Robert J. King

A mitochondrial marker for red algal intraspecific relationships

Intraspecific studies of red algae have relied on nuclear or plastid markers rather than mitochondrial data to address questions of systematics, biogeography or population genetics. In this study, primers were developed that spanned the noncoding intergenic region between the mitochondrial cytochrome oxidase subunit 2 and cytochrome oxidase subunit 3 genes. These primers were demonstrated to be successful on a variety of red algae in different orders: Gracilariales, Bonnemaisoniales and Ceramiales (families: Delesseriaceae, Ceramiaceae and Rhodomelaceae). Amplification products were between 450 and 320 bp in length, with variation in length shown among geographically distant isolates within a species. The region was variable within a single species, as shown for Bostrychia moritziana and B. radicans, and within populations of Caloglossa leprieurii. In the latter species, four mitochondrial haplotypes were observed in isolates from a single locality in Woolooware Bay, New South Wales, Australia. Analysis of hybrids between different mitochondrial haplotypes of B. moritziana revealed that the mitochondria are maternally inherited in this species. This is the first report of a mitochondrial marker that is variable within red algal populations and may lead to a better understanding of the population ecology of these important marine organisms.

Marine, Brackish, and Freshwater Fish Communities in the Vegetated and Bare Shallows of an Australian Coastal River

Fish communities found in the marine, brackish, and freshwater shallows of the Clarence River (New South Wales, Australia) were sampled over a 12-mo period to compare communities inhabiting bare and vegetated substrata. A total of 26,107 fish representing 57 species and 36 families was collected biomonthly suing a 6-mm mesh siene. Permanent residents (i.e., species found throughout the year) were primarily gobies (Gobiidae) and gudgeons (Eleotridae). Juveniles of many species found as adults in other parts of the river were seasonally abundant in the shallows. Most numerous was a small ambassid, the glassy perchlet (Ambassis jacksoniensis), the majority of which was collected from marine and brackish regions of the river. Firetail gudgeons (Hypseleotris compressus) were caught in highest numbers at the freshwater sites. Cluster analysis suggested that fish communities in widely separated vegetated sites were more similar to each other than to those in adjacent bare sites. Shallow vegetated (Zostera capricorni) habitats in the marine region of the river had greatest diversity and highest abundances of fish, particularly during recruitment periods. At these times, juveniles of many commercially important species were captured, including yellowfin bream (Acanthopagrus australis), sea mullet (Mugil cephalus), flat-tail mullet (Liza agentea), tarwhine (Rhabdosargus sarba), luderick (Girella tricuspidata), silver biddy (Gerres subfasciatus), and sand whiting (Sillago ciliata). The vegetated (Vallisneria gigantea) sites in the brackish region also had significantly more species and individuals during recruitment periods (spring) than bare sites. Although freshwater vegetated sites consistently had more individuals than freshwater bare sites, there were no significant differences in species richness between vegetated and bare habitats. The need to conservatively manage shallow-water fish habitats is stressed. *** DIRECT SUPPORT *** A01BY073 00004

LOW MOLECULAR WEIGHT CARBOHYDRATE PATTERNS IN THE BANGIOPHYCEAE (RHODOPHYTA)

The qualitative and quantitative occurrence of low molecular weight carbohydrates (LMWCs) in the Bangiophyceae is surveyed. Members of the orders Erythropeltidales (Sahlingia and Erythrotrichia), Compsopogonales (Compsopogon), and Bangiales (Bangia and Porphyra) all contain floridoside and, in some cases, D‐ and L‐isofloridoside, making the group chemotaxonomically the most homogeneous; L‐isofloridoside occurred only in the Bangiales. The Porphyridiales showed great variation in carbohydrate types: Chroodactylon contained only sorbitol, Porphyridium contained only floridoside, Dixoniella and Rhodella species exhibited only mannitol, and Rhodosorus showed digeneaside and sorbitol, whereas Stylonema contained floridoside, D‐isofloridoside, digeneaside, and sorbitol. The extensive variation in LMWC composition within and between the different orders suggests that the enzyme systems for each might have developed repeatedly in different Bangiophyceae and Florideophyceae. Nonetheless, a phylogenetic tree based on small‐subunit ribosomal RNA supports the LMWC patterns in the Bangiophyceae.

A rapid method to score plastid haplotypes in red seaweeds and its use in determining parental inheritance of plastids in the red alga Bostrychia (Ceramiales)

A method for the rapid identification of plastid haplotypes in red algae is presented. This method is based on single-stranded conformation polymorphism (SSCP) to detect variation in DNA sequence of PCR-amplified plastid-encoded ribulose bisphosphate carboxylase/oxygenase large and small subunit intergenic spacer. The method detects variation within a population of the mangrove red alga Caloglossa leprieurii from New South Wales, Australia. Three haplotypes (plastid variants) were easily scored in our samples, and a spatial variation of haplotypes within the intertidal is suggested. This method also reveals maternal inheritance of plastids in crosses between isolates of Bostrychia radicans and in multiple crosses between isolates of B. moritziana. Fluorescence microscopy shows that plastids are contained within the spermatia of these species and that bi- or paternal inheritance of plastids is possible. SSCP is a simple and rapid method for the detection of plastid variation (haplotypes) within populations of red algae that should also be applicable to brown seaweeds.

Inter- and intraspecific genetic variation in Caulerpa (Chlorophyta) based on nuclear rDNA ITS sequences

Caulerpa (Chlorophyta, Ulvophyceae) is a common marine tropical-subtropical genus of about 70 species, inhabiting the eulittoral zone on rocks and corals as well as mangroves. The genus is particularly diverse and abundant along southern Australian coasts, where it is thought to have originated. Here, we compare sequences from the nuclear ribosomal cistron among five species of Caulerpa, including nine populations of Caulerpa filiformis from two biogeographic regions : five from Australia and four from South Africa. Species relationships were well resolved by internal transcribed spacer (ITS) sequences and supported by high bootstrap values as follows: (C. geminata (C. simpliciuscula (C. trifaria (C. scalpelliformis (C. filiformis – Australia, C. filiformis – South Africa ))))). Nucleotide divergence within C. filiformis was low with four and five nucleotide differences present in ITS1 and ITS2 respectively. Unexpectedly, the 5.8S rRNA gene showed eight nucleotide differences between the Australian and So...

EVOLUTIONARY DIVERGENCE IN THE RED ALGAE CALOGLOSSA LEPRIEURII AND C. APOMEIOTICA

Morphological comparisons, hybridization experiments, and molecular phylogenetic analyses using the RUBISCO spacer region were undertaken on 12 populations of Caloglossa leprieurii (Montagne) J. Agardh in order to clarify their relationships. In addition, data from one population of the morphologically similar but asexual species, C. apomeiotica (West et Zuccarello), were included in the assessment. Three morphological types were recognized on the basis of the number of cell rows at nodes of the main axis opposite to the lateral branch and blade width: single/ broad (with three mating groups), multiple/broad (three mating groups) and multiple/slender (one mating group). In the molecular analyses, C. leprieurii was resolved as two clusters that correspond phenetically to the single and multiple cell row types. Both the morphological and molecular data indicate that the asexual species was derived from sexual plants of the multiple cell row type. The reproductive compatibility correlates with genetic distance rather than geographical distance. Sympatric mating groups are completely incompatible and have 10–21 nucleotide changes in the examined region, whereas mating groups that produce abnormal progeny or pseudocystocarps are allopatrically distributed with 5–7 nucleotide changes. The present data suggest that the two populations, one with single and the other with multiple cell rows, which are sympatrically distributed in southeastern Japan, have probably evolved by allopatric speciation. The single/broad type that is restricted to the western Pacific, may have diverged genetically between eastern and western Australia, with subsequent dispersal from the western population as far as Japan.

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheKm values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD+. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aKm value of 1.2 mM and a high requirement of Mg2+ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedKm values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD+. For the non-specific HK, only theKm values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.

Mannitol in the red algal genus Caloglossa (Harvey) J. Agardh

Summary Analytical data on the occurrence of the polyhydric alcohol mannitol in the red algal genus Caloglossa (Ceramiales, Delesseriaceae) were obtained by 13 C NMR and HPLC techniques. Six species were isolated in different geographic regions and kept in laboratory cultures. In all isolates intracellular mannitol concentrations ranging from 25.4 to 579.3 mmol kg -1 DW were measured. In field material of Caloglossa leprieurii from New South Wales, Australia the mannitol content appears to be influenced by salinity of the natural habitat: plants from freshwater (0 % 0 ) contained 3.2 mmol kg -1 DW, plants from estuarine (26–35 % 0 ) and marine (35 % 0 ) habitats exhibited 40 and 202.6 mmol kg -1 DW respectively. Cultured material of Caloglossa apomeiotica was subjected to osmotic stresses ranging from 5.3–70 % 0 . The mannitol content linearly increased with increasing salinities. The possible function of mannitol as a compatible solute in Caloglossa is discussed. The occurrence of these high mannitol concentrations and the absence of typical red algal carbohydrates are discussed with reference to the chemotaxonomy of the genus.

Physiological responses of Caloglossa leprieurii (Ceramiales, Rhodophyta) to salinity stress

Marine, estuarine and freshwater isolates of Caloglossa leprieurii (Montagne) J. Agardh exhibit a high salinity tolerance, reflected by broad cell viability and growth rate. Osmotic adjustment is shown to rely to a large extent on ion‐transport systems, with K+ and Cl‐ accumulated in osmotically‐ significant quantities and active Na+ extrusion. The ion concentrations contribute a large proportion (67–94%) to internal osmotic pressure. The concentration of the organic osmolyte mannitol in all populations was strongly salinity dependent. Mannitol made a lower contribution to the internal osmotic pressure, when compared to ion concentrations, but nonetheless represented an important proportion of the internal osmolality. The response of the three isolates is discussed in relation to the salinity of their respective environments.

Ion compartmentation in the red alga Caloglossa leprieurii in response to salinity changes: freeze‐substitution and X‐ray microanalysis

The elemental content of cell compartments in the euryhaline red macroalga Caloglossa leprieurii (Montague) J. Agardh subjected to different salinities, was determined by X-ray microanalysis of freeze-substituted thin sections. Duty are given for three compartments; cell wall, vacuole and cytoplasm. The most abundant elements detected were potassium, sodium and chlorine, with a Sulphur peak in the cell wall indicating the presence of sulphated polysaccharides. With salinity upshock the cytoplasm is maintained low in K, Na and Cl, whereas in the vacuoles they increased. High levels of K and Na located in the cell wall reflect cation binding to negatively charged polysaccharides. Hyposaline shock resulted in decreased K, Na and Cl levels in all compartments, though K was retained in the cytoplasm. Results are discussed with regard to the role of ion compartmentation in the salt tolerance of this alga, and the cation exchange properties of the cell wall are emphasized.