Plant material

Several plants morphologically resembling Alyssum rossetii were sampled in July 2022 at the locality between Pic de Qüenca and Rocablanca near Alós d'Isil in the province of Lleida in the Pyrenees (684PQU, Fig. 1 and Appendix 1). They were used for phylogenetic analyses (ITS of nrDNA), genome size measurements (by flow cytometry), chromosome counting and morphological comparisons. Five living individuals were transferred to the Botanical Garden of P. J. Šafárik University in Košice and kept there until the anthesis in April 2023 (Fig. 2). Phylogenetic analyses and morphological evaluation included A. rossetii from the type locality in the Alps, and other perennial Alyssum species (A. cacuminum, A. fastigiatum, A. montanum) and annual species (A. alyssoides, A. collinum, A. granatense and A. simplex) previously reported from the Pyrenees and adjacent regions (Appendix 1). In addition, three species of the Gamosepalum lineage from Turkey, previously resolved as closest to A. rossetii (Španiel & al. 2023a), and Odontarrhena muralis (Waldst. & Kit.) Endl. and O. tortuosa (Waldst. & Kit. ex Willd.) C. A. Mey. (both as outgroups) were also included in the phylogenetic inferences. ITS sequences of nrDNA were generated for the present study or taken from GenBank and published studies (Appendix 1).

In contrast, Alyssum taxa occurring in France and Spain but neither in the Pyrenees nor in their foothills (annual A. minutum Schltdl. ex DC., perennial A. flexi-caule Jord., A. gallaecicum (S. Ortiz) Španiel, Marhold & Lihová, A. loiseleurii P. Fourn., A. orophilum Jord. & Fourr., and A. rhodanense Jord. & Fourr.) were not included in the present study. Their phylogenetic relationships and morphological variation were analysed and discussed in detail in previous studies (Zozomová-Lihová & al. 2014; Španiel & al. 2019; Cetlová & al. 2021).

Chromosome counting and flow cytometry

Chromosome numbers were determined in mitotic metaphases of root tip cells. The root tips were taken from two adult plants identified as Alyssum rossetii collected at the locality 684PQU (Appendix 1) in the Pyrenees and kept in the Botanical Garden of P. J. Šafárik University in Košice. The root tips were pre-treated in a 0.002 M aqueous solution of 8-hydroxyquinoline for approximately 18 hours at 4° C, fixed in a mixture of 96 % ethanol and 98 % acetic acid (3:1) for 1–24 hours at 4° C, and macerated in 1-N HCl at 60° C for 6 min. Between each step, the root tips were washed in distilled water. Permanent squashes were prepared using the cellophane square method (Murín 1960) and stained in a 7 % Giemsa stock solution in Sørensen phosphate buffer for 1 h. The squashes were washed with distilled water, dried and observed in a drop of immersion oil using a Leica DM 1000 microscope equipped with HDCE-X5 camera and ScopeImage 9.0 software.

Flow cytometry using the AT-selective fluorochrome DAPI was employed to determine the relative genome size and ploidy level of six plants sampled in situ at the locality 684PQU in the Pyrenees, including the same individuals used for chromosome counting. Solanum lycoper-sicum L. “Stupické polní rané” (2C = 1.96 pg; Doležel & al. 1992) was used as an internal standard. The analyses were performed using a Partec Cyflow ML instrument equipped with an HBO-100 mercury arc lamp (Partec, Münster, Germany) at the Plant Science and Biodiversity Centre of the Slovak Academy of Sciences in Bratislava following the protocol described in Španiel & al. (2011a).

Ploidy levels of the other species were known from previous studies (Španiel & al. 2010, 2011a, 2018a, 2018b; Zozomová-Lihová & al. 2014; Cetlová & al. 2021; Appendix 1).

Phylogenetic analyses

The ITS region of nrDNA was amplified using the universal primers ITS4 and ITS5 (White & al. 1990) following the protocol of Melichárková & al. (2019). Purified PCR products were either sequenced directly or cloned if intraindividual polymorphisms were detected after direct sequencing, indicating the presence of multiple ITS copy variants within a genome. Molecular cloning was performed as described in Melichárková & al. (2017). Sequencing was carried out at Eurofins Genomics (Konstanz, Germany). The ITS sequences were edited and aligned in Geneious software v. R10.2.6 (Biomatters Ltd, Auckland, New Zealand). Two data partitions (for the non-coding ITS1+ITS2 and the coding 5.8S regions) were defined and the best-fit models of nucleotide substitutions were assessed separately for each of them in jModelTest version 2.1.10 (Darriba & al. 2012) using the Akaike information criterion. The phylogenetic tree was constructed using maximum-likelihood (ML) inference in GARLI v. 2.01 (Zwickl 2006) run at the CIPRES Science Gateway (Miller & al. 2010). Settings for ML phylogenetic computations followed Melichárková & al. (2017). All newly generated ITS sequences were submitted to GenBank (accession numbers OR016717–OR016761; Appendix 1).

Fig. 2.

Alyssum rossetii from the Pyrenees in cultivation. – A: flowering plant; B: inflorescence. – Slovakia, Košice, Botanical Garden of P. J. Šafárik University, 28 Apr 2023, photographs by L. Mártonfiová; voucher specimens deposited in SAV.

Study of morphology and examination of herbaria

Morphology of the Pyrenean plants resembling Alyssum rossetii was evaluated on the basis of samples collected in situ and five ex situ flowering individuals in the Botanical Garden of P. J. Šafárik University in Košice. The examined morphological characters include those that unambiguously differentiate A. rossetii from taxa of the A. montanum–A. repens group and were identified in the previous study (Španiel & al. 2018b): presence/absence and shape of wings and teeth on the four inner (longer) filaments of stamens, presence/absence of purplish coloration on sepals, length of trichome rays of stellate trichomes on the lower surface of middle stem leaves and density (number per 0.5 mm²) of stellate trichomes on the lower and upper surface of middle stem leaves. Due to the small number of investigated individuals and the ex situ origin of the flowers, we did not carry out a thorough multivariate morphometric comparison of the Pyrenean and Alpine plants of A. rossetii. Values of morphological characters of almost all other taxa except A. alyssoides and A. granatense, were taken from previous studies (Zozomová-Lihová & al. 2014; Cetlová & al. 2019). For the latter two species, several individuals from the populations listed in Appendix 1 were examined (characters on fruits). Characters of indumentum on leaves and silicules were observed and measured using a stereomicroscope (Olympus SZ61) at magnification 80× and QuickPHOTO Micro 3.2 software. The photo of the trichome of A. rossetii was taken using a scanning electron microscope (JEOL JSM-6390LV) at the Geological Institute of the Slovak Academy of Sciences in Banská Bystrica.

In order to find other potential localities of plants similar to Alyssum rossetii in the Pyrenees, we examined herbarium collections in BC, BCN and MA.

Chromosome number, ploidy level and relative genome size

A diploid chromosome number (2n = 16) was determined in two plants of Alyssum rossetii from the locality 684PQU in the Pyrenees (Fig. 1). The total relative genome size (2C) of these two and additional four individuals was 0.916 ± 0.005 which corresponds to the monoploid relative genome size (Cx) of 0.458 ± 0.002 (mean ± standard deviation in arbitrary units).

Phylogenetic analyses

The ITS alignment included 107 sequences from 48 individuals and was 629 bp long. It comprised 134 variable sites and a total of 62 different sequence variants (ribotypes) were identified. Intragenomic variation was frequently observed, within both diploid and polyploid accessions, with up to seven different ribotypes detected per individual. In contrast, all four analysed individuals of Alyssum rossetii from the Alps (560FEU) had a single ribotype that was identical to that found in the plants from the Pyrenees (four individuals from 684PQU). In the ML tree (Fig. 3), the ribotype of A. rossetii was placed in the Gamosepalum lineage, which was clearly distinct from the clade comprising the other Alyssum species studied: annuals and the nested perennial A. montanum–A. repens complex, in congruence with the known phylogeny of the genus (see Introduction).

Fig. 3.

Maximum-likelihood (ML) tree based on ITS sequence data of studied Alyssum taxa. Population codes follow Appendix 1. Bootstrap support values (> 50%) are indicated.

Morphology and herbarium data

Individuals found in the Pyrenees (684PQU), tentatively identified as Alyssum rossetii, have green sepals with a darker coloration at the apex, however not as dark and purplish as in the plants from the type locality of A. rossetii. Wings and teeth on the four inner (longer) filaments of stamens are only slightly developed or absent (same as in the plants from the type locality of A. rossetii). Lower surface of middle stem leaves is covered by 31–45 stellate trichomes per 0.5 mm² with rays 0.11–0.13 mm long; upper surface of middle stem leaves is covered by 20–31 stellate trichomes per 0.5 mm². These values are within the range of those previously measured in plants from the type locality of A. rossetii. On the other hand, individuals of A. rossetii, considering plants from both the Pyrenees and Alps, clearly differ from other perennial or annual species growing in the Pyrenees and adjacent regions, as presented in the Identification key below.

We found no herbarium specimens from the Pyrenees morphologically similar to Alyssum rossetii in the herbarium collections of BC, BCN and MA, and no earlier collections from the locality of 684PQU studied here.

Identification key of Alyssum species occurring in the Pyrenees and adjacent regions

The value ranges of the quantitative characters represent the 5^th and 95^th percentiles; asterisks (*) indicate mean values of three random counts per leaf surface or silicule valve. Indumentum of leaves should be inspected on leaves placed in a middle part of flowering stems, not on non-flowering shoots (therefore the term “middle stem leaf” is used throughout the key). Overall geographic ranges and habitats of species are reported according to Maire (1967), Küpfer & Nieto Feliner (1993), Jalas & al. (1996), Zozomová-Lihová & al. (2014), Španiel & al. (2018b) and Cetlová & al. (2019, 2021).