Journal of Applied Phycology 9: 129–135, 1997. 129c 1997 Kluwer Academic Publishers. Printed in Belgium.

Ceramialean epiphytism in an intertidal Gracilaria chilensis (Rhodophyta)bed in southern Chile

Alejandro H. Buschmann, Claudia A. Retamales & Claudia FigueroaDepartamento de Acuicultura, Universidad de Los Lagos, Casilla 933, Osorno, Chile(Phone: (56)-64-205274; fax: (56)-64-239517)

Received 15 January 1997; revised 14 April 1997; accepted 16 April 1997

Key words: abundance, ceramiales, Chile, epiphytism, Gracilaria, microscopic stages, recruitment

Abstract

The cultivation of the agarophytic red alga Gracilaria has become an activity of major importance in several parts ofthe world. However, Gracilaria cultivation in Chile still faces problems such as epiphytism. We report ceramialeanepiphytism abundance, recruitment patterns and the microscopic stages fixed on the Gracilaria thalli in an intertidalbed (Metri bay) of southern Chile. Using a factorial field experiment, we analyze the effect of intertidal elevationand the use of epiphyte free inoculum on the abundance of ceramialean epiphytic algae. This evidence is used toprovide management and control recommendations for ceramialean epiphytes.

The results show a summer recruitment and increase in abundance of ceramialean epiphytes. A seasonal andspatial pattern of abundance of microscopic stages of this epiphytic algae was also found, showing a significantincrease from the apical (new tissues) to the central parts of the thalli (older tissues). Apical tissues are freeof epiphytic propagules. The epiphyte-free tissue zone decreases significantly from winter to summer. Also, asignificantly higher density of microscopic stages of ceramialean epiphytes was found on the thalli collected inthe farming area during the summer at low tidal levels. The experimental results indicated that the abundance ofepiphytes was greater at the lower tidal levels than at the higher distribution limits of Gracilaria and it was notpossible to control epiphytism by manual cleaning.

Introduction

The cultivation of the agarophytic red alga Gracilariahas become of major importance in several parts ofthe world, such as Asia, South America and south-ern Africa (Santelices & Doty, 1989). Chile is oneof the major world producers with a total productionof 60 000 wet t during 1992, of which over 80% wasproduced by cultivation (Buschmann et al., 1995). Therapid development of Gracilaria cultivation techniquesis related to desirable biological characteristics, suchas the capacity to anchor the thallus in soft substra-ta, a high regeneration capacity, high growth ratesand good agar yield (Santelices et al., 1984, Pizarro,1986; Santelices & Ugarte, 1987). The developmentof this activity produced returns to the country inexcess of US$ 40 million in 1995, considering bothdry seaweed and agar exportations. Nevertheless, dur-

ing the past years several problems have arisen whichrequire basic research to determine technological solu-tions. One of these problems is epiphytism by red,brown and green algae (Pizarro, 1986; Gonzalez et al.,1993; Buschmann et al., 1995). The epiphytism levelsfound in commercial Gracilaria beds imply lower algalgrowth rates, increased loss of stocking biomass andproduction of raw material with lower value due to thepresence of the nuisance algae (Kuschel & Buschmann,1990; Buschmann & Gomez, 1993; Buschmann et al.,1994).

Post-harvest epiphyte control methods seem to be ineffective in obtaining an epiphytic-free product, as theyare time-consuming and cost-demanding (Buschmannet al., 1995). For this reason, farmers prefer preven-tive methods to manage epiphyte abundance. Fletcher(1995) pointed out several methods that have beingused to control epiphyte abundance, such as physical

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removal from the host, reducing the light intensity withnetting or changing the light quality, drying of culturesystems, changing water circulation, preventive chem-ical methods using hypoclorite solutions, copper basedpaints, manipulation of pH and nutrient regimes andbiological control methods. Most of these methods areonly suitable for tank cultures and are difficult to applysuccessfully in open culture areas, as is the case ofGracilaria in Chile. In open systems, biological con-trol of epiphytes in suspended (Brawley & Fei, 1987)and bottom (Buschmann et al., 1994) cultures havebeen tested, but its effectiveness on a full commercialscale still needs to be demonstrated.

The knowledge accumulated regarding Gracilar-ia epiphytism has been mainly descriptive, and thereis a need for experimental studies on detailed eco-logical processes that can sustain recommendationsto control epiphytes (Fletcher, 1995). In this study,we evaluate the ceramialean epiphytic loads, preva-lence of infections, recruitment and microscopic stageabundance patterns in an intertidal Gracilaria chilensisBird McLachlan et Oliveira bed in southern Chile. Wehypothesized that epiphytic loads fluctuate seasonallyand that variations can be explained by recruitment andsporeling abundance of ceramialean algae. By using afactorial field experiment, the effect of intertidal eleva-tion and epiphyte-free host inoculum on the abundanceof ceramialean epiphytic algae was assessed. Theseresults were used to propose management recommen-dations regarding the control of epiphytes.

Materials and methods

Study site

This study was carried out in an intertidal sandy beachat Metri Bay (41� 360S, 72� 420W), 30 km southeastof Puerto Montt (Figure 1). The tidal amplitude atthe site ranged between 5 to 7 m, leaving extensiveintertidal areas available for the establishment of com-mercial Gracilaria farms. The study was undertaken ina ca 1 ha experimental farm planted with G. chilensisbetween 0 to 2.5 m above the mean low water lev-el (MLW). Intertidal cultures systems have a biomassproduction potential that ranges between 60 to 75 t(wet) ha�1 y�1 if no nuisance invertebrates or epi-phytic algae appear in the culture areas (Buschmannet al., 1995). The principal epiphytes described forthis area are brown filamentous species, mainly Giffor-dia sp., which appear in the spring, different groups of

Figure 1. Experimental farming area in Metri, 30 km south-east ofPuerto Montt in southern Chile.

ulvoids (Ulva sp., Enteromorpha sp. and Rhizocloniumsp.) and red species belonging to the Ceramiales (twospecies of Polysiphonia, Ceramium rubrum (Hudson)C. Ag. and Callithamnion sp.).

Methods for assessment of macroscopic abundanceand recruitment

From June 1997 to June 1993, samples were takenat low (0.7–1.0 m above MLW), intermediate (1.4–1.5 m) and at the highest (above 2.0 m) Gracilariadistributional limits at Metri Bay. Five samples weretaken at each tidal level by removing all the harvestablebiomass of Gracilaria in a 0.25 m2 quadrat placed ran-domly along a 20 m transect running parallel to theshore. The harvested Gracilaria was brought to thelaboratory and all the epiphytes were removed man-ually, classified and the ceramialean species pooledtogether. Both Gracilaria and the epiphytic cerami-alean species were wet weighed on a Sartorius balance(� 0.01 g accuracy) to determine the epiphytic load.

The prevalence of ceramialean epiphytes, definedas the frequency at which they appear in the field,was also estimated. This variable is important becauseit provides an indication of the dispersion of theepiphytes within a farm. For measuring the preva-lence, direct field observations were undertaken on thepresence of ceramialean epiphytes in thirty 0.25 m2

quadrats. The quadrats were laid randomly in a 100-m

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transect parallel to the seashore, at the three tidal levelsas described above.

For measuring the recruitment of ceramialean juve-niles, three ceramic plates and three stones wereinstalled on the sandy beach at the mid-intertidal lev-el (between 1.4 to 1.6 m above MLW) each month,during a year. Two types of substrata (ceramic platesand stones) were used as the recruitment success candepend on their selection (Foster & Sousa, 1985). Aftertwo months the plates were brought to the laboratoryand the ceramialean cover recorded by placing a trans-parent plastic sheet with 49 randomly placed dots. Theperiod of two months was long enough to allow themacroscopic development of ceramialean recruits, buttoo short for other colonizing seaweeds to develop andmonopolize the substrata. The plates and stones wereinstalled 3 cm above the bottom to minimize sand depo-sition and avoid the activity of Tegula atra (Lesson),a common gastropod grazer here (Buschmann et al.,1994).

Methods for the assessment of microscopic stages

To determine the presence of microscopic stages ofceramialean epiphytes on Gracilaria, thalli were col-lected at the three tidal levels and brought to the lab-oratory. In the laboratory, 25 thalli from each tidalheight (low, mid and high tidal level) with a mini-mal length of 12 cm were haphazardly selected andobserved under a binocular microscope. Thus, it waspossible to count the ceramialean microscopic stagesattached on one side of the thallus surface. For eachobservation, all the sporelings present within a segmentof 1 mm Gracilaria thallus were counted to obtain thenumber of germlings per mm of host thallus. As epi-phytic algae were absent from the apical portion of thethalli, the distance from the apical end of the thallito the first point of appearance of epiphytic propaguleattached to the thalli was determined. Due to the highvariance in the observations, a high replicate numberper treatment (n� 15) was preferred over a month-ly sampling. For this reason, data were collected ontwo occasions: in winter (June; low epiphyte abun-dance season) and during the summer (January; highepiphyte abundance season).

Experimental methods

A factorial experiment was implemented to establishthe effect of macroscopic epiphytes abundance (withand without macroscopic epiphytes) at two tidal lev-

els (0.8 and 1.5 m above MLW). Each experimentalquadrat (6) measured 1.5� 2.0 m but only the cen-ter, with a dimension of 1� 1 m, was used to mini-mize border effects. In each quadrat, two plastic tubeswere installed and to each of them three bundles ofGracilaria (100 g each) were tied with a plastic string,as described in earlier studies (Santelices & Ugarte,1987; Buschmann et al., 1995). Poliethylene tubeswere placed in the quadrats and prior to their instal-lation, half of the Gracilaria bundles were manual-ly cleaned in order to eliminate all the macroscopicepiphytes. Bundles with and without epiphytes wheredistributed randomly inside the experimental quadrats.

One month after installation and during a threemonth period, 6 bundles per treatment, were taken tothe laboratory. Each sample was separated in 15 ran-dom subsamples and in each of these the presence ofceramialean epiphytes was determined under a stere-omicroscope. Based on these data, it was possible todetermine the frequency of epiphytes for both treat-ments. The data were then analyzed by using a two-way ANOVA with time as a repeated measurement,using SYSTAT (Wilkinson et al., 1992) after ensuringthe normality and homoscedasticity of variances.

Results

Macroscopic abundance and recruitment

The results show a seasonal abundance variation ofceramialean algae appearing in September and macro-scopically disappearing in April–May (Figure 2A). Theepiphytic ceramialean load at the higher intertidal lev-els can be higher (over twice the Gracilaria biomass),but their occurrence is restricted to short periods inlate summer as compared to the lower intertidal levels(Figure 2A). The macroscopic prevalence of cerami-alean epiphytes varies greatly during the winter andspring, but increases constantly from values around20% in winter, reaching 100% during the summer andstart to decline the following autumn (Figure 2B). Therecruitment of ceramialean epiphytes occurred only onthe plates installed in the summer months (Januaryto March) (Figure 3). During the rest of the year noceramialean algae developed on plates or stones.

Microscopic stages

The abundance of microscopic stages of cerami-alean epiphytes on Gracilaria increased significantly

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Figure 2. (A) Ceramialean epiphyte seasonal loads (g epiphytes g�1

Gracilaria 100; mean � 1 SE) variation at three intertidal height.(B) Ceramialean prevalence (%) at the three intertidal height studied.

Figure 3. Ceramialean cover (%; mean � 1SE) of recruits on sub-strates (ceramic plates and stones) installed in the Gracilaria bedduring an annual cycle.

(Table 1) from the apical (new tissues) to the cen-tral parts of the thalli (older tissues) during the winter(Figure 4). The number of microscopic stages alsoincreased significantly during the summer in compari-

Table 1. Summary of ANOVA showing the effects of thedistance from the apical tissues (0–2.5; 2.5–5.0; 5.0–7.5 and>7.5 cm), at three tidal levels of the Gracilaria bed (low,mid, high) and two sampling periods (winter, summer).

Treatment d.f. F-value Probability

Distance (D) 1 8.66 P<0.01

Tidal level (TL) 2 159.62 P<<0.01

Sampling Periods (SP) 1 775.66 P<<0.01

D�TL 6 0.76 P>0.05

TL�SP 2 171.82 P<<0.01

D�SP 3 1.32 P>0.05

D�TL�SP 6 1.36 P>0.05

Figure 4. Ceramialean microscopic stages density (number m m�1

of Gracilaria thallus; mean �1SE) over the Gracilaria thalli foundat different distances from the apical tissue during the winter (A)and during the summer (B).

son to the winter (Table 1). During summer, the incre-ment of the number of microscopic stages occurredpreferentially on the mid and lower intertidal levels,situation noted by a significative interaction betweentidal level and period of sampling (Table 1). At thelow tidal level the abundance of microscopic stages ofceramialean increased significantly to values above 10individuals per mm of Gracilaria thallus during thesummer (Figure 4B).

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Figure 5. Epiphyte-free thalli (mm; mean � 1SE) of Gracilariathalli during the winter and summer at the three tidal levels: low,mid and high.

Table 2. Summary of ANOVA showing the effects of macro-scopical epiphyte removal (clean and unclean Gracilaria)and tidal level (high and low) on ceramialean epiphyte preva-lence. The time (months) of observation was used as a repeat-ed measurement.

Treatment d.f. F-value Probability

A: Between Subjects

Epiphtye Removal (E) 1 1.239 0.282

Tidal Level (TL) 1 4.151 0.059

E�TL 1 5.157 0.037

B: Within Subjects

Month (M) 2 2.889 0.070

M�E 2 0.117 0.890

M�TL 2 1.653 0.207

M�E�TL 2 0.871 0.428

The apical tissues were free of visible epiphyt-ic propagules and the epiphyte-free Gracilaria tis-sues decreased significantly from 22–50 mm to 5–18 mm from winter to summer, respectively (Figure 5).Furthermore, the free epiphytic-free tissues show aninverse pattern when winter and summer epiphytic-free epiphyte Gracilaria tissues are compared at dif-ferent tidal levels (Figure 5). During summer, the dis-tance from the apical end of the Gracilaria thalli to theappearance of the first epiphytic propagule was shorterat the low tidal levels than at higher tidal levels (Fig-ure 5). An inverse situation occurred during the winter,when the longest distance free of epiphytic tissues wasregistered at the lower tidal levels.

Experimental results

The experimental results indicated that ceramialeanabundance was marginally higher (Table 2) at the low-

Figure 6. Experimental results of the ceramialean prevalence (%;mean� 1SE) testing the effect of epiphyte removal and tidal level(HTL = high tidal level, LTL = low tidal level), during a three-monthexperimental period.

er tidal levels than at the higher distribution limits ofthe Gracilaria farm (Figure 6). The initial cleaning ofthe macroscopic epiphyte seaweeds did not significant-ly affect the frequency of ceramialean algae (Table 2).Also, a significant interaction was found (Table 2),indicating that the effect of the tidal height was differ-ent for plants with and without epiphytes (Figure 6).Time as a repeated measurement did not show sig-nificant differences or interacted significantly with thetidal level where the experiment was installed (Table 2).

Discussion

The recent review of Fletcher (1995) on Gracilariaepiphytes indicates that, in general, epiphyte abun-dance increases during spring and summer. In subti-dal farming areas in northern Chile, Polysiphonia sp.appears abundantly (>73 g kg�1 of Gracilaria) and is

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reproductive during early spring (October), presentingsenescent tissues in late spring (November) (Pizarro,1986). In subtidal estuarine cultivation areas of south-ern Chile, ceramialean epiphytes are the most commongroup, with highest abundance during summer (West-ermeier et al., 1991). Another epiphytism pattern wasreported for a suspended culture where the most com-mon epiphytes were again algae belonging to Cerami-ales which presented a marked seasonal variation, withtwo maximal values, one in summer and the other inspring (Westermeier et al., 1993). Nevertheless, recentpublished data indicate seasonal patterns can differin intensity and time of occurrence between differentculture areas (Buschmann et al., 1995) it seems thatceramialean epiphytism temporal abundance patternsare common for different latitudes, environments andculture systems in the Chilean coast.

Notwithstanding the agreement to the general abun-dance patterns described above, it is important toemphasize from a management point of view, the vari-ability of the patterns according to intertidal height(this study), depth (Westermeier et al., 1991) and thepresence of microenvironments with different humid-ity conditions (Buschmann et al., 1994). Also, a highvariability in epiphytism patterns (between years) hasbeen shown by Pizarro and Santelices (1993) in north-ern Chile, associated with longterm variations in envi-ronmental conditions such as surface water tempera-ture and light intensity. This must be considered by thefarmers, as a particular factor could be responsible forenhancing the abundance of epiphytes in part of or thecomplete Gracilaria bed.Although the high abundanceof ceramialean epiphytes seems restricted to the sum-mer, their prevalence in the field occurs during mostof the year (see also Buschmann et al., 1995). Underfavourable environmental conditions, regrowth of set-tled individuals could therefore produce an increase inthe epiphytic load.

Another issue to be considered before starting afarming operation is to select unpolluted areas andaway from possible sources of nuisance organisms.Previous studies indicated that different culture areas insouthern Chile present different degrees of epiphytismand caused by various species even when environmen-tal conditions appear to be similar (Buschmann et al.,1995). However, significant differences also exist with-in a culture area, which means that each farmer shouldmanage the epiphytes differently (for example, plant-ing at different tidal heights or depths). The increas-ing amount of propagules found colonizing Gracilar-ia thalli at lower tidal heights recommends that these

areas should be avoided if ceramialean epiphytism is aproblem.

The spore recruitment is restricted to the summer,which explains the higher abundances of microscopicstages and the reduced apical thalli free of epiphytesfound during this season. It has been suggested that thebank of microscopic propagules is more important forthe survival of perennial species, like Ceramiales, thanfor fugitive forms (Santelices et al., 1995b). We sug-gest that spore recruitment and regrowth of previouslyfixed ceramialean thalli explain the summer epiphyticbiomass increase. Therefore, during the summer sporerecruitment, host algae should be constantly harvestedto reduce the available substrata to ceramialean propag-ules. This result seems different for other epiphytes, asindicated for green algae, where regrowth seems to beunimportant to explain the epiphytic loads and wherethe spore production period is more extended duringthe year (Buschmann et al., in press).

The removal of epiphytes from the inoculum priorto outplanting has been recommended (Santelices &Doty, 1989; Fletcher, 1995). However, our data showthat manual cleaning of Gracilaria used for planti-ng an artificial bed did not reduce the prevalence ofceramialean epiphytes. The existence of penetratingrhizoids in the ceramialean epiphytes (Gonzalez et al.,1993) and the importance of regrowth may explainour results. Nevertheless, these results could be differ-ent if the epiphyte abundance is determined by sporerecruitment, as is the case of green algae (Buschmannet al., in press). The current harvesting methodologyof Gracilaria favours epiphytic prevalence and load,because the portions of the plants remaining after thecutting are the basal more infected portions of the thal-lus. For this reason we suggest that it is possible toprevent the infection of Gracilaria by harvesting priorto recruitment. Westermeier et al. (1993) found thatwhen the alga is harvested manually, the epiphytismlevels by Ceramiales is reduced in ropes with high algalinoculum (1.2 kg m�2), as compared with those at low-er biomass (0.6 kg m�2). This last evidence indicatesthat high plant densities and the resulting self-shaddingcan be an important strategy for controlling cerami-alean epiphytism.

Strain selection has been mentioned to be impor-tant in producing host types that can have broader tol-erances to epiphytes (Dawes, 1987). Santelices andUgarte (1990) detected inter-population differences inGracilaria as regards their susceptibility to epiphytesand Buschmann (1990), detected differences in theepiphytism loads when two different origin inoculum

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were planted in the same cultivation site. Neverthe-less, recent studies have shown the existence of intra-individual differences and instability of some clones(Santelices et al., 1995a) which suggests that the selec-tion of an epiphyte resistant strain could prove to be ahard challenge and requires further attention.

Acknowledgements

This study was possible thanks the financial supportof IFS (Grant number 1600/2). The first author alsowish to acknowledge the help of Pedro Vergara. Theauthors are thankful to Juan Correa,Bernabe Santelicesand anonymous reviewers for valuable criticism andsuggestions on the manuscript. This paper was writtenduring a visiting period of the first author to the LagunaUniversity in Spain, supported by INTERCAMPUSprogram and wish to thank the help of the staff ofthe Plant Biology Department, specially M. C. Gil-Rodrıguez and M. C. Hernandez-Gonzalez.

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