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European Journal of Protistology 47 (2011) 224–230

ynamics in space and time of four testate amoebae (Difflugia spp.)o-existing in the zooplankton of a reservoir in southern China

o-Ping Hana, Tian Wanga, Lei Xua, Qiu Qi Lina, Henri J. Dumonta,b,∗

Institute of Hydrobiology, Jinan University, Guangzhou 510632, ChinaDepartment of Biology, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium

eceived 30 December 2010; received in revised form 18 April 2011; accepted 18 April 2011

bstract

We studied a long time series of the dynamics in space and time of four species of Difflugia (thecamoebae) that co-exist in theelagic plankton of Liuxihe Reservoir, an oligo-mesotrophic impoundment in southern China, during 8–9 months (“summer”orm March to November), and retreat to the benthos during the rest of the year (“winter”). We discuss the reasons for the winteretreat, and suggest that predator evasion may be involved, although temperature-linked physiological effects (like the rate ofas bubble production) appear more probable. Clear diel vertical migration of Difflugia was not observed, but patchiness was

ommon. We found no evident lake edge-effects in the spatial pattern either, but the abundances were strongly influenced byrophic conditions and increased by up to one order of magnitude in the upstream, eutrophic sections of the reservoir.

2011 Elsevier GmbH. All rights reserved.

bae; T

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eywords: Zooplankton; Lake trophy; Pelagic space; Testate amoe

ntroduction

The pioneers of limnology in the late 19th centurylready knew that testate amoebae of the genus Difflugiat times make up a sizeable fraction of the limnetic plank-on of European lakes (review and references in Schönborn962; Hutchinson 1967; Arndt 1993). Not much was addedo this qualitative body of knowledge until Schönborn1962) detailed the seasonal cycle of Difflugia hydrostaticaacharias, 1897 in Lake Stechlin and adjacent lakes, Ger-any, and Meisterfeld (1991) studied the vertical distribution

nd migration of Difflugia hydrostatica (of which D. limnet-ca Levander, 1900 was declared a junior synonym by Ogdennd Meisterfeld 1989) in two sand pits in the lower Rhine val-

∗Corresponding author at: Department of Biology, Ghent University,edeganckstraat 35, B-9000 Ghent, Belgium. Tel.: +32 92645253;

ax: +32 53684708.E-mail address: Henri.Dumont@ugent.be (H.J. Dumont).

toaiflV1e

932-4739/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.ejop.2011.04.003

ime series; Vertical distribution

ey, Germany. In spite of these two studies, the importancef pelagic testates continued to be ignored by most workers,ho kept concentrating on the “big three” of the zooplankton,

he rotifers, copepods and cladocerans.From the tropics and subtropics, records are even more

necdotal; the thrust of many older papers is largelyaxonomic-faunistic, and the ecology is reduced to someotes on natural history at best. Thus, many brief suggestionsn the occurrence of some species in pelagic environ-ents may be found, but little substantial information. The

nly country where tropical–subtropical testate communi-ies have been well studied is Brazil. After an initial phasef exploratory work in the early and middle 20th century,n impressive suite of papers was published, mainly deal-ng with the tropical–subtropical Upper Parana River and itsoodplain (Lansac-Tôha et al. 2000; Lansac-Tôha et al. 2001;

elho and Lansac-Tôha 1996; Velho et al. 1996; Velho et al.999; Velho et al. 2001; Velho et al. 2003; Lahr and GomezSouza 2011). Somewhat earlier, Green (1975) had com-

nal of Protistology 47 (2011) 224–230 225

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B.-P. Han et al. / European Jour

ented on rhizopod communities found in various watersf the Matto Grosso area. He still thought that many of theccurrences in the pelagic zooplankton were accidental, butansac-Tôha et al. (2004) convincingly showed that testatesere not only consistently present in the pelagic of lakes,ut often were second in importance in the community to theotifers only.

In East Asia, the phase of taxonomic descriptions is not yetver, but it is clear that several regionally endemic Difflugiaccur in the basins of the Yangtze and Pearl Rivers (Qin et al.011). At least two of the four Chinese species dealt withn the present paper have, for example, been described onlyecently (Hu et al. 1997; Yang et al. 2004; Yang et al. 2005;ang and Shen 2005). In addition to showing a restricted

ange, these species appear to be planktonic rather than lit-oral or benthic.

In spite of all recently accumulated knowledge on testatemoebae in lake zooplankton, including the species foundn China, there is still a dearth of long time series on theirynamics, and such information is all but lacking from thearm belt of our planet. If temperature were the controlling

actor, it could be predicted that the long winter gap in theimnetic appearance of Difflugia found by Schönborn (1962)n Germany would gradually shorten and finally disappearnder tropical conditions. We set out to test this assumptionn Liuxihe Reservoir, a more than 50-year-old impoundmentn Guangdong Province, South China, over a time period of 6ears. We also made observations to ascertain that the speciesnvolved are limnetic indeed, by looking at their vertical andorizontal distribution, and by examining the benthos duringinter.

aterial and Methods

iuxihe reservoir

The reservoir on Liuxi River (=He), an affluent of the Pearliver, is situated at ca. 235 m asl, at 23◦45′N, 113◦46′E, at

he confluence of two tributaries, the Yuxi and the Lutiantreams just north of the tropic of Cancer (Fig. 1). It has aarm-temperate to subtropical climate, with a strong influ-

nce of the summer monsoon. Photoperiod does not varyuch, but the amount of sunshine and precipitation received,

nd temperature show important yearly amplitudes. About5% of precipitation, on average some 1500 mm per annum,s concentrated in the “monsoon months” May till Septemberet may vary by a factor two or more between years. Wateremperature at the surface of the reservoir fluctuates between2 ◦C and 30 ◦C, that of the atmosphere between ca 6 ◦C (Jan-ary) and 35 ◦C (July). The shape of the reservoir is typically

endritic-elongate with the main channel deep and canyon-ike and depth maximum 73 m when full and at the foot ofhe dam. Surface area is ca 15 km2, and average depth is ca1 m. The reservoir provides drinking water and electricity,

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ig. 1. Situation and map of Liuxihe Reservoir, Guangdong, China,ith the sampling stations indicated.

nd its water level is continually changing according to theeeds of its clients.

amples for temporal and spatial dynamics

In all, five stations (S1–3, S5, S7) were surveyed (Fig. 1);amples for dynamics were mainly taken at S7 (near the dam),t monthly intervals, from January 2006 till December 2009.etween February 2004 and April 2006, similar samples wereollected, but studied for D. tuberspinifera Hu et al., 1997nly, so that for this species 6 years of observations are avail-ble. All samples were compounded over the entire waterolumn by pumping water to a 5-l sampler, at 2-m intervalsrom 0.5 m to 20 m. A single sample therefore represented5–60 l in all, but all results are expressed as specimens peraverage) litre. The water was filtered through a 50 �m meshlankton net and samples were preserved in formalin anddentified and counted under the microscope. In 2009 (Aprilill October), two series of stations were surveyed, one in theiverine shallow and narrow (width 50–100 m) upper part ofhe reservoir (S1–3), and another (stations 5 and 7) in theower, lake-like part of the reservoir (Fig. 1). The depth at7 (when the reservoir is full) is about 55 m; at S1–3, it was5–17 m.

ertical samples

Vertical distribution and, if present, migration werehecked on several occasions in 2009, e.g. on 15 and 29ugust, by a team equipped with two samplers for pump-

ng water from a series of layers between the surface and2-m depth so as to obtain all vertical samples in the short-st time possible. Samples were taken at four metre intervals

0 m, 4 m, . . ., 32 m) at 00:00, 6:00, 12:00 and 18:00 h. On9 July, another cycle had been performed to specificallytudy D. tuberspinifera. The sampling depths were the same

2 nal of Protistology 47 (2011) 224–230

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Fig. 2. Temporal dynamics of Difflugia tuberspinifera from 2004t

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26 B.-P. Han et al. / European Jour

s before, but six instead of four samplings were performedcross 24 h. From each depth, a total of 30 l were pumpedp and filtered through a 50-�m mesh plankton net; samplesere preserved in formalin and identified and counted under

he microscope.

ittoral-pelagic transect

On August 12th 2008, at a site close to Station 5 (S5), aeries of samples was taken at increasing distances from thehore: 1 m, 3 m, 5 m, . . ., 21 m, the latter being part of theelagic water column. At each site, samples were taken fromurface to bottom at intervals according to depth. At eachampling site, around 30 l of water were thus pumped up andltered through a 50-�m mesh plankton net. Two sampleeries were collected, one in the morning at ca 9:00 am, andne in the evening at around 7:00 pm.

enthic phase

On 12 November 2010, we ascertained the winter retreatf Difflugia to the lake bottom by qualitatively sampling theurface of the sediment at Station 3 (S3), having first verifiedhat not a single specimen was left in the plankton. We used50-�m mesh-size plankton net that was gently lowered to

he sediment surface, just enough to stir up and collect theop, largely organic fraction. The samples were taken to theaboratory at once, and examined alive under a compound

icroscope.

esults

elagic Difflugia

At least 6 species of Difflugia have been recorded fromhe pelagic space of Liuxihe Reservoir, viz Difflugia tuber-pinifera Hu et al., 1997, Difflugia biwae Kawamura, 1918,ifflugia mulanensis Yang et al., 2005, Difflugia hydrostaticaacharias, 1897 (a smaller species that first became common

n 2008), Difflugia cf ampullula Playfair, 1917 and Difflugiaf gassowkii Ogden, 1983. The first four of them occurred inumbers sufficient for further analysis.

At S7, all four showed a cycle that was recurrent in itseneral aspect: amoebae first appeared in spring (March,ometimes February) and disappeared in autumn (Septem-er to November) after a peak in summer (April to August)Figs 2, 3). In the details of the cycle, considerable vari-tion occurred in both the timing of the yearly peak, andhe abundance of the two most common species, D. tuber-pinifera and D. biwae. The peaks of both taxa tended to

e shifted somewhat in time, but on the whole, there waso clear temporal segregation. Amoebae start disappearinground 16 ◦C and reappear at ca 12 ◦C. In 2009, the sum-er peak was somewhat anomalous and occurred as early

FD

ill 2009 at station 7, Liuxihe Reservoir.

s April. Had no data for D. tuberspinifera, been availableor 2004–2005, it would have seemed as if the species wasecreasing in abundance over the years. Now, it rather looksike it was exceptionally abundant in 2006. However, a com-letely different pattern emerged at the upstream stations in009 (Fig. 4). Summer peaks here were up to an order ofagnitude higher than in the main reservoir. Furthermore,

nly three species were present during the first years. Dif-ugia hydrostatica appearing in numbers only in 2008 and009.

nfluence of distance from the shore

Testing for a possible effect of the shore and/or lake bot-om as a permanent source of Difflugia in the water column,e carried out a 1-day sampling in mid-summer (12 August008), with one sampling run in the morning, and another onen the evening (Fig. 5). Two species (D. biwae and D. hydro-tatica) were sufficiently common to be analysed further. Weound no significant trend, i.e. nearness of the shore or lakeottom did neither increase nor decrease the abundance of

ig. 3. Temporal dynamics of Difflugia biwae, D. mulanensis, and. hydrostatica at station 7, Liuxihe Reservoir, from 2006 till 2009.

B.-P. Han et al. / European Journal of Protistology 47 (2011) 224–230 227

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Fig. 4. Temporal dynamics of four pelag

ertical distribution

On 19 July 2009, the vertical distribution of D. tuber-pinifera (Fig. 6) was almost perfectly random throughout

ig. 5. Horizontal distribution of Difflugia hydrostatica and D.iwae at increasing distances from the shore.

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ugia at five sations in Liuxihe reservoir.

he diel cycle: the average catch was 16 specimens per litrever any vertical sample series, and 15.53 per depth levelcross time. The number of specimens per individual samplel) varied from 3.5 (midnight, 20 m) to 35 (8 am, 8 m).

On 15 August 2009 (Figs 7–9), there was evidence ofatchiness, with total numbers being higher at 6 am (first sam-

ling run) than at any time afterwards in D. tuberspinifera and. hydrostatica, and at 12 pm in D. biwae. There was further

vidence for a slight concentration of all species in or near

ig. 6. Diel vertical distribution of Difflugia tuberspinifera in Liux-he on 19 July 2009.

228 B.-P. Han et al. / European Journal of Protistology 47 (2011) 224–230

Fig. 7. Diel vertical distribution of Difflugia hydrostatica in LiuxiheReservoir on 15 August 2009 (distribution on 25 August not shownbecause similar). Average abundance at 6 am was 21 individuals perlitre, at 12 am 6 per litre.

Fig. 8. Diel vertical distribution of Difflugia tuberspinifera on 15and 25 August 2009.

Fig. 9. Diel vertical distribution of Difflugia biwae on 15 and 25A

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ugust 2009 (D. mulanensis not shown because similar).

he 8-m stratum. On the whole, there was no vertical segre-ation and no sign of diurnal vertical migration in any of themoeban species.

On 25 August 2009, evidence for patchiness was eventronger (first sampling run) in three out of the four species,ith again a concentration at and around 8 m (Fig. 9), except

n D. mulanensis (data not shown) that tended to aggregatet the surface and somewhat less below 8 m, thus creating airror-image of the other three species. Again, Difflugia did

ot perform any diel vertical migration.

enthic phase

D. tuberspinifera, D. biwae, and D. mulanensis were abun-ant in the benthos. Most individuals were alive and active,nd a few cases of feeding and excreting were observed.

iscussion

The yearly recurrence of a pelagic–benthic cycle in theifflugia investigated here seems beyond dispute. That these

hecamoebae are intrinsically benthic, but for some reason,ike bottom anoxia, gave up the bottom for a “forced” pelagicife, is refuted by the fact that there was no gradient from theittoral towards the open water.

The interpretation of the year-to-year differences inlanktonic peak height of the different species is not straight-orward, and compounded by patchiness (variation at single

tations) and local conditions (variation between stations). Auch higher abundance of Difflugia in the upstream, river-

ne part of the reservoir appears beyond doubt, however. Lin

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2007) showed that there is a general increase in zooplanktonere, as well as in chlorophyll (phytoplankton), by a factorwo to five. This is less than in the thecamoebae, but stilluggests that trophy, a higher abundance of food, could behe main reason, since the riverine section is the zone where

ost particles of riverine origin begin to sediment, nutrientsre liberated, and phytoplankton blooms.

Some observations are consistent with a degree of avoid-nce between the four co-existing amoebae. The bestvidence is for 2009 and is expressed as a relative displace-ent of the population peaks of D. tuberspinifera (peakingainly in S1–3 and early in summer) and D. hydrostatica

peaking mainly in S1–3 late in summer), and D. biwae-ulanensis (peaking mainly in S5–7) (Fig. 4). Such an

voidance could be caused by competition for a commonesource (e.g. shell-building material); however, given theredatory nature of the Difflugia’s involved (see Han et al.011), it could also be that the larger species prey on themaller ones.

Like in the case of the German lakes studied by Schönborn1962), the pelagic phase largely coincided with summer, andtemperature sinking to 16 ◦C seemed to set the scene fordisappearance from the pelagic space, while a tempera-

ure of 12 ◦C in spring signaled the time of reappearance. Allhis is quite similar to what happened in German lakes, the

ain difference being that in Liuxihe Reservoir, the Difflugia-ree period was reduced to 3–4 months, while in Germany itmounted to 6–8 months. This shortening suggests that inruly tropical conditions, with a permanently high tempera-ure, pelagic Difflugia might well become aseasonal. Foodeems to be much less of a determinant than temperature,ince at the time of their retreat from the pelagic, Difflugiaould still find plenty of small particulate and live inverte-rate food here (see e.g. Lin 2007 for an overview of theycle of most planktonic invertebrates in Liuxihe Reservoir,nd Han et al. 2011 for a discussion of the food and feedingabits of the Difflugia). In fact, Lin (2007) showed that manyclassical” components of the zooplankton (rotifers, clado-erans and copepods) reach their annual maximum just beforer during winter. Some of these (the rotifers Asplanchna andloesoma, all cyclopoid copepods) are predators of Difflu-ia (see Han et al. 2008; Han et al. 2011; Wang 2010; Wangt al. 2011 for details). An ecological factor may thereforee involved, the Difflugia retreating to the lake bottom atimes when predation pressure in the water column is high-st. Arguing against the importance of this effect is the lackf a vertical migration, which is often a predator avoidanceovement (Dumont and De Meester 1990; Lampert 1993;an and Straskraba 2000). Predators are indeed present and

ctive in Liuxihe Reservoir, but perhaps not in sufficientumbers to control the abundance or the thecamoebae. Theccasional (slight) concentration found at around 8 m simply

eflects the fact that phytoplankton congregates there, as wells algivorous invertebrates such as rotifers and ciliates, andhis abundance of food may in its turn also have attractedmoebae (Han et al. 2008).

rotistology 47 (2011) 224–230 229

An alternative, physiological explanation linked to tem-erature is that at and below 16 ◦C, the metabolism of themoebae may slow down to a point where they can no longeraintain the gas bubbles and/or fat reserves that allow them

o keep afloat. Both hypotheses are not mutually exclusive,nd could in fact reinforce each other. The subject clearlyeserves further study.

cknowledgements

We thank Prof. Louis Beyens (Antwerp University) and theate Prof. Stanley Dodson (Wisconsin, USA) for stimulatingiscussions and help with the literature. Support from Chi-ese NSF grants (U0733007 and 30970467) is appreciateds well.

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