121

J. Black Sea/Mediterranean Environment Vol. 23, No. 2: 121-155 (2017)

RESEARCH ARTICLE Protists (Ciliophora, Gromiida, Foraminifera) in the Black Sea meiobenthic communities Nelli G. Sergeeva1*, Derya Ürkmez2, Igor V. Dovgal1, Murat Sezgin3 1Institute of Marine Biological Research of Russian Academy of Sciences, 2, Nakhimov ave., Sevastopol, 299011, RUSSIA 2Scientific and Technological Research and Application Center, Sinop University, Sinop, TURKEY 3Department of Hydrobiology, Faculty of Fisheries, Sinop University, Sinop, TURKEY *Corresponding author: nserg05@mail.ru Abstract

The aim of this work is to present a view on eukaryotic life under oxic and permanent anoxic conditions associated with hydrogen sulfide in bottom sediments of the Black Sea with emphasis on unicellular eukaryotes (protists). It was shown that benthic Ciliophora, Gromiida and Foraminifera are present at all studied depths. Thus, obtained results showed that benthic protists are the constant and integral components of the meiobenthic communities under varying conditions of the Black Sea. Unevenness has been detected in spatial and bathymetric distribution of the protozoans and metazoans. The contribution of benthic protists to meiobenthos at different parts of the Black Sea under conditions ranging from normoxia to anoxia enriched with hydrogen sulfide (to the 300 m water depth) was investigated in the present study.

Keywords: Hard-shelled foraminifera, soft-shelled foraminifera, Gromiida, Ciliophora, meiobenthos, abundance Received: 29.05.2017, Accepted: 20.07.2017 Introduction The Black Sea is a unique marine water body where the dissolved oxygen disappears at a depth of about 200 m while hydrogen sulfide is present at all greater depths (Zaitsev 2008). There is a thin intermediate layer of up to 10-20 m with coexistence of oxygen and hydrogen sulfide. It has been considered that only some organisms which are characteristic for the oxygenated layer of marine waters might be found in this zone (Zaitsev 2008). The knowledge about the presence of oxygen only in trace amounts or its absence limited hydrobiological investigations concerning eukaryotic life under sulfidic conditions in the deeper

122

parts of the Black Sea. As a result, some authors excluded the idea that eukaryotic life can exist in the bottom sediments of the sulfidic and anoxic Black sea waters (Zaitsev et al., 2007, 2008; Zaitsev and Polikarpov 2008). Accordingly, current investigations on the Black Sea benthos have been focusing on coastal communities. For various reasons, researchers generally do not take into account the single-celled eukaryotic organisms as the component of marine benthic communities. For many years, gromiid and large allogromiid (soft-shelled) foraminifera were considered difficult by non-specialists to taxonomically classify and even they were not recognized as protists (Gooday et al. 2000; Nyholm and Gertz 1973). This was also observed in the study of the Black Sea benthic communities. However, in recent years, we obtained extensive data on the Black Sea meiobenthos by not ignoring various benthic protists, as they were also recorded in meiobenthos along the shelf and continental slope of the Black Sea in environments with decreased oxygen levels (Brennan et al. 2013; Revkov and Sergeeva 2004; Sergeeva 2003, 2016; Sergeeva et al. 2012a, 2013, 2015; Sergeeva and Anikeeva 2014; Sergeeva and Gulin 2007; Sergeeva and Zaika 2013; Ürkmez et al. 2015, 2016a, b). The aim of the present study was first to understand the response of the main benthic protozoan taxa to oxygen depletion, and secondly, to analyze the change in the contribution of the basic representatives of benthic protists in the meiobenthos of the Black Sea both under shallow oxygenated conditions and also in the deep-water permanently extreme environments. The other important task of the study was to show the poorly known bottom protists of the Black Sea including Ciliophora, Gromiida and Foraminifera (hard-shelled or soft-shelled - monothalamous) as components of the bottom communities and to attract the attention of scientists to the necessity of more comprehensive benthic studies for the assessment of the significant role of various size-ecological organism groups in the functioning of benthic ecosystems of this unique basin. Thus we focused on the contribution of the mentioned group of organisms to the meiobenthic communities in different regions of the Black Sea with examples from the shelf at oxygenated zone and at the upper slopes along the oxic/anoxic interface zone taking into account certain characteristics (bathymetric and spatial distribution, abundance, and ratio of these organisms in total meiobenthos). It is known that the representatives of benthic fauna are evaluated in the composition of bottom communities depending on their size and they are accepted as micro- (of size 10 to 100 µm), meio- (100 to 1000 µm) and macrobenthos (more than 1 mm) (Mare 1942). We considered all meiobenthic organisms, both protozoans and metazoans including permanent as well as temporary meiobenthos that represent juvenile stages of macrobenthos (Bougis 1950; Chislenko 1961; Giere 2008; Higgins and Thiel 1988; Luth and Luth 1997, 1998; Mare 1942; Sergeeva 2004; Sergeeva and Gulin 2007; Sergeeva et al. 2012, 2013; Soltwedel 2000).

123

In this work, the information on the contribution of most numerous and common benthic protists to the bottom communities of the Black Sea is provided. These protists include Ciliophora, Gromiida and Foraminifera (hard-shelled or soft-shelled - monothalamous). Here we discuss only the relative abundance of these representatives of protozoans in total meiobenthos compared to that of metazoans, without considering their biomass.

Materials and Methods During several research programs of meiobenthos in the Black Sea, we collected extensive data on protozoans as a component of meiobenthic communities. Samples were collected at various parts of the sea, from shallow bays to the layer of deep-water permanent hypoxia, lying on the waters containing hydrogen sulfide. The changes in the distribution of benthic protozoans and metazoans and the proportion of main benthic protist taxa in the deep-water meiobenthos along oxic/anoxic interface (75-300 m) were studied in the outlet area of the Istanbul Strait (Bosporus) in the Black Sea, the northwestern region of the Crimean Peninsula and the southern part of the Black Sea in Sinop area (90-203 m). Also, the contribution of protozoans and metazoans to the meiobenthos at oxygenated zone of the northeastern, northwestern, southern and southeastern parts of the Black Sea near the Kerch Strait (60-110 m), Karkinitsky Gulf (10-36 m), İğneada Bay (5-20 m) and Sinop coasts (3-10 m) was studied. Samples of bottom sediments for biological studies were collected at the oxic/anoxic interface of the SW and NW parts of the Black Sea using a multiple corer (MuC), push corer (PsC) and gravity corer (GrC), devices that obtain virtually undisturbed sediment samples. Sediments were obtained directly from the MUC tubes (ø = 9.6 cm), PsC (ø =7.3 cm), and GrC (ø =7.3 cm). Two-three sediment core tubes were collected at each station from independent MuC or PsC casts. Sediment cores were sectioned as subsamples of one centimeter intervals for the upper 7-10 cm of the cores. Sampling in oxic zones at the western and southern coasts of the Black Sea was conducted using PsC (mouth area = 12.56 cm2) and bottoms of oxic/anoxic interface at the southern Black Sea (Sinop, Turkey) coasts were sampled using a remote operated vehicle (ROV) ‘Hercules’ and the material was taken using push cores (ø = 6.4 cm). Samples were collected at 18 stations in the outlet area of the Istanbul Strait and from 25 stations on the open slope of the northwestern Crimean Peninsula at a water depth ranging between 75-300 m during the 15/1 cruise of RV ‘Maria S. Merian’ in April - May 2010 (Figures 1 and 2). Meiobenthos was studied on the basis of samples of bottom sediments obtained using push-corer and multiple-corer in two areas located on the open shelf at the western part of Crimean peninsula (Figure 2). Area I covered 6 stations (84-212 m) and Area II was represented by 19 stations (101-375 m).

124

Figure 1. Meiobenthic sampling stations at the outlet area of the Istanbul Strait in the

Black Sea (R/V “Maria S. Merian”, April 2010)

Figure 2. Meiobenthic sampling stations at the outer western Crimean shelf located on Area I and Area II (RV ‘Maria S. Merian’, May, 2010)

125

The meiobenthos of oxygenated zone were studied near the Kerch Strait outlet area of the Black Sea (60-109 m water depth) at 17 stations during cruise 75 of the RV ’Professor Vodyanitsky’ in July 2013 (Ukraine) (Figure 3A). Bottom sediment samples were collected using GrC (ø =12.7 cm) at the northeastern part. The upper layer of 5 cm height was cut off from core columns for biological analysis. Benthic protozoans and metazoans were studied in the oxic zone of the Black Sea in Karkinitsky Gulf (NW, the Crimean Peninsula) at 10 stations with a depth range of 10-46 m (NW) during cruise 70 of RV ‘Professor Vodyanitsky’ in 2011 (Figure 3B). Bottom grab ‘Ocean-25’ was used in this area for collection of the materials. A sediment core (ø = 4.8 cm) was used in three replicates and the upper 5 cm layer was cut off for the surface monoliths of bottom sediments obtained by the core.

Figure 3. Meiobenthic sampling stations: A - in the near Kerch Strait of the Black Sea (cruise 75, RV ‘Professor Vodyanitsky’, August 2013), B- in Karkinitsky Gulf (B) (cruise

70, RV ‘Professor Vodyanitsky’, August 2011) Materials from the southern Black Sea coasts (Turkey) were obtained during three different cruises (Figure 4). Meiobenthic samples were collected from oxygenated waters of İğneada Bay (8 stations, 5-20 m) in November 2012 and from the coast of Sinop Bay (8 stations, 3-10 m) monthly between August 2009 and July 2010. Oxic/anoxic interface off Sinop shores (90-203 m) was sampled in August 2011 during the expedition NA012 of the EV ‘Nautilus’ (Dive numbers: NA012-001, NA012-002, NA012-003). Scuba divers used push cores to collect the material in İğneada and Sinop Bays, whereas remote operated vehicle “Hercules” was used to obtain the sediment samples at the oxic/anoxic transition zone off Sinop shores. We preserved all subsamples of sediments in 75% ethanol, which is known to preserve morphological structures of fauna without distortion. We avoided prior fixation in formalin in order not to damage calcareous taxa. The sediments were washed under distilled water through sieves with a mesh size of 1 mm and 63 μm.

126

All protozoans and metazoans isolated as ‘live’ (stained) and larger than 63 μm size were categorized as meiobenthos. The material retained on the sieves was stained by Rose Bengal solution before being sorted in water under a stereomicroscope. The ‘live’ (intensely stained) organisms were identified to higher taxa. The “operational” definition of meiobenthos which include organisms that pass through a sieve of 1 mm mesh size and retained on a 63 µm mesh size sieve was accepted. Also certain specimens of meiobenthos that reached 2 mm in size, mainly larger foraminifers (hard- and soft-shelled), gromiids, ciliophorans and nematodes were evaluated. All the isolated organisms were counted and identified to higher taxa level (type, class, order, and genus). Due to the lack of sufficient knowledge in taxonomy of protists, it was possible sometimes to identify down to genus level at most.

Figure 4. Meiobenthic sampling stations A – İğneada Bay at the southwestern Black Sea

(Turkey, November 2012), B – Sinop Bay at the southern Black Sea (Turkey, August2009-July 2010), C – Oxic / anoxic interface off Sinop shores, Turkey, August

2011 (S: suboxic, A: anoxic, O: oxic stations)

We studied the protists and metazoans in detail by 100-1000х magnification, with a light microscope Olympus СХ41 and a Nikon Eclipse Ni-U advanced research microscope (with DIC attachment) equipped with a digital camera. The average total abundance of bottom fauna found at all core levels from 0 to 5 cm depth was studied. Density of meiobenthos was calculated per m2 of the bottom area, on the

127

basis of the number of meiobenthic organisms found in upper 5 cm of the cores. Oxygen concentrations in the bottom-water along the interface zone at the outer western Crimean shelf and at the Istanbul Strait of the Black Sea ranged from normoxic (175 μmol O2L-1) and hypoxic (less than < 63 μmol O2 L-1) to even anoxic/sulfidic conditions within a few kilometers range (Holtappels et al. 2011; Lichtschlag et al. 2015). Results Our data suggest that some benthic eukaryotes (protozoan and metazoan) are tolerant to hypoxia and anoxia associated with hydrogen sulfide. 1. The deep-water in the outlet area of the Istanbul Strait (Bosporus) of the Black Sea In April 2010, investigations were conducted at oxic/anoxic interface along a depth range of 80-300 m to understand the response of main benthic protists to oxygen depletion and to assess their contribution to meiobenthos in the deep-waters of the Istanbul Strait. Distribution of the abundance of metazoans and benthic protists in total meiobenthos was not regular along the studied depth gradient (Figure 5).

Figure 5. Distribution of the abundance (103ind.m-2) of metazoans and protozoans along

the studied depth gradient near the Istanbul Strait in 2010 Benthic protists were recorded at all the stations studied (75-300 m) and an unevenness was observed in their vertical distribution (Figure 6). In 2009, two peaks of total abundance were recorded at 88-103 (Sergeeva and Mazlumyan 2013) and 250 m in the Istanbul Strait area and one strong peak was recorded at 196 m in 2010. On one hand, it can be explained by micro-distribution depending on the availability of food resources, the number of macrobenthic fauna and their bioturbation activities that can be observed at the oxygenated zone. On the other

128

hand, there was a trend of increase in numbers at depths of 200-250 m under anaerobic conditions with a high concentration of hydrogen sulfide. We assume that some forms of protists are adapted to such conditions and can reach to large quantities.

Figure 6. The abundance (103ind.m-2) of protists along the studied depth gradient in the

outlet area of the Istanbul Strait in different years. A: November 2009 (in agreement with Sergeeva et al. 2015), B: April 2010.

Gromiida was a permanent component of the meiobenthos at all studied depths of the outlet area of the Istanbul Strait of the Black Sea in November 2009. The relative share of Gromiida was 30% in total meiobenthos. Ciliophora made a prominent contribution at 103, 250, 300 m depths and its relative share ranged between 15-90 %. The relative share of Foraminifera was 3-88 % and presented a main contribution at depths of 82, 160 and 190 m (Sergeeva et al. 2015). In 2010, the ratio of protozoans and metazoans in meiobenthos, as well as of main taxa in protozoan communities changed with increasing water depth and with reduction in dissolved oxygen concentration in the environment (Figure 7). The contribution of the single-celled representatives to the total abundance of meiobenthos was 5-12%. The hard-shelled foraminifera accounted for 19% of the total number of protists in the oxygenated zone, but their contribution did not exceed 3% under hypoxia and anoxia. These forms were distributed at depths of 160-173 m. The hard-shelled foraminifera had a significant contribution (19%) to the total number of protists only at the upper oxygenated zone. The soft-shelled foraminifera, unlike hard-shelled forms, made a significant contribution (27-16%) to protozoan communities under anoxic conditions associated with hydrogen sulfide. Soft-shelled foraminifera had the greatest contribution (60%) among the protists at the oxic zone (80-116 m) and by the upper boundary of the hydrogen sulfide zone (135 m). A similar trend of increase was observed among gromiids (4-27%) and ciliates (22-63%) in their contribution to the community under hypoxic and anoxic conditions. Thus, the soft-shelled Foraminifera, Gromiida and Ciliophora had the greatest share as main components of the protozoan community and meiobenthos in the Istanbul Strait area (Figure 7).

129

Figure 7. Share of protozoa and metazoa in total meiobenthos (A), share of main taxa of benthic protists in protozoan communities (B) along the studied depth gradient at the

Istanbul Strait area of the Black Sea (April, 2010) (F: Foraminifera)

130

2. The deep-water of the outer western Crimean shelf

Bacterial mats were observed on bottom sediments at two stations of Area I (425/163m and 426/175m) and at four stations of Area II (378/155m, 398/144m, 405/151m and 412/124m). The structure of core columns, from their surface to a depth of 5 cm, was heterogeneous. These differences were prominent in the columns obtained at same depths and they contained light gray, sometimes with a greenish tinge, dark gray, brown and black sediment layers. The structure of the sediments ranged from viscous and runny to hard consistency with varying values of hydrogen sulfide recorded by sensory devices. Live bivalve mollusks or just shells of dead mollusks with varying degrees of destruction were detected on the surface of different columns obtained at the same station. Populations of Modiolula phaseolina and great number of ferromanganese nodules were observed in some sediment columns taken from the same station at the spots with oxidized surface layer, whereas no live mollusks or ferromanganese nodules were seen in other sediment columns. Measurements of oxygen concentrations revealed four zones with different oxygen regimes within a distance of more than 30 km at bottom-water layer of the outer western Crimean shelf along the transect from 95 to 218m water depths. Oxic zone was situated at <130m, where oxygen concentrations at the bottom-water layer were more than 63 µmol L-1; oxic/hypoxic zone between 130-142m, where oxygen concentrations ranged from >63 to >0 µmol L-1; hypoxic/anoxic zone- between 142-170 m water depths where O2 concentrations were 63-0 µmol L-1, and anoxic/sulfide zone at deeper than 170 m water depths where sulfide was present in anoxic bottom waters (Holtappels et al. 2012; Lichtschlag et al. 2015). Area I covers the methane escape spots from the bottom surface. A visual overview of this area was conducted before sediment sampling using a remote operated vehicle ‘Medusa’ and a mosaic coloring was noticed on the bottom surface. These spotted areas with different structure and color were particularly chosen for the sampling of meiobenthos. In particular, the surface sediment had a general gray-brown color, and in some zones there were large spots covered with white bacterial mold. Taking into account the heterogeneity of the sediment, one column of bottom sediment sample was taken using push-cores from different zones of all the stations. The only exception was the station located at 212 m water depth, where only one sample was taken. The bathymetric distribution of meiobenthos (metazoans and benthic protozoans) was not even at the top 5 cm layer of the columns taken from bottom sediments at two areas (I and II) of the Crimean shelf. A sharp increase in the average number of multicellular and unicellular organisms was observed in the hypoxic/anoxic zone of Area I (Figures 8 and 9). Unlike the Istanbul Strait region, the contribution of benthic protozoans to the total abundance of meiobenthos increased from 8 to 62% along the oxygen gradient at the transition zone from normoxia to hypoxia and anoxia at Area I of the Crimean shelf (Figure 9).

131

Furthermore, gromiids and soft-shelled Foraminifera dominated at the oxygenated conditions, whereas the protozoans were totally composed of Ciliophora (81-97%) at the hypoxic / anoxic environments.

Figure 8. Distribution of the metazoan and protozoan abundance (103 ind.m-2) along the

studied depth gradient at the outer western Crimean shelf, Area I (May 2010)

Figure 9. Share of protozoa and metazoa in total meiobenthos (A), share of main taxa of

benthic protists in protozoan communities (B) along the studied depth gradient at the outer western part of Crimean shelf, Area I (May 2010) (F: Foraminifera)

132

We observed trends of reduction with depth in the number of multi-cellular organisms at Area II. Moreover, the density of protozoa under acute hypoxia and anoxia was similar or higher than that of under oxygenated conditions (Figures 10 and 11). Exactly the same overall trend of increase was observed in the shares of the total structure of meiobenthos (from 3 to 47%) along the oxygen gradient at Area 2 of the Crimean shelf (Figure 10). However, the ratio of studied taxa was different. Here the hard-shelled foraminifera and gromiids provided a significant contribution (6-44% and 11-42%, respectively) under oxygenated conditions. On the other hand, the proportion of soft-shelled foraminifera increased (from 29 to 48%) at the hypoxic/anoxic zone, while ciliates dominated (93%) under anoxic conditions.

Figure 10. Distribution of the metazoan and protozoan abundance (103ind.m-2) along the studied depth gradient at the outer western part of the Crimean shelf, Area II (May 2010) The study area is located at the northeastern part of the Black Sea within the oil and gas structures of the Kerch shelf. The first data on the diversity, and the vertical and spatial distribution of meiobenthos was obtained in 2013. The meiobenthic community was composed of 17 higher taxa, including Ciliophora, Foraminifera and Gromiida (Sergeeva 2013). Here we carried out a comparative analysis of the density of protozoans and metazoans in total, and the contribution of main benthic protists to the meiobenthic community at studied depths.

133

Figure 11. Contribution of Protozoa and Metazoa to total meiobenthos (A), share of the main taxa of benthic protists in protozoan communities (B) on the studied depth gradient

at the outer western part of the Crimean shelf (May 2010) (F: Foraminifera)

The values of the abundance of the meiobenthos, including the benthic protozoans, do not reach high values such as in deep water areas at the northwestern and southwestern part of the Black Sea (see above). Nevertheless, we also observed a trend of uneven distribution of the benthic protists at the oxygenated zone (Figure 12). The proportion of the total number of protozoa in meiobenthos reached to 17-40%, but only three taxa (Figure 13B) showed a significant share in the community. Gromiids or Ciliophores were prevailing groups (Figure 13).

134

Figure 12. Distribution of the metazoan and protozoan abundance (103ind.m-2) along the

studied depth gradient at the oxygenated zone of the Crimean shelf, near Kerch Strait (NE Black Sea, August 2013)

Figure 13. Contribution of protozoa and metazoa in total meiobenthos (A), share of the main taxa of benthic protists to protozoan communities (B) at the studied depth gradient of oxygenated zone at the Crimean shelf, near Kerch Strait (NE Black Sea, May 2013)

(F: Foraminifera)

135

3. NE part of the Black Sea (Crimean coast of Kerch Strait) The distribution of hard-shelled foraminifera was random and they were found only as single specimens (less than 1%). Gromiids presented a main share in the community of protists at 60-90 m depths, and share of the other groups did not exceed 18-20% of the total number of protists. One of the two stations (depth 109 m) was represented exclusively by gromiids, and only gromiids and soft-shelled foraminifera were recorded at another station (depth 249 m). It should be noted that the first morphotypes of soft-shelled foraminifera found at the Crimean shelf near Kerch Strait were similar in terms of morphological characteristics to the specimens that were found in the Istanbul Strait area. The studies on species identifications continue, but they are considered to be as the representatives of the genus Nemogulmia or Stepherdella. 4. The Karkinitsky Gulf (NW Black Sea) The contribution of the benthic protozoans to total meiobenthos was evaluated separately in three parts of the studied area: the inland (10-18m), middle (26-31m) and outer (32-46m) parts. During the study, the temperature of the upper water masses in this Gulf (0-8m) was 22.42-22.05°C. There was a temperature drop from 22°C to 8°C in the thermocline layer (8-26m). In the inland part of the Gulf, the temperature of the bottom (8m) water was high as 24.56°C, indicating a weak hydrodynamic activity in this area. An influx of cold water (probably from the northwestern part) was observed at the mid-northern part of the bay, where the upper mixed layer was limited to 0-5m, and the thermocline layer was only 5m (with a temperature range of 22-12°C). Distribution of salinity was characterized by a relatively uniform increase in the direction from the inland part (from 16.23 to 18.42‰) to the outer zone. We also observed a significant decrease in the oxygen concentration to 5.39 ml/L in stagnant water at the inland part of the Gulf, while its concentration was about 6-7 ml/L in other areas of the Gulf (Sergeeva et al. 2012b). The proportion of protozoans and multicellular components in the meiobenthic communities of Karkinitsky Bay was unusual. The share of protists and metazoans in meiobenthos of the inland and outer parts of the Gulf were found to be similar (26 and 74%, respectively), whereas the contribution of protists and metazoans in the middle part was only 12% and 88%, respectively (Figure 14). Soft-shelled foraminifera dominated (61-93%) in the cenosis of benthic protists along the Gulf, increasing in the direction from inland to the seaward zone. The role of individual taxa was different at the studied gulf areas.

136

Figure 14. Contribution of Protozoa and Metazoa in total meiobenthos (A), share of the main taxa of benthic protists to protozoan communities (B) at the oxygenated zone of the

different parts of Karkinitsky Gulf (May 2013) (F: Foraminifera)

5. İğneada Bay (SW Black Sea, Turkey) İğneada Bay is located at the western part of the Black Sea coast of Turkey and has a border wih a Bulgarian Marine Protected Area (MPA). As a result of a preliminary meiobenthos study conducted at 8 stations on 3 transects (5, 10, 20m), 12 higher taxa were recorded. Protozoans were represented at all the stations except one (St. I-1-2) and their percentage ranged between 0-19% with the highest share at St. I-2-1 (5 m) which is located at the middle of the bay presented with a biotope of fine sand+silt (Figure 15). Protozoans of İğneada Bay were composed of soft-shelled and hard-shelled foraminifera and ciliophores were not recorded. Hard-shelled foraminifera was the third dominant taxon (Ürkmez et al. 2016b) in the studied area and made its highest contribution to meiobenthos at two stations along transect 2, St. I-2-1 (19.5%) and St. I-2-2 (7.5%). Soft-shelled foraminifera was found only at two stations with 5 (St. I-3-1) and 10 m (St. I-2-2) depth.

137

Figure 15. Share of protozoa and metazoa at the stations of İğneada Bay, SW Black Sea,

Turkey (November 2012)

6. Sinop coasts (Southern Black Sea, Turkey) A comprehensive meiobenthic sampling campaign by the Turkish scientists has been conducted at the central Black Sea coast of Turkey (Sinop Bay). Material was collected monthly at 8 stations (4 transects) from depths of 3 and 10 m between August 2009 and July 2010. Hard-shelled and soft-shelled foraminifera were found at the majority of the stations in most of the sampling months. Furthermore, hard-shelled foraminiferans were among the dominant taxa following nematodes and harpacticoids in all the samples (Ürkmez et al. 2016a). Ciliophorans were particularly recorded at stations A1, B1 and C1 with a biotope of coarse sand. The mean abundances of autumn (September, October and November), spring (March, April and May), winter (December, January, and February) and summer (June, July and August) were calculated to examine the abundance of protozoans. It was clearly seen that the density of protozoans was higher at stations with 10 m depth, particularly at stations A2 and C2 with coarse sand. Protozoan densities ranged between 10-371x103ind.m-2 during autumn, 7-275x103 ind. m-2 in winter, 10-185x103 ind. m-2 in spring and 10-166 x103 ind. m-

2 in summer. The highest value was recorded at St. A2 in autumn (371x103 ind. m-2). The contribution of protozoans to meiobenthos was prominent at station D1 in summer, spring and winter (64, 61, 75%), at station A2 in autumn (70%) and winter (69%) and at station C2 in winter (Figure 16). Percentages of 3 taxa in protozoan community are given in Figure 17. Hard-shelled foraminifera was the

138

most contributing taxon to protozoans particularly at stations C1, D1, A2, C2 and D2 during four seasons. Contribution of soft-shelled foraminifera was prominent at stations A1, B1, C1 and B2, and the highest percentages were recorded in spring (82% at st.A1) and summer (72%, st. B1). Ciliophora was found mainly at three stations with 3 m depth presenting with a biotope of coarse sand (A1, B1, C1) and its contribution was also higher in spring and summer.

Figure 16. Protozoan and metazoan abundances (103ind.m-2) at the oxygenated stations of Sinop Bay (Southern Black Sea, Turkey) during four seasons, 2009-2010 (A,B,C,D

are transects; 1 indicates stations with 3 m, 2 indicates stations with 10 m)

Figure 17. Contribution of soft-shelled and hard-shelled foraminifera and ciliophora to meiobenthic protozoans at the oxygenated stations of Sinop Bay (Southern Black Sea,

Turkey) during four seasons, 2009-2010 (F: Foraminifera; A,B,C,D are transects; 1 indicates stations with 3 m, 2 indicates stations with 10 m)

139

Oxic/anoxic interface off the Sinop Peninsula was sampled at three depths representing three different zones (oxic zone, suboxic zone, anoxic zone) at depths of 90m, 120.5m and 203m (Brennan et al. 2013). Respective oxygen concentrations were 69.61 µM, 1.44 µM and 0 µM and they were recorded by an optode mounted on the ROV. The suboxic core was composed of soft sediment with shells and anoxic core was full of fine sand. Three replicates of samples were only collected at the anoxic zone and the other two zones were represented with one sample. Meiobenthic assemblages were composed of 13 major taxa and ciliophorans were not found in the community structure of the area (Ürkmez et al. 2015). Density of protozoans (composed only of hard-shelled foraminifera) was highest at the oxic zone (90 m), however their share was only 1% compared to metazoans. Soft-shelled foraminifera had a share of 10% and recorded only at the suboxic zone in total meiobenthos assemblage of the studied area (Figure 18).

Figure 18. Share of protozoans and metazoans in the meiobenthic community structure

of the oxic/anoxic interface off the Sinop Peninsula, August 2011 (Southern Black Sea, Turkey) (F: Foraminifera)

Discussion The study of hard-shelled foraminiferans in the Black Sea has been the focus of repeated researches (Golemansky 1999a; Mikhalevich 1968; Vorobyova 1999; Yanko and Vorobyova 1991; Temelkov 2008, 2010; Temelkov et al. 2006; Öksüz et al. 2011; Öksüz et al. 2013; Öksüz et al. 2016a; 2016b; Űrkmez et al. 2016b). The first 10 species of hard-shelled foraminifera were found during the 1880s (Pereyaslavtseva 1886) in this area and the number increased to 26 species by 1968 (Mikhalevich, 1968). Currently, there are more than 100 species known for

140

this unique basin (Temelkov et al. 2006; Yanko 1990; Yanko and Troitskaya 1987). Soft-shelled species of foraminiferans with organic-walled shells (‘allogromiid’) or agglutinated forms of bottom micro particles or detritus (‘saccamminids’) are an important component of marine foraminiferal assemblages, particularly in fine-grained sediments of deep sea at high latitude regions. The organic-walled forms belong to the suborder Allogromiina. Despite their high abundance and ecological importance, the taxonomy and diversity of these groups of foraminifera are very poorly known (Gooday et al. 2002). First information about findings of non-calcareous foraminifera in the Black Sea appeared in the 1960s (Valkanova 1964; Mikhalevich 1968). The data on the distribution of soft-shelled foraminifera in the Black Sea emerged in the late 20th century (Sergeeva and Kolesnikova 1996) which was the beginning of more comprehensive further studies of this group of protists (Anikeeva and Gooday 2016; Gooday et al. 2011; Sergeeva 2016; Sergeeva et al. 2010; 2014). The rapid accumulation of data on soft-shelled foraminifera linked to the systematic collection of meiobenthic material from the Black Sea using modern bottom samplers. It is shown that the number of allogromiids is higher than that of hard-shelled foraminifera in many habitats. Allogromiids are more resistant to hypoxia, they are found closer to the boundary of sulfide water and penetrate deeper into the sediment (Sergeeva et al. 2014). Representatives of soft-shelled foraminiferans and gromiids in Bulgarian coastal zone were noticed by Valkanov (1970) and Golemansky (1999a; 2007a). However, these components are generally not taken into consideration when assessing the role of benthic communities in marine ecosystems. Nevertheless, it is known that foraminiferans are found on or in sediments and on solid substrates at all depths (Fenchel 1987). At present, the delicate soft-walled foraminifera in the meiobenthic communities of the Black Sea are composed of 39 forms from the families Allogromiidae, Saccamminidae and Psammosphaeridae which are identified to species (14) or genus level (25) as well as with undescribed species (4 species of the genus Nodellum, 6–Vellaria, 4–Bathyallogromia, 3–Tinogullmia, 2–Psammophaga, 1–Psammosphaera, 1–Congieria, and 4–Nemogullmia-like). More than 50 of their representatives from various coastal and deep waters of the Black Sea are in the collections of IMBR (Institute of Marine Biological Research) RAS and require further detailed study (Sergeeva 2016). Fourteen allogromiid species are known from the Black Sea up to now: Hipocrepinella hirudinea Heron-Allen et Erland, 1932, Psammophaga simplora Arnold, 1982, P. cf. riemanni Gooday, 1990, Vellaria pellucida Gooday, 1992, V. sacculus Gooday, 1992, Lagynis pontica Golemansky, 1999, Tinogullmia lukyanovae Gooday et al., 2006, Goodayia rostellatum Sergeeva and Anikeeva 2008, Nellya rugosa Gooday et al. 2011,

141

Cedhagenia saltatus Gooday et al., 2011, Ovammina opaca Dahlgren, 1962, Guanduella podensis Temelkov, 2010, Krymia fusiformis Anikeeva, Sergeeva and Gooday, 2013 and Bellarium rotundus Anikeeva, Sergeeva and Gooday, 2013 (Sergeeva et al. 2015; Anikeeva and Gooday 2016). First data on this group of Foraminifera was from the Azov Sea and the hyper-saline Siwash Bay. According to our still incomplete data, these foraminiferans are represented by three species in these areas, namely Bellarium rotundus, Vellaria cf. pellucida, Psammophaga simplora and not less than by 10 morphotypes (Sergeeva 2016). Several new species and morphotypes of soft-shelled foraminiferans have been described from coastal and deep-waters of the Black Sea (Anikeeva and Gooday 2016; Gooday et al. 2011; Sergeeva et al. 2010) only recently. Since the morphological characters are not always sufficient to establish the phylogenetic relationships among allogromiids, some recent species descriptions include the analysis of molecular data (Pawlowski et al. 2002; Gooday et al. 2004). Gromiids are bottom protists which can reach to a size of 30 mm in diameter. They are ubiquitously distributed in environments ranging from polar seas to tropical coral reefs, and in areas including the Mediterranean, the coasts of North America, northwestern Europe, New Zealand, and Antarctica and they live at shallow waters or large depths (Piña-Ochoa et al. 2010, Matz et al. 2008; Gooday et al. 2000). Deep-sea gromiids are likely to play an important role in carbon cycling at bathyal eutrophic regions (Rothe et al. 2009; 2011). Based on their general morphology, the amoeboid water protists with a monothalamous (single-chambered) proteinaceous test and filose pseudopodia of the genus Gromia Claparède and Lachman, 1859 were initially classified under Foraminifera, but because of their non-granular, filose pseudopodia, they were later included to filopodia-bearing protists (the Filosea). The morphological resemblance between Gromia and Foraminifera may be due to a common ancestor (Longet et al. 2004). Molecular phylogeny showed that foraminifera and Gromia are separate branches of Rhizaria (Burki et al. 2002; Rothe et al. 2011) and Gromia represents a sister group to Foraminifera. Like soft-shelled foraminiferans, representatives of the genus Gromia have also been mentioned in the works concerning the Black Sea benthic communities (Golemansky 1999b, 2007a; Sergeeva et al. 2012; Sergeeva and Mazlumyan 2015). These protists have been found at different depths of the Black Sea, from the coastal areas down to the deep-water zone where permanent strong hypoxic and anoxic conditions occur (10-250m depth). Vertical distribution of Gromia at the oxic/anoxic interface of the Istanbul Strait showed some marked characteristics and several peaks were recorded in their abundance at depths of 75, 88, 122 and 250 m. Most of the individuals inhabited upper layer of the bottom sediment at the point of maximum observable abundance (75, 88, 250 m), and deeper layer (7-9 cm) was inhabited by Gromia only at a depth of 122 m. It was

142

notable that Gromia was not found at the middle layer (3-5 cm) of the investigated bottom sediment (Sergeeva and Mazlumyan 2015). The discovered gromiids in the Black Sea available in our collection are in spherical, elongate and ellipsoidal forms, with a size ranging from 230-600μm to more than 2mm. As a preliminary finding, our studies showed the presence of at least one gromiid species, Gromia cf. oviformis Dujardin, 1835 and seven morphotypes, which may be new to science. Ciliates were represented in aerobic habitats of the Black Sea both as plankton and benthos. The total number of species found in pelagic aerobic waters (29) was low compared to the number of benthic species (476) (Azovsky and Mazei 2003a, 2005; Polikarpov et al. 2003). Approximately 300 species of Ciliata have been observed in the Bulgarian Black Sea sector. Most of them were benthic, and particularly psammal inhabitants (Golemansky 2007b). As a result of the investigations conducted at the Northeastern (Caucasian) region of the Black Sea at depths ranging between 0 to 432 m, the highest diversity of ciliates was found at oxygenated conditions at 3-10 m and they were not found at the zone containing H2S (deeper than 100 m) (Azovsky and Mazei 2003b, 2005). Earlier studies of the oxic/anoxic chemocline of the Black Sea water column revealed only two ciliate species: a representative of Mesodiniidae, and a small unidentified ciliate with symbiotic bacteria on its surface (Sazhin et al. 1991). Wylezich and Jürgens (2011) studied the species composition of protists inhabiting pelagic suboxic and sulfidic waters of the Black Sea using molecular tools. They reported high diversity of ciliates and flagellates in hypoxic and particularly in sulfidic water layers. Many taxa of protists in the Black Sea redoxcline were also observed in other anoxic and sulfidic marine systems (Stoeck et al. 2003, 2007). The authors emphasized the importance of anoxic, sulfidic waters as habitats for high protist diversity and the significance of these groups as a major trophic link in the hypoxic system of marine basins. The first findings of benthic free-living ciliates and data on their bathymetric distribution at depths ranging from 120 to 2075m near the Dnieper Canyon and the Sorokin Trough (the eastern part of the Black Sea) were described from the samples of near bottom water, sediment surface detritus, and the upper layer (0-1cm) of sediment. Hydrogen sulfide was registered beginning from 160-170m. The ciliates found in the mentioned samples were the representatives of the genera Chilodonella Strand 1928, Trachelocerca Ehrenberg 1834, Tracheloraphis Dragesco 1960 and Loxophyllum Dujardin 1841 (Zaika 2008, Zaika and Sergeeva 2009). Epibiont peritrich and suctorian ciliates were first observed on oligochaetes, nematodes and harpacticoids in deep waters of the Black Sea under

143

hypoxic/anoxic conditions (Sergeeva and Dovgal 2014; 2016) and more than 30 morphological species were recognized among collected materials (Sergeeva and Zaika 2008). Ciliate abundances revealed peaks at depths of 120, 160-190, and 240m (Sergeeva et al. 2008). These peaks coincide with the depths presenting with hydrogen sulfide. Additionally, there are pelagic peaks in the intensity of bacterial chemosynthesis and bacteria production (Sorokin and Sorokina 2008) at the same depths. Therefore, peaks in the abundance of ciliates may be related with food accumulations. The highest number of ciliate morphotypes (8) was recorded at 240m depth, where a lower peak of abundance was observed (Zaika and Sergeeva 2009). Thus, our results showed that benthic protists were the constant and integral components of the meiobenthic communities under different conditions of the Black Sea. We detected an unevenness in spatial and bathymetric distribution of the protozoans and metazoans. Conducted studies on main taxa of meiobenthic communities at different depths and regions of the Black Sea allow us to give an overall picture of the distribution and the reaction of protists to the environment, establishing a link between different taxa and suggesting their important role in benthic ecosystems. As the water depth increases in the Black Sea, the biodiversity and abundance of macrobenthos decreases and the contribution of protozoa to benthic ecosystems becomes higher. Changes observed in the taxonomical composition and contribution of different taxa of protists to total meiobenthos depend on various factors. It can be explained by the specific characteristics of microdistribution and the availability of food resources, the number of macrobenthic fauna and their bioturbation activities, and changes in oxygenated conditions switching to anaerobic conditions containing a high concentration of hydrogen sulfide. We assume that some forms of protists are adapted to such conditions and can reach large quantities. Protozoans are significant components of benthic fauna since they are an important food source for bottom invertebrates. They are consumers of bottom bacteria, microalgae, terrestrial pollens and marine filamentous fungi; they may also prey upon unicellular and multicellular organisms as predators. Anaerobic bacteria decompose organic material under conditions of hydrogen sulfide in the Black Sea, and the final products are sulfates. These bacteria, in turn, are a food source of many benthic protists. In addition, the representatives of the above-mentioned groups are in the diet of many bottom invertebrates inhabiting various habitats (Figure 19). Obviously, they play a particular role in terms of oxygen depletion, so that they are able to enter into a symbiotic relationship (Figure 20) and included in the cycle of organic matter in benthic ecosystems at these environments, where life of other organisms are limited. Figure 20 shows the shells of a gromiid that is filled (preliminarily determined) with Cryptobiontes-like and Symbiodinium-like (Dinophyta) organisms.

144

Figure 19. Examples of food sources for benthic protists (scales = 50 µm) A-C: Soft-shelled foraminifera consuming bacteria and microalgae:

A- Vellaria pellucida Gooday 1992 B- Krymia fusiformis Anikeeva, Sergeeva and Gooday 2013

C- Bellarium rotundus Anikeeva, Sergeeva and Gooday, 2013 D-Е: Protists as food for multicellular predators:

D- Free-living Nematoda ingesting inner content of Gromia sp. E- Free-living Nematoda ingesting inner content of Psammophaga simplora

(allogromiids) F-G: Soft-shelled foraminiferans as predators:

F- Allogromiida g. sp. assimilating free-living nematodes G- Psammosphaeridae g. sp. assimilating free-living nematodes

H, I: Unidentified ciliate with diatoms

145

Figure 20. General form of Gromia sp. (A) examples for symbionts (scale bar =100

µm); (B-D) and their packaging in the shell of Gromia sp. (scale bar = 20 µm) These data point out the need to study the species diversity of protists inhabiting different habitats. Undoubtedly, it is clear that specific species of ciliates, foraminiferans and gromiids can be found in addition to widespread forms in these extreme habitats. Based on the data obtained, we conclude that the benthic protists are an important component of the bottom communities and it is necessary to investigate their role in the functioning of the benthic ecosystem of the Black Sea. In this context, we studied certain characteristics of protozoan communities (bathymetric and spatial distribution, abundance, and ratio of main taxa) as a component of whole meiobenthic assemblages. The existing and original data on main taxa of benthic protists and changes in their contribution to meiobenthic communities under conditions ranging from normoxia to anoxia was summarized for the first time for the Black Sea. Future research should focus on their species diversity and understanding the role of each species or group of

146

protists in benthic ecosystems, links between different taxa and their relationship with the environment. The presented data suggest that eukaryotic life is present in anoxic Black Sea waters and hydrogen-sulfide sediments which have been considered as a “toxic” environment. This result is consistent with the data about occurrence of anaerobic metabolism in many protozoans (Fenchel 1987; Massana and Pedros-Alio 1994). It is possible now to refuse the existing view about the lifelessness of the Black Sea at depths containing hydrogen sulfide by direct observations of active protozoans and metazoans, living in the deep-water hydrogen sulfide zone (Sergeeva et al. 2014). Additionally, two species of epibiont peritrich and suctorian ciliates living on metazoan benthic animals have recently been described at a depth of 253 m under anoxic conditions enriched with hydrogen sulfide pollution (Sergeeva and Dovgal 2014, 2016). Further studies will be necessary to reveal the factors regulating the abundance of benthic protists and metazoans at the oxic-anoxic interface of the Black Sea, to identify the origin of specific deep-water communities, their diversity and role. Under hypoxia, many aerobic organisms have difficulties in their life, so it leads to changes in the structure of benthic communities and a reduced biological diversity. Extreme conditions also lead to disappearance of a number of aerobic benthic forms. Our data suggest that the oxic/anoxic transition zone supports diversity and density of some meiofaunal taxa and also protists (Gromiida, Ciliophora and Foraminifera), which are the most abundant representatives of eumeiobenthos in samples from deeper, anoxic/sulfidic areas of the Black Sea. Acknowledgements The work has been conducted within the frameworks of the EU FP7 projects HYPOX 226213 in 2009-2010 and COCONET in 2011, EU ENV-BLACK SEA Project: MSFD (Marine Strategy Framework Directive) Guiding Improvements in the Black Sea Integrated Monitoring System – MISIS – Project No: 07.020400/2012/616044/SUB/D2 in 2012-2014, a joint project between TUBITAK (the Scientific and Technological Research Council of Turkey) and NASU (the National Academy of Sciences of Ukraine) (Project No. 108Y340) in 2009-2010, a project funded by the Expedition Council of the National Geographic Society in 2011 and the National Programs of NAS Ukraine and the RAS Russia (2013 and 2015-2016). We are grateful to Dr. Sofia A. Mazlumyan of IBSS for taking part in benthic sampling campaign. We are grateful to Professor Antje Boetius for providing an opportunity to participate in RV ‘M.S. Merian’ cruise 15/1 and to all participants of this cruise for their help. Robert Ballard, Michael L. Brennan and Tufan Turanlı are acknowledged for providing opportunity to take part in the Black Sea expedition of E/V Nautilus and thanks are due to the crew for sampling the material. We thank the anonymous reviewers for detailed comments that helped to improve the paper considerably. We sincerely thank all colleagues of IBSS NASU, IMBI RAS and Sinop University, Faculty of Fisheries, Department of Marine Biology for their cooperation in the material collecting and its processing. Special thanks to our colleagues L.F.

147

Lukyanova, Dr. Melek Ersoy Karaçuha and İbrahim Öksüz for processing of bottom sediment samples. This paper is dedicated to the memory of a brilliant Turkish scientist, Professor Dr. Murat Sezgin, who suddenly passed away at age 43 in July 28, 2017, in a deplorable traffic accident. He was the dean of the Faculty of Fisheries (Sinop University, Turkey), an active team leader of the benthos ecology group who made significant contributions to the studies of meiobenthos ecology in Turkey creating a fruitful cooperation with colleagues in other Black Sea countries. He will be missed by the marine scientific community of the Black Sea and Turkey. Karadeniz’in Meiobentik Komunitelerinde Protistler (Ciliophora, Gromiida, Foraminifera) Öz Bu çalışmanın amacı, Karadeniz’in oksik ve hidrojen sulfur içeren sürekli anoksik koşullar altındaki bentik bölgelerinde var olan ökaryotik yaşam hakkında görüş sunmaktır. Çalışmada tek hücreli ökaryotik organizmalar (protistler) üzerinde durulmuştur. Buna göre, Ciliophora, Gromiida ve Foraminifera’nın çalışılan tüm derinliklerde bulunduğu gösterilmiştir. Elde edilen sonuçlar, bentik protistlerin Karadeniz’in farklı çevresel koşulları altında meiobentik komunitelerin sürekli bileşenleri olduğunu göstermektedir. Meiobentos içerisinde protozoa ve metazoa komunitelerinin hem alansal hem de batimetrik dağılımının düzenli olmadığı tespit edilmiştir. Çalışma kapsamında, oksijenli koşullar ve hidrojen sülfür içeren oksijensiz koşullar (300 m derinliğe kadar) arasında değişim gösteren ortamlarda bentik protistlerin meiobentosa yaptığı katkı incelenmiştir. References Anikeeva, O.V., Gooday, A.J. (2016) Taxonomic composition and distribution of soft-walled monothalamid foraminifera in the area of Zernov’s Phyllophora Field (NW Black Sea). Marine Biology Research 12 (6): 640-646. Anikeeva, O.V., Sergeeva, N.G., Gooday, A.J. (2013) Two new genera and species of the monothalamous foraminifera from coastal waters of the Black Sea. Marine Biodiversity 43(4): 473-479. Azovsky, A. I., Mazei, Yu. A. (2003a) A conspectus of the Black Sea fauna of benthic ciliates. Protistology 3(2): 72–91. Azovsky, A. I., Mazei, Yu. A. (2003b) Ciliates of coarse ground on the Northeastern Black Sea coast. Zoologichesky Zhurnal 82(8): 899-912 (In Russian). Azovsky, A.I., Mazei, Yu. A. (2005) Distribution and community structure of benthic ciliates in the Northeastern part of the Black Sea. Protistology 4(2): 83-90.

148

Bougis, B. (1950) Mèthode pour l’etude quantitative de la microfaune de fonde marine (meiobenthos). Vie Milieu 1: 23-38. Brennan, M. L., Davis, D., Roman, C., Buynevich, I., Catsambis, A., Kofahl, M., Ürkmez, D., Vaughn, J. I., Merrigan, M., Duman, M. (2013) Ocean dynamics and anthropogenic impacts along the southern Black Sea shelf examined through the preservation of premodern shipwrecks. Continental Shelf Research 53: 89-101. Burki, F., Berney, C., Pawlowski, J. (2002) Phylogenetic position of Gromia oviformis Dujardin inferred from nuclear-encoded small subunit ribosomal DNA. Protistology 153: 251-260. Chislenko, L.L. (1961) On the existence of dimensional gap in the marine fauna of the littoral and sublittoral. In: Proceedings of Academy of Sciences of the USSR, New Series 37, pp. 431-435 (In Russian). Fenchel, T. (1987) Ecology of Protozoa: the Biology of Free-living Phagotrophic Protists. Springer-Verlag, Madison. 197 pp. Giere, O. (2008) Meiobenthology: The Microscopic Motile Fauna of Aquatic Sediments. Springer-Verlag, New York. 528 pp. Golemansky, V.G. (1999a) Lagynis pontica n. sp., a new monothalamous rhizopod (Granuloreticulosea: Lagynidae) from the Black Sea littoral. Acta Zoologica Bulgarica 51: 3-13. Golemansky, V.G. (1999b) Second observation of Paramphitrema pontica Valk.1970 (Rhizopoda: Gromiida) and supplement to their morphomentry. Acta Zoologica Bulgarica 51(1): 3-13. Golemansky, V.G. (2007a) Testate amoebas and monothalamous foraminifera (Protozoa) from the Bulgarian Black Sea coast. In: Biogeography and Ecology of Bulgaria (eds., V. Fet, A. Popov), Springer, Dodrecht, pp.555-570. Golemansky V.G. (2007b) Biodiversity and ecology of the Bulgarian Black Sea invertebrates. In: Biogeography and Ecology of Bulgaria (eds., V. Fet, A. Popov), Springer, Dodrecht, pp. 537-553. Gooday, A.J., Anikeeva, O.V., Pawlowski, J. (2011) New genera and species of monothalamous foraminifera from Balaclava and Kazach’ya Bays (Crimean peninsula, Black Sea). Marine Biodiversity 41: 481-494. Gooday, A.J., Anikeeva, O.V., Sergeeva, N.G. (2006) Tinogullmia lukyanovae sp. nov.- a monothalamous, organic-walled foraminiferan from the coastal Black Sea. Marine Biological Association of the United Kingdom 86: 43-49.

149

Gooday, A.J., Bernhard, J.M., Levin, L.A., Suhr, S.B. (2000) Foraminifera in the Arabian Sea oxygen minimum zone and other oxygen-deficient settings: taxonomic composition, diversity, and relation to metazoan faunas. Deep-Sea Research II 47: 25-54. Gooday, A. J., Holzmann, M., Guiard, J., Cornelius, N., Pawlowski, J. (2004) A new monothalamous foraminiferan from 1000 to 6300 m water depth in Weddell Sea: morphological and molecular characterization. Deep-Sea Research II 51: 1603-1616. Gooday, A., Hori, S., Todo, Y. (2002) Soft-walled, monothalamous benthic foraminiferans in the Pacific, Indian and Atlantic Oceans: aspects of biodiversity and biogeography. Deep-Sea Research Part I: Oceanographic Research Papers 52(1): 33-53. Higgins R.P., Thiel, H. (1988) Introduction to the Study of Meiofauna. Smithson. Inst. Press, Washington, D.C., London, 488 pp. Holtappels M., Janssen, F., Lichtschlag A., et al. (2012) Highly dynamic oxygen conditions and increased denitrification in Crimean Shelf sediments (Black Sea). EGU General Assembly, Vienna, Austria: 11447. Holtappels, M., Lichtschlag, A., Ihsan Y., Boetius, A.,Cagatay,N., Gaute, L., Kuypers, M. (2011) Coupled denitrification – sulfide oxidation stimulated by the inflow of oxic Mediterranean waters into the anoxic layers of the Black Sea. Geophysical Research Abstracts, 13, EGU2011-9125. Lichtschlag, A., Donis, D., Janssen, F., Jessen, G.L., Holtappels, M., Wenzhöfer, F., Mazlumyan, S., Sergeeva, N., Waldmann, C., Boetius, A. (2015) Effects of fluctuating hypoxia on benthic oxygen consumption in the Black Sea (Crimean Shelf). Biogeosciences Discuss. 12: 6445-6488. Longet, D., Burkli, F., Flakowski, J., Berney, C., Polet, S., Fahnri, J., Pawlowski, J. (2004) Multigene evidence for close evolutionary relations between Gromia and Foraminifera. Acta Protozoolica 43: 303-311. Luth, C., Luth, U. (1997) A benthic approach to determine long-term changes of the oxic/anoxic interface in the water column of the Black Sea. In: The Responses of Marine Organisms to Their Environments, Proceedings of the 30th European Marine Biology Symposium, Southampton Oceanography Centre, Southampton, U.K., (eds., L.E. Hawkins, S. Hutchinson), pp. 231-242. Luth, U., Luth, C. (1998) Benthic meiofauna and macrofauna of a methane seep area south-west of the Crimean Peninsula, Black Sea. In: (eds.) MEGASEEPS – Methane Gas Seeps Exploration in the Black Sea, vol 14, (eds., U. Luth, C. Luth,

150

H. Thiel), Berichte aus dem Zentrum für Meeres- und Klimatoforsch, Hamburg, pp.113-126. Mare, M.F. (1942) A study of a marine benthic community with special reference to the microorganisms. Marine Biological Association of the United Kingdom 25: 517-554. Massana, R., Pedros-Alio, C. (1994) Role of anaerobic ciliates in planktonic food webs: abundance, feeding, and impact on bacteria in the field. Applied and Environmental Microbiology 60(4): 1325-1334. Matz, M.V., Frank, T. M., Marshall, N. J., Widder, E. A., Johnsen, S. (2008) Giant deep-sea protist produces bilaterian-like traces. Current Biology 18: 1849-1854. Mikhalevich, V.I. (1968) Phylum Protozoa. Class Sarcodina. In: The Determinant of the Fauna of the Black and Azov Seas, I. (ed., V.A. Vodyanitsky), Naukova Dumka, Kiev, pp. 9-21 (In Russian). Nyholm, K.G., Gertz, I. (1973) To the biology of the monothalamous foraminifer Allogromia marina sp. nov. Zoon. 1: 89-93. Öksüz, İ., Sezgin, M., Kırcı-Elmas, E., Gündoğdu, A. (2011) Seasonal changes of Ammonia (Foraminifera) inhabiting the southern Black Sea (Sinop Bay, Turkey). In: Proceedings of the 3rd Bi-annual Black Sea Scientific Conference, Odessa, Ukraine, p. 62. Öksüz, İ., Sezgin, M., Meriç, E. (2013) Live agglutinated foraminifera from surface sediments of Sinop coasts (Southern Black Sea). In: Proceedings of the 4th Biannual Black Sea Scientific Conference: Black Sea-Challenges Towards Good Environmental Status, Constanta, Romania, pp. 90-91. Öksüz, İ., Sezgin, M., Ürkmez, D. (2016a) The use of living benthic foraminifera as ecological indicators in Sinop Bay (Black Sea, Turkey). In: Proceedings of Meioscool 2016, Plouzane, France, pp. 47-48. Öksüz, İ., Sezgin, M., Ürkmez, D., Ersoy-Karaçuha, M. (2016b) The relationship between sediment grain size and the distribution of living benthic foraminifera in Sinop Bay (Black Sea, Turkey). In: Proceedings of the 16th International Meiofauna Conference, Heraklion, Crete/Greece, pp.104-105. Pawlowski, J., Fahrni, J.F., Brykczynska, U., Habura, A., Bowser, S.S. (2002) Molecular data reveal high taxonomic diversity of allogromiid foraminifera in Explorers Cove (McMurdo Sound, Antarctica). Polar Biology 25: 96-105.

151

Pereyaslavtseva, S.M. (1886) Protozoa of the Black Sea. In: Note Novorossiysk Society of Naturalists, Odessa X, 39 p. (In Russian). Piña-Ochoa, E., Høgslund, S., Geslin, E., Cedhagen, T., Revsbech, N.P., Nielsen, L.P., Schweizer, M., Jorissen, F., Rysgaard, S., Risgaard-Petersen, N. (2010) Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida. Proceedings of the National Academy of Sciences of the United States of America 107(3): 1148-1153. Polikarpov, I.G., Saburova, M.A., Manzhos, L.A., Pavlovskaya, T.V., Gavrilova, N.A. (2003) Biologic diversity of the microplankton in near shore Black Sea waters (Sevastopol area, 2001-2003). In: Modern State of Biodiversity in Crimea Nearshore Waters, (eds., V.N. Eremeev, A.V. Gaevskaya), Ecosi-Hydrophysika, Sevastopol, pp.16-42. Revkov, N.K., Sergeeva, N.G. (2004) Current state of the zoobenthos at the Crimean shores of the Black Sea. In: International Workshop of the Black Sea Benthos, 18-23 April 2004, (eds., B. Ozturk, V.O. Mokievsky, B. Topaloglu), TÜDAV, Istanbul, pp. 189-217. Rothe, N., Gooday, A.J., Cedhagen, T., Fahrni, J., Hughes, J. A., Page, A., Pearce, R.B., Pawlowski, I.J. (2009) Three new species of deep-sea Gromia (Protista, Rhizaria) from the bathyal and abyssal Weddell Sea, Antarctica. Zoological Jour. of the Linnean Society 157: 451-469. Rothe, N., Gooday, A.J., Cedhagen, T., Hughes, J.A. (2011) Biodiversity and distribution of the genus Gromia (Protista, Rhizaria) in the deep Weddell Sea (Southern Ocean). Polar Biology 34: 69-81. Sazhin, A.F., Subkov, M.V., Drabkova, V.G. (1991) Heterotrophic microplankton of lower part of oxygenated waters during spring 1988. In: Variability of the Black Sea Ecosystem: Natural and Anthropogenic Factors, (ed., M.E. Vinogradov), Nauka, Moscow, pp. 204-210. Sergeeva, N.G. (2003) Meiobenthos of the Crimean fluffy bottom bed. In: Modern Condition of Biological Diversity in Near-shore Zone of the Crimea (the Black Sea sector), (eds., V.N. Eremeev, A.V. Gaevskaya), Ekosi-Gidrophizika, Sevastopol, pp. 248-251 (In Russian). Sergeeva, N.G. (2004) Structure and distribution of meiobenthos in the region of methane gas seeps from the Black Sea bottom. Hydrobiology Journal 40(6): 45-56.

152

Sergeeva, N.G. (2013) Meiobenthos. In: Geological, Geo-environmental, Hydro-acoustic and Hydroecological Investigations of the Shelf and Continental Slope of the Ukrainian Sector of the Black Sea, (ed., A.Y. Mitropolsky-K.), pp.119-124. Sergeeva, N.G. (2016) Soft-walled benthic Foraminifera of the Black Sea and Azov Sea. In: Proc. All-Russian Scien.-Pract. Conf. Marine Biological Research: Achievements and Perspectives, Sevastopol, (ed., A.V. Gaevskaya), 2, pp. 141-145. Sergeeva, N.G., Anikeeva, O.V. (2014) Soft-walled foraminifera under normoxia/hypoxia conditions in the shallow areas of the Black Sea. Chapter IX. In: Foraminifera. Aspects of Classification, Stratigraphy, Ecology and Evolution, (ed., M. Dan Georgescu), pp. 227-247. Sergeeva, N.G., Anikeeva, O.V., Gooday, A.J. (2010) Soft-shelled monothalamous foraminifera from the oxic/anoxic interface (NW Black Sea). Micropalaeontology 56, 393-407. Sergeeva, N.G., Dovgal, I.V. (2014) First finding of epibiont peritrich and suctorian ciliates (Ciliophora) on oligochaetes and harpacticoid copepods from the deep-water hypoxic/anoxic conditions of the Black Sea. Ecologica Montenegrina 1(1): 49-54. Sergeeva, N.G., Dovgal, I. (2016) Loricophrya bosporica n. sp. (Ciliophora, Suctorea) epibiont of Desmoscolex cf. minutus (Nematoda, Desmoscolecida) from oxic/anoxic boundary of the Black Sea Istanbul Strait’s outlet area. Zootaxa 4061(5): 596-600. Sergeeva, N.G., Gooday, A.J., Mazlumyan, S.A., Kolesnikova, E.A, Lichtschlag, A., Kosheleva, T.N., Anikeeva, O.V. (2012a) Meiobenthos of the oxic/anoxic interface in the Southwestern region of the Black Sea: abundance and taxonomic composition. In: Anoxia: Paleontological Strategies and Evidence for Eukaryotes Survival, (eds., A.V. Altenbach, J.M. Bernhard, J. Seckbach), Springer, pp. 369-401. Sergeeva, N.G., Gulin, M.B. (2007) Meiobenthos from active methane seepage area in NW Black Sea. Marine Ecology 28: 152-159. Sergeeva, N.G., Kolesnikova, E.A. (1996) Results of the study of the Black Sea meiobenthos. Ecologiya Morya 45: 54-62 (In Russian). Sergeeva, N.G., Kolesnikova, E.A., Kharkevich, Kh.O., Melnikov, V.V. (2012b) Taxonomic diversity of meiobenthos in the soft-bottom sediments of the Karkinitsky Gulf (NW, Black Sea, In: Extended Abstracts of the II International

153

Scientific-practical Conference "Biodiversity and Steady Development". Simferopol (Ukraine), (ed., N.V. Bagrov), pp. 35-39 (In Russian). Sergeeva, N.G., Mazlumyan, S.A. (2013) Foraminifera under conditions of hypoxia/anoxia at the Istanbul Strait’s (Bosporus) outlet area to the Black Sea. In: Proceedings of the IGCP- 610 First Plenary Meeting and Field Trip, Tbilisi, Georgia, pp. 122-125. Sergeeva, N.G, Mazlumyan, S.A. (2015) Deep-water hypoxic meiobenthic protozoa and metazoa taxa of the Istanbul Strait’s (Bosporus) outlet area of the Black Sea. Ecologica Montenegrina 2(3): 255-270. Sergeeva, N.G., Mazlumyan, S. A., Cagatay, N., Lichtschlag, A. (2013) Hypoxic meiobenthic communities of the Istanbul strait’s (Bosporus) outlet area of the Black Sea. Turkish Journal of Fisheries and Aquatic Sciences 13: 33-41. Sergeeva, N.G., Mazlumyan, S.A., Lichtschlag, A., Holtappels, M. (2014) Benthic protozoa and metazoa living under anoxic and sulfide conditions in the Black Sea: direct observations of actively moving Ciliophora and Nematoda. International Journal of Marine Science 4(42): 1-11. Sergeeva, N.G., Zaika, V.E. (2008) Ciliophora in hydrogen sulfide zone of the Black Sea. Marine Ecological Journal 7(1): 80-85. (In Russian, with summary in English). Sergeeva, N.G., Zaika, V.E. (2013) The Black Sea meiobenthos in permanently hypoxic habitat. Acta Zoologica Bulgarica 65(2): 139-150. Sergeeva, N.G., Zaika, V.E., Anikeeva, O.V. (2015) On overview on distribution and abundance of meiobenthic foraminifera in the Black Sea. Ecologica Montenegrina 2(1): 124-141. Sergeeva, N.G., Zaika, V.E., Lichtschlag, A. (2008) Preliminary data on the presence of diverse benthic ciliate species in deep anoxic Black Sea. In: 5th international conference “Environmental Micropaleontology, Microbiology and Meiobenthology”, Chennai, India, pp. 279-282. Soltwedel, T. (2000) Metazoan meiobenthos along continental margins: a review. Progress Oceanography 46: 59-84. Sorokin, Y.I., Sorokina, O.V. (2008) Primary production and bacterioplankton dynamics in the Black Sea during the cold season. Marine Ecol. J. 7(2): 65-75.

154

Stoeck, T., Taylor, G.T., Epstein S.S. (2003) Novel eukaryotes from the permanently anoxic Cariaco Basin (Caribbean Sea). Applied Environmental Microbiol. 69(9): 5656-5663. Stoeck, T., Zuendorf, A., Breiner, H.W., Behnke, A. (2007) A molecular approach to identify active microbes in environmental eukaryote clone libraries. Microb Ecol 53: 328-339. Temelkov, B.K. (2008) Ecological characteristics of the foraminiferal fauna (Protozoa: Foraminifera) of the Bulgarian South Black Sea area. Acta Zoologica Bulgarica 2: 275-282. Temelkov, B.K. (2010) Guanduella podensis n. sp. and Psammosphaera sp. - foraminifera from the Bulgarian Black Sea coast. In: Biotechnol. and Biotechnol. EQ. Second Balkan Conference on Biology, Plovdiv, 50 years University of Plovdiv, pp. 618-622. Temelkov, B.K., Golemansky, V.G., Todorov, M.T. (2006) Updated check-list of the recent foraminifera from the Bulgarian Black Sea coast. Acta Zoologica Bulgarica 58(1): 17-36. Ürkmez, D., Brennan, M., Sezgin, M., Bat, L. (2015) A brief look at the free-living Nematoda of the oxic/anoxic interface with a new genus record (Trefusia) to the Black Sea. Oceanological and Hydrobiological Studies 44(4): 539-551. · Ürkmez, D., Sezgin, M., Ersoy Karaçuha, M., Öksüz, İ., Katağan, T., Bat, L., Dağlı, E., Şahin, F. (2016a) Within-year spatio-temporal variation in meiofaunal abundance and community structure, Sinop Bay, the Southern Black Sea. Oceanological and Hydrobiological Studies 45(1): 55-65. Ürkmez, D., Sezgin, M., Ersoy Karaҁuha, M., Öksüz, I. (2016b) Meiobenthic assemblages from the southwestern coast of the Black Sea, İgneada (Turkey). Biologia 71/9: 1017-1026. Valkanova, Ch. (1964) Über eine neue Rhizopodengattung–Rhabdogromia. Zoologischer Anzeiger 172(4): 258-261. Valkanov, A. (1970) Beitrag zur Kenntnis der Protozoen des Schawarzen Meeres. Zoologischer Anzeiger 184(3/4): 241-290. Vorobyova, L.V. (1999) Meiobenthos of Ukrainian Shelf of the Black and Azov Seas. Naukova Dumka, Kiev, 300 pp. (In Russian). Wylezich, C., Jürgens, K. (2011) Protist diversity in suboxic and sulfidic waters of the Black Sea. Environmental Microbiology 13(11): 2939-2956.

155

Yanko, V. (1990) Stratigraphy and paleogeography of marine Pleistocene and Holocene deposits of the southern seas of the USSR. Memorie della Società Geologica Italiana 44: 167-187. Yanko, V., Troitskaya, T. (1987) Late Quaternary foraminifera of the Black Sea. Nauka, Moscow, 111 pp. (In Russian). Yanko, V.V., Vorobyova, L.V. (1991) Foraminifera of the Bosphorus and the Black Sea. Ekologiya Morya 39: 47-51 (In Russian). Zaika, V.E. (2008) Is there animal life at the Black sea great depths? Marine Ecological Journal 7: 5-11 (In Russian). Zaika, V.E., Sergeeva, N.G. (2009) The vertical distribution of the deep-water ciliates in the Black Sea. Marine Ecological Journal 8(1): 32-36. Zaitsev, Y.P. (2008) An Introduction to the Black Sea Ecology. Smil Edition and Publishing Agency Ltd., Odessa, Ukraine, 228 pp. Zaitsev, Y.P., Polikarpov, G.G. (2008) Recently discovered new biospheric pelocontour function in the Black Sea reductive bathyal zone. Journal of the Black Sea/ Mediterranean Environment 14(3): 151-165. Zaitsev, Y.P., Polikarpov, G.G., Egorov, V.N., Alexandrov B.G., Garkusha O.P., Kopytina N.I., Kurilov, A.V., Nesterova, D.A., Nidzvetskaya, L.M., Nikonova, S.E., Polikarpov I.G., Popovichev, V.N., Rusnak, E.M., Stokozov, N.A., Teplinskaya, N.G., Teren`ko, L.M. (2007) Accumulation of the remnants of oxybiotic organisms and a bank of living spores of higher fungi and diatoms in bottom sediments of the hydrogen-sulphide bathyal zone of the Black Sea. Rep. Nat. Acad. Sci. Ukraine 7: 159-164. Zaitsev, Y.P., Polikarpov, G.G., Egorov, V.N., Gulin, S.B., Kopytina, N.I, Kurilov, A.V., Nesterova, D.A., Nidzvetskaya, L.M., Polikarpov, I.G., Stokozov, N.A., Teplinskaya, N.G., Teren`ko, L.M. (2008) Biological diversity of oxybionts (in the form of viable spores) and anaerobes in bottom sediments of the hydrogen sulfide bathyal zone of the Black Sea. Rep. Nat. Acad. Sci. Ukraine 5: 168-173.