Zoodiversity_01_2021.indb UDC 594.38 (477.8) SHELL BANDING AND COLOR POLYMORPHISM OF THE INTRODUCED SNAIL CEPAEA NEMORALIS (GASTROPODA, HELICIDAE) IN LVIV, WESTERN UKRAINE N. V. Gural-Sverlova1*, R. I. Gural1, T. V. Rodych2 1State Museum of Natural History, NAS of Ukraine Teatralna st., 18, Lviv, 79008 Ukraine E-mail: sverlova@pip-mollusca.org 2Lviv National Academy of Arts Kubiyovych st., 38, Lviv, 79011 Ukraine E-mail: rodych.taras@gmail.com *Corresponding author N. V. Gural-Sverlova (https://orcid.org/0000-0002-3892-5338) R. I. Gural (https://orcid.org/0000-0002-1546-1956) Shell Banding and Color Polymorphism of the Introduced Snail Cepaea nemoralis (Gastropoda, Helicidae) in Lviv, Western Ukraine. Gural-Sverlova, N. V., Gural, R. I., Rodych, T.V. — Th e shell coloration was studied in more than three thousand adults of the grove snail, Cepaea nemoralis (Linnaeus, 1758), from 15 colonies discovered in 2019–2020 in Lviv City and in the immediate vicinity of its south- ern administrative border (Zubra village). In most colonies, relatively light phenotypes prevailed: most oft en P00000, less oft en Y00000 or Y00300. In total, more than a third of the collected specimens had the phenotype P00000. Great variability of the phenotypic and genetic structure of this introduced species in the study area was demonstrated, which usually does not have a clear connection with the locations of colonies or with the shading of habitats. Th is may be due to both random factors (founder eff ect, gene drift in recently formed colonies), and the relative youth of the colonies. In the future, the obtained data can be used to monitor possible long-term changes in the phenotypic composition of the investigated colonies to assess the potential eff ect of selective factors on this composition, fi rst of all, climatic selection outside the natural range of C. nemoralis. K e y w o r d s : terrestrial mollusks, grove snail, introduction, phenotypic composition, Ukraine. Zoodiversity, 55(1): 51–62, 2021 DOI 10.15407/zoo2021.01.051 52 N. V. Gural-Sverlova, R. I. Gural, T. V. Rodych Introduction Cepaea nemoralis (Linnaeus, 1758) is a species of Western European origin (Boettger, 1926; Taylor, 1914), the natural range of which also partially includes Northern and Central Europe. In Ukraine, few reliable fi ndings of this species are known, most of which were made in the west of the country and only in recent years (Gural-Sverlova et al., 2020). In 2019–2020, 15 previously unknown colonies of C. nemoralis were found on the territory of Lviv and in the immediate vicinity of the city's administrative borders. Many of these colonies were quite abundant, which made it possible for the fi rst time to quantitatively analyze the variability of the phenotypic composition within the city. Until now, studies of a similar focus and scale have been carried out only in Northern and Central Europe (Cameron et al., 2009, 2014), i. e. in regions with a less continental climate compared to Ukraine, and in Eastern Europe only for the related species Cepaea hortensis (O. F. Müller, 1774) in Lviv (Sverlova, 2005; Sverlova et al., 2006). S ince many alternative coloring traits (phenes) in C. nemoralis and C. hortensis are inherited (Murray, 1975), the study of their spatial and temporal variability allows one to study population genetic processes, which can be especially interesting for introduced species. Th erefore, the main tasks of this publication were: 1) as detailed description of the phenotypic composition in all found colonies as possible — as a basis for its subsequent monitoring; 2) a ssessment of the scale and peculiarities of the present phenotypic and genetic vari- ability of C. nemoralis within the city; 3) analysis of the possible connection of this variability with the character of habitats inhabited by snails. Material and methods Adult specimens of C . nemoralis (with a refl ected apertural margin of the shell) were collected in 2019– 2020 at 14 sites within the administrative boundaries of Lviv (sites 1–14) and one additional site o n its southern outskirts (Zubra village, site 15). Th e locations of the collecting sites are shown in fi gure 1, and their descriptions are given below. Th e sites were numbered in the direction from the western border of Lviv to its central part, and then from the center to the south. At a low abundance of snails, the samples also included their empty shells with well-preserved coloration, which made it possible to accurately determine the phenotypes. In total, the shell ground color and banding pattern were scored for more than three thousand specimens of C. nemoralis. S ite 1 — H orodotska Street near the exit to the ring road, between 49°49'14.7" N 23°54'56.6" E and 49°49'17.0" N 23°55'09.6" E, length about 180 m, forest belt along the highway, indep endent samplings in June (coll. Rodych, along the entire length of the site, designated as “1a”) and in July 2020 (coll. Gural-Sverlova, in the central and eastern parts of the site, designated as “1b”). Since some individuals from the fi rst sample could be included in the second, the second sampling was not taken into account in table 1 in the “Total” column. Table 2 shows the mean frequencies of phenotype groups in two samples. At all other sites, the repeated sampling of the same snails or empty shells was excluded. Site 2 — Liubinska Street, in front of the old airport terminal, 49°48'59.5" N 23°57'19.6" E, a round lawn slightly less than 20 m in diam eter with groups of ornamental shrubs planted on it, mainly low-gro wing juni- pers, two samplin gs in July 2020, coll. G ural-Sverlova. Reconstruction and gardening of the site were carried out about 10 years ago. Th is may be the age of the discovered colony. Site 3 — Liubinska Street, on the former territory of a military unit, 49°49'08.5" N 23°57'36.0" E, length about 30 m, shaded plot with dense woody vegetation, several samplings in May 2020, coll. Rodych. Site 4 — Syhnivka Street, household plot near the building No. 48, 49°49' 31.4" N 23°57'27.9" E, tall orna- mental shrubs (Kerria japonica), wild grapes on a wire fence, several samplings in July 2020, coll. Gural-Sverlo- va. Seven adults and several juveniles were found along a fragment of the fence about 4 m long. No C. nemoralis has yet been found at neighboring sites. Th e nearest colony (Site 5) is at a distance of about 200 m. Site 5 — Li utneva Street, around the mansion No. 33, 49°49'33.0"  N 23°57'43.1"  E, length about 30 m, various ornamental plants along the fence, several samplings in July 2020, coll. Gural-S verlova. Apparently, t he colony was formed most recently, and snails are just beginning to penetrate the household plots of neighboring mansions. Site 6 — near the inte rsection of Horodotska Street with Ivan Bahrianyi Street and Rivna Street, be- twe en 49°49'48.4" N 23°58'35.2 “E, 49°49'54.1" N 23°58'29.9" E, 49°49'54.9" N 23°58'33.8" E and 49°49'51.2" N 23°58'36.6"  E, length about 150 m, household plots of mansions and roadsides with diff erent vegetation and lighting, 28.06.2020, coll. Gural-Sverlova. Site 7 — near the int ersection of General Chuprynka Street with Osyp Makovei Street and Lonhyn Tsehelskyi Street, between 49°49'21.5" N 24°00'00.1" E, 49°49'21.5" N 24°00'03.4" E, 49°49'19.3" N 23°59'58.7" E and 49°49'18.5" N 24°00'02.8" E, length about 60 m, plots with diff erent vegetation and lighting near mansions and apartment buildings, several samplings in Juny 202 0, coll. Gural-Sverlova. Site 8 — Chervona Street, bet ween 49°49'25.3" N 24°00'24.6" E and 49°49'30.9" N 24°00'25.0" E, length slightly more than 100 m, plots with diff erent vegetation and lighting near mansions and multi-storey building No. 26 on Princess Olga Street, several samplings in July 2020, coll. Gural-Sverlova. Site 9 — Dm ytro Vitovskyi Street, near buildings No. 30 and 34 and on the adjacent edge of the Citadel Park, between 49°49'47.1"  N 24°01'35.8"  E, 49°49'48.8"  N 24°01'32.5"  E and 49°49'47.8"  N 24°01'30.6"  E, length up to 53Shell Banding and Color Polymorphism of the Introduced Snail Cepaea nemoralis… 80 m, most of the site is a steep southern slope shaded by tall trees, multiple samplings in June 2020, coll. Gural-Sverlova and Gural. Th e sit e is separated from the subsequent one by a hill with the Citadel Park, in the upper part of which C. nemoralis has not yet been found. Site 10 — Kalicha Hora Street, around the former villa “Zasvittia” (No. 24), between 49°50'05.4" N 24°01'37.6" E and 49°50'07.7" N 24°01'33.6"  E, length about 70 m, on haw- thorns planted along the brick fence of the villa and at greened plots near neighboring apartment buildings, multiple samplings in August 2019 and May–June 2020, coll. Gural- Sverlova and Gural. In a previous publication (Gural-Sverlova e t al., 2020), the site was des- ignated as “Lviv-3”. When analyzing the ma- terial collected on it in 2019 (Gural-Sverlova et al., 2020: table 1), several individuals of C. hortensis with a pink shell and a dark lip, locally found at the same site, were mistaken for C. nemoralis. In this article, they have been excluded from the calculations. Site 11 — Volodymyr Hnatiuk Street, near the monument to the dead law enforcement offi cers, 49°50'29.8" N 24°01'21.0" E, two small fragments of a lawn 10–15 m long and up to 5 m wide with groups of low-growing coniferous ornamental plants, mainly junipers, several samplings in late June and early July 2020, coll. Gural-Sverlova and Gural. Th e monument was opened at the end of 1999, aft er which the area around it was landscaped. Th erefore, the age of the discovered colony may be about 20 years. Site 12 — between Zelena Street and Krymska Street, on the edge of the Snopkiv Park, between 49°4 9'28.6" N 24°02'41.0" E, 49°49'27.8" N 24°02'56.9" E and 49°49'26.8" N 24°02'45.6" E, length about 200 m, mostly household plots of mansions and adjacent ope n areas with tall grass (Gural-Sverlova et al., 2020: fi g. 2A), multiple samplings in July 2019 and from May to July 2020, coll. Gural-Sverlova and Gural. In a previous publication (Gural-Sverlova et al., 2020), the site was designated as “Lviv-2”. Site 13 — between Ivan Chmola Street (mostly) and Luhanska Street (several individuals), around the territory of the former garden center, between 49°48'52.1"  N 24°01'30.2"  E, 49°48'52.6"  N 24°01'12.6"  E, 49°48'42.9" N 24°01'20.5" E and 49°48'41.6" N 24°01'29.3" E, length about 250 m, mostly in places shaded by tall trees (Gural-Sverlova et al., 2020: fi g. 1A), multiple samplings from June to September 2019 and from April to September 2020, coll. Gural-Sverlova and Gural. In a previous publication (Gural- Sverlova et al., 2020), the site was designated as “Lviv-1”. Site 14 — Khutorivka Street near the entrance to the Shuvar market, wasteland next to the garden center, between 49°47'58.8"  N 24°02'07.4"  E and 49°47'58.9"  N 24°02'02.6"  E, length about 70 m, open plot with tall grass, the remains of grapes on a wire fence, 23.06.2020, coll. Gural-Sverlova. Site 15 — Pustomyty District, Zubra villa ge (territory adjacent to the southern outskirts of Lviv), between 49°46'42.7"  N 24°03'08.4"  E and 49°46'43.4"  N 24°03'01.4"  E, length up to 100 m, wasteland between houses with tall grass, on one edge with sparse bushes and young trees (Gural-Sverlova et al., 2020: fi g. 1C), several samplings in May-June 2019 and June 2020, coll. Gural-Sverlova. In order to avoid the infl uence of random factors of the collection on the calculation results, we excluded from them the sites where less than 50 alive adult snails or their empty shells were collected. Th ese were sites 4 and 5, apparently only recently colonized by C. nemoralis, as evidenced by both the low abundance of snails and the extremely limited space at which they were recorded (see descriptions of the corresponding sites). To analyze the possible infl uence of the nature of the biotopes inhabited by snails on the phenotypic composition of their colonies, all the studied sites (with the exception of the above-mentioned sites 4 and 5) were divided into three groups, designated as 1) “open”, with good insolation, most of the territory of which was not shaded by trees or tall bushes, less oft en with their complete absence; 2) “shaded”, completely or almost completely shaded by tall trees, oft en with dense tree-shrub undergrowth; 3) “mosaic”, at which, in a relatively small area, places with diff erent vegetation and shading alternated, and whic h therefore could not be attributed to any of the previous types. In addition to shading by trees, we also took into account the shading of sites by houses (especially multi-storey ones), solid (not wire) fences, etc. Although the territory of the former garden center (site 13) also had open plots, snails were collected mainly in places shaded by tall trees along the edge of this territory and near it. Th erefore, site 13 was assigned to the group of shaded biotopes. Phenotypes were scored based on the ground color of the shells and the banding pattern of their ulti- mate whorl according to the standard method (Clarke, 1960). Spiral dark bands were designated by Arabic Fig. 1. Th e locations of the collecting sites in Lviv. 54 N. V. Gural-Sverlova, R. I. Gural, T. V. Rodych numerals from 1 to 5, counting them from the apex to the base of the shell. Th e absence of band(s) was indicated as “0” in place of the corresp onding numeral(s). Th e fusion of adjacent bands was indicated with parenthe- ses. Th e bands were considered to be fused if they were fully or partial ly merged for no less than a quarter of a whorl before the aperture. Th e presence of additional weak, oft en blurry bands (modifi cation), oc- casionally appearing on shells with a genetically determined phenotype 00000, 00300, or 00345, was indicated by square brackets. During further sorting of phenotypes into groups (see below), such bands were not taken into account. Barely noticeable traces of bands, sometimes visible only near the shell aperture, were not specially designated in the formulas of phe- notypes. Th e shell ground color was designated as Y — yellow, P — pink, B — brown. White or nearly white shells wit hout yellow pigment have traditionally been classifi ed as yellow. In the subsequent analysis of the phenotypic composition of the stu died colonies of C. nemoralis, the phenotypes distinguished by the shell banding (ta ble 1) were co mbined into 4 groups: 1) unbaded — in this study were represented exclusively by the phenotype 00000; in general may in- clude also some genetically unbanded shells with 1–2 unclear bands (modi- fi cations); 2) mid-banded — the same for the phenotype 00300: 3) three-banded — al l shells with three lower bands, discrete or fused, occasionally with one or two fuzzy upper bands (modifi cations); 4) fi ve-banded — the phenotype 12 345 and its variations with the absence or fusion of some bands. Since in C. nemoralis the absence/presence of bands on the shell and its ground color can be inherited linked (Murray, 1975), when comparing the phenotypic composition of the st udied colonies, these groups were considered in combination with the ground color (table 2). To formally assess the intensity of shell pigmentation in colonies, all phenotypes were divided into 3 groups: 1) “light”, including phenotypes Y00000, Y00300, P00000, P00300 and their modifi cations; 2) “me- dium”, including all yellow and pink three-banded shells as well as fi ve-ba nded shells without band fusion; 3) “dark”, including brown shells (in Lviv, represented exclusively by the phenotype B00000) as well as yellow and pink fi ve-banded shells with fused bands. Shells from the fi rst group are characterized not only by light, but also relatively homogeneous coloration. Th e second group is characterized by a combination of contrast- ing dark and light fragments (dark discrete bands separated by narrow light gaps, as well as a combination of a light top and dark bottom in th ree-banded shells). In the third group, the coloration of the fi ve-banded shells becomes not only darker, but also more homogeneous (less banded) due to the band fusion. To assess the variability of the phenotypic composition of C. nemor ali s between Lviv, the inbreeding coeffi cient Fst was used, calculated based on the frequencies of phenotypic man ifestation of some inherited traits (Cameron et al., 2009) or the frequencies of the corresponding alle les. In the fi rst case, the frequencies of unbanded shells as well as the shells with diff erent ground color were calculated from the total sample size, the frequencies of shells with a central band from the number of banded shells, and the frequencies of three-banded shell from the number of multi-banded shells (Cameron et al., 2009, 2014), which corresponds to the known data on the character of inheritance of the mentioned traits (Murray, 1975). Allele frequencies were calculated conditionally, u sing the Hardy-Weinberg formula for an ideal panmictic population. Th e phenotypic similarity of the studied samples, presented in the form of a dendrogram in fi gure 2, was calculated by the formula: Fig. 2. Th e similarity of the phenotypic (top) and genetic (bottom) composition of the studied samples. Open habitats are denoted by light circles, shaded by black, and mosaic by black and white. 55Shell Banding and Color Polymorphism of the Introduced Snail Cepaea nemoralis… r = 11qp + 22qp + ... + mmqp ; where p1, p2 … pi are the frequencies of phenotype groups at one site; q1, q 2 … qi — frequencies of the same groups at another site. Th e freq uencies of 9 phenotype groups, distinguished by the combination of the ground color and the number of bands on the shell (table 2), were used for calculations. Genetic similarity was assessed by the mean value obtained for the allele frequencies of four genes responsible for the ground color of the shell, the absence of all bands on it, the absence of all bands except for the central one, and the absence of the two upper bands. Th en, the average was calculated from the four values obtained. It is considered that the diameter of the panmictic unit for C. nemora lis is 50–60 m (Lamotte, 1951, cited according to Jones et al ., 1977), according to other data — up to 100 m (Schnetter, 1950) in the absence of natural or anthropogenic (Sverlova, 2002 a) barriers preventing the free locomotion of snails. Th e sizes of the sites studied by us sometimes exceeded the mentioned values by several times, but the snails inhabiting them had a common origin, could freely move within the sites, and were reliably spatially isolated from other known colonies of C. ne- moralis in Lviv. We have used the term “colony” to denote such aggregations of snails, although in most cases (with the exception of sites 4, 5 and possibly 3) they could be considered already established populations. Th e materials used in this study were partially deposited in the malacological collection of the State Museum of Natural History of the National Academy of Sciences of Ukraine in Lviv. Photos of live snails and their shells with diff erent coloration are posted on one of the museum's Internet resources (Gural-Sverlova, Gural, 2012–2020). Results In total, in 15 studied c olonies of C. nemoralis a large variety of phenotypes was re- corded (table 1), most of which were of hereditary nature, and some rare coloration vari- ants should obviously be considered as modifi cations. Th e latter refers primarily to shells with poorly developed, indistinct bands, indicated by square brackets (see Material and methods). More than a third of all collected specimens had one phenotype (P00000). At all sites, polymorphism in the shell ground color was recorded (table 2), although in the Zubra (site 15) only a single individuals had pink shells (table 1). On the other hand, at site 2, more than 90 % of snails had pink shells , which was associated with a very high pro- portion of the phenotype P00000 (about three quarters of all collected snails). In general, snails with a yellow or pink ground color of the shell predominated in the studied colonies equally oft en (table 2). Brown shells, represented by one phenotype B00000, were found at only three sites (table 2), although at site 9, a third of the collected snails had such shell ground color. At the same site, a large variability of the intensity of the brown color was observed — from very dark to almost white with a slight brownish tint in few specimens (Gural-Sverlova, Gural, 2012–2020). At the other two sites, only individuals with dark brown shells were found (Gural-Sverlova et al., 2020, fi g. 2, F). At site 14, not only all brown shells were unbanded, but all unbanded ones were brown (table 2), which may indicate a completely linked inheritance of these two dominant traits. Th e only exception was fi ve unbanded shells of pink or yellow color, which we classifi ed as modifi cations from the phenotypes P00300 and Y00300 (table 1). On the periphery of whorls, each of these shells had a light spiral band (Gural-Sverlova, Gural, 2012–2020). A similar band, much lighter than the ground color, is sometimes clearly visible under the dark central band in the phenotype 00300, but not found in 00000. In one of the above- mentioned shells, faint traces of the missing central dark band were also noticeable near the aperture (Gural-Sverlova, Gural, 2012–2020). In addition to yellow, pink, and brown shells, white shells without traces of yellow, pink, or brown pigment were also found in Lviv (Gural-Sverlova, Gural, 2012–2020), which were combined with yellow ones in the calculations (see Material and methods). In most samples, they were either absent or were represented by a few individuals. However, at site 6, 20 % of the snails in the sample had white shells. And among specimens classifi ed as Y00300, those were almost 30 %. In contrast to the ground color, a number of the samples were monomorphic in one of the three genes responsible for the absence (dominant trait) of all bands on the shell, two upper and 56 N. V. Gural-Sverlova, R. I. Gural, T. V. Rodych T ab le 1 . Th e p he no ty pi c co m po si ti on o f t he sa m pl es o f C . n em or al is fr om th e st ud ie d si te s Ph en ot yp es N 1 a N 1 b N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 N 1 0 N 1 1 N 1 2 N 1 3 N 1 4 N 1 5 T ot al ** Y 00 00 0 4 – – 1 – – – 17 – 12 – – 13 5 3* 23 78 Y 00 30 0 28 0 34 4 27 + 9 47 – 1 4 4 – 7 11 7 60 9 56 9 Y 0[ 2] 30 0 – – – – – – – – – – – – – 1 – – 1 Y 00 30 [5 ] 2 – – – – – 1 – – – – – – 2 – – 5 Y 00 3[ 4] 0 – – – 1 – – – – – – – – – – – – 1 Y 00 34 5 39 7 – – – – – 9 2 – 2 – 2 12 3 – 69 Y 00 3( 45 ) 24 4 – – – – – 1 5 1 10 – 2 11 2 – 56 Y 00 (3 45 ) – – – – – – – – – – 1 – – – – – 1 Y 00 04 5 1 – – – – – – – – – – – – – – – 1 Y [1 ][ 2] 34 5 2 – – – – – – – – – – – – – – – 2 Y 12 34 5 – – 10 6 – 1 10 11 7 1 9 3 4 10 6 – 13 18 1 Y 10 34 5 – – – 1 – – – – – – – – + 1 – – 2 Y (1 2) 34 5 – – – – – – – 1 1 – – – – 14 – – 16 Y 1( 23 )4 5 – – – – 1 – 1 – 1 – – – – 3 – 1 7 Y 12 3( 45 ) – – 2 8 – – 2 – 1 4 3 4 – 38 – 3 65 Y (1 23 )4 5 – – – – – – + – – – – – – 3 – – 3 Y (1 2) 3( 45 ) – – 4 2 – + 11 – 1 7 8 1 1 87 – 1 12 3 Y 1( 23 )( 45 ) – – – – – – – – – – – – – 1 – + 1 Y (1 23 )( 45 ) – – 2 – 2 – 3 – 1 – 2 1 1 35 – – 47 Y (1 2) (3 45 ) – – – – – – – – – – – 1 – – – – 1 Y 1( 23 45 ) – – – – 1 – – – – – – – – – – – 1 Y (1 23 45 ) – – 1 – – – 1 – – 1 2 – – 3 – – 8 P0 00 00 59 0 99 29 4 37 – – – 1 19 2 33 48 6 73 2* 1 11 06 P0 03 00 18 6 31 19 5 1 9 8 – 1 1 5 – 9 63 16 – 32 3 P0 03 0[ 5] – – – – – – – – – – – – – 1 – – 1 P0 03 [4 ]0 – – – – – – – – – – – – – 1 – – 1 P( 12 )3 00 – – – – – – – – – – – – – 1 – – 1 P0 03 45 18 4 – – 1 – – 3 3 – + – 6 12 7 – 50 P0 03 (4 5) 18 4 – – – – – + 1 1 7 – 1 14 1 – 43 P0 0( 34 )5 – – – – – – – – – – – – – 1 – – 1 P[ 1] 03 45 1 – – – – – – – – – – – – – – – 1 P[ 1] [2 ]3 (4 5) 2 – – – – – – – – – – – – – – – 2 P1 23 45 – – 8 1 1 1 1 2 3 – 2 2 5 30 – 1 57 57Shell Banding and Color Polymorphism of the Introduced Snail Cepaea nemoralis… T ab le 1 (c on ti nu ed ) Ph en ot yp es N 1 a N 1 b N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 N 1 0 N 1 1 N 1 2 N 1 3 N 1 4 N 1 5 T ot al ** P1 20 45 – – – – – – – – – – – – – 1 – – 1 P1 03 45 – – – – – – – – – – – – 1 – – – 1 P( 12 )3 45 – – – 1 – – 1 1 – – – – – 5 – – 8 P1 (2 3) 45 – – – – – + – – – – – – 1 – – – 1 P1 23 (4 5) – – 29 – – – 1 2 – – 1 2 – 19 – – 54 P( 12 )3 (4 5) – – 11 5 – – 1 2 2 – 7 3 1 37 – 1 70 P1 (2 3) (4 5) – – 1 – – – – – – – – – – 1 – – 2 P( 12 3) (4 5) – – 3 – – – 2 – 2 – 2 – – 32 – – 41 P1 (2 34 5) – – – – – – – – – – 1 – – 1 – – 2 P( 12 34 5) – – – – – – – – – – 1 – – 1 – – 2 B0 00 00 – – – – – – – – – 17 3 – – – 13 – 33 T ot al 11 67 18 3 38 8 95 7 20 90 50 51 51 10 3 65 60 73 2 10 7 53 30 39 N o te . * U nb an de d m od ifi ca tio ns fr om 0 03 00 ; * * w ith ou t N 1b ; + o nl y in ju ve ni le s. two lower bands, or only two upper bands (Murray, 1975), which leads to the formation of unbanded, mid-banded and three-banded shells respectively. In most cases, such samples did not contain specimens with a dominant allele of one of the genes listed above, and occasionally two of them (table 3). At site 10, three adults were found with weakly pigmented (light brown or almost pink) or colorless and transparent (hyalosonate) bands. Th e lip of these shells was also not typically light for C. nemoralis — from pinkish to almost wholly white (Gural-Sverlova, Gural, 2012–2020). One juvenile with a shell diameter of no more than 1 cm and with hyalosonate bands was also found at site 8. Among the groups of phenotypes distin- guished by the combination of the ground color and the number of bands (table 2), one of the four lightest variants of shell coloration (see Material and methods) prevailed in almost all cases, most oft en P00000, less oft en Y00000 or Y00300. Th e to- tal frequency of the above mentioned phenotypes and P00300, on average, was slightly higher at open sites, the shells with “medium” intensity and clear- ly expressed heterogeneity of coloration — at sites with heterogeneous vegetation and alternating open and shaded places, and the darkest pheno- types — in shaded habitats, although these diff er- ences did not always reach a statistically signifi cant level (table 4). Th e average frequencies of yellow, unbanded and mid-banded shells did not show statistically signifi cant diff erences between open, shaded and “mosaic” habitats, although the latter had more yellow banded shells (table 4). When constructing dendrograms of pheno- typic and genetic similarity of the studied colonies (fi g. 2), the greatest similarity was oft en demon- strated by samples collected in diff erent parts of the city and in diff erent types of habitats. In the fi rst case, all samples were divided into three main groups: 1) with a pronounced predominance of Y00300 (more than half of the specimens in the samples) and the absence of Y00000 and P00000 (sites 6 and 14); 2) with high frequencies Y00000 — from 22 to 43 % in comparison with 0–1 % in other samples (sites 7, 9, 12 and 15); 3) others, most of- ten with a predominance of P00000. In the second case, six sites located in the central part of the city (sites 9 and 10) and slightly to the south (sites 7, 8, 12 and 13) were united into one group. Only at these sites, with the exception of site 7, were col- lected the samples that were not monomorphic for any of the three genes responsible for the complete 58 N. V. Gural-Sverlova, R. I. Gural, T. V. Rodych or partial absence of bands on the shells of C. nemoralis (table 3). In both cases, a great sim- ilarity showed plots 2 and 11, where snails live under similar conditions, at small isolated open plots with regularly mowed lawns and groups of low-growing junipers (see Material and methods). Th e calculated values of the inbreeding coeffi cient Fst are given in table 5. For all the examined inherited traits, they were higher when used in the calculations the frequencies of their phenotypic manifestation, rather than the allele frequencies of the corresponding genes. Th e smallest contribution to the phenotypic and genetic variability of C. nemoralis within Lviv is made by such traits as the brown color of the shell (a rare trait) and the com- plete absence of bands on it (a common trait). Discussion Th e fi rst attempt to introduce C. nemoralis to Lviv was made at the end of the 19th century (Łomnicki, 1899). Th is, however, did not lead to the formation of established colo- nies in the places where snails were released, which would have survived to this day (Gural- Sverlova et al., 2020). At the turn of the 20th and 21st centuries, only one colony of this species was known in one of the city parks, characterized by a low abundance and poor phenotypic composition (Sverlova, 2002 b; Sverlova et al., 2006), by now almost completely extinct (Gural-Sverlova, Savchuk, 2019). A number of the colonies of C. nemoralis found in Lviv in 2019 and 2020 and described in this article, obviously, have no relation to it, which is confi rmed by a richer phenotypic composition, fi rst of all, by the presence of snails with unbanded and/or fi ve-banded shells at all sites (Gural-Sverlova et al., 2020). Th erefore, recently discovered colonies should be considered as a result of a later in- troduction (or, more likely, several independent introductions) together with ornamental T a b l e 2 . Percentages of phenotype groups at the sites with diff erent degrees of shading Phenotype groups Open Shaded Mosaic N 2 N 11 N 12 N 14 N 15 mean N 1 N 3 N 9 N 13 mean N 6 N 7 N 8 N 10 mean Ground color and banding Y-0 – – 21.7 – 43.4 13.0 0.2 1.1 23.5 0.7 6.4 – 34.0 – – 8.5 Y-1 1.0 – 11.7 58.9 17.0 17.7 21.4 29.5 7.8 16.4 18.8 53.3 – 2.0 3.9 14.8 Y-3 – – 6.7 4.7 – 2.3 5.8 – 2.0 3.1 2.7 – 20.0 13.7 12.6 11.6 Y-5 4.9 15.4 10.0 – 34.0 12.8 – 17.9 25.5 39.8 20.8 31.1 24.0 23.5 23.3 25.5 P-0 75.8 73.8 10.0 – 1.9 32.3 52.3 38.9 3.9 10.0 26.3 – 2.0 37.3 32.0 17.8 P-1 4.9 – 15.0 16.8 – 7.3 16.4 5.3 2.0 9.0 8.2 8.9 – 2.0 4.9 3.9 P-3 – – 11.7 7.5 – 3.8 3.9 – 2.0 3.7 2.4 – 6.0 7.8 6.8 5.2 P-5 13.4 10.8 13.3 – 3.8 8.3 – 7.4 – 17.3 6.2 6.7 14.0 13.7 13.6 12.0 B-0 – – – 12.1 – 2.4 – – 33.3 – 8.3 – – – 2.9 0.7 Number of groups 5 3 8 5 5 5.2 6 6 8 8 7.0 4 6 7 8 6.3 Ground color Yellow 5.9 15.4 50.0 63.6 94.3 45.8 27.4 48.4 58.8 60.0 48.6 84.4 78.0 39.2 39.8 60.4 Pink 94.1 84.6 50.0 24.3 5.7 51.7 72.6 51.6 7.8 40.0 43.0 15.6 22.0 60.8 57.3 38.9 Brown – – – 12.1 – 2.4 – – 33.3 – 8.3 – – – 2.9 0.7 Number of bands Unbanded 75.8 73.8 31.7 12.1 45.3 47.7 52.5 40.0 60.8 10.7 41.0 – 36.0 37.3 35.0 27.1 Mid- banded 5.9 – 26.7 75.7 17.0 25.1 37.8 34.7 9.8 25.4 26.9 62.2 – 3.9 8.7 18.7 Th ree- banded – – 18.3 12.1 – 6.1 9.7 – 3.9 6.8 5.1 – 26.0 21.6 19.4 16.7 Five- banded 18.3 26.2 23.3 – 37.7 21.1 – 25.3 25.5 57.1 27.0 37.8 38.0 37.3 36.9 37.5 N o t e . Th e maximum values are bold and underlined. 59Shell Banding and Color Polymorphism of the Introduced Snail Cepaea nemoralis… plants, which is confi rmed by the fi ndings of two colonies near garden centers, working (site 14) or recently closed (site 13). Already Boettger (1926) drew attention to the close connection between the expansion of the range of C. nemoralis and the garden culture. It is impossible to determine the exact age of most of the colonies found by us, in some cases it can be indirectly estimated at 10–20 years (see descriptions of sites 2 and 11 in Material and methods). Th is is in good agreement with the end of a strong economic decline in Ukraine in the 1990s and the beginning of an active and almost uncontrolled import of garden and ornamental plants from other European countries. I t is considered that Fst values may depend on the time of colonization of cities by C . nemoralis ( Cameron et al., 2009, 2014). Our results (table 5) are comparable with the data obtained in other regions of Europe for cities, the active colonization of which by C . nemoralis began only in the last decades (Cameron et al., 2009, 2014). However, the high values of this index calculated for Lviv may be associated not only with the relative youth of the studied colonies, but also with their complete isolation from each other and, possibly, with diff erent origins. I n the future, the phenotypic and genetic composition of such iso- lated colonies can become more similar only if they change i n the same direction under the infl uence of climatic selection, the infl uence of which theoretically should increase outside the natural range. Th e mosaic distribution of the frequencies of the main phenotype groups over the study area, which oft en l eads to a greater phenotypic similarity of distant rather than neighbor- ing colonies of C . nemoralis in Lviv (fi g. 2), is quite ex pected for colonies that arise not as a result of own locomotor activity of mollusks, but solely as a result of the transfer of a cer- tain number of snails or their eggs by humans. In our opinion, the similarity of the genetic composition of almost all colonies located in t he central part of Lviv and a little further south of it is more interesting (see Results).Th e most likely reasons for this could be the following: T a b l e 3 . Samples monomorphic in one trait Inherited pheno- typic traits Number of samples monomorphic by Site numbers its presence its absence Unbanded – 1 (3) (4), (5), 6 Mid-banded – 2 7, 11 Th ree-banded 2 5 (6) 1 –3, (5), 6, 11, 14, 15 Any of them 2 6 (8) 1–3, (4), (5*), 6*, 7, 11*, 14, 15 N o t e . In parentheses, taking into account two small samples at sites 4 and 5; an asterisk denotes sites, the samples from which were monomorphic in two traits. T a b l e 4 . Diff erences in the percentages of some traits and phenotype groups at the sites with diff erent shading Traits or phe- notype groups Open (O) Shaded (Sh) Mosaic (M) Diff erences(Mann-Whitney test) min–max mean min –max mean min–max mean O–Sh O –M M–Sh Some inherited phenotypic traits Yellow 5.9–94.3 45.8 27.4–60.0 48.6 39.2–84.4 60.4 10 8 6 Unbanded 12.1–75.8 47.7 10.7–60.8 41.0 0–37.3 27.1 8 6 3 Mid-banded* 0–86.2 36.1 25.0–79.6 47.7 0–62.2 20.5 8 6.5 3 Intensity of shell coloration (phenotype groups) Light 58.3–81.7 70.4 36.1–90.3 59.6 36.0–62.2 45.0 8 1** 6 Medium 4.6–35.0 17.2 5.9–25.7 12.4 12.2–52.0 33.9 9 3 1** Dark 6.7–18.5 12.5 0–56.9 28.0 12.0–29.1 21.1 6 4 7 N o t e . *Calculated from the number of banded shells, ** signifi cant at p = 0.05. 60 N. V. Gural-Sverlova, R. I. Gural, T. V. Rodych 1) all the mentioned sites, or at least part of them, could have been colonized by individuals from a common origin, for example, from a large garden center located near the city center (near site 13), which worked there from the 20th century until 2018; 2) these sites were colo- nized by a larger number of founder individuals (including also egg clutches located among the roots of seedlings) with a greater genetic and phenotypic diversity, which prevented the accidental disappearance or monomorphization of some hereditary traits. Indirect confi rmation of the fi rst hypothesis could be the discovery at some of these sites also individuals of C. hortensis with a shell coloration not typical for Lviv: yellow banded (sites 9, 10, 13), pink in combination with a dark lip (about 20 % at site 10, as well as one anatomically verifi ed specimen at site 7), which su ggests a possible joint introduction of these two species. Numerous colonies of C. hortensis in Lviv, formed by the descendants of mollusks that were introduced to Western Ukraine already in the 20th century (Sverlova et al., 2006), are represented by only three variants of shell colora tion: yellow unbanded, white unbanded, and white banded. On the other hand, at some sites there are the coloration variants that are absent near the garden center: brown shells in C . nemoralis (sites 9 and 10), pink shells with a dark lip in C. hortensis (see above). Th us, this garden center is hardly worth considering as a potential origin of colonization of all central sites. Unfortunately, we were not able to fi nd out which company (or companies) planted junipers and other ornamental plants at sites 2 and 11. Th erefore, we do not know whether the great similarity of the phenotypic and genetic composition of these two colonies (fi g. 2) is the result of their common origin or habitation in similar conditions. In contrast to the urbanized biotopes of southeastern Poland (Ożgo, 2005), the colo- nization of which by C. nemoralis began already at the end of the 19th century (Bąkowski, 1880), we were unable to fi nd statistically signifi cant diff erences in the frequencies of yel- low shells at the sites with diff erent degrees of shading. Th e diff erences in the frequencies of unbanded and mid-banded shells also did not reach a statistically signifi cant level. More signifi cant were the diff erences in the frequencies of the phenotype groups distinguished by the intensity of shell pigmentation and irr espective of the character of inheritance of certain phenotypic traits (table 5). At present, when analyzing pot ential selective changes in the phenotypic composition of C. nemoralis, the frequencies of the phenotypic manifestation of three alleles responsible for the yellow ground color of the shell, the complete absence of bands on it, and the for- mation of shells with one central band among banded shells are oft en used (Silvertown et al., 2011). However, with this approach, shells with diff erent intensities of coloration, from very light to very dark, inevitably fal l into each of the groups. Th e latter, for example, may include dark brown shells among unbanded and mid-banded, fi ve-banded shells with fused bands among yellow ones. Also, this does not take into account the possible linked inheritance of the ground color of the shell and the complete absence of bands on it (Murray, 1975), whi ch may result in the fact that shells with a lighter (yellow) ground color are relatively more oft en banded, and pink and especially brown shells — unbanded (Schilder, Schilder, 1957; Sverlova, 2007). It is possible that the observed ratio is also infl uenced by selection, acting against both the lightest and darkest coloration variants (Sverlova, 2004), and which can increase in introduced populations (Sverlova, 2007), up to complete linkage of the yellow color with banded, and pink color with unbanded shells (Gural-Sverlova et al., 2020; Sverlova, 2007). Th is approach is especially problematic for introduced and relatively young colo- nies, the initial phenotypic composition of which is largely dependent on random factors (founder eff ect as well as gene drift at low snail numbers in recently formed colonies). Even with strong selection, climatic or visual (Jones et al., 1977), further changes in such colonies can occur in diff erent ways, depending on the initial ratio of phenotypes. Th at is why we believe that it is more expedient to consider the frequencies not of single darkest or lightest phenotypes, but of their aggregates (see Material and methods). 61Shell Banding and Color Polymorphism of the Introduced Snail Cepaea nemoralis… It is very likely that the weak rel ationship between the intensity of shell coloration and the degree of shading of habitats in the Lviv colonies of C. nemoralis studied by us is caused not only with the above-mentioned random factors, but also with the relative youth of these colonies. In this case, over time, this relationship may intensify which can be confi rmed or ref uted only by further long-term studies, similar to studies of C. hortensis in Lviv (Gural- Sverlova, Gural, 2018). However, it cannot be ruled out that the more continental climate of Western Ukraine in comparison with the natural range of C. nemoralis will facilitate the selection of relatively light phenotypes even in shaded habitats, as is now observed in C. hortensis. According to Kirchhoff 's law of thermal radiation, light-colored shells, like any other light-colored surfaces, must not only heat up more slowly in the sun but also cool more slowly as a result of their own thermal radiation (Arnason, Grant, 1976; Sverlova, 2004). Th erefore, it is assumed that the dark-colored phenotypes of Cepaea should have a selective advantage in cooler, but at the same time relatively stable climatic conditions: in forests, on coasts, etc (Sverlova, 2004). And a lighter coloration can prevent not only overheating of shells in the sun but also their cooling too quickly with a sharp decrease in ambient temperature (Arnason, Grant, 1976). Th erefore, snails with light-colored shells can theoretically receive a selective advantage also with sharper temperature fl uctuations characteristic of a more continental climate (Sverlova et al., 2006). Сonclusions Our studies have shown not only the presence in Lviv of a number of relatively young colonies of the introduced species C. nemoralis, but also a great variability of the phenotypic composition within the city, which usually does not have a clear connection with the location of colonies or with the character of habitats. In the future, the obtained data can be used to monitor possible changes in the phenotypic composition of the investigated colonies to assess the potential eff ect of selective factors on this composition, fi rst of all, climatic selection outside the natural range of C. nemoralis. References Arna son, E., Grant, P. R. 1976. Climatic selection in Cepaea hortensis at the northern limit of its range in Ice- land. Evolution, 30, 499–508 . 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