Phylogeny, species delimitation and population structure of the steppe-inhabiting land snail genus Helicopsis in Eastern Europe Author Balashov, Igor A. Author Neiber, Marco T. Author Hausdorf, Bernhard text Zoological Journal of the Linnean Society 2020 2020-12-22 193 1108 1125 journal article 3658 10.1093/zoolinnean/zlaa156 c61c4597-0700-4f1d-bf39-ff3059c60d42 0024-4082 5636863 SYSTEMATICS OF HELICOPSIS Our phylogenetic analysis of 16S rDNA sequences ( Fig. 2 ; Supporting Information, Fig. S1 ) confirms that Helicopsis forms a clade with Xerolenta , Xeromunda , Xeropicta and Pseudoxerophila ( Hausdorf & Bössneck 2016 ; Neiber et al. , 2017 ) and shows that also Kalitinaia Hudec & Lezhawa, 1967 from the Caucasus region belongs in this clade named Helicopsini. The delimitation of Helicopsis was considered questionable, because it is characterized only by plesiomorphic character states ( Hausdorf, 1996 ). Actually, the molecular phylogeny based on mitochondrial 16S rDNA sequences ( Fig. 2 ; Supporting Information, Fig. S1 ) revealed that H . subcalcarata from Turkey and Helicopsis sp. from Cyprus , which is closely related to H . cypriola (Westerlund, 1889) , do not belong to Helicopsis . Helicopsis subcalcarata is sister to the remaining Helicopsini and, thus, a separate genus is required for this species. Helicopsis sp. from Cyprus is closely related to the eastern Mediterranean genus Pseudoxerophila . Amore detailed analysis of the relationships of these species is in preparation. Figure 4. Neighbour-net network of Helicopsis from Ukraine based on Jaccard distances obtained from AFLP data. Coloration of clusters corresponds to the STRUCTURE solution for K = 2. Red corresponds to H . filimargo , yellow to H . lunulata . DNA voucher numbers for specimens are given at the tips of the network. Coloured dots at the tips of the network correspond to cluster assignments (on majority-rule basis) of specimens in the STRUCTURE solutions for K = 5 (inner) and K = 7 (outer). For locality data and the distribution of clusters, see also Supporting Information, Table S1 and Figure 6, respectively. Helicopsis gittenbergeri from Greece formed together with H . aelleni and H . persica from Iran the well-supported sister-group of the Helicopsis species from Eastern and Central Europe [as in the phylogenetic analysis of Hausdorf & Bössneck (2016) ]. The monophyly of the Eastern and Central European Helicopsis species , as well as the monophyly of the Central European Helicopsis group including H.striata , H. austriaca and H. hungarica , are well supported in the 16S rDNA tree ( Fig. 2 ; Supporting Information, Fig. S1 ) and the tree based on the concatenated COI , 12S rDNA and 16S rDNA sequences ( Fig. 3 ). The populations from Eastern Europe, which are the focus of this study, form two highly supported clades in these trees, for which the oldest names are H. lunulata (Krynicki, 1833) and H . filimargo (Krynicki, 1833) . The analyses of the concatenated sequences show that H. filimargo is sister to the Central European clade, while H . lunulata is sister to all other Central and Eastern European species ( Fig. 3 ). Figure 5. Assignment of specimens of Helicopsis from Ukraine to clusters resulting from the admixture analysis of AFLP data with STRUCTURE. A, solution for K = 2. B, solution for K = 5. C, solution for K = 7. Numbers below the plots refer to populations (see Fig. 6; Supporting Information, Table S1). Letters above the plots refer to mitochondrial clades: a, H . filimargo (clade A); b, H . filimargo (clade B); l, H . lunulata . The H . lunulata clade includes mainly populations with depressed conical shells previously identified as H. striata . However, individuals with disc-like shells, identified as H. instabilis based on shell characters and originating from the Lviv region, the type locality of this nominal species, also belonged to this clade. The same is true for the disc-like specimens from Romania . Finally, specimens also identified as H. ( filimargo ) arenosa from Yelanets Steppe Nature Reserve in the Mykolaiv region belong to this clade. Balashov (2016: 471 , fig. 264) thought that the specimens at this locality represent sympatric populations of H. striata and H. filimargo arenosa . However, both, specimens identified as H. striata and specimens identified as H. filimargo arenosa belong to the same clade. Beside specimens from Romania , Ukraine and adjacent Russia , an individual of an introduced population from north-eastern Germany (see Zettler et al. , 2006 ) also belongs in this clade. The disc-like shells of this population agree with shells described as Helicella ? depulsa from the surroundings of Varna in Bulgaria by Pintér (1969) , which is similar to H. instabilis . Helicella ? depulsa has been classified as a subspecies or a synonym of Xerolenta obvia ( Welter-Schultes, 2012 ) . Specimens from the surroundings of Varna have to be examined to clarify the identity of the nominal taxon. Figure 6. Distribution of clusters obtained from the admixture analysis of AFLP data of Helicopsis from Ukraine with STRUCTURE. A, solution for K = 2. B, solution for K = 5. C, solution for K = 7. Population numbers are given next to the bar plots (Supporting Information, Table S1). The bar plots show the proportional assignment of an individual to a cluster (see also Fig. 5). The clustering of the various forms that share the discussed mitochondrial haplotype clade in the network based on the nuclear AFLP markers ( Fig. 4 ) and in a STRUCTURE analysis of these data ( Fig. 5 ) demonstrated that they actually represent a single species. This species is distinct from H. striata , with which most populations of this clade were identified. Helicopsis striata is more closely related with the Eastern Alpine H. austriaca and H. hungarica from the Pannonian Basin, than with the Helicopsis species from Eastern Europe ( Fig. 2 ; Supporting Information, Fig. S1 ) and is apparently restricted to the Northern steppes and the northwestern Pannonian Basin in Central Europe. There are several names for forms that belong to the discussed species. The most often used name is H. instabilis (Rossmässler, 1838) , given for a disc-like form from Lviv in Western Ukraine , but also used for disc-like forms from Romania . However, the oldest and, thus, valid name of the species is H. lunulata (Krynicki, 1833) , given for a depressed conical form from Odessa [ lectotype see Hausdorf (1990 : pl. 2 fig. 4); paralectotype see Sysoev & Schileyko (2009 : fig. 113A)]. Our records show that H. lunulata occurs in Transylvania, in the Podolian Upland and the Black Sea Lowland and in the Donetsk Upland and the adjacent Central Russian Upland ( Fig. 1 ; Supporting Information, Table S1 ), from where a sequence belonging to the H. lunulata clade has already been reported by Sychev & Snegin (2016) from Zasosna. Unfortunately, no AFLP data were available from these eastern specimens. Thus, we cannot completely exclude the possibility that these specimens belong to H. filimargo with a mitochondrial introgression, although there is no evidence for this. There are records of H. instabilis and H. striata from Moldova ( Balashov et al. , 2013b ) and Bulgaria ( Damjanov & Likharev, 1975 ), indicating that H. lunulata is probably also distributed in these countries. However, it has to be checked whether all populations identified as H. instabilis so far actually belong to H. lunulata . Beside populations with disc-like shells corresponding to H. instabilis , there are also populations with depressed conical shells in Transylvania that are often classified as an endemic species, H. cereoflava ( Grossu, 1983 ; Welter-Schultes, 2012). Considering the large intraspecific variability observed in H. lunulata , it is possible that all Transylvanian populations of Helicopsis belong to this species. However, typical H. cereoflava should be studied genetically to prove this hypothesis. The H. filimargo clade is found in Ukraine east of the Dnieper River, from Crimea and the adjacent eastern part of the Black Sea Lowland to the Donetsk Upland and the Central Russian Upland ( Fig. 1 ). This group comprises even more diverse morphological forms than H. lunulata . It includes, beside the nominal species listed from Crimea and the Black Sea Lowland, H. filimargo , H. arenosa and H. retowskii and the recently described supposed endemics from the Donetsk Upland, H. luganica , H. martynovi and H. subfilimargo , plus some populations classified as H. striata from eastern Ukraine . Helicopsis filimargo and H. arenosa have also been recorded from Dobrogea in Romania ( Grossu, 1983 ) and the latter species was also found in Bulgaria ( Damjanov & Likharev, 1975 ). However, given the lack of diagnostic morphological characters distinguishing H. filimargo and H. lunulata , it has to be checked genetically whether the populations from Romania and Bulgaria actually belong to H. filimargo or whether they represent forms of H. lunulata . The H. filimargo clade is subdivided into two geographically restricted clades in the mitochondrial tree ( Fig. 2 ; Supporting Information, Fig. S1 ). One occupies most of the range, whereas the other is restricted to southernmost Crimea ( Fig. 1 ). There are a few populations along the mountains where both haplotypes occurred together. The specimens from southernmost Crimea are concentrated in one cluster of the network based on nuclear AFLP markers ( Fig. 4 ). However, there are also specimens from the rest of the range with the other mitochondrial subclade among them. This indicates that differentiation between the two subgroups is also evolving in the nuclear genome, but is not yet advanced. This is also corroborated by the STRUCTURE analyses ( Fig. 5 ). Thus, we do not classify them as subspecies. The STRUCTURE analyses shows little admixture between H. filimargo and H. lunulata ( Fig. 5 ), supporting the species status of the two taxa. The only exception was the specimen from the introduced population in north-eastern Germany . However, this specimen is older and not preserved in 100% alcohol, so that the slightly different AFLP pattern might be an artefact resulting from DNA degradation. The specific distinctness of H. filimargo and H. lunulata is also corroborated by the isolation by distance analysis that shows that the distances between individuals of the two taxa are larger than expected based on their geographical distances and the relationship of genetic distances with geographical distances found within the two taxa ( Fig. 7 ). An individual from Teleshovka in the Belgorod region in the Central Russian Upland represented another clade in the mitochondrial tree ( Fig. 2 ; Supporting Information, Fig. S1 ), which can be identified as H. hungarica based on sequences of the species from Hungary and Austria ( Duda et al. , 2018 ). Actually, a sequence of the same haplotype group has already been reported as H. striata from Gubkin in the Belgorod Region by Sychev & Snegin (2016) and identified as H. hungarica by Duda et al. (2018) . The specific identity of the specimens from the Central Russian Upland with the Pannonian H. hungarica should be confirmed with non-mitochondrial genetic markers. Our results, based on mitochondrial DNA sequences ( Figs 1–3 ) and nuclear AFLP markers ( Figs 4–7 ), demonstrate that the native populations from Ukraine and adjacent regions can be classified into two species, H. lunulata and H. filimargo . This result resembles the suggestion of Balashov (2016) to classify the Ukrainian Helicopsis populations into two species, H. striata and H. filimargo . However, the delimitation and the distribution of the species in the two classifications are different. Although H. lunulata includes mainly populations with depressed conical shells formerly assigned to H. striata , it also comprises populations with disc-like shells formerly assigned to H. instabilis , which was included in H. filimargo by Balashov (2016) . Moreover, Balashov (2016) classified some H. lunulata as H. filimargo arenosa and supposed that there are syntopic co-occurrences of H. striata and H. filimargo . We have not found syntopic co-occurrences of H. lunulata and H. filimargo , although we found some overlap of the ranges of the two species in the Donetsk Upland and the adjacent Central Russian Upland so that co-occurrences are possible.