Circumscription of the order Ectocarpales (Phaeophyceae): bibliographical synthesis and molecular evidence

Phylogenetic analyses of 27 brown algae including the type genera of the orders Chordariales, Dictyosiphonales, Ectocarpales sensu stricto, and Scytosiphonales, using partial SSU + LSU combined rDNA sequence data, supports a broadly circumscribed order Ectocarpales. This order is redefined to include taxa possessing an exserted, pedunculated pyrenoid. Previous then taxa were placed in the Ectocarpales sensu stricto, the Chordariales, the Dictyosiphonales, or the Scytosiphonales. Algae either lacking a pyrenoid, and sometimes included in the Ectocarpales (Tilopteridales, Ralfsiales sensu Nakamura), or which possess a non-pedunculated pyrenoid (such as those placed in the recently proposed order Scytothamnales, as well as Asteronema, Asterocladon and Bachelotia), are excluded from the Ectocarpales.     

PHYLOGENETIC RELATIONSHIPS OF THE BROWN ALGAL ORDERS ECTOCARPALES, CHORDARIALES, DICTYOSIPHONALES, AND TILOPTERIDALES (PHAEOPHYCEAE) BASED ON RUBISCO LARGE SUBUNIT AND SPACER SEQUENCES

Phylogenetic relationships among 23 species of morphologically simple brown algae belonging to the Ectocarpales sensu stricto , Chordariales, Dictyosiphonales, and Tilopteridales sensu stricto , Phaeophyceae (Fucophyceae), were analyzed using chloroplast-encoded RUBISCO large subunit gene sequences ( rbc L) and the associated RUBISCO spacer sequences. Comparison of the observed and expected sequence divergence at the three codon positions of rbc L showed that the level of mutational saturation within the brown algae is minor. Thus, rbc L is well suited for phylogenetic studies in this group. Unweighted parsimony analyses and a neighbor-joining distance analysis were performed using unambiguously aligned rbc L sequences from the above four orders, one marine raphidophyte and two Tribophyceae (Xantophyceae). Polyphyly of Tilopteridales sensu lato (i.e. including Dictyosiphonales) is verified; we therefore recommend the use of Tilopteridales in the strict sense. The Ectocarpales, Chordariales, and Dictyosiphonales are paraphyletic with respect to each other, forming a highly interwoven clade. A separate parsimony analysis of the RUBISCO spacer as well as a combined rbc L and spacer analysis supported the close relationship among the latter three orders, adding to the evidence that they should be subsumed into the Ectocarpales sensu lato.     

A RIBOSOMAL DNA PHYLOGENY SUPPORTS THE CLOSE EVOLUTIONARY RELATIONSHIPS AMONG THE SPOROCHNALES,DESMARESTIALES, AND LAMINARIALES (PHAEOPHYCEAE)1

We report six complete 18S ribosomal DNA (rDNA) sequences representing five brown algal orders: Sporochnus comosus C. A. Agardh (Sporochnales), Chorda tomentosa Lyngbye (Chordaceae, Laminariales), Saccorhiza polyschides (Lightfoot) Batters (Phyllariaceae, Laminariales), Desmarestia ligulata (Lightfoot) Lamouroux (Desmarestiales), Ectocarpus siliculosus (Dillwyn) Lyngbye (Ectocarpales), and Scytosiphon lomentaria (Lyngbye) J. G. Agardh (Scytosiphonales). These sequences were compared with published laminarialean (Alaria marginata Postel et Ruprecht [Alariaceae] and Macrocystis integrifolia Bory [Lessoniaceae]) and fucalean (Fucus gardneri Silva) rDNA sequences for phylogeny inference using both the distance-matrix and parsimony methods. The inferred 18S phylogenies clustered Sporochnus, Desmarestia, Chorda, Saccorhiza, Alaria, and Macrocystis in an assemblage. This Sporochnales–Desmarestiales–Laminariales (S-D-L) complex was consistently separated from the Ectocarpales, Scytosiphonales, and Fucales by bootstrap analyses. The inferred phylogenies are consistent with several possible evolutionary processes leading to this S-D-L complex. Members in this assemblage lack eyespots in their sperm, and their sperm have the atypical brown algal flagellation: shorter anterior and longer posterior flagella. In addition, they are oogamous with a heteromorphic alternation of generations between a microscopic gametophyte and a macroscopic sporophyte. Members of the S-D-L complex can be separated into different phylogenetic lines based on the presence/absence of eyespots in their meiospores. Our findings support the contention that the Sporochnales, Desmarestiales, and Laminariales are closely related. In addition, our rDNA tree suggests that the Laminariales is paraphyletic.     

A NEW BROWN ALGAL ORDER,ISHIGEALES (PHAEOPHYCEAE), ESTABLISHED ON THE BASIS OF PLASTID PROTEIN‐CODING rbcL,psaA, AND psbA REGION COMPARISONS1

The brown algal family Ishigeaceae currently includes a single genus, Ishige Yendo, with two species. The relationship of the family to other brown algal lineages is less studied in terms of their plastid ultrastructure and molecular phylogeny. We determined the sequences of rbcL from four samples of the two Ishige species and nine putative relatives and the psaA and psbA sequences from 37 representatives of the brown algae. Analyses of individual and combined data sets resulted in similar trees; however, the concatenated data gave greater resolution and clade support than each individual gene. In all the phylogenies, the Phaeophyceae was well resolved, the Ectocarpales being placed in a terminal position and the Ishigeaceae ending up in a basal position. From our ultrastructural study, we concluded that the pyrenoid is absent in the Ishigeaceae, despite the presence of a rudimentary pyrenoid in I. okamurae. These results suggest that the Ishigeaceae is an early diverging brown lineage. Our molecular and morphological data, therefore, lead us to exclude the Ishigeaceae from the Ectocarpales s.l., which have an elaborate pyrenoid, and to propose its own order Ishigeales ord. nov. The Ishigeales is distinguished by oligostichous structure of thalli, phaeophycean hairs formed within cryptostomata, unilocular sporangia transformed from terminal cortical cells, and plurilocular sporangia lacking sterile terminal cells. This study is the first to document the utility of the psaA and psbA sequences for brown algae and also the first report on the multigene phylogeny of the Phaeophyceae based on three protein‐coding plastid genes.     

A MOLECULAR ANALYSIS OF ANALIPUS AND RALFSIA (PHAEOPHYCEAE) SUGGESTS THE ORDER ECTOCARPALES IS POLYPHYLETIC1

We report partial 18S ribosomal DNA sequences of Analipus japonicus (Harvey) Wynne and Ralfsia fungiformis (Grunnerus) Setchell et Gardner. These sequences were compared with the corresponding sequences of 13 brown algae representing six phaeophycean orders: Dictyotales, Ectocarpales, Fucales, Laminariales, Sphacelariales, and Syringodermatales. These 15 brown algae included 10 ectocarpoids representing nine ectocarpacean (sensu Gabrielson et al. 1989) families: Chordariaceae, Dictyosiphonaceae, Ectocarpaceae, Elachistaceae, Heterochordariaceae, Leathesiaceae, Punctariaceae, Ralfsiaceae, and Scytosiphonaceae. We addressed the controversial taxonomic placement of A. japonicus and R. fungiformis in the Ectocarpales by analysis of DNA sequences. Neighbor-joining and maximum parsimony-inferred phylogenies provided evidence that A. japonicus and R. fungiformis are not closely associated with the other representatives of the Ectocarpales. Bootstrap analyses suggest polyphyly of the order Ectocarpales (sensu Gabrielson et al.). However, our analysis failed to resolve the phylogenetic relationship between A. japonicus and R. fungiformis. Our results suggest that the ectocarpoids are just as distantly related to A. japonicus and R. fungiformis as they are to members of the advanced orders Desmarestiales, Dictyotales, Fucales, Laminariales, Sphacelariales, and Syringodermatales.     

Fatty acids of some algae from the Bohai Sea.

The fatty acid compositions of 22 species of marine macrophytes, belonging to the Ceramiales, Cryptonemiales, Nemalionales, Laminariales, Chordariales, Scytosiphonales, Desmarestiales, Dictyosiphonales, Fucales, Dictyotales and Ulvales and collected from the Bohai Sea, were determined by capillary gas chromatography. The contents of polyunsaturated fatty acids (FAs) in the Bohai Sea algae, in comparison with the same species from the Yellow Sea were found to be lower. Red algae had relatively high levels of the acids 16:0, 18:1(n-7), 18:1(n-9), 20:5(n-3) and 20:4(n-6), and those examined were rich in C(20) PUFAs, these chiefly being arachidonic and eicosapentaenoic acids. The major FAs encountered in the Phaeophyta were 14:0, 16:0, 18:1(n-9), 18:2(n-6), 18:3(n-3), 18:4(n-3), 20:4(n-6) and 20:5(n-3). C(18)PUFAs are of greater abundance in the brown algae than in the red algae examined. All three green algae from the Ulvales had similar fatty acid patterns with major components, 16:0, 16:4(n-3), 18:1(n-7), 18:2(n-6), 18:3(n-3), and 18:4(n-3). They contained 16:3(n-3) and more 16:4(n-3), were rich in C(18)PUFAs, chiefly 18:3(n-3) and 18:4(n-3) and had 18:1(n-7)/18:1(n-9) ratios higher than 1.     

Plastid Genome of <Emphasis Type="Italic">Dictyopteris divaricata</Emphasis> (Dictyotales,Phaeophyceae): Understanding the Evolution of Plastid Genomes in Brown Algae

Dictyotophycidae is a subclass of brown algae containing 395 species that are distributed worldwide. A complete plastid (chloroplast) genome (ptDNA or cpDNA) had not previously been sequenced from this group. In this study, the complete plastid genome of Dictyopteris divaricata (Okamura) Okamura (Dictyotales, Phaeophyceae) was characterized and compared to other brown algal ptDNAs. This plastid genome was 126,099 bp in size with two inverted repeats (IRs) of 6026 bp. The D. divaricata IRs contained rpl21, making its IRs larger than representatives from the orders Fucales and Laminariales, but was smaller than that from Ectocarpales. The G + C content of D. divaricata (31.19%) was the highest of the known ptDNAs of brown algae (28.94–31.05%). Two protein-coding genes, rbcR and rpl32, were present in ptDNAs of Laminariales, Ectocarpales (Ectocarpus siliculosus), and Fucales (LEF) but were absent in D. divaricata. Reduced intergenic space (13.11%) and eight pairs of overlapping genes in D. divaricata ptDNA made it the most compact plastid genome in brown algae so far. The architecture of D. divaricata ptDNA showed higher similarity to that of Laminariales compared with Fucales and Ectocarpales. The difference in general features, gene content, and architecture among the ptDNAs of D. divaricata and LEF clade revealed the diversity and evolutionary trends of plastid genomes in brown algae.     

Utility of rbcS gene as a novel target DNA region for brown algal molecular systematics

The usefulness of molecular phylogenetic studies has increased remarkably as the quantity and quality of available DNA sequences has increased. When compared with the progress that has occurred in angiosperms and animals, there have been relatively few target DNA regions identified for use in taxonomic studies of brown algae. Therefore, in this study, we developed a new set of primers to amplify Rubisco small subunit (rbcS) gene sequences and determined the rbcS gene sequences of various species of brown algae including those belonging to Dictyotales, Ectocarpales, Fucales and Sphacelariales. The level of sequence variations in the rbcS gene varied according to the brown algal lineages. When focusing on the relationship of species within the genus Sargassum, the rbcS gene sequences provided useful information regarding the phylogenetic relationship among sections of the subgenus Bactrophycus. Based on the broad applicability and phylogenetic utility of the rbcS gene, we suggest that the sequence be used as a new target region for the molecular systematics of brown algae.     

UNIVERSAL PRIMERS AMPLIFY A 23S rDNA PLASTID MARKER IN EUKARYOTIC ALGAE AND CYANOBACTERIA1

The challenge in the development of universal algal primers lies in the genetic diversity contained within the vast array of evolutionary lineages present in this informally named group of organisms. A comparative genomics approach was used previously to identify conserved primers flanking a region of the plastid genome. Our present research illustrates the feasibility of amplifying and sequencing this marker across multiple algal lineages. We present a preliminary framework of 107 novel sequences of this region from 62 red algae, 19 green algae, 14 brown algae, 8 cyanobacteria, 2 diatoms, 1 xanthophyte, and 1 euglenoid, and illustrate levels of divergence of the marker for well‐represented groups in a neighbor‐joining analysis. This ～410 nt region distinguishes most species included in the analysis. The remarkable universality of these primers suggests potential for their use in assays of environmental samples in which they could be used to simultaneously detect a number of different algal lineages.     

Molecular phylogeny of small brown algae,with special reference to the systematic position of Caepidium antarcticum (adenocystaceae,ectocarpales)

Nuclear ribosomal DNA (3′-SSU, ITS, 5′-LSU) and plastid-encoded (rbcL and Rubisco spacer) sequences were determined in Caepidium antarcticum and compared to homologous sequences of relatives from Ectocarpales, Scytothamnales, and other brown algae. Plastidial sequences confirmed a previous conclusions from nuclear ribosomal sequences that some taxa with stellate plastids (Asterocladon and relatives) form the closest outgroup to the Ectocarpales as yet identified. To reconcile nomenclature with the clades resolved in recent molecular studies, we propose a subdivision of the Ectocarpales in five families. Plastidial sequences support the recent proposal of Adenocystaceae, and all sequences suggest that Caepidium should be included in this family. As a further result, Geminocarpus was shown to belong to the same clade as Pylaiella and a number of other brown algae with an isomorphic life history and discoid plastids. We recognise this clade, whose correct name is Acinetosporaceae, as another family in the Ectocarpales. We also propose to unite a number of genetically related taxa, which were formely classified in different families, in an extended Chordariaceae. The remaining species of the Ectocarpales belong to Scytosiphonaceae and to Ectocarpaceae, the latter containing only Ectocarpus and Kuckuckia.     

RESOLVING EVOLUTIONARY RELATIONSHIPS AMONG THE BROWN ALGAE USING CHLOROPLAST AND NUCLEAR GENES1

The brown algae are one of the largest and most important groups of primary producers in benthic coastal marine environments. Despite their biological importance, consensus regarding their taxonomic or evolutionary relationships remains elusive. Our goal was to produce a taxon‐rich two‐gene (rbcL and LSU rDNA) phylogeny. Key species were sequenced to represent each order and family in the analyses across all 19 orders and ～40 families, including selected outgroups Schizocladiophyceae and Xanthophyceae. Our results are in sharp contrast to traditional phylogenetic concepts; the Ectocarpales are not an early diverging clade, nor do the Fucales diverge early from other brown algae. Rather, Choristocarpus is sister to the remaining brown algae. Other groups traditionally considered to have primitive features are actually recently diverged lineages, turning traditional phylogenetic concepts upside down. Additionally, our results allow for the assessment, in the broadest context, of many of the historical and more recent taxonomic changes, resulting in several emended groups along with proposals for two new orders (Onslowiales, Nemodermatales) and one new family (Phaeosiphoniellaceae).     

Molecular phylogeny of Punctaria mageshimensis reveals evidence for its transfer to Spatoglossum as S. mageshimense (Dictyotales,Phaeophyceae)

Taxonomy of the little‐studied brown algal species Punctaria mageshimensis (Ectocarpales s.l.) was reexamined by molecular phylogeny and morphology. In the genetic analyses of newly collected specimens using plastid rbcL and psaA gene sequences, the specimens morphologically referable to P. mageshimensis were phylogenetically distant from Ectocarpales s.l. and were included in the clade of Spatoglossum (Dictyotales). Morphological reexamination of the type specimen and newly collected specimens confirmed its systematic position in Dictyotales: Branched thallus; cushion‐shaped rhizoidal holdfast occasionally forming secondary holdfast at the bottom of the thallus; many discoidal plastids without pyrenoid per cell; tetrasporangium‐like reproductive structures with dark, homogeneous cell content; occurrence of hair tufts. Genetically P. mageshimensis was most related to a reported sequence of Spatoglossum asperum, but P. mageshimensis was considerably different from S. asperum as well as other known Spatoglossum species in the deep habitat and in having scarcely‐branched lanceolate and considerably thickened thallus. In conclusion, we propose the transfer of P. mageshimensis to Spatoglossum as S. mageshimense comb. nov.     

MOLECULAR EVIDENCE FROM nrDNA ITS SEQUENCES THAT LAMINARIOCOLAX (PHAEOPHYCEAE, ECTOCARPALES SENSU LATO) IS A WORLDWIDE CLADE OF CLOSELY RELATED KELP ENDOPHYTES

Marine brown algae living as endophytes in macroalgae are morphologically simple and their taxonomy is particularly difficult. A molecular phylogeny for endophytic taxa isolated from kelps and red algae, and for putative epiphytic and free-living relatives, was inferred from partial small subunit and complete internal transcribed spacer nuclear ribosomal DNA sequences. It has revealed the following results. (1) Three species of endophytes isolated from members of the Laminariales are closely related. They form a clade together with the epi-endophytic species Laminariocolax tomentosoides (Farlow) Kylin. Members of the clade possess uniseriate plurilocular sporangia, and they may form erect filaments. Laminariocolax eckloniae sp. nov., occurring in the South African host Ecklonia maxima (Osbeck) Papenfuss, is described. The new combinations, Laminariocolax aecidioides (Rosenvinge) comb. nov. and L. macrocystis (Peters) comb. nov., are proposed for two taxa previously classified in Gononema and Streblonema , respectively. (2) The genus Laminariocolax occurs worldwide in temperate areas, and the phylogeny of the taxa studied is in agreement with biogeographic distribution. (3) Laminariocolax belongs to the Ectocarpales sensu lato. The genus is more closely related to Chordaria than to Dictyosiphon, Ectocarpus, or Scytosiphon . (4) Two brown endophytes ( Streblonema spp.), isolated from red algae, are closely related to each other and may form a sister clade to Laminariocolax .     

Phylogenetic relationships of the Raphidophyceae and Xanthophyceae as inferred from nucleotide sequences of the 18S ribosomal RNA gene

Some earlier studies suggested an evolutionary relationship between the Raphidophyceae (chloromonads) and Xanthophyceae (yellow-green algae), whereas other studies suggested relationships with different algal classes or the öomycete fungi. To evaluate the relationships, we determined the complete nucleotide sequences of the 18S ribosomal RNA gene from the raphidophytes Vacuolaria virescens, Chattonella subsalsa, and Heterosigma carterae, and the xanthophytes Vaucheria bursata, Botrydium stoloniferum, Botrydiopsis intercedens, and Xanthonema debile. The results showed that the Xanthophyceae were most closely related to the Phaeophyceae. A cladistic analysis of combined data sets (nucleotide sequences, ultrastructure, and pigments) suggested the Raphidophyceae are the sister taxon to the Phaeophyceae-Xanthophyceae clade, but the bootstrap value was low (40%). The raphidophyte genera were united with high (100%) bootstrap values, supporting a hypothesis based upon ultrastructural features that marine and freshwater raphidophytes form a monophyletic group. We examined the relationship between Vaucheria, a siphoneous xanthophyte alga, and the öomycetes, and we confirmed that Vaucheria is a member of the class Xanthophyceae. Partial nucleotide sequences of the 18S rRNA gene from eight xanthophytes (including Bumillariopsis filiformis, Heterococcus caespitiosus, and Mischococcus sphaerocephalus) produce a phylogeny that is not congruent with the current morphology-based classification scheme.     

KINETICS OF BLUE-LIGHT STIMULATION AND CIRCADIAN RHYTHMICITY OF LIGHT-SATURATED PHOTOSYNTHESIS IN BROWN ALGAE: A SPECIES COMPARISON1

The time courses of photosynthetic rates in red light, with and without additional blue light, were investigated and compared in 20 species of brown algae. Species could be separated into two groups on the basis of the rhythmicity of their photosynthesis in red light and the kinetics of their responses to blue-light pulses. One group, which consisted of members of the Ectocarpales, Chordariales, and Dictyosiphonales, was characterized by strong and persistent circadian rhythmicity in red light. The photosynthetic responses of these species to blue-light pulses started within 10–30 s of the beginning of blue-light treatment and mostly contained at least two distinct kinetic components. An early component, which reached a maximum about 5–10 min after the blue-light pulse, was always detectable. Later components were seen as separate peaks or shoulders after an additional 10–20 min. The decay of the response in this group of species was mostly slow, with half-lives of between 0.5 and 1.5 h. In the second group of species, consisting of members of the Dictyotales, Laminariales, and Fucales, photosynthesis in red light was usually non-rhythmic, although circadian rhythms with a weak amplitude or of transient occurrence were observed in some plants of some species. The increase in photosynthesis in response to a blue-light pulse was not detectable until 70–330 s after the start of blue-light treatment, and the response itself had only a single component, with a maximum after about 10 min and half-life of 10–20 min. The lengths of the lag-phases were positively correlated with the times taken to reach the peak in this group, although the lag-phases and the half lives sometimes varied with time in individual plants. Two members of the Sphacelariales (Sphacelaria, Cladostephus) did not fit into either of the two groups because their photosynthesis was rhythmic, but their responses had long lag-phases, a single component, and moderately long half-lives. The differences in the kinetics of the photosynthetic response to blue-light pulses, which have been described for the two main groups of species, are thought to indicate that there are two distinct mechanisms by which light-saturated photosynthesis responds to blue light in brown algae. Since in some species the maximal photosynthesis after a blue-light pulse and the rate of photosynthesis in continuous blue light also varied in a circadian pattern, the response to blue light itself may be under circadian control.     

GENOMIC INSIGHTS INTO EVOLUTIONARY RELATIONSHIPS AMONG HETEROKONT LINEAGES EMPHASIZING THE PHAEOPHYCEAE1

Heterokonts comprise a large and diverse group of organisms unified by the heterokont biflagellate condition. Monophyly of many of these lineages is well established, but evolutionary relationships among the various lineages remain elusive. Among these lineages, the brown algae (Phaeophyceae) are a monophyletic, taxonomically diverse, and ecologically critical group common to marine environments. Despite their biological and scientific importance, consensus regarding brown algal phylogeny and taxonomic relationships is missing. Our long‐term research goal is to produce a well‐resolved taxon‐rich phylogeny of the class to assess evolutionary patterns and taxonomic relationships among brown algal lineages and their relationship to other closely related heterokont groups. To accomplish this goal and augment existing loci for phaeophycean‐wide systematic studies, we generated expressed sequence tags (ESTs) from several major brown algal lineages and from the heterokont lineage representing the closest sister group to brown algae. To date, we have successfully constructed cDNA libraries for two lineages (Choristocarpus tenellus Zanardini and Schizocladia ischiensis E. C. Henry, Okuda et H. Kawai) and in the library test phase obtained up to 1,600 ESTs per organism. Annotation results showed a gene discovery rate of 45%–50% for each library revealing 500–700 unique genes from each organism. We have identified several potential genes for phylogenetic inference and used these loci for preliminary molecular clock analyses. Our molecular clock analysis suggests that the basal divergence in brown algae occurred around the time of the pennate‐centric diatom divergence. Here we report this analysis and other uses of ESTs in brown algal phylogenomics and the utility of these data for resolving the phylogeny of this group.     

A new measure to study phylogenetic relations in the brown algal order Ectocarpales: The “codon impact parameter”

We analyse forty-seven chloroplast genes of the large subunit of RuBisCO, from the algal order Ectocarpales, sourced from GenBank. Codon-usage weighted by the nucleotide base-bias defines our score called the codon-impact-parameter. This score is used to obtain phylogenetic relations amongst the 47 Ectocarpales. We compare our classification with the ones done earlier.     

Ribosomal DNA phylogeny of the Bangiophycidae (Rhodophyta) and the origin of secondary plastids

We sequenced the nuclear small subunit ribosomal DNA coding region from 20 members of the Bangiophycidae and from two members of the Florideophycidae to gain insights into red algal evolution. A combined alignment of nuclear and plastid small subunit rDNA and a data set of Rubisco protein sequences were also studied to complement the understanding of bangiophyte phylogeny and to address red algal secondary symbiosis. Our results are consistent with a monophyletic origin of the Florideophycidae, which form a sister-group to the Bangiales. Bangiales monophyly is strongly supported, although Porphyra is polyphyletic within Bangia. Bangiophycidae orders such as the Porphyridiales are distributed over three independent red algal lineages. The Compsopogonales sensu stricto, consisting of two freshwater families, Compsopogonaceae and Boldiaceae, forms a well-supported monophyletic grouping. The single taxon within the Rhodochaetales, Rhodochaete parvula, is positioned within a cluster containing members of the Erythropeltidales. Analyses of Rubisco sequences show that the plastids of the heterokonts are most closely related to members of the Cyanidiales and are not directly related to cryptophyte and haptophyte plastid genomes. Our results support the independent origins of these secondary algal plastids from different members of the Bangiophycidae.     

Differentiation between Bacillus thuringiensis strains by gyrB PCR-Sau3Al fingerprinting

gyrB DNA fragments of seven Bacillus thuringiensis local collection family representatives were amplified by PCR and sequenced. Several differences in their corresponding sequences were evidenced. Both in silico and in vitro restriction maps of gyrB sequences and fragments respectively confirmed that EcoRI and Sau3AI could be used to differentiate between B. thuringiensis strains. However, the phylogeny analysis showed that only the gyrB PCR-Sau3AI allows a strains classification that correlates very well with that obtained on the basis of the sequences analysis. Thus, these finds show that gyrB PCR- Sau3AI digestion could be considered as an efficient, rapid, and easy method to make a distinction, not only between strains belonging to the Bacillus cereus group, but also between those belonging to B. thuringiensis.