Revealing Hidden Diversity and Community Dynamics of Land Snails through DNA Barcoding: Implications for Conservation and Ecological Studies

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Therefore, understanding the characteristics of their communities is vital for ecological studies and the development of effective conservation strategies. In this study, we employed DNA barcoding (COI gene) to identify land snails inhabiting the Hyrcanian Forest. Furthermore, we examined the variations in their community composition along elevational gradients. Snail samples were collected from three distinct elevations in three different forest locations within the Hyrcanian area of Iran. Through our comprehensive analysis, we identified a total of 10 OTUs, which were further classified into seven families and nine genera. Remarkably, five of these genera had never been reported in the study region before. By employing statistical analyses such as ANOVA and PERMANOVA, we determined significant differences in the features of snail communities across different elevations. Interestingly, we observed a decline in OTU richness with increasing elevation; however, the maximum abundance of snails was found at higher elevations. The unique climatic conditions and spatial distribution of precipitation from lowlands to highlands, as well as from west to east, make the Hyrcanian forests an ideal case study area for understanding the dynamics of land snail communities. In summary, this study provides novel insights into the land snail communities thriving in the Hyrcanian forests. The findings from our research can contribute significantly to the development of effective conservation management strategies for forest ecosystems. By understanding the factors influencing the distribution and composition of land snail communities, we can make informed decisions to protect and preserve these valuable organisms and the balance they maintain within their habitats. Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences land snail forest DNA barcoding community composition elevational incline Conservation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Land snails belong to the Mollusca phylum, with more than 24,000 recognized species standing as one of the most diverse groups of animals on Earth (Brown, and Lydeard 2010 ; Chavhan et al., 2015 ). These ancient species due to their position in the food web as decomposers and their role as an important calcium source for other animals like birds, snakes, and mammals, play a vital role in ensuring the sustainability of their habitat (Rull et al., 2019 ; Dempsey et al., 2020 ). This group of gastropods inhabits a variety of terrestrial environments, including leaf litter, soil, rocks, and trees. Different species have specific habitat preferences based on factors such as moisture, temperature, and soil composition (Jurickova et al., 2008 ). Soil chemical parameters, particularly calcium availability, play an important role in their population density and shell formation (Millar and Waite, 2002 ; Hotopp, 2002 ). Additionally, land snails’ dispersal and activity are influenced by elevation. Many studies have shown a decrease in land snail species richness towards high elevations, accompanied by changes in abundance and species composition (Aubry et al., 2005 ; Wronski and Hausdorf, 2008 ; Liew et al., 2010 ; Dourson and Langdon, 2012 ). Their specific habitat needs and limited mobility make them reliable indicators for evaluating environmental conditions and habitat quality. Furthermore, land snails serve as an ideal model for ecological genetic studies because of their ecological responses to environmental challenges like climate change, introduced species, and habitat fragmentation (Shimizu and Ueshima, 2000 ). Gaining a comprehensive understanding of the correlation between snail communities and environmental factors, especially in spatial dimensions like altitudinal gradients, provides conservation managers with valuable insights for protecting biodiversity and habitat conservation (Mathias et al., 2001 ; Goodacre, 2002 ; Arnaud et al., 2003 ; Jackson and Blois 2015 ; Nicolai and Ansart, 2017 ). Land snails are one of the indispensable components of forest ecosystems. In recent years due to several reasons like the presence of invasive or non-native species and, climate change the global forest cover has faced a drastic reduction (da Silva et al., 2019 ). since land snails serve as a bioindicator that offers valuable insights into the overall health and diversity of their ecosystem, evaluating their biodiversity and community characteristics in forest ecosystems yields valuable information for effective forest conservation. In this regard, the Hyrcanian forest of Iran provides a fascinating case study. This forest is the most important remnant of the Cenozoic forests and a refuge for ancient species in the last ice age (Tarkhnishvili et al., 2012 ; Dufresnes et al., 2016 ; Ahmadzadeh et al. 2020 ; Amiri et al., 2021 ; Saberi-Pirooz et al., 2021 ) and also a unique habitat for endemic land snails. While some studies have identified land snail species in the Hyrcanian forest, there is limited research on community composition and ecological analyses of these snails, with approximately 40 species reported thus far (Issel, 1865 ; Forcart, 1935 ; Starmuhlner and Edlauer, 1957 ; Yasini, 1976; Eliazian et al., 1979 ; Mansourian, 2005; Ahmadi, 2012 ). Furthermore, the identification of snails in these studies was primarily based on morphological characteristics. Despite the extensive research conducted on land snail identification using morphological features in various regions, there remain numerous species with an uncertain systematic status. This lack of clarity can pose significant challenges for ecological studies (Tattersfield et al., 2001 ). Land snail morphological identification, which relies on conchology (the study of shells), is prone to frequent misidentification, particularly for small species, due to shell variations influenced by habitat-specific environmental factors (Nekola and Coles, 2010 ; Smith and Hendricks, 2013 ). In addition, land snails, like numerous other organisms, can possess cryptic species that exhibit close morphological resemblance despite significant genetic differentiation. Additionally, the expertise required to accurately recall species names and identify morphological aspects of land snails is limited among biologists. (Waugh, 2007 ; Zeng et al., 2017 ; Mouahid et al., 2018 ). Exclusively relying on morphological characteristics for species identification can lead to misidentification, significantly impacting biodiversity evaluation crucial for conservation management (Bickford et al., 2007 ). To address these challenges, integrating morphological identification with other methods, such as DNA barcoding and molecular techniques, can improve the efficiency of land snail species identification. In the past decade, DNA barcoding has emerged as a rapid and highly accurate method for species identification, surpassing traditional morphology-based approaches (Ezzine et al., 2018 ; Galan et al., 2018 ; Nantarat et al., 2019 ). In this study, our objective was to identify terrestrial snails inhabiting the Hyrcanian forest by employing the DNA barcode method, targeting the COI gene. Following that we assessed their abundance, species richness, and composition along a gradient of elevation, comprising three distinct elevational levels within our study area. By conducting this analysis, we aimed to gain insights into the distribution patterns and ecological dynamics of land snails across different elevations within the forest. We also attempted to determine whether soil parameters were correlated with the presence of land snails. Eventually, we aimed to contribute to a deeper understanding of the ecological dynamics, species composition, and environmental interactions of land snails within this unique forest ecosystem. Such knowledge can be invaluable for conservation efforts, ecosystem management, and the preservation of biodiversity in the Hyrcanian forest and similar habitats. Material and Methods Study area Hyrcanian forests which are known as one of the most important biomes of the world extend from the Talish region in the southeast of the Republic of Azerbaijan to the Golestan Province in the northeast of Iran, with an area of approximately 50000 km 2 . These forests stretch from sea level to elevations of 2800 meters, three altitudinal belts were recognized across the Hyrcanian forests, i.e. lowland, sub-montane, and montane forests (Hamzeh’ee et al ., 2008; Siadati et al., 2010 ). Spatial analysis of seasonal precipitation shows that the Hyrcanian region has different patterns in the western and eastern parts and annual rainfall decreases from west to east (Akhani et al., 2010 ). The ecological coefficient of drought in the region is insignificant, as a whole, the Hyrcanian climate in the east is the warm Mediterranean, and in the central and western parts is temperate and semi-temperate Mediterranean, and from time to time temperate and dry (Talebi et al., 2014 ). This special natural world heritage is a unique source of biodiversity for the Northern Hemisphere which has universal importance beyond political boundaries (Gutleb and Wieser, 2002 ). Furthermore, these forests provide valuable ecosystem services that are vital for local people and these benefits depend on the biodiversity of these forests (Scharnweber et al., 2007 ; Tohidfar et al ., 2016). Sampling Snail collecting was carried out in Spring 2021. Due to land snails being nocturnal, sampling was performed on cloudy days (Sallam and El-Wakeil 2012 ). Three locations were selected for sampling along with Caspian Hyrcanian forests in Kordkoy (Forest 1), Amol (Forest 2), and Tonekabon (Forest 3). At each location, three sampling sites were specified in an elevation slope approximately 170m to 1600m height from sea level (Elevation 1, Elevation 2, and Elevation 3 that demonstrate the highest elevation to lowest elevation in each location respectively) (Fig. 1 and Table 1 ). Three plots of 3m×3m were applied over an area of about 150m 2 in each elevation (Clergeau et al., 2011 ; Horsak et al., 2013 ; Nurinsiyah et al., 2016 ). Snail species were collected by hand and then relaxed in the water for a day. Subsequently, they were preserved in 96% ethanol. A total of 679 samples were collected from 27 plots across nine elevations. Furthermore, topsoil was collected from each plot and transported in a plastic bag to the laboratory for measurement of some physicochemical parameters. In the following F1, F2, and, F3 refer to Forest 1, Forest 2, and Forest 3, and also E1, E2, and E3 refer to Elevation 1, Elevation 2, and Elevation 3 respectively. Table 1 Locations of the sampling sites Location Elevation (m) Latitude Longitude Code Kordkoy 1097 36.701583 N 54.10025 E F1-E1 Kordkoy 609 36.714306 N 54.105917 E F1-E2 Kordkoy 169 36.743389 N 54.117028 E F1-E3 Amol 560 36.358169 N 52.313897 E F2-E1 Amol 423 36.369101 N 52.320888 E F2-E2 Amol 225 36.396889 N 52.3405 E F2-E3 Tonekabon 1611 36.480142 N 50.856114 E F3-E1 Tonekabon 833 36.591972 N 50.835333 E F3-E2 Tonekabon 492 36.662722 N 50.812583 E F3-E3 Laboratory procedures Land snail samples were assigned into morphospecies in the laboratory. The identification of individuals was done by available morphological keys identification (Eliazian et al., 1979 ; Dourson and Dourson, 2006 ; Perez et al., 2008 ; Getz et al., 2018 ). A total of 16 morphospecies were distinguished from the collected samples within the study area. For the molecular investigation, 37 specimens (a minimum of two samples from each morphospecies) were chosen. Total DNA of selected samples was extracted using a high-salt method (Sambrook and Russell, 2001). A fragment of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene was used for the barcoding study. The primers employed in this study encompassed LCO 1490/HCO 2198 (Folmer et al., 1994 ) and HCO2198-JJ/LCO1490-JJ (Astrin and Stüben, 2008 ). Polymerase chain reactions (PCRs) were applied in a total volume of 25 µl, including 10.5 µl of ddH 2 O, 12.5 µl of Mastermix Red, (Amplicon, Copenhagen, Denmark) 0.5-1 µl of each primer (10 pmol/µl) and 1 µl of template DNA. PCR cycling for LCO 1490/HCO 2198 was run under the following condition: 94˚C for 5 min, 94˚C for 30 S (repeated for 35 cycles), 48˚C for 30 S, 72˚C for 1 min, and finally, an extension cycle at 72˚C for 5 min and the PCR condition for HCO2198-JJ/LCO1490-JJ was 94˚C for 1 min, 94˚C for 30 S (repeated for 5 cycles), 47˚C for 90 S, 72˚C for 1 min, 94˚C for 30 S (repeated for 35 cycles), 51˚C for 90 S, 72˚C for 1 min and a final extension cycle at 72˚C for 5 min. PCR products quality were surveyed using 1% agarose gel stained with Safe-Red. Then the acceptable amplicons were sent to Pishgam Company (Tehran, Iran) for purification and sequencing. Measurement of soil Physico-chemical properties Soil samples were obtained from the central area of each plot using a spade, reaching a depth of up to 10 cm, to assess the physicochemical properties of the soil. The collected soil samples from three plots of each elevation were combined. Six factors including soil pH, %saturation moisture, total calcium (Ca), exchangeable calcium (Ca ++ ), total magnesium (Mg), and exchangeable magnesium (Mg ++ ) measured using standard methods by Research Institute of Forest and Rangeland (Tehran, Iran). Genetic analysis DNA sequences of 36 individuals were edited using Geneious Prime v. 2022.1.1 ( www.geneious.com ). All newly obtained sequences were deposited to GenBank (Table 2 ). The sequences were checked in NCBI ( https://blast.ncbi.nlm.nih.gov/ ) by the Standard Nucleotide Basic Local Alignment Search Tool (BLASTn) (Altschul et al., 1990 ) and also Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert, 200; https://www.boldsystems.org/ ). Alignments were obtained with MAFFT 7 online version (Katoh et al., 2002 ). The best-fit evolutionary model was determined GTR + I + G model using MrModeltest V.2.3 (Nylander, 2004) according to Akaike’s Information Criterion (Akaike, 1974 ). Phylogenetic trees were constructed using both Bayesian Inference (BI) and maximum likelihood (ML) methods. Enchytraeidae sp. was considered as an outgroup. MrBayes was accomplished with MrBayes version 3.2. (Ronquist et al., 2012 ) under two independent runs (four chains for each run) for 5×10 6 generations and sampling trees every 100 generations. Finally, 10% of the trees were cast off as burn-in. Tracer V.1.7 (Rambaut et al., 2018 ) was used to assess the efficiency of runs. Maximum Likelihood tree was programmed with RAxML v.8.2 (Stamatakis, 2014 ) under the GTRGAMMA model with 1000 pseudoreplicates. Uncorrected genetic distances ( p -distances) among species were calculated by MEGA11 (Tamura et al., 2021 ). Table 2 List of land snails identified in this study Code ID OTU Family Genus Species Location Code Accession number ES3422 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F3-E1 OR189157 ES3432 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F3-E3 OR189158 ES3388 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F3-E3 OR189159 ES3317 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F1-E1 OR189150 ES3353 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F1-E3 OR189156 ES3303 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F1-E1 OR189151 ES3337 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F1-E2 OR189152 ES3302 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F2-E3 OR189149 ES3350 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F1-E3 OR189155 ES3338 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F1-E2 OR189153 ES3293 OTU 1 Cyclophoridae Caspicyclotus Caspicyclotus sieversi F2-E2 OR189147 ES3342 OTU 2 Pomatiidae Pomatias Pomatias Rivulare F1-E3 OR189154 ES3299 OTU 2 Pomatiidae Pomatias Pomatias Rivulare F2-E1 OR189148 ES3260 OTU 2 Pomatiidae Pomatias Pomatias Rivulare F3-E1 OR189145 ES3276 OTU 2 Pomatiidae Pomatias Pomatias Rivulare F2-E2 OR189146 ES3419 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E1 OR189170 ES3421 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E1 OR189171 ES3426 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E1 OR189173 ES3435 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E3 OR189176 ES3439 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E3 OR189180 ES3438 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E3 OR189179 ES3364 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E3 OR189161 ES3436 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F2-E3 OR189177 ES3379 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E3 OR189168 ES3437 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E3 OR189178 ES3371 OTU 3 Helicidae Caucasotachea Caucasotachea Leuaranea F3-E3 OR189162 ES3387 OTU 4 Hygromiidae Pyrenaearia - F3-E3 OR189169 ES3375 OTU 4 Hygromiidae Pyrenaearia - F3-E3 OR189165 ES3376 OTU 4 Hygromiidae Pyrenaearia - F3-E3 OR189166 ES3377 OTU 5 Hygromiidae Pyrenaearia - F3-E3 OR189167 ES3440 OTU 6 Helicidae Theba - F3-E3 OR189181 ES3373 OTU 6 Helicidae Theba - F3-E3 OR189164 ES3434 OTU 7 Hygromiidae Dioscuria - F3-E3 OR189175 ES3431 OTU 8 Pupillidae Pupilla - F1-E3 OR189174 ES3425 OTU 8 Pupillidae Pupilla - F3-E2 OR189172 ES3360 OTU 9 Orculidae Schileykula - F3-E3 OR189160 ES3328 OTU 10 Oxychilidae Oxychilus Oxychilus filicum - F1-E2 OR189163 Species-delimitation To infer hypothetical candidate species, two widely-used statistical methods of species delimitation were employed (automatic barcode gap discovery (ABGD (Puillandre et al., 2012 )) and Bayesian implementation of the Poisson tree processes (bPTP (Zhang et al., 2013 ))). ABGD analysis is a clustering method based on the pairwise distance between species sequences. It was performed on ABGD Web Server ( https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html ) using Kimura (K80) distance with the default Ts/Tv ratio (2.0) and the following conditions: range of prior intraspecific divergence from 0.01(Pmin) to 0.1 (Pmax) with 20 steps in between, X (relative gap width): 0.05 and Nbin: 10 (Mason et al., 2020 ). bPTP is a tree-based method that required a non-ultrametric phylogenetic tree as input. Analysis was carried out on the bPTP web server (Species delimitation server (h-its.org)) using a rooted RAxML tree for 500,000 MCMC generations, with 0.1 of samples conservatively discarded as burn-in (Mason et al., 2020 ). Statistical Analyses Statistical analysis was performed using R v. 4.0.3 (R Core Team., 2020). Two-way analysis of variance (two-way ANOVA), followed by two-sided Tukey HSD tests, was conducted to assess the differences in total snail density, total OTUs diversity, and Shannon-Wiener diversity index (biodiversity metrics) among different elevations (three levels) in each forest. The statistical analyses were conducted using the "aov()" function in R. Assumptions of homogeneity of variances (tested with Levene's test, p > 0.05) and normality of residuals (tested with Shapiro-Wilk test, p > 0.05) were checked using the R package v. 1.2-7 in R (Fox et al ., 2007). To assess the dissimilarities among snail communities at different elevations within each forest (β-diversity), we utilized the betapart package (Baselga et al., 2018 ) in R with the Jaccard dissimilarity index. β-diversity was employed to measure how species compositions among samples have changed across the area (Graham and Fine, 2008 ). This approach offers a theoretical framework for partitioning total dissimilarity into nestedness (species gains or losses) and turnover components (species replacement) (Baselga, 2010 ). Biodiversity metrics were calculated to assess the snail communities using various measures. The Shannon-Wiener diversity index (H) was calculated using the vegan package (v. 1.17-8) in R. Additionally, the R package Picante (Kembel et al., 2010 ) was utilized to calculate phylogenetic diversity (PD) and mean pairwise distance (MPD) between pairs of communities. To examine the spatial compositional differences in snail compositions, permutational multivariate analysis of variance (PERMANOVA) was employed. Permutations of residuals (n = 9999) were performed under the reduced model, following the same factorial design as the univariate analysis. The Sorensen dissimilarity matrices were used for the analysis. In cases where significant differences were found, the SIMPER routine was applied to determine the proportional contribution of each species and main group to the observed pairwise dissimilarity in composition. The SIMPER results included species with a cumulative dissimilarity cut-off of 70% (Clarke, 1993 ). Both the PERMANOVA and SIMPER analyses were conducted using the vegan package. Additionally, Canonical Correspondence Analysis (CCA) was employed as a multivariate statistical approach to investigate the relationship between species and environmental variables. The analysis utilized a dataset containing the Operational OTUs present in each forest, and six soil variables were selected as explanatory factors. Before conducting the analysis, the environmental data underwent initial screening using the Principal Component Analysis (PCA) methodology. The entire variation in the environmental data was explained by the initial six principal components (PCs). Prominent variables were identified by considering the highest absolute loading values within each PC. Subsequently, two parameters were chosen for further analysis after the screening process. Results Molecular and phylogenetic analyses Based on Cytochrome oxidase I (COI) (596bp) sequences of 37 individuals, three sequences could be assigned to species named Pomatias rivulare, Oxychilus filicum , and Caucasotachea leucoranea and six of them were assigned to five genera named Pyrenaearia , Theba , Dioscuria , Pupilla , and Schileykula . one Sequences weren’t assigned with any sequences in NCBI and BOLD data sets which means it’s the first barcoding study on this species. Based on shell features this OTU was identified as Caspicyclotus sieversi (see Table 2 ). The p -distance between OTUs ranged from 0.016 to 0.35. Our criterion for categorization of sequences to OTU was more than eight percent genetic distances among sequences (Table 3 ). Species delimitation with bPTP resulted in 10 putative species for the dataset and most of them were supported with high values (> 80%). The ABGD analysis yielded 7–17 hypothetical OTU using about 0.1–0.01 maximum intraspecific divergence (Fig. S1 ). Furthermore, the phylogenetic tree revealed similar terminals (Fig. 2 ). Totally, 10 OTUs were recognized in this study which is categorized into seven families named Cyclophoridae, Pomatiidae, Helicidae, Hygromiidae, Pupillidae, Orculidae, and Oxychilidae. Table 3 Uncorrected genetic distances (p - distances) between land snail OTUs based on COI gene OTU 1 OTU 2 OTU 3 OTU 4 OTU 5 OTU 6 OTU 7 OTU 8 OTU 9 OTU 10 OTU 1 OTU 2 0.27 OTU 3 0.31 0.31 OTU 4 0.32 0.29 0.2 OTU 5 0.31 0.31 0.2 0.09 OTU 6 0.32 0.28 0.21 0.11 0.11 OTU 7 0.31 0.28 0.19 0.08 0.09 0.11 OTU 8 0.3 0.3 0.23 0.2 0.19 0.2 0.18 OTU 9 0.32 0.33 0.26 0.27 0.25 0.26 0.26 0.23 OTU 10 0.31 0.28 0.23 0.19 0.21 0.19 0.19 0.21 0.25 Comparison of land snail abundance and OTUs diversity Exploring the abundance of land snails between three forests shows a considerable difference in snail abundance in Forest 3. As the bar chart illustrates (Fig. 3 ) there is a bit of decline in abundance from Forest 1 to Forets 2 then it rises by far in Forest 3. In addition, comparing three elevations in each forest demonstrate that lowlands have less abundance than the highest elevations in all forests. Comparing OTUs richness in all forests indicated the number of OTUs had decreased in high elevations (Fig. 4 ). Overall, the highest elevation in Forest 3 (F3-E1) shows the most abundance and the maximum number of OTUs found in the lowest elevation of this forest. The results of ANOVA indicated significant effects of elevations, forests, and interaction (Table 4 ). Based on the total OUTs diversity and also Shannon-Wiener diversity dataset, t-test analysis showed that there was a significant difference between high elevation (E1) and lowland (E3) in Forest 2. Additionally, the highest elevation in Forest 1 exhibited significant differences when compared to the mid and low elevations within this forest (E2 and E3). Furthermore, the density of OTUs in the lower elevation of Forest 3 (E3) differed significantly from both the high elevation (E1) and mid-elevation (E2) in this forest, also Forest 3 was different from the other two locations in the density of OTUs. In addition, the results indicate a significant different between highlands and lowlands in Forest 2 and Forest 3 (See Table S1 ). Table 4 Summary of statistical analysis of ANOVA including Density of total snails, diversity of OTUs, and Shannon-Wiener diversity index Data set ANOVA Forest Elevation Forest: Elevation Density of total snails F 2,18 = 170.67 p = 1.99e-12 F 2,18 = 152.22 p = 5.26e-12 F 4,18 = 87.89 p = 1.48e-11 Diversity of total OTUs F 2,18 = 19.44 p = 3.18e-05 F 2,18 = 49.78 p = 4.63e-08 F 4,18 = 12.44 p = 5.00e-05 Shannon-Wiener diversity index F 2,18 = 10.693 p = 0.00087 F 2,18 = 55.988 p = 1.87e-08 F 4,18 = 3.928 p = 0.01831 Investigation of β-diversity partitioning figured out that the spatial turnover portion occurred between three elevations in Forest 3. However, Forest 2 and Forest 1 had different results, and nestedness was also detected. Generally, the β-diversity index was between 0.5 to 0.8 in all datasets (Table 5 ). All forests showed an increase in PD values as elevation increased. Conversely, the MPD values remained relatively consistent across the three elevations within each forest. The result of the Shannon-Wiener diversity index is shown in Table S2. Table 5 β-diversity, phylogenetic diversity (PD), mean pairwise distance (MPD) for each forest and every elevation Location Date Set Beta diversity PD MPD Turnover Nestedness Total β-diversity Forest 1 (E1 and E2) (E1 and E3) (E2 and E3) 0.00 0.25 0.40 0.50 0.50 0.35 0.50 0.75 0.75 E1: 0.16 E2: 0.42 E3: 0.59 0.20 0.22 0.24 Forest 2 (E1 and E2) (E1 and E3) (E2 and E3) 0.00 0.44 0.36 0.66 0.17 0.16 0.66 0.61 0.53 E1: 0.15 E2: 0.30 E3: 0.30 0.00 0.10 0.10 Forest 3 (E1 and E2) (E1 and E3) (E2 and E3) 0.70 0.76 0.75 0.90 0.06 0.06 0.8275 0.82 0.82 E1: 0.46 E2: 0.60 E3: 0.67 0.23 0.25 0.25 Assemblage composition The PERMANOVA indicated significant effects of elevation levels (F 2,3 = 3.8246, p = 0.004), forests (F 1,3 = 7.5693, p = 0.001), and their interactions (F 1,3 = 6.0400, p = 0.001) on the OTUs composition OTUs. SIMPER analysis indicated that three OTUs including OUT 1, OUT 2, and OUT 3 were the most responsible groups for the observed significant difference between all of the comparisons (elevation levels and forests). The list of OTUs that have differences between elevations and forests is shown in Table 6 . Table 6 Output from SIMPER analysis of the difference in OTUs composition between three forests and three elevations SIMPER Data set Responsible species Forest 1*Forest 2 Forest 1*Forest 3 Forest 2*Forest 3 Elevation 1*Elevation 2 Elevation 1*Elevation 3 Elevation 2*Elevation 3 OTU 1, OTU 2, OTU3 OTU 1, OTU 2, OTU3, OTU 8, OTU 10 OTU 1, OTU 2, OTU3, OTU 8 OTU 1, OTU 2, OTU3, OTU 8 OTU 1, OTU 2, OTU3, OTU 4, OTU 5 OTU 1, OTU 2, OTU3, OTU 4, OTU 8, OTU 10 Relationship between snail communities and environmental variables The measurement of soil parameters (pH, Ca, Mg, Ca ++ , Mg ++ . and %saturation moisture) are shown in Table S3. The range of pH values were varied from 6.1 (F1-E1) to 7.7 (F3-E1). According to our results, Forest 2 had the highest Ca content (44 mg) observed in E3, and Forest 1 at E2 had the lowest amount (16 mg). The Ca ++ content (6.09%) was found in F2-E1 and the minimum amount (0.79%) was recorded in F3-E2. Forest 1 exhibited the highest Mg content (44 meq/lit) at E1, whereas F3-E1 had the lowest amount (4 meq/lit). The highest Mg ++ content (2.21%) was observed in F3-E1, and the lowest amount (0.49%) was recorded in F2-E3. The highest content of %saturation moisture (= 95) was recorded in F3-E3 and the lowest amount (= 51.9) was observed in F3-E1. To determine the environmental factors responsible for shaping the composition of snail communities in each forest, CCA was conducted using OTUs data and the selected environmental parameters after PCA analysis. According to PCA analysis, %saturation moisture and Ca in Forest 1, %saturation moisture and Mg in Forest 2, and in Forest 3, Ca and Mg have more contribution to each forest. In Forest 1, the selected variables explained 88% of the total variation in OTUs composition. Following that in Forest 2 and Forest 3, the physicochemical variables explained 53% and 69% of the total variation in OTU composition. The comparison of variations explained by constrained and unconstrained axes revealed > 82% efficiency of the constrained axes 1 and 2 in all three forests (Fig. 5 ). Discussion This study is the first genetic investigation on terrestrial snail diversity in the Hyrcanian forest (in the northern part of Iran), which lead to the identification of 10 OTUs in the area based on COI gene sequences. Furthermore, land snails abundance and OTUs diversity were compared in an elevational incline and the results showed that elevation affected snail communities. In addition, the survey on soil physicochemical parameters displayed a correlation between some soil features and land snails' presence. Identifying land snails In our current research, both morphological and molecular approaches were employed to identify land snails and assess their species diversity. A total of 679 individuals were investigated, and 37 sequences were analyzed, leading to the recognition of seven families, nine genera, and four species (see Table 2 ). The ABGD and bPTP methods were used to analyze species delimitation, resulting in the identification of approximately 7 to 17 potential species and also 10 putative species through ABGD and bPTP respectively. By considering our findings on morphology, species delimitations, genetic distances, and the phylogenetic tree, we have determined 10 OTUs within the collected samples. Among 10 OTUs identified in our research four of them including OUT 1, OUT 2, OUT 3, and OUT 10 refer to Caspicylotus sieversi, Pomatias rivulare, Caucasotachea leuaranea , and Oxychilus filicum respectively reported in the previous studies from the region (Issel 1865 ; Forcart 1935 ; Starmuhlner and Edlauer 1957 ; Yassini 1976 ; Eliazian et al., 1979 ; Mansoorian, 2005 ; Ahmadi; 2012 ). It is worth noting that the remaining six OTUs which referred to five genera named Pyrenaearia , Theba , Dioscuria , Pupilla , and Shileykula are the first documented occurrences of these genera in the Hyrcanian Forest of Iran. All of the aforementioned studies, just relied on morphological approaches, however in this study we used molecular methods too. We believe that recognition of snails using morphological approach may lead to misidentification because of their morphological variety and cryptic diversity. Therefore we suggest that a combination of molecular approaches and morphological taxonomy should be employed as the primary method for species identification, aiming to achieve more dependable results in land snail identification. Although land snails exhibit substantial mitochondrial diversity and are among the most diverse animals, there have been limited barcoding studies conducted on them, with the majority of investigations focusing on marine gastropods (Meyer and Paulary, 2005; Kelly et al., 2007 ; Campbell et al., 2008 ). Consequently, there is a lack of comprehensive genetic data available in the GenBank dataset for barcoding studies and the COI gene dataset alone cannot currently serve as a practical method for identifying land snail species. However, it can serve as a reliable tool for assessing their biodiversity (Davison et al., 2009 ; Ranasinghe et al., 2022 ). Statistical analysis The ANOVA analysis revealed significant differences in the total density of land snails among different elevations within each forest. In addition, evaluating the abundance in three elevations at each forest indicates the abundance of snails in the highest elevation is more than in the lowlands (Fig. 6 (A)). A possible explanation for this difference is precipitation patterns and humidity levels in this area. Highlands in Hyrcanian forests are more humid, and also precipitation increases at higher elevations (Talebi et al., 2014 ). In line with our findings, Tattersfield et al ( 2006 ) found out land snail abundance increases in high-elevation sets. In addition, De Chavez and De Lara ( 2010 ) observed a positive relationship between land snail abundance and elevation in the Mount Makiling rainforest in the Philippines based on their observation. Although, in some cases has been obtained different results. Tattersfield et al., ( 2001 ) expressed that land snail abundance in Forest habitats in mountain Kenya decreases with enhancing elevation and also Liew et al. ( 2010 ) concluded that there is no relationship between land snail abundance and elevation. Comparing OTUs richness in each forest displays that the number of OTUs gradually increases from high elevation to low elevation (Fig. 6 (B)). Additionally, the ANOVA revealed a significant distinction in Shannon-wiener diversity and total diversity of OTUs between three elevations across all forests. Similarly, the PD analyses confirm this pattern, showing an inverse relationship between PD values and increasing elevation. This pattern follows the common law for the most group of organisms in all environments (Gaston, 2000 ). As elevation affects many climatic variations including temperature and precipitation it can play an important role in habitat suitability. Precipitation enhancement, the longer period of cold temperatures besides less productivity in highlands cause a decrease in the amount of available energy in these areas. Thus the species-energy relationship (the relationship between energy availability and species richness) may clarify this pattern between species richness and elevation. Receiving a specified content of energy can preserve a specified number of species which means higher elevations shelter fewer species than lowlands (Gaston, 2000 ; Whittaker et al., 2001 ; Willis and Whittaker, 2002 ; Aubry et al., 2005 ). Several investigations reported an increase in land snails species richness and species density in an elevation slope; Tattersfield et al. ( 2001 ) achieve the same result for forest habitats in Kenya, they remarked rainfall as the most important factor for land snail communities, as well as Aubry et al. ( 2005 ) observed the same pattern in south-eastern France. Based on their research different factors such as climate altitudinal slope and also land cover heterogeneities can explain this pattern. Liew et al. ( 2010 ) reported a decline in land snail and slugs species density with elevation as a result of lessen nich diversity in high elevations and also historical events which lead to snail upward migration. However, Tattersfield et al. ( 2006 ) observed an increase in land snail richness and species density in elevation in Mwanihana Forest in Tanzania. Comparing three forests altogether revealed that Forest 3 which is located in the western part of the study area was different from the two other forests with higher OTUs richness and abundance. Considering that land snails are highly dependent on humidity, the observed differences can be explained by the decreasing precipitation pattern along the southern Caspian Sea shoreline from east to west. In addition, ecological dryness declined from the east toward the west of the Hyrcanian Forests and the climate in the western part is more humid than the eastern part (Shimizu and Ueshima, 2000 ; Talebi et al., 2014 ). Based on the PERMANOVA analysis there were significant differences in snail composition across various elevations and among the three forests under investigation. These findings highlight the intricate ecological patterns and distinct species assemblages within each forest ecosystem. In general, some OTUs were exclusively identified in certain forests, indicating unique ecological characteristics that allow them to thrive in specific habitats. Five OTUs including OTU 4, OTU 5, OTU 6, OTU 7, and OTU 9 were observed only in Forest 3 and Forest 1 exhibited the exclusive presence of OTU 10. Furthermore, three OTUs (OTU 1, OTU 2, and OTU 3) were observed, across all forests. The SIMPER analysis highlighted these three OTUs are responsible for observed differences among forests and elevation levels. Additionally, the β-diversity partitioning results showed that the spatial turnover component played a dominant role in driving dissimilarity within Forest 2 and Forest 3 (Table 5 ). This finding implies that species replacement exerts a more substantial influence on the observed variation than differences in species richness. Conversely, in Forest 1, dissimilarity was primarily attributed to nestedness, indicating that dissimilarities were influenced by disparities in species richness. Environmental variable The result of PCA revealed that Ca, Mg, and %saturation moisture have more contribution in physicochemical soil factors. These factors exhibited positive trends with most of OTUs (see CCA results, Fig. 5 ). Because these environmental factors play a crucial role in shaping the distribution and diversity of snail populations. Calcium and magnesium are essential for land snails' shell development and overall functions. Evidence from previous studies consistently supports the notion that land snail abundance and species richness tend to be higher in areas characterized by elevated levels of calcium (Ca), magnesium (Mg), and moisture (Silvan et al., 2000 ; Hotopp, 2002 ). Snails flourish in regions with ample soil levels of these minerals, resulting in larger populations and more diverse species (Nekola and Smith, 2000 ; Millar and Waite, 2002 ). Moisture availability also plays a significant role in determining snail abundance and richness. Areas with higher moisture content in the soil create favorable conditions for snails, as it provides them with a suitable microhabitat (Getz and Uetz, 1994 ; Morecroft et al ., 2002). Conclusion This survey represents the first barcoding study conducted on land snail species within the Hyrcanian Forest, resulting in the identification of 10 OTUs across the region. Notably, this study reports the occurrence of five genera, namely Pyrenaearia , Theba , Dioscuria , Pupilla , and Schileykula , for the first time within this geographical area. Furthermore, the examination of land snail communities along a gradient of elevation revealed significant alternation in their abundance, richness, and community composition. Soil physicochemical parameters analyses revealed that Ca, Mg, and %saturation moisture are the most important factors which impact snail distribution. This foundational study offers new insights into the snail community within the Hyrcanian forest, providing valuable data that can be utilized for future research in this globally significant world heritage site and similar habitats elsewhere. Further barcoding studies in different parts of the region is recomeded for future studies to uncover additional species as well as consider other environmental factors such as climatic variables can contribute to a better understanding of land snail communities along elevational slopes. These efforts will play a significant role in offering valuable insights into the conservation of snail biodiversity and the preservation of their habitats. Declarations Data availability statement The datasets generated during the current study are available in the [NCBI] repository, [ACCESSION NUMBERs are represented in table 2 in the article]. Additionally, the datasets generated in this study are accessible upon request from the corresponding author ( [email protected] ) in accordance with reasonable terms. Acknowledgments We wish to express our gratitude to everyone who contributed to the completion of this article. Special thanks go to Reihaneh Saberi-Pirooz for her invaluable assistance in data analysis and the preparation of the paper. Her expertise and dedication significantly enhanced the quality of our research. Furthermore, we would like to express our profound appreciation to Khashayar Allahbedashti for his unwavering support and significant contributions during the sampling phase of this study. References Ahmadi, E. (2012). Identification of snails in citrus orchards in Mazandaran province, Iran. Journal of Field Crop Entomology. 2(1), 57–64. (In Persian). Ahmadzadeh, F., Shahrokhi, G., Saberi-Pirooz, R., Oladi, M., Taati, M., Poyarkov, N. A., & Rödder, D. (2020). Alborz Heritage: geographic distribution and genetic differentiation of the Iranian Paradactylodon (Amphibia: Hynobiidae). Amphibia-Reptilia, 41(4), 519–534. Akaike, H. (1974). A new look at the statistical model identification. IEEE Transactions on automatic control, 19(6), 716–723. Akhani, H., Djamali, M., Ghorbanalizadeh, A., & Ramezani, E. (2010). Plant biodiversity of Hyrcanian relict forests, N Iran: an overview of the flora, vegetation, palaeoecology, and conservation. Pakistan Journal of Botany, 42(1), 231–258. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Amiri, N., Vaissi, S., Aghamir, F., Saberi-Pirooz, R., Rödder, D., Ebrahimi, E., & Ahmadzadeh, F. (2021). Tracking climate change in the spatial distribution pattern and the phylogeographic structure of Hyrcanian wood frog, Rana pseudodalmatina (Anura: Ranidae). Journal of Zoological Systematics and Evolutionary Research, 59(7), 1604–1619. Arnaud, J. F., Madec, L., Guiller, A., & Deunff, J. (2003). Population genetic structure in a human-disturbed environment: a case study in the land snail Helix aspersa (Gastropoda: Pulmonata). Heredity, 90(6), 451–458. Astrin, J. J., & Stüben, P. E. (2008). Phylogeny in cryptic weevils: molecules, morphology and new genera of western Palaearctic Cryptorhynchinae (Coleoptera: Curculionidae). Invertebrate systematics, 22(5), 503–522. Aubry, S., Magnin, F., Bonnet, V., & Preece, R. C. (2005). Multi-scale altitudinal patterns in species richness of land snail communities in south‐eastern France. Journal of Biogeography, 32(6), 985–998. Baselga, A. (2010). Partitioning the turnover and nestedness components of beta diversity. Global ecology and biogeography, 19(1), 134–143. Baselga, A., Orme, D., Villeger, S., De Bortoli, J., Leprieur, F., Baselga, M.A. (2018). Package ‘betapart. Beck, M. W. (2017). ggord: Ordination Plots with ggplot2. R package version 1.0. 0. Bickford, D., Lohman, D. J., Sodhi, N. S., Ng, P. K., Meier, R., Winker, K., … Das, I. (2007). Cryptic species as a window on diversity and conservation. Trends in ecology & evolution, 22(3), 148–155. Brown, K. M., & Lydeard, C. (2010). Mollusca: gastropoda. In Ecology and classification of North American freshwater invertebrates (pp. 277–306). Academic Press. Campbell, D. C., Johnson, P. D., Williams, J. D., Rindsberg, A. K., Serb, J. M., Small, K. K., & Lydeard, C. (2008). Identification of ‘extinct freshwater mussel species using DNA barcoding. Molecular Ecology Resources, 8(4), 711–724. Chavhan, A., Pawar, S. and Jadhao, R. (2015). Study of Biodiversity of Terrestrial Snail in Selected Locality of Amravati City, Central India. Indian Journal of Applied Research, 5(8), 713–714. Clarke, K. R. (1993). Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology, 18(1), 117–143. Clergeau, P., Tapko, N., & Fontaine, B. (2011). A simplified method for conducting ecological studies of land snail communities in urban landscapes. Ecological research, 26(3), 515–521. da Silva, L. P., Heleno, R. H., Costa, J. M., Valente, M., Mata, V. A., Gonçalves, S. C., … Ramos, J. A. (2019). Natural woodlands hold more diverse, abundant, and unique biota than novel anthropogenic forests: a multi-group assessment. European Journal of Forest Research, 138(3), 461–472. Davison, A., Blackie, R. L., & Scothern, G. P. (2009). DNA barcoding of stylommatophoran land snails: a test of existing sequences. Molecular Ecology Resources, 9(4), 1092–1101. De Chavez, E. R. C., & De Lara, A. V. (2010). Diversity and spatial distribution patterns of macro land snails in Mount Makiling Forest Reserve, Philippines. Asia Life Sciences-The Asian International Journal of Life Sciences, 20(1), 183–197. Dempsey, Z. W., Goater, C. P., & Burg, T. M. (2020). Living on the edge: comparative phylogeography and phylogenetics of Oreohelix land snails at their range edge in Western Canada. BMC evolutionary biology, 20(1), 1–13. De Salle R, Egan MG, Siddall M (2005). The unholy trinity: taxonomy, species delimitation and DNA barcoding. Philosophical Transactions of the Royal Society B: Biological Sciences, 360, 1905. Dufresnes, C., Litvinchuk, S. N., Leuenberger, J., Ghali, K., Zinenko, O., Stöck, M., & Perrin, N. (2016). Evolutionary melting pots: a biodiversity hotspot shaped by ring diversifications around the Black Sea in the Eastern tree frog (Hyla orientalis). Molecular Ecology, 25(17), 4285–4300. Dourson, D. and Dourson, J. (2006). Land snails of the great smoky mountains (Eastern Region). Appalachian Highlands Science Learning Center, Purchase Knob, Great Smokey Mountains National Park. Dourson, D. C., & Langdon, K. (2012). Land Snails of Selected Rare High Elevation Forests and Heath Balds of the Great Smoky Mountains National Park. Journal of North Carolina Academy of Science, 128(2), 27–32. Eliazian, M., Tamiji, Y., Akbarzadeh, M., & Hagh-Nazari, J. (1979). Snails from the northern parts of Iran (Caspian Area). Archives of Razi Institute, 31(1), 29–36. Ezzine, I. K., Dimassi, N., Pfarrer, B., Said, K., & Neubert, E. (2018). New records of the endemic Sicilian land snail species Marmorana (Murella) muralis (OF Müller, 1774) from the north of Tunisia (Pulmonata, Gastropoda). ZooKeys, (775), 131. Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenkoek, R. (1994). Molecular Marine Biology and Biotechnology. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates, 3, 294–299. Forcart, L. (1935). Die Mollusken der nordpersischen Provinz Masenderan und ihre tiergeographischen Bedeutung. Akademische Verlagsgesellschaft. Galan, G. L., Mendez, N. P., & dela Cruz, R. Y. (2018). DNA barcoding of three selected gastropod species using cytochrome oxidase (COI) gene. Annales of West University of Timisoara. Series of Biology, 21(1), 93–102. Gaston, K. J. (2000). Global patterns in biodiversity. Nature, 405(6783), 220–227. Getz, L. L., Chichester, L. F., & Burch, J. B. (2018). Land mollusks of the Northeastern United States and Southeastern Canada. Getz, L. L., & Uetz, G. W. (1994). Species diversity of terrestrial snails in the southern Appalachian mountains, USA. Malacological Review, 27(1–2), 61–74. Goodacre, S. L. (2002). Population structure, history and gene flow in a group of closely related land snails: genetic variation in Partula from the Society Islands of the Pacific. Molecular Ecology, 11(1), 55–68. Graham, C. H., & Fine, P. V. (2008). Phylogenetic beta diversity: linking ecological and evolutionary processes across space in time. Ecology letters, 11(12), 1265–1277. Gutleb, B., & Wieser, C. (2002). Ergebnisse einer zoologischen Exkursion in den Nordiran, 2001: Arthropoda (Lepidoptera, Trichoptera, Neuroptera, Heteroptera, Coleoptera, Opiliones, Araneae, Decapoda) und Vertebrata (Amphibia, Reptilia, Aves, Mammalia). Hamzeh'ee, B., Naqinezhad, A., Attar, F., Ghahreman, A., Assadi, M., & Prieditis, N. (2008). Phytosociological survey of remnant Alnus glutinosa ssp. barbata communities in the lowland Caspian forests of northern Iran. Phytocoenologia, 117–132. Hotopp, K. P. (2002). Land snails and soil calcium in central Appalachian Mountain forest. Southeastern Naturalist, 1(1), 27–44. Horsak, M., Jurickova, L., and Picka, J. (2013). Mollusks of the Czech and Slovak Republics. Issel, A. (1865). Catalogo del molluschi raccolti della Missione Italiana in Persia. Mem. Delle realeacademia della scienze di Torino, Ser, 2. Jackson, S. T., & Blois, J. L. (2015). Community ecology in a changing environment: Perspectives from the Quaternary. Proceedings of the National Academy of Sciences, 112(16), 4915–4921. Jurickova, L., Horsák, M., Cameron, R., Hylander, K., Míkovcová, A., Hlaváč, J. Č., & Rohovec, J. (2008). Land snail distribution patterns within a site: the role of different calcium sources. European Journal of Soil Biology, 44(2), 172–179. Katoh, K., Misawa, K., Kuma, K. & Miyata, T. (2002) MAFFT: a novel method for rapid multiple sequence alignments based on fast Fourier transform. Nucleic Acids Research, 30 (14), 3059–3066. Kelly RP, Sarkar IN, Eernisse DJ, Desalle R (2007) DNA barcoding using chitons (genus Mopalia ). Molecular Ecology Notes, 7, 177–183. Kembel, S. W., Cowan, P. D., Helmus, M. R., Cornwell, W. K., Morlon, H., Ackerly, D. D., … Webb, C. O. (2010). Picante: R tools for integrating phylogenies and ecology. Bioinformatics, 26(11), 1463–1464. Liew, T. S., Schilthuizen, M., & bin Lakim, M. (2010). The determinants of land snail diversity along a tropical elevational gradient: insularity, geometry, and niches. Journal of Biogeography, 37(6), 1071–1078. Mansoorian, A. (2005). Terrestrial Molluscs of Golestan, and Mazandaran provinces, northern Iran. (in Persian). Mason, K., Fehér, Z., Bamberger, S., Reier, S., Szekeres, M., Sattmann, H., … Haring, E. (2020). New insights into and limitations of the molecular phylogeny in the taxon-rich land snail genus Montenegrina (Mollusca: Gastropoda: Clausiliidae). Journal of Zoological Systematics and Evolutionary Research, 58(3), 662–690. Mathias, A., Kisdi, È., & Olivieri, I. (2001). Divergent evolution of dispersal in a heterogeneous landscape. Evolution, 55(2), 246–259. Meyer, C. P., & Paulay, G. (2005). DNA barcoding: error rates based on comprehensive sampling. PLOS biology, 3(12), e422. Millar, A. J., & Waite, S. (2002). The relationship between snails, soil factors and calcitic earthworm granules in a coppice woodland in Sussex. Journal of Conchology, 37(5), 483–504. Mouahid, G., Clerissi, C., Allienne, J. F., Chaparro, C., Al Yafae, S., Mintsa Nguema, R., … Moné, H. (2018). The phylogeny of the genus Indoplanorbis (Gastropoda, Planorbidae) from Africa and the French West Indies. Zoologica Scripta, 47(5), 558–564. Nantarat, N., Sutcharit, C., Tongkerd, P., Wade, C. M., Naggs, F., & Panha, S. (2019). Phylogenetics and species delimitations of the operculated land snail Cyclophorus volvulus (Gastropoda: Cyclophoridae) reveal cryptic diversity and new species in Thailand. Scientific Reports, 9(1), 1–12. Nekola, J. C., & Coles, B. F. (2010). Pupillid land snails of eastern North America. American Malacological Bulletin, 28(2), 29–57. Nekola, J. C., & Smith, T. M. (2000). Terrestrial gastropod richness patterns in Wisconsin carbonate cliff communities. MALACOLOGIA-PHILADELPHIA-, 41(1), 253–270. Nicolai, A., & Ansart, A. (2017). Conservation at a slow pace: terrestrial gastropods facing fast-changing climate. Conservation Physiology, 5(1), cox007. Nurinsiyah, A. S., Fauzia, H., Hennig, C., & Hausdorf, B. (2016). Native and introduced land snail species as ecological indicators in different land use types in Java. Ecological Indicators, 70, 557–565. Perez, K. E., Cordeiro, J. R., & Coppolino, M. L. (2008). A guide for terrestrial gastropod identification. In American Malacological Society Annual Meeting, Carbondale, Illinois. Puillandre, N., Lambert, A., Brouillet, S., & Achaz, G. J. M. E. (2012). ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology, 21(8), 1864–1877. Rambaut, A., Drummond, A. J., Xie, D., Baele, G., & Suchard, M. A. (2018). Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67(5), 901–904. Ranasinghe, U. G. S. L., Eberle, J., Thormann, J., Bohacz, C., Benjamin, S. P., & Ahrens, D. (2022). Multiple species delimitation approaches with COI barcodes poorly fit each other and morphospecies–An integrative taxonomy case of Sri Lankan Sericini chafers (Coleoptera: Scarabaeidae). Ecology and Evolution, 12(5), e8942. Ratnasingham, S., Hebert, P.D.N., 2007. BOLD: The Barcode of Life Data System ( http://www.barcodinglife . org). Mol. Ecol. Notes 7, 355–364. R Core Team. (2020). R: A Language and Environment for Statistical Computing (Version 3.4. 0). R Foundation for Statistical Computing. Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D. L., Darling, A., Höhna, S., … Huelsenbeck, J. P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61(3), 539–542. Rull, V., Vegas-Vilarrúbia, T., Huber, O., & Señaris, C. (Eds.). (2019). Biodiversity of Pantepui: The Pristine" Lost World" of the Neotropical Guiana Highlands. Academic Press. Saberi-Pirooz, R., Rajabi‐Maham, H., Ahmadzadeh, F., Kiabi, B. H., Javidkar, M., & Carretero, M. A. (2021). Pleistocene climate fluctuations as the major driver of genetic diversity and distribution patterns of the Caspian green lizard, Lacerta strigata Eichwald, 1831. Ecology and Evolution, 11(11), 6927–6940. Sallam, A., & El-Wakeil, N. (2012). Biological and ecological studies on land snails and their control. Integrated Pest Management and Pest Control-Current and Future Tactics, (1). Scharnweber, T., Rietschel, M., & Manthey, M. (2007). Degradation stages of the Hyrcanian forests in southern Azerbaijan. Archiv für Naturschutz und Landschaftsforschung, 46(2), 133–156. Shimizu, Y., & Ueshima, R. (2000). Historical biogeography and interspecific mtDNA introgression in E uhadra peliomphala (the Japanese land snail). Heredity, 85(1), 84–96. Siadati, S., Moradi, H., Attar, F., Etemad, V., Hamzeh’ee, B. E. H. N. A. M., & Naqinezhad, A. (2010). Botanical diversity of Hyrcanian forests; a case study of a transect in the Kheyrud protected lowland mountain forests in northern Iran. Phytotaxa, 7(1), 1–18. Silvan, N., Laiho, R., & Vasander, H. (2000). Changes in mesofauna abundance in peat soils drained for forestry. Forest Ecology and Management, 133(1–2), 127–133. Smith, U. E., & Hendricks, J. R. (2013). Geometric morphometric character suites as phylogenetic data: extracting phylogenetic signal from gastropod shells. Systematic Biology, 62(3), 366–385. Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9), 1312–1313. Starmuhlner, F., & Edlauer, A. (1957). Ergebnisse der österreichischen Iran-Expedi-tion 1949/50: Beiträge zur Kenntnis der Molluskenfauna des Iran. Sitz. Ber. öst. Akad. Wiss., Math.-naturw. Kl., Abt, 1(166), 9. Talebi, K. S., Sajedi, T., & Pourhashemi, M. (2014). Forests of Iran. A treasure from the past, a hope for the future, 10. Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: molecular evolutionary genetics analysis version 11. Molecular biology and evolution, 38(7), 3022–3027. Tarkhnishvili, D., Gavashelishvili, A., & Mumladze, L. (2012). Palaeoclimatic models help to understand current distribution of Caucasian forest species. Biological Journal of the Linnean Society, 105(1), 231–248. Tattersfield, P., Warui, C. M., Seddon, M. B., & Kiringe, J. W. (2001). Land-snail faunas of afromontane forests of Mount Kenya, Kenya: Ecology, diversity and distribution patterns. Journal of Biogeography, 28(7), 843–861. Tattersfield, P., Seddon, M. B., Ngereza, C., & Rowson, B. (2006). Elevational variation in diversity and composition of land-snail faunas in a Tanzanian forest. African Journal of Ecology, 44(1), 47–60. Waugh J (2007) DNA barcoding in animal species: progress, potential and pitfalls. Bioessays, 29, 188–197. Whittaker, R. J., Willis, K. J., & Field, R. (2001). Scale and species richness: towards a general, hierarchical theory of species diversity. Journal of Biogeography, 28(4), 453–470. Willis, K. J., & Whittaker, R. J. (2002). Species diversity–scale matters. Science, 295(5558), 1245–1248. Wronski, T., & Hausdorf, B. (2008). Distribution patterns of land snails in Ugandan rainforest support the existence of Pleistocene forest refugia. Journal of Biogeography, 35(10), 1759–1768. Yassini, I. (1976). Contribution to the study of some terrestrial mollusks of Mazandaran Gilan and Azerbaijan province. Journal of Environmental Studies, 6(6). (In Persian). Zeng, X., Yiu, W. C., Cheung, K. H., Yip, H. Y., Nong, W., He, P., … Hui, J. H. L. (2017). Distribution and current infection status of Biomphalaria straminea in Hong Kong. Parasites & vectors, 10(1), 1–12. Zhang, J., Kapli, P., Pavlidis, P., & Stamatakis, A. (2013). A general species delimitation method with applications to phylogenetic placements. Bioinformatics, 29(22), 2869–2876. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3084235","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":221613111,"identity":"992e2382-0853-4d43-8003-b1e6fe3ec794","order_by":0,"name":"Sima Mohammadi","email":"","orcid":"","institution":"Environmental Sciences Research Institute, Shahid Beheshti University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sima","middleName":"","lastName":"Mohammadi","suffix":""},{"id":221613112,"identity":"bbe9acf7-d939-4412-b98c-9dfdcb8d9108","order_by":1,"name":"Faraham Ahmadzadeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie3RMQrCMBTG8a8U4tLiWgi0V4gIdejQqyiCXSLiIgoOTrp5HHGsPNAlBwi4qEN3l0IXsSAiLlE3h/wghBf4w4MAlvWH0hysvtz6dJ2Ts3i85qZE4JW44ueEBc/ESMAtLnqepM1Vfz+rtoTmKndoakhSsE5b7jM3UMXg6CtCoLrYKfNiMZeMGLSMj86SAA3sTAsKNEoub+RFelSOqzqJPidezIdLCoSWDH6diC+SCR+uM9FSRZv7KvNaqrf4tNiGyzJJw0P/fK22SRgeiK6mBNHpffaAr37HsizLMrkDHoFLtd3L6UYAAAAASUVORK5CYII=","orcid":"","institution":"Environmental Sciences Research Institute, Shahid Beheshti University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Faraham","middleName":"","lastName":"Ahmadzadeh","suffix":""}],"badges":[],"createdAt":"2023-06-19 20:44:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3084235/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3084235/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3389/fevo.2024.1329581","type":"published","date":"2024-04-16T12:24:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":40839667,"identity":"c42baec3-1ea3-4dce-a86a-a9015bc4fb10","added_by":"auto","created_at":"2023-07-31 18:46:43","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":271483,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of study areas and sampling sites\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3084235/v1/82d15b5b47b67a06b8485660.jpeg"},{"id":40839668,"identity":"6c693d3c-b0a7-4cd4-9f3c-cf07a52b77a5","added_by":"auto","created_at":"2023-07-31 18:46:43","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":281615,"visible":true,"origin":"","legend":"\u003cp\u003eThe phylogenetic tree of Hyrcanian land snails using the COI gene. BI and Ml trees show the same tree topology, therefore only the ML tree is illustrated. The number at the branches are posterior probabilities for the Bayesian (above) and bootstrap supports for ML (below). Only values greater than 0.9 and 90 are shown.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3084235/v1/c3d2398c7a7c5e11333c54f2.jpeg"},{"id":40840138,"identity":"92b7bea8-f91a-44dd-8ddf-ddf5e759b6f0","added_by":"auto","created_at":"2023-07-31 18:54:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4235,"visible":true,"origin":"","legend":"\u003cp\u003eLand snails abundance within each site and between forests\u003c/p\u003e","description":"","filename":"Onlinedrawingimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3084235/v1/b4d5d73de5caec7b8767e0cf.png"},{"id":40839666,"identity":"c339e621-aece-48c4-b28b-1296f1c45a5c","added_by":"auto","created_at":"2023-07-31 18:46:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4597,"visible":true,"origin":"","legend":"\u003cp\u003eOTUs richness and diversity within each forest\u003c/p\u003e","description":"","filename":"Onlinedrawingimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3084235/v1/c71f70c4e6f2b9ea90df7ce3.png"},{"id":40840139,"identity":"b8ca291d-e108-45bb-8bf0-70a0b8be554e","added_by":"auto","created_at":"2023-07-31 18:54:43","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":168196,"visible":true,"origin":"","legend":"\u003cp\u003eThe CCA ordination plot illustrates the connection between environmental parameters and the structure of the land snails community in Forest 1, Forest 2, and Forest 3\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3084235/v1/75ae307410bcb3196f50cda0.jpeg"},{"id":40839670,"identity":"de6cde73-d259-46a6-972f-595138ab938c","added_by":"auto","created_at":"2023-07-31 18:46:43","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":514740,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of land snails abundance (A) and OTUs richness (B) within each site\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3084235/v1/329767a66ba2963fdc374446.jpeg"},{"id":54764435,"identity":"fbde0b9f-034f-48ff-8263-6cc3926d6e3e","added_by":"auto","created_at":"2024-04-16 12:24:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":768924,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3084235/v1/60f516e8-a4b1-4100-b67c-bd7821a25dc7.pdf"},{"id":40839672,"identity":"62cf0701-a76c-4772-afef-cbc8d7021ffc","added_by":"auto","created_at":"2023-07-31 18:46:43","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":346811,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3084235/v1/6d994e20bd9e69ba0ac306ed.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Revealing Hidden Diversity and Community Dynamics of Land Snails through DNA Barcoding: Implications for Conservation and Ecological Studies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLand snails belong to the Mollusca phylum, with more than 24,000 recognized species standing as one of the most diverse groups of animals on Earth (Brown, and Lydeard \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chavhan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These ancient species due to their position in the food web as decomposers and their role as an important calcium source for other animals like birds, snakes, and mammals, play a vital role in ensuring the sustainability of their habitat (Rull et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dempsey et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This group of gastropods inhabits a variety of terrestrial environments, including leaf litter, soil, rocks, and trees. Different species have specific habitat preferences based on factors such as moisture, temperature, and soil composition (Jurickova et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Soil chemical parameters, particularly calcium availability, play an important role in their population density and shell formation (Millar and Waite, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Hotopp, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Additionally, land snails\u0026rsquo; dispersal and activity are influenced by elevation. Many studies have shown a decrease in land snail species richness towards high elevations, accompanied by changes in abundance and species composition (Aubry et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Wronski and Hausdorf, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Liew et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Dourson and Langdon, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTheir specific habitat needs and limited mobility make them reliable indicators for evaluating environmental conditions and habitat quality. Furthermore, land snails serve as an ideal model for ecological genetic studies because of their ecological responses to environmental challenges like climate change, introduced species, and habitat fragmentation (Shimizu and Ueshima, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Gaining a comprehensive understanding of the correlation between snail communities and environmental factors, especially in spatial dimensions like altitudinal gradients, provides conservation managers with valuable insights for protecting biodiversity and habitat conservation (Mathias et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Goodacre, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Arnaud et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Jackson and Blois \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nicolai and Ansart, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLand snails are one of the indispensable components of forest ecosystems. In recent years due to several reasons like the presence of invasive or non-native species and, climate change the global forest cover has faced a drastic reduction (da Silva et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). since land snails serve as a bioindicator that offers valuable insights into the overall health and diversity of their ecosystem, evaluating their biodiversity and community characteristics in forest ecosystems yields valuable information for effective forest conservation.\u003c/p\u003e \u003cp\u003eIn this regard, the Hyrcanian forest of Iran provides a fascinating case study. This forest is the most important remnant of the Cenozoic forests and a refuge for ancient species in the last ice age (Tarkhnishvili et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dufresnes et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ahmadzadeh et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Amiri et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Saberi-Pirooz et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and also a unique habitat for endemic land snails. While some studies have identified land snail species in the Hyrcanian forest, there is limited research on community composition and ecological analyses of these snails, with approximately 40 species reported thus far (Issel, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1865\u003c/span\u003e; Forcart, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1935\u003c/span\u003e; Starmuhlner and Edlauer, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1957\u003c/span\u003e; Yasini, 1976; Eliazian et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Mansourian, 2005; Ahmadi, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the identification of snails in these studies was primarily based on morphological characteristics. Despite the extensive research conducted on land snail identification using morphological features in various regions, there remain numerous species with an uncertain systematic status. This lack of clarity can pose significant challenges for ecological studies (Tattersfield et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Land snail morphological identification, which relies on conchology (the study of shells), is prone to frequent misidentification, particularly for small species, due to shell variations influenced by habitat-specific environmental factors (Nekola and Coles, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Smith and Hendricks, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In addition, land snails, like numerous other organisms, can possess cryptic species that exhibit close morphological resemblance despite significant genetic differentiation. Additionally, the expertise required to accurately recall species names and identify morphological aspects of land snails is limited among biologists. (Waugh, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zeng et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mouahid et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Exclusively relying on morphological characteristics for species identification can lead to misidentification, significantly impacting biodiversity evaluation crucial for conservation management (Bickford et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). To address these challenges, integrating morphological identification with other methods, such as DNA barcoding and molecular techniques, can improve the efficiency of land snail species identification. In the past decade, DNA barcoding has emerged as a rapid and highly accurate method for species identification, surpassing traditional morphology-based approaches (Ezzine et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Galan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nantarat et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, our objective was to identify terrestrial snails inhabiting the Hyrcanian forest by employing the DNA barcode method, targeting the COI gene. Following that we assessed their abundance, species richness, and composition along a gradient of elevation, comprising three distinct elevational levels within our study area. By conducting this analysis, we aimed to gain insights into the distribution patterns and ecological dynamics of land snails across different elevations within the forest. We also attempted to determine whether soil parameters were correlated with the presence of land snails. Eventually, we aimed to contribute to a deeper understanding of the ecological dynamics, species composition, and environmental interactions of land snails within this unique forest ecosystem. Such knowledge can be invaluable for conservation efforts, ecosystem management, and the preservation of biodiversity in the Hyrcanian forest and similar habitats.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eHyrcanian forests which are known as one of the most important biomes of the world extend from the Talish region in the southeast of the Republic of Azerbaijan to the Golestan Province in the northeast of Iran, with an area of approximately 50000 km\u003csup\u003e2\u003c/sup\u003e. These forests stretch from sea level to elevations of 2800 meters, three altitudinal belts were recognized across the Hyrcanian forests, i.e. lowland, sub-montane, and montane forests (Hamzeh\u0026rsquo;ee \u003cem\u003eet al\u003c/em\u003e., 2008; Siadati et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Spatial analysis of seasonal precipitation shows that the Hyrcanian region has different patterns in the western and eastern parts and annual rainfall decreases from west to east (Akhani et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The ecological coefficient of drought in the region is insignificant, as a whole, the Hyrcanian climate in the east is the warm Mediterranean, and in the central and western parts is temperate and semi-temperate Mediterranean, and from time to time temperate and dry (Talebi et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This special natural world heritage is a unique source of biodiversity for the Northern Hemisphere which has universal importance beyond political boundaries (Gutleb and Wieser, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Furthermore, these forests provide valuable ecosystem services that are vital for local people and these benefits depend on the biodiversity of these forests (Scharnweber et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Tohidfar \u003cem\u003eet al\u003c/em\u003e., 2016).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSampling\u003c/h2\u003e \u003cp\u003eSnail collecting was carried out in Spring 2021. Due to land snails being nocturnal, sampling was performed on cloudy days (Sallam and El-Wakeil \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Three locations were selected for sampling along with Caspian Hyrcanian forests in Kordkoy (Forest 1), Amol (Forest 2), and Tonekabon (Forest 3). At each location, three sampling sites were specified in an elevation slope approximately 170m to 1600m height from sea level (Elevation 1, Elevation 2, and Elevation 3 that demonstrate the highest elevation to lowest elevation in each location respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Three plots of 3m\u0026times;3m were applied over an area of about 150m\u003csup\u003e2\u003c/sup\u003e in each elevation (Clergeau et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Horsak et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nurinsiyah et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Snail species were collected by hand and then relaxed in the water for a day. Subsequently, they were preserved in 96% ethanol. A total of 679 samples were collected from 27 plots across nine elevations. Furthermore, topsoil was collected from each plot and transported in a plastic bag to the laboratory for measurement of some physicochemical parameters. In the following F1, F2, and, F3 refer to Forest 1, Forest 2, and Forest 3, and also E1, E2, and E3 refer to Elevation 1, Elevation 2, and Elevation 3 respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLocations of the sampling sites\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElevation (m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLatitude\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLongitude\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKordkoy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.701583 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.10025 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF1-E1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKordkoy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e609\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.714306 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.105917 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF1-E2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKordkoy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.743389 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.117028 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF1-E3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.358169 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.313897 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF2-E1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e423\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.369101 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.320888 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF2-E2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.396889 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.3405 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF2-E3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTonekabon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1611\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.480142 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.856114 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF3-E1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTonekabon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.591972 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.835333 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF3-E2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTonekabon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e492\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.662722 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.812583 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory procedures\u003c/h2\u003e \u003cp\u003eLand snail samples were assigned into morphospecies in the laboratory. The identification of individuals was done by available morphological keys identification (Eliazian et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Dourson and Dourson, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Perez et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Getz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A total of 16 morphospecies were distinguished from the collected samples within the study area. For the molecular investigation, 37 specimens (a minimum of two samples from each morphospecies) were chosen. Total DNA of selected samples was extracted using a high-salt method (Sambrook and Russell, 2001). A fragment of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene was used for the barcoding study. The primers employed in this study encompassed LCO 1490/HCO 2198 (Folmer et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and HCO2198-JJ/LCO1490-JJ (Astrin and St\u0026uuml;ben, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Polymerase chain reactions (PCRs) were applied in a total volume of 25 \u0026micro;l, including 10.5 \u0026micro;l of ddH\u003csub\u003e2\u003c/sub\u003eO, 12.5 \u0026micro;l of Mastermix Red, (Amplicon, Copenhagen, Denmark) 0.5-1 \u0026micro;l of each primer (10 pmol/\u0026micro;l) and 1 \u0026micro;l of template DNA. PCR cycling for LCO 1490/HCO 2198 was run under the following condition: 94˚C for 5 min, 94˚C for 30 S (repeated for 35 cycles), 48˚C for 30 S, 72˚C for 1 min, and finally, an extension cycle at 72˚C for 5 min and the PCR condition for HCO2198-JJ/LCO1490-JJ was 94˚C for 1 min, 94˚C for 30 S (repeated for 5 cycles), 47˚C for 90 S, 72˚C for 1 min, 94˚C for 30 S (repeated for 35 cycles), 51˚C for 90 S, 72˚C for 1 min and a final extension cycle at 72˚C for 5 min. PCR products quality were surveyed using 1% agarose gel stained with Safe-Red. Then the acceptable amplicons were sent to Pishgam Company (Tehran, Iran) for purification and sequencing.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eMeasurement of soil Physico-chemical properties\u003c/h2\u003e \u003cp\u003eSoil samples were obtained from the central area of each plot using a spade, reaching a depth of up to 10 cm, to assess the physicochemical properties of the soil. The collected soil samples from three plots of each elevation were combined. Six factors including soil pH, %saturation moisture, total calcium (Ca), exchangeable calcium (Ca\u003csup\u003e++\u003c/sup\u003e), total magnesium (Mg), and exchangeable magnesium (Mg\u003csup\u003e++\u003c/sup\u003e) measured using standard methods by Research Institute of Forest and Rangeland (Tehran, Iran).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGenetic analysis\u003c/h2\u003e \u003cp\u003eDNA sequences of 36 individuals were edited using Geneious Prime v. 2022.1.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://orcid.org/0000-0002-8000-2916\" target=\"_blank\"\u003ewww.geneious.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.geneious.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All newly obtained sequences were deposited to GenBank (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The sequences were checked in NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by the Standard Nucleotide Basic Local Alignment Search Tool (BLASTn) (Altschul et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) and also Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert, 200; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.boldsystems.org/\u003c/span\u003e\u003cspan address=\"https://www.boldsystems.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Alignments were obtained with MAFFT 7 online version (Katoh et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The best-fit evolutionary model was determined GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G model using MrModeltest V.2.3 (Nylander, 2004) according to Akaike\u0026rsquo;s Information Criterion (Akaike, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). Phylogenetic trees were constructed using both Bayesian Inference (BI) and maximum likelihood (ML) methods. \u003cem\u003eEnchytraeidae\u003c/em\u003e sp. was considered as an outgroup. MrBayes was accomplished with MrBayes version 3.2. (Ronquist et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) under two independent runs (four chains for each run) for 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e generations and sampling trees every 100 generations. Finally, 10% of the trees were cast off as burn-in. Tracer V.1.7 (Rambaut et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) was used to assess the efficiency of runs. Maximum Likelihood tree was programmed with RAxML v.8.2 (Stamatakis, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) under the GTRGAMMA model with 1000 pseudoreplicates. Uncorrected genetic distances (\u003cem\u003ep\u003c/em\u003e-distances) among species were calculated by MEGA11 (Tamura et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of land snails identified in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCode ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFamily\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGenus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLocation Code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAccession number\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189157\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3388\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189159\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3317\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3353\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189156\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3303\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189151\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189152\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF2-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189149\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189155\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3338\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclophoridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF2-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189147\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePomatiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePomatias\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePomatias Rivulare\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189154\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePomatiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePomatias\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePomatias Rivulare\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF2-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePomatiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePomatias\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePomatias Rivulare\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189145\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePomatiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePomatias\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePomatias Rivulare\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF2-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189170\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189173\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189176\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189179\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189161\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3436\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF2-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189177\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189168\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189178\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaucasotachea Leuaranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHygromiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePyrenaearia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189169\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHygromiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePyrenaearia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189165\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHygromiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePyrenaearia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189166\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHygromiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePyrenaearia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189167\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTheba\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3373\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHelicidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTheba\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189164\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3434\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHygromiidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDioscuria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189175\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePupillidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePupilla\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189174\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3425\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePupillidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePupilla\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189172\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3360\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrculidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSchileykula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF3-E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES3328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOxychilidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eOxychilus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eOxychilus filicum -\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF1-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR189163\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSpecies-delimitation\u003c/h2\u003e \u003cp\u003eTo infer hypothetical candidate species, two widely-used statistical methods of species delimitation were employed (automatic barcode gap discovery (ABGD (Puillandre et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)) and Bayesian implementation of the Poisson tree processes (bPTP (Zhang et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2013\u003c/span\u003e))). ABGD analysis is a clustering method based on the pairwise distance between species sequences. It was performed on ABGD Web Server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html\u003c/span\u003e\u003cspan address=\"https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using Kimura (K80) distance with the default Ts/Tv ratio (2.0) and the following conditions: range of prior intraspecific divergence from 0.01(Pmin) to 0.1 (Pmax) with 20 steps in between, X (relative gap width): 0.05 and Nbin: 10 (Mason et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ebPTP is a tree-based method that required a non-ultrametric phylogenetic tree as input. Analysis was carried out on the bPTP web server (Species delimitation server (h-its.org)) using a rooted RAxML tree for 500,000 MCMC generations, with 0.1 of samples conservatively discarded as burn-in (Mason et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using R v. 4.0.3 (R Core Team., 2020). Two-way analysis of variance (two-way ANOVA), followed by two-sided Tukey HSD tests, was conducted to assess the differences in total snail density, total OTUs diversity, and Shannon-Wiener diversity index (biodiversity metrics) among different elevations (three levels) in each forest. The statistical analyses were conducted using the \"aov()\" function in R. Assumptions of homogeneity of variances (tested with Levene's test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and normality of residuals (tested with Shapiro-Wilk test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were checked using the R package v. 1.2-7 in R (Fox \u003cem\u003eet al\u003c/em\u003e., 2007).\u003c/p\u003e \u003cp\u003eTo assess the dissimilarities among snail communities at different elevations within each forest (β-diversity), we utilized the betapart package (Baselga et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) in R with the Jaccard dissimilarity index. β-diversity was employed to measure how species compositions among samples have changed across the area (Graham and Fine, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This approach offers a theoretical framework for partitioning total dissimilarity into nestedness (species gains or losses) and turnover components (species replacement) (Baselga, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiodiversity metrics were calculated to assess the snail communities using various measures. The Shannon-Wiener diversity index (H) was calculated using the vegan package (v. 1.17-8) in R. Additionally, the R package Picante (Kembel et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) was utilized to calculate phylogenetic diversity (PD) and mean pairwise distance (MPD) between pairs of communities.\u003c/p\u003e \u003cp\u003eTo examine the spatial compositional differences in snail compositions, permutational multivariate analysis of variance (PERMANOVA) was employed. Permutations of residuals (n\u0026thinsp;=\u0026thinsp;9999) were performed under the reduced model, following the same factorial design as the univariate analysis. The Sorensen dissimilarity matrices were used for the analysis. In cases where significant differences were found, the SIMPER routine was applied to determine the proportional contribution of each species and main group to the observed pairwise dissimilarity in composition. The SIMPER results included species with a cumulative dissimilarity cut-off of 70% (Clarke, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Both the PERMANOVA and SIMPER analyses were conducted using the vegan package.\u003c/p\u003e \u003cp\u003eAdditionally, Canonical Correspondence Analysis (CCA) was employed as a multivariate statistical approach to investigate the relationship between species and environmental variables. The analysis utilized a dataset containing the Operational OTUs present in each forest, and six soil variables were selected as explanatory factors. Before conducting the analysis, the environmental data underwent initial screening using the Principal Component Analysis (PCA) methodology. The entire variation in the environmental data was explained by the initial six principal components (PCs). Prominent variables were identified by considering the highest absolute loading values within each PC. Subsequently, two parameters were chosen for further analysis after the screening process.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMolecular and phylogenetic analyses\u003c/h2\u003e \u003cp\u003eBased on Cytochrome oxidase I (COI) (596bp) sequences of 37 individuals, three sequences could be assigned to species named \u003cem\u003ePomatias rivulare, Oxychilus filicum\u003c/em\u003e, and \u003cem\u003eCaucasotachea leucoranea\u003c/em\u003e and six of them were assigned to five genera named \u003cem\u003ePyrenaearia\u003c/em\u003e, \u003cem\u003eTheba\u003c/em\u003e, \u003cem\u003eDioscuria\u003c/em\u003e, \u003cem\u003ePupilla\u003c/em\u003e, and \u003cem\u003eSchileykula\u003c/em\u003e. one Sequences weren\u0026rsquo;t assigned with any sequences in NCBI and BOLD data sets which means it\u0026rsquo;s the first barcoding study on this species. Based on shell features this OTU was identified as \u003cem\u003eCaspicyclotus sieversi\u003c/em\u003e (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003cem\u003ep\u003c/em\u003e-distance between OTUs ranged from 0.016 to 0.35. Our criterion for categorization of sequences to OTU was more than eight percent genetic distances among sequences (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Species delimitation with bPTP resulted in 10 putative species for the dataset and most of them were supported with high values (\u0026gt;\u0026thinsp;80%). The ABGD analysis yielded 7\u0026ndash;17 hypothetical OTU using about 0.1\u0026ndash;0.01 maximum intraspecific divergence (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Furthermore, the phylogenetic tree revealed similar terminals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Totally, 10 OTUs were recognized in this study which is categorized into seven families named Cyclophoridae, Pomatiidae, Helicidae, Hygromiidae, Pupillidae, Orculidae, and Oxychilidae.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUncorrected genetic distances (p - distances) between land snail OTUs based on COI gene\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOTU 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOTU 4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOTU 5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOTU 6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOTU 7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eOTU 8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eOTU 9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eOTU 10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTU 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComparison of land snail abundance and OTUs diversity\u003c/h2\u003e \u003cp\u003eExploring the abundance of land snails between three forests shows a considerable difference in snail abundance in Forest 3. As the bar chart illustrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) there is a bit of decline in abundance from Forest 1 to Forets 2 then it rises by far in Forest 3. In addition, comparing three elevations in each forest demonstrate that lowlands have less abundance than the highest elevations in all forests. Comparing OTUs richness in all forests indicated the number of OTUs had decreased in high elevations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall, the highest elevation in Forest 3 (F3-E1) shows the most abundance and the maximum number of OTUs found in the lowest elevation of this forest.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of ANOVA indicated significant effects of elevations, forests, and interaction (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Based on the total OUTs diversity and also Shannon-Wiener diversity dataset, t-test analysis showed that there was a significant difference between high elevation (E1) and lowland (E3) in Forest 2. Additionally, the highest elevation in Forest 1 exhibited significant differences when compared to the mid and low elevations within this forest (E2 and E3). Furthermore, the density of OTUs in the lower elevation of Forest 3 (E3) differed significantly from both the high elevation (E1) and mid-elevation (E2) in this forest, also Forest 3 was different from the other two locations in the density of OTUs. In addition, the results indicate a significant different between highlands and lowlands in Forest 2 and Forest 3 (See Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of statistical analysis of ANOVA including Density of total snails, diversity of OTUs, and Shannon-Wiener diversity index\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eData set\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eANOVA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForest\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eElevation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eForest: Elevation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDensity of total snails\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003csub\u003e2,18\u003c/sub\u003e = 170.67 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.99e-12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003csub\u003e2,18\u003c/sub\u003e = 152.22 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.26e-12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003csub\u003e4,18\u003c/sub\u003e = 87.89 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.48e-11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiversity of total OTUs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003csub\u003e2,18\u003c/sub\u003e = 19.44 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.18e-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003csub\u003e2,18\u003c/sub\u003e = 49.78 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.63e-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003csub\u003e4,18\u003c/sub\u003e = 12.44 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.00e-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShannon-Wiener diversity index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003csub\u003e2,18\u003c/sub\u003e = 10.693 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003csub\u003e2,18\u003c/sub\u003e = 55.988 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.87e-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003csub\u003e4,18\u003c/sub\u003e = 3.928 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01831\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eInvestigation of β-diversity partitioning figured out that the spatial turnover portion occurred between three elevations in Forest 3. However, Forest 2 and Forest 1 had different results, and nestedness was also detected. Generally, the β-diversity index was between 0.5 to 0.8 in all datasets (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All forests showed an increase in PD values as elevation increased. Conversely, the MPD values remained relatively consistent across the three elevations within each forest. The result of the Shannon-Wiener diversity index is shown in Table S2.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eβ-diversity, phylogenetic diversity (PD), mean pairwise distance (MPD) for each forest and every elevation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDate Set\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eBeta diversity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMPD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTurnover\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNestedness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal β-diversity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForest 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(E1 and E2)\u003c/p\u003e \u003cp\u003e(E1 and E3)\u003c/p\u003e \u003cp\u003e(E2 and E3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003cp\u003e0.25\u003c/p\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003cp\u003e0.50\u003c/p\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003cp\u003e0.75\u003c/p\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE1: 0.16\u003c/p\u003e \u003cp\u003eE2: 0.42\u003c/p\u003e \u003cp\u003eE3: 0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003cp\u003e0.22\u003c/p\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForest 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(E1 and E2)\u003c/p\u003e \u003cp\u003e(E1 and E3)\u003c/p\u003e \u003cp\u003e(E2 and E3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003cp\u003e0.44\u003c/p\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003cp\u003e0.17\u003c/p\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003cp\u003e0.61\u003c/p\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE1: 0.15\u003c/p\u003e \u003cp\u003eE2: 0.30\u003c/p\u003e \u003cp\u003eE3: 0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003cp\u003e0.10\u003c/p\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForest 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(E1 and E2)\u003c/p\u003e \u003cp\u003e(E1 and E3)\u003c/p\u003e \u003cp\u003e(E2 and E3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003cp\u003e0.76\u003c/p\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003cp\u003e0.06\u003c/p\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.8275\u003c/p\u003e \u003cp\u003e0.82\u003c/p\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE1: 0.46\u003c/p\u003e \u003cp\u003eE2: 0.60\u003c/p\u003e \u003cp\u003eE3: 0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003cp\u003e0.25\u003c/p\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssemblage composition\u003c/h2\u003e \u003cp\u003eThe PERMANOVA indicated significant effects of elevation levels (F\u003csub\u003e2,3\u003c/sub\u003e= 3.8246, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), forests (F\u003csub\u003e1,3\u003c/sub\u003e= 7.5693, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), and their interactions (F\u003csub\u003e1,3\u003c/sub\u003e= 6.0400, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) on the OTUs composition OTUs. SIMPER analysis indicated that three OTUs including OUT 1, OUT 2, and OUT 3 were the most responsible groups for the observed significant difference between all of the comparisons (elevation levels and forests). The list of OTUs that have differences between elevations and forests is shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOutput from SIMPER analysis of the difference in OTUs composition between three forests and three elevations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSIMPER\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eData set\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResponsible species\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForest 1*Forest 2\u003c/p\u003e \u003cp\u003eForest 1*Forest 3\u003c/p\u003e \u003cp\u003eForest 2*Forest 3\u003c/p\u003e \u003cp\u003eElevation 1*Elevation 2\u003c/p\u003e \u003cp\u003eElevation 1*Elevation 3\u003c/p\u003e \u003cp\u003eElevation 2*Elevation 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTU 1, OTU 2, OTU3\u003c/p\u003e \u003cp\u003eOTU 1, OTU 2, OTU3, OTU 8, OTU 10\u003c/p\u003e \u003cp\u003eOTU 1, OTU 2, OTU3, OTU 8\u003c/p\u003e \u003cp\u003eOTU 1, OTU 2, OTU3, OTU 8\u003c/p\u003e \u003cp\u003eOTU 1, OTU 2, OTU3, OTU 4, OTU 5\u003c/p\u003e \u003cp\u003eOTU 1, OTU 2, OTU3, OTU 4, OTU 8, OTU 10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRelationship between snail communities and environmental variables\u003c/h2\u003e \u003cp\u003eThe measurement of soil parameters (pH, Ca, Mg, Ca\u003csup\u003e++\u003c/sup\u003e, Mg\u003csup\u003e++\u003c/sup\u003e. and %saturation moisture) are shown in Table S3. The range of pH values were varied from 6.1 (F1-E1) to 7.7 (F3-E1). According to our results, Forest 2 had the highest Ca content (44 mg) observed in E3, and Forest 1 at E2 had the lowest amount (16 mg). The Ca\u003csup\u003e++\u003c/sup\u003e content (6.09%) was found in F2-E1 and the minimum amount (0.79%) was recorded in F3-E2. Forest 1 exhibited the highest Mg content (44 meq/lit) at E1, whereas F3-E1 had the lowest amount (4 meq/lit). The highest Mg\u003csup\u003e++\u003c/sup\u003e content (2.21%) was observed in F3-E1, and the lowest amount (0.49%) was recorded in F2-E3. The highest content of %saturation moisture (=\u0026thinsp;95) was recorded in F3-E3 and the lowest amount (=\u0026thinsp;51.9) was observed in F3-E1.\u003c/p\u003e \u003cp\u003eTo determine the environmental factors responsible for shaping the composition of snail communities in each forest, CCA was conducted using OTUs data and the selected environmental parameters after PCA analysis. According to PCA analysis, %saturation moisture and Ca in Forest 1, %saturation moisture and Mg in Forest 2, and in Forest 3, Ca and Mg have more contribution to each forest. In Forest 1, the selected variables explained 88% of the total variation in OTUs composition. Following that in Forest 2 and Forest 3, the physicochemical variables explained 53% and 69% of the total variation in OTU composition. The comparison of variations explained by constrained and unconstrained axes revealed\u0026thinsp;\u0026gt;\u0026thinsp;82% efficiency of the constrained axes 1 and 2 in all three forests (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study is the first genetic investigation on terrestrial snail diversity in the Hyrcanian forest (in the northern part of Iran), which lead to the identification of 10 OTUs in the area based on COI gene sequences. Furthermore, land snails abundance and OTUs diversity were compared in an elevational incline and the results showed that elevation affected snail communities. In addition, the survey on soil physicochemical parameters displayed a correlation between some soil features and land snails' presence.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIdentifying land snails\u003c/h2\u003e \u003cp\u003eIn our current research, both morphological and molecular approaches were employed to identify land snails and assess their species diversity. A total of 679 individuals were investigated, and 37 sequences were analyzed, leading to the recognition of seven families, nine genera, and four species (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The ABGD and bPTP methods were used to analyze species delimitation, resulting in the identification of approximately 7 to 17 potential species and also 10 putative species through ABGD and bPTP respectively. By considering our findings on morphology, species delimitations, genetic distances, and the phylogenetic tree, we have determined 10 OTUs within the collected samples.\u003c/p\u003e \u003cp\u003eAmong 10 OTUs identified in our research four of them including OUT 1, OUT 2, OUT 3, and OUT 10 refer to \u003cem\u003eCaspicylotus sieversi, Pomatias rivulare, Caucasotachea leuaranea\u003c/em\u003e, and \u003cem\u003eOxychilus filicum\u003c/em\u003e respectively reported in the previous studies from the region (Issel \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1865\u003c/span\u003e; Forcart \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1935\u003c/span\u003e; Starmuhlner and Edlauer \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1957\u003c/span\u003e; Yassini \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Eliazian et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Mansoorian, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ahmadi; \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). It is worth noting that the remaining six OTUs which referred to five genera named \u003cem\u003ePyrenaearia\u003c/em\u003e, \u003cem\u003eTheba\u003c/em\u003e, \u003cem\u003eDioscuria\u003c/em\u003e, \u003cem\u003ePupilla\u003c/em\u003e, and \u003cem\u003eShileykula\u003c/em\u003e are the first documented occurrences of these genera in the Hyrcanian Forest of Iran. All of the aforementioned studies, just relied on morphological approaches, however in this study we used molecular methods too. We believe that recognition of snails using morphological approach may lead to misidentification because of their morphological variety and cryptic diversity. Therefore we suggest that a combination of molecular approaches and morphological taxonomy should be employed as the primary method for species identification, aiming to achieve more dependable results in land snail identification.\u003c/p\u003e \u003cp\u003eAlthough land snails exhibit substantial mitochondrial diversity and are among the most diverse animals, there have been limited barcoding studies conducted on them, with the majority of investigations focusing on marine gastropods (Meyer and Paulary, 2005; Kelly et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Campbell et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Consequently, there is a lack of comprehensive genetic data available in the GenBank dataset for barcoding studies and the COI gene dataset alone cannot currently serve as a practical method for identifying land snail species. However, it can serve as a reliable tool for assessing their biodiversity (Davison et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ranasinghe et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe ANOVA analysis revealed significant differences in the total density of land snails among different elevations within each forest. In addition, evaluating the abundance in three elevations at each forest indicates the abundance of snails in the highest elevation is more than in the lowlands (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (A)). A possible explanation for this difference is precipitation patterns and humidity levels in this area. Highlands in Hyrcanian forests are more humid, and also precipitation increases at higher elevations (Talebi et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In line with our findings, Tattersfield et al (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) found out land snail abundance increases in high-elevation sets. In addition, De Chavez and De Lara (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) observed a positive relationship between land snail abundance and elevation in the Mount Makiling rainforest in the Philippines based on their observation. Although, in some cases has been obtained different results. Tattersfield et al., (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) expressed that land snail abundance in Forest habitats in mountain Kenya decreases with enhancing elevation and also Liew et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) concluded that there is no relationship between land snail abundance and elevation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eComparing OTUs richness in each forest displays that the number of OTUs gradually increases from high elevation to low elevation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (B)). Additionally, the ANOVA revealed a significant distinction in Shannon-wiener diversity and total diversity of OTUs between three elevations across all forests. Similarly, the PD analyses confirm this pattern, showing an inverse relationship between PD values and increasing elevation. This pattern follows the common law for the most group of organisms in all environments (Gaston, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). As elevation affects many climatic variations including temperature and precipitation it can play an important role in habitat suitability. Precipitation enhancement, the longer period of cold temperatures besides less productivity in highlands cause a decrease in the amount of available energy in these areas. Thus the species-energy relationship (the relationship between energy availability and species richness) may clarify this pattern between species richness and elevation. Receiving a specified content of energy can preserve a specified number of species which means higher elevations shelter fewer species than lowlands (Gaston, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Whittaker et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Willis and Whittaker, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Aubry et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral investigations reported an increase in land snails species richness and species density in an elevation slope; Tattersfield et al. (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) achieve the same result for forest habitats in Kenya, they remarked rainfall as the most important factor for land snail communities, as well as Aubry et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) observed the same pattern in south-eastern France. Based on their research different factors such as climate altitudinal slope and also land cover heterogeneities can explain this pattern. Liew et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) reported a decline in land snail and slugs species density with elevation as a result of lessen nich diversity in high elevations and also historical events which lead to snail upward migration. However, Tattersfield et al. (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) observed an increase in land snail richness and species density in elevation in Mwanihana Forest in Tanzania.\u003c/p\u003e \u003cp\u003eComparing three forests altogether revealed that Forest 3 which is located in the western part of the study area was different from the two other forests with higher OTUs richness and abundance. Considering that land snails are highly dependent on humidity, the observed differences can be explained by the decreasing precipitation pattern along the southern Caspian Sea shoreline from east to west. In addition, ecological dryness declined from the east toward the west of the Hyrcanian Forests and the climate in the western part is more humid than the eastern part (Shimizu and Ueshima, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Talebi et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the PERMANOVA analysis there were significant differences in snail composition across various elevations and among the three forests under investigation. These findings highlight the intricate ecological patterns and distinct species assemblages within each forest ecosystem. In general, some OTUs were exclusively identified in certain forests, indicating unique ecological characteristics that allow them to thrive in specific habitats. Five OTUs including OTU 4, OTU 5, OTU 6, OTU 7, and OTU 9 were observed only in Forest 3 and Forest 1 exhibited the exclusive presence of OTU 10. Furthermore, three OTUs (OTU 1, OTU 2, and OTU 3) were observed, across all forests. The SIMPER analysis highlighted these three OTUs are responsible for observed differences among forests and elevation levels. Additionally, the β-diversity partitioning results showed that the spatial turnover component played a dominant role in driving dissimilarity within Forest 2 and Forest 3 (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This finding implies that species replacement exerts a more substantial influence on the observed variation than differences in species richness. Conversely, in Forest 1, dissimilarity was primarily attributed to nestedness, indicating that dissimilarities were influenced by disparities in species richness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEnvironmental variable\u003c/h2\u003e \u003cp\u003eThe result of PCA revealed that Ca, Mg, and %saturation moisture have more contribution in physicochemical soil factors. These factors exhibited positive trends with most of OTUs (see CCA results, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Because these environmental factors play a crucial role in shaping the distribution and diversity of snail populations. Calcium and magnesium are essential for land snails' shell development and overall functions. Evidence from previous studies consistently supports the notion that land snail abundance and species richness tend to be higher in areas characterized by elevated levels of calcium (Ca), magnesium (Mg), and moisture (Silvan et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Hotopp, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Snails flourish in regions with ample soil levels of these minerals, resulting in larger populations and more diverse species (Nekola and Smith, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Millar and Waite, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Moisture availability also plays a significant role in determining snail abundance and richness. Areas with higher moisture content in the soil create favorable conditions for snails, as it provides them with a suitable microhabitat (Getz and Uetz, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Morecroft \u003cem\u003eet al\u003c/em\u003e., 2002).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis survey represents the first barcoding study conducted on land snail species within the Hyrcanian Forest, resulting in the identification of 10 OTUs across the region. Notably, this study reports the occurrence of five genera, namely \u003cem\u003ePyrenaearia\u003c/em\u003e, \u003cem\u003eTheba\u003c/em\u003e, \u003cem\u003eDioscuria\u003c/em\u003e, \u003cem\u003ePupilla\u003c/em\u003e, and \u003cem\u003eSchileykula\u003c/em\u003e, for the first time within this geographical area. Furthermore, the examination of land snail communities along a gradient of elevation revealed significant alternation in their abundance, richness, and community composition. Soil physicochemical parameters analyses revealed that Ca, Mg, and %saturation moisture are the most important factors which impact snail distribution. This foundational study offers new insights into the snail community within the Hyrcanian forest, providing valuable data that can be utilized for future research in this globally significant world heritage site and similar habitats elsewhere. Further barcoding studies in different parts of the region is recomeded for future studies to uncover additional species as well as consider other environmental factors such as climatic variables can contribute to a better understanding of land snail communities along elevational slopes. These efforts will play a significant role in offering valuable insights into the conservation of snail biodiversity and the preservation of their habitats.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available in the [NCBI] repository, [ACCESSION NUMBERs are represented in table 2 in the article].\u0026nbsp;Additionally, the datasets generated in this study are accessible upon request from the corresponding author (\u003cu\[email protected]\u003c/u\u003e) in accordance with reasonable terms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe wish to express our gratitude to everyone who contributed to the completion of this article. Special thanks go to Reihaneh Saberi-Pirooz for her invaluable assistance in data analysis and the preparation of the paper. Her expertise and dedication significantly enhanced the quality of our research. Furthermore, we would like to express our profound appreciation to Khashayar Allahbedashti for his unwavering support and significant contributions during the sampling phase of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmadi, E. (2012). Identification of snails in citrus orchards in Mazandaran province, Iran. Journal of Field Crop Entomology. 2(1), 57\u0026ndash;64. (In Persian).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmadzadeh, F., Shahrokhi, G., Saberi-Pirooz, R., Oladi, M., Taati, M., Poyarkov, N. A., \u0026amp; R\u0026ouml;dder, D. (2020). Alborz Heritage: geographic distribution and genetic differentiation of the Iranian Paradactylodon (Amphibia: Hynobiidae). Amphibia-Reptilia, 41(4), 519\u0026ndash;534.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkaike, H. (1974). A new look at the statistical model identification. IEEE Transactions on automatic control, 19(6), 716\u0026ndash;723.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkhani, H., Djamali, M., Ghorbanalizadeh, A., \u0026amp; Ramezani, E. (2010). Plant biodiversity of Hyrcanian relict forests, N Iran: an overview of the flora, vegetation, palaeoecology, and conservation. Pakistan Journal of Botany, 42(1), 231\u0026ndash;258.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403\u0026ndash;410.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmiri, N., Vaissi, S., Aghamir, F., Saberi-Pirooz, R., R\u0026ouml;dder, D., Ebrahimi, E., \u0026amp; Ahmadzadeh, F. (2021). Tracking climate change in the spatial distribution pattern and the phylogeographic structure of Hyrcanian wood frog, Rana pseudodalmatina (Anura: Ranidae). Journal of Zoological Systematics and Evolutionary Research, 59(7), 1604\u0026ndash;1619.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArnaud, J. F., Madec, L., Guiller, A., \u0026amp; Deunff, J. (2003). Population genetic structure in a human-disturbed environment: a case study in the land snail Helix aspersa (Gastropoda: Pulmonata). Heredity, 90(6), 451\u0026ndash;458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAstrin, J. J., \u0026amp; St\u0026uuml;ben, P. E. (2008). Phylogeny in cryptic weevils: molecules, morphology and new genera of western Palaearctic Cryptorhynchinae (Coleoptera: Curculionidae). Invertebrate systematics, 22(5), 503\u0026ndash;522.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAubry, S., Magnin, F., Bonnet, V., \u0026amp; Preece, R. C. (2005). Multi-scale altitudinal patterns in species richness of land snail communities in south‐eastern France. Journal of Biogeography, 32(6), 985\u0026ndash;998.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaselga, A. (2010). Partitioning the turnover and nestedness components of beta diversity. Global ecology and biogeography, 19(1), 134\u0026ndash;143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaselga, A., Orme, D., Villeger, S., De Bortoli, J., Leprieur, F., Baselga, M.A. (2018). Package \u0026lsquo;betapart.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeck, M. W. (2017). ggord: Ordination Plots with ggplot2. R package version 1.0. 0.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBickford, D., Lohman, D. J., Sodhi, N. S., Ng, P. K., Meier, R., Winker, K., \u0026hellip; Das, I. (2007). Cryptic species as a window on diversity and conservation. Trends in ecology \u0026amp; evolution, 22(3), 148\u0026ndash;155.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown, K. M., \u0026amp; Lydeard, C. (2010). Mollusca: gastropoda. In Ecology and classification of North American freshwater invertebrates (pp.\u0026nbsp;277\u0026ndash;306). Academic Press.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampbell, D. C., Johnson, P. D., Williams, J. D., Rindsberg, A. K., Serb, J. M., Small, K. K., \u0026amp; Lydeard, C. (2008). Identification of \u0026lsquo;extinct freshwater mussel species using DNA barcoding. Molecular Ecology Resources, 8(4), 711\u0026ndash;724.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChavhan, A., Pawar, S. and Jadhao, R. (2015). Study of Biodiversity of Terrestrial Snail in Selected Locality of Amravati City, Central India. Indian Journal of Applied Research, 5(8), 713\u0026ndash;714.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClarke, K. R. (1993). Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology, 18(1), 117\u0026ndash;143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClergeau, P., Tapko, N., \u0026amp; Fontaine, B. (2011). A simplified method for conducting ecological studies of land snail communities in urban landscapes. Ecological research, 26(3), 515\u0026ndash;521.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva, L. P., Heleno, R. H., Costa, J. M., Valente, M., Mata, V. A., Gon\u0026ccedil;alves, S. C., \u0026hellip; Ramos, J. A. (2019). Natural woodlands hold more diverse, abundant, and unique biota than novel anthropogenic forests: a multi-group assessment. European Journal of Forest Research, 138(3), 461\u0026ndash;472.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavison, A., Blackie, R. L., \u0026amp; Scothern, G. P. (2009). DNA barcoding of stylommatophoran land snails: a test of existing sequences. Molecular Ecology Resources, 9(4), 1092\u0026ndash;1101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Chavez, E. R. C., \u0026amp; De Lara, A. V. (2010). Diversity and spatial distribution patterns of macro land snails in Mount Makiling Forest Reserve, Philippines. Asia Life Sciences-The Asian International Journal of Life Sciences, 20(1), 183\u0026ndash;197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDempsey, Z. W., Goater, C. P., \u0026amp; Burg, T. M. (2020). Living on the edge: comparative phylogeography and phylogenetics of Oreohelix land snails at their range edge in Western Canada. BMC evolutionary biology, 20(1), 1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Salle R, Egan MG, Siddall M (2005). The unholy trinity: taxonomy, species delimitation and DNA barcoding. Philosophical Transactions of the Royal Society B: Biological Sciences, 360, 1905.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDufresnes, C., Litvinchuk, S. N., Leuenberger, J., Ghali, K., Zinenko, O., St\u0026ouml;ck, M., \u0026amp; Perrin, N. (2016). Evolutionary melting pots: a biodiversity hotspot shaped by ring diversifications around the Black Sea in the Eastern tree frog (Hyla orientalis). Molecular Ecology, 25(17), 4285\u0026ndash;4300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDourson, D. and Dourson, J. (2006). Land snails of the great smoky mountains (Eastern Region). Appalachian Highlands Science Learning Center, Purchase Knob, Great Smokey Mountains National Park.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDourson, D. C., \u0026amp; Langdon, K. (2012). Land Snails of Selected Rare High Elevation Forests and Heath Balds of the Great Smoky Mountains National Park. Journal of North Carolina Academy of Science, 128(2), 27\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEliazian, M., Tamiji, Y., Akbarzadeh, M., \u0026amp; Hagh-Nazari, J. (1979). Snails from the northern parts of Iran (Caspian Area). Archives of Razi Institute, 31(1), 29\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEzzine, I. K., Dimassi, N., Pfarrer, B., Said, K., \u0026amp; Neubert, E. (2018). New records of the endemic Sicilian land snail species Marmorana (Murella) muralis (OF M\u0026uuml;ller, 1774) from the north of Tunisia (Pulmonata, Gastropoda). ZooKeys, (775), 131.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolmer, O., Black, M., Hoeh, W., Lutz, R., \u0026amp; Vrijenkoek, R. (1994). Molecular Marine Biology and Biotechnology. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates, 3, 294\u0026ndash;299.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForcart, L. (1935). Die Mollusken der nordpersischen Provinz Masenderan und ihre tiergeographischen Bedeutung. Akademische Verlagsgesellschaft.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalan, G. L., Mendez, N. P., \u0026amp; dela Cruz, R. Y. (2018). DNA barcoding of three selected gastropod species using cytochrome oxidase (COI) gene. Annales of West University of Timisoara. Series of Biology, 21(1), 93\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaston, K. J. (2000). Global patterns in biodiversity. Nature, 405(6783), 220\u0026ndash;227.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGetz, L. L., Chichester, L. F., \u0026amp; Burch, J. B. (2018). Land mollusks of the Northeastern United States and Southeastern Canada.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGetz, L. L., \u0026amp; Uetz, G. W. (1994). Species diversity of terrestrial snails in the southern Appalachian mountains, USA. Malacological Review, 27(1\u0026ndash;2), 61\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodacre, S. L. (2002). Population structure, history and gene flow in a group of closely related land snails: genetic variation in Partula from the Society Islands of the Pacific. Molecular Ecology, 11(1), 55\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraham, C. H., \u0026amp; Fine, P. V. (2008). Phylogenetic beta diversity: linking ecological and evolutionary processes across space in time. Ecology letters, 11(12), 1265\u0026ndash;1277.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGutleb, B., \u0026amp; Wieser, C. (2002). Ergebnisse einer zoologischen Exkursion in den Nordiran, 2001: Arthropoda (Lepidoptera, Trichoptera, Neuroptera, Heteroptera, Coleoptera, Opiliones, Araneae, Decapoda) und Vertebrata (Amphibia, Reptilia, Aves, Mammalia).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamzeh'ee, B., Naqinezhad, A., Attar, F., Ghahreman, A., Assadi, M., \u0026amp; Prieditis, N. (2008). Phytosociological survey of remnant Alnus glutinosa ssp. barbata communities in the lowland Caspian forests of northern Iran. Phytocoenologia, 117\u0026ndash;132.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHotopp, K. P. (2002). Land snails and soil calcium in central Appalachian Mountain forest. Southeastern Naturalist, 1(1), 27\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorsak, M., Jurickova, L., and Picka, J. (2013). Mollusks of the Czech and Slovak Republics.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIssel, A. (1865). Catalogo del molluschi raccolti della Missione Italiana in Persia. Mem. Delle realeacademia della scienze di Torino, Ser, 2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJackson, S. T., \u0026amp; Blois, J. L. (2015). Community ecology in a changing environment: Perspectives from the Quaternary. Proceedings of the National Academy of Sciences, 112(16), 4915\u0026ndash;4921.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJurickova, L., Hors\u0026aacute;k, M., Cameron, R., Hylander, K., M\u0026iacute;kovcov\u0026aacute;, A., Hlav\u0026aacute;č, J. Č., \u0026amp; Rohovec, J. (2008). Land snail distribution patterns within a site: the role of different calcium sources. European Journal of Soil Biology, 44(2), 172\u0026ndash;179.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh, K., Misawa, K., Kuma, K. \u0026amp; Miyata, T. (2002) MAFFT: a novel method for rapid multiple sequence alignments based on fast Fourier transform. Nucleic Acids Research, 30 (14), 3059\u0026ndash;3066.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelly RP, Sarkar IN, Eernisse DJ, Desalle R (2007) DNA barcoding using chitons (genus \u003cem\u003eMopalia\u003c/em\u003e). Molecular Ecology Notes, 7, 177\u0026ndash;183.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKembel, S. W., Cowan, P. D., Helmus, M. R., Cornwell, W. K., Morlon, H., Ackerly, D. D., \u0026hellip; Webb, C. O. (2010). Picante: R tools for integrating phylogenies and ecology. Bioinformatics, 26(11), 1463\u0026ndash;1464.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiew, T. S., Schilthuizen, M., \u0026amp; bin Lakim, M. (2010). The determinants of land snail diversity along a tropical elevational gradient: insularity, geometry, and niches. Journal of Biogeography, 37(6), 1071\u0026ndash;1078.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMansoorian, A. (2005). Terrestrial Molluscs of Golestan, and Mazandaran provinces, northern Iran. (in Persian).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMason, K., Feh\u0026eacute;r, Z., Bamberger, S., Reier, S., Szekeres, M., Sattmann, H., \u0026hellip; Haring, E. (2020). New insights into and limitations of the molecular phylogeny in the taxon-rich land snail genus Montenegrina (Mollusca: Gastropoda: Clausiliidae). Journal of Zoological Systematics and Evolutionary Research, 58(3), 662\u0026ndash;690.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMathias, A., Kisdi, \u0026Egrave;., \u0026amp; Olivieri, I. (2001). Divergent evolution of dispersal in a heterogeneous landscape. Evolution, 55(2), 246\u0026ndash;259.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyer, C. P., \u0026amp; Paulay, G. (2005). DNA barcoding: error rates based on comprehensive sampling. PLOS biology, 3(12), e422.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMillar, A. J., \u0026amp; Waite, S. (2002). The relationship between snails, soil factors and calcitic earthworm granules in a coppice woodland in Sussex. Journal of Conchology, 37(5), 483\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMouahid, G., Clerissi, C., Allienne, J. F., Chaparro, C., Al Yafae, S., Mintsa Nguema, R., \u0026hellip; Mon\u0026eacute;, H. (2018). The phylogeny of the genus Indoplanorbis (Gastropoda, Planorbidae) from Africa and the French West Indies. Zoologica Scripta, 47(5), 558\u0026ndash;564.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNantarat, N., Sutcharit, C., Tongkerd, P., Wade, C. M., Naggs, F., \u0026amp; Panha, S. (2019). Phylogenetics and species delimitations of the operculated land snail Cyclophorus volvulus (Gastropoda: Cyclophoridae) reveal cryptic diversity and new species in Thailand. Scientific Reports, 9(1), 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNekola, J. C., \u0026amp; Coles, B. F. (2010). Pupillid land snails of eastern North America. American Malacological Bulletin, 28(2), 29\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNekola, J. C., \u0026amp; Smith, T. M. (2000). Terrestrial gastropod richness patterns in Wisconsin carbonate cliff communities. MALACOLOGIA-PHILADELPHIA-, 41(1), 253\u0026ndash;270.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicolai, A., \u0026amp; Ansart, A. (2017). Conservation at a slow pace: terrestrial gastropods facing fast-changing climate. Conservation Physiology, 5(1), cox007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNurinsiyah, A. S., Fauzia, H., Hennig, C., \u0026amp; Hausdorf, B. (2016). Native and introduced land snail species as ecological indicators in different land use types in Java. Ecological Indicators, 70, 557\u0026ndash;565.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez, K. E., Cordeiro, J. R., \u0026amp; Coppolino, M. L. (2008). A guide for terrestrial gastropod identification. In American Malacological Society Annual Meeting, Carbondale, Illinois.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuillandre, N., Lambert, A., Brouillet, S., \u0026amp; Achaz, G. J. M. E. (2012). ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology, 21(8), 1864\u0026ndash;1877.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRambaut, A., Drummond, A. J., Xie, D., Baele, G., \u0026amp; Suchard, M. A. (2018). Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67(5), 901\u0026ndash;904.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRanasinghe, U. G. S. L., Eberle, J., Thormann, J., Bohacz, C., Benjamin, S. P., \u0026amp; Ahrens, D. (2022). Multiple species delimitation approaches with COI barcodes poorly fit each other and morphospecies\u0026ndash;An integrative taxonomy case of Sri Lankan Sericini chafers (Coleoptera: Scarabaeidae). Ecology and Evolution, 12(5), e8942.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatnasingham, S., Hebert, P.D.N., 2007. BOLD: The Barcode of Life Data System (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.barcodinglife\u003c/span\u003e\u003cspan address=\"http://www.barcodinglife\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. org). Mol. Ecol. Notes 7, 355\u0026ndash;364.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team. (2020). R: A Language and Environment for Statistical Computing (Version 3.4. 0). R Foundation for Statistical Computing.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRonquist, F., Teslenko, M., Van Der Mark, P., Ayres, D. L., Darling, A., H\u0026ouml;hna, S., \u0026hellip; Huelsenbeck, J. P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61(3), 539\u0026ndash;542.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRull, V., Vegas-Vilarr\u0026uacute;bia, T., Huber, O., \u0026amp; Se\u0026ntilde;aris, C. (Eds.). (2019). Biodiversity of Pantepui: The Pristine\" Lost World\" of the Neotropical Guiana Highlands. Academic Press.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaberi-Pirooz, R., Rajabi‐Maham, H., Ahmadzadeh, F., Kiabi, B. H., Javidkar, M., \u0026amp; Carretero, M. A. (2021). Pleistocene climate fluctuations as the major driver of genetic diversity and distribution patterns of the Caspian green lizard, Lacerta strigata Eichwald, 1831. Ecology and Evolution, 11(11), 6927\u0026ndash;6940.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSallam, A., \u0026amp; El-Wakeil, N. (2012). Biological and ecological studies on land snails and their control. Integrated Pest Management and Pest Control-Current and Future Tactics, (1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScharnweber, T., Rietschel, M., \u0026amp; Manthey, M. (2007). Degradation stages of the Hyrcanian forests in southern Azerbaijan. Archiv f\u0026uuml;r Naturschutz und Landschaftsforschung, 46(2), 133\u0026ndash;156.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimizu, Y., \u0026amp; Ueshima, R. (2000). Historical biogeography and interspecific mtDNA introgression in E\u003cem\u003euhadra peliomphala\u003c/em\u003e (the Japanese land snail). Heredity, 85(1), 84\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiadati, S., Moradi, H., Attar, F., Etemad, V., Hamzeh\u0026rsquo;ee, B. E. H. N. A. M., \u0026amp; Naqinezhad, A. (2010). Botanical diversity of Hyrcanian forests; a case study of a transect in the Kheyrud protected lowland mountain forests in northern Iran. Phytotaxa, 7(1), 1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilvan, N., Laiho, R., \u0026amp; Vasander, H. (2000). Changes in mesofauna abundance in peat soils drained for forestry. Forest Ecology and Management, 133(1\u0026ndash;2), 127\u0026ndash;133.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith, U. E., \u0026amp; Hendricks, J. R. (2013). Geometric morphometric character suites as phylogenetic data: extracting phylogenetic signal from gastropod shells. Systematic Biology, 62(3), 366\u0026ndash;385.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9), 1312\u0026ndash;1313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStarmuhlner, F., \u0026amp; Edlauer, A. (1957). Ergebnisse der \u0026ouml;sterreichischen Iran-Expedi-tion 1949/50: Beitr\u0026auml;ge zur Kenntnis der Molluskenfauna des Iran. Sitz. Ber. \u0026ouml;st. Akad. Wiss., Math.-naturw. Kl., Abt, 1(166), 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalebi, K. S., Sajedi, T., \u0026amp; Pourhashemi, M. (2014). Forests of Iran. A treasure from the past, a hope for the future, 10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura, K., Stecher, G., \u0026amp; Kumar, S. (2021). MEGA11: molecular evolutionary genetics analysis version 11. Molecular biology and evolution, 38(7), 3022\u0026ndash;3027.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTarkhnishvili, D., Gavashelishvili, A., \u0026amp; Mumladze, L. (2012). Palaeoclimatic models help to understand current distribution of Caucasian forest species. Biological Journal of the Linnean Society, 105(1), 231\u0026ndash;248.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTattersfield, P., Warui, C. M., Seddon, M. B., \u0026amp; Kiringe, J. W. (2001). Land-snail faunas of afromontane forests of Mount Kenya, Kenya: Ecology, diversity and distribution patterns. Journal of Biogeography, 28(7), 843\u0026ndash;861.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTattersfield, P., Seddon, M. B., Ngereza, C., \u0026amp; Rowson, B. (2006). Elevational variation in diversity and composition of land-snail faunas in a Tanzanian forest. African Journal of Ecology, 44(1), 47\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaugh J (2007) DNA barcoding in animal species: progress, potential and pitfalls. Bioessays, 29, 188\u0026ndash;197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhittaker, R. J., Willis, K. J., \u0026amp; Field, R. (2001). Scale and species richness: towards a general, hierarchical theory of species diversity. Journal of Biogeography, 28(4), 453\u0026ndash;470.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillis, K. J., \u0026amp; Whittaker, R. J. (2002). Species diversity\u0026ndash;scale matters. Science, 295(5558), 1245\u0026ndash;1248.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWronski, T., \u0026amp; Hausdorf, B. (2008). Distribution patterns of land snails in Ugandan rainforest support the existence of Pleistocene forest refugia. Journal of Biogeography, 35(10), 1759\u0026ndash;1768.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYassini, I. (1976). Contribution to the study of some terrestrial mollusks of Mazandaran Gilan and Azerbaijan province. Journal of Environmental Studies, 6(6). (In Persian).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng, X., Yiu, W. C., Cheung, K. H., Yip, H. Y., Nong, W., He, P., \u0026hellip; Hui, J. H. L. (2017). Distribution and current infection status of Biomphalaria straminea in Hong Kong. Parasites \u0026amp; vectors, 10(1), 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, J., Kapli, P., Pavlidis, P., \u0026amp; Stamatakis, A. (2013). A general species delimitation method with applications to phylogenetic placements. Bioinformatics, 29(22), 2869\u0026ndash;2876.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"land snail, forest, DNA barcoding, community composition, elevational incline, Conservation","lastPublishedDoi":"10.21203/rs.3.rs-3084235/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3084235/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLand snails play a crucial role in maintaining ecosystem sustainability within their habitats. Therefore, understanding the characteristics of their communities is vital for ecological studies and the development of effective conservation strategies. In this study, we employed DNA barcoding (COI gene) to identify land snails inhabiting the Hyrcanian Forest. Furthermore, we examined the variations in their community composition along elevational gradients. Snail samples were collected from three distinct elevations in three different forest locations within the Hyrcanian area of Iran. Through our comprehensive analysis, we identified a total of 10 OTUs, which were further classified into seven families and nine genera. Remarkably, five of these genera had never been reported in the study region before. By employing statistical analyses such as ANOVA and PERMANOVA, we determined significant differences in the features of snail communities across different elevations. Interestingly, we observed a decline in OTU richness with increasing elevation; however, the maximum abundance of snails was found at higher elevations. The unique climatic conditions and spatial distribution of precipitation from lowlands to highlands, as well as from west to east, make the Hyrcanian forests an ideal case study area for understanding the dynamics of land snail communities. In summary, this study provides novel insights into the land snail communities thriving in the Hyrcanian forests. The findings from our research can contribute significantly to the development of effective conservation management strategies for forest ecosystems. By understanding the factors influencing the distribution and composition of land snail communities, we can make informed decisions to protect and preserve these valuable organisms and the balance they maintain within their habitats.\u003c/p\u003e","manuscriptTitle":"Revealing Hidden Diversity and Community Dynamics of Land Snails through DNA Barcoding: Implications for Conservation and Ecological Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-31 18:46:38","doi":"10.21203/rs.3.rs-3084235/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b2d206b6-9276-432f-b4a5-ec56bc0132e5","owner":[],"postedDate":"July 31st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":23561993,"name":"Earth and environmental sciences/Ecology"},{"id":23561994,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2024-04-16T12:24:03+00:00","versionOfRecord":{"articleIdentity":"rs-3084235","link":"https://doi.org/10.3389/fevo.2024.1329581","journal":{"identity":"frontiers-in-ecology-and-evolution","isVorOnly":true,"title":"Frontiers in Ecology and Evolution"},"publishedOn":"2024-04-16 12:24:03","publishedOnDateReadable":"April 16th, 2024"},"versionCreatedAt":"2023-07-31 18:46:38","video":"","vorDoi":"10.3389/fevo.2024.1329581","vorDoiUrl":"https://doi.org/10.3389/fevo.2024.1329581","workflowStages":[]},"version":"v1","identity":"rs-3084235","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3084235","identity":"rs-3084235","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}