MOLECULAR SYSTEMATICS OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) WITH EMPHASIS ON SOUTHERN AFRICA1

Southern Africa has economically exploited populations of terete gracilarioids on the cool temperate west coast and numerous species of endemic and Indo‐Pacific tropical Gracilariaceae on the south and east coasts. Gross morphological characters have been the main means of identification, and incorrect applications have led to a number of misidentifications. In this study, small subunit rDNA and RUBISCO spacer sequences were used to determine phylogenetic relationships. Whereas rDNA sequences successfully differentiate major groups within the family as well as species belonging to the Gracilariopsis and the Curdiea/Melanthalia clade, RUBISCO spacer sequencing was required to distinguish between species of Gracilaria. The southern African gracilarioid complex (stringy, terete, elongate members of the Gracilariaceae) was resolved into three species: Gracilaria gracilis, Gracilariopsis longissima, and Gracilariopsis funicularis. South African Gracilaria protea was shown to be conspecific with tropical Indian Ocean G. corticata. Apart from G. gracilis and G. corticata, South African Gracilaria species were differentiated into a temperate‐tropical terete grouping and a temperate‐tropical flattened grouping.     

Molecular Systematics and Phylogenetics of Gracilariacean Species from the Mediterranean Sea

Mediterranean reports of Gracilariaceae species, in particular those assigned to the G. verrucosa complex, were re-examined with the use of molecular tools, in order to verify their systematic position and better understand their distribution. Within this complex, we recognized four distinct taxa: Gracilariopsis longissima, Gracilaria gracilis, Gracilaria longa and a possible new species. The rbcL gene sequences, together with those of other terete Mediterranean entities, were included in a broad molecular phylogeny of the family. The reproductive characters of the studied taxa do not fit completely with published hypothesis on the generic and intrageneric relationships, suggesting that the anatomy of some subgroups should be better characterized.     

Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content

Select species of the agarophyte Gracilaria were studied from southeastern Australia. The morphology and anatomy of species is described and molecular relations are inferred based on plastid and mitochon‐drial DNA sequence data. Agar yields and qualities are determined for each species. Gracilaria chilensis, found in Tasmania and Victoria, is morphologically and molecularly similar to G. chilensis from New Zealand and Chile and has low agar yields of 11–16%. Gracilaria cliftonii from Victoria, has high crude agar yield (52%) and is molecularly uniform. Gracilaria perplexa sp. nov., known only from Botany Bay, New South Wales, has an agar yield of 39%. The agar of G. perplexa is unusual in requiring the addition of 0.1 mol L^?1 NaCl for alcohol precipitation and is cold‐water (25°C) soluble because of the very high sulfate ester content. Molecular phylogeny shows that G. perplexa is closely related to Gracilaria preissiana from western Australia, but differs from the latter in its reduced branching and narrower more terete axes.     

SINGLE LOCUS MICROSATELLITES IN GRACILARIALES (RHODOPHYTA): HIGH LEVEL OF GENETIC VARIABILITY WITHIN GRACILARIA GRACILIS AND CONSERVATION IN RELATED SPECIES1

Four single locus microsatellites identified in the red alga Gracilaria gracilis (Stackhouse) Steentoft, Irvine, et Farnham (Rhodophyta) were examined for allelic diversity at different spatial and taxonomic levels. First, because simple morphological diagnostic characters are often missing within the Gracilariaceae, we developed a simple and rapid method based on rDNA ITS size variation in order to verify the taxonomic status of the samples used in this study. All European (including Mediterranean samples), Argentinian, and Namibian samples used in our study were confirmed to be a homogenous G. gracilis group. By contrast, our results on rDNA ITS sizes showed that Gracilaria from Japan, initially identified as G. gracilis, was different from the rest of the G. gracilis group. Secondly, microsatellite polymorphism and conservation at the species level was tested on the worldwide collection of G. gracilis and within a single population. The loci Gv1AAG and Gv1AAC showed no allelic variation, whereas two others, Gv1CT and Gv2CT, were highly polymorphic. All microsatellite loci were conserved within G. gracilis, except in the sample from Japan. The taxonomic status of G. gracilis from Japan is thus questionable. This study revealed a high level of within-population polymorphism (52 alleles for Gv1CT and 12 for Gv2CT). Moreover, the combination of these two loci was shown to be very powerful for identifying individuals within a population, that is, 93% of the individuals were characterized by a unique genotype. Finally, conservation of the four loci was tested in taxonomically related species of Gracilaria (G. chilensis, G. pacifica, and G. tikvahiae) and two Gracilariopsis species (Gs. sp. and Gs. longissima). The results suggest that the polymorphic locus Gv2CT may provide a valuable genetic marker within the different species of the Gracilariaceae.     

57 Systematics of hydropuntia montagne (gracilariaceae,gracilariales, rhodophyta) based on comparative morphology and rbcL sequence analysis

The taxonomic validity of the genus Hydropuntia Montagne (1843) (including Polycavernosa) within the Gracilariaceae (Gracilariales, Rhodophyta) is controversial. Morphological characters that define species of Hydropuntia are said to be variable and to overlap with those of Gracilaria. Here we present a global phylogenetic study of the family based on a Bayesian analysis of a large rbcL DNA sequence dataset indicating that the genus Hydropuntia forms a well supported monophyletic clade within the family, and recognize Hydropuntia as a genus distinct from Gracilaria. We also conducted smaller phylogenetic analyses in which thirty four Hydropuntia rbcL sequences resulted in two major clades within the genus, comprising a Caribbean clade and an Indo‐Pacific clade. Diagnostic reproductive stages that separate these two clades will be illustrated.     

PHYLOGENY AND SYSTEMATICS OF THE MARINE ALGAL FAMILY GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) BASED ON SMALL SUBUNIT rDNA AND ITS SEQUENCES OF ATLANTIC AND PACIFIC SPECIES1

We sequenced the small subunit rDNA and internal transcribed spacer region of Gracilariaceae from the tropical Atlantic and Pacific, with emphasis on flattened or compressed species. Sequence comparisons confirmed three main lineages of Gracilariaceae: Curdiea/Melanthalia, Gracilariopsis/Gracilariophila, and Gracilaria. The Curdiea/Melanthalia diverged early in the family. Gracilariopsis was paraphyletic, because at least one Gracilariophila species evolved from it. The Atlantic Gracilariopsis were monophyletic and separated from the Pacific lineages. The Gracilaria included all species referable to its own species and to Hydropuntia, which was paraphyletic, formed by distantly related lineages. The new combination Gracilaria pauciramosa (N. Rodríguez Ríos) Bellorin, M. C. Oliveira et E. C. Oliveira is proposed for Polycavernosa pauciramosa N. Rodríguez Ríos. Recognition of subgenera within Gracilaria, based on spermatangial arrangement, was not supported. Instead, infrageneric groups were delineated by geographic origins and combinations of reproductive characters. Most Pacific species with either “textorii” or “verrucosa” type spermatangia were deeply separated from Atlantic species. Within the Atlantic Gracilaria, a lineage encompassing mostly tropical cylindrical species with “henriquesiana” type spermatangia and distinctive cystocarp anatomy was recognized. A lineage was also retrieved for cold water stringy species with verrucosa type spermatangia. Several species from the western Atlantic are closely related to Gracilaria tikvahiae McLachlan with nearly identical morphology. On the other hand, most flattened species from the tropical Atlantic were closely related despite their diverse morphologies. The interpretation of our data in addition to the literature indicates that more populations from the Indo‐Pacific must be studied before a general picture of Gracilariaceae evolution can be framed.     

Phylogeny of Gracilariaceae (Rhodophyta): evidence from plastid and mitochondrial nucleotide sequences

Gracilariaceae are mostly pantropical red algae and include ~230 species in seven genera. Infrafamilial classification of the group has long been based on reproductive characters, but previous phylogenies have shown that traditionally circumscribed groups are not monophyletic. We performed phylogenetic analyses using two plastid (universal plastid amplicon and rbcL) and one mitochondrial (cox1) loci from a greatly expanded number of taxa to better assess generic relationships and understand patterns of character distributions. Our analyses produce the most well‐supported phylogeny of the family to date, and indicate that key characteristics of spermatangia and cystocarp type do not delineate genera as commonly suggested. Our results further indicate that Hydropuntia is not monophyletic. Given their morphological overlap with closely related members of Gracilaria, we propose that Hydropuntia be synonymized with the former. Our results additionally expand the known ranges of several Gracilariaceae species to include Brazil. Lastly, we demonstrate that the recently described Gracilaria yoneshigueana should be synonymized as G. domingensis based on morphological and molecular characters. These results demonstrate the utility of DNA barcoding for understanding poorly known and fragmentary materials of cryptic red algae.     

The Gracilariaceae Germplasm Bank of the University of São Paulo,Brazil—a DNA barcoding approach

The University of São Paulo Gracilariaceae Germplasm Bank has 50 strains collected mostly in Brazil, but also elsewhere in the world. This bank has been used as a source of material for research developed locally and abroad. With over 200 species, some of which have high economic value, the family Gracilariaceae has been extensively studied. Nonetheless, taxonomic problems still persist by the existence of cryptic species, phenotypic plasticity, and broad geographic distribution. In the case of algae kept in culture for long periods of time, the identification is even more problematic as a consequence of considerable morphological modification. Thus, the use of molecular markers has been shown to be an efficient tool to elucidate taxonomic issues in the group. In this work, we sequenced the 5′-end of the cox1 gene for 41 strains and the universal plastid amplicon (UPA) plastid region for 45 strains, covering all 50 strains in the bank. In addition, the rbcL for representatives of the cox1/UPA clusters was sequenced for 14 strains. The original species identification based on morphology was compared with the molecular data obtained in this work, resulting in the identification of 13 different species. Our analyses indicate that cox1 and UPA are suitable markers for the delineation of species of Gracilariales in the germplasm bank. The addition of DNA barcode tags to the samples in the Gracilariaceae germplasm bank and the molecular identification of the species will make this bank even more useful for future research as the species can be easily traced and confirmed.     

An integrative approach to the evolution of shrimps of the genus Palaemon (Decapoda,Palaemonidae)

Shrimps of the genus Palaemon Weber, 1795 comprise of 86 species with a wide morphological and ecological variability along the tropical and temperate regions. Studies based on molecular data have indicated that despite a recent taxonomic rearrangement, it may remain not monophyletic. On the other hand, cladistic, morphological analyses have suggested the presence of synapomorphies, implying a natural status for the genus. In this work, a broad taxonomic and molecular sampling is applied to verify whether Palaemon is a monophyletic taxon and, based on the recovered phylogeny, identify geographical and morphological patterns related to the lineages. Partial sequences of 16S rRNA, histone H3 and 18S rRNA from 60 species of Palaemon and 15 species from other genera of Palaemonidae were analysed. In addition, previously used characters as well as novel diagnostic characters were scrutinized. The present phylogeny indicates that the species of Palaemon fall into three distinct lineages and that the colonization of America and Europe likely occurred multiple times. Morphological characters allow for the identification of at least four monophyletic groups in Palaemon; two of which are monospecific at the moment. Based on the present results, it may become necessary to establish two new genera (to accommodate Palaemon concinnus and Palaemon mercedae, respectively), as well as re‐erect the genus Alaocaris Holthuis, 1949 for Palaemon antrorum, potentially including a further six American species.     

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.     

SYSTEMATICS OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYTA): A CRITICAL ASSESSMENT BASED ON RBCL SEQUENCE ANALYSES1

Generic concepts in the economically important agarophyte red algal family Gracilariaceae were evaluated based on maximum parsimony, Bayesian likelihood, and minimum evolution analyses of the chloroplast‐encoded rbc L gene from 67 specimens worldwide. The results confirm the monophyly of the family and identify three large clades, one of which corresponds to the ancestral antiboreal genera Curdiea and Melanthalia, one to Gracilariopsis, and one to Gracilaria sensu lato, which contains nine distinct independent evolutionary lineages, including Hydropuntia. The species currently attributed to Hydropuntia comprise a single well‐supported clade composed of two distinct lineages. The two most basal clades within Gracilaria sensu lato deserve generic rank: a new genus centered around G. chilensis Bird, McLachlan et Oliveira and G. aff. tenuistipitata Chang et Xia and a resurrected Hydropuntia encompassing primarily Indo‐Pacific (G. urvillei [Montagne] Abbott, G. edulis [S. Gmelin] P. Silva, G. eucheumatoides Harvey, G. preissiana [Sonder] Womersley, and G. rangiferina [Kützing] Piccone) and western Atlantic species (G. cornea J. Agardh, G. crassissima P. et H. Crouan in Mazé et Schramm, G. usneoides [C. Agardh] J. Agardh, G. caudata J. Agardh, and G. secunda P. et H. Crouan in Mazé et Schramm). Cystocarpic features within the Gracilaria sensu lato clades appear to be more phylogenetically informative than male characters. The textorii‐type spermatangial configuration is represented in two distinct clusters of Gracilaria. The rbc L genetic divergence among the Gracilariaceae genera ranged between 8.46% and 16.41%, providing at least 2.5 times more genetic variation than does the 18S nuclear rDNA. rbc L also resolves intrageneric relationships, especially within Gracilaria sensu lato. The current number of gracilariacean species is underestimated in the western Atlantic because of convergence in habit and apparent homoplasy in vegetative and reproductive anatomy.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

Anatomical characterization of Strobilanthes (Acanthaceae) species from the northern Western Ghats of India and its implication for identification at vegetative state

A comprehensive study of stem, leaf and petiole anatomy of 10 species of Strobilanthes from northern Western Ghats of India was carried out to identify characteristics which would enable species identification when flowering material is unavailable. In Strobilanthes, some species bloom annually, others are plietesials, i.e. they grow without blooming for several years and then produce huge quantities of flowers, release seeds and die. Therefore, alternative methods, such as anatomical characters, are essential to distinguish Strobilanthes species in their vegetative stage. We collected ten species of Strobilanthes for anatomical characterization. Under the bright‐field microscope, stem cross‐sections of different species were found to be undulate, quadrangular, quadrangular‐winged or terete. Study of the stem revealed a distinct outer and inner cortex, the distribution of cystoliths (CaCO₃ crystals), raphides (CaC₂H₂O₅ crystals) and sclereids which varied from species to species. Study of leaf anatomy showed structural variation and vascular bundle shapes that differed between the species. Leaf epidermal characters under light and scanning electron microscopy exhibited variation in characters such as stomatal index, stomatal length and width, stomatal type and presence of glandular and non‐glandular trichomes. The petiole anatomy was species‐specific, especially with respect to vascular bundle structure and the distribution of structures such as sclereids, cystoliths, sphaeraphides and tannin cells varied. Hence, unique anatomical features of the stem, leaf and petiole could be used as taxonomic characters to identify Strobilanthes species in a vegetative state.     

Gracilaria vermiculophylla: A western Pacific species of Gracilariaceae (Rhodophyta) first recorded from the eastern Pacific

We report Gracilaria vermiculophylla (Ohmi) Papenfuss from the Pacific coast of North America based on a morphoanatomical revision and comparison of sequences of the nuclear gene coding for the small subunit of ribosomal RNA and the internal transcribed spacers of populations from Baja California, Mexico and Hiroshima, Japan. It is the first convincing report of this species out of its center of distribution in western north Pacific, where it has been considered as a synonym of former ‘G. verrucosa’ records. G. vermiculophylla also occurs in central California as indicated by internal transcribed spacer sequences of a previously unknown Gracilaria Greville material. In the northeastern Pacific G. vermiculophylla is characterized by a robust somewhat vermiform, well‐branched cylindrical thallus, with gradual cell size transition from cortex to medulla, deep spermatangial conceptacles, regular chains of carposporangia and carposporangial initials, downwardly oriented tubular nutritive cells, but rarely with upwardly oriented tubular nutritive cells. In some features of its cystocarp anatomy G. vermiculophylla is related to Gracilariopsis E. Y. Dawson or Hydropuntia Montagne and it is a relevant species for discussions about Gracilariaceae genera.