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Planktothrix populations in subalpine lakes: selectionfor strains with strong gas vesicles as a function of lakedepth, morphometry and circulation
DOMENICO D’ALELIO, ANDREA GANDOLFI , ADRIANO BOSCAINI, GIOVANNA FLAIM,
MONICA TOLOTTI AND NICO SALMASO
IASMA Research and Innovation Centre, Fondazione Edmund Mach, S. Michele a ⁄ Adige, Trento, Italy
SUMMARY
1. The genus Planktothrix (Cyanobacteria) usually produces concentrated populations of
filaments in the summer metalimnion of thermally stratifying lakes. This has been
associated with the action of gas vesicles, cellular structures providing positive buoyancy.
At the end of the summer, filaments are carried by convective mixing deeper into the water
column where some gas vesicles collapse as a result of high hydrostatic pressure. They
then lose their buoyancy, sink and are lost from the euphotic zone.
2. The resistance of gas vesicles to hydrostatic pressures is critical for the survival of
Planktothrix in deep lakes. However, comparative observations on populations from lakes
of a range of depths and hydrodynamic regimes are still needed to examine the
relationships between the adaptive trait (i.e. the ‘critical’ pressure at which each gas vesicle
collapses) with the environmental factor (i.e. the maximum hydrostatic pressure).
3. To explore the adaptation of Planktothrix populations to the depth of winter circulation
in different systems, we collected 276 strains of P. cf. rubescens from eight lakes (zmax = 24–
410 m) in Northern Italy during summer 2009 and we analysed the multicopy gene gvpC
coding for a protein that crucially influences the critical pressure.
4. The strains analysed clustered into two main groups having gas vesicles with a mean
critical pressure of 1.1 and 0.9 MPa, respectively. The proportion of the stronger strains
was generally positively related to lake depth, although the overall pattern was
complicated by individual lake morphology and hydrology. The relative frequency of
stronger filaments was (i) greatest in deep basins with concave slopes and (ii) least in one
deep, but permanently stratified lake.
5. The simultaneous presence of ‘weaker’ and ‘stronger’ filaments could allow for a rapid
adaptive response to changes in hydrostatic pressures, related to changes in the amplitude
of vertical circulation characterising deep lakes.
Keywords: adaptation, cyanobacteria, gas vesicles, Planktothrix, selective processes
Introduction
The microcystin-producing Planktothrix Anagnostidis
& Komarek, 1988 (Christiansen et al., 2003; Komarek
& Anagnostidis, 2005) is a widespread planktonic
filamentous cyanobacterium adapted to deep oligo-
mesotrophic and thermally stratified lakes, where
it produces concentrated metalimnetic populations
during summer and autumn (e.g. Halstvedt et al.,
2007; Kaiblinger et al., 2007; Salmaso, 2010).
The ecological success of Planktothrix species
depends largely on gas vesicles, which provide the
cells with buoyancy (Walsby, Avery & Schanz, 1998;
Correspondence: Domenico D’Alelio, IASMA Research and
Innovation Centre, Fondazione Edmund Mach, Via E. Mach, 1,
I-38010, S. Michele a ⁄ Adige, Trento, Italy
E-mail: domenico.dalelio@iasma.it
Freshwater Biology (2011) 56, 1481–1493 doi:10.1111/j.1365-2427.2011.02584.x
� 2011 Blackwell Publishing Ltd 1481
Walsby & Schanz, 2002; Walsby, Schanz & Schmid,
2006). By regulating their buoyancy in response to the
vertical light field, Planktothrix filaments are able to
dwell in the metalimnion of lakes that are thermally
stratified in summer. The gas vesicles, which are
hollow cylindrical structures, can withstand moderate
pressure but collapse flat at a certain ‘critical pressure’
(pc) and no longer provide buoyancy. The main sources
of pressure that together cause gas vesicles to collapse
are cell turgor pressure, usually 0.2–0.4 MPa (during
winter and summer, respectively), and hydrostatic
pressure of the water column, which approximately
increases by 0.01 MPa per metre. The critical pressure
of individual gas vesicles within any cell varies around
a well-defined mean value, the ‘mean critical pressure’
(mean pc), which varies in different cyanobacteria from
0.3 MPa to more than 1.1 MPa (Walsby, 1994). Strains
with gas vesicles of the highest critical pressure are
found in the deepest lakes (e.g. Walsby et al., 1998).
The critical pressure of a gas vesicle varies inversely
with its cylindrical diameter, and hence, narrow gas
vesicles withstand higher pressures though provide
less buoyancy (Hayes, Buchholz & Walsby, 1992;
Bright & Walsby, 1999). It has been suggested that
differences in the width, and hence the critical
pressure, of gas vesicles are explained by differences
in the amino acid sequence of the structural proteins
forming the gas vesicle (Walsby, 1994; Kinsman,
Walsby & Hayes, 1995; Oliver & Ganf, 2000). GvpA
proteins (encoded by the gvpA gene) form the ribs
sustaining the main cylinder and show little variation.
GvpC proteins (encoded by gvpC) form an outer layer;
they show several variants that are correlated with
variations in gas vesicle width and strength. In
Planktothrix species, there are three principal variants
of gvpC – gvpC16, gvpC20 and gvpC28 – encoding GvpC
proteins of molar mass 16, 20 and 28 kDa, respectively
(Beard et al., 1999, 2000). The gene variants gvpC16 and
gvpC28 (the shorter and the longer, respectively)
originate from gvpC20 by the deletion of a 99 bp
section and by the insertion of a 219 bp section,
respectively (Beard et al., 1999, 2000). The gas vesicles
constructed from the three different GvpC variants of
increasing molar mass have been shown to be of
different widths and have decreasing pc – in the region
of approximately 1.1, 0.9 and 0.7 MPa (cf. Bright &
Walsby, 1999). Moreover, the contemporary presence
of multiple gvpC variants in Planktothrix has an effect
on the relative strength of the gas vesicles: for
instance, strains having both the gvpC16 and gvpC20
variants and those having only gvpC28 ones were
demonstrated to be able to produce gas vesicles of 1.1
and 0.7 mean pc, respectively. Strains of the first type
are dominant in deep Lake Zurich (zmax = 136 m),
while those having the sole gvpC28 variant have been
detected only in shallower lakes of Northern Europe
(zmax = 8–67 m) (Beard et al., 2000). The correlation
between genotype and mean pc is so high that the
genotype can be used as diagnostic of critical pressure
(Beard et al., 1999, 2000).
We have investigated the relationship between
Planktothrix strains of different gas vesicle critical
pressure and mixing depth in a set of lakes in Northern
Italy. Although the correlation between genotype
(gvpC sequence) and phenotype (mean pc) has been
demonstrated only for the Planktothrix population in
Lake Zurich, we assumed that it also holds for
populations in Italian lakes. In order to test the
hypothesis that the strength of gas vesicles and mixing
depth in winter are related, we (i) isolated 276 strains of
Planktothrix from the summer phytoplankton commu-
nity of eight Italian subalpine lakes differing in
maximum depth, morphometry and dynamics of
vertical water circulation; (ii) amplified portions of
the gvpC gene variants by means of diagnostic PCRs
and used the amplicons as markers of gas vesicle
phenotype and (iii) related the relative abundance of
strains belonging to different genotypes ⁄phenotypes to
lake characteristics. We also considered the role of lake
morphometry in gas vesicle selection: for instance, a
concave shape to the lake basin yields a relatively high
volume of water (along with the entrained plankton)
that can be circulated towards the lake bottom, while
the reverse is true of conical lakes. This aspect could be
crucial to the dynamics of gas vesicles selection in
Planktothrix populations. Furthermore, we hypothe-
sised that filaments with weaker gas vesicles would be
more abundant in lakes where the littoral zone is
sufficient for a large portion of the Planktothrix popu-
lation to remain above the critical pressure depth and
thus avoid a loss of buoyancy during annual mixing.
Methods
Study sites
We investigated eight natural lakes in Northern Italy
(Fig. 1). Lakes Como, Garda, Iseo, Maggiore and the
1482 D. D’Alelio et al.
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northern basin of Lake Lugano are large and deep;
Idro, Levico and Pusiano are smaller and shallower.
The large Lakes Como, Maggiore and Garda are
oligomictic, with spring mixing involving an epilim-
nion never <100 m and complete circulation occurring
irregularly during exceptionally cold winters (Buzzi,
2002; Ambrosetti, Barbanti & Carrara, 2010; Salmaso,
2010). The other two large and deep lakes, Iseo and
Lugano (northern basin), tend to be meromictic
because of very strong chemical gradients through
the water column favouring permanent stratification.
Nonetheless, they underwent complete winter mixing
in 2005 and 2006 (Salmaso et al., 2007; Holzner et al.,
2009), and the mixed depth was never <80 m in the
following years (for Iseo: Letizia Garibaldi pers.
comm.; for Lugano: Marco Simona pers comm., see
also http://www.cipais.org/html/lago-lugano-stato.
asp). The mean depth of the mixed layer in these
five deep lakes during the period 2005–09 ranged
from 120 to 211 m (Table 1). Lake Idro is smaller but
nevertheless deep and has not undergone a complete
overturn since the 1960s (Garibaldi et al., 1996, 1997;
Garibaldi personal comm.). The mixed layer depth is
always around 50 m in Lake Idro (Table 1). The
shallow Lakes Levico and Pusiano are dimictic and
monomictic, respectively (Copetti et al., 2006; Perini
et al., 2009). The study lakes have different morpho-
metrics and steepness, which are reflected in their
‘volume development’, Vd (Hakanson, 1981), a para-
meter estimating the departure of the shape of a lake
basin from a cone (Vd = 1). Lake volume development
was calculated as three times the ratio between mean
depth (zmean) and maximum depth (zmax) (Hakanson,
1981). The volume of the littoral zone at a specific
depth was calculated by the formula: ðh=3ÞðA1þ A2þffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
A1 � A2p
Þ � A2h, where ‘h’ is the vertical depth of a
stratum, A1 the area of the upper surface and A2 the
area of the lower surface of the stratum whose volume
is to be determined (Wetzel, 2001).
Sampling and culturing
All samples were taken in summer 2009. For each
lake, temperature and oxygen profiles were taken at
Fig. 1 Location of the study sites in Northern Italy.
Table 1 Geographic, morphometric and hydrological characteristics of the lakes studied
Lake
Latitude
N
Longitude
E
Altitude
h
(m)
Area
A
(km2)
Maximum
depth
zmax
(m)
Mean
depth
zmean
(m)
Volume
V
(km3)
Relative
depth
zr
(%)
Volume
development
Vd
Renewal
time s(year)
Mixed
depth†
(m)
Como 46�00¢ 09�15¢ 198 146 410 154 22.05 3 1.13 4.5(1) 190(6,13)
Maggiore 45�58¢ 08�39¢ 193 213 370 178 37.50 2 1.44 4.1(1) 120(6,7)
Garda 45�42¢ 10�43¢ 65 368 350 133 49.00 1 1.14 26.6(2) 211(6,8)
Lugano* 45�58¢ 08�59¢ 271 28 288 171 4.07 4 1.78 12.4(3) 163(9,10)
Iseo 45�44¢ 10�04¢ 186 62 251 123 7.06 2 1.47 4.1(1) 148(11,12)
Idro 45�46¢ 10�32¢ 368 11 120 65 0.09 3 1.62 1.2(4) 50(12)
Levico 46�00¢ 11�17¢ 440 1 38 11 0.01 3 0.88 1.1(5) 38
Pusiano 45�48¢ 09�16¢ 259 4 24 14 0.07 1 1.75 0.7(4) 24
*Refers to the Northern basin of Lake Lugano. †Average of the maximum depth of the mixed layer over the period 2005–09. (1)Ambrosetti,
Barbanti & Sala (2003). (2)Ambrosetti & Barbanti (1997). (3)Barbieri & Polli (1992). (4)Ambrosetti & Barbanti (1992). (5)Perini et al. (2009).(6)Salmaso & Mosello (2010 and references therein). (7)Ambrosetti et al. (2010). (8)Salmaso (unpublished) (9)Holzner et al. (2009). (10)Marco
Simona (pers. comm.) and website (in Italian): http://www.cipais.org/html/lago-lugano-stato.asp. (11)Salmaso et al. (2007). (12)Letizia
Garibaldi (pers. comm.) (13)Working Group Lake Como (2006). The mixed layer depths in shallow Lakes Levico and Pusiano were
considered equivalent to the respective lake depth according to Perini et al. (2009) and Copetti et al. (2006), respectively.
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1 m intervals with an Ocean Seven 316 multiparameter
probe (Idronaut, Milan, Italy) or a SD4a probe
(Hydrolab, Austin, TX, U.S.A.) in order to identify
the metalimnion. The phytoplankton community was
than sampled: (i) at discrete depths (every 10 m from
0 to 60 m in deep lakes; every 5 m from 0 to 20 m in
shallow lakes) with a Niskin bottle; (ii) along an
integrated water column (from the base of the meta-
limnion up to the surface of the water column) with a
Schroder sampler and a 40-lm plankton net. The use
of multiple devices was intended to maximise the
number of Planktothrix filaments collected. Single
Planktothrix filaments were isolated by Pasteur micro-
pipette and washed at least three times with sterile
lake water; monoclonal cultures were incubated in
standard BG-11 medium (Stanier et al., 1971) at 20 �C,
30 lmol photon m)2 s)1 and 12 : 12 h = L : D photo-
cycle. Filaments were identified by morphological
criteria according to Komarek & Anagnostidis (2005).
For molecular analyses, a 1 mL aliquot of a dense
culture of each strain was mixed with proteinase K
(0.2 mg mL)1), incubated for 15 min at 55 �C and
subsequently for 15 min at 80 �C; the supernatant was
collected after brief centrifugation, as described by
Beard et al. (2000).
PCR amplification and sequencing
The gvpC region of each strain was characterised by a
single PCR containing three Planktothrix-specific oli-
gonucleotides, as described in Beard et al. (2000): a
forward primer, GVPC9 (ACGCCAACAGGGAGT
TCAAGAACG) and two alternative reverse primers,
GVPC1B (TGGGAACTCCCCCAAACTGTCTG) and
GVPC11 (GGCTACCCACAAACGCTCGGTG), with
the latter specific for the gvpC28 variant (Fig. 2a). Each
amplification was performed in a total volume of
25 lL, containing 5 lL of the cell lysate as a template,
1.5 mMM MgCl2, 200 lMM of dNTP, 1 lMM of each primer,
1· Gold Buffer (Applied Biosystems, Foster City, CA,
U.S.A.) and 1 U of AmpliTaq Gold (Applied Biosys-
tems). PCRs were subjected to an initial denaturation
step at 94 �C for 4 min, followed by 30 amplification
cycles at 94 �C for 45 s, 65 �C for 1 min and 72 �C for
1 min, and by a final elongation step of 72 �C for
5 min, in a Master Cycler Gradient (Eppendorf AG,
Hamburg, Germany). Amplicons were separated by
electrophoresis on a 2% w/v agarose gel prepared in
Tris ⁄Borate ⁄EDTA buffer, stained with ethidium
bromide, and their size was estimated under UV
light by comparison with a known DNA standard
(MassRuler� DNA Ladder Mix and MassRuler� Low
Range DNA, Fermentas International Inc., Burlington,
ON, Canada).
To check for correspondence between observed and
expected size for different amplicons, selected bands
were cut from the gel, purified with the MinElute Gel
Extraction kit (Qiagen, Hilden, Germany) and
sequenced in both directions; the same primers as in
the PCR (with either GVPC1B or GVPC11 depending
on the observed band size) were used with the BigDye
Terminator Cycle Sequencing technology (Applied
Biosystems), according to the manufacturers’ proto-
cols and recommendations. After purification in
A C20 A
A C16 A
A AC28
303 bp
402 bp
181 bp
C9 C1B
C9 C1B
C9 C11
181 bp
402 bp303 bp
1 2 3 4(b)(a)
Fig. 2 Gas vesicle gene gvpC in Planktothrix rubescens. (a) Portion of the gas vesicle gene cluster with alternating gvpA and gvpC genes
(modified from Beard et al., 2000); boxed regions indicate open reading frames, lines below boxes indicate the length of the fragment
amplified by the primers GVPC9, GVPC1B and GVPC11 specific for gvpC (Beard et al., 2000). (b) Electrophoresis gel (2%, w ⁄ v) of PCR
products for the amplification of gvpC in Planktothrix strains from Lake Como: lane 1, strain FEM_DD219, single amplicon of size
402 bp representing the variant gvpC20; lane 2, strain FEM_DD220, two amplicons of size 402 and 303 bp representing the variants
gvpC20 and gvpC16, respectively; lane 3, FEM_DD221, two amplicons of size 402 and 181 bp representing the whole variant gvpC20 and
a portion of gvpC28; lane 4, size standard.
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automation using the Millipore Montage SEQ96
Sequencing Reaction Cleanup kit (Millipore Corporate,
Billerica, MA, U.S.A.), products were run on an
Automated Capillary Electrophoresis Sequencer
3730XL DNA Analyser (Applied Biosystems). Forward
and reverse chromatograms were assembled using
ChromasPro (Version 1.49 Beta 2) (Technelysium Pty
Ltd, Tewantin, QLD, Australia).
Data analysis
Text format sequences were first automatically
aligned using the application CLUSTALLUSTALW (Thompson,
Higgins & Gibbson, 1994) included in the program
BioEdit 7.01 (Hall, 1999); the final alignment was then
improved by hand. Regression analysis (Sokal &
Rohlf, 1995) was used to relate different lake morpho-
metrics characteristics to the frequency of different gas
vesicles types. All procedures were run in Statistica 9.1
for Windows (Statsoft Inc., Tulsa, OK, U.S.A.).
Results
Vertical temperature and oxygen profiles were con-
sidered as markers for the localisation of the meta-
limnion and the associated phytoplankton community
(Fig. 3). The survival rate of the different isolates in
laboratory conditions was not identical. For instance,
Fig. 3 Vertical profiles of temperature and dissolved oxygen concentration of the lakes studied. Profiles to 100 m or to the bottom
(for shallow lakes) are presented. The name of the lake and the sampling date are indicated on each graph. For Lake Maggiore, only
the first 50 m of the water column was sampled.
Differential selection in Planktothrix 1485
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for lakes Como, Idro, Lugano and Pusiano, only
strains isolated from integrated samples were brought
successfully into culture (Table S1). Ultimately, cul-
tures of 276 monoclonal strains of Planktothrix yielded
a biomass sufficiently dense for genetic analyses. All
these strains were identified as Planktothrix cf. rubes-
cens according to standard taxonomic keys (included
in Komarek & Anagnostidis, 2005) – e.g. the arrange-
ment of trichomes (long and straight filaments of cells
showing dense aggregates of gas vesicles), the shape
of apical cells (rounded and sometimes bearing
calyptra), colour (absence of green pigmentation and
tendency to red water discoloration in dense cultures)
and morphometrics (cell width ‡ 6 lm) (Fig. 4).
Fingerprinting analysis of gvpC
Three nucleotide fragments, accounting for about 130,
300 and 400 base pairs (bp), were amplified by PCRs
in the cultures. Nucleotide sequences were derived
from amplicons detected in selected cultures from
Lake Como (Fig. 2b). The �130 bp amplicon corre-
sponded to a 128 bp portion of gvpC28 of the
P. rubescens strain PCC7821 (GenBank accession num-
ber AJ253128; Beard et al., 2000) coding for the gas
vesicle protein GvpC28, the � 300 bp fragment corre-
sponded to a 303 bp portion of gvpC16 in the strains
BC-Pla 9401, BC-Pla 9401 ⁄a and BC-Pla 9736
(GenBank accession numbers AJ132354, AJ494990
and AJ238352, respectively; Beard et al., 1999) and
the � 400 bp amplicon corresponded to a 402 bp
portion of gvpC20 in the P. rubescens strain BC-Pla 9303
(GenBank acc. n. AJ132357; Beard et al., 1999).
Based on the combinations of the gvpC variants
detected by gel electrophoresis, the isolates clustered
into four groups. For simplicity, here we indicate the
genotype showing the sole gene variant gvpC20 as
‘C20’, the one showing solely the gene variant gvpC16
as ‘C16’, the one showing gvpC16 and gvpC20 as
‘C16 + C20’ and the one showing gvpC20 and gvpC28
as ‘C20 + C28’. The vast majority of strains belonged to
the genotypes C20 and C16 + C20, two strains to the
type C16 (FEM_DD265 and )281, Lake Pusiano) and
only one strain to the type C20 + C28 (strain
FEM_DD221, Lake Como) (Table S1; Fig. 5). The type
C20 was dominant in lakes Idro (38 of 40 strains) and
Levico (38 of 51), while C16 + C20 was dominant in
Lakes Iseo (40 of 43), Lugano (23 of 24), Maggiore
(13 of 14) and Pusiano (28 of 41) (Fig. 5). In Lakes
Garda and Como, strains were almost evenly divided
between the genotypes C20 and C16 + C20 (Fig. 5).
No significant correlation was found between the
frequency distribution of the gas vesicles genotypes
and the depth at which strains were sampled.
Nucleotide diversity in selected strains (Table S1)
was investigated in the amplicons referring to gvpC16
and gvpC20, the most frequent variants in our cultures.
Besides an insertion ⁄deletion between positions 183
and 281 in our alignment (available upon request),
differentiating the variant gvpC20 from gvpC16, three
single-nucleotide polymorphisms (SNPs) were found
in the gvpC20 variant. Similar to the strain BC-Pla 9303
(GenBank acc. n. AJ132357), all our sequences showed
a guanine instead of the cytosine present in other
P. rubescens strains (e.g. BC-Pla 9736, AJ238352) at
position 155. The other two SNPs were derived from a
point mutation in one sequence each: a transition in
the gvpC20 of strain FEM_DD389 (GenBank acc. n.
HM595037) (C versus T at positions 168) and a
transversion in the gvpC20 of strain FEM_DD212
(T versus A at position 401) (GenBank acc. n.
HM595039). Non-synonymous substitutions in the
referring amino acid sequence were induced by SNPs
at positions 155 (G instead of C fi Arg instead of
Thr) and 401 (T instead of A fi Phe instead of Tyr).
An ambiguity as an incompletely specified base
(Y = thymine or cytosine, at position 184) was also
(a)
(b)
(c)
Fig. 4 Light microscopy microphotographs of Planktothrix cf.
rubescens from North Italy lakes. Cluster of living filaments (a)
Extremities of a single filament with a (b) caliptra end and a (c)
round end.
1486 D. D’Alelio et al.
� 2011 Blackwell Publishing Ltd, Freshwater Biology, 56, 1481–1493
found in strains FEM_DD129 and FEM_DD137
(GenBank acc. n. HM595038), probably because of
the simultaneous presence of mutated and unmutated
copies of gvpC20 within the same genome. This
nucleotide ambiguity resulted in the contemporary
presence of two potential amino acid sequences
having either Arg or Cys in the same position.
Gas vesicle genotype frequencies versus lake
characteristics
Gas vesicle genotype frequencies were compared to
hydrodynamic and morphometric characteristics of
the lake basins (Table 1). A weak correlation between
the relative frequency of the two most frequent
genotypes (the ratio [C20] ⁄ [C16 + C20]) and the mixed
depth (averaged over the period 2005–09) was
observed (r = 0.65, P = 0.06, n = 7; Fig. 6a). Lake
Pusiano, an outlier in our data set, was excluded in
this correlation. The portion of C20 strains (i.e. the
weaker ones; Table 2) was higher in lakes in which the
mixed depth during winter (over the period 2005–09)
was within 50 m (namely, Levico and Idro). On the
other hand, the fraction of strains C16 + C20 (i.e. the
relatively stronger ones, Table 2) was higher in those
lakes in which, over the same time period, the average
mixed depth during winter exceeded 100 m (Como,
Garda, Iseo, Lugano, Maggiore). Since, in these deep
lakes, the mixed depth was higher than the critical
depths of both genotypes under examination
(Table 2), every change in [C20] ⁄ [C16 + C20] should
have been caused by factors other than the maximum
depth of the mixed layer (e.g. lake volume develop-
ment, proportion of the littoral zone within the gas
vesicles critical depth). In fact, the higher the Vd, the
more concave is the lake basin and the greater is the
volume of the pelagic compared to the littoral at
distinct depths, calculated in this study for mono-
basin lakes (i.e. Garda, Lugano, Maggiore and Zurich)
(Fig. 7). Thus, for these lakes, we correlated the ratio
[C20] ⁄ [C16 + C20] to volume development Vd (an inte-
grated index for lake morphometry), also including
data from Beard et al. (1999) for a population of
Planktothrix from Lake Zurich (zmax = 136 m). The
correlation was statistically well supported (r = 0.95,
P = 0.002, n = 6) (Fig. 6b): strains of genotype C20
were almost absent in lakes with a Vd approaching 2
(i.e. with a more concave basin), while they were
almost in the same proportion as strains of type
C16 + C20 in lakes with a Vd approaching 1 (with a
conical basin). Finally, we correlated [C20] ⁄ [C16 + C20]
with the proportion of the lake volume accounted for
by the littoral zone within 70 m of the surface (the
critical depth for strains of type C20) and we found a
strong correlation (r = 0.89, P = 0.06, n = 4), although
there are few data because reliable volumetric indices
were available for mono-basin lakes only (Fig. 6c).
20406080
100A
bund
ance
(%)
20406080
20406080
20406080
20406080
20406080
20406080
20406080
0
Maggiore zmax = 370 m
V d = 1.44n = 14
Luganozmax = 288 m
V d = 1.78n = 24
Iseozmax = 251 m
V d = 1.47n = 43
Como zmax = 410 m
V d = 1.13n = 47
Gardazmax = 350 m
V d = 1.14n = 16
Idrozmax = 120 m
V d = 1.62n = 40
Levicozmax = 38 m
V d = 0.88n = 51
Pusianozmax = 24 m
V d = 1.75n = 41
C16 C20 C16 +C 20 C20 + C28
Gas vesicle genotypes
Fig. 5 Planktothrix cf. rubescens strains belonging to different gas
vesicle genotypes from lakes in Northern Italy. Grey and black
boxes represent the percentage (%) of strains in the groups C20
and C16 + C20, which are able to produce weaker and stronger
gas vesicles, respectively; white boxes represent the percentage
of strains in the groups C16 and C20 + C28; ‘zmax’ and ‘Vd’ are,
respectively, the maximum depth and the volume development
of each lake; ‘n’ is the number of strains analysed in the corre-
sponding lake.
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The frequency of C20 strains was greater in lakes with
a higher proportion of littoral zone within the top
70 m (the absolute critical depth of these strains).
Discussion
Natural populations of Planktothrix cf. rubescens living
in lakes of the Italian subalpine district were repre-
sented by two main genotypes producing gas vesicles
with different resistance to the hydrostatic pressure.
The filaments with stronger gas vesicles were more
frequent in those lakes where the mixed layer depth
during winter–spring is higher than the maximum
depth at which each of the two kinds of gas vesicles
can resist. The filaments with stronger gas vesicles
were even more frequent in lakes with highly concave
basins – i.e. in which vertical mixing involves a larger
volume of water. Although we isolated a limited
number of strains per lake (n £ 50), we are confident
that our sampling was representative of Planktothrix
populations actually present at that time. In each lake,
Planktothrix dominated the phytoplankton in the
metalimnion at the time of our sampling. Each
population could be considered (i) concentrated in a
narrow water layer and (ii) homogeneously distri-
buted horizontally within this layer.
We explain the higher frequency of Planktothrix
strains with stronger gas vesicles (higher critical
pressure, or pc) in deep lakes undergoing annual
circulation (Fig. 6a) as an effect of the strong positive
selection brought about by vertical mixing in these
basins. Strong natural selection for strains with higher
pc is expected in deeper lakes because the higher
hydrostatic pressure at greater depths will collapse
more of the gas vesicles with lower pc; therefore,
filaments lose their buoyancy and ability to remain
and grow in the metalimnion. The selection of gas
vesicle pc is not constant over the entire annual cycle
(Walsby et al., 1998). During summer, Planktothrix is
restricted to the metalimnion. This settling is explained
by buoyancy regulation in relation to irradiance
(Walsby et al., 2004). For a population living in a
metalimnion spanning, during summer, 5–15 m
depth, the combination of hydrostatic pressure
(<0.15 MPa) and turgor pressure (<0.4 MPa) would
produce a total pressure (�0.55 MPa) that is less than
the lowest pc (>0.6 MPa) (Table 2) and, therefore, not
sufficient to cause gas vesicle collapse and cell death.
Filaments become entrained in the surface mixed layer
1 1.2 1.4 1.6 1.8 20.01
0.1
1
LuIsMa
Ga
Co(b)
[C20
]/[C16
+ C
20]
Vd
Zü
50 100 150 200 250
0.1
1
10
[C20
]/[C
16+
C20]
Mixed depth (m)
Le
Id
Co
Ga
Ma
Is
Lu
(a)
10 20 30 40 50
0.1
1(c)
ZüGa
Lu
[C20
] /[C
16 +
C20
]
Littoral zone within 70 m (%)
Ma
Fig. 6 Gas vesicle selection versus lake characteristics (note
Log ⁄ Log scales). Correlation between the relative frequency of
the two gas vesicle genotypes as the ratio ‘[C20 strains] ⁄ [C16 +
C20 strains]’ and (a) the depth of the mixed layer during
winter (average over the time period 2005–09; see also Table 1),
(b) the volume development (Vd) and (c) the portion of the
littoral zone within 70 m (expressed as % of volume). Open
circles are data points in all graphs, dashed lines represent the
power functions y = 11 000x)2 (r = 0.65, P = 0.06), y = 1.3x)6.5
(r = 0.95, P = 0.002) and y = 0.0006x2 (r = 0.89, P = 0.06)
approximating the relationships in (a), (b) and (c), respectively.
In the three graphs, ‘Co’ refers Lake Como, ‘Ga’ to L. Garda,
‘Id’ to L. Idro, ‘Is’ to L. Iseo, ‘Le’ to L. Levico, ‘Lu’ to L. Lugano,
‘Ma’ to L. Maggiore, ‘Pu’ to L. Pusiano, ‘Zu’ to L. Zurich.
1488 D. D’Alelio et al.
� 2011 Blackwell Publishing Ltd, Freshwater Biology, 56, 1481–1493
during the autumn and are then mixed progressively
deeper as the lake cools. After exposure to deep
mixing during winter, selection occurs during spring.
In fact, filaments that retain their gas vesicles, and
thereby their buoyancy, will float to the newly
reformed metalimnion and will form the inoculum
for the next season’s growth. Clearly, in very shallow
lakes, all of the gas vesicles can survive, even those
with the lowest critical pressure. Some of these will
survive even in the deepest lakes, because they happen
to remain in the top few metres at the shallow fringes
of the lake. Three factors (maximum depth, depth of
mixing and the proportion of surface water mixed to
various depths) determine whether or not a filament
retains sufficient gas vesicles to provide buoyancy (i.e.
gas vesicles selection). In general, the deeper the lake
and the greater the degree of circulation, the more
likely only gas vesicles of high pc will survive.
Functional genetics of gas vesicles
The strength of the gas vesicles in the genus Plankto-
thrix, estimated by measuring the pressure necessary
to collapse them, has been related to the peculiar
configuration of composite gene clusters including
numerous copies of the main genes, gvpA and gvpC
(Beard et al., 1999, 2000). Because of insertions and
deletions in sequence repeats, gvpC shows different
and sometimes co-occurring variants (namely, gvpC16,
gvpC20, gvpC28) coding for proteins with fairly differ-
ent molecular weights. These variants are effectively
transcribed in single filaments (Becker, Hayes &
Walsby, 2005), even though no study on potential
post-transcriptional regulations of these genes at
different pressures has been carried out. However,
owing to the congruence between genetic and pheno-
typic data collected to date, the pattern of the gas
vesicle gene is a marker for the strength of gas vesicles
in Planktothrix. In the strains we sampled, the gene
Vd > 1
Vd < 1
Vd = 1
(a)
(b)
Vd
Litto
ral z
one
(%)
45
5
25
1.81.0 1.4
(c)
Zü
Ga
Ma Lu
55
35
15
1.1 1.3 1.5 1.6 1.71.2
Fig. 7 Schematic lake morphometry parameters. (a) The shape
of a basin in relation to the volume development (Vd): the solid,
dotted and dashed line drawings represent conical, convex and
concave basins for which Vd is 1, <1 or >1, respectively. (b) The
extent of the littoral zone in three basins with different shape but
the same maximum depth: the unbroken and the dashed grey
areas represent, respectively, the portion of littoral and pelagic
zones within a total depth. The ratio between littoral and pelagic
zones is higher or lower in lake with convex or concave basins,
respectively. (c) The relationship between the portion of littoral
zone (expressed as % of volume at a generic depth) and the
volume development: open triangles and circles indicate data
points for the littoral zones within the depths of 70 and 100 m.
‘Ga’ refers to Lake Garda, ‘Lu’ to L. Lugano, ‘Ma’ to L. Maggiore
and ‘Zu’ to Lake Zurich.
Table 2 Correspondence between Planktothrix gas vesicles genotypes, phenotypes and critical depth in summer (cell turgor pres-
sure = 0.4 MPa) and winter (cell turgor pressure = 0.2 MPa). Modified from Bright & Walsby (1999) and Beard et al. (2000)
gvpC
variants
Gas vesicles
genotype*
Mean pc of
genotype class (MPa)
Min–max pc
(MPa)
Summer critical
depth (m)
Winter critical
depth (m)
C20 GV1 or -2 0.9 0.8–0.9 40–50 60–70
C16 + C20 GV3 1.1 1.0–1.2 60–80 80–100
C20 + C28 GV4 0.9 0.8–1.0 40–60 60–80
C28 GV5 or -6 0.7 0.6–0.7 20–30 40–50
*sensu Beard et al. (2000).
Differential selection in Planktothrix 1489
� 2011 Blackwell Publishing Ltd, Freshwater Biology, 56, 1481–1493
gvpC was present in all the three variants mentioned
previously. The vast majority of monoclonal strains
had either only gvpC20 or both gvpC16 and gvpC20.
Following the nomenclature used by previous
authors, the former strains should belong to geno-
types GV1 and GV2 sensu Beard et al. (1999, 2000), in
which the ratio gvpA : gvpC20 was 1 : 1 and 4 : 3,
respectively, as detected by PCR (Beard et al., 2000)
and quantitative PCR (Becker et al., 2005). This gene
configuration was suggested to be at the basis of the
production of gas vesicles with mean pc of 0.9 MPa
(critical depth £ 70 m) detected in both genotypes.
Moreover, the few GV1 strains (n = 3), detected only
in Lake Zurich, showed a resistance to pressure
comparable to GV2s (Table 2; Bright & Walsby,
1999). For this reason, we considered all strains
showing the sole variant gvpC20 as belonging to the
same genetic ⁄phenotypic unit, without distinguishing
between GV1 and GV2. On the other hand, strains
showing both gvpC16 and gvpC20 should belong to the
genotype GV3 sensu Beard et al. (1999, 2000). Here, the
ratio gvpA : gvpC16 : gvpC20 was about 6 : 3 : 1, and
the high frequency of the short variant gvpC16 was
thought to promote narrow gas vesicles with mean pc
of 1.1 MPa, therefore resisting depths £ 100 m
(Table 2) (Beard et al., 2000; Becker et al., 2005). Only
one of our 276 strains had both gvpC20 and gvpC28,
which should belong to the genotype GV4 sensu Beard
et al. (2000), also producing gas vesicles with mean pc
of 0.9 MPa (critical depth £ 80 m). We found no
strains having the sole long allele gvpC28, whose
presence in the genotypes GV5 and GV6 sensu Beard
et al. (2000) was related to the production of wide gas
vesicles with mean pc of 0.7 MPa (critical depth £ 50 m).
However, this gene arrangement seems to be present
most frequently in green strains of Planktothrix
(namely, P. cf. agardhii; see also Beard et al., 2000),
while we isolated only reddish Planktothrix strains
(namely, P.cf. rubescens). Finally, we found two strains
showing only gvpC16, which cannot be associated with
any known genotype but, following the criteria
mentioned previously, they would be characterised
by very narrow and strong gas vesicles.
Gas vesicle selection versus circulation
Lakes Iseo, Lugano and the shallower Idro represent
interesting case studies that provide clues to the effect
of circulation on gas vesicle selection in Planktothrix.
Lake Idro shows a permanent chemolimnion at 50 m
(Garibaldi et al., 1996, 1997; Letizia Garibaldi pers.
comm.). A survey conducted during winter 2010
confirmed this vertical stratification, with convective
mixing limited to the first 50 m of the water column
(unpublished data from our group). In this lake,
filaments with a maximum critical depth of 70 m
(genotype C20) are more frequent than those with a
maximum critical depth of 100 m (genotype
C16 + C20). However, the depth of this lake
(zmax = 120 m) would suggest natural selection in
favour of the C16 + C20 genotype. We explain this
apparent contradiction by the limited winter mixing
that could have entrapped most Planktothrix filaments
for many years within a shallow layer. This layer
(0–50 m depth) was always less than the critical depth
at which the weaker filaments usually lose their
buoyancy (70 m). The consequent relaxed selection
(i.e. persistent absence of selective pressure to resist
collapse) could have favoured the dominance of the
weaker filaments rather than the stronger ones
(Fig. 6a). Conversely, a long-lasting stratification
persisted until winter 2005 in Lakes Iseo and Lugano
N-Basin, but it collapsed because of the deep overturn
events occurring in 2005 and 2006 (Salmaso et al.,
2007; Holzner et al., 2009): these bottleneck events
could have promoted the selection of stronger fila-
ments (max critical depth = 100 m) and the removal
of weaker filaments (max critical depth = 70 m)
(Fig. 6a). On the other hand, in the years following
the 2005–06 overturns, the mixed depth was never less
than 80 m (for Iseo: Letizia Garibaldi pers. comm.; for
Lugano: Marco Simona pers. comm.; see also http://
www.cipais.org/html/lago-lugano-stato.asp), and
thus, higher than the critical pressure for weaker
filaments. This condition should have reduced recruit-
ment of weaker filaments. Unfortunately, we have no
information about the frequency of gas vesicle geno-
types before the 2005 winter mixing to compare with
the situation we depict.
Differential selection in large and deep lakes
Como, Garda, Iseo, Lugano and Maggiore are among
the deepest European lakes, and the depth of the
mixed layer averaged over the 5 years preceding our
study was never <120 m in these basins (Table 1).
In all these lakes, while the depth of mixing is strongly
influenced by climatic features and water-column
1490 D. D’Alelio et al.
� 2011 Blackwell Publishing Ltd, Freshwater Biology, 56, 1481–1493
stability, the proportion of surface water mixed to
various depths depends on lake morphometry. The
most important features are the shape of the basin
(slope steepness) and concavity of the lake bed, both
of which are reflected in ‘volume development (Vd)’.
In this study, we found a significant correlation
between Vd and the abundance of gas vesicle geno-
types in Planktothrix populations living in large and
deep lakes (Fig. 6b). In Lakes Como and Garda
(Vd < 1.20), the abundance of genotypes C20 and
C16 + C20 was approximately equal, as in deep Lake
Zurich (zmax = 136 m; Vd = 1.03) (Beard et al., 1999;
Bright & Walsby, 1999). The congruency between
physical and biological data in these three systems
suggests the existence of similar environmental
stressor versus adaptive response mechanisms. Con-
versely, in Lakes Maggiore, Iseo and Lugano
(Vd > 1.4), the genotype C16 + C20 was dominant with
respect to C20. The trade-off between the two geno-
types seems to lay in the range 1.2 < Vd < 1.4 and it
could be explained mechanistically. For instance, the
genotype C20 loses buoyancy at depths > 70 m
(Table 2). The survival rate of C20 filaments should
be proportional to the probability that they are not
transported below that depth. Theoretically, this
probability increases with an increase in the propor-
tion of littoral domain within 70 m from the surface.
This condition could be maximal in the very conical
Lake Zurich (Vd = 1.03), where the volume of fringes
within 70 m accounts for 44% of the lake volume
above that depth (Fig. 7). This percentage decreases to
22% in Lake Garda (Vd = 1.14) and 11% in Lake
Maggiore (Vd = 1.44). Remarkably, the decrease in C20
strains with an increase in Vd appears to accord with a
decline in the proportion of the lake volume
accounted for by the littoral zone (at depths below
70 m) with an increase in the same Vd (compare
Figs 6c and 7). Conversely, an increase in C16 + C20
strains seems to be directly related to higher Vd. Our
results suggest that lake morphometry, by determin-
ing the proportion of surface water mixed to different
depths, is likely to influence gas vesicle selection in
those lakes where the depth of the mixed layer
exceeds the critical depth of the strongest gas vesicles.
Differential selection in shallow lakes
Finally, the two shallower lakes (Levico and Pusiano)
showed contrasting patterns in the frequencies of
Planktothrix gas vesicle genotypes. The clear domi-
nance of C20 filaments (max critical depth = 70 m) in
Lake Levico agrees with the weak selective pressure
determined by its relatively restricted depth
(zmax = 38 m). On the other hand, the high abundance
of filaments of genotype C16 + C20 (max critical
depth = 100 m) in Lake Pusiano cannot be similarly
explained. The first bloom of Planktothrix in this lake
occurred in 2001 (Legnani et al., 2005), probably after a
colonisation of strains from nearby Lake Como. Thus,
it is probable that a ‘founder effect’ still characterises
the present population of Planktothrix in Lake Pusiano
(zmax = 24 m). If the founder population was richer in
stronger (genotype C16 + C20) than in weaker fila-
ments (genotype C20), then, without selection, there
would probably be a shift towards weaker genotypes
if there is a cost of being ‘unnecessary strong’. If so, it
would be interesting to follow the future development
of Planktothrix in Lake Pusiano in order to track the
frequency of different genotypes.
In conclusion, our data support the hypothesis that
the strength of gas vesicles is an adaptive trait under
strong and weak selection in lakes with deep and
shallow winter circulation, respectively. Moreover,
lake morphometry further influences these selective
processes. In addition, multiannual fluctuations of
phases of deep circulation and permanent stratifica-
tion could drive cycles of ‘stronger’ and ‘weaker’
filaments of Planktothrix. We conclude that the simul-
taneous presence of ‘weaker’ and ‘stronger’ filaments
could allow adaptive responses to changes in hydro-
static pressures related to changes in the regime of
vertical circulation.
Acknowledgments
This work was funded by the Autonomous Province
of Trento (Italy), project ACE-SAP (Alpine Ecosystems
in a Changing Environment: Biodiversity Sensitivity
and Adaptive Potential) (regulation number 23,
12th June 2008, of the University and Scientific
Research Service). The authors thank A.E. Walsby
and A. Hildrew for their positive criticism and
fundamental suggestions that improved the quality
of this work and one anonymous reviewer for useful
comments and corrections. The authors also thank
Marco Simona (Scuola Universitaria Professionale
della Svizzera Italiana, Switzerland), Fabio Buzzi
(Environmental Protection Agency, Lombardia),
Differential selection in Planktothrix 1491
� 2011 Blackwell Publishing Ltd, Freshwater Biology, 56, 1481–1493
Letizia Garibaldi (University of Milano-Bicocca, Mi-
lan), Rosario Mosello (Institute of Ecosystem Study,
CNR-ISE, Verbania-Pallanza) and the Fire Depart-
ment of Riva del Garda (Trento) for providing logistic
support. We are grateful to Nicola Merlo and Andrea
Zampedri (IASMA Research and Innovation Centre,
San Michele all’Adige) and to Mario Contesini (CNR-
ISE) for their support in the field activities. Massimo
Pindo and the technical staff of FEM (IASMA
Research and Innovation Centre) Sequencing Platform
are also acknowledged.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Table S1. Strains of Planktothrix rubescens isolated
from lakes in North Italy and analysed in this paper
As a service to our authors and readers, this journal
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(Manuscript accepted 19 January 2011)
Differential selection in Planktothrix 1493
� 2011 Blackwell Publishing Ltd, Freshwater Biology, 56, 1481–1493