Tracking the invasive history of the green alga Codium fragile ssp. tomentosoides

The spread of nonindigenous species into new habitats is having a drastic effect on natural ecosystems and represents an increasing threat to global biodiversity. In the marine environment, where data on the movement of invasive species is scarce, the spread of alien seaweeds represents a particular problem. We have employed a combination of plastid microsatellite markers and DNA sequence data from three regions of the plastid genome to trace the invasive history of the green alga Codium fragile ssp. tomentosoides. Extremely low levels of genetic variation were detected, with only four haplotypes present in the species' native range in Japan and only two of these found in introduced populations. These invasive populations displayed a high level of geographical structuring of haplotypes, with one haplotype localized in the Mediterranean and the other found in Northwest Atlantic, northern European and South Pacific populations. Consequently, we postulate that there have been at least two separate introductions of C. fragile ssp. tomentosoides from its native range in the North Pacific.     

Mismatch of morphological and molecular identifications in native and invasive subspecies of Codium fragile (Bryopsidophyceae,Chlorophyta)

Several subspecies are defined within Codium fragile, including the invasive C. fragile ssp. fragile, first reported in New Zealand in 1973. An endemic subspecies, C. fragile ssp. novae‐zelandiae, is also found throughout New Zealand. The two subspecies exhibit morphological and molecular variation, although these have never been evaluated together. We compared variation between subspecies at locations in Auckland, identifying subspecies using rps3‐rpl16 DNA sequence data, and assessing gross morphological differences, anatomical utricle characters and morphometrics. The taxonomic utility of the morphometric data sets was assessed by linear discriminant analysis. Utricle characters and measurements varied within individual thalli and between different preservation methods. The phenotypes of both subspecies were highly variable and influenced by environment. Accurate subspecies delimitation using morphological data was not possible; the discriminant analyses performed no better than chance for all combinations of the morphological data. Specimens from New Zealand, Canada, Australia and Ireland were sequenced using both the rps3‐rpl16 and tufA plastid markers. The tufA elongation factor was shown to be a good candidate for differentiating subspecies of C. fragile. This marker is twice the length of the rps3‐rpl16 spacer, shows greater variation between ssp. fragile and novae‐zelandiae, and is less prone to sequencing error. A simple restriction enzyme digest of the tufA amplicon can distinguish ssp. fragile and ssp. novae‐zelandiae. Our study expands the known range of the ssp. fragile in New Zealand, including the first record of this subspecies from the west coast of Auckland, and points to a need to re‐evaluate morphological and molecular criteria for subspecies currently defined within C. fragile.     

温度与盐度对刺松藻生长和营养盐吸收的影响

随着养殖业迅猛发展,海洋富营养化问题日益加剧。利用大型海藻所具有的超吸收氮磷营养盐的能力,可有效治理和修复富营养化海域。从福建省连江县罗源湾海域养殖筏架采取经济价值较高的刺松藻,为分析其在海水养殖区的生长条件,在实验室条件下,研究温度（18、25、32 ℃）和盐度（10、20、30）对刺松藻生长和营养盐吸收的影响。研究结果表明,刺松藻生长受温度影响极显著(P<0.01),受盐度及二者相互作用影响显著(P<0.05);在25 ℃和盐度20的条件下,刺松藻特定生长率（specific growth rate,SGR）最高,达89.45%;在32 ℃和盐度30的条件下,刺松藻对营养盐的吸收效果最好,其中对NH+4-N的吸收效果最佳。研究结果为生物修复种群的选择和刺松藻的人工养殖提供了一定的参考。     

Tracking biological invasions in space and time: elucidating the invasive history of the green alga Codium fragile using old DNA

With the advent of 'ancient DNA' studies on preserved material of extant and extinct species, museums and herbaria now represent an important although still underutilized resource in molecular ecology. The ability to obtain sequence data from archived specimens can reveal the recent history of cryptic species and introductions. We have analysed extant and herbarium samples of the highly invasive green alga Codium fragile , many over 100 years old, to identify cryptic accessions of the invasive strain known as C. fragile ssp. tomentosoides , which can be identified by a unique haplotype. Molecular characterization of specimens previously identified as native in various regions shows that the invasive tomentosoides strain has been colonizing new habitats across the world for longer than records indicate, in some cases nearly 100 years before it was noticed. It can now be found in the ranges of all the other native haplotypes detected, several of which correspond to recognized subspecies. Within regions in the southern hemisphere there was a greater diversity of haplotypes than in the northern hemisphere, probably as a result of dispersal by the Antarctic Circumpolar Current. The findings of this study highlight the importance of herbaria in preserving contemporaneous records of invasions as they occur, especially when invasive taxa are cryptic.     

Typification of species of Codium (Bryopsidales, Chlorophyta) described by Okamura

Type specimens of six species of Codium described by Okamura are illustrated, including the holotype of Codium barbatum and lectotypes of Codium coactum, Codium intricatum, Codium pugniforme, Codium saccatum, and Codium subtubulosum. All species are currently recognized except C. pugniforme, which is indistinguishable from the previously described Codium spongiosum Harvey, Codium subtubulosum was abandoned by Okamura in favor of the previously described Codium divaricatum Holmes, but because of the prior existence of C. divaricatum (C. Agardh) Biasoletto, C. subtubulosum is the correct name for this species.     

A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower

We present a method for isolation of chloroplast and mitochondrial DNA from sunflower seedlings. The protocol includes: organelle isolation, deoxyribonuclease treatment, lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple samples of organelle DNA from a small amount of tissue.     

Assembly of the protoplasm of Codium fragile (Bryopsidales, Chlorophyta) into new protoplasts

The cell organelles of the coenocytic alga Codium fragile (Sun) Hariot aggregated rapidly and protoplasts were formed when its protoplasm was extruded out in seawater. Continuous observation showed that there were long and gelatinous threads connecting the cell organelles. The threads contracted, and thus the cell organelles aggregated into protoplasmic masses. The enzyme digestion experiments and Coomassie Brilliant Blue and Anthrone stainings showed that the long and gelatinous threads involved in the formation of the protoplasts might Include protein and saccharides as structure components. Nile Red staining Indicated that the protoplast primary envelope was non-lipid at first, and then lipid materials Integrated Into its surface gradually. The fluorescent brightener staining Indicated that the cell wall did not regenerate in the newly formed protoplasts and they all disintegrated within 72 h after formation. Transmission electron microscopy of the cell wall of wild C. fragile showed electron-dense material embedded in the whole cell wall at regular intervals. The experiments indicated that C. fragile would be a suitable model alga for studying the formation of protoplasts.     

Unusual characteristics of Codium fragile chloroplast DNA revealed by physical and gene mapping

Summary A complete physical map of the Codium fragile chloroplast genome was constructed and the locations of a number of chloroplast genes were determined. Several features of this circular genome are unusual. At 89 kb in size, it is the smallest chloroplast genome known. Unlike most chloroplast genomes it lacks any large repeat elements. The 8 kb spacer region between the 16 S and 23 S rRNA genes is the largest such spacer characterized to date in chloroplast DNA. This spacer region is also unusual in that it contains the rps12 gene or at least a portion thereof. Three regions polymorphic for size are present in the Codium chloroplast genome. The psbA and psbC genes map closely to one of these regions, another region is in the spacer between the 16 S and 23 S rRNA genes and the third is very close to or possibly within the 16 S rRNA gene. The gene order in the Codium genome bears no marked resemblance to either the consensus vascular plant order or to that of any green algal or bryophyte genome. Present address: Department of Biology, Texas A&M University, College Station, TX 77843; USA     

INTRON REVEALED BY NUCLEOTIDE SEQUENCE OF LARGE SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE FROM CODIUM FRAGILE (CHLOROPHYTA): PHYLOGENETIC ANALYSIS1

The coding sequence for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Codium fragile (Suringar) Hariot chloroplast DNA is 1428 bp in length and contains a 1813-bp group II intron. The only other organisms in which introns have been found in the rbcL gene are Euglena and Astasia. The Codium intron likely had a separate origin from the Euglena and Astasia introns, based on comparisons of intron sizes and sequences. Phylogenetic analyses of rbcL nucleotide and amino acid sequences place Codium between Chlorella and two Chlamydomonas spp., indicating that the Chlorophyceae may be polyphyletic.     

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.     

Cultivation of the green alga, Codium fragile (Suringar) Hariot, by artificial seed production in Korea

Codium fragile (Suringar) Hariot is an edible green alga farmed in Korea using seed stock produced from regeneration of isolated utricles and medullary filaments. Experiments were conducted to reveal the optimal conditions for nursery culture and out-growing of C. fragile. Sampling and measurement of underwater irradiance were carried out at farms cultivating C. fragile at Wando, on the southwestern coast of Korea, from October 2004 to August 2005. Growth of erect thalli and underwater irradiance were measured over a range of depths for three culture stages. During the nursery cultivation stage (Stage I), growth rate was greatest at 0.5 m depth (0.055 ± 0.032 mm day⁻¹), where the average midday irradiance over 60 days was 924 ± 32 μmol photons m⁻² s⁻¹. During the pre-main cultivation stage (Stage II), the greatest growth rate occurred at a depth of 2 m (0.113 ± 0.003 mm day⁻¹) with an average irradiance of 248 ± 116 μmol photons m⁻² s⁻¹. For the main cultivation stage (Stage III) of the alga, thalli achieved the greatest increase in biomass at 1 m depth (7.2 ± 1.0 kg fresh wt m⁻¹). These results suggest that optimal growth at each cultivation stages of C. fragile could be controlled by depth of cultivation rope.     

Life history of Cladophora surera sp. nov. (Cladophorales, Ulvophyceae)

The life history of Cladophora surera sp. Nov., described from the south of the province of Buenos Aires, Argentina, was found to be diplobiontic and isomorphic with haploid gametophytic (n= 24) and sporophytic (2n= 48) plants. Other freshwater species, namely C, suhriana Kutz, and C. callicoma Kütz. have also been reported to be diplobiontic and isomorphic but differing from C. surera by their ploidy level. Plants exhibit great morphological variation, as the number of branches/mm² tends to increase with higher water velocity, thus adopting morphotypes that resemble very different species, that is, C. vagabunda (L.) Hoek, C. glomerata (L.) Kutz., C, laetevirens (Dillw.) Kutz., C. brasiliana Martens, C. dalmatica Kütz., C. vadorum (Aresch.) Kütz. and C. rivularis (L.) Hoek., but clearly differing from them by other morphological parameters; apical cell diameters, The ploidy level 24/48 has not been established for a species of Cladophora. Autogamy is the normal conjugation method and can take place even inside the gametangia before the gametes are released. Isogametes conjugate in a slow behavioral anisogamy, in which the contents of one migrate to the other. Tetra-flagellate zoospores result from meiosis.     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Variability in the rbcL Introns of Caulerpalean Algae (Chlorophyta, Ulvophyceae)

rbcL gene have been reported to date. Four new cases from Caulerpales, Ulvophyceae are described here. In the genus Caulerpa, the presence of an intron was unstable even in the infraspecific taxa. Based on comprehensive comparisons of the inserted positions, lengths of introns and so on, the presence of at least three kinds of introns, which probably have independent origins, was suggested in Caulerpales. Received 12 May 2000/ Accepted in revised form 12 October 2000     

Isolation of functional chloroplasts from the sacoglossan mollusc Elysia viridis Montague

A novel technique has been developed in which functional chloroplasts have been isolated and purified from the symbiotic sacoglossan mollusc Elysia viridis , Montague. This entailed the progressive filtration and gel-filtration of a hyaluronidase-incubated homogenate. Isopycnic centrifugation of the resultant crude chloroplast suspension reduced the cytoplasmic/mitochondrial and microbody contamination by approx. 70 and 90% respectively. Purified chloroplast suspensions were capable of linear rates of ¹⁴CO₂ fixation for at least 25 min at 50 μmol m² s⁻¹ (PPFD), even though approx. 54% of assimilate was being released into the bathing medium. Values for CO₂ fixation and assimilate release were similar to those obtained for the intact mollusc.     

Ultrastructure of the biflagellate gametes of Collinsiella cava (Ulvophyceae, Chlorophyta)

The fine structure of the biflagellate gametes of Collinsiella cava (Yendo) Printz was investigated in detail to clarify the species's taxonomic and phylo‐genetic position. Gametes are covered by small square scales with no distinct substructure. The chloroplast of the gamete includes an eyespot comprised of two layers of globules, and a pyrenoid that is traversed by one or a few thylakoids. Basal bodies overlap at their proximal ends and are offset in a counterclockwise orientation. Each basal body has a small bipartite terminal cap, a prominent proximal sheath comprised of two unequal subunits and a circular element situated at the cartwheel portion. A distal fibre, a connecting fibre and linkage between proximal sheaths connect the two basal bodies. Microtubular roots are comprised of two dexter (d) roots, subtended by the system I fibre, and two sinister (s) roots. Gametes have a single rhizoplast which extends parallel to one of the two d roots and extends to the mating structure. The ultrastructure of Collinsiella gametes is very similar to that of Mono‐stroma and other members of the Ulotrichales, Ulvophyceae, and we concluded that the genus Collinsiella should be treated as a member of the Monostromat‐aceae. The planozygote has four basal bodies, eight microtubular roots and two eyespots always situated at the same face of the cell. From observations of the planozygotes, the position of the mating structure relative to the flagellar apparatus is not consistent, but converse, between two mating types. A comparison of the location of the mating structure in Chlamydomonas and other green algae is presented.