DNA studies

DNA studies

Report on DNA studies for taxonomic work

Hartmut Nordsieck (X.2020, supplemented III.2022)


DNA studies only for taxonomic work are rare. Mostly such studies are made within projects with impressive targets in order to get financial support from the giver institutions. In these projects only one or few species of a group are treated, and related species appear only as outgroups. Thus, DNA-analysed spots of knowledge are generated with large unknown ranges between, not very useful for ordinary taxonomic work.
During the work on European Clausiliidae I was aware that until now several groups of greater importance have not or only incompletely been investigated by DNA studies, though they exhibit severe taxonomic problems. For five of these groups I initiated DNA analyses carried out at my request by molecular working groups (mainly biome-id), financed with own funds saved for this purpose. In all these groups taxonomic work led to problems which could not be solved by morphology alone.
The first task was the subdivision of the genus Macrogastra (with Julica), a group which is widely distributed and common in non-meridional Europe (Project 1). The second task was the tribe subdivision of the subfamily Alopiinae, which is the most speciose group of European Clausiliidae and widespread in southern Europe (Project 2). The third task was the genus system of the tribe Delimini which is highly diverse and like other tribes of Alopiinae distributed mainly in southern Europe (Project 3).The fourth task was the analysis of the relations between the genera of the tribe Clausiliini, also common in non-meridional Europe, and to Graciliaria, a genus from the southern Carpathians, with unclear systematic position (Project 4). The fifth task was the system of the tribe Baleini, especially of the genera from Europe proper, some common in the whole of Europe (Project 5).
The restricted funds did allow only the examination of a limited number of species and the use of few markers. Therefore, the results of these analyses should be regarded as first approaches; they have to be expanded by future research. Nonetheless they were useful by leading to a better understanding of the phylogeny of the groups.
This is a report on the results. The full information on the work will be given in the written publication on European Clausiliidae.

Project 1: Macrogastra and Julica (Clausiliinae)

DNA studies of the Macrogastra group (JulicaMacrogastra) have been carried out by two working groups, the molecular identification team biome-id (Barco & Knebelsberger w. c.) and the Austrian Barcode of Life = ABOL group (Kruckenhauser w. c.). For the trees of the latter mtDNA markers (COI, 16S, 12S), for those of biome-id mtDNA and nDNA markers (COI, H3) have been used.
The results of biome-id were presented by a COI-H3 consensus tree (all but one species and two Clausilia species included, outgroup Cochlodina laminata, Fig. 1). The Clausilia species come out in a clade which is the sister group of a well-supported clade including Julica schmidtii and all Macrogastra species. Within this clade J.schmidtii is basal to the Macrogastra species which are united in a well-supported clade. Within the Macrogastra clade Mplicatula is basal to the other species which form a weakly supported polytomy with the following species: MventricosaMrolphiiMtumidaMbadiaMdensestriata, and a well-supported clade including Mborealis and a likewise strongly supported subclade Mattenuata + Masphaltina.
The polytomy is resolved, if one considers the results of the ABOL group (not figured here). In the COI-16S consensus tree presented by this group Mrolphii is basal to a clade including Mtumida and the other species (MattenuataMasphaltinaMborealisMbadiaMdensestriata), while Mventricosa is in another clade. In the COI-16S-12S consensus tree presented (in which Mrolphii was not included) Mventricosa is basal to the same clade including Mtumida and the other named species. In that clade again, Mtumida is basal to the other species. It is weakly supported (73%), while the subclade with the other species is well-supported (89%). Within that subclade Mattenuata + Mborealis and Mbadia + Mdensestriata come out as sister groups, the first (Mattenuata group) strongly supported (100%).
The results are the following:
In the COI-H3 tree of biome-id (Fig. 1) and also in the COI-16S-12S tree of the ABOL group (99% support) Julica and Macrogastra appear as a monophyletic group. In the COI-H3 tree Julica represents the sister group of the Macrogastra species; this concurs with its different genital morphology (Nordsieck 1963). Therefore, it is ranked as a genus of its own.
In the COI-H3 tree the Macrogastra species form a monophyletic group; thus they are assembled in one genus.
Within this genus Mplicatula is the sister group of all other species and therefore separated in a subgenus M. (Pyrostoma). This concurs with its male copulatory organs (parepiphallus) different from all other M. species (Nordsieck 2006).
Among the remaining species Mattenuata, Masphaltina and Mborealis belong to a strongly supported monophyletic group. The species Mdensestriata and Mbadia are related to that group, because in the COI-16S-12S tree of the ABOL group they form a well-supported clade with this group.
The remaining species of MacrogastraMtumidaMrolphii and Mventricosa, are obviously less closely related to that group. Mtumida is in the trees of the ABOL group basal to the named clade within a weakly supported larger clade. This is supported by the male copulatory organs of Mtumida, which except of their shortened loop resemble those of the Mattenuata group.

Fig. 1

Project 2: Alopiinae

Tribe systems of the Alopiinae based on morphological (mainly genital) characters were proposed by me in several papers since the sixties of the past century (Nordsieck 1969, 1972, 1997).
The DNA analysis of several genera of the Alopiinae by Uit de Weerd & Gittenberger (2013: tree fig. 3) had the following result: Macedonica, until now classified with the Cochlodinini, was basal to the rest of the subfamily, which came out as monophyletic. This rest was split into two groups, which appeared as sister groups: Cochlodina + Delimini and Alopiini (+ Montenegrina) + Medorini. Except of Macedonica, Alopiinae could thus be subdivided into Cochlodinini, Delimini, Alopiini (+Montenegrina) and Medorini (as proposed by Bouchet et al. 2017). The weak points of this system, however, were the following: 1. Macedonica, represented by only one species, was without tribe affiliation; 2. Montenegrina did not fit within the Alopiini, neither by shell nor by genital characters; 3. Medora, type genus of the Medorini, and Strigilodelima were not included in the analysis.
Therefore an expanded DNA analysis of the Alopiinae (three markers: COI, H3, ITS1 of rRNA gene) was carried out by the molecular identification team biome-id (Barco & Knebelsberger w. c.). In contrast to that of Uit de Weerd & Gittenberger, several species of the genus Macedonica and one each of the genera Medora and Strigilodelima were included.
The result (consensus tree Fig. 2) was the following: Macedonica species form a well-supported clade, basal to the other groups. The rest is distributed among three clades: clade 1 = Cochlodina, clade 2 (well-supported) = Delimini, and clade 3 (well-supported), in which several subclades appear as follows: HerillaMontenegrinaStrigilodelimaAlopia, and Medorini (CristatariaAgathylla-MuticariaMedora-Albinaria group). Herilla and Alopia are obviously less related as assumed because of their similar morphology; the same is true for Medora and Agathylla. A comparison with the consensus tree of Uit de Weerd & Gittenberger shows that a well-supported clade with the subclades Herilla and AlopiaMontenegrina) = Alopiini sensu Bouchet et al. is not present in the expanded tree of biome-id.
Therefore the following tribe system is proposed: new tribe for Macedonica, Cochlodinini, Delimini, and Alopiini (sensu lato), with unnamed subgroups of HerillaMontenegrinaStrigilodelimaAlopia and former Medorini of about the same rank. To erect tribes for each of these subgroups would certainly be an oversplitting. The system shows that the secondary plicae type of lunellar (lunella more or less reduced, palatal plicae left, clausilium plate notched) has evolved within Alopiinae several times independently.

Fig. 2

Recently, this analysis was once more expanded by an inclusion of the genus Triloba, until now assumed to be closely related to Herilla. The result was surprising: Triloba is basal to all other Alopiinae in the combined gene tree. This means that also for Triloba a new tribe is required.

Project 3: Delimini (Alopiinae)

The genera within the Delimini, tribe of the Alopiinae, were based on morphological characters, mainly of the genital organs, and geographical distribution (Nordsieck1979, 2002, 2013). With increasing knowledge, however, there arose problems with the delimitation of the genera, because several morphological characters revealed as unsuited for that purpose.
There are no shell characters which can be used for defining genera.
What concerns the genital characters, only few are suited for defining genera. By the different delimitation of the proximal part of penis three groups can be distinguished: CharpentieriaSiciliaria group (SiciliariaPapillifera), and Delima group (DelimaDilatariaBarcania). It is distinct in Charpentieria and absent in the Delima group; it is more or less distinct in the Siciliaria group. A distinct epiphallar thickening at the transition epiphallus-penis is present in most subgenera of Siciliaria (except Gibbularia) and absent in all other genera; this character could be regarded as an autapomorphy of this genus.
Until now, DNA studies, in which Delimini species are treated (Scheel & Hausdorf 2012, Uit de Weerd & Gittenberger 2013), are incomplete, because only certain species or species groups were included. In the tree of Uit de Weerd & Gittenberger (fig. 3) Charpentieria is basal to the other genera; the Delima group (DelimaDilataria) and the Siciliaria group (SiciliariaPapillifera) are sister groups. The expanded tree of Alopiinae of biome-id (see Project 2) had a similar result: Charpentieria basal, Siciliaria and Delima + Papillifera as sister groups.
Therefore a DNA analysis of the Delimini (three markers: COI, H3, ITS1) was carried out by the molecular identification team biome-id (Barco & Knebelsberger w. c.). In contrast to that of Uit de Weerd & Gittenberger and of Project 2 more species (17) and species of all genus taxa (except of Barcania) are included.
The result (consensus tree Fig.3) was the following:
Charpentieria and Papillifera appear as monophyletic.
The Siciliaria group (SiciliariaPapillifera) comes out as one clade, but without support. Within that clade S. (Gibbularia) and Papillifera form one subclade with weak support. Siciliaria species from N. W. Sicily are found in different positions: species of S. (Siciliaria) in a well-supported subclade and a species of another group, S. (Sicania), as lineage of its own. Species of other subgenera of Siciliaria (PiceataPaestanaMauritanica) are represented by one species each as own lineages. The two species of S. (Stigmatica), however, are found in different positions, Sstigmatica as own lineage and Sincerta in a clade with S. (Siciliaria) species from N. W. Sicily.
The Dilataria and Delima species have a basal position within the clades; Delima species appear in two different clades.
In the following, a DNA analysis with the marker 16S has been carried out for comparison. It has been expanded by species taxa from GenBank and for a second tree by further species of Delima and Siciliaria (Stigmatica).
The result (tree Fig. 4) was the following:
The Dilataria species is basal within the tree again.
Like in the consensus tree Charpentieria and Papillifera appear as monophyletic. The support for the Charpentieria clade is canceled, because in contrast to the consensus tree Dmontenegrina is found within that clade.
A group with Siciliaria without Papillifera but with most Delima species appears as one clade, with good support. Within this clade the included Delima species form a strongly supported subclade. Siciliaria is thus paraphyletic. S. (Piceata) and a subclade with S. (Siciliaria) species from N. W. Sicily (including Sincerta like in the consensus tree) come out in a strongly supported subclade. Another but weakly supported subclade is formed by species of S. (Stigmatica) (without Sincerta), S. (Paestana), S. (Sicania) and S. (Mauritanica). The S. (Stigmatica) species appear in two different lineages, one with Sstigmatica, the other with all other species. S. (Gibbularia) is represented as a subclade of its own.
Delima species appear in three different positions: DlaevissimaDmontenegrina, both in basal positions, and the other Delima species in the clade with Siciliaria. Therefore, it is probable that Delima is at least diphyletic, both southern species not much related and the northern ones monophyletic in a subclade.

Fig. 3

Fig. 4

In an expanded 16S DNA analysis further species taxa of S. (Stigmatica) are included. D. montenegrina is no more part of the Charpentieria clade, but a lineage of its own. The clade with Siciliaria and the northern Delima species persists, Siciliaria remains thus paraphyletic. The S. (Stigmatica) species form two clades like in the former tree, S. lamellata even a lineage of its own.
A further expansion of the 16S analysis by inclusion of 11 additional taxa led to the following results:
In comparison to the foregoing analysis Papillifera, the northern group of Delima = Dalmatica and Siciliaria come out in a clade, in which the species classified until now within Siciliaria form the following subclades: Siciliaria (S.), with S. piceata; Gibbularia; the stigmatica group of Stigmatica, S. lamellata as lineage of its own; S. paestana as lineage of its own and a clade with Mauritanica, Sicania and the remaining Stigmatica species.
S. scarificata from Marettimo Island clusters with Mauritanica.
S. ernae from Campania clusters with the remaining Stigmatica species, not with S. paestana.
S. honii from the coasts of southern Italy clusters with S. vulcanica, not with S. gibbula.
Within the Siciliaria (S.) subclade the northern and the southern subspecies of S. tiberii form a clade with S. calcarae, with S. leucophryna as sister group.
In the meantime (III. 2022) the 16S tree was expanded by inclusion of several further species.
The conclusions for the system of the Delimini are the following:
As a first step the genus system compared with that of Nordsieck (2007) is changed as follows:
Charpentieria and Siciliaria are separated as genera, as already proposed by Nordsieck (2013).
Delima (Piceata) is transferred to Siciliaria, as also proposed in that paper.
Mauritanica is confirmed as belonging to Siciliaria (as already stated in Nordsieck 2007).
S. (Siciliaria) is divided into two subgenera, S. (Siciliaria) and S. (Sicania), though this is not supported by morphological differences.
S. (Stigmatica) is also divided into two subgenera, S. (Stigmatica) and S. (Paestana). Apparently, Sincerta does not belong to S. (Stigmatica), but near to S. (Siciliaria).
Delima species are provisionally distributed among two subgenera, D. (Delima), including the southern species, and D. (Dalmatica) (name chosen from several group names of O. Boettger), including the northern species. The position of D. (Dugiana), which is not part of the analysis, is not yet clear.
In the meantime (III. 2022) the 16S tree was expanded by inclusion of further species.
The conclusions for the system of the Delimini are the following:
As a first step according to these analyses the genus system compared with that of Nordsieck (2007) is changed as follows:
Charpentieria and Siciliaria are separated as genera, as already proposed by Nordsieck (2013).
Delima (Piceata) is transferred to Siciliaria, as also proposed in that paper.
Mauritanica is confirmed as belonging to Siciliaria (as already stated in Nordsieck 2007).
S. (Siciliaria) is divided into two subgenera, S. (Siciliaria) and S. (Sicania), though this is not supported by morphological differences.
S. (Stigmatica) is also divided into two subgenera, S. (Stigmatica) and S. (Paestana). S. incerta does not belong to S. (Stigmatica), but to S. (Siciliaria).
Thus, Siciliaria would comprise the following subgenera: S. (Gibbularia), S. (Mauritanica), S. (Paestana), S. (Piceata), S. (Sicania), S. (Siciliaria), and S. (Stigmatica).
Delima species are classified within three subgenera, D. (Delima) and D. (Albanica), including the southern species D. laevissima and D. montenegrina, respectively, and D. (Dalmatica) (name chosen from several group names proposed by O. Boettger), including the northern species. D. amoena does not belong to D. (Dalmatica), but to S. (Stigmatica). The position of D. (Dugiana), which is not part of the analysis, is not yet clear.
As a second step a division of Siciliaria and Delima into several genera, which could better reflect the phylogenetic relationships of the species groups treated here as subgenera, may be necessary. Such a division was already proposed by de Mattia et al. (2021), but their system includes only a part of the groups concerned. The genera would be the following (type species in brackets):
Albanica (semilabiata = montenegrina), Charpentieria (dyodon), Dalmatica (conspurcata = blanda), Delima (laevissima), Dilataria (succineata), Gibbularia (gibbula), Mauritanica (tristrami), Paestana (paestana), Papillifera (solida), Piceata (piceata), Sicania (crassicostata), Siciliaria (grohmanniana), Stigmatica (stigmatica). In none of the trees Stigmatica appears as monophyletic, so that it probably has to be subdivided once more. Further investigations are needed to confirm this genus system.

Project 4: Clausiliini (Clausiliinae)

In the Clausiliini, tribe of the Clausiliinae, the morphological analysis revealed a directed evolution of the male copulatory organs to a shortening of their length and a reduction of penis and its retractor, correlated with a change of mode and duration of copulation (Nordsieck 1963, 1966, 2007). It was desirable to see if the evolution concluded from morphology could be confirmed by DNA analyses. In this analysis the systematic position of the genera with more plesiomorphic male copulatory organs, related to or included in the Clausiliini, GraciliariaRuthenicaPseudofusulus, and Julica, is of special interest.
Graciliaria has a pseudoepiphallus like the Clausiliini, but male end ducts like other groups of Clausiliinae with normal male copulatory organs = former Mentissoideinae. It could represent the earliest branch of the lineage leading to the Clausiliini.
Ruthenica exhibits male end ducts of normal length in which the epiphallus is reduced and indistinguishable from the distal part of vas deferens, thus an incipient parepiphallus. It could represent an early branch of the tribe.
The male end ducts of Pseudofusulus and Julica are like those of the remaining Clausiliini, but with a long parepiphallus which is difficult to delimit from the pseudoepiphallus, thus in comparison with the remaining genera of the tribe with more plesiomorphic end ducts. Both could represent basal branches with respect to other genera of the tribe. The basal position of Julica within the Macrogastra group has already been shown in Project 1 (tree Fig. 1).
The DNA analysis of the Clausiliini (marker: 16S), which was carried out by the molecular identification team biome-id (Barco & Knebelsberger w. c.) (tree Fig. 5, outgroup Idyla bicristata, a species of former Mentissoideinae), had the following result:
Graciliaria is together with the Clausiliini in a highly supported clade.
Within this clade the Macrogastra group (of Project 1, but with Erjavecia) and the remaining Clausiliini come out as sister groups.
Within the Macrogastra group, which is highly supported, Julica and M. (Pyrostoma) have a basal position, like in the tree of Project 1 (Fig. 1).The other Macrogastra species and Erjavecia form a polytomy which has to be resolved.
Graciliaria is basal to the remaining Clausiliini, but this is not supported. In another tree (with Delimini species as outgroup, not figured) Graciliaria is basal to the Clausiliini as a whole.
Pseudofusulus comes out as a lineage basal to the remaining Clausiliini.
Within the remaining Clausiliini Ruthenica is basal to the Clausilia species. In the other tree (with Delimini species as outgroup), it appears in a tritomy with Macrogastra group and Clausilia group.
The Clausilia species form a highly supported clade. Within this clade Neostyriaca and Clausilia sensu stricto appear as sister groups. The species of the latter form a highly supported subclade.
Within Clausilia sensu stricto there are three subclades or lineages, respectively, Clausilia rugosa + CpumilaCdubia and Cbidentata + Ccruciata. The latter subclade is also highly supported. In the other tree (with Delimini species as outgroup) C. dubia is basal to the other subclades.
An analysis of the group with the same marker and the same outgroup, which was expanded by the inclusion of some species of Acrotomini, former Mentissoideinae (one Akramowskia species, two Acrotoma species) (tree not figured), yielded a similar tree. The Acrotomini form a highly supported clade with Graciliaria + Clausiliini; both come out as sister groups. Again Graciliaria is together with the Clausiliini in a highly supported clade. This genus is now basal to the Macrogastra group, which is better resolved (with Erjavecia nested within).
If as outgroup the Acrotomini species are chosen, Idyla forms a less supported clade with Graciliaria + Clausiliini, with Idyla basal to the latter.
The analysis with two other markers (COI, H3), but with the same outgroup, also expanded by the inclusion of the Acrotomini species (tree Fig. 6), had the following result:
Like in the 16S tree the Acrotomini form a highly supported clade with Graciliaria + Clausiliini, and Graciliaria is together with the Clausiliini in a highly supported clade. Now Graciliaria is basal to the Clausiliini as a whole (like in the 16S tree with Delimini species as outgroup). The Clausiliini form a polytomy, including two highly supported subclades, Macrogastra group (with Erjavecia) and Clausilia (C.). In this respect the tree is less informative than the 16S tree.
If as outgroup the Acrotomini species are chosen, Idyla forms an unsupported clade with Graciliaria + Clausiliini, with Idyla basal to the latter.
The conclusions from the trees are the following:
The results of the morphological analysis of the Clausiliini, especially concerning the evolution concluded from the genital anatomy, are largely confirmed.
Graciliaria is closely related to the Clausiliini, but is basal in all trees to the Clausiliini or certain groups within. It is related neither to the Acrotomini, which are similar in genital morphology, nor to Idyla, which includes a subgenus (Strigilidyla) similar in shell morphology. It can remain in a tribe of its own: Graciliariini.
Ruthenica belongs to the Clausiliini, where it has in the 16S analyses a position basal to the Clausilia group. Therefore, a transfer from the Graciliariini, with which I have formerly classified it (Nordsieck 2007), to the Clausiliini, already assumed from the DNA analysis of Uit de Weerd & Gittenberger (2013), is necessary.
The classification of Pseudofusulus with the Clausiliini is confirmed. Because it is an independent lineage in the trees, it remains to be regarded as a genus of its own.
For the classification of the Macrogastra group (JulicaMacrogastra) see Project 1. The position of Julica and Mplicatula (Pyrostoma) distant from the remaining Macrogastra species is made plain by all trees.
Erjavecia appears in all trees as closely related to Macrogastra. From the trees alone it could be regarded as a subgenus of Macrogastra.
As already concluded from a DNA study of Clausilia (Strobeliella) by Hausdorf & Nägele (2016), the former genus Neostyriaca should be classified with the genus Clausilia. The Neostyriaca group (NeostyriacaLombardiellaStrobeliella), in the trees only represented by Ncorynodes, is in the 16S analyses the sister group of Clausilia (C.).
According to the trees a further subdivision of Clausilia (C.) because of the special genital anatomy of some species (CdubiaCrugosa, Nordsieck 1990) is not appropriate, because both species are nested within that subclade (Cdubia can also be basal to the other species).

Fig. 5

Fig. 6

Most recently the analyses have been expanded by the inclusion of Erjaveciella approximans, member of the Fusulus group of Clausiliini, and Elia moesta (Mentissoideini, former Mentissoideinae).
In the 16S analysis including only Graciliaria and Clausiliini species (outgroup Idyla bicristata) the Erjaveciella species comes out within the highly supported clade basal to all other genera inclusive of Graciliaria (tree Fig. 7). If the Acrotomini group is included, it clusters with Acrotoma, out of Graciliaria and the other Clausiliini species. In  the COI-H3 analysis E. approximans forms a weakly supported clade with Ruthenica within the Clausiliini.
In the COI-H3 analyses Elia moesta is basal to Graciliaria and the Clausiliini, with which it forms a highly supported clade. In the 16S analyses E. moesta appears within the Clausiliini, nested within the Clausilia clade with high support.
As already shown by the genital anatomy Erjaveciella, which belongs to the Fusulus group, has an isolated systematic position. According to the 16S analysis that group could be separated as a tribe of its own (Fusulini Lindholm 1924), according to the COI-H3 analysis it could also remain in the Clausiliini as an isolated subgroup. Until now, Fusulus interruptus has not been included in DNA analyses. Further investigations are needed to decide on the rank of the Fusulus group within the Clausiliinae.
The included groups of former Mentissoideinae, Acrotomini and Idyla, are obviously less related to the group Graciliaria + Clausiliini than Elia (Mentissoideini). The latter is deemed to be more closely related to that group, because in all analyses it forms with it highly supported clades.

Fig. 7

Project 5: Baleini (Clausiliinae)

The Baleini, tribe of the Clausiliinae, are distributed in the Caucasian regions and Europe proper, mainly in southeastern Europe. The Caucasian genera have a deeply ending spiral lamella in common; (except of Micropontica) they exhibit the plicae type of lunellar. Both characters are regarded as plesiomorphic (Nordsieck 2007). Among the genera of Europe proper only the Vestia group (VestiaPavlovicia ) has a deeply ending spiral lamella. Because of the reduced CA, however, this character is not recognizable in Balea. The Vestia group, Strigillaria and Balea are characterized by special morphological characters (Vestia group shell, Strigillaria and Balea genital characters, see article on genital studies).The genera of the Laciniaria group have some apomorphies of the shell in common and may represent a monophyletic group. Thus, from morphology it could be concluded that the Caucasian Baleini and the Vestia group have a basal position in phylogeny, while the other genera of Europe proper represent evolutionary lineages with special apomorphic characters.
Only few DNA studies include Baleini species from Europe proper.
In the trees of Van Moorsel, Schilthuizen, Piel & Gittenberger in Van Moorsel (2001: figs. 4-5) Balea = Alinda and Bulgarica = Strigillaria came out as only distantly related. Gittenberger et al. (2006) realized a large genetic distance between Balea (Alinda) and B. (Balea). The separation of Balea and Alinda as independent genera was thus supported. In the tree of Uit de Weerd & Gittenberger (2013: fig. 3) two groups with different positions appeared, Vestia + Balea and Laciniaria + Alinda. This means that the first two genera are only distantly related to the two latter ones.
The DNA analysis of European Baleini species of nearly all genera (marker: 16S), which at my request was carried out by the molecular identification team biome-id (Barco & Knebelsberger w. c.) (expanded by inclusion of some Caucasian species, outgroup Idyla bicristata, a species of former Mentissoideinae), had the following result:
The examined species form a clade with high support, i. e., the Baleini appear as monophyletic. Within this clade, two subclades come out, one with MentissaLaciniariaAlinda and Pseudalinda, which corresponds to the Laciniaria group (see above), and one with StrigillariaBalea and Vestia . The Caucasian species form a strongly supported clade which is basal to the second group, but without support. Within the first group Mentissa and the remaining genera are sister groups. These genera form a well-supported subclade, with Pseudalinda basal and Laciniaria and Alinda as sister groups. Pseudalinda comes out as paraphyletic with respect to the two other genera; this is in contrast to its special shell characters which speak for the monophyly of the genus. The inclusion of Alinda wagneri within Alinda shows that it is no Pseudalinda species as assumed by former authors. Within the second group one Strigillaria group, S. (Bulgarica + Denticularia), is basal to the remaining groups, which appear as two sister clades, one clade containing the other Strigillaria group = S. (Strigillaria) and Balea, the other clade Vestia, all clades with high support. The separate position of both Strigillaria groups corresponds to their considerable differences in genital morphology (see article on genital studies) and supports a separation of Strigillaria and Bulgarica as genera of their own. In the Vestia clade the V. (Brabenecia) species is basal to the V. (Vestia) species, which agrees with the genital differences of both subgenera (vagina, penis) and their different modes of reproduction (ovipary vs. (ovo)vivipary).
The inclusion of three further Bulgarica species and a Pavlovicia species led to a slight modification of the tree (tree Fig. 8). Within the Baleini clade, three subclades come out, one with the Caucasian species, one with Mentissa, Laciniaria, Alinda and Pseudalinda and one with Pavlovicia, Vestia, Balea and the Strigillaria group. Within the second clade Mentissa and the other genera appear as sister groups. These other genera form a highly supported subclade, with Pseudalinda and Laciniaria + Alinda as sister groups; Pseudalinda is now monophyletic. The third clade is highly supported, with three subclades forming a tritomy: Pavlovicia, Vestia and Balea + Strigillaria group. Within the latter Balea is basal to the Strigillaria group, but without support. The Strigillaria group with high support contains Strigillaria and Bulgarica as sister groups, the latter, also with high support,  B. thessalonica and the other four species as sister groups. Thus, Pavlovicia is confirmed as a genus of its own. Within Bulgarica the subdivision into two subgenera, B. (Denticularia) and B. (Bulgarica) is confirmed.

Fig. 8


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