Alison R. Sherwood

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

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.

A molecular method for identification of the morphologically plastic invasive algal genera Eucheuma and Kappaphycus (Rhodophyta, Gigartinales) in Hawaii

A paucity of diagnostic morphological characters for identification and high morphological plasticity within the genera Eucheuma and Kappaphycus has led to confusion about the distributions and spread of three introduced eucheumoid species in Hawaii. Entities previously identified as E. denticulatum, K. alvarezii, and K. striatum have had profound negative effects on Oahu’s coral reef ecosystems. The use of molecular tools to aid identification of algal species has been promising in other morphologically challenging taxa. We used three molecular markers (partial nuclear 28S rRNA, partial plastid 23S rRNA, and mitochondrial 5′ COI) and followed a DNA barcoding-like approach to identify Eucheuma and Kappaphycus samples from Hawaii. Neighbor-joining analyses were congruent in their separation of Eucheuma and Kappaphycus, and the resulting clusters were consistent with those revealed for global comparisons with the mitochondrial cox2-3 spacer and GenBank data. Based on these results, new insights were revealed into the distribution of these groups in Hawaii.

The Hawaiian Rhodophyta Biodiversity Survey (2006-2010): a summary of principal findings

BackgroundThe Hawaiian red algal flora is diverse, isolated, and well studied from a morphological and anatomical perspective, making it an excellent candidate for assessment using a combination of traditional taxonomic and molecular approaches. Acquiring and making these biodiversity data freely available in a timely manner ensures that other researchers can incorporate these baseline findings into phylogeographic studies of Hawaiian red algae or red algae found in other locations.ResultsA total of 1,946 accessions are represented in the collections from 305 different geographical locations in the Hawaiian archipelago. These accessions represent 24 orders, 49 families, 152 genera and 252 species/subspecific taxa of red algae. One order of red algae (the Rhodachlyales) was recognized in Hawaii for the first time and 196 new island distributional records were determined from the survey collections. One family and four genera are reported for the first time from Hawaii, and multiple species descriptions are in progress for newly discovered taxa. A total of 2,418 sequences were generated for Hawaiian red algae in the course of this study - 915 for the nuclear LSU marker, 864 for the plastidial UPA marker, and 639 for the mitochondrial COI marker. These baseline molecular data are presented as neighbor-joining trees to illustrate degrees of divergence within and among taxa. The LSU marker was typically most conserved, followed by UPA and COI. Phylogenetic analysis of a set of concatenated LSU, UPA and COI sequences recovered a tree that broadly resembled the current understanding of florideophyte red algal relationships, but bootstrap support was largely absent above the ordinal level. Phylogeographic trends are reported here for some common taxa within the Hawaiian Islands and include examples of those with, as well as without, intraspecific variation.ConclusionsThe UPA and COI markers were determined to be the most useful of the three and are recommended for inclusion in future algal biodiversity surveys. Molecular data for the survey provide the most extensive assessment of Hawaiian red algal diversity and, in combination with the morphological/anatomical and distributional data collected as part of the project, provide a solid baseline data set for future studies of the flora. The data are freely available via the Hawaiian Algal Database (HADB), which was designed and constructed to accommodate the results of the project. We present the first DNA sequence reference collection for a tropical Pacific seaweed flora, whose value extends beyond Hawaii since many Hawaiian taxa are shared with other tropical areas.

Molecular assessment of ulva spp. (ulvophyceae, chlorophyta) in the hawaiian islands

Sequences of the nuclear internal transcribed spacer 1 (ITS1) region and the chloroplast rbcL gene were obtained from 86 specimens of Ulva (including “Enteromorpha”) from five of the main Hawaiian Islands. These 86 specimens were divided into 11 operational taxonomic units (OTUs) based on analyses of primary sequence data and comparisons of ITS1 secondary structure. Of the 11 OTUs, six have not previously been reported from anywhere in the world. Only three represented exact sequence matches to named species (Ulva lactuca L., syn. U. fasciata Delile; U. ohnoi Hiraoka et Shimada); two others represented exact sequence matches to unnamed species from Japan and New Zealand. Of the 12 species names currently in use for Hawaiian Ulva, only one, U. lactuca (as U. fasciata), was substantiated. General morphology of the specimens did not always correspond with molecular OTUs; for example, reticulate thallus morphology, previously considered diagnostic for the species U. reticulata Forssk., was expressed in thalli assigned to U. ohnoi and to one of the novel OTUs. This finding confirms a number of recent studies and provides further support for a molecular species concept for Ulva. These results suggest that Ulva populations in tropical and subtropical regions consist of species that are largely unique to these areas, for which the application of names based on types from temperate and boreal European and North American waters is inappropriate. Ulva ohnoi, a “green tide” species, is reported from Hawaii for the first time.

MOLECULAR PHYLOGENY OF THE GREEN ALGAL ORDER PRASIOLALES (TREBOUXIOPHYCEAE, CHLOROPHYTA)1

The systematics of the Prasiolales was investigated by phylogenetic inference based on analyses of the rbcL and 18S rRNA genes for representatives of all four genera currently attributed to this order (Prasiococcus, Prasiola, Prasiolopsis, Rosenvingiella), including all type species. The rbcL gene had higher sequence divergence than the 18S rRNA gene and was more useful for phylogenetic inference at the ranks of genus and species. In the rbcL gene phylogeny, three main clades were observed, corresponding to Prasiola, Prasiolopsis, and Rosenvingiella. Prasiococcus was nested among species of Prasiola occurring in subaerial and supralittoral habitats. Trichophilus welckeri Weber Bosse, a subaerial alga occurring in the fur of sloths in Amazonia, was closely related to Prasiolopsis ramosa Vischer. The species of Prasiola were grouped into three well‐supported clades comprising (i) marine species, (ii) freshwater and terrestrial species with linear blades, and (iii) terrestrial species with rounded or fan‐shaped blades. Sequence divergence was unexpectedly low in the marine group, which included species with different morphologies. For the 18S rRNA gene, the phylogenetic analyses produced several clades observed for the rbcL gene sequence analysis, but, due to very little sequence variation, it showed considerably lower resolution for inference at the species and genus levels. Due to the low support of some internal branches, the results of the analyses did not allow an unambiguous clarification of the origin and the early evolution of the Prasiolales.

Assessing the phylogenetic position of the Prasiolales (Chlorophyta) using rbcL and 18S rRNA gene sequence data

Abstract Partial sequences of the genes coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the nuclear ribosomal small subunit (18S rRNA) were used to determine the phylogenetic position of the order Prasiolales among other members of the Chlorophyta. Sequences were obtained for seven representatives of the Prasiolales (Prasiola fluviatilis, P. furfuracea, P. linearis, two collections of P. mexieana, P. meridionalis and Rosenvingiella sp.) together with two Ulvophyceae, for which sequences were not previously available. Sequences of the representative Prasiolales were aligned with those of 17 green algal representatives for the rbcL gene and 20 for the 18S rRNA gene. Parsimony and distance analyses of the rbcL and 18S rRNA sequences indicate that the Prasiolales is a well-defined order. Parsimony and distance analyses of the rbcL gene give moderate support to an alliance with the Ulvophyceae, but support for the remaining nodes of the trees is very low to negligible. Both parsimony and distance analysis of the 18S rRNA gene provide moderate support for a relationship with the Trebouxiophyceae, which has been postulated in past studies based on cellular and ultrastructural characters. Although a large polytomy was determined by parsimony analysis containing many of the lineages within the Chlorophyta, the analyses of the 18S rRNA gene are more resolved than the rbcL gene analyses. Sequence divergence values within the Prasiolales for the rbcL gene are low (0–6.1%) compared to values among the other green algal sequences used in our analyses, confirming the cohesive nature of the order. Divergence values among the Prasiolales for the 18S rRNA gene are also low (0.4–3.8%).

A proposal for a new red algal order, the Thoreales

Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well‐supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate‐like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate‐like.

SYSTEMATICS of THE BATRACHOSPERMALES (RHODOPHYTA) - A SYNTHESIS

Recent molecular and morphological data necessitate a major taxonomic revision of the Batrachospermales, an order of red algae, distributed in freshwater habitats throughout the world. This article is a synthesis of available information with some targeted additional sequence data, resulting in a few relatively conservative taxonomic changes to begin the process of creating a natural taxonomy for the Batrachospermales. To increase the information content of our taxonomic categories, and in particular to reduce paraphyly, we describe one new genus (Kumanoa) and a new section in Batrachospermum (section Macrospora), and we amend the circumscriptions of the family Batrachospermaceae (to include Lemaneaceae and Psilosiphonaceae), the genus Batrachospermum (to exclude the sections Contorta and Hybrida, raised to genus level as Kumanoa), and the sections Aristata, Helminthoidea, and Batrachospermum of Batrachospermum. We also provide a new name, B. montagnei, for the illegitimate B. guyanense, and recognize an informal paraphyletic grouping of taxa within Batrachospermum, the “Australasica Group.” This taxonomic synthesis increases the level of monophyly within the Batrachospermales but minimizes taxonomic change where data are still inadequate.

Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty‐five accessions of the species‐rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species‐level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0–3 bp). Comparisons to previously published mitochondrial cox2–3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2–3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.

Taxonomy and distribution of Trentepohlia and Printzina (Trentepohliales, Chlorophyta) in the Hawaiian Islands

F. Rindi, A.R. Sherwood and M.D. Guiry. 2005. Taxonomy and distribution of Trentepohlia and Printzina (Trentepohliales, Chlorophyta) in the Hawaiian Islands. Phycologia 44: 270–284. The taxonomy and distribution of the species of Trentepohlia and Printzina in the Hawaiian Islands were studied by examination of field collections and herbarium specimens from Bernice P. Bishop Museum, Honolulu (BISH). Eight taxa attributable to these genera were recorded in the field: Printzina bosseae var. samoensis, P. effusa, P. lagenifera, Trentepohlia abietina, T. abietina var. tenue, T. arborum, Trentepohlia cf. flava and T. umbrina. The record of Trentepohlia aurea, based on some specimens deposited in BISH, is rejected. Growth and reproduction of some entities (P. bosseae var. samoensis, P. effusa, P. lagenifera and T. arborum) were examined in culture. In the Hawaiian Islands these algae grow primarily on tree bark and are rarely found on other types of substrata. Reproduction takes place by production of presumptive biflagellate gametangia in P. lagenifera, T. abietina and Trentepohlia cf. flava and by release of zoosporangia in P. bosseae var. samoensis and T. arborum; in P. effusa, both types of reproductive structures were found. Vegetative growth was the only form of reproduction found in culture for the species for which isolation was possible. The taxonomic position of the species collected and implications for the conservation of the Trentepohliaceae in tropical regions are discussed.