J Appl Phycol (2018) 30:1157–1166
https://doi.org/10.1007/s10811-017-1305-1
Factors affecting morphological transformation and secondary
attachment of apexes of Chondracanthus chamissoi
(Rhodophyta, Gigartinales)
Catalina Y. Rodríguez 1 & Ricardo D. Otaíza 1,2
Received: 28 April 2017 / Revised and accepted: 1 October 2017Published online: 7 October 2017
# Springer Science+Business Media B.V. 2017
Abstract Fragments of Chondracanthus chamissoi have the
capacity of secondary attachment. In the laboratory, apexes of
upright branches of C. chamissoi placed 1–2 mm and parallel
to horizontal substrata underwent morphological transforma-
tion prior to becoming attached, changing from thick and
blunt to elongated and cylindrical and from straight to curved
towards the substratum, where they eventually produced sec-
ondary attachment discs. When transformation occurred,
signs could already be observed after 3 days of incubation.
Several factors that could affect transformation and attach-
ment were tested. The vertical or horizontal orientation of
the fragments, the angle of incidence of light, and the phase
of the life cycle had no effect on transformation, whereas the
presence of reproductive structures had a negative effect com-
pared to vegetative fragments. Low light intensity and low
water flow favored the transformation response. Calcareous
substrata (bivalve shells and coralline crusts) produced the
highest frequency of transformation and other solid substrata
(glass slides, rock, and sea-squirt tunic) followed in the rank-
ing, but fleshy seaweeds (Ulva sp., Sarcothalia crispata, and
C. chamissoi) failed to produce any response. Thus, the ex-
perimental upright branches developed features and reactivity
similar to basal branches when the former were placed in
conditions similar to those where the latter occur in the natural
environment. Vegetative reproduction has been incorporated
* Ricardo D. Otaíza
rotaiza@ucsc.cl
1
Departamento de Ecología, Facultad de Ciencias, Universidad
Católica de la Santísima Concepción, Concepcion, Chile
2
Centro de Investigación en Biodiversidad y Ambientes Sustentables
(CIBAS), UCSC, Concepcion, Chile
in cultivation techniques of C. chamissoi, but it could also be
used in repopulation or restoration programs of this species.
Keywords Apex transformation . Secondary attachment .
Vegetative reproduction . Light intensity . Calcium . Substrate
selection
Introduction
In seaweeds, reproduction occurs mainly via gametes and
spores. These unicellular structures are released to the water
column, where they disperse and eventually become attached
to a substratum and grow into a new individual (Hoffmann
1987; Santelices 1990; Clayton 1992; Norton 1992). Also,
whole thalli or fragments can produce secondary attachment
structures, such as rhizoids (Perrone and Cecere 1997) or sec-
ondary attachment discs (Pacheco-Ruíz et al. 2005; Otaíza and
Fonseca 2011), becoming fixed to the substratum and thus
generating new individuals (Destombe and Oppliger 2011).
Both spores and fragments have been used in cultivation
techniques of red seaweeds. For example, spores may be in-
oculated on oyster shells or nets for cultivation of species of
Porphyra/Pyropia (Redmond et al. 2014). Fragments have
been used in various ways. Free floating fragments of different
species have been used in tank cultivation of Chondrus
crispus (Neish et al. 1977; Redmond et al. 2014). Fragments
of Gracilaria chilensis are seeded by partly burying them in
sediments (Santelices and Doty 1989; Buschmann et al.
1995), and fragments of species of Eucheuma/Kappaphycus
are intertwined on ropes for cultivation on shallow waters
(Doty 1987). Secondary attachment of fragments to different
substrata has also been proposed for cultivation techniques for
Gelidium and Chondracanthus species (Salinas 1991;
Macchiavello et al. 2003; Pacheco-Ruíz et al. 2005). For the
1158
latter strategy, knowledge of the factors that affect secondary
attachment can be used to improve cultivation methods.
In Chile, Chondracanthus chamissoi (C. Agardh) Kützing
(Rhodophyta, Gigartinaceae) has been harvested for the ex-
traction of carrageenan and, more recently, an increasing in-
terest in cultivation of this species has arisen as it is now also
exported to Asia as food for humans (González et al. 1997;
Hoffmann and Santelices 1997; Bulboa and Macchiavello
2006; Ávila et al. 2010). Geographically, it ranges from
Paita, Perú (5° S) to Ancud, Chile (42° S) (Ramírez and
Santelices 1991; Hoffmann and Santelices 1997), although
recent molecular evidence suggests that it is also present in
the Northwest Pacific and France (Yang et al. 2015). Locally,
it occurs in low intertidal pools and extends to a depth of 15 m
(Hoffmann and Santelices 1997) growing mainly on rocks,
mollusk shells (Vásquez and Vega 2001; Macchiavello et al.
2003; Fonck et al. 2008), and on patches of the sea-squirt
Pyura chilensis (Pers obs.). It has a triphasic Polysiphonia
type, life-cycle with alternating isomorphic generations of ga-
metophytes and sporophytes (Hoffmann and Santelices 1997;
Ávila et al. 2010). Thalli consist of usually 15–20 cm long,
narrow blades with some lateral branches, and numerous short
pinnules growing distichously along the margins of the main
axes and lateral branches (Hommersand et al. 1993; Hoffmann
and Santelices 1997) (Fig. 1a). The lowest branches of the
main axes, unlike the rest of the branches, are cylindrical
and curve towards the substratum (Fig. 1b), where they form
secondary attachment discs. The tips of the upright branches
and pinnules can also form secondary attachment discs when
they come in contact with some hard substrata (Macchiavello
et al. 2003; Fonck et al. 2008; Sáez et al. 2008; Otaíza and
Fonseca 2011). It has been described that calcareous substrata
enhance the production of secondary attachment discs in
C. chamissoi (Macchiavello et al. 2003; Fonck et al. 2008;
Sáez et al. 2008; Otaíza and Fonseca 2011), and also in
C. squarrulosus (Pacheco-Ruíz et al. 2005) and Gelidium
spp. (Salinas 1991; Juanes and Puente 1993; Santelices and
Varela 1994).
Our preliminary observations showed that before the
secondary attachment structures are produced, the tip of
branches and pinnules of C. chamissoi undergo a morpho-
logical transformation when placed in proximity to the
substratum. Additionally, some substrata did not elicit a
response. The aim of this study was to test the effect of
some factors that may affect secondary attachment of
C. chamissoi. First, we evaluated whether the angle of in-
cidence of light and the orientation of the fragments had an
effect on the morphological transformation of the apexes.
Then, we compared among fragments of different types of
free living individuals and of reproductive state of the thal-
li to reduce variability in the following experiments.
Finally, we evaluated the effect of light intensity, water
flow, and type of substratum on the attachment process in
J Appl Phycol (2018) 30:1157–1166
C. chamissoi. The most relevant factors may be used to
favor attachment of fragments in culture.
Materials and methods
Thalli of Chondracanthus chamissoi were collected from a
natural bed at Ramuntcho (36°45′06″S 73°11′04″ W),
Región del Biobío, in south central Chile, between April and
June, 2014, and immediately taken to the Marine Biology
Station of the Universidad Católica de la Santísima
Concepción, located in the nearby town of Lenga. On each
occasion, thalli were sorted and selected for different
experiments.
Six different experiments were conducted, all with a similar
setup. Straight fragments, 50 mm long, obtained from lateral
branches that included the apex, were cut each from different
C. chamissoi plants. Except when indicated, these fragments
had no visible reproductive structures, but the remaining blade
allowed the identification of the type of free-living individual
(tetrasporophyte, or male or female gametophyte). In the ex-
perimental setup, these fragments were kept in position by
trapping their base between two 25 × 75 mm glass-slides
(for microscopy) held together by pieces of PVC tubes and
using cellulose tissue as padding to prevent the border of the
glass-slide from severing the fragment. During the experi-
ments, the fragments were put in 20 × 30 × 25 cm glass
aquaria with fresh, circulating seawater at a temperature of
12 ± 1 °C, photoperiod of 12:12 (light:darkness), irradiance
of 50–60 μmol photons m−2 s−1 obtained from natural light
supplemented with cool white, day light tubes illuminating
only from above, except when indicated otherwise, and per-
manent aeration.
The first three experiments were aimed at defining some of
the conditions for the following three experiments (see first
and second column in Table 1). In the first experiment, we
evaluated the effect of the orientation of the fragments and the
angle of light incidence on the fragment to assess potential
effects of the orientation of the fragments with respect to in-
cidence of light. Eight replicate fragments, cut from different
female plants, were placed in one of two orientations: verti-
cally (with the tip directed upwards) or horizontally, in com-
bination with two angles of incidence of light: frontally on the
tip or laterally over the surface of the fragment. Thus, four
treatments were included combining the two positions with
the two directions of incidence of light. At all times, the frag-
ments were kept away from the bottom and sides of the aquar-
ium. Black shading net was used to prevent reflection of light
from the internal lateral surfaces and bottom of the aquarium.
In the second experiment, we compared fragments of different
phases of the life cycle. Eight replicate fragments from each of
the three types of free-living individuals (tetrasporophytes and
male and female gametophytes) were setup as indicated
J Appl Phycol (2018) 30:1157–1166
Fig. 1 Chondracanthus chamissoi from Ramuntcho, south central Chile.
a Blade of C. chamissoi detached from the basal disc, showing basal
branches curved towards the substratum [→] and the upright branches
of different order with pinnules on the margins. b Basal portion of
C. chamissoi showing the basal disc [→] and the basal portion of an
upright blade. Some cylindrical branches that curve towards the
substratum [►] originated from the basal disc, while others [>] are
above, except that the fragments were placed horizontally at
1–2 mm from the surface of rocky (granite) boulders. In the
third experiment, we compared vegetative and reproductive
fragments. Only female thalli were used in two treatments:
fragments with or without visible cystocarps (reproductive
and vegetative, respectively), with 16 replicates of each con-
dition. Fragments were setup horizontally over boulders as in
the second experiment.
These initial experiments were evaluated every 3 days for
15 days. On each occasion, all apexes were individually
checked and photographed from the side, including a ruler
as reference. Three aspects were used to describe the transfor-
mation of each apex: a curvature in the new growth at the tip
of the fragment, the orientation of this new growth (towards
the substratum or in another direction), and a change in shape
of this new growth, becoming clearly cylindrical (Fig. 1c, d).
After 15 days in the experiment, the frequency of transforma-
tion of apexes was calculated. Given that the second and third
experiments included boulders as substrata, the frequency of
apexes that became attached to the surface of the substratum
was also evaluated.
1159
produced from the most basal portion of the upright blade. c Image of a
whole blade of C. chamissoi placed on a concrete brick (not from this
study) where transformation and attachment reaction can be seen in the
apexes of most of the pinnules. d Tips of two fragments showing apex
after 15 days in cultivation where the change in morphology and
curvature of the tip can be seen. The scale in a and b is 10 mm; the
scale in d is 1 mm
Photographs were digitally analyzed (ImageTool 3.0,
UTHSCSA, USA). Growth of the apex over the 15-day
period was evaluated as the increase in length in the seg-
ment between the tip of the apex and the base of the nearest
pinnule (used as the reference point, usually 4–8 mm) iden-
tified in the first photograph. Relative growth rate of each
apex was calculated with the equation: RGR = 100 × ln (Lt/
L0)/t, where RGR is the relative growth rate, Lt is the
length of the segment at day 15 of the experiment, L0 is
the initial length of the segment, t is the time period,
15 days in these cases, and it was expressed as % day−1.
Also, a curvature index was obtained as the ratio between
the arc and the chord of the distal most 3 mm of the apex
after 15 days of the experiment; a value of 1 identified a
straight apex.
Based on the results of these initial experiments, two new
experiments were set up to evaluate the effect of light intensity
and water flow on the transformation of apexes of
C. chamissoi. The setup was the same as the initial experi-
ments, except that 20 replicate fragments were used in each
treatment, only sporophytic branches without reproductive
1160 J Appl Phycol (2018) 30:1157–1166

Table 1 Summary of the results of the six experiments done in this
study in relation to transformation and attachment of apexes of
Chondracanthus chamissoi. We indicate the experimental design
(factors, treatments, and number of replicates in each treatment) and the
results of the response variables (specific growth rate, curvature index,
frequency of transformation of the apexes, and frequency of attachment)
at the end of each experiment. Results of statistical analyses are also
indicated (ANOVA for the specific growth rate and curvature index, χ2
for the frequency of curvature, of transformation, and of attachment; ns
non-significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Treatment Replication Specific Curvature Frequency of Frequency of

growth rate index transformation (%) attachment (%)

Experiment 1: angle of incidence of light, fragment orientation

Light from above—horizontal fragment n=8 3.1 ± 0.3 ns 1.00 ± 0.00 ns 0 0
Light from above—vertical fragment 4.4 ± 0.5 1.00 ± 0.00 0 0
Light from the side—horizontal fragment 3.7 ± 0.3 1.00 ± 0.00 0 0
Light from the side—vertical fragment 3.5 ± 0.4 1.00 ± 0.00 0 0
Experiment 2: phase of the life cycle
Female gametophyte n=8 3.3 ± 0.3 ns 1.06 ± 0.02 ns 75.0 ns 38 ns
Male gametophyte 3.4 ± 0.4 1.05 ± 0.01 87.5 38
Tetrasporophyte 4.6 ± 0.6 1.09 ± 0.02 100 50
Experiment 3: reproductive state
Vegetative n = 16 4.2 ± 0.2*** 1.13 ± 0.03*** 93.3*** 33*
Reproductive 3.2 ± 0.2 1.01 ± 0.01 14.3 0
Experiment 4: light intensity
Low (8 μmol photons m−2 s−1) n = 20 4.1 ± 0.3*** 1.20 ± 0.03** 100* 50*
High (80 μmol photons m−2 s−1) 6.7 ± 0.4 1.08 ± 0.02 68.4 11
Experiment 5: water-flow
Low (0.2 mL min−1) n = 20 4.1 ± 0.2* 1.14 ± 0.03* 100 ns 50*
High (14 mL min−1) 4.9 ± 0.3 1.06 ± 0.02 75.0 12
Experiment 6: type of substratum (first version)
Bivalve shell n=8 5.8 ± 0.5 ns 1.12 ± 0.04*** 100*** 86*
Granite rock 5.0 ± 0.2 1.14 ± 0.01 100 29
Sea-squirt tunic 4.8 ± 0.4 1.16 ± 0.02 100 25
Glass slide 4.7 ± 0.3 1.09 ± 0.02 85.7 14
Chondracanthus chamissoi 5.4 ± 0.3 1.01 ± 0.01 0 0
Experiment 6: type of substratum (second version)
Granite rock n=8 4.6 ± 0.2 ns 1.14 ± 0.01*** 100 38**
Corallinaceae 4.6 ± 0.2 1.13 ± 0.01 100 67
Ulva sp. 5.4 ± 0.4 1.06 ± 0.01 0 0
Sarcothalia crispata 5.2 ± 0.2 1.02 ± 0.01 0 0
Chondracanthus chamissoi 5.2 ± 0.2 1.01 ± 0.01 0 0

structures were included, and they were placed horizontally on
the substratum. In experiment 4, two levels of light intensity
were used: 10 and 80 μmol photons m−2 s−1. Likewise, in
experiment 5, two levels of water flow were also used: 0.2
and 14 mL min−1. In both of these experiments, granite boul-
ders were used as substrata.
In the last experiment, experiment 6, we evaluated the ef-
fect of the type of substratum on the transformation and at-
tachment of the apexes of C. chamissoi. The experiment was
done twice with some different substrata. In the first version,
the substrata used were bivalve shells (Venus antiqua), granite
rock, sea-squirt tunic (Pyura chilensis), glass-slides for
microscopy, and thalli of C. chamissoi. In the second version,
the substrata used were granite rock, encrusting coralline algae
(Corallinaceae) growing on rock, blades of Ulva sp., blades of
Sarcothalia crispata, and, again, thalli of C. chamissoi. Eight
replicate fragments were used for each type of substratum in
each version, and they were placed horizontally at a distance
of 1–2 mm over the substrata. Only sporophytic branches
without reproductive structures were used.
These last three experiments were evaluated every 3 days,
for 12 days. The response variables were the same and were
evaluated in the same way as for the second and third exper-
iments, except that they considered a period of 12 days.
J Appl Phycol (2018) 30:1157–1166
Statistical analyses
The specific growth rate and the curvature index were ana-
lyzed with one- or two-way ANOVA, depending on the ex-
periment, with prior testing of normality using Shapiro-Wilk’s
test and homoscedasticity using Cochran’s test (Underwood
1997; Zar 1999; Quinn and Keough 2002). When necessary,
logarithmic transformations were successful to comply with
the assumptions of ANOVA. Significant results were explored
with the a posteriori Tukey’s HSD test (Zar 1999). In all anal-
yses, the significance level considered was α = 0.05.
The frequency of transformation and frequency of apexes
that attached to the substratum were analyzed with Pearson’s
χ2, applying Yates correction for continuity (Zar 1999) and
grouping some classes when necessary. When a fragment be-
came detached or discolored during an experiment, data from
the fragment were not considered in the analyses and the de-
grees of freedom were corrected.
To determine whether the morphological transformation of
an apex favored its attachment to the substratum, data of cur-
vature index and occurrence of attachment by the end of each
experiment were analyzed with logistic regressions. The prob-
ability of error in the regression was determined with the like-
lihood ratio after the evaluation of goodness of fit with
Hosmer and Lemeshow’s test (Hosmer and Lemeshow
1989; Quinn and Keough 2002).
Results
Growth
All of the apexes used exhibited growth during the experi-
ments, indicating that the incubation conditions were appro-
priate. Specific growth rates varied between 3.1 ± 0.3 and
6.7 ± 0.4% day−1 (Table 1). These values are within the range
described for C. chamissoi (González and Meneses 1996;
Bulboa and Macchiavello 2001; Bulboa et al. 2008; Fonck
et al. 2008; Sáez et al. 2008; Ávila et al. 2010; Bulboa et al.
2010) and C. squarrulosus (Cabello-Pasini et al. 2003).
Significant differences between treatments in specific growth
rates were obtained in three of the experiments. In the third
experiment, growth was greater in vegetative than reproduc-
tive apexes (F1,27 = 19.68; p = 0.0001); in the fourth experi-
ment, it was greater in higher than lower light intensity
(F1,33 = 27.42; p < 0.0001), and in the fifth experiment, it
was greater in higher than lower water flow (F1,28 = 5.20;
p = 0.03) (Table 1).
Morphological transformation of the apex
The first experiment, which included orientation of the apexes
and the angle of light incidence, was different from the rest
1161
because apexes were distant from any substrata. These apexes
grew, but showed no response associated with morphological
transformation, remaining straight, and without a change of
shape (Table 1).
In the remaining experiments, many apexes underwent
morphological transformation. No significant differences in
the curvature index or in the frequency of transformation were
detected in experiment 2 among apexes of the three free-living
types of thalli (F2,21 = 1.06; p = 0.36 for the curvature index,
and χ22 = 2.32, p = 0.87, for the frequency of transformation).
In contrast, in experiment 3, vegetative apexes showed a sig-
nificantly greater curvature index (F1,27 = 22.89; p = 0.0001)
and greater frequency of transformation than reproductive
apexes (χ21 = 18.29, p < 0.0001). Based on these results, the
remaining three experiments were set up using branches with-
out reproductive structures, obtained from sporophytic thalli.
Morphological transformation of the apexes occurred in
both levels of light intensity (experiment 4). Apexes in lower
light intensity showed a significantly greater curvature index
(F1,33 = 12.07; p = 0.0014) and frequency of transformation
(χ21 = 4.08, p = 0.044) than apexes in higher light intensity.
As for light intensity, morphological transformation of the
apexes was observed in both levels of water flow (experiment
5). The curvature index was significantly greater in apexes
exposed to lower water flow (F1,28 = 5.36; p = 0.028), al-
though at the end of the experiments, the frequency of trans-
formed apexes did not differ between treatments (χ21 = 2.16,
p = 0.14). This suggests that water flow does not affect the
frequency of transformation, but that it does affect the degree
to which apexes curve.
Morphological transformation was different in apexes
placed in close vicinity with different types of substrata (ex-
periment 6). In the first version of this experiment, transfor-
mation occurred in all apexes placed in close vicinity to bi-
valve shells, granite rock, and sea-squirt tunics, and in most of
the apexes placed close to glass, but no transformation was
observed in the apexes placed close to thalli of C. chamissoi
(Table 1). At the end of this experiment, there were significant
differences among treatments in the curvature index
(F4,32 = 8.23; p < 0.0001) as well as in the frequency of
transformation (χ21 = 26.73, p < 0.0001). In the second ver-
sion of experiment 6, transformation was observed in all apex-
es placed in close vicinity to granite rock and calcareous cor-
alline crusts, but in none of the apexes placed on Ulva sp.,
S. crispata, or C. chamissoi. Differences among treatments at
the end of the experiment were significant for the curvature
index (F4,27 = 35.67; p < 0.0001) and for the frequency of
transformation (χ21 = 17.03, p < 0.0001).
Our results show that the morphological transformation
was initiated very soon after the experiments were set up.
The change in shape could already be noted at the first eval-
uation, 3 days from the start of the experiment, in at least some
treatments of all the experiments that included a substratum
1162 J Appl Phycol (2018) 30:1157–1166

(Fig. 2). In experiments where rocky or calcareous substrata
were used, the curvature index tended to increase steadily in
most treatments (Fig.2a–d). In contrast, where fleshy sea-
weeds were used as substrata, some apexes showed a curva-
ture, but it was not as immediate as in other treatments, mostly
after 9–12 days (Fig. 2e, f). Also, these apexes did not curve
towards the substrata, and were not considered to be
transformed.
Attachment
Attachment of the fragments to a substratum during the ex-
periments occurred only in treatments where the apexes be-
came morphologically transformed (Table 1). In experiment 2,
there were no significant differences in attachment among the
three types of thalli (χ22 = 0.34, p = 0.99), whereas in exper-
iment 3, more apexes became attached in vegetative than in
reproductive fragments (χ21 = 4.83, p = 0.028). There was
attachment of fragments to the substrata in both treatments
of experiments 4 and 5, although the frequency of attachment
was significantly greater in apexes in lower light intensity
(χ21 = 6.63, p = 0.010) and in lower water flow (χ21 = 5.45,
p = 0.0196).
Attachment occurred on some types of substrata, but not on
others. Considering all of the experiments, more than 80% of
the apexes that were transformed, and none that were not
transformed, became attached during the experiments (15 or
12 days). Experiments 2 to 5 used only rocky substrata, and
attachment occurred. In the first version of experiment 6, at-
tachment occurred in most fragments placed over bivalve
shells and also in some placed on rock, sea-squirt tunics, and
glass, but it was not observed in the apexes placed on thalli of
C. chamissoi (see Table 1). Thus, there were significant dif-
ferences in the frequency of attachment among these substrata
(χ24 = 10.38, p = 0.034). In the second version of this exper-
iment, attachment was frequent on calcareous algae and rock,
but did not occur in the treatments with fleshy seaweeds (Ulva
sp., S. crispata and C. chamissoi; χ24 = 8.78, p = 0.003).
The curvature index was a good predictor of attachment.
Logistic regressions relating the occurrence of attachment dur-
ing the experiment with the curvature index were significant
for all the experiments. In the experiments where substrata
were used, more apexes became attached as the curvature
index increased (Table 2, see an example in Fig. 3).
Discussion
The tips of branches of Chondracanthus chamissoi have the
capacity for secondary attachment. González et al. (1997)

Fig. 2 Curvature index (mean ± 1.25 a Female gametophyte

1.25 b Vegetative thalli
standard error) for apexes of Male gametophyte
1.20 1.20 Reproductive thalli
Chondracanthus chamissoi in Tetrasporophyte
different experiments. a 1.15 1.15
Experiment 2, comparing the
phase of the life cycle of the 1.10 1.10
fragments. b Experiment 3,
comparting reproductive state of 1.05 1.05
the fragments. c Experiment 4, 1.00
1.00
comparing two levels of light 0 3 6 9 12 15 0 3 6 9 12 15
intensity. d Experiment 5,
comparing two levels of water 1.25 c Low light intensity
1.25 d Low water flow
flow. e and f Experiment 6 (first High light intensity High water flow
1.20 1.20
and second version, respectively)
Curvature index

comparing different types of 1.15 1.15

substrata
1.10 1.10

1.05 1.05

1.00 1.00
0 3 6 9 12 15 0 3 6 9 12 15

1.25 e Rock 1.25 f Rock

Bivalve shell Coralline crusts
1.20 1.20
Glass slides Ulva sp.
Sea-squirt tunic Sarcothalia crispata
1.15 1.15
C. chamissoi C. chamissoi
1.10 1.10

1.05 1.05

1.00 1.00
0 3 6 9 12 15 0 3 6 9 12 15
Time (days)
J Appl Phycol (2018) 30:1157–1166 1163

Table 2 Results of the logistic regressions used to relate the attachment Our results also indicate that the transformation of the
of apexes of Chondracanthus chamissoi and the curvature index in the
apexes was affected by some of the experimental factors tested
experiments where transformation of apexes was observed. The χ2
statistic and the probability are indicated and not by others. Results of experiment 1 indicate that the
angle of incidence of light on the fragments and orientation of
Experiment Logistic regression results the apexes were not related to the morphological transforma-
Exp. 2. phase of the life cycle (χ21 = 22.50, p < 0.0001)
tion and that, in the absence of contact with a substratum or
Exp. 3. reproductive state (χ21 = 25.22, p < 0.0001)
other stimuli (e.g., the presence of calcium), transformation
did not occur. These apexes grew normally during the exper-
Exp. 4. light intensity (χ21 = 31.57, p < 0.0001)
iment (Table 1), and their morphology did not change.
Exp. 5. water-flow (χ21 = 22.35, p < 0.0001)
Therefore, the responses observed in the remaining experi-
Exp. 6.1. type of substratum (χ21 = 11.68, p = 0.001)
ments were not due to these two factors or other conditions
Exp. 6.2. type of substratum (χ21 = 18.88, p < 0.0001)
of the experimental setup.
Transformation was not affected by the type of free-living
thalli (experiment 2) (Sáez et al. 2008), but the reproductive
proposed that drifting fronds of C. chamissoi could reattach
state (experiment 3) was important (Table 1). Non-
and generate new plants and, thus, be particularly important
reproductive branches may have been younger or energy
for the maintenance of the populations. Subsequent studies
may have been allocated mainly to growth, producing a rapid
reported that thalli of C. chamissoi reattached to different sub-
transformation response. In contrast, branches with develop-
strata in the field (Macchiavello et al. 2003) and in the labo-
ing reproductive structures may have allocated energy mainly
ratory (Fonck et al. 2008) and described the formation of
to the local production of spores to the detriment of growth
secondary attachment discs (Sáez et al. 2008; Otaíza and
(Salinas 1991; Destombe and Oppliger 2011) and the trans-
Fonseca 2011) as had been done for C. squarrulosus
formation response. In fact, these reproductive apexes had the
(Pacheco-Ruíz and Zertuche-González 1999; Pacheco-Ruíz
second lowest growth rate among the treatments in the differ-
et al. 2005). It has also been shown that the secondary attach-
ent experiments (see Table 1).
ment discs can produce new upright shoots (Pacheco-Ruíz
Earlier experimental results had shown that fragments from
et al. 2005; Fonck et al. 2008; Sáez et al. 2008).
different parts of the thallus showed different production of
Our results expand on the results reported by some of the
attachment structures. Otaíza and Fonseca (2011) found that
above mentioned authors. They indicate that secondary at-
apexes of basal branches produced these structures in greater
tachment of C. chamissoi can include a morphological trans-
proportion than those of lateral branches and pinnules. Apart
formation of the apexes. Initially, the tips of the experimental
from the apexes, Fonck et al. (2008) indicated that secondary
fragments were straight, thick, and blunt but, once they were
discs could be produced throughout the surface of the thallus
placed experimentally close to appropriate substrata, the
fragment, which explained the formation of multiple second-
growing tips rapidly became elongated and cylindrical, and
ary discs found in some of their experimental fragments
they tapered and curved, bringing the apexes in contact with
(Fonck et al. 2008; Sáez et al. 2008). Our observations of
the substratum (Fig. 1c, d). Thus, if apexes are short of direct
C. chamissoi indicate that, besides the apex of branches and
contact with the substratum, they can elongate and also be-
pinnules, attachment structures can be formed from the tip of
come attached.
pinnules located on the sides of the blades and also from the
tip of spines that are sometimes present in rows on the surface
1
of some blades. This could also explain the report of multiple
secondary discs found by Fonck et al. (2008) and Sáez et al.
Attachment

(2008). Secondary attachment has also been reported from the

tips of branches of Solieria chordalis (Floc’h et al. 1987).
Similarly, Perrone and Cecere (1997) showed that secondary
attachment structures were formed on the tips of branches that
contacted the substratum, and also from regenerating surfaces
0 of experimental cuts on the fragment in Solieria filiformis.
1 1.1 1.2 1.3 1.4 1.5 Thus, attachment structure formation in C. chamissoi appears
Curvature index to happen from the tip of branches, pinnules, and spines (see
Fig. 3 Occurrence of attachment of apexes of Chondracanthus also Salinas 1991) where rapid growth from an apical meri-
chamissoi in relation to the curvature index in experiment 3 (under high stem is occurring.
and low light intensities). All 40 apexes used in that experiment are
shown (some points overlap). Attachment occurred only in a few
Experiments 4 and 5 indicated that low light intensity and
apexes under high light intensity and in 50% of those in low light low water flow favored morphological transformation and at-
intensity tachment of the apexes (Table 1). Similar responses have been
1164
reported for other seaweeds. Low light intensity favored the
formation of attachment structures (e.g., Buggeln 1981;
D’Antonio and Gibor 1985; Perrone and Cecere 1997;
Fonck et al. 2008), whereas high light intensity promoted
upright axes (Buggeln 1981; Salinas 1991). On the other
hand, rhizoid-like processes may be induced by the reduction
of some nutrients (Buggeln 1981), which can happen with low
water flow, and rhizoid initiation may be stimulated by ab-
sence of water flow (Salinas 1991). In seaweeds like
C. chamissoi, the basal part of an attached individual is sub-
jected to lower light intensity than the upright branches due to
self-shading and also to lower water flow due to proximity to
the ocean floor. Therefore, growth of basal branches towards
the substratum is consistent with the conditions where they
grow. Interestingly, low light intensity and low water flow
induced transformation of the apex of upright branches, giving
them morphological and functional characteristics of basal
branches. Therefore, in the natural environment, tips of
drifting thalli or fragments will be induced to undergo mor-
phological transformation when they settle or become trapped
in spots with reduced light and water flow, such as in crevices
or under boulders.
The results of experiment 6 showed that the type of sub-
stratum had a strong effect on the frequency of morphological
transformation and attachment of the apexes of C. chamissoi
(Table 1). All of the apexes tested on calcareous substrata
(bivalve shells and coralline algae) were morphologically
transformed and most became attached during the
experiments. This result is in agreement with other studies.
Fonck et al. (2008) found that a greater proportion of the thalli
attached to shell gravel than to stones in laboratory experi-
ments, while Macchiavello et al. (2003) showed that attach-
ment to shells was frequent in the field, in agreement with
result of C. squarrulosus (Pacheco-Ruíz et al. 2005). Even
the addition of dissolved calcium to the growth medium fa-
vored attachment of branches of C. chamissoi (Otaíza and
Fonseca 2011). A similar effect of calcium has been described
for other red seaweeds (Salinas 1991; Juanes and Puente
1993; Santelices and Varela 1994; Perrone and Cecere
1997). Calcium also has positive effects in the attachment of
spores of red seaweeds (Santelices and Aedo 2006). In the
remaining solid substrata (rock, glass, and sea-squirt tunics),
apexes also showed a high frequency of transformation, but a
more reduced frequency of attachment during the experiment.
Except for glass, the solid substrata used represent natural
substrata present where C. chamissoi is generally found
(Vásquez and Vega 2001; Macchiavello et al. 2003; Pers.
obs.). In contrast to the solid substrata, the living tissue of
the fleshy seaweeds Ulva sp., S. crispata, and C. chamissoi
failed to produce any response. This is in agreement with the
fact that C. chamissoi does not grow as an epiphyte on fleshy
seaweeds. Also, apexes of C. chamissoi attaching to their own
thalli would produce an entanglement of the blades, which
J Appl Phycol (2018) 30:1157–1166
would likely have effects on the morphology of the thalli.
This has not been observed for C. chamissoi, but this type of
morphology has been described in Solieria chordalis (Floc’h
et al. 1987). Thus, C. chamissoi can reattach to a wide range of
solid substrata, both natural and artificial (Table 1, see also
Bulboa et al. 2005; Otaíza and Fonseca 2011), but show a
distinct selective response against fleshy seaweeds.
Our results in experiment 6 also indicated that the apexes of
C. chamissoi have a remarkable capacity to perceive stimuli
from the surrounding environment and display a rapid
response. Fonck et al. (2008) found that almost 90% of the
fragments of C. chamissoi placed in aquaria with shell frag-
ments formed secondary attachment discs in 20 days, but
many were already attached at the moment of the first control,
after 5 days of incubation. Similarly, Sáez et al. (2008) found
that an average of 54% of the fragments placed in aquaria with
shells formed secondary attachment discs in 20 days, but some
were already attached at day 5 of their experiments. Otaíza
and Fonseca (2011) found that after 8 days of incubation,
more than 80% of the apexes had at least initiated formation
of these structures and 12% had already attached to the sub-
stratum in calcium addition treatments. Our results show that
initiation of transformation was already visually detectable
during the first control, after 3 days of incubation (Fig. 2).
Similar values have been recorded for other species. Floc’h
et al. (1987) found that many fragments of Solieria chordalis
produced attachment discs after 2 weeks in culture. Khou et al.
(2007) found that Caulerpa filiformis can produce rhizoids in
5 days. Perrone and Cecere (1997) found that when explants
of Solieria filiformis were forced into contact with the substra-
tum, rhizoids attached within 5 days. In responses to light,
growth reactions can be very fast, and can take only a few
minutes to initiate a change (Buggeln 1981). An attachment
response that requires a few days to develop may be too slow
to be considered adaptive, but it must be considered that frag-
ments may become progressively stimulated to attach while
adrift (Khou et al. 2007) such that, after being fully stimulated,
a short quiet period may be enough for attachment to occur.
We cannot offer a mechanism to explain the transformation
and attachment response in C. chamissoi. Different environ-
mental variables may represent cues for, or inhibitors to, the
response. The nature of these cues may be either physical
(e.g., light intensity or light wave length, Lüning 1990), chem-
ical (e.g., concentration of calcium, Salinas 1991), or biolog-
ical (e.g., biofilms, Joint et al. 2007; Singh and Reddy 2014;
exudates of fleshy seaweeds, Santelices and Varela 1993).
The factors that favor transformation of the apexes appar-
ently stimulate the transformation of branches and pinnules
into basal branches. The reverse may also occur: basal
branches may be transformed into upright thalli if exposed
to high light intensity and water flow.
The capacity of apexes of C. chamissoi to becoming fixed
to the substratum by producing secondary discs that can
J Appl Phycol (2018) 30:1157–1166
generate new shoots has different consequences in the life
history of this organism. When plants fixed to the substrata
produce new attachment structures (mainly from basal
branches), new adhesion points of are added to the thallus,
consolidating the attachment system of the plant (Pacheco-
Ruíz et al. 2005; Otaíza and Fonseca 2011). When this occurs
repeatedly, it may lead to the expansion of natural patches, and
be considered a mechanism of vegetative propagation. On the
other hand, when drifting fragments come in contact with
appropriate substrata, the ability to produce secondary discs
that generate new upright shoots, i.e., new individuals, must
be considered as part of a mechanism of vegetative reproduc-
tion (Floc’h et al. 1987; González et al. 1997; Macchiavello
et al. 2003; Pacheco-Ruíz et al. 2005; Fonck et al. 2008; Sáez
et al. 2008). In either situation, if the parental tissue is dam-
aged by adverse seasonal conditions, the basal discs may also
be considered as a mechanism of resistance and perennation
(Pacheco-Ruíz et al. 2005).
Cultivation techniques using fragment attachment have
been proposed for C. chamissoi (Macchiavello et al. 2003;
Bulboa et al. 2005; Bulboa and Macchiavello 2006; Bulboa
et al. 2013). Results obtained here may be used to favor at-
tachment of fragments in cultivation. The capacity of the
apexes to grow and bend towards the substratum indicates that
tips of branches and pinnules do not require to be in immediate
contact with the substratum to trigger the attachment response.
Also, younger, non-reproductive thalli will attach more read-
ily. Moreover, low light levels and slow water flow may be
used to induce the transformation response and increase the
number of attachment points. Alternatively, a pretreatment of
fragments in these conditions before seeding onto their sub-
strata may also enhance attachment. Finally, many solid and
particularly calcareous substrata may be appropriate, but
fleshy seaweeds fouling the substrata may interfere with at-
tachment. These recommendations may be also considered for
repopulation or restoration programs for this species.
Acknowledgements We are grateful to P. Neill for suggestions on the
manuscript. This study was part of the thesis presented by the first author
as partial fulfillment of the Marine Biology Program at the Universidad
Católica de la Santísima Concepción.
Funding information Funds for this research were provided by the
Facultad de Ciencias, UCSC.
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