Pigment-specific rates of phytoplankton growth and

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Pigment-specific rates of phytoplankton growth and microzooplankton grazing in a subtropical lagoon 1

CENTRO INTERDISCIPLINARIO DE CIENCIAS MARINAS (IPN-CICIMAR), DEPARTAMENTO DE PLANCTON Y ECOLOGIA MARINA, AVENUE INSTITUTO 2

POLITECNICO NACIONAL S/N, COL. PLAYA PALO DE SANTA RITA, CP 23090, APDO POSTAL, 592 LA PAZ, BAJA CALIFORNIA SUR, ME´XICO AND CENTRO DE ´ GICAS DEL NOROESTE, S. C., MAR BERMEJO 195, COL. PLAYA PALO DE SANTA RITA, CP 23090 LA PAZ, BAJA CALIFORNIA SUR, MEXICO INVESTIGACIONES BIOLO

*CORRESPONDING AUTHOR: [email protected]

Received March 13, 2006; accepted in principle July 10, 2006; accepted for publication September 18, 2006; published online September 21, 2006 Communicating editor: K.J. Flynn

Phytoplankton growth and microzooplankton grazing rates were evaluated in one station in Bahıá Concepcioń, located in the middle region of the Gulf of California, Me´xico. We used high-performance liquid chromatography (HPLC) estimations of phytoplankton pigment signatures to evaluate the annual variation of taxon-specific grazing and growth rates obtained with the dilution technique. Chlorophyll-a (Chl-a) concentrations varied widely (0.34–3.32 mg L1) and showed two maxima, during late spring and autumn, associated with the transition between mixed and stratified conditions. Phytoplankton growth rates varied seasonally with the lowest rates during summer (range: 0.01– 2.55 day1 for Chl-a; 0.00–3.84 day1 for Chl-b; 0.26–3.29 day1 for fucoxanthin; 0.00–6.27 day1 for peridinin; 0.00–4.35 day1 for zeaxanthin). Microzooplankton grazing was an important loss process (range: 0.0–1.89 day1 for Chl-a; 0.00–3.12 day1 for Chl-b; 0.26–3.29 day1 for fucoxanthin; 0.00–2.03 day1 for peridinin; 0.00–3.51 day1 for zeaxanthin). Average grazing rates accounted ,68–89% of estimated average phytoplankton pigment-specific growth rates. The analysis of pigment signatures indicates that diatoms and dinoflagellates were the dominant groups, and contrary to expectation for typical subtropical lagoons, the specific growth rates in Bahıá Concepcioń showed a pronounced seasonal variability, linked to transitional hydrographic conditions. Our results indicate a close coupling between the community microzooplankton grazing and phytoplankton growth rates, without selective feeding behavior. These results suggest that microzooplankton play a critical role and may significantly modify the availability and efficiency of transfer of energy to higher trophic levels.

INTRODUCTION Microzooplankton in a broad sense include diverse assemblages of protists and metazoans of different size (80% of the daily grazing production (Gallegos, 1989; Dagg, 1995; Lehrter

et al., 1999; Irigoien et al., 2005). Several studies indicate that microzooplankton tend to dominate mesozooplankton as primary consumers, in both oligotrophic and euritrophic waters (Sherr and Sherr, 1992; Calbet and Landry, 2004), and their relevance as part of microbial loop has been demonstrated for diverse marine ecosystems (Odate and Imai, 2003; Bo¨ttjer and Morales, 2005). However, the role of micrograzers in coastal areas is less clear, although several studies of coastal ecosystems have demonstrated the importance of microzooplankton both as consumers of phytoplankton and as prey for

doi:10.1093/plankt/fbl051, available online at www.plankt.oxfordjournals.org The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Downloaded from http://plankt.oxfordjournals.org/ at Centro de Investigaciones Biológicas del Noroeste, S.C. on February 25, 2015

´ N2 AND D. LO ´ PEZ-CORTE´S2 RICARDO PALOMARES-GARCIÁ1*, J. J. BUSTILLOS-GUZMA


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(Strom and Welschmeyer, 1991; Waterhouse and Welschmeyer, 1995). Garcıá-Pa´manes and Lara-Lara (Garcıá-Pa´manes and Lara-Lara, 2001) used the dilution method to estimate the microzooplankton grazing and growth rates for the central region of the Gulf of California but based only in the spectrophotometer estimation of chlorophyll-a (Chl-a); thus, these authors did not distinguish among different phytoplankton groups. Although many studies on phytoplankton dynamics have been conducted in Bahıá Concepcioń, this is so far the first study about microzooplankton growth and grazing rates. In the present study, we determine the seasonal variations in these vital rates associated with individual phytoplankton taxa. The use of the HPLC technique to determine the functional phytoplankton assemblage may have potential bias. Kruskopf and Flynn (Kruskopf and Flynn, 2005) alert about the relative weak relationship between Chl-a and C-biomass, particularly at low nutrient concentrations. Therefore, Chl-a alone would not be considered as a robust indicator of the relative growth rate. We recommend that the use of pigment markers must be considered only as a suitable reference but not strictly in a quantitative sense.

METHODS Twenty-two grazing experiments were conducted at approximately bi-weekly intervals over a 1-year period. Grazing experiments were conducted at a station located in the middle part of Bahıá Concepcioń, Me´xico (2643¢N; 11149¢W), from March 2001 to March 2002. We obtained samples of seawater for pigments determinations, dissolved nutrients (NO3, NO2, PO43 and SiO32), salinity and temperature at standard depths (surface, 5, 10, 15, 20 and 25 m). Water temperature was recorded using a Khalsico thermometer (±0.1C), and salinity and nutrients were measured using standard methods (Strickland and Parsons, 1972). Variation in the vertical density gradient was calculated to estimate the stability of the column water (Peterson et al., 1988): Density gradient ¼

4 t 4z

ð3Þ

Where 4 t is the difference between surface and bottom 4 t and D z is the difference between surface and bottom depth.

Sampling protocol All carboys, bottles and tubes were precleaned by soaking in 10% HCl and rinsing in distilled water. For each experiment, two 22-L polyethylene carboys were filled

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mesozooplankton (Gifford, 1991; Verity et al., 1993; Kiørboe and Nielsen, 1994). Bahıá Concepcioń is one of the best-studied bays in the Gulf of California (2633¢–2653¢N; 11142¢–11256¢W). It is ,45-km long and 10 km at the widest part. The depth in the central channel is ,30 m (Obeso-Nieblas, 1996). The seasonal wind pattern is predominantly NW during autumn and winter (cold period) and SE during spring and summer (warm period). Seasonal solar irradiation variability is associated with strong changes in the vertical stability of the water column. NW winds produced a well-mixed water column during autumn and winter, and the decrease of these winds starts the transition period from cold and well-mixed water to stratified and warm conditions. As reported earlier (Lo´pez-Corte´s et al., 2003a), the largest phytoplankton bloom in Bahıá Concepcioń has been observed during this transition period associated with the entrance of nutrient-rich waters from the Gulf of California. During spring, the trade winds change to SE, decreasing the turbulence in the water column and increasing the air temperature. Such conditions produce a strong thermocline (Reyes Salinas, 1994). During autumn, a short transition period completes the cycle, the NW wind forces water from the Gulf of California to enter into the bay (Lo´pez Corte´s et al., 1991), rapidly cooling and mixing the water column (Lechuga-Deve´ze and Morquecho-Escamilla, 1998). Those hydrographic conditions produce significant changes in the nutrient flux and promote differential increase of specific phytoplankton groups. Although the dominance of diatoms and dinoflagellate is a typical community phytoplankton feature in Bahıá Concepcioń (Lo´pez-Corte´s et al., 2003a), other phytoplanktonic groups such as Chlorophyceae, Prasinophyceae and Cyanophyte can be seasonally abundant (Martıńez-Lo´pez and Ga´rate-Liza´rraga, 1994, 1997; Lo´pez-Corte´s et al., 2003a). The phytoplankton biomass has a strong seasonal variability and reaches the annual maximum during both transitional hydrographic periods that occur between cool and warm periods. Our goal was to determine the growth and grazing rates associated with specific groups of phytoplankton by coupling the seawater dilution method (Landry and Hasset, 1982), with the high-performance liquid chromatography (HPLC) technique. The HPLC technique for the estimation of pigment ‘biomarkers’ has been used by several authors around the world mostly to determine oceanic phytoplankton community structure (Higgins and Mackey, 2000; Lo´pez-Corte´s et al., 2003b) and to investigate the natural diets of macrozooplankton (Gieskes and Kraay, 1986) and more recently as a tool to estimate the microzooplankton grazing and phytoplankton growth rates of several autotrophic groups

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Pigment analysis Pigments were identified and quantified using the HPLC technique, using known pigment standards and following the method of Vidussi et al. (Vidussi et al., 1996). Main pigments were identified through retention times and spectral characteristics and quantified using the pigment response factor (Mantoura and Repeta, 1997) obtained from commercial pigment standards (International Agency for 14C determinations, Denmark). In this study, peridinin, fucoxanthin, Chl-b and zeaxanthin were used as signatures of dinoflagellates, diatoms, Chl-b containers (Chlorophyceae and Prasinophyceae) and cyanobacteria, respectively. The apparent pigment-specific growth rates from changes in the concentration of Chl-a and other selected pigments were calculated in individual bottles using a exponential growth model (Landry and Hasset, 1982). This method assumes that, under decreased grazing pressure, the phytoplankton assemblage in each treatment grows at a constant rate, which is a linear function of grazer density. The change in density of phytoplankton (P), in time (t) is: Pt ¼ P0 expðk gÞt

ð3Þ

GRAZING AND GROWTH RATES

1 t

ln

Pt P0

¼kg

where k is the instantaneous growth rate of phytoplankton (day1) and g is the instantaneous mortality rate of phytoplankton due to grazing (day1). The term ln(Pt/ P0)/t is the apparent phytoplankton growth rate. The apparent phytoplankton growth rate was plotted as a function of the dilution factor, and the y-intercept of the regression line is the theoretical phytoplankton growth rate (k), in absence of grazing pressure, and the negative slope is the grazing coefficient, g. Both the parameters are calculated using least squares linear regression analysis, with confidence intervals calculated for k, g and the regression coefficient (r). In coastal waters, we can observe a non-linear adjust when plots net growth rate versus dilution level (Gallegos, 1989). In a non-linear instance, we used the three-point extrapolation procedure (Gallegos, 1989) that considered only the extrapolation of the straight line that connects the average growth rates (m) of the two highest dilutions (5 and 10%); microzoplankton grazing rate (g) was then estimated as m-net growth in undiluted bottles. The estimated g and k values were used to calculate the plankton doubling time, the potential primary production and the percentage of the production removed by the microzooplankton, following an indirect approach (Gifford, 1988; Garcıá-Pa´manes and Lara-Lara, 2001; Verity et al., 2002). However, there are two important caveats in our approach that may limit our interpretations about the estimated in situ growth and grazing rates. First, as we mentioned before, there exists a relative weak relationship between Chl-a concentration and C-biomass. Second, it is well known that Chl-a varies mostly as function of nitrogen, whereas carotenoid varies mostly as function of carbon. We made the assumption that this does not introduce significant errors to estimate in situ growth and grazing rates. The second one involves the potential source of variability introduced by the Chl versus carotenoid-specific rates, because Chl-a varies in the function of nitrogen and carotenoids depend of carbon content.

RESULTS Hydrographic conditions Seasonal variations of hydrographic conditions and nutrients (NO3, NO2, PO4; and SiO3) in the water column are shown in Figs 1 and 2. Temperature ranged from 16 to 31C, with colder temperatures in winterspring and warmer during summer. Salinity ranged from 34.2 to 35.9 at the surface and from 34.1 to 35.8 below 15 m, with

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with sea surface water or with water collected with 8-L Niskin bottles from 10-m depth. Seawater from half of these carboys was filtered through Whatman GF/F filters to obtain particle-free seawater for the dilution series (Li, 1986). The remaining unfiltered seawater was gently filtered through a nitex screen of 200 mm. Dilution series were established by combining prescreened ‘whole’ with particle-free seawater in 2.8-L polycarbonate bottles. Three bottles were filled by each of the following fractions of whole seawater: 1.0, 0.5, 0.2, 0.1 and 0.05. The 100% of dilution (filtered seawater) was used as control. Two additional bottles were filled with the same dilution proportions and sampled to estimate initial pigment levels. All the treatments (with three replicates) were incubated under in situ conditions at 3-m depth for 24 h, near the water collection sites, without nutrient addition. After 24 h, bottles were transported to the laboratory and filtered in duplicate using GF/F filters. Samples were kept frozen in liquid nitrogen until further analysis. Water samples were filtered for the determinations of pigment signatures with GF/F filters and extracted with acetone 100% under dark and cool conditions (4C, 24 h). Additionally, for each dilution, 500 mL of each replicate was preserved in 2% lugol-seawater solution for microplankton reference.

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Fig. 1. Monthly variation of the temperature (A), salinity (B) and dissolved oxyigen (C) during the 2001–02 annual cycle in Bahıá Concepcioń, Gulf of California.

higher salinity during the stratification period. Dissolved oxygen in the surface layer varied between 4 and 6 mL O2 L1 through the year, with the lowest concentration in summer and the highest values in winter. The profiles of temperature and salinity (Figs 1A and B) showed evidence of four different periods; a colder period, characterized by a well-mixed water column (January, February, March and December of 2001 and January and February of 2002); two transition periods April–early-May of 2001 (spring–summer) and October–November of 2001 (summer-autumn) and one warm period when the temperature rose rapidly and showed intense stratification (end of May, June, July and August of 2001).

During the mixed period, dissolved oxygen values (Fig. 1C) were very similar through the water column, whereas, when the thermocline was well developed (density gradient > 0.08), the bottom layers showed the lowest values (