Contrasting evolutionary origins of two mountain endemics: Saxifraga wahlenbergii (Western Carpathians) and S. styriaca (Eastern Alps)

Abstract

Background: The Carpathians and the Alps are the largest mountain ranges of the European Alpine System and important centres of endemism. Among the distinctive endemic species of this area is Saxifraga wahlenbergii, a Western Carpathians member of the speciose genus Saxifraga. It was frequently considered a taxonomically isolated Tertiary palaeopolyploid and palaeoendemic, for which the closest relatives could not yet be traced. A recently described narrow endemic of the Eastern Alps, S. styriaca, was hypothesized to be closely related to S. wahlenbergii based on shared presence of peculiar glandular hairs. To elucidate the origin and phylogenetic relationships of both species we studied nuclear and plastid DNA markers based on multiple accessions and analysed the data in a wide taxonomic context. We applied Sanger sequencing, followed by targeted next-generation sequencing (NGS) for a refined analysis of nrITS variants to detect signatures of ancient hybridization. The ITS data were used to estimate divergence times of different lineages using a relaxed molecular clock.

Results: We demonstrate divergent evolutionary histories for the two mountain endemics. For S. wahlenbergii we revealed a complicated hybrid origin. Its maternal parent belongs to a Western Eurasian lineage of high mountain taxa grouped in subsect. Androsaceae and is most likely the widespread S. androsacea. The putative second parent was most likely S. adscendens, which belongs to the distantly related subsect. Tridactylites. While Sanger sequencing of nrITS only showed S. adscendens-related variants in S. wahlenbergii, our NGS screening revealed presence of sequences from both lineages with clear predominance of the paternal over the maternal lineage.

Conclusions: Saxifraga styriaca was unambiguously assigned to subsect. Androsaceae and is not the sister taxon of S. wahlenbergii. Accordingly, the similarity of the glandular hairs observed in both taxa rests on parallelism and both species do not constitute an example of a close evolutionary link between the floras of the Western Carpathians and Eastern Alps. With the origin of its paternal, S. adscendens-like ITS DNA estimated to ca. 4.7 Ma, S. wahlenbergii is not a relict of the mid-Tertiary climate optimum. Its hybrid origin is much younger and most likely took place in the Pleistocene.

Keywords: Alps; Carpathians; Endemics; European mountains; Hybrid speciation; ITS genomic screening; Molecular clock; NGS; Phylogenetics; Tertiary relict.

Fig. 1

Photographs of Saxifraga wahlenbergii (a) and S. styriaca (b–d). a and b Habitus. c Flowers after anthesis. One of the tiny petals is marked by an arrow. D Rosette leaves. Photographs by S. Wróbel (a; Poland, Western Carpathians, Tatry, Wyżnia Świstówka, 14 June 2008) and N. Tkach (b–d; Austria, Lower Tauern, Rettlkirchspitze, M. Röser 11,356 & N. Tkach, 16 June 2018)

Fig. 2

Distribution of Saxifraga wahlenbergii in the Western Carpathians and S. styriaca in the Eastern Alps modified from Köckinger (2003) and Jasičová & Futák (1985). Topographic map from OpenStreetMap contributors, https://maps-for-free.com/

Fig. 3

Maximum likelihood phylogenetic tree of Saxifraga sect. Saxifraga (arrow) and representatives of other sections of Saxifraga based on nrITS Sanger sequences (reduced dataset; see text for details). Micranthes nivalis was chosen as outgroup. Maximum likelihood and maximum parsimony bootstrap support values as well as posterior probabilities of Bayesian inference ≥50% are indicated on the branches. The subsections mentioned in the main text are labelled on the right-hand side

Fig. 4

Maximum likelihood phylogenetic tree of Saxifraga sect. Saxifraga (arrow) and representatives of other sections of Saxifraga based on the plastid trnL–trnF and rpl32–trnL Sanger sequences (reduced dataset; see text for details). Micranthes nivalis was chosen as outgroup. Maximum likelihood and maximum parsimony bootstrap support values as well as posterior probabilities of Bayesian inference ≥50% are indicated on the branches. The subsections mentioned in the main text are labelled on the right-hand side

Fig. 5

Maximum likelihood phylogenetic tree of Saxifraga sect. Saxifraga (arrow) and representatives of other sections of Saxifraga based on nrITS Sanger sequences and NGS reads (reduced dataset). Micranthes nivalis was chosen as outgroup. Maximum likelihood and maximum parsimony bootstrap support values as well as posterior probabilities of Bayesian inference ≥50% are indicated on the branches. The Arabic numerals behind taxon names indicate different provenances listed in Additional file 1: Table S1. Letters A–D mean different variants of NGS sequences, if present, followed by the number of NGS reads (size). Taxon names without such letter and without number of reads are Sanger sequences. Numbering of the main clades is according to Table 1. The subsections mentioned in the main text are labelled on the right-hand side. The clade 10 is not resolved

Fig. 6

(a and b) Divergence time estimations for Saxifragaceae with focus on Saxifraga sect. Saxifraga (arrow) using an uncorrelated lognormal relaxed clock model applied for Sanger and NGS nrITS2 sequence data with the software BEAST v. 1.8.4. The numbers 1 to 17 refer to the main clades characterised in Table 1. Node heights indicate median ages. Horizontal node bars show the 95% posterior density probability date ranges. Four calibration points (see main text) are marked by circles. The time line with the main geological epochs is shown as horizontal axis in million years. Labelling behind taxon names as in Fig. 5

Fig. 6

(a and b) Divergence time estimations for Saxifragaceae with focus on Saxifraga sect. Saxifraga (arrow) using an uncorrelated lognormal relaxed clock model applied for Sanger and NGS nrITS2 sequence data with the software BEAST v. 1.8.4. The numbers 1 to 17 refer to the main clades characterised in Table 1. Node heights indicate median ages. Horizontal node bars show the 95% posterior density probability date ranges. Four calibration points (see main text) are marked by circles. The time line with the main geological epochs is shown as horizontal axis in million years. Labelling behind taxon names as in Fig. 5

Fig. 7

SEM photomicrographs of leaf trichomes in Saxifraga wahlenbergii (a, b) and S. styriaca (c). See Methods for the specimens studied