As flutuações de temperatura no ambiente marinho, decorrentes de processos naturais e de atividades antrópicas, como a flutuação das marés, o resfriamento de usinas e o aquecimento global afetam a dinâmica ecológica e as respostas fisiológicas de organismos marinhos, especialmente das macroalgas bentônicas. Macroalgas que ocorrem nos limites das faixas entre-marés podem estar sujeitas a fortes variações de temperatura em curto-prazo, como por exemplo: Sargassum e Pyropia. Sob aquela perspectiva, o objetivo deste projeto foi prover subsídios para a compreensão do efeito da temperatura e dos mecanismos de tolerância e sensibilidade das espécies do mediolitoral paulista Sargassum stenophyllum (Fucales, Ochrophyta) e Pyropia spiralis (Bangiales, Rhodophyta) mediante a análise da performance fotossintetizante, variação na composição química e respostas antioxidantes, além de fornecer informação sobre o seu potencial como produto funcional. As respostas das algas modelos foram avaliadas sob temperaturas de 15, 20, 25 (controle), 30 e 35 ºC ao longo de sete dias, em condições controladas de laboratório. As espécies de estudo mantiveram proporções semelhantes na dissipação de energia e taxa de crescimento, e poucas alterações no perfil químico (p. ex. pigmentos, carboidratos, carotenoides e aminoácidos) entre os 15 e 30 ºC, o qual pode ser compreendido como faixa de tolerância. Em contraste, existiu sensibilidade no talo das macroalgas estudadas na temperatura de 35 ºC, tendo diminuição na fotossíntese e crescimento desde o terceiro dia do período experimental. Em alta temperatura, acima de 30 ºC, tanto os filoides de S. stenophyllum quanto o talo da P. spiralis podem ter liberado substâncias químicas, como substâncias fenólicas, evidenciado pela mudança de cor na água do mar. Em condição de baixa temperatura (15 ºC), P. spiralis mostrou acúmulo de aminoácidos tipo-micosporinas. Em termos gerais, as espécies de estudo são promissoras como produto funcional envolvido na área de alimentação devido à sua composição nutricional e propriedades antioxidantes / Temperature fluctuations in the marine environment, due to natural processes and anthropogenic activities, caused by tidal fluctuation, cooling of energy power plants, and global warming affect the ecological dynamics and physiological responses of marine organisms, mainly in benthic macroalgae. Seaweeds inhabiting the boundaries of the intertidal zone may be subjected to strong short-term temperature variations, such as Sargassum and Pyropia. On this perspective, the aim of this project was to provide subsidies for understand the effect of temperature and the mechanisms of tolerance and sensitivity of the intertidal species Sargassum stenophyllum (Fucales, Ochrophyta) and Pyropia spiralis (Bangiales, Rhodophyta) from São Paulo, through the analysis of photosynthetic performance, chemical variation, and antioxidant responses, as well as, to provide information about its potential as functional product. The responses of the studied seaweeds were evaluated under temperatures of 15, 20, 25 (control), 30 and 35 °C over seven days in laboratory controlled conditions. The species displayed similar proportions in energy dissipation and growth rate, and few changes in the chemical profile (e.g. pigments, carbohydrates, carotenoids, and amino acids) between 15 and 30 ºC, which can be identificated as thermal tolerance range. In contrast, algae were sensitive at 35 ºC, with decrease in photosynthesis and growth since the third day of the experimental period. Under high temperature, above 30 °C, S. stenophyllum phylloids and P. spiralis thalli might have released chemical compounds, such as phenolic compounds, evidenced by color change of the seawater. Under low temperature condition (15 °C), P. spiralis showed accumulation of mycosporin-like amino acids. In general, the studied species are promising as a functional product involved in the feed area due to its nutritional composition and antioxidant properties

The Rhodophyta (red algae) are an ancient crown group of the Eukarya (ca. 1400-1500 million years), comprised of 5000 - 6000 species. Gametophytes of taxa excluding the speciose Class Florideophyceae are typically of very simple unicellular, filamentous or foliose morphologies. These simple morphologies are often homoplasious (resulting from convergent or parallel evolution) and can be indistinguishable among distinct taxa, leading to cryptic species. As a result, historical morphology-based taxonomy is often not congruent with evolutionary history. Intraspecific genetic variation is not yet characterized for non-Florideophyceae taxa. Here the intraspecific genetic variation was characterized for a locally endemic, morphologically distinct bangiophyte red alga, Bangia maxima Gardner using inter simple sequence repeat (ISSR) patterns from 91 individual filaments across seven local populations. A high degree of genetic variation was observed over very small distances (< 25 cm) and very little genetic exchange was observed between populations. It is possible that B. maxima is a true endemic species and its population dynamics may differ from other Bangia species. Metrics of sequence-based identification rely on genetic divergence among isolates to distinguish taxonomic units independent of morphology. Such metrics are especially useful for morphologically simple or cryptic species. The mitochondrial cytochrome oxidase c subunit 1 gene has been proposed for the Florideophyceae. An evaluation of this gene as a metric for non-Florideophyceae taxa was undertaken and limited utility was demonstrated in most lineages of Rhodophyta due to poor or inconsistent amplification and conflicts with nuclear and plastid phylogenies. Patterns of genetic divergence among taxa are used to infer evolutionary relationships. The nuclear ribosomal small subunit (nSSU rRNA) is the taxonomically broadest pool of gene sequence data for the Rhodophyta. The use of stochastic models of nucleotide evolution is the most common approach to inferring phylogenies using this gene, ignoring much of its evolutionary information as different characters that contribute to secondary structure (e.g. paired nucleotides) are treated independently. The incorporation of structural information leads to more biologically realistic evolutionary models increasing phylogenetic resolution. Parametric models incorporating structural information were used here to more fully resolve phylogenies for all known Rhodophyta lineages. Novel phylogenetic topologies were observed and well supported for each Class within the Rhodophyta resulting in a number of formally proposed or suggested taxonomic revisions. These include phylogenetic resolution of Rhodophyta Classes, support for the introduction of 11 genera within the Bangiales and support for various taxonomic revisions within the Florideophyceae previously proposed but not yet fully adopted. As structure evolves more slowly than its constituent sequence, secondary structure elements can further resolve evolutionary relationships, especially in lineages as old as the Rhodophyta. A novel encoding of secondary structure elements and subsequent multivariate analysis was performed for all known Rhodophyta nSSU rRNA gene sequences, reinforcing phylogenetic results. Computer programs developed for these analyses are publicly available. The analyses presented here significantly advanced understanding of the evolutionary distribution of cryptic species within the Rhodophyta. Furthermore, useful methods for the characterization of such species are presented, as is a demonstration of the utility of biologically realistic sequence models parameterizing nSSU rRNA structure in resolving ambiguous phylogenetic relationships. Most importantly, this work also represents a significant improvement toward taxonomy congruent with evolutionary history for the Rhodophyta.

The Rhodophyta (red algae) are an ancient crown group of the Eukarya (ca. 1400-1500 million years), comprised of 5000 - 6000 species. Gametophytes of taxa excluding the speciose Class Florideophyceae are typically of very simple unicellular, filamentous or foliose morphologies. These simple morphologies are often homoplasious (resulting from convergent or parallel evolution) and can be indistinguishable among distinct taxa, leading to cryptic species. As a result, historical morphology-based taxonomy is often not congruent with evolutionary history. Intraspecific genetic variation is not yet characterized for non-Florideophyceae taxa. Here the intraspecific genetic variation was characterized for a locally endemic, morphologically distinct bangiophyte red alga, Bangia maxima Gardner using inter simple sequence repeat (ISSR) patterns from 91 individual filaments across seven local populations. A high degree of genetic variation was observed over very small distances (< 25 cm) and very little genetic exchange was observed between populations. It is possible that B. maxima is a true endemic species and its population dynamics may differ from other Bangia species. Metrics of sequence-based identification rely on genetic divergence among isolates to distinguish taxonomic units independent of morphology. Such metrics are especially useful for morphologically simple or cryptic species. The mitochondrial cytochrome oxidase c subunit 1 gene has been proposed for the Florideophyceae. An evaluation of this gene as a metric for non-Florideophyceae taxa was undertaken and limited utility was demonstrated in most lineages of Rhodophyta due to poor or inconsistent amplification and conflicts with nuclear and plastid phylogenies. Patterns of genetic divergence among taxa are used to infer evolutionary relationships. The nuclear ribosomal small subunit (nSSU rRNA) is the taxonomically broadest pool of gene sequence data for the Rhodophyta. The use of stochastic models of nucleotide evolution is the most common approach to inferring phylogenies using this gene, ignoring much of its evolutionary information as different characters that contribute to secondary structure (e.g. paired nucleotides) are treated independently. The incorporation of structural information leads to more biologically realistic evolutionary models increasing phylogenetic resolution. Parametric models incorporating structural information were used here to more fully resolve phylogenies for all known Rhodophyta lineages. Novel phylogenetic topologies were observed and well supported for each Class within the Rhodophyta resulting in a number of formally proposed or suggested taxonomic revisions. These include phylogenetic resolution of Rhodophyta Classes, support for the introduction of 11 genera within the Bangiales and support for various taxonomic revisions within the Florideophyceae previously proposed but not yet fully adopted. As structure evolves more slowly than its constituent sequence, secondary structure elements can further resolve evolutionary relationships, especially in lineages as old as the Rhodophyta. A novel encoding of secondary structure elements and subsequent multivariate analysis was performed for all known Rhodophyta nSSU rRNA gene sequences, reinforcing phylogenetic results. Computer programs developed for these analyses are publicly available. The analyses presented here significantly advanced understanding of the evolutionary distribution of cryptic species within the Rhodophyta. Furthermore, useful methods for the characterization of such species are presented, as is a demonstration of the utility of biologically realistic sequence models parameterizing nSSU rRNA structure in resolving ambiguous phylogenetic relationships. Most importantly, this work also represents a significant improvement toward taxonomy congruent with evolutionary history for the Rhodophyta.

O gênero Porphyra (Rhodophyta) apresenta uma considerável importância econômica, sendo extensivamente cultivado e consumido como alimento. O gênero é representado por mais de 70 espécies e apresenta ampla distribuição geográfica, desde regiões tropicais até polares. Sua taxonomia, baseada em poucos caracteres da fase macroscópica do seu ciclo de vida, é ainda bastante problemática. Para tentar entender melhor a taxonomia e a história evolutiva de Porphyra foram utilizadas metodologias de biologia molecular e características da fase conchocelis do ciclo de vida. Verificou-se que caracteres da fase microscópica podem ser utilizados para complementar os conhecimentos taxonômicos tradicionais. Para tentar elucidar a posição filogenética do gênero Porphyra na divisão Rhodophyta e, dentro do gênero, entre espécies do Atlântico, o gene nuclear que codifica para o RNA ribossomal da subunidade pequena do ribossomo (rDNA 18S) foi amplificado através de PCR, clonado e completamente sequenciado. Foram utilizadas três espécies de Porphyra da Nova Escócia (Canadá) e duas de São Paulo (Brasil). As sequências obtidas foram alinhadas com as de alguns eucariontes e de outras algas vermelhas, incluindo uma sequência publicada de \"Porphyra umbilicalis\" da França. As árvores filogenéticas foram construídas através dos métodos de parcimônia, distancia e máxima verossimilhança. As analises mostraram que o gênero Porphyra é monofilético para as cinco espécies estudadas e constitui um dos ramos mais antigos dentro das algas vermelhas já analisados. O gênero Porphyra, subclasse Bangiophycidae, apresentou uma diferença substancial em relação aos gêneros da subclasse Florrideophycidae, sustentando assim, a divisão de Rhodophyta em duas subclasses pela taxonomia tradicional. Entre os eucariontes, Porphyra divergiu ao mesmo tempo que o nuclemorfo de Cryptomonas. O alto grau de divergência genética encontrada entre espécies de Porphyra, além de indicações do registo fóssil, na literatura, sugerem que o gênero é bastante primitivo dentro das algas vermelhas. Surpreendentemente, a sequência publicada para \"Porphyra umbilicalis\" apresentou mais de 99% de identidade com uma espécie de Palmaria que pertence à subclasse Florideophycidae; neste caso, a biologia molecular serviu para comprovar a identificação errônea do exemplar cuja sequência foi publicada. Durante a análise filogenética, verificou-se a ocorrência de um intron do grupo ICI nos genes rDNA 18S de Porphyra spiralis var. amplifolia. Esse intron ocorre na mesma posição que os introns do grupo IC1 nos rDNA 18S dos fungos Pneumocystis carinii, Protomyces inouyei e da alga verde Chlorella ellipsoidea, e apresenta identidade de sequências nos domínios P1 e P2, fora da região conservada, com o intron de Pn. Carinii. Três variantes, diferindo do tamanho da seqüencia do domínio P1, foram observados em três populações com distribuição geográfica diferente. O variante maior pode se auto-processar (\"self-splice\") in vitro. Quadros abertos de leitura estão presentes nos introns, mas não correspondem a nenhum gene conhecido. Introns estão presentes no rDNA 18S de outras espécies de Porphyra, que também podem apresentar variantes do rDNA 18S sem introns / The red algas genus Porphyra has considerable economic importance, and some species are extensively cultivated for human food. The genus is represented by more than 70 species, and occurs worldwide. Its taxonomy, based mainly on morphological characters of the macroscopic phase of its life-cycle is still unsettled. Alternatives to try to understand better the taxonomy and evolutive history of the genus were ascertained. It was verified that characters of the microscopic, filamentous phase, of the life-cycle of Porphyra may be used to complement the traditional taxonomic studies. To try to elucidate the phylogenetic position of Porphyra relative to the other red algae, and within the genus, among isolates from different locations, nuclear-encoded small-subunit ribosomal RNA genes (18S rDNAs) were PCR-amplified, cloned and completely sequenced. Three species of Porphyra from Nova Scotia and two species from Brasil were aligned with 18S sequences of other eukaryotes, including one published sequence of \"Porphyra umbilicalis\" from France. Phylogenetic trees were constructed by parsimony, distance and maximum-likelihood procedures. Analysis of our data revealed that these Porphyra species represented one of the deepest branches so far discovered within red algae. There was a great degree of primary sequence difference between Porphyra (subclass Bangiophycidae), and the other red algae belonging to the subclasses Florideophycidae. These results support the division of red algae into two subclasses by traditional taxonomy. Among eukaryotes Porphyra diverges at the same point as the Cryptomonas nucleomorph. The great among of sequence divergence, and the fossil record suggest that Porphyra, my indeed, be a very primitive red alga. Surprisingly, the 18S RNA sequence of the French \"Porphyra umbilicalis\" does not fit in our Porphyra category; instead, it has more than 99% identity with a species of Palmaria belonging to the subclass Florideophycidae. Therefore it was concluded that \"P. umbilicalis\" with the published sequence was actually a Palmaria palmate that was misidentified. During the phylogenetic analysis it was found that a group IC1 intron occurs in nuclear 18S rRNA genes of Porphyra spiralis var. amplifolia. This intron occurs at the same position of the group IC1 introns in 18S rDNAs of the fungus Pneumocystis carinii, Protomyces inouyei and the green alga Chlorella ellipsoidea, and shares primary-structural identity with the Pn. Carinii intron in domains P1 and P2, outside the conserved core. Three size-variants, differing in amount of optimal sequence in P1, exist and are differentially distributed in geographically distinct populations. The largest variant can self-splice in vitro. Open reading frames are present, but do correspond to known genes. Introns are present in the 18S rDNAs of several other Porphyra species, that may also have intronless rDNA copies

O gênero Porphyra (Rhodophyta) apresenta uma considerável importância econômica, sendo extensivamente cultivado e consumido como alimento. O gênero é representado por mais de 70 espécies e apresenta ampla distribuição geográfica, desde regiões tropicais até polares. Sua taxonomia, baseada em poucos caracteres da fase macroscópica do seu ciclo de vida, é ainda bastante problemática. Para tentar entender melhor a taxonomia e a história evolutiva de Porphyra foram utilizadas metodologias de biologia molecular e características da fase conchocelis do ciclo de vida. Verificou-se que caracteres da fase microscópica podem ser utilizados para complementar os conhecimentos taxonômicos tradicionais. Para tentar elucidar a posição filogenética do gênero Porphyra na divisão Rhodophyta e, dentro do gênero, entre espécies do Atlântico, o gene nuclear que codifica para o RNA ribossomal da subunidade pequena do ribossomo (rDNA 18S) foi amplificado através de PCR, clonado e completamente sequenciado. Foram utilizadas três espécies de Porphyra da Nova Escócia (Canadá) e duas de São Paulo (Brasil). As sequências obtidas foram alinhadas com as de alguns eucariontes e de outras algas vermelhas, incluindo uma sequência publicada de \"Porphyra umbilicalis\" da França. As árvores filogenéticas foram construídas através dos métodos de parcimônia, distancia e máxima verossimilhança. As analises mostraram que o gênero Porphyra é monofilético para as cinco espécies estudadas e constitui um dos ramos mais antigos dentro das algas vermelhas já analisados. O gênero Porphyra, subclasse Bangiophycidae, apresentou uma diferença substancial em relação aos gêneros da subclasse Florrideophycidae, sustentando assim, a divisão de Rhodophyta em duas subclasses pela taxonomia tradicional. Entre os eucariontes, Porphyra divergiu ao mesmo tempo que o nuclemorfo de Cryptomonas. O alto grau de divergência genética encontrada entre espécies de Porphyra, além de indicações do registo fóssil, na literatura, sugerem que o gênero é bastante primitivo dentro das algas vermelhas. Surpreendentemente, a sequência publicada para \"Porphyra umbilicalis\" apresentou mais de 99% de identidade com uma espécie de Palmaria que pertence à subclasse Florideophycidae; neste caso, a biologia molecular serviu para comprovar a identificação errônea do exemplar cuja sequência foi publicada. Durante a análise filogenética, verificou-se a ocorrência de um intron do grupo ICI nos genes rDNA 18S de Porphyra spiralis var. amplifolia. Esse intron ocorre na mesma posição que os introns do grupo IC1 nos rDNA 18S dos fungos Pneumocystis carinii, Protomyces inouyei e da alga verde Chlorella ellipsoidea, e apresenta identidade de sequências nos domínios P1 e P2, fora da região conservada, com o intron de Pn. Carinii. Três variantes, diferindo do tamanho da seqüencia do domínio P1, foram observados em três populações com distribuição geográfica diferente. O variante maior pode se auto-processar (\"self-splice\") in vitro. Quadros abertos de leitura estão presentes nos introns, mas não correspondem a nenhum gene conhecido. Introns estão presentes no rDNA 18S de outras espécies de Porphyra, que também podem apresentar variantes do rDNA 18S sem introns / The red algas genus Porphyra has considerable economic importance, and some species are extensively cultivated for human food. The genus is represented by more than 70 species, and occurs worldwide. Its taxonomy, based mainly on morphological characters of the macroscopic phase of its life-cycle is still unsettled. Alternatives to try to understand better the taxonomy and evolutive history of the genus were ascertained. It was verified that characters of the microscopic, filamentous phase, of the life-cycle of Porphyra may be used to complement the traditional taxonomic studies. To try to elucidate the phylogenetic position of Porphyra relative to the other red algae, and within the genus, among isolates from different locations, nuclear-encoded small-subunit ribosomal RNA genes (18S rDNAs) were PCR-amplified, cloned and completely sequenced. Three species of Porphyra from Nova Scotia and two species from Brasil were aligned with 18S sequences of other eukaryotes, including one published sequence of \"Porphyra umbilicalis\" from France. Phylogenetic trees were constructed by parsimony, distance and maximum-likelihood procedures. Analysis of our data revealed that these Porphyra species represented one of the deepest branches so far discovered within red algae. There was a great degree of primary sequence difference between Porphyra (subclass Bangiophycidae), and the other red algae belonging to the subclasses Florideophycidae. These results support the division of red algae into two subclasses by traditional taxonomy. Among eukaryotes Porphyra diverges at the same point as the Cryptomonas nucleomorph. The great among of sequence divergence, and the fossil record suggest that Porphyra, my indeed, be a very primitive red alga. Surprisingly, the 18S RNA sequence of the French \"Porphyra umbilicalis\" does not fit in our Porphyra category; instead, it has more than 99% identity with a species of Palmaria belonging to the subclass Florideophycidae. Therefore it was concluded that \"P. umbilicalis\" with the published sequence was actually a Palmaria palmate that was misidentified. During the phylogenetic analysis it was found that a group IC1 intron occurs in nuclear 18S rRNA genes of Porphyra spiralis var. amplifolia. This intron occurs at the same position of the group IC1 introns in 18S rDNAs of the fungus Pneumocystis carinii, Protomyces inouyei and the green alga Chlorella ellipsoidea, and shares primary-structural identity with the Pn. Carinii intron in domains P1 and P2, outside the conserved core. Three size-variants, differing in amount of optimal sequence in P1, exist and are differentially distributed in geographically distinct populations. The largest variant can self-splice in vitro. Open reading frames are present, but do correspond to known genes. Introns are present in the 18S rDNAs of several other Porphyra species, that may also have intronless rDNA copies