Environmental Optima for Seven Strains of Pseudochattonella (Dictyochophyceae,Heterokonta)

The ichthyotoxic flagellate Pseudochattonella has formed recurrent blooms in the North Sea, Skagerrak and Kattegat since 1998. Five strains of Pseudochattonella farcimen and two strains of P. verruculosa were examined in an assay comparing the light response of specific growth rates over a range of temperatures and salinities to get further knowledge on the autecology of members of this genus. Temperature optima were lower in P. farcimen (9°C–15°C) than in P. verruculosa (12°C–20°C). P. farcimen also showed a somewhat lower salinity optimum (18–26) than P. verruculosa (20–32). All strains showed light‐dependent growth responses reaching saturation between 18 and 52 μmol · photons · m^?2 · s^?1 at optimal temperature and salinity conditions. Compensation point estimates ranged from 4.2 to 15 μmol · photons · m^?2 · s^?1. Loss rates increased with temperature and were lowest at salinities close to optimal growth conditions. Blooms of P. farcimen have been recorded in nature under conditions more similar to those minimizing loss rates rather than those maximizing growth rates in our culture study.     

A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

The ichthyotoxic genus Pseudochattonella forms recurrent extensive blooms in coastal waters in Japan, New Zealand and Northern Europe. It comprises of two morphologically similar species, P. verruculosa and P. farcimen, which complicates visual species identification and enumeration of live and fixed material. Primers designed previously could not quantitatively distinguish species in mixed assemblages. To address this issue we developed two primer sets: one revealed itself to be genus specific for Pseudochattonella and the other species‐specific for P. verruculosa. By subtracting cell estimates for P. verruculosa from combined results we could calculate cell numbers for P. farcimen. This approach has overcome the challenges posed by the very limited sequence availability and low gene variability between the two species. The qPCR assay was extensively tested for specificity, efficiency and sensitivity over an entire growth cycle in both single and mixed assemblages. Comparison of cell abundance estimates obtained by qPCR assay and microscopy showed no statistically significant difference until stationary and death phases. The assay was also tested on environmental samples collected during a small Pseudochattonella bloom in Denmark in March–April 2015. It was impossible to distinguish P. farcimen and P. verruculosa by light microscopy but qPCR showed both species were present. The two methods provided nearly identical cell numbers but the assay provided discrimination and enumeration of both species.     

Proposal of Pseudochattonella verruculosa gen. nov., comb. nov. (Dictyochophyceae) for a formar raphidophycean alga Chattonella verruculosa, based on 18S rDNA phylogeny and ultrastructural characteristics

Chattonella verruculosa Y. Hara et Chihara was re‐examined by molecular methods and microscopic examination. The 18S rDNA phylogenetic analysis clearly indicated that C. verruculosa is a member of the Dictyochophyceae, with a specific affinity to Florenciella parvula. The morphological features in C. verruculosa– namely the proximal helix with two gyres and many scattered DNA‐containing areas in the chloroplasts – display the evolutionary link to the Dictyochophyceae, instead of the Raphidophyceae. Similarly, unique pyrenoid morphologies are shared between C. verruculosa and the dictyochophycean algae. Combining the molecular data and morphological characteristics, C. verruculosa is transferred to Pseudochattonella gen. nov. of the class Dictyochophyceae as Pseudochattonella verruculosa (Y. Hara et Chihara) Hosoi‐Tanabe, Honda, Fukaya, Inagaki et Sako comb. nov.     

Verrucophora farcimen gen. et sp. nov. (Dictyochophyceae,Heterokonta)—a bloom‐forming ichthyotoxic flagellate from the Skagerrak,Norway1

Since 1998, a heterokont flagellate initially named Chattonella aff. verruculosa has formed recurrent extensive blooms in the North Sea and the Skagerrak, causing fish mortalities. Cells were isolated from the 2001 bloom off the south coast of Norway, and monoalgal cultures were established and compared with the Chattonella verruculosa Y. Hara et Chihara reference strain NIES 670 from Japan. The cells in Norwegian cultured isolates were very variable in size and form, being large oblong (up to 34 μm long) to small rounded (5–9 μm in diameter) with two unequal flagella, numerous chloroplasts, and mucocysts. The SSU and partial LSU rDNA sequences of strains from Norway and Japan were compared and differed by 0.4% (SSU) and 1.3% (LSU), respectively. Five strains from Norway were identical in the LSU rDNA region. Phylogenetic analyses based on heterokont SSU and concatenated SSU + LSU rDNA sequences placed C. aff. verruculosa and the Japanese C. verruculosa within the clade of Dictyochophyceae, with the picoflagellate Florenciella parvula Eikrem as the closest relative. Ultrastructure, morphology, and pigment composition supported this affinity. We propose the name Verrucophora farcimen sp. et gen. nov. for this flagellate and systematically place it within the class Dictyochophyceae. Our studies also show that C. verruculosa from Japan is genetically and morphologically different but closely related to V. farcimen. The species is transferred from the class Raphidophyceae to the class Dictyochophyceae and renamed Verrucophora verruculosa. We propose a new order, Florenciellales, to accommodate V. farcimen, V. verruculosa, and F. parvula.     

A high-throughput method for measuring growth and loss rates in microalgal cultures

A miniaturized and low-cost assay for algal growth and loss rates, and estimation of compensation light was developed and optimized. Microalgal cultures were grown in white 96-well microplates to estimate specific growth rates at six temperatures, five salinities and eight light levels. Data from black 24-well microplates at six temperatures, five salinities and five light conditions were used in addition to estimate loss rates and compensation light. Absorption and reflection of light were different in the white and black microplates. Growth rates were estimated from daily in vivo fluorescence (IVF) measurements using a microplate reader fitted with a fluorometer. To validate the microplate algal growth assay, IVF was compared with cell counting by flow cytometry. Maximal growth rate for the test alga Pseudochattonella farcimen (Heterokonta) was estimated to 0.52?±?0.05 day^?1 at optimal temperatures ranging from 9 to 14°C and salinities 18–26 psu. Lowest value of compensation light as photosynthetic photon flux density (PPFD) was 4.2?±?1.2 μmol photons m^?2 s^?1, and lowest saturation light, 34.1?±?3.7 μmol photons m^?2 s^?1, was observed in the temperature range 5–11°C and salinity range 23–28 psu. Minimum loss rate was obtained at temperatures 5–8°C and salinities 26–31 psu. Blooms of P. farcimen have been recorded in nature under conditions similar to those minimizing loss rates rather than maximizing growth rates in this study. The microalgal assay described here allows for a large number of conditions to be tested, and accurate optimal conditions for growth and loss rates to be obtained.     

Variation in scope for growth: a test of food limitation among intertidal mussels

Wellington Harbour supports large populations of the mussels Aulacomya maoriana, Mytilus galloprovincialis and Perna canaliculus that are almost entirely absent from nearby coastal locations in Cook Strait. We calculated scope for growth (SFG) using ambient Cook Strait water over a broad temporal scale and a broad range of seston conditions to determine if negative SFG explains this phenomenon. Although all three mussel species had positive mean SFG values, variation in SFG was high and negative values often occurred: A. maoriana 19.1 J g⁻¹ h⁻¹, 43% of mussels showed negative SFG; M. galloprovincialis 1.26, 52% negative SFG; P. canaliculus 45.6, 27% negative SFG. Negative SFG was most often due to negative absorption efficiency caused by metabolic faecal loss that is characteristic of mussels feeding in environments with low seston quality. From our ecophysiology data we constructed a model to estimate SFG based on physiological responses to the narrow range of seston conditions typical of Cook Strait (Model One), and a model to estimate SFG based on physiological responses of mussels to the broad range of seston conditions typical of Wellington Harbour and Cook Strait (Model Two). We used seston data collected over an 18-month period from sites in Wellington Harbour and Cook Strait to derive 159 estimates of species-specific mussel SFG from both models. Both models produced higher estimates of SFG for mussels in the Harbour compared with those at Cook Strait sites. This was consistent with elevated particulate concentrations in the Harbour than at Cook Strait sites, and in agreement with previous studies. For Cook Strait mussels, both models produced negative estimates of net energy balance for long periods of time (several months), whereas for Harbour mussels negative SFG estimates were generally short in duration. We conclude that our short-term laboratory-based determinations of SFG and our long-term bioenergetics modelling estimates do not conclusively support the hypothesis of food limitation for three coexisting taxa of mussels in the intertidal region of Cook Strait, New Zealand. Handling editor: P. Viaroli     

Ancylostoma ceylanicum,a re-emerging but neglected parasitic zoonosis

Although Ancylostoma ceylanicum is known to be an endemic and widely distributed hookworm of dogs and cats in Asia, its contribution to human morbidity as a potentially zoonotic hookworm remains largely unexplored. Since its discovery by Lane (1913) as a ‘new parasite’ of humans a century ago, the hookworm has been regarded as a ‘rare’ and ‘abnormal’ parasite and largely overlooked in surveys of human parasites. Recent molecular-based surveys in Asia, however, have demonstrated that A. ceylanicum is the second most common hookworm species infecting humans, comprising between 6% and 23% of total patent hookworm infections. In experimentally induced infections, A. ceylanicum mimics the clinical picture produced by the anthroponotic hookworms of ‘ground itch’ and moderate to severe abdominal pain in the acute phase. Natural infections with A. ceylanicum in humans have been reported in almost all geographical areas in which the hookworm is known to be endemic in dogs and cats, however for the majority of reports, no clinical data are available. Much like the anthroponotic hookworm species, patent A. ceylanicum adults can isolate within the jejunum to produce chronic infections that on occasion, may occur in high enough burdens to produce anaemia. In addition, the hookworm can act much like Ancylostoma caninum and be found lower in the gastrointestinal tract leading to abdominal distension and pain, diarrhoea and occult blood in the faeces accompanied by peripheral eosinophilia. Whether A. ceylanicum is capable of producing both classical hookworm disease and evoking morbidity through an uncontrolled allergic response in some individuals remains unascertained. Future investigations combining the use of molecular diagnostic tools with clinical and pathological data will shed further light on its role as a human pathogen. The control of this zoonosis necessitates an integrated and inter-sectorial “One Health” approach be adopted in communities where large numbers of dogs share a close relationship with humans.     

Evaluation of the rust fungus Puccinia rapipes for biological control of Lycium ferocissimum (African boxthorn) in Australia: Life cycle,taxonomy and pathogenicity

Fungal plant pathogens are increasingly recognised as being among the most effective and safe agents in classical weed biological control programs worldwide. Suitability of the rust fungus P. rapipes as a classical biological control agent for Lycium ferocissimum (African boxthorn) in Australia was assessed using a streamlined agent selection framework. Studies with P. rapipes were undertaken to elucidate its life cycle, confirm its taxonomic placement and determine its pathogenicity to L. ferocissimum and seven closely-related Solanaceae species that occur in Australia. Field surveys in the native range of South Africa, experiments in a containment facility in Australia and DNA sequencing confirmed that P. rapipes is macrocyclic and autoecious, producing all five spore stages on L. ferocissimum. The stages not previously encountered, spermogonia and aecia, are described. Sequencing also confirmed that P. rapipes is sister to Puccinia afra, in the ‘Old World Lineage’ of Puccinia species on Lycieae. Two purified isolates of the fungus, representing the Eastern and Western Cape distributions of P. rapipes in South Africa, were cultured in the containment facility for use in pathogenicity testing. L. ferocissimum and all of the Lycium species of Eurasian origin tested ‒ Lycium barbarum (goji berry), Lycium chinense (goji berry ‘chinense’) and Lycium ruthenicum (black goji berry) – were susceptible to both isolates of P. rapipes. The Australian native L. australe and three more distantly related species in different genera tested were resistant to both isolates. The isolate from the Western Cape was significantly more pathogenic on L. ferocissimum from Australia, than the Eastern Cape isolate. Our results indicate that P. rapipes may be sufficiently host specific to pursue as a biological control agent in an Australian context, should regulators be willing to accept damage to the Eurasian goji berries being grown, albeit to a limited extent, in Australia.     

Patterns of Occurrence of Sharks in Sydney Harbour,a Large Urbanised Estuary

Information about spatial and temporal variability in the distribution and abundance of shark-populations are required for their conservation, management and to update measures designed to mitigate human-shark interactions. However, because some species of sharks are mobile, migratory and occur in relatively small numbers, estimating their patterns of distribution and abundance can be very difficult. In this study, we used a hierarchical sampling design to examine differences in the composition of species, size- and sex-structures of sharks sampled with bottom-set longlines in three different areas with increasing distance from the entrance of Sydney Harbour, a large urbanised estuary. During two years of sampling, we obtained data for four species of sharks (Port Jackson, Heterodontus portusjacksoni; wobbegong, Orectolobus maculatus; dusky whaler, Carcharhinus obscurus and bull shark, Carcharhinus leucas). Only a few O. maculatus and C. obscurus were caught, all in the area closest to the entrance of the Harbour. O. maculatus were caught in all seasons, except summer, while C. obscurus was only caught in summer. Heterodontus portusjacksoni were the most abundant species, caught in the entrance location mostly between July to November, when water temperature was below 21.5°C. This pattern was consistent across both years. C. leucas, the second most abundant species, were captured in all areas of Sydney Harbour but only in summer and autumn when water temperatures were above 23°C. This study quantified, for this first time, how different species utilise different areas of Sydney Harbour, at different times of the year. This information has implications for the management of human-shark interactions, by enabling creation of education programs to modify human behaviour in times of increased risk of potentially dangerous sharks.     

The genome of ‘Candidatus Phytoplasma solani’ strain SA-1 is highly dynamic and prone to adopting foreign sequences

Bacteria of the genus ‘Candidatus Phytoplasma’ are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as ‘Ca. P. solani’, have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of ‘Ca. P. solani’ strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322 bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The ‘Ca. P. solani’ SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.     

Control of whitefly, Trialeurodes vaporariorum and cotton aphid, Aphis gossypii in glasshouses by two isolates of the fungus, Verticillium lecanii

The abilities of two isoiates of the entomopathogenic fungus, Verticillium lecanii (one isolated from whitefly and one from aphids), to control both aphids and whitefly on glasshouse cucumbers was investigated. Control of homologous hosts was better than that of heterologous hosts. A single spray of a commercial substrate-containing formulation of the ‘whitefly’ isolate controlled established whitefly populations satisfactorily on sprayed foliage and also when the whitefly moved up to younger untreated foliage. In addition, because the substrate permitted growth and sporulation of the fungus on leaf surfaces, whitefly populations introduced after a V. lecanii application were also controlled. Control of whitefly by the ‘aphid’ isolate alone was unsatisfactory although control could be obtained in conjunction with the hymenopterous parasite Encarsia formosa. Control of Aphis gossypii by the ‘aphid’ isolate was obtained by spraying a commercial formulation containing a substrate but not by spraying pure spore suspensions. The ‘whitefly’ isolate did not control aphids satisfactorily. The difference between the two isolates in pathogenicity for aphids and whitefly was supported by laboratory infectivity experiments.     

Non-monophyly and cryptic lineages between two morphoforms of Megalurothrips usitatus (Thysanoptera: Thripidae) in Peninsular Malaysia: Insights from morphological and molecular data

The DNA barcode data revealed two lineages of M. usitatus. Lineage I, consisted of the typical M. usitatus morphoform ‘a’ from the field collected and laboratory reared specimens, and the reference sequences from the GenBank whereas Lineage II consisted of both M. usitatus morphoforms ‘a’ and ‘b’ from the field collected specimens. The interspecific distances between both lineages ranged from 8.78 to 9.63%, suggesting the presence of cryptic and non-monophyly lineages between two morphoforms of M. usitatus in Peninsular Malaysia.     

Association of ‘Candidatus Phytoplasma cynodontis’ with Bermuda grass white leaf disease and its new hosts in Qassim province,Saudi Arabia

Typical symptoms of phytoplasma such as whitening of the leaves, shortening of the stolons on Bermuda grass, variegated leaves, yellows, stunting, little leaves and yellows on Giant reed, Cooba and sand olive shrub were observed in Qassim province, Saudi Arabia, during the autumn season of 2015. When tested for phytoplasma by universal primers P1/P7 followed by R16mF2/R16mR2, products of approximately 1400?bp (as expected) were amplified from 16 plants with symptoms but not from symptomless plants. Based on sequencing, phylogenetic analysis and virtual restriction fragment length polymorphism patterns of the 16S rDNA F2nR2 fragments of seven Qassim phytoplasma isolates, bermuda grass isolates 170, 175 and 177, giant reed isolate 180, sand olive isolates 181 and 182 and cooba isolate 185, the associated phytoplasma was identified as a member of ‘Candidatus Phytoplasma cynodontis’ which belong to the 16SrXIV-A subgroup. The 16S rDNA gene sequences of seven Qassim phytoplasma isolates exhibited over 99.2% identity with members of ‘Ca. Phytoplasma cynodontis’ group of phytoplasmas. This is the first report of characterization of ‘Ca. phytoplasma cynodonties’ (16SrXIV) associated with Cynodon dactylon in Saudi Arabia and its new hosts, Dodonaea angustifolia, Arundo donax and Acacia salicia.     

Ecological fits, mis-fits and lotteries involving insect herbivores on the invasive plant, Bunias orientalis

Exotic plants bring with them traits that evolved elsewhere into their new ranges. These traits may make them unattractive or even toxic to native herbivores, or vice versa. Here, interactions between two species of specialist (Pieris rapae and P. brassicae) and two species of generalist (Spodoptera exigua and Mamestra brassicae) insect herbivores were examined on two native crucifer species in the Netherlands, Brassica nigra and Sinapis arvensis, and an exotic, Bunias orientalis. Bu. orientalis originates in eastern Europe and western Asia but is now an invasive pest in many countries in central Europe. P. rapae, P. brassicae and S. exigua performed very poorly on Bu. orientalis, with close to 100% of larvae failing to pupate, whereas survival was much higher on the native plants. In choice experiments, the pierid butterflies preferred to oviposit on the native plants. Alternatively, M. brassicae developed very poorly on the native plants but thrived on Bu. orientalis. Further assays with a German Bu. orientalis population also showed that several specialist and generalist herbivores performed very poorly on this plant, with the exception of Spodoptera littoralis and M. brassicae. Bu. orientalis produced higher levels of secondary plant compounds (glucosinolates) than B. nigra but not S. arvensis but these do not appear to be important factors for herbivore development. Our results suggest that Bu. orientalis is a potential demographic ‘trap’ for some herbivores, such as pierid butterflies. However, through the effects of an evolutionary ‘lottery’, M. brassicae has found its way through the plant’s chemical ‘minefield’.     

Cyst and radionuclide evidence demonstrate historic Gymnodinium catenatum dinoflagellate populations in Manukau and Hokianga Harbours, New Zealand

Between May 2000 and February 2001, a major bloom of the toxic dinoflagellate Gymnodinium catenatum (a causative organism of Paralytic Shellfish Poisoning, PSP) affected over 1500 km of coastline of New Zealand’s North Island. As this was the first record of this species in New Zealand, we aimed to resolve whether this represented a recent introduction/spreading event or perhaps an indigenous cryptic species stimulated by environmental/climatic change. To answer this question, we analysed for G. catenatum resting cysts in ²¹⁰Pb dated sediment cores (18–34 cm long; sedimentation rates 0.34–0.69 cm per year) collected by SCUBA divers from Manukau Harbour, where the species was first detected, and from Hokianga Harbour, where the highest shellfish toxicity was recorded, while using Wellington Harbour as a well-monitored control site. The results of this study conclusively demonstrate that abundant G. catenatum has been in northern New Zealand at least since the early 1980s, increasing up to 1200 cysts/g around the year 2000, but with low cyst concentrations possibly present since at least 1937. In contrast, Wellington Harbour cores contained only very sparse G. catenatum cysts (8 cysts/g), present only to a depth of 7 cm (surface mixed layer depth), reflecting an apparent recent range expansion of this dinoflagellate in New Zealand, possibly stimulated by unusual climatic conditions associated with the 2000 La Nina event. The significant increases since the early 1980s also of Protoperidinium cysts at Hokianga Harbour and of Gonyaulax, Protoperidinium and Protoceratium cysts at Manukau Harbour suggest a broad scale environmental change has occurred in Northland, New Zealand.     

Genetic analysis of hybrid seed formation ability of Brassica rapa in intergeneric crossings with Raphanus sativus

A hybridization barrier leads to the inability of seed formation after intergeneric crossings between Brassica rapa and Raphanus sativus. Most B. rapa lines cannot set intergeneric hybrid seeds because of embryo breakdown, but a B. rapa line obtained from turnip cultivar ‘Shogoin-kabu’ is able to produce a large number of hybrid seeds as a maternal parent by crossings with R. sativus. In ‘Shogoin-kabu’ crossed with R. sativus, developments of embryos and endosperms were slower than those in intraspecific crossings, but some of them grew to mature seeds without embryo breakdown. Intergeneric hybrid seeds were obtained in a ‘Shogoin-kabu’ line at a rate of 0.13 per pollinated flower, while no hybrid seeds were obtained in a line developed from Chinese cabbage cultivar ‘Chiifu’. F₁ hybrid plants between the lines of ‘Shogoin-kabu’ and ‘Chiifu’ set a larger number of hybrid seeds per flower, 0.68, than both the parental lines. Quantitative trait loci (QTLs) for hybrid seed formation were analyzed after intergeneric crossings using two different F₂ populations derived from the F₁ hybrids, and three QTLs with significant logarithm of odds scores were detected. Among them, two QTLs, i.e., one in linkage group A10 and the other in linkage group A01, were detected in both the F₂ populations. These two QTLs had contrary effects on the number of hybrid seeds. Epistatic interaction between these two QTLs was revealed. Possible candidate genes controlling hybrid seed formation ability in QTL regions were inferred using the published B. rapa genome sequences.     

Phylogeny and taxonomy of the ‘manna lichens’

We present ataxonomic revision of the ‘manna lichens’ based on morphological, chemical, ecological and molecular data. A large number of herbarium specimens and fresh collections were examined. Phylogenetic analyses were performed using nuclear ribosomal (nrITS, nrLSU) and mitochondrial small subunit (mtSSU) sequences. Some notable phenotypic characters were plotted on the phylogenetic tree, and the analysis reveals that some of these characters are useful for genus and species level distinction of certain ‘manna lichens.’ Phylogeny of the Megasporaceae was revised using a combined data set of nrLSU and mtSSU and performing parsimony and Bayesian analyses. Five genera (Aspicilia, Circinaria, Lobothallia, Megaspora and Sagedia) are recognized. Further, the relationships of five presumably closely related genera of ‘manna lichens’, namely Agrestia (vagrant), Aspicilia (crustose) Circinaria (crustose), Chlorangium (vagrant) and Sphaerothallia (vagrant) with different growth forms were analysed. The analyses revealed that ‘manna lichens’ do not form a monophyletic group but occur in different clades within the genus Circinaria. The genera Agrestia, Chlorangium and Sphaerothallia are assigned as new synonyms under the genus Circinaria and no vagrant or erratic species remain in the genus Aspicilia. The analyses also show that five new erratic, vagrant and crustose species can be recognized. In this study two ‘manna lichens’, viz. Circinaria rostamii sp. nov. (Azerbaijan, Iran and Turkey), and Circinaria gyrosa sp. nov. (Armenia, Azerbaijan, Iran, Turkey, Turkmenistan and Spain) are described as new to science. Three potentially new species with crustose and erratic forms need additional study. Aspicilia fruticolosofoliacea is reduced to synonymy under C. alpicola, and a lectotype is designated for C. aspera. Thirteen new combinations in Circinaria are presented. The phenomenon of vagrancy is briefly discussed, and the biogeography of the ‘manna lichens’ is outlined. Illustrations, distribution maps, and an identification key to the species are provided.     

Organogenesis from root explants of commercial populations of <Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims and a wild passionfruit species, <Emphasis Type="Italic">P. cincinnata</Emphasis> Masters

Root explants of a wild passionfruit species (Passiflora cincinnata) and three P. edulis commercial populations (‘FB 100’, ‘FB 200’, and ‘FB 300’) were incubated on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyladenine (BA) to induce shoot organogenesis. Shoots elongated in liquid medium with 2.89 μM gibberellic acid (GA₃) under agitation were rooted in coconut fiber and acclimatized followed by transfer to a greenhouse into pots containing mixture of coconut fiber and Plantmax^® (1:1). Explant samples were collected during organogenesis and submitted to light and scanning electron microscopy (SEM). Root explants of P. cincinnata responded earlier than those of P. edulis. However, on the third assessment, at 90 days, the genotype ‘FB 200’ showed shoot number significantly higher than ‘FB 100’ and ‘FB 300’, not differing from P. cincinnata. Organogenesis in P. cincinnata and P. edulis occurred via direct pathway, which was confirmed by anatomical studies and SEM. Flow cytometric analysis revealed no variation in DNA content of regenerated plantlets among all genotypes. Nuclear DNA (2C) values (pg) in regenerants of P. cincinnata (2.99 pg) and P. edulis (3.26–3.28 pg) were consistent with DNA amounts of seed-derived control plants.