Botanical Studies (2009) 50: 205-215

4

Current address: CICS- Centro de investigacao em Ciencias

da Saude, Universidade da Beira Interior, Av. Infante D.

Henrique, 6200-506 Covilha, Portugal. E-mail: ecairrao@

fcsaude.ubi.pt.

*

Corresponding author. E-mail: fmorgado@bio.ua.pt; Tel:

234-370300; Fax: 234-426408.

INTRODUCTION

The genus Fucus is widely distributed along the Iberian

Peninsula. Five species of Fucus have been described

for Spain and four for Portugal - Fucus spiralis L., F.

vesiculosus L., F. ceranoides L. and F. serratus L. (Perez-

Ruzafa et al., 1993).

Phenotypic variation of Fucus ceranoides, F. spiralis and

F. vesiculosus in a temperate coast (NW Portugal)

E. CAIRRAO

1,2,4

, M.J. PEREIRA

1

, F. MORGADO

1,

*, A.J.A. NOGUEIRA

1

, L. GUILHERMINO

2,3

,

and A.M.V.M. SOARES

1

1

Departamento de Biologia, Universidade de Aveiro, 3800-193 Aveiro, Portugal

2

Centro Interdisciplinar de Investigacao Marinha e Ambiental, Laboratorio de Ecotoxicologia, Rua dos Bragas No177,

4050-123, Porto, Portugal

3

Instituto de Ciencias Biomedicas de Abel Salazar, Departamento de Estudos de populacoes, Laboratorio de

Ecotoxicologia, Universidade do Porto, 4009-003 Porto, Portugal

(Received July 25, 2008; Accepted October 2, 2008)

ABSTRACT.

Brown algae includes several species of Fucus, reported both in the tidal and intertidal zones

of cold and temperate regions. Environmental parameters induce wide biological variability in intertidal algae,

manifested by alterations at several levels, and this has lead to the failure of some reports to discriminate be-

tween closely related taxa, particularly Fucus species. As the genus Fucus is widely represented on the Portu-

guese coast, the biometric parameters of three species (F. spiralis, F. vesiculosus and F. ceranoides) collected

from several sampling sites in Portugal, were studied over twelve months. Environmental parameters (water

temperature, pH, dissolved oxygen, salinity, phosphorous - orthophosphate and total phosphate, nitrate, nitrite

and ammonia) were analysed. The objective of this study was to understand how environmental parameters

influence and establish morphological variation in the Fucus species. Canonical Correspondence Analysis

(CCA), which helps define the relationships between morphological and physicochemical variables, was car-

ried out for each species in order to determine which physicochemical parameter most affects the morphology

of Fucus. The variable biometric that strongly separates the three Fucus species is the number of receptacles

per thalli, and this parameter was highly correlated with F. ceranoides. The two others species were distin-

guished principally by the height of the bigger receptacle, the midrib height of the holdfast, the height of the

smaller receptacle, and the midrib width of the holdfast. The CCA analysis also showed that the dominant fac-

tor influencing morphometric parameters was salinity, being always in strict correlation with water tempera-

tures and orthophosphate. For F. ceranoides, physicochemical parameters (especially a higher concentration of

orthophosphate and lower salinity) seem to influence morphological parameters, mainly in the raised number

of receptacles per thalli. Salinity was the most important environmental parameter to distinguishing F. spiralis

and F. vesiculosus in northern Portugal.

Keywords: Canonical Correspondence Analysis; Environmental parameters; Fucus; Morphology; Northwest-

ern coast of Portugal.

Fucus shows a high level of variability in various

characteristics (biological, biochemical, physiological,

morphological, and life history), and these differ with

geographical distribution (Kalvas and Kautsky, 1998;

Pearson et al., 2000). This variation is principally related

to light intensity (Major and Davison, 1998; Nygard and

Ekelund, 2006), temperature (Major and Davison, 1998;

Pearson et al., 2000), salinity (Ruuskanen and Back, 1999a

b; Scott et al. 2001), coastal exposition (Ruuskanen and

Back, 1999a; Hurd, 2000; Engelen et al., 2005), predation

(Ruuskanen and Back, 1999a; Alstyne and Pelletreau,

2000), pH (Hurd, 2000), concentration of nutrients

(Alstyne and Pelletreau, 2000; Bergstrom et al., 2003),

hybridization (Mallet, 2005), and introgression (Coyer et

al., 2006).

mORPhOlOgy

206

Botanical Studies, Vol. 50, 2009

The onset of reproductive periods is dependant on

temperature and the day/night ratio (Hurd, 2000; Berger

et al., 2001). Reproductive success depends on salinity

(Andersson et al., 1994). Reproduction is not continuous,

but features one or more reproductive peaks, depending

on the region, and this quality also varies between species

(Berger et al., 2001).

One important characteristic of the species of the genus

Fucus is the evident zonation pattern among species.

Initially, this was considered exclusively controlled by

the availability of nutrients in the water (Schonbeck

and Norton, 1979). However, Hurd and Dring (1990)

showed that different species have different assimilation

rates, suggesting other factors may be responsible for

the zonation observed. Further studies showed that a

complex web of physiological and biological interactions

alongside the duration of periods of emersion i.e.

desiccation tolerance (Beer and Kautsky, 1992) and local

hydrodynamics can account for these phenomena (Hurd

and Dring, 1990). According to Karez and Chapman

(1998), competitive capacity is inversely related to

position on the rocky substratum which can be seen in the

fact that F. vesiculosus is more competitive than F. spiralis,

despite the inverse localization.

For a long time, morphological variation was attributed

to the high plasticity of the species in adapting to

changes in environmental conditions (Chapman, 1995).

However, Scott et al. (2001) showed that the expected

patterns according to this hypothesis are not always

observed, and several distributions can be better described

as a geographic mosaic. This concept is based on the

existence of stable polymorphisms (formae) within the

species, which would be organized according to a patchy

distribution of factors affecting the population (Scott

et al., 2001). However, recent DNA sequence data and

other molecular methods are beginning to show that the

maintenance of morphological and genetic differences

observed in Fucus species is paradoxical in face of

potential interspecific gene flow (Billard et al., 2005). This

supports the need to better understand the relationship

between environmental parameters and morphologic

variation, in an attempt to know even more about their

biodiversity. The three taxa F. vesiculosos , F. spiralis and

F. ceranoides are closely related, possibly as the result of a

recent evolutionary radiation (Serrao et al., 1999).

In order to understand how environmental parameters

influence morphological variation for each species in situ,

we evaluated the morphological variation of the Fucus

species F. ceranoides, F. spiralis var. platycarpus Batters,

and F. vesiculosus and their relation to environmental

parameters. The main environmental parameters that

determine the morphological variation for each species

and their geographic distribution were investigated as

well. We also evaluated the mainly biometric parameters

that distinguish these species. This study also investigated

whether the position the algae occupy in the rocky

substratum was in accordance with Karez and Chapman

(1998).

mATERIAlS AND mEThODS

Sampling sites

Three species of Fucus were collected from 11 sites

along the northwestern Portuguese coast, between

the Minho River Estuary and Aveiro¡¦s Lagoon. These

sampling sites were selected for their similarly low wave

exposure,. The Fucus species were identified according to

Flora Phycologica Iberica (Marti et al., 2001) as Fucus

ceranoides, F. spiralis var. platycarpus, and F. vesiculosus

var. vesiculosus. A global positioning system (GPS,

Magelan 2000XL) was used to determine sampling site

coordinates (Table 1).

Table 1. Localisation and coordinates of the three species of Fucus.

Sampling site Fucus species

Localisation

Coordinates

S1

F. ceranoides

Seixas-Minho river estuary

(41¢X53¡¦, 45" N; 8¢X49¡¦, 47" W)

S2

F. ceranoides

Caminha-Mouth of Minho river estuary (41¢X52¡¦, 10" N; 8¢X51¡¦, 48" W)

S3

F. spiralis var. platycarpus Carreco-coastal zone

(41¢X44¡¦, 63" N; 8¢X52¡¦, 53" W)

S4

F. vesiculosus var. vesiculosus Areosa-coastal zone

(41¢X41¡¦, 71" N; 8¢X50¡¦, 88" W)

S5

F. spiralis var. platycarpus Cabedelo-Mouth of Lima river estuary

(41¢X41¡¦, 04" N; 8¢X49¡¦, 88" W)

F. vesiculosus var. vesiculosus

S6

F. spiralis var. platycarpus Sao Bartolomeu do Mar-coastal zone

(41¢X34¡¦, 50" N; 8¢X47¡¦, 82" W)

S7

F. ceranoides

Esposende-Cavado river estuary

(41¢X31¡¦, 85" N; 8¢X46¡¦, 85" W)

S8

F. ceranoides

Azurara-Ave river estuary

(41¢X24¡¦, 84"N; 8¢X44¡¦, 85¡¦¡¦ W)

S9

F. spiralis var. platycarpus Cabo do Mundo-coastal zone

(41¢X13¡¦, 62" N; 8¢X42¡¦, 92" W)

S10

F. spiralis var. platycarpus Boa Nova-coastal zone

(41¢X12¡¦, 23" N; 8¢X42¡¦, 77" W)

S11

F. vesiculosus var. vesiculosus Barra Harbour-Aveiro Lagoon

(40¢X38¡¦, 69" N; 8¢X39¡¦, 66" W)

CAIRRAO et al. ¡X Phenotypic variation of

Fucus

spp.

207

Over a period of twelve months Fucus species

and superficial water samples were collected and

physicochemical parameters were measured monthly in

diurnal ebb low tide conditions during the crescent moon

phase, between October 2001 and September 2002. In

January, Fucus specimens at two sites (S9 and S10) could

not be collected due to bad weather and sea conditions.

The geographical location of each sampling site is

provided in Table 1. The most northerly site (S1) was

located in Seixas, 3 km from the mouth of the Minho

River Estuary. Site S2 was located in the mouth of the

Minho River Estuary. Carreco (S3) was situated on the

coastal zone, between Caminha and Viana do Castelo.

Areosa (S4) was situated in the coast, a region very similar

to S3, but rockier. Cabedelo, in the mouth of the River

Lima Estuary (S5) is very close to Viana do Castelo. Site

S6, on the coastal zone, was in Sao Bartolomeu do Mar.

S7 and S8 were in the estuaries of Cavado and Ave Rivers,

respectively. Site S9 (Cabo do Mundo) was located in

the coastal zone, at the mouth of a small watercourse

which periodically releases both domestic and industrial

effluents into the sea, being located north of an oil refinery.

Site S10 (Boa Nova) also located in the coastal zone, is

contaminated with products from an oil refinery, from the

harbour activities of the sea port of Leixoes, and from

marine traffic. The sampling site situated in Barra Harbour,

in the Aveiro Lagoon (S11), was exposed to the type of

pollution associated with a comercial sea port. Other

sources of pollution in the lagoon are agricultural runoff

and urban and industrial effluents (even though the main

releases are now directly introduced into the sea).

Sampling Technique

At each selected sampling site, 20 specimens, i.e., the

minimum number recommended for a statistical analysis

(Dytham, 1999), of each of the existing varieties, identified

according to Flora Phycologica Iberica (Marti et al.,

2001) in the field, were randomly collected from a one

meter square area at the lower shore level. Each specimen,

always with receptacles, was carefully collected to prevent

biomass loss, especially at the holdfast, which in most

cases was fixed to the rocky substrata. Specimens were

transported in a thermic container and were later stored at

4oC. The measurement of morphological parameters was

carried out within 48 h to minimise possible damage to

biological material from predation and dehydration.

Environmental parameters

The measured physical-chemical parameters included:

water temperature, pH, percentage saturation of dissolved

oxygen, and salinity and these were measured in situ,

using a Multi-Line WTW apparatus. Superficial water

samples were collected monthly at each sampling

site. In the laboratory, samples were filtered (0.45 £gm

porosity cellulose filters) and analysed immediately, for

the determination of nitrate (NO

3

¡Ã

-N), nitrite (NO

2

¡Ã

-N),

phosphates (orthophosphate and total phosphate), and

ammonia (NH

3

).

The concentration of nitrate was determined using the

salicylate method according to the technique described by

Rodier (1996) in a spectrophotometer JENWAY 6505 UV/

VIS (Essex, United Kingdom).

Nitrite, ammonia and phosphate were analysed using

a HACH DR/2000 spectrophotometer (Loveland, CO,

USA) and following the Ferrous Sulphate method 8153,

the Salicylate method 8155, and the Ascorbic Acid method

8048, respectively, in accordance with the Hach Water

Analysis Handbook.

morphological parameters

Sixteen biometric parameters were selected to be

used as criteria for the evaluation of the influence of

environmental parameters upon the studied species,

according to Ruuskanem and Back (1999a, b). These

parameters were: wet weight (WW; g), total length of the

frond (TLF; cm), stipe width (SW; mm), midrib width

(MW; mm) and height (MH; mm), stipe distance to

the border of membrane (SDM; cm), distance of oldest

dichotomy to the base (DOD; cm), midrib height (MHH)

and width (MWH) of the holdfast (mm), length, width, and

height of the largest (LBR, WBR and HBR, respectively)

and smallest (LSR, WSR and HSR, respectively)

receptacles (mm), and the number of receptacles per

thalli (NREC). Monthly measurements were made using

a precision balance (KERN PB, accuracy = 0.001 g), a

millimetre ruler (accuracy = 0.5 mm) and a digital caliper

(accuracy = 0.005 mm).

Statistical Analysis

Morphometric parameters were analysed by one-way

Analysis of Variance (ANOVA), using the SigmaStat

software package, version 1.00 (San Rafael, CA,

USA). The Tukey multicomparison test was used to

determine significant differences among the means of

the Fucus species. Probability levels lower than 5% were

considered significant (P<0.05). All physicochemical

and morphometric data were log(10) transformed prior

to the Canonical Correspondence Analysis (CCA) using

a CANOCO 4.5 Statistical Package (Wales, United

Kingdom) (Van den Brink and Ter Braak, 1999).

RESUlTS

Environmental parameters

Seasonal and spatial variations were observed regarding

the physicochemical conditions in the study area, between

October 2001 and September 2002. Table 2 shows the

average, maximum and minimum values of environmental

parameters (water temperature, salinity, percentage

saturation of dissolved oxygen, pH, orthophosphate, total

phosphate, ammonia, nitrite and nitrate).

208

Botanical Studies, Vol. 50, 2009

CAIRRAO et al. ¡X Phenotypic variation of

Fucus

spp.

209

(SDM; cm), distance of oldest dichotomy to the base

(DOD; cm), midrib height (MHH) midrib width (MWH)

of the holdfast (mm), length, width, and height of the

largest (LBR, WBR and HBR, respectively) and smallest

(LSR, WSR and HSR, respectively) receptacles (mm),

and the number of receptacles per thalli (NREC) in each

of the existing varieties. Only TLF, SW, MW, MH, WSR,

HBR, HSR were significantly different for all the species

analysed (P<0.05, one-way ANOVA). The SDM and WW

had similar values for the species analysed. The NREC,

WBR, DOD, MWH and MHH were significantly different

between the F. ceranoides and the other two Fucus species

analysed, and LBR and LSR were significantly different

between the F. vesiculosus var. vesiculosus and the other

two Fucus species analysed (P<0.05, one-way ANOVA).

Environmental and morphometric parameters

of Fucus species

To evaluate the relationships between morphological

and physicochemical variables, a Canonical

Correspondence Analysis (CCA) was performed separately

for each variety and for all physicochemical variables, but

only the most relevant are presented. The advantage of the

Spatial distribution of the species

Fucus species were found fixed to rocky substratum,

their distribution on the Portuguese coast being apparently

first determined by the presence or absence of rocky

substrata. Thus, their distribution was not homogeneous in

the studied area. The species Fucus ceranoides appeared

in four stations as the only species of this genus: S1, S2,

S7 and S8. The species Fucus spiralis var. platycarpus

was found at S3, S6, S9 and S10, as the only species of

the genus, and at S5 (in the Lima River Estuary) with F.

vesiculosus, this last being found in the lower coastal zone.

The species Fucus vesiculosus var. vesiculosus was also

found at S4 and S11, as the only species of the genus.

morphometric parameters of Fucus species

The three taxa analysed are closely related, and in

order to better morphologically discriminate between

these Fucus species, several morphometric parameters

were analysed. Table 3 shows the average maximum and

minimum values of sixteen biometric parameters: wet

weight (WW; g), total length of the frond (TLF; cm), stipe

width (SW; mm), midrib width (MW; mm), midrib height

(MH; mm), stipe distance to the border of membrane

Table 3. Morphometric data of the three species of Fucus.

F. ceranoides

F. spiralis var. platycarpus

F. vesiculosus var. vesiculosus

Average Maximum Minimum Average Maximum Minimum Average Maximum Minimum

TLF

23.51 35.82 14.18

28.16 55.56 10.13

33.54 49.57 24.45

SW

7.69 10.06 5.56

12.56 15.82 9.53

11.77 14.98

8.67

MW

1.12 1.86 0.68

1.73 2.77 1.17

1.53 2.02

0.98

MH

0.46 0.67 0.32

0.63 0.91 0.33

0.53 0.76

0.34

NREC

113.18 340.25 18.65

47.25 137.70 7.30

77.78 263.15 19.85

LBR

21.26 30.99 12.64

27.73 35.86 18.36

21.41 34.48 15.67

LSR

9.31 19.51 6.75

10.67 18.28 6.68

10.53 61.65

6.74

WBR

12.98 17.81 7.82

17.06 26.71 11.85

15.46 26.85 10.73

WSR

4.29 6.71 3.15

6.40 9.44 4.64

5.61 7.57

4.06

HBR

2.56 4.20 1.54

5.44 8.74 2.15

4.01 5.95

2.10

HSR

1.40 2.84 0.87

2.44 4.39 1.38

1.88 2.81

1.30

SDM

2.21 4.39 0.34

2.43 4.59 0.41

2.02 3.97

0.78

DOD

1.62 5.09 0.42

4.68 19.18 0.67

5.10 13.41

1.89

MWH

1.66 2.32 0.94

2.82 3.95 1.89

2.61 3.87

1.65

MHH

0.85 1.15 0.59

1.68 2.67 1.08

1.61 2.61

0.95

WW

39.73 129.78 7.64

48.78 145.67 6.42

61.05 172.10 17.97

WW: wet weight (g); TLF: total length of the frond (cm); SW: stipe width (mm); MW: midrib width (mm); MH: midrib height (mm);

SDM: stipe distance to the beginning of membrane (cm); DOD: distance of oldest dichotomy (cm); MHH: midrib height of the

holdfast (mm); MWH: midrib width of the holdfast (mm); LBR: length of the bigger receptacle (mm); WBR: width of the bigger

receptacle (mm); HBR: height of the bigger receptacle (mm); LSR: length of the smaller receptacle (mm); WSR: width of the

smaller receptacle (mm); HSR: height of the smaller receptacle (mm) and NREC: number of receptacles per thalli.

210

Botanical Studies, Vol. 50, 2009

The Figure 2 depicts a CCA ordination diagram that

shows the relationship between the biometric parameters

of each species of Fucus and the environmental

variables of the sample sites. For Fucus ceranoides

(Figure 2a), F. spiralis var. platycarpus (Figure 2b) and

F. vesiculosus var. vesiculosus (Figure 2c) two sample

groups are observed between biometric parameters

and environmental variables. In Figure 2a, axes 1 and

2 (eigenvalues, respectively, of 0.73 and 0.65; the sum

of all canonical eigenvalues is 0.60) explain the 18.8%

of cumulative percentage variance in species data and

the 53.9% of cumulative percentage variance of the

species-environment relationships; both are statistically

significant (Monte Carlo Permutation Test P = 0.005).

For F. ceranoides (Figure 2a) two sample groups are

clearly distinguished, and one of these correlated groups

between variables showed orthophosphate as the main

physicochemical variable correlated to the sampling sites

S1, S2, S7 and S8, where this species occurred. The second

general grouping included the other physicochemical

variables.

In Figure 2b, axes 1 and 2 (eigenvalues, respectively,

of 0.68 and 0.33; the sum of all canonical eigenvalues is

0.30) explain the 12.5% of cumulative percentage variance

in species data and the 85.4% of cumulative percentage

variance of the species-environment relationships; both

are statistically significant (Monte Carlo Permutation

Test P = 0.005). In Figure 3c, axes 1 and 2 (eigenvalues,

respectively, of 0.73 and 0.64; the sum of all canonical

eigenvalues is 0.60) explain the 19.2% of cumulative

percentage variance in species data and the 54.1%

of cumulative percentage variance of the species-

environment relationships; both are statistically significant

(Monte Carlo Permutation Test P = 0.005). For F. spiralis

(Figure 2b) and for F. vesiculosus (Figure 2c) two sample

correlated groups between variables are not clearly

delimited, but a similar pattern was observed.

Figure 3a, 3b and 3c display data obtained by CCA

for different values of water temperature, salinity, and

orthophosphate, respectively. In this analysis we tried to

delimit several correlated groups between variables in

order to conclude whether the geographic localisation

of each species is determined principally by those three

environmental variables. Axes 1 and 2, in Figure 3

(eigenvalues, respectively, of 0.73 and 0.64; the sum

of all canonical eigenvalues is 0.60) explain the 19.2%

of cumulative percentage variance in species data and

the 54.1% of cumulative percentage variance of the

species-environment relationships; both are statistically

significant (Monte Carlo Permutation Test P = 0.005).

For water temperatures (Figure 3a) two sample groups

were clearly distinguished, one of these correlated groups

between variables corresponded to F. ceranoides, and

the second general grouping included F. spiralis and F.

vesiculosus. For salinity (Figure 3b) three sample groups

were observed, and each was associated with one species

of Fucus, according to the gradient of this parameter. For

orthophosphate (Figure 3c) two distinct sample groups

biplot is that it displays both the variables (environmental

parameters as arrows) and the cases (the morphometric

measurements as dots) in one plane in this multivariate

analysis. The correlation of variables (arrows) to the

first axis are of most importance, and cases (projected

perpendicular to the arrows) closest to the tip of the arrows

show the best correlation between case and parameter

(Kent and Coker, 1992; Van den Brink and Ter Braak,

1999).

The first CCA (Figure 1) included all biometric

parameters and the three species of Fucus. Axes 1 and 2

(eigenvalues, respectively, of 0.74 and 0.64; the sum of

all canonical eigenvalues is 1.20) explain the 20.0% of

cumulative percentage variance in species data and the

52.4% of cumulative percentage variance of the species-

environment relationships; both are statistically significant

(Monte Carlo Permutation Test P = 0.005). In this figure,

the variable biometric that strongly separated these three

species is the number of receptacles per thalli (NREC),

and this is strongly correlated to F. ceranoides. The two

others species are distinguished principally by height of

the bigger receptacle (HBR), midrib height of the holdfast

(MHH), height of the smaller receptacle (HSR), and

midrib width of the holdfast (MWH).

Figure 1. Correlation biplot of biom etric varia ble s ba sed

on CCA (Canonic al Cor re spondence Analysis). W W: wet

weight (g), T LF: tota l length of the frond (cm), SW: stipe

width (mm), MW: midrib width (m m), MH: m id rib height

(mm), SDM: stipe distance to the beginning of membrane

(cm), DOD: distance of oldest dichotomy (cm), MHH: midrib

height of the holdfast (mm), MWH: midrib width of the hold-

fast (mm), LBR: length of the bigger receptacle (m m), WBR:

width of the bigger receptacle (m m), HBR: height of the big-

ger receptacle (m m), LSR: lengt h of the smaller recept acle

(m m), WSR: width of t he sm aller rece pt acle (m m), HSR:

height of the smaller receptacle (mm) and NREC: number of

receptacles per thalli.

CAIRRAO et al. ¡X Phenotypic variation of

Fucus

spp.

211

were observed, one of these correlated groups between

variables corresponded to F. ceranoides, and the second

general grouping included F. spiralis and F. vesiculosus.

DISCUSSION

Environmental parameters

With respect to physical and chemical parameters,

water temperature showed seasonal variation. Over the

sampling period, mean values for water temperatures were

higher at the northern sites (S1 and S2; 14.8 and 14.0oC,

respectively), and the more southerly site (S11) did not

register the highest temperature (16.2oC maximum) as

expected. The highest temperature (16.4oC) was observed

at S8, possibly due to different levels of coastal exposure;

the estuaries have more sheltered sites associated with the

entry of warmer riverine inputs.

Surface salinity was lower at sites situated further

upstream in the estuaries (S1, S7 and S8; 6.6, 4.5 and

8.4., respectively) and variable at the mouths of the

rivers (S2 and S5). These fluctuations were mainly due

to the ratio between fluvial discharge and flux, and to

extention of tidal excursion (Pardal, 1998). Salinity values

for coastal zones did not indicate large fluctuations, with

the exception of S9 in November, where a very low value

(10.5.) was measured. This variation could be related to

an increase in the flux of the stream following a period of

high precipitation.

Dissolved oxygen attained its highest values at

sampling site S4. The occurrence of a high biomass of

green macro-algae could be, in part, responsible for

these high values, as green macro-algae contribute to the

increase in dissolved oxygen through their photosynthetic

activity (Lyngby et al., 1999).

The pH values can vary with numerous factors, one of

the most obvious being photosynthetic activity (Pardal,

1998; Hurd, 2000). Site S4 registered the highest pH

value, which can be explained based on the large quantity

of algae existing at this site (as testified to by the values

obtained for the percentage saturation of dissolved oxygen

for the same site).

Photoautotrophic organisms use nutrients in the

synthesis of organic matter via the photosynthetic process;

the concentration of nutrients in the medium can vary

as a result of biological production (Flindt et al., 1999).

A high variation in the concentration of phosphates

(orthophosphate and total phosphate) was observed during

the sampling period for all of the sites. This may be

explained by point source or non point source discharges

of urban and industrial effluents, frequent in estuarine

environments and coastal zones (Pardal, 1998; Azeiteiro et

al., 1999). In accordance with this, the highest mean values

of orthophosphate and total phosphate were observed at S7

(0.23 and 0.58 mg/L) and S8 (1.25 and 1.53 mg/L).

Figure 2. Correlation biplot of a) Fucus ceranoides, b) F.

spiralis var. platycarpus and c) F. vesiculosus var. vesicu-

losus based on CCA (Canonical Correspondence Analysis).

W W: wet weight (g), TLF: total length of t he frond (cm),

SW: stipe width (mm), MW: midrib width (mm), MH: midrib

height (mm), SDM: stipe distance to the beginning of mem-

brane (cm), DOD: distance of oldest dichotomy (cm), MHH:

mid rib he ig ht of the holdfa st (m m), M WH: mid rib width

of the holdfast (mm), LBR: length of the bigger re cepta cle

(m m), WBR: widt h of the bigger re cept acle (m m), HBR:

height of t he bigge r rece pta cle (mm), LSR: lengt h of the

smaller receptacle (mm), WSR: width of the smaller recep-

tacle (mm), HSR: height of the smaller receptacle (mm) and

NREC: number of receptacles per thalli.

212

Botanical Studies, Vol. 50, 2009

The three nitrogenous components quantified in this

study showed higher concentrations during autumn and

the beginning of winter. These values may be due to an

increase in runoff from farmlands and an increase in the

release of urban and industrial effluents, principally in the

estuarine environments (S7 and S8) and in Cabo do Mun-

do (S9), situated near a watercourse where urban effluents

are released (INAG, 2000).

morphometric parameters of Fucus species

The three taxa F. ceranoides, F. spiralis, and F.

vesiculosus are closely related, possibly as the result

of a recent evolutionary radiation (Serrao et al., 1999).

Phylogenetic studies have used nuclear rDNA-SSU

and LSU sequences to survey fucoid genera at the

family level and more variable nrDNA-ITS sequences

to explore relationships at the genus level (Rousseau

et al., 1997; Leclerc et al., 1998; Serrao et al., 1999).

However, only some of the most recent genetic analysis,

like microsatellite loci, allow establishing the differences

between closely related taxa of the Fucus species (Billard

et al., 2005). The morphometric parameters used in this

work allow differentiating these three closely related taxa

of Fucus species along the Northwestern Portuguese coast,

between the Minho river estuary and the Aveiro¡¦s Lagoon.

The total length of the frond, stipe width, midrib width

and height, length and height of the largest receptacle, and

width of the smallest receptacle are significantly different

for all the species analysed. The number of receptacles

per thalli, width of the largest receptacle, distance of

the oldest dichotomy to the base and midrib height and

width of the holdfast allow us to distingush between the

F. ceranoides and the others two Fucus species analysed,

and the length of the largest and smallest receptacles

allow us to differentiate morphologically between the

F. vesiculosus var. vesiculosus and the other two Fucus

species analysed. In contrast, several phenotypic (Burrows

and Lodge, 1951; Perez-Ruzafa et al., 1993) and genetic

(Serrao et al., 1999) studies failed to differentiate between

F. ceranoides, F. spiralis, and F. vesiculosus. However,

several recent genetic studies showed a clear separation

of these three Fucus species (Billard et al., 2005), and

these species could be identified as three different genetic

and morphometric species. This work also showed that F.

spiralis is more similar to F. vesiculosus. This similarity

was mainly due to an incomplete reproductive isolation

between these two Fucus species (Billard et al., 2005).

Canonical Correspondence analysis also indicated

that F. ceranoides was morphologically discriminable,

principally by a raised number of receptacles per thalli.

The two others species were discriminable principally

by the height of the bigger receptacle, the midrib height

F igure 3. Correlat ion biplot of a) wat er te mpe ratu re s, b)

sali nity and c) ort ophosphate s base d on CCA (Ca nonical

Corresponde nce Analysis. W W: wet weight (g), TLF: total

length of the frond (cm), SW: stipe width (mm), MW: m idrib

width (mm), MH: midrib height (mm), SDM: stipe distance

to the beginning of membrane (cm), DOD: distance of oldest

dichotomy (cm), MHH: midrib height of the holdfast (mm),

MW H: midrib width of the holdfast (mm), L BR: length of

the bigger receptacle (m m), WBR: width of the bigger recep-

tacle (mm), HBR: height of the bigger receptacle (mm), LSR:

length of t he smaller receptacle (mm), WSR: width of the

smaller receptacle (mm), HSR: height of the smaller recep-

tacle (m m) and NREC: number of receptacles per thalli.

CAIRRAO et al. ¡X Phenotypic variation of

Fucus

spp.

213

of the holdfast, the height of the smaller receptacle, and

the midrib width of the holdfast. This morphological

variability is clearly evident and highlights the relevance

of physiological and genetic differences. Our results

indicate that in northwestern Portugal these species are

principally identified visually and metrically by these five

parameters.

Fucus ceranoides¡¦ physicochemical parameters

(especially orthophosphate and salinity) seem to influence

its morphological parameters, mainly in the raised number

of receptacles per thalli. For F. ceranoides (Figure 2c)

two sample groups are clearly distinguishable, and

this grouping shows a correlation between the raised

concentration of orthophosphate and the sampling sites S1,

S2, S7 and S8, where this species occurred and the salinity

was lower. The correlation between orthophosphate and

F. ceranoides was also observed in Figure 3c, where

two sample groups were also observed, according to the

concentration of orthophosphate. Kraufvelin et al. (2006)

observed that nutrient enrichment may have adverse

or beneficial effects on reproduction, in rocky shore

communities, and these effects were mainly dependent

on the species involved. Our results showed that the

reproduction of F. ceranoides may also be affected by the

higher environmental concentrations of orthophosphate,

observed at the sampling sites S1, S2, S7 and S8, where

this species occurred. Another possible explanation

is that low salinity areas are affected by freshwater

runoff bringing sewage, inducing higher environmental

concentrations of orthophosphate. Our results showed that

F. ceranoides always has more receptacles than the other

two Fucus species since they have more dichotomies and

thus more reproductive tips per thalli, and F. ceranoides

occurred only at low salinity sites. The effect of low

salinity on reproduction is corroborated by Ruuskanen and

Back (1999a) whose studies in the Gulf of Finland suggest

that salinity is the main environmental factor behind

seasonal variations in reproduction.

A similar pattern was seen in F. spiralis var. platycarpus

and in F. vesiculosus, but the two sample groupings are not

clearly delimited, indicating that all the physicochemical

parameters influence morphological parameters.

Regarding the remaining Figure 3a, 3b and 3c (water

temperature, salinity and orthophosphate, respectively)

a consistent pattern seems to emerge. Fucus ceranoides

is present at sites with higher concentrations of

orthophosphate and lower salinity while the opposite

was true for the other two Fucus species. Geographic

localisation of this species is determined principally

by three environmental variables. However, salinity is

the main parameter distinguishing F. spiralis from F.

vesiculosus in northern Portugal.

The vertical distribution of the species in the study area

agrees with the description in the available literature, F.

spiralis var. platycarpus is the variety which inhabits the

upper eulittoral zone, and F. vesiculosus is observed in the

lower eulittoral zone (Hurd and Dring, 1990; Karez and

Chapman, 1998; Cairrao et al., 2004).

In conclusion, our results clearly indicate that the

dominant fact influencing morphometric parameters is

salinity, being always in strict correlation with water

temperatures and orthophosphate. In addition, for all

species of Fucus studied, morphological variability is

clearly evident, and several biometric parameters analysed

seem to establish the differences between these closely

related taxa of the Fucus species.

Acknowledgements. This work was supported by

Fundacao para a Ciencia e Tecnologia (FCT) through

project CONTROL (contract: PDCTM/C/MAR/

15266/1999).

lITERATURE CITED

Alstyne, Van K.L. and K.N. Pelletreau. 2000. Effects of nutrient

enrichment on growth and phlorotannin production in Fu-

cus gardneri embryos. Mar. Ecol. Prog. Ser. 206: 33-43.

Andersson, S., L. Kautsky, and A. Kalvas. 1994. Circadian and

Lunar gamete release in Fucus vesiculosus in the atidal Bal-

tic Sea. MEPS 110: 195-201.

Azeiteiro, M., J.C. Marques, and P. Re. 1999. Zooplankton An-

nual cycle in the Mondego river estuary (Portugal). Arquiv-

os do Museu Bocage. Vol. III, 8: 239-264.

Beer, S. and L. Kautsky. 1992. The recovery of net photosyn-

thesis during rehydration of three Fucus species from the

Swedish west coast following exposure to air. Botanica Ma-

rina 35: 487-491.

Berger, R., T. Malm, and L. Kautsky. 2001. Two reproductive

strategies in Baltic Fucus vesiculosus (Phaeophycea). Eur. J.

Phycol. 36: 265-273.

Bergstrom, L., L. Berger, and L. Kautsky. 2003. Negative direct

effects of nutrient enrichment on the establishment of Fucus

vesiculosus in the Baltic Sea. Eur. J. Phycol. 38: 41-46.

Billard, E., C. Daguin, G. Pearson, E. Serrao, C. Engel, and M.

Valero. 2005. Genetic isolation between three closely re-

lated taxa: Fucus vesiculosus, F. spiralis and F. ceranoides

(Phaophyceae). J. Phycol. 41: 900-905.

Burrows, E.M. and S.M. Lodge. 1951. Autecology and species

problem in Fucus. J. Mar. Biol. Assoc. UK 30: 161-175.

Cairrao, E., M. Couderchet, A.M.V.M. Soares, and L. Guilher-

mino. 2004. Glutathione-S-transferas e activity of Fucus

spp. as a biomarker of environmental contamination. Aquat.

Toxicol. 70: 277-286.

Chapman, A.R.O. 1995. Functional ecology of fucoid algae:

twenty-three years of progress. Phycologia 34: 1-13.

Coyer, J.A., G. Hoarau, M.P. Oudot-Le, W.T. Stam, and J.L.

Olsen. 2006. A mtDNA-based phylogeny of the brown algal

genus Fucus (Heterokontophyta; Phaeophyta). Mol. Phylog.

Evol. 39: 209-222.

Dytham, C. 1999. Choosing and Using Statistics: A Biologists

Guide. Blackwell Science, Oxford.

214

Botanical Studies, Vol. 50, 2009

Engelen, A.H., P. Aberg, J. Olsen, W.T. Stam, and A. Breeman.

2005. Effects of wave exposure and depth on biomass, den-

sity and fertility of the fucoid seaweed Sargassum polyc-

eratium (Phaeophyta, Sargassaceae). Eur. J. Phycol. 40(2):

149-158

Flindt, M.R., M.A. Pardal, A.I. Lillebo, I. Martins, and J.C.

Marques. 1999. Nutrient cycling and plant dynamics in es-

tuaries: A brief review. Acta Oecol. 20: 237-248.

Hurd, C.L. 2000. Water motion, marine macroalgal physiology,

and production. J. Phycol. 36: 453-472.

Hurd, C.L. and M.J. Dring. 1990. Phosphate uptake by intertidal

algae in relation to zonation and season. Mar. Biol. 107:

281-289.

INAG. 2000. Planos das bacias Hidrograficas dos rios Luso ¡V

Espanhois ¡V Sintese. Caracterizacao e Diagnostico. Instituto

da Agua, Direccao de servicos de recursos Hidricos, Divi-

sao de recursos Subterraneos.

Kalvas, A. and L. Kautsky. 1998. Morphological variation in Fu-

cus vesiculosus populations along temperature and salinity

gradients in Iceland. Mar. Biol. Assoc. UK 78: 985-1001.

Karez, R. and A.R.O. Chapman. 1998. A competitive hierarchy

model integrating roles of physiological competence and

competitive ability does not provide a mechanistic expla-

nation for the zonation of three intertidal Fucus species in

Europe. OIKOS 81: 471-494.

Kent, M. and P. Coker. 1992. Vegetation description and analy-

sis: a practical approach. Wiley, New York.

Kraufvelin, P., M.E. Frithjof, C. Hartvig, and T.L. Bokn. 2006.

Nutrient addition to experimental rocky shore communities

revisited: delayed responses, rapid recovery. Ecosystems 9:

10076-1093.

Leclerc, M.C., B. Veronique, L. Guillaume, and B. De Reviers.

1998. Low divergence in rDNA ITS sequences among five

species of Fucus (Phaeophyceae) suggests a very recent ra-

diation. J. Mol. Evol. 46: 115-120.

Lyngby, J.E., S. Mortensen, and N. Ahrensberg. 1999. Bioassess-

ment techniques for monitoring of eutrophication and nutri-

ent limitation in coastal ecosystems. Mar. Pollt. Bull. 36:

212-223.

Major, K.M. and I.R. Davison. 1998. Influence of temperature

and light on growth and photosynthetic physiology of Fu-

cus evanescens (Phaeophyta) embryos. Eur. J. Phycol. 33:

129-138.

Mallet, J. 2005. Hybridization as an invasion of the genome.

Trends Ecol. Evol. 20(5): 229-237.

Marti, M.C.B., T.G. Garcia, A.G. Garreta, I. Perez-Ruzafa,

M.A.R. Siguan, and J.R. Lluch. 2001. Flora Phycologica

Iberica. Vol. I. Fucales. In A.G. Garreta (ed.), Universidad

de Murcia, Murcia.

Nygard, C.A. and N.G.A. Ekelund. 2006. Photosynthesis and

UV-B tolerance of the marine alga Fucus vesiculosus at dif-

ferent sea water salinities. J. Appl. Phycol. 18: 461-467.

Pardal, M.A.C. 1998. Impacto da Eutrofizacao nas Comunidades

Macrobentonicas do braco Sul do Estuario do Mondego

(Portugal). Ph.D. Thesis, Universidade de Coimbra, Coim-

bra.

Pearson, G.A., L. Kautsky, and E. Serrao. 2000. Recent evolu-

tion in Baltic Fucus vesiculosus: reduced tolerance to emer-

sion stress compared to intertidal (North Sea) populations.

Mar. Ecol. Prog. Ser. 202: 67-79.

Perez-Ruzafa, I., T. Gallardo, and R. Gomez-Cancio. 1993. Nu-

merical taxonomy of taxa of the genus Fucus in the Iberian

Peninsula. Hydrobiologia 260/261: 81-90.

Rodier, J. 1996. L¡¦analyse de l¡¦eau: eaux naturelles, eaux resid-

uaires, eaux de mer. 8 Ed. Dunod, Paris.

Rousseau, F., M.C. Leclerc, and B. de Reviers. 1997. Molecu-

lar phylogeny of Fucales (Phaeophyceae) based on partial

large-subunit rDNA s equence c om paris ons. P hycol. 6:

436-438.

Ruuskanen, A. and S. Back. 1999a. Morphological variation of

northern Baltic Sea Fucus vesiculosus L. Ophelia 50: 43-59.

Ruuskanen, A. and S. Back. 1999b. Does environmental stress

affect fertility and frond regeneration of Fucus vesiculosus.

Ann. Bot. Fennici. 36: 285-290.

Schonbeck, M.W. and T.A. Norton. 1979. The effects of brief

periodic submergence on intertidal fucoid algae. Estuar. Cstl

Mar. Sci. 8: 205-211.

Scott, G.W., S.L. Hull, S.E. Hornby, F.G. Hardy, and N.J.P.

Owe ns. 2001. P henotypic variat ion in Fuc us s pir alis

(Phaeophycea): morphology, chemical Phenotype and their

relationship to the environment. J. Phycol. 36: 43-50.

Serrao, E., L.A. Alice, and S.H. Brawley. 1999. Evolution of

the Fucaceae (Phaeophyceae) inferred from nrDNA-ITS. J.

Phycol. 35: 382-94.

Va n den Brink, P.J . a nd C.J.F. Ter Braak. 1999. P ri ncipal

response curves: Analysis of time dependent multivariate

responses of biological comm unit y to s tress . E nviron.

Toxicol Chem. 18: 138-148.

CAIRRAO et al. ¡X Phenotypic variation of

Fucus

spp.

215