The seagrass genus Halophila Thouars has more than twenty described species and is predominately distributed over a wide geographic range along the tropical and the warm temperate coastlines in the Indo-West Pacific Oceans. A brief history of the Halophila taxonomic development is presented. Based on reproductive and vegetative morphology, the genus is divided into eight sections including three new sections: section Australes, section Stipulaceae and section Decipientes. A rewritten taxonomic description of the type species for the genus Halophila,H. madagascariensis Steudel ex Doty et B.C. Stone, is provided. The lectotype of H. engelmannii Asch. as well as neotypes of H. hawaiiana Doty et B.C. Stone and H. spinulosa (Br.) Asch. are designated. Furthermore, H. ovalis ssp. bullosa, ssp. ramamurthiana and ssp. linearis together with H. balforurii have been recognised as distinct species. Nomenclature, typification, morphological description and botanical illustrations are presented for each taxon. Recent molecular phylogenetic surveys on certain Halophila taxa are also discussed. Field surveys for the deep water Halophila in West Pacific regions are suggested. Morphological studies combined with molecular investigations for the Halophila on the east coast of Africa and the West Indian Ocean are urgently needed and highly recommended.

Halophila balfourii Solereder has long been treated as a synonym of Halophila stipulacea (Forrsk.) Asch., although it was named more than a century ago. Microscopic (optical microscope and scanning electron microscope) studies on all available herbarium materials of these two species have reconfirmed that the unique papillose leaf epidermis is only presented in H. balfourii but not in H. stipulacea. The pattern of seed testa reticulate is significantly different between these two species. Furthermore, H. balfourii is predominately restricted to the Rodriguez and Mauritius Islands while membranous leafed H. stipulacea is widely distributed in the Red Sea, the Indian Ocean and the Mediterranean Sea as well as East Africa coasts. Based on distinctive characteristics of the leaf and seed coat, and its geographic distribution, it is recommended to reinstate H. balfourii as an independent species and not as a synonym of H. stipulacea.

The widespread adoption of new methodologies, especially molecular techniques, has dramatically changed our understanding of how species of seagrasses and macroalgae are classified and distributed. One consequence of this new paradigm is increased uncertainty regarding biogeographic studies based on pre-molecular species records. The question “how does one delineate an individual species?” has changed and differing interpretations may alter how previous assessments are viewed. In some instances, specimens previously regarded as a single species have been shown to represent multiple genetic lineages. An extreme example is that of the red alga Portiera hornemannii, now thought to include 21 cryptic species in the Philippines alone, and possibly up to 96 species in the wider Indo-Pacific. Reworking and reclassification of species based on DNA analyses have sunk many species into one, or combined or reorganized genera. These changes in taxonomic concepts have implications for conservation and biogeographical assessments, but our understanding of many groups is still in its infancy and requires further work. In order to address these mounting challenges, significant investment and a commitment to taxonomic research will be required in the coming decades.

Seagrasses are unique angiosperms that carry out growth and reproduction submerged in seawater. They occur in at least three families of the Alismatales. All have chloroplasts mainly in the cells of the epidermis. Living in seawater, the supply of inorganic carbon (C-i) to the chloroplasts is diffusion limited, especially under unstirred conditions. Therefore, the supply of CO2 and bicarbonate across the diffusive boundary layer on the outer side of the epidermis is often a limiting factor. Here we discuss the evidence for mechanisms that enhance the uptake of C-i into the epidermal cells. Since bicarbonate is plentiful in seawater, a bicarbonate pump might be expected; however, the evidence for such a pump is not strongly supported. There is evidence for a carbonic anhydrase outside the outer plasmalemma. This, together with evidence for an outward proton pump, suggests the possibility that local acidification leads to enhanced concentrations of CO2 adjacent to the outer tangential epidermal walls, which enhances the uptake of CO2, and this could be followed by a carbon-concentrating mechanism (CCM) in the cytoplasm and/or chloroplasts. The lines of evidence for such an epidermal CCM are discussed, including evidence for special 'transfer cells' in some but not all seagrass leaves in the tangential inner walls of the epidermal cells. It is concluded that seagrasses have a CCM but that the case for concentration of CO2 at the site of Rubisco carboxylation is not proven.

Premise of the study: Few studies have investigated the effects of substrates on the accumulation and precipitation of magnesium, calcium, and sulfur in plants. Acacia stipuligera and A. robeorum growing in their natural habitats with different substrates show different accumulation and precipitation patterns of these elements. Here, we compared the accumulation and precipitation of magnesium, calcium, and sulfur in A. stipuligera and A. robeorum grown in different substrates proposed for mine-site rehabilitation and expected the differences in substrates to have significant effects on the accumulation and precipitation of these elements in the two species.Methods: Saplings were grown in sandy topsoil or in a topsoil-siltstone mixture in a glasshouse. Phyllode magnesium, calcium, and sulfur concentrations of 25-wk-old plants were measured. Precipitation of these elements in phyllodes and branchlets was investigated by means of scanning electron microscopy and energy-dispersive x-ray spectroscopy.Key results: Phyllode magnesium, calcium, and sulfur concentrations were generally significantly greater in A. robeorum than in A. stipuligera. The two species responded in unique ways to the substrate, with A. stipuligera having similar phyllode magnesium and calcium concentrations in both substrates, but greater sulfur concentration in the topsoil-siltstone mixture, while A. robeorum showed lower phyllode magnesium, calcium, and sulfur concentrations in the topsoil-siltstone mixture. For both substrates, mineral precipitates were observed in both species, with A. robeorum having more mineral precipitates containing magnesium, calcium, and sulfur in its phyllodes than A. stipuligera did.Conclusions: The accumulation and precipitation patterns of magnesium, calcium, and sulfur are more species-specific than substrate-affected.

SEAGRASSES are a group of some 72 species of marine flowering plants found in the world’s shallow coastal oceans (Green and Short 2003, Short et al. 2011). There is now scientific consensus that they create an important marine habitat not only by themselves, but also as a component of more complex ecosystems within marine coastal zones. Seagrasses contribute to the health of coral reefs and mangroves, salt marshes and oyster reefs (Dorenbosch et al. 2004; Duke et al. 2007; Heck et al. 2008; Unsworth et al. 2008). Seagrasses have high primary productivity and are a basis of many marine food webs through direct herbivory and the through a detrital cycle (Hemminga and Duarte, 2000). They have enormous value in providing nutrients (N and P) and organic carbon to other parts of the oceans, including the deep sea, and they contribute significantly to carbon sequestration (Suchanek et al. 1985; Duarte et al. 2005). Armed with this knowledge today it is interesting to remember that it is only just over a hundred years since scientists first began speculating on the roles and values of seagrass in the marine environment, with the first focus occurring in Europe on eelgrass (Zostera marina). Many at the time discounted seagrass as an important primary producer (den Hartog 1980). It was not until after the 1930s, when vast areas of Zostera marina were lost in the northern hemisphere from a wasting disease that scientists and governments started to understand and investigate the value of seagrass to coastal ecosystems (Milne and Milne 1951). The loss of Zostera marina led to obvious declines in migratory waterfowl, crustaceans, finfish and shellfish populations (Thayer et al. 1984) In response to those concerns about ongoing losses of Zostera marina and other seagrass species, a meeting of scientists in Fairbanks, Alaska in early 1973 decided to coordinate seagrass research globally. This led to the first International Seagrass Workshop being organized and held in Leiden, The Netherlands, later that year. The report of that conference (McRoy and Helfferich 1977) makes interesting reading, looking back from the perspective of the 21st century. There is only one contribution from the southern hemisphere (Larkum 1977) with Australia referred to as a “little known region”. What we now know as the centre of seagrass biodiversity, Insular Southeast Asia and the broader Indo Pacific region, receives no mention at all. A significant and long-lasting outcome of the Leiden meeting was the birth of the journal “Aquatic Botany”.

Biomineralization is widespread in the plant kingdom. The most common types of biominerals in plants are calcium oxalate crystals, calcium carbonate, and silica. Functions of biominerals may depend on their shape, size, abundance, placement, and chemical composition. In this review we highlight advances in understanding physiological and ecological significance of biomineralization in plants. We focus on the functions of biomineralization in regulating cytoplasmic free calcium levels, detoxifying aluminum and heavy metals, light gathering and scattering to optimize photosynthesis, aiding in pollen release, germination, and tube growth, the roles it plays in herbivore deterrence, biogeochemical cycling of carbon, calcium, and silicon, and sequestering atmospheric CO2.

Proteaceae species in south-western Australia occur on phosphorus- (P) impoverished soils. Their leaves contain very low P levels, but have relatively high rates of photosynthesis. We measured ribosomal RNA (rRNA) abundance, soluble protein, activities of several enzymes and glucose 6-phosphate (Glc6P) levels in expanding and mature leaves of six Proteaceae species in their natural habitat. The results were compared with those for Arabidopsis thaliana. Compared with A.thaliana, immature leaves of Proteaceae species contained very low levels of rRNA, especially plastidic rRNA. Proteaceae species showed slow development of the photosynthetic apparatus (delayed greening'), with young leaves having very low levels of chlorophyll and Calvin-Benson cycle enzymes. In mature leaves, soluble protein and Calvin-Benson cycle enzyme activities were low, but Glc6P levels were similar to those in A.thaliana. We propose that low ribosome abundance contributes to the high P efficiency of these Proteaceae species in three ways: (1) less P is invested in ribosomes; (2) the rate of growth and, hence, demand for P is low; and (3) the especially low plastidic ribosome abundance in young leaves delays formation of the photosynthetic machinery, spreading investment of P in rRNA. Although Calvin-Benson cycle enzyme activities are low, Glc6P levels are maintained, allowing their effective use.Proteaceae species in south-western Australia occur on phosphorus- impoverished soils. Their leaves contain very low phosphorus levels, but have relatively high rates of photosynthesis and activities of enzymes involved in primary metabolism, suggesting they do not compromise their metabolic machinery in order to save P. In contrast, we show that low ribosome abundance contributes to the high phosphorus-use efficiency of these Proteaceae species in three ways: less ribosomes means less P investment; the rate of growth and, hence, demand for phosphorus is decreased; young leaves show a delay in the formation of the photosynthetic machinery, exhibiting very low plastidic ribosome abundance and spreading investment of phosphorus in ribosomes over a longer time. Commentary:

This chapter briefly describes conventional and microwave-assisted chemical fixation methods, as well as cryo-specimen preparation techniques for studying the cellular and organelle ultrastructure of plant tissues under transmission electron microscopy. The general methods and procedures for the plant specimen preparation (including fixation, dehydration, resin infiltration, and embedding) are similar to those for animal tissues. However, certain special characteristic features of plant tissues such as thick cellulosic cell wall, waxy substance in the cuticle, large amount of gases in the intercellular spaces, and the presence of vacuoles have created fixation and resin filtration difficulties. Specific modifications of the protocols used for animal tissues are therefore required, such as the application of vacuum during the initial fixation and resin infiltration stage to remove gases from the tissues and resin. Microwave-assisted procedure can reduce specimen preparation time, but both conventional and microwave-assisted chemical fixation procedures produce artifacts. Cryo-specimen preparation involves with high-pressure freezing and freeze-substitution can minimize artifact formation, but their application to highly vacuolated, thick-walled plant cells is limited.

Seagrasses are among the most productive habitats in the marine realm, performing several crucial physical and biological ecosystem services. One group of seagrasses is the family Zosteraceae, which includes three to four genera and >20 species inhabiting temperate waters of both the northern and southern hemisphere. Species delineation depends on the type of data used, ranging from morphological to molecular. The main goal of this study was to better understand the evolution and divergence within the family, using a broad taxon sampling (>90 individuals) representing all species across the entire biogeographical range in both hemispheres and a four-locus approach (ITS1, matK, rbcL, psbA-trnH). The concatenated four-locus analysis supported earlier studies showing four genera in the family: Phyllospadix, Zostera, Nanozostera and Heterozostera. Four species were resolved within the genus Zostera, four within Nanozostera and two within Heterozostera. No distinction was revealed between H. nigracaulis (Australia) and H. chiliensis (Chile), suggesting a very recent introduction to Chile. A time-calibrated phylogeny using the rbcL gene revealed an early divergence of Zostera-Nanozostera/Heterozostera at 14.4 Ma, followed by a late Miocene radiation of Nanozostera-Heterozostera at 6.4 Ma, and the H. polychalymas-H. nigracaulis/tasmanica/chiliensis split at 2.3 Ma. Zostera asiatica diverged from other species of Zostera at 4.6 Ma. Phylogenetic analyses indicated that matK was the most informative single locus, whereas psbA-trnH (a widely used barcoding locus) was unable to resolve any entities within the Zosteraceae. A commonly used barcoding combination for plants, rbcL/matK, distinguished all genera, but was unable to resolve several species.

Proteaceae species in south-western Australia occur on severely phosphorus (P)-impoverished soils. They have very low leaf P concentrations, but relatively fast rates of photosynthesis, thus exhibiting extremely high photosynthetic phosphorus-use-efficiency (PPUE). Although the mechanisms underpinning their high PPUE remain unknown, one possibility is that these species may be able to replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. For six Proteaceae species, we measured soil and leaf P concentrations and rates of photosynthesis of both young expanding and mature leaves. We also assessed the investment in galactolipids, sulfolipids and phospholipids in young and mature leaves, and compared these results with those on Arabidopsis thaliana, grown under both P-sufficient and P-deficient conditions. In all Proteaceae species, phospholipid levels strongly decreased during leaf development, whereas those of galactolipids and sulfolipids strongly increased. Photosynthetic rates increased from young to mature leaves. This shows that these species extensively replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. A considerably less pronounced shift was observed in A. thaliana. Our results clearly show that a low investment in phospholipids, relative to nonphospholipids, offers a partial explanation for a high photosynthetic rate per unit leaf P in Proteaceae adapted to P-impoverished soils.

Background and aims Strawberry (Fragaria x ananassa) is a high-value crop worldwide. Fusarium oxysporum f. sp. fragariae causes rapid wilting and death of strawberry plants and severe economic losses worldwide. To date, no studies have been conducted to determine colonisation of either susceptible or resistant strawberry plants by F. oxysporum f. sp. fragariae, or whether plant colonisation by F. oxysporum f. sp. fragariae differs between susceptible and resistant cultivars.Methods Colonisation of strawberry plants by a pathogenic isolate of F. oxysporum f. sp. fragariae was examined both on the root surface and within root tissue of one resistant cv. Festival and one susceptible cv. Camarosa using light and scanning electron microscopy from 4 h to 7 d post inoculation (pi).Results Resistant cv. Festival significantly impeded the spore germination and penetration from 4 to 12 hpi and subsequent growth and colonisation by this pathogen until 7 dpi compared with susceptible cv. Camarosa. At 7 dpi, fungal colonisation in resistant cv. Festival remained mainly confined to the epidermal layer of the root, while in susceptible cv. Camarosa, hyphae not only had heavily colonised the cortical tissue throughout but had also colonised vascular tissues.Conclusions This study demonstrates for the first time that resistance of a strawberry cultivar to F. oxysporum f. sp. fragariae is a result of impedance of pathogen growth and colonisation both on the plant surface and within host tissues. Resistance mechanisms identified in this study will be of high value for breeding programmes in developing new disease-resistant cultivars to manage this serious strawberry disorder.

BACKGROUND AND AIMS:Formation of calcium oxalate crystals is common in the plant kingdom, but biogenic formation of calcium sulfate crystals in plants is rare. We investigated the morphologies and elemental compositions of crystals found in phyllodes and branchlets of Acacia robeorum, a desert shrub of north-western Australia.METHODS:Morphologies of crystals in phyllodes and branchlets of A. robeorum were studied using scanning electron microscopy (SEM), and elemental compositions of the crystals were identified by energy-dispersive X-ray spectroscopy. Distributional patterns of the crystals were studied using optical microscopy together with SEM.KEY RESULTS:According to the elemental compositions, the crystals were classified into three groups: (1) calcium oxalate; (2) calcium sulfate, which is a possible mixture of calcium sulfate and calcium oxalate with calcium sulfate being the major component; and (3) calcium sulfate · magnesium oxalate, presumably mixtures of calcium sulfate, calcium oxalate, magnesium oxalate and silica. The crystals were of various morphologies, including prisms, raphides, styloids, druses, crystal sand, spheres and clusters. Both calcium oxalate and calcium sulfate crystals were observed in almost all tissues, including mesophyll, parenchyma, sclerenchyma (fibre cells), pith, pith ray and cortex; calcium sulfate · magnesium oxalate crystals were only found in mesophyll and parenchyma cells in phyllodes.CONCLUSIONS:The formation of most crystals was biologically induced, as confirmed by studying the crystals formed in the phyllodes from seedlings grown in a glasshouse. The crystals may have functions in removing excess calcium, magnesium and sulfur, protecting the plants against herbivory, and detoxifying aluminium and heavy metals.

Precipitation of calcium in plants is common. There are abundant studies on the uptake and content of magnesium, strontium and barium, which have similar chemical properties to calcium, in comparison with those of calcium in plants, but studies on co-precipitation of these elements with calcium in plants are rare. In this study, we compared morphologies, distributional patterns, and elemental compositions of crystals in tissues of four Acacia species grown in the field as well as in the glasshouse. A comparison was also made of field-grown plants and glasshouse-grown plants, and of phyllodes of different ages for each species. Crystals of various morphologies and distributional patterns were observed in the four Acacia species studied. Magnesium, strontium and barium were precipitated together with calcium, mainly in phyllodes of the four Acacia species, and sometimes in branchlets and primary roots. These elements were most likely precipitated in forms of oxalate and sulfate in various tissues, including epidermis, mesophyll, parenchyma, sclerenchyma (fibre cells), pith, pith ray and cortex. In most cases, precipitation of calcium, magnesium, strontium and barium was biologically induced, and elements precipitated differed between soil types, plant species, and tissues within an individual plant; the precipitation was also related to tissue age. Formation of crystals containing these elements might play a role in regulating and detoxifying these elements in plants, and protecting the plants against herbivory.

Sulfides in sediments and hydrogen sulfide (H2S) intrusion in plant tissues were investigated for six species of seagrass in Shark Bay, Western Australia, at two sites with elevated salinities of 42 and 45 psu. H2S intrusion ranged from <20% to 100% in roots and rhizomes, indicating a high degree of sulfide intrusion in some cases, although this did not vary consistently between larger, long-lived species and smaller, less persistent species. There were significant differences in accumulation of total sulfur (TS) among species. Anatomy of rhizomes and roots showed species-specific differences in aerenchyma, the air channels that allow oxygen to diffuse down to the roots and sediments, and tissues with thickened cell walls that could present a barrier to diffusion of H2S, suggesting that morphology may influence sulfide intrusion and sulfur accumulation. Sulfide concentrations in seagrass sediments were far lower in Shark Bay than in Florida Bay, a subtropical embayment where sulfide toxicity has been implicated in seagrass dieback. Despite significant H2S intrusion into tissues of some Shark Bay seagrasses, there was no evidence of any deleterious effects in the current conditions.

Endophytic contamination reduces efficiency in micropropagation and causes plant-culture losses. Micropropagation of black kangaroo paw (Macropidia fuliginosa (Hook.) Druce) contributes both to the needs of the Western Australia’s nursery industry and towards conservation of this unique Australian wildflower, because the seeds are difficult to germinate. Plant preservative mixture (PPM), a proprietary mixture of two broad-spectrum isothiazolone biocides, has recently been used as a prophylactic anti-bacterial agent in plant-tissue culture. Its efficacy for eradicating endogenous bacterial contaminants in M. fuliginosa was demonstrated. Plantlets of M. fuliginosa were artificially infected with Sinorhizobium meliloti, a non-sporing bacterium isolated from rhizome tissues of red kangaroo paw (Anigozanthos rufus). Histological studies using light and electron microscopy revealed the presence of bacterial cells in intercellular spaces within the leaf mesophyll and in the lumen of xylem vessels after infection. Bacterial cells were also found in intercellular spaces of callus, the latter induced from the stem base of infected shoots with 0.05 mg L–1 thidiazuron. The eradication protocol involved infiltration of infected axillary buds and basal-stem calli with 5 mL L–1 PPM under vacuum. Quantitation by high-performance liquid chromatography (HPLC) revealed that callus tissue absorbed significantly more PPM than did axillary buds. Indexation of plantlets raised from PPM-treated tissues indicated successful eradication of the endophyte from basal-stem calli, and from shoots regenerated from them.