Spatial and temporal variability of planktonic archaeal abundance in the Humboldt Current System off Chile

ARTICLE IN PRESS Deep-Sea Research II 56 (2009) 1073–1082

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Spatial and temporal variability of planktonic archaeal abundance in the Humboldt Current System off Chile ˜ ones a,b,, He´ctor A. Levipan a, Homero Urrutia c Renato A. Quin a b c

´fica en el Pacı´fico Sur-Oriental (FONDAP-COPAS), Universidad de Concepcio ´n Oceanogra ´n, Casilla 160-C, Concepcio ´n, Chile Centro de Investigacio ´ficas, Universidad de Concepcio ´n, Casilla 160C, Concepcio ´n, Chile Departamento de Oceanografı´a, Facultad de Ciencias Naturales y Oceanogra ´gicas, Universidad de Concepcio ´n, Casilla 160-C, Concepcio ´n, Chile Departamento de Microbiologı´a, Facultad de Ciencias Biolo

a r t i c l e in fo

abstract

Article history: Accepted 13 September 2008 Available online 5 November 2008

The latest advances in the field of microbial ecology have shown that planktonic Archaea are one of the most abundant unicellular microorganisms of the oceans. However, no information is available on the contribution this group makes to the prokaryote assemblages that inhabit the eastern South Pacific Ocean. Here, we describe the relative abundance and vertical distribution of planktonic Archaea off northern and central-southern Chile. Data come from several cruises and a 45-month time series at a station located on the shelf off central-southern Chile. Both the taxonomic composition of the prokaryote community and its relative abundance were determined using quantitative dot blot 16S-rRNA hybridizations. Total Archaea in central-southern Chile made up 6–87% of the prokaryote rRNA in the water column and did not present evidence of any seasonal pattern. Crenarchaea were the most abundant archaeal group at this site and were significantly associated with the ammonium concentration (r2 ¼ 0.16, p ¼ 0.0003, n ¼ 80). Archaeal abundance in the time series was usually greater in the deeper layer (450 m), with contributions reaching up to 90% of the prokaryote rRNA on certain occasions, and decreasing towards the surface. Important increments in the relative abundance of total Archaea were observed on given dates at the surface of the time-series station off central-southern Chile. Off northern Chile, total Archaea normally contributed from 10% to 50% of the prokaryote rRNA found between 10 and 1000 m, and were generally important in the mesopelagic realm. Our results indicate that Archaea constitute an important fraction of the prokaryote assemblage in the water column of the Humboldt Current System, especially in the oxygen minimum zone. & 2008 Elsevier Ltd. All rights reserved.

Keywords: Planktonic Archaea Oxygen minimum zone Benthic boundary layer Hypoxia Upwelling Humboldt Current System

1. Introduction The continental shelf located between central-southern and northern Chile is part of one of the most productive marine ecosystems of the world (Daneri et al., 2000; Troncoso et al., 2003). This shelf is characterized by the presence of a subsurface low-oxygen water mass (Equatorial Subsurface Water, ESSW), geographically different upwelling zones, and the important ˜ o Southern influence of interannual variations due to El Nin Oscillation (ENSO). An oxygen minimum zone (OMZ) that is associated with ESSW acts as an important regulator for both ˜ ones, 1999; Gonza´lez and aerobic respiration (Eissler and Quin ˜ ones, 2002) and pelagic biota distribution (Boyd et al., 1980; Quin Morales et al., 1996; Escribano and Hidalgo, 2000; Gonza´lez and ˜ ones, 2000). Quin  Corresponding author at: Departamento de Oceanografı´a and Centro de Investigacio´n Oceanogra´fica en el Pacı´fico Sur-Oriental (FONDAP-COPAS), Universidad de Concepcio´n, Casilla 160-C, Concepcio´n, Chile. Tel.: +56 41 220 7426; fax: +56 41 222 5400. ˜ ones). E-mail address: [email protected] (R.A. Quin

0967-0645/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr2.2008.09.012

It is widely known that prokaryotes make up a major portion of the biomass in marine planktonic environments (Cole et al., 1988), playing a key role in the carbon flux as reported for the Humboldt Current System (HCS) (Troncoso et al., 2003; Cuevas et al., 2004). Molecular biology techniques have revealed that Archaea are abundant and distributed virtually in all environments (e.g., oceanic, freshwater, soil; for review, see Chaban et al., 2006). Indeed, uncultured Archaea are one of the most abundant unicellular groups in the ocean, especially in the mesopelagic zone (Massana et al., 1997, 2000; Fuhrman and Ouverney, 1998; Karner et al., 2001). In this context, Karner et al. (2001) suggest that the global oceans harbor approximately 1.3  1028 archaeal cells, which is roughly equivalent to 42% of the global abundance of bacteria in the oceans. Three major phyla have been proposed as constituting the Archaea domain although only two are presently accepted (Boone and Castenholz, 2001): Crenarchaea and Euryarchaea (Woese and Olsen, 1986). The third phylum (candidate), called Korarchaea, is a more distant and deeper branch of Archaea whose existence has been suggested based only on DNA sequences from environmental surveys (Barns et al., 1994). A fourth group, the Nanoarchaea, have

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been proposed as a new archaeal phylum (Huber et al., 2002, 2003). However, as in the case of Korarchaea, its position in the archaeal tree is controversial (Robertson et al., 2005). The 16S-rDNA sequences from picoplanktonic Archaea can be classified in four marine groups: marine group I falls within the Crenarchaea whereas marine groups II, III, and IV fall within the Euryarchaea (Giovannoni and Stingl, 2005; Schleper et al., 2005). In marine temperate regions, marine group I Crenarchaeota dominates at depths X75 m, depending on the site studied, and Euryarchaea (group II) can be important in shallow waters (Massana et al., 1997; DeLong et al., 1999; Murray et al., 1999a; Massana et al., 2000; Pernthaler et al., 2002). Nonetheless, marine group I frequently represents the bulk fraction throughout the water column (Massana et al., 1998; Murray et al., 1998, 1999b; Karner et al., 2001). To the best of our knowledge, no information is currently available regarding the contribution of planktonic Archaea to the prokaryotic assemblages inhabiting the eastern South Pacific Ocean. Here, we describe the vertical distribution of planktonic Archaea and its temporal fluctuations using vertical profiles of relative abundance obtained during several cruises off Chile and a nearly 4-year-long time series at a shelf station that is sampled monthly.

central-southern Chile onboard the R/V Kay-Kay (University of Concepcio´n) and off northern Chile onboard the R/V Vidal Gormaz (Chilean Navy). The samples were collected at different depths with Niskin bottles and kept onboard (at in situ surface temperature and in complete darkness) in carboys pre-washed with 10 N HCl until arrival at the marine coastal laboratory. These samples were used to analyze the taxonomic composition at the domain level by quantitative dot blot hybridization. Water samples were also collected for analyses of phosphate, silicate, nitrite, nitrate, ammonium, dissolved oxygen, and chlorophyll. Temperature, salinity, and oxygen profiles were taken with a CTDO (model SBE-19, Sea-Bird Electronics Inc.). PO4, SiO4, NO2-N, and NO3-N were determined using an ALPKEM FlowSolution IV autoanalyzer (OI Analytical, College Station) according to Strickland and Parsons (1972). NH4-N was determined as described by Holmes et al. (1999) using a Turner Designs fluorometer (model 10-AU). The oxygen measurements taken with the CTDO were calibrated using dissolved oxygen determinations by the Winkler method (Carpenter, 1965) in discreet samples. Phytoplanktonic standing stock was estimated through chlorophyll-a (Chl-a) measurements following Holm-Hansen et al. (1965). 2.2. Dot blot hybridizations of rRNA

2. Material and methods 2.1. Field sampling and environmental variables The study was conducted at two upwelling sites in the HCS off Chile and the data comes from several cruises and a 45-month time series at a station located off central-southern Chile (Fig. 1, Table 1). Water samples of at least 7 L were collected off

RNase-inactivation was achieved by treating all solutions with diethylpyrocarbonate (DEPC, Sigma Chemical Co.) as described by Sambrook et al. (1989). The seawater was pre-filtered through 25 mm and concentrated by vacuum filtration (o10 cm Hg) using cellulose ester filters (pore size 0.22 mm; GSWP04700, Millipore Corp.). Subsequently, the microorganisms retained on the filters were resuspended in pre-filtered (0.22 mm; GSWP04700, Millipore Corp.) seawater containing Tween 20 (final concentration

Fig. 1. Study area in the eastern South Pacific. Sampling stations are indicated by black dots.

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Table 1 Location of the sampling stations. Zone

Station

Location

Depth (m)

Latitude S

Longitude W

Sampling date (d/m/y) or (y)

Sampling

Northern Chile Northern Chile Northern Chile Northern Chile Northern Chile Central-southern Chile Central-southern Chile

EW4 EW9 1 4 6 18a 40

Iquique El Loa Mejillones Mejillones Mejillones Concepcio´n Concepcio´n

1200 950 95 106 886 88 1200

19159.860 20159.870 23102.540 22158.550 23104.690 36130.80 36120.00

70138.960 70132.510 72127.150 70123.240 70137.600 73107.70 73144.00

21 & 23/09/2005 24 & 26/09/2005 28/09/2005 29/09/2005 30/09/2005 2002–2005 2004

Water column Water column Only BBL Only BBL Only BBL Water column Water column

BBL: benthic boundary layer. a Time-series station.

Table 2 Names, specificities, target positions, sequences, and melting temperature (Tm) of the probes used. Probe

Target group

Target sitea

Sequence (50 –30 )

Tm (1C)

Reference

EUB338 ARCH915 CREN499 EURY498

Bacteria Archaea Crenarchaea Euryarchaea

16S, 16S, 16S, 16S,

GCT GCC TCC CGT AGG AGT GTG CTC CCC CGC CAA TTC CT CCA GRC TTG CCC CCC GCT CTT GCC CRG CCC TT

51.5 61.8 61.6 46.1

Amann et al. (1990) Stahl and Amann (1991) Burggraf et al. (1994) Burggraf et al. (1994)

a

338–355 915–934 499–515 498–510

Escherichia coli numbering.

0.05% v/v) using a vortex and an ultrasonic water bath at 4 1C for 7 min. These suspensions were harvested by centrifuging at 11,500g for 15 min at 4 1C. The pellets from each depth were washed once with phosphate-buffered saline (PBS: 120 mM NaCl and 2.7 mM KCl in 10 mM phosphate buffer, pH 7.6) and then processed for rRNA extraction following the method described by Summers (1970) with the following modifications: 15 mL of mutanolysin (5000 U mL1, Sigma) was added in each reaction to obtain a final concentration of 150 U mL1 and lysozyme (Sigma) was used at a final concentration of 1 mg mL1. Four microliters of rRNA extracts were prepared for formaldehydeagarose gel electrophoresis as described by Rosen and VillaKomaroff (1990). An RNA ladder (1.5 mg) between 0.24 and 9.5 Kb was loaded in each electrophoresis (Gibco-BLR). Dilution series were carried out based on the rRNA extracts and from a standard of 16S-rRNA (Escherichia coli MRE600, Roche Diagnostics). The dilutions were denatured in denaturing solution at 65 1C for 15 min, and immediately transferred and kept on ice for 15 min more. The denaturing solution was composed of formamide, formaldehyde, and MOPS solution (5: 1.62: 1; MOPS Sol.: 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.2). Then, denatured dilution series were loaded on nylon membranes (Hybond-N, Amersham BioSciences) by vacuum filtration for 10 min using a dot blotting apparatus (Bio-Rad), immobilized by baking at 80 1C for 2.5 h, and maintained at 20 1C until assay. Hybridization was carried out at 44 1C according to the protocol of Raskin et al. (1994a, b) by using 50 end digoxigenin-labeled oligodeoxynucleotide probes (Thermo Electron Corp., Thermo Biosciences). The hybridization temperature was optimized with respect to the NaCl concentration and the percentage of formamide present in the hybridization cocktail with the following control strains: E. coli DH5a, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Photobacterium phosphoreum CECT 4172. These bacterial strains were also used as negative controls for all archaeal probes. The probes (Table 2) were detected by enzymatic reaction between alkaline phosphatase (conjugated to an anti-digoxigin antibody) and a chemiluminescent substrate (CSPD) following manufacturer recommendations (Roche Diagnostics). The hybridization signals on the membranes were transferred to fluorescence-sensitive films (ECL, Amersham BioSciences),

which were finally digitized on a grey scale and processed to quantify the 16S-rRNA using Quantity One software (Bio-Rad, version 4.2). The quantification of the prokaryotic rRNA was carried out with a calibration curve constructed using an internal standard of 16S-rRNA (E. coli MRE600) as described by Levipan et al. (2004). The relative abundance of each archaeal group is expressed as a percentage of prokaryote rRNA. The rRNA concentrations determined for any given probe were expressed as a percentage of prokaryote rRNA (i.e., archaeal rRNA plus bacterial rRNA) quantified with the general probes (in our work: EUB338 and ARCH915) as suggested by Raskin et al. (1994b) and Massana et al. (1998). It is important to remark that, in this article, the expressions ‘‘archaeal abundance’’, ‘‘crenarchaeal abundance’’, and ‘‘euryarchaeal abundance’’ do not refer to the numerical abundance (cell counts) of these groups but to their relative abundance in relation to total prokaryote rRNA as determined by dot blot hybridization.

3. Results 3.1. Archaeal abundance off central-southern Chile Total Archaea represented an important fraction of the prokaryote community collected at the time-series station located on the shelf off central-southern Chile (Sta. 18; 361300 S–731070 W), typically contributing between 6% and 87% of the prokaryote rRNA extractable from the water samples (Fig. 2). The generally important archaeal presence found below 40–50 m depth included some contributions reaching to 90% of the prokaryote community (Fig. 2). In fact, the average annual abundance of total Archaea in the OMZ under 50 m depth was nearly 50% in 2003 and 2005 (Fig. 3). A slightly lower abundance was observed in 2004 (Fig. 3). The archaeal abundance tends to decrease towards the surface (Fig. 2), showing average annual values of less than 33% in the first 10 m depth (Fig. 3). On several occasions during the timeseries sampling (e.g., May–July 2003, May–June 2005) (Fig. 2) archaeal abundance at the surface reached values as high as those found in the deeper portions of the water column, including contributions to the total prokaryote rRNA as high as 87% (August 2005) (Fig. 2). The variability observed in archaeal abundance

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1076

10

-100 -90

20

-80 30 40

-60 -50

50

-40 60

% of rRNA

Depth (m)

-70

-30 -20

70

-10 80

Station 18

-0

A S N D J F A M J JA S O N D JA M J JA S O N D J F M A M JA S O N D 2002

2003

2004

2005

Time (mo/yr) Fig. 2. Time series of total abundance of planktonic Archaea from a station located on the continental shelf (Station 18) in the coastal upwelling area off central-southern Chile. This station is located approximately 18 nautical miles from the coast and has an approximate depth of 90 m. The symbol (~) indicates dates and sampling depths (10, 30, 50, 80, and 85 m). Data are averages of two sub-samples; the standard errors of the means were typically o16%. Total archaeal abundance is expressed as a percentage of archaeal rRNA relative to prokaryotic rRNA (i.e., archaeal rRNA [ARCH915] plus bacterial rRNA [EUB338]). The typical range was between 6% and 87%.

% of rRNA 0

10 20 30 40 50 60 70 80 90

0

10 20 30 40 50 60 70 80 90

0

10 20 30 40 50 60 70 80 90

0 2003 (St18) 10

20

2004 (St18)

2005 (St18) Total Archaea Crenarchaea Euryarchaea

Depth (m)

30

40

50

60

70

80

90 Fig. 3. Mean annual abundance of total Archaea, Crenarchaeota, and Euryarchaeota (relative to archaeal rRNA plus bacterial rRNA) in the coastal upwelling area off centralsouthern Chile. Values correspond to monthly averages of samples from the time-series station (Station 18). Error bars correspond to standard errors of the means. The cross-hatched area shows the water column normally associated with the OMZ.

during the 45-month long time series at Sta. 18 did not reflect a seasonal pattern (Fig. 2). Crenarchaea was the dominant archaeal group in the water column during the entire study period (Fig. 3) except at the end of 2002, when Euryarchaea became dominant (data not shown).

In 2004, total Archaea at the oceanic station 40 (Sta. 40; 361200 S-731440 W) off central-southern Chile represented, on average, 40% of the prokaryote community towards 1000 m depth. This contribution decreased towards the surface until reaching values roughly between 10% and 23% in the first 100 m

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% of rRNA

0

O2 (mL L-1)

Temp (°C)

0 10 20 30 40 50 60 70 80 90

6

3

9

12 15

1077

Nitrate (µM)

0 1 2 3 4 5 6 0 10 20 30 40 50

2004 (St40)

100 200

Depth (m)

300 400 500 600 700 800

Total Archaea Crenarchaea Euryarchaea

900 1000

(A)

(B)

(C)

33.6 34.0 34.4 34.8 0.0 Salinity (psu)

(D) 0.3

0.6

Nitrite (µM)

0.9 0

1

2

3

Phosphate (µM)

Fig. 4. Vertical profiles of archaeal abundance and environmental variables measured at the oceanic station off central-southern Chile (Station 40). (A) Mean abundance of total Archaea, Crenarchaeota, and Euryarchaeota. Data correspond to the averages of samples collected at the oceanic station 40 on four sampling dates (24 February 2004, 02 June 2004, 10 August 2004, 25 November 2004). Station 40 is located approximately 40 nautical miles from the coast and has a total depth of 1200 m. Water samples were obtained from the following depths: 15, 30, 100, 300, 750, and 1000 m. Climatic conditions impeded more comprehensive annual sampling at Station 40. Error bars show standard errors of the means. The hatched area shows the typical vertical location of the OMZ. (B–D) Environmental variables are expressed as mean7SD.

(Fig. 4A). The abundance of Euryarchaea increased with depth (Fig. 4A) contributing about 22% of the total prokaryote and 50% of the archaeal rRNA at 1000 m depth. Crenarchaeota were the dominant archaeal group in the first 100 m depth, although its abundance diminished slightly from this depth down to the bottom of the OMZ (i.e. about 300 m depth) (Fig. 4A). No significant differences were observed between the abundances of Crenarchaea and Euryarchaea in the deep layer (i.e. below 300 m) at the oceanic station (Sta. 40) (Fig. 4A).

(Fig. 5B, D) and, as at station EW4, the crenarchaeal abundance experienced an important decrease (Fig. 5D) or was not detectable with increasing depth (Fig. 5B). In contrast with the increment of Euryarchaeota with depth found at the oceanic station off centralsouthern Chile (Sta. 40) (Fig. 4A), Euryarchaea were not detected below 60 or 200 m depth off northern Chile (Fig. 5). The total archaeal abundance in the benthic boundary layer (BBL) represented 56710.5% (7SE) of the total prokaryote rRNA extractable from the water samples collected off Mejillones.

3.2. Archaeal abundance off northern Chile

3.3. Associations between archaeal distribution and environmental variables

As in the central-southern region, total Archaea off northern Chile represented an important fraction of the picoplankton during the sampling dates. The oceanographic station off Iquique (EW4) revealed that total archaeal abundance was normally between 10% and o50% of the prokaryote rRNA in the first 60 m depth (Fig. 5A, C). Below this depth, total archaeal abundance was about 30% of the prokaryote rRNA (Fig. 5A, C), excepting lower abundances observed at 750 m depth (10.4%) (Fig. 5A). Generally Crenarchaeota was the dominant archaeal group in the first 60 m depth at EW4 station (Fig. 5A, C), although its abundance dropped sharply below this depth (Fig. 5C) or was not even detectable (Fig. 5A). The abundance of total Archaea in the first 60 m depth at the station off El Loa River (EW9) was similar to that of the EW4 station, i.e., between 10% and 50% (Fig. 5B, D). Nevertheless, on one of the sampling dates, the total Archaea abundance in the OMZ (50 to 350 m depth; data not shown) was in the lower limit of the reported range, as compared to the surface and deeper layers of the water column (Fig. 5B). In the surface stratum at station EW9, Crenarchaeota were the dominant archaeal group

The archaeal rRNA concentration as determined by the ARCH915 general probe was not significantly associated (p40.05) with the environmental variables (temperature, salinity, dissolved oxygen, phosphate, silicate, nitrite, nitrate, ammonium, chlorophyll). However, the concentration of crenarchaeal rRNA was positively associated with the ammonium concentration (r2 ¼ 0.16, p ¼ 0.0003, n ¼ 80) (Fig. 6). Bacterial rRNA concentrations were positively associated with temperature (r2 ¼ 0.11, p ¼ 0.0167, n ¼ 114) in a narrow thermal range (i.e., 10–15 1C), as found in the coastal water column off central-southern Chile. A positive association was also observed between bacterial rRNA and Chl-a (r2 ¼ 0.29, p ¼ 0.005, n ¼ 81). No associations were observed between bacterial rRNA and the remaining environmental variables. In spite of the lack of association between the abundances of the different archaeal groups evaluated and the dissolved oxygen concentration, an important percentage of the prokaryote rRNA corresponded to archaea in the OMZ off Concepcio´n (Sta. 18 and

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% of rRNA 0

% of rRNA

10 20 30 40 50 60 70 80 90

0

10 20 30 40 50 60 70 80 90

0

20

Total Archaea

20

Crenarchaea

40

Euryarchaea 60

40 60

80 Depth (m)

0

80

400 100 500 120 600 140 700 160

800

180

900 E W4

1000

0

10 20 30 40 50 60 70 80 90

0

E W4

200

0 10 20 30 40 50 60 70 80 90 0

50

20

100 40

Depth (m)

150 200

60

250

80

400

500

500 600

600 700

700

800 900

EW9

800

EW9

Fig. 5. Vertical profiles of total Archaea, Crenarchaeota, and Euryarchaeota abundances off northern Chile. EW4 ¼ oceanographic station off Iquique, EW9 ¼ oceanographic station off El Loa River. See Table 1 for details on station location. Data are averages of two sub-samples and error bars show standard errors of the means. Sampling dates: (A) 21 September 2005, (B) 24 September 2005, (C) 23 September 2005, and (D) 26 September 2005.

Sta. 40), where the dissolved oxygen concentration was typically p0.5 mL L1 during the study period (e.g., Fig. 4C). In northern Chile, the vertical abundance of Archaea did not show a clear increase within the OMZ (see, for example, Fig. 5B), although this result should be taken cautiously due to sample size (only four profiles) and the possible influence of the El Loa River at station EW9.

4. Discussion High archaeal abundance was found in the deep layer of the water column (below 50 m depth) at the coastal time-series station (Sta. 18) off central-southern Chile. The total Archaea decreased towards the surface, averaging o33% of the prokaryote rRNA in the first 10 m depth (Fig. 3). Murray et al. (1999a)

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101

CREN499

100

10-1

10-2

10-3

10-4 10-2

10-1

100

101

NH4 (µM) Fig. 6. Relationship between ammonium and crenarchaeal rRNA (lineal scale) as estimated using CREN499 probe (r2 ¼ 0.16, p ¼ 0.0003, n ¼ 80).

observed that archaea can reach, on given occasions, between 20% and 50% of the total rRNA in the Santa Barbara Channel at depths over 20 m. The general vertical distribution pattern found at Sta. 18 agreed with reports from other oceanic regions where the Archaea abundance increases with depth and/or low oxygen (e.g., Massana et al., 1997; DeLong et al., 1999; Murray et al., 1999a; Karner et al., 2001; Sinninghe Damste´ et al., 2002; Teira et al., 2004, 2006a, b; Schubert et al., 2006). In fact, the water layer below 50 m off central Chile was characterized by having, commonly, dissolved oxygen concentrations of o0.5 mL L1 (for a detailed description, see Sobarzo et al., 2007). It is interesting to note that the depth of the euphotic zone in the study area has been estimated between 50 and 65 m (Montecino et al., 2004) and, therefore, the microbial biosynthetic processes are not driven by light. Both at the coastal (Sta. 18) and oceanic (Sta. 40) stations off central Chile, archaeal abundance increased with depth, especially within the OMZ (Figs. 3 and 4). No significant differences were observed at this station for total archaeal abundance from the lower limit of the OMZ (about 300 m depth) down to the bottom of the water column (Fig. 4). The latter agrees with the observations of Karner et al. (2001), who found that Archaea (Crenarcheota) represented 39% of the DAPI counts in the mesopelagic zone of the northern Pacific, and with the observed archaeal dominance (450% of prokaryotic abundance) in different water masses below 100 m depth in the North Atlantic (Herndl et al., 2005). Similarly, high concentrations of crenarchaeol and other tetraether lipids have been measured in the northwestern Arabian Sea indicating that crenarchaea are widely distributed and abundant in the water column, especially, in the core of the OMZ at 500 m depth (Sinninghe Damste´ et al., 2002). Furthermore, archaeal richness can also increase with depth (e.g., Madrid et al., 2001; Bano et al., 2004) or with low oxygen (Vetriani et al., 2003). Off northern Chile, total Archaea normally contributed from 10% to 50% of the prokaryote rRNA found between 10 and 1000 m, and they were generally important in the mesopelagic realm (Fig. 5).

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Crenarchaea were frequently the dominant archaeal group in the water column of the HCS off Chile (Figs. 3 and 5), in agreement with observations from other marine ecosystems (Massana et al., 1998; Murray et al., 1998; Karner et al., 2001). Nevertheless, Euryarchaea were the dominant archaeal group off central-south Chile (Sta. 18; austral summer) at the end of 2002, probably, due to a strong contribution of methanogen-like archaea from sediments during upwelling periods in the study area, as discussed elsewhere (Levipan et al., 2007a). The abundances of Euryarchaeota and Crenarchaeota in the mesopelagic realm off central-southern Chile were similar (Fig. 4A). Off northern Chile, Euryarchaeota were detectable down to 60 or 200 m depth (Fig. 5), which is consistent with evidence that euryarchaeal clones can be important or dominant phylotypes in surface waters (Massana et al., 1997, 2000; Murray et al., 1999a; Pernthaler et al., 2002). However, Crenarchaeota were not detectable below 60 or 200 m depth (Fig. 5A, B), or represented a minor fraction of the total Archaea detected in the water column (Fig. 5C, D). This result suggests that the taxonomic composition of the archaeal assemblages may differ between the northern and central-southern zones of the HCS, especially regarding the portion of the water column below about 60 m. Research using a cloning sequencing approach (or fingerprinting approaches, e.g., DGGE) can be very useful in clarifying this issue. For instance, Stevens and Ulloa (2008) used a cloning sequencing approach to report important vertical changes in the taxonomic composition of the bacterial community inhabiting the water column off Iquique. Moreover, these authors suggest the existence of novel and currently uncultivated bacterial lineages within the OMZ. It would not be surprising if pronounced vertical taxonomic variability also occur within the archaeal assemblage. Unusually high contributions of archaeal abundance to the prokaryote assemblage were observed throughout nearly all the water column at the coastal station off central-southern Chile (Sta. 18) on given occasions (Fig. 2) without showing a seasonal pattern. For instance, the major abundance of total Archaea in the whole water column reached values as high as 8077% (see August 2005) (Fig. 2). Although some studies indicate that the relative proportion of Archaea in the water column tend to be high in winter and low in summer for areas where light-limitation occur in winter (Massana et al., 1998; Murray et al., 1998), this was not always the case at Sta. 18, where the levels of photosynthetically active radiation are seasonally distributed, presenting maximum values in spring–summer and low values in winter (Montero et al., 2007). Due to the coastal nature of the time-series station off centralsouthern Chile (Sta. 18), this site is influenced by river-discharge and events such as upwelling (typically more frequent in austral spring–summer) or mixing/turbulence (typically more frequent in austral winter) (Sobarzo et al., 2007). During upwelling, ESSW (salinity 434.5; dissolved oxygen o1 mL L1) can occupy the entire water column below 20 m (Sobarzo et al., 2007). However, the variability of archaeal abundance (Fig. 2) was not strictly associated with these events. For instance, upwelling events taking place between November and December 2004 (Castro et al., 2004) were not associated with high archaeal abundance at the surface, which was likely due to the low abundance of this group in the suboxic portion of the water column adjacent to the bottom during this period (Fig. 2). Vertical transport from the benthic boundary layer was the most probable cause for the high archaeal abundance at the surface off central-southern Chile (e.g., August 2005) (Fig. 2), which occurred mostly through coastal upwelling and, to a lesser degree, through mixing/turbulence caused by storms. The total Archaea rRNA concentration was not associated significantly (p40.05) with the environmental variables at the

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coastal station (Sta. 18). Only the concentration of crenarchaeal rRNA was positive and significantly associated with the concentration of ammonium (po0.05) (Fig. 6). Recently, a re-assessment of the role of the nitrification process in the world oceans has begun and organisms such as Crenarchaea, able to growth chemolithoautotrophically using ammonia (Francis et al., 2005; Ko¨nneke et al., 2005; Wuchter et al., 2006), can have profound effects on net community production processes and can impact the coupling of C–N–P cycles as well as the net oceanic sequestration of atmospheric carbon dioxide (Nicol and Schleper, 2006). In this context, we suggest that at least a minor fraction of the crenarchaeal assemblage off Concepcio´n may have potential autotrophic growth. On some occasions, only a minor contribution of Archaea to the total prokaryote abundance was observed in the OMZ off centralsouth Chile (Fig. 2), and once in the northern zone (Fig. 5B). Therefore, although oxygen-depleted conditions are likely to favor the presence of archaea in the ESSW, it is clear that oxygen is not the only environmental factor involved. There is evidence indicating that specific prokaryotic populations accumulate in defined strata in the water column, presumably in response to optimal physical, chemical, or nutritional requirements as well as biotic interactions including competition, predation (Massana et al., 1997), and viral infection (Winter et al., 2004). On the other hand, the bacterial rRNA concentration was significantly associated with the Chl-a concentration (r2 ¼ 0.29, p ¼ 0.005). This indicates that the strong relationship found in the HCS between carbon synthesis and prokaryote carbon consumption (Troncoso et al., 2003), referred to by these authors as ‘‘bacterial’’ carbon consumption, certainly involves members of the Bacteria domain. In fact, total bacteria were normally the dominant prokaryote group in the water column of the HCS off Concepcio´n, representing the complementary percentage to the annual average abundance of Archaea reported in Fig. 3. In general, the bacterial domain was also the dominant prokaryote group in the water column off northern Chile (Fig. 5). Archaea constitute an important metabolic component of the carbon cycle in the Humboldt Current System’s OMZ through temporal pulses of heterotrophy (Levipan et al., 2007b). However, at present, no clear consensus exists about the relative importance of heterotrophic and autotrophic metabolic pathways in the growth of marine Archaea (Ouverney and Fuhrman, 2000; Wuchter et al., 2003, 2006; Teira et al., 2004, 2006b; Herndl et al., 2005; Biddle et al., 2006; Ingalls et al., 2006; Nicol and Schleper, 2006). Moreover, the identification of genes that encodes nearly all the tricarboxylic acid cycle and several components of the modified 3-hydroxypropionate cycle in Crenarcheum symbiosum provide strong evidence for mixotrophy (Hallam et al., 2006). Consequently, the main pathway of carbon incorporation used by Archaea still remains a key issue to be elucidated. It is important to highlight the recent study by Levipan et al. (2007b) on archaeal production in the OMZ off central-southern Chile. These authors reported that total prokaryote production (i.e., bacteria+archaea) in the OMZ off central-southern Chile could reach values as high as 910 mg C m3 h1 with archaeal secondary production corresponding to, on average, 42.478.5% (7SE) of the total prokaryote production, although its contribution could reach nearly 97% (Levipan et al., 2007b). Bacterial and archaeal assemblages can reach secondary production values in the OMZ as high as 600 and 300 mg C m3 h1, respectively. The high estimates of archaea secondary production in the OMZ reported by Levipan et al. (2007b) and the abundances and vertical distribution patterns described in this research, strongly suggest that Archaea are an important component of the metabolism of the water column in the HCS.

Finally, our results also emphasize the complexity of the microbial realm in the HCS. Since the mid-1990s, a body of evidence has emerged indicating that the microbial food web is very important in this ecosystem (e.g., Gonza´lez et al., 1998; ˜ ones, 1999; Montecino and Quiroz, 2000). This Eissler and Quin point of view challenged the classic notion that the HCS behaves as a typical upwelling ecosystem with most energy and matter flowing up through a short classic food chain (sensu Steele, 1974), and in which the carbon generated via photosynthesis is efficiently transferred through chain-forming-diatoms to higher trophic levels. Today, studies on prokaryote assemblages have shown substantial biomass and very high secondary production (McManus and Peterson, 1988; Pantoja et al., 1989; Pacheco and Troncoso, 1998; Troncoso et al., 2003; Cuevas et al., 2004; Levipan et al., 2007b), indicating that a significant fraction of the primary productivity is channeled through the microbial food web. Estimates of metabolic activity and organic matter decomposition have also shown that the microbial realm is extremely active in the oxic and suboxic portion of the water column of the HCS (Pantoja et al., 2004; Gonza´lez et al., 2007). Carbon budgets at specific sites also highlight the microbial food web as a crucial component for the flux of carbon and energy in the HCS (e.g., Gonza´lez et al., 1998; Vargas et al., 2007). In conclusion, Archaea are a highly relevant structural component of the prokaryote community in the water column of both northern and central-southern Chile that emphasize the need to understand the biogeochemical role played by this group in the eastern South Pacific.

Acknowledgments This research was funded by the COPAS Center (FONDAP no. 150100007, CONICYT, Chile) and by FONDECYT Grant no. 1000373 (CONICYT, Chile). We are grateful to Ariel Pacheco, Gerdhard Jessen, Oscar Chiang and Karol Espejo for their field support. We acknowledge the COPAS scientists and technical staff, especially Ruben Escribano and Marı´a Ange´lica Varas, for their work in obtaining the wind, CTDO, and nutrient data. We thank the captain and crew of the R/V Kay-Kay for their valuable assistance and support. References Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., Stahl, D.A., 1990. Combination of 16S rRNA-targeted oligonucleotides probes with flow cytometry for analyzing mixed microbial populations. Applied and Environmental Microbiology 56, 1919–1925. Bano, N., Ruffin, S., Ransom, B., Hollibaugh, J.T., 2004. Phylogenetic composition of Arctic Ocean archaeal assemblages and comparison with Antarctic assemblages. Applied and Environmental Microbiology 70, 781–789. Barns, S.M., Fundyga, R.E., Jeffries, M.W., Pace, N.R., 1994. Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proceedings of the National Academy of Sciences of the United States of America 91, 1609–1613. Biddle, J.F., Lipp, J.S., Lever, M.A., Lloyd, K.G., Sørensen, K.B., Anderson, R., Fredricks, H.F., Elvert, M., Kelly, T.J., Schrag, D.P., Sogin, M.L., Brenchley, J.E., Teske, A., House, C.H., Hinrichs, K.-U., 2006. Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. Proceedings of the National Academy of Sciences of the United States of America 103, 3846–3851. Boone, D.R., Castenholz, R.W., 2001. Bergey’s Manual of Systematic Bacteriology. Springer, New York. Boyd, C.M., Smith, S.L., Cowles, T.J., 1980. Grazing patterns of copepods in the upwelling system off Peru. Limnology and Oceanography 25, 583–596. Burggraf, S., Mayer, T., Amann, R., Schadhauser, S., Woese, C.R., Stetter, K.O., 1994. Identifying members of the domain Archaea with rRNA-targeted oligonucleotides probes. Applied and Environmental Microbiology 60, 3112–3119. Carpenter, J.H., 1965. The Chesapeake Bay Institute technique for the Winkler dissolved oxygen method. Limnology and Oceanography 10, 141–143. Castro, L., Daneri, G., Escribano, R., Farı´as, L., Gonza´lez, H., Morales, C., Pizarro, O., Rosales, S., 2004. Monitoreo de las condiciones bio-oceanogra´ficas en la VIII

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