Allium serbicum Visiani & Pančić (1865: 479

Bogdanović, Sandro, Anačkov, Goran, Salmeri, Cristina, Galdo, Gianpietro Giusso Del & Brullo, Salvatore, 2024, Cytotaxonomical remarks on Allium serbicum (Amaryllidaceae), a misappreciated species of the Balkan flora, Phytotaxa 674 (1), pp. 97-110 : 99-108

Allium serbicum Visiani & Pančić (1865: 479 , Tab. 22, fig. 1). Figs. 1 , 2 , 4

Type: — SERBIA. Mokra gora Serb. merid. in rupestrib. calcareis, Jul, J. Pančić s.n. (lectotype: BEOU 11931!), second-step designated here ( Fig. 2 ).

— A. tenuiflorum Hayek (1932: 57) non Tenore (1811: 165)

— A. pallens Stearn (1978: 1619) non Linnaeus (1762: 427)

Bulb ovoid, 13–14 × 7–8 mm, outer tunics fibrous, grey-brown, inner tunics membranaceous, whitish. Stem erect, (13) 18–40 cm tall, glabrous, cylindrical, covered by leaf sheaths for 1/3–1/2 of total length. Leaves 3(−4), green, glabrous, blade not rigid, slightly semi-cylindrical, compact, undulate at margine, without ribs, 10−20 cm long, 1−2 mm wide. Spathe persistent, with two valves unequal, opposite, divaricated to reflexed at flowering, with the appendage longer than the inflorescence, the larger one 5-nerved and 2.5−3 cm long, the smaller one 3-nerved and 0.5−1.5 cm long. Inflorescence compact, globose, up to 2 cm in diameter, 10−20(25)-flowered; pedicels erect, subequal, ca. 5 mm long. Perigone campanulate, with tepals equal, white to pinkish-white, tinged with pink, elliptical, rounded at the apex, 4.8−5.3 × 2.3−2.4 mm, with midrib purplish-green. Stamens included in the perigone or slightly exerted, with simple filament, white, subequal, 1.8−3.5 mm long, connate below into an annulus 0.7−0.8 mm high; anthers white tinged with pink, oblong, 1.4 × 0.8 mm, rounded at the apex. Ovary globose, markedly contracted at the base, greenishyellow, tuberculate above, 2.3 × 2.1−2.2 mm; style white, ca. 1 mm long. Capsule 3-valved, subglobose, green, 3.8−4 × 4−4.2 mm.

Distribution and ecology: —According to literature data ( Visiani & Pančić 1865, Anačkov 2009, Stojanović et al. 2017) and field investigation Allium serbicum is localized in few stands of West Serbia and in one nearby place of East Bosnia and Herzegovina ( Fig. 3 ). It grows in calcareous screes ( Fig. 4 ) more or less stabilized from 500 up to 900 m of altitude, in habitats characterized by several endemic species such as Pseudo-fumaria alba ( Miller, 1768: no. 3) Lidén (1986: 32) subsp. leiosperma ( Conrath, 1888: 50) Lidén (1986: 32) , Reichardia macrophylla Visiani & Pančić in Pančić (1874: 460), Stachys anisochila Visiani & Pančić (1870: 13) , Scabiosa fumarioides Visiani & Pančić (1865: 466) , Micromeria croatica (Person, 1806: 130) Schott (1857: 93) , and some other species with wider range Achnatherum calamagrostis P. Beauvois (1812: 19) , Athamanta turbith ( Linnaeus, 1756: 14) Brotero (1804: 435) , Clinopodium thymifolium Kuntze (1891: 516) , Satureja montana Linnaeus (1753: 568) , Epilobium dodonaei Villars (1779: 45) , Aurinia corymbosa Grisebach (1843: 271) etc.

Leaf anatomy: —The leaf cross section of Allium serbicum shows almost semi-circular outline markedly undulate at the margin. The epidermis shows regular small cells, covered by a well-developed cuticle. The surface of the leaf is covered with needle-like waxy structures. The stomata are numerous and distributed along the whole leaf perimeter, they are partially indented and framed by a thickened wax ring. The palisade tissue is arranged in two well distinct stratifications, the external one is constituted of elongated unistratified cell uniformly distributed in the whole perimeter, the inner one is constituted of small and rounded pluristratified cells (2−3 layers). The spongy tissue is compact, constituted by cell of different size, with many secretor canals under the palisade tissue. The vascular bundles are 10 (in the largest leaves), 7 of which variable in size are distributed in the adaxial face, while only 3 of small-size are in the abaxial face ( Fig. 5 ).

Karyology: — Allium serbicum has a diploid chromosome number, with 2 n = 16. Its chromosome complement ( Fig. 6A ) is characterized by nearly metacentric chromosomes, with two submetacentric pairs. Specifically, the karyogram ( Fig. 6B ) shows the chromosomes arranged in five typical metacentric (m) pairs (I, II, III, V, VI), one meta-submetacentric (msm) pair (IV), having an arm ratio between 1.30 and 1.67 (Tzanoudakis et al. 1983), and two submetacentric (sm) pairs (VII, VIII). Microsatellites were detected on the short arms of the meta-submetacentric pair and of one submetacentric pair ( Fig. 7 ). Thus, the chromosome formula can be expressed as 2 n = 2x = 16: 10 m + 2 msmsat + 2 sm + 2 smsat. Chromosomes have on average a total length varying from 12.4 ± 1.3 µm of the longest one to 6.5 ± 0.5 µm of the shortest one, with a mean length (MCL) of 9.03 ± 1.8 µm and a length coefficient of variation (CV CL) of 19.86. Chromosome arm ratio (AR) shifts from 1.07 to 2.26, with a mean centromeric asymmetry (MCA) equal to 14.12%. The relative chromosome length ranges from 8.58% to 4.49% ( Table 2).

Seed testa micromorphology: —In the genus Allium the seed morphology provides useful systematic characters at various taxonomic levels ( Celep et al. 2012, Salmeri et al. 2016, Lin & Tan 2017, Baasanmunkh et al. 2021, Yusupov et al. 2022). From this literature, it is clear that the seed testa ornamentations are conservative and stable features, which provide important information on this genus also from a phylogenetic point of view. The seeds of A. serbicum show a semi-ovoid outline (3.2–3.6 × 1.8–1.9 mm), with a rugose surface ( Fig. 8A ). Observations under SEM at high magnification (800×) show irregularly polygonate testa cells, 27–43 μm long. The anticlinal walls appear flat and straight, covered by strap-like sculptures forming a widened intercellular region. The anticlinal walls of the seedbed are characterized by a short connection in the form of the U-Ω connection model according to Fritsch et al. (2006). A similar form of undulation is also present in taxa that prefer thermophilic and xerophilic habitats ( Anačkov 2009). The periclinal walls are slightly raised, with several granulose verrucae, distributed along the edges or sometime in the whole surfaces ( Fig. 8B ). These ornamentations are very similar to those ones observed in other species of Allium sect. Codonoprasum ( Češmedžiev & Terzijski 1997, Neshati & Fritsch 2009, Celep et al. 2012, Brullo et al. 2013, Salmeri et al. 2016, Özhatay et al. 2018).

Pollen grain micromorphology: —According to SEM investigations, the pollen grains are monad, monosulcate, and heteropolar with bilateral symmetry ( Fig. 8C ). The pollen shape is peroblate, with 29.40–32.00 µm long axis (LA) and 16.40–20.80 µm long short axis (SA). The sulcus is quite narrow and extends from distal to proximal portion of the pollen grain and the exine ornamentation is rugulate ( Fig. 8D ). According to the palynological investigation on the genus Allium ( Nair & Sharma 1965, Diez 1987, Güler & Pehlivan 2006, Bogdanović et al. 2008, Neshati et al. 2009, Koçyiğit 2014, Yildiz 2023), A. serbicum shows a pollen morphology very similar to other taxa of the sect. Codonoprasum .

Conservation status: —Based on field investigations, Allium serbicum is currently know from seven locations where it grows in populations usually constituted by a few mature individuals or smaller groups, arranged in small tuffs dispersed over large scree surfaces. Extent of Occurrence (EOO) values were calculated for the species, which is 547.3 km 2, while the Area of Occupancy (AOO) value is 8 km 2. Namely, these values would have been different and lower if the microhabitat had not been found in the valley of the Mileševka River in 2009. The assessment was carried out according to standard criteria in the application made for the IUCN categorization of plant taxa for Serbia ( Niketić 2022). Namely, intensive monitoring of the main population, located at the locus classicus, revealed a drastic reduction of the population, ranging between 30 and 50% for the previous 10 years. For the next period, given the established protection measures, it is expected that the reduction in the next 10 years will be between 10 and 20%, and maybe even less. The total number of mature individuals does not exceed 1000 and often not all individuals have formed fruits and seeds. There are also negative effects of expansion in the environment, especially fluctuations in the size of the space and occupied area, as well as the number of mature individuals. The habitat colonized by this species are quite natural and do not seem influenced by human activities except the removal of stones for construction. Since the implementation of protection measures, it has been observed that the population decline is so pronounced. According to the IUCN (2024), this species for its rarity, low number of mature individuals and restricted population distribution should be included in the category as CR: B2b(i,ii,iii,iv,v)c(i,ii,iv).

Additional specimens seen:— BOSNIA AND HERZEGOVINA. Dobrun-Razdoline , 43 ⁰45ʹ28ʺN, 19 ⁰24ʹ04ʺE, 507 m, 7 August 2022, S. Bogdanović s.n. (ZAGR!) ; Dobrun-Razdoline , in cult., 16 June 2023, S. Brullo s.n. (CAT!) . SERBIA. West Serbia: Beli Rzav Gorge , entrance to the gorge, 43 ⁰46ʹ27ʺN, 19 ⁰24ʹ44ʺE, 527 m, July 2003, B. Zlatković s.n. (BUNS!) ; Mokra Gora, Kotroman, Beli Rzav Gorge , entrance to the gorge, 22 October 2004, B. Zlatković s.n. (BUNS!) ; Mokra Gora, Beli Rzav Gorge , srubs on limestone, limestone screes, 43 ⁰46ʹ25ʺN, 19 ⁰27ʹ44ʺE, 514 m, 27 July 2014, G. Anačkov s.n. (BUNS!) ; Mokra Gora, Beli Rzav Gorge , 43 ⁰46ʹ28ʺN, 19 ⁰27ʹ39ʺE, 523 m, 5 July 2015, G. Anačkov s.n. (BUNS!) ; Mokra Gora, Beli Rzav Gorge , 43 ⁰46ʹ40ʺN, 19 ⁰27ʹ39ʺE, 513 m, 31 July 2009, G. Anačkov & B. Božin s.n. (BUNS!) ; Prijepolje, Mileševka Gorge , on rocks, 43 ⁰21ʹ45ʺN, 19 ⁰43ʹ50ʺE, 657 m, 29 July 2009, G. Anačkov & B. Božin s.n. (BUNS!) .

Taxonomic relationships: —From the taxonomic point of view, Allium serbicum must be treated as a species morphologically quite isolated from the other species of A. sect. Codonoprasum . For its globose inflorescence, it was attributed by various authors ( Stearn 1978, Gregory et al. 1998, Govaerts et al. 2005 –2014, Raab-Straube 2023, POWO 2024) to A. pallens species usually linked to synanthropic habitats and showing a wider Mediterranean distribution. According to Brullo et al. (2003a), A. pallens is well differentiated from A. serbicum in having a robust and bigger size, leaves 4–6, fistulous and semi-cylindrical, spathe valves longer, 6–10-nerved, inflorescence bigger, with more than 50 flowers, pedicels unequal 5–25 mm long, perigone with smaller tepals (4–4.5 mm long), usually white, stamens always exerted from perigone, anther yellow, shorter (1.2–1.3 mm long), ovary ellipsoid, smooth, longer (3–4 mm), capsule bigger (4–4.5 mm). Besides, all populations of A. pallens are characterized by a tetraploid chromosome number with 2 n = 4 x = 32 ( Brullo et al. 2003a). As concerns the relationships between A. serbicum and A. tenuiflorum , species by some authors considered synonyms ( Hayek 1933, Tatić 1975, Anačkov 2009), they represent two completely distinct taxa. Indeed, based on the investigation carried out on A. tenuiflorum by Brullo et al. (2003b, 2013), it clearly differs from A. serbicum for habit, inflorescence, spathe valves, floral morphology, leaf anatomy, seed testa micromorphology and karyotype. In particular, A. tenuiflorum is characterized by outer bulb tunics coriaceous, blackish-brown, leaves 4–6, 5-ribed, manifestly pentagonal in cross section, spathe valves longer with more nerves (5 in smaller one, 7 in longer one), inflorescence lax and hemispherical, tepals narrower and apiculate, anthers yellow, shorter (1 mm), apiculate at the apex, annulus longer (1–1.3 mm), ovary more or less cylindrical, longer (3–3.2 mm), capsule longer (4–4.5 mm). Besides, the seed testa of A. tenuiflorum shows more elongated cells (50–61 µm long), with anticlinal walls depressed, covered by dense and regular strip-like sculptures, and periclinal walls highly convex, with markedly tubercles arranged in 1–2 rows. Both species have a diploid chromosome complement, with 2 n = 16, but the karyotype structure is rather different (see Brullo et al. 2013 Fig. 3C, D ). In fact, A. tenuiflorum shows a more symmetric karyotype ( Fig. 7 ), mainly characterized by median chromosomes and only one submetacentric pair (vs. 2 sm pairs in A. serbicum ). Moreover, many populations of A. tenuiflorum revealed the presence of supernumerary chromosomes, with 0 up to 6 B-chromosomes ( Brullo et al. 2003b, 2013).

On the whole, A. serbicum due to some its morphological peculiarities, especially regarding the leaf anatomy, type of inflorescence and floral traits, seems to have no close taxonomical relationships with Mediterranean species of the A. sect. Codonoprasum . For globose inflorescence, and short spathe valves with few ribs, it shows only some weak affinity with the species belonging to group of A. staticiforme Smith in Sibthorp & Smith (1809: 225), but it differs significantly from these ones in having large tepals exceeding 4 mm in length and stamens included in the perigone ( Brullo et al. 1995, 2017, Koçyiğit et al. 2023).

Previously, Clementi et al. (2015) designated as lectotype of A. serbicum the illustration cited in the protologue by Visiani & Pančić (1865, Tab. 22, fig. 1), preferring it to the isotype belonging to original material kept in Pančić’s herbarium mentioned by themselves “Mokra gora Serb[ia] merid[ionalis] in rupestrib[us] calcareis, Jul[io], J. Pančić s.n. (BEOU 11931!)”, since in their opinion it was not preserved in good conditions. The choice of this lectotype is not in conformity with the Article 9.12 of the ICN ( Turland et al. 2018) and therefore must not be followed. Applying the Article 9.17 of the ICN, the specimen at the left of the herbarium sheet preserved at BEOU 11931! is here chosen as a lectotype of A. serbicum in second-step typification ( Fig. 2 ).

Figures

FIGURE 1. Diagnostic features of Allium serbicum. A. Habit. B. Inflorescence. C. Flowers. D. Open perigone and stamens. E. Anther. F. Ovaries. G. Capsule. H. Spathe valves. Illustration by S. Brullo based on living material from type locality.

FIGURE 2. Lectotype of Allium serbicum is indicated by black star from BEOU 11931!.

FIGURE 3. Distribution map of Allium serbicum (red dots) on the Balkan Peninsula.

FIGURE 4. Phenological features of Allium serbicum from Beli Rzav (Serbia). A. Natural habitat. B. Individuals in natural habitat. C.–D.Inflorescences. (Photos A, C by B. Zlatković, B by G. Anačkov, D by U. Buzurović).

FIGURE 5. Leaf cross section of Allium serbicum from the type locality. Drawn by S. Brullo.

FIGURE 6. Chromosome complement (2n = 16) of Allium serbicum. Mitotic metaphase plate from type locality (A), arrows indicate satellited chromosomes, and karyogram (B).

FIGURE 7. Comparison of karyotype structure between Allium serbicum and A. tenuiflorum based on the arm ratio of chromosome pairs ordered by decreasing size.

FIGURE 8. SEM micrographs of the seed coat and pollen grain of Allium serbicum. A. Seed (dorsal face, ×35). B. Seed coat (dorsal face, ×800). C. Pollen grain (equatorial view, ×3.300). D. Exine ornamentation (×10.000). Photos from material coming from the type locality (BUNS).