Anita S. Klein

DETERMINING THE AFFINITIES OF SALT MARSH FUCOIDS USING MICROSATELLITE MARKERS: EVIDENCE OF HYBRIDIZATION AND INTROGRESSION BETWEEN TWO SPECIES OF FUCUS (PHAEOPHYTA) IN A MAINE ESTUARY

The high degree of morphological plasticity displayed by species of the brown algal genus Fucus L. is well documented. Such variation is especially pronounced for those estuarine taxa lacking holdfasts (termed ecads) that often bear little resemblance to the attached species from which they are derived. To better understand the systematics of salt marsh fucoids, we developed a suite of four microsatellite‐containing loci capable of distinguishing between F. vesiculosus L. and F. spiralis L. The genetic markers were used to determine the relationships of the fucoid ecads F. vesiculosus ecad volubilis (Hudson) Turner and a muscoides‐like Fucus in the Brave Boat Harbor (ME, USA) estuary. Ecad populations had 2‐ to 3‐fold higher levels of heterozygosity than attached populations of F. vesiculosus and F. spiralis. Further, ecads were “intermediate” between F. vesiculosus and F. spiralis in their allele frequencies and genotype composition. Our data indicate that populations of muscoides‐like Fucus in Brave Boat Harbor mainly consist of F1 hybrids between F. vesiculosus and F. spiralis, whereas F. vesiculosus ecad volubilis may arise through backcrosses between F. vesiculosus and other fertile hybrids. Finally, our data support the hypothesis that introgression has occurred between attached populations of F. vesiculosus and F. spiralis.

Identification of north-western Atlantic Porphyra (Bangiaceae, Bangiales) based on sequence variation in nuclear SSU and plastid rbcL genes

Abstract Six species of Porphyra have commonly been recognized in the north-western Atlantic from Long Island Sound to the Canadian Maritimes: P. amplissima, P. leucosticra, P. linearis, P. miniata, P. purpurea, and P. umbilicalis. Distinguishing them with certainty has been problematic. A DNA-based system of molecular identification was developed using partial sequences of the nuclear small subunit ribosomal RNA gene (SSU) or the plastid ribulose-1,5-bisphosphate carboxylase–oxygenase large subunit gene (rbcL). Multiple samples of each taxon were surveyed for intraspecific variation. Intraspecific SSU divergences for Porphyra ‘leucosticta’, P. ‘miniata’, P. ‘umbilicalis’, and P. ‘purpurea’ ranged from 0% to 1%. There was more variation for P. ‘amplissima’ (0–2.1%) and P. ‘linearis’ (0–3.5%); however, each taxon was monophyletic. No intraspecific differences were observed for these taxa in rbcL (one to eight samples per taxon). These sequences were compared with P. yezoensis U51, introduced to Maine, and with P. ‘dioica’, a north-east Atlantic Porphyra easily confused with P. ‘purpurea’. To discriminate between P. ‘purpurea’, P. ‘umbilicalis’, and P. ‘leucosticta’, SSU variation was used to design primers for the Allele-Specific Polymerase Chain Reaction™. With molecular tools, we could classify over 80% of the monostromatic specimens surveyed, but the residue of unidentifiable specimens may indicate the existence of further monostromatic species in the north-west Atlantic. Porphyra ‘purpurea’ was found to occur further south than previously recorded. A morphologically cryptic Porphyra was discovered at Herring Cove, Nova Scotia, Canada.† Phylogenetic analyses using SSU or rbcL sequences showed ‘soft incongruence’ between gene trees, i.e. the topologies of the phylograms were similar but not identical, with only weak to moderate bootstrap support for the nodes that differed. Both trees strongly supported a clade including P. ‘purpurea’, P. ‘umbilicalis’, P. ‘linearis’, and P. ‘dioica’. Porphyra sp. Herring Cove was allied with the remaining Porphyra taxa in the SSU tree. The rbcL phylogeny was less well resolved, consisting of a polytomy of a P. ‘purpurea’–P. ‘umbilicalis’–P. ‘linearis’–P. ‘dioica’ clade, Porphyra sp. Herring Cove, a clade comprising P. ‘amplissima’ and P. ‘miniata’, and a P. ‘suborbiculata’–P. ‘leucosticta’–P. yezoensis clade.

Endophytic fungal DNA, the source of contamination in spruce needle DNA

DNA isolated and amplified from higher plants may originate from symbiotic microbes occupying plant tissues. A recent report on the phylogeny of Picea contained sequence data that upon later analysis proved to originate from filamentous ascomycetes. Isolates of endophytic fungi from Picea foliage collected from the same location as the original samples were examined to identify the source of the contaminating DNA. The ITS region of isolates was screened by Southern blotting using an oligonucleotide probe homologous to a unique portion of the reported ‘spruce’ sequences. This study identifies a DNA sequence originally attributed to Picea engelmannii (Engelmann spruce) as Hormonema dematioides, a ubiquitous foliar endophyte of conifers. Infections of plants by endophytic fungi are common and their presence is not revealed by external symptoms. Plant molecular researchers should be aware of the potential for this type of DNA contamination.

Species-specific nuclear and chloroplast single nucleotide polymorphisms to distinguish Picea glauca, P. mariana and P. rubens

Abstract Picea rubens (red spruce) and P. mariana (black spruce) are closely related species which are difficult to differentiate morphologically. They are sympatric with P. glauca (white spruce) in the northern portion of their ranges. In order to identify potential interspecific polymorphisms, the chloroplast trnK intron and rpl33-psaJ-trnP region were sequenced, and the nuclear-encoded ITS region of the rDNA repeat was partially sequenced. Thirteen chloroplast and 12 nuclear candidate interspecific single nucleotide polymorphisms (SNPs) were identified. The species-specificity of several SNPs was determined by surveying DNAs amplified from trees representing range-wide provenance tests; these included 46 red spruce from 11 provenances, 84 black spruce from 30 provenances and 90 white spruce from 22 provenances. Two SNPs (1 chloroplast and 1 nuclear), which distinguish black spruce from red and white spruce, were consistent among 96–100% of the trees surveyed. Five SNPs (4 chloroplast and 1 nuclear), which distinguish white spruce from red and black spruce, were consistent among 100% of surveyed trees. These species-specific SNPs were used to identify anonymous spruce samples in a blind test, and their utility for small amounts of tissue, as little as single needles, was demonstrated. Scoring these SNPs is much less labor intensive than previous molecular methods for taxa differentiation (restriction fragment length polymorphisms or random amplified polymorphic DNAs), therefore they can be applied to large population studies.

Phylogenetic relationships of the Hamamelidaceae inferred from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

Intergeneric relationships in the Hamamelidaceae have long been controversial. In this study, sequences of the internal transcribed spacers of nuclear ribosomal DNA were used to reconstruct the phylogeny for the Hamamelidaceae. Three major clades were recognized in the ITS-based phylogenetic tree: (1) Mytilaria-Exbucklandia-Rhodoleia, (2) Disanthus, and (3) the Hamamelidoideae. Within the Hamamelidoideae there were three well-supported lineages: (1) Corylopsis-Loropetalum-Tetrathyrium-Maingaya-Matudaea, (2) Eustigmateae sensu Endress, plus Molinadendron-Dicoryphinae, and (3) Hamamelis-Fothergilleae sensu Endress, excluding Matudaea and Molinadendron. The Exbucklandioideae sensu Endress were not monophyletic, nor were the tribes in the Hamamelidoideae in their current circumscriptions except for the Corylopsideae. Strap-shaped petals, apetaly, and wind pollination have evolved three times independently in the Hamamelidaceae s.s. (Hamamelidaceae minus Altingioideae), suggesting that homoplasy should be considered in future classifications of the family.

PORPHYRA BIRDIAE SP. NOV. (BANGIALES, RHODOPHYTA): A NEW SPECIES FROM THE NORTHWEST ATLANTIC

The red algal genus Porphyra Roth (Bangiales, Rhodophyta) includes approximately 140 recognized species (Yoshida et al. 1997; Silva 1999). Several recent investigations in diverse geographic regions have resulted in newly described species and/or range extensions, including the North Pacific (Lindstrom and Cole 1990a, 1992a, 1992c; Stiller and Waaland 1993, 1996; Hwang and Lee 1994), North Atlantic (Coll and Cox 1977; Kornmann and Sahling 1991; Brodie and Irvine 1997), South Africa (Griffin et al. 1999) and New Zealand (Nelson et al. 1998; Broom et al. 2002). There appears to be a consensus among the authors of these studies that the genus is understudied and that the number of reported species represents an underestimation of the species present. In the Northwest Atlantic, Bird and McLachlan (1992) indicate “it is becoming apparent that the limits of some species of Porphyra have been too broadly interpreted, and these taxa are in fact ‘form-species’ comprising a number of similar entities.” Very little taxonomic work has been conducted on Porphyra from the Atlantic coast of North America and only eight species have been reported (Schneider and Searles 1991; Bird and McLachlan 1992, Broom et al. 2002): P. amplissima Kjellman, P. miniata (C. Agardh) C. Agardh, P. linearis Greville, P. leucosticta Thuret in LeJolis, P. purpurea (Roth) C. Agardh, P. umbilicalis Kutzing, P. rosengurttii Coll et Cox, and P. suborbiculata Kjellman. Six of the eight species were originally described from Europe in the late 1700’s and 1800’s (Brodie and Irvine in press). In early work by Collins (1900), he lists three species from New England that are Algae Volume 17(4): 203-216, 2002

Analysis of Porphyra Membrane Transporters Demonstrates Gene Transfer among Photosynthetic Eukaryotes and Numerous Sodium-Coupled Transport Systems

Membrane transporters play a central role in many cellular processes that rely on the movement of ions and organic molecules between the environment and the cell, and between cellular compartments. Transporters have been well characterized in plants and green algae, but little is known about transporters or their evolutionary histories in the red algae. Here we examined 482 expressed sequence tag contigs that encode putative membrane transporters in the economically important red seaweed Porphyra (Bangiophyceae, Rhodophyta). These contigs are part of a comprehensive transcriptome dataset from Porphyra umbilicalis and Porphyra purpurea. Using phylogenomics, we identified 30 trees that support the expected monophyly of red and green algae/plants (i.e. the Plantae hypothesis) and 19 expressed sequence tag contigs that show evidence of endosymbiotic/horizontal gene transfer involving stramenopiles. The majority (77%) of analyzed contigs encode transporters with unresolved phylogenies, demonstrating the difficulty in resolving the evolutionary history of genes. We observed molecular features of many sodium-coupled transport systems in marine algae, and the potential for coregulation of Porphyra transporter genes that are associated with fatty acid biosynthesis and intracellular lipid trafficking. Although both the tissue-specific and subcellular locations of the encoded proteins require further investigation, our study provides red algal gene candidates associated with transport functions and novel insights into the biology and evolution of these transporters.

Porphyra: Complex Life Histories in a Harsh Environment: P. umbilicalis, an Intertidal Red Alga for Genomic Analysis

Porphyra encompasses a large group of multicellular red algae that have a prominent gametophytic phase. The complex, heteromorphic life history of species in this genus, their remarkable resilience to high light and desiccation, ancient fossil records, and value as human food (e.g., laver, nori), make Porphyra a compelling model for genome sequencing. Sequencing of the nuclear genome of Porphyra umbilicalis from the northwestern Atlantic is currently in process. The ∼270 Mb genome of this alga is much larger than that of the unicellular acidophilic Cyanidioschyzon merolae (16.5 Mb), the only rhodophyte for which there is a fully sequenced genome, and is approximately twice as large as the Arabidopsis genome. Future analyses of the P. umbilicalis genome should provide opportunities for researchers to (1) develop an increased understanding of the ways in which these algae have adapted to severe physiological stresses, (2) elucidate the molecular features of development through the complex life history, and (3) define key components required for the transition of growth from a single cell to a multicellular organism.

Distribution, morphology, and genetic affinities of dwarf embedded Fucus populations from the Northwest Atlantic Ocean

Abstract Dwarf embedded Fucus populations in the Northwest Atlantic Ocean are restricted to the upper intertidal zone in sandy salt marsh environments; they lack holdfasts and are from attached parental populations of F. spiralis or F. spiralis×F. vesiculosus hybrids after breakage and entanglement with halophytic marsh grasses. Dwarf forms are dichotomously branched, flat, and have a mean overall length and width of 20.3 and 1.3 mm, respectively. Thus, they are longer than Irish (mean 9.3 mm) and Alaskan (mean 15.0 mm) populations identified as F. cottonii. Reciprocal transplants of different Fucus taxa in a Maine salt marsh confirm that F. spiralis can become transformed into dwarf embedded thalli within the high intertidal zone, while the latter can grow into F. s. ecad lutarius within the mid intertidal zone. Thus, vertical transplantation can modify fucoid morphology and result in varying ecads. Microsatellite markers indicate that attached F. spiralis and F. vesiculosus are genetically distinct, while dwarf forms may arise via hybridization between the two taxa. The ratio of intermediate to species-specific-genotypes decreased with larger thalli. Also, F. s. ecad lutarius consists of a mixture of intermediate and “pure” genotypes, while dwarf thalli show a greater frequency of hybrids.

Phylogenetic relationships in the Hamamelidaceae: Evidence from the nucleotide sequences of the plastid genematK

The Hamamelidaceae is a family that bridges the basal elements of the Rosidae and the “lower” Hamamelidae, thus a better understanding of the phylogeny of the family is important for clarifying evolutionary patterns in the diversification of eudicots. However, subfamilial as well as tribal relationships in the Hamamelidaceae have been controversial. Nucleotide sequences of the chloroplast genematK were used to study the intergeneric relationships of the family. In the phylogenetic trees, constructed using parsimony analysis, the clade containingAltingia andLiquidambar (Altingioideae) is sister to a clade that includes all other Hamamelidaceae.Exbucklandia andRhodoleia form a clade, suggesting a close relationship between the two genera.Disanthus is sister to the monophyletic Hamamelidoideae. The paraphyletic arrangement ofDisanthus, Mytilaria andExbucklandia with respect to the Hamamelidoideae does not support the combination of these genera in one subfamily. In the Hamamelidoideae, thematK phylogeny supports the monophyly of several previously recognized groups with modifications, including the tribes Eustigmateae (incl.Molinadendron), Fothergilleae (excl.Molinadendron andMatudaea), and the subtribe Dicoryphinae. However, the Hamamelideae as traditionally circumscribed is polyphyletic. Apetaly has evolved three times independently in the Hamamelidoideae.