Allium albanicum (Amaryllidaceae), a new species from Balkans and its relationships with A. meteoricum Heldr. & Hausskn. ex Halacsy Author Brullo, Salvatore Author Brullo, Cristian Author Cambria, Salvatore Author Giusso del Galdo, Giampietro Author Salmeri, Cristina text PhytoKeys 2019 119 117 136 http://dx.doi.org/10.3897/phytokeys.119.30790 journal article http://dx.doi.org/10.3897/phytokeys.119.30790 1314-2003-119-117 70286A0A687DFF8EEB50FFFBFF94FF9C 2646780 Allium albanicum Brullo, C. Brullo, Cambria, Giusso & Salmeri sp. nov. Figs 1 , 7 B-D Allium meteoricum auct. fl. Albania non Halacsy, Consp. Fl. Graec. 3(1): 250. 1904, Syn. Type. ALBANIA. Devoli river, near Berat, serpentines, ca. 700 m elev., 40°43'12.00"N , 20°32'18.00"E , 26 June 2017, S. Cambria s.n. (Holotype: CAT; Isotypes: CAT, FI, G). Diagnosis. Allio meteoricum similis sed bulbis minoribus tunicis exterioribus brunneis, scapo ad 1/4 longitudinem vaginis foliorum tecto, spathae valvis in dimidio inferiore connatis, appendice usque ad 2,5 mm longa, majore 3-5 nervata, minore 3 nervata, tepalis albo-roseis, minoribus, apice erosis, filamentis staminorum minoribus, luteis superne, annulo breviore, antheris viridulis- pallide luteis, apice rotundatis, ovario luteo leviter apice rugoso, poris nectariferis majoris, capsula majore subgloboso-obovata, differt. Description. Bulb ovoid, 8-10 x 5-10 mm, with outer tunics coriaceous, brownish, the inner membranous, whitish. Stem 14-28(30) cm tall, cylindrical, flexuous, 1-1.5 mm in diameter, glabrous, erect, covered for 1/4 of its length by the leaf sheaths. Leaves 3, rather flat, glabrous, green, ribbed, up to 10 cm long and 1-2.2 mm wide, denticulate at margins. Spathe persistent, with 2 valves subequal, 8-12 mm long, shorter than the inflorescence, fused to half of their length, with an appendage 1-2.5 mm long, the larger 3-5-nerved, the smaller 3-nerved. Bostryces 12. Inflorescence laxly hemispheric, 2-3 cm in diameter, many flowered, with unequal pedicels 6-25 mm long. Perigon cylindrical-urceolate, with tepals of equal length, white tinged with pink, mid-vein greenish-purple, the inner ones linear-elliptical, the outer ones sublanceolate, rounded and slightly eroded at the apex, 5.5-6.5 mm long and 1.7-2 mm wide. Stamens included, with simple filament yellowish above and whitish below, the outers 1.7-2.1 mm long, the inners 2.5-3.2 mm long, below connate into an annulus 0.5-0.6 mm high. Anthers greenish-pale yellow, elliptical, 1-1.1 x 0.6 mm, rounded at the apex. Ovary subglobose-ovoid, yellow, slightly wrinkled at the apex, 1.5-1.7 x 1.4-1.7 mm, with large nectariferous pores, long about half the ovary. Style white, 2.7-2.8 mm long, stigma capitate. Caspule trivalved, subglobose-obovate, 4-4.5 mm, with evident nectariferous pores. Figure 1. Allium albanicum Brullo, C. Brullo, Cambria, Giusso & Salmeri sp. nov. A Habit B Flower C Perigon and stamens open D Anther E Ovaries F Capsule G Spathe valves. Drawing by S. Brullo based on living material coming from the type locality. 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 2 n = 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 2 n = 2 x = 16: 4 m + 4 msat + 4 msm + 2 msmsat + 2 smsat. 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 2 n = 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. Figure 2. Chromosome complement (2 n = 2 x = 16) of Allium albanicum . A Mitotic metaphase plate from type locality; arrows indicate satellited chromosomes B idiogram. 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 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 ). Figure 3. Comparison of karyotype morphometric data between Allium albanicum and A. meteoricum . A Variation of centromeric index for each chromosome pair B Variation of the main karyomorphometric parameters and symmetry indices ( LC longest chrom., SC shortest chrom.; MCL mean chromosome length; MLA mean long arm; MSA mean short arm; other abbreviations see Table 2 ). Figure 4. Leaf cross section of Allium albanicum from living material coming from the type locality. Drawing by S. Brullo. Seed micromorphology. As emphasised by numerous authors ( Pastor 1981 , Cesmedziev and Terzijski 1997 , Fritsch et al. 2006a , Neshati and Fritsch 2009 , Celep et al. 2012 , Salmeri et al. 2016 , Lin and Tan 2017 , Oezhatay et al. 2018 , Brullo 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 x ) showed a semi-ovoid shape (3.5-4.0 x 2.4-2.5 mm), with a rather rugose surface (Fig. 5A, B ). The seeds observed at high magnification (600 x and 1200 x ) revealed irregularly polygonal testa cells, having a size of 40-80 x 17-40 µm (Fig. 5 C-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 x ) revealed a semi-globose shape and a smaller size (2.2-2.5 x 1.9-2.0 mm), with less pronounced surface roughness (Fig. 6A, B ). The seeds observed at high magnification (600 x and 1200 x ) also showed irregularly polygonal testa cells, but with a larger size (60-120 x 15-50 µm ) (Fig. 6 C-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. Figure 5. SEM micrographs of the seed coat of Allium albanicum . A Seed (dorsal face, 30 x ) B Seed (ventral face, 30 x ) C Seed coat (central part of dorsal face, 600 x ) D Seed coat (central part of ventral face, 600 x ) E Seed coat (central part of dorsal face, 1200 x ) F Seed (central part of ventral face, 1200 x ). Photos from material of type locality (CAT). Figure 6. SEM micrographs of the seed coat of Allium meteoricum . A Seed (dorsal face, 30 x ) B Seed (ventral face, 30 x ) C Seed coat (central part of dorsal face, 600 x ) D Seed coat (central part of ventral face, 600 x ) E Seed coat (central part of dorsal face, 1200 x ) F Seed (central part of ventral face, 1200 x ). Photos from material of type locality (CAT). 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 ). Plants grow in shrub vegetation differentiated by some serpentinicolous plants, such as Acantholimon albanicum O.Schwarz & F.K.Mey, Centaurea salonitana Vis., Centranthus longiflorus Steven, Festucopsis serpentini (C.E. Hubb.) Melderis, Forsythia europaea Degen & Bald., Iberis umbellata L., Salvia ringens Sibth. & Sm. etc. According to literature ( Pils 2016 , Barina 2017 ) and herbarium investigations, A. albanicum seems to have a scattered distribution in Albania, though its effective geographic range might be better defined only through further field surveys. Based on Brullo et al. (2001) , 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. Figure 7. Phenological features of Allium albanicum and A. meteoricum . A Growing habitat of A. albanicum in the locus classicus (Albania) B Individuals of A. albanicum from the locus classicus C A. albanicum cultivated material in Botanical Garden of Catania D Leaf of A. albanicum , cultivated material E, F Individual of A. meteoricum , from Meteora (Greece). Photos by S. Cambria. Additional specimens examined. ALBANIA. In humidis collinis serpentinum ad Renci distr. Scutari, 11 June 1897, Baldacci 85a (BM, G); In humidis collinis serpentinii ad Renci distr. Scutari, June 1897, Baldacci 355 (WU); Nordost Albanien, auf Felsen in der subalp. Region des Pastrik ca. 1200 m elev., 31 July 1914, Dorfler 593 (WU); Nord Albania, Umgebung von Shkodra Abhaenge des kleinen Bordans alt. Serpentin, 8 June 1916, Janchen s. n. (WU); Hasi Pastrik an Felsen des westlichen Auslaeufers , ca. 1200 m elev., 22 July 1918. Dorfler 908 (BM, K, W, WU). Examined specimens of Allium meteoricum . GREECE, Thessalia superior in collibus circa monasteria Meteora supra Kalabaka (Aeginium veterum), substrata diluviali e saxis conglomeratis, 15/16 July 1885 Heldreich s. n ., sub Allium meteoricum Heldreich & Haussknecht sp. nova (WU Herbarium Halacsy); this specimen, already quoted by Brullo et al. 2001 as a type of A. meteoricum , is more completely reported and here correctly designated as lectotype of Allium meteoricum . Thessalia, Trikkala at Meteora above Kalambaki, sunny rocks, 29/06/2018, S. Brullo & S. Cambria s. n. (CAT); for other examined specimens, see Brullo et al. (2001) . 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 cylindrical-urceolate, stamens not exserted, ovary with evident nectariferous pores, the populations of A. albanicum were previously referred to as A. meteoricum (Halacsy 1904, Hayek 1932 , Bornmueller 1933 , Stearn 1978 , 1980 , 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 2 n = 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 ). Based on literature ( Stearn 1978 , 1980 , Tzanoudakis 1983 , Tzanoudakis and Vosa 1988 , Brullo et al. 2001 ), A. meteoricum was included in the sect. Scorodon Koch, but as highlighted by Brullo et al. (2018) , this traditional section is actually an assemblage of various and well-differentiated phylogenetic lineages (see Fritsch and Friesen 2002 , Friesen et al. 2006 , Nguyen et al. 2008 , Hirschegger et al. 2010 , Li et al. 2010 ). In particular, the sect. Scorodon s.str., typified by A. moschatum L., now belongs to the subgen. Polyprason Radic , 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 .