A comparison of HPLC pigment signatures and electron microscopic observations for oligotrophic waters of the North Atlantic and Pacific Oceans

Pergamon

Deep-Sea

0967-%45(95)ooo!&x

Revearch

II, Vol. 43. No. 2-3. pp. 517-537. 1996 Copyright 0 1996 Elwicr science Ltd Printed in Great Britain. All rights merwd 0967+X5/96 S15.OO+O.M)

A comparison of HPLC pigment signatures and electron microscopic observations for oligotrophic waters of the North Atlantic and Pacific Oceans ROBERT A. ANDERSEN,* ROBERT R. BIDIGARE,t MAUREEN D. KELLER* and MIKEL LATASAt (Received26

October 1994; in revisedform 11 May 1995; accepted 10 August 1995)

Abstract-The use of HPLC pigment analysis has become a primary tool for investigating the taxonomic composition of natural phytoplankton populations. In this study, we compare, for the first time, the taxonomic composition based upon HPLC pigment signatures with direct electron microscopic taxonomic identifications from two sets of open ocean oligotrophic field samples. Electron microscopic observations at sites in the Atlantic and Pacific Oceans (Hydrostation S and Station ALOHA, respectively) agree with taxonomic partitioning based upon HPLC algorithms in the upper water-column samples, but there is increasing disagreement between the two methods in deeper water samples. This disparity probably results from depth-dependent changes in cellular pigment content and accessory pigment-to-chlorophyll ratios. At both locations, the eukaryotic ultraplankton was similar in taxonomic composition, at least at the class level, and the Prymnesiophyceae and the newly described Pelagophyceae were the two most abundant groups of eukaryotes. Copyright 0 1996 Elsevier Science Ltd

INTRODUCTION Photosynthetic pigment analysis is commonly used to identify the presence of different algal groups .(see review by Millie et al., 1993). More recently, photosynthetic pigment distributions have been employed to estimate primary production rates bio-optically (Bidigare et al., 1992). High-performance liquid chromatographic (HPLC) methods can be employed to separate known pigments quantitatively, thereby providing a pigment signature for each sample (Wright et al., 1991). Specific algal groups, most commonly taxonomic classes, are characterized by specific photosynthetic pigments (Table 1). For example, members of the Class Prymnesiophyceae produce chlorophyll a, chlorophylls cl + c2 or c2 + ~3, and numerous carotenoids including b-carotene, diatoxanthin, diadinoxanthin, fucoxanthin, 19’-hexanoyloxyfucoxanthin (Jeffrey, 1989; Bjerrnland and Liaaen-Jensen, 1989). With algorithms, one can fractionate the HPLC pigment signature and assign the relative proportions to specific taxa (Gieskes et al., 1988; Everitt et al., 1990; Letelier et al., 1993; Bidigare and Ondrusek, in press). Radiolabeling (14C) of taxon-specific pigments can be used to determine growth rates for various algal classes (Gieskes and Kraay, 1989). The combined use of these techniques enables one to estimate both chlorophyll a biomass and growth of individual taxonomic groups. For example, the * Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, ME 04575, U.S.A. t Department of Oceanography, University of Hawaii, Honolulu, HI 96822, U.S.A. 517

518 Table

R. A. Andersen et al. 1.

Summary

Algal Group Prochlorophytes

of photosynthetic pigment distributions among marine phytoplankton. carotenoidpigments in bold are diagnostic markers

Chlorophyll and

Major Pigments Present Divinyl chlorophylls u and b, monovinyl chlorophyll b, zeaxanthin, a-carotene, chlorophyll

Pelagophytes

c-like pigment Monovinyl chlorophyll a, zeaxanthin, /?-carotene, phycoerythrin, phycocyanin, allophycocyanin, Monovinyl chlorophyll a, chlorophylls cr and ~2,fucoxantbin + diadinoxanthin, diatoxanthin, b-carotene Monovinyl chlorophyll a, chlorophylls c, + c2 or c2+ cj, 19’~hexanoytoxyfkoxantbin, fucoxanthin, diadinoxanthin, diatoxanthin, B-carotene Monovinyl chlorophyll a, chlorophylls c2 and +,l!Y-butanoyloxyfucoxantbin,

Chrysophytes Cryptophytes

diatoxanthin fucoxanthin, diadinoxanthin, B-carotene Monovinyl chlorophyll a, chlorophylls cl and cl, fucoxanthin + violaxantbin, B-carotene Monovinyl chlorophyll a, chlorophyll c2, alloxanthin, phycoerythrin or

Cyanobacteria Diatoms Prymnesiophytes

Dinoflagellates Prasinophytes Chlorophytes

phycocyanin, crocoxanthin, monadoxanthin, a-carotene Monovinyl chlorophyll a, chlorophyll ~2,peridinin’, dinoxanthin, diadinoxanthin, diatoxanthin, b-carotene Monovinyl chlorophylls a and b, prasinoxantbiut, chlorophyll c-like pigment (Mg 3,8 DVPa& zeaxanthin, neoxanthin, violaxanthin, a- and p-carotene Monovinyl chlorophylls a and b, lutein, neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, E- and p-carotene

*Some species possess fucoxanthin-related pigments instead of peridinin. %ome species possess lutein (e.g. Pyramimonas), siphonein or siphonaxanthin instead of prasinoxanthin.

recent discovery of Prochlorococcus, with its unique divinyl chlorophyll a pigment (Chisholm et al., 1988, 1992), has allowed investigators to assess its contribution to phytoplankton biomass and primary productivity in oceanic waters (Goericke and Welschmeyer, 1993). When applied to dilution experiments (cf. Landry and Hassett, 1982), HPLC pigment analysis can provide taxon-specific growth and grazing rates of phytoplankton (Burkill et al., 1987; Strom and Welschmeyer, 1991). Interpretation of data resulting from these techniques requires one to assume that the pigment markers found in mono-specific cultures representing major algal classes (e.g. Table 1) are the same as those found in the various algal classes from natural phytoplankton populations. The pigment marker approach for estimating the biomass of specific phytoplanktonic groups is based upon detailed pigment analysis of select species grown in culture (e.g. Foss et al., 1984; Liaaen-Jensen, 1985; Hooks et al., 1988; Bidigare et al., 1990; Goericke and Repeta, 1992). This approach assumes that: (i) the species of the taxonomic group that grow in the sample area produce the same pigments in approximately the ratios as those in culture, and (ii) any unknown algal groups in the sample represent an insignificant portion. These assumptions cannot be validated internally (using HPLC), and they can be tested only partially by light microscopy or flow cytometry because some taxonomic characters rely on electron microscopic observations. We undertook this study to compare the taxonomic composition at the class level as estimated by HPLC pigment signatures and the taxonomic composition as observed by direct electron microscopic observations. From each sample, we prepared one subsample for HPLC pigment analysis, one subsample for epifluorescence microscopic analysis, and one subsample for transmission electron microscopic analysis. In this paper we report good

Comparison of HPLC pigment siguatures and electron microscopic observations

519

agreement between the HPLC estimated taxonomic composition and the taxonomic composition determined by direct electron microscopic observations, especially for samples collected at shallower depths.

MATERIALS

AND METHODS

Sample collection

Water samples were collected at six depths (20,70,90, 110, 120, 150 m) using 12 1Tefloncoated Niskin bottles at the Bermuda Atlantic Time-series Study (BATS) Hydrostation S (32”10’N, 64”3O’W) on 27 July 1992, and at six depths (20,60,90,120,143,200 m) using 12 1 PVC bottles at the Hawaii Ocean Time-series (HOT) Station ALOHA (24”45’N, 158”OO’W) on 15 April 1993. Three subsamples of seawater were taken from each depth for HPLC analysis, electron microscopy and epifluorescence microscopy. The 90 and 150 m samples collected at Hydrostation S were not examined by electron microscopy.

Pigment analysis

Seawater samples (3-10 1 each) were filtered through 25 mm Whatman GF/F glass fiber filters. Filters were stored under liquid nitrogen prior to analysis to improve extraction efficiency and minimize pigment alterations. Filters were placed in 3 ml acetone followed by the addition of an internal standard (50 ~1 canthaxanthin in acetone). The samples were subsequently disrupted by sonication (O’C, in the dark) and allowed to extract for 24 h (- 20°C in the dark). Since GF/F filters retain a significant amount of seawater following filtration (N 0.2 ml per 25 mm filter), the final acetone concentration in the pigment extracts was calculated to be -94% (acetone:water, vol:vol). Prior to analysis, pigment extracts were vortexed and centrifuged to remove cellular and filter debris. Acetone extracts were analyzed for pigments by HPLC using a modified version of the method described by Wright et aE.(199 1). The composition of “Solvent B” (cf. Wright et al., 199 1) was changed to 85: 15, acetonitrile:water (vol:vol) in order to improve separations of the polar pigments and the lutein-zeaxanthin pigment pair. With the exception of chlorophyll a-related pigments (see below), eluting pigments were detected by absorbance spectroscopy (436 nm) and quantified by peak area. Pigment concentrations (ng I- ’ seawater filtered) were calculated using internal (canthaxanthin) and external standards provided as part of the U.S. JGOFS pigment intercalibration exercise (Latasa et al., in press). Peaks were identified by comparing their retention times with those of pure standards. The HPLC method employed is not capable of physically separating monovinyl chlorophyll a from divinyl chlorophyll a nor monovinyl chlorophyll b from divinyl chlorophyll b. Concentrations of monovinyl chlorophyll a and divinyl chlorophyll a were determined by monitoring the “monovinyl chlorophyll a plus divinyl chlorophyll a” peak at two wavelengths (436 and 450 nm) and computing their respective concentrations via the dichromatic equations given in Latasa et al. (in press). Since a divinyl chlorophyll b standard was not available for instrument calibaration, “total” chlorophyll b concentrations (i.e. monovinyl chlorophyll b plus divinyl chlorophyll b) were estimated with the response factor determined for monovinyl chlorophyll b.

520

R. A.

Andersen

et al.

Pigment biomass algorithms

Chlorophyll a contributions provided by the major eukaryotic phytoplankton groups (i.e. prymnesiophytes, pelagophytes (sensu Andersen et al. (1993), referred to chrysophytes in Letelier et al. (1993)), dinoflagellates and diatoms) sampled at the BATS and HOT stations were calculated using HPLC-determined pigment concentrations and the algorithms given in Table 2 (after Letelier et al., 1993). Divinyl chlorophyll a concentrations were used directly as prochlorophyte chlorophyll contributions. Chlorophyll a contributed by “other” algae was estimated as the difference between total chlorophyll a and that contributed by prochlorophytes, prymnesiophytes, pelagophytes, dinoflagellates and diatoms. Possible contributions by prasinophytes, chrysophytes, chlorophytes, cryptophytes and cyanobacteria are included in the “other” algae category. Electron microscopy

The phytoplankton contained in approximately 5-6 1 of seawater was fixed immediately at sea with unbuffered glutaraldehyde (final concentration = 1.O%) at room temperature. After a minimum of 1 h, the cells were collected on one or more 1 pm Poretics filters and stored in screw-top test tubes containing filtrate and fixative. In the laboratory, the test tube with filters and fixative were centrifuged at high speed using a clinical centrifuge, rinsed three times with seawater, post-fixed for 1 h in 1% osmium tetroxide in 0.05 M cacodylate buffer (pH 7.2), rinsed with distilled water, dehydrated through a graded ethanol series with two final dehydration steps in 100% propylene oxide, and finally infiltrated and embedded using Spurr’s resin. The filters were embedded between two glass microscope slides coated with Teflon spray. Cells that fell from the filters during preparative steps were embedded in beem capsules. Serial thin sections were collected on slot grids for both filter and cell pellets, and for select samples, serial thick sections (ca 350-500 nm) were collected. The sections were stained with uranyl acetate and lead citrate and examined using a Zeiss 902A electron microscope. At least 100 eukaryotic cells were examined for each sample, and each cell was identified, if possible, at least to taxonomic class. In cases where cells could not be identified

Table 2.

Pigment algorithms (Letelier et al., 1993) used for partitioning chlorophyll a biomass among the major phytoplankton groups Equation

Algal Group Prochlorophytes Prymnesiophytes Pelagophytes Dinoflagellates Diatoms Other algae

[Chl alpro= [divinyl chlorophyll a] 01 alpry,,,= 1.3 x [19’-hex],,, [Chl a],1 = 0.9 x [19’-but],,, [Chl aldin,, = 1.5 x [peridinin] [Chl a],_+iat= 0.8 ([fucox] - (0.02 [19’-hex],, [Chl &hers where,

= 01

altotal -

01

+0.14

[19’-but],,))

alpro+ prym+ pel+ dino

+ diat

[ 19’-hexl,,,, = VW- Cl)=(W-h4t,t,~ - (119’-butlt,l,l 4) [19’-but],, = (p/V’- c)) * (119’-butltotal - (i19’-hexlt,t,l J/P)) P = [19’-hex],,,/[l9’-but],, = 54.27 C= [19’-hex]+ / [19’-but],, = 0.14

Comparison of HPLC pigment signatures and electron microscopic observations

521

in that section, the same cell was observed in serial sections. If examination of serial sections failed to provide identifiable features, the cell was recorded as unidentified. After all samples were examined, the relative abundances (%) of major algal groups were calculated. For ecological interest, the number of heterotrophic eukaryotes was recorded for each sample.

Epifluorescence microscopy

Size-fractionated subsamples were enumerated by epifluorescence microscopy using methods similar to those of Murphy and Haugen (1985) and Shapiro and Haugen (1988). A 250 ml whole water sample was taken at each depth and preserved immediately with 50% glutaraldehyde to a final concentration of 0.5%. After refrigeration for 1 h to harden the cells, the sample was divided into fractions, one of which was stained with the fluorochrome proflavine hemisulfate (5 pg ml- ’final concentration; Haas, 1982). Stained subsamples (100 ml each) were filtered serially through 8 pm and 3 pm Nuclepore polycarbonate filters. The > 8 pm and 3-8 pm size fractions were enumerated for chlorophyll-containing cells using a Zeiss Axioskop microscope equipped for epifluorescence. Unstained subsamples (50 ml each) were filtered serially through 3 pm and 0.2 pm Nuclepore polycarbonate filters and chlorophyll-containing cells were enumerated.

RESULTS

AND DISCUSSION

Pigment distributions

The HPLC pigment concentrations (ng 1-l) determined for Hydrostation S and Station ALOHA are given in Table 3. The quantitatively important pigments detected were monovinyl chlorophyll a, divinyl chlorophyll II, chlorophyll b, chlorophyll cl +2, chlorophyll c-like pigments (including chlorophyll cs), 19’-butanoyloxyfucoxanthin, fucoxanthin, 19’hexanoyloxyfucoxanthin, zeaxanthin, diadinoxanthin and a-carotene. Concentrations of peridinin, violaxanthin, lutein and b-carotene were low (< 4 ng l-l), and prasinoxanthin and alloxanthin concentrations were below the limit of HPLC quantification ( < 0.1 ng l- ‘). These results suggest that chlorophytes, prasinophytes, chrysophytes, cryptophytes and dinoflagellates were not important biomass components when these time-series stations were sampled (cf. Table 1). The dominant light-harvesting pigments (i.e. the accessory chlorophylls and fucoxanthin-related carotenoids) measured at Station ALOHA and Hydrostation S displayed distinct subsurface maxima located between 90 and 150 m. Concentrations of the photoprotective carotenoids, violaxanthin, zeaxanthin and p-carotene, were highest in the upper portion of the water column. Chlorophyll a biomass was partitioned into contributions by diatoms, prymnesiophytes, pelagophytes, dinoflagellates, prochlorophytes and “other” algae (Figs 1 and 2). For both Hydrostation S and Station ALOHA, two generalities can be made regarding chl biomass distributions. First, the proportion of diatom and dinoflageliate chl biomass was highest in the upper 80 m of the Water column. Second, the proportion of pelagophyte chl biomass increased with increasing depth. For prochlorophytes at Hydrostation S and prymnesiophytes at Station ALOHA, chl biomass contributions increased with increasing depth. The per cent chl associated with prochlorophytes at Station ALOHA and prymnesiophytes at Hydrostation S did not vary systematically with depth.

3.4 13.0 19.5 19.3 30.8 7.4

5.9 7.1 11.8 41.4 22.2 3.9

S
Hydrostation 20m 70m 90m 110m 120m 150m

Station ALOHA 20m 2.2 60m 4.8 90m 10.5 120m 48.5 143 m 29.2 200m 4.6

Chl c

Chl C*

Depth

0.8 I.1 1.8 2.8 1.4 0.3

0.5 1.6 2.1 1.4 1.3
Per

3.9 6.5 17.6 69.7 44.3 10.3

2.0 10.5 21.1 47.8 45.5 14.9

But

6.8 7.5 6.2 17.0 8.1 1.5

1.2 4.0 4.6 5.2 6.3 1.4

Fuc

9.5 13.3 36.6 96.3 64.0 13.4

8.0 29.2 54.2 61.4 76.2 23.2

Hex

0.8 0.7 0.9 0.9 0.1
1.0 2.3 2.0 0.6 0.3 <0.1

Vi0

3.9 3.1 4.6 8.0 4.6 0.9

3.1 4.6 6.1 4.9 4.9 1.5

Ddx

0.6 0.3 0.2 0.3 to.1
to.1
Lut

41.4 47.4 59.6 42.5 18.1 1.7

5.9 28.4 43.6 41.1 34.3 5.1

Zea

5.3 6.2 37.8 151.9 116.4 21.8

1.0 11.8 38.1 148.1 167.9 64.9

Chl b

3.8 5.5 15.0 29.7 18.2 2.8

0.3 3.1 17.4 34.1 36.3 11.9

a-Car

2.3 1.2
<0.1

2.0 3.6
/?-Car

27.7 29.7 87.3 142.6 76.2 11.5


DVA

41.5 51.1 89.8 214.1 137.6 22.8

27.2 79.9 124.7 135.8 144.2 42.8

MVA

69.2 80.8 177.1 356.7 213.8 34.3

27.2 98.5 199.4 248.8 250.9 71.1

Chl a

Table 3. Concentrations ofphotosynthetic pigments (ng I-‘) determinedat Hydrostation Sand Station ALOHA (chic’ = chlorophyllc-like pigments; chic = chlorophyll cl + 2; Per = peridinin; But = 19’-butanoyloqfucoxanthin; Fur = fuco.xanthin; Hex = 19’-hesanoylo.~yfucoxanthin; Vio = violaxanthin; Ddx = diadinoxanthin; Lut = lutein; Zea = zeasanthin; Cl11b = divinyl chlorophyll b plus monovinyl chlorophyll b; u-Car = u-carotene; p-Car = p-carotene; DVA = divinyl chlorophyll a; MVA = monovinyl chlorophyll a: Chl a = divinyl chlorophyll a plus monovinyl chlorophyll a)

Comparison of HPLC pigment signatures and electron microscopic observations

523

’ -r)lvdroatation S

%[Chl a] Fig. 1. Per cent of total chlorophyll a biomass contributed by prochlorophytes, prymnesiophytes, pelagophytes, diatoms, dinoflagellates and “other” algae at Hydrostation S (27 July 1992).

0

Station

ALOHA

. -22.5- -

. -250 0

I. 10

%[TChl alpro %[TChl ajprym s %rChl ajpel V I. I 20 30

.

I 40

%[TChl aldiat %rChl a]dino %[TChl ajother . I . 50

60

%[Chl a] Fig. 2. Per cent of total chlorophyll a biomass contributed by prochlorophytes, prymnesiophytes, pelagophytes, diatoms, dinoflagellates and “other” algae at Station ALOHA (15 April 1993).

Enumeration of cells by epifluorescence microscopy

The picoeukaryotes (defined here as cells <3 pm) were numerically dominant as they comprised 50-90% of the total eukaryotic phytoplankton (Table 4). The ultraphytoplankton (defined here as cells <8 pm) accounted for virtually ail of the cells ( > 98%) at both locations. Ceils greater than 8 pm (e.g. diatoms and dinoflagellates) were rare. Picoeukaryote abundance maxima at Station ALOHA during April 1993 (1.3 x lo6

524

R. A. Andersen et al.

at Hydrostation S and Station Table 4. Vertical distribution of different sizefractions of eukaryoticphytoplankton ALOHA. Whole water samples were sequentially filtered through 8, 3 and 0.2 pm Nucleporefilters. Cell abundances represent those cells retained on each filter. Numbers in parentheses represent percentage of toial eukaryotic cells in each size fraction

Abundance of eukaryotic phytoplankton (cells per liter) Depth

Total Eukaryotes

Hydrostation S (27 July 1992) 20 m 5.41 x 104(100) 70 m 6.78 x lo4 (100) 90m 2.54 x lo4 (100) 1lOm 7.12 x lo5 (100) 120m 5.92 x 10’(100) 150m 1.88 x 105(100) Station ALOHA (15 April 1993) 20 m 4.61 x lo5 (100) 60m 5.09 x 105(100) 3.86 x lo5 (100) 90m 120m 1.48 x 106(100) 143 m 1.04x 106(100) 200 m 3.13 x lo4 (100)

c 3pm Eukaryotes

3-8 pm Eukaryotes

2.12 x 5.51 x 4.26 x 1.40 x

104(83.4) 105(77.4) lo5 (72.0) 105(74.5)

1.17 x 3.39 x 4.24x 1.61 x 1.62 x 4.77 x

3.81 x 4.57 x 2.67 x 1.33 x 8.76 x 2.70 x

lo5 (82.7) 105(89.8) lo5 (69.3) 106(89.9) 10’(84.2) lo4 (86.3)

7.99x 5.18 x 1.12 x 1.51 x 1.64x 4.32 x

4.24 x lo4 (78.4) 3.39 x lo4 (50.0)

lo4 (21.6) 104(50.0) lO’(l6.6) lo5 (22.6) lo5 (27.3) lo4 (25.5)

104(17.3) lo4 (10.2) lo5 (29.0) 105(10.1) lO’(15.8) lo3 (13.7)

> 8 pm Eukaryotes

1.1 x102(<1.0) 6.0x 10’(
0 (0) 0 (0) 6.48 x lo3 (1.7) 0 (0) 0 (0) 0 (0)

cells 1-l) and Hydrostation S during July 1992 (5.5 x 10’cells 1-l) were located at depths of 110-120 m. Similar picoeukaryote distributions were reported for Station ALOHA (April 1991) by Campbell and Vaulot (1993) and the OFP site near Bermuda (July 1989) by Olson et al. (1990). Observations of cells by electron microscopy

Marine phytoplankton are difficult to fix for electron microscopy, and successful fixations usually include simultaneous or near simultanteous additions of glutaraldehyde and osmium tetroxide (Vesk and Puttock, 1980). However, simultaneous fixation of seawater samples requires either a concentration of cells before fixation or a large volume of chemical fixatives. We did not concentrate cells prior to fixation because we did not want to disrupt delicate cells with centrifugation or filtration forces. Since we could not use large volumes of osmium tetroxide, we compromised by (i) using a large volume of glutaraldehyde to fix unconcentrated samples, (ii) concentrating the cells after they were fixed to minimize disruption, and (iii) post-fixing the cells with osmium tetroxide. This method preserved all of the different types of phytoplankton, but the preservation of cellular features was not ideal. The green algae and cyanobacteria, which ordinarily fix well using glutaraldehyde alone, were best preserved, and the chromophyte algae, which usually require simultaneous fixation protocols, were not well preserved. Also, osmium “blacks” (osmium precipitations) occurred during post-fixation, probably due to incomplete removal of the unbound glutaraldehyde. Most cells could be identified to class based upon chloroplast, mitochondrion, flagellar (when present) and cell surface features. On a few occasions it was difhcult to classify an alga into a green algal class or a chromophyte class when critical

Comparison of HPLC pigment signatures and electron microscopic observations

525

features were absent. Thus, even though cellular preservation was not ideal, this method preserved cells to the extent that they could be identified at the class level in most cases. The electron microscopic observations revealed an apparently broad biodiversity of species and taxonomic groups. The prasinophyte algae were preserved better than the other eukaryotic algal groups, and they could be identified by their scaly surfaces, chloroplast lamellar arrangement and the presence of starch grains within the chloroplast (Fig. 3(a)(e)). The chromophyte algae were poorly preserved and cells were often ruptured. The prymnesiophytes were identified most easily by their chloroplast structure, i.e. typically two chloroplasts with lamellae consisting of three thylakoids and without a girdle lamella (Fig. 4(a) and (b)). Organic scales were commonly observed, but coccoliths were observed only once. The low coccolith abundances may be the result of dissolution caused by the use of unbuffered glutaraldehyde to fix the samples. Diatoms were the best preserved of the chromophytes, and they were easily identified by the presence of siliceous frustules (Fig. 4(c) and (d)). Both centric and pennate diatoms were present, but it was not always possible to distinguish the two types if the raphes were not present in the serial sections. The pelagophytes typically had one chloroplast with a distinctive girdle lamella, and they possessed either a thick organic wall or a thin theta (Fig. 5(a)-(c)). The basal body of one pelagophyte cell showed the presence of doublet microtubules (Fig. 5(b)), a feature that seems to be typical for this group (Heimann et al., 1995). The chrysophytes Sen,sustrict0 were rarely observed, but could be identified by the presence of a transitional helix in the flagellar region (Fig. 5(d), Hibberd, 1979). The dinoflagellates were almost always poorly preserved, but the structure of the chromosomes, the alveolate/thecate cell surface and the presence of massive microtubular bands were distinctive features for this group (Fig. 6(a) and (b). Dinoflagellates with chloroplasts often contained food vacuoles as well, indicating a mixotrophic existence (Fig. 6(a)). Although the study tabulated chloroplast-bearing eukaryotic cells, numerous other cell types were observed. These included many types of heterotrophic flagellates, aplastidic dinoflagellates, ciliates (Fig. 7(a)), a pelagophyte with virus particles, an unidentifable cell filled with viral particles (Fig. 7(b)), bacteria (Fig. 7(c)), prokaryotic algae (Fig. 7(d)-(f)), and a helizoon amoeba (Fig. 8). Heterotrophic eukaryotic cells were enumerated, and the relative abundances of heterotrophic ( = nonphotosynthetic) and phototrophic (= chloroplast-containing) eukaryotes are tabulated in Table 5. There were no apparent depth-dependent variations in the relative abundances of phototrophs and heterotrophs, and on average eukaryotic phototrophs were 2-4 times more numerous than eukaryotic heterotrophs. The ratio of phototropic to heterotrophic cells in our study adds additional support to light microscopic studies that show that heterotrophs are abundant in the open oceans. A previous study in the North Atlantic Ocean showed that heterotrophic flagellates and ciliates accounted for about 15-25% of the particulate matter and biomass (Sieracki et al., 1991). Comparison of HPLC pigment distributions and electron microscopic observations

The electron microscopic observations were quantitatively limited to eukaryotic algae because many prokaryotes, especially Prochlorococcus spp., passed through the filters (1 pm pore size). Therefore, the prochlorophyte chlorophyll a concentrations (i.e. divinyl chlorophyll a) were subtracted from the total chlorophyll a concentrations, and the remaining “monovinyl chlorophyll u” concentrations were partitioned via the pigment

526

R.A.Andemtnetal. Table 5. Relative abumkances of phototrophic and heterotrophic eukaryotic cells measured at Hydrostation S and Station ALOHA. Abundances are given as a percentage of total eukaryotic cells Depth

Phototrophs

Heterotrophs

Hydrostation S 20m 70m 1lOm 120m

75 82 74 78

25 18 26 22

Station ALOHA 20m 60m 90m 120m 143m 200m

74 66 63 80 69 62

26 34 37 20 31 38

algorithms (Table 2) into relative contributions by prymnesiophytes, pelagophytes, dinoflagellates, diatoms and “other” algal groups. The monovinyl chlorcphyll a associated with “other” algae includes possible contributions by prasinophytes, chrysophytes, chlorophytes, cryptophytes and cyanobacteria. For electron microscopy, the “other” category included eukaryotes only, including prasinophytes, chrysophytes, etc., as well as species that could not be identified and may have belonged to one of the four specific groups. The relative numbers of prymnesiophytes, pelagophytes, dinoflagellates, diatoms and “other” algae identified by electron microscopy are compared to the relative abundances of the same groups estimated via the pigment algorithms (Figs 9 and 10). These comparisons should be regarded as “first order” since more “exact” comparisons would require detailed measurements of cell volumes and pigment concentrations per cell, probably at the species level. Such measurements are not readily feasible for natural phytoplankton populations. Nonetheless, we consider our comparisons to be “reasonably” good since differences in cell sizes and pigment concentrations at each depth are probably compensated, at least in part, by the averages of many cells. The prymnesiophytes and pelagophytes were found to be the most abundant eukaryotic algae using both methods, accounting for approximately 40-80% of the eukaryotes (Figs 9 and 10). The pigment estimates and the electron microscopic observations of prymnesiophytes were similar at the shallower depths, but the pigment estimates were higher than the electron microscopic observations in deeper water samples. It is possible that some of these differences may be accounted for by (i) prymnesiophytes that could not be identified and were counted as “other” algae, and (ii) depth-dependent variations in accessory pigment-to-chlorophyll ratios (see Jeffrey and Wright, 1994). The relative abundances of pelagophytes based on pigment and microscopical data are similar for most samples. The recent description of this “new” algal class and its pigment composition (Andersen et al., 1993) has allowed its representatives to be identified by HPLC in

Comparison of HPLC pigment signatures and electron microscopic observations

Fig, 3. Green algae. (a) A scaly green alga showing the nucleus (N). chloroplasts (P) and scale covering (arrow). Hydrostation S, 70 m. Scale bar = 200 nm. (b) Section through a scaly green alga showing the nucleus, chloroplast and pyrenoid (P). Hydrostation S, 70 m. Scale bar = 200 nm. (c) Green algal scales resembling those of Dolicomustix (arrow). Hydrostation S, 20 m. Scale bar = 120 nm. (d) Tangential section of a Bathycoccus cell showing the scale structure. Hydrostation S, 70 m. Scale bar = 70 nm. (e) Square-shaped flagellar scales, probably belonging to a Pyramimonus cell, surrounding two flagellar axonemes. Hydrostation S, 20 m. Scale bar = 120 nm.

527

528

R. A. Andersen et al.

Fig. 4. Prymnesiophytes and diatoms. (a) Typical scaly prymnesiophyte cell with two chloroplasts connected to the nucleus. Hydrostation S, 70 m. Scale bar = 200 nm. (b) Prymnesiophyte cell showing the Golgi body (G) and storage vacuole (V). Hydrostation S, 70 m. Scale bar = 500 nm. (c) A longitudinal section of a centric diatom with several chloroplasts, a mitochondrion (M) and a heavily silicified frustule (arrows). Station ALOHA, 200 m. Scale bar = 500 nm. (d) A cross-section of a pennate diatom showing the raphe canals (arrowheads) in the frustule (arrows). Station ALOHA, 60 m. Scale bar = 500 nm.

Comparison of HPLC pigment signatures and electron microscopic observations

:> Fig. 5. Pelagophytes and a chrysophyte. (a) A pelagophyte cell with a reduced basal body. Hydrostation S, 70 m. Scale bar = 200 nm. (b) An enlargement of (a) showing a nascent basal body with doublet microtubules rather than triplet microtubules. Rotated 180”. Scale bar = 70 nm. (c) Typical Pelugococcus cell. Cell wall is coated with osmium “blacks”. Station ALOHA, 120 m. Scale bar = 200 nm. (d) A colorless chrysophyte flagellate having a transitional helix (arrow). Hydrostation S, 20 m. Scale bar = 200 nm.

529

530

R. A. Andersen

et al.

Fig. 6. Dinoflagellates. (a) Typical phototrophic dinoflagellate cell. Note the food vacuole (arrow) that appears to contain a eukaryotic cell that in turn engulfed a prokaryotic cell. Station ALOHA, 20 m. Scale bar = 1 mm. (b) Complex microtubular roots extending from the basal bodies (B). Hydrostation S, 70 m. Scale bar = 200 nm.

Fig. 7. Ciliate, virus-infected cell and prokaryotic cells. (a) Ciliate. Hydrostation S, 20 m. Scale bar = 2.5 mm. (b) Virus-infected cell. Station ALOHA, 200 m. Scale bar = 200 nm. (c) Typical bacterial cell. Hydrostation S, 70 m. Scale bar = 200 nm. (d) Photosynthetic prokaryote. Hydrostation S, 70 m. Scale bar = 120 nm. (e) Photosynthetic prokaryote. Hydrostation S, 70 m. Scale bar = 200 nm. (f) Photosynthetic prokaryote. Hydrostation S, 70 m. Scale bar = 120 nm.

Comparison of HPLC pigment signatures and electron microscopic observations

Fig. 7.

531

532

R. A. Andersen et

al.

Fig. 8. Heliozoon amoeba showing the microtubule clusters (arrows), which support the pseudopodia, arising from the nuclear envelope. Hydrostation S, 120 m. Scale bar = 500 nm.

Comparisonof HPLC pigmentsignaturesand electronmicroscopicobservations (b) Rlsgophytes

(a) Prymnesiophytes

70

120

20

70

110

Depth (m)

Depth (ml

(19 Dinoflagellates

(d) Diatoms

kGG!!L

20

110

70

533

II

_

110

120

Depth (ml

$!I

120

_

20

70

110

120

Depth (rn)

M Other Algae

20

70

110

120

Depth (m)

Fig. 9. Comparisonof the per cent monovinylchlorophylla (solidbars)and the EM-determined per cent of algal groups(whitebars) at HydrostationS for the depthsof 20, 70, 110,and 120m: (a) prymnesiophytes,(b) pelagophytes,(c)dinoflagellates, (d) diatoms,and (e) other algalgroups.

subsequent field studies (Bidigare and Ondrusek, in press). This algal group was not formally recognized in earlier interpretations of pigment data collected at the BATS (Michaels et al., 1994) and HOT (Letelier el al., 1993) time-series stations, as 19’butanoyloxyfucoxanthin was thought to be associated with chrysophyte phytoplankton (cf. Bjermland et al., 1989; Vesk and Jeffrey, 1987; Bidigare, 1989). If these samples are representative of the open-oceanic habitat, then pelagophytes may be one of the most abundant groups of eukaryotic algae. In an attempt to statistically compare electron microscopic (EM) and HPLC data, abundances of the dominant eukaryotic algae (i.e. prymnesiophytes and pelagophytes) were estimated by multiplying the EM-determined per cent abundances (Figs 9 and 10) by the total eukaryotic phytoplankton counts given in Table 4. These abundances were then compared to concentrations of chl contributed by prymnesiophytes and pelagophytes by performing geometric mean model II regression analyses. It should be noted that these comparisons are only an approximation. In addition to methodological errors, natural variability of pigment content per cell (i.e. that caused by photoadaptation and changes in cell volume) probably account for the scatter about the regression lines. With the exception

534

R. A. Andersen

et al.

(b) Pelagophytes

(a) Prymnesiophytes 80

80 E

60

8

40

$

20

I

0 z

8

00)

8 r

a c

O a

Depth (m)

Depth (ml

(c) Dinoflagellates

(d) Diatoms

(el Other Algae

E

60

E

40

g

20 r-l

z 8 8 z c

?? 3 r

t4

Depth (ml

Fig. 10. Comparison of the per cent monovinyl chlorophyll a (solid bars) and the EM-determined per cent of algal groups (white bar) at Station ALOHA for depths of 20,60,90,120,143, and 200 m: (a) prymnesiophytes, (b) pelagophytes, (c) dinoflagellates, (d) diatoms, and (e) other algal groups.

of a prymnesiophyte “outher”, significant correlations were obtained (Fig. 11). The resulting slopes correspond to average chl concentrations of 0.37 and 0.18 pg chl per prymnesiophyte and pelagophyte cell, respectively. By comparison, the value obtained for the prymnesiophytes falls within the range determined for Emiliania huxleyi (0.18-0.40 pg chl cell-‘; Schofield et al., 1990) and was close to the average found for six species of 19’hexanoyloxyfucoxanthin-containing prymnesiophytes (mean f standard deviation = 0.27 kO.28) by Jeffrey and Wright (1994). The lower chl content estimated for the pelagophytes is consistent with their smaller size (Simon et al., 1994). The relative abundance of dinoflagellates estimated by electron microscopy was consistently higher than that determined by HPLC. Similar observations have been made elsewhere (Jeffrey and Hallegraeff, 1987; Latasa et al., 1992; Bidigare and Ondrusek, in press). One possible explanation is that some oceanic dinoflagellates may have pigments that are different from those found in the coastal species used in algorithm construction. For example, there are several distinct pigment types in the dinoflagellate group, including those dominated by peridinin, fucoxanthin or 19’-hexanoyloxyfucoxanthin, and those which

Comparison of HPLC pigment signatures and electron microscopic observations

535

??

0

5-h

0-p

_’

Oe+O

0

.

0

y = (0.000373)x

+ 1.33 (Pfym, r = 0.65)

y = (0.000180)x

+ 2.65 (Pelag,

I 2e+5

, 4et5

I 6e+5

r = 0.86)

8e+5

Abundance (cells/L) Fig. 11. Cross plot of chlorophyll a contributed by prymnesiophytes and pelagophytes versus their respective cell abundances. The equations and linear fits were determined by geometric mean model II regression analysis. The prymnesiophyte outlier was not used in the regression analysis. Chlorophyll II contributions were calculated using the equations given in Table 2, and class-specific cell abundances were calculated as the product of EM-determined per cent abundances (Figs 9 and 10) and total eukaryotic phytoplankton counts (Table 4).

possess phycobilipigments (see Bjarrnland and Liaaen-Jensen, 1989). It is not possible to distinguish peridinin and non-peridinin dinoflagellates based upon ultrastructure, so we could not test this hypothesis. CONCLUSIONS Electron microscopic observations provided direct identification of specific taxonomic groups and these observations generally agreed with inferred taxonomic identities based upon HPLC pigment analysis. Electron microscopy is difficult and time-consuming, and a small percentage of cells cannot be identified with certainty. HPLC analysis does not provide direct evidence on a cell-by-cell basis, but it is quicker and it does include several orders of magnitude more cells than is possible to identify in the electron microscope. The general agreement of the two methods, although not perfect, suggests that HPLC data provide a good first approximation concerning the taxonomic composition of the phytoplankton. Furthermore, when new groups such as the pelagophytes are discovered, algorithms can be adjusted (even years later) to distinguish these new groups. Electron microscopic observations and HPLC data for eukaryotic algae are reasonably similar, and we suggest that pigment analysis can be used as a means for approximating the taxonomic composition of the phytoplankton. Acknowledgements-The authors thank the crews of the R.V. Wecoma and R.V. Weatherbird II, as well as HOT and BATS personnel for their valuable assistance and support. This work was supported by NSF grant BSR9012892 to RAA, RRB and MDK and NSF grant OCE-9315311 to RRB. Mike1 Latasa was supported by a postdoctoral fellowship from the Consejo Superior de Investigaciones Cientificas (Spain). This is JGOFS contribution no. 205.

R. A. Andersen et al.

536

REFERENCES Andersen R. A., G. W. Saunders, M. P. Paskind and J. P. Sexton (1993) Ultrastructure and 18s rRNA gene sequence for Pelagomonas calceolata gen. et sp. nov. and the description of a new algal class, the Pelagophyceae classis nov. Journal of Phycology, 29.701-7 15. Bidigare R. R. (1989) Photosynthetic pigment composition of the brown tide alga: Unique chlorophyll and carotenoid derivatives. In: Novelphytoplanhton blooms, E. Cosper, E. J. Carpenter and M. Bricelj, editors, Coastal and Estuarine Studies, Vol. 35, Springer, Berlin, pp. 57-75. Bidigare R. R. and M. E. Ondrusek (in press) Spatial and temporal variability of phytoplankton pigment distributions in the central equatorial Pacific Ocean. Deep-Sea Research I. Bidigare R. R., M. C. Kennicutt II, M. E. Ondrusek, M. D. Keller and R. R. L. Guillard (1990) Novel chlorophyll-related compounds in marine phytoplankton: Distributions and geochemical implications. Energy and Fuels, 4, 653-657.

Bidigare R. R., B. B. Prezelin and R. C. Smith (1992) Bio-optical models and the problems of scaling. In: Primary productivity and biogeochemical cycles in the sea, P. G. Falkowski and A. D. Woodhead, editors, Plenum Press, New York, pp. 175-212. Bjemland T. and S. Liaaen-Jensen (1989) Distribution of carotenoids in relation to chromophyte phylogeny and systematics. In: The chromophyte algae: Problems andperspectives, J. C. Green, B. S. C. Leadbeater and W. L. Diver, editors, Oxford Science Press, Oxford, U.K., pp. 37-60. Bjernland T., S. Liaaen-Jensen and J. Throndsen (1989) Carotenoids of the marine chrysophyte Pelagococcus subviridis. Phytochemistry, 28, 3347-3353.

Burkill P. H., R. C. F. Mantoura, C. A. Llewellyn and N. J. P. Owens (1987) Microzooplankton grazing and selectivity of phytoplankton in coastal waters. Marine Biology, 93, 581-590. Campbell L. and D. Vaulot (1993) Photosynthetic picoplankton community structure in the subtropical North Pacific Ocean near Hawaii (Station ALOHA). Deep-Sea Research I, 40, 2043-2060. Chisholm S. W., R. J. Olson, E. R. Zettler, R. Goericke, J. B. Waterbury and N. A. Welschmeyer (1988) A novel free-living prochlorophyte abundant in the oceanic euphotic zone. Nature, 344, 340-343. Chisholm S. W., S. Frankel, R. Goericke, R. J. Olson, B. Palenik, E. Urbach, J. B. Waterbury and E. R. Zettler (1992) Prochlorococcus marinus nov. gen. nov. sp.: An oxyphototrophic marine prokaryote containing divinyl chlorophyll a and b. Archives of Microbiology, 157, 297-300. Everitt D. A., S. W. Wright, J. K. Volkman, D. P. Thomas and E. J. Lindstrom (1990) Phytoplankton community composition in the western equatorial Pacific determined from chlorophyll and carotenoid pigment distributions. Deep-Sea Research, 37, 975-997. Foss P., R. R. L. Guillard and S. Liaaen-Jensen (1984) Prasinoxanthin-A chemosystematic marker for algae. Phytochemistry, 23. 1629-1633. Gieskes W. W. C. and G. W. Kraay (1989) Estimating the carbon-specific growth rate of the major algal species groups in eastern Indonesian waters by 14C labeling of taxon-specific carotenoids. Deep-Sea Research, 36, 1127-l 139. Gieskes W. W. C., G. W. Kraay, A. Nontji, D. Setiapermana and Sutomo (1988) Monsoonal alternation of a mixed and a layered structure in the phytoplankton of the euphotic zone of the Banda Sea (Indonesia): A mathematical analysis of algal fingerprints. Netherlands Journal of Sea Research, 22, 123-137.

Goericke R. and D. J. Repeta (1992) The pigments of Prochlorococcus marinus: The presence of divinyl chlorophyll a and b in a marine prochlorophyte. Limnology and Oceanography, 37, 425-433. Goericke R. and N. A. Welschmeyer (1993) The marine prochlorophyte Prochlorococcus contributes significantly to phytoplankton biomass and primary production in the Sargasso Sea. Deep-Sea Research I, 40. 22832294.

Haas L.W. (1982) Improved epifluoresccnce microscope technique for observing planktonic organisms. Annals of the Institute of Oceanogrography,

58, 261-266.

Heimann K., R. A. Andersen and R. Wetherbee (1995) The flagellar development cycle of the uniflagellate Pelagomonas calceolata (Pelagophyceae). Journal of Phycology, 31, 577-583. Hibberd D. J. (1979) The structure and phylogenetic significance of the flagellar transition region in the chlorophyll c-containing algae. BioSystems, 11, 243-261. Hooks C. E., R. R. Bidigare, M. D. Keller and R. R. L. Guillard (1988) Coccoid eukaryotic marine ultraplankters with ‘four different HPLC pigment signatures. Journal of Phycology, 24, 571-580. Jeffrey S. W. (1989) Chlorophyll c pigments and their distribution in the chromophyte algae. In: The chromophyte

Comparison of HPLC pigment signatures and electron microscopic observations

537

algae:problems

andperspectives, J. C. Green, B. S. C. Leadbeater and W. L. Diver, editors, Oxford Science Press, Oxford, U.K., pp. 13-36. Jeffrey S. W. and G. M. Hallegraeff (1987) Phytoplankton pigments, species and light climate in a complex warm core eddy of the East Australian Current. Deep-Sea Research, 34, 479-495. Jeffrey S. W. and S. W. Wright (1994) Photosynthetic pigments in the Haptophyta. In: The haptophyte algae, J. C. Green and B. S. C. Leadbeater, editors, Claredon Press, Oxford, U.K., pp. 11l-132. Landry M. R. and R. P. Hassett (1982) Estimating the grazing impact of marine micro-zooplankton. Marine Biology, 67, 283-288.

Latasa M., M. Estrada and M. Delgado (1992) Plankton-pigment relationships in the northwestern Mediterranean during stratification. Marine Ecology Progess Series, 88, 61-73. Latasa M., R. R. Bidigare, M. E. Ondrusek and M. C. Kennicutt II (in press) HPLC analysis of algal pigments: A comparison exercise among laboratories and recommendations for improved analytical performance. Marine Chemistry.

Letelier R. M., R. R. Bidigare, D. V. Hebel, M. E. Ondrusek, C. D. Winn and D. M. Karl (1993) Temporal variability of phytoplankton community structure at the U.S.-JGOFS time-series Station ALOHA (22”45’N, 158?JO’W)based on HPLC pigment analysis. Limnology and Oceanography, 38, 1420-1437. Liaaen-Jensen S. (1985)Carotenoids of lower plants-Recent progress. Pure and Applied Chemistry, 57,649-658. Michaels A. F., A. H. Knap, R. L. Dow, K. Gundersen, R. J. Johnson, J. Sorensen, A. Close, G. A. Knaur, S. E. Lohrenz, V. Asper, M. Tuel and R. R. Bidigare (1994) Seasonal patterns of ocean biogeochemistry at the U.S. JGOFS Bermuda Atlantic Time-series Study site. Deep-Sea Research I, 41, 1013-1038. Millie D. F., H. W. Paerl and J. P. Hurley (1993) Microalgal pigment assessments using high-performance liquid chromatography: A synopsis of organismal and ecological applications. Canadian Journal of Fisheries and Aquatic Sciences, 50, 25 13-2526.

Murphy L. S. and E. M. Haugen (1985) The distribution and abundance of phototrophic ultraplankton in the North Atlantic. Limnology and Oceanography, 30, 47-58. Olson R. J., S. W. Chisholm, E. R. Zettler, M. A. Altabet and J. A. Dusenberry (1990) Spatial and temporal distributions of prochlorophyte picoplankton in the North Atlantic Ocean. Deep-Sea Research. 37, 10331051.

Schofield O., R. R. Bidigare and B. B. Prezelin (1990) Chromatic photoadaptation and enhancement effects on wavelength-dependent quantum yield and productivity in the diatom Chaetoceros grucile and the prymnesiophyte Emiliania huxleyi. Marine Ecology Progress Series, 64, 175-l 86. Shapiro L. P. and E. M. Haugen (1988) Seasonal distribution and temperature tolerance of Synechococcus in Boothbay Harbor, Maine. Estuarine and Coastal Shelf Science, 26, 517-525. Sieracki M. E., P. G. Verity and D. K. Stoecker (1991) Plankton community response to sequential silicate and nitrate depletion during the 1989 North Atlantic spring bloom. Deep-Sea Research II, 40, 213-225. Simon N., R. G. Barlow, D. Marie, F. Partensky and D. Vaulot (1994) Characterization of oceanic photosynthetic picoeukaryotes by flow cytometry. Journnl of‘Phyco/ogy, 30, 922-935. Strom S. L. and N. A. Welschmeyer (1991) Pigment-specific rates of phytoplankton growth and microzooplankton grazing in the open subarctic Pacific Ocean. Limnology and Oceanography, 36, 50-63. Vesk M. and S. W. Jeffrey (1987) Ultrastructure and pigments of two strains of the picoplanktonic alga Pelagococcus subviridis (Chrysophyceae). Journal of Phycology, 23, 322-336. Vesk M. and C. F. Puttock (1980) Fixation of marine unicellular algae. Micron, 11, 505-506. Wright S. W., S. W. Jeffrey, R. F. C. Mantoura, C. A. Llewellyn, T. Bjemland, D. Repeta and N. Welschmeyer (1991) Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Marine Ecology Progress Series, 77, 183-196.