New Records for Tardigrada Species from the High Mountain Region in Turkey (Mount Verçenik, Rize)

Phylum Tardigrada, which has been quite a little studied both around the world and Turkey, belongs to members of a metazoan. In this study eight moss and lichen samples were collected and investigated from a variety of elevations of the Verçenik Mountain in Rize (Turkey). In total 62 specimens, 11 exivium and 10 eggs of tardigrades were found. Five species were identified, and they belong to genera: Acanthechiniscus, Pseudechiniscus, Macrobiotus, Paramacrobiotus and, Ramazzottius. Three of them are new records for Turkish fauna i.e. Acanthechiniscus victor (Ehrenberg, 1853), Pseudechiniscus ramazzottii ramazzottii Maucci, 1952 and Macrobiotus spectabilis Thulin, 1928. Including these three species, the tardigrade fauna of Turkey increased to 54. In this paper, we present the previous study a full list of the known high mountain and alpine tardigrade species with their localities. All specimens are deposited in the Aquatic Animals Research Laboratory at Ankara University.


Introduction
Tardigrades were first observed by German scientist Johann A. E. Goeze in 1773. Three years later Italian scientist Lazzaro Spallanzani (1776), gave them a name Tardigrada (meaning "slow stepper"). From the first discovery to date 1246 tardigrade species have been reported around the world (Degma and Guidetti 2007; Vicente and Bertolani 2013; ).
Very limited papers have been published on Turkish Tardigrada up to now (Kaczmarek et al. 2012). However, Maucci conducted quite intensive studies on Turkish tardigrade in 1973, 1978, 1979, 1981. Later, only Morgan (1977, Binda (1988), Kaczmarek et al. (2012) and Ürkmez et al. (2018) reported a few more tardigrade species from this region and up to now only 51 species which are belonging to 18 genera have been recorded from Turkey.
Lichens and mosses resistant to drought, which melt under the snow, grow under the snow, or in rock cracks, are encountered in Alpine zone. These plants which are durable to the drought may be exposed to the fundamental physical variances at short ranges because of the interaction between temperature and ground and also soil moisture which changes according to the effect of sun and wind (Atay et al. 2009). Tardigrades are one of the invertebrates adapting to these short-term physical changes and conformance to adverse environmental conditions.
Our present study is the first high-altitude study to date on Turkish Tardigrada. Verçenik Mountain is located in the northern part of Turkey in Kaçkar Mountain Regions and originated from Alpide orogeny (Okay 2008). Up to now, zoogeographic and taxonomic information of tardigrades has been presented which is identified from alpine and subalpine zones in Turkey.

Materials and Methods
In August 2016, eight moss and lichen samples were collected on various altitudes of the Verçenik Mountain in Rize (Figure 1). But the tardigrades were found only in five of them (list of samples are presented in Table 1). All fresh samples were put into the small paper envelopes and allowed to dry slowly.
In the laboratory rehydrated samples were filtrated by the sieve of 25 and 400 µm, and the remaining residual was taken to a petri dish (Nelson 2002;Convey and McInnes 2005). Then tardigrades and their eggs were placed in a separate petri dish for identification on a stereomicroscope. All specimens were mounted on microscopic slides in Hoyer's medium, four adults and five eggs were prepared for Scanning Electron Microscopy (SEM) (JEOLJSM-6060 LV) analysis, following the protocols by Guidetti et al. (2000). The identification of specimens was carried out by analyzing morphological characters on the Phase-contrast microscope (Zeiss Axio ImagerM1) and SEM. Species were identified using a key to the world fauna of Tardigrada (Ramazzotti and Maucci 1983) and several original papers (Dastych 1988;Bertolani and Rebecchi 1993;Stec et al. 2018). All materials are deposited at the Aquatic Animals Research Laboratory at Ankara University.

Results
A total of 62 specimens, 10 eggs, and 11 exivium were isolated and 5 species were identified. Three of them are new records for the Turkish Tardigrade fauna. A list of the identified Turkish high mountain and alpine tardigrade, with their localities and zoogeographical and taxonomic comments, are in Table 2. It was found in Erzurum/Pasinler (3,4). But according to Dastych (1988), it classified as submontane and distributed in Holarctic (6). It was reported in Bursa/Uludağ Mountain at 2200 m (2,3) but it was found between 0 and 400 m in the Faroe Islands by Trygvadóttir and Kristensen (2013). Echiniscus testudo (Doyère, 1840)

2000-2400 m
It is a cosmopolitan species and Holarctic. Its distribution is most of the Palearctic biogeographic region (6, Jørgensen et al. 2007, McInnes et al. 2017. It was only observed in Erzurum and Ağrı from Turkey (2,3). In contrast, it was found between 0 and 200 m in the Faroe Islands by Trygvadóttir and Kristensen. (2013). Ramazzottius oberhaeuseri (Doyére, 1840) 2000 m It is a cosmopolitan, known from numerous locations in Europe from Greenland, the Arctic and Antarctic, North and South America, Africa, Afghanistan and New Zealand (Ramazzotti andMaucci 1983, McInnes et al. 2017).
In this study, it was found from rock mosses and lichen in Rize/Verçenik Mountain. According to Ramazzotti (1956), it is classified as an alpine. Although these species have been reported at low altitude, it is still classified as alpine species by Ramazzotti (1956). It was found Bozdağ/İzmir in Turkey (1,2,3). Maucci, 1952 Known only from two localities in Turkey (2,3). Cosmopolitan (6), however records of this species need to be verified (Kaczmarek and Michalczyk 2009).
It was reported in Ağrı and originally described from Turkey (3). On the other hand, it occurred at lowland altitudes (13 m asl) in Cyprus (Tekatlı and Altındağ 2017). Mesobiotus harmsworthi (Murray, 1907) 1450 m Cosmopolitan species, known from many localities throughout the world (6), but the species' geographic range is unknown (Kaczmarek et al. 2012).

Taxonomic accounts
New records for Turkey were marked by an asterisk (*).
Remarks: Species new for Turkey and most abundant in studied samples (Figure 2). It is considered an alpine species by Ramazzotti (1956). The specimens obtained correspond perfectly to the characterization of this species by Ramazzotti and Maucci (1983) and Dastych (1988). It has been recorded in many countries (Italy, Romania, Switzerland, Poland, Alps, Spitsbergen, Iceland, USSR, USA, Canada, Greenland) (McInnes 1994). Material examined: 12 specimens and 2 exivium collected from two rock moss.
Remarks: It is a new record for Turkey. It was observed from rock mosses at an altitude of 2308 meters in Rize/Verçenik Mountain (Figure 4). According to Dastych (1988), it is classified as a mountain (mesoalpine) species. It is known from few localities from Siberia, Poland, Italy, France, Finland, Russia, USA, Argentina, China and Norway (Ramazzotti and Maucci 1983;McInnes 1994;Zhang and Sun 2014). The found specimens correspond perfectly to the characterization of this species presented by Dastych (1973) and Maucci and Pilato (1974). Genus: Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar and Wolf, 2009 Subgenus Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar and Wolf, 2009
Remarks: It is one of the species of more common tardigrades and widespread everywhere, and present in different habitats, including terrestrial moss (Ramazzotti and Maucci 1983). In the study of Dastych (1988), on polish tardigrades, this species found in an area up to 2000 meters high. In this study, it was obtained from rock mosses at an altitude of 2131 m ( Figure 5). In Turkey, it was collected between 250-1140 m altitude in Ankara, Antalya, Burdur, Çanakkale, İzmir and Van (Maucci 1973(Maucci , 1975(Maucci , 1980Kaczmarek et al. 2012). But this genus has been revised by some researchers Marley et al. 2018). Family: Ramazzottiidae Sands, McInnes, Marley, Goodall-Copestake, Convey, and Linse, 2008 Genus: Ramazzottius Binda and Pilato, 1986 5. Ramazzottius oberhaeuseri (Doyère 1840) Material examined: 11 specimens and 3 eggs collected from two rock moss samples.

Discussion
Alpine, subalpine, boreo-alpine, and nival regions are one of the few studied regions, as access to these regions is both difficult and land conditions are appropriate only during a certain period of summer. So far, the number of Tardigrada obtained in studies on these regions in the world is quite limited.
The alpine regions are located above the last tree line (Pechlaner 1971). Some researchers have splitthe tardigrade species into groups according to altitudinal ranges and localities (Ramazzotti and Maucci 1983;Dastych 1987). Ramazzotti and Maucci (1983), defined tardigrade species obtained from 500 m and above areas as alpine species, while Dastych (1988), defined the lower limit as 1000 m. In addition, studies are showing that the alpine region starts at 400 m and above (Trygvadottir and Kristensen 2013). The alpine zone varies according to the region in Turkey.
Subalpine regions begin at about 1800 m. After approximately 2000 m, it continues as typical alpine regions (Atay et al. 2009). The alpine zone for Verçenik Mountain starts at 2000 m (Gürbüzer 2018). Therefore, our samples can be classified as alpine species. Previously obtained alpine species from Turkey were compared with this study (Table 2).
In the studies conducted so far, not only the distribution, abundance and diversity of the elevation according to species, but also the species obtained from different regions in the same alpine zone have been studied (Collins and Bateman 2001;Trygvadottir and Kristensen 2013). According to Dastych (1980); increasing the number of individuals together with the height decreased; In his research in 1987, he stated that species diversity increased as the height increased. This result was supported by Rodríguez-Roda (1951). Additionally, Guil et al. (2009) cited that the abundance of a tardigrade is the highest at 1000-2000 m asl.; the lowest is 0-500 m asl. But other researchers have found that the abundance is not more than 0-500 m. asl (Utsugi 1997;Collins and Bateman 2001). Furthermore, Nichols (1999) stated that there is no relationship between height and tardigrade distribution in the study of the distribution of tardigrade on Dugger Mountain in Alabama. In addition, Kathman and Cross (1991) found in their research that there is no effect of height on tardigrades in the Mountains of Vancouver in Canada. Kaczmarek et al. (2011) cited that, even not linear, increasing of height revealed the abundance of a tardigrade. It is possible to reach tardigrade which is not very common in algae samples obtained at 2000-and 4000-meters altitude (Ramazzotti and Maucci 1983).
It is known that temperature, humidity, food availability, competition, predation, and parasitism are effective on tardigrade populations (Dastych 1982;Ramazzotti and Maucci 1983;Collins and Bateman 2001). Although there are limited studies on the effect of biotic and abiotic factors on the animal, some researchers have tried to understand whether the altitude has a role in this living organism. There have been contradictory studies on this subject, but some researchers have found a correlation between height and diversity (Kaczmarek et al. 2011).
Most of the Tardigrade species obtained in Turkey at 2000 meters and above are observed to be tychoalpine species (an organism that is found at every altitude) compared to other areas. There is no study available on these creatures obtained associated with altitude. Until now, the relationship between the species obtained up to 2000 meters and above and altitude has not been determined when compared with other studies. The reason for this difference is thought to be due to the environmental conditions and microhabitat where the samples are exposed to, rather than the effect of the altitude on the habitat where the samples are taken.
Many studies on the Phylum Tardigrada have focused on limnoterrestrial tardigrades, which are easy to access and have a lot of diversity. Since transportation and weather conditions are difficult in Alpine regions, very few studies have been done so far. The data obtained is not enough to illuminate the distribution and diversity of the tardigrades in these regions. In the future, studies in high-altitude regions will give us more information about the distribution and characteristics of these animals.