Diatoms in Kamchatka’s Hot Spring Soils

: Diatoms inhabiting terrestrial habitats that are a ﬀ ected by thermal activity remain poorly studied, despite signiﬁcant interest in the biodiversity of hot springs. The Kamchatka peninsula is characterized by the presence of 30 active volcanoes associated with hydrotherms. Our study involved a survey of diatom diversity in soils around the Malki, Upper Paratunka, and Dachnie thermal springs on the Kamchatka peninsula. A total of 49 diatom taxa were found. The genera Pinnularia , Planothidium , Fragilariforma , Epithemia , Halamphora , Gomphonema , Nitzschia , Aulocoseira , Sellaphora , Surirella , and Navicula were the most common. Pinnularia cf . subcapitata and Planothidium lanceolatum were dominant in all springs. Diatom communities in the soils near the thermal springs included both aquatic and terrestrial species, which may reﬂect the transitional nature of habitats at the borders of hot springs and soils. To gain a better understanding of the diversity of diatom communities in soils near thermal springs, broader worldwide studies are necessary.


Introduction
The ecology and biodiversity of microorganisms inhabiting extreme habitats have attracted significant attention in recent years because they may shed light on the origins of life on Earth and potentially on other planets [1][2][3][4][5][6][7][8][9][10]. Terrestrial hydrothermal systems (including hot or thermal springs heated by geothermal groundwater discharging at the land surface) are of particular interest and likely represent the oldest ecosystems on the Earth s surface [11,12].
Hot spring water is characterized by constantly elevated water temperatures that exceed ambient ground temperatures and sometimes reach boiling point. In addition to its high temperature, the water in these springs is rich in minerals; therefore, such springs are often regarded as mineral springs [1]. Only limited groups of organisms can survive in such harsh environments, and diatoms are one of these groups. Some researchers have investigated the biodiversity of diatom algae in hot springs [13][14][15][16][17]. Achnanthidium exiguum (Grunow) Czarnecki and Achnanthidium saprophilum (H.Kobayashi & Mayama) Round & Bukhtiyarova are the most distributed species in diatom communities in the thermo-mineral springs of Galicia, NW Spain [15]. Diatomella balfouriana Greville, Achnanthidium exiguum, and Anomoeoneis sphaerophora Pfitzer are the dominant species in hot springs in northern Thailand [17]. Pinnularia Ehrenberg was commonly found in hot springs in New Zealand, with Navicula Bory and Anomoeoneis Pfitzer being the distributed taxa common to Kenyan hot springs [13]. However, very little is known about diatoms inhabiting the soils surrounding the The Malki thermal springs (area 1) are situated on the left bank of the river Kluchevka (N 53 • 19 20 E 157 • 32 18 ). There are six groups of vents at the small thermal site, which is covered by gravel [25].
The Upper Paratunka thermal springs (area 2) (N 52 • 49 20 E 158 • 09 28 ) are situated on the left slope of the Paratunka river valley, 2.5 km above the river Karimshina confluence and ca. 16 km south-west of Paratunka and the village Paratunka. The springs are located at an 80 m altitude in a small fold of the hill of Goriachaya s slope that separates the valleys of Paratunka and Karimshina [25].
The Dachnie thermal springs (area 3) comprise the largest group in the Mutnovskie springs, which are situated in the Mutnovski volcano valley. The Mutnovski geothermal deposit is one of the largest in the world [20]. The Dachnie hot springs are located in the fumarole field, with discharging gases heating the cold stream waters. The physical and chemical characteristics of the hot springs studied are summarized in Table 1 [20,21,26,27]. Table 1. Physical and chemical characteristics of the hot springs studied [20,21,26,27].

Studied Area
Temperature, • C pH Total Mineralization, g/L

Samples Collections
Eight composite wet soil samples (each from five 5 cm 3 soil blocks) were collected in August 2009 from eight sites ( Table 2). Samples were taken from soil near the springs with metal cylinders according to the standard method for sampling terrestrial diatoms [28], and then were dried in the shade and put into sterile plastic bags. The soil samples were treated according to previously described methods [28]. In one case (site 6), mud samples from the thermal bath were collected. In the laboratory, diatom cells were extracted from 1 g of each sample by rinsing the soil with 10 mL of deionized water and 10 mL of nitric acid. This mixture was boiled to a double volume reduction for water evaporation and, after cooling to room temperature, was washed four times with deionized water. To minimize damage during acid removal and washing of the diatom frustules, they were settled by sedimentation instead of centrifugation. Suspensions of cleaned frustules were dried on glass coverslips and mounted on permanent slides using Naphrax following standard procedures [29]. Valves were examined in a bright field (BF) at × 1000 magnification using Ergaval and Zeiss Axio Imager A2 light microscopes (Carl Zeiss, Jena, Germany) equipped with oil immersion objectives with differential interference contrast (DIC) and Axio Cam MRсcameras. Soil samples and permanent slides were deposited in the Bashkortostan Collection of Algae and Cyanobacteria (BCAC) (WDCM 1023, Ufa, Russia). For species identification, relevant references [30][31][32][33][34] and recent publications [35][36][37][38][39] were used. For descriptions of the ecology of species, standard flora and related literature were used [40][41][42][43][44][45][46][47].
For floristic similarity estimation purposes, the Sørensen-Czekanowski coefficient was used: where К is the floristic similarity coefficient, с is the number of species common to the two floras, a is the number of species in the flora of the first area, and b is the number of species in the flora of the second area [48]. For the comparison and visualization of the similarities between diatom communities from the Malki, Upper Paratunka, and Dachnie thermal springs, we used a Venn diagram [49,50]. The Venn diagram was created using the web-based InteractiVenn tool [51].
The abundance of species was estimated on a coverslip according to the method of R.R. Kabirov [52]. During microscopic observation, five transects were examined: four along the perimeter and one through the center. The abundance of algae was assessed on a 15-point scale on a slide: 1 point, 1-3 diatom valves found on the transect (1-15 on a slide); 2 points, 4-10 diatom valves (4-80 on a slide); 3 points, more than 10 diatom valves (more than 50 on a slide). After observation of five transects, the sum of the abundance points was calculated. Thus, the minimum abundance was 1 point (1-15 on a slide) and the maximum was 15 points (more than 50 on a slide).
Representatives from the class Bacillariophyceae were the most diverse and accounted for 48 diatom taxa (98% of the total). The genera Pinnularia and Eunotia (5 species/taxa each), Navicula (4 species), and Encyonema and Gomphonema (3 species/taxa each) contributed the most assemblages. The majority of the taxa identified have been described in the literature as benthic (18 species, 37% of the total species), indifferent to salinity (31 species, 63%), and alkaliphilic (24 species, 49%). Widely distributed and cosmopolitan species prevailed (38 species, 78%).
In all samples studied, we found representatives of the genera Encyonema, Navicula, Pinnularia, and Planothidium. The number of species ranged from 3 to 12. The highest number of species (12) was identified in sample 2 collected from the Malki thermal springs soils, and the lowest (3) was found in sample 3 from the Upper Paratunka thermal springs. An average of ±3 species were identified in the samples. The diatom flora of the Malki hot springs soils (27 taxa, 55%) was the most diverse, while at Dachnie thermal springs, 25 taxa (51%) were found, and 16 taxa (32%) were found in the soils from the Upper Paratunka springs. Only five species (Encyonema silesiacum, Navicula cincta, Pinnularia microstauron ( Figure 3V), P. cf. subcapitata ( Figure 3W), and Planothidium lanceolatum) were found in all three hot spring systems (Table 3, Figure 4), which resulted in low similarities between the local flora. The samples from Malki and Upper Paratunka thermal springs showed maximum similarities ( Figure 4) and values of the Czekanowski-Sørensen coefficient (41.9%). The similarity between the Dachnie and Upper Paratunka thermal springs was 39.0% (Figure 4). The Malki and Dachnie hot springs were less similar (26.9%) (Figure 4). Epithemia operculata ( Figure 3G), Fragilariforma virescens, Halamphora normanii, Nitzschia hantzschiana ( Figure 3R), Pinnularia cf. subcapitata and Planothidium lanceolatum were the most abundant in the diatom communities in soils associated with hot springs. These species had the maximum abundance scores (14-15 points) ( Table 3).

Discussion
In the diatom communities of the Malki, Upper Paratunka, and Dachnie thermal springs, representatives of the genera and species common to such environments were identified ( Table 3).
The wide distribution of the genus Pinnularia (Table 3) in the thermal springs has been discussed in previous studies [13]. Pinnularia is abundant in slightly acidic freshwater bodies [42], and also was typical for diatom communities in terrestrial ecosystems [40].
In all of the springs studied, at pH levels from 3.5 to 9.4, Pinnularia cf. subcapitata dominated (Table 3). Pinnularia subcapitata belongs to acidophilic group of species (Table 3), and Pinnularia subcapitata var. elongata has been identified in samples from Bekanbeushi Wetland (Eastern Hokkaido, Japan) in a territory of the Kuril-Kamchatka island arc [53]. Like most cosmopolitan species, this taxon is possibly adapted for life in a wide range of ecological conditions.
Another representative of genus Pinnularia-P. microstauron, which was abundant in all springs studied (Table 3), has been recorded at pH 6.7-7.0 and temperature 40 • C in Stolbchaty Cape hydrothermal springs (Kunashir Island) [54]. Thus, our results indicate that P. microstauron exists in wider ranges of pH and temperature than has been reported previously for the thermal springs of the Kuril-Kamchatka island arc.
The results of our study confirm that P. borealis also belongs to the ecologically resistant taxa. In the samples we obtained from the Upper Paratunka and Dachnie springs, this diatom was found at pH 3.2-9.4 and temperature 30.0-69.5 • C. In a previous study of diatoms from hydrothermal springs on Kunashir Island, P. borealis was identified in Stolbchaty Cape springs and in Alekhin Bay springs at pH 4-5, 50-55 • C [54]. This taxon has also been found in the sulfidic Cuntis hot springs in Galicia (NW Spain), which are characterized by deep hydrothermal flows and temperatures of 48-54 • C [15].
The wide distribution of P. borealis, P. subcapitata, and P. microstauron in terrestrial habitats [40] confirms the ecological resistance of these species and explains their frequency in thermal spring ecotones.
Planothidium lanceolatum, which dominated in all of the springs we investigated, belongs to the most common and abundant species of diatoms in the springs and streams of Türkmen Mt., Turkey [36]. This species was found as a dominant or abundant species in ten Sakhalin and Kuril Islands hot springs at temperatures from 24 to 71 • C, and has been characterized as indifferent to the salinity rheophilic diatom in the Sakhalin hydrothermal springs [54]. Planothidium lanceolatum was previously found to be dominant in alpine springs with cold water, low conductivity, and slightly alkaline pH, and also in the Font de Bleix thermo-mineral spring in Auvergne, France, which has high concentrations of nitrates and calcium [17]. In addition, this diatom has been identified in hot spring diatoms in northern Thailand. A number of ecological factors, such as concentration of silicon dioxide, pH, conductivity, water temperature, and total hardness influence the relative abundance of P. lanceolatum [16].
We discovered that Encyonema silesiacum was relatively frequent in diatom communities in the Malki, Upper Paratunka, and Dachnie thermal springs. This species has been found in the Stolbchaty Cape springs at pH 6.7-7.0 and temperature 40 • C (Kunashir Island) [54]. Encyonema silesiacum has also been observed in Sardinia, Italy, in the San Saturnino thermo-mineral spring, which has a medium-high conductivity and low nutrient content [17]. Our findings indicate a greater thermal and pH amplitude for E. silesiacum than has previously been recorded [54]. Similar to representatives of the genus Pinnularia, this taxon is cosmopolitan in terrestrial ecosystems [40].
Another species that was found in all of the thermal springs we studied was Navicula cincta. Navicula is one of the most common genera in springs [55][56][57][58]. Navicula species related to N. cincta are rare in springs, but many new species from this group have been described [58]. Navicula is a benthic-epiphytic, alkaliphilous species with cosmopolitan distribution [46], and is also common in thermo-mineral springs [15,17]. Navicula cincta prefers mostly eutrophic, electrolyte-rich waters and periodically wet habitats [58], and it is widely distributed in soils [40], so the discovery of this species in soils near hot springs was expected.
The dominance of Fragilariforma virescens and Epithemia operculata in the diatom communities in the Malki and Upper Paratunka hot springs at 39.5-69.5 • C and pH 3.5-8.3 indicates the environmental plasticity of these species. Fragilariforma virescens prefers freshwater littoral-benthic habitats and is distributed in arcto-alpine regions [46]. This diatom belongs to the characteristic species found in algal communities in an ephemeral sandstone stream in the Bohemian Switzerland National Park, Czech Republic [59]. Epithemia operculata has been recorded in both freshwater [60] and terrestrial habitats [40].
Of particular interest in this investigation is our finding of the typical freshwater taxa Aulacoseira distans. This may indicate the transitional characters of the studied habitats between aquatic and terrestrial environments. Aulacoseira is one of the most distributed freshwater-centric diatom genera [44]. In previous studies of Kunashir Island, A. distans var. distans was recorded in Alekhin Bay hydrothermal springs [55].
The abundance of Nitzschia hantzschiana, Halamphora normanii, and Gomphonema sp. found in our study confirms existing data regarding the distribution these taxa in thermo-mineral springs [15,17,54].
The Gomphonema species were abundant in the sulfureous spring on the coast of Alekhin Bay on Kunashir Island [54]. Gomphonema is also member of diatom communities in the Outariz thermo-mineral springs in Galicia, NW Spain [15], and have frequently been found in thermo-mineral springs in Auvergne and Sardinia [17].
Nitzschia hantzschiana has been recorded in Guitiriz thermo-mineral springs in Galicia, NW Spain, in conditions of lower conductivity and temperature [15]. Halamphora normanii is a common species in the Auvergne and Sardinia thermo-mineral springs [17]. Both taxa are common in terrestrial ecosystems [40].
In this work, Diatoma moniliformis and Surirella brebissonii were abundant in the Malki hot springs in low pH conditions (Table 3, Figure 4), but according to existing literature, these taxa are alkaliphilic. However, we studied soil and mud samples, the pH of which could differ from the pH of water in the thermal springs. Diatoma moniliformis has been recorded in the alluvial Middle Fork Flathead River within the Nyack Flood Plain, Montana, at downwelling and neutral exchange sites [61]. Surirella brebissonii has been recorded in hydrothermal springs on Sakhalin Island [54].
In our study, Eunotia curtagrunowii and Odontidium hyemale were recorded in the Malki and Dachnie thermal springs at 30-65.9 • C and pH 3.2-8.3. The diatoms from the genus Eunotia prefer soft and acidic water bodies [41]. Eunotia curtagrunowii has also been recorded in soils in the Low Beskids, Poland [47], and it may inhabit the soils near the thermal springs that we studied. Odontidium hyemale has been found in the Tovare rheocrenic spring in the Brenta mountain range in the Western Dolomites [62].
Sellaphora rectangularis and Navicula leptostriata are abundant in the Upper Paratunka thermal springs diatom communities at 39.5 • C and pH 8.3 [41]. According to published data, Sellaphora is a widespread genus in alkaline waters and brackish waters close to a neutral pH. Navicula leptostriata has been recorded in freshwater bodies on Bering Island at pH 5.2-9.6 (Kamchatka) [38].
In the present work, Eunotia exigua, Frustulia krammeri, Caloneis lancettula, and Neidium dubium were recorded in the Dachnie thermal springs at 30.0-50.0 • C and pH 3.2-9.4. Eunotia exigua is typical in hot springs with a low pH [54,63]. Frustulia krammeri, like Navicula leptostriata, has been found in Bering Island freshwater habitats at neutral and alkaline pH [38]. Caloneis lancettula is a common diatom in freshwater bodies and is a good indicator of an elevated trophic level [34]. It has been found in streams and springs in the territory of Turkey [36]. The genus Neidium is common in neutral and slightly acidic waters, and this genus is characterized by a low abundance [42].
The species composition of diatoms that we found in soils associated with the Malki, Upper Paratunka, and Dachnie thermal springs of Kamchatka was similar to the species composition of periphyton communities described in the same springs in previous studies [20,21]. In these habitats, boreal species that are adapted to high temperatures prevail [21]. Surprisingly, in the aquatic and terrestrial habitats of the studied area, thermophilic species did not play significant roles in this work. Hannaea arcus, Encyonema silesiacum, Gomphonema sp., Planothidium lanceolatum, Frustulia krammeri, and others appear to be well adapted to a wide range of temperatures and habitats, as they have been recorded in a wide range of cold water habitats and hot springs in Kamchatka, Kuril, and Sakhalin Islands in previous works [54] and in our study.
In our study, dominance of cosmopolitan diatom taxa was the basic feature of the soils near the Malki, Upper Paratunka, and Dachnie thermal springs. The same results were obtained for diatom flora of the Malki, Upper Paratunka [21], and Dachnie thermal springs [20] in previous studies. The springs we investigated are characterized by a range of abiotic factors such as high temperature, mineralization, and soil moisture, which affect the species diversity of diatoms. The prevalence of diatom species that are benthic, indifferent to salinity, or alkaliphilic in the soils near the Malki, Upper Paratunka, and Dachnie thermal springs might be a consequence of environmental conditions. The diatom flora of the hot springs in Sakhalin Island have the same characteristics [54]. The diatoms were characterized by predominance of taxa that are benthic, indifferent to salinity, or acidophilic. These features may reflect the peculiarities of the ecological conditions in the thermal springs of the Kuril-Kamchatka volcanic arc.
The territory that we studied is a kind of ecotone between aquatic and terrestrial habitats; therefore, typical terrestrial species should be expected in soils associated with the hot springs. We found numerous soil taxa such as Gomphonema brebissonii, Gomphonema sp., Hantzschia cf. abundans, Navicula cincta, N. minima, Pinnularia borealis, P. cf. subcapitata, and Tabellaria flocculosa, together with typical aquatic species like Aulacoseira distans, Decussiphycus placenta, Hannaea arcus, Nitzschia recta, Odontidium hyemale, Surirella brebissonii, and others. Such aerophilic diatoms, which are also regarded as common in soil like Pinnularia borealis, have also been observed in water samples from the Malki, Upper Paratunka [21], and Dachnie thermal springs [20]. In soil samples, however, the number of terrestrial species is more numerous. This phenomenon can be explained by the transitional nature of the habitat at the border of the hot springs and soils. To gain a better understanding of the diversity of diatom communities in soils near thermal springs, broader worldwide studies are necessary.