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versión On-line ISSN 0717-6538
Gayana (Concepc.) v.74 n.1 Concepción 2010
Gayana 74(1): 66-73, 2010 ISSN 0717-652X
First record of flamentous fungi in the coastal upwelling ecosystem off central Chile
Primer registro de hongos flamentosos en el ecosistema de surgencia costero frente a Chile central
Marcelo H. Gutiérrez 1,4 , Silvio Pantoja 2,4 , Renato A. Quiñones 2,4 & Rodrigo R. González3
1Programa de Postgrado en Oceanografía, Departamento de Oceanografía, Universidad de Concepción, Chile.
2Departamento de Oceanografía y Centro de Investigación Oceanográfca en el Pacífco Sur-Oriental, Universidad de Concepción, Chile.
3Programa de Biotecnología Marina, Facultad de Ciencias Naturales y Oceanográfcas, Universidad de Concepción, Chile.
4Programa de Investigación Marina de Excelencia, PIMEX-Nueva Aldea, Facultad de Ciencias Naturales y Oceanográfcas, Universidad de Concepción, Chile. *Email: firstname.lastname@example.org
We report for the frst time the presence of flamentous fungi in the water column and sediment in the coastal upwelling ecosystem off central Chile, using molecular tools and epifuorescence microscopy. Positive amplifcations of SSU 18s rDNA with specifc fungal primers were obtained for surface waters and sediments of this coastal ecosystem. Molecular richness obtained from denaturing gradient gel electrophoresis showed a higher number of fungal genotypes in nearshore than offshore sites and in summer than winter. Fungal structures identifed by epifuorescence microscopy in the water column were present as individual flaments or as aggregates of hyphae. We show for the frst time vertical water column profles of fungal biomass in the marine ecosystem. Fungal biomass reached up to 5 µg CL-1 in surface waters during summer and their vertical patterns agreed with those of chlorophyll-a and with the general distribution of microplankton biomass in the ocean. The presence of viable fungi in the coastal ocean encourages us to decipher their role in the processing of marine organic matter and to evalúate their inclusión in the actual paradigm of the microbial loop and in the biogeochemistry of the oceans.
Keywords: Fungi, upwelling ecosystem, central south Chile.
Este estudio reporta por primera vez la presencia de hongos flamentosos en la columna de agua y sedimentos del ecosistema de surgencia costero de Chile central. La detección de hongos fue realizada utilizando herramientas moleculares y microscopía de epifuorescencia. Productos de amplifcación positivos del gen 18s rADN fueron obtenidos para muestras de agua y sedimento superfcial de este ecosistema costero. El análisis de riqueza molecular de los productos de PCR, realizado por electroforesis en gel con gradiente denaturante, mostró un mayor número de genotipos de hongos en las estaciones más cercanas a la costa y durante el verano. Estructuras de hongos fueron identifcadas por microscopía de epifuorescencia y fueron observadas como flamentos individuales o como agregados de hifas. Nuestros resultados muestran los primeros perfles verticales de biomasa de hongos en el ecosistema marino. La biomasa de hongos alcanzó valores de hasta 5 µg C L-1 en aguas superfciales durante el verano y su estructura vertical fue similar a la observada para clorofla-a y consistente con la distribución vertical general descrita para la biomasa microplactónica en el océano. La presencia de hongos viables en el océano costero plantea la necesidad de descifrar su rol en el procesamiento materia orgánica y evaluar su incorporación en el paradigma actual del anillo microbiano y en los ciclos biogeoquímicos del océano.
Palabras clave: Hongos, ecosistema de surgencia, centro sur de Chile.
The Humboldt Current System oíf Chile is one the most productive marine ecosystems of the world, with annual primary production rates cióse to 1 kg C m2 (Daneri et al. 2000) and daily rates as high as 25 mg C m2 during the productive season in the coastal upwelling área oíf Concepción (Montero et al. 2007). High productivity during austral spring and summer is fuelled by upwelling events (Daneri et al. 2000) that bring up cold waters with high nutrient concentration toward the ocean’s surface. A major fraction of photosynthetic carbón produced in the Humboldt Current System off Chile is degraded by the microbial community (González et al. 1998, Troncoso et al. 2003, Cuevas et al. 2004, Montero et al. 2007). Most studies have attributed to bacteria the major role in processing organic matter inthe microbial loop off central Chile (e. g. Troncoso et al. 2003, Cuevas et al. 2004). However, there is evidence that archaea are also responsible for an important fraction of microbial secondary production (Levipán et al. 2007, Quiñones et al. 2009).
A noticeable increase in lithogenic opal in the water column off Concepción suggests an important input of terrigenous material during winter due to the discharge of the nearby Itata and Biobio rivers (Sánchez et al. 2009). The succession of upwelling and river discharge during the annual cycle and the drastic changes in environmental conditions (e. g. temperature, salinity, oxygen) (Cáceres & Arcos 1991, Strub et al. 1998, Daneri et al. 2000, Figueroa & Moffat 2000, Sobarzo et al. 2007), result in a heterogeneous environment with temporal changes in the quality and abundance of organic matter. This could sustain a diverse microbial community in this coastal upwelling ecosystem.
Fungi are important in terrestrial environments for their role in processing detrital organic matter from plants (Carlile et al. 2001) but have also been detected in several freshwater and coastal marine ecosystems (e. g. Hyde et al. 1998, Wong et al. 1998, Fell & Newell 1998, Raghukumar 2005, Gulis et al. 2006, Gessner et al. 2007), where they could play a major role in the detrital food web (e. g. Hyde et al. 1998, Raghukumar et al. 2005). However, their role in processing marine organic matter and in the biogeochemical cy cíes in the ocean is poorly known, when compared with the terrestrial environment (Fell & Newell 1998, Clipson 2006).
Here we report for the frst time the presence of flamentous fungi in the marine ecosystem off central Chile and their spatial distribution in the water column and sediment. Althoughouranalysis of the spatial and temporal distribution of fungi is preliminary, it strongly suggests that they are regular inhabitants of the upwelling ecosystem off Central Chile, and that they play a biogeochemical role that we have begun to unravel.
materials and methods
The study was conducted in the coastal zone off central Chile, in the área adjacent to the discharge of the Itata river (Fig. 1), one the main rivers in central-southern Chile, with typical annual average runoffs of 300 m3 s1 (Quiñones & Montes 2001).
Seawater and marine sediments were sampled during three cruises conducted in the coastal zone adjacent to the Itata river discharge (Fig. 1) in austral winter and summer, onboard the R/V Kay Kay II. Additional water samples were collected in summer from a continental shelf océanographic station located at 18 NM from the coast of Concepción in central Chile (Fig. 1). Temperature, oxygen and chlorophyll-a data at Station 18 were derived from the Time Series Coastal Station 18 (www.copas.udec.cl/eng/research/serie/) of the Center for Oceanographic Research in the eastern South Pacific (FONDAP-COPAS Center) of the University of Concepción. Temperature and oxygen at Station A were obtained using CTDO casts (Seabird 19 plus) and chlorophyll-a was determined by fluorometry (Parsons et al. 1984) in a Turner Designs® fluorometer.
The water column at Station A was sampled on August 3-4, 2007 (Winter) at 5, 15, 30, 50, 110 m depth and at 5 m in Station C, and during the Summer (December 16, 2008) at Station 18 (7, 30, 50, 80 m depth). Samples were collected withNiskinbottles and stored insterile containers, whichwere protected from sunlight until they arrived to the laboratory. Water samples were fltered through 0.45 um sterile cellulose ester flters for molecular analyses (1 L) and through 0.7 um glass fber for chlorophyll-a determination (1 L). Filters were stored in liquid nitrogen until processing. Aliquots of 50 mL seawater were poisoned with formaldehyde (2% final concentrations) and stored (4 °C at darkness) for fungi epifuorescense detection and counting.
Surface sediment samples were collected with a modifed Van Veen grab and stored in liquid nitrogen for molecular analysis. Surface sediment sampling was carried out at Stations B and C on August 16-18, 2006 (winter) and at Stations A, B, C on January 18-19 2007 (summer).
Dna extraction and PCR AMPLIFICATION DNA
was extracted from the flters and sediments using the Power Soil DNA Kit (MO BIO Laboratories, Inc). One uL of the témplate DNA from the sediments was subjected to standard PCR using fungal primers NS1 and GC-Fung, which amplify a suitable segment of SSU 18s rDNAforDenaturing Gradient Gel Electrophoresis (DGGE) analysis (May et al. 2001). PCR parameters were initial denaturation for 2 min at 94°C, 35 amplifcation cycles of 94°C for 1 min denaturation, 1 min annealing at 50.3°C, and 72°C for 1 min extensión, and a fnal extensión of 5 min at 72°C. The amplifcation reaction mixture (50 uL) included 200 uM each DNTP, 3.5 uM MgCl2, 0.4 uM of each primer, and one unit of taq DNA polymerase (GoTaq® Flexi DNA Polymerase, Promega). All amplifcations were performed with an Eppendorf Mastercycler gradient thermocycler (Eppendorf ®).
DNA from seawater was preamplified with fungal general primers NS1 and ITS4 (White et al. 1990). PCR parameters were adjusted to an initial denaturation of 2 min at 95°C, 30 amplification cycles of 94°C for 1 min denaturation and 55°C for 4 min annealing/extension, and a final extensión of 5 min at 72°C. The reaction mixture was prepared as described earlier. One uL of the obtained amplifications producís was subjected to standard PCR with NS 1 and GC-Fung primers for DGGE analysis as previously described.
Denaturing gradient gel electrophoresis
An 8% acrylamide/bisacrylamide (37.5:1) gel with a 20-60% denaturing gradient (1 mm thick, lxTAE buffer, 20 cm x 20 cm) was used. Following polymerization, the gel was placed in the buffer chamber of a DCode Universal Mutation Detection System Unit (Bio-Rad) in lxTAE buffer at 60°C PCR producís (~ 40 uL) were loaded into the gel with 10 uL of 2x gel-loading dye and electrophoresis proceeded at 100 V and 60°C for 10 h. The gel was stained in a 0.5 mg L1 lx TAE/ethidium bromide solution and photographed on a UV transilluminator PCR producís of Sacharomyces cerevisiae strain were used as positive control. Molecular richness was estimated as band number in each DGGE profle.
Abundance of fungal hyphae in seawater was estimated by epifuorescence microscopy using an adaptation of the Calcofuor White stain method (Damare & Raghukumar 2008, Cathrine & Raghukumar 2009, Rasconi et al. 2009). Aliquots of 5 to 30 mL seawater were fltered on 0.22-um mesh black polycarbonate flters (Millipore Corp.). Filters with the retained material were stained directly with 600 uL of aqueous 0.1% Calcofuor White, covering the complete flter área.
Figure 1. Location of the sampling stations in the área adjacent to the Itata river mouth (stations A, B, C) and at the COPAS Center Time Series Station 18.
Figura 1. Ubicación de las estaciones de muestreo en el área adyacente a la desembocadura del río Itata (estaciones A, B, C) y la Estación 18 de la Serie de Tiempo del Centro COPAS.
The complete effective área of the flters was examined under an epifuorescence microscope (Axioscop 2 Plus, Cari Zeiss Ltd.) equipped with Filter set 49 (Cari Zeiss Ltd., 365 nm excitation and 445-450 nm emission band pass). The stained fungal flaments were recognized by the presence of septate hyphae, easily distinguishable from other filamentous materials such as detritus. All hyphae identifed on the flters were counted at 1000X and their length and width recorded. Considering a tubular shape of hyphae, a cylinder volume was used as morphological approximation to estímate biovolume of fungal flaments (Cathrine & Raghukumar 2009). Fungal carbón was estimated using the conversión factor 1 (im3 = 1 pg C (Van Veen & Paúl 1979). The average coeffcient of variation for fungal carbón estimated for replicate samples was lower than 20%.
Results and Discussion
Saprotrophic fungi are the main degraders of plant detritus in terrestrial ecosystems (Carlile et al. 2001), and it is considered that they are better adapted than bacteria to acquiring nutrients in poor soil (De Boer et al. 2005). Fungi are also present in freshwater (e. g. Gulis et al. 2006) and have been detected in marine environments such as coastal waters (Gao et al. 2010), deep-sea sediments (Damare et al. 2006), hypersaline waters (Buchalo et al. 2000, Kis-Papo et al. 2003), methane hydrates (Lai et al. 2007), oxygen defcient ecosystems (Cathrine & Raghukumar 2009), mangroves and salt marshes (Hyde et al. 1998, Raghukumar 2005), and hydrothermalvents (Le Calvez et al. 2009). Here, we demónstrate for the frst time the presence of flamentous fungi in the coastal upwelling ecosystem off central Chile.
Molecular evidence of fungí
DNA from seawater and sediments of the área adjacent to the Itata river mouth showed positive amplifcations of SSU 18s rDNA with specifc fungal primers (Fig. 2a), evidencing the occurrence of flamentous fungi inhabiting waters and sediments of the coastal upwelling ecosystem off central south Chile.
The coastal upwelling ecosystem off Concepción is infuenced by freshwater discharge from the Itata and Biobío rivers (Sobarzo et al. 2007), which may supply terrigenous organic matter to the coastal zone (Sánchez et al. 2009). Considering that fungi have been found to be responsible for degrading mainly terrigenous detritus in marine/terrestrial ecotones (Hyde et al. 1998, Raghukumar 2005), we associated the presence of fungi in our study área to the availability of terrestrial remains from rivers. In agreement with this idea, our results of molecular richness (Table 1), analyzed by DGGE, showed a higher number of fungal genotypes in surface waters (5 m, Fig. 2b) and sediment (Fig. 2c) near the Itata river mouth and the coast (stations B, C) than in offshore áreas (station A). Recently, a similar trend of higher molecular richness inshore than offshore, was shown by Gao et al. (2010) for waters off the Hawaiian coast.
Figure 2. Positive PCR producís for témplate rDNA extracted from surface seawater (5m) and surface sediments (a) and denaturing gradient gel electrophoresis patterns in seawater (b) and sediments (c) for stations A, B and C in the área adjacent to the Itata river mouth.
Figura 2. Productos de PCR positivos desde rADN extraído de agua (5m) y sedimento superfcial (a) y patrón de electroforesis en gel con gradiente denaturante en agua de mar (b) y sedimentos (c) para las estaciones A, B y C en el área adyacente a la desembocadura del río Itata.
Since rivers could also discharge spores or remains of fungal tissues from freshwater and terrestrial environments (Kohlmeyer & Kohlmeyer 1979) to the ocean, one could argüe that the observed genotype patterns of fungí in coastal waters and sediments may include resistant propagules or surviving mycelium that have been transponed to the marine environment. However, our results showed a higher number of molecular fungal types in sediments from stations B and C during the summer, when the Itata river fow is at its minimum (Sánchez et al. 2009), than in winter (Table 2, Fig. 2b,c). The seasonal variability observed in the DGGE profles of surface sediments suggests that warm conditions and the increment in marine organic matter available during the productive season, allow the development of a more diverse community of fungi. It has been found that environmental conditions and the availability of substrates in freshwater are the main factors triggering seasonal changes in fungal diversity, abundance, and activity (Gulis et al. 2006, Gessner et al. 2007).
Tabla 1. Riqueza molecular estimada a partir del número de bandas en los perfles de DGGE (n.d. = no determinado)
Detection of fungí by epifluorescence microscopy
Direct observation of fungi retained on flters and stained with Calcofuor White revealed the presence of septate hyphae and spores (Fig. 3). The identifed fungal flaments, ranging from 1 to 3 um in diameter and from 10 to > 200 um length, were observed as individual flaments or aggregates (Fig. 3) that could reach up to 20 um on diameter and over 50 um long, similar to fungi detected in deep-sea sediments in the Indian Basin (Damare & Raghukumar 2008) and to water-stable aggregates associated to mycorrhizal fungi in soils (e. g. Tisdall & Oades 2006). Organic aggregates in the water column represent growth habitats for microorganism (Kiørboe & Jackson 2001). Since fungi produce a wide range of extracellular enzymes to hydrolyze organic polymers (Carlile et al. 2001), the combined activity of fungi and prokaryotes in microbial hotspots could result in a highly effcient micro-bioreactor able to process particulate and dissolved organic matter during sedimentation of particles in the ocean.
It has been shown that both the transport of organic carbón to the sediment water interface and the sequestering of carbón in the ocean are infuenced by marine snow formation (Azam & Long 2001). Therefore, if the contribution of fungi to the formation of organic aggregates in the water column proves to be an ubiquitous phenomenon, they should be involved in the biogeochemical cycles of organic matter at macroscale.
Vertical distribution of fungí in the water column
Here we show, for the frst time, vertical water column profles of fungal biomass in the marine ecosystem. Fungal biomass ranged from 0.03 to 0.12 ug C L1 in the top 15 m, decreasing toward bottom waters to valúes between 0.006 and 0.01 ug C L1 in Station A near the Itata river during the winter, and~6 ug C L1 at 7 m and between 0.19 and 0.44 ug C L1 below 30 m at Station 18 during the summer (Fig. 4a). Fungal biomass vertical structures (Fig. 4a) agree with those of chlorophyll-a (Fig. 4b), with the general distribution of microplankton biomass in the ocean (e. g. Lalli & Parsons 1996), and with the distribution of physical parameters of the water column (e. g. temperature and oxygen; Fig. 4c,d). This pattern suggests that fungi are active in the water column and respond, like most microbial heterotrophs, to the higher biological activity and organic matter availability in surface waters of the ocean during the productive season (summer).
Our fndings show evidence of fungi in coastal upwelling ecosystem and suggest that fungal biomass and molecular richness vary seasonally in coastal waters off central Chile. We are currently investigating the implications of the presence of an active fungal community in the ocean on carbón cycling, as well as the role of fungi in the current paradigm of the microbial loop.
Figure 3. Filamentous fungi observed in the water column of Station A as single hyphae (a) and as aggregates (b). Scale bars correspond to 10 µm.
Figure 4. Vertical profles of fungal biomass (a), chlorophyll-a (b), temperature (c) and oxygen (c) in station A during austral Winter 2007 and in station 18 during austral Summer 2008.
Figura 4. Perfles verticales de biomasa de hongos (a), clorofla-a (b), temperatura (c) y oxígeno (d) en la estación A durante el invierno austral de 2007 y en la estación 18 durante el verano austral de 2008.
We thank E. Tejos, for his valuable help during feld and laboratory work. This research was funded by the Programa de Investigación Marina de Excelencia Pimex-Nueva Aldea (Universidad de Concepción) funded by Celulosa Arauco y Constitución S. A., and partially by the COPAS Center (Project N° 150100007). We are thankful to the COPAS Time Series team, the R/VKay Kay II crew for their valuable help during feld work and the reviewers for their valuable comments.
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