An Unexpected Occurrence of Alyssum rossetii (Brassicaceae) in the Pyrenees, a New Species for the Spanish flora

Abstract: Alyssum rossetii is recorded for the first time in Spain from a single locality in the Pyrenees. After the type locality in the Valpelline valley (Pennine Alps, Italy), it is only the second known locality of this species. Morphology, diploid chromosome number (2n = 16), relative genome size, and ITS sequences confirm that the Pyrenean plants are conspecific with the Alpine ones and should be classified as A. rossetii. Phylogenetic analyses placed A. rossetii within the clade of A. sect. Gamosepalum comprising species from Turkey and demonstrated that A. rossetii is only distantly related to other Alyssum species occurring in the Pyrenees and adjacent regions: A. alyssoides, A. cacuminum, A. collinum, A. fastigiatum, A. granatense, A. montanum, and A. simplex. An identification key to all Alyssum species known from the Pyrenees is presented. Citation: Š aniel S Mártonfiová L & Zozomová-Lihová J 2024: An unex ected occurrence of Al ssum rossetii (Brassicaceae) in the Pyrenees, a new species for the Spanish flora. – Willdenowia 53: 297–307 Version of record first published online on 29 January 2024.


Introduction
The genus Alyssum L. includes about 114 species distributed mainly in Eurasia (Španiel & al. 2015).They are divided into two main phylogenetic clades (Flynn 2013;Rešetnik & al. 2013;Li & al. 2015;Cetlová & al. 2021).One of them includes almost all annual species and a nested monophyletic group of perennials belonging to the A. montanum-A.repens species complex with the centre of diversity in southern Europe (Zozomová-Lihová & al. 2014;Španiel & al. 2011b, 2017a, 2017b).The other of the two clades includes two annual taxa (A.dasycarpum Stephan ex Willd.and A. macropodum Boiss.& Balansa) and perennials, most of which were formerly classified in A. sect.Gamosepalum (Hausskn.)T. R. Dudley (hereafter referred to as the Gamosepalum lineage) and occur mainly in Asia.The Gamosepalum lineage comprises only a few taxa that occur in eastern Europe (e.g. A. doerfleri Degen, A. lenense Adams, A. pulvinare Velen., A. tayge teum Heldr.), including one (A.doerfleri) that was also recently discovered in southern Italy (Bernardo & al. 2018).
Alyssum rossetii Španiel & al. is a recently discovered and described perennial species known from a single site in the Valpelline valley (Pennine Alps, Italy) (Španiel & al. 2018b).The population is relatively small and until now no other localities have been recorded.Recent mo-lecular studies revealed that it belongs to the Gamose palum lineage (Španiel & al. 2023a, 2023b) and is phylogenetically placed closest to A. aurantiacum Boiss., A. misirdalianum Orcan & Binzet and A. praecox Boiss., all species known only from Turkey (Španiel & al. 2023a).The origin of such a large geographic disjunction (Alps versus Anatolia) is not clear.It might be a consequence of colonization events from Anatolia via the Balkans during glacial periods followed by range fragmentation and extinction in the intervening areas or a rare case of recent (Pleistocene) long-distance dispersal (Španiel & al. 2023a).All other perennial Alyssum species that occur in southwestern Europe belong to the A. montanum-A.re pens complex.Alyssum rossetii differs from this species complex by a larger genome size and several morphological characters (Španiel & al. 2018b).
During a field trip in the Pyrenees in 2022, taxonomically unidentified plants resembling Alyssum rossetii were discovered by one of the authors on a rocky slope between Pic de Qüenca and Rocablanca near Alós d'Isil.The site is 620 km distant from the only known (and type) locality of A. rossetii in the Alps.According to available distribution data (Anthos 2023) and the most recent taxonomic treatments (Zozomová-Lihová & al. 2014;Španiel & al. 2015;Cetlová & al. 2019), seven species of Alyssum have been reported so far from the Pyrenees and adjacent regions.
Four of these species are annual herbs: A. alyssoides (L.) L., A. collinum Brot., A. granatense Boiss.& Reut.and A. simplex Rudolphi.The other three taxa are perennial and belong to the A. montanum-A.repens complex: A. cacu minum Španiel & al., A. fastigiatum Heywood and A. mon tanum L. (Zozomová-Lihová & al. 2014).Alyssum cacu minum is endemic to the Pyrenees.Its populations were previously recognized under the name A. cuneifolium Ten., but recent studies revealed that this Apennine endemic is genetically and morphologically clearly different from the Pyrenean and Balkan plants for which this name was previously used (Zozomová-Lihová & al. 2014;Španiel & al. 2019).Alyssum fastigiatum was originally described as an endemic of Sierra de Cazorla, but molecular as well as morphological studies revealed that this name should be applied to all Iberian populations previously treated as A. cussed 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).
The aim of the present study is to clarify the taxonomic identity of the Pyrenean plants resembling Alys sum rossetii by inspecting genetic, karyological and morphological variability.More specifically, we aimed to (1) inspect their phylogenetic and morphological affinity to the Alpine A. rossetii and other relevant taxa of Alyssum, (2) determine their chromosome number and relative genome size, and (3) establish an identification key to Alys sum species occurring in the Pyrenees and adjacent areas.

Plant material
Several plants morphologically resembling Alyssum ros setii 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. fasti giatum, 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 mu ralis (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).

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. mon tanum-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 mm 2 ) 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).

Morphology and herbarium data
Individuals found in the Pyrenees (684PQU), tentatively identifi ed 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. ros setii.Wings and teeth on the four inner (longer) fi laments 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 mm 2 with rays 0.11-0.13mm long; upper surface of middle stem leaves is covered by 20-31 stellate trichomes per 0.5 mm 2 .These values are within the range of those previously measured in plants from the type locality of A. rossetii.On the other hand, individuals  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. (2019Cetlová & al. ( , 2021)) Španiel & al. 2023b), whereas the centre of distribution and species diversity is in Asia (Irano-Turanian region).However, genetic variation patterns and phylogenetic relationships are poorly known in the Gamosepalum lineage (Rešetnik & al. 2013;Li & al. 2015).Further studies employing high-resolution genetic markers will be needed to understand the evolution and biogeographic history of the Gamosepalum lineage, to explain the lack of genetic variation within A. rossetii, its extreme disjunction, as well as the scattered occurrence of other species in Europe.All investigated morphological characters of the plants from the locality 684PQU in the Pyrenees, preliminarily identified as Alyssum rossetii, were within the range of morphological variation previously detected in plants from the type locality of A. rossetii from the Pennine Alps (560FEU; Španiel & al. 2018b).The only exception is the dark coloration at the apex of sepals, which was not as pronounced and purplish as in the plants from the type locality.Nevertheless, this difference may be caused by their cultivation in the botanical garden.We assume that the darker coloration is more pronounced in situ (as observed in the plants from the Alps) due to high elevation and more intense sunlight.Plants in situ in the Alps and Pyrenees grow under direct sunlight in 2200-2320 m a.s.l. and flower in mid-June.In contrast, the investigated indi-viduals were kept in the botanical garden in 250 m a.s.l. in partial shade and flowered in April.
Alyssum rossetii occurs at the type locality in the Pennine Alps at a single high-mountain site between La Tsa and Pas des Feuilles (population 560FEU).It grows on a steep south-facing slope in a subalpine-alpine grassland, especially on microsites with open soil and sparse vegetation along a footpath, but also in juniper bushes and in clumps with other alpine plants which include several calcicolous species.The bedrock in this region consists of the dominant paragneiss (kinzigits) with numerous small outcrops of calcareous rocks -ancient silicate-rich marbles (Španiel & al. 2018b).The habitat of A. rossetii in the Pyrenees between Pic de Qüenca and Rocablanca (684PQU) is similar.Here, plants of A. rossetii grow on south-facing rocky slopes on microsites with sparse vegetation and open soil, but also directly in rock crevices.The bedrock in this area consists of limestone, slate and siltstone (Sanz López & Palau Ramírez 1996).The Pyrenean population of A. rossetii is of similar size (few dozens individuals) to the Alpine population and occupies a relatively small area of approximately 100 m 2 .The thorough research of herbarium specimens of Alyssum from the Pyrenees in the collections of BC, BCN and MAD revealed no other localities of this species.A detailed investigation in the broader surroundings of the locality has not yet been carried out.It should be noted that a distantly related Odontarrhena serpyllifolia (previously treated under Alyssum) grows only a few metres from the site of A. rossetii, but at first glance differs from A. rossetii by compound inflorescences.
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.