Variability character of conchological features in the mollusc Gibbulinopsis signata (Mousson, 1873) (Gastropoda: Pupillidae) in Northwestern Uzbekistan

: Analysis of the results shows that mollusc of the species Gibbulinopsis signata (Mousson, 1873) from the northwestern region of Uzbekistan exhibits significant variability in conchological characteristics, including variability in shell size, shape, and colour, as well as variations in aperture shape and fittings. Among these characteristics, shell height is found to be the most variable and is recognised as an ecological and biological indicator of mollusc’s adaptability to habitat conditions. The results of molecular analysis, using the sequences of the gene 18S of ribosomal DNA showed that all three populations belonged to the same species


Introduction
Despite more than 150 years of research on the molluscan fauna of Central Asia, the first information about the molluscan fauna of the northwestern part of Uzbekistan was only published in 2020 (Avazmetova, 2020;Pazilov & Avazmetova, 2020). Therefore, any research carried out on the molluscan fauna (fauna, zoogeography, ecology, and research) of this region is of significant scientific and practical importance.
During the research on the species composition of G. signata, the variability of conchological signs in their populations was observed, which serves as the subject of this article. The main goal of this work is to study the inter-population variation of conchological features of the common species G. signata.

Material and study area
The study was conducted in agricultural areas located in the northwestern part of Uzbekistan, specifically in the Balitov, Bukantov, and Tomditov mountains (Fig.  1). The materials used in this study were collected during the spring (April, May) and autumn (September, October) periods of 2020-2021 years. A total of 132 samples of G. signata were collected. The materials were collected during humid weather in the morning between 7:00 and 10:00 am, as terrestrial molluscs are typically active during this time and can be easily found.
During the collection of live molluscs, they were placed in a jar filled with cold water and covered with a rubber pad. After one day, they were removed from the water and stored in 40% alcohol. After six days, the alcohol concentration was increased to 70%, and after 14 days, it was further increased to 75%. The collected materials were stored in 50-100 ml glass bottles in the malacology collection of Gulistan State University, Uzbekistan.

Morphology
The species composition of G. signata was determined using the methods described by Schileyko (1978Schileyko ( , 1984 and Likharev & Wiktor (1980). The conchological characteristics of the molluscs were studied, and 30 sexually mature individuals were randomly selected from each group of molluscs to study the signs of conchological changes. Shell measurements were performed using the method described by Schileyko (1978Schileyko ( , 1984, and the following parameters were measured: shell height (SH), width (SW), aperture height (AH), aperture width (AW), and last whorl height (LWH).
To conduct statistical analysis of the morphometric indicators for each local population, the following characteristics were calculated: arithmetic means (±), coefficient of variation (SV), and determination (r2). To compare the general variability of the under-researched shells, methods described by Terentev & Rostova (1977), Rostova (1978), and Lakin (1980) were used. Biometric processing of the data obtained from the study of shells was conducted using SPSS Statistics 17.0 and Microsoft Excel 7.0 to determine the conchological variability of molluscs.

DNA isolation, PCR and sequencing
The specimens of molluscs were analysed using molecular genetic methods. For each population of snails, a part of the foot was used for the molecular study. DNA extraction was performed using the Qiamp DNA mini kit (Qiagen, Germany), following the manufacturer's instructions. PCR was carried out in a 25 µl volume using 1хPCR buffer, 0,2 µM each dNTP, 0,25 µl (1,25 units) of Taq polymerase (Qiagen, Germany), 2 µl of extracted DNA solution and 25 pmol of each of the primers F (5'-CTGGTTGAT(CT)CTGCCAGT-3') and R (5'-CTGAGATCCAACTAGGAGCTT-3') for amplification of the domain of 18S rDNA (Winnepenninckx et al., 1998). Amplicons were analysed by electrophoresis in a 1,5% agarose gel containing ethidium bromide. The PCR products were directly sequenced in both directions with the primers used for DNA amplification (Synthol Company, Moscow). The obtained sequences were compared with sequences of terrestrial mollusks available in GenBank. The sequences have been deposited in Genbank with accession numbers ON584276, ON584352, and ON584384.

Morphometrics
The variability of shell characters was studied in three populations. Balitov, at high altitudes of 146 m above sea level. The shells are light brown and the mouth is whole with wide edges. There are 2 teeth in the mouth, with parietal curved teeth connected by an angular bulge (Fig. 2C).
It is worth noting that, besides the aforementioned changes, the variability of morphometric features of G. signata has also been investigated, including shell height (SH), shell width (SW), aperture height (AH), aperture width (AW), last whorl height (LWH), which can be diagnostically quantified. The variability of these morphometric markers among the three populations of G. signata is as follows. Statistical analysis of the first population revealed an arithmetic mean value of 4,12 mm for formed shell height under Bokantov conditions (Table 1), with minimum and maximum values of 3,70-4,60 mm. The variant coefficient of shell height was 5,74%, indicating relatively low variability. The shell width also exhibited low variability, with a variant coefficient of 6,80%. However, the height and width of the shell mouth had higher variability, with variant coefficients of 14,17-13,57%. According to accepted classification, these characters are moderately variable and dependent on external environmental factors.
Among the studied traits, AH (3) showed relatively strong determination in the first population (Fig. 3), indicating that its variability depends largely on the external environment. On the other hand, 1 -SH, 2 -SW, and 5 -LWH were found to be less variable and more genetically determined.
In the second population, the statistical analysis revealed that the mean shell height of molluscs in The first population consists of dark brown cylindrical shell molluscs found among the tall shrubs under the rocks in Bukantov, which is 764 m above sea level. The mouth is whole with 5 teeth, with the parietal tooth connected to the angular bulge and the columellar tooth underdeveloped. There are 2 soft wrinkles, the bottom being long and the top relatively short, and the soft bulge is well-developed ( Fig. 2A).
The second population consists of molluscs with round-cylindrical shells living among the tall shrubs in Tomditov, around the village of Ajritki, which is 922 m above sea level. The shells have 6 folds and are medium brown and glossy. The mouth has angular teeth and 2 soft bulges (Fig. 2B).
The third population consists of molluscs with straight cylindrical shells living in the stems of shrubs in  Tomditov was 3,53 mm, while it was 4,12 mm in Bokantov (Table 1). The shell height was found to be 0,59 mm lower in Tomditov than in Bokantov, with a variance ratio of 10,18%. Notably, in Bokantov, this ratio was 5,74%. Thus, the height of the shell was found to be two times less in Tomditov's conditions than in Bokantov's. In the second population (Fig. 2), as in the first, the most strongly determined trait was MW (3), while anterior fold height (PH) was one of the less variable and moderately variable characters.
The data suggest that the conchological variability of G. signata is best reflected in the shape, colour, oral reinforcement, and size of the shell (Table 1).

Molecular analysis
Based on comparative molecular taxonomic studies of the nucleotide sequence of 18S rDNA regions, two nucleotide differences were found between the 1st population (Bukantov), the 2nd population (Tomditov) and the 3rd population of G. signata (Balitov) collected from different mountains in the northwestern part of Uzbekistan. These differences were explained by the substitution of G-guanine instead of A-adenine at 458 nucleotides and T-thymine nucleotides instead of Aadenine at 562 nucleotides (Fig. 4). The total difference between the populations of G. signata (Bukantov) and the populations of G. signata (Tomditov) and G. signata (Balitov) was 0,3%. The similarity between the 2nd population of G. signata (Tomditov) and the 3rd population of G. signata (Balitov) was 100%. These sequences were compared with the nucleotides of the species Cochlicopa lubrica (Cochlicopidae) (MN022682) from the GenBank database, and the difference between them was 4,2 % (Fig. 4).

Discussion
The conchological variability of terrestrial molluscs has been studied by numerous foreign and local scientists, including Harvey (1976), Haase et al. (2020), and Pazilov & Avazmetova (2020). For example, studies have focused on the factors influencing shell changes in terrestrial molluscs, particularly in the genus Cepaea species, examining shell polymorphism (Harvey, 1976) and shell size in natural populations (Bengston et al., 1979). Other studies have explored the conchological polymorphism of shell molluscs, including shell colour and size (Smet & Rompu, 1984), as well as the dependence of shell colour on temperature, activeness, and drought resistance (Staikou, 1999). Additionally, shell Historia naturalis bulgarica 45 (2023) polymorphism has been determined through the megalab database (Cameron & Cook, 2012), shell circumference and habitat dependence of Theba pisana have been studied (Johnson, 1980), and micro-geographic variation in Littorina striata shell has been examined (Wolf et al., 1997).
malacologists (Pazilov & Azimov, 2003). Variability in shell shape and size, colour, and mouth and mouth fittings can be observed. The species G. signata is highly drought tolerant and often forms large clusters with a density of up to 100 individuals per square metre at heights of up to 3500 metres above sea level during unfavorable times of the year. It lives under rocks, bushes, and pebbles in semi-desert and mountaindesert areas, and is widespread in Central Asia, the Eastern Caucasus, northern Iran, and Afghanistan (Matyokin, 1959).
G. signata is known to inhabit under bedding, rocks, and rock crevices. Due to its small size, the shell of G. signata provides some protection from accidental falls into uncomfortable conditions, which may occur due to sudden changes in air temperature and humidity. In such situations, the snails may experience significant water loss, causing them to become inactive and cling to rocks. The cylindrical shells of G. signata, which live in very dry rocky crevices of Bokantov, Tomditov, and Balitov mountains, provide a clear example of this behaviour.
The flexibility of terrestrial mollusks is evident in the colour of their shells. In most taxa, the assimilation of new adaptation zones, such as open dry spaces, leads to a change in shell colour. For instance, G. signata living under rocks and boulders in the open air tend to have light brown shells, while those living among bushes and under rocks have dark brown shells, as observed in Bukantov. This difference is attributed to the fact that mollusks living in open areas are more exposed to sunlight than those living under rocks among bushes.
Moreover, the shell structure of G. signata varies across its range. In particular, different levels of advanced aperture structures can be seen within the studied type. Additionally, the number of oral teeth in G. signata varies depending on their habitat. For instance, those living under rocks between bushes in Bukantov have five teeth in their mouth, while those living on the stems of semi-shrubs in Balitov at a height of 146 m above sea level have two teeth in their mouth.
The research results indicate that molluscs which live among rocks between bushes and never climb on plant stems (even during the wettest times of the year) have well-developed mouth teeth. Apparently, these developed oral teeth serve to clear soil particles from the surface of their feet when they retract into their shells. On the other hand, molluscs living in the lower part of semi-shrub plants do not have mouth teeth, sug-gesting that there is no need for their development in this habitat.

Conclusion
The conchological signs of molluscs can vary in the shape and size of their shells, colour variability, as well as in the shape of the mouth and mouth aperture. This trait can be considered a moderate biological indicator, while a high level of variability in these traits indicates a balance of growth processes in the ontogenetic process (Pazilov & Azimov, 2003). In the north-western part of Uzbekistan, the optimal ratio of shell size and energy consumption in mollusc movement can be observed, which is crucial during recurrent high drought conditions. Molecular analysis was performed on specimens from three populations (1st, 2nd, and 3rd) of the mollusc G. signata in Bukantov, Tomditov, and Balitov mountains in Uzbekistan. The results showed that all populations belonged to G. signata, and the sequences obtained from these snails were identical (99,7-100%). Further studies using more specific markers of ribosomal and mitochondrial genes are proposed to identify intraspecific boundaries of variability within these populations.