# Microbial diversity in a Pacific Ocean transect from the Arctic to Antarctic circles

Amy J. Baldwin, Joseph A. Moss, Joseph D. Pakulski, Philippe Catala, Fabien Joux, Wade H. Jeffrey

► To cite this version:

Amy J. Baldwin, Joseph A. Moss, Joseph D. Pakulski, Philippe Catala, Fabien Joux, et al. Microbial diversity in a Pacific Ocean transect from the Arctic to Antarctic circles. Aquatic Microbial Ecology, 2005, 41, pp.91-102. [https://doi.org/10.1007/s00258-005-0102-9](https://doi.org/10.1007/s00258-005-0102-9)

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# Microbial diversity in a Pacific Ocean transect from the Arctic to Antarctic circles

Amy J. Baldwin<fnref n="1" />, Joseph A. Moss<fnref n="1" />, Joseph D. Pakulski<fnref n="1" />, Phillipe Catala<fnref n="2" />, Fabien Joux<fnref n="2" />, Wade H. Jeffrey<fnref n="1" />,*

**ABSTRACT:** Microbial diversity in surface waters was examined along a ~15 400 km transect of the Pacific Ocean from 70°N to 68°S latitude between late August and early November 2003. Comparative microbial diversity was determined using terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR amplified 16S and 18S rDNA restriction digested with *CfoI* and *MspI*. Bacterial numbers and total chlorophyll were greatest at higher latitudes in both hemispheres, with a smaller peak in equatorial waters. Flow cytometry analysis indicated a strong peak in *Prochlorococcus* from approximately 30°N to 30°S. Richness at each station was relatively low, with ~11 prokaryotic peaks per sample and ~12 eukaryotic peaks per community. Overall, prokaryotic populations appeared more diverse, with 181 total terminal restriction fragments (T-RFs) generated, while eukaryotic populations produced a total of 135 T-RFs. Prokaryotic and eukaryotic similarity dendrograms revealed 4 distinct cluster groups relating to regions sub-Arctic/Arctic, temperate, tropical and sub-Antarctic/Antarctic. T-RFLP patterns suggest that microbial communities may be influenced by ambient water temperature, with mid-latitudinal and equatorial communities more similar in composition to each other than to cold water communities. Global distribution of prokaryotic communities revealed an average inter-group similarity of ~52%, while eukaryotic communities showed ~51% similarity, implying that Pacific planktonic communities appear to be fairly homogeneous in composition. Several T-RFs were ubiquitously distributed and contributed significantly to each cluster group, while several T-RFs were observed to be endemic to particular oceanic regions. Within-group similarities of >70% were attributed to 12–14 T-RFs and 8–11 T-RFs in prokaryotic and eukaryotic profiles, respectively, suggesting that a small number of phylogenetic groups were responsible for each cluster group.

**KEY WORDS:** Microbial diversity · Pacific Ocean · T-RFLP · 16S rDNA · 18S rDNA · Biogeographical distribution

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## INTRODUCTION

In order to understand planktonic diversity and the roles of microorganisms in global biogeochemical cycling, we must recognize the distribution patterns of planktonic phylotypes and their controlling environmental factors. The introduction of molecular technologies has revolutionized our view of the microbial world, resulting in a dramatic increase in the number

of microbial diversity studies. Before the 1990s, investigations of microbial diversity were driven primarily by culture-based methodologies until Giovannoni et al. (1990) reported the results of a phylogenetically analyzed clone library from the Sargasso Sea. Using 16S rRNA genes, the novel clade SAR11 and the oxygenic phototrophs (cyanobacteria, *Synechococcus*, *Prochlorophytes*) were revealed as dominant members of the resident bacterioplankton community (Giovannoni et

<footnote n="1">¹Center for Environmental Diagnostics and Bioremediation, University of West Florida, Building 58, 11000 University Parkway, Pensacola, Florida 32514 (850) 474-2472, USA</footnote>

<footnote n="2">²Observatoire Océanologique de Banyuls, Laboratoire d'Océanographie Biologique, Université Pierre et Marie Curie, CNRS UMR7621, BP 44, 66651 Banyuls-sur-Mer Cedex, France</footnote>
al. 1990). Continual advancement and application of molecular-based technologies has revealed the presence of marine microorganisms that were not detectable by earlier culture based methods (Giovanonni et al. 1990, Fuhrman et al. 1993, DeLong 1998). Molecular methods have revolutionized our understanding of marine microbial communities in terms of the composition, phylogeny, function and physiology within the environment, and they provide further insight into the overall biogeochemical capabilities of microbial communities. These studies contribute greatly to the ever-expanding database of known microbial DNA sequences (Benson et al. 2004), thus aiding further studies of microbial diversity. While commonly used SSU rRNA (small subunit ribosomal RNA) gene 'markers' have limitations, they are an effective means for providing an initial phylogenetic framework from which physiological and ecological impacts can be determined and for garnering an overall view of microbial diversity from single or multiple habitats.

Diversity studies of marine bacterioplankton have revealed that ~80% of marine bacteria fall within only 9 phylogenetic groups which form clusters of related genes (not single lineages) and appear to be globally distributed (Giovanonni & Rappé 2000). Of these 9 groups, only 2 have had representatives that have been successfully cultured, indicating the substantial need for molecular-based approaches, especially when examining large-scale distribution patterns of marine bacterioplankton.

In contrast to bacterial phylogenetics using SSU rRNA genes, molecular-based protistan diversity studies have lagged behind due to well-established morphology-based taxonomy. Many protistan groups are of sufficient size to allow for identification based on morphological characteristics alone, although there are concerns about the reliability of morphology for classification purposes (Darling et al. 2000, Norris & de Vargas 2000). Molecular-based methods enable a more thorough characterization of protistan diversity over a wider range of sampling locations in comparison to culture-dependent techniques and microscopy. These morphology and culture-independent approaches are revealing higher levels of protistan diversity in a variety of ecosystems (Diez et al. 2001, Moon-van der Staay et al. 2001, Gast et al. 2004, Countway et al. 2005).

Most recent microbial diversity studies have been PCR based. Methods such as denaturing gradient gel electrophoresis (DGGE), cloning, sequencing and terminal restriction fragment length polymorphism (T-RFLP) allow for the identification of individual species (operational taxonomic units [OTU]) in a heterogenous community based on phylogenetic markers such as

16S rDNA and 18S rDNA (Ward et al. 1990, Diez et al. 2001). T-RFLP is a technique wherein a gene of interest (e.g. 16S or 18S rRNA) is amplified from a heterogenous community using a fluorescently labeled primer. Following digestion with restriction endonucleases, variably sized terminal fragments can be differentiated with high resolution (±1 base) through use of an automated DNA sequencer allowing researchers to generate 'fingerprints' of complex microbial communities (Liu et al. 1997). This readily applied technique has been shown to be effective in the comparison of microbial assemblages in various marine and terrestrial environments (Urakawa et al. 2001, Mumey & Stahl 2003, Gomez et al. 2004). Both prokaryotic and eukaryotic diversity have been documented in specific oceanic regions, but not on global scales. Herein we present a T-RFLP-based broad characterization of both the prokaryotic and eukaryotic components of the microbial communities in surface waters collected from the central Pacific Ocean from the Arctic to Antarctic circles during a ship of opportunity cruise: Production Observations Through Another Trans-latitudinal Oceanic Expedition (POTATOE).

## MATERIALS AND METHODS

**Sample collection.** Samples were collected at 40 stations along a ~15 400 km transect of the Pacific Ocean on the RVIB 'Nathaniel B. Palmer' from 70°N to 68°S latitude between late August and early November 2003 (Fig. 1). Daily surface water samples were collected before sunrise at ~370 km intervals via bucket casting or Go-Flo bottles (General Oceanics) attached to a CTD rosette. Continuous measurements of surface salinity and temperature along the entire north-south transect were collected by a SeaBird SBE21 underway CTD. Samples for phylogenetic analysis (1 l) were filtered onto 0.2 µm 47 mm Supor 200 filters (Pall), folded loosely, placed into 2 ml microcentrifuge tubes and stored at -80°C until further processing off ship.

**Bacterial production.** Bacterial production was measured via the incorporation of ³H-leucine (Smith & Azam 1992). Sample water (20 ml) was amended with ³H-leucine (final concentration 10 nM). Aliquots of 5 ml were placed into 4 WhirlPak bags, one of which contained 0.5 ml of 100% TCA as a killed control, and incubated for 4 h in the dark in flowing seawater tables. Incubations were terminated by the addition of TCA to a final concentration of 5%. Samples were processed using the microfuge method of Smith & Azam (1992).

**Chlorophyll a concentration and bacterial cell abundance.** Chlorophyll samples were collected in duplicate 500 ml volumes on GF/F filters (Millipore),
<figure><img src="image_2.png" /><figcaption>Fig. 1. Transect of sampling sites during the POTATOE cruise, August to November 2003</figcaption></figure>

extracted in 90% acetone and quantified fluorometrically (Welschmeyer 1994). Bacterial cell concentrations were measured by epifluorescence microscopy of filtered-formalin preserved (2% final concentration, stored at 4°C) DAPI-stained samples (Porter & Feig 1980).

**Nutrient analysis.** Samples for inorganic nutrients were collected in 10% HCl and sample rinsed polyethylene bottles and frozen (–80°C) immediately. Nutrient concentrations were measured post-cruise by automated methods (US EEPA 1983).

**Flow cytometry analysis of phytoplankton.** Samples (3 ml) for flow cytometric analysis of phytoplankton were fixed with 2% formaldehyde (final concentration) in cryotubes held at room temperature for 10 to 15 min and then stored at –80°C until analysis. The samples were thawed at room temperature and analyzed on a flow cytometer (FACSCalibur, Becton Dickinson) equipped with a 488 nm, 15 mW Argon laser. Samples were run at ~60 µl min⁻¹ for 5 min. The data were collected, stored and displayed on 4 decade scales using CELLQuest software (Becton Dickinson). Orange fluorescence (from phycoerythrin) was collected through a 585/42 nm filter and red fluorescence (from chlorophyll) through a 650 nm LP filter. According to their flow cytometric signatures, 4 phytoplanktonic groups were distinguished. *Synechococcus* cells were easily recognized by their orange fluorescence. *Prochlorococcus* had lower red fluorescence and right angle-light side scatter (SSC) signals than *Synechococcus*. Eukaryotic phytoplankton had higher fluorescence and SSC signals than *Synechococcus* and were divided into 2 size-classes (small and large cells) tentatively identified as pico- and nanophytoplankton cells. The red fluorescence was used as a threshold parameter.

The settings (voltage of SSC and fluorescence photomultiplier tubes) were fixed and cell characteristics were normalized to fluorescent beads signals (1.0 µm; Polysciences).

**Nucleic acid extraction.** Samples were extracted for community analysis using the autoclave DNA extraction method of Simon et al. (2004). This consisted of crushing a frozen filter inside the microfuge tube, adding 0.5 ml 1× TE (10 mM Tris-HCl, pH 7.5; 1 mM EDTA), vortexing to loosen cells bound to filter pieces, and autoclaving in a pre-heated chamber (AMSCO 3024-S isothermal sterilizer) under the following conditions: 10 s purge, sterilization at 121°C for 1 min and fast exhaust. The lysate was removed and placed onto a Centricon YM-100 spin column (Millipore) to concentrate and purify

the DNA. The column was spun in a refrigerated centrifuge at 2630 rpm (1500 × g) for 20 min. The column was subsequently washed twice with 0.75 ml 1× TE and spun for 30 min. Columns were inverted and purified DNA was collected.

**PCR amplification of 16S rDNA.** Purified DNA samples were quantified using a GeneQuant pro (Amersham Biosciences) spectrophotometer. Ten to 100 ng of DNA were used per 50 µl PCR reaction. Genes encoding for 16S rRNA were amplified for T-RFLP analysis with the universal eubacterial fluorescently labeled (6-carboxy-fluorescein [6-FAM]) forward primer 27F-(5'-6-FAM-AGAGTTTGATC(A/C)TGGCTCAG-3') and the reverse primer 1492R (5'-GG(C/T)TACCTTGTTACGACTT-3'), producing an amplicon of ~1500 bp (Lane 1991).

PCR was performed in triplicate for each sample with the following final reagent concentrations in 50 µl: 0.2 µM each primer, 0.2 mM each TaKaRa Ex Taq dNTPs (Takara Bio), 1× Ex Taq buffer (2 mM Mg²⁺), 0.2 mg ml⁻¹ BSA (10 mg ml⁻¹; Promega), 1.25 U µl⁻¹ Ex Taq polymerase, 10 to 100 ng DNA template and brought to a final volume of 50 µl with sterile diethylpyrocarbonate (DEPC)-treated water (Fisher Scientific). PCR amplification was performed under the following thermal conditions: initial denaturation at 95°C for 1.5 min, 30 cycles at 95°C for 30 s, 50°C for 1 min, 72°C for 1.5 min, and a final extension at 72°C for 7 min with a hold at 4°C.

PCR products used for T-RFLP analysis were purified from a 0.8% agarose gel run in 1× TAE buffer with 0.05 µg ml⁻¹ ethidium bromide. PCR products were isolated from the gel and purified with the QIAprep spin kit (QIAGEN) according to the manufacturer's instructions. Of each purified sample, 10 µl were then digested with the restriction endonucleases CfoI and
*MspI* (New England Biolabs) in separate 40 µl reactions at 37°C for 4 h. Digested DNA was precipitated for 1 h at –20°C with 0.1 volumes 3 M NaOAc (pH 5.2) and 2.5× volume of cold 95% ethanol (molecular biology grade). DNA was pelleted at 13 000 rpm (9500 × g) at 4°C for 1 h. The DNA pellet was desalted with 70% ethanol, air-dried and re-suspended in 20 µl sterile DEPC-treated water.

**PCR amplification of 18S rDNA.** Purified and quantified DNA samples were amplified for analysis by T-RFLP of the 18S rRNA gene using the fluorescently labeled forward primer (6-FAM)-EUK-A (5'-6-FAM-AACCTGGTTGATCCTGCCAGT-3') and the reverse primer EUK-570R (5'-GCTATTGGAGCTGGAATTAC-3'), which yielded an amplicon of ~600 bp (Countway et al. 2005). PCR was performed in triplicate for each sample, with the following final reagent concentrations in 50 µl: 0.5 µM each primer, 0.2 mM each TaKaRa *Ex Taq* dNTPs, 1× *Ex Taq* buffer (2 mM Mg²⁺), 0.2 mg ml⁻¹ BSA (10 mg ml⁻¹, Promega), 1.25 U µl⁻¹ *Ex Taq* polymerase, 10 to 100 ng template DNA and brought to a final volume of 50 µl with sterile DEPC-treated water.

PCR amplification was performed under the following thermal conditions: initial denaturation at 95°C for 2 min, 35 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 5 min with a hold at 4°C. Triplicate PCR products were pooled then cleaned and concentrated using the UltraClean PCR Clean-up Kit (Mo Bio Labs) according to the manufacturer's protocol. Concentrated PCR products (~1000 ng) were digested with 5 U Mung bean nuclease (New England Biolabs) to eliminate the effect of pseudo-terminal restriction fragments (T-RFs) on the T-RFLP analysis (Egert & Frederich 2003). Mung bean digested PCR products were again purified and concentrated using the UltraClean PCR Clean-up Kit. Concentrated PCR products (50 µl) were gel purified from a 0.8% agarose gel run in 1× TAE buffer with 0.05 µg ml⁻¹ ethidium bromide using the UltraClean 15 DNA Purification Kit (Mo Bio Labs; Countway et al. 2005). Of each sample, 5 µl were digested in a total volume of 20 µl with the restriction endonucleases *CfoI* and *MspI* in separate reactions at 37°C for 4 h. Digested DNA was precipitated as described above for 16S rDNA samples and re-suspended in 10 µl sterile DEPC-treated water.

**T-RFLP analysis.** Digested DNA (0.25 to 3 µl) was combined with 0.25 to 0.75 µl of DNA standard (MapMarker 1000, Bioventures) and denatured after the addition of 2 volumes of deionized formamide at 95°C for 5 min. Fragments were separated on an automated DNA sequencer (3100 Avant genetic analyzer, Applied Biosystems) in GeneScan mode (15 kV, 60°C for 40 min) using a 36 cm capillary and performance optimized polymer 4 (POP 4, Applied Biosystems). Fluorescence

intensities of major peaks from community profiles were adjusted by increasing or decreasing template volumes and/or injection times to fall within 6500 to 8000 fluorescence units to ensure the comparability of profiles. The sizes of the 5'-T-RFs and corresponding fluorescence intensities (peak areas) were calculated using DAX® software (Data Acquisition and Analysis v7.0). A 150 fluorescence unit threshold value was used to separate true signals from background noise.

The richness of each community was estimated from the number of unique T-RFs in each restriction. In order to compare T-RFLP profiles, T-RFs from each community were binned according to fragment length (±1 bp). Relative abundance was estimated for each T-RF by dividing the peak area by the total peak area and multiplying by 100.

Resultant richness and abundance data were analyzed for comparison of communities. Similarity dendrograms were constructed using Primer5® software (Primer-E) applying the Bray-Curtis coefficient onT-RF relative peak abundances (log transformed) with the complete linkage similarity coefficient. SIMPER analysis (Primer5® software) of microbial community T-RFLP profiles was used to compare similarities within assemblages, dissimilarities between assemblages and to identify individual T-RFs responsible for clustered assemblages.

## RESULTS

### Surface water temperature, chemistry, chlorophyll concentrations and bacterial abundance

Surface water temperatures were ~10°C in the northern Pacific, peaking between 35°N and 22°S to 27°C and decreasing to ~0.3°C in the southern part of the ocean (Fig. 2A). A peak in nitrate concentrations was observed in the sub-Arctic/Arctic Pacific HNLC (High Nutrient Low Chlorophyll) Zone, where iron limitation results in the accumulation of unutilized nitrate (Chisholm & Morel 1991). A peak was also seen at the Equatorial Upwelling and the New Zealand Continental Shelf, where the spring bloom was underway (Fig. 2A). Chlorophyll concentration was greatest at 54°N (5.09 µg l⁻¹), with average values in the northern Pacific of ~2.42 µg l⁻¹; a substantial decrease in concentration was observed in the mid- to lower latitudes (~0.51 µg l⁻¹), and maximum concentration of 4.29 µg l⁻¹ was found in the southern waters at 40°S (Fig. 2B). Bacterial abundance and production rates also showed latitudinal patterns, exhibiting bacterial cell concentrations of ~2.70 × 10⁸ cells l⁻¹ in the northern Pacific through 30°N, a decline from 30°N through 30°S to ~1.50 × 10⁸ cells l⁻¹, and average cell numbers
<figure><img src="image_3.png" /><figcaption>Fig. 2. Latitudinal trends in (A) surface orthophosphorus (P), nitrate (NO₃), silica (Si) and temperature; and (B) bacterial abundance (Bact), surface chlorophyll concentration (Chl a), and bacterial ³H-leucine incorporation (Leu). Positive latitude values represent Northern Hemisphere locations and negative latitudes represent the Southern Hemisphere</figcaption></figure>

of ~1.10 × 10⁸ cells l⁻¹ throughout southern Pacific waters (Fig. 2B). Bacterial production in the northern Pacific was ~39.40 pmol leucine (Leu) l⁻¹ h⁻¹, decreased to ~14.50 pmol Leu l⁻¹ h⁻¹ in the mid- to lower latitudes and peaked at 36°S with 87.50 pmol Leu l⁻¹ h⁻¹ in the southern waters (Fig. 2B).

Flow cytometry

Flow cytometry analysis revealed the presence of *Prochlorococcus* between 35°N and 35°S (average abundance of ~4.38 × 10⁷ cells l⁻¹) with a maximum value at the equator (1.10 × 10⁸ cells l⁻¹; Fig. 3A). *Synechococcus* were present throughout the transect, with ~5.75 × 10⁶ cells l⁻¹ in northern Pacific waters, a slight increase in average abundance throughout the mid- to

<figure><img src="image_4.png" /><figcaption>Fig. 3. Flow cytometric abundances of (A) *Prochlorococcus*, (B) *Synechococcus*, (C) picoeukaryotes and (D) nanoeukaryotes. Positive latitude values represent Northern Hemisphere locations and negative latitudes represent the Southern Hemisphere</figcaption></figure>
lower latitudes (~7.60 × 10⁶ cells l⁻¹), and a peak in
abundance at 33° S (~6.50 × 10⁷ cells l⁻¹; Fig. 3B). Pho-
tosynthetic picoeukaryote abundance followed latitu-
dinal patterns as would be expected, with average
abundance in the northern Pacific waters of ~7.18 × 10⁶
cells l⁻¹, decreased abundance in the mid- to lower lat-
itudes (~1.47 × 10⁶ cells l⁻¹) and a slight elevation at
30° S of ~2.42 × 10⁶ cells l⁻¹ throughout the southern
polar waters (Fig. 3C). Photosynthetic nanoeukaryote
abundance also followed latitudinal trends with abund-
dances in the northern Pacific and temperate/tropical
regions of the Pacific of ~2.11 × 10⁶ and ~3.38 × 10⁵
cells l⁻¹, respectively and ~7.44 × 10⁶ cells l⁻¹ south
from 30° (Fig. 3D).

Microbial community structure analysis: restriction
endonuclease production of 5'-terminal restriction
fragments

Prokaryotic and eukaryotic 5' T-RFLP profiles were
generated following separate digestions of PCR pro-
ducts from each location with the endonucleases CfoI
and MspI. The number of T-RFs from each sample
obtained through MspI restriction of amplified 16S
rDNA was greater than that determined with CfoI gen-
erated fragments, with 8 to 38 and 4 to 20 T-RFs,
respectively. PCR amplified 18S samples restricted
with CfoI and MspI produced similar T-RF quantities of
5 to 23 and 6 to 25 T-RFs, respectively.

Ribotype richness

No latitudinal pattern of ribotype richness or com-
munity diversity between or amongst prokaryotic and
eukaryotic profiles was found; however, the prokary-
otic population appeared to have a higher ribotype
richness with 181 total T-RFs produced, while eukary-
otic populations produced 135 total T-RFs along the
transect (data not shown). It is possible that this greater
ribotype richness of the prokaryotic population is due
to the lack of nuclease digestion prior to the T-RFLP
analysis.

Cluster analysis of T-RFLP profiles

To assess similarity among prokaryotic and eukary-
otic communities, T-RFLP patterns from each sampling
location were implemented as a 'community finger-
print' based on the presence or absence of T-RFs and
the relative amount of DNA for each T-RF. Communi-
ties analyzed through the use of hierarchical clustering
of Bray-Curtis similarities indicated strong geographic

integrity for 16S T-RFLP profiles but slightly less for
the 18S profiles. Hierarchical analysis of 16S T-RFLP
profiles indicated the presence of 4 distinct clusters —
sub-Arctic/Arctic (Group I), tropical (Group II), tem-
perate (Group III) and sub-Antarctic/Antarctic (Group
IV)—apportioned according to oceanic regions, sug-
gesting a relationship with temperature and its envi-
ronmental correlates (Fig. 4). According to SIMPER
analysis, prokaryotic cluster groups contained high
within-group average similarities (~55 to 67%) and
comparable inter-group similarity (average similarity
42 to 57%). Groups I and II exhibited 44% similarity,
Groups I and III 51% similarity, Groups I and IV, II and
IV, and III and IV exhibited 47, 42 and 44% similarity,
respectively, while Groups II and III had the highest
degree of similarity at 57%.

<figure><img src="image_5.png" /><figcaption>Fig. 4. 16S rDNA similarity dendrogram based on the presence/absence and relative abundances of *CfoI* and *MspI* restricted terminal restriction fragments (T-RFs)</figcaption></figure>
Hierarchical analysis of 18S T-RFLP profiles produced 4 major clusters following the same oceanic groupings as 16S profiles—sub-Arctic/Arctic (Group IV), tropical (Group II), temperate (Group I) and sub-Antarctic/ Antarctic (Group III) (Fig. 5), although the clusters were less congruent in their geographic constitution when compared to prokaryotic cluster assemblages. Eukary- otic assemblages had markedly high within-group average similarities (~50 to 62%), with Groups III and IV showing the highest inter-group similarity (average similarity 53%) compared to all other inter-group assessments. Groups I and II, Groups I and III and Groups I and IV exhibited 48, 47 and 49% similarity, re- spectively, while Groups II and IV and Groups II and III exhibited 49 and 48% similarity, respectively. Groups II and III had the highest degree of similarity at 53%. Eukaryotic communities appeared to exhibit the same

<figure><img src="image_6.png" /><figcaption>Fig. 5. 18S rDNA similarity dendrogram based on the presence/absence and relative abundances of *CfoI* and *MspI* restricted terminal restriction fragments (T-RFs)</figcaption></figure>

degree of within cluster similarity (51%) over latitudinal gradients as the prokaryotic communities (52%).

SIMPER analysis of profiles was used to determine the magnitude and identity of the T-RFs responsible for similarities within and dissimilarities between each group. Bacterioplankton community analysis revealed that 12 to 14 T-RFs were responsible for >70% of each of the 4 cluster's within-group similarities, while eukaryotic community analysis revealed 8 to 11 T-RFs as the contributors to >70% of the 4 cluster's within-group similarities (Tables 1 & 2). These results, coupled with the order-level resolution of the T-RFLP analysis, indicate that a small number of phylogenetic groups contributed to each distinct assemblage. In addition to being major contributors to within-group similarities, several of these T-RFs were present in most or all of the 16S T-RFLP profiles, suggesting a cosmopolitan distribution. The *CfoI*-derived 910 bp peak was consistently and substantially present in all profiles, contributing 26 to 92% of the total community 16S rDNA and showing only lower relative abundance (10 to 35% of the total) in northern Pacific waters. *CfoI*-derived T-RFs 367, 564 and *MspI*-derived T-RFs 145 and 489 were also present at all latitudes, but contributed only 3 and 6% (367 and 564 bp) and 6 and 9% (145 and 489 bp) to the overall community structure (Table 1). Phylogenetic group(s) comprising the 489 bp peak showed high abundance (32%) in Group II, while the phylogenetic group(s) representing the 145 bp peak had an abundance of 25% in Group IV (Table 1).

Complementary patterns were observed in the eukaryotic samples along the transect. Eukaryotic phylogenetic groups representing fragments at 172 and 433 bp (*CfoI*-restricted) were present in all samples and contributed greatest to Group II (36 and 25%) and least to Groups I and III (8 and 9% for 172 bp and 8% for 433 bp; Table 2). *MspI*-derived T-RFs 280 and 382 were also present in all samples, with T-RF 280 showing the greatest contribution to Group II (47%) and the least to Groups I and III (8 and 9%), and T-RF 382 showing an average contribution of 13% to all groups (Table 2). *CfoI*-restricted 419 and 426 bp T-RFs were present throughout the transect profiles, although their respective abundances were typically low (<3%). Of the T-RFs commonly present within eukaryotic community profiles, the *MspI*-derived 232, 240 bp T-RFs showed a considerable increase in abundance in sub-Antarctic/Antarctic T-RFLP profiles from sub-Arctic/Arctic and temperate/tropical profiles (1 to 4%). As with prokaryotic profiles, several T-RFs varied in their presence and/or absence, with no apparent trends observed with respect to other T-RFs and/or latitude (data not shown).

Differences within prokaryotic clustering were primarily accounted for by T-RFs that were more
<table><caption>Table 1. Mean relative abundance (Abund., average values as percentage) and percent contribution (Contrib., to the within-group similarity) of 16S rDNA 5'-terminal restriction fragments (T-RFs) contributing to >70% of within-group similarity. Average similarities: Group I = 58%; Group II = 67%; Group III = 60%; Group IV = 55%</caption><thead><tr><th colspan="3">Group I</th><th colspan="3">Group II</th><th colspan="3">Group III</th><th colspan="3">Group IV</th></tr><tr><th>T-RF (bp)</th><th>Abund.</th><th>Contrib.</th><th>T-RF (bp)</th><th>Abund.</th><th>Contrib.</th><th>T-RF (bp)</th><th>Abund.</th><th>Contrib.</th><th>T-RF (bp)</th><th>Abund.</th><th>Contrib.</th></tr></thead><tbody><tr><td>910 Cfol</td><td>31</td><td>8</td><td>910 Cfol</td><td>64</td><td>11</td><td>910 Cfol</td><td>74</td><td>12</td><td>910 Cfol</td><td>67</td><td>12</td></tr><tr><td>90 Cfol</td><td>24</td><td>8</td><td>439 MspI</td><td>35</td><td>9</td><td>439 MspI</td><td>25</td><td>9</td><td>145 MspI</td><td>25</td><td>9</td></tr><tr><td>93 Cfol</td><td>14</td><td>7</td><td>489 MspI</td><td>32</td><td>9</td><td>145 MspI</td><td>24</td><td>9</td><td>489 MspI</td><td>10</td><td>7</td></tr><tr><td>145 MspI</td><td>12</td><td>6</td><td>178 Cfol</td><td>11</td><td>7</td><td>489 MspI</td><td>17</td><td>8</td><td>367 Cfol</td><td>5</td><td>6</td></tr><tr><td>55 Cfol</td><td>12</td><td>6</td><td>668 Cfol</td><td>16</td><td>6</td><td>367 Cfol</td><td>6</td><td>6</td><td>439 MspI</td><td>6</td><td>5</td></tr><tr><td>367 Cfol</td><td>7</td><td>6</td><td>145 MspI</td><td>8</td><td>6</td><td>137 MspI</td><td>3</td><td>5</td><td>518 Cfol</td><td>10</td><td>5</td></tr><tr><td>489 MspI</td><td>11</td><td>5</td><td>367 Cfol</td><td>4</td><td>5</td><td>542 MspI</td><td>5</td><td>4</td><td>495 MspI</td><td>11</td><td>4</td></tr><tr><td>96 Cfol</td><td>4</td><td>5</td><td>450 MspI</td><td>3</td><td>4</td><td>178 Cfol</td><td>4</td><td>4</td><td>306 MspI</td><td>6</td><td>4</td></tr><tr><td>542 MspI</td><td>3</td><td>4</td><td>435 MspI</td><td>2</td><td>4</td><td>90 Cfol</td><td>2</td><td>4</td><td>93 Cfol</td><td>4</td><td>4</td></tr><tr><td>137 MspI</td><td>2</td><td>4</td><td>137 MspI</td><td>2</td><td>4</td><td>88 MspI</td><td>3</td><td>4</td><td>55 Cfol</td><td>1</td><td>4</td></tr><tr><td>88 MspI</td><td>6</td><td>4</td><td>167 MspI</td><td>1</td><td>4</td><td>450 MspI</td><td>2</td><td>4</td><td>497 MspI</td><td>10</td><td>4</td></tr><tr><td>487 MspI</td><td>3</td><td>3</td><td>474 MspI</td><td>2</td><td>3</td><td>474 MspI</td><td>4</td><td>3</td><td>564 Cfol</td><td>1</td><td>3</td></tr><tr><td>85 MspI</td><td>6</td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td>85 MspI</td><td>1</td><td>3</td></tr><tr><td>437 MspI</td><td>18</td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td>542 MspI</td><td>2</td><td>3</td></tr></tbody></table>

<table><caption>Table 2. Mean relative abundance (Abund., average values as percentage) and percent contribution (Contrib., to the within-group similarity) of 18S rDNA 5'-terminal restriction fragments (T-RFs) contributing to >70% of within-group similarity. Average similarities: Group I = 50%; Group II = 54%; Group III = 62%; Group IV = 56%</caption><thead><tr><th colspan="3">Group I</th><th colspan="3">Group II</th><th colspan="3">Group III</th><th colspan="3">Group IV</th></tr><tr><th>T-RF (bp)</th><th>Abund.</th><th>Contrib.</th><th>T-RF (bp)</th><th>Abund.</th><th>Contrib.</th><th>T-RF (bp)</th><th>Abund.</th><th>Contrib.</th><th>T-RF (bp)</th><th>Abund.</th><th>Contrib.</th></tr></thead><tbody><tr><td>382 MspI</td><td>29</td><td>13</td><td>382 MspI</td><td>39</td><td>12</td><td>382 MspI</td><td>74</td><td>12</td><td>172 Cfol</td><td>16</td><td>12</td></tr><tr><td>196 Cfol</td><td>13</td><td>11</td><td>433 Cfol</td><td>41</td><td>25</td><td>172 Cfol</td><td>25</td><td>9</td><td>382 MspI</td><td>18</td><td>11</td></tr><tr><td>172 Cfol</td><td>8</td><td>8</td><td>172 Cfol</td><td>30</td><td>36</td><td>280 MspI</td><td>24</td><td>9</td><td>433 Cfol</td><td>14</td><td>11</td></tr><tr><td>280 MspI</td><td>8</td><td>8</td><td>280 MspI</td><td>13</td><td>47</td><td>433 Cfol</td><td>17</td><td>8</td><td>280 MspI</td><td>12</td><td>11</td></tr><tr><td>433 Cfol</td><td>7</td><td>8</td><td>223 MspI</td><td>5</td><td>54</td><td>368 MspI</td><td>6</td><td>6</td><td>596 Cfol</td><td>8</td><td>7</td></tr><tr><td>598 MspI</td><td>2</td><td>5</td><td>426 Cfol</td><td>2</td><td>61</td><td>419 Cfol</td><td>3</td><td>5</td><td>419 Cfol</td><td>3</td><td>6</td></tr><tr><td>600 Cfol</td><td>2</td><td>5</td><td>378 MspI</td><td>8</td><td>66</td><td>232 MspI</td><td>5</td><td>4</td><td>279 Cfol</td><td>2</td><td>6</td></tr><tr><td>267 Cfol</td><td>1</td><td>4</td><td>199 Cfol</td><td>6</td><td>71</td><td>598 MspI</td><td>4</td><td>4</td><td>196 Cfol</td><td>7</td><td>5</td></tr><tr><td>286 MspI</td><td>3</td><td>4</td><td></td><td></td><td></td><td>373 MspI</td><td>2</td><td>4</td><td>223 MspI</td><td>1</td><td>4</td></tr><tr><td>430 Cfol</td><td>1</td><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>279 Cfol</td><td>1</td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

dominant at the lower latitudes (e.g. MspI-derived 439 bp peak) and those T-RFs that were dominant in sub-Arctic/Arctic regions, such as the Cfol-derived 55, 90, and 93 bp T-RFs and the MspI-derived 85, 88 and 437 bp T-RFs (Table 1), where Cfol peaks 55, 90 and 93 showed an inverse relationship in abundance with the universally distributed 910 bp peak (data not shown). Several T-RFs were found solely in samples from specific biomes of the Pacific. Cfol-derived T-RFs 511 and 668, and MspI-derived 312 and 507 T-RFs, were either present at low frequencies or absent in high latitudinal zones, more abundant in the mid-latitudinal and equatorial waters and absent from sub-Antarctic/Antarctic waters (Fig. 6A). Cfol-derived T-RFs 365, 518, 575 and 585 and MspI-derived T-RFs 92 and 540 were present only in samples from the sub-Arctic/Arctic and/or sub-Antarctic/Antarctic (Fig. 6A).

Distinction between the 'tropical' assemblage and all other latitudinal groupings (I, III and IV) was ascribed mainly to the presence of the Cfol-derived 668 bp peak. Using the Restriction Mapper program (Version 3; www.restrictionmapper.org) representatives of the Prochlorococcus clade produced terminal fragment lengths of 641 to 648 when digested with Cfol. This may offer a putative identification of this T-RF as a representative of the Prochlorophytes, as this peak is seen only in the temperate/tropical regions and coincides with the appearance of Prochlorococcus (~4.38 × 10⁷ cells l⁻¹) as seen in our flow cytometry results (Fig. 3). Several less abundant T-RFs (~57% of all T-RFs) were present sporadically and showed no observable trends with respect to latitude.

Similar patterns in eukaryotic T-RFLP profiles were apparent, although less latitudinally congruent than
<figure><img src="image_7.png" /><figcaption>Fig. 6. Comparison of T-RFs representative of polar vs. tropical/temperate species of (A) prokaryotes and (B) eukaryotes. Other prokaryotic T-RFs Cfol 511, MspI 312 and 507 appeared only in temperate/tropical waters. Prokaryotic T-RFs Cfol 518, 575, 585, and MspI 92 and 540 appeared only in the polar latitudes. Eukaryotic T-RF Cfol 226 appeared only in the polar latitudes. Eukaryotic T-RFs Cfol 212, 285, 605 and MspI 114, 118, and 274 appeared in temperate/tropical waters (data not shown). Positive latitude values represent Northern Hemisphere locations and negative latitudes represent the Southern Hemisphere</figcaption></figure>

prokaryotic profiles. The Cfol-derived 226 peak and the MspI-derived 260 peak appeared only in the polar latitudes (Fig. 6B), while Cfol-derived 212, 217, 285 and 605 T-RFs and MspI-derived 114, 118 and 274 T-RFs appeared only in temperate/tropical regions (Fig. 6B). Several T-RFs (62% of the total T-RFs generated) contributed only a small fraction to the total peak area of a community and appeared infrequently (<3 locations).

Overall, analysis of both the prokaryote and eukaryote profiles revealed several T-RFs that were present in all 4 biogeographic regions, although sometimes represented at only 1 latitude, lending the possibility that the phylogenetic groups representing these T-RFs

are indeed globally distributed. Prokaryotic profiles showed that 25% of the T-RFs generated from both Cfol and MspI restrictions fall within this global distribution. Eukaryotic profiles showed that 16% of Cfol- and MspI-generated T-RFs were globally distributed. In contrast, 8% of the prokaryotic T-RFs and 9% of the eukaryotic T-RFs constituted those groups found in only particular oceanic regions, suggesting a more endemic distribution.

## DISCUSSION

Due to their small size, microorganisms are extremely abundant and disperse easily, leading some to believe that they are cosmopolitan in their distribution (Finlay 2002). Baas-Becking (1934) stated that 'everything is everywhere, and the environment selects', suggesting that local environmental conditions drive community composition. If we assume theories of cosmopolitanism to be true, then how do we explain the absence and/or presence of diverse species in the molecular analysis of microbial communities? Finlay (2002) suggests that some organisms may be extremely rare or present only in some cryptic form (e.g. cysts or spores) and hence not detected by broad detection methods (e.g. T-RFLP). In a comparison of microbial assemblages from the Arctic and Antarctic, Wilkinson (2001) proposed that universal distribution may be cell-size related, with ubiquitously distributed organisms maintaining an average size of <100 µm, suggesting that overall global species richness is moderate.

A few studies have supported the hypothesis of universal distribution of microorganisms (Finlay & Clarke 1999, Zwart et al. 2002), while others support the existence of endemic microorganisms (Whitaker et al. 2003). If microorganisms are truly endemic, our present day view of global microbial diversity and richness may be a gross underestimation. One way to determine the true distribution of microbial phylotypes is to use molecular-based methods to examine communities on large geographical scales. While by no means excluded from inherent biases (Suzuki & Giovannoni 1996), PCR-based molecular techniques offer a fast and reliable approach to assessing diversity over these large spatial scales. Utilizing T-RFLP technology, we were able to assess the diversity of the dominant portions of microbial communities encompassing a range of the Pacific Ocean from the Arctic to the Antarctic circles. Microbial diversity studies have traditionally been limited to smaller spatial scales and isolated environments, making this study unique in its scope.

T-RFLP analysis of Pacific Ocean microbial communities indicated 4 major groupings (clusters) representing
tative of the sub-Arctic/Arctic Pacific, temperate Pacific, tropical Pacific and the sub-Antarctic/Antarctic Pacific Ocean. High within-group similarities for both prokaryotic and eukaryotic communities were driven by a relatively low number of T-RFs (8 to 14). This could be the result of several different phylogenetic clades sharing identical terminal restriction sites, resulting in the same length terminal fragment. Microbial community analysis revealed many phylogenetic groups that were ubiquitously distributed as well as those isolated to specific habitats in both prokaryotic and eukaryotic fractions. For our purposes, a ubiquitous ribotype (T-RF) is one appearing in all 4 regions of the Pacific Ocean, while an endemic ribotype is seen only in specific habitat types (e.g. the polar regions or the temperate/tropical regions). We did not observe prokaryotic populations restricted solely to the sub-Arctic/Arctic or sub-Antarctic/Antarctic, but rather populations existing in both polar regions. It has been suggested by Hollibaugh (1994) that there may be phylogenetic differences between bacterioplankton communities in response to varying types of organic matter present in the local environment due to the requirement for specialized metabolic capabilities in utilizing local substrates. It is possible that organisms may be ubiquitously dispersed throughout the Pacific, but in a non-functioning state awaiting local conditions to become favorable. In comparison to our findings of Pacific Arctic bacterioplankton communities, Ferrari & Hollibaugh (1999) analyzed samples from the Canadian Arctic using DGGE and identified 5 major clusters with similarities greater than 80%. Their findings suggested that bacterioplankton communities in this area of the Arctic are the result of responses to local environmental conditions.

Several populations of microbes were found to be restricted to the temperate and tropical latitudes, possibly in response to higher temperatures and lower nutrient resources, although we did observe a peak in nitrate at the Equatorial Upwelling zone, where we also saw an increase in eukaryotic phytoplankton (pico- and nano-), *Prochlorococcus*, and *Synechococcus* from the immediate northern waters. Distinctions between the temperate and tropical assemblages and the polar assemblages resulted from the presence of a few peaks. Prokaryotic communities were dominated by ~4 T-RFs in these regions, with the most dominant T-RF being the CfoI-derived 668 bp peak. The appearance of this T-RF coincided with flow cytometry determined *Prochlorococcus* abundances observed at the temperate/tropical latitudes. Campbell et al. (1994) observed the ubiquity of *Prochlorococcus* in the latitudinal band from 40°N to 40°S in the central Pacific Ocean, where they showed that it comprises a significant portion of the photosynthetic biomass. *Synecho-

*coccus* was represented in all regions of the Pacific Ocean as shown by flow cytometry analysis. These cyanobacterial groups are an important food source for planktonic heterotrophic flagellates and ciliates (Yahel et al. 1998) and greatly contribute to the total primary production in tropical regions (Li et al. 1983). We are unable to say absolutely that the CfoI-derived 668 bp T-RF is representative of *Prochlorococcus* given that T-RFLP does not allow complete identification of T-RFs, but based on preliminary computational restriction digestion with the Restriction Mapper program, we are able to relate this T-RF with *Prochlorococcus*. We were, however, unable to determine the T-RF that may be representative of *Synechococcus*. Eukaryotic mid- and lower latitude communities were dominated by ~7 T-RFs endemic to this region, of which the CfoI-derived 285 bp T-RF was the most dominant.

Prokaryotic communities showed high similarity across the transect, although not all communities shared the same members throughout. This similarity may be explained by the findings of earlier investigators in that most marine bacteria belong to only 9 phylogenetic groups (Giovannoni & Rappé 2000). Eukaryotic communities also showed high similarity across the transect, but a decreased number of T-RFs were generated in comparison to prokaryotic populations. This may be due to the selection of primers used to amplify eukaryotic DNA from the mixed assemblage, under-sampling, the addition of a nuclease digestion step, or the fact that prokaryotic organisms are smaller and more abundant than eukaryotic organisms. We could hypothesize that the eukaryotic fraction contains fewer members than the prokaryotic fraction based on the ecological principle that the number of individuals in a community is inversely proportional to their size (Fenchel 1993). It is also possible that some of the prokaryotic T-RFs actually represent eukaryotic plastids. Rappé et al. (1997, 1998) reported significant numbers of eukaryotic plastids from 16S rRNA gene clone libraries off the Atlantic and Pacific coasts of the United States. It is therefore possible that the absence of pre-filtration in our samples resulted in some 16S T-RFs representing eukaryotic plastids.

The selection of restriction enzyme(s) used for T-RFLP also has a substantial effect on the resolution of the microbial community and the phylogenetic groups detected. This is due to the possibility that different phylogenetic groups may have enzyme-specific terminal-end cut sites at the same location on the PCR amplicon, resulting in the same size terminal fragment. Our selection and analysis using 2 restriction enzymes offer higher resolution than a single enzyme; however, we are aware that some T-RFs may represent more than one phylogenetic group, resulting in an underestimation of the richness of Pacific Ocean
microbial communities. Since the major goal of this
study was to provide a broad spatial overview of
microbial diversity in the Pacific Ocean, the use of
different and/or more restriction enzymes would not
necessarily increase the power of the analysis in
contrast to the additional time and monetary expen-
ditures required.

T-RFLP only recognizes the dominant members of a
community, resulting in a somewhat skewed version of
'true' diversity. Low abundance members in a commu-
nity may be overlooked by molecular-based methods
due to sampling biases (Finlay 1998). Sampling size
may influence the true diversity of a community by
under-representing or excluding particular community
members from small sampling sizes. Prokaryotic com-
munities are probably less affected by our sample size
(1 l) but eukaryotic organisms may require a larger
sampling volume to include all representatives of the
local community. Although another potential bias is
the method used for DNA extraction, the protocol used
was developed, in part, to improve DNA extraction
from organisms which are difficult to lyse. It is
assumed that our method is sufficiently robust to
include all members of the microbial community (see
Simmon et al. 2004), although we cannot eliminate the
possibility that this method excludes the cryptic forms
(e.g. spores) present as a 'seed bank', thus overlooking
these phylogenetic groups which may be ubiquitously
present but dormant (Finlay 2002). Although we are
aware of the potential biases in our analysis, their
elimination is not yet possible and will continue to be a
drawback to these types of molecular approaches and
large geographical surveys.

The seasonal variability along this transect may also
contribute to the observed segregation of both the
prokaryotes and eukaryotes into the 4 distinct biogeо-
graphical regions, as sampling in the Northern Hemi-
sphere was completed during the late summer and
Southern Hemisphere sampling occurred during the
Austral spring. The results represent a 'snap shot' of
the microbial diversity along a 15 400 km geographical
region during a sampling time window that was as
short as logistically possible.

The results herein represent a broad overview of
microbial diversity on a geographic scale never before
available. Discernable patterns are apparent and there
is evidence that some phylotypes co-exist, while some
increase in abundance only in the absence of other
species. In order to obtain a more detailed view of the
phylogenetic composition of these Pacific Ocean
microbial communities, possibly link community struc-
ture to function, and present a more complete picture
of ubiquity versus endemicism in the Pacific Ocean,
cloning and sequencing must be employed, which was
beyond the scope of this study.

*Acknowledgements.* We thank the captain, crew and Raytheon Polar Services personnel of the RVIB 'Nathaniel B. Palmer' for their service and assistance at sea, and R. Stevens for his assistance with sample collection. Nutrient analyses were provided by J. Macauley at the University of West Florida Wetlands Research Laboratory. We are grateful to the National Science Foundation Office of Polar Programs for their efforts to facilitate our participation in this ship of opportunity cruise. This research was supported by National Science Foundation award OPP 0127022 to W.H.J.

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