the studied species, Caulerpa racemosa showed the greatest effectiveness in eradicating A. aegypti larvae with an LC₅₀ = 43.5 ppm, followed by Padina boryana with an LC₅₀ = 51.93 ppm. Both species proved to be excellent candidates as a source of larvicidal agents and could be used commercially in mosquito control programs as eco-friendly biopesticides. The combined activity of different mixtures against mosquito larvae was expressed as the coeffective factor (C.F.). C.F. values showed that the joint activity of insect growth regulator Dudim in combination with Caulerpa racemosa and Padina boryana extracts produced degrees of potentiation effects and degrees of additive effects were obtained with Dudim in combination with Turbinaria ornata extract.     

Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta,Bryopsidales) from the Dry Tortugas,Florida

Morphological and molecular evidence is provided to further document the status of the enigmatic taxon known as Caulerpa floridana W.R. Taylor from White Shoal, Dry Tortugas, Florida. DNA sequencing of three historical herbarium specimens (WRT329, WRT345 and WRT349) housed at the University of Michigan Herbarium (MICH) demonstrated the molecular separation of this species based on the reconstruction of 931 nucleotides of the chloroplast gene tufA. Caulerpa floridana is sister to the western Atlantic endemic C. ashmeadii Harvey and an unknown Caulerpa taxon from the Florida Middle Grounds. Caulerpa floridana most reliably differs from C. ashmeadii by the presence of a sharp, unequivocal apiculus at the tip of each pinnule. A morphological review of southwestern Atlantic records of C. floridana from Brazil excludes these reports as representative of the species.     

Genetic polymorphism in Caulerpa taxifolia (Ulvophyceae) chloroplast DNA revealed by a PCR‐based assay of the invasive Mediterranean strain

Abstract An invasive, cold‐tolerant strain of the tropical green alga Caulerpa taxifolia was introduced recently in the Mediterranean Sea and along the Californian coast. We screened 50 aquarium and open‐sea C. taxifolia specimens for the presence/absence of an intron located in the rbcL gene of chloroplast DNA. We also reanalysed a total of 229 sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA, combining previously published sequences from different studies with 68 new sequences to complement rbcL data. The introduced Mediterranean strain was found to be characterized by the absence of the rbcL intron and by the occurrence of a particular monomorphic ITS type. A PCR assay based on rbcL gene was developed to detect new introductions of the invasive strain of C. taxifolia. This rapid and inexpensive test could be useful to assist environment managers in the preservation of coastal marine ecosystems.     

MEDITERRANEAN CAULERPA TAXIFOLIA AND C. MEXICANA (CHLOROPHYTA) ARE NOT CONSPECIFIC

In 1984, Caulerpa taxifolia (Vahl) C. Agardh was reported along the coast of Monaco. Over the past decade it has spread along 60 km of the Mediterranean coastline and presently represents a potential risk to biodiversity. Several explanations have been advanced regarding the presence of C. taxifolia in the Mediterranean. One hypothesis maintains that the alga was introduced accidentally into the sea at Monaco, where it has been used as a decorative alga in aquaria. Caulerpa taxifolia has not been reported in earlier marine floras of the Mediterranean, and its sudden appearance has suggested that it may be a recent introduction. Another hypothesis proposes that C. taxifolia and Caulerpa mexicana Sonder ex Kützing are morphological variants of one another and hence conspecific taxa. Caulerpa mexicana has been found in the eastern Mediterranean since at least 1941. In order to establish the taxonomic identities of these taxa, individuals from five populations of C. taxifolia and four populations of C. mexicana were collected from within and outside of the Mediterranean. Comparative DNA sequence analysis of the nuclear ribosomal cistron, including the 3′-end of the 18S, ITS1, 5.8S, and ITS2 regions, show clear phylogenetic separation of the two taxa using parsimony and maximum likelihood analyses. Separation is maintained whether the analyses are based on just the more conserved 18S data or just the fast- evolving spacers. The two species are thus not conspecific. For specimens of uncertain identity (i.e. taxifolia–mexicana intermediates), a PCR diagnostic amplification can easily be performed because the ITS1 in C. taxifolia is 36 nucleotides shorter than the ITS1 in C. mexicana. Whether or not C. taxifolia has been present for a longer period of time in the marine flora, either as a cryptic endemic species or as the result of one or more introductions, represents an additional hypothesis that will require identification of biogeographic populations from throughout the world, as well as a population-level study of the Mediterranean region.     

NEW PYRENOID FORMATION IN THE BROWN ALGA,SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

The Scytosiphon lomentaria (Lyngbye) Link cell characteristically has only one chloroplast with a prominent protruding pyrenoid. We observed the appearance of a new pyrenoid in each chloroplast during first mitosis in zygotes of S. lomentaria, using the freeze substitution technique. At first, a pyrenoid matrix appeared within the outermost stroma, in which thylakoid triplets and ribosomes were absent. At this time, the surface of this part remained smooth. The old pyrenoid was covered with a pyrenoid cap on the cytoplasmic side, whereas there was no pyrenoid cap on the new pyrenoid before protrusion. Irregularly shaped membranous sacs containing fine granular materials associated with the cytoplasmic side of the new pyrenoid. The sacs fused with each other and changed conformation and finally transformed into the pyrenoid cap. The new pyrenoid gradually protruded toward the cytoplasm, and the new pyrenoid cap became curved along the surface of pyrenoid. Cytokinesis occurred, and each chloroplast had two prominent protruding pyrenoids in two‐celled zygotes. We examined immunolocalization of β‐1,3‐glucans within the pyrenoid cap with a monoclonal antibody, using EM. Gold particles indicating localization of β‐1,3‐glucans were detected in vacuoles but never in the pyrenoid cap. This observation suggests that the pyrenoid cap in brown algae contains no photosynthetic products such as polysaccharide.     

PHYLOGENETIC RELATIONSHIPS OF CAULERPA (CHLOROPHYTA) BASED ON COMPARATIVE CHLOROPLAST ULTRASTRUCTURE1,2

The ultrastructure of chloroplasts from 28 of the 73 species of Caulerpa Lamouroux (Chlorophyta, Caulerpales) has been studied to aid in interpreting phylogenetic relationships among the 12 recognized sections. Variations of systematic value include pyrenoid occurrence and fine structure, thylakoid architecture and amount of photosynthate storage. Comparisons of field and culture specimens indicate these characters are consistent. Chloroplast thylakoids are grouped into bands, with the distribution of bands differing among species. In the most common arrangement, bands are evenly distributed throughout the chloroplast. A few species show lateral displacement of bands whereas others have a majority of bands arranged at one end of the chloroplast. Starch is stored cither as one or two large grains (> 1 μm diam.) or numerous small grains (< 0.5 μm diam.). Electron-transparent regions are common in other species in which chloroplasts rarely store starch. Simple, embedded pyrenoids are present in several species of section Sedoideae. An opaque region occurs in chloroplasts of C. elongata which may represent an intermediate stage in the evolutionary loss of the pyrenoid. It is suggested that the chloroplast of Caulerpa evolved, from a large, complex, pyrenoid-containing organelle housing both photosynthetic and amylogenic functions, to a small, structurally simpler one, specialized for photosynthesis alone. A phylogeny of the 12 sections of Caulerpa is constructed, based on chloroplast evolution which agrees with an earlier morphology-based hypothesis on the origin and evolution of Caulerpa.     

PLASTID DNA RESTRICTION ANALYSIS LINKS THE HETEROMORPHIC PHASES OF AN APOMICTIC RED ALGAL LIFE HISTORY1

Gymnogongrus sp. (Phyllophoraceae) from Nova Scotia, Canada, identified tentatively as G. devoniensis (Greville) Schotter, grows in association with an Erythrodermis-like that forms chains of tetrasporangia or bisporangia. The crust resembles tetrasporophytic phases of other Gymnogongrus species, but in culture both it and the G. devoniensis gametophytes cycle independently by apomictic reproduction. A method was developed for extracting organelle DNA from this carrageenophyte genus involving purification of nucleic acids by binding to hydroxylapatite. Plastid DNA from G. devoniensis and bisporangial Erythrodermis-like crusts was compared with that of G. devoniensis and G. crenulatus (Turner) J. Agardh from France and of G. furcellatus (C. Agardh) J. Agardh from Chile. Plastid genomes of all Gymnogongrus species and the Erythrodermis-like crust were approximately 175 kb long. A single 3.5-kb plasmid DNA species was found in G. devoniensis and the Erythrodermis-like bisporophyte but not in other samples. Digestion of plasted DNA with several restriction endonucleases produced identical patterns in G. devoniensis and the Erythrodermis-like bisporophyte from the same location, indicating clearly that these entities represent two phases of an uncoupled life history. These results were confirmed with heteologous probes. A restriction fragment length polymorphism was identified between two Nova Scotian G. devoniensis populations. There was no similarity in restriction patterns between G. devoniensis from Nova Scotia, G. devoniensis from France. G. crenulatus or G. furcellatus, suggesting that molecular taxonomic methods could be important in delineating members of this morphologically variable genus. Further study is necessary to determine whether either Nova Scotian G. devoniensis or French G. devoniensis corresponds to type populations of G. devoniensis from Devon, England.     

Immunocytochemical Localization of Phosphoribulokinase in Microalgae

Employing immunogold electron microscopy, the subcellular location of the Calvin cycle enzyme phosphoribulokinase (PRK) was determined for two diverse species of microalgae. In both the red alga Porphyridium cruentum and the green alga Chlamydomonas reinhardtii, PRK was distributed throughout the thylakoid-containing chloroplast stroma. In contrast, the next enzyme in the pathway, ribulose 1,5-bisphosphate carboxylase/oxygenase, was predominantly pyrenoid-localized in both species. In Porphyridium, the chloroplast stroma abuts the pyrenoid but in Chlamydomonas and other green algae, the pyrenoid appears encased in a starch sheath. Unique inclusions found in the pyrenoid of Chlamydomonas were immunolabelled by anti-PRK and thus identified as regions of chloroplast stroma. It is postulated that such PRK-containing stromal inclusions in the pyrenoids of Chlamydomonas and perhaps other green algae provide a means for exchange of Calvin cycle metabolites between pyrenoid and stroma.     

Nutrient availability in the sediment and the reciprocal effects between the native seagrass Cymodocea nodosa and the introduced rhizophytic alga Caulerpa taxifolia

Two reciprocal experiments testing for the effects of nutrient addition in the sediment and competitive interactions between the native seagrass Cymodocea nodosa (Ucria) Ascherson and the introduced alga Caulerpa taxifolia (Vahl) C. Agardh were performed. This study was conducted for 13 months (August 1995 until September 1996) in a bay on the south coast of Elba Island (Italy). Each experiment consisted of the manipulation of the level of nutrients (addition vs. control) and the manipulation of the neighbours (presence vs. removal). Response variables were blade density and size for one experiment and shoot density and leaf length of seagrass in the other. Results indicated that the presence of Caulerpa taxifolia did not affect significantly Cymodocea nodosa shoot density and the increased nutrient availability in the sediment did not alter this pattern. Neither the removal of the canopy of the seagrass nor the fertilization of the sediment has influenced significantly the density of the alga. Both species, where co-occurring, show larger size than where the neighbour is removed. Hence, results of this study suggest that the two species on the long term are likely to coexist and that the high nutrient supply of the sediment would not enhance the probability of success neither of the seagrass nor of the alga. Predictions made on the basis of short-term results, that high nutrient loads of the substratum would have represented an even more suitable condition for C. taxifolia to colonize C. nodosa beds and that on the long-term the alga has a high probability of success, did not occur.     

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.     

Phycoerythrin is absent from the pyrenoid of Porphyridium cruentum: photosynthetic implications

The thylakoid lamellae which traverse the pyrenoid of the unicellular red alga Porphyridium cruentum (Agardh) Nägeli appear to lack phycobilisomes. We have confirmed by immuno-electron microscopy that phycoerythrin (PE), an important structural component of the phycobilisomes of red algae, is absent from the pyrenoid. To characterize pyrenoid thylakoids further, electron-microscopic cytochemical methods were employed to detect photosystem activity. Photosystem (PS) I activity was demonstrated in both stromal and pyrenoid thylakoids by the photooxidation of 3,3-diaminobenzidine. In contrast, the localization of photoreduced distyryl nitroblue tetrazolium demonstrated that PSII activity was restricted to stromal thylakoids. The observed partitioning of PE and PSII activity within the plastid may be related to another observation, that being the localization of nearly all ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) within the pyrenoid of this alga. It is possible that the pyrenoid of P. cruentum functions as a specific metabolic compartment where CO₂ fixation is enhanced by the absence of photosynthetic O₂ evolution.Abbreviations DAB 3,3-diaminobenzidine-4HCl - DS-NBT distyryl nitroblue tetrazolium chloride - EF exoplasmic face - LSU large subunit of RuBisCO - PE phycoerythrin - PS photosystem - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Drs. Jacqueline Fleck (CNRS, Strasbourg) and Robert MacColl (New York State Department of Health, Albany) for providing us with the antibodies used in this study. We also thank Dr. C.E. Smith for use of the Zeiss MOP-3 digital analyser and Dr. Geneviève Bricheux for kindly providing Lowicryl-embedded samples of P. cruentum. Aatrex^® was kindly donated by Ciba-Geigy. This research was supported by the Natural Sciences and Engineering Research Council of Canada (grant No. A-2921).     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.     

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.     

SPECIES-SPECIFIC DIFFERENCES OF PYRENOIDS IN CHLORELLA (CHLOROPHYTA)1

The structure of the pyrenoid supports the separation of Chlorella species into two groups based on cell wall chemistry and suggests evolutionary relationships. Chlorella species with a glucan-type wall exhibit quite diverse pyrenoid structures, which may indicate that these species are not closely related. Those species with glucosamine cell walls (C. kessleri, C. sorokiniana, C. vulgaris) are virtually identical in pyrenoid morphology, indicating a closer evolutionary relationship. In the species with glucosamine walls, the thylakoid that penetrates into the pyrenoid matrix, is unijormly double-layered. Pyrenoids in the species with glucan walls show various features: 1) a pyrenoid matrix only, 2) a pyrenoid traversed by a few discs of double thylakoids with many adhering pyrenoglobuli, 3) a pyrenoid penetrated with tubelike structures or 4) a pyrenoid penetrated with many single undulating thylakoids. The pyrenoid structure of the symbiotic Chlorella in Paramecium bursaria resembles those of free-living Chlorella with glucosamine walls.