Underestimated diversity in one of the world’s best studied mountain ranges: The polyploid complex of Senecio carniolicus (Asteraceae) contains four species in the European Alps

Senecio carniolicus (Asteraceae) is an intricate polyploid complex distributed in the European Alps (di-, tetra- and hexaploids) and Carpathians (hexaploids only). Molecular genetic, ecological, and crossing data allowed four evolutionary groups within S. carniolicus to be identified. Here, we establish that these four groups (two vicariant diploid lineages, tetraploids and hexaploids) are also morphologically differentiated. As a consequence, we draw taxonomic conclusions by characterizing four species, including the more narrowly circumscribed S. carniolicus (lectotypified here), the taxonomically elevated S. insubricus comb. nov. (lectotypified here), and the two newly described species S. disjunctus and S. noricus.


Senecio carniolicus
Senecio carniolicus is widely distributed in the Eastern European Alps and the Carpathians. The Alpine distribution area ranges from the Alpi Lepontine and the Prealpi Luganesi at the border between Switzerland and Italy to the easternmost central Alps of Austria , Sonnleitner et al. 2010. It occurs almost exclusively on siliceous bedrock (Ellenberg 1996) and thrives in a variety of alpine habitats, such as grasslands and dwarf shrub communities, stabilized scree slopes and rock crevices (Pignatti 1982), moraines and pioneer swards (Beger in Hegi 1928) as well as wind-exposed fellfields with strong freeze-thawing dynamics (Franz 1986). The altitudinal distribution ranges from timberline up to the nival zone (Reisigl & Pitschmann 1958).
The presence of ploidy level variation in S. carniolicus , Hülber et al. 2009) suggests that the morphological types might not merely be habitatinduced modifications of the same species, but instead correspond to different cytotypes. In the Alps, three main cytotypes (di-, tetra-and hexaploids) are found, while in the Carpathians only hexaploids occur . Cytotypes co-occur in major parts of the distribution area , Sonnleitner et al. 2010 sometimes within a few decimetres (Hülber et al. 2009(Hülber et al. , 2015. Nevertheless, intermediate ploidy levels were only found in ca. 1% of individuals in a comprehensive sample of about 5000 individuals (Sonnleitner et al. 2010), indicating strong crossing barriers under natural conditions. This is supported by molecular genetic differentiation among cytotypes (Hülber et al. 2015, M. Winkler et al. unpubl.) and, within the diploids, between two longitudinally vicariant groups (Escobar García et al. 2012); by poor seed sets and very low hybrid viability in crosses between (eastern) diploids and polyploids, though not between polyploids (Sonnleitner et al. 2013); and by microhabitat differentiation between the three main cytotypes (Sonnleitner et al. 2010, Hülber et al. 2015. Consequently, there is strong evidence that S. carniolicus in its present taxonomic circumscription contains four ecologically differentiated, genetically distinct, and partly reproductively isolated groups: western diploids, eastern diploids, tetraploids and hexaploids.
Here, we establish that these four groups are morphologically differentiated. As a consequence, we draw taxonomic conclusions by formally describing two new species and by redefining, at the species level, the circumscriptions of S. carniolicus subsp. carniolicus and S. carniolicus subsp. insubricus. In addition, we provide diagnostic characters and the geographic distribution of these four species as well as a determination key.

Material and Methods
We sampled S. carniolicus in its current wide circumscription on 28 collecting sites evenly distributed over its distribution area (collecting sites 1, 2, 4, 10, 15, 18, 20, 21, 22, 23, 26, 40, 41, 46, 63, 64, 65, 66, 72, 77, 79, 80, 81, 87, 92, 96, 97, 100 in Sonnleitner et al. 2010), collecting one flowering shoot and one vegetative rosette per individual. As the collection was done without prior knowledge of the cytotype, sample sizes differ across the sites. Plant material was preserved in 75% alcoholic aqueous solution until preparation. Additionally, several specimens were taken from each population to be deposited in the herbarium WU. All individuals were ploidy-checked (Sonnleitner et al. 2010). From the alcohol-preserved material, one fully developed rosette leaf and one cauline leaf from the middle of the flowering stalk were pressed and dried. The number of ray and disk flowers of one fully FLATSCHER et al.

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts anthetic capitulum was counted and flowers were mounted on sticky paper. Involucre width and length as well as the height of the flowering shoot and of the synflorescence were measured directly on the alcohol material. Values presented in the species descriptions correspond to the 10% and 90% quantiles, supplemented by extreme values scored on specimens listed under "Additional specimens examined".

Results and Discussion
The polyploid complex of S. carniolicus is an example of underestimated species diversity in the generally well-explored european Alps. It comprises a group of closely related taxa that are not only genetically and ecologically distinct, but exhibit also clear morphological differences. Our field experience showed that flowering individuals can be assigned to one of the four groups with high accuracy. Morphological separation of the two diploid taxa is mainly based on higher indumentum density and a lower number of flowering heads per synflorescence in western diploids, and coincides with complete geographical separation and the absence of recent gene flow (Escobar García et al. 2012). Polyploids differ from diploids in a taller growth and a longer corolla of ray flowers. In comparison to hexaploids, tetraploids are characterized by a stronger degree of the leaf dissection, i.e., deeply incised rosette leaves and presence of distinct secondary lobes in stem leaves.
The congruence of morphological differences with genetic divergence (Escobar García et al. 2012, Hülber et al. 2015, ecological differentiation and the presence of crossing barriers , Hülber et al. 2009, Sonnleitner et al. 2010, 2013 highlight the distinctness of these evolutionary lineages. On this basis we propose splitting S. carniolicus into four taxa, which together constitute the S. carniolicus agg. Separation at the species level seems most appropriate, because the four entities meet several requirements of different species concepts. Most importantly, reproductive isolation between diploids and polyploids is almost complete. No intermediate cytotypes were encountered in the broad area of co-occurrence of western diploids and hexaploids (Sonnleitner et al. 2010), and artificial crossings between eastern diploids and polyploids failed almost completely (Sonnleitner et al. 2013). Tetra-and hexaploids hybridize upon hand pollination (Sonnleitner et al. 2013), but intermediate cytotypes are nevertheless rare in nature (Sonnleitner et al. 2010). This is likely due to ecological differentiation particularly in areas of sympatry of tetraploids and hexaploids (Hülber et al. 2015). Specifically, tetraploids are most common on northern slopes, which are rarely occupied by hexaploids, and-in contrast to the other three entities-also extend to intermediate to slightly basic soils. Therefore, polyploids usually do not form mixed populations, but only narrow contact zones in areas of ecological overlap (Hülber et al. 2015). The four entities can therefore be regarded as functional biological species, which hybridize only occasionally. Occupation of different ecological niches or "adaptive zones" (Van Valen 1976), as demanded by the ecological species concept (Coyne & Orr 2004), further supports distinction at the species level.
Ecology:-Alpine meadows and dwarf shrub communities, with a tendency towards northexposed slopes, sometimes also on stony, more shallow soils than S. carniolicus; usually on siliceous bedrock, but more frequently found on intermediate to slightly basic substrates than the other species; ca. 1870-3080 m.
Distribution:-The species occurs in two disjunct distribution areas (Fig. 5D); the western partial range spans from the Alpi Bergamasche to the Ortler/Ortles and Adamello massifs (Südtirol/Trentino/Brescia, Italy; Graubünden, Switzerland) and the eastern partial range extends from the easternmost Hohe Tauern eastwards (Salzburg/Kärnten/Steiermark/ Austria). It remains to be investigated based on a broader sampling whether the morphological differences between individuals from the two partial distribution areas are constant enough to allow for the taxonomic recognition at the subspecific level.
Etymology:-The epithet refers to the distribution pattern of the species, whose range is split into two disjunct partial areas.

Determination key to the S. carniolicus aggregate and its closest relatives
This key includes all Alpine species of the Incani-clade sensu Pelser et al. (2003) with the exception of the morphologically very divergent S. abrotanifolius Linnaeus (1753: 869).
Thus, in addition to the S. carniolicus aggregate this key also contains S. incanus, which was formerly often considered conspecific with S. carniolicus (e.g., Chater & Walters 1976, Aeschimann et al. 2004, Fischer et al. 2008, and the morphologically clearly distinct and thus taxonomically uncontroversial Western Alpine species S. persoonii De Notaris (1844: 229) and S. halleri Dandy (1970: 625)  6 Rosette leaves deeply (more than half the distance to the midrib) incised, lateral lobes longer than wide, always divided (usually with one or two secondary lobes), adult leaves of eastern (Austrian) populations glabrous, those of western (Italian and Swiss) populations pubescent

S. disjunctus
-Rosette leaves shallowly lobed (less than half the distance to the midrib) to dentate or rarely almost entire, lateral lobes at most as long as wide, only occasionally with secondary lobes, hairy to slightly tomentose at least when young, older leaves often glabrescent

Acknowledgements
This paper presents the results of the Master thesis of Ruth Flatscher, who passed away at the age of 28 after an extended period of illness. Ruth was an extraordinarily gifted PhD student at the Universities of Innsbruck and Vienna; her premature death not only ended the life of a promising young scientist but also deprived her friends and colleagues of a very special human being. We deeply miss her.
We thank Marco Caccianiga, Božo Frajman, Christian Gilli, Manfred Schmucker and Daniela Stawik for help with collecting and Walter Gutermann for insightful advice and discussion on the manuscript. Marianne Magauer determined the pollen diametres. We are grateful to the Austrian Science Fund (project P 20736-B16 to P.S. followed by G.M.S), the Commission of Interdisciplinary Ecological Studies (KIOES) of the Austrian Academy of Sciences (grant P2008-01 to G.M.S.) and the Association for the Advancement of Plant Sciences (supporting R.F.) for funding our research. We are grateful to John Spillmann for providing us with photos of specimens of S. insubricus stored in ZT. An anonymous reviewer and the subject editor Alexander Sennikov provided helpful comments that substantially improved the manuscript.