www.elsevier.com/locate/jembe

Journal of Experimental Marine Biolog

Biomechanical properties and holdfast morphology of coenocytic

algae (Halimedales, Chlorophyta) in Bocas del Toro, Panama

Kim Anderson a, Lisa Close b, Robert E. DeWreede c,*, Brandon J. Lynch d,

Carlos Ormond e, Matt Walker f

a University of Central Oklahoma,100 N. University Drive, Edmond, OK 73034, United Statesb University of California, Davis; School of Veterinary Medicine, One Shields Avenue, Davis, CA 95616, United Statesc University of British Columbia, Department of Botany, 3529-6270 University Blvd., Vancouver, BC, Canada V6T 1Z4

d University of Arizona, Tucson, AZ 85721, United Statese The University of British Columbia, Vancouver, BC, Canada V6T 1Z4

f Texas A and M University-Kingsville, 700 University Blvd. MSC 114, Kingsville, TX 78363 * 361.593.2111, United States

Received 15 April 2005; received in revised form 4 July 2005; accepted 11 July 2005

Abstract

For attached marine organisms, specific biomechanical properties may result in detachment or in tissue loss, when sufficient

tensile force is applied. Algae experience such forces through water movement, which may thus act to limit size, abundance,

and species composition, of populations of algae.

Coenocytic construction is uncommon in the algae, but it occurs relatively more frequently in green algae found in

shallow subtidal sediments associated with coral reefs, e.g., at our study site of Isla Colon, Bocas del Toro, Panama. We

studied the biomechanical properties of some tropical coenocytic algae (Udotea flabellum (Ellis et Solander) Lamouroux,

Penicillus capitatus Lamarck, P. pyriformis A. and E.S. Gepp, and Halimeda gracilis Harvey) anchored in sediments. We

compare our results with published data on other coenocytic algae, as well as with multicellular algae. Our results show that

properties of sand-dwelling coenocytes, such as mean force to dislodge (4.9–12.7 N), mean force to break (6.6–22.1 N), and

mean strength (1.0–7.0 MN m�2), are all within the range reported for temperate, multicellular, algae. In contrast, the

coenocytes differed markedly from the temperate non-coenocytes in the consequences of applied tensile force: coenocytes

were removed whole, while most temperate algae attached to rocks break within the thallus. Some multicellular algae can

regrow from tissue left on the substratum; three of the four coenocytic species we examined had rhizoids connecting closely

adjacent (0.1–0.15 m) individuals, and these rhizoids may serve to regrow a new individual. While our experiments indicated

that sufficient tensile force results in dislodgment, calculations using the experimentally determined variables led us to

conclude that water velocities sufficient to dislodge individuals are unlikely to occur. Since dislodgment is usually fatal for

algae, the role of the holdfast is a critical one. All of the species we investigated had similar holdfast morphology, a mass of

rhizoids which entrained sand, the entire unit forming a hemispherical to cylindrical mass. Despite the consistency in holdfast

0022-0981/$ - s

doi:10.1016/j.jem

* Correspondi

E-mail addre

y and Ecology 328 (2006) 155–167

ee front matter D 2005 Elsevier B.V. All rights reserved.

be.2005.07.005

ng author. Tel.: +1 604 822 6785; fax: +1 604 822 6089.

ss: dewreede@interchange.ubc.ca (R.E. DeWreede).

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167156

form, and the initial prediction that this was an optimal form for anchoring these algae, our data suggest that this is not the

case.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Algae; Biomechanics; Coenocyte; Detachment; Holdfast

1. Introduction

Green algae, some of which are both coenocytic

and calcified, are common in tropical shallow water

marine habitats, where they function as primary

producers and generate calcium carbonate sediments.

Unlike most temperate zone marine macroalgae,

which are predominantly multicellular, attached to

solid substrata (e.g., rocks), and not calcified, tropi-

cal coenocytes are unicellular, are frequently

anchored in sediments such as coral rubble, sedi-

ments, and mangrove detritus, and many are calci-

fied. Despite the internal structural simplicity

(connected filaments lacking separating walls), gen-

era such as Caulerpa, Halimeda, Udotea and Peni-

cillus attain a complex morphology consisting of a

rhizoidal holdfast(s), a stipe or stolon, and an

astounding morphological variation across species

and genera. For these algae, holdfast morphology

and sediment cohesiveness are important determi-

nants of the maximum tensile force they are able

to withstand. This contrasts with algae attached to

rock surfaces, where adhesion of the algal tissue to

rock is critical. At our two study sites in the vicinity

of Isla Colon, Bocas del Toro (N 98 24V 53.53, W828 19V 49.90), Panama, Udotea, Penicillus, and

Halimeda co-exist, often intermixed in habitats ran-

ging from shallow wave exposed coral sand to finer

sediment areas adjacent to the red mangrove, Rhizo-

phora mangle Linnaeus.

Among the many environmental factors determin-

ing the abundance, size, and overall morphology of

algae are forces generated by water motion, e.g., tidal

currents and wave impact (e.g., Shaughnessy et al.,

1996; Neushul, 1972; Blanchette, 1997; Denny et al.,

1997; Duggins et al., 2003). When such forces are

sufficiently great, algae may be removed in their

entirety from the rocks and sediments (Duggins et

al., 2003; Collado-Vides et al., 1998), or break within

the thallus, e.g., losing surface area to decrease drag

(Carrington, 1990; Shaughnessy et al., 1996; Blanch-

ette, 1997; Milligan and DeWreede, 2004). While

there have been many studies detailing biomechanical

properties, and effects of wave impact, on temperate

algae attached to rocks (Koehl and Wainwright, 1977;

Armstrong, 1987; Carrington, 1990; Gaylord et al.,

1994; Johnson and Koehl, 1994; Gaylord and Denny,

1997; Milligan and DeWreede, 2000; Duggins et al.,

2003; Milligan and DeWreede, 2004), few studies

have examined such features for tropical sand-dwell-

ing coenocytes (Collado-Vides et al., 1998; Padilla,

1989). We undertook this research to compare biome-

chanical properties between coenocytic and multicel-

lular algae and to examine the biomechanical

properties of sand-dwelling coenocytes in relation to

water movement they might reasonably be expected to

encounter.

To date, detailed biomechanical properties (relating

to attachment) of calcified coenocytes have only been

reported for a single species, Udotea flabellum (Col-

lado-Vides et al., 1998). Here, by investigating these

properties for three additional species, and also for

one of the same species but at a geographically distant

site, we seek to place the limited prior data into a

broader context and thus reveal any patterns that may

exist. Furthermore, given the structural differences

between multicellular and coenocytic algae, our

more extensive knowledge of the former may not be

applicable to coenocytes. For example, it has been

reported that some multicellular algae survive forces

generated through water movement by loosing thallus

tissue (e.g., Blanchette, 1997; Milligan and

DeWreede, 2004); it seems unlikely that such a

mechanism is a viable one for coenocytic algae,

given that repeatedly torn tissue could result in a

significant loss of cytoplasm. As noted by Ross et

al. (2005), Vroom et al. (2003) for Halimeda tuna,

and Vroom and Smith (2003) more generally, some

coenocytes are able to tolerate severe injury, whereas

others die as a result.

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167 157

For algae in general, holdfast morphology varies

from a hemispherical rhizoidal mass, to creeping

rhizomes, to solid disks. The holdfast shape of

the individuals we examined and tested was con-

sistently bowl-shaped to cylindrical, unless attached

to coral fragments; in which case the holdfast forms

a flat rhizoidal mass along the upper surface of the

coral. The bowl/cylinder form arises as the algal

filaments enclose the sediments and form a cohe-

sive mass. Some tropical sand-dwelling coenocytic

algae (e.g., some species of Udotea) have subsur-

face lateral rhizoids arising from the holdfast area

that connect to other individual(s) of the same

species (Fig. 1; and Littler and Littler, 2000);

these lateral rhizoids may give rise to new indivi-

duals, at least in culture (Hillis-Colinvaux, 1972).

If these rhizoidal connections occur generally, and

if they are able to give rise to new individuals in

situ, a dislodged individual may die, but a new

one may be regenerated. We examined the algae

we studied for the presence of these rhizoidal

connections.

Collado-Vides et al. (1998) described biomecha-

nical properties of Udotea flabellum, found at

Fig. 1. Halimeda gracilis, Udotea flabellum, and Penicillus pyr-

iformis; scale bar=4 cm. Lateral rhizoids connect two individuals of

U. flabellum and two of P. pyriformis, such rhizoids were not found

for H. gracilis neighbors.

Puerto Morelos, Mexico. The authors reported that

95% of U. flabellum individuals are removed from

the substratum in their entirety (i.e., do not break)

after applying a mean tensile force of 8.6 N (95%

C.I.=1.6), break primarily at the stipe–holdfast

junction in the laboratory (19.3 N; 95% C.I.=2.1),

and have a mean strength of 4.8 MN (95%

C.I.=0.63). Here, we report the biomechanical prop-

erties of four species of coenocytic green algae,

including Udotea flabellum. We tested the hypoth-

eses detailed below, extend the number of coenocy-

tic species for which biomechanical properties are

known, and compare these properties for the differ-

ent species we tested.

Based on the species studied to date (Collado-

Vides et al., 1998; Padilla, 1989), we hypothesized

that when sufficient tensile force is applied, coeno-

cytic individuals will not break within the thallus but

will be pulled whole from the substratum. If the

preceding holds, then the force to break an indivi-

dual exceeds the force required to remove an entire

individual. Both these predictions differ from what is

common in multicellular algae attached to rocky

surfaces, which frequently break first within the

stipe and stipe/holdfast region (e.g., Shaughnessy et

al., 1996; Utter and Denny, 1996; Johnson and

Koehl, 1994; Carrington, 1990) or in the blade

(Blanchette et al., 2002; Blanchette, 1997); hold-

fast/substratum breaks occur less frequently (Milli-

gan and DeWreede, 2000, 2004; but see Duggins et

al., 2003).

The holdfast is crucial in anchoring algae, and

we predicted that the tensile force required to

remove algae from the substratum will increase as

holdfast volume increases. For multicellular algae

attached to rocky substrata, this relationship is

equivocal, as a positive correlation between hold-

fast biomass and tenacity was reported for the

kelp Agarum fimbriatum, but no such correlation

was found for Costaria costata (Duggins et al.,

2003).

Drag on algae increases with water velocity and

blade surface area; in flexible organisms, blade

surface area will change with increasing water velo-

city, and this change is encapsulated by the coeffi-

cient of drag. We predicted that the force required

to remove algae from the substratum increases as

blade surface area increases. Such a relationship

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167158

was found for both Agarum fimbriatum and Cost-

aria costata (Duggins et al., 2003). In contrast, we

predicted no correlation of strength with the tensile

force needed to remove a coenocytic individual,

with blade surface area, or with holdfast volume.

Finally, given the consistency of the cylindrical to

bowl shape of the holdfasts in all the coenocytes

we studied, we predicted that these shapes provide

maximum tenacity, compared to other shapes con-

structed from an equal mass of tissue.

2. Materials and methods

2.1. Study area

Our research was done using the facilities of the

Institute for Tropical Ecology and Conservation

(ITEC) in Isla Colon, Bocas del Toro, Panama. Our

study sites were at Lime Point (N 9 24V, W 82 19V) andConch Point (N 9 22V, W 82 17V).

We attempted to measure water motion over the

course of this study, but we were unsuccessful.

However, we judge Lime Point as relatively wave

exposed compared to Conch Point (about 6 km

from Lime Point). Near shore and shallow waters

(b0.50 m) at Lime Point are characterized by

patches of Thalassia testudinum Koenig (turtle

grass), Syringodium filiforme Kuetzing (manatee

grass), and sand. The shallower sandy areas have

Penicillum capitatus and Udotea flabellum, as well

as species of Caulerpa (e.g., C. racemosa (Forsskal)

J. Aghard and C. sertularioides (S.G. Gmelin)

Howe), all species intermixing with the sea grasses.

Sites closer to the reef edge, and deeper (N1 m)

have a greater abundance of Udotea, and less of

Penicillus, and an increased abundance of live coral.

Here, the urchins Diadema antillarum Philippi and

Echinometra lucunter lucunter Linnaeus are first

seen, and the latter increase in abundance over the

reef crest where live coral and greater water depths

(0.5 m to 5 m) occur.

The second study site, Conch Point, is along a

more wave-protected shore lined with mangroves

(Rhizophora mangle). Water depth here ranges

from 0.5 to 1 m at the mangrove edge and

increases to approximately 7 m at some 20 m

from shore. The substratum immediately adjacent

to the mangrove edge is populated with sea grass

(Thalassia testudinum) and green algae, and in

deeper water with various coral species and

sponges. The mangroves are inhabited by small

groups of juvenile fish, and the sea grass beds

and coral clusters are inhabited by small stingrays,

other fish, feather duster worms (e.g., Anamobaea

spp.), brittle stars (Ophiuroidea spp.), various urch-

ins (e.g., Echinometra and Tripneustis), and other

invertebrates. Along the mangroves, sand comprised

60.8% of the substratum, followed by algae

(21.9%), sea grass (8%), coral rubble (4.9%), live

coral (3.5%), and sponge (0.9%). The density of

the algae investigated in this study varied from 161

per square meter (Halimeda gracilis), 1.7 indivi-

duals per square meter for Udotea flabellum, to 0.4

per square meter for Penicillus spp.

The two sites from which we collected data

differed in wave exposure during our stay, with

Lime Point more wave exposed than Conch Point.

That this is a persistent difference is re-enforced by

the observations that Lime Point has a much longer

fetch (reaching to the open waters of the Carib-

bean), lacks the mangroves which border the reef

at Conch Point, and a coarser surface sediment than

at Conch Point. We assume that they can attain, if

rarely, 16–20 m s�1 (Jones and Demetropoulos,

1968, Denny et al., 1985).

2.2. Species tested

We studied four of the most common species of

green coenocytic algae in the shallow subtidal zone

at Bocas del Toro, Udotea flabellum (Ellis et Solan-

der) Lamouroux, Penicillus capitatus Lamarck, P.

pyriformis A. and E.S. Gepp, and Halimeda gracilis

Harvey. All four species are patchy, and Penicillus

spp. and H. gracilis both occur intermixed in places

with U. flabellum. Given this distribution, we

assume that for a given site, these algae are all

exposed to similar water movement. All of the

experimental species tested were not visibly

damaged and non-reproductive; however, some

Halimeda gracilis developed gametangia after

August 1, 2004 (a night with a full moon). At

this time about 2% of the individuals turned white

and showed the bright green gametangia typical of

the species.

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167 159

2.3. Species size distribution and holdfast connections

The size class distribution for each species was

obtained by measuring, from substratum to blade

tip, about 100 in situ individuals. To ascertain whether

individuals were connected by underground lateral

rhizoids, we carefully removed 15–20 pairs (within

10–15 cm of each other) from the substratum.

2.4. Biomechanical procedures

Biomechanical procedures were as described in

Collado-Vides et al. (1998). In brief, thalli were

clamped in situ either between wooden blocks

lined with neoprene (and held together with a

clamp), or with the industrial clamp itself. The

blocks or clamps were attached by 16 kg test line

to a spring device (tensiometer—Bell and Denny,

1994; Milligan and DeWreede, 2000), which mea-

sured the force required to detach or break the

thallus. Since most individuals detached whole,

this process was repeated in the laboratory to deter-

mine break location (e.g., blade, stipe/holdfast junc-

tion, etc.) and force-to-break. Strength was

determined by measuring the cross-sectional area

of the break using calipers and the formula for

the closest shape (e.g., an oval or a circle), and

dividing the force-to-break (in newtons) by the

cross-sectional area (m2); the result is given in

mega-newtons (=106 N). Each spring scale used

was calibrated using weights of known mass.

Areas of blades and volumes of holdfasts were

determined using techniques appropriate to their

structure, e.g., the single flat surface of Udotea

blades and the small discs comprising the dbladeTof Halimeda. Blade surface area of Udotea was

estimated by cutting a 4 cm by 4 cm piece from a

Udotea blade, weighing it, and then weighing the

remainder of the blade to determine its (approximate)

surface area. For Penicillus, the relevant surface area

(the projected surface area) was obtained by flatten-

ing the fibrils comprising the blade, tracing the out-

line on a piece of paper and cutting along the

outline. By weighing a 4 cm by 4 cm piece of

paper and then the cut-out paper, the approximate

surface area of each Penicillus blade was calculated.

The surface area of Halimeda was determined by

filling a 4 cm by 4 cm piece of paper with the

individual ddiscs,T weighing the filled paper, and

then weighing the complete blade (comprised of

many discs) and converting this weight to surface

area. Holdfast volume for all species was determined

by measuring water displacement in a graduated

cylinder.

We calculated approximate coefficient of drag

(Cd) values for two species, Halimeda gracilis

and Penicillus capitatus. For Udotea flabellum,

we used previously calculated Cd values (Col-

lado-Vides et al., 1998). We assumed that values

for P. capitatus were similar to those for P. pyr-

iformis, based on the similar size, level of calcifi-

cation, and morphology of these two species.

Values for Cd were determined using a Pesolan0–600 g scale, a PVC pipe to hold the scale, a

connecting fish-line, a lead sinker, and by attaching

the seaweed to the end of the line; this equipment

held the seaweed over the side of the boat and

about 35 cm below the surface of the water. These

tests were done at slack tide, and always in the

same direction, thus minimizing effects of residual

water flow. Following a series of measurements at

known velocities, control values were obtained by

repeating the procedure without the seaweed. Velo-

cities were determined by timing the boat run over

a known distance (250 m) marked on the shore by

flagging tape tied to coconut trees; we were able to

measure drag forces at velocities ranging from 1.1

to 3.3 m s�1.

We indirectly tested the relationship between hold-

fast tenacity and shape by forming wooden blocks

into different holdfast shapes (bowl, short cylinder,

cylinder, and inverted mushroom—the first two are

most similar to the holdfast morphology we encoun-

tered), and measuring their resistance to tensile forces

with and without a coating of the dloopT surface of

Velcro (n) (the Velcro was used to simulate rhizoidal

filaments).

3. Results

3.1. Size class distribution

Size class distribution for each of the species we

tested is given in Fig. 2A–E and indicates that the

maxima differ among species. The most common

A. B.Udotea flabellum: Size Class Distribution

(Lime Point)

0

2

4

6

8

10

12

14

16

18

Above-ground Length (cm)

Per

cent

in S

ize

Cla

ss

Udotea flabellum: Size Class Distribution (ConchPoint)

0

5

10

15

20

25

1 2 3 4 5 6 7 8 9

3 4 5 6 7 8 9

10 11 12 13 14 15 16 171 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

10 11 12

13 14 15 16 17

Above-ground Length (cm)

Per

cent

in S

ize

Cla

ss

C. D.Halimeda gracilis: Size Class Distribution (Conch Point)

0

5

10

15

20

25

30

Above-ground Length (cm)

Penicillus capitatus: Size Class Distribution (Lime Point)

0

5

10

15

20

25

30

35

40

1 2Above-ground Length (cm)

Per

cent

in S

ize

Cla

ss

E. Penicillus pyriformes: Size Class Distribution

(Conch Point)

0

5

10

15

20

25

30

35

Above-Ground Length (cm)

Per

cent

in S

ize

Cla

ssP

erce

nt in

Siz

e C

lass

Fig. 2. (A) Size class distribution of Udotea flabellum at Lime Point; (B) size class distribution of Udotea flabellum at Conch Point; (C) size

class distribution of Halimeda gracilis at Conch Point; (D) size class distribution of Penicillus capitatus at Lime Point; (E) size class distribution

of Penicillus capitatus at Conch Point. N =100 for all species.

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167160

size class for Udotea flabellum at Lime Pt. is the 8–

8.9 cm class, as it is for Halimeda gracilis. The

other species are generally shorter, with both Peni-

cillus species predominating in the 4–4.9 cm size

class. The absence of the smallest size classes is

real, not an artifact of search techniques.

3.2. Morphological variables

The morphological variables (e.g., blade surface

area and holdfast volume) varied for the different

species (Table 1). Udotea flabellum had the largest

mean blade surface areas and holdfast volumes, and

Penicillus spp. the smallest. Halimeda gracilis dif-

fers from the other species tested in that its dbladeTconsists of a series of calcified, discoid segments,

each disc joined to its neighbor by non-calcified and

adhering filaments.

We found rhizoidal connections for three of our

test species, but not for Halimeda gracilis (Fig. 1);

however, we were only able to find these connections

for two or three individuals of each species (we

excavated 10–15 neighboring pairs for each species).

This absence could indicate that we broke such con-

nections, as they are extremely fragile, or that they

occur infrequently.

3.3. Biomechanical measurements

We found that 100% of Udotea flabellum and

98% of Penicillus spp. individuals tested came out

of the sediment as complete thalli when sufficient

Table 3

Correlations of biomechanically related morphological features for

Udotea flabellum at Lime Pt., Bocas del Toro, Panama

Lime Pt.

Correlation of: With: Significance of correlation

Strength Force to NS p =0.612 r2=0.009

Table 1

Summary of means (with 95% confidence interval) of blade surface

area in square meters (m2) and holdfast volume in milliliters (ml) for

the species tested

Species Site Blade surface

area (m2)

Holdfast

volume (ml)

Udotea flabellum Lime Pt. 0.0049

(0.0017)

6.84 (1.94)

Udotea flabellum Conch Point 0.0023

(0.0034)

4.17 (5.83)

Penicillus capitatus Lime Pt. 0.00031

(0.000072)

0.96 (0.273)

Penicillus pyriforme Conch Point 0.00043

(0.000061)

1.17 (0.144)

Halimeda gracilis Conch Point 0.00261

(0.00058)

2.03 (0.702)

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167 161

tensile force was applied. Halimeda gracilis, how-

ever, always broke at the dpads,T immediately adja-

cent to the location where the clamp was applied.

The force needed to break these pads was so small

that we were unable to obtain a reading on our

instruments. However, when the clamp was placed

on the stipe of H. gracilis, the entire plant was

pulled from the substratum.

Results of the biomechanical measurements

(removal force, break force, and strength) are in

Table 2. For the species we tested, Udotea flabel-

lum requires the greatest mean force to remove,

requires the greatest force-to-break, but has inter-

Table 2

Summary of mean removal force, break force, and strength in mega-

newtons per square meter (MN/m2); values in parentheses are 95%

confidence intervals

Species Site Removal

force

(newtons)

Break

force

(newtons)

Strength

(MN/m2)

Udotea flabellum Lime Pt. 12.7 (3.0) 22.1 (3.4) 3.5 (0.8)

Udotea flabellum Conch Point 8.6 (2.8) 22.3 (3.2) 5.7 (1.3)

Udotea flabelluma Puerto

Morelos

8.6 (1.6) 19.3 (2.1) 4.8 (0.6)

Penicillus

capitatus

Lime Pt. 5.0 (0.6) 8.1 (0.9) 7.0 (1.6)

Penicillus

pyriforme

Conch Point 4.9 (0.4) 6.6 (0.9) 4.5 (0.5)

Halimeda gracilis Conch Point 7.1 (1.7)b 12.3 (1.8) 1.0 (0.2)

a Collado-Vides et al., 1998.b Clamp placed at stipe, as individual discs broke at forces too low

to measure.

mediate strength. The values measured for U. fla-

bellum at Bocas del Toro are similar to those

reported by Collado-Vides et al. (1998) for Puerto

Morelos in the Mexican Caribbean (Table 2). Tables

3 and 4(A–D) show that for each species we tested,

the force required to remove it from sediments is

always significantly less than the force required to

break it.

Estimates of the coefficient of drag (Cd) were

obtained at four water velocities, for Penicillus and

Halimeda; the Cd measured at the highest velocity

(3.3 m s�1) was used, in conjunction with surface

area and the force required to pull an individual

from the substratum, to calculate the water velocity

at which sufficient drag was generated to pull it

from the sediments (Table 5). The highest water

velocities so calculated were for the most weakly

attached individuals, of Penicillus capitatus (42.7 m

s�1), and Udotea flabellum (26.1 m s�1). The low-

est water velocity to dislodge was 5.0 m s�1 for

Halimeda gracilis; in fact, this species required the

remove

Blade

surface

area

NS p =0.670 r2=0.007

Force to

break

NS p =0.259 r2=0.045

Holdfast

volume

NS p =0.355 r2=0.349

Holdfast

Volume

Force to

remove

NS p =0.116 r2=0.086

Blade Surface

Area

Force to

remove

S p b0.0001 r2=0.522

Holdfast

volume

S p =0.0006 r2=0.349

Means

significantly

different?

(dtT-test):Force to remove

compared to

force to break

S p b0.0001

NS—not significant at p =0.05; S=significant at a probability level

(=p) of b0.05; r2 is the coefficient of determination.

Table 4

Correlations of biomechanically related morphological features for Udotea, Halimeda, and Penicillus, at Bocas del Toro, Panama

Correlation of: With: Significance of Correlation

A. Conch point: Udotea flabellum

Blade surface area Force to remove S p b0.0001 r2=0.756

Holdfast volume S p b0.0001 r2=0.776

Means significantly different? (dtT-test):Force to remove compared to force to break S p b0.0001

B. Conch point: Halimeda sp.

Blade Surface Area Force to remove S p =0.035 r2=0.280

Holdfast volume NS p =0.954 r2=0.0003

Means significantly different? (dtT-test):Force to remove compared to force to break S p b0.0001

C. Lime point: Penicillus capitatus

Blade surface area Force to remove S p =0.008 r2=0.2000

Holdfast volume NS p =0.463 r2=0.0169

Means significantly different? (dtT-test):Force to Remove compared to Force to break S p =6.13E-8

D. Conch point: Penicillus pyriforme

Blade Surface Area Force to remove NS p =0.209 r2=0.0556

Holdfast volume NS p =0.136 r2=0.0777

Means significantly different? (dtT-test):Force to Remove compared to Force to break S p =0.036

NS—not significant at p =0.05; S—significant at a probability level (=p) of b0.05; r2 is the coefficient of determination. Correlations tested

were the same as in Table 3, but only significantly different comparisons (for any species) are listed below.

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167162

lowest water velocity for dislodgment for each of

the three categories in Table 5.

To determine tenacity of different holdfast shapes,

we tested four wooden blocks carved into holdfast

Table 5

Calculation of the water velocity in meters per second (m s�1) req

Species Uf Lime Pt Uf Conch Pt

Minimum RF (N) 3.2 2.4

Cd 0.021 0.021

Area (m2) 0.00044 0.00065

V to remove (m s�1) 26.1 18.5

Mean RF (N) 12.7 11.6

Cd 0.021 0.021

Area (m2) 0.0049 0.0023

V to remove (m s�1) 15.5 21.7

Maximum RF (N) 34.4 27.2

Cd 0.021 0.021

Area (m2) 0.016 0.013

V to Remove (m s�1) 14.4 14.0

Species are Uf—Udotea flabellum; Hg—Halimeda gracilis; Pc—Penici

velocity required to remove an individual is based on field measurements

species from the substratum (minimum, medium, and maximum removal

thallus surface area (area) in square meters (m2 ).

shapes, each with and without a Velcro cover. The

addition of Velcro significantly increased the resis-

tance to tensile forces (Table 6), apparently due to a

better adherence to the sediment particles. Of the four

uired to remove (V to remove) a species from the substratum

Hg Conch Pt Pc Lime Pt. Pp Conch Pt.

1.7 3.1 2.8

0.073 0.12 0.12

0.00043 0.000027 0.000067

10.4 42.7 25.7

7.1 5.0 4.9

0.100 0.12 0.12

0.0026 0.0003 0.0004

7.3 15.8 13.4

12.1 10.0 9.4

0.13 0.12 0.12

0.0071 0.0009 0.0011

5.0 11.8 11.8

llus capitatus; Pp—Penicillus pyriforme. The calculation of water

of the minimum, medium, and maximum force found to remove a

force in newtons (N)), coefficient of drag (Cd) at 3.3 m s� 1 , and

Table 6

Mean removal force (N), and 95% confidence interval (95% C.I.) of

wooden models of holdfasts, with and without Velcro

Shape No Velcro With Velcro

Mean (N) 95% C.I. Mean (N) 95% C.I.

Bowl 142.0 36.6 222.0 41.6

Cylinder 142.0 36.6 176.7 38.0

Mushroom 202.5 40.9 280.0 46.5

Short cylinder 198.0 36.6 290.0 41.6

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167 163

shapes, the dmushroom and dshort cylinderT morphol-

ogies had significantly greater resistance to removal

than did either the dbowlT or dcylinderT shape.

4. Discussion

4.1. Detachment and force-to-break.

Almost all individuals of Udotea flabellum and

Penicillus spp. pulled out of the substratum whole

rather than breaking; these results were similar to

those found for U. flabellum in Puerto Morelos

(Collado-Vides et al., 1998), but contrast with the

pattern seen for temperate algae attached to rocks.

For algae that leave tissue on the substratum as a

result of a break (e.g., holdfast portions, holdfast and

stipe, etc.), these dremaindersT provide an opportunity

for regrowth; however, little is known about this

capacity in algae, including the tropical coenocytes.

The relationship between location of a break and

capacity for regrowth provides an interesting area

of future research with important ecological ramifi-

cations for individual survivorship in response to

water motion.

The breaking force of the algae we tested varies

from about 1.8 times greater than the removal force

(Udotea flabellum) to 1.2 times (Penicillus pyri-

forme). As noted above, the exception is Halimeda

gracilis, which broke initially at the dpads,T with

minimal force; however, after clamping below the

pads, these same individuals pulled out completely

from the substratum. In contrast, and as discussed

above, multicellular algae attached to rocks frequently

break within the thallus.

The force required to break a seaweed must be

clearly distinguished from its strength; the former is

measured in newtons, strength in newtons per unit

area. Break force can thus be increased by adding

tissue to a potential break-point or by increasing

strength. Measured break forces for algae vary widely;

for temperate algae, we measured forces ranging from

means of 2.6 N for Mastocarpus papillatus, to 92.6 N

for Egregia menziesii (personal observations). The

coenocytes we report on here had mean break forces

ranging from 6.6 N (Penicillus pyriforme; range: 1.6–

16.9 N) to 22.3 N (Udotea flabellum; range: 8.0–48.8

N). We conclude that the force necessary to break

coenocytes is similar to that of multicellular algae

attached to rocks, and that the coenocytic nature of

the species we tested does not significantly affect this

property.

4.2. Relationship between holdfast volume and

removal force

We predicted that larger holdfasts have a greater

resistance to tensile forces, and we expected a positive

and significant correlation. However, none of the

species showed a significant correlation between

holdfast volume and force-to-remove. In multicellular

algae, this relationship is also equivocal (Duggins et

al., 2003). The absence of such a correlation for algae

anchored in sediments may be because holdfast tena-

city is determined by a combination of factors such as

sediment shear strength, holdfast surface area, and

rhizoid strength. Thus, localized compaction of sedi-

ments may result in a smaller holdfast dplugT but onewith similar tenacity as a larger volume holdfast in

looser sediments; this would obscure relationships

between holdfast volume and tenacity. These factors

are addressed for vascular plants (those with flexible

stems and fibrous roots) by Ennos (1993) and Russell

(1977), but we know of no such data for algae

anchored in sediments.

4.3. Blade surface area and holdfast tenacity

As the blade surface area (=BSA) of algae

increases, so does drag generated by water flow and

we anticipated that attachment tenacity would increase

with BSA. We found a significant correlation between

BSA and the force required to remove an individual

from the substratum for all species except Penicillus

pyriforme. The mechanism behind this correlation is

not known but, except for Udotea, it does not result

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167164

from an increase in holdfast volume as blade surface

area increases.

A similar significant and positive correlation

between BSA and attachment tenacity was found for

two temperate subtidal brown algae attached to rocks,

Agarum fimbriatum and Costaria costata (Duggins et

al., 2003). For A. fimbriatum Duggins et al. (2003)

also found a significant and positive relationship

between force to dislodge and holdfast dry mass,

but this was absent for Costaria costata at 3 of the

4 sites they studied. Once again, the question of

mechanism arises for C. costata, as it did above for

the coenocytic sand-dwelling algae. For C. costata, an

increase in detachment force as thallus wet mass

increases does not translate to a positive relationship

between dislodgment force and holdfast dry mass.

What accounts for the increased attachment force? Is

it a tighter adherence due to greater penetration by

holdfast rhizoids into the substratum, or is it due to the

generation of a stronger dglueT with age? Once again,

the biomechanical properties give rise to additional

research questions.

4.4. The relationship between strength and

morphology

None of the species we tested showed a significant

relationship between strength and any of the other

measured variables, hence, we conclude that our

hypothesis (of no correlation between strength and

our other measured variables) is supported. Our

results do indicate that mean strength differs signifi-

cantly among species, ranging from a low of 1 MN

m�2 in Halimeda gracilis to a high of 7 MN m�2 for

Penicillus capitatus. These values are comparable to

those for multicellular species, e.g., 6.7 MN m�2 for

Mastocarpus papillatus (Carrington, 1990), and 0.8–

1.3 MN m�2 for three non-calcified multicellular

algae and also for three non-calcified coenocytes

(Padilla, 1993); for calcified coenocytic and multi-

cellular algae Padilla (1993) found mean values of

1.4–2.9 MN m�2. Even the large kelps from tempe-

rate waters show similar strengths, e.g., 3.5 MN m�2

for Alaria marginata and 1.7 MN m�2 for Egregia

menziesii (DeWreede, personal observations). The

similarity in strength for a wide range of morpholo-

gical types of algae argues for a fundamental con-

straint on altering strength, possibly a function of the

biological materials available for wall composition.

This, despite the fact that temperate algae tend to

break rather than dislodge whole, presumably re-

enforcing selection for greater strength.

Padilla (1985) found that calcified tropical algae (a

coenocyte and two multicellular species) were signif-

icantly stronger than similar non-calcified algae; all of

the algae we tested were calcified. In a biomechanical

context calcification thus acts to strengthen the thallus

against tensile forces, in addition to other possible

functions such as influencing herbivory (Padilla,

1985, 1989; Littler and Littler, 1980). However,

these results raise the question that if greater strength

assures that an individual coenocyte is removed whole

from the substratum, rather than breaking, of what

advantage is this?

4.5. Holdfast form and tenacity

The consistency in holdfast form (a short cylinder

or hemispherical bowl) arising from the rhizoids and

the sediment they entrain suggests that this morphol-

ogy ensures minimal dislodgment, compared to other

possible holdfast shapes. In contrast to available lit-

erature on vascular plant roots (e.g., Ennos, 1993;

Stokes et al., 1996; Mickovski, 2002), we found no

literature addressing the question of doptimal formT forholdfasts of sand-dwelling coenocytes. For macroal-

gae attached to a rocky substratum, Vogel (1988)

concluded that b. . . the common response seems to

be a tapering disk of attachment that is increasingly

flexible toward the periphery—the whole thing dis-

torts a little to avoid stress concentrations at an edge.QAny comparison of algal holdfasts with vascular plant

roots must be undertaken with the caveat that while

algal holdfasts function only in anchorage (but see

Chisholm et al., 1996), vascular plant roots also

absorb nutrients. This latter function may alter root

structure and morphology compared to that if only

biomechanical constraints apply.

Both algae and vascular plant anchoring systems

are subjected to two major forces, vertical tensile

forces (as are generated during grazing) and lateral

forces (as from wind in vascular plants, and moving

water in algae). Ennos (1993) points out that to neu-

tralize lateral loads (vascular) plants are expected to

have at least one rigid element that will resist with its

bending resistance, while the surrounding soil resists

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167 165

with its compressive resistance. Vertical forces, how-

ever, are b. . . best resisted by a large number of thin

fibrous roots which have a large surface area over

which tension can be quickly transferred to the soilQ(Stokes et al., 1996; Ennos, 1990). The plug formed in

our coenocytic algae by the adherence of sand and

filaments may serve both functions, resisting tensile

forces by the frictional resistance between the holdfast

plug and the surrounding sand, and lateral forces by

the rigidity of the plug itself.

4.6. Water velocities and detachment

Water movement has been shown to be an impor-

tant physical variable affecting both size and abun-

dance of algae (Denny et al., 1985; Gaylord et al.,

1994). Carrington (1990) concluded that the interplay

of stipe diameter, blade surface area, and water velo-

city determined the maximum size of Mastocarpus

papillatus blades. Similarly, Blanchette (1997) and

Blanchette et al. (2002) showed that water motion

affected the size of Fucus gardneri blades and survi-

val of Egregia menziesii, respectively. Also, Milligan

and DeWreede (2000) found that holdfast attachment

strength was critical for survival of Hedophyllum

sessile, and that only more strongly attached indivi-

duals survived winter storms. However, for Udotea

flabellum, Collado-Vides et al. (1998) showed that

water velocities sufficient to remove this species

would be rarely encountered.

The morphological and biomechanical variables

we measured were used to calculate the water velocity

required to remove species from the substratum. As

discussed, on-site evidence led us to conclude that

Lime Point is more wave exposed than Conch Point.

These observations coincide with the lower mean

removal forces found for Udotea flabellum at Conch

Point and for Penicillus pyriforme (Conch Point)

compared to Penicillus capitatus from Lime Point.

Halimeda gracilis is only found at Conch Point and

exhibited the lowest removal force of any of the algae

we tested. However, at Lime Point (Fig. 2A), the size

class distribution of Udotea is shifted to the right

compared to Conch Point (Fig. 2B), suggesting that

blade surface area does not limit these algae.

If the full range of water velocities suggested by

Denny et al. (1985) and Gaylord (1999) occurs at our

study sites, none of the species we tested are immune

from the possibility of dislodgment. Based on our

calculations, all four species were consistent in that

individuals with the largest blade surface area required

the lowest water velocity for removal. For Halimeda

gracilis, all three categories (Table 5) are predicted to

be dislodged at velocities from 5 to 11 m s�1. Since

H. gracilis was abundant at Conch Point, we conclude

that water velocities exceeding 11 m s�1 had not

occurred at this location since the establishment of

this population, but that the higher wave impacts

observed at Lime Point may contribute to the absence

of this species at this site.

The importance of water movement in structuring

these populations of algae cannot be answered in full.

The smaller individuals of each species required velo-

cities of at least 10 m s�1 (Halimeda gracilis), and the

other species in excess of 18 m s�1, for dislodgment.

In Bocas del Toro higher winds occur in January–

February, with a brief increase in storms during July.

If reproduction in these species is limited to a parti-

cular time, then it is possible that the juveniles occur

during the season in which storms are most likely, and

that these grow to adults in the calmer weather. For

Panama, time specific, and synchronous, reproduction

has been reported by Clifton (1997) for each of the

genera studied by us; the most frequent reproductive

activity occurred from April to June, but varied within

this time frame for a specific species. We found no

juveniles in our July (2004) sampling, except for a

small percentage (b3%) in the population of Penicil-

lus pyriformes. In the absence of growth rate data on

any of these species, or of longevity, the importance of

waves in eliminating the larger size classes is

unknown. However, it can be concluded that for all

but H. gracilis, water velocities necessary to dislodge

the individuals we encountered (those that are left?)

are highly unlikely to occur.

4.7. Dislodgment and survival

For all of the species we tested, individuals were

pulled from the sand with less force than is required to

break that same individual. Is it advantageous to these

individuals that they retain their morphological integ-

rity even though they loose their attachment? With

one exception, we know of no evidence that any of the

tropical sand-dwelling coenocytic algae are able to re-

attach after removal from the substratum. The excep-

K. Anderson et al. / J. Exp. Mar. Biol. Ecol. 328 (2006) 155–167166

tion is among species of Halimeda (Walters et al.,

2002) where field and laboratory evidence points to

re-attachment and subsequent growth of vegetative

fragments, e.g., individual discs. We suggest that the

small force required to detach Halimeda gracilis frag-

ments contributes to this phenomenon. As was con-

cluded by Collado-Vides et al. (1998) for Udotea

flabellum, removal of H. gracilis from the substratum

results in its death, as re-attachment is unlikely. We

propose that the same applies to individuals of Peni-

cillus spp.

However, it appears that death of an individual

may not result in the death of the genet. As we use

the term here, an dindividualT can be either a ramet

(if individuals are connected via rhizoids), or the

entire genet (if there are no connections). We looked

for and found rhizoidal connections between indivi-

duals (of the same species) of Penicillus capitatus,

P. pyriforme, and Udotea flabellum, but these con-

nections were not found for Halimeda gracilis. Lit-

tler and Littler (2000) suggest that these rhizoids

function to proliferate ramets of a genet and may

also give rise to a new individual after dislodgment

of an earlier one. In this case, the small force

required to detach individuals of Halimeda, and

the absence of regenerating dstolons,T is offset by

the vegetative reproduction made possible by the

reattachment and subsequent growth of fragments.

Penicillus and Udotea apparently lack the ability to

regenerate from fragments, but require a greater

force for detachment than does Halimeda, and may

be able to regenerate at least one individual from the

dstolonT left behind.

5. Conclusions

Coenocytic construction is unusual in the algae,

but it occurs relatively more frequently in green algae

found in shallow subtidal sediments associated with

coral reefs. The combination of this relatively unique

construction and poor anchorage in sediments led us

to investigate their attachment and other biomechani-

cal properties. We compared the biomechanical prop-

erties we found to those of non-coenocytic temperate

algae already in the published literature. Our results

show that properties of coenocytes, such as force to

dislodge, force to break, and strength, are all within

the range reported for temperate, multicellular, algae.

In contrast, the sand-dwelling coenocytes differed

markedly from the latter in the consequences of

applied tensile force: coenocytes were removed

whole, while most temperate algae attached to rocks

break within the thallus. Thus, it appears that loosing

tissue to survive hydrodynamic forces is not an option

for species of Udotea and Penicillus. Some temperate

algae can also regrow from the tissue left on the

substratum, but whether coenocytes, in situ, can

regrow from remnant underground rhizoids is not

known. Nevertheless, three of the four species we

examined had such rhizoids, and the one that did

not (Halimeda gracilis) is capable of growing from

detached thallus fragments. What has not been tested

is the attachment and breaking force of coenocytes

attached to solid surfaces such as coral.

Since dislodgment is usually fatal for algae, the

role of the holdfast is a critical one. All of the species

we investigated had similar holdfast morphology, a

mass of rhizoids which entrained sand, the entire unit

forming a hemispherical to cylindrical mass. Despite

the consistency in holdfast form, and the initial pre-

diction that this was an optimal form for anchoring

these algae, our preliminary data suggest this is not

the case. Our data suggest that a longer cylinder

(longer than we commonly encountered in these

algae) provides more resistance to tensile forces.

The problems of optimal holdfast morphology for

sand-dwelling algae, and how this relates to both

sediment coherence and drag forces encountered, are

worthy of a detailed investigation.

Acknowledgements

A portion of this study was supported by a Dis-

covery Grant (NSERC 589872) to R. DeWreede. We

also thank the staff and director of ITEC for use of a

boat and laboratory facilities. [AU]

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