REPUBLIC OF AZERBAIJAN

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ABSTRACT of the dissertation for the degree of Doctor of Philosophy

THE FAUNA AND ECOLOGY OF TESTATE AMOEBAE IN FRESH WATER BODIES OF THE LANKARAN NATURAL AREA

Speciality: 2401.01 – “Zoology”

Field of science Biology

Applicant: Elyana Nail Tahirova

Baku – 2021 The work was performed at the laboratory of Protozoology of the Institute of Zoology of Azerbaijan National Academy of Sciences.

Scientific supervisor: Doctor of biological sciences, associate professor Natalia Yuryevna Snegovaya

Official opponents: Doctor of biological sciences, associate professor Giyas Nagi Guliyev Doctor of biological sciences, associate professor Namig Janali Mustafayev PhD in Biological Sciences Elnara Eldar Jafarova

Dissertation council FD 1.09 of Supreme Attestation Commission under the President of the Republic of Azerbaijan operating at the Institute of Zoology of Azerbaijan National Academy of Sciences

Chairman of the Doctor of biological sciences, Dissertation council: associate professor Elshad Ilyas Ahmadov ______

Scientific secretary of PhD in Biological Sciences, the Dissertation council: associate professor Gular Aydin Huseynzade ______

Chairman of the Doctor of biological sciences, prof., scientific seminar: corresponding member of ANAS Ilham Khayyam Alekperov ______

2 INTRODUCTION Research rationale and the state of knowledge. As it is known, testate amoebae, as one of the groups of free-living protozoa, play an important role in biological processes that occur in freshwater bodies. Testate amoebae are active consumers of detritus, bacteria and many groups of microscopic algae, including cocci and diatoms. In addition, testate amoebae are involved, on the one hand, in the biological self-purification of fresh water, and on the other, in the processes of the formation and decomposition of organic matter in freshwater bodies. These free-living protozoa are highly regarded for their ability to be used as an indicator of environmental conditions. In recent years, work towards studies of this group of protozoa has expanded, but there are still many places in the world, where such researches are not carried out at all. In addition, freshwater bodies are the focus of environmental science and it is not possible to determine their significance. Lakes and reservoirs in both developed and developing countries are important resources for environmental quality, economic development and social well-being. However, natural or anthropogenic decrease to freshwater biodiversity affects ecosystem processes and worsens human health due to a reduction of ecosystem services. Taking into account all of the above, it becomes clear that protistologists are very interested in the study of this important group of unicellular animals, which are of great importance from a practical point of view. In Azerbaijan, the degree of knowledge of freshwater testate amoebae is still at an extremely low level. To date, only 2 studies have been carried out that are directly related to this group. One of these studies was carried out in the 90s of the XX century by T.Zakhidov. 1 And the second study, carried out by N.Snegovaya 2 was devoted to the fauna of testate amoebae of the water bodies of

1Zakhidov, T.F. Testate amoebas of some soils of the Sheki-Zagatala region of Azerbaijan: / Abstract of the dissertation for the degree of Doctor of Philosophy / – Baku, – 1995. – 24 p.(in Russian) 2Snegovaya N.Yu. Supplementation to the fauna of testate amoebae (Rhizopoda, Testacea, Protozoa) of the water bodies of Absheron (Family Arcellidae, Centropyxidae) // – Baku: J.Bilgi, – 2001. №3, – p. 30-35 (in Russian). 3

Absheron peninsula. Recent studies carried out by modern cytological methods have revealed a rich species diversity and high specificity of the fauna of freshwater testate amoebae in Azerbaijan. Based on the above, we have studied the fauna and ecology of testaceans living in freshwater bodies of the Lankaran region. The purpose and objectives of the research. The main goal of this work was to study the fauna, the quantitative development of freshwater testate amoebae, the morphology of rare species and the structure of their communities, as well as their relationship to certain environmental factors. To achieve this goal, it was envisaged to consider the following issues: 1. Study of the fauna, species and quantitative development of freshwater testate amoebae in water bodies with varying levels of saprobity. 2. Study of ecological features (connections with various ecological factors) of freshwater testate amoebae of the Lankaran natural area and their food relationships with other hydrobionts. 3. Study of the possibilities of using some species of testate amoebae in bioassay of organic pollution degree of fresh waters. 4. Carrying out a comparative analysis of the similarity of the fauna of freshwater testate amoebae in the Lankaran natural area with the fauna of freshwater testate amoebae in other regions of Azerbaijan. Research methods. Methods of glycerin-gelatin and impregnation with silver nitrate were used to identify species of testate amoebae.3,4 An Olympus light microscope, a JCM-6000 scanning electron microscope (SEM) and a FlowCam densitometer were used to qualitatively and quantitatively analyze the collected materials. Biometric parameters for morphometric analysis of species were calculated using the statistical analysis program SigmaStat-12.5. The collection of specimens of testate amoebae in benthic samples under

3 Alekperov, I.Kh. Collecting and studying methods of free-living ciliates and testate amoebas. / I.Kh.Alekperov, E.S.Asadullayeva, T.F.Zakhidov. – Russia, Saint-Petersburg: «Saygon», – 1996. – 41p. (in Russian) 4Alekperov, I.Kh., Musaev, M.A., Zakhidov, T.F. A new method for studying testate amoebae by impregnation with silver nitrate // Moscow: J. Zoology, – 1994. vol.73, №1, – p.148-151 (in Russian). 4 laboratory conditions was carried out by the flotation method. The similarity degree of the species composition of testate amoebae in the studied water bodies was determined using of Bray-Curtis cluster analysis, and the dominant species were revealed by means of calculating the frequency of occurrence. Defending statements: 1. Species and quantitative composition of the fauna of testate amoebae, and regularity of their distribution in the water bodies of Lankaran natural area. 2. Adaptation of testate amoebae to some environmental factors (salinity, temperature, gas regime, pH level, etc.). 3.The role of testate amoebae in biocenoses of various water bodies. 4. Possibilities and prospects of using testate amoebas as a test object for determining and subsequent biomonitoring of the degree of organic pollution of fresh waters. Scientific novelty of the research. For the first time in Azerbaijan, a scanning electron microscope was used at the international level to study testate amoebae. We have registered 8 species and subspecies of testate amoebae for the first time. Of these, 2 species (Difflugia alekperovi Snegovaya and Tahirova, 2015; Awerintzewia minuta Snegovaya, Tahirova, 2018) are new to science. 4 species were recorded for the first time in the Caucasus (D.microclaviformis Ogden, 1983; D.petricola Cash, 1909; D.biconcava Ertl, 1964 and D.serbica Ogden and Živković, 1983), and 2 subspecies were recorded for the first time in the fauna of Azerbaijan (Arcella discoides ssp. scutelliformis Playfair, 1918 and D.corona ssp. tuberculata Vuchetich, 1973). In addition, for the first time, one genus was registered for the fauna of Caucasus (Awerintzewia Schouteden, 1906). The theoretical and practical significance of the work. The 60 morphological descriptions of characteristic and rare species of testate amoeba presented in the dissertation, made on the basis of micrographs, can be further used by young researchers as a guide and they can be included in the curricula of the Faculty of Biology. Indicator species of saprobity that we have identified can be used as a test object for biomonitoring of fresh waters.

5

Approbation and application of the work. The main provisions of the dissertation were heard and discussed at the annual report meetings of the Laboratory of Protozoology and the Scientific Council of the Institute of Zoology of ANAS, the Scientific Seminars of the Institute, as well as the following Republican and International scientific-practical conferences: - XVIII International Scientific Conference of Doctoral Students and Young Researchers (Baku, 2013); - IV International Scientific Conference of Young Scientists and Researchers "Innovation Problems of Modern Biology" dedicated to the 91st anniversary of Great Leader Heydar Aliyev (Baku, 2014); - International multidisciplinary forum "Academic Science Week-2015", dedicated to the 70th anniversary of the National Academy of Sciences of Azerbaijan (Baku, 2015) - International Youth Forum “Integration processes of world science in the XXI century” (Ganja, 2016); - XIX International Scientific Conference of Young Researchers "Biological Diversity of the Caucasus and Southern Russia", dedicated to the 75th anniversary of Professor Gairbek Abdurrahmanov (Makhachkala, 2017); - Conference “Wildlife Ecology and Inventory Techniques Course - 2” supported by TUBITAK (Turkey, Afyon, 2018); - XXI International Scientific Conference of Young Researchers "Biological Diversity of the Caucasus and Southern Russia", dedicated to the 25th anniversary of Ingush State University and the 80th anniversary of the corresponding member of the Russian Academy of Ecology T.Tochiyev (Magas, Ingushetia, 2019); - II International Scientific Conference of Young Scientists and Specialists "Multidisciplinary approach to solving modern problems of fundamental and applied sciences" (Baku, 2020); On the basis of research materials, 10 articles (4 of them in journals included in international citation databases) and 7 conference papers reflecting the main content of the dissertation were published in the republic and abroad. Name of the organization where the dissertation work was performed. The research was carried out in the Protozoology 6

Laboratory of the Institute of Zoology of the Azerbaijan National Academy of Sciences. The structure and volume of the dissertation. The dissertation is in the amount of 205545 characters, including introduction (8111 characters), 6 chapters (181164 characters), conclusion (12591 characters), result (2541 characters), practical recommendations (1138 characters). The dissertation also contains a list of 233 titles of literature in Azerbaijani, Russian and other languages. The dissertation covers 10 tables, 66 figures and 9 graphs.

CHAPTER I. Testate amoebae: their position in the system of eukaryotes, a study level in the world and in Azerbaijan The chapter provides information on the study of the fauna and ecology of testate amoebae in various countries around the world and in Azerbaijan from the 19th century to the present, as well as the position and modern taxonomy of these protozoans in the eukaryotic system. In this study, we have based on a higher hierarchical classification of testate amoebae (from kingdom to orders) proposed by Adl (2012), 5 M.Rugiero, et al. (2015) 6 and adopted by the International Committee for high taxonomic units. It is difficult to determine how many species of testate amoebae there are in the world today. In addition, a number of factors, such as the small size of the organisms, the complexity of obtaining original images, the confusing nomenclature, the lack of phylums and precisely designed catalogs, etc., are the main reasons for the difficulties in research in this area. However, according to the literature, more than 1900 testate amoebae species and subspecies have been recorded so far.7 At the same time, the chapter discusses the biology of testate

5Adl, S.M. The revised classification of eukaryotes / S.M. Adl, A.G.Simpson, C.E. Lane [et al.] // The Journal of Eukaryotic Microbiology, – London: – 2012, vol. 59, – pp. 429–493. 6 Ruggiero, M. A Higher Level Classification of All Living Organisms / M.Ruggiero, D.Gordon, T.Orrell [et al.] // PLoSOne, – San Francisco: – 2015, vol. 10, No 6: e0130114. 7 Qin, Y. Diversity, distribution and biogeography of testate amoebae in China: implications for ecological studies in Asia / Y.Qin, S.Xie, H.G.Smith [et al.] // European Journal of Protistology, – Munich: – 2011. vol. 47, – p.1-9. 7 amoebae and the cell structure, the structure and forms of the shells and pseudopods, which belong to important taxonomic characteristics. The availability of shell gives the cell's cytoplasm a relatively stable shape and allows it to differentiate into various parts from a functional point of view.

CHAPTER II. Research materials and methods The material was processed in the Protozoology Laboratory of the Institute of Zoology of the National Academy of Sciences of Azerbaijan. The studies were conducted in various freshwater bodies of the Lankaran natural area in 2013-2018 years. Lankaran natural area is located in the south-east of Azerbaijan. The region, which differs sharply from other regions of Azerbaijan by its nature, is the second humid subtropical region in the South Caucasus. Lankaran natural area is bounded on the north by the Mugan plain, the eastern border is the shoreline of the Caspian Sea. It is separated from the northwest of Iran by the water line of the Talish Mountains and the Astarachay. The northern end of the Talysh Mountains is in 39°27" north latitude, and the southern end is in 38°22" latitude. The highest point in the west is in 65°37" north longitude, and in the north 66°32" longitude. The length of the Lankaran zone from north to south is 115 km, and from west to east is 96 km8. The region includes 6 administrative districts: Lankaran, Astara, Masalli, Lerik, Yardimli and Jalilabad. Water samples were collected seasonally from various freshwater bodies in the above-mentioned regions (Figure). The region is also located in a humid subtropical zone and has a dense river network. The region's rivers are fed by rain and groundwater and flow directly into the Caspian Sea. Samples were collected from lakes, reservoirs (Khanbulanchay, Vilashchay, Goytapa, Babaser reservoirs), as well as rivers (Astarachay, Tangarudchay, Vilash, Lankaranchay, Bashari river, Goytapachay).

8 Geography of the Republic of Azerbaijan. Regional geography / Institute of Geography named after H.A.Aliyev, ANAS - Baku: "Europe" Publishing House, - vol. 3. - 2015. - 400 p. 8

Figure. Sampling points on water basins (with GPS coordinates). Goytepe reservoir - 39.11727, 48.58978, Haziabad - 39.06848, 48.56888. Gullutepe - 39.03971, 48.56437, Tatyanoba - 39.04185, 48.58978, Masalli - 39.02771, 48.64746, Vilaschay reservoir - 39.00317, 48.58652, Boradigah - 38.9295, 48.72299, Rudakenar (Bababulagi) - 38.87989, 48.70067, Rudakenar - 38.86891, 48.68282, Babaser reservoir - 38.94223, 4869484, Porsova - 38.97889, 48.40129, Perimbel - 38.91988, 48.12629. Çayuzu - 38.88675, 48.18466, Avash - 38.86644, 48.09677, Lerik - 38.77335, 48.41554, Rvarud - 38.69666, 48.48978, Siyov - 38.66835, 48.59802, Basharichay - 38.70867, 48.75409, Rvo - 38.71558, 48.7462, Lankaran - 38.76265, 48.8507, Xanbulanchay reservoir - 38.65816, 48.77122, Vilvan - 38.82152, 48.7405, Azfilial (Khanbulan) - 38.67334, 48.79655, Astara - 38.47079, 48.86718, Pensar - 38.61526, 48.81773, Tangarud - 38.57662, 48.81738, Mashkhan - 38.55165, 48.81225, Gilakeran - 38.47872, 48.85843, Astarachay - 38.44183, 48.87792. 9

In addition, in order to carry out a comparative analysis of testate amoebae fauna of different regions, samples were collected from freshwater reservoirs and lakes in Goygol, Guba-Khachmaz regions of Azerbaijan, as well as in the Nakhchivan Autonomous Republic. Horiba U-52 (Japan) multi-parameter device was used in hydrological studies to conduct quality analysis in all studied water bodies. This chapter gives a brief description of some of the water reservoirs studied and the results of our auxiliary hydrological analyzes. More than 2000 water samples were collected and processed throughout the study. Samples were collected in polyethylene containers. The collected material was delivered to the laboratory as soon as possible for processing. Besides Bogorov chamber, a FlowCam (USA) densitometer was used to quantitate the shells. A flotation method was used to collect testate amoebae easily with microcapillaries in benthic specimens. For quality analysis of the collected material, we used light microscope Olympus - CX41RF (Tokyo, Japan) as well as JEOL, JCM- 6000 (Tokyo, Japan) scanning electron microscope (SEM). All descriptions and micro-images of testate amoebae were taken on the basis of permanent and temporary preparations, in vivo observation, GXCAM digital camera and scanning electron microscope. In addition, a simplified method of impregnation with silver-nitrate was used for more detailed and accurate processing of the collected material. SigmaStat-12.5 statistical software was used in the calculation of biometric parameters during morphometric analysis of species. The main indicators taken into account in the morphometric analysis are 1) shell length; 2) shell width; 3) shell height; 4) the height of the shell aperture; 5) the width of the shell aperture. During the analysis, measurements were performed on at least 10 individuals of the same species. For the analysis of the ecological structure of the testate amoebae communities of the studied water bodies, we have calculated the main ecological indices. Indices and graphs were calculated using the Biodiversity Professional-2.0 computer software. Thus, to measure the biodiversity of some of the water bodies studied, the Simpson Diversity Index was calculated by the following formula:

10

( ) = 𝑆𝑆 ( ) (1) ∑𝑖𝑖=1 𝑛𝑛𝑖𝑖 𝑛𝑛−1 - where n is the total number of individuals of a particular 𝐷𝐷 𝑁𝑁 𝑁𝑁−1 species, N - the total number of individuals of all species. Bray-Curtis cluster analysis was used to determine the level of similarity of the species composition of the testate amoebae of the studied water bodies, and the results were presented graphically in the form of a dendrogram. Also, the similarity between the fauna of testate amoebae of different regions of Azerbaijan was compared with the Czekanowski-Sorensen9 index (2) and the results are given in tabular form: = × 100% (2) - where C is the total2𝐶𝐶 number of species common for 2 communities, A is the number𝐾𝐾 𝐴𝐴+ of𝐵𝐵 species in the first community, B is the number of species in the second community. The dominant species of testate amoebae were identified by calculating the frequency of occurrence (3). According to the frequency of occurrence, the species are constant (occurrence in more than 50% of collected samples), secondary (occurrence in 25- 50% of collected samples) and random (occurrence in less than 25% of collected samples), calculated by the following formula:

pF = m/n×100 (3)

- where m is the number of samples of individuals of a certain type, n is the total number of samples. According to the classification of W.Tischler (1955),10 all species are divided into 4 groups: 1) subrecedent - n/N< 1 %, 2) recedent - n/N- 1-2%, 3) subdominant - n/N - 2-5 %,

9 Czekanowski, J. Application of Statistical Methods to Anthropology. / J.Czekanowski – Warsaw: Warsaw Scientific Society, – 1913, – 178 p. (in Polish). 10Tischler, W. Synocology of terrestrial animals. / W.Tischler. – Stuttgart: Gustav Fischer Verlag, – 1955, – 414 p. (in German) 11

4) dominant - n/N > 5% - where n - the number of specimens of a particular species, N - the total number of all specimens.

CHAPTER III. Species composition of testate amoebae of fresh water bodies of the Lankaran natural area This chapter provides a complete species composition and distribution of testate amoebae in the investigated water bodies of the Lankaran natural area, as well as a morphological description of the species photographed by light and scanning electron microscopes. The morphological descriptions of 60 characteristic species, including new species, are given in the chapter. In total, 126 species of testate amoebae were recorded in the freshwater bodies of the natural area, most of which are representatives of the families Difflugiidae (70 species), Arcellidae (17 species) and Centropyxidae (18 species). For the first time, 8 species and subspecies of testate amoebae were recorded. Of these, 2 species (Difflugia alekperovi Snegovaya and Tahirova, 2015; Awerintzewia minuta Snegovaya and Tahirova, 2018) were first described for science, and 4 species (D.microclaviformis Ogden, 1983; D.petricola Cash, 1909; D.biconcava Ertl, 1964 and D.serbica Ogden and Živković, 1983) are new to the Caucasus, and 2 subspecies (Arcella discoides ssp. scutelliformis Playfair, 1918 and D.corona ssp. tuberculata Vuchetich, 1973) are new to fauna of Azerbaijan.

CHAPTER IV. Seasonal dynamics of species and quantitative composition of testate amoebae in the studied water bodies We have studied seasonal dynamics of species and quantitative composition of testate amoebae in water bodies of 6 districts of the Lankaran natural area. The testate amoebae species recorded in the studied water bodies belong to the following families, including 70 species to Difflugiidae family, 18 species to Centropyxidae family, 17 species to Arcellidae family, 6 species to Trigonopyxidae family, 2 species to Lesquereusidae family, 2 species to Plagiopyxiidae family, 12

1 species to Heleoperidae family, 3 species to Pseudodifflugiidae family, 3 species to Euglyphiidae family, 3 species to Cyphoderiidae family, 1 species to Cryptodifflugiidae family. Due to the species composition of testate amoebae, closed stagnant water reservoirs are richer than rivers. Among the studied water reservoirs, the pond near the Azfilial settlement (Khanbulan) takes the first place with 56 species. Among the rivers studied, Lankaranchay ranks first in terms of species composition with 24 species. The highest species diversity was recorded in the water bodies of Lankaran region - 105 species. 42 species of testate amoebae were registered in Jalilabad region, 38 species in Masalli region, 36 species in each of Astara and Yardimli regions, and 37 species of testate amoeba in Lerik region. It is known that the difference in the temperature range in which testate amoebae are found affects their total number and the regularity of the seasonal distribution of species composition. The highest quantitative growth of testate amoebae was observed in all studied reservoirs during the warm season. As a results of our research in Lankaran region, the highest amount of testate amoebae was observed in the water reservoir near Azfilial settlement (7.4 thousand individuals/dm2), and the lowest quantitative increase was observed in the water body in Gumbashi area (4.6 thousand individuals/dm2). The lowest value of the total number was observed in winter in all surveyed water bodies and the maximum number of testate amoebae in this season was observed in Dashtatuk water body (0.9 thousand individuals/dm2) and the minimum number in the pond near the Gumbashi settlement (0.3 thousand individuals/dm2). The same pattern was observed in other regions. During the years of our study (2013-2018), the highest number of species and biomass of testate amoebae were recorded in July 2014 (7.4 thousand individuals/dm2 and 57.1 mg/dm2), and the lowest in January 2015 (0.1 thousand individuals/dm2 and 3.2 mg/dm2). Throughout our research, 2 groups were distinguished according to the frequency of occurrence of species in different freshwater reservoirs: constant and secondary species. Thus, there

13 were totally 16 constant species in different water bodies of Lankaran, Astara, Jalilabad districts, 14 in Masalli district, 13 and 12 in Yardimli and Lerik districts, respectively. A similar pattern was observed for secondary species. These species are found in 25-50% of the collected samples. The largest number of species belonging to this group was registered in Lankaran region - 10 species (Arcella dentata, A.gibbosa, Cyclopyxis kahli, C.elongata, Difflugia giganteacuminata, D.globulosa, D.claviformis, D.pyriformis, D.lanceolata, D.microclaviformis). The lowest number of species was registered in Yardimli region with 2 species (C.elongata, D.pyriformis). Also, the biodiversity of 3 different mountain rivers (Lankaranchay, Vilash and Bashari rivers) included in the territory of Lankaran natural area according to the fauna of testate amoeba was calculated by Simpson's diversity index (Table). Thus, a total of 38 species belonging to 6 families were recorded in the samples collected from the rivers (Table). Table Simpson’s index of diversity in investigated rivers. Rivers Species Total number of Ds (Simpson number individuals diversity index) Lankaranchay 24 476 0.955 Basharu river 15 263 0.797 Vilash river 13 182 0.652

Lankaranchay ranks first in terms of the number of species (24 species). 15 species of testate amoebae were recorded in the Bashari river and 13 species in the Vilash river. In general, all three rivers have similar figures for the diversity index. However, Lankaranchay differs relatively from the other two rivers in terms of biodiversity index (0.955). This is due to the fact that Lankaranchay has a large population and a large number of species. The high species diversity in this river is a sign of the large number of sustainable species and the existence of a stable ecosystem. In other words, there are many environmental niches. This means that the environment is suitable for most species and that environmental variability is unlikely to damage the ecosystem as a 14 whole. The low diversity index in the other two rivers (0,797 and 0,652) indicates that the number of sustainable species in the fauna of these rivers is lower than in Lankaranchay, as well as the food chain in these rivers is simpler.

CHAPTER V. Ecological characteristics of testate amoebae in the studied water bodies Testate amoebae react to changes of environmental conditions by changes in the species composition and quantity of communities, as well as the morphological structure of the shell. In particular, changes in water regime parameters lead to a restructuring of the testate amoebae communities (species composition, quantity, biomass, genera and species dominance). These protozoans’ shells are morphologically variable and are resistant to decomposition. The shell mediates between the organism and the environment, and reflects environmental impacts on the population. We studied a number of issues related to the ecology of testate amoebae, the temperature, pH level, salinity and attitude of different species to dissolved oxygen. During the research, we studied the regularity of the distribution of testate amoebae in various ecological groups. Thus, the smallest species diversity of testate amoebae was recorded in plankton in the studied water reservoirs and there were observed from 2 species (water body in the village of Avash, Yardimli district (38°52'40''N, 48°02'27'E)) up to 21 species (water body in the settlement Azfilial, Lankaran district (38°40'56ʺN, 48°46'58''E)). In general, the species richness in plankton was recorded only in the Lankaran region, which is explained by the shallowness of the water bodies of the area. Of the total number of species recorded in the study area, only 14 species (Cyclopyxis euristoma, Centropyxis aculeata, C.elongata, Difflugia acuminata. D.corona, D.difficilis, D.elegans, D.gramen, D.litophila, D.pyriformis, D.urceolata, D.oblonga, Pontigulasia compressoidea, Cyphoderia ampulla) were found in water bodies in all studied areas. The number of species in the periphyton was higher than in 15 plankton. The largest number of species was recorded in shallow water bodies, which are richer in aquatic plants. High species diversity was recorded in Azfilial settlement of Lankaran region - 24 species and in Bajiravan village of Jalilabad region - 19 species. The lowest number of species in the periphyton was recorded in the standing water reservoir in Avash village of Yardimli region - 7 species. The eurybiont group of testate amoebae, which is found simultaneously in two or more biotopes and is shown as an facultative species, represented by such species as D.gramen, D. urceolata, D.oblonga, D.litophila, D.corona, P.compressoidea, C.aculeata, etc. The greatest diversity of this group was observed in shallow water bodies (water bodies in Bajiravan, Haziabad and Chayuzu villages). This means that testate amoebae are organisms that lead a sedentary life, so their migration from one biotope to another is easier in shallow water bodies. The highest species diversity was observed in the benthos in all investigated water bodies. Most of the recorded species are benthic species. Only in the Goytapa reservoir we recorded 2 species of testate amoebae in the benthos (D.gramen, C.aculeata). The most important abiotic factor in the aquatic environment is temperature. The fauna of testate amoebae of Lankaran natural area can be divided into 3 ecological complexes depending on temperature: 1. The group of eurythermic species that is found mainly throughout the year - Arcella vulgaris, Centropyxis aculeata, C.marsupiformis, C.plagiostoma, Difflugia oblonga, D.corona, D.elegans, D.gramen and others. 2. The group of thermophilic species found only in the high temperature range from early summer to mid-autumn - Difflugia difficilis, D.claviformis, Pontigulasia bigibbosa, P.compressa, Cyphoderia ampulla, etc. 3. The group of stenothermic cryophilic species whose maximum development occurs at a sufficiently low temperature (4-9°C) - Difflugia lobostama, D.globularis. The active reaction (pH) of the environment has a strong effect on the testate amoebae. The species richness of testate amoebae, 16 mainly in freshwater, was observed by us in the pH range of 7.5-8.5. Most of the species we have recorded (many representatives of the genera Difflugia, Arcella and Centropyxis) belong to euryionic species. Only a few species of the genus Arcella were recorded at a pH of 7.2. The pH did not exceed 8.5 in Gilakaran lake, water bodies in Mashkhan, Tangarud, Rvarud, Pensar, Vilvan and Azfilial settlements. The maximum pH value in rivers was 9.1. No testate amoeba individuals were recorded at pH below 7.2. The salinity and gas regime of water also affect the species composition and distribution of testate amoebae. In most of the studied water bodies, the salinity did not exceed 1.5 ‰. The optimal increase in the quantity and quality of testate amoebae was recorded at a salinity of 0.03-0.5 ‰. Only in the samples taken from the Goytapa reservoir, the salinity was 3 ‰, and only 2 small species of Difflugia gramen and Centropyxis aculeata were recorded in these samples. About 100 testate amoeba species were recorded in water bodies with salinity ranged from 0.03 to 0.5 ‰. Therefore, these recorded species can be considered real freshwater species. In our research, we observed that the salinity of water has an inhibitory effect on the size of the shells. For example, in saline waters there are individuals with small shells only. If large species such as A.polypora, D.corona, D.tuberculata, C.discoides are recorded at a salinity of 0.03-0.5 ‰, then in waters with a salinity higher than 0.5 ‰, the species with the shell not exceeding 90 mkm in size (A. vulgaris, D. lobostoma, D. litophila, D. distenda, C. kahli, etc.) were found. By measuring water-soluble oxygen, we divided the recorded species into two groups, euryoxybionts and oxyphylls. A constant supply of dissolved oxygen in reservoirs ensures the development of oxyphilic species. D.corona belongs to such species. Lower concentrations of dissolved oxygen (25-50%) in the water bodies we studied were observed in samples taken from the depth. Eurioxybiont species with low oxygen concentrations such as Pontigulasia bigibbosa, Centropyxis aculeata, Difflugia difficilis, D.oblonga and others can be an example. Among the freshwater bodies we have studied in the Lankaran

17 natural region, the Khanbulanchay and Vilashchay freshwater bodies are of great agricultural importance. During the research, we recorded 28 species of testate amoebae in the Khanbulanchay water reservoir and 17 species in the Vilashchay reservoir. Based on our long-term observations and literature data11, it was determined that 15 of these species are indicators of different saprobial zones (Graph 1). Khanbulanchay reservoir 14.4% 21.4%

o-β-mesosaprobes β-α-mesosaprobes β-mesosaprobes 35.7% 28.5% α-mesosaprobes

14.3% Vilashchay reservoir 57.1%

β-α-mesosaprobes β-mesosaprobes 28.5% α-mesosaprobes

Graph 1. The ratio of indicator species to different degrees of saprobity in the Khanbulanchay and Vilashchay reservoirs.

Only 14 of all species recorded in the Khanbulanchay reservoir are considered to be indicator species. In the Vilashchay reservoir, this figure is 7. Graph 1 shows the percentage of testacean species that are an indicator of different saprobial zones of Khanbulanchay and Vilashchay reservoirs. As can be seen from Graph 1, the majority of testate amoeba species in the Khanbulanchay reservoir, which are indicators of saprobity, are representatives of the β-mesosaprobic zone (35.7%). The Vilashchay reservoir can also be referred to the β-α-mesosaprobic

11Vikol, M.M. Rhizopods (Rhizopoda, Testacea) of water bodies of the Dniester basin / M.M.Vikol. – Kishinev : Shtiinta, – 1992, – 128 p. (in Russian). 18 zone according to the indicator species. Thus, the rate of this zone was high (57.1%). It is known that one of the important environmental factors is the trophic factor. In addition to studying the species composition of these protozoans, their feeding, vital activity, and their relationship with other hydrobionts were also studied during the research. Thus, because testate amoebae are one of the free-living groups of protozoans that are constantly present in water biocenoses, their important role as an important link in the food chain is obvious. The choice of food by testate amoebae depends primarily on the ratio of the size of the food particles to the size and shape of the individual's body. Usually, the size of prays of testate amoebae is limited by diameter of shell aperture. According to our observations, the endoplasm of individuals of such genera as Difflugia, Arcella and Euglypha often contains 5-10 small diatoms (Navicula sp.). In addition, it is known that representatives of the genus Difflugia often use diatoms to build their own shells and the composition and number of these cells on the shell of amoebae directly depends on the biotope and the presence of diatoms in the environment. Representatives of diatom algae species such as Campylodiscus, Navicula, Fragilaria, Diploneis were recorded in the water reservoirs studied by us. During our research, Difflugia corona was found in water bodies all year round and it prefers species of aquatic plants as food. It crawls in search of food and feeds on more by microscopic algae. In general, representatives of the genus Difflugia are the primary consumers of algae as the main saprophytes. Difflugia acuminata is found among the filamentous algae in most water storage reservoirs we have studied. This species is mainly benthic and lives in detritus. D.lobostoma has been found on various surfaces in water bodies, most likely it is a predatory species. Although D.oblonga is found in benthic detritus throughout the year, it has not been recorded during periods of high water levels. D.tuberculata is also found among water macrophytes and algae throughout the year. We also analyzed the dominance of testate amoebae in the

19 studied water bodies of different regions of Lankaran natural area. Thus, the percentage of the dominant species group was recorded in the range of 12-26% in 6 districts of the region. Species such as Difflugia corona, D.acuminata, Centropyxis aculeata were registered as dominant species in the freshwater bodies of all studies areas, according to the classification of W.Tischler. A number of species of the genus Arcella (Arcella gibbosa, A.vulgaris, A.polypora, A.discoides, A.hemisphaerica) are considered as subdominant species.

CHAPTER VI. Comparison of the species composition of testate amoebae of the Lankaran natural area with the fauna of other regions of Azerbaijan The first study of any group of animals always begins with faunistic work. It is not enough to study the structure of the fauna to understand its features and its subsequent faunagenesis. As a result of accurate comparative analysis of the structure of the fauna of different regions, their differences and specificity become clear. For such initial comparisons, data on the fauna of certain animal groups in neighboring regions are usually used. In order to clarify the features, specificity and differences of the fauna of testate amoeba of the Lankaran natural area, we conducted a comparative analysis of the similarities between the testacean fauna of different regions of Azerbaijan. The most faunistically studied regions of Azerbaijan - the north-eastern part (Khachmaz), the western part (Goygol) and the Absheron Peninsula were taken as comparable areas (Graph 2). We used a cluster analysis of the faunistic similarity of Bray – Curtis to assess the similarities and differences between the fauna of testate amoebae of compared regions. When comparing the combined cluster of species composition of Lankaran natural region and the north-eastern part of Azerbaijan (Khachmaz) with the Absheron Peninsula, the similarity of fauna reaches 52.98%. When comparing the species composition of freshwater testate amoebas of the western part of Azerbaijan with the combined groups of the above regions, the lowest similarity percentage was 42.9%. 20

Graph 2. Cluster analysis of faunistic similarity of testate amoebae of different regions of Azerbaijan (the results of Bray-Curtis analysis).

We also compared the similarity between the fauna of testate amoebae of different regions of Azerbaijan with the Czekanowsky- Sorensen index. The results of this comparison were consistent with the results of the cluster analysis. It should be noted that additional research is needed in the future to accurately compare the similarity of the testacean fauna of Azerbaijan. Along with the comparison of the fauna of testate amoeba of different regions of Azerbaijan, we compared the species composition of freshwater testate amoebae of the studied regions of Lankaran natural area. The results of the comparison are given in Graph 3. As can be seen from the dendrogram, the greatest similarity was observed between the species composition of testate amoebae of Lerik and Yardimli regions and was 65.6%. The similarity between Jalilabad and Masalli regions was 57.5%. The similarity between the combined clusters of the above-mentioned regions and the cluster of species composition of testate amoebae of Astara region was 56.3%. The least similarity was registered between the 5 districts listed above (Masalli, Jalilabad, Lerik, Yardimli and Astara) and Lankaran district and was equal to 44.8%.

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Graph 3. Cluster analysis of the faunistic similarity of testate amoebae of the studied regions in Lankaran Natural Area.

In order to conduct a comparative analysis of the fauna of testate amoebae in different regions of Azerbaijan, besides the Lankaran natural region, we also conducted researches in the Nakhchivan AR (Lake Batabat - N39°32ʹ27.39ʺ; E45°47ʹ09.17ʺ; 2120 m above sea level) and Goygol region (Maralgol - N40°22ʹ39.11ʺ; E46 °18ʹ52.41ʺ; 1920 m above sea level). 1 new species (Awerintzewia minuta Snegovaya and Tahirova, 2018) was firstly described for science during the analysis of samples collected from the above-mentioned high mountain lakes in July 2016, and 1 genus (Awerintzewia Schouteden, 1906) and 1 new species (Difflugia serbica Ogden and Živković, 1983) were first recorded for Caucasian fauna. 12.

CONCLUSION The final section summarizes the generalized results and comparative analysis of faunistic and ecological researches conducted in about 30 freshwater bodies of Lankaran natural area.

12Snegovaya, N.Y., Tahirova, E.N. The new and rare species of testate amoebae (Amoebozoa: Arcellinida) from mountain lakes of Azerbaijan // – Saint Petersburg: Protistology, vol.12, № 1, – 2018, – pp. 38-46. 22

RESULT 1. In the freshwater bodies of Lankaran natural area 126 species of testate amoebae belonging to 11 families were registered. They are as follows: 17 species of Arcellidae, 18 species of Centropyxidae, 2 species of Plagiopyxidae, 6 species of Trigonopyxidae, 70 species of Difflugiidae, 2 species of Lesquereusidae, 1 species of Heleoperidae, 3 species of Pseudodifflugiidae, 3 species of Euglyphiidae, 3 species of Cyphoderiidae, 1 species of Cryptodifflugiidae [ 2, 4, 7, 11, 12, 15]. 2. 8 species and subspecies were registered by us for the first time. Of these, 2 species (Difflugia alekperovi Snegovaya and Tahirova, 2015; Awerintzewia minuta Snegovaya and Tahirova, 2018) were first described for science, and 4 species (D.microclaviformis Ogden, 1983; D.petricola Cash, 1909; D.biconcava Ertl, 1964 and D.serbica Ogden and Živković, 1983) for the Caucasus, and 2 subspecies (Arcella discoides ssp. scutelliformis Playfair, 1918 and D.corona ssp.tuberculata Vuchetich, 1973) were first recorded for the fauna of Azerbaijan [2, 6, 13, 14]. 3. Representatives of such families as Arcellidae (13%), Centropyxidae (14%) and Difflugiidae (55%) dominated in all studied water bodies [1, 11]. 4. The maximum abundance of testate amoebae was recorded in the summer in a pond near the Azfilial settlement (7,4 thous.ind/dm2), and the minimum total number was noted in the pond near the Porsova village (Yardimli region) (1000 ind/dm2). In winter, the minimum total number of spesies was 0,1 thousand ind/dm2 (in the Tangarud river and a pond near the Porsova village [14]. 5. A list of testate amoebas-indicator species of different saprobial zones has been compiled. In general, 15 of the recorded species of testate amoebae are bioindicators of different saprobial zones. According to the results obtained, by the ratio of indicator species Khanbulanchay reservoir belongs to the β-mesosaprobic zone depending on the degree of organic pollution, and Vilashchay reservoir belongs to the β-α-mesosaprobic zone. 6. Comparison of the testaceans fauna of north-eastern, western parts of Azerbaijan and Absheron peninsula with Lankaran natural

23 area by the cluster analysis showed that the highest similarity (56.17%) was recorded between Lankaran and Khachmaz regions. The similarity analysis of the species composition of testate amoebae fauna of 6 regions of the Lankaran natural area showed that the high resemblance is between the fauna of Lerik and Yardimli districts (65.6%) and the least similarity is between the Lankaran and combined cluster of other five regions (44.8%) [16, 17].

PRACTICAL RECOMMENDATIONS Based on the results achieved, we have the following practical recommendations: 1. The bioindicator species of testate amoebae registered by us can be used as a test object for biomonitoring of freshwater bodies. 2. Bioindicator species of testate amoebae, along with other species of other hydrobiont groups can be used to prepare a common multi-test for biomonitoring and assessment of the quality of fresh waters in Azerbaijan. 3. At present in modern schools there is an increase in the processes of biological education and upbringing. All these, in turn, require the use of modern methods. The present work can be used in this regard. When teaching the course of zoology "Protozoa" in high and secondary schools, it is recommended to provide the information presented in the dissertation on self-regulation of the protozoa, determining with their help the degree of pollution and their role in the processes of water bodies cleaning. At the same time, this would create an idea of such concepts as “saprobity”, “degree of saprobity”, “indicator organisms”.

List of publications on dissertation 1. Tahirova, E.N. Preliminary information on testate amoeba fauna of some freshwater bodies of Lankaran // Materials of the XVIII Republican scientific conference of doctoral students and young researchers, vol. 1, – Baku: – 19 – 20 December, – 2013, – pp. 153- 155 (in Azerbaijani). 2. Tahirova, E.N. To the study of the fauna of testate amoebas

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(Rhizopoda, Testacea) of fresh waters of the Lankaran natural area // - Baku: Proceedings of the Institute of Zoology of ANAS, – 2014. vol. 32, №1, – pp. 208-216 (in Russian). 3. Tahirova E.N. The study of testate amoebae (Testacea) by generally accepted methods // IV International Scientific Conference of Young Scientists and Researchers on "Innovation Problems of Modern Biology" dedicated to the 91st anniversary of the great leader Heydar Aliyev, – Baku: – 16 – 17 May, – 2014, – pp. 90-91 (in Azerbaijani). 4. Tahirova, E.N. Species composition of freshwater testate amoebae in Lankaran region // – Baku: Proceedings of ANAS (Biological and Medical Sciences), “Elm”, –2014, vol.69, №3, – pp.33-37 (in Russian). 5. Tahirova, E.N. Morphological description of testate amoebae (Testacea) of freshwater basins of Lerik region (South-east Azerbaijan) // - Nakhchivan: News of Nakhchivan Section of ANAS (The Series of Natural and Technical Sciences), “Tusi”, – 2015. vol. 11, – pp. 245-252 (in Azerbaijani). 6. Snegovaya, N.Y., Tahirova, E.N. A new species of testate amoebae of the genus Difflugia from the freshwaters of Azerbaijan (Rhizopoda, Testacea, Difflugiidae) // – Kiev: Vestnik zoologii, – 2015. vol. 49, № 2, – pp. 99-104. 7. Tahirova, E.N. Species composition of testate amoebae of freshwater basins of Astara region of South-Eastern Azerbaijan // Baku: Proceedings of Young Scientists, – 2015. №12, – pp. 117- 121 (in Azerbaijani). 8. Tahirova, E.N. The study of testate amoebae (Testacea) in Azerbaijan by modern methods // Academic Science Week-2015 International Multidisciplinary Forum dedicated to the 70th anniversary of ANAS, – Baku: – 2 – 4 November, – 2015, – pp. 414-415 (in Azerbaijani). 9. Tahirova, E.N. Trophic relationships of testate amoebae (Protozoa, Testacea) with other groups of hydrobionts // International Youth Forum “Integration processes of world science in the XXI century”, – Ganja: “Khazar University” Publishing House, – 10 – 14 October, – 2016, – pp. 170-171 (in Azerbaijani). 10. Tahirova, E.N. The influence of some environmental factors on the

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species composition of testate amoeba // - Ganja: "Scientific News", ANAS Ganja Branch, – 2016. vol. 65, №3, – pp. 22-28 (in Russian). 11. Tahirova, E.N. Biodiversity of testate amoebae (Rhizopoda, Testacea) in freshwaters of Lankaran region // XIX International Scientific Conference With Elements of the Scientific School of Young Students "Biodiversity of the Caucasus and Southern Russia", – Makhachkala: – 4 – 7 November, – 2017, vol. 2, – pp. 516-518 (in Russian). 12. Alekperov, I.Kh., Snegovaya, N.Y., Tahirova, E.N. The cadastre of free-living ciliates and testate amoebae of Azerbaijan // –Saint Petersburg: Protistology, – 2017. vol. 11, № 2, – pp. 57-129. 13. Snegovaya, N.Y., Tahirova, E.N. The new and rare species of testate amoebae (Amoebozoa: Arcellinida) from mountain lakes of Azerbaijan // – Saint Petersburg: Protistology, – 2018. vol. 12, №1, – pp. 38-46. 14. Tahirova, E.N. The fauna of testate amoebae in freshwater basins of Jalilabad and Yardimli districts in the Lankaran natural area (Rhizopoda, Testacea) // Baku: Proceedings of ANAS (Biological and Medical Sciences), “Elm”, – 2018. vol. 73, №1, – p. 98-103. 15. Tahirova, E.N. Species composition of testate amoebae in mountain rivers of Lankaran Natural Area (South-Eastern Azerbaijan) // International Scientific Conference "Biodiversity of the Caucasus and Southern Russia". – Magas: – 15 – 18 November, – 2019, – pp. 435-438. 16. Tahirova, E.N. Comparative analysis of testate amoeba fauna of Lankaran natural region with other regions of Azerbaijan // II International Scientific Conference of Young Scientists and Specialists “Multidisciplinary Approach to Solving Modern Problems of Fundamental and Applied Sciences”. – Baku: – 3 – 6 March, – 2020, – pp.141-143 (in Azerbaijani). 17. Tahirova, E.N., Snegovaya, N.Y. A comparative analysis of freshwater testate amoebae species composition between the south-eastern part of Azerbaijan and other regions of Azerbaijan // – Saint Petersburg: Amurian Zoological Journal, – 2020. vol. XII, №2, – pp. 189-200.

26 The defense will be held on ______2021 at ____ at the meeting of the Dissertation council FD 1.09 of Supreme Attestation Commission under the President of the Republic of Azerbaijan operating at the Institute of Zoology of Azerbaijan National Academy of Sciences.

Address: AZE 1004, Baku, A.Abbaszade 115, passage 1128, block 504.

Dissertation is accessible at the Institute of Zoology of Azerbaijan National Academy of Sciences Library.

Electronic versions of dissertation and its abstract are available on the official website of the Institute of Zoology of Azerbaijan National Academy of Sciences.

Abstract was sent to the required addresses on ______2021.

27 Signed for print: 12.03.2021 Paper format: 60x841/16 Volume: 38789 characters Number of hard copies: 20

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