Investigation of the dinoflagellate community of lake Tovel by genetic analysis 1
of environmental samples. 2
3
M. D’Andrea, N. Daugbjerg and G. Flaim. 4
5
Introduction 6
Dinoflagellates constitute a diverse and ubiquitous assemblage in marine and 7
freshwater ecosystems. They play an important part in aquatic food web as their 8
nutritional modes are auto-, mixo- or heterotrophic. Hence, the dinoflagellate 9
assemblage comprises both primary producers and predators. Due to recent 10
advances in the field of molecular biology (particularly DNA sequence 11
determination) the taxonomy of dinoflagellates is currently under major revisions 12
(SAUNDERS et al. 1997, DAUGBJERG et al. 2000). Phylogenetic studies on 13
dinoflagellates published in the last decade include mostly marine species (FLØ 14
JØRGENSEN et al. 2004, GAST et al. 2004). Community studies of marine 15
dinoflagellates based on environmental DNA samples are few in number whereas 16
DNA studies on freshwater dinoflagellate communities are virtually in the 17
making. 18
The experimental site chosen for this study is Lake Tovel: an Italian alpine lake 19
famous for its past red summer blooms caused by a dinoflagellate identified as 20
Glenodinium sanguineum March. (MARCHESONI 1941). Previous publications
21
based on material collected in Lake Tovel have focused on Glenodinium 22
sanguineum while essentially ignoring the many other dinoflagellates known to
exist in the lake. Recent limnological surveys have revealed that the number of 24
dinoflagellate taxa present in Lake Tovel has increased and there was a shift in 25
species composition during the last 50 years (FLAIM et al. 2003, FLAIM et al. 26
2004). Given the inherent difficulty of properly identifying naked dinoflagellates 27
by light microscopy especially from fixed samples and due to the difficulty of 28
establishing in vitro cultures, we applied a molecular approach directly on 29
environmental samples to further describe the dinoflagellate community 30
inhabiting the lake. Recently, several molecular techniques proved to be good 31
tools for natural community investigation especially on marine prokaryotes (DÌEZ 32
et al. 2001). Here we have used restriction analysis length polymorphisms (RFLP) 33
of PCR products combined with a clone library construction. This approach has 34
previously been applied for screening of eukaryotic communities based on SSU 35
(Small Subunit Unity) rDNA (ROMARI & VAULOT 2004). However, this gene 36
fragment is not useful for investigations on dinoflagellate diversity due to its high 37
level of genetic conservation. 38
In order to elucidate the dinoflagellate community in Lake Tovel we developed an 39
approach based on PCR-RFLP of environmental samples using a group specific 40
primer designed to amplify the LSU (Large Subunit Unity) rDNA of 41
dinoflagellates only (HANSEN & DAUGBJERG 2004). 42
43
Keywords 44
Lake Tovel, dinoflagellate community study, environmental samples, LSU rDNA, 45
RFLP. 46
Methods 47
Lake Tovel is situated in the Trentino province (northern Italy) at an altitude of 48
1178 m a.s.l. The lake is divided in two basins: a north-eastern one (zmax = 39 m)
49
and a much smaller south-western (zmax = 5 m) basin called Red Bay because of
50
its past red blooms. The lake is usually frozen from December to April and it is 51
classified as oligotrophic with phosphorus as the limiting nutrient (CORRADINI 52
et al. 2001). 53
Water samples from the centre of the Red Bay were collected at 0-1m with the 54
Ruttner bottle, they were promptly filtered in the lab with a5µm SVPP filter 55
(Millipore) and frozen at –20°C. The following samples will be analysed here: 56
19th August 2003 (summer), 22nd October 2003 (autumn), 18th November 2003 57
(winter-last sampling), 11th May 2004 (spring- ice melting). 58
Total genomic DNA was extracted from the SVPP filters using the CTAB method 59
(DOYLE & DOYLE 1987) but with a few modification as outlined in 60
D’ANDREA et al. (submitted). Extracted DNA was amplified first using primers 61
D1Rf and dino-specific (HANSEN & DAUGBJERG 2004) and then re-amplified 62
in semi-nested using the forward primer D3Af (DAUGBJERG et al.2000). PCR 63
reactions (total reaction volume 25µl) were performed with the HotMaster Taq 64
(Eppendorf) with 200µM of dNTPs and 0.5µM of each primer. The PCR 65
conditions are outlined in D’ANDREA et al. (submitted). The amplified PCR 66
fragments were checked on a 1% agarose gel stained with ethidium bromide. PCR 67
products were then cloned on a PCR TOPO-XL vector (Invitrogen) and one 68
hundred colonies grown on LB agar plates were picked up and reamplified. The 69
products of reamplification were digested with the restriction enzymes Taq I and 70
Sau 3A I, Cfr 13 I, Alu I. The reaction conditions were as suggested by the 71
manufacturer (Amersham Biosciences) and the fragments were separated on 2% 72
agarose gel stained with ethidium bromide.
73
74
Results 75
In order to assess the diversity of the dinoflagellate community as revealed by 76
each restriction enzyme, some samples were digested with the four enzymes. 77
Fig.1 shows the patterns found for some colonies isolated from the October 78
sample and treated with Taq I or Cfr 13 I. 79
Taq I proved to be the most informative enzyme because it permitted identifying 80
more ribotypes then the others: in 100 colonies from October sample Taq I 81
distinguished 7 profiles instead of 5 recognised by Alu I and 4 by Sau 3A I and 82
Cfr 13 I. Taq I phylogenetic assignments proved to be the least ambiguous 83
because they were usually repeated with at least 2 of the other enzymes. 84
Ribotype differences are significant only within the dominant pattern “E” which is 85
divided into 8 subgroups when considering the 4 enzymes. However in the 86
August sample (data not shown) this heterogeneity is less evident: within the Taq 87
I dominant pattern “A” only 2 subgroups were recorded by Cfr 13 I and Sau 3A 88
while Alu I did not distinguish any subgroup. For these reasons we choose Taq I 89
for a preliminary rapid screening by RFLP on the samples collected over the year 90
in the Red Bay. 91
Fig. 2 illustrates the relative occurrence of Taq I ribotypes in environmental 92
samples from different seasons as percentage of colonies which show a particular 93
profile: 10 patterns designated as A to L, were recorded considering all the 94
samples analysed. 95
Taq I ribotypes are interpreted as separate genera or groups of similar species 96
(D’ANDREA et al. submitted); when using an enzymes pool we can reach a 97
species distinction or intraspecific separation. 98
Seasonal changes in the community are evident and reflect the natural succession 99
of species caused by encystment and excystment or by the proliferation of one 100
species in a particular period. In August we obtained the highest biodiversity of 101
the four samples considered, in fact almost all the patterns were found even if in 102
low quantities. 103
We found a dominant pattern which changes over the months: A in summer (89% 104
of colonies), E in autumn and winter, F in spring. The presence of a dominant 105
ribotype agrees with microscopical observations of live material but is probably 106
overestimated because of the competitive nature of the PCR which amplifies 107
more frequently the more concentrated DNA molecules and can fail to amplify 108
less abundant ones (DÍEZ et al. 2001). The emerging trend suggests an August 109
maxima of almost all ribotypes with one dominant; in October and November 110
some species disappeared or are less abundant while one autumnal-winter 111
ribotype (E) became dominant and a new ribotype (L) was revealed; at ice melt a 112
spring group (F) is dominant while the summer ribotype A begins to increase in 113
abundance. 114
This temporal distribution tends to reflectthe seasonality of dinoflagellates and 115
turbulence in the Red Bay (FLAIM et al. submitted, Verh. Internat. Verein. 116
Limnol.). Despite the impossibility of seeing all of the low density groups, our 117
approach reflected the community trend expected from live observations and 118
allowed to identify more patterns than the morphotypes observed by light 119
microscopy. A finer identification of the dinoflagellates present in the lake was 120
achieved overcoming difficulties in distinguishing similar species by gross 121
morphological features seen with light microscopy. 122
This work can be regarded as one of the first attempts to use PCR - RFLP of LSU 123
rDNA for freshwater dinoflagellate community studies on environmental samples. 124
We show that this molecular approach is an easy and reliable tool for the 125
screening of the seasonal variability among the dinoflagellate group. 126
The comparison of LSU sequences of different ribotypes with the published 127
database is in progress in order to see if any environmental sequence is similar to 128
an already sequenced dinoflagellate. However we would expect to discover that 129
some environmental ribotypes represent unknown dinoflagellates, peculiar to this 130
habitat. 131
The study of the spatial and temporal distribution of dinoflagellates, which starts 132
with this work, will also be useful for a long term monitoring of these organisms 133
in other Trentino lakes. 134
135 136 137
Acknowledgements 138
This work was supported by the Autonomous Province of Trento, Italy (SALTO 139
grant WP 5). Authors thanks V.Pinamonti and G.Leonardi for sampling. 140
141
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Formattato: Inglese (Regno Unito)
Authors' addresses: 181
M. D’ANDREA *, G. FLAIM, Istituto Agrario di San Michele all’Adige, 38010 182
San Michele all’Adige (TN), Italy. 183
N. DAUGBJERG, Botanical Institute, University of Copenhagen, DK 2100 184
Copenaghen Ø, Denmark. 185
*Corresponding author. E-mail: maudand@yahoo.it
186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203
Formattato: Inglese (Regno Unito)
FIGURES 204
Fig. 1. 205
Agarose gel showing a comparison of the ribotypes revealed by 19 colonies 206
isolated from the Oct’03 sample after digestion with the enzymes Taq I(up) and 207 Cfr 13 I(down). 208 209 210 211 212 213 214 215 216 217 218 219 220 221
Fig. 2. 222
Relative occurrence of Taq I ribotypes in Red Bay environmental samples 223
collected over the year: percentage of the colonies analysed showing each pattern 224 (A to L). 225 % occurrence 0 20 40 60 80 100 a b c d e f g h i l AUG 226 % occurrence 0 20 40 60 80 100 a b c d e f g h i l OCT 227 % occurrence 0 20 40 60 80 100 a b c d e f g h i l NOV 228 % occurrence 0 20 40 60 80 100 a b c d e f g h i l MAY 229
