Gerald T. Kraft

AMPLIFICATION OF THE POLYMORPHIC 5.8S rRNA GENE FROM SELECTED AUSTRALIAN GIGARTINALEAN SPECIES (RHODOPHYTA) BY POLYMERASE CHAIN REACTION1

We have initiated comparative studies of ribosomal RNA (rRNA) gene structure to explore its potential to provide taxonomically useful data within the large red algal order Gigartinales. In southern Australia, this group is extremely diverse and includes large numbers of endemic taxa, many of potential economic importance. The 5.8S rRNA gene occurs in the middle region of the ribosomal DNA (rDNA) cistron and is flanked by two internal transcribed spacers (ITSs). These spacers contain regions of DNA, which are highly consented at the generic level and above, interspersed with highly divergent sequences. The 5.8S and associated ITS s of 11 species of Gigartinales (including five species of the largest Australian endemic marine algal genus, Mychodea), plus five taxa belonging to other orders, were amplified by the polymerase chain reaction. The size of the 5.8S rDNA and its flanking ITSs varied not only within and between genera, but also at the species level. However, this rDNA sequence appears to be relatively constant within populations find may be useful as a populational marker.

Marine and estuarine benthic green algae (Chlorophyta) of Lord Howe Island, south-western Pacific

A taxonomic survey of marine and estuarine benthic green algae has been conducted at Lord Howe Island, the site of the world’s southernmost (at 31.5˚S) consolidated coral reef. Thirty-two genera and 71 species are described from habitats ranging from upper intertidal to 20–30-m depths. Sixteen of the species are based on Lord Howe types, and 11 are described as new. Twelve species are currently known only from the Island. Particularly well represented in the flora are the genera Enteromorpha (six species, one variety), Chaetomorpha (four species), Cladophora (13 species),Codium (five species), Caulerpa (seven species, two varieties) and Bryopsis (three species). The island is further distinctive in having two representatives of some relatively obscure genera (Cladophoropsis, Rhipiliopsis, Pseudochlorodesmis) and in being the highest-latitude collecting locality for several species. Affinities of the Lord Howe flora are primarily tropical Indo-Pacific, with very few elements otherwise restricted to southern Australia and New Zealand.

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.

BETA/KAPPA‐CARRAGEENANS AS EVIDENCE FOR CONTINUED SEPARATION OF THE FAMILIES DICRANEMATACEAE AND SARCODIACEAE (GIGARTINALES, RHODOPHYTA)

The Dicranemataceae consists of five species in four genera of macroscopic red algae endemic to the southern half of Australia plus a single species from southern Japan. Investigations of the nonfibrillar wall components of five of the six species show that all are composed mainly of hybrid (or mixed) beta (β)/kappa(κ)‐type carrageenans. Detailed studies of Tylotus obtusatus (Sonder) J. Agardh show that it produces the largest dry‐weight percentage of β‐carrageenan yet recorded. Monosaccharide composition, total sulfate content, sulfation pattern revealed by infrared and 13C‐nuclear magnetic resonance spectroscopy and a positive specific optical rotation ([α]D+ 54°) are indicative of a low‐sulfate‐containing carrageenan with gelling properties similar to those of agar and furcellaran. β‐carrageenan is recorded in only five other red algal species belonging to relatively unrelated families, and we conclude that its uniform occurrence in the highly specialized family Dicranemataceae has phylogenetic significance. Chemical and anatomical examination of the genus Sarcodia, which produce lambda‐type carrageenan in both its gametophytic and tetrasporophytic phases, suggests that, despite the recent proposal to incorporate the Dicranemataceae into the Sarcodiaceae, the two families should continue to be separated.

Sulfated galactans from Australian specimens of the red alga Phacelocarpus peperocarpos (Gigartinales, Rhodophyta)

Polysaccharides from the red alga Phacelocarpus peperocarpos were extracted with hot water, clarified, and precipitated with 2-propanol. The native preparation was highly sulfated (36.2% w/w). Alkali modification decreased the sulfate content by 2.0% w/w. The alkali-modified polysaccharide is composed mostly of galactose (Gal. 51 mol%) and 3,6-anhydrogalactose (AnGal, 41 mol%), with minor amounts of a mono-O-methylgalactose (MeGal, 1 mol%), xylose (Xyl, 6 mol%), and glucose (Glc, 1 mol%). The FTIR spectrum of the alkali-modified polysaccharide resembled kappa-carrageenan with absorption at 930 cm-1 (indicative of AnGal) and 850 cm-1 (Gal 4-sulfate). However, an additional, major band of absorption occurred sulfate ester substitution at O-6 of at 820 cm-1, indicating the presence of equatorial sulfate ester substitution at O-6 of Gal residues. A combination of linkage and 13C NMR spectroscopic analyses showed that the polysaccharide was composed predominantly of a novel repeating-unit, O-beta-D-galactopyranosyl 4,6-disulfate)-(1-->4)-3,6-anhydro-alpha-D-galactopyranose. Minor structural variations also occurred, including alternative patterns of sulfation and the presence of terminal Xylp. The location of the terminal Xylp residues was not certain but evidence supported their attachment at O-3 of some 4-linked Galp residues. The cell-wall galactans remain unchanged during the life cycle of the alga.

Cell wall polysaccharides from Australian red algae of the family Solieriaceae (Gigartinales, Rhodophyta): highly methylated carrageenans from the genus Rhabdonia

The hot water-soluble polysaccharides from Rhabdonia coccinea and R. verticillata were characterised by a combination of constituent sugar analysis, sulphate and pyruvate content assays, infrared (IR) spectroscopy, linkage analysis, and C-13-nuclear magnetic resonance (NMR) spectroscopy. These revealed unique polysaccharides belonging to the red algal galactan family. The polysaccharides had IR spectra resembling that of l-carrageenan, but were rich in 6-O-methylgalactose (ca. 31 mol% and 17 mol% for R. coccinea and R. verticillata, respectively). Data from C-13-NMR spectroscopy provided evidence that the polysaccharides were carrageenans rather than agarocolloids. The preparations contained mainly l-carrageenan, partially methylated at C(O)6 of the 3-linked galactose residue. The polysaccharide from R. verticillata also contained significant quantities of 3-O-methylgalactose and pyruvate. The unusual sugar 3-O-methylgalactose occurred primarily as main-chain 4-linked residues, with a small proportion in the form of terminal residues. Other structural variations occurred in the polysaccharides of both species.

TWO NEW AUSTRALIAN SPECIES OF PREDAEA (NEMASTOMATACEAE, RHODOPHYTA) WITH TAXONOMIC RECOMMENDATIONS FOR AN EMENDED NEMASTOMATALES AND EXPANDED HALYMENIALES1

Two new species of the red algal genus Predaea are described from Australia. The first, Predaea aurora Kraft et G.W. Saunders, sp. nov., is unusual in its cool‐temperate distribution and in a number of anatomical attributes, most notably the rhizoid‐like habit of the nutritive filaments associated with the auxiliary cells. The second species, Predaea tumescens Kraft et G.W. Saunders, sp. nov., inhabits a coral reef habitat more typical of the genus but nonetheless differs in a number of anatomical details from other reported species. Nuclear small subunit rDNA sequences have confirmed the affinity of P. aurora to other species currently included in this genus. Molecular analyses further indicate that Predaea belongs in the Nemastomataceae and that the Nemastomataceae and Schizymeniaceae are sister taxa in an independent clade of “lineage 4” florideophyte algae. As such, a proposal is made to resurrect the Nemastomatales Kylin emend. G.W. Saunders et Kraft to accommodate these two families. Within the Schizymeniaceae, the Australian‐endemic Platoma australicum and Platoma foliosum were only distantly related to the other included Platoma, Schizymenia, and Titanophora spp. We therefore propose Wetherbeella G.W. Saunders et Kraft, gen. nov., to accommodate these two species. An additional outcome of our molecular analyses is that the genus Tsengia is not a member of the Nemastomataceae (where it is currently placed) but rather forms an independent lineage in the Halymeniales that we now designate as the Tsengiaceae G.W. Saunders et Kraft, fam. nov. Finally, the South African Nemastoma lanceolatum J. Agardh is transferred to Tsengia.

A REVISION OF THE SYSTEMATICS OF THE NIZYMENIACEAE (GIGARTINALES, RHODOPHYTA) BASED ON POLYSACCHARIDES, ANATOMY, AND NUCLEOTIDE SEQUENCES1

The Australian endemic family Nizymeniaceae, based on Nizymenia australis Sonder, consists of three species in the two genera Nizymenia (1 sp.) and Stenocladia (2 spp.). We have reassessed the generic composition of the family based on evidence from nonfibrillar wall polysaccharides, vegetative anatomy, and the nucleotide sequences of an internal transcribed spacer, ITS 2, of the nuclear ribosomal cistron. Investigation of the polysaccharides by constituent sugar analysis, sulfate content determination, and methylation analysis, combined with gas chromatography‐mass spectrometry and infrared analysis, showed that the polysaccharides elaborated by the three species were branched, highly sulfated xylogalactans. These polysaccharides also contained significant amounts of mono‐O‐methyl galactose (5–8 mol% of total sugars), mainly 4‐O‐methyl galactose. Although no discrete chemical structures could be assigned to the polysaccharides, the analyses showed that those from Nizymenia australis and Stenocladia australis (Sonder) Silva were more alike than either was to that from S. furcata (Harvey) J. Agardh. This polysaccharide affinity was echoed by a suite of vegetative anatomical features. However, the only likely synapomorphy was the presence of refractive, thick‐walled medullary rhizines in both N. australis and S. australis. The ITS 2 sequences were inferred from direct sequencing of the products of polymerase chain reaction amplification. Comparison of the ITS 2 sequences of its three species with those of two outgroups indicated that the family Nizymeniaceae is monophyletic but that interspecific relationships within the family could not be resolved. We conclude that there is insufficient evidence to separate any of the species from the rest at the genus level. Therefore, all three species are consolidated into the genus Nizymenia. This necessitates nomenclatural changes of Stenocladia australis to Nizymenia conferta (Harvey) Chiovitti, Saunders, et Kraft comb. nov.

Phylogenetic analyses of anatomical and nuclear SSU rDNA sequence data indicate that the Dasyaceae and Delesseriaceae (Ceramiales, Rhodophyta) are polyphyletic

The aim of the current investigation was to test the general convention that the Dasyaceae, Delesseriaceae and Rhodomelaceae are all monophyletic families of the red algal order Ceramiales. Phylogenetic relationships among 45 ceramialean taxa were determined, including eight ceramiacean, 18 dasyacean, nine delesseriacean and eight rhodomelacean species, plus two of uncertain ceramialean affinities, based on 34 anatomical characters and nuclear small subunit (SSU) rDNA sequences. Results from our ‘total-evidence’ approach were consistent with the notion that the Dasyaceae, Delesseriaceae and Rhodomelaceae have evolved from a common ancestor within a paraphyletic Ceramiaceae. Our data indicate, however, that the Rhodomelaceae alone is monophyletic at the family level, with both the Dasyaceae and Delesseriaceae polyphyletic. In particular we resolved two independent and well-supported lineages for the included Dasyaceae, viz., a Dasya group and a Heterosiphonia group, which were as distinct from one another as they were from the Rhodomelaceae and the various lineages of Delesseriaceae. The molecular data alone were equivocal on the issue of monophyly of the Dasyaceae. We therefore advocate conservative taxonomic revisions as an interim step towards eventual resolution of familial-level taxonomy in the Ceramiales. Based on our results, the Heterosiphonioideae H.-G. Choi, Kraft, I.K. Lee et G.W. Saunders subfam. nov. is proposed for Heterosiphonia and five closely related genera, and the Dasyoideae Schmitz et Falkenberg is emended for the remaining taxa. Although the Dasyoideae is a natural group, it is in need of a thorough systematic reinvestigation at the generic level. Our analyses indicate that the genus Dasya is polyphyletic or paraphyletic in excluding Dasysiphonia, Eupogodon and Rhodoptilum and that Heterosiphonia japonica also has affinities to this group, taxonomic issues that will be addressed in detail elsewhere.

Nuclear small-subunit rDNA sequences from Ballia spp. (Rhodophyta): proposal of the Balliales ord. nov., Balliaceae fam. nov., Ballia nana sp. nov. and Inkyuleea gen. nov. (Ceramiales)

Abstract Nuclear small-subunit rDNA sequences from three species of Ballia (Ceramiales, Rhodophyta) native to southern Australia have been investigated, initially to assess the tribal placement of the genus within the Ceramiaceae. In distance and parsimony analyses, however, Ballia callitricha was strongly positioned in the ‘two-cap-layered pit-plug assemblage’ of the Florideo-phyceae as a distinct lineage, sister to the Acrochaetiales, Batrachospermales, Nemaliales and Palmariales. These taxa are only remotely related to the florideophyte ‘membrane-only pit-plug lineage’ that includes the Ceramiales. As B. callitricha is the type species of the genus Ballia and is distinct in anatomical and molecular features from all recognized orders in the two-cap-layer lineage, a new order and family, Balliales H.-G. Choi, Kraft & G.W. Saunders ord. nov. and Balliaceae H.-G. Choi, Kraft & G.W. Saunders fam. nov., are proposed to accommodate B. callitricha, B. pennoides, B. sertularioides, and a new species, B. nana Kraft & G.W. Saunders sp. nov., the latter known from a single collection in the Melbourne region of southeast Australia. Ballia ballioides and B. mariana, on the other hand, were solidly allied to the membrane-only taxa, although their position within this lineage, particularly with reference to the Ceramiales, was equivocal in our analyses. Nevertheless, these two species are clearly distinct from B. callitricha in morphological and molecular characters, and both are transferred to the new genus Inkyuleea H.-G. Choi, Kraft & G.W. Saunders, as I. ballioides (Sonder) H.-G. Choi, Kraft & G.W. Saunders comb. nov. (the type species) and I. mariana (Harvey) H.-G. Choi, Kraft & G.W. Saunders comb. nov. Inkyuleea differs in a number of reproductive characteristics from existing members of the Ceramiales, the possible significance of which is discussed.