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Abstract

The chain-forming toxic dinoflagellate Gymnodinium catenatum Graham is a known
causative organism of paralytic shellfish poisoning (PSP). During the mid-1970's the
geographic extent of G. catenatum plankton blooms increased dramatically, causing
toxic episodes in Spain, Portugal, Mexico, Venezuela, Argentina, Uruguay, Japan,
Korea and southern Australia (Tasmania). Studies of the distinctive microreticulate
resting cysts of G. catenatum in Tasmania suggest it was introduced to southern
Tasmania during the early 1970's, possibly via ballast water from either Japanese,
Korean or Spanish populations. The cysts of this species have now been widely
reported, even from areas where G. catenatum has rarely, or never, been detected in
the plankton. The reported cyst diameter varies considerably (17-63 p,m diameter),
displaying a bimodal size distribution at some localities, suggesting that cysts may
belong to a species complex of two or more related species.
This work examines the distribution, and morphological and genetic variation, of the
Gymnodinium catenatum species complex to resolve three distinct microreticulate cystforming
species. Resolution of the species complex allowed the elucidation of
population genetic relationships between strains of the toxic "true G. catenatum"
isolated from Japan, Spain and Portugal, and Australia, to examine the hypothesis that
Tasmanian G. catenatum was introduced to Australia (Tasmania) from one of these
two potential source populations. Mapping the distribution of G. catenatum by examining sediments for the
microreticulate cysts is hampered by their low abundance in coastal sediments and the
low proportion of intact and viable specimens. Cyst concentration methods (sodium
polytungstate density centrifugation) and PCR-based genetic identification methods
were developed to improve cyst survey detection limits. Sediment surveys of 105
sampling sites at 17 estuarine and coastal locations demonstrated that microreticulate
cysts are widely distributed in Australian coastal sediments. Two distinct morphotypes
were noted: a "small-form" cyst (17-28 p,m) widely distributed in temperate and
tropical Australian estuaries and a "large-form" typical of G. catenatum ( 37-62 p.m)
which was restricted to the coasts of south-eastern Tasmania, southern Victoria, Port
Lincoln (South Australia) and the Hawkesbury Estuary (NSW). Germination
experiments showed that the smaller cyst-type belonged to a species that was
morphologically and genetically distinct from both G. catenatum and the European
rnicroreticulate cyst species, G. nolleri Ellegaard et Moestrup. This new species is
described herein as Gymnodinium microreticulatum sp. nov. Bolch et Hallegraeff. The possible origin and evolution of G. catenatum was investigated by examining the
genetic relationships among 27 species of gymnodinoid, prorocentroid and peridinoid
dinoflagellates using partial sequences of the large sub-unit ribosomal RNA gene. The
phylogenies constructed confirmed earlier findings from small sub-unit (SSU) RNA
studies. The relationships of the 21 free-living gymnodinoids correlated with their
morphological and cytological features with the loop-apical-grooved species forming
four clusters delineated by chloroplast structure and arrangement, and resting cyst
morphology. The G. catenatum complex (G. catenaturn, G. nolleri and G.
microreticulatum) formed a distinct monophyletic lineage arising from the base of this
group. Within the complex, G. microreticulatum diverged earliest, followed by G.
nolleri and G. catenatum. Comparison of nuclear volumes of the three species suggests
that the group may have evolved by polyploidy from a G. microreticulatum-like
ancestor. The abundant LSU-rDNA sequence variation among G. microreticulaturn
isolates contrasted the absence of verifiable sequence variation among G. catenaturn
from Australia, Hong Kong, Japan, Spain and Uruguay Analysis of allozymes and large-subunit ribosomal RNA sequences failed to reveal
genetic polymorphism among G. catenatum strains from Australia, Japan, Spain and
Portugal. However, reproductive compatibility analysis demonstrated extensive intrapopulation
compatibility and an outbreeding, multiple-group mating system. Mating
success analysis (by cyst production) and variation in post-meiotic progeny viability
from inter-population crossing experiments indicated that Japanese and Spanish strains
were more closely related to each other than to Australian strains. Mating studies were
supported by genetic studies using RAPD-PCR. Genetic variation was partitioned
primarily within populations (87%), consistent with a sexually outbreeding species, as
confirmed by mating studies. The G. catenatum strains could be clearly separated into
regional clusters: Australia, Japan and Spain/Portugal. The Spanish/Portuguese and
Japanese clusters were most closely related with the Australian cluster more distant and
almost equally related to the others. The similarity between Japanese and Spanish G.
catenatum compared to Australian strains suggests recent dispersal between these two
populations. The source population for Australian G. catenatum remains unclear,
however, the data support a secondary relocation of Tasmanian G. catenatum to
mainland Australia, possibly via a domestic shipping vector. Geographic and temporal
clustering of Tasmanian strains by isolation location and bloom year indicates that
genetic exchange between neighbouring estuaries is limited and that Tasmanian G.
catenatum blooms are composed of localised, estuary-bound sub-populations.
Re-examination of the dinoflagellate fossil record in light of recent molecular
phylogenetic data suggests that the G. catenatum complex evolved near the start of the
Cretaceous period [circa 150 million years ago (Mya)]. Using a G. catenatum complex "molecular clock" based on LSU-rDNA sequences, it is estimated that the common
ancestor of the complex (probably a G. microreticulatum-like species) evolved around
130-140 Mya and that the G.nolleri and G. catenatum lineages diverged about 16-19
Mya. The known modern distributions of the three species suggest a European
evolutionary origin of G. catenation followed by a geologically recent global dispersal.
Considering the lack of rDNA variation among the five populations examined, this
dispersal is conservatively estimated to have occurred well within the last 25 thousand
years. Whether dispersal has been by natural processes or assisted by human means
(or a combination of both) is not yet clear.
This study demonstrates that PCR-fingerprinting methods, such as RAPD-PCR, can
discriminate fine-scale genetic population structure and discriminate global population
clusters. Comparative genetic studies of more G. catenation populations would allow a
better assessment of global relationships and may identify population genetic dines that
correlate with G. catenatum natural dispersal corridors, or discontinuities that imply
trans-oceanic transfer and human introduction.

Item Type:	Thesis - PhD
Authors/Creators:	Bolch, Christopher John Stanley
Keywords:	Dinoflagellate blooms, Toxic marine algae, Algal blooms, Paralytic shellfish poisoning
Copyright Holders:	The Author
Additional Information:	Includes notes in pocket. Thesis (Ph.D.)--University of Tasmania, 1999. Includes bibliographical references
Item Statistics:	View statistics for this item

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