Introduction

Interspecific hybridization has enormous evolutionary potential and can result in homoploid or polyploid hybrid speciation in addition to introgressive hybridization (Arnold 1997). Homoploid hybrid speciation appears to be rare and has been documented more or less convincingly in fewer than 30 cases (Yakimowski & Rieseberg 2014; Kadereit 2015; Schumer & al. 2014; Nieto Feliner & al. 2017). In contrast, polyploid hybrid speciation may account for 2% to 15% of speciation events in flowering plants (Otto & Whitton 2000; Wood & al. 2009).

Viola L. (Violaceae) is a genus of approximately 525 (Ballard & al. 2014) or 580 to 620 species (Wahlert & al. 2014) distributed in temperate regions and montane areas in the tropics worldwide (Ballard & al. 2014). Several species have long been the subject of intensive “bio-systematic” studies (Briggs & Walters 2016), including studies of hybrid speciation based mainly on chromosome numbers, morphology, ecology and geographical distribution (Clausen 1926, 1927, 1931; Valentine 1950, 1958; Moore & Harvey 1961; Schmidt 1961, 1962, 1963, 1964; Harvey 1966; Küpfer 1971; Merxmüller 1974; Merxmüller & Lippert 1977; Erben 1985).

Several of these studies examined the Italian species of the informally named Calcarata complex (Merxmüller 1974; Merxmüller & Lippert 1977; Pignatti 1994; Erben & Raimondo 1995; Fenaroli & Moraldo 2003). The unranked taxon Calcaratae was first introduced by Becker (1910). In his treatment of Viola in the 2nd edition of Die natürlichen Pflanzenfamilien (Becker 1925), which is the last comprehensive treatment of the entire genus, Becker characterized his V. sect. Melanium Ging. [unranked] Elongatae W. Becker [unranked] Crenatifoliae W. Becker [unranked] Calcaratae W. Becker as violets with large flowers with long or short spurs, often basally dentate sepals and pinnate to dentate stipules. Gams (1925) characterized V. [unranked] Calcaratae as having long and creeping primary axes with leaves and flowers on lateral axes. Becker (1925) divided V. [unranked] Calcaratae into V. [unranked] Eucalcaratae W. Becker with long and acute spurs and V. [unranked] Altaicae W. Becker with long or short but obtuse spurs. These two groups were claimed to be distributed mostly in the Alps and southern European mountains, the Mediterranean islands (V. [unranked] Eucalcaratae) and from North Africa to East Asia (V. [unranked] Altaicae).

Following Becker (1925), Viola [unranked] Eucalcaratae contains V. calcarata L. from the Alps, V. bertolonii Pio emend. Merxm. & W. Lippert from Italy and V. corsica Nyman from Corsica, while V. [unranked] Altaicae contains V. altaica Ker Gawl. from southeastern Europe through the Caucasus to China, V. dichroa Boiss. from Turkey, V. arsenica Beck from southeastern Europe and V. eugeniae Parl., V. nebrodensis Presl and V. pseudogracilis Strobl from Italy as well as V. munbyana Boiss. & Reut. from North Africa.

In the course of time, the use of the name Viola [unranked] Calcaratae, as the Calcarata complex (V. calcarata-Gruppe; Schmidt 1964), was limited to mostly Italian species, and the complex was considered by Schmidt (1964) to contain taxa with large flowers. Many of the species considered to belong to the Calcarata complex or found to be associated with it by us, have flowers > 2 cm, and sometimes up to 4 cm wide (Valentine & al. 1968; Pignatti 2017). Pignatti (2017) characterized the complex as often having dimorphic leaves where the lower and upper cauline leaves differ in shape, complex stipules (pinnately or palmately divided), and thin and rather long (20–30 cm) ascending axes. According to Pignatti (2017) the complex occurs at higher elevations in Italy and on Sicily, Sardinia, Elba and Corsica. Following Schmidt (1964) and Pignatti (1994, 2017), the Italian Calcarata complex contains the following 15 species: V. aethnensis Parl., V. bertolonii, V. calcarata, V. corsica, V. culminis F. Fen. & Moraldo, V. dubyana Burnat ex Gremli, V. etrusca Erben, V. eugeniae, V. ferrarinii Moraldo & Ricceri, V. merxmuelleri Erben, V. nebrodensis, V. pseudogracilis, V. tineorum Erben & Raimondo, V. ucriana Erben & Raimondo and V. valderia All. Several of these species were treated by Fiori (1924) as varieties of V. calcarata. These species were grouped by Pignatti (2017) with V. argen-teria Moraldo & Forneris, V. cenisia L., V. comollia Massara and V. magellensis Porta & Rigo ex Strobl, which all have more or less undivided stipules, in V. sect. Melanium group I [unranked] Heterophyllae Pignatti. It is conceivable that some of the species newly described by Ricceri & al. (2018) also belong to this complex. Schmidt (1964) suspected that the Italian Calcarata complex species might be closely related to species from the Balkans and Greece.

The Calcarata complex clearly belongs to Viola sect. Melanium, a group of more than 120 species (Valentine & al. 1968; Merxmüller & Lippert 1977; Erben 1984, 1985, 1986, 1989; Erben & Raimondo 1995; Ballard & al. 1999; Erben 2000; Fenaroli & Moraldo 2003; Blaxland 2004; Moraldo & al. 2011; Marcussen & al. 2015; Margini & Scoppola 2015; Ricceri & al. 2018; Perrino & al. 2018). Viola sect. Melanium is widely distributed in western Asia and Europe, with its centre of diversity in the hills and mountains of southern Europe, especially the Balkan Peninsula and the Apennines (Erben 1996). The only species of V. sect. Melanium to occur in the New World is V. bicolor Pursh from eastern North America (Clausen & al. 1964). Previous studies have recovered V. sect. Melanium as monophyletic (Ballard & al. 1999: two species; Yockteng & al. 2003: 20 species; Marcussen & al. 2015: three species; Slomka & al. 2015: 25 species), but very little phylogenetic resolution within the section was obtained using ITS sequences (Yockteng & al. 2003; Slomka & al. 2015).

In preparation of a detailed study of hybrid speciation in the Calcarata complex using restriction-site associated DNA sequencing (RADseq), we set out to investigate whether this Calcarata complex, which is not well-characterized morphologically, can be resolved as monophyletic using Sanger sequencing data, and whether species from outside Italy and the listed Mediterranean islands also belong to this Calcarata complex. For this purpose, we inferred a phylogeny of Viola sect. Melanium using 92 species and DNA sequence data from the nuclear ribosomal internal transcribed spacer region (ITS), the nuclear ribosomal external transcribed spacer region (ETS) and the plastid trnS–trnG intergenic spacer region.

Material and methods

Results

The sequence alignments were 491 bp (ITS), 471 bp (ETS) and 628 bp (trnS–trnG, trnS gene) long. The combined nuclear dataset and the plastid dataset contained 132 and 134 accessions, respectively. The ML and BI phylogenies from the combined nuclear dataset and from the plastid dataset showed supported topological differences. In general, the ML/BI plastid phylogenies were far less resolved than the nuclear phylogenies. Apart from the absence of a few clades in the ML phylogeny, the ML and BI topologies did not differ. Accordingly, the phylogenetic trees shown (Fig. 1, 2) represent the BI topology with ML bootstrap support (BS) added. Branches with BS < 50 or posterior probabilities (PP) < 0.95 were collapsed.

Viola sect. Melanium was recovered as monophyletic in all analyses. In the following, we will describe our results with reference to the Calcarata complex as described in the introduction. Most species of the Calcarata complex group in four larger clades in the BI analysis of the nuclear dataset (Fig. 1). Viola aethnensis subsp. aethnensis, V. corsica subsp. limbarae Merxm. & W. Lippert, V. dubyana, V. merxmuelleri and V. valderia were recovered outside these clades in larger polytomies. In the following description, clades with species of the Calcarata complex are given capital letters in the nuclear dataset (Fig. 1) and numbers in the chloroplast dataset (Fig. 2). These clades are also listed in Table 1.

Clade A (PP 1, BS 86) contains Viola nebrodensis, V. ucriana, V. tineorum, V. bertolonii and V. ferrarinii of the Calcarata complex together with V. cornuta L. from the Pyrenees, V. grisebachiana Vis. and V. orphanidis Boiss. both from the Balkan peninsula and V. paradoxa Lowe from Madeira. In this clade, three species of the Calcarata complex from Sicily, V. nebrodensis, V. ucriana and V. tineorum, form a supported subclade (PP 1, BS 76). Of the Calcarata complex species of clade A, V. ferrarinii and V. bertolonii fall into clade 2 (PP 1, BS 64) in the BI analysis of the plastid data (Fig. 2; Table 1), and V. nebrodensis and V. ucriana form a supported clade (1) (PP 1, BS 70; Table 1). Clade B (PP 0.98, BS 62) contains V. eugeniae subsp. levieri (Parl.) A. F. W. Schmidt, V. eugeniae subsp. eugeniae, V. pseudogracilis subsp. cassinensis (Strobl) Merxm. & A. F. W. Schmidt, V. aethnensis subsp. splendida (W. Becker) Merxm. & W. Lippert, V. corsica subsp. ilvensis (W. Becker) Merxm., V. aethnensis subsp. calabra (A. Terracc.) Peruzzi and V. pseudogracilis subsp. pseudogracilis of the Calcarata complex plus the Italian V. magellensis and the Greek V. rausii Erben, V. graeca (W. Becker) Halacsy and V. sfikasiana Erben. Of the Calcarata complex species of clade B, V. corsica subsp. ilvensis is part of clade 2 in the BI analysis of the plastid data (Fig. 2; Table 1). All other Calcarata complex taxa are part of large polytomies in this analysis. Clade C (PP 1, BS 72) contains V. corsica subsp. corsica and V. etrusca of the Calcarata complex together with V. montcaunica Pau from Spain. Of the Calcarata complex species of clade C, V. corsica subsp. corsica is part of clade 2 in the BI analysis of the plastid data (Fig. 2a; Table 1) and V. etrusca and V. montcaunica are supported sister to each other (clade 3; PP 0.99, BS 68; Fig. 2; Table 1). Clade D (PP 1, BS 88) contains V. culminis, V. calcarata subsp. calcarata, V. calcarata subsp. cavillieri (W. Becker) Merxm. & W. Lippert, V. calcarata subsp. zoysii (Wulf.) Merxm. and V. calcarata subsp. villarsiana (Roem. & Schult.) Merxm. of the Calcarata complex. They group together with the widespread V. lutea Huds. subsp. lutea and V. beckiana Fiala from the southern Balkan peninsula. Of the Calcarata complex taxa of clade D, V. calcarata subsp. villarsiana and V. calcarata subsp. cavillieri are part of clade 2 in the BI analysis of the plastid data (Fig. 2; Table 1). Viola culminis is part of a supported clade (4) together with V. comollia in the plastid phylogeny (PP 1, BS 60; Fig. 2; Table 1), and the remaining Calcarata complex taxa of clade D are part of large polytomies in this analysis.

In summary, species of the Calcarata complex fall into four larger clades in the BI analysis of the nuclear dataset. Interestingly, some species from these four clades are part of the only larger supported clade (2) in the plastid phylogeny, which also contains Viola corsica subsp. limbarae, which is not part of any larger clade in the nuclear phylogeny. All species recovered in clade 2 of the plastid phylogeny are part of the Calcarata complex.

The ITS substitution rate calculated using the crown group age of Viola sect. Melanium estimated by Marcussen & al. (2015) is 0.57 × 10^-9 substitutions/site/year.

Discussion

Considering the phylogenetic topologies obtained by us from the analysis of nuclear (ITS/ETS) and plastid (trnS–trnG) sequences (Fig. 1, 2), it is obvious that neither the Italian Calcarata complex as understood by Schmidt (1964) and Pignatti (1994, 2017) nor Becker's (1925) Viola [unranked] Calcaratae was recovered as a monophyletic group. In the nuclear dataset, most taxa of the Italian Calcarata complex – except V. aethnensis subsp. aethnensis, V. corsica subsp. limbarae, V. dubyana, V. merxmuelleri and V. valderia – fall into four larger clades which, however, all also contain additional species that had not previously been considered part of the Calcarata complex. In the plastid dataset, some species of all four nuclear clades fall into clade 2, which contains only Calcarata complex species. However, the majority of Calcarata complex species are found in unresolved positions in a large polytomy.

Table 1.

Clades recovered in the analyses of the ITS/ETS and trnS–trnG datasets. Species of the Calcarata complex are marked in bold script. Placement of species of ITS/ETS clades in trnS–trnG clades and of species of trnS–trnG clades in ITS/ETS clades is indicated in parentheses.

Fig. 1.

Bayesian inference (BI) phylogeny of Viola sect. Melanium based on the combined nuclear dataset (ITS/ETS). Values above branches are Bayesian posterior probability and maximum likelihood bootstrap values (PP/BS). Only posterior probabilities ≥ 0.95 and bootstrap values ≥ 50 are shown. A hyphen (-) represents posterior probabilities < 0.95 or bootstrap values < 50. Absence of clades is indicated by n.p. (not present). Capital letters refer to clades discussed in the text. Species in bold script have been considered part of the Calcarata complex in the past.

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The question arises as to why the Calcarata complex/Viola [unranked] Calcaratae is not resolved as monophyletic and why, more generally, phylogenetic resolution is rather poor in both our nuclear and plastid phylogenies, as also found by others (e.g. Ballard & al. 1999;Yockteng & al. 2003; Wahlert & al. 2014; Slomka & al. 2015). The lack of resolution could be caused by insufficient sequence divergence (Maddison 1989), incomplete lineage sorting, or hybridization (Doyle 1992; Maddison 1997). In this case, we postulate that hybridization is the major problem in reconstructing phylogenetic relationships in Viola in general and in V. sect. Melanium in particular. First, there exist a large number of studies which have either documented interspecific hybridization in the genus and in V. sect. Melanium (Clausen 1927; Fothergill 1938; Erben 1985, 1996; Pettet 1964; Kakes 1979; Krahulcová 1996; Marcussen & al. 2001; Conesa & al. 2008) or hybrid speciation (Erben 1996; Küpfer 1971; Merxmüller & Lippert 1977; Pignatti 1994; Erben & Raimondo 1995; Erben 1996; Fenaroli & Moraldo 2003; Siuta & al. 2005; Marcussen & al. 2012), and the role of hybridization in the origin of supraspecific lineages in Viola, suspected previously by Ballard & al. (1999), has been convincingly demonstrated by Marcussen & al. (2015). An example of hybrid speciation in V. sect. Melanium inferred by nuclear-plastid incongruence may be highlighted by V. corsica subsp. ilvensis. This taxon has been hypothesized by Pignatti (1994) to have originated from hybridization between V. bertolonii, with which it groups in clade 2 of the plastid phylogeny (Fig. 2), and V. eugeniae or V. pseudogracilis, with which it groups in clade B in the nuclear phylogeny (Fig. 1). Second, evidence for hybridization may also be provided by the limited amount of ITS sequence divergence in the section. If indeed, as estimated by Marcussen & al. (2015), Viola is of Oligocene origin and the crown group age of V. sect. Melanium is between 12.76 and 15.26 (means) ma, the ITS substitution rate calculated by us is 0.57 × 10^-9 substitutions/site/year. Such a rate clearly falls outside the known range of ITS substitution rates for herbaceous annual or perennial plant species of 1.72 × 10^-9 to 8.34 × 10^-9 substitutions/site/year and is similar to the lowest rates otherwise found only in woody plants (Kay & al. 2006). The low substitution rate found by us is then perhaps best explained by hybridization across the entire section, resulting in homogenization of younger ribotypes leading to the exclusion of older ribotypes.

Fig. 2.

Bayesian inference (BI) phylogeny of Viola sect. Melanium based on the plastid dataset (trnS–trnG). Values above branches are Bayesian posterior probability and maximum likelihood bootstrap values (PP/BS). Only posterior probabilities ≥ 0.95 and bootstrap values ≥ 50 are shown. A hyphen (-) represents bootstrap values < 50. Numbers refer to clades discussed in the text. Species in bold script have been considered part of the Calcarata complex in the past.

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Based on our findings, we hypothesize that hybridization has been so frequent in the evolution of Viola sect. Melanium that tree building methods such as ML and BI are not suitable for the reconstruction of phylogenetic relationships (Posada & Crandall 2001).

Irrespective of the likely great importance of hybridization in the evolution of Viola sect. Melanium, we will briefly examine (1) those species of the Italian Calcarata complex that did not fall into the four major nuclear clades, (2) those species that fell into these nuclear clades but had not been explicitly associated with the Calcarata complex or V. [unranked] Calcaratae before, and (3) those species that fell into the plastid clade but had not been explicitly associated with the Calcarata complex or V. [unranked] Calcaratae before. The four major nuclear clades found by us do not correspond to the major morphological groups among Italian Calcarata complex species identified by Pignatti (2017).

As for the five taxa not falling into the four larger clades of Calcarata complex species in our nuclear phylogeny, i.e. Viola aethnensis subsp. aethnensis, V. corsica subsp. limbarae, V. dubyana, V. merxmuelleri and V. valderia, their relationship to the Calcarata complex has never been doubted from a morphological point of view (Pignatti 2017). In the case of V. aethnensis subsp. aethnensis and V. corsica subsp. limbarae, other subspecies of these two species group in the four major nuclear clades found by us, and V. corsica subsp. limbarae falls into clade 2 of our plastid phylogeny.

Species not strictly associated with the Italian Calcarata complex or Viola [unranked] Calcaratae are V. cornuta, V. grisebachiana, V. orphanidis and V. paradoxa in clade A, V. graeca, V. magellensis, V. rausii and V. sfikasiana in clade B, V. montcaunica in clade C and V. beckiana and V. lutea subsp. lutea in clade D. Of these 11 species, the widespread V. lutea subsp. lutea (clade D) has been considered closely related to V. calcarata by Reiche & Taubert (1895), and Becker (1925) included V. dubyana, a species of the Calcarata complex, in his V. [unranked] Luteae W. Becker, which also included V. lutea. Viola lutea was found in a clade containing Calcarata complex species also by Hildebrandt & al. (2006). Viola beckiana (clade D), a species from serpentine or calcareous rocks in the southern Balkan peninsula, is large-flowered and has palmately or pinnately divided stipules (Valentine & al. 1968). The Spanish V. montcaunica (clade C) and V. cornuta (clade A; both with 2n =22), of which the former was described as essentially a smaller form of the latter by Valentine & al. (1968), have palmately divided stipules like V. valderia (see above) and share relatively large flowers and long spurs with species of the Calcarata complex. Viola magellensis was included in V. sect. Melanium group I [unranked] Heterophyllae, which also includes the Calcarata complex, by Pignatti (2017). Of the Greek species (all clade B), V. graeca has large flowers and long spurs, V. rausii has long spurs and dimorphic leaves and V. sfikasiana has dimorphic leaves, all characters found in at least part of the Calcarata complex. Viola orphanidis and V. cornuta, grouping with species of the Calcarata complex in clade A, are similar to each other in leaf and stipule shape. Following Gams (1925), Becker included V. orphanidis, a species from the Balkans, in V. lutea, and V. lutea had previously been linked to the Calcarata complex (see above). Viola grisebachiana (clade A) from the Balkans is acaulescent according to Valentine & al. (1968) and has no obvious similarities to the Calcarata complex. Finally, V. paradoxa (clade A) from Madeira has been postulated to be a close relative of V. calcarata by Lowe (1868); the species has long axes, dimorphic leaves and large flowers (Short 1994). In the plastid clades, only V. montcaunica (clade 3) and V. comollia (clade 4) fell into supported clades with Calcarata complex species (Fig. 2). Of these, V. montcaunica has been discussed above, and V. comollia was included in V. sect. Melanium group I [unranked] Heterophyllae by Pignatti (2017).

With the exception of Viola grisebachiana, a relationship of all those species that have not been associated with the Calcarata complex but falling into nuclear Calcarata complex clades seems possible on the basis of their morphology or on the basis of their classification by earlier authors. However, in the absence of clear morphological characters defining the Calcarata complex – all characters used by Pignatti (2017) to characterize the Calcarata complex can also be found in other species of V. sect. Melanium (Valentine & al. 1968) – association of non-Calcarata species with the Calcarata complex based on morphological characters is not fully convincing.

In summary, tree-building methods such as ML and BI do not appear to be suitable for the reconstruction of phylogenetic relationships in Viola and V. sect. Melanium because of rampant interspecific hybridization. Reconstruction of relationships instead requires both larger DNA sequence datasets and tree-building methods that take hybridization into account (e.g. Wen & al. 2018).