How do algae form multicellular groups?

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Abstract
Background: Theory suggests that how groups are formed can have a significant influence on the evolution of cooperation, and whether cooperative groups make the major evolutionary transition to a higher-level individual. The formation of clonal groups, by remaining with parents (subsocial group formation), leads to a greater kin selected benefit of cooperation, compared with formation of groups by aggregating, with potential non-relatives (semisocial group formation). Freshwater algae form multicellular groups in response to the presence of predators, but it is not clear whether they form groups by remaining together or by aggregation. Organisms: The freshwater algae Chlorella sorokiniana, Chlorella vulgaris, and Scenedesmus obliquus, and the freshwater crustacean predator Daphnia magna. Results: Fluorescence microscopy and time-lapse photography revealed that, in response to predator supernatant/live predators, these algae form groups by both remaining with parents and aggregation. Additionally, different algal species form mixed-species multicellular groups in response to predation. Conclusion: The observation of aggregation, even between species: (1) emphasizes the likelihood of direct fitness benefits of forming groups to avoid predation; and (2) strengthens the across-species correlation between the method of group formation and whether multicellularity is facultative or obligate.
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How do algae form multicellular groups?
Stefania E. Kapsetaki, Alexander Tep and Stuart A. West
Department of Zoology, University of Oxford, Oxford, UK
ABSTRACT
Background: Theory suggests that how groups are formed can have a significant influence on
the evolution of cooperation, and whether cooperative groups make the major evolutionary
transition to a higher-level individual. The formation of clonal groups, by remaining with
parents (subsocial group formation), leads to a greater kin selected benefit of cooperation,
compared with formation of groups by aggregating, with potential non-relatives (semisocial
group formation). Freshwater algae form multicellular groups in response to the presence of
predators, but it is not clear whether they form groups by remaining together or by aggregation.
Organisms: The freshwater algae Chlorella sorokiniana, Chlorella vulgaris, and Scenedesmus
obliquus, and the freshwater crustacean predator Daphnia magna.
Results: Fluorescence microscopy and time-lapse photography revealed that, in response to
predator supernatant/live predators, these algae form groups by both remaining with parents
and aggregation. Additionally, different algal species form mixed-species multicellular groups in
response to predation.
Conclusion: The observation of aggregation, even between species: (1) emphasizes the likeli-
hood of direct fitness benefits of forming groups to avoid predation; and (2) strengthens the
across-species correlation between the method of group formation and whether multicellularity
is facultative or obligate.
Keywords: predation, Chlorophyceae, induced defence, aggregation, multicellularity.
INTRODUCTION
There have been at least eight independent major transitions to obligate multicellularity on
Earth (Maynard Smith and Szathmary, 1995; Bonner, 1998; Grosberg and Strathmann, 1998, 2007; Bourke, 2011; Fisher et al.,
2013). All of these transitions from single cells to an obligate multicellular lifestyle arose from
daughter cells remaining attached to their parent cell after division (Raven, 1998; Kirk, 2005;
Grosberg and Strathmann, 2007; Michod, 2007; Fisher et al., 2013). This pathway towards social group
formation is also known as ‘subsocial’, a term first used to describe the social lifestyle of
insects (Michener, 1969; Bourke, 2011). The high degree of relatedness and minimal conflict between
members of such a group can favour extreme levels of cooperation, alignment of interests,
Correspondence: S.E. Kapsetaki, Department of Zoology, University of Oxford, South Parks Road, The
Tinbergen Building, Oxford OX1 3PS, UK. email: stefania.kapsetaki@zoo.ox.ac.uk
Consult the copyright statement on the inside front cover for non-commercial copying policies.
Evolutionary Ecology Research, 2017, 18: 663–675
© 2017 Stefania E. Kapsetaki
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and interdependence between members, which are defining features of major transitions in
individuality (Hamilton, 1964; Maynard Smith and Szathmary, 1995; Boomsma, 2007, 2009; Fisher et al., 2013; West et al.,
2015). In contrast, other species, such as slime moulds and Pseudomonas biofilms, only form
multicellular groups facultatively, under certain conditions, and have not made the major
transition to obligate multicellularity (West et al., 2015). The formation of these facultative
multicellular groups often occurs via cells aggregating together. Because these cells are not
necessarily related, group formation via aggregation can lead to more potential for conflict.
Many freshwater algae form multicellular groups in response to predators (Solari et al., 2015;
Kapsetaki et al., 2016). However, it is not known if these algae form groups by daughter cells
remaining with their parents, or by potentially unrelated cells aggregating together. For
example, Boraas et al. (1998) and Lurling and Van Donk (2000) suggested that group formation
in Chlorella vulgaris and Scenedesmus obliquus was via daughter cells remaining within the
parent cell wall after division, similar to multicellular filament formation in the bacteria
Flectobacillus sp. (Corno and Jürgens, 2006), and subsocial palmelloid formation in
Chlamydomonas induced by the predator Brachionus (Lurling and Beekman, 2006; Harris, 2009). In
contrast, Chlamydomonas forms groups by aggregation in response to the predator
Peranema (Sathe and Durand, 2016) and S. obliquus forms predator-induced groups within 1 hour,
which is faster than its division time, indicating aggregation (Kapsetaki et al., 2016).
In this study, we determine how three algal species, Chlorella sorokiniana, C. vulgaris, and
S. obliquus, form groups in response to the presence of predators. We dyed algae of the same
species with two different fluorescent dyes, and then exposed them to either live Daphnia or
the supernatant from cultures in which Daphnia had been growing. We have previously
shown in all three of these algal species that live Daphnia and/or the supernatant from
Daphnia cultures induces group formation (Kapsetaki et al., 2016). The appearance of
dichromatic groups, composed of individuals dyed with each colour, would indicate at least
some aggregation. We examine group formation caused by both Daphnia and the
supernatant from Daphnia cultures, so that we can distinguish between the behaviour of
the algae and any aggregation or breaking up of groups that could have been caused by the
movement of Daphnia. To further validate our findings, we use an additional technique,
time-lapse photography, to observe how single cells form multicellular groups.
MATERIALS AND METHODS
Strains
We maintained the algae Chlorella sorokiniana 211/8K (non-axenic from CCAP), Chlorella
vulgaris 211/11B (axenic from CCAP), and Scenedesmus obliquus 276/3A (non-axenic
from CCAP) in Bolds Basal media at 20C under a light/dark cycle of 16 : 8 hours using
fluorescent illumination. We added 500 µg·mL
1 of the antibiotic rifampicin to 1-mL
samples of the C. sorokiniana and S. obliquus cultures, and diluted them 1: 300 after 24
hours in Bolds Basal media (Kapsetaki et al., 2016), to eliminate bacteria in the cultures. We
maintained the cultures in 1-litre Erlenmeyer flasks shaking at 220 rpm, a light/dark cycle of
16: 8 hours using fluorescent illumination, and a temperature of 20C before using these
cultures in experiments.
As predators, we used Daphnia magna (Sciento, UK), which we fed 5 mL S. obliquus
(106 cells · mL1) every 4–5 days. We maintained the Daphnia in 500-mL jars at 20C with a
light/dark cycle of 16:8 hours.
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Fluorescence experiments
Same species
We tested how algae form groups by dyeing two cell cultures of the same species with two
different fluorescent dyes, mixing them, and then inducing group formation by adding live
predators or predator supernatant. We followed a modified version of the manufacturer’s
recommended staining procedure (Thermo Fisher Scientific, CellTracker Fluorescent
Probes). We centrifuged the exponentially growing C. sorokiniana at 100 g for 10 minutes
and resuspended the pellet in CD-CHO Medium (Gibco, Carlsbad, CA). We then split this
culture in equal volumes and added the fluorescent dye CellTracker Green BODIPY (final
concentration 20 µ) to one culture and CellTracker Violet BMQC (final concentration
20 µ) to the other culture. We diluted stock dyes in 10 m DMSO. We covered the
two cultures with aluminium foil and left them shaking at 170 rpm overnight at room
temperature, centrifuged both cultures at 100 g for 10 minutes, and resuspended them in
Bolds Basal media to remove the dyes.
We sonicated the two algal cultures (10 one-second pulses, amplitude 20%) to break up
any groups that may have formed during the dyeing process, diluted both cultures to
106 cells · mL1, and then mixed them together in a 1: 1 volume ratio. We added 4.04 mL of
the dyed algae in 50-mL falcon tubes to either 0.96 mL of filtered Bolds Basal media
(referred to as media in the remainder of this manuscript), 5 adult Daphnia, or 0.96 mL
filtered liquid from the Daphnia culture (predator supernatant; final concentration of three
individuals per millilitre). The filter we used in all experiments had a pore diameter of 0.22
µ. We define ‘predator supernatant’ as anything present in the predator culture that could
pass through the 0.22-µ filter. This filtered liquid may contain products released from the
predators, and/or products from grazed/ungrazed S. obliquus. We replicated each treatment
three times. We kept the falcon tube caps loose to allow for oxygenation and randomized
the tubes on a rack in an incubator at 20C with a light/dark cycle of 16 : 8 hours using
fluorescent illumination. After 0 and 24 hours, we tilted the falcon tubes five times to mix
the culture, and collected 20-µL samples.
We constructed fluid tunnel slides by placing a cover slip onto two strips of Scotch
double-sided tape on a microscope slide and pipetting the 20-µL algal samples between the
cover slip and the slide. We sealed the coverslip with nail varnish, and imaged the samples
using a Zeiss Axio Zoom V16 fluorescence stereoscope (Carl Zeiss, Oberkochen, Germany).
As excitation/emission spectra for the violet and green dye, we used 405 nm/475 nm and
488 nm/538 nm, respectively. We took nine images per replicate (9 ×3=27 images per
treatment), and quantified the proportion of cells in monochromatic groups (number of
algal cells in monochromatic groups/total number of algal cells) and dichromatic groups
(number of algal cells in dichromatic groups/total number of algal cells). In many cases, the
exact number of cells in a three-dimensional group, especially in large groups, was difficult
to determine from the two-dimensional images (e.g. Fig. 1), as many cells were ‘hidden in
the background’. We counted what we observed in the two-dimensional images.
We followed the same procedure for C. vulgaris and S. obliquus, but in the case of
S. obliquus we obtained samples at 48 hours instead of 24 hours, as predator-induced group
formation had previously been observed at this time point (Kapsetaki et al., 2016).
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Different species
To assess whether different species of algae group together, we followed the same
experimental procedure as above, except that in the initial steps we dyed a culture of
C. sorokiniana with the green dye and a culture of C. vulgaris with the violet dye. In the
combination C. sorokiniana with C. vulgaris, we obtained samples at 0 and 24 hours;
in C. sorokiniana with S. obliquus and C. vulgaris with S. obliquus, we collected samples at
0 and 48 hours.
Fig. 1. Representative images of dichromatic groups within (A) and between (B) species. (A)
Green- and violet-dyed Chlorella sorokiniana form a dichromatic group in the presence of Daphnia.
(B) Green-dyed Chlorella sorokiniana and violet-dyed Chlorella vulgaris form a mixed-species
dichromatic group in the presence of Daphnia.
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Time-lapse photography
We also tested how C. sorokiniana forms groups using time-lapse photography. We added
4.04 mL of C. sorokiniana (initial concentration 106 cells · mL1) to 0.96 mL of filtered
D. magna water (final concentration of three individuals per millilitre) in a 50-mL falcon
tube. We maintained the tube at 20C with a light/dark cycle of 16 : 8 hours using fluorescent
illumination. After 10 hours, we diluted the culture using Bolds Basal media to a final
concentration of 4 ×105 cells ·mL1 and transferred 1 mL of the diluted culture onto a
24-well plate. We placed the 24-well plate at room temperature under a phase-contrast
microscope (Nikon ELWD 0.3, 20× magnification, LWD) and set the digital camera (Nikon
D300, Japan), which was attached to the microscope, to take photos every minute for a total
of 96 hours. We assembled the photos into a movie of 4 frames per second using ‘Time
Lapse Assembler’ (v.1.5.3).
From the end of the movie, we randomly chose a cell in a multicellular group and tracked
it back in time, stopping at the first instant at which it joined this group. We noted whether it
joined the group by aggregation (attaching to a group or pair) or by remaining attached to a
mother cell after division. We defined a multicellular group as 3 cells in close proximity
that could not be distinguished as separate cells. We tracked 50 cells in total, each from a
different randomly selected group. Using these 50 cells, we measured the proportion that
joined their group by aggregation, the remaining cells joining their group as a result of
division from their parent cell. However, we were not able to distinguish whether this was
just division as part of their normal life cycle or actual group formation. We also measured
the time these cells spent with their parent cell after division.
We followed the same experimental procedure for C. vulgaris and S. obliquus.
Statistical analysis
We performed statistical analyses using R v.3.2.3 (R Development Core Team, 2017). To compare the
proportion of cells in monochromatic groups between the media, predator supernatant, and
live predators treatments in the fluorescence experiments, we used generalized linear models
(glm), specifying the family as quasibinomial to account for overdispersion of the data. We
performed the same test to compare the proportion of cells in dichromatic groups between
the three treatments.
We tested whether group formation was the result of random aggregation in the
fluorescence experiments. Random group formation would lead to the proportion of each
colour of cells in groups following a binomial distribution. We used the regression method
of Green et al. (1982) to compare the observed variance (Vo) with that expected from a
binomial distribution. The observed variance is given by s2(1 r2), where s2 is variance in the
number of green cells per group and r is the regression coefficient in the relationship
between the number of green cells in a group and group size. The expected variance (Ve) is
given by αp(1 p), where α is group size and p is the expected proportion of green cells.
Specifically, p is (b + rα)/α, where b represents the intercept. We tested whether the observed
variance was significantly higher than binomial. Under the null hypothesis of random
aggregation, the residual statistic, χ2=(Vo/Ve)/(N 2), should come approximately from a
chi-squared distribution with N 2 degrees of freedom, where N is the number of groups
sampled (Green et al., 1982).
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RESULTS
Within-species group formation
Consistent with previous results, we found that all three algal species – C. sorokiniana,
C. vulgaris, and S. obliquus – formed groups in response to predators/predator supernatant
(glm, media vs. predator supernatant and live predators: C. sorokiniana, F=131.29,
P<0.001, df =7; C. vulgaris, F=82.07, P<0.001, df =7; S. obliquus, F=8.79, P=0.02,
df =7) (Fig. 2). The statistical analysis for each treatment pair is presented in Table S1
(www.evolutionary-ecology.com/data/3099Appendix.pdf).
When we added predators or predator supernatant, the proportion of cells in dichromatic
groups, which indicates at least some aggregation, was between 7.1% and 70.8% (Fig. 2).
For all three algal species, the proportion of cells in dichromatic groups was higher with
predator supernatant or live predators than when just media was added (glm across the
three treatments: C. sorokiniana, F=13.08, P=0.006, df =6; C. vulgaris, F=39.79, P<
0.001, df =6; S. obliquus, F=20.26, P=0.002, df =6; glm, media vs. predator supernatant
and live predators: C. sorokiniana, F=20.22, P=0.002, df =7; C. vulgaris, F=53.14,
P<0.001, df =7; S. obliquus, F=25.14, P=0.001, df =7) (Fig. 2).
We also found that the distribution of green cells in groups showed significantly
more than binomial variation in all three algal species when exposed to either predator
supernatant or live predators, except for C. sorokiniana upon exposure to live predators
(Table 1). Binomial variation would have been consistent with completely random group
aggregation, and so our finding of greater than binomial variation suggests some tendency
to form groups with algae of the same colour.
Time-lapse experiments of C. sorokiniana in the presence of predator supernatant
revealed that of the 50 observed cells, each belonging to a different group, 47 had joined
their group by aggregation. In C. vulgaris and S. obliquus, 31 and 22 of the observed cells
respectively had joined their group by aggregation when exposed to predator supernatant.
The remaining cells (3 in C. sorokiniana, 19 in C. vulgaris, and 28 in S. obliquus) joined their
group as a result of division from their parent cell, although we could not distinguish
whether this was simply division as part of their life cycle or actual group formation.
These cells spent on average 31.8 ±20.6 hours (mean ±SEM), 10.8 ±2.6 hours, and
51.6 ±4.4 hours respectively with their parent cell after division.
Between-species group formation
We found that all three combinations of algal species – C. sorokiniana with C. vulgaris,
C. sorokiniana with S. obliquus, and C. vulgaris with S. obliquus – formed multicellular
groups in response to predators or predator supernatant (glm, media vs. predator
supernatant and live predators: C. sorokiniana with C. vulgaris, F=96.12, P<0.001, df =7;
C. sorokiniana with S. obliquus, F=33.02, P<0.001, df =7; C. vulgaris with S. obliquus,
F=57.21, P<0.001, df =7) (Fig. 3).
After adding predators or predator supernatant, the proportion of cells in dichromatic
groups (suggesting some between-species group formation) was between 14.8% and 46.8%
(Fig. 3). In all three algal species combinations, the proportion of cells in dichromatic
groups was higher with predator supernatant or live predators than when just media was
added (glm across the three treatments: C. sorokiniana with C. vulgaris, F=11.10,
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P=0.009, df =6; C. sorokiniana with S. obliquus, F=15.19, P=0.004, df =6; C. vulgaris
with S. obliquus, F=36.90, P<0.001, df =6; glm, media vs. predator supernatant and live
predators: C. sorokiniana with C. vulgaris, F=23.88, P=0.001, df =7; C. sorokiniana with
S. obliquus, F=27.42, P=0.001, df =7; C. vulgaris with S. obliquus, F=67.91, P<0.001,
df =7) (Fig. 3).
Furthermore, in all three algal species combinations the distribution of green cells in
groups showed significantly more than binomial variation when exposed to predator
Fig. 2. Within-species group formation. The proportion of cells in groups is plotted in the absence
of predators (media), the presence of predator supernatant, and the presence of live predators. The
cells are divided between those in groups containing only violet- or green-dyed cells (monochromatic),
and those in groups containing a mixture of violet- and green-dyed cells (dichromatic). The different
panels show results for the three different algae species: (A) C. sorokiniana after 24 hours;
(B) C. vulgaris after 24 hours; and (C) S. obliquus after 48 hours. The values of the y-axes differ
between panels. The error bars are standard errors of the mean for each of these two colour com-
binations. For all species, we found that the presence of live predators or predator supernatant led
to increased group formation and increased proportion of cells in dichromatic groups, suggesting a
role of aggregation.
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supernatant or live predators (Table 1). This suggests a greater than random propensity to
form groups with members of the same species.
DISCUSSION
We found group formation in all three algal species – Chlorella sorokiniana, C. vulgaris, and
Scenedesmus obliquus – in response to live predators/predator supernatant (Fig. 2), con-
sistent with previous results using these and other algae species (reviewed in Kapsetaki et al., 2016). In
all three species, when we dyed algae two different colours and mixed them, we found that
they formed dichromatic groups, suggesting that some group formation is via individuals
aggregating together (semisocial group formation; Figs. 1A, 2). This result was supported
by direct observation in all three species, with time-lapse photography, where we observed
individuals coming together. In each of these species, the distribution of dyed cells in groups
showed greater than binomial variation, and so group formation was not only due to
random aggregation (Table 1). This suggests that either some group formation is via off-
spring remaining with their parents (subsocial group formation) or that there is some spatial
clustering of cells (Table 1). Finally, we found that individuals of these three species also
form groups with each other, leading to mixed-species groups, again emphasizing the role of
group formation via aggregation (Figs. 1B, 3).
Table 1. Comparison of the proportion of green-dyed cells in groups relative to a random binomial
variance
Algae Predators Vo/Ve
Number of
groups sampled χ2-value P-value
Within species
Chlorella sorokiniana supernatant 4.81 4 9.62 0.008
live 1.63 3 1.63 0.201
Chlorella vulgaris supernatant 7.49 36 254.71 <0.001
live 68.20 6 272.81 <0.001
Scenedesmus obliquus supernatant 23.75 221 5201.83 <0.001
live 55.57 54 2889.77 <0.001
Between species
C. sorokiniana and C. vulgaris supernatant 6.22 32 186.78 <0.001
live 17.90 12 179.03 <0.001
C. sorokiniana and S. obliquus supernatant 17.48 24 384.61 <0.001
live 24.00 28 624.07 <0.001
C. vulgaris and S. obliquus supernatant 66.78 163 10752.68 <0.001
live 39.29 58 2200.58 <0.001
Note: Analyses are shown for when the different coloured cells (green and violet) are the same or different species,
and when group formation was induced either by predators or predator supernatant. A value of observed/expected
variance (Vo/Ve) >1 would imply overdispersion, where groups tend to show a bias to one of the two colours. In
all cases within species and between species, except C. sorokiniana in the presence of live predators, mixing was
non-random.
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Previous studies have suggested that cell division is necessary for group formation
(Lampert et al., 1994; Trainor, 1998). Chlorella sorokiniana, C. vulgaris, and S. obliquus acquire energy
from sunlight and nutrients in their environment, leading to an increase in cell size, after
which the parent cell divides into daughter cells inside the cell wall (Nilshammar and Walles, 1974;
Trainor et al., 1976; Boraas et al., 1998; Trainor, 1998; Yamamoto et al., 2005). Then, in response to predation, as
reported in C. vulgaris and S. obliquus (Boraas et al., 1998; Lurling and Van Donk, 2000), daughter cells
fail to break free from the parent cell wall, leading to group formation. As stated clearly by
Fig. 3. Between-species group formation. The proportion of cells in groups is shown in the absence of
predators (media), the presence of predator supernatant, and the presence of live predators. The cells
are divided between those in groups containing only violet- or green-dyed cells (monochromatic), and
those in groups containing a mixture of violet- and green-dyed cells (dichromatic). The different
panels show results for the three different algal species combinations: (A) C. sorokiniana with
C. vulgaris after 24 hours; (B) C. sorokiniana with S. obliquus after 48 hours; and (C) C. vulgaris with
S. obliquus after 48 hours. The values of the y-axes differ between panels. Error bars represent
standard errors of the mean for each of these two coloured types. In all three combinations, we found
that the presence of live predators or predator supernatant led to increased group formation and
increased proportion of cells in dichromatic groups, indicating between-species group formation.
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Lürling (2001), in Scenedesmus ‘ . . . colony formation is not clogging of individual cells, but
the result of a reproductive process’. In contrast to this assumption, we have found that a
significant fraction of group formation is via aggregation (Figs. 1, 2). Our results do not
exclude the possibility that some group formation occurs through remaining with parents,
because group formation is not purely random (Table 1), and with time-lapse photography
we observed some cells forming groups by division. However, we could not determine
whether this was just division as part of their life cycle or actual group formation (e.g.
3099Appendix – Movie S1). By further analysing our time-lapse data, we found that in all
three algal species, daughter cells spent more time on average with their parent cells after
division in the presence than in the absence of predator supernatant (3099Appendix – Time-
lapse analysis), although these two treatments were not conducted simultaneously. These
observations support the idea of some group formation by remaining with parent cells.
Bonner (1998) suggested that group formation by remaining with parents is more likely to
have evolved in aquatic species, whereas we are more likely to see group formation via
aggregation in terrestrial species (Bonner, 2003; Velicer and Vos, 2009). Group formation by aggrega-
tion has been considered more difficult in water because cells disperse easier in water than
on land (Bonner, 2009; Bourke, 2011). How can we explain the group formation by aggregation that
we have observed in non-motile aquatic species (Yamamoto et al., 2005)? These algae seem to
move at random in the liquid culture, consistent with Brownian motion. In the presence of
predator supernatant only, we saw cells dividing and the daughter cells dispersing, cells
dividing and the daughter cells remaining with their parent cell, and several cases where a
group formed both by cells remaining with parents and by aggregation (3099Appendix –
Movies S2 and S3).
Not only did algae form groups via aggregation, but they also grouped with other species
(Fig. 3). This would be expected if rapid group formation provided a direct benefit in
defence against predators. Between-species multicellular aggregates have been observed
previously in C. vulgaris with the bacteria Bacteroidia, Flavobacteria, Beta-proteobacteria,
Gamma-proteobacteria, and filamentous blue-green algae (Gutzeit et al., 2005; Lee et al., 2013; Quijano
et al., 2017), between different species of Chlamydomonas (Sathe and Durand, 2016), and in
Dictyostelium amoebae (Kaushik et al., 2006; Sathe et al., 2010, 2014). Examples of mixed-species
multicellular groups also exist in bacterial biofilms, such as Pseudomonas suringae with
Pseudomonas agglomerans, and Acinetobacter with Pseudomonas putida (Monier and Lindow, 2005;
Hansen et al., 2007), where groups may provide protection against grazing by predators (Matz and
Kjelleberg, 2005; Chavez-Dozal et al., 2013; Friman et al., 2013).
We found that group formation was not random, either within or between species
(Table 1). There are a number of possible explanations for this. First, some group formation
could be via remaining with parent cells. For example, the algae Chlamydomonas can form
groups by both remaining with parents and aggregating (Lurling and Beekman, 2006; Harris, 2009; Sathe
and Durand, 2016). Second, clumping of the same clone/species might occur just through spatial
clustering after division (i.e. limited dispersal in a structured population). Third, individuals
might discriminate who they form groups with, as has previously been observed in
Dictyostelium amoebae (Mehdiabadi et al., 2006).
In conclusion, across species there is a correlation between the method of group forma-
tion and whether multicellularity is facultative or obligate (Grosberg and Strathmann, 2007; Fisher et al.,
2013, 2016). All the known major transitions to obligate multicellularity have arisen via off-
spring remaining with their parent cell (Fisher et al., 2013). In contrast, transitions to facultative
multicellularity have occurred via both aggregation and remaining with parents (Fisher et al.,
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2013). Consequently, our finding that facultative group formation in algae is via aggregation
strengthens the across-species correlation between the method of group formation and
whether multicellularity is facultative or obligate.
DATA ACCESSIBILITY
The data for this paper are available on Dryad (doi: 10.5061/dryad.vb665).
ACKNOWLEDGEMENTS
We thank Roni McGowan, Lindsay Turnbull, and Nick Hardberd for providing temporary lab space,
Kevin Foster for allowing us to use the fluorescent stereoscope, Isabel Frost and Jamie Wheeler for
valuable assistance in using the fluorescent stereoscope, and Roberta Fisher and David Crosse for
helpful comments on the manuscript. This research was funded by the European Research Council
(to S.A.W.), the HSBC (to A.T.), the Alexander S. Onassis Public Benefit Foundation, and the A.G.
Leventis Foundation (to S.E.K.).
REFERENCES
Bonner, J.T. 1998. The origins of multicellularity. Integr. Biol. Issues, News, Rev., 1: 27–36.
Bonner, J.T. 2003. Evolution of development in the cellular slime molds. Evol. Dev., 5: 305–313.
Bonner, J.T. 2009. The Social Amoebae: The Biology of Cellular Slime Molds. Princeton, NJ:
Princeton University Press.
Boomsma, J.J. 2007. Kin selection versus sexual selection: why the ends do not meet. Curr. Biol.,
17: R673–R683.
Boomsma, J.J. 2009. Lifetime monogamy and the evolution of eusociality. Phil. Trans. R. Soc. Lond.
B: Biol. Sci., 364: 3191–3207.
Boraas, M.E., Seale, D.B. and Boxhorn, J.E. 1998. Phagotrophy by a flagellate selects for colonial
prey: a possible origin of multicellularity. Evol. Ecol., 12: 153–164.
Bourke, A.F.G. 2011. Principles of Social Evolution. Oxford: Oxford University Press.
Chavez-Dozal, A., Gorman, C., Erken, M., Steinberg, P.D., McDougald, D. and Nishiguchi, M.K.
2013. Predation response of Vibrio fischeri biofilms to bacterivorus protists. Appl. Environ.
Microbiol., 79: 553–558.
Corno, G. and Jürgens, K. 2006. Direct and indirect effects of protist predation on population
size structure of a bacterial strain with high phenotypic plasticity. Appl. Environ. Microbiol., 72:
78–86.
Fisher, R.M., Cornwallis, C.K. and West, S.A. 2013. Group formation, relatedness, and the
evolution of multicellularity. Curr. Biol., 23: 1120–1125.
Fisher, R.M., Bell, T. and West, S.A. 2016. Multicellular group formation in response to predators in
the alga Chlorella vulgaris. J. Evol. Biol., 29: 551–559.
Friman, V.P., Diggle, S.P. and Buckling, A. 2013. Protist predation can favour cooperation within
bacterial species. Biol. Lett., 9 (5): 20130548.
Green, R.F., Gordh, G. and Hawkins, B.A. 1982. Precise sex ratios in highly inbred parasitic wasps.
Am. Nat., 120: 653–665.
Grosberg, R.K. and Strathmann, R.R. 1998. One cell, two cell, red cell, blue cell: the persistence of a
unicellular stage in multicellular life histories. Trends Ecol. Evol., 13: 112–116.
Grosberg, R.K. and Strathmann, R.R. 2007. The evolution of multicellularity: a minor major
transition? Annu. Rev. Ecol. Evol. Syst., 38: 621–654.
Gutzeit, G., Lorch, D., Weber, A., Engels, M. and Neis, U. 2005. Bioflocculent algal–bacterial
biomass improves low-cost wastewater treatment. Water Sci. Technol., 52: 9–18.
How do algae form multicellular groups? 673
07:11:07:12:17
Page 674
Page 674
Hamilton, W.D. 1964. The genetical evolution of social behaviour. I. J. Theor. Biol., 7: 1–16.
Hansen, S.K., Haagensen, J.A.J., Gjermansen, M., Jørgensen, T.M., Tolker-Nielsen, T. and Molin, S.
2007. Characterization of a Pseudomonas putida rough variant evolved in a mixed-species
biofilm with Acinetobacter sp. strain C6. J. Bacteriol., 189: 4932–4943.
Harris, E.H. 2009. The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and its
Laboratory Use. San Diego, CA: Academic Press.
Kapsetaki, S.E., Fisher, R.M. and West, S.A. 2016. Predation and the formation of multicellular
groups in algae. Evol. Ecol. Res., 17: 651–669.
Kaushik, S., Katoch, B. and Nanjundiah, V. 2006. Social behaviour in genetically heterogeneous
groups of Dictyostelium giganteum. Behav. Ecol. Sociobiol., 59: 521–530.
Kirk, D.L. 2005. A twelve-step program for evolving multicellularity and a division of labor.
Bioessays, 27: 299–310.
Lampert, W., Rothhaupt, K.O. and von Elert, E. 1994. Chemical induction of colony formation in a
green alga (Scenedesmus acutus) by grazers (Daphnia). Limnol. Oceanogr., 39: 1543–1550.
Lee, J., Cho, D.-H., Ramanan, R., Kim, B.-H., Oh, H.-M. and Kim, H.-S. 2013. Microalgae-
associated bacteria play a key role in the flocculation of Chlorella vulgaris. Bioresour. Technol.,
131: 195–201.
Lürling, M. 2001. Grazing-associated infochemicals induce colony formation in the green alga
Scenedesmus. Protist, 152: 7–16.
Lurling, M. and Beekman, W. 2006. Palmelloids formation in Chlamydomonas reinhardtii: defence
against rotifer predators? Ann. Limnol. – Int. J. Limnol., 42: 65–72.
Lurling, M. and Van Donk, E. 2000. Grazer-induced colony formation in Scenedesmus: are there
costs to being colonial? Oikos, 88: 111–118.
Matz, C. and Kjelleberg, S. 2005. Off the hook – how bacteria survive protozoan grazing. Trends
Microbiol., 13: 302–307.
Maynard Smith, J. and Szathmary, E. 1995. The Major transitions in Evolution. Oxford: W.H. Free-
man Spektrum.
Mehdiabadi, N.J., Jack, C.N., Farnham, T.T., Platt, T.G., Kalla, S.E., Shaulsky, G. et al. 2006. Social
evolution: kin preference in a social microbe. Nature, 442: 881–882.
Michener, C.D. 1969. Comparative social behavior of bees. Annu. Rev. Entomol., 14: 299–342.
Michod, R.E. 2007. Evolution of individuality during the transition from unicellular to multicellular
life. Proc. Natl. Acad. Sci. USA, 104: 8613–8618.
Monier, J.-M. and Lindow, S.E. 2005. Spatial organization of dual-species bacterial aggregates on
leaf surfaces. Appl. Environ. Microbiol., 71: 5484–5493.
Nilshammar, M. and Walles, B. 1974. Electron microscope studies on cell differentiation in
synchronized cultures of the green alga Scenedesmus. Protoplasma, 79: 317–332.
Quijano, G., Arcila, J.S. and Buitrón, G. 2017. Microalgal-bacterial aggregates: applications and
perspectives for wastewater treatment. Biotechnol. Adv., 35: 772–781.
Raven, J. 1998. Book review: David L. Kirk. Volvox: Molecular-Genetic Origins of Multicellularity
and Cellular Differentiation. Developmental and Cell Biology Series, editors J.D.L. Bard, P.W.
Barlow, P.B. Green and D.L. Kirk. Cambridge University Press, Cambridge, 1998, xvi + 381 pp.
Eur. J. Phycol., 33: 275–280.
R Development Core Team. 2017. R: A Language and Environment for Statistical Computing.
Vienna, Austria: R Foundation for Statistical Computing.
Sathe, S. and Durand, P.M. 2016. Cellular aggregation in Chlamydomonas (Chlorophyceae) is
chimaeric and depends on traits like cell size and motility. Eur. J. Phycol., 51: 129–138.
Sathe, S., Kaushik, S., Lalremruata, A., Aggarwal, R.K., Cavender, J.C. and Nanjundiah, V. 2010.
Genetic heterogeneity in wild isolates of cellular slime mold social groups. Microb. Ecol., 60:
137–148.
Sathe, S., Khetan, N. and Nanjundiah, V. 2014. Interspecies and intraspecies interactions in social
amoebae. J. Evol. Biol., 27: 349–362.
Kapsetaki et al.674
07:11:07:12:17
Page 675
Page 675
Solari, C.A., Galzenati, V.J. and Kessler, J.O. 2015. The evolutionary ecology of multicellularity: the
volvocine green algae as a case study. In Evolutionary Transitions to Multicellular Life: Principles
and Mechanisms (I. Ruiz-Trillo and A.M. Nedelcu, eds.), pp. 201–223. Dordrecht: Springer.
Trainor, F.R. 1998. Biological Aspects of Scenedesmus (Chlorophyceae) – Phenotypic Plasticity.
Stuttgart: J. Cramer.
Trainor, F.R., Cain, J.R. and Shubert, L.E. 1976. Morphology and nutrition of the colonial green
alga Scenedesmus: 80 years later. Bot. Rev., 42: 5–25.
Velicer, G.J. and Vos, M. 2009. Sociobiology of the myxobacteria. Annu. Rev. Microbiol., 63:
599–623.
West, S.A., Fisher, R.M., Gardner, A. and Kiers, E.T. 2015. Major evolutionary transitions in
individuality. Proc. Natl. Acad. Sci. USA, 112: 1–8.
Yamamoto, M., Kurihara, I. and Kawano, S. 2005. Late type of daughter cell wall synthesis in
one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxiophyceae). Planta, 221:
766–775.
How do algae form multicellular groups? 675
07:11:07:12:17
Page 676
Page 676
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    Noted biologist and author John Tyler Bonner has experimented with cellular slime molds for more than sixty years, and he has done more than anyone else to raise these peculiar collections of amoebae from a minor biological curiosity to a major model organism--one that is widely studied for clues to the development and evolution of all living things. Now, five decades after he published his first pioneering book on cellular slime molds, Bonner steps back from the proliferating and increasingly specialized knowledge about the organism to provide a broad, nontechnical picture of its whole biology, including its evolution, sociobiology, ecology, behavior, and development.The Social Amoebaedraws the big lessons from decades of research, and shows how slime molds fit into and illuminate biology as a whole.Slime molds are very different from other organisms; they feed as individual amoebae before coming together to form a multicellular organism that has a remarkable ability to move and orient itself in its environment. Furthermore, these social amoebae display a sophisticated division of labor; within each organism, some cells form the stalk and others become the spores that will seed the next generation. InThe Social Amoebae, Bonner examines all these parts together, giving a balanced, concise, and clear overview of slime mold biology, from molecules to cells to multicells, as he advances some unconventional and unexpected insights.
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