Hydrobiologia 230: 79-92, 1992. O 1992 Kluwer Academic Publishers. Printed in Belgium.

The interstitial ciliated protozoa of a Mediterranean microcommunity

Giovanni Santangelo & Pia Lucchesi Dipartimento Scienze Ambiente e Territorio, via Volta 4, 56100 Pisa, Italy

Received 14 June 1990; in revised form 19 February 1991; accepted 26 March 1991

Key words: ciliated protozoa, Mediterranean sea, interstitial environment, diversity, interspecific inter- actions

Abstract

The main features of an interstitial ciliate community, living in the coastal sand of the Mediterranean sea, were analyzed during a one-year survey, carried out on 113 samples. The community was composed of 56 species, 3 1 of which belong to 8 'resident' genera. Total density varied from 0 to 410 individuals cm- and followed a Spring-Summer and an Autumn-Winter trend, not related to temperature or to any single abiotic variable (interstitial dissolved 0,, grain sand size, salinity), although the density of some taxa was related to one abiotic factor. During Spring, diversity increased by a synchronous bloom involving the whole community. Some taxa, such as the predator Lacrymaria and its prey Frontonia, were significantly associated. The finding of the simultaneous bloom of congeneric species, like that of the genus Remanella suggests that they respond to the same environmental factors, and avoid interspecific competition.

Introduction

The role of ciliated protozoa in the marine environment has been discussed by many authors (Bamforth, 1985; Fenchel, 1967, 1987a; Porter et al., 1985); these eucariotes feed at various trophic levels, i.e. bacteria, algae, flagellates and other ciliates, shunting the bacteria-flagellate step of the microbial loop (Sherr & Sherr, 1987).

In spite of their fundamental role, little research has been carried out to characterize ciliates as a component of the marine interstitial micro- community. The main work on this topic is still Fenchel's, published twenty years ago (Fenchel, 1969). The extreme difficulty in species determi- nation, the impossibility of directly fixing living samples and, as a consequence, the problematic collection of qualitative and quantitative data,

make such an ecological investigation an awk- ward task.

We attempted to set up a quantitative and qualitative study on ciliate taxocoenosis, by sampling fixed sand volumes from which ciliates were extracted, identifying and counting them by a standardized procedure. In order to estimate the biological diversity, widely neglected in ciliates, the Patil-Taillie index (Patil & Taillie, 1976; Dennis et al., 1979) was calculated. We tried to obtain a picture of taxonomic structure, diversity and succession in the microcommunity by analyz- ing a wide number of samples collected over the whole year. Associations of different ciliate taxa and between ciliates and flagellates were also ana- lyzed and some possible predatory - prey inter- actions were pointed out. Abiotic variables, such as dissolved oxygen, salinity, and temperature

were recorded directly in the sand-layer in order to characterize the main features of the interstitial environment in which ciliates live.

Materials and methods

Sampling collection and analysis

A total of 113 samples, each consisting of 9.44 cm3, were collected on 14 occasions over one year within a semi-circular area of about 2 m2, located about 10 metres from the coastal line, at 'Marina di Levante' Viareggio (Liguria Sea 43". 51 ' Lat. Nord, 10" .15' .05" Long. Est). All samples were collected at the same hour in the morning, in shallow water (50 cm depth), as fol- lows: a core of sand was collected by pressing a 2 cm diameter glass tube 6 cm into the sediment. It was then closed at both ends with a cork and the core of sand was divided into two different levels (0-3 and 3-6 cm).

The samples were taken to the laboratory in a thermal bag and analyzed quantitatively within 6 hours.

To homogenize ciliate distribution, the sand samples from each depth level were shaken and two subsamples of 1 cm3 were sucked off by pipette. Uhlig's 'sea-water ice' method (Uhlig, 1964) for extracting interstitial microfauna was used; sand was filtered with a 250 pm mesh nylon net. Ciliates were counted and identified; diatoms and flagellates were estimated as abundance classes (3 classes are present: 1-20; 20-40; > 40). The quantitative observations were carried out under the stereo microscope while the qualita- tive, on living and stained cell, were performed under the optic microscope. Corliss' classification of ciliates was followed (Corliss, 1979); species descriptions by Kahl (1930-1935), Dragesco (1960, 1965, 1986), Curds (1975), Ricci et al. (1982) and Wright (1983) were utilized.

Abiotic data

solved oxygen and salinity were measured in the interstitial water sucked off by inserting a syringe fitted with a filter, 3 cm into the sand layer; tem- perature was measured at the same depth. A refractometer (Hand American Optical Corpo- ration) was utilized to determine salinity values.

Sand grain size was analyzed on samples dialyzed in distilled water overnight, oven-dried at 150 "C and screened with 2000, 840, 420, 250, 149, 75 pm mesh seives. The fractions were weighed and results plotted cumulatively. The median grain size (Md+ was read directly on the graph (Gray, 1981).

Population analysis

The ciliate community structure was analyzed by applying the Patil and Taillie diversity index (Patil & Taillie 1976), comprehensive of a family diver- sity index:

where S is the number of taxa, ni is the relative abundance of the ith biological group, and AP represents the measure of biotic diversity as a P function. For a given community, this index is a decreasing function of beta. The values of beta range from - 1 to 1 and include the three indices more frequently used by ecologists : taxa richness (P = - l), Shannon (P-+ O), and Simpson (P =

+ 1) indices. The closer Patil's index comes to the beta axis, the more taxonomically homogeneous is the population.

Ciliates were divided in two different groups according to how frequently they were found in samples: those found in more than 35% of samples were considered to be 'resident' and the others 'non-resident'. The 35% threshold value was chosen to allow a statistical evaluation of the densities by means of presence-absence chi- square test (Pielou & Pielou, 1967) and correla- tion coefficient (r).

Abiotic data (temperature, salinity, dissolved oxy- gen, and sand grain-size) were analyzed. Dis-

Results

Abiotic data

Salinity, temperature and dissolved oxygen in the interstitial environment were recorded throughout the year at each sampling. Salinity shows two sharp minima in May (l8%,) and in March (26%,), while, in the other months it ranges between 32 and 36%,. The lowest temperature value occurred in December (9.9 "C) and the highest in July (25 "C). There were two different seasons: a warm one from June (2 1 " C) to October (22 " C) and a cold one from November (14.5 " C) to May (17 "C) (Fig. 1).

The samples were also analyzed for their grain-

size composition; the sand was medium grain sized (Gray 198 l), (Mdq ranges between 270 and 430 pm), poorly sorted (ranges between 1 and 2) and homogeneous throughout the year, except the second September samples, composed by coarse sand (Mdq = 590 pm).

The ciliate community structure

Kinetofragminophora were the most numerous ciliates, with the highest number of taxa throughout the year, while Oligohymenophora outstripped them in the December (D), January (Ja) and March (M) samplings.

Polyhymenophora are the least common reach-

ABIOTIC DATA

samples

April 1988

March 1989

Fig. 1. Dissolved oxygen, temperature and salinity values recorded in the interstitial environment during the whole year.

8 2

ing their maximal density and highest taxonomic richness in the April sampling (A). The taxa rich- ness of the three classes is reported in Fig. 2 and in Tables 1 and 2.

The species of about 92% of ciliates was identi- fied but individuals of only 46% of the species determined were counted (Table 2); so quantita- tive data of genera rather than of species were used in calculating diversity indexes.

As mentioned under Methods (3), taxa present in more than 35% of the samples, (i.e. the perma- nent framework of the community) are considered

as 'resident', while the others as 'non-resident'. There are 3 1 species in the 'resident' community belonging to eight genera, six of which are Kineto- fragminophora and two Oligohymenophora. Twenty-four 'non-resident' genera were also identified.

Trachelocerca binucleata and Trachelocerca schulzei, were the most abundant and frequent Trachelocercidae in the resident population. Only one of the four species identified of the genus Lacrymaria was present over the whole year (Table 2). Five species of the Remanella genus

POLYHYMENOPHORA

I I I I I I 1

Apr May Jun Jul Aug Sep Oct Nov Dic Jan k b Mar

Fig. 2. Total density of ciliates and percentage of the different classes found in each sampling.

t- t - m d N

m m - w e 0 N

d - d N w

" " ' D - m

Table 2. List of species found in the sampled area. (Number of individuals in 6 cm3; S = Shallow (0-3 cm); D = Deep (3-6 cm) into the sand bottom; + = presence).

Siephanopogon sp. Trachelocerca binucleata

grucilis

minuta

multinucleata

schulzei

lacrymariae

SP. Trachelonema minlma

SP. Tracheloraphis marginatus

prenanli

SP. Kentrophoros sp. Remanella margaritfera

minula

obtusa

ruxosa

sp. Helicoprodo~ sp. Prorodon sp. Coleps pulcher

spl sp2

Lacrj~maria ucufo

coronata marina

multim~cleara

SP. Enchelyodo~i sp. Trochelius ovum

SP. Mesodmiurn sp. Amphileptus sp. Lorophyllum sp. Chilodontopsis voral;

SP. Frontor~ia arenaria

microsloma fa

SP. Clalhrostoma sp. Pleuronema nlarinum

coronarum

SP. Schizocaljptra

Cyclidium

Condylostomo sp. Stenlor sp. Spirostomum sp. urost~,la sp. Holoxticho sp. Oxytrica sp Gastrostyla sp. Aspidisca sp. Euploles

D~ophrys

Swedmarkia Other species

Table 3. The relationship between the densities of some 'resident' taxa and abiotic parameters was tested on the basis of the correlation coefficent (r).

Temperature Salinity Dissolved 0, Grain-size

0.4734 Trachelocerca 14

0.0437

0.467 Trachelonema 14

0.0458

0.4604 Tracheloraphis 14

0.0488

0.4361 Remanella 14

0.0595

0.3 158 Lacrymaria 14

0.1357

were found in the same sampling showing the same abundance. Finding species of the same genus blooming at the same time agrees with the diversified feeding strategy that Fenchel (1968, 1987a) describes for the genus Remanella, four species of which feed on non-overlapping food size classes.

The 'resident' population density follows two trends: a Spring-Summer one, in which the high- est densities are reached, and an Autumn-Winter one, in which density falls remarkably (Fig. 2). 'Resident' population was more numerous in the deep sand-layer (3-6 cm) (Tables 1-2).

The relationship between abiotic and biotic parame- ters

The relationship between abiotic variables and total ciliate densities was tested by the correlation coefficient (r) which was not significant for both the total and 'resident' population. However, the abiotic parameters and the densities of single taxa were significantly correlated (P < 0.05), as fol- lows: temperature values and density of Trachelocerca, Tracheloraphis and Trachelonema;

Table 4. The association between Frontonia, Lacrymaria and Pleuronema was analized by Pielou's coexistence test.

Lacrymaria absent present

Frontonia

Frontonia absent present

Pleuronema

45

Lacrymaria absent present

absent '7;

Pleuronema present

83 30 113

Significant: positive association

Significant: negative association

Significant: positive association

dissolved oxygen and Lacrymaria density. Salinity resulted negatively correlated with the density of this last taxon. Remanella density and temperature values were nearly significative (P = 0.059). All data are reported in Table 3. A sharp depression of salinity, of total density and species richness co-occur in the same sampling of May (Ma).

The behaviour of the dgerent taxa

The total densities of ciliates, flagellates and diatoms are reported in Fig. 3a. It is worth

remembering here that the latter two groups are generally considered as one of the main food- sources for ciliates.

Flagellate and diatom densities showed similar values in most of the samplings; those collected during the Spring and in January (Ja) were the richest. Ciliates, by contrast, are numerous in the Spring- Summer samples and scarce in Autumn and the first half of Winter. In the first September sampling ( S , ) the total ciliate density is strongly affected by a remarkable blooming of some species of the genus Remanella (Fig. 3a, b); Remanella are considered (Fenchel, 1968) 'omni- vorous' species.

samples

D c i l i a t e s

C] Flagellates

R ~ i a t o m s

Fig. 3. Densities found in the sampling collected during the whole year: Flagellates, diatoms and ciliates (a), Remanella (b), Frontonia (c), Pleuronema (d) .

a~emanel la

a Flagellates

Diatoms

samples

Frontonia

Flagellates

g ~ i a t oms

samples

I leu ronerna

samples Fig. 3d.

The two Frontonia species found, described by Fenchel (1968) as diatom grazers, reach their highest densities during the Spring, following the diatom density trend (Fig. 3c).

The density of ~leuronema,'which, according to Fenchel (1968, 1969) feeds on flagellates and diatoms, but mostly on bacteria, oscillates irregu- larly and reaches highest values in the sampling ( S , ) in which Remanella density is maximal too (Fig. 3d, b).

In the community examined the eight 'resident' genera were tested on the basis of the presence- absence chi-square test (Pielou & Pielou, 1967) to evaluate a possible intergeneric association. Lacrymaria resulted positively associated (P < 0.001) with Pleuronema and negatively with Frontonia (P < 0.025); Frontonia and Pleuronema resulted negatively associated (Table 4). It is worth mentioning here that Lacrymaria feed on

both Frontonia and Pleuronema (Fenchel, 1968). Other predator-prey associations such as Trachelocerca gracilis, Helicoprorodon, Remanella (Fenchel, 1968), were found not significant.

The densities of some other taxa, mostly belonging to the family of Trachelocercidae, whose frequency can not be tested by Pielou's Chi-square, resulted significantly correlated with each other and with Lacrymaria, Coleps and Frontonia, according to the correlation coeficient (r), but the meaning of these correlations remains obscure.

The diversity analysis

Although diversity indeces are a useful tool for describing and comparing the taxonomic assem- blage of populations, they have seldom been used

4 - 1 0 1

I Beta

-1 i, Beta 1

0 6 -1-1 - 1 Beta

I 1 Bet a

Bet. k t .

depth 0 - 3 c m

- - - - - 3 - 6 c m

i - 1 1

I Beta

- - 1 0 1

Bet*

Fig. 4. The Patil and Taillie indexes, calculated for each sampling and in the two sand-layers, are graphically represented.

Table 5. The comparison of the diversity values (Shannon index) between the two sand depths resulted not significant on the basis of Wilcoxon Matched-pairs Signed-ranks Test.

N Sample Shannon index (H) Difference Signed-ranks

1 12 April '88 1.33 1.82 - 0.49 - 11 2 25 May '88 1.58 1.62 - 0.04 - 1 3 16 June '88 1.68 1.87 - 0.19 - 5.5 4 7 July '8 8 2.08 1.89 0.19 5.5 5 25 July '88 1.61 1.91 - 0.3 - 7 6 8 August '88 1.11 0.66 0.45 10 7 7 September '88 1.2 0.54 0.66 13 8 27 September '88 1.78 0.96 0.82 14 9 18 October '88 1.94 1.86 0.08 3

10 18 November '88 1.61 1.82 - 0.21 - 8 11 19 December '88 0.63 1.06 - 0.43 - 9 12 11 January '89 1.37 1.94 - 0.57 - 12 13 22 February '89 1.11 1.27 - 0.16 - 4 14 29 March '89 1.79 1.86 - 0.07 - 2

Sum of positive ranks (T) = 45.5 From table of Critical Values of Tin the Wilcoxon Matched-pairs Signed-ranks Test P > 0.05

for ciliate communities because of the difficulty encountered in identifying and counting the num- ber of ciliates in each taxon.

We utilized Patil and Taillie's index, our values are reported in Fig. 4. Taxa richness ranges between 4 and 14, the Shannon index ranges from 0.54 to 2.08. This index, calculated on samples collected in the two different sand layers, and tested by the Wilcoxon matched-pairs signed- ranks test (Siegel, 1956), was not significant. Nevertheless a remarkable difference between the two strata was found in the samples collected in June (Jn), January (Ja) and March (M) due to a difference in taxonomic richness (Table 5). In the September samples (S,, S,) the diversity differ- ences between the two strata, shown in Fig. 4, is presumably related to the total density difference reported in paragraphs 2 and 3. In the samples of 7 July (J, ) and 18 October ( 0 ) the Patil and Taillie index curves completely overlap.

The dominance of one taxon is clearly shown in sample (S,) .

Discussion

In the present research we attempted an analysis of a marine ciliate interstitial taxocoenosis, based

on qualitative and quantitative data, in order to delineate some of the main features of this com- ponent of the interstitial community. Little research on this topic has been carried out till now and no background information is available on Mediterranean interstitial ciliate population den- sity and diversity.

The general picture emerging from the abiotic variables describes an environment in which tem- perature and dissolved oxygen vary sharply with time, while salinity and sand grain-size are more constant.

The community studied is clearly characterized by two components, one 'resident' and one 'non- resident', termed thus according to their fre- quencies. The 'resident' community consists of eight genera only, whose densities vary remark- ably with time. Among these we found about half of the taxa Fenchel(1969) found in the Baltic sea (Helsingor beach).

Diversity, rather neglected in studies on ciliates, was analyzed by the Patil and Taillie index. Diver- sity increases with density at large. The gaps in density among the taxa dwelling in the same sample is responsible for the low values of the Shannon index (H) or general diversity index.

The H values (0.54-2.08) are comparable to

that found in a fresh water benthic ciliate com- munity by Madoni (1989) (0-2.08), indicating a more heterogeneous community in our sandy marine beach, were 56 species were found in com- parison to 46 sampled by Madoni.

Some taxa bloomed when taxonomic richness and total population density reached their highest values; it is worth stressing that different species of the same genus bloom at the same time, presumably responding to the same environ- mental factors and behaving like a 'guild' inside which competition is avoided (Fenchel, 1968, 1987b). The ecological meaning of analysis based on taxonomic levels higher than species, the only possible one for rich populations of ciliates, is sustained by Heip et al. (1988) and Warwick (1988).

Seasonal variation in total ciliate density fol- lows Spring-Summer and Autumn-Winter trends; these two trends do not overlap the thermal curve. A positive correlation between temperature increase and growth rate of ciliates, under lab conditions, was found by Fenchel (1969) and Giese (1973), but, on the basis of our data, no correlation was found between the den- sity of the ciliate community and temperature increase. By contrast, the density of some taxa and temperature increase were positively corre- lated; the increase in only a few taxa strongly depresses community diversity.

All these results suggest that other constraints, i.e. predation, competition, or some environ- mental factor (such as dissolved oxygen that reaches higher values at lower temperature) could balance temperature induced numerical increase. Lacrymaria and Pleuronema show a significant, positive association giving strength to the hypoth- esis of a feeding interaction.

Although we made an effort to evidentiate inter- relationships between abiotic and biotic parame- ters, their synergism presumably covers the single interactions and the rarity of some events does not allow a statistical analysis. For example, the sharp fall in salinity found in May depressed den- sity and species richness, but did not affect gen- eral diversity. Hydrodynamism is certainly one of the main sources of perturbation, affecting coloni-

zation and re-colonization rates and the vertical distribution pattern of ciliates.

Acknowledgements

We are particularly grateful to Professor R. Nobili for his critical revision of the manuscript and to Professor T. Fenchel for his useful suggestions. Thanks are also due to Dr. E. Philpot for her revision of the English text.

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