1 1
Molecular phylogenetics and morphology of Gambierdiscus yasumotoi from tropical eastern
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Australia
3 4
Shauna Murray*1,2, Paolo Momigliano*3,4, , Kirsten Heimann3,5, David Blair3
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*These authors contributed equally
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1
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NSW 2007, Australia
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2
Sydney Institute of Marine Sciences, Chowder Bay Rd, Mosman NSW Australia
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3
School of Marine and Tropical Biology, James Cook University, QLD 4810, Australia
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4
Department of Biological Sciences, Macquarie University, North Ryde 2019 NSW,
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Australia
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5
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4810, Australia
Plant Functional Biology and Climate Change Cluster, University of Technology, Sydney
Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook University, QLD
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Corresponding author:
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Paolo Momigliano
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email address:
[email protected]
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phone: +61 2 9850 8331
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2 22
Abstract
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Here the occurrence of the species Gambierdiscus yasumotoi is reported for the first time
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along a latitudinal gradient spanning more than 1550 km of the Australian Great Barrier Reef
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(GBR), a region with endemic ciguatera fish poisoning. Gambierdiscus yasumotoi was found
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at three tropical and sub tropical coral reef sites, Raine Island (northern GBR) , Nelly Bay
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(central GBR) and Heron Island (southern GBR), indicating a wide-ranging distribution in
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tropical and subtropical eastern Australia. Specimens from Australia broadly fitted the
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original description of Gambierdiscus yasumotoi, but differed in some aspects, showing some
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similarities to G. ruetzleri. Molecular phylogenetic analyses based on nuclear rRNA gene
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sequences and morphological analyses showed specimens to be intermediate between the two
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species Gambierdiscus yasumotoi and Gambierdiscus ruetzleri. The full intraspecific
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diversity of these two species appears to be incompletely known, and these two species may
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represent a species complex. Strains of this species from other sites around the world have
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been found to produce an as yet unknown toxin, possibly an analogue of maitotoxin.
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Keywords: Gambierdiscus, Ciguatera, Great Barrier Reef, Phylogenetics
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3 38
1 Introduction
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The genus Gambierdiscus Adachi and Fukuyo (Gonyaulacales, Dinoflagellata, Alveolata) is
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one of more than 550 known genera of dinoflagellates. Species of this genus are the main
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causative agents of Ciguatera Fish Poisoning (CFP), caused by the consumption of fish
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contaminated with toxins produced by these dinoflagellates, which are biomagnified along
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the food web. CFP is the most common non-bacterial seafood poisoning disease in the world
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(Fleming et al., 1998) and mainly occurs in tropical countries. Cases have been reported in
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coral reef areas in Australia (Gillespie et al., 1985; Lewis, 2006), the Pacific and Indian
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oceans as well as the Caribbean (Tosteson, 2004). In Australia, there were more than 1,400
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documented cases between 1965 and 2010, including two fatalities (Gillespie et al., 1985;
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Hamilton et al., 2010).
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Gambierdiscus are epibenthic on seagrass, macroalgae, sand and coral rubble, however, they
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can also occur in the plankton (Nakahara et al., 1996; Parsons et al., 2011). Species of this
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genus have the plate formula: Po, 3', 7", 6c,?s, 5’’', 1p, 2”” (Chinain et al., 1999; Faust, 1995;
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Fraga et al., 2011; Holmes, 1998; Litaker et al., 2009) and are distinguished from one another
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based on characters such as surface patterns, cell flattening (apical or dorso-ventral), size and
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shapes of the plates and of the cells.
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The type species, Gambierdiscus toxicus, was first identified as recently as 1979 (Adachi and
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Fukuyo, 1979), and until 15 years ago, only 3 species were known in the genus. It was
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recognised that the type description of Gambierdiscus toxicus probably incorporated several
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species, and G. toxicus was redescribed more narrowly, based on a lectotype (Litaker et al.,
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2009). Currently, twelve species of Gambierdiscus are recognised: Gambierdiscus australes,
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G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus, G. polynesiensis,
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G. ruetzleri, G. toxicus, G scabrosus, G. yasumotoi, and G. excentricus (Chinain et al., 1999;
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Faust, 1995; Fraga et al., 2011; Holmes, 1998; Litaker et al., 2009; Nishimura et al., 2014).
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At least five additional unnamed clades that are clearly genetically distinguishable have been
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found, indicating that further species may be distinguished in future (Litaker et al., 2009;
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Richlen et al., 2008; Xu et al., 2014).
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Published reports initially indicated the presence of only a single species, Gambierdiscus
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toxicus in Australia, from tropical regions of Queensland (Holmes et al., 1990; Holmes et al.,
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1991; Holmes et al., 1994), and more recently, from New South Wales (Murray, 2010).
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Cultures of this taxon from sites in Queensland were made in the 1990s (Babinchak et al.,
4 70
1994; Holmes et al., 1990; Holmes et al., 1991; Holmes et al., 1994) and subsequently lost.
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Since these initial reports, our understanding of the morphological and genetic diversity
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present in the genus Gambierdiscus has increased significantly (Chinain et al., 1999; Fraga et
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al., 2011; Litaker et al., 2009; Nishimura et al., 2014; Richlen et al., 2008; Xu et al., 2014),
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and it is therefore necessary to revisit initial sites in order to assess the presence of these
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species. Recently, a second species of Gambierdiscus, G. carpenteri, has been reported from
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several sites in Australia for the first time (Kohli et al., 2014a; Kohli et al., 2014b).
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The species Gambierdiscus yasumotoi was originally described from Singapore waters
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(Holmes, 1998), and was found to produce an uncharacterised toxin that was lethal to mice,
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producing symptoms similar to those of maitotoxin (Holmes, 1998). This species has since
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been reported from additional sites around the world, including from the Pacific and
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Caribbean waters. Strains isolated from New Zealand were found not to produce known
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analogues of ciguatoxins (CTXs) or maitotoxin (MTX) 1, but were likely to produce an as yet
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uncharacterised analogue of maitotoxin (Rhodes et al., 2014a).
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In this study the presence of Gambierdiscus yasumotoi is shown for the first time along a
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latitudinal gradient of more than 1550 km in waters of the northern, central and southern
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Great Barrier Reef (GBR), Queensland, Australia. Gambierdiscus yasumotoi is described
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from Australia based on scanning electron microscopy of field material, as well as scanning
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electron microscopy and phylogenetic analysis of a culture of G. yasumotoi isolated from the
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central GBR region. The occurrence of two other species of Gamberdiscus is also
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documented: G. carpenteri and G. cf belizeanus.
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2 Materials and Methods
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2.1 Sample collection.
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Between the 3rd-8th December 2003, eight samples were taken at low tide at sites on Raine
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Island (11° 45’.501 S, 144° 02’.309 E), a remote uninhabited tropical coral cay in the
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northern GBR, in the vicinity of the tower end of the beach (Fig 1), as part of annual
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sampling coordinated by the Raine Island Corporation. On 15th June 2003, four samples were
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collected from Heron Island (23°25’ S, 151° 55’ E), a tropical coral cay in the southern GBR
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(Fig 1). Samples were taken from encrusting macroalgae in shallow (less than 30 cm deep)
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tidal pools at low tide.
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5 101
Macroalgal samples were obtained from central GBR waters on low tide in the area between
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Townsville (Pallarenda at the mouth of Three Mile Creek, 19° 12' 34'' S, 146° 46' 36'' E) and
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Magnetic Island (Nelly Bay, 1910’ S, 14650’ E; Great Barrier Reef Marine Park Authority
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Permit G06/20234.1) in August 2004 and March 2008 (Fig 1). Macroalgal material was
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washed in filtered (0.45 µm) seawater to flush epiphytic dinoflagellates off their surface and
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the wash water was sequentially concentrated by filtration through 53 and 20 µm nylon mesh
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filters.
108 109
2.2 Culture establishment.
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Dinoflagellates were isolated from Pallarenda and Nelly Bay macroalgal wash waters at the
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North Queensland Algal Identification/Culturing Facility (NQAIF) at James Cook University,
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Townsville, Australia using the microcapillary‐capturing‐technique (Andersen and Kawachi,
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2005) at 10X magnification on an inverted light microscope (Olympus CKK 41, Olympus
114
Australia Ltd, Mt Waverley VIC 3149). Cells were dispensed in autoclaved and filtered (0.45
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μm) seawater. Cells were allowed to swim for ten minutes and were then recaptured. This
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procedure was repeated ten times to ensure that nano‐ and pico‐plankton were no longer in
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the vicinity of the cell to be isolated. Cultures of NQAIF116 (Pallarenda) and NQAIF210
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(Nelly Bay) were established in L1 medium (Guillard and Hargraves, 1993) prepared in
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natural seawater and maintained at 24°C, a 12:12 h photoperiod and light intensity of 45
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μmol photons m‐2s‐1 in a Contherm cross‐flow phytoplankton growth chamber. Cultures were
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subcultured in L1 medium every 4 weeks
122 123
2.3 Microscopy.
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Samples were immediately examined using a dissecting light microscope. Individual cells
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were isolated onto 5 µm millipore filtersor 1 cm round glass coverslips that had been treated
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with poly-L-lysine (Marchant and Thomas, 1983), to make preparations to observe in the
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scanning electron microscope. Cells were dehydrated in a series of increasing ethanol
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concentrations (15, 30, 50, 70, 90, and 100%), followed by 50% and 100%
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hexamethyldisilizane (HMDS). The filters were air dried before being sputter coated with
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gold or gold/palladium. They were observed using a Philips 505 scanning electron
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microscope (Philips, Eindhoven, the Netherlands) at 10–15kV and a JEOL JSM-5410LV
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scanning electron microscope (JEOL, Sydney, Australia).
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6 134
2.4 DNA extraction and sequencing.
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DNA was extracted from cultures NQAIF210 and NQAIF116 using a modified Chelex®
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protocol (Walsh et al., 1991) as outlined in Momigliano et al. (2013). The near-complete
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nuclear 18S rRNA gene, and the D1-D3 and D8-D10 regions of the 28S rRNA gene were
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amplified and sequenced using the primers listed in Table S1. PCR reactions were set up as
139
follows: 1µl of template DNA, 800 µM each DNTPs, 10 pmol of each primer, 1X Green
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GoTaq® Flexi Buffer and 1 unit GoTaq® Flexi Polymerase (Promega, Inc.). PCR reactions
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underwent an initial denaturation of 3 min at 94 °C, followed by 35 cycles of 30 s
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denaturation (94 °C), 30 s annealing (see Table S1 for details), and 1 min extension (72 °C),
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and a final extension of 5 min at 72 °C. PCR products were cleaned by isopropanol
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precipitation and sequenced using a commercial service (Macrogen, Inc.). All sequences
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were deposited in GenBank (accession numbers: KM272970-272974).
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2.5 Sequence alignment and phylogenetic analysis.
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Overlapping fragments of the D1-D3 and D8-D10 regions of the 28S and the near-complete
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18S genes were assembled using the software Chromas Pro (Technelysium Pty Ltd.). The
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sequences obtained from culture NQAIF210 and NQAIF116 were aligned with available
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sequences of Gambierdiscus spp. (for a list of the strains used see Table S2) using Clustal W
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(Thompson et al., 1994) and the alignments were visually refined using BioEdit (Hall, 1999).
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Phylogenetic analysis was performed on the datasets including the D8-D10 region of the 28S
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rRNA and the near complete 18S rRNA gene. Substitution models were selected for each
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dataset in jModelTest2 (Darriba et al., 2012), using Bayesian Information Criterion as a
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measure of the relative quality of the models. Phylogenetic reconstructions were carried out
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using both a Maximum Likelihood (ML) and Bayesian Inference (BI) approach.
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ML trees were produced in PhyML 3.1 (Guindon et al., 2010), using a TrN+G substitution
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model with 4 gamma categories (alpha= 0.46) and a TIM2+G model with 4 gamma
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categories (alpha=0.46) for the D8-D10 region of the 28S gene and the 18S dataset
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respectively, tree improvement by using the best of nearest neighbour interchanges (NNI)
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and subtree pruning and regrafting (NPR), starting with 5 random trees. Bayesian analysis
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was performed in MrBayes 3.2.2 (Ronquist and Huelsenbeck, 2003). The substitution model
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used for the D8-D10 region of the 28S gene was the same as in the ML analysis. However as
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MrBayes does not implement the TIM2 substitution model, we used the next-best available
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model, the HKY+G model. The number of Markov Chain Monte Carlo generations was set to
7 166
2 000 000, but set to automatically stop when the standard deviation of split frequencies fell
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below 0.01. Trees were sampled every 100 generations, and convergence of the 2
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independent runs was checked by analysing the stability of each parameter estimate in the
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software Tracer v1.6.
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In addition, for each locus p-distances for all available strains belonging to the G.
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yasumotoi/ruetzleri clade were estimated in the R statistical environment using the ape
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package (Paradis et al., 2004). The distance matrices were further analysed using Principal
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Coordinates Analysis (PCoA) implemented in the package ade4 (Dray and Dufour, 2007).
174 175
3 Results
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3.1 Phylogenetic analyses.
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The final alignments of the D8-D10 region of the 28S rRNA gene based on 45 sequences
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included 929 unambiguously aligned nucleotides and 348 variable sites, of which 286 were
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parsimony informative. The 18S alignment included 36 sequences, 1746 unambiguously
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aligned nucleotides and 696 variable sites of which 603 were parsimony informative. Both
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phylogenies show strong support for all terminal branches (Figs 2 & 3), and largely support
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previously published phylogenies of the genus Gambierdiscus. Small differences in the trees
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presented here relative to previously published phylogenies are due to the fact that out-group
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taxa were not used in phylogenetic reconstruction and disappear if the trees are rooted with
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the most basal Gambierdiscus clade (G. yasumotoi clade).
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NQAIF210 grouped with high support within the G. ruetzleri/G. yasumotoi clade - Clade I in
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Nishimura et al. (2013) in both phylogenies (Figs 2 & 3 ). NQAIF210 could not be
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unambiguously identified as either G. yasumotoi nor G. ruetzleri based on 18S sequences. In
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the phylogeny reconstructed using the D8-D10 region of the 28S gene, NQAIF210 grouped
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with high support with the New Zealand strain CAWD210, and both NQAIF210 and
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CAWD210 grouped with G. ruetzleri with moderate bootstrap support (Fig 3). The culture
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NQAIF116 consistently grouped with high support within the G. carpenteri clade (Figs 2 &
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3). Representations of p-distance matrices in reduced space (Fig 4) showed that NQAIF210
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and CAWD210 have identical sequences for the D1-D3 region, and have small p-distances
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for the D8-D10 region but the analysis also failed to show any clear grouping of NQAIF210
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with other previously identified strains in this clade, from which NQAIF210 is roughly
8 197
equidistant (Fig. 4) . However, it must be remembered that all sequences of G. yasumotoi
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utilized in the phylogenies and p-distance analyses are clones of the same strain (Gyasu), and
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therefore they cannot be used as a proxy of intra-specific variation. Furthermore, p-distances
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within this clade, and between putative species within it, are very small when compared to
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between-species distances elsewhere in the genus. P-distances between G. yasumotoi and G.
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ruetzleri are as little as 0.01 (D1-D3 and D8-D10 regions of the 28S rRNA gene) and 0.004
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(near complete 18S gene).
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Interestingly, p-distances among clones of the same strain of G. yasumotoi are comparable to
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those observed between clones of G. yasumotoi and strains of G. ruetzleri (Fig 4, particularly
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4A, 4C and 4E).
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3.2 Morphological analyses
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The morphology of Gambierdiscus yasumotoi from field samples and from the cultured strain
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is shown (Figs 5,6,7). Cells from field samples were laterally compressed in ventral view, and
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oval to round in lateral view, with a depth of 57 µm (range 54- 59 µm), width 48 µm (range
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46-50 µm) and length 56 µm (range 53-59 µm) (Figs 6,7). The depth to width ratio was 1.18,
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while the length to width ratio was 1.16. Cultured cells were slightly smaller, more globular
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and less obviously laterally compressed, with a depth of 49 µm (range 44- 54 µm), width 45
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µm (range 40-49 µm) and length 51 µm (range 49-54 µm) (Fig 5). The depth to width ratio
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was 1.08, while the length to width ratio was 1.13 (Table 1).
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The thecal plates were smooth with many small round pores, approximately 0.3 µm diameter
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(Figs 5b, 7a,b). The epicone had the plate formula of Gambierdiscus, with three apical plates
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and seven precingular plates (Figs 5a, 6b,g,h,i). The apical pore plate was tear drop shaped,
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while the pore was long and fish hook shaped at the cell apex, approximately 8-9 µm long,
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with 33-45 pores surrounding (Figs 5b, 6g, 7a). The hypocone consisted of five postcingular
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plates, two antapical plates and one posterior intercalary plate (Figs 5f, g, i, 6f).
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The cingulum was deeply excavated and descending, displaced about 1-2 cingular widths
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(Figs 5c, 6a, b, c, 7b, c, d). Prominent cingular lists were present in all cells,(Figs 5i, 6a, c,
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7b, c, d). In cultured cells, the cingulum was positioned slightly higher than the mid point of
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the cell in left lateral view, approximately in the mid point of the cell in dorsal view, and just
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below the mid point of the cell in right lateral view (Figs 5c, d, e). In cells from field samples,
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the cingulum was positioned higher, approximately 1/3 of the way down the cell from the
9 228
apex in left lateral view, and slightly above the mid point of the cell in right lateral view (Figs
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6a, c, f).
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The sulcus has been reported to consist of seven plates, the Sdp, Sda, Ssp, Ssa, t, Sma and
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Smp (Litaker et al., 2009). Of these, the Ssp, Ssa, Sdp, t and Sda plates were clearly visible in
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this study (Fig. 7b, c, d). The tiny, oval shaped Sma plate, which is adjacent to the Sda plate,
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was also visible in several images (Fig. 7c, d). The Smp plate could not be observed in our
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images. Prominent sulcal lists were present in all cells from the field (Figs 5i, 6a, c, 7b, c, d),
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and some cultured cells (Fig. 5c), but absent in other cultured cells (Fig 5h). The sulcal lists
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consisted of a long list on the left side of the Ssa and Ssp plates, and a shorter list on the right
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side of the Sdp plate (Fig. 7b, c, d). The 2'''' plate was fork shaped and invaded the sulcus
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(Figs 5g, h, i, 6a).
239
Substantial plate overlaps, particularly in the hypothecal cells, were found in some cells in the
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cultured material (Fig 5g).
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Two other species of Gambierdiscus were found in field samples: Gambierdiscus cf
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belizeanus, and Gambierdiscus carpenteri. These species were only partially documented
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from the sites, and the morphological information about them is shown in the Supplementary
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Figs.
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4 Discussion
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Gambierdiscus yasumotoi has previously been reported from sub-tropical Japan (Nishimura
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et al., 2013),the Persian Gulf and the Red Sea (Saburova et al., 2013), Singapore (Holmes,
248
1998), the Mexican Caribbean (Hernández-Becerril and Almazán Becerril, 2004), and even
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the northern coast of New Zealand (Rhodes et al., 2014a), but not from Australian waters.
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Here the occurrence of G. yasumotoi at three sites along a latitudinal gradient spanning more
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than 1550 km of the Australian GBR is reported, a region with endemic CFP.
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Records of G. yasumotoi in tropical and sub-tropical regions in the Indian, and Pacific
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Oceans, both in the northern and southern hemispheres, suggest that this species has a
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circum-global distribution in tropical and sub-tropical waters. Interesting is the occurrence of
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G. yasumotoi in northern New Zealand, reported by Rhodes et al. (2014a), as sea surface
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temperatures (SST) fall well below 15 º C in this region, the lowest thermal limit for any
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species of Gambierdiscus reported to date (Kibler et al., 2012). However, as (Rhodes et al.,
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2014a) noted, the specimens were collected in summer, when SST was 23 ºC. It is possible
10 259
that the occurrence of G. yasumotoi in New Zealand is seasonal, with cells being transported
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from tropical and sub-tropical Australia during the warmer months via the East Australian
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Current (EAC) and the Tasman Front. The EAC is known to affect phytoplankton
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communities in the eastern Australian coast, transporting tropical species into sub-tropical
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and temperate areas (Armbrecht et al., 2013).
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4.1 Comparison between Gambierdiscus yasumotoi and G. ruetzleri
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Interestingly, most of the strains of G. yasumotoi which have been recently isolated have
266
intermediate morphologies between G. yasumotoi and the more recently described G.
267
ruetzleri (Table 1). According to the original description of G. ruetzleri, the features that
268
distinguish this species from G. yasumotoi are: size (G. ruetzleri is significantly smaller and
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has a cell width less than 42 µm); a smaller, narrower and differently shaped 2’’’’ plate; a
270
greater depth to width ratio, meaning they are proportionally narrower; a smaller length to
271
width ratio, and a smaller epitheca to hyptheca ratio, as the cingulum is closer to the centre of
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the cell, rather than closer to the cell apex (Litaker et al., 2009).
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There was some variability in these features in both individual cells from the culture, and
274
between cells found in field samples as compared to cultured cells (Table 1). Both the field
275
and cultured material had L:W and D:W ratios that were more similar to those in the original
276
description of Gambierdiscus yasumotoi than that of G. ruetzleri (Table 1). However, the
277
epitheca to hypotheca ratios appeared to differ between the field and cultured material. For
278
the cultured strain, this ratio seems to be smaller than that reported for G. yasumotoi, but the
279
difference is not nearly as pronounced as in the original description of G. ruetzleri (Litaker et
280
al., 2009). In previous studies, the D:W and L:W ratios of material from New Zealand were
281
most similar to those of G. ruetzleri. However in overall cell size, these specimens more
282
closely resembled G. yasumotoi (Rhodes et al. 2014a, Table 1). In the material from Kuwait
283
(Saburova et al., 2013), the D:W ratio and epitheca:hypotheca ratio appeared to be most
284
similar to those of Gambierdiscus ruetzleri, while the cell width and overall cell size were
285
most similar to those of G. yasumotoi.
286
In terms of the shape of the 2’’’’ plate, variability was found in the Australian samples. In
287
some cells this appeared to have an almost horizontal posterior side (Figs 5f, 6a), similar to
288
the shape as described for G. yasumotoi. Other cells had a 2’’’’ plate with a 45° angle on the
289
posterior side of this plate (Fig. 6c), a shape more similar to that of G. ruetzleri (Litaker et al.,
290
2009).
11 291
Several specimens of Gambierdiscus yasumotoi with aberrant plate pattern have been
292
observed among cultured cells (Saburova et al., 2013) and cells with substantial plate
293
overlaps were found in this study (Fig 5g), which would tend to distort the plate size or
294
shape. For this reason, interpreting species-level differences based on minor differences in
295
plate sizes or shapes could be misleading.
296 297
In our molecular analysis, NQAIF210 grouped with high support within the G.
298
yasumtoi/ruetzleri clade, but it was not clearly grouped with either based on 18S gene
299
sequences (Figs 3,4). The phylogeny constructed using the D8-D10 region of the 28S rRNA
300
gene suggests that NQAIF210 and the G. cf. yasumotoi isolated from New Zealand constitute
301
a highly supported sub-clade, and the two strains had identical sequences for the D1-D3
302
region of the same gene (Fig. 3). The genetic data support the morphological observations,
303
suggesting that these strains are indeed very similar. Interestingly, while both strains were
304
morphologically closer to the original description of G. yasumotoi, in the D8-D10 phylogeny
305
they grouped with high support with G. ruetzleri (Fig. 3). There are now reports of a number
306
of strains, including NQAIF210 (this study) , IR4G and Go3 (Nishimura et al., 2013) and
307
CAWD210 (Rhodes et al., 2014a), which always group within the G. yasumotoi /G. ruetzleri
308
clade, but cannot be clearly identified based on molecular and morphological data as either
309
species.
310 311
Our analyses of p-distances among strains (Fig 4) belonging to the Gambierdiscus yasumotoi
312
clade suggest that caution is needed in trying to delineate species boundaries within this
313
group. Firstly, the erection of G. ruetzleri as a new species was based on a comparison with a
314
single strain of G. yasumotoi, the strain used in the original description (Litaker et al., 2009).
315
The estimates of within-species p-distances for G. yasumotoi are based on multiple sequences
316
from this same strain, and therefore it could be considered a form of pseudo-replication to use
317
such differences to recognise G. yasumotoi and G. ruetzleri as distinct, well supported entities
318
in the phylogenetic analysis. Furthermore, our PCoA analyses of p-distance matrices for 18S
319
and 28S rRNA gene sequences seems to suggest that p-distances among clones of G.
320
yasumotoi are in some cases comparable to p-distances between strains of G. yasumotoi and
321
G. ruetzleri (Fig. 4). In addition, the genetic differences between these two species are less
322
than those determined for within-species divergence for other Gambierdiscus species
323
(Nishimura et al., 2013; Rhodes et al., 2014a). Morphological and genetic distances similar
324
to, or even larger than the ones reported between putative species within the G.
12 325
yasumoti/ruetzleri clade have been described within species of other dinoflagellate genera,
326
such as Coolia (Fraga et al., 2008; Momigliano et al., 2013; Rhodes et al., 2014b) and
327
Amphidinium carterae (Murray et al., 2012). Within the G. yasumotoi/ruetzleri clade, the
328
discovery of a number of strains which show intermediate morphologies and cannot be
329
unambiguously identified as either G. yasumotoi or G. ruetzleri (Nishimura et al., 2013;
330
Rhodes et al., 2014a) throws into doubt the status of these as distinct species. It is possible
331
that G. yasumotoi and G. ruetzleri are conspecific, and the minor genetic and morphological
332
differences reported are at the population level, or that they form an as yet unresolved species
333
complex.
334 335
4.2Toxicity
336
In the original description of Gambierdiscus yasumotoi, methanol extracts were found to be
337
toxic to mice with symptoms similar to those of maitotoxins (MTX) (Holmes, 1998). The
338
strain of Gambierdiscus yasumotoi from New Zealand was found by LC-MS analysis not to
339
produce known analogues of MTX or of the monitored analogues of CTXs at detectable
340
levels (Rhodes et al., 2014a). However, a putative MTX analogue which had the same mass
341
as MTX-3 (Holmes and Lewis, 1994; Lewis et al., 1994) was found (Rhodes et al., 2014a). A
342
study using the human erythrocyte lysis assay (HELA) showed significant toxicity in
343
Gambierdiscus ruetzleri (Holland et al., 2013). Experiments which used specific inhibitors
344
of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic
345
cell extracts containing MTX, were consistent with MTX as the primary hemolytic
346
compound produced in those studies (Holland et al., 2013). MTX was generally considered
347
not to be related to CFP symptoms, which are thought to be solely caused by ciguatoxins
348
(CTX) (Lewis and Holmes, 1993). However, the propensity for MTX to accumulate in fish
349
flesh following exposure to Gambierdiscus spp has been re-examined experimentally, and its
350
role in fish toxicity remains incompletely known(Kohli et al., 2014c). A strain of G.
351
yasumotoi was recently found to increase mortality and the expression of stress related genes
352
in copepods (Lee et al., 2014)
353 354 355
Acknowledgements
13 356
We thank the Raine Island Corporation for funding the field trip to Raine Island (SM), the
357
Australian Biological Resources Study for additional funding (SM) and the University of
358
Sydney Australian Centre for Microscopy and Microanalysis for access to the scanning
359
electron microscopes (SM). We also thank the Marine and Tropical Sciences Research
360
Facility for funding field work and laboratory work in the central GBR (KH, DB, PM).
361 362
Author contributions
363
Conceived and designed the experiments: PM, SM, KH, DB. Performed the experiments:
364
PM, SM, KH. Analysed the data: PM, SM. Contributed reagents/materials/analytical tools:
365
KH, SM, DB. Wrote the paper: PM, SM. Provided feedback on the manuscript: KH, DB.
366 367 368
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503
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504 505 506 507 508 509 510 511 512 513 514 515 516 517 518
19 519
Figures Legends
520 521
Figure 1. Map of the sampling sites in Great Barrier Reef (GBR) region, east coast,
522
Queensland, Australia. Inset box, showing the location of the sampling area within Australia.
523
The three regions in which sampling took place: Raine Island in the far northern GBR; Nelly
524
Bay and Pallarenda, Townsville, in the central GBR; Heron Island in the southern GBR.
20
525 526 527
Figure 2. Maximum Likelihood phylogeny of the genus Gambierdiscus based on near-
528
complete 18S gene sequences. Branch support values (ML/BI) represent bootstrap support
529
based on 1000 pseudo-replicate datasets and Bayesian clade credibility values respectively.
530
Branches with 100/100 support are indicated by an asterisk. Scale represents number of
531
changes per nucleotide. Strains isolated in this study are shown in bold.
21
532 533
Figure 3. Maximum Likelihood phylogeny of the genus Gambierdiscus based on the D8-D10
534
region of the 28S rRNA gene. Branch support values (ML/BI) represent bootstrap support
535
based on 1000 pseudo-replicate datasets and Bayesian clade credibility values respectively.
536
Branches with 100/100 support are indicated by an asterisk. Scale represents number of
537
changes per nucleotide. Strains isolated in this study are shown in bold.
538 539 540 541 542 543 544 545
22 546 547
548 549
Figure 4. Principal Coordinate Analyses (PCoA) based on p-distance matrices among
550
sequences within the G. yasumotoi/ G. ruetzleri clade. PCoA based on near complete 18S
551
gene sequences (A & B) and on the D8-D10 (C & D) and D1-D3 (E & F) regions of the 28S
552
rRNA gene. Clones from the same strain are represented by partially transparent symbols
553
with the same colour, allowing visualization of density of points via colour saturation .
554
Eigenvalues of the plotted axes are shown in black.
23
555 556
Figure 5. Scanning electron microscope images of Gambierdiscus yasumotoi from the
557
cultured strain, NQAIF210. Scale bars are shown. A. Apical view, showing the epithecal
558
plate pattern and shape of the epicone. B. The apical pore plate, showing the comma and
559
pores. C. Cell in left lateral view. D. Cell in dorsal view. E. Cell in dorsal view. F. Antapical-
560
ventral view, showing antapical plates and part of the sulcus. G. Antapical view, showing
561
substantial plate overlaps. H. Ventral view, showing the sulcus and a lack of sulcal lists. I.
562
Antapical/lateral view, showing the sulcal lists.
563 564
24
565 566
Figure 6. Scanning electron microscope images of Gambierdiscus yasumotoi from the field
567
material from Raine Island and Heron Island. Scale bars are 10 µm. A. Cell in ventral/lateral
568
view, showing sulcal lists. B. Cell in apical/ventral view, showing part of the epicone. C. Cell
569
in ventral view. D. Cell in right lateral view. E. Cell in left lateral view. F. Cell in left lateral
570
view. G. Cell in apical view, showing the epicone, including the apical pore plate and
571
comma. H. Cell in apical view, showing the epicone. I. Cell in apical view, showing the
572
epicone.
573 574 575
25
576 577
Figure 7. Scanning electron microscope images of Gambierdiscus yasumotoi from the field
578
material from Raine Island and Heron Island, showing the apical pore region, and sulcus.
579
Scale bars represent 10 µm (A) and (B).A. The apical pore plate. B. The sulcal plates,
580
showing the list, and ssa and ssp. C. Showing the sma and sda plates. D. Showing the sma
581
plate and sulcal list.
582
26
Tables Table 1. Cell sizes and comparison of other morphological features of G.yasumotoi, from our data and previous reports.
site
G.
G.
G. cf
G.
G.
G. yasumotoi
G. yasumotoi
G. ruetzleri
yasumotoi
yasumotoi
yasumotoi
yasumotoi
yasumotoi
(Holmes 1998),
(Litaker et al
(Litaker et al
(this study
(this study
(Rhodes et
(Saburova
(Saburova
type description
2009
2009)
field cells)
culture)
al in 2014a
et al 2013
et al 2013
Heron
Townsville northern
Kuwait
Jordan
Island,
New
Raine
Zealand
Singapore
Island Depth
Mean =
Mean = 49
Mean =
Mean=62.9 Mean= 62.6
Mean = 50
Mean = 56.8
Mean = 45.5
57.2
Range 44-
54.8
± 4.4
(n=17)
(n=14)
Range 41-55
Range 54-
54
± 5.7
(n=30)
Mean = 56
Mean= 51
Mean =
Mean=
Mean= 62.9
Mean = 53
Mean= 62.4
Mean = 51.6
Range 53-
Range 49-
59.8 ± 7.5
61.7 ± 6.2,
± 6.8, N=7
(n=17)
(n=14)
45-59.5
59
54
Mean=48
Mean=45
Mean =
Mean=
Mean=
Mean = 44
Mean = 51.7
Mean=37.5
Range 46-
Range 40-
42.5 ± 4.1
54.1 ± 5.1,
54.7± 1.5,
38-50
(n=14)
30.9-42.2
50
49
N=9
N=3
± 6.1, N =7
Range 43-61
59 Length
Width
N = 20
45-63
27
Epitheca
~0.5
~1.0
D:W ratio 1.18
1.08
L:W ratio
1.13
~1.0
0.5-0.6
~1.0
1.29
1.28
1.14
1.13
1.1
1.35
1.41
1.14
1.15
1.20
1.2
1.45
to hypotheca ratio
1.16
28
