Alliumalbanicum (Amaryllidaceae), a new species from Balkans and its relationships with A.meteoricum Heldr. & Hausskn. ex Halácsy

Abstract A new species, Alliumalbanicum, is described and illustrated from Albania (Balkan Peninsula). It grows on serpentines or limestone in open rocky stands with a scattered distribution, mainly in mountain locations. Previously, the populations of this geophyte were attributed to A.meteoricum Heldr. & Hausskn. ex Halácsy, described from a few localities of North and Central Greece. These two species indeed show close relationships, chiefly regarding some features of the spathe valves, inflorescence and floral parts. They also share the same diploid chromosome number 2n =16 and similar karyotype, while seed testa micro-sculptures and leaf anatomy reveal remarkable differences. There are also several morphological features that allow them to be differentiated at specific level. The inclusion of both species into a newly described section Pseudoscorodon of the subgen. Allium is proposed. An analytic key to the species, included in the new section, is also provided.


Introduction
One of the richest and largest genera of Monocotyledons is Allium L. and it is almost exclusively widespread in the northern hemisphere, where it is represented by ca. 1200 taxa (Govaerts et al. 2018). This genus is characterised by a high rate of endemism, especially observable in North America, Asia and the Mediterranean area, which represent the main centres of diversity.
In the context of cytotaxonomical research on the genus Allium in the Mediterranean territories, especially regarding the subgen. Allium (Bogdanović et al. 2008, 2011a, 2011b, Brullo et al. 1997a, 1997b, 1999, 2001, 2003a, 2003b, 2004, 2007, 2008a, 2008b, 2014, Özhatay et al. 2018), a peculiar population occurring in Albania, previously attributed to A. meteoricum Heldr. & Hausskn. ex Halácsy, is examined. Allium meteoricum s. str. was described from Meteora in Central Greece by Halácsy (1904) and later also recorded from Assopos, Greece (Tzanoudakis 1983, Tzanoudakis andVosa 1988, Brullo et al. 2001). Extensive morphological investigations, carried out on herbarium material and living specimens coming from Albania (Devoli river near Berat on serpentines) and Greece (Meteora on sandstones), allowed us to verify that the Albanian populations are very different from those of Meteora, which is the locus classicus of A. meteoricum. Detailed analyses regarding the chromosome complement and karyotype structure, seed testa micro-morphology and leaf anatomy provided relevant discriminant features. Based on these data, the Albanian populations were referred to a species new to science, named Allium albanicum.

Materials and methods
Plant morphology was analysed on 20 living mature plants. Qualitative and quantitative morphological characters, considered as diagnostic in Allium, were analysed and scored (Table 1) on fresh material. Comparison of A. albanicum with A. meteoricum was based on living plants coming from the type locality of both species, collected by S. Cambria in Albania (June 2017) and by S. Brullo and C. Cambria in Greece (June 2018), as well as on several herbarium specimens (BM, CAT, G, K, W, WU) in order to check the correct sample identification. Literature data were also considered. Collected specimens are preserved in CAT.
For the karyological study, living bulbs were collected and potted at the Botanical Garden of Catania University. Root tips were pre-treated with 0.3% (w/v) colchicine water solution for 3 h at room temperature and then fixed overnight in fresh Farmer's fixative (3:1 v/v, absolute ethanol: glacial acetic acid). Root tips were hydrolysed in 1N HCl at 60 °C for 7 min, washed and stained with Feulgen for 1 h. Microphotographs of good quality metaphase plates were taken with a Zeiss Axioskop2 light microscope equipped with an Axiocam MRc5 high resolution digital camera. Chromosome number and karyotype details were analysed from 10 well spread metaphase plates from 5 individuals, the mean values being used for the karyotype characterisation. Metaphase chro-  Brullo (2002). The chromosome types were named according to the position of the centromere: r = 1-1.3 (m) median, r = 1.3-1.7 (msm) median-submedian, r = 1.7-3 (sm), r = 3-7 (st) subterminal (Tzanoudakis 1983). All measured karyomorphometric parameters are given in Table 2. Karyotype symmetry indices followed Paszko (2006) and Peruzzi and Eroğlu (2013). Leaf anatomy was studied on living materials coming from the type locality and cultivated in the Botanical Garden of Catania University. Leaf blades of maximum size, in their optimal vegetative development, usually before the flowering stage, were taken from the middle part and fixed in Carnoy. Leaf cross sections were double stained with ruthenium red and light green, analysed and photographed with a light microscope (Zeiss Axioskop2 and Axiocam MRc5 digital camera).
Phenology. Flowering and fruiting from June to July. Etymology. The epithet refers to the Latin "Albanicum", coming from Albania, the country where the species grows.
Karyology. The investigated specimens of A. albanicum from the type locality revealed a diploid chromosome number with 2n = 16. The karyotype obtained from somatic metaphase plates ( Fig. 2A) is mostly characterised by nearly metacentric chromosomes; specifically, the mean karyogram ( Fig. 2B) reveals 4 typical metacentric (m) pairs (III, V, VI, VIII), 3 meta-submetamentric (msm) pairs (I, II, VII), having an arm ratio between 1.30 and 1.67 and one submetacentric (sm) pair (IV). Microsatellites were detected on the short arms of two metacentric chromosome pairs, one meta-submetacentric pair and the submetacentric one. Thus, the chromosome formula can be expressed as 2n = 2x = 16: 4 m + 4 m sat + 4 msm + 2 msm sat + 2 sm sat . Chromosomes have a total length varying from 8.90 ± 2.5 µm of the longest chromosome to 5.16 ± 0.8 µm of the shortest one, while the relative length ranges from 8.01% to 4.73%. As already emphasised by Tzanoudakis (1983) and Brullo et al. (2001), A. meteoricum also has a diploid chromosome complement with 2n = 16 (cf. Brullo et al. 2001, Fig. 6A), which is characterised by 5 metacentric chromosome pairs, two of which microsatellited on the short arm, 2 msm pairs and one submetacentric microsatellited pair (cf. Brullo et al. 2001, fig. 8A). Chromosomes vary in total length from 7.29 µm of the longest chromosome to 4.03 µm of the shortest one, while the relative chromosome length ranges from 7.8% to 4.3%. Table 2 shows the mean values for all measured karyomorphometric parameters and symmetry indices of A. albanicum and A. meteoricum from the type locality. Abbreviations: LA = long arm; SA = short arm; TAL = total absolute length; TRL = total relative length; AR = arm ratio; CI = centromeric index; CA = centromeric asymmetry; Type=chromosome nomenclature; sat = satellited; TCL = total chromosome length; MCL = mean chromosome length; d-value = difference between Long arms and Short arms; DRL% = difference of relative length; S% = Relative length of shortest chromosome; MAR = mean arm ratio; MCI = mean centromeric index; Cv CL = coefficient of variation of chromosome length; Cv CI = coefficient of variation of centromeric index; MCA = mean centromeric asymmetry.  Leaf anatomy. The leaf cross section of A. albanicum shows a flat outline, with some dorsal ribs. The epidermis is formed by small cells covered by a well-developed cuticle externally more thickened. Stomata are numerous and distributed along the whole leaf perimeter. The palisade tissue is regular and compact, arranged in one layer  Table 2). of long cylindrical cells, more developed on the adaxial face. The spongy tissue is rather compact and slightly lacunose, in the peripheral part many secretory canals occur. The maximum number of vascular bundles is 20, 11 of which are very small and are localised on the adaxial face, while on the abaxial face, there is one large central vascular bundle and 4 smaller ones for each side (Fig. 4).
Seed micromorphology. As emphasised by numerous authors (Pastor 1981, Češmedžiev and Terzijski 1997, Fritsch et al. 2006a, Neshati and Fritsch 2009, Celep et al. 2012, Salmeri et al. 2016, Lin and Tan 2017, Özhatay et al. 2018, the micro-sculptures of the seed testa in the Allium species represent a very stable and conservative character, showing usually relevant taxonomical and phylogenetical implications. Seeds of A. albanicum at low magnification (30×) showed a semiovoid shape (3.5-4.0 × 2.4-2.5 mm), with a rather rugose surface (Fig. 5A, B). The seeds observed at high magnification (600× and 1200×) revealed irregularly polygonal testa cells, having a size of 40-80 × 17-40 µm (Fig. 5C-F). The anticlinal walls appeared flat, rather straight and partly covered by strip-like sculptures forming a widened intercellular region, not or just a little lacerate. The periclinal walls were flat, with few flat and smooth or slightly knobby verrucae, usually arranged along the margin surrounding a central one. Conversely, the seeds of A. meteoricum at low magnification (30×) revealed a semi-globose shape and a smaller size (2.2-2.5 × 1.9-2.0 mm), with less pronounced surface roughness (Fig. 6A, B). The seeds observed at high magnification (600× and 1200×) also showed irregularly polygonal testa cells, but with a larger size (60-120 × 15-50 µm) (Fig. 6C-F). The anticlinal walls appeared flat, rather straight and partly covered by strip-like sculptures forming a widened intercellular region, partially lacerate. The periclinal walls were weakly protruding with several knobby verrucae distributed over the whole surface.
Ecology and distribution. The investigated population of A. albanicum, previously reported as sub A. meteoricum (Pils 2016, Barina 2017, was collected on serpentinic substrata of open stands characterised by rocky outcrops at ca. 700 m of elevation (Fig. 7).    Dimopoulos et al. (2013) and personal herbarium surveys, A. meteoricum is a Greek endemic, circumscribed to northern and central Greece and further populations reported in other Greek sites or different territories cannot be referred to this species. Therefore, the remaining Albanian populations referred to as A. meteoricum should also be checked in detail as regards their taxonomic attribution.
Discussion. For its general habit and some features such as flat leaves, spathe valves very short, 3-5 nerved, briefly appendiculate, umbel laxly subglobose, perigon cylindricalurceolate, stamens not exserted, ovary with evident nectariferous pores, the populations of A. albanicum were previously referred to as A. meteoricum (Halacsy 1904, Hayek 1932, Bornmüller 1933, Stearn 1978, Meyer 2011, Vangjeli 2015, Pils 2016, Barina 2017. In light of in-depth taxonomical investigations carried out on living and herbarium material, the analysed Albanian populations are well differentiated from those of A. meteoricum coming from the locus classicus, formerly studied by Brullo et al. (2001). Table 1 summarises the most relevant morphological characters differentiating the two species, which mainly consist in the different size and colour of bulbs and tepals, length of the scape covered by the leaf sheaths, shape of spathe valves, colour and size of stamens, ovary and capsule and the shape of nectariferous pores. In particular, A. meteoricum differs from A. albanicum in having larger bulbs with blackish-brown outer tunics, stem covered up to 1/2 of its length by the leaf sheaths, free spathe valves, with appendage up to 4 mm long, tepals purplish-pink, up to 7.5 mm long, smooth at the apex, staminal filaments longer, whitish, annulus longer, anthers yellow, apiculate at the apex, ovary green, smooth, with much smaller nectariferous pores and smaller capsule. Other relevant differences concern the leaf anatomy, since the leaf cross-section of A. meteoricum (cf. Brullo et al. 2001, fig. 11A) is characterised by a thinner cuticle, cells of palisade tissue with uniform size along the entire perimeter, spongy tissue markedly lacunose in the centre and with few vascular bundles in the abaxial face.
According to previous research data (Stearn 1978, Tzanoudakis 1983, Brullo et al. 2001, A. meteoricum and A. albanicum share the same diploid chromosome complement with 2n = 16 and their karyotypes are prevalently constituted by more or less metacentric chromosomes (arm ratio less than 1.67), except for one submetacentric pair, microsatellited in the short arm. The chromosome formulae are also rather similar, with some differences regarding the proportion of m and msm chromosomes, which are, respectively, 10 and 4 in A. meteoricum, contrary to 8 and 6 in A. albanicum and the number of recognisable satellited chromosomes, consisting in three pairs of chromosomes for A. meteoricum (vs. 4 pairs for A. albanicum). The high morphological chromosome homogeneity and karyotype symmetry, rather common in closely allied Allium species, accounts for the overall karyological similarity between the two species, with no statistically significant differences in their karyomorphometric parameters (Fig. 3).
In particular, the sect. Scorodon s.str., typified by A. moschatum L., now belongs to the subgen. Polyprason Radić, which groups rhizomatous species (Friesen et al. 2006, Fritsch et al. 2006b), rather than to subgen. Allium, to which A. meteoricum and A. albanicum clearly belong. Effectively, there are several species previously included within the sect. Scorodon s.l. which require a taxonomic reassessment, consisting in the recognition of a distinct new section of the subgen. Allium, herein proposed and named as sect. Pseudoscorodon.
Description. Bulb solitary or bulbilliferous, leaves glabrous to hairy, never thread-like, spathe valves persistent and usually shorter than the inflorescence, at least 3-nerved, stamen filaments flattened and widened in the lower part, the inner ones often uni-bicuspidate, ovary with well-developed nectariferous pores, bordered by a membranous plica, partly covering the nectariferous pore.
In order to highlight the morphological similarities and differences amongst the species of the new section, the following analytic key is provided.
Key to the species referable to the sect. Pseudoscorodon