Seasonal abundances and community structure of benthic rhizopods in shallow lagoons of the southern Baltic Sea

Europ. J. Protistol, 36, 103-115 (2000) May4,2000 http://www.urbanfischer.de/journalslejp

European Journal of

PROTISTOLOGY

Seasonal Abundances and Community Structure of Benthic Rhizopods in Shallow Lagoons of the Southern Baltic Sea Tobias

Garstecki l .2 .'~

and Hartmut Arndt1,2

Zoolog isches Institut, Universitat zu K61n, Allgemeine Okologie und Limnologie, Weyertal 119, D-50923 K61n , Germany; Fax (49)221-4705932; e-mail [email protected] 21nstitut fur Okologie, Ernst-Moritz-Arndt-Universitat Greifswald, Schwedenhagen 6, D-18565 Kloster/Hiddensee, Germany 1

Summary In order to assess the quantitative importance and community structure of benthic rhizopods in brackish lagoons of the southern Baltic Sea, their abundance, biovolume, and taxonomic composition were studied by using a liquid cultivation method. Seasonal dynamics in the superficial sediment of station Bak (Hiddensee Island) were investigated during eleven sampling campaigns between March and November 1996. Additional samples from three sediment layers were taken at two other stations. Rhizopod abundances at station Bak ranged from 2,80010,900 cells cm-l • A seasonal trend with a summer maximum and minima in early spring and late autumn was observed. 46 morphotypes of rhizopods were found, 27 of which were identified to species. Gymnamoebia and Schizopyrenida always dominated the rhizopod community numerically,whereas testate and naked filose rhizopods sometimes contributed the major part to community biovolume. Typical marine and freshwater rhizopod s coexisted in the stud y area. In addition to bacterivorou s forms, high contributions of herbivores and omnivores were found. Our results suggest that the hitherto little-studied rhizopods are a major component of benthic protistan commun ities of shallow coastal waters. The high contributions of herbivorous and omn ivorou s forms to the rhizopod community indicate a complex trophic role of rhizopod s within the benthic microbial food web. Key words: Amoebae; Benthos; Microbial food web; Protozoa; Sediment

Introduction The func tional importance of the micro bial foo d web for the carbo n and nutrie nt turnover of pelagic "corresponding author © 2000 by Urban & Fischer Verlag

ecosystems is now firmly established [e. g. 6, 24]. Recent findings show that a similar foo d web exists in coastal sediments, fuelled by sedimen ting detritus and microp hytob enthos primary production [8, 22, 23]. Similar to the water column, heterotrophic protists act as bacterivores, herbivores, and omnivore s within the benthic microbial food we b. While rhizopods other than for aminiferan s generally play a min or role w ithin the p rotozo opl ankton [4, 17], th eir imp ortance in th e benthos is still unclear. Studies focussing on rhi zop od abun dance and distribut ion in marine sediments have only recen tly been published [2, 15, 19]. Abundances in th e order of 103-104 ind oem? found du rin g th ese studies suppo rt th e hyp othesis th at - in cont rast to th e wa ter column - rhizo pods are an impo rt ant compo nent of th e benthic microb ial foo d web. H ow ever, conclusive statements about th e qu antitative imp ortance of rhizo po ds within a given ecosys tem require comparative data on other p rotistan groups, whic h have only rarely been present ed [e. g. 25]. Micro - and nanozo ob enthic rhizo po ds are a polyph yletic and hetero geneou s gro up. T hey include Schizo pyre nida, Gymnamoebia, Testacealob osia, Aeo nchulinida, Testaceafilosia, G ranul oreticulos ea, and amoe bo id Proti sta incert ae sedis. The few qu antitative accounts published have focussed on naked lobose rhizopod s belon ging to th e Gymnamoebia and Schizopyrenid a. The taxon om ic reso lution of th ese studies was restricted to a distinction between broad morphological categories [2, 19]. In co nt rast, faun istic stu dies have shown tha t rhizopo d communities may comprise a considerable species richness [27, 28, 48, 49, 60]. T he tr ophic ro les of natural rhi zopod communities are likely to vary with th eir taxo no mic compos itio n. Bakterivory, herb ivor y, and omnivo ry have been experi 0932-4739/0 0/36/01-103 $ 12.00/0

104

T. Garstecki and H. Arndt

mentally demonstrated in a variety of rhizopod spe cies [11, 16, 33]. A high taxonomic resolution of quantitative field data is necessa ry to derive hypotheses about the role of rhizopods within the benthic microbial food web using functional group approaches [e. g. 45]. This study was carried out in order to provide new information about the ecological role of rhizopods within the benthic microbial food web. We have assessed the quantitative import ance (absolutely and in comparison to other protistan groups), seasonal distribution and taxono mic composition of a micro- and nanozoobenthic rhizopod community in sha llow lagoons of the Sou thern Baltic. In order to qu antify the rela tive importance of rhizopods, their community biovolumes were compared to those of ciliates and heterotrophic flagellates w hich were enumerated in the same samples by cooperating colleagues [20]. Based on the taxonomic composition of the community, we have estimated the relative importance of different functional groups, and of marine versus freshwater species, within the benthic rhizopod community.

5km

Material and Methods Study area: Th e stud y was carried out in shallow, nontidal, brackish lagoons at th e German coast of the Baltic Sea (Fig. 1). Rhizopod community structure and seasonal dyn amics in the superficial sediment were investigated at station Bak throughout 1996. Additional samples from three sediment layers and from the water column were taken during five samplin g campaigns in 1996 and 1997 at stations Rassower Strom and Kirrbucht. In this article, abundance and biomass totals from station Bak and data about the community struc tur e of all three stations are presented. The stations varied in salinity, depth, trophic status and sediment type (Table 1, Fig. 2). Sam pling: At each sampling, duplicate sediment cores (100 mm internal diameter) with approx imately 10 ern overlying water were obtained by using a manua l corer (Bak and Kirrbu cht) or a ship-operated multicorer (Rassower Strom) . From these cores, subsamples were taken for sediment characteristics, direct microscopical observation, enrichment cultures , and for the enumeration of amoebae, respectively. At station Bak, subsamples (0- 3 mm depth) were taken with a cut off plastic-syringe with an 11 mm internal diameter. Three subsamples were taken from each core and pooled to reduce the small-scale spatial heterogeneity within individual cores. At stations Rassower Strom and Kirrbucht, the flocculent surface layer and some of the overlying wat er were pipet ted from the sediment surface and sampled prior to sediment sampling. Part s of the resulting slur ry were kept in a graduated glass cylinder at 20°C for 24 h. From the compacti on of the particulate material in the cylinder, correction factors for the dilution of the flocculent surface layer during sampling were calculated . Sediment subcores were taken from the 100-mm cores in plastic tubes with a 24 mm internal diameter. They were subsequently cut into the 0-5 mm and 5-10 mm layers. Because of the greater diameter of these subcores, no subs ample replication and pooling were carried out with samples from Rassower d Strom and Kirrbucht. Subsamples were resuspended in defined volumes of autoclaved seawater and kept at 4 °C in the dark until further processing (generally within 1-6 hours after sampling).

Fig. 1. Location of sampling stations. Table 1. Characterization of sampling sites. Ranges are given in parenth eses. (n. det.: not determined; OC: organ ic carbon content)

Water depth em] Salinity [PSU] Troph ic statu s Water column chi a [pg 1-1] Seston [mg 1-1] Sediment type Sediment OC [%]

Biik

Rassower Strom

Kirrbucht

0.5 8 (6-10) mesotrophic n.det. n. det. muddy sand 4

3.8 9 (8- 12) mesotrophic 2.7 (1.3-4.5) 7 (4.5-11) sandy mud 4

0.7 5 (2-10) polytrophic 24 (12-33) 60 (20-1 00) muddy sand 1

Benthic Rhizopods

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Fig.2. Water temperature and salinity at station Bak.

Different subsampling schemes were used in order to allow a comparison of rhizopod data with data on other protistan groups which were obtained from the same samples by live-counting as part of two independent sampling programs for station Bak and the other two stations. Since the aerobic sediment layer usually extended down to a depth of 3-5 mm [26], the 0-3 mm subsamples from station Bak and the 0-5 mm subsamples from stations Rassower Strom and Kirrbucht represented the oxic sediment stratum whereas the 5-10 mm subsamples from the latter stations represented the upper anoxic stratum. Sediments deeper than 10 mm were not sampled because preliminary sampling yielded negligible abundances of rhizopods in these generally anoxic sediments . Taxonomic composition of the rhizopod community: The morphotype composition of the sediments was determined in fresh samples and in enrichment cultures under a Zeiss Axioplan microscope with phase contrast and interference contrast optics. Enrichment cultures from approximately 1 ml of the diluted subsamples were incubated at 18 °C in 7.5 ml modified Feyns-Erdschreiber-medium (MErd, [39]) for 10-20 d. Cell dimensions (length and breadth of cells, diameter of nucleus and nucleolus, proportion of hyaloplasm and granuloplasm) were measured with an ocular micrometer under 400x magnification . Thickness of attached rhizopods was measured with a calibrated stage drive (interval 1 urn),

105

20-50 cells of each species were measured, depending on their frequency of occurrence. Specific individual biovolumes were estimated from cell dimensions (without subpseudopodia) applied to geometric models (Table 3). In addition to these parameters, the speed and locomotive behaviour of moving cells and the morphology of floating forms were recorded . Whenever possible, modern identification keys [39,40,41] or original descriptions were used for the identification of rhizopods. Enumeration of rhizopods: For the enumeration of amoebae, a modified version of the liquid aliquot method (LAM) [15,57] was employed. 36 aliquots (10 pi) from the enumeration subsample of each core were inoculated into the wells of tissue culture plates (Falcon) containing 2 ml of MErd-mediurn. The plates were incubated in the dark at 18 °C and screened for the presence of rhizopod morphotypes after 12 and 24 days under an inverted microscope (Nikon Diaphot) at 200x magnification. The abundance of rhizopods of each morphotype in the subsamples was estimated from their frequency of occurrence in the wells assuming a Poisson distribution: P(k) = A.k I k! r.. e-A with A.= N IT where P(k) is the probability of a culture to originate from k cells in the inoculum, A. is the parameter of the Poisson distribution, N is the actual number of cells in the inoculum, and T is the number of cultures . Although not tested explicitly, the Poisson distribution is the most conservative assumption if on average there are only few cells per well [58]. Based on this distribution, expected frequencies of cultures originating from one, two and more inoculated cells were calculated for different cell abundances in the inoculum. From the proportion of cultures originating from one, two or more cells, it was possible to calculate a calibration curve relating the number of cultures in which a species was found to the number of cells of this species in the inoculum. Cell abundance (N) for each sample was calculated from the number of cells in the inoculum according to the following formula :

N [Ind. crn']

=(N ::. fd ::. 1000) I (36 ::. Va[pi])

where N is the number of cells in the inoculum, fd is the factor of sample dilution, and Va is the inoculum volume pipetted into each of the 36 wells.

Table 2. Geometric models and formulas for biovolume calculations from cell dimensions of rhizopods. Subpseudopodia were not considered for biovolume calculations . d... diameter of cell; d gp... diameter of the granuloplasmatic hump; h = height of cell; he = height of cylinder; h, = height of spherical segment; 1 = length of cell; l, = length of semi-cylinder; V = volume of cell; w = width of cell. Rhizopod group

Shape

Remarks

Formula

Limax amoebae, Stygamoeba Thecamoebidae, Paraflabellula Paramoebidae, Vexilliferidae

Semi-cylinder Spherical segment

Ie =0.8 ::' 1

V =0.5 1t r.. h2 ::. l,

Chrysamoeba, Massisteria

Vannellidae, Cochliopodidae Aconchulinida, Diplophrys

Phryganella

Testaceafilosia, Granuloreticulosea

Flat cylinder with ovoid cross-section Flat cylinder with ovoid cross section + spherical segment Sphere Hemisphere Rotation ellipsoid

V = 1t I 6 ::. h ::. (0.75 r.. J2 + h 2) V =0.25 1t ::. 1::. W

Flat ellipse: he =0.5 ::. h Spherical segment : h, =0.5 ::. h and d = d gp Dimensions of cytoplasm only Dimensions of cytoplasm only Dimensions of cytoplasm only

:f

h

V =0.25 1t ::. 1::. w ::. he + 1t I 6 ::. h, ::. (0.75 ::. d g/ + h/)

V = 1t I 6 ::. d3 V =1t I 12 ::. d 3 V =1t I 6 ::. I ::. h ::. W

106

T. Garstecki and H. Arndt

Methodological investigations: The potential problem of an overestimation of vegetative cell abundance due to excystment during incubation was addressed in three ways: 1) The taxonomic resolution of the analysis allowed a discrimination between cyst-forming species and others without a documented ability to form cysts. 2) At one sampling occasion (station Bak, 12 September 1996), parts of each subsample were kept in seawater-HCl (2% final concentration) for 12 h to kill vegetative cells. This procedure is commonly used in studie s of soil protozoa [10, 54]. After neutralization, the pretreated subsamples were incubated and scored as usual. All cells found in these cultures were attributed to excystment. 3) From the crude enrichment cultures described in the previous section, strains of the common potentially cyst-forming species were isolated . These cultures were subjected to a variety of adverse conditions : gradual heating to 35 °C and subsequent desiccation, gradual cooling to 5 °C and starving conditions, gradual (within 14 d) decrease of salinity to < 2 PSU, and gradual (within 3 d) decrease of pH to < 3. They were regularly screened for the presence of cysts during the follow ing 21 d. Subsequently, standard conditions were restored and those cultures where no vegetative cells had been found after 21 d were checked for their reappearance after another 7 d.

Results

Abundance and biovolume totalsat station Bak In the superficial sediment layer of station Bak, total rhizopod abun dances ranged from 2,800-10,900 cells ern" (Fig. 3). The corresponding biovolume estimate ranged from 5.1-28.5 :, 106 pm 3 crrr". Maximal abundances were found in late summer. Rhizopod biovolurnes peaked in spring and summer. Apart from a relative decrease between Ap ril and July 1996 tha t was caused by a shift of biovolume dominance from cells> 40 pm to cells < 30 pm equal spherical diameter (data not shown), biovol ume estima tes generally showed similar trends as rhizopod abundance.

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Fig. 3. Abundance and biovolume of rhizopods in sediment samples (0-3 mm) at station Bak. (Error bars: 1/2 range, N =2).

Taxonomic composition of the rhizopod fauna During the whole investigation, 46 amoeboid morphotypes were found, 27 of which could be attributed to described species (Table 3). These organisms represented a variety of taxonomic groups including Schizopyrenida (2 morphotypes), Gymnamoebia (19), Testacealobosia (6), Aconchulinida (4), Testaceafilosia (4), the G ranuloreticulosea (2), and Protista incertae sedis (4). Accordingly, the rhizopod fauna consisted of several feeding types [45] and occupied a broad size range. Individual biovolumes ranged from 20-45. 000 prrr' . Expressed as equivalent spherical diame ter, most of the rhizopods were in the range of 10-20 pm. Both marine and typical freshwater rhizopods occurred in the study area, but no shift towards dominance of the latter was found at the less saline Kirrbucht station or at station Bak when salinity was low (see Fig. 2). Apart from a few rare species, most rhizopods were found at all sampling stations. While only a small minority of the species identified are known to encyst, some of the forms that could not be identified to species belong to genera which include at least some cyst-forming species (e.g. Vahlkampfia, Hartmannella, Cochliopodium, and members of the Testaceafilosia) . In addition to the species listed in Table 3, a number of unidentified limax amoebae (amoebae with an elongate, semi-cylindrical cell shape), Vannellidae, Paramoebidae, Granuloreticulosea, and small « 5 prn) or non-motile, naked rhizopods were found. Among the testate rhizopods, empty tests of a number of marine interstitial forms (e. g. Pseudocorythion spec., Micropsammella spec.) were recorded.

Contribution of taxonomic groups The main taxonomic groups contributed differently to the combined abundance and biovol ume of the rhi-

zopod community, and they showed different seasonal trends. Whereas Gymnamoebia and Schizopyrenida dominated abundances (49 and 22% mean abundance contribution, respectivel y), larger Testaceafilosia (mainl y Cyphoderia ampulla and Trinema lineare), Testacealobosia (mainly Phryganella spec.), and Aconchulinida (mainly Lithocolla globosa) dominated biovolume totals during the first part of the sampling programme (Fig. 4). During late spring and summer, biovolume dominance shifted towards the Gymnamoebia and Schizopyrenida. Granuloreticulosea were generally of minor importance « 1% mean biovolume contribution). Stygamoeba polymorpha (Protista incertae sedis) reached abundances of up to 1030 cells ern? and thus was the only rhizopod of uncertain taxonomic position which contributed significan tly to th e rhizopod community. The contribution of taxonomic

Benthic Rhizopods

107

Table 3. Commented list of those morphotypes identified to species or genus. Reference to the identification literature is made because most taxonomic keys do not include all described species. n. det. = not determined; F = freshwater; M = marine; C =common (present in most samples); R = regularly (present in > 10% of samples); 0 = rare (present in < 10% of samples). Species

Biovol.

[pm']

Typical habitat

Cysts

Occurrence

Identification

X X

C R R

[39] [39] [41] [41] [41] [39]

Previous Baltic records

150 1,100 120 n.det. 13,400 n. det .

M F/M F F F/M M

480 1,300 150 4,200 980 1,300 110 210

M M M F F F/M M F/M

C R C R R R C

[38] [38] [39] [41] [41] [39] [49] [39]

720 5,600 670 110 300

M M F/M M F/M

R C R R

[50] [39] [39] [39] [39]

600 120 n. det,

M M F

0

[42] [50] [41]

[e.g.52]

970 480 90 1,100 n. det, 33,000

F F F F F F

[9,40] [40] [40] [40] [29] [14]

[e. g. 60]

n.det. n.det. 11,000 n. det .

F F/M F/M F

X

310 3,500 45,000 n.det.

F F/M F/M F/M

X ? ? ?

80 240

F F/M

n.det. 70

M F

Limax amoebae

Vahlkampfia damariscottae Vahlkampfia spec. Hartmannella vermiformis Cashia limacoides Saccamoeba spec. Rhizamoeba spec.

0 0 0

Thecamoebidae & Vannellidae

Thecamoeba orbis Thecamoeba hilla Vannella aberdonica Vannella simplex Vannella platypodia Vannella spec. Platyamoeba langae Platyamoeba spec.

0

[60] [60] [60] [60]

Paramoebidae & VexillHeridae

Mayorella smalli Mayorella gemmifera Mayorella (Dactylamoeba ?) spec. Vexillifera minutissima Vexillifera spec.

0

Other naked lobose amoebae

Paraf/abellula reniformis Stygamoeba polymorpha Acanthamoeba spec.

?

X

C

0

Testacealobosia

Cochliopodium bilimbosum Cochliopodium minus Cochliopodium spec. Gocevia spec. ? Arcella vulgaris Phryganella spec. ? Aconchulinida

Nuclearia spec. Pinaciophora rubicunda Lithocolla globosa Arachnula spec. Testaceafilosia Lecythium spec. ?

Trinema lineare Cyphoderia ampulla Paulinella chromatophora

X ? ? X

0 C R

0 0

R

X

0 0

R

0

[41] [41] [41] [41]

R R R

0

[12] [29] [29] [29]

R R

[35] [14]

0

[21] [30]

[e.g.27]

[47] [53]

[e. g. 27] [e. g. 27] [43]

Granuloreticulosea

Gymnophrys cometa Apogromia spec. O t her amoeboid protists

Diplophrys marina Chrysamoeba radians Massisteria marina

20

M

R 0

[44]

[32]

108

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Fig. 4. Abundance and biovolume of major taxonomic groups of rhizopods in sediment samples (0-3 mm) from station Bak, (Error bars: 1/2 range, N =2).

gro ups to the rh izopod community at Rassower Strom and Kirrbucht was similar to that of station Bak except for the fact that G ranuloreticulosea and Aconchulinida were of minor importance (Fig. 5). Gymnamoebia were the most important group in terms of species richness and abundance. Vannellidae and Paramoebidae were the dominant gymnamoebian fami lies (Fig. 6). They reache d mean abundance contributions of 47% and 36%, respectively. The T hecamoebidae were present in all samples at low abundance (6%

mean contribution), whereas abundance contributions of Vexilliferidae and Flabellulidae (the latt er rep resent ed by Paraf/abellula reniformis onl y) were low during spring and summer and increased in autumn (4% and 1% mean contribution).

Methodological investigations Of the spec ies identified as po tential cyst-formers (Tab. 2), on ly Vahlkampfia spec., Stygamoeba polymer-

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11 0

T. Garstecki and H. Arndt •

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~ Paramoebldae

100 ~ 80

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60

:::J

go

iii

40

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20

~ Sediment (0-5 mm) 100

Fig. 6. Relative contrib ution of important families to the abundance and biovolume of Gymnamoebia in sediment samples (0-3 mm) from station Bak,

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pha, Cochliopodium minus and the Testaceafilosia contributed more th an 5% to total rhizopod abundance or biovolume in any samples. Their combine d contribution to total rhizopod abundance an d biomass in samples fro m station Bilk is shown in Fig . 7. This contribu tion was wi thi n the same range at stations R assow er Strom and Kirrbucht (data not shown). Stygamoeba polymorpha contri buted up to 25% to total rhizopod abundance. Testaceafilosia dominated the potentially cyst-forming fraction of the rhizopod fauna in terms of biovolume. In three of the 48 subsamples treated with HCI prior to inoculation on 12 September, 1996, Acanthamoeba spec. was fo und after incubation. No other rhizopod species was found. Acanthamoeba was never detected in any of the untreated enumeration cultures. It should be noted that relatively high biovolu m e contributions of potentially cyst-forming species were found at this sampling occasion (Fig. 7). When cultures of Acanthamoeba spec., Stygamoeba polymorpha, Cochliopodium minus, and Vahlkampfia spec. were subjected to encystment conditions, only Acanthamoeba spec. p roduced clearly dis ti nguis hable res ting cysts an d reappeared in treatments w here no vegetative cells were found after 21 d . C ult ures of

iii

20 0 r--

j

Jul .

Sept.

Jan.

Apr.

Fig. 8. Con tribution of herbivorous and omnivoro us species (according to [45]) to the combined biovolume of Gymnamoebia in the flocculent surface layer and in two sediment layers at station Rassower Strom. Data from all campaigns during which all three layers were sampled are included.

Cochliopodium minus always contained a high proportion (average 90%) of inactive, rounded cells without visible cyst walls or cytoplasmatic irregularities, but these cells did not enable the cultures to recolonize those treatments (pH < 3 and heat) where no vegetative cells were found after 21 d . The same was true for inac tive, ro unded cells of Vahlkampfia spec. which were only observed in acid and heat treatments. These cells did not resemble the typical vahlkampfiid cysts that were reg u larly fo und in stock cultures of Vahlkampfia spec.. C ultures of Stygamoeba polymorpha d id not form cysts or cys t- like cells and irreversibly d isap peared in acid and h eat treatments.

Benthic Rhizopods

Discussion Quantitative importance and distribution of rhizopods Our results show that rhizopods form a numerically important component of the micro- and nanozoobenthos of the study area, and probably of shallow coastal waters in general. Our findings agree with recent data from other coastal waters which were obtained usin g the same methods: mean annual abundances of 870 and 2,200 cells cm' (G ymnamoebia and Schizopyrenida only) were reported from sand y and silt y sediment s, respectively, of the Scottish Cl yde Sea [IS). Abundances of naked rhizopods of 1,020-45,600 and 17,600-40,600 cells g' sediment were found in sediments of Hiroshima Bay and of marine and brackish habitats at Bermuda, respectively [2, 19]. These figures are even high er than ours: expressed on a weight basis, our abundances would have been in the range of 5,600-21,800 cells g-l because sediment density at station Bak was approximately 2 g cnr", A comparison of rhizopod biovolume to those of ciliates and heterotrophic flagellate s shows the relative importance of rhizopods in the study area (Table 4). The latter groups were quantified in the same sediment samples as rhizopods [20). On average, rhizopods made up 45% of the total protistan biovolume in the superficial sediment layer of station Bak, The abundance data given in this paper refer to one station and one sediment layer only, and only to a limited part of the seasonal cycle. However, the abundance figures from two other stations in the study area, Rassower Strom and Kirrbucht, generally agree with those of station Bak (Garstecki & Arndt, unpublished results). In addition, the y allow some extrapolations: While similar abundance ranges were found in the superficial sediments, ran ges were wider and maximum abundances were higher in the flocculent surface layer. Maxima of 24,000 cells crrr" (Rassower Strom) and 17,000 cells crrr" (Kirrbucht) were found in spring 1996. They may have been a response to phytoplankton sedimentation, as found in earlier studies in the same area [5]. The 5-10 mm layer abundances at Rassower Strom and Kirrbucht tended to be lower and less variabl e than those in the sup erficial sediment layer. In

111

agreement with the low rhizopod densities found at station Bak in March and November, very low abundances of rhizopods occurred during winter in all sediment samples from Rassower Strom and Kirrbucht, In conclusion, a tendency towards lower and less variable abundances deeper in the sediment and a seasonal trend with maxima during the warm season emerges from the combined data of all three stations. A similar vertical distribution, but no consistent seasonal pattern was reported previously from the Cl yde Sea, Scotland [15]. Studies on the seasonality of benthic heterotrophic nanoflagellates have yielded differing patterns as well : While summer maxima and winter minima of benthic flagellate abundances were found in our study area and at the majority of stations investigated during a study of North Sea sediment s [20, 31], no such pattern wa s observed in a freshwater littoral zone [59). The seasonal trend of benthic abundances that was found in our study area suggests that the benthic microbial food web in such shallow waters directly responds to seasonal variations of organic input from the water column.

Rhizopod community structure The benthic rhizopod community of our study area comprises representatives of all major rhizopod groups and of freshwater as well as marine species . Only 13 of 27 morphotypes identified to species have been reported from th e Baltic before (Table 3). Three species (Hartmannella vermiformis, Cashia limacoides, and Chrysamoeba radians) have been found in freshwater habitats only, and three marine species (Platyamoeba langae, Stygamoeba polymorpha, and Diplophrys marina) are new to Europe. The difference in salinity between stations Bak and Rassower Strom (8-10 PSU) and station Kirrbucht (5 PSU) had no influence on the faunal composition of rhizopods. G ymnamoebia were the species-richest group at all stations, followed by Testacealobosia. However, in addition to the two Vahlkampfia-species, some of the unidentified limax rhizopods showed eruptive movement and thus may belong to the Schizopyrenida as well. Large testate rhizopods are probably undersampled with the liquid aliquot method, which may explain the low species yield of this group in our study as compared to other

Table 4. Contributions [%] of rhizopods, ciliates and heterotrophic flagellates to total heterotrophic protistan biovolume in sediment samples (0-3 mm) from station Bak, Ciliates and heterotrophic flagellates were enumerated by live-counting [20].

Rhizopods Ciliates Flagellates

17. Mar.

13. Apr.

27. Apr.

13.May

33 7

82

64 9 27

45

60

8 10

9 46

25. May

10.Jun.

24.Jun.

39

27 41 32

23 31 46

29 32

112

T. Garstecki and H. Arndt

faunistic surveys [e. g. 27]. The same is true for foraminiferans which were occasionally found in fresh samples. Although many rhizopod species are easily recognizable under the light microscope, the taxonomic analysis was limited by the methodology to our disposal. The genera VannellalPlatyamoeba and Mayorellal Dactylamoeba were distinguished based on the morphology of floating and locomotive cells only. Although there are well-established criteria for this kind of distinction, electron-microscopical examination of the cell surface is considered necessary to reach a final conclusion [e. g. 41]. If these strict criteria would be applied, our species identifications within the above mentioned genera would only be preliminary. Generally, taxonomy of marine naked rhizopods is still a growing field, and a variety of new species have been described during the last few years [e. g. 3, 55, 56]. In the light of this situation it appears likely that some of the morphotypes that could not be identified to species during our study were actually undescribed species. The Cochliopodidae (Testacealobosia), another group that is quantitatively important in the Baltic, have received little attention since the Seventies [9,37]. The sometimes high biovolume contributions of filose amoebae and Testacealobosia found during our study indicate that these groups are equally or even more important than naked lobose amoebae in Baltic coastal sediments. Since most of the Testacealobosia and Aconchulinida found were brackish or freshwater species, their high contributions may be a peculiarity of the brackish Baltic community. In contrast, a speciesrich fauna of testate rhizopods (excluding foraminiferans) has been reported from the int erstitial of other coastal waters as well [e. g. 27, 36]. Further research into their quantitative importance and functional role appears warranted. The fact that different taxonomic groups showed different seasonal trends during our study (Fig. 4-6) suggests that they respond specificly to environmental conditions such as available food resources. The high biovolumes of Testacealobosia and Aconchulinida in spring were comprised of many large and herbivorous species which may have fed on sedimenting phytoplankton [see 45]. They were the main cause of the exceptionally high biovolume contributions of rhizopods to the benthic heterotrophic protistan community in the spring samples. The taxonomic resolution of earlier quantitative studies on rhizopods has been restricted to the distinction between broad morphological categories. The high contribution of Vannellidae and Paramoebidae to the abundance and biovolume of Gymnamoebia in our samples is in agreement with the dominance of amoe-

bae with subpseudopodia and discoidal or fan-shaped amoebae within the rhizopod communities in sediments of Hiroshima Bay (japan) and of some inshore aquatic habitats at Bermuda [2, 19]. Compared to the above mentioned studies, Hartmannellida were conspiciously rare in our samples . In contrast to earlier findings [15, 19], Gymnamoebia or Schizopyrenida smaller than 10 pm contributed little to total rhizopod abundances or biovolume although they were regularly found . However, our sediments were coarser than most of those sampled during the above mentioned studies and the presence of a highly productive microphytobenthos in these shallow waters may have shifted the rhizopod community structure towards larger herbivorous and omnivorous forms [see 26, 45]. Although the liquid aliquot method selectively favours bacterivorous species, we found high contributions of non-bacterivorous Gymnamoebia throughout the study, and especially at the sediment surface (Fig. 8). The high contributions of non-bacterivores within the rhizopod community suggest that rhizopods fulfill various trophic roles within the benthic microbial food web, rather than being restricted to bacterivory.

Methodology Rhizopods were also included in earlier quantitative accounts of the marine micro- and nanozoobenthos [e. g. 1,5,7,23,25,34]. Except Mare [34] who used serial dilution cultures and estimated abundances of 2,300-6,800 cells g-t in muddy subtidal sediments of the British Channel, most of these workers reported very low rhizopod densities. This was probably due to the enumeration methods employed: Naked rhizopods are hardly visible in fresh sediment samples because they are often closely surface-associated and move relatively slowly. After fixation, they tend to assume irregular body shapes, or to round up [46]. Therefore, they are easily overlooked or misidentified in live-counted or fixed samples . Cultivation methods, particularly the liquid aliquot method which yielded higher abundances than the MPN method [54] and plate counting methods in direct comparison [15], stand out as the most appropriate enumeration methods for marine naked rhizopods. According to our results, the liquid aliquot method is suitable for sediments from brackish waters as well. Al though a number of rhizopods with a potential ability to form cysts was found in our samples, the results of our encystment trials do not indicate that excystation during incubation contributed significantly to the abundances determined by LAM. The same appears to be true for the larger Testaceafilosia (R. MEISTERFELD, pers . comm.). This is relevant from the methodological

Benthic Rhizopods

point of view because a high rate of excys tme nt would have caused ove restima tio ns of vegetative cell abundance in th e inoculum. However, some lim itations of th e method have to be considered in order to interpret th e results properl y : Our abundan ce figures are likel y to be minimum estimates of th e actual abunda nces becaus e any clumping of sedime nt and assoc iated rhizop od s in th e inoc ulates lead s to underestimat ions of actual abunda nces und er the assumption of a Poisson distributio n, an d becau se th e culture co nd itio ns we re certa inly not app ro p riate for all spec ies in th e inoculum. For example, th e hartmannellid rhizopod Cashia limacoides was fr equentl y observed in fr esh samples, but never in LAM cultures. The same may have been true for lar ge herbivorous and omnivorou s speci es although so me of th em regularl y gre w in the cultures, possibly feed ing on - and cont rolling - bacteri vorou s protists. Withou t th ese methodolo gical shortcomings, abundance and biovolume co ntributions of rhizo pods to the mic ro- an d nanozoob enthos at sta tio n Bak w ould have been even higher. In order to judge th e relative importa nce of taxa of differing siz e, we calcul ated biovolumes from live cell dimensions. The accuracy of this approach is limited by the vari ability of cell morphology and th e accuracy of the cell th ickness estima te. Biovolume sta nda rd deviations in th e ord er of 20-30% we re frequ ently observed. However, th e range of rhizopo d sp ecies biovolu mes (20-45.000 prrr' ) found du ring thi s study was sufficiently wi de in co mparison to th e erro rs in biovolume estimation to allow some general comparison s on th e basis of live cell dimensi ons. Alternative meth od s which use fixed cells [e.g. 46] are prone to cell shrinkage and loss upon fixation, given w hat is known from ciliates [18]. The advance of laser- scanning mic ro scop y ma y help to solve the problem of rhizo po d biovolume estima tio n in the future. In spite of th e method olo gical p roblems involved in th e bio volume estima tion, quanti fication, and communit y ana lys is of rhizopod s, it is o bvious that th ey fo rm a significant co mponent of protistan co mmunities in co astal sediments. Work is in progress in our laboratory to invest igat e the ir fun ct ional role within the benthic microbial food web. Acknowledgements: Th is research represents partial fulfilment for th e PhD requirements of Tobias Garstecki. Part of the study was carried out within the interdisciplinary "O EKO BO D " project supported by the Federal Ministry for Edu cation and Research. We wish to th ank No rbert H iilsmann, Ralf Meisterfeld, and Alexander P. Mylnikov who gave useful taxonomic advice, Andrew Rogerson for his introduction to th e liquid aliquot method, and Stephen A. Wickham and two anonymous reviewers for helpful comments on th e manuscript.

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