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Protistology 5 (2/3), 213-230 (2007/8)
Protistology
Species diversity of heterotrophic flagellates in Rdeisky reserve wetlands
Denis V. Tikhonenkov
Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, Russia Summary
The active and cryptic species diversity, abundance, biomass, community structure of heterotrophic flagellates were studied in the acid boggy lakes in the Rdeisky State Nature Reserve in March and July, 2005. Ninety species and forms of heterotrophic flagellates were found. The most abundant groups are chrysomonads, kinetoplastids, euglenids, the most abundant species are Paraphysomonas sp., Spumella sp., Bodo designis, Goniomonas truncata, Astasia sp., Protaspis simplex. In different biotopes, the population of heterotrophic flagellates ranged from 114 to 7457 ind./cm3; their biomass, from 17.1 x 10-3 to 1833.5 x 10-3 |ig/cm3. Within one lake, sphagnobiont communities are characterized by higher integral community characteristics (species diversity and richness, abundance, biomass) than communities in other habitats. This fact can be explained by a higher habitat heterogeneity and a large number of potential ecological niches for the heterotrophic flagellates. The total pattern of flagellates' distribution is extremely mosaic. Distinctions between microbiotopes are greater than those between different lakes. "Basic" communities (those of sphagnobionts and overrotten plant residues) and "derivative" communities (those of bottom sediments and peat) are distinguished in each lake. The "basic" communities are characterized by maximal abundance and species richness. The derivative communities are simplified variants of the "basic" ones, with lower abundance and species richness, and the absence of characteristic species. The same tendencies are traced at the level of both cryptic and active species diversity. Cryptic species diversity is considerable higher than the active one, but represents the latter in many respects.
Key words: Heterotrophic flagellates, species diversity, community structure, Rdeisky reserve
Introduction
Heterotrophic flagellates (HF) are the major link between the microbial communities and the meta-zoan part of aquatic food webs (Lee et al., 1999) and an obligatory component of all types of water ecosystems. HF are especially abundant in the boggy sites, where their communities are the most specific (Mazei et al., 2005b). Though HF are extremely important in functioning of boggy lakes and bog ecosystems
(Mazei et al., 2005b), information on their species diversity and abundance in sphagnous biotopes is very scarce (Zolotarev and Zhukov, 1994; Simek et al., 1998; Tikhonenkov et al., 2006a).
Bogs and boggy habitats play a significant role in the formation of the hydrological routines of the surrounding area and the environmental conditions in general (Bogdanovskaja-Gienef, 1969; Boch and Mischenko, 2000). The investigation of their biodiversity is currently very important. In particular, this
© 2007 by Russia, Protistology
concerns the biotopes that are not much exposed to anthropogenic influence and whose communities may be used as a model for ecosystems of the given type. The study of such coenoses is necessary for the development of biological environment monitoring.
The aim of our study was to investigate species diversity and the community structure of heterotrophic flagellates in the boggy lakes of Rdeisky reserve.
Material and Methods
The samples were collected in 11 acid boggy lakes (pH 4.4-5.1) in the Rdeisky State Nature Reserve territory on March and July, 2005. These lakes belong to one of the largest European bog systems, Polisto-Lovatskaja system, which has an international significance (subzone of the south taiga, western part of the Valdai Hills, Polist' and Lovat' rivers watershed (Novgorodskaja oblast, Russia)). Polisto-Lovatskaja system is a large body of a non-broken bog with boggy lakes.
The HF communities of lakes Bolshoe Goret-skoe, Chudskoe, Domshinskoe, Kornilovskoe, Ma-loe Goretskoe, Mezhnitskoe, Ostrovistoe, Poddom-shinskoe, Rdeiskoe, Rogovskoe, Russkoe were investigated. Water samples with Sphagnum sp. stems were taken in the sphagnum quagmires of the lakes' catchment area and in marginal zones of all lakes; samples of bottom sediments were also taken at a depth of 1.5-1.8 m in Domshinskoe and Ostrovistoe lakes. Two samples were collected at each station.
Only non-empty (HF-containing) samples taken in July were used for active and cryptic species diversity comparison. March samples were used for a more elaborate investigation of HF species richness.
Samples of the sphagnum and the sediments with water were placed in 50 ml flasks and maintained at approximately 3°C during transportation to the laboratory. The 5 ml sample volume was analyzed in two replicates in Petri dishes in order to study the species composition. Active species diversity was studied directly after transportation. For studying cryptic species diversity, the same samples were enriched with suspension of Pseudomonas fluorescens bacteria and maintained in darkness at 25°C in the thermostat; cryptic species diversity was studied on the 3rd, 6th, 9th days of exposition (V0rs, 1992). For specific identification of some forms, clonal cultures were used.
Quantitative and qualitative analyses of HF in fresh and enriched samples with living cells was performed, permitting one to identify small HF on the species level.
The HF cell volumes were defined by means of approximation to the simple geometrical figures with the use of our own organism's measurements and the
data published (Larsen and Patterson, 1990; V0rs, 1992; Zhukov, 1993; Patterson and Simpson, 1996; Kosolapova and Mylnikov, 2001; Mylnikov et al., 2002; Tikhonenkov, 2006). The findings were used for HF weight computation (the density was accepted to equal 1g/cm3).
Light microscopic observations were made with the aid of Biolam-I microscope (Russia) equipped with phase contrast and water immersion objectives at a total magnification of x770, and Reichert (Austria) microscope with interference contrast and glycerin immersion objectives (1000x). The microscopes were equipped with analogous video camera AVT HORN MC - 1009/S, connected to a video recorder Panasonic NV-HS 850. Image acquisition in VHS and S-VHS mode with subsequent digitization of the image and preservation of video film fragments as AVI-format files was carried out for a more precise identification of the HF discovered.
The systems of eukaryotes and protists by Lee et al. (2002) and Adl et al. (2005) were used.
The abundance (ind./cm3), biomass (^g/cm3) of organisms, Margalef species richness index (Margalef, 1957), Shannon species diversity index (Shannon and Weaver, 1949), Shannon equitabil-ity (Pielou, 1966) were applied in order to obtain the integral characteristic of community structure. Mann-Whitney U Test was used for an estimation of reliability of distinctions in abundance, biomass and species diversity between the different variants of community. Marked tests are significant at P <0.05.
The classification of communities (R-analysis) by their species structure (qualitative data) was conducted with the aid of cluster analysis (complete linkage) based on the Hacker and Dice indices similarity matrix.
D(X,Y)
a + min(b, c)'
a - number of common species in X and Y samples, b and c - number of the species observed only in one sample.
The abundance biomass comparison (ABC) method was used to characterize size community structure (Warwick et al., 1987). In stable non-broken communities (with prevalence of large forms), the curve of abundance is considered to lie below the biomass curve, and in broken ones (with prevalence of small forms), above it. The ABC-index was calculated to characterize interposition of these curves (Meire and Dereu, 1990).
W
ABC = ]T(Bi - Ni)/W,
i=i
B. and N. - cumulative percents of biomass and abundance of i the first species in order, W - total species number.
Positive values of the index indicate prevalence of relatively large forms, and negative values, prevalence of small ones. Values close to nought indicate mixed size structure, with domination of both large and small forms.
All computations were performed with the aid of the program packages STATISTICA 5.5 (StatSoft, Inc., 1999) PAST (Hammer et al., 2001) and ECOS 1.3 (Azovskii, 1993).
Results
Ninety species and forms of heterotrophic flagellates were found in the biotopes investigated (Table 1). Seven species (Bicosoeca campanulata,
Cercomonas acutus, Notosolenus alatellus, N. urceo-latus, Quadricilia rotundata, Rhynchobodo thaenia-ta, Salpingoeca napiformis) are new for the Russian freshwater protistan fauna. Morphological features of the HF species identified corresponded to those reported earlier (Zhukov, 1971; Skuja, 1956; Larsen and Patterson, 1990; V0rs, 1992; Zhukov, 1993; Patterson and Simpson, 1996; Lee and Patterson, 2000; Al-Quassab et al., 2002; Mylnikov and Kosolapova, 2004), with the exception of Metromonas sp., which was different from the Metromonas species described earlier.
Metromonas sp. (Figs 1, 2)
Description: Cell flattened and rigid, comma-shaped in profile, 4-7 (5.5) ^m long and 2.5-4.5 (3) wide with smooth margins. Posterior part of the cell is the widest one, small rostrum is situated in the
Species and forms Russkoe Mezhnitskoe Domshinskoe Poddomshinskoe Kornilovskoe Ostrovistoe Maloe Goretskoe Bol'shoe Goretskoe Rogovskoe Rdeiskoe Chudskoe
AMOEBOZOA (Lühe, 1913) Cavalier-Smith, 1998
*Incertae sedis AMOEBOZOA
Quadricilia rotundata (Skuja, 1948) V0rs, 1992 -/+ -/+
*Incertae sedis AMOEBOZOA: Spongomonadida (Hibberd, 1983) Karpov, 1990
**Phalansterium Stein, 1878
Phalansterium solitarium Sandon, 1924 +/+ -/+
**Spongomonadidae Karpov, 1990
Spongomonas uvella Stein, 1878 -/+ -/+ -/+ -/+ -/+ -/+ -/+
Spongomonas sp. -/+
OPISTHOKONTA (Cavalier-Smith, 1987) Adl et al., 2005
*Choanomonada Kent, 1880
**Monosigidae Zhukov and Karpov, 1985
Codonosiga botrytis Kent, 1880 -/+ -/+ -/+ -/+ -/+ -/+ +/+
Monosiga ovata Kent, 1880 -/+ -/+ -/+ -/+
**Salpingoecidae Kent, 1880
Salpingoeca amphora Kent, 1880 -/+
S. amphoridium Clark, 1868 +/-
S. massarti De Saedeleer, 1927 -/+
S. napiformis Kent, 1880 -/+ -/+
S. schilleri (Schiller, 1953) Starmach, 1968 +/-
Table 1. List of heterotrophic flagellates found in this study
Salpingoeca sp. -/+
RHIZARIA Cavalier-Smith, 2002
*Cercozoa (Cavalier-Smith, 1998) Adl et al., 2005
**Cercomonadida (Poche, 1913) Mylnikov, 1986
***Cercomonadidae (Kent, 1880) Mylnikov and Karpov, 2004
Cercomonas acutus (Skuja, 1948) Mylnikov and Karpov, 2004 -/+
C. aff. agilis (Moroff, 1904) Mylnikov and Karpov, 2004 -/+ -/+ -/+ -/+ -/+ -/+
C. angustus (Skuja, 1948) Mylnikov and Karpov, 2004 -/+
C. bodo (Meyer, 1897) Mylnikov and Karpov, 2004 -/+ -/+
C. crassicauda (Dujardin, 1841) Mylnikov and Karpov, 2004 -/+ -/+
C. granulifera (Hollande, 1942) Mylnikov and Karpov, 2004 -/+ -/+
C. laciniagerens (Krassiltschik, 1886) Mylnikov and Karpov, 2004 -/+ -/+ -/+ -/+
C. longicauda Dujardin, 1841 -/+ -/+ -/+
C. ovatus (Klebs, 1892) comb. nov. non C. ovata Tong, V0rs and Patterson, 1997 -/+
C. plasmodialis Mylnikov, 1992 -/+
C. radiatus (Klebs, 1892) Mylnikov and Karpov, 2004 -/+ -/+
Cercomonas sp. +/- -/+ +/+ -/+ -/+ -/+ +/+ +/+
Helkesimastix faecicola Woodcock et Lapage, 1914 -/+ -/+ -/+ -/+ -/+
***Heteromitidae (Kent, 1880) Mylnikov and Karpov, 2004
Heteromitaglobosa Stein, 1878 -/+ -/+ -/+ +/+ -/+ -/+
H. minima (Hollande, 1942) Mylnikov and Karpov, 2004 -/+ -/+ -/+ -/+ -/+ -/+ -/+ +/+
H. reniformis (Zhukov, 1978) Mylnikov and Karpov, 2004 -/+
Heteromita sp. -/+ -/+ -/+ -/+
Incertae sedis Heteromitidae
Allantion tachyploon Sandon, 1924 -/+ -/+ +/+ -/+ -/+ -/+
Protaspis simplex V0rs, 1992 -/+ +/+ -/+ +/+ +/+ -/+ -/+ -/+ +/+
ARCHAEPLASTIDA Adl et al., 2005
*Chloroplastida Adl et al., 2005
**Chlorophyta (Pascher, 1914) Lewis and Mc Court, 2004.
***Chlorophyceae Christensen, 1994
Polytomapapillata Pascher, 1927 +/-
CHROMALVEOLATA Adl et al., 2005
*Cryptophyceae (Pascher, 1913) Schoenichen, 1925
**Cryptomonadales Pascher, 1913
Cryptomonas sp. -/+ +/+
**Goniomonadales Novarino and Lucas, 1993
Goniomonas truncata (Fresenius, 1858) Stein, 1887 -/+ +/+ +/- +/+ +/+
*Stramenopiles (Patterson, 1989) Adl et. al., 2005
**Bicosoecida (Grassé, 1926) Karpov, 1998
Bicosoeca campanulata (Lackey, 1942) Bourrelly, 1953 -/+
B. cylindrica (Lackey, 1939) Bourrelly, 1951 -/+ -/+ -/+ +/+
B. exilis Penard, 1921 +/-
B. paropsis Skuja, 1956 -/+
Cyathobodo sp. -/+
Pseudodendromonas vlkii (Vlk, 1938) Bourrelly, 1953 -/+
**Chrysophyceae Pascher, 1914
Paraphysomonas sp. -/+ -/+ +/+ -/+ +/+ +/+ -/+ -/+ -/+ +/+ +/+
Spumella sp. -/+ -/+ +/+ -/+ +/+ +/+ -/+ -/+ +/+ +/+
Stokesiella acuminata (Stokes, 1888) Lemmermann, 1910 -/+
***Chromulinales Pascher, 1910
Anthophysa vegetans (O.F. Mueller, 1773) Stein, 1878 -/+
Dendromonas laxa (Kent, 1871) Blochmann, 1895 -/+
**Dictyochophyceae Silva, 1980
***Pedinellales Zimmermann, M0estrup and Hällfors, 1984
Ciliophrys infusionum Cienkowsky, 1876 -/+ -/+
Pteridomonas pulex Penard, 1890 -/+
*Alveolata Cavalier-Smith, 1991
**Dinozoa (Cavalier-Smith, 1981) Cavalier-Smith and Chao, 2004
***Dinoflagellata (Bütschli, 1885) Adl et al., 2005
****Dinophyceae Pascher, 1914
*****Gymnodiniphycidae Fensome et al., 1993
Amphydinium sp. -/+
**Apicomplexa (Levine, 1980) Adl et al., 2005
***Colpodellida (Cavalier-Smith, 1993) Adl et al., 2005
Colpodella angusta (Dujardin, 1841) Simpson et Patterson, 1996 +/-
Colpodella sp. -/+
*Incertae sedis Alveolata
Colponema loxodes Stein, 1878 -/+
EXCAVATA (Cavalier-Smith, 2002) Simpson, 2003
*Jakobida (Cavalier-Smith, 1993) Adl et al., 2005
**Histionidae Flavin and Nerad, 1993
Histiona aroides Pascher, 1943 -/+ +/- -/+ -/+
Reclinomonas americana Flavin et Nerad, 1993 -/+ -/+ +/- -/+ -/+
*Euglenozoa (Cavalier-Smith, 1981) Simpson, 1997
**Euglenida (Bütschli, 1884) Simpson, 1997
***Euglenea (Bütschli, 1884) Busse and Preisfeld, 2002
Astasia parva Pringsheim, 1942 -/+ -/+
Astasia sp. +/- +/-
***Heteronematina Leedale, 1967
Ploeotia costata (Triemer, 1986) Farmer and Triemer, 1988 -/+
P. oblonga Larsen and Patterson, 1990 -/+
Ploeotia sp. -/+
Urceolus sp. -/+
***Sphenomonadina Leedale, 1967
Notosolenus alatellus Larsen and Patterson, 1990 -/+
N. apocamptus Stokes, 1884 -/+ -/+
N. urceolatus Larsen and Patterson, 1990 -/+
Petalomonas minor Larsen and Patterson, 1990 -/+
P. minuta Hollande, 1942 +/- -/+ -/+
P. pusilla Skuja, 1948 +/- +/+ -/+ -/+
Petalomonas sp. 1 -/+
Petalomonas sp. 2 +/- +/+ +/- -/+ +/+
Sphenomonas teres (Stein, 1878) Klebs, 1893 -/+ -/+
**Kinetoplastea Honigberg, 1963
***Metakinetoplastina Vickerman (in Moreira, Lopez-Garicia and Vickerman, 2004)
****Neobodonida Vickerman (in Moreira, Lopez-Garicia and Vickerman, 2004)
Dimastigella mimosa Frolov, Mylnikov and Malysheva, 1997 -/+ -/+
D. trypaniformis Sandon, 1928 -/+ -/+
Rhynchobodo taeniata V0rs, 1992 +/-
Rhynchomonas nasuta (Stokes, 1888) Klebs, 1892 -/+ +/+ -/+
****Parabodonida Vickerman (in Moreira, Lopez-Garicia and Vickerman, 2004)
Procryptobia sorokini (Zhukov, 1975) Frolov, Karpov and Mylnikov, 2001 +/-
****Eubodonida Vickerman (in Moreira, Lopez-Garicia and Vickerman, 2004)
Bodoangustatus (Dujardin, 1841) Butschli, 1883 -/+
B. crassus Skuja, 1956 +/-
B. curvifilis Griessmann, 1913 -/+
B. designis Skuja, 1948 -/+ +/- +/+ +/+ -/+ -/+ -/+ +/+ +/+
B. minimus Klebs, 1893 -/+ -/+ -/+
B. saltans Ehrenberg, 1832 -/+ -/+ +/+ +/+ -/+ -/+ +/+ -/+
B. spora Skuja, 1956 -/+
Bodo sp. 1 -/+ -/+
Bodo sp. 2 -/+
Incertae sedis EUKARYOTA
Metromonas simplex (Griessmann, 1913) Larsen and Patterson, 1990 -/+
Metromonas sp. -/+ -/+ -/+
Phyllomitus amylophagus Klebs, 1893 -/+ -/+
P. apiculatus Skuja, 1948 +/- +/+ -/+ -/+ +/+
Ancyromonas Kent, 1880
Ancyromonas contorta (Klebs, 1893) Lemmermann, 1910 -/+
A. sigmoides Kent, 1880 -/+ +/- +/- +/-
Spironemidae Doflein, 1916
Hemimastix sp. -/+
Kathablepharidae V0rs, 1992
Kathablepharis sp. -/+
* - **** - unformalized ranks of taxa; ".../"- active species richness; "/..."- cryptic species richness; "+"- presence of the species in the samples; "-"- absence of the species in the samples.
anterior end, from under which two nonacronematic flagella emerge (Fig. 1a). Anterior flagellum is shorter than the cell body, curved to the dorsal cell surface, 3.5-4 long. Posterior flagellum is about 1.5-2 times (10-12 ^m) of the cell length. Nucleus is situated in the anterior part of the cell near the flagellar basal bodies (Fig. 1b, d, 2b). The lateral contractive vacuole is located posteriorly (Fig. 1c, 2c, e ). The cell is attached to the substrate by a curved distal part of the posterior flagellum moving with a pendulum action circumscribing a half-round. The organism may also glide actively, during gliding the cell body becomes straight and elongates a little (7x2.5 ^m) (Fig. 1d, 2d, e, f). At the same time, earlier inactive minor flagellum begins to operate actively. The recurrent flagellum is trailing behind the cell. The posterior part of the cell is cocked, only the anterior end of the cell contacts the substrate during gliding (Fig. 1d, 2d, e). Sometimes binuclear cells with two anterior flagella are observed (Fig. 1c), usually such cells were dividing. Cysts were not found.
Remarks: A large food vacuole is often observed at the abflagellar end of the cell, perhaps indicating that this organism is predatory. Other representatives of the genus Metromonas are known to be predators (Larsen and Patterson, 1990). A similar species, Metromonas simplex (Griessmann, 1913) Larsen, Patterson, 1990, also noted in boggy lakes (Mylnikov
and Kosolapova, 2004; Tikhonenkov, 2006), is distinguished by an abovate or pear-shaped cell profile and small, poorly visible free anterior flagellum (Larsen and Patterson, 1990; Al-Qassab et al., 2002). Metromonas sp. differs from Metromonas grandis Larsen and Patterson, 1990 by a smaller cell body size, the comma-shape of the cell, a remarkably long anterior flagellum, the absence of the slight protrusion (nipple) at the abflagellar end, and not folded or ridged margins (Larsen and Patterson, 1990; Lee and Patterson, 2000).
Fig. 1. Metromonas sp. a-c - attached cells, d - gliding cell. Abbreviations: AF - anterior flagellum; PF - posterior flagellum; FV - food vacuole; CV - contractive vacuole; N - nucleus. Scale bar 5 |im.
Fig. 2. Metromonas sp. a-c, f - attached cells, d, e - gliding cells. Abbreviations: AF - anterior flagellum; PF - posterior flagellum; FV - food vacuole; CV - contractive vacuole; N - nucleus. Scale bar 5 |im.
The organism under discussion is very similar to Metromonas sp. by Mylnikov and Kosolapova (2004), differing only in a smaller cell size. The latter species was found in boggy lake with acid reaction, too. It is very likely that we observed the same new species of heterotrophic flagellates as Mylnikov and Kosolapova (2004).
Active species diversity
The greatest numbers of the species in the biotopes investigated belong to kinetoplastids (6 species), cer-comonads (5 species), euglenids (4 species). The most common species are Spumella sp. (observed in 100% locations), Bodo designis (77.8), Cercomonas sp. (77.8), Paraphysomonas sp. (77.8), Protaspis simplex (66.7). The most abundant groups are chrysomonads, ki-netoplastids, euglenids and species Paraphysomonas sp., Spumella sp., Bodo designis, Goniomonas trunca-ta, Astasia sp., Protaspis simplex, which form about 95% of the total abundance.
Fig. 3. Abundance (N) of heterotrophic flagellates (ind./cm3) in different communities. Whiskers - minimums and maximums; boxes - quartiles 25-75%; triangles - medians. 1 - Kornilovskoe lake, peat at the border with water surface; 2 - Kornilovskoe lake, sphagnum quagmire; 3 - Domshinskoe lake, sphagnum and Carex sp. at the border with water surface; 4 - Ostrovistoe lake, sphagnum quagmire; 5 - Ostrovistoe lake, bottom sediments (1.5 m depth); 6 - Rdeiskoe lake, overrotten plant residues; 7 - Rdeiskoe lake, bottom sediments (1.8 m depth); 8 - Chudskoe lake, peat at the border with water surface; 9 - Chudskoe lake, sphagnum quagmire.
B
0.075 -
0.0075
0.0025
Fig. 4. Biomass (B) of heterotrophic flagellates (|ig/cm3) in different communities. Whiskers - minimums and maximums; boxes - quartiles 25-75%; triangles - medians. The stations' order as in Fig. 3.H
Abundance of heterotrophic flagellates in different biotopes was in the range of 114 - 11875 ind./ cm3, whereas biomass was in the range of 17.1 x 10-3 -2241.8 x 10-3 |g/cm3. Biomass and abundance of flagellates increase in communities of sphagnobionts of Kornilovskoe lake (st. 2) and overrotten plant residues of Rdeiskoe lake (st. 6). On the whole, the greatest abundance and biomass of HF were observed in phytophilous communities, and the lowest, in marginal zones at the border of sphagnum quagmires and water surface as well as in the bottom sediments (Fig. 3, 4). The distinction between these groups is significant (P < 0.05; Mann-Whitney criterion), but inauthentic inside them.
The Margalef species richness index indicates that the HF communities of sphagnum quagmires of Ostrovistoe and Chudskoe lakes are the most species-rich (Fig. 5). Sphagnum biotopes are defined by the highest species richness of HF independently of lake type. The distinction between sphagnobionts
species diversity and others communities is significant (P < 0.05; Mann-Whitney criterion).
The values of Shannon species diversity index indicate the greatest complexity of sphagnobionts communities (Kornilovskoe, Domshinskoe, Ostrovistoe, Chudskoe lakes). The distinctions are significant (P=0.02) (Fig. 6). The bottom sediments (st. 7) and marginal zones (st. 6, 8) communities are characterized by the lowest species diversity (P < 0.05; Mann-Whitney criterion). Decrease of community species diversity indicates the simplification of species structure.
High evenness of species structure is noted almost in all the biotopes. It is explained by the absence of a single dominant, except in the phytophilous communities of Kornilovskoe and Rdeiskoe lakes (Fig. 7). These communities are characterized by the presence of pronounced dominants, Paraphysomonas sp. and Spumella sp., which formed a 83.5% (Kornilovskoe lake) and 66.5% (Rdeiskoe lake) of total HF community abundance.
Fig. 5. The Margalef species richness (M) of heterotrophic flagellates in different communities. Whiskers - minimums and maximums; boxes - quartiles 25-75%; triangles - medians. The stations' order as in Fig. 3.
The method of abundance biomass comparison (Warwick, 1984) has been used for characterization of the size structure of HF community. The communities of almost all the sites investigated are characterized by high positive values of ABC index, with the exception of the communities formed in the bottom sediments of Rdeiskoe lake (-1.2) and Kornilovskoe lake quagmire (0.3) (Fig. 8). Community at station 7 has low species richness with the prevalence, in terms of abundance, of Histiona aroides, having a small biomass. Low value of ABC index at station 2 is associated with the prevalence of small chryso-monads Spumella sp. and Paraphysomonas sp. The maximal values of ABC index and medium-sized parameters of individuals were noted in those communities where large forms of HF dominated in terms of both abundance and biomass. This method is considered to be a rather sensitive indicator of natural disturbance of environmental and anthropogenic stresses (Azovskii et al., 1998). The ABC-method has proved earlier to be a good indicator of stressful
influence with reference to HF communities (Mazei et al., 2005a). Hence, the value of ABC-index is positive under stable conditions, in absence of stressful factors. It points to the prevalence of large forms in community (probably, K-strategists, or violents and patients) and, probably, to its non-broken state. Values of this parameter are negative in coenoses, forming under the stressor influence, which testifies to the prevalence of small species in the community (probably, r-strategists, or explerents) and probably indicates its broken state or a rather high structural lability.
Cryptic species diversity
Cercomonads (19 species), euglenids (14), kineto-plastids (11) are the dominant groups in terms of the number of species. All other taxa are characterized by the twice as small species richness. The most common species are Heteromita minima (observed in 100% locations), Bodo saltans (80), Paraphysomonas
Fig. 6. The Shannon species diversity (H) of heterotrophic flagellates in different communities. Whiskers - minimums and maximums; boxes - quartiles 25-75%; triangles - medians. The stations' order as in Fig. 3.
sp. (80), Spumella sp (70), Bodo designis (60), Allantion tachyploon (50), Protaspis simplex (50), Spongomonas uvella (50). Community species richness increased more than twofold after samples enrichment. Cercomonads, euglenids, kinetoplastids have actively developed after exposition. Out of these taxa, nineteen species not observed during active specific diversity investigation were identified.
Patterns of community species richness changes are similar to those revealed at the level of active species diversity (Fig. 9). The values of Margalef species richness index are maximal in the communities of sphagnum quagmires and overrotten plant residues of Rdeiskoe lake.
The communities formed in peat at the border with water surface and in bottom sediments are characterized by the minimal species richness. The distinction between these groups is significant (P < 0.05; Mann-Whitney criterion). It was noted again that sphagnobiont communities are richer in terms of species richness.
Active vs. cryptic species diversity and HF community distribution
The overall HF distribution pattern is extremely mosaic. A great number of rare species calls forth a high variability of species composition both from lake to lake and within one lake. However, distinctions between microbiotopes are greater than those between different lakes. Even inside different replicates of the same sample the differences are very large. Hacker-Dice replicates similarity in active species diversity does not exceed 0.58 (0.39 on average), but that in cryptic species diversity, 0.73 (0.43 on average). However, the distinction between these similarity values is not significant (P > 0.05; Mann-Whitney criterion). Interestingly, in the active community species richness of the marginal zones of Kornilovskoe lake no common species were observed in two replicates of the same sample. However, cryptic species richness of these replicates was similar, about 57% (D = 0.73).
In the classification by active and cryptic species
4 5 6 7 Stations
Fig. 7. The HF community evenness (E) in different biotopes. Whiskers - minimums and maximums; boxes - quartiles 25-75%; triangles - medians. The stations' order as in Fig. 3.
Stations
Fig. 8. The ABC-index values in different coenoses. The stations' order as in Fig. 3.
1 23456789 10
Stations
Fig. 9. Cryptic Margalef species richness (M) of heterotrophic flagellates in different communities. Whiskers - minimums and maximums; boxes - quartiles 25-75%; triangles - medians. 1 - Kornilovskoe lake, peat at the border with water surface; 2 -Kornilovskoe lake, sphagnum quagmire; 3 - Domshinskoe lake, bottom sediments (1.8 m depth); 4 - Domshinskoe lake, sphagnum and Carex sp. at the border with water surface; 5 - Ostrovistoe lake, sphagnum quagmire; 6 - Ostrovistoe lake, bottom sediments (1.5 m depth); 7 - Rdeiskoe lake, overrotten plant residues; 8 - Rdeiskoe lake, bottom sediments (1.8 m depth); 9 - Chudskoe lake, peat at the border with water surface; 10 - Chudskoe lake, sphagnum quagmire.
compositions, the allocated groupings differed mainly in species richness values. In the active species composition (Fig. 10), the community of Ostrovistoe lake quagmire (st. 4), the richest in term of species diversity, is separated from all the other communities. Sphagnum quagmires' and overrotten plant residues' communities are grouped together (st. 2, 3, 6, 9) and differ from the less rich coenoses formed in the bottom sediments (st. 5, 7) and in the peat (st. 1, 8). In the cryptic species diversity community classification (Fig. 11), sphagnobionts' and overrotten plant residues' coenoses are similar again and characterized by high values of species richness (st. 2, 4, 5, 7, 10). The bottom sediments and peat communities (st. 1, 3, 6, 8, 9) differ from them in term of species richness, but bottom sediments' coenoses of Ostrovistoe lake (st. 6) are separated from all the other communities and include some characteristic species (Cercomonas laciniagerens, Heteromita sp. (clone PS-5F), Phyllomitus amylophagus).
Thus, "basic" communities (sphagnobionts and overrotten plant residues' coenoses) and "derivative" communities (bottom sediments and peat coenoses) are distinguished in each lake. The "basic" communities are characterized by maximal abundance and species richness. The "derivative" communities represent simplified variants with lower abundance and species richness, and also the absence of characteristic species.
The same tendency is traced at the level of both cryptic and active species diversity.
Discussion
The HF communities in the boggy lakes investigated are characterized by large species diversity, highly heterogeneous structure and unique species composition. During this relatively small-scale and short-term exploration, one HF species probably new for science was found, and seven species new for Russian freshwater protistan fauna were observed. Representatives
1
0.9 0.8 -0.7 -
•t 0.6 h
cd
m 0.5 0.4 0.3 0.2 0.1 H
Fig. 10. The similarity of active species richness in different coenoses. The stations' order as in Fig. 3.
of very small taxa such as spongomonads, pseudoden-dromonads and phalansterids, were often identified in the present study. These HF groups, in general seldom mentioned in protozoological research, are probably characteristic of boggy habitats (Lemmermann, 1914; Zolotarev and Zhukov, 1994; Mylnikov and Kosolapova, 2004). It is extremely important since endemism, on the whole, is not characteristic of HF (Patterson and Lee, 2000). HF have a very broad occurrence, they are ubiquitous and cosmopolite. The same species were observed in plankton and benthos, marine and freshwater habitats (Fenchel, 1986, 1987; Arndt et al., 2000; Skuja, 1956; Mylnikov, 1978; Hamar, 1979; Mylnikov and Zhgarev, 1984; Patterson and Lee, 2000; Kosolapova and Mylnikov, 2001; Kopylov, 2003).
In some samples with low values of Eh-level from bottom sediments (2-4m depth) no HF species were observed. Free-living anaerobic flagellates mostly belonging to groups lacking mitochondria, such as diplomonads, retortomonads and trichomonads, can dwell under these conditions (Mylnikov, 1991). They
are the most common flagellates in anoxic habitats (Lackey, 1932, 1938; Mylnikov, 1983; Brugerolle and Müller, 2000). But anaerobic flagellates are very sensitive to increase in oxygen concentration (Mylnikov, 1985) and could perish during transportation of samples to the laboratory.
Abundance and biomass of the HF from the boggy lakes coenoses investigated are similar and correspond to the values reported earlier from boggy habitats (Simek et al., 1998; Kosolapova, 2001; Kosolapova, 2005; Mazei et al., 2005a). The maximal values of the HF abundance in the biotopes investigated are, on the average, higher than in fresh-water planktonic communities of non-acid lakes and ponds (Travnik, 1989; Carlough and Meyer, 1990; Pace et al., 1990; Sanders et al., 1992; Pick and Hamilton, 1994; Mathes and Arndt, 1995; Kosolapova, 2001; Mazei et al., 2005a). However, these values are much lower than those reported from eutrophic benthic coeno-ses of anaerobic sediments, where the abundance can amount to 2400000 ind/cm3 (Finlay et al., 1988). The HF abundance correlates positively with the trophic
64527 10 3 89 1
1 -j
0.9 -0.8 -0.7 -
•a 0.6 - ____
.1 0.5 - --
Gn __
-1- -
0.3 -0.2 " 0.1 -
Fig. 11. The similarity of cryptic species richness in different coenoses. The stations' order as in Fig. 9.
status of the biotope (Sanders et al., 1992; Auer and Arndt, 2001). The minimal values of biomass (peat at the border with water surface) correspond to those in mesotrophic reservoirs (Auer and Arndt, 2001), whereas maximal values (sphagnum quagmires) correspond to the biomass of HF communities in anaerobic bottom sediments (Finlay et al., 1988).
Species diversity of HF in the boggy habitats investigated is higher than in the boggy sites of the Yaroslavl oblast (30-85 species) (Mazei et al., 2005b; Kosolapova, 2005; Tikhonenkov, 2006; Tikhonenkov et al., 2006a; Mylnikov and Kosolapova, 2004) and Karelia (57 species) (Tikhonenkov and Mazei, 2005). The number of the HF species observed is similar to that in benthic communities of eutrophic ponds (Finlay et al., 1988). A greater species diversity is noted only in coastal marine sediments (Larsen and Patterson, 1990; Tikhonenkov et al., 2006b).
Within one lake, sphagnobiont communities are characterized by a higher species richness than those from bottom sediments and peat communities, which confirms the previous research (Mazei et al., 2005a, b). This fact can be explained by a higher habitat heterogeneity and a large number of potential ecological niches, since HF are an extremely heterogeneous ecological group, exhibiting a large variety of life forms, food strategies and various ranges of tolerance to environmental factors (Zhukov, 1993).
Cryptic species richness is considerably higher than the active one in the communities investigated. The number of species increased 2.9 times after samples enrichment by bacteria and thermostat exposition. Only 9 species (Bicosoeca exilis, Bodo crassus, Polytoma papillata, Procriptobia sorokini, Salpingoeca amphoridium, S. schilleri, Rhynchobodo thaeniata, Astasia sp., Colpodella angusta) identified in active species richness have not been observed during cryptic species richness investigation. Apparently, these species are very sensitive to an increase in bacterial abundance or bacterial metabolism products. For example, high concentration of bacterial food causes clogging of collar tentacules and death of choanofla-gellates (Mylnikov, pers. com.).
In spite of the considerable differences in HF species composition in enriched and non-enriched samples, cryptic species diversity of the communities investigated represents the active one in many respects, and local coenoses have a similar distribution patterns at cryptic and active species diversity levels.
Acknowledgements
The author would like to thank Dr. Yu. A. Mazei and Dr. A. P. Mylnikov for critical comments on different aspects of this study, and Dr. V. T. Komov, Dr. A. V. Krylov and M. S. Kulikovsky for help in the
material collection. This study was supported by the Russian Foundation for Basic Research (grant numbers 04-04-48338 and 05-04-48180).
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Address for correspondence: Denis Victorovich Tikhonenkov. Institute for Biology of Inland Waters, Russian Academy of Sciences. BOROK, Yaroslavskaya obl. 152 742 RUSSIA. E-mail: tikhon@ibiw.yaroslavl.ru or tik-ho-denis@yandex.ru
Editorial responsibility: Sergey Karpov
