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Changes in the ciliate assemblage along a fluvial system related to physical, chemical and geomorphological characteristics
ARTICLE IN PRESS European Journal of
PROTISTOLOGY European Journal of Protistology 43 (2007) 67–75 www.elsevier.de/ejop
Changes in the ciliate assemblage along a fluvial system related to physical, chemical and geomorphological characteristics Paolo Madoni, Sonia Braghiroli Department of Environmental Sciences, University of Parma, via G.P. Usberti 33/A, 43100 Parma, Italy Received 4 August 2006; received in revised form 21 September 2006; accepted 26 September 2006
Abstract Samples were collected monthly from the water–sediment interface at six stations along the Mincio River (northern Italy) during a 1-year study of the ciliated protozoan communities. Four stations were located upstream of the Mantua lakes in the hyporhithron fluvial zone and two stations were located in the potamon fluvial zone between the Mantua lakes and the confluence with the Po River. A total of 133 species of active trophic ciliates belonging to 76 genera were found. Community structures revealed in this data were analysed using some statistical methods (similarity index, and categorical principal component analysis (CATPCA)) and this allowed the determination of differences between stations and between ciliate communities characteristic of stations. Species typical of the ecotypes located in both rhithron and potamon fluvial zones were defined. The saprobic index and valency analysis methods were used to quantify organic input and to follow changes in saprobicity along the river. A change in the ciliate communities was observed between stations located upstream and stations located downstream of the town of Mantua. The former were composed mainly of b-mesosaprobic species, typical of the hill zone of running waters, while in the latter increased numbers of a-mesosaprobic species are associated with the higher anthropogenic pressures. Our results reiterate the high sensitivity shown by ciliated protozoa as indicators of organic load in watercourses. r 2006 Elsevier GmbH. All rights reserved. Keywords: River ecosystem; Ciliated protozoa; Comparative ecological analysis; Water quality; Saprobicity
Introduction In the last few years, various studies have addressed the composition, distribution, and dynamics of ciliate communities as well as their dependence on the saprobicity of watercourses (Gracia et al. 1989; Grolie`re et al. 1990; Bereczky and Nosek 1993; Madoni 1993, 1994, 2005; Packroff and Zwick 1998; Madoni and Bassanini 1999; Madoni and Zangrossi 2005). Furthermore, studies on the distribution of ciliates related to Corresponding author. Tel.: +39 521 905622; fax: +39 521 905402.
E-mail address: [email protected] (P. Madoni). 0932-4739/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2006.09.004
river zonation revealed the existence of a correlation between ciliate community structure and abiotic characteristics in the various sections of the river (Madoni 1983, 1984a). In this way, fluvial zones such as epi-, meta-, hyporhithron, and potamon should be characterized by typical ciliate communities unless modified by organic pollution that reduces or eliminates the effects of the characteristics of longitudinal zonation. In Europe, methods widely used for the biological evaluation of the quality of running waters are essentially based upon the presence of indicator organisms. The major part of these methods belongs to two main groups: saprobic system and extended biotic index.
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The saprobic system of water quality was founded by Kolkwitz and Marsson (1908, 1909) and was soon widely used in central and eastern Europe in different versions proposed by Pantle and Buck (1955), Zelinka and Marvan (1961), Liebmann (1962), and Sladecek (1973). Since ciliate protozoans constitute a quantitatively important component both of the plankton and benthos of freshwater environments and play a significant role in the decomposition process, they can be useful as biological indicators of organic pollution of streams and rivers. Even though macroinvertebrates have proved to be the most practical indicators for evaluating the quality of running waters, the sensitivity of ciliates, both to changes in organic load and to trophic conditions of the sediments, suggests that they should be used alongside macroinvertebrates in the biological monitoring of watercourses (Madoni 1994). In this work, a 1-year study of ciliated protozoa collected at six stations along the Mincio river (northern Italy) was performed. The main aims were: (1) to define the typical species of the communities of the studied watercourse, (2) to compare the ciliate taxocenoses at the six sampling stations in relation to the different physical, chemical and geomorphological characteristics, bringing further knowledge on the distribution of ciliated protozoa in relation to river zonation, and (3) to evaluate the water quality of the river by means of the ciliate taxocenoses.
Fig. 1. Hydrographic map of the Mincio river, showing the locations of the six sampling stations.
Materials and methods
Sampling of ciliates
Study area
At each station samples were collected monthly from September 2004 to August 2005. Benthic ciliates were collected from the substrate surface with a pump aspirator (4-cm diameter) already used in previous studies and specifically designed for sampling in streams characterized by hard river-bed substrates and low water level. At each station four samples were collected along the line of the river cross-section and pooled. Samples were examined in the laboratory and abundance ratios were determined within 5 h after collection. The ciliates were classified according to Foissner et al. (1991, 1992, 1994, 1995) and Foissner and Berger (1996). When necessary, samples were prepared for microscopical examination by silver carbonate staining. Estimates of ciliate population density were based on the enumeration from subsamples extracted with an automatic micropipette and expressed in number of ciliates per 1 cm2 of bottom surface. The most convenient drop size and number of replicate counts were selected each time according to the sub-sampling technique described by Madoni (1984b). Usually, sub-samples of 100 ml in volume were taken and 10 replicates of this volume were counted. Evaluation of water saprobicity (saprobic
The Mincio river arises as the outflow from Lake Garda (northern Italy) and, after 73 km, flows into the river Po. In its first reach (or section) (hyporhithron) the river flows through morainic hills in which the substrate type is pebbles and gravel. In its middle reach, between the villages of Goito and Formigosa, the river flows across a plain and enlarges forming the Mantua lakes. In its final reach, the Mincio river flows slowly in a canalized bed and receives purified effluents of sewage treatment from the town of Mantua. Six sampling stations were defined (Fig. 1): the first was located at Peschiera del Garda, 300 m downstream from the Garda Lake in a section in which the watercourse has been canalized and flows very slowly. The second was located at Monzambano village downstream of the entry point of purified sewage from the treatment plant of Peschiera del Garda. Stations 3 and 4 were located in a reach of the upper Mincio scantily influenced by human activities. Stations 5 and 6 were located in the lower Mincio belonging to the potamon fluvial zone.
ARTICLE IN PRESS P. Madoni, S. Braghiroli / European Journal of Protistology 43 (2007) 67–75
index S of Pantle and Buck (1955), saprobic levels of Zelinka and Marvan (1961), quality classes (DIN 38410 1990; LAWA 1991; Friedrich et al. 1995)) were made using Sladecek’s list of ciliates revised by Foissner et al. (1995). A selection of physical, chemical and bacteriological measurements were also made on water samples taken in parallel with the biological samples. In order to describe the relationships between stations and species, statistical treatment of data was carried out using categorical principal component analysis (CATPCA). This procedure simultaneously quantifies categorical variables—and in general variables containing positive integer data—while reducing the dimensionality of the data, thus values of the frequency of occurrence of the species were considered. The principal component analysis (PCA) was applied to nine selected non-ciliate parameters to determine the relationships between stations and environmental conditions. Nonciliate data were transformed to logarithmic values. These analyses were performed using the program SPSS 11.0.4. Further details can be found in the on-line SPSS Manual.
pristine conditions in the upper Mincio. Values of dissolved oxygen were quite high throughout the watercourse with saturation levels ranging from 92% at station 6 to 97% at station 1. During the annual cycle, 133 species of ciliated protozoa belonging to 76 genera were identified at the six stations studied (Table 2). The orders represented by the highest numbers of species were Hypotrichida (25), Hymenostomatida (14), Cyrtophorida (12) and Peritricha (12). Twenty-one species were found at all six stations (although not in every month of the year). Among these ciliates Cinetochilum margaritaceum was observed in the highest number of samples (49 out of 72); this species was present in April at stations 5 and 6, with abundances of 114 and 108 ind. cm2, respectively, when it co-dominated the ciliate community together with Carchesium polypinum. Other ciliates were observed in relatively high numbers of samples: Holosticha pullaster was present in 41 out of 72 samples and was the dominant species in July at station 4 with 30 ind. cm2; Strobilidium caudatum was observed in 33 samples and reached its highest density at station 3 in autumn with 32 ind. cm2; Pleuronema coronatum was present in 29 out of 72 samples but with low density values (o10 ind. cm2). There were some species that were observed in a low number of samples but with high density in at least one of them. Among these ciliates, Carchesium polypinum reached a peak of 102 and 114 ind. cm2 at stations 5 and 6, respectively, in April; Coleps hirtus was the dominant species at station 6 in July with 84 ind. cm2, and Urocentrum turbo reached a peak of 72 ind. cm2 at station 1 in July. Finally, 35 ciliate species were observed in only one sample with very few individuals. These sporadic species are not listed in Table 2. Mean values of number of species of the ciliate communities at the six stations are shown in Fig. 2. The two-way non-parametric ANOVA (Friedman Test) indicated a highly significant difference among stations
Results and discussion The mean values of selected non-ciliate parameters of water samples at the stations along the Mincio river are summarized in Table 1. Significant differences appear between the stations located in the upper Mincio and those located in the lower Mincio. In particular, at stations 5 and 6 higher values of COD, suspended solids, conductivity, ammonium and total P were measured. High values of ammonium, phosphorus and Escherichia coli were registered at station 2 also, due to the inflow of sewage effluents. A reduction in the values of these parameters, observed at the following stations 3 and 4, was due to the self-purification process favoured by the
Table 1.
69
Mean values (ranges in parenthesis) of selected non-ciliate parameters measured at the six sampling stations Stations
1
COD (mg 1 ) Suspended solids (mg l1) Water flow (m s1) Conductivity (mS cm1) N-NH4 (mg N l1) N-NO3 (mg N l1) Total N (mg N l1) Total P (mg P l1) Oxygen concentr. (%) E. coli (UFC/100 ml)
1
2
3
4
5
6
9 (4–14) 3.28 (2–6) 0.36 (0.11–0.78) 230 (185–260) 0.1 (0.02–0.19) 0.51 (0.20–0.90) 0.58 (0.38–1.00) 0.07 (0.05–0.16) 97 (82–114) 44 (0–200)
11 (4–19) 4.94 (2–13) 0.53 (0.32–1.30) 290 (240–375) 0.17 (0.05–0.57) 1.25 (0.50–2.40) 1.73 (0.50–3.20) 0.13 (0.06–0.44) 96 (75–108) 317 (100–600)
11 (5–17) 4.53 (2–9) 0.20 (0.14–0.41) 346 (254–388) 0.1 (0.02–0.25) 2.11 (0.90–4.07) 2.26 (0.08–4.20) 0.11 (0.05–0.29) 96 (82–117) 253 (100–1000)
9 (4–21) 4.11 (2–9) 1.00 (0.5–1.61) 284 (234–335) 0.11 (0.03–0.24) 1.23 (0.50–1.80) 1.58 (0.50–2.40) 0.1 (0.05–0.27) 93 (77–106) 259 (50–800)
16 (10–34) 15.17 (3–35) 0.43 (0.27–0.70) 426 (350–521) 0.18 (0.03–0.30) 1.87 (0.10–3.10) 2.43 (1.30–3.00) 0.15 (0.09–0.49) 95 (78–125) 310 (11–1000)
14 (10–21) 14.5 (2–35) 0.23 (0.18–0.46) 436 (350–529) 0.19 (0.03–0.46) 1.90 (0.70–3.30) 2.55 (1.18–4.30) 0.16 (0.10–0.47) 92 (78–110) 178 (60–400)
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Table 2. List of ciliate species collected at the six stations and number of samples in which each species was found
Table 2. (continued ) Species
Species
Station
Acineria uncinata Acineta flava Acineta tuberosa Actinobolina radians Amphileptus claparedii Amphileptus pleurosigma Amphileptus procerus Aspidisca cicada Aspidisca lynceus Carchesium polypinum Chilodonella uncinata Chlamydonella alpestris Cinetochilum margaritaceum Climacostomum virens Codonella cratera Coleps hirtus Coleps nolandi Ctedoctema acanthocryptum Cyclidium glaucoma Cyclidium heptatrichum Dexiotricha granulosa Dileptus margaritifer Epistylis plicatilis Euplotes affinis Euplotes patella Frontonia acuminata Frontonia angusta Frontonia leucas Halteria grandinella Histriculus vorax Holophrya discolor Holophrya teres Holosticha kessleri Holosticha monilata Holosticha multistilata Holosticha pullaster Homalozoon vermiculare Kahlilembus attenuatus Lacrymaria olor Lembadion lucens Lembadion magnum Litonotus alpestris Litonotus crystallinus Litonotus cygnus Litonotus lamella Loxodes magnus Loxodes striatus Mesodinium acarus Mesodinium pulex Monilicaryon monilatus Nassula ornata Nassula picta Obertrumia aurea Oxytricha haematoplasma Oxytricha setigera Paraurostyla weissei
1
2
3
4
5
6
4
6 2 2 1
4
1
2
1 1 1 2 1
1
2
1 1
5 2 3
7 6 2 5
9
8
2 1 4 2
3 5
11
1 2 10
1 3 1 7 1 4 1
1 8 2 1 8
1 6 1
4 3 1 5 2
1
1 2 1 3 3
2 2 3 3
7 1 5 6
4 1 5 7
2 1
3
3 1 1 3 5 1 4 1
2 1 9 6 4 2 5
3 5 1 1 1
3 2 1 3
1 1 1 3 3 8 1 4 5 2 1 1 1 2 2 2 6 1 1 1 1
3 1 7 1 3 1
1 2 4
1 1 1
4 2 3 8 2 1 5 6 2 3 8 1 3 1 2 2 3
1 1 1
1 1 1
3 1 2 8
1 2 3
2 2 3
1 6 1 2 4 2
1 1 1 2
1 1 2 1
1 2 2
2
1 1 1
5 2 2 2 2 1 1 1 2 2 4 1 5 2 1 2 2 2
1
1 4
Station
Phascolodon vorticella Philasterides armatus Pleuronema coronatum Pseudochilodonopsis fluviatilis Pseudochilodonopsis piscatoris Pseudomicrothorax agilis Pseudovorticella monilata Spirostomum minus Spirostomum teres Stentor muelleri Stentor roeselii Sterkiella histriomuscorum Stokesia vernalis Strobilidium caudatum Strobilidium humile Strombidium viride Stylonychia mytilus-complex Stylonychia pustulata Tachysoma pellionellum Tokophrya quadripartita Trithigmostoma cucullulus Trithigmostoma steini Trochilia minuta Urocentrum turbo Uroleptus piscis Uroleptus rattulus Urostyla grandis Urotricha agilis Urotricha farcta Urotricha globosa Vaginicola ingenita Vorticella aquadulcis-complex Vorticella campanula Vorticella convallaria-complex Vorticella natans Vorticella picta Zoothamnium arbuscola Zosterodasys transversa
1
5
2 3 1
2
4 1 1 2
3
4 1 2 3 2
4
6 2
1 7 1 2
6 2 1 3
1
1 1 1
5
2 1
1 2 1
1 1 3 4
2 1 5 1
6
7
6
2
2
1 2 1
1 3 1
1
1 1 3
2 2 2 2 3 3 2
5 1 3 1 4
1 2 2 3 5 5 3
4 4 1 1
1
3
4 1 1 2 1 1 1 1 3
2 1 1
1 3 5 3 1
1 2 2
1 5 3 3 2 1 2
1
1 3 1 6
1
1 1
2
1
4
2
1
2
3
3 1
3 1 1 1 1 1 2
Sporadic species (those found in only one sample) are omitted.
(w2 ¼ 18.150; df ¼ 5; n ¼ 12; p ¼ 0.003). In general, the taxocenoses at the upper Mincio stations were slightly richer in species than at the lower Mincio stations. In particular, station 3 reached the highest mean number of species (16) with a range from 9 in March to 23 in December. The large range in the number of species recorded at the six sampling stations in different months can be caused by seasonal changes in environmental conditions that limited, or supported, growth of ciliate species (Madoni 1984a). For example, the minimum numbers of species at stations 4, 5, and 6, and the maximum number of ciliate species at station 3 were registered in December; in the same month, stations 4, 5,
ARTICLE IN PRESS P. Madoni, S. Braghiroli / European Journal of Protistology 43 (2007) 67–75
Number of species
25 20 15 10 5 0
1
2
3 4 Stations
5
6
Fig. 2. Mean number of ciliate species recorded at the six sampling stations during the annual cycle. Bars indicate maximum and minimum numbers of species at each station.
ciliates in the polluted ones. The same spatial distribution was observed in other watercourses (Grabacka 1988; Kasza 1988; Madoni and Bassanini 1999; Madoni 2005). In order to evaluate the water quality of the river at the six sampling stations, the values of both the saprobic index S and the saprobicity levels (oligosaprobic, b-mesosaprobic, a-mesosaprobic and polysaprobic) were determined using the ciliate taxocenoses. The results of the saprobic evaluations are reported in Figs. 4 and 5. The highest biological quality was reached at station 3 (Pozzolo) throughout the studied period. At this station, in fact, values of S ranged from 2.25 (August) to 2.55 (July) corresponding to a b-mesosaprobic level (quality class II in the Saprobic System). At the other stations mean values of S were slightly higher, ranging from 2.41 at station 1 to 2.57 at station 4, and 4
50
Saprobic index S
40 Percentage
71
30 20
3
2
10 0
1 1
2
3
4
5
6
1
2
3 4 Stations
Sampling stations A
O
C
Fig. 3. The percentage contribution of algivorous (A), bacterivorous (B), carnivorous (C), and omnivorous (O) ciliates to the ciliate community at the six sampling stations through the annual cycle.
and 6 were affected by higher values of total nitrogen and by lower numbers of E. coli, on the contrary, at station 3 a lower content of nitrogen and higher values of bacterial counts were registered. By grouping the ciliated protozoa into trophic categories it was found that of the 133 species found in the river Mincio, 52 were bacterivorous, 39 algivorous, 25 omnivorous and 17 carnivorous. Algivorous ciliates formed the largest group in terms of relative abundance at stations 3 and 4 (Fig. 3). Nevertheless, in the polluted waters of station 2, bacterivorous ciliates numerically dominated (44%) over algivores (24%), and this was observed also at the lower river stations 5 and 6. The effect of organic load on the structure of the ciliate communities thus appears to be well depicted by the trophic categories, algivorous ciliates being dominant in the unpolluted reaches of the river and bacterivorous
6
Fig. 4. Mean saprobic index values at the six sampling stations. Bars indicate the maximum and the minimum values. The difference between means was shown to be statistically significant by the non-parametric Friedman Test (w2 ¼ 13.289; n ¼ 12; df ¼ 5; p ¼ 0.021).
6 5 Saprobic levels
B
5
4
ο β
3
α
2
Ρ
1 0
1
2
3
4
5
6
Sampling stations
Fig. 5. Mean saprobic levels at the six sampling stations, as defined by the ciliate communities.
ARTICLE IN PRESS P. Madoni, S. Braghiroli / European Journal of Protistology 43 (2007) 67–75
the corresponding saprobic level was b-a-mesosaprobic (quality class II–III). The graph of Fig. 5 also points out the sensitivity of ciliates in indicating changes in the organic load along the watercourse: the good water characteristics demonstrated at station 1 were partially lost because of the inflow of sewage effluents at station 2 (prevalence of the a-ms level). The clear prevalence of the b-ms level at the following stations 3 and 4 indicates the good self-purification capability of the river in the reach between stations 2 and 4. This is corroborated by the values of some chemical parameters such as COD, N-NH4, N-NO3 (Table 1). Finally, the gradual decrease of the biological quality in the lower Mincio was marked by the progressive increase of the a-ms and polysaprobic levels, together with the slight decrease of the b-ms level. In order to estimate the degree of similarity among the ciliate taxocenoses of the six stations studied, Sorensen’s similarity index was calculated (Table 3). The ciliate communities of stations 1, 3, and 4 located in the upper Mincio turned out to be quite similar (S460%). Station 2 showed high similarity to the adjacent station 1, but low similarity values to other stations; this was due to the presence, even if sporadic, of anaerobic ciliates such as Saprodinium dentatum (July), Plagiopyla nasuta (April) and other ciliates, probably coming from sewage effluents, such as Euplotes aediculatus, Sterkiella histriomuscorum and Zoothamnium procerius, that were recorded only at station 2. The ciliate community of station 1 showed high similarity values with all the stations; this may be due to the fact that at this sampling site the river presents intermediate characteristics between hyporhithron and potamon (cf. Study area). In general, the lowest similarity values were observed between upperand lower Mincio stations, even if the similarity between stations 5 and 6 was high (72%). In order to define the relationships between stations and species, a categorical principal component analysis (CATPCA) was carried out on the data. The graph in Fig. 6 illustrates the arrangement of both species and stations in a bidimensional plane. The arrangement of the species along the first dimension (horizontal axis), is related to their presence in one or more stations: on the left side the species which were observed sporadically in Table 3. Sorensen’s similarity index (%) among stations applied to the ciliated protozoa communities Station
1
2
3
4
5
6
1 2 3 4 5 6
—
75 —
68 63 —
71 56 60 —
75 59 54 56 —
67 60 52 48 72 —
5 4
6 5
3 Dimension 2
72
2 1 0
3
−1 −2
1
−3 −4 −2
−1
0
1 Dimension 1
2
3
2 4
4
Fig. 6. Bidimensional topographic representation of the standardized distances among stations (1–6), ciliate species (o), and among ciliates and stations as results from the categorical principal component analysis.
one or two stations, on the right side the species which were found in all six stations. The arrangement of the species along the second dimension (vertical axis) is related to their distribution along the river: upper Mincio stations (lower part of the diagram) and lower Mincio stations (upper part of the diagram). The species located in the upper part of the diagram, which showed a higher affinity with stations 5 and 6, were: Carchesium polypinum, Codonella cratera, Coleps hirtus, Paraurostyla weissei, Phascolodon vorticella, Stentor roeseli, Strobilidium humile. These species can be considered typical of the potamon fluvial zone. Species found only in the upper Mincio and ciliate species which were found at all six stations group together in the lower part of the diagram: among the former there are some ciliates such as Euplotes affinis, Mesodinium acarus, Pseudomicrothorax agilis, Trochilia minuta, and Zosterodasys transversa that can be considered typical of the hyporhithron fluvial zone, whilst ciliates found at all stations can be considered common species whose preferred habitat type is found in all the fluvial zones. The PCA, performed on physical, chemical and bacteriological data collected in the six stations (Table 1), confirmed the above results. The scatter diagram of Fig. 7 reveals the degree of affinity between stations 5 and 6 that, in turn, are clearly rather separate from those stations which are located in the upper river. The physical, chemical and bacteriological conditions at station 1 are found to be distant from those at the lower river stations. The first component explains 66.8% of the variance and it groups together all variables except the water flow. The second component (14.3% variance explained) includes the variable water flow. From the statistical analyses, it clearly emerges that the structure of the ciliate communities is strongly affected by the physical, chemical and geomorphological
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73
Table 4. Occurrence in other watercourses of the 21 common species found in the Mincio river Species
Fig. 7. Bidimensional topographic representation of the six sampling stations as defined by principal component analysis applied to non-ciliate data (variance explained: PC 1 ¼ 66.8%, PC 2 ¼ 14.3%).
characteristics of the several reaches of the river. In fact, station 1, even though located in the hyporhithron fluvial zone, suffered effects from its close proximity to Lake Garda, still retaining some forms typical of lentic waters such as Askenasia volvox, Codonella cratera, Halteria grandinella, Urocentrum turbo, and Urotricha furcata, which made it similar to communities located at the lower Mincio stations in the potamon fluvial zone. Likewise, the ciliate community at station 2, even though it maintained a structure similar to those of the other stations located in the hyporhithron fluvial zone, was affected by the organic load entering from sewage effluents, harbouring ciliate species indicative of elevated saprobicity such as Carchesium polypinum, Epistylis plicatilis, Euplotes aediculatus, Histriculus vorax, Microthorax pusillus, Plagiopyla nasuta, Saprodinium dentatum, Sterkiella histriomuscorum, Vorticella convallaria, and Zoothamnium procerius. Some of these ciliate species probably drifted down with the sewage effluent. The arrangement of stations 3 and 4 along the second component (vertical axis) is related to their different water flow. This can explain the different number of ciliate species found at these two stations. Previous studies on the distribution of ciliates related to river zonation (Madoni 1983, 1984a) revealed the existence of species typical of eight identified ecotypes. In the present study, 18 out 21 ciliate species defined as common in this watercourse, and 4 out of 5 species typical of the hyporhithron fluvial zone were also found as common and typical species in the hyporhithron zone of the river Taro (Madoni and Zangrossi 2005). Among these 21 common species (Table 4), 12 were also found in the sandy hyporheic zone of a lowland stream (Ladberger Mu¨hlenbach) (Cleven 2004), 9 were common to the epirhithron zone of a Spanish river (Henares)
Acineria uncinata Aspidisca cicada Aspidisca lynceus Cinetochilum margaritaceum Cyclidium glaucoma Euplotes patella Frontonia acuminata Frontonia angusta Holosticha kessleri Holosticha multistilata Holosticha pullaster Lacrymaria olor Lembadion lucens Litonotus cygnus Litonotus lamella Pleuronema coronatum Strobilidium caudatum Stylonychia mytilus-complex Uroleptus piscis Urostyla grandis Vorticella picta
River He
La
+
+ + + +
+ +
+
+ + + + + + +
+ + + +
+ +
St
Ta
Sa
Ll
+ + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + +
+ + + + + +
+ + + +
+ +
+
+ + +
+
+ + + + + + + +
He: River Henares, Spain; La: Ladberger Mu¨hlenbach, Germany; St: River Stirone, Italy; Ta: River Taro, Italy; Sa: River Sava, Slovenia; Ll: River Llobregat, Spain (References are given in the text).
(Sola et al. 1996), 20 were dominant in the metarhithron fluvial zone of the river Stirone (Madoni and Bassanini 1999), 14 were found along the river Llobregad (Gracia et al. 1989), and 13 were found in a branch of the river Sava located in the lower rhithral area (Primc-Abdija et al. 1996). In the last watercourse, Trochilia minuta was the dominant species and this corroborates our findings about its allocation as a typical species in the hyporhithron fluvial zone.
Conclusions The 1-year study of the ciliate populations along the Mincio River, has enabled the characterization of the biocenoses of the studied watercourse. The typical species of the ecotype located in the potamon zone, characterized by slow flow in a canalized bed, were defined. The ciliate species typical of the hyporhithron fluvial zone were also defined. Among the 133 species of ciliated protozoa found in this watercourse, only 21 species were common to all six studied stations. These species were commonly observed in all fluvial zones of other watercourses, thus they could be considered cosmopolitan inhabitants of running waters.
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Changes in the water quality produce quantitative and qualitative changes in the structure of the ciliate communities. These changes involve a decrease in the percentage of algivorous ciliates and an increase in the number of bacterivorous forms with the increase of the degree of saprobicity along the river. Even though we can define the ciliate communities typical of the different fluvial zones, gradual changes in the physical, chemical and geomorphological characteristics along the river, produce a gradual change in the community structure. The common species form the basic structure of the community, while the species which gradually replace one another along the river, are indicators of that particular fluvial section.
Acknowledgments The authors would like to express their thanks to Dr. Franco Sartore for his valuable assistance in the statistical processing of the data. This work was supported by grants (FIL) from Italian Ministry of University and Scientific and Technological Research.
References Bereczky, M.C., Nosek, J.N., 1993. The influence of ecological factors on the abundance of different ciliated protozoa populations in the Danube River, I: investigation of the ecological amplitude. Acta Protozool. 32, 1–16. Cleven, E.-J., 2004. Seasonal and spatial distribution of ciliates in the sandy hyporheic zone of a lowland stream. Eur. J. Protistol. 40, 71–80. DIN 38410, 1990. Deutsche Einheitsverfahren zur WasserAbwasser- und Schlammuntersuchung: Biologisch-o¨kologische Gewa¨sseruntersuchung (Gruppe M): Bestimmung des Saprobienindex (M2). Foissner, W., Berger, H., 1996. A user-friendly guide to the ciliates (protozoa, Ciliophora) commonly used by hydrobiologists as bioindicators in rivers, lakes, and waste waters, with notes on their ecology. Freshwater Biol. 35, 375–482. Foissner, W., Blatterer, H., Berger, H., Kohmann, F., 1991. Taxonomische und O¨kologische Revision der Ciliaten des Saprobiensystems, Band I: Cyrtophorida, Oligotrichida, Hypotrichida, Colpodea. Bayerische Landesamt fu¨r Wasserwirstschaft, Mu¨nchen, 471p. Foissner, W., Berger, H., Kohmann, F., 1992. Taxonomische und O¨kologische Revision der Ciliaten des Saprobiensystems, Band II: Peritrichia, Heterotrichida, Odontostomatida. Bayerische Landesamt fu¨r Wasserwirstschaft, Mu¨nchen, 502p. Foissner, W., Berger, H., Kohmann, F., 1994. Taxonomische und O¨kologische Revision der Ciliaten des Saprobiensystems, Band III: Hymenostomata, Prostomatida, Nassulida. Bayerische Landesamt fu¨r Wasserwirstschaft, Mu¨nchen, 548p.
Foissner, W., Berger, H., Blatterer, H., Kohmann, F., 1995. Taxonomische und O¨kologische Revision der Ciliaten des Saprobiensystems, Band IV: Gymnostomatida, Loxodes, Suctoria. Bayerische Landesamt fu¨r Wasserwirstschaft, Mu¨nchen, 540p. Friedrich, G., Coring, E., Ku¨chenhoff, B., 1995. Vergleich verschiedener europa¨ischer Untersuchung- und Bewertungsmethoden fu¨r Fliessgewa¨sser. Essen, Landesumweltamt NRW, 140S. Grabacka, E., 1988. A regulated river ecosystem in a polluted section of the Upper Vistula, 7: bottom Ciliata. Acta Hydrobiol. 30, 73–80. Gracia, M., Castellon, C., Igual, J., Sunyer, R., 1989. Ciliated protozoan communities in a fluvial ecosystem. Hydrobiologia 183, 11–31. Grolie`re, C.A., Chakli, R., Sparagano, O., Pepin, D., 1990. Application de la colonization d’un substrat artificiel par les cilie´s a` l’e´tude de la qualite´ des eaux d’une rivie`re. Eur. J. Protistol. 25, 381–390. Kasza, H., 1988. A regulated river ecosystem in a polluted section of the Upper Vistula, 2: hydrochemistry. Acta Hydrobiol. 30, 15–22. Kolkwitz, R., Marsson, M., 1908. O¨kologie der pflanzlichen Saprobien. Ber. Dt. Bot. Ges. 26A, 505–519. Kolkwitz, R., Marsson, M., 1909. O¨kologie der tierischen Saprobien. Int. Rev. Hydrobiol. 2, 126–152. LAWA, 1991. Die Gewa¨ssergu¨terkarte der Bundesrepublik Deutschland 1990. La¨nderarbeitsgemmenschaft Wasser, Berlin. Liebmann, H., 1962. Handbuch der Frischwasser- und Abwasserbiologie. Fischer, Jena, p. 588. Madoni, P., 1983. Relationship between ciliated Protozoa and ecological zones in some water courses of the River Po basin. Int. J. Ecol. Environ. Sci. 9, 87–98. Madoni, P., 1984a. Ecological characterization of different types of watercourses by the multivariate analysis of ciliated protozoa populations. Arch. Hydrobiol. 100, 171–188. Madoni, P., 1984b. Estimation of the size of freshwater ciliate population by a subsampling technique. Hydrobiologia 111, 201–206. Madoni, P., 1993. Ciliated protozoa and water quality in the Parma River (Northern Italy): long-term changes in the community structure. Hydrobiologia 264, 129–135. Madoni, P., 1994. Evaluation of water quality in streams by comparing ciliate and macroinvertebrate community structure. Verh. Int. Verein. Limnol. 25, 1954–1957. Madoni, P., 2005. Ciliated protozoan communities and saprobic evaluation of water quality in the hilly zone of some tributaries of the Po River (northern Italy). Hydrobiologia 541, 55–69. Madoni, P., Bassanini, N., 1999. Longitudinal changes in the ciliated protozoa communities along a fluvial system polluted by organic matter. Eur. J. Protistol. 35, 391–402. Madoni, P., Zangrossi, S., 2005. Ciliated protozoa and saprobical evaluation of water quality in the Taro River (northern Italy). Ital. J. Zool. 72, 21–25. Packroff, G., Zwick, P., 1998. The ciliate fauna of an unpolluted German foothill stream, the Breitenbach, 2:
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quantitative aspects of the ciliates (Ciliophora; Protozoa) in fine sediments. Eur. J. Protistol. 34, 436–445. Pantle, R., Buck, H., 1955. Die biologische U¨berwachung der Gewa¨sser und Darstellung Ergebnisse. Gas Wasserfach. 96, 604–624. Primc-Abdija, B., Abdija, I., Mestrov, M., Radanovic, I., 1996. Composition of ciliate fauna and its seasonal changes in fluvial drift. Aquat. Sci. 58, 224–240.
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PROTISTOLOGY European Journal of Protistology 43 (2007) 67–75 www.elsevier.de/ejop
Changes in the ciliate assemblage along a fluvial system related to physical, chemical and geomorphological characteristics Paolo Madoni, Sonia Braghiroli Department of Environmental Sciences, University of Parma, via G.P. Usberti 33/A, 43100 Parma, Italy Received 4 August 2006; received in revised form 21 September 2006; accepted 26 September 2006
Abstract Samples were collected monthly from the water–sediment interface at six stations along the Mincio River (northern Italy) during a 1-year study of the ciliated protozoan communities. Four stations were located upstream of the Mantua lakes in the hyporhithron fluvial zone and two stations were located in the potamon fluvial zone between the Mantua lakes and the confluence with the Po River. A total of 133 species of active trophic ciliates belonging to 76 genera were found. Community structures revealed in this data were analysed using some statistical methods (similarity index, and categorical principal component analysis (CATPCA)) and this allowed the determination of differences between stations and between ciliate communities characteristic of stations. Species typical of the ecotypes located in both rhithron and potamon fluvial zones were defined. The saprobic index and valency analysis methods were used to quantify organic input and to follow changes in saprobicity along the river. A change in the ciliate communities was observed between stations located upstream and stations located downstream of the town of Mantua. The former were composed mainly of b-mesosaprobic species, typical of the hill zone of running waters, while in the latter increased numbers of a-mesosaprobic species are associated with the higher anthropogenic pressures. Our results reiterate the high sensitivity shown by ciliated protozoa as indicators of organic load in watercourses. r 2006 Elsevier GmbH. All rights reserved. Keywords: River ecosystem; Ciliated protozoa; Comparative ecological analysis; Water quality; Saprobicity
Introduction In the last few years, various studies have addressed the composition, distribution, and dynamics of ciliate communities as well as their dependence on the saprobicity of watercourses (Gracia et al. 1989; Grolie`re et al. 1990; Bereczky and Nosek 1993; Madoni 1993, 1994, 2005; Packroff and Zwick 1998; Madoni and Bassanini 1999; Madoni and Zangrossi 2005). Furthermore, studies on the distribution of ciliates related to Corresponding author. Tel.: +39 521 905622; fax: +39 521 905402.
E-mail address: [email protected] (P. Madoni). 0932-4739/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2006.09.004
river zonation revealed the existence of a correlation between ciliate community structure and abiotic characteristics in the various sections of the river (Madoni 1983, 1984a). In this way, fluvial zones such as epi-, meta-, hyporhithron, and potamon should be characterized by typical ciliate communities unless modified by organic pollution that reduces or eliminates the effects of the characteristics of longitudinal zonation. In Europe, methods widely used for the biological evaluation of the quality of running waters are essentially based upon the presence of indicator organisms. The major part of these methods belongs to two main groups: saprobic system and extended biotic index.
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The saprobic system of water quality was founded by Kolkwitz and Marsson (1908, 1909) and was soon widely used in central and eastern Europe in different versions proposed by Pantle and Buck (1955), Zelinka and Marvan (1961), Liebmann (1962), and Sladecek (1973). Since ciliate protozoans constitute a quantitatively important component both of the plankton and benthos of freshwater environments and play a significant role in the decomposition process, they can be useful as biological indicators of organic pollution of streams and rivers. Even though macroinvertebrates have proved to be the most practical indicators for evaluating the quality of running waters, the sensitivity of ciliates, both to changes in organic load and to trophic conditions of the sediments, suggests that they should be used alongside macroinvertebrates in the biological monitoring of watercourses (Madoni 1994). In this work, a 1-year study of ciliated protozoa collected at six stations along the Mincio river (northern Italy) was performed. The main aims were: (1) to define the typical species of the communities of the studied watercourse, (2) to compare the ciliate taxocenoses at the six sampling stations in relation to the different physical, chemical and geomorphological characteristics, bringing further knowledge on the distribution of ciliated protozoa in relation to river zonation, and (3) to evaluate the water quality of the river by means of the ciliate taxocenoses.
Fig. 1. Hydrographic map of the Mincio river, showing the locations of the six sampling stations.
Materials and methods
Sampling of ciliates
Study area
At each station samples were collected monthly from September 2004 to August 2005. Benthic ciliates were collected from the substrate surface with a pump aspirator (4-cm diameter) already used in previous studies and specifically designed for sampling in streams characterized by hard river-bed substrates and low water level. At each station four samples were collected along the line of the river cross-section and pooled. Samples were examined in the laboratory and abundance ratios were determined within 5 h after collection. The ciliates were classified according to Foissner et al. (1991, 1992, 1994, 1995) and Foissner and Berger (1996). When necessary, samples were prepared for microscopical examination by silver carbonate staining. Estimates of ciliate population density were based on the enumeration from subsamples extracted with an automatic micropipette and expressed in number of ciliates per 1 cm2 of bottom surface. The most convenient drop size and number of replicate counts were selected each time according to the sub-sampling technique described by Madoni (1984b). Usually, sub-samples of 100 ml in volume were taken and 10 replicates of this volume were counted. Evaluation of water saprobicity (saprobic
The Mincio river arises as the outflow from Lake Garda (northern Italy) and, after 73 km, flows into the river Po. In its first reach (or section) (hyporhithron) the river flows through morainic hills in which the substrate type is pebbles and gravel. In its middle reach, between the villages of Goito and Formigosa, the river flows across a plain and enlarges forming the Mantua lakes. In its final reach, the Mincio river flows slowly in a canalized bed and receives purified effluents of sewage treatment from the town of Mantua. Six sampling stations were defined (Fig. 1): the first was located at Peschiera del Garda, 300 m downstream from the Garda Lake in a section in which the watercourse has been canalized and flows very slowly. The second was located at Monzambano village downstream of the entry point of purified sewage from the treatment plant of Peschiera del Garda. Stations 3 and 4 were located in a reach of the upper Mincio scantily influenced by human activities. Stations 5 and 6 were located in the lower Mincio belonging to the potamon fluvial zone.
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index S of Pantle and Buck (1955), saprobic levels of Zelinka and Marvan (1961), quality classes (DIN 38410 1990; LAWA 1991; Friedrich et al. 1995)) were made using Sladecek’s list of ciliates revised by Foissner et al. (1995). A selection of physical, chemical and bacteriological measurements were also made on water samples taken in parallel with the biological samples. In order to describe the relationships between stations and species, statistical treatment of data was carried out using categorical principal component analysis (CATPCA). This procedure simultaneously quantifies categorical variables—and in general variables containing positive integer data—while reducing the dimensionality of the data, thus values of the frequency of occurrence of the species were considered. The principal component analysis (PCA) was applied to nine selected non-ciliate parameters to determine the relationships between stations and environmental conditions. Nonciliate data were transformed to logarithmic values. These analyses were performed using the program SPSS 11.0.4. Further details can be found in the on-line SPSS Manual.
pristine conditions in the upper Mincio. Values of dissolved oxygen were quite high throughout the watercourse with saturation levels ranging from 92% at station 6 to 97% at station 1. During the annual cycle, 133 species of ciliated protozoa belonging to 76 genera were identified at the six stations studied (Table 2). The orders represented by the highest numbers of species were Hypotrichida (25), Hymenostomatida (14), Cyrtophorida (12) and Peritricha (12). Twenty-one species were found at all six stations (although not in every month of the year). Among these ciliates Cinetochilum margaritaceum was observed in the highest number of samples (49 out of 72); this species was present in April at stations 5 and 6, with abundances of 114 and 108 ind. cm2, respectively, when it co-dominated the ciliate community together with Carchesium polypinum. Other ciliates were observed in relatively high numbers of samples: Holosticha pullaster was present in 41 out of 72 samples and was the dominant species in July at station 4 with 30 ind. cm2; Strobilidium caudatum was observed in 33 samples and reached its highest density at station 3 in autumn with 32 ind. cm2; Pleuronema coronatum was present in 29 out of 72 samples but with low density values (o10 ind. cm2). There were some species that were observed in a low number of samples but with high density in at least one of them. Among these ciliates, Carchesium polypinum reached a peak of 102 and 114 ind. cm2 at stations 5 and 6, respectively, in April; Coleps hirtus was the dominant species at station 6 in July with 84 ind. cm2, and Urocentrum turbo reached a peak of 72 ind. cm2 at station 1 in July. Finally, 35 ciliate species were observed in only one sample with very few individuals. These sporadic species are not listed in Table 2. Mean values of number of species of the ciliate communities at the six stations are shown in Fig. 2. The two-way non-parametric ANOVA (Friedman Test) indicated a highly significant difference among stations
Results and discussion The mean values of selected non-ciliate parameters of water samples at the stations along the Mincio river are summarized in Table 1. Significant differences appear between the stations located in the upper Mincio and those located in the lower Mincio. In particular, at stations 5 and 6 higher values of COD, suspended solids, conductivity, ammonium and total P were measured. High values of ammonium, phosphorus and Escherichia coli were registered at station 2 also, due to the inflow of sewage effluents. A reduction in the values of these parameters, observed at the following stations 3 and 4, was due to the self-purification process favoured by the
Table 1.
69
Mean values (ranges in parenthesis) of selected non-ciliate parameters measured at the six sampling stations Stations
1
COD (mg 1 ) Suspended solids (mg l1) Water flow (m s1) Conductivity (mS cm1) N-NH4 (mg N l1) N-NO3 (mg N l1) Total N (mg N l1) Total P (mg P l1) Oxygen concentr. (%) E. coli (UFC/100 ml)
1
2
3
4
5
6
9 (4–14) 3.28 (2–6) 0.36 (0.11–0.78) 230 (185–260) 0.1 (0.02–0.19) 0.51 (0.20–0.90) 0.58 (0.38–1.00) 0.07 (0.05–0.16) 97 (82–114) 44 (0–200)
11 (4–19) 4.94 (2–13) 0.53 (0.32–1.30) 290 (240–375) 0.17 (0.05–0.57) 1.25 (0.50–2.40) 1.73 (0.50–3.20) 0.13 (0.06–0.44) 96 (75–108) 317 (100–600)
11 (5–17) 4.53 (2–9) 0.20 (0.14–0.41) 346 (254–388) 0.1 (0.02–0.25) 2.11 (0.90–4.07) 2.26 (0.08–4.20) 0.11 (0.05–0.29) 96 (82–117) 253 (100–1000)
9 (4–21) 4.11 (2–9) 1.00 (0.5–1.61) 284 (234–335) 0.11 (0.03–0.24) 1.23 (0.50–1.80) 1.58 (0.50–2.40) 0.1 (0.05–0.27) 93 (77–106) 259 (50–800)
16 (10–34) 15.17 (3–35) 0.43 (0.27–0.70) 426 (350–521) 0.18 (0.03–0.30) 1.87 (0.10–3.10) 2.43 (1.30–3.00) 0.15 (0.09–0.49) 95 (78–125) 310 (11–1000)
14 (10–21) 14.5 (2–35) 0.23 (0.18–0.46) 436 (350–529) 0.19 (0.03–0.46) 1.90 (0.70–3.30) 2.55 (1.18–4.30) 0.16 (0.10–0.47) 92 (78–110) 178 (60–400)
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Table 2. List of ciliate species collected at the six stations and number of samples in which each species was found
Table 2. (continued ) Species
Species
Station
Acineria uncinata Acineta flava Acineta tuberosa Actinobolina radians Amphileptus claparedii Amphileptus pleurosigma Amphileptus procerus Aspidisca cicada Aspidisca lynceus Carchesium polypinum Chilodonella uncinata Chlamydonella alpestris Cinetochilum margaritaceum Climacostomum virens Codonella cratera Coleps hirtus Coleps nolandi Ctedoctema acanthocryptum Cyclidium glaucoma Cyclidium heptatrichum Dexiotricha granulosa Dileptus margaritifer Epistylis plicatilis Euplotes affinis Euplotes patella Frontonia acuminata Frontonia angusta Frontonia leucas Halteria grandinella Histriculus vorax Holophrya discolor Holophrya teres Holosticha kessleri Holosticha monilata Holosticha multistilata Holosticha pullaster Homalozoon vermiculare Kahlilembus attenuatus Lacrymaria olor Lembadion lucens Lembadion magnum Litonotus alpestris Litonotus crystallinus Litonotus cygnus Litonotus lamella Loxodes magnus Loxodes striatus Mesodinium acarus Mesodinium pulex Monilicaryon monilatus Nassula ornata Nassula picta Obertrumia aurea Oxytricha haematoplasma Oxytricha setigera Paraurostyla weissei
1
2
3
4
5
6
4
6 2 2 1
4
1
2
1 1 1 2 1
1
2
1 1
5 2 3
7 6 2 5
9
8
2 1 4 2
3 5
11
1 2 10
1 3 1 7 1 4 1
1 8 2 1 8
1 6 1
4 3 1 5 2
1
1 2 1 3 3
2 2 3 3
7 1 5 6
4 1 5 7
2 1
3
3 1 1 3 5 1 4 1
2 1 9 6 4 2 5
3 5 1 1 1
3 2 1 3
1 1 1 3 3 8 1 4 5 2 1 1 1 2 2 2 6 1 1 1 1
3 1 7 1 3 1
1 2 4
1 1 1
4 2 3 8 2 1 5 6 2 3 8 1 3 1 2 2 3
1 1 1
1 1 1
3 1 2 8
1 2 3
2 2 3
1 6 1 2 4 2
1 1 1 2
1 1 2 1
1 2 2
2
1 1 1
5 2 2 2 2 1 1 1 2 2 4 1 5 2 1 2 2 2
1
1 4
Station
Phascolodon vorticella Philasterides armatus Pleuronema coronatum Pseudochilodonopsis fluviatilis Pseudochilodonopsis piscatoris Pseudomicrothorax agilis Pseudovorticella monilata Spirostomum minus Spirostomum teres Stentor muelleri Stentor roeselii Sterkiella histriomuscorum Stokesia vernalis Strobilidium caudatum Strobilidium humile Strombidium viride Stylonychia mytilus-complex Stylonychia pustulata Tachysoma pellionellum Tokophrya quadripartita Trithigmostoma cucullulus Trithigmostoma steini Trochilia minuta Urocentrum turbo Uroleptus piscis Uroleptus rattulus Urostyla grandis Urotricha agilis Urotricha farcta Urotricha globosa Vaginicola ingenita Vorticella aquadulcis-complex Vorticella campanula Vorticella convallaria-complex Vorticella natans Vorticella picta Zoothamnium arbuscola Zosterodasys transversa
1
5
2 3 1
2
4 1 1 2
3
4 1 2 3 2
4
6 2
1 7 1 2
6 2 1 3
1
1 1 1
5
2 1
1 2 1
1 1 3 4
2 1 5 1
6
7
6
2
2
1 2 1
1 3 1
1
1 1 3
2 2 2 2 3 3 2
5 1 3 1 4
1 2 2 3 5 5 3
4 4 1 1
1
3
4 1 1 2 1 1 1 1 3
2 1 1
1 3 5 3 1
1 2 2
1 5 3 3 2 1 2
1
1 3 1 6
1
1 1
2
1
4
2
1
2
3
3 1
3 1 1 1 1 1 2
Sporadic species (those found in only one sample) are omitted.
(w2 ¼ 18.150; df ¼ 5; n ¼ 12; p ¼ 0.003). In general, the taxocenoses at the upper Mincio stations were slightly richer in species than at the lower Mincio stations. In particular, station 3 reached the highest mean number of species (16) with a range from 9 in March to 23 in December. The large range in the number of species recorded at the six sampling stations in different months can be caused by seasonal changes in environmental conditions that limited, or supported, growth of ciliate species (Madoni 1984a). For example, the minimum numbers of species at stations 4, 5, and 6, and the maximum number of ciliate species at station 3 were registered in December; in the same month, stations 4, 5,
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Number of species
25 20 15 10 5 0
1
2
3 4 Stations
5
6
Fig. 2. Mean number of ciliate species recorded at the six sampling stations during the annual cycle. Bars indicate maximum and minimum numbers of species at each station.
ciliates in the polluted ones. The same spatial distribution was observed in other watercourses (Grabacka 1988; Kasza 1988; Madoni and Bassanini 1999; Madoni 2005). In order to evaluate the water quality of the river at the six sampling stations, the values of both the saprobic index S and the saprobicity levels (oligosaprobic, b-mesosaprobic, a-mesosaprobic and polysaprobic) were determined using the ciliate taxocenoses. The results of the saprobic evaluations are reported in Figs. 4 and 5. The highest biological quality was reached at station 3 (Pozzolo) throughout the studied period. At this station, in fact, values of S ranged from 2.25 (August) to 2.55 (July) corresponding to a b-mesosaprobic level (quality class II in the Saprobic System). At the other stations mean values of S were slightly higher, ranging from 2.41 at station 1 to 2.57 at station 4, and 4
50
Saprobic index S
40 Percentage
71
30 20
3
2
10 0
1 1
2
3
4
5
6
1
2
3 4 Stations
Sampling stations A
O
C
Fig. 3. The percentage contribution of algivorous (A), bacterivorous (B), carnivorous (C), and omnivorous (O) ciliates to the ciliate community at the six sampling stations through the annual cycle.
and 6 were affected by higher values of total nitrogen and by lower numbers of E. coli, on the contrary, at station 3 a lower content of nitrogen and higher values of bacterial counts were registered. By grouping the ciliated protozoa into trophic categories it was found that of the 133 species found in the river Mincio, 52 were bacterivorous, 39 algivorous, 25 omnivorous and 17 carnivorous. Algivorous ciliates formed the largest group in terms of relative abundance at stations 3 and 4 (Fig. 3). Nevertheless, in the polluted waters of station 2, bacterivorous ciliates numerically dominated (44%) over algivores (24%), and this was observed also at the lower river stations 5 and 6. The effect of organic load on the structure of the ciliate communities thus appears to be well depicted by the trophic categories, algivorous ciliates being dominant in the unpolluted reaches of the river and bacterivorous
6
Fig. 4. Mean saprobic index values at the six sampling stations. Bars indicate the maximum and the minimum values. The difference between means was shown to be statistically significant by the non-parametric Friedman Test (w2 ¼ 13.289; n ¼ 12; df ¼ 5; p ¼ 0.021).
6 5 Saprobic levels
B
5
4
ο β
3
α
2
Ρ
1 0
1
2
3
4
5
6
Sampling stations
Fig. 5. Mean saprobic levels at the six sampling stations, as defined by the ciliate communities.
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the corresponding saprobic level was b-a-mesosaprobic (quality class II–III). The graph of Fig. 5 also points out the sensitivity of ciliates in indicating changes in the organic load along the watercourse: the good water characteristics demonstrated at station 1 were partially lost because of the inflow of sewage effluents at station 2 (prevalence of the a-ms level). The clear prevalence of the b-ms level at the following stations 3 and 4 indicates the good self-purification capability of the river in the reach between stations 2 and 4. This is corroborated by the values of some chemical parameters such as COD, N-NH4, N-NO3 (Table 1). Finally, the gradual decrease of the biological quality in the lower Mincio was marked by the progressive increase of the a-ms and polysaprobic levels, together with the slight decrease of the b-ms level. In order to estimate the degree of similarity among the ciliate taxocenoses of the six stations studied, Sorensen’s similarity index was calculated (Table 3). The ciliate communities of stations 1, 3, and 4 located in the upper Mincio turned out to be quite similar (S460%). Station 2 showed high similarity to the adjacent station 1, but low similarity values to other stations; this was due to the presence, even if sporadic, of anaerobic ciliates such as Saprodinium dentatum (July), Plagiopyla nasuta (April) and other ciliates, probably coming from sewage effluents, such as Euplotes aediculatus, Sterkiella histriomuscorum and Zoothamnium procerius, that were recorded only at station 2. The ciliate community of station 1 showed high similarity values with all the stations; this may be due to the fact that at this sampling site the river presents intermediate characteristics between hyporhithron and potamon (cf. Study area). In general, the lowest similarity values were observed between upperand lower Mincio stations, even if the similarity between stations 5 and 6 was high (72%). In order to define the relationships between stations and species, a categorical principal component analysis (CATPCA) was carried out on the data. The graph in Fig. 6 illustrates the arrangement of both species and stations in a bidimensional plane. The arrangement of the species along the first dimension (horizontal axis), is related to their presence in one or more stations: on the left side the species which were observed sporadically in Table 3. Sorensen’s similarity index (%) among stations applied to the ciliated protozoa communities Station
1
2
3
4
5
6
1 2 3 4 5 6
—
75 —
68 63 —
71 56 60 —
75 59 54 56 —
67 60 52 48 72 —
5 4
6 5
3 Dimension 2
72
2 1 0
3
−1 −2
1
−3 −4 −2
−1
0
1 Dimension 1
2
3
2 4
4
Fig. 6. Bidimensional topographic representation of the standardized distances among stations (1–6), ciliate species (o), and among ciliates and stations as results from the categorical principal component analysis.
one or two stations, on the right side the species which were found in all six stations. The arrangement of the species along the second dimension (vertical axis) is related to their distribution along the river: upper Mincio stations (lower part of the diagram) and lower Mincio stations (upper part of the diagram). The species located in the upper part of the diagram, which showed a higher affinity with stations 5 and 6, were: Carchesium polypinum, Codonella cratera, Coleps hirtus, Paraurostyla weissei, Phascolodon vorticella, Stentor roeseli, Strobilidium humile. These species can be considered typical of the potamon fluvial zone. Species found only in the upper Mincio and ciliate species which were found at all six stations group together in the lower part of the diagram: among the former there are some ciliates such as Euplotes affinis, Mesodinium acarus, Pseudomicrothorax agilis, Trochilia minuta, and Zosterodasys transversa that can be considered typical of the hyporhithron fluvial zone, whilst ciliates found at all stations can be considered common species whose preferred habitat type is found in all the fluvial zones. The PCA, performed on physical, chemical and bacteriological data collected in the six stations (Table 1), confirmed the above results. The scatter diagram of Fig. 7 reveals the degree of affinity between stations 5 and 6 that, in turn, are clearly rather separate from those stations which are located in the upper river. The physical, chemical and bacteriological conditions at station 1 are found to be distant from those at the lower river stations. The first component explains 66.8% of the variance and it groups together all variables except the water flow. The second component (14.3% variance explained) includes the variable water flow. From the statistical analyses, it clearly emerges that the structure of the ciliate communities is strongly affected by the physical, chemical and geomorphological
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Table 4. Occurrence in other watercourses of the 21 common species found in the Mincio river Species
Fig. 7. Bidimensional topographic representation of the six sampling stations as defined by principal component analysis applied to non-ciliate data (variance explained: PC 1 ¼ 66.8%, PC 2 ¼ 14.3%).
characteristics of the several reaches of the river. In fact, station 1, even though located in the hyporhithron fluvial zone, suffered effects from its close proximity to Lake Garda, still retaining some forms typical of lentic waters such as Askenasia volvox, Codonella cratera, Halteria grandinella, Urocentrum turbo, and Urotricha furcata, which made it similar to communities located at the lower Mincio stations in the potamon fluvial zone. Likewise, the ciliate community at station 2, even though it maintained a structure similar to those of the other stations located in the hyporhithron fluvial zone, was affected by the organic load entering from sewage effluents, harbouring ciliate species indicative of elevated saprobicity such as Carchesium polypinum, Epistylis plicatilis, Euplotes aediculatus, Histriculus vorax, Microthorax pusillus, Plagiopyla nasuta, Saprodinium dentatum, Sterkiella histriomuscorum, Vorticella convallaria, and Zoothamnium procerius. Some of these ciliate species probably drifted down with the sewage effluent. The arrangement of stations 3 and 4 along the second component (vertical axis) is related to their different water flow. This can explain the different number of ciliate species found at these two stations. Previous studies on the distribution of ciliates related to river zonation (Madoni 1983, 1984a) revealed the existence of species typical of eight identified ecotypes. In the present study, 18 out 21 ciliate species defined as common in this watercourse, and 4 out of 5 species typical of the hyporhithron fluvial zone were also found as common and typical species in the hyporhithron zone of the river Taro (Madoni and Zangrossi 2005). Among these 21 common species (Table 4), 12 were also found in the sandy hyporheic zone of a lowland stream (Ladberger Mu¨hlenbach) (Cleven 2004), 9 were common to the epirhithron zone of a Spanish river (Henares)
Acineria uncinata Aspidisca cicada Aspidisca lynceus Cinetochilum margaritaceum Cyclidium glaucoma Euplotes patella Frontonia acuminata Frontonia angusta Holosticha kessleri Holosticha multistilata Holosticha pullaster Lacrymaria olor Lembadion lucens Litonotus cygnus Litonotus lamella Pleuronema coronatum Strobilidium caudatum Stylonychia mytilus-complex Uroleptus piscis Urostyla grandis Vorticella picta
River He
La
+
+ + + +
+ +
+
+ + + + + + +
+ + + +
+ +
St
Ta
Sa
Ll
+ + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + +
+ + + + + +
+ + + +
+ +
+
+ + +
+
+ + + + + + + +
He: River Henares, Spain; La: Ladberger Mu¨hlenbach, Germany; St: River Stirone, Italy; Ta: River Taro, Italy; Sa: River Sava, Slovenia; Ll: River Llobregat, Spain (References are given in the text).
(Sola et al. 1996), 20 were dominant in the metarhithron fluvial zone of the river Stirone (Madoni and Bassanini 1999), 14 were found along the river Llobregad (Gracia et al. 1989), and 13 were found in a branch of the river Sava located in the lower rhithral area (Primc-Abdija et al. 1996). In the last watercourse, Trochilia minuta was the dominant species and this corroborates our findings about its allocation as a typical species in the hyporhithron fluvial zone.
Conclusions The 1-year study of the ciliate populations along the Mincio River, has enabled the characterization of the biocenoses of the studied watercourse. The typical species of the ecotype located in the potamon zone, characterized by slow flow in a canalized bed, were defined. The ciliate species typical of the hyporhithron fluvial zone were also defined. Among the 133 species of ciliated protozoa found in this watercourse, only 21 species were common to all six studied stations. These species were commonly observed in all fluvial zones of other watercourses, thus they could be considered cosmopolitan inhabitants of running waters.
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Changes in the water quality produce quantitative and qualitative changes in the structure of the ciliate communities. These changes involve a decrease in the percentage of algivorous ciliates and an increase in the number of bacterivorous forms with the increase of the degree of saprobicity along the river. Even though we can define the ciliate communities typical of the different fluvial zones, gradual changes in the physical, chemical and geomorphological characteristics along the river, produce a gradual change in the community structure. The common species form the basic structure of the community, while the species which gradually replace one another along the river, are indicators of that particular fluvial section.
Acknowledgments The authors would like to express their thanks to Dr. Franco Sartore for his valuable assistance in the statistical processing of the data. This work was supported by grants (FIL) from Italian Ministry of University and Scientific and Technological Research.
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