Verbascum albidiflorum Ranjbar & Nouri (Scrophulariaceae) is described and illustrated as a new species of V. [sect. Bothrosperma Murb.] subsect. Singuliflora Murb. from Kermanshah Province, W Iran. It is morphologically most similar to V. alceoides Boiss. & Hausskn. and the two species are compared with respect to gross morphology, leaf anatomy and pollen morphology. The chromosome number 2n = 38 is reported for the first time in V. albidiflorum and 2n = 48 is reported in V. alceoides, both from Kermanshah Province, Iran.
Introduction
The genus Verbascum comprises some 360 species worldwide (Heywood 1993), 42 species in Iran (Huber-Morath 1981; Sharifnia & Assadi 2011) and 49 species in the Flora iranica (Huber-Morath 1981), which includes parts of Afghanistan, Iraq, Pakistan, Talish and Turkmenistan. The main centre of Verbascum diversity and evolution is Turkey and Iran (Zohary 1973; Huber-Morath 1978). Among species distributed in Iran, 15 are endemic (Huber-Morath 1981; Sharifnia 2007). In recent years the relationships between Verbascum species based on morphological characteristics (Karavelioğulları & al. 2004, 2008; Vural & Aydoğdu 1993; Al-Hadeethy & al. 2014; Cabi & al. 2011; Parolly & Eren 2008; Parolly & Tan 2007; Kaynak & al. 2006; Sharifnia & Assadi 2007), anatomical characteristics (Petković & al. 1997; Kheiri & al. 2009), cytological characteristics (Ghaffari 2008; Malik & al. 2011; Marhold & Breitwieser 2009) and pollen-morphological characteristics (Pehlivan & al. 2008; Kheiri & Khayami 2006) have been somewhat explored in various studies. The present study focuses on the morphological, leaf-anatomical, pollen-morphological and karyological analysis of a species of Verbascum apparently endemic to W Iran, in the Zagros Mountains in Kermanshah Province. These plants have been cross-checked with the keys provided by Murbeck (1933), the floras of the neighbouring countries and other taxonomic literature (Boissier 1879; Fedtschenko 1955; Ferguson 1972; Huber-Morath 1978, 1981) and finally compared with herbarium material. These studies have allowed us to recognize the plants as a new and distinctive species, which is described here as V. albidiflorum Ranjbar & Nouri, and to consider V. alceoides Boiss. & Hausskn., which is distributed from NE Iraq to W Iran (Huber-Morath 1981; Fig. 5), as the most similar species and possible nearest relative.
All Iranian species of Verbascum belong to V. sect. Bothrosperma Murb., in which has been recognized two main groups: “A” and “B” (Huber-Morath 1978, 1981; Sharifnia & Assadi 2011). In Flora iranica (Huber-Morath 1981), V. alceoides Boiss. & Hausskn. is in group A, characterized by having a solitary flower in the axil of each bract, and subgroup b, characterized by being covered, at least partially, with stellate and dendroid hairs. In Flora of Turkey (Huber-Morath 1978), V. alceoides is in V. [sect. Bothrosperma] subsect. Singuliflora Murb.
Material and methods
Material
The material used for this study represents four populations (Table 4). Verbascum albidiflorum and the collection of V. alceoides referred to here as “V. alceoides 35707” were collected during field exploration in Kermanshah Province, Iran, during May 2014 and were deposited in the BASU herbarium (herbarium codes according to Thiers 2015+). The holotype of V. alceoides (at G), from NE Iraq, was also studied. In addition, micromorphological data from a further collection of V. alceoides, also from NE Iraq and here referred to as “V. alceoides 235”, were taken from Al-Hadeethy & al. (2014); this collection was selected in that work because it is the nearest to the type locality of V. alceoides.
Morphology
The present study is mainly based on herbarium material examined at B, BASU, E, G, JE, LD, P, W and WU. At first glance, Verbascum albidiflorum seemed to be similar to V. alceoides. Measurements, colours and other details given in the description are based on living material, alcohol-preserved specimens, and data derived from field notes. Morphological characters were surveyed, including stem shape and size, leaf shape and size, type of leaf pubescence, calyx colour and shape, corolla colour and size, and capsule width.
Leaf anatomy
Micromorphological characters, especially those related to hairs and stomata, can be considered as useful taxonomic features in species of Verbascum (Al-Hadeethy & al. 2014). The importance of anatomical studies was emphasized by Lersten & Curtis (2001), and it seems that epidermis peels have also proved to be diagnostically helpful. In this study, mature leaves from dried specimens were chosen and softened in a mixture of distilled water/ glycerine/ethanol 70% (1:1:1) for 2 weeks. Indumentum and epidermis were removed from the middle part of the leaves and petiole using commercial razor blades, stained by methyl blue and carmine and mounted on slides using Canada balsam. Hairs and stomata on the leaves were examined using an Olympus BX-51 photomicroscope at 400× magnification and photographed with an Olympus digital camera.
Pollen morphology
Pollen samples were obtained from herbarium specimens and prepared using the standard method of acetolysis (Erdtman 1960). They were mounted in unstained glycerin jelly and observations were made with a Nikon Type-2 microscope. The following measurements were made on 25 pollen grains from each specimen: polar axis (P), equatorial diameter (E), distance between colpi, colpus length, pore length, pore width and shape index (= P/E).
Karyology
The chromosome number of mitotic stages was studied in Verbascum alceoides and V. albidiflorum, both collected from Kermanshah Province, Iran, during May 2014, and deposed in the BASU herbarium. Flower buds at an appropriate stage of development were fixed in 96 % ethanol, chloroform and propionic acid (6:3:2) for 24 hours at room temperature and then stored in 70 % alcohol at 4 °C until used. Anthers were squashed and stained with 2% acetocarmine. All slides were made permanent by Venetian turpentine. Photographs were taken on an Olympus BX-51 photomicroscope initially at 1000× magnification. Chromosome counts were made from well-spread metaphases in intact cells, by direct observation and from photomicrographs.
Results and Discussion
Verbascum albidiflorum Ranjbar & Nouri, sp. nov. — Fig. 1.
Holotype: Iran, Kermanshah Province, Gahvareh to Khosro Abad, along road from Naylak to Beryakhani, eastern road from Khosro Abad to Kerend-e Gharb, 34°18′10.6″N, 46°23′26.7″E, 1450 m, 12 May 2014, S. Nouri 35706 (BASU!; isotypes: BASU!, W!).
Diagnosis — Verbascum albidiflorum Ranjbar & Nouri (Fig. 1) is most similar to V. alceoides Boiss. & Hausskn. (Fig. 2) but differs from V. alceoides in having stems 70–80 (vs 16–22) cm tall; basal leaf petiole 10–14 (vs 2–3) cm long; bracts 4–12 (vs 4–5) mm long; corolla milk-coloured or yellowish white (vs yellow) with dense purplish red spots in throat (vs unspotted); and filament hairs white to grey (vs purple-violet) (see Table 1).
Description — Herbs perennial, slightly woody at base, 70–80 cm tall. Stems several, robust, erect, unbranched or pyramidally branched from 15–20 cm above base toward apex, light green to somewhat purplish brown at base, cylindric, densely leafy, densely covered with short-stalked stellate and simple eglandular hairs, densely pubescent at base. Leaves alternate, mostly congested at base in a dense persistent rosette; basal leaves: petiole 10–14 cm long; leaf blade dark green when fresh, purplish brown when dried, oblong to elliptic, 20-26 × 3.4-4.4 cm, loosely yellowish white tomentose on both surfaces, with stellate hairs and glandular hairs on both surfaces, sometimes with dendroid hairs, veins indistinctly pinnately reticulate, more densely covered with hairs, base gradually attenuate into petiole, margin clearly obtuse-crenulate, apex acute or acuminate; cauline leaves decreasing in size toward flowering part of stem; lower cauline leaves: petiole 3.5–4 cm long; leaf blade 13-18 × 3.3-4.3 cm, margin crenulate to serrulate; upper cauline leaves subsessile; leaf blade elliptic, 4.5-7 × 2-2.4 cm, margin slightly crenulate to entire. Inflorescence racemose or loosely paniculate, 30–35 cm long; lateral branches erect, loose, green, elongating to 25–30 cm long, many-flowered, with pale white or grey eglandular stellate hairs; internodes 1.5–2 cm at inflorescence base; inflorescence leaves ovate to elliptic, 8-30 × 3-6 mm, similar to floral bracts, apex acute or acuminate; bracts green when fresh, purplish brown when dried, linear-lanceolate, 4-12 × 1-1.5 mm, densely covered with stellate hairs, sparsely covered with glandular and dendroid hairs, margin entire, apex acute to acuminate. Flowers solitary at each node (i.e. each bract embracing 1 flower), subsessile, ebracteolate, horizontal to ascending. Calyx green to greenish white, broadly ovoid or infundibuliform, 7-8 × 5.5-6 mm, lobed for ½ or more of length, densely tomentose with stellate hairs outside; lobes lanceolate-ovate, 3–5 mm long, subequal, distal ½ with deciduous hairs, apex acute and mucronulate. Corolla milk-coloured or yellowish white when fresh, pale yellow when dried, with dense purplish red spots in throat, rotate, 31–35 mm in diam.; tube 1–1.5 mm long; lobes orbicular, 17-19 × 8-10 mm, subequal, often 2 smaller than others, stellate tomentose outside. Stamens 5, fertile, subequal; filaments free, bluish purple, 9–10 mm long, glabrous at base, remainder densely covered with clavate villous hairs, with white to grey glandular hairs near anthers, 2 anterior filaments more sparsely haired than others; anthers yellowish white, 1.5–2 mm long, connate with connective, base decurrent on anterior anthers and reniform on others. Ovary 2-locular, ovoid to globose, densely stellate hairy; style terminal, filiform, 7–8 mm long, pubescent with stellate hairs at base; stigma clavate, 2-3 × c. 0.5 mm, apex semiglobose. Capsule olive-brown, shining, ovoid or ellipsoid to globose, 7-8 × 6-7 mm, scarcely longer than calyx, pubescent-velutinous, densely or rarely sparsely covered with stellate hairs to glabrescent, dehiscing by a longitudinal groove, apex shortly beaked. Seeds numerous, greenish to light brown when immature, rugose, uniformly traversely sulcate.
Distribution and ecology — The new species is a rare endemic of W Iran, in the Zagros Mountains between Gahvareh and Khosro Abad in Kermanshah Province, about 100 km E of the Iraq border (Fig. 5). It is an Irano-Turanian geoelement. It grows on hills of clay and stone in open Quercus forest at altitudes of 1400–1500 m. In addition, some other plant species occurring in the habitat included Aegilops triuncialis L., Cousinia dalahuensis Attar & Ghahr., Gundelia tournefortii L., Hedysarum criniferum Boiss., Onobrychis melanotricha Boiss., Poa trivialis L. and Quercus brantii Lindl.
Conservation status — Verbascum albidiflorum is known only from the type locality in Kermanshah Province. The estimated area of occupancy is less than 2 km2 and the known number of mature individuals is fewer than five. It can therefore be categorized as Critically Endangered (CR) according to IUCN criterion D (IUCN 2012).
Etymology — The specific epithet albidiflorum means “whitish flowered”.
Fig. 1.
Verbascum albidiflorum — A & B : habitat and habit, plants with inflorescences; C: basal leaves showing surfaces and margin; D: cauline leaves, parts of inflorescence, partly opened flower and flower buds; E: as D but with fully opened flower; F: fully opened flower, apical view; G: inside of flower showing stamens. — Scale bars: A = 5 cm; B–F = 3 cm; G = 5 mm. — Type locality, 12 May 2014, photographed by S. Nouri.
Fig. 2.
Verbascum alceoides 35707 — A: habitat and habit, plant with inflorescence; B: part of inflorescence with fully opened flowers and flower bud; C: cauline leaves upper surface. — Scale bars: A-C = 3 cm. — See Table 4 for locality, 2 May 2014, photographed by S. Nouri.
Table 1.
Diagnostic morphological characters of Verbascum albidiflorum and the holotype of V. alceoides.
Table 2.
Diagnostic anatomical characters (hairs and stomata) on leaves of Verbascum albidiflorum and V. alceoides 235. Dimensions are in µm. Mean is shown in brackets.
Table 3.
Quantitative analysis of the pollen morphology of Verbascum albidiflorum and V. alceoides 35707. Dimensions are in µm. Mean is shown in brackets.
Table 4.
Origin of material used in this study.
Fig. 3.
Photomicrographs of leaf epidermis, leaf hairs, petiole epidermis and pollen grains in Verbascum albidiflorum — A: surface section of leaf with anomocytic stomata in lower epidermis; B: surface section of leaf with anomocytic stomata in upper epidermis; C: transverse section of petiole with cavities in lower epidermis; D: eglandular and glandular hairs on leaf with cavities in lower epidermis; E: dendroid glandular hairs with multicellular base on leaf with cavity in lower epidermis; F: glandular hair on leaf; G: stellate hair with unicellular base; H: stellate hair with tricellular base; I: stellate hair with multicellular base; J: dendroid hair; K: pollen grain, equatorial view; L: pollen grain, polar view. — Scale bars: A & E = 10 µm; B & F = 20 µm; C & D = 50 µm; G, H, I & J = 100 µm; K & L = 3 µm.
Leaf anatomy
Results from leaf-hair morphology in Verbascum albidiflorum showed that the leaves are covered with eglandular and glandular hairs (Fig. 3D–J). There are three types of hairs in this species; (1) eglandular stellate hairs with a unicellular or multicellular single stalk by which they are attached to the surface of the leaf (Fig. 3G–I); (2) eglandular many-branched dendroid hairs (Fig. 3J); and (3) glandular hairs that are simple or rarely branched (Fig. 3E & F). The glandular hairs have a short neck and a broad 4-celled head. For comparison with V. albidiflorum using hair and stomata characters, the collection V. alceoides 235 was used (Table 2). Both collections have glandular and eglandular hairs on the lower and upper surfaces of the leaves and there is no difference in the diameter of the glandular hairs, but they are different in the length of the neck. Verbascum alceoides 235 has a multicellular short neck ranging from 5–6.25 µm long, which is often shorter than that of V albidiflorum which ranges from 4–12 µm long. Also the terminal cell of the glandular hair neck has a round shape in V. albidiflorum but is oblong in V. alceoides 235.
Fig. 4.
Representative prophase in a mitotic cell. — A: Verbascum albidiflorum, 2n = 38; B: V. alceoides 35707, 2n = 48. — Scale bars: A & B = 1 µm.
The study of stomata characters carried out on Verbascum albidiflorum and V. alceoides 235 indicated that they are uniformly amphistomatic (i.e. with stomata on both lower and upper leaf surfaces) and have the anomocytic type of stomata. The shape of the guard cells on the lower and upper surfaces is reniform in both V. albidiflorum and V. alceoides 235. There are three types of subsidiary cells in the leaves of V albidiflorum: (1) stomata completely surrounded by 3 subsidiary cells, which are of more or less equal size or one of them is distinctly smaller (Fig. 3B); (2) stomata completely surrounded by 4 subsidiary cells, which are equal or unequal in size (Fig. 3A); and (3) stomata surrounded by 5 subsidiary cells, which are equal or unequal in size. Often the stomata are smaller on the upper surface with 3 subsidiary cells (Fig. 3B). Stomata with 4 subsidiary cells and 3 subsidiary cells with equal percentages (46.66) have the highest density and stomata with 5 subsidiary cells have the lowest density (6.66) on both leaf surfaces. All the subsidiary cells in leaves are monocyclic with ridged walls. The stomatal index on both surfaces in V. albidiflorum is larger than in V. alceoides 235. There are differences in length and width of stomata on the lower and upper leaf surfaces: the stomata length in V. alceoides 235 is larger than in V. albidiflorum. In addition, cavities were seen under the lower epidermis of the leaf blades and petioles of V. albidiflorum (Fig. 3C–E); these anatomical features might have taxonomic value.
Pollen morphology
The present study showed that most pollen grains of Verbascum albidiflorum (Fig. 3K & L) and V. alceoides 35707 are radially symmetrical, isopolar, oblate-spheroidal or sub-spheroidal or sub-prolate and tricolporate. Observations by light microscopy of seven pollen characters of V. albidiflorum and V. alceoides 35707 are reported here (Table 3). The pollen grains in V. alceoides 35707 are larger than in V. albidiflorum.
Karyology
Few studies have been conducted on the chromosomes of Verbascum species (e.g. Mori 1957; Arts-Damler 1960; Nilsson & Lassen 1971; Koktay 1974; Dane & Yilmaz 2002) and, as has been pointed out (Yılmaz & Dane 2011), they have very small chromosomes. So far in Iran, only one chromosome number, 2n = 2x = 30, has been reported for two Verbascum species: V. sinuatum (Ghaffari 2008) and V. speciosum (Cartier 1983). We are reporting the mitotic chromosome number 2n = 38 for V. albidiflorum (Fig. 4A) and, for the first time, 2n = 48 for V. alceoides 35707, from W Iran (Fig. 4B).
Acknowledgements
We are indebted to the staff of the herbaria B, E, G, JE, LD, P, W and WU for their cooperation. The field work in Iran was supported by grants provided by the Bu-Ali Sina University. We are grateful to Nicholas Turland (B) for helpful discussion and comments and substantial improvement of the text. We also thank Manfred A. Fischer (WU) and an anonymous reviewer for their comments on an earlier draft of this paper.
