Heterotrophic flagellates from the saline lakes of the Crimean Peninsula

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Heterotrophic flagellates from the saline lakes of the Crimean Peninsula

Kristina I. Prokina

Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok 109, Nekouz District, Yaroslavl Region, 152742 Russia

kristin892@mail.ru

Received: 29.03.2020 Accepted: 15.04.2020 Published online: 06.05.2020

DOI: 10.23859/estr-200329 UDC 593.1 (574.5)

ISSN 2619-094X Print ISSN 2619-0931 Online

The species composition of heterotrophic flagellates has been studied in saline inland water bodies of the Crimean Peninsula. Twenty-nine species and forms belonging to 20 genera and 5 supergroups have been found. All identified species are new to the studied lakes. Rhynchomonas nasuta, Neobodo designis, Petalo-monas poosilla, Percolomonas cosmopolitus and Cafeteria sp. are the most common species. Euryhaline species form about a half of the species list. The number of the flagellate species was the lowest in the samples obtained in hypersaline ponds characterized by the salinity exceeding 100%o. There are no clear patterns observed in the cluster formation at the dendrogram of taxonomic similarity of the studied lakes.

Prokina, K.I., 2020. Heterotrophic flagellates from the saline lakes of the Crimean Peninsula. Ecosystem Transformation 3 (2), 3-10.

Introduction

Saline continental water bodies are among the most extreme water habitats of the Crimean Peninsula due to high water temperature and low oxygen content in addition to high water salinity (Shadrin et al., 2017); since such water bodies are often characterized by the shallow depths (1-3 m), they warm up to 35-40 °C in the summer. It is also noted that the alternation of dry and rainy periods preconditions the significant seasonal and interannual salinity variations (Balushkina et al., 2009). Saline lakes of the Crimean Peninsula were repeatedly studied by protistologists (Dagaeva, 1927; Goryacheva and Zhukov, 1976; Kulagin, 1888; Meyer, 1916; Nevrova and Shadrin, 2005, 2008; Pavlovskaya et al., 2009; Senicheva and et al., 2008; Zagorodnaya et al., 2008). However, the data on the species composition of heterotrophic flagellates are absent; and the species list is absent as well in the only known study of the flagellates of hyperhaline lakes of Crimea, namely, Sasyk-Sivash, Saki, and Moynaki lakes (Goryacheva and Zhukov, 1976).

Materials and Methods

Samples from the saline lakes of the Crimean Peninsula were taken by the author and A.P. Mylnikov in July 2017 and July 2018. All studied reservoirs were shallow, well warmed up, the water salinity ranged from 10 up to 204%o during the sampling period. The studied water bodies could be divided into continental and marine by the origin. Lakes of continental origin were the Lake Marfovskoe, located in the central part of the Kerch Peninsula, as well as nameless saline lakes and puddles in its vicinity. Such reservoirs have a chloride-sulfate-carbonate ionic composition (Nevrova and Shadrin, 2005). Lakes of marine origin (estuary type) included Lake Chokrak and Lake Sivash (originated from the Sea of Azov), lakes Kuchuk-Adjigol& and Sasyk-Sivash (originated from the Black Sea). Such lakes were fed mainly by filtering the seawater through the dam, as well as by atmospheric precipitation, surface runoff, and seawater overflow during a storm. Lake Sivash was an exception, its western part was directly connected to the Sea of Azov through the Henichesk

Strait; the salinity of the lake increased significantly eastwards. One to three samples were taken at each study site; the data on the species composition were then pooled for the analysis. The detailed information on the sampling sites is presented in Table 1.

Water samples, partly with the bottom sediments, were placed into 15 mL plastic tubes and delivered alive at 4 °C in the dark to the laboratory. Under laboratory conditions, the samples were transferred into the Petri dishes, enriched with a bacterial suspension of Pseudomonas fluorescens Migula, 1895 (0.15 mL of bacterial suspension per 5 mL of sample) and reared in the incubator for 10 days in the dark at a temperature of 22 °C. Live flagellates were checked throughout the exposure time to reveal hidden species diversity according to the generally accepted methodology (V0rs, 1992). The species identification has been performed under AxioScope A1 light microscope (Carl Zeiss, Germany) at phase contrast, DIC, and water immersion lenses (total magnification *1120).

The dendrogram of the species similarity in the studied water bodies has been plotted in the PAST program using the single linkage Bray-Curtis similarity index (Hammer et al., 2001). The up-to-date eukaryotic system was applied for the taxonomy (Adl et al., 2019).

Results and Discussion

In the studied saline lakes of the Republic of Crimea, 29 species of heterotrophic flagellates from 20 genera and 5 supergroups were identified, two species belong to eukaryotes of an uncertain systematic position (Table 2). Rhynchomonas nasuta, Neobodo designis, Petalomonas poosilla, Percolomonas cosmopolites, and Cafeteria sp. have been noted the most often (in 30% or more of the studied lakes). Seventeen species (58.6%) were rare, i.e. noted for a single sample only.

The number of the flagellate species per sample averaged 5.4, which was 2-3 times lower than in the samples obtained in the Black Sea (Prokina et al., 2017, 2018). Our data are consistent with the studies of other protist groups, when a significantly lower species richness of diatoms has been noted in saline lakes compared with coastal marine areas (Nevrova and Shadrin, 2005). A total of six species identified in saline lakes within the present study were not previously found in the marine area of the Crimean Peninsula; these species were Salpingoeca rosetta, Halocafeteria seosinensis, "Protist 4", Percolomonas cosmopolitus, Phyllomitus undulatus, and Pleurostomum flabellatum (Prokina et al., 2017, 2018). Most of the flagellate species have been found

Table 1. Characteristics of the studied saline lakes of the Crimean Peninsula.

No. Water body, sediment type Date, dd.mm.yyyy Coordinates Salinity, %o T, °C

Table 2. Distribution of heterotrophic flagellates in the studied saline lakes of the Crimean Peninsula. * - hierarchical ranks of taxa are given from higher rank to lower one. The numbering of reservoirs is given in accordance with Table 1.

Water bodies* (water salinity, %o)

Domain AMORPHEA Adl et al., 2012 Supergroup Obazoa Brown et al., 2013

*Apusomonadida Karpov et Mylnikov, 1989

Amastigomonas caudata Zhukov, 1975

A. mutabilis (Griessmann, 1913) Molina et Nerad, 1991

*Opisthokonta Cavalier-Smith, 1987

**Holozoa Lang et al., 2002 *Filasterea Shalchian-Tabrizi et al., 2008

Ministeria vibrans Tong, 1997

***Choanoflagellata Kent, 1880-1882

****Craspedida Cavalier-Smith, 1997 *Salpingoecidae Kent, 1880-1882 sensu Nitsche et al., 2011

Salpingoeca infusionum Kent, 1880-1882 S. rosetta Dayel et al., 2011

****Acanthoecida Cavalier-Smith, 1997 *****Acanthoecidae Norris, 1965 sensu Nitsche et al,

Savillea parva (Ellis, 1929) Loeblich, 1967

Domain DIAPHORETICKES Adl et al., 2012 Supergroup SAR Burki et al., 2008 *Stramenopiles Patterson, 1989 **Bigyra Cavalier-Smith, 1998 ***Opalozoa Cavalier-Smith, 1991 ****Bicosoecida Grasse, 1926

Bicosoeca maris Picken, 1841

Caecitellus parvulus (Griessmann, 1913) Patterson et al., 1998

Cafeteria ligulifera Larsen et Patterson, 1990

C. roenbergensis Fenchel et Patterson, 1988

Cafeteria sp.

Halocafeteria seosinensis Park et al., 2006 Pseudobodo tremulans Griessmann, 1913

+ +

+ +

Water bodies* (water salinity, %o)

**Gyrista Cavalier-Smith, 1998 ***Ochrophyta Cavalier-Smith, 1986 ****Diatomista Derelle et al., 2016 *****Dictyochophyceae Silva, 1980 ******Pedinellales Zimmermann et al., 1984

Ciliophrys infusionum Cienkowski, 1876

Supergroup Cryptista Adl et al., 2018

*Cryptophyceae Pascher, 1913 **Cyathomonadacea Pringsheim, 1944

Goniomonas pacifica Larsen et Patterson, 1990

G. truncata (Fresenius, 1858) Stein, 1878 - -- -- -- Incertae sedis EUKARYA Formal group Excavates [ex-supergroup Excavata Cavalier-Smith, 2002]

*Discoba Simpson, 2009 **Euglenozoa Cavalier-Smith, 1981 ***Kinetoplastea Honigberg, 1963 Incertae sedis Kinetoplastea

Bordnamonas tropicana Larsen et Patterson, 1990

**Metakinetoplastina Vickerman, 2004 *****Neobodonida Vickerman, 2004

Neobodo curvifilus (Griessmann, 1913) Moreira et al., 2004

N. designis (Skuja, 1948) Moreira et al., 2004

Rhynchomonas nasuta (Stokes, 1888) Klebs, 1893

Bodo saltans Ehrenberg, 1838

*****Eubodonida Vickerman, 2004

***Euglenida Butschli, 1884 ****Heteronematina Leedale, 1967

Notosolenus apocamptus Stokes, 1884

N. urceolatus Larsen et Patterson, 1990

Petalomonas poosilla (Skuja, 1948) Larsen et Patterson, 1990

+ +

+ +

Water bodies* (water salinity, %o)

**Heterolobosea Page et Blanton, 1985

***Tetramitia Cavalier-Smith, 1993 ****Eutetramitia Hanouskovâ et al., 2018 *****Percolomonadidae Cavalier-Smith, 2008

Percolomonas cosmopolitus (Ruinen, 1938) Fenchel et Patterson, 1986

*****Tulamoebidae Kirby et al., 2015

Pleurostomum flabellatum Ruinen, 1938

Clade Ancyromonadida Cavalier-Smith, 1998

Ancyromonas sigmoides (Kent, 1880-1882) Heiss et al., 2010

Incertae sedis EUKARYOTA

Phyllomitus undulatus Stein, 1878 "Protist-4"

Total species number

+ +

by the author both in saline continental water bodies and in marine biotopes of the Crimean Peninsula.

When comparing the saline lakes of other regions of Russia and of the world with the studied saline water bodies of the Republic of Crimea, the latter were characterized by a low species diversity of heterotrophic flagellates: 74 species were recorded in the saline lakes of the Orenburg Region (Russia), including 41 species found in hyperhaline lakes with salinity of 92-291%o (Plotnikov et al., 2011); 46 species, in the hyperhaline habitats of Western Australia (60-150%) (Patterson and Simpson, 1996). The resulting low species diversity of the flagellates in the study area comparing with other regions may be explained by the incompleteness of our studies and the insufficient number of samples taken.

The hyperhaline Lake Sivash (15 species) and the nameless saline lake in the Lyubimovka microdistrict, the city of Sevastopol (10 species) turned out to have the richest species composition, from 2 to 6 species have been found in the other studied lakes. Here, no decrease in the species richness of the flagellates of saline habitats has been found in regard to the water sa I inity increase, as it was shown earlier (Patterson and Simpson, 1996; Plotnikov et al., 2011; Post et al., 1983).

Two flagellate species (Halocafeteria seosinensis and Pleurostomum flabellatum), found within the study, are extremely halophilic according to the

known distribution (Park et al., 2006, 2007; Plotnikov et al., 2011); our data testify for their confinement to the hyperhaline biotopes. About a half of the heterotrophic flagellates identified down to the species level (13 species) were previously noted not only in saline continental and marine biotopes, but also in fresh waters, so they can be considered euryhaline. About the same number of species (14 species) were previously found in the sea waters only.

According the Venice system (Romanenko, 2004), the studied lakes can be divided into brackish (mesogaline (5-18%) and polyhaline (18-30%)) and hyperhaline (exceeding 40%) by their degree of mineralization. Moreover, hyperhaline lakes with salinity up to 100% had a higher flagellate species diversity and the greatest number of the species found there exclusively comparing to all other lakes. Meantime, hyperhaline lakes with salinity exceeding 100% were characterized by the lowest species number per sample (Table 3). Among brackish lakes, a greater number of species were noted in polyhaline lakes.

Regard must be paid to the species found within the present study in hyperhaline lakes only; these species are also characteristic of the hyperhaline habitats of the Orenburg Region (Russia), as well as Australia and Korea, namely, these are Halocafeteria seosinensis, Percolomonas cosmopolitus, and Pleurostomum flabellatum (Park et al., 2006, 2007, 2007; Patterson and Simpson, 1996; Plotnikov et al., 2011; Post et al.,

Comparison of the species richness of heterotrophic flagellates of different types of bottom sediments and fouling communities (Table 4) evidences on the maximum species diversity, the highest average species number and the number of unique species per sample in the samples obtained at the sites characterized by silty and sandy sediments, while the minimum vales of these parameters have been noted in the samples obtained at clay sediments and in periphyton samples.

Several small clusters are visible on the dendrogram of taxonomic similarities (Fig. 1) when analyzing the species composition of the flagellates in saline continental reservoirs of the Crimean Peninsula. One cluster includes the lakes located in Marfovka village (two sites within Lake Marfovskoe

and the unnamed lake located nearby); another cluster comprises the reservoirs with similar salinity (Lake Kuchuk-Adjigol& and a puddle in the village of Marjevka). However, there is no general relationship between the formation of the flagellate species composition and the lake salinity, which is probably due to a significant change in the salinity of these lakes during the year.

Conclusions

In the saline lakes of the Republic of Crimea, 29 species of heterotrophic flagellates have been identified, including two flagellates unidentified down to the species level. The species diversity of the studied saline lakes is significantly lower than that of the Black Sea and saline water bodies of the Orenburg Region (Russia), Australia, and Korea, according to the literature data. There is no direct pattern trend of a decrease in the species richness of flagellates in regard to the water salinity increase, which may be due to a low number of studied reservoirs and samples analyzed. Nevertheless, there is a sharp decrease in the number of flagellate species in the water bodies characterized by the water salinity over 100%o, which is consistent with previously published studies of the flagellate composition in hyperhaline biotopes. There are no clear patterns in the formation of the species composition of the flagellate communities in the water bodies with different salinity.

Табл. 3. Species number of heterotrophic flagellates in the studied saline lakes of different types.

Tuno of lake water salinity Total species Species number in the Species found exclusively for the iype of |ake, water sa|inity number samp|e iake type

lake type

Mesohaline, 5-18%o Polyhaline, 18-30% Hyperhaline, 40-100% Hyperhaline, >100%

Табл. 4. Species number of heterotrophic flagellates in different types of bottom sediments and periphyton.

Sediment/sample type Total species number Species number in the sample Species found exclusively for the lake type

Silt 19 6.3 9 (47.4%)

Sand 10 10.0 5 (50%)

Clay 8 2.7 1 (12.5%)

Periphyton 6 6.0 2 (33.3%)

Fig. 1. Dendrogram of the species similarity of heterotrophic flagellates (%) of the studied saline lakes on the Crimean Peninsula. The numbering of reservoirs is given in accordance with Table 1.

Acknowledgments

The author is grateful to A.P. Mylnikov for the help in collecting material and advice on the species identification and to D.V. Tikhonenkov for the invaluable comments given during the manuscript preparation. The study was supported by the Russian Foundation for Basic Research (grant no. 20-04-00583) and performed within the framework of the State Task (registration no. AAAA-A18-118012690098-5).

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