Phylogenetic affinities and systematic position of the Italian populations of Gymnospermium (Berberidaceae)

Gymnospermium Spach is a small genus of tuberous herbs in family Berberidaceae, characterized by the fruits with exposed seeds when the membranous pericarp splits (from the Greek “gymnos”, naked, and “sperma”, seed). Together with the related genera Leontice L. and Caulophyllum Michx. it is part of tribe Leonticeae (Spach) Kosenko, which is characterized by succulent staminodia, eu-reticulate pollen exine, utricular gynoecia and a chromosome base number of x = 8 (with exceptions). Although Gymnospermium is the most diverse genus in Leonticeae, it only includes around twelve specific or subspecific taxa inhabiting semiarid steppes, montane shrublands and mesic forests across Eurasia. These taxa are mostly allopatric diploid endemics forming three distinct geographic groups in eastern China, central-western Asia and the Balkan peninsula (1,2). In spite of such a vast distribution range, the degree of morphological differentiation in the group is low and this has caused different interpretations of the limits, taxonomic status and number of species. In 2014 populations of Gymnospermium were discovered by the first two authors of this work in a narrow forest area of the Maddalena mountains, a massif of the southern Apennines (Salerno province). Biogeographically, this finding allowed to extend much to the west the western limit of the genus range. Based on macro-morphological characters, the Italian populations were first identified as G. scipetarum E. Mayer et Pulević, a species endemic to central Albania and south Montenegro. Other closely related species are G. maloi Kit Tan & Shuka, endemic to south Albania, and G. peloponnesiacum (Phitos) Strid, the third Balkan species endemic to the Peloponnese (3). The discovery of Gymnospermium in Italy prompted further studies on the species-level systematics of this little-known group, about which neither taxonomic investigations nor phylogenetic analyses have been conducted to date. We aimed at filling this gap using a combined molecular and morphological approach and performing a phylogenetic analysis of the genus based on a taxon sampling including all the Balkan taxa plus other species from Europe, western and central Asia and the Far East. Both nrDNA (ITS-5.8S) and cpDNA (trnL-F) markers were used to infer interspecific relationships and better understand the affinities of the Italian populations. Rate of variation of both markers was relatively low, so that not all the nodes were resolved in the Bayesian and Maximum Parsimony trees. This result matches the low degree of morphological differentiation among species, despite their often wide geographic isolation. However, the Albanian and Italian accessions were retrieved as a monophyletic group by both markers, and the populations from the southern Apennines resulted closer to G. maloi than to G. scipetarum. The Italian accessions, however, were characterized by a few Single Nucleotide Positions (SNPs) and formed a distinct, well supported group in the combined ITS- trnLF analysis. The morphometric study revealed a somewhat intermediate position of the Italian plants between the two above species, from which they differ by a peculiar combination of characters. Karyological analysis revealed the chromosome number 2n=14, as in G. maloi (2), unlike most of the other investigated species of Gymnospermium which have 2n =16. In the light of these results, the Italian populations are provisionally referred to a new species which is currently under description