Turkish Journal of Botany Turk J Bot (2016) 40: 87-96 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1407-71

New species and a synonym of the Hesperis () from Turkey

Ahmet DURAN, Özlem ÇETİN* Department of Biology, Faculty of Science, Selçuk University, Selçuklu, Konya, Turkey

Received: 18.07.2014 Accepted/Published Online: 12.04.2015 Final Version: 01.01.2016

Abstract: Hesperis tosyaensis A.Duran, a new species from northern Anatolia, is described and illustrated. It grows under open Quercus forest. The new species is distinct from the closely allied H. ozcelikii A.Duran by the following features: stem erect, 45–90 cm tall, hispid, hairs 2–2.5 mm, and fruit glabrous. Genetic differentiation between closely related Hesperis L. taxa and Matthiola W.T.Aiton was examined by molecular analyses of the ISSR. The chromosome number for Hesperis tosyaensis is 2n = 14. Information is also presented on its ecology and conservation status. In addition, Hesperis aspera E.Fourn. is treated as a synonym of Hesperis bicuspidata (Willd.) Poir.

Key words: Hesperis, new species, ISSR, Cruciferae, systematics

1. Introduction on the infrageneric and infraspecific classification of the Hesperis L. (Brassicaceae) is represented by many taxa at genus Hesperis, ecological and geographic variations of the junctions of the Irano-Turanian, Mediterranean, and some Hesperis taxa cause some confusion in Euro-Siberian phytogeographic regions; these regions and nomenclature, especially among meet in Anatolia. Towards the outer boundaries of each L., H. bicuspidata (Willd.) Poir., H. pendula DC., and phytogeographic region, Hesperis is represented by fewer certain other taxa. Hesperis bicuspidata is localized in the taxa, which is also indicated by the number of Hesperis taxa Irano-Turanian phytogeographical region and the species in various floras. There are 14 species in Europe, 11 species has a widespread distribution in Turkey. H. bicuspidata in Iran, 9 species in Romania, 5 species in Iraq, 3 species in grows in different habitats with different features. Hesperis Italy, and 1 species in Palestine (Săvulescu, 1955; Ball, 1964; bicuspidata specimens were collected from different Zohary, 1966; Dvořák, 1968, 1980; Pignatti, 1982; Duran localities in Turkey. and Hamzaoğlu, 2004; Duran and Ocak, 2005; Duran, 2005; Hesperis aspera, which is endemic to Turkey and Parolly and Tan, 2006; Duran, 2008, 2009). is known only from Tosya (Kastamonu Province), was The genusHesperis is represented by 31 species in the described as a new species by Fournuer (1866). Our flora of Turkey (Cullen, 1965; Davis et al., 1988). Five new examinations of type specimens and herbarium specimens species have been described from Turkey and 10 species of H. aspera have revealed that it is a synonym of H. are treated as synonymous (Duran and Ocak, 2005; Parolly bicuspidata. Based on our study, H. aspera is treated as a and Tan, 2006; Duran, 2008, 2009; Duran et al., 2011). synonym of H. bicuspidata. According to the latest study, Hesperis is represented by 27 Some interesting Hesperis specimens were collected species recorded from Turkey (Duran, 2012). from the Tosya district (Kastamonu). Herbarium studies The genus Hesperis in Turkey has been studied in terms and field observations showed that our specimen is a of morphology, floristic features, palynology, molecular new taxon. Diagnostic morphological characters from characteristics, anatomy, and chemotaxonomy (Duran, closely similar Hesperis species are discussed in this study. 2005; Duran and Ocak, 2005; Duran, 2008; Ünal et al., Ecology, conservation status, and biogeography of the new 2008; Yücel et al., 2008; Aras et al., 2009; Büyükkartal et al., species are presented. Genetic differentiation of the new 2009; Duran, 2009; Pınar et al., 2009; Kırımer et al., 2010). species and related species were analyzed using NTSYSpc Despite the fact that many studies have been performed 2.1 software and dendrograms were obtained. * Correspondence: [email protected] 87 DURAN and ÇETİN / Turk J Bot

2. Materials and methods performed in a mix containing 2.5 μL of PCR buffer, 3 μL

2.1. Morphology of 25 mM MgCl2, 1 μL of primer, 0.5 μL of dNTP mix, 0.3 We followed the descriptions of taxa according to the μL of Taq DNA polymerase, 4 μL of each diluted DNA, and Flora of Turkey and the eastern Aegean Islands and 13.7 units of PCR water. After a predenaturation step of 3 other floras (Halácsy, 1900; Hayek, 1927; Busch, 1939; min at 94 °C, amplification reactions were cycled 40 times Săvulescu, 1955; Tzvelev, 1959; Ball, 1964; Cullen, 1965; at 94 °C for 1 min, at annealing temperature (Table 1) for 1 Zohary, 1966; Dvorák, 1968; Dvorák, 1980; Pignatti, 1982; min, and at 72 °C for 1 min in an Eppendorf Mastercycler Davis et al., 1988; Tan and Iatrou, 2001) were examined. gradient thermocycler. A final extension was performed The specimens present in the AEF, ANK, BM, E, EGE, G, for 10 min at 72 °C. Amplified fragments were visualized GAZI, HUB, ISTF, K, KNYA, P, and WU herbaria were under a UV transilluminator and photographed using examined. All measurements were made on 10 randomly a gel documentation system (Vilbert Lourmat, Infinity selected specimens. Measurements were made of model). All amplified fragments were treated as dominant parts using a ruler and an ocular micrometer under a genetic markers. Each DNA band generated was visually microscope. Localities of studied taxa are given in the scored as an independent character or locus. Analyses Appendix. were performed with the NTSYS-pc 2.1 software package (Rohlf, 2001). The dendrogram was constructed using SM 2.2. Karyology coefficient and UPGMA clustering methods. Karyological observations were carried out on root tips obtained from germinating seeds. Root tips were 3. Results pretreated for 16 h in α-monobromonaphthalene at 4 °C Hesperis bicuspidata (Willd.) Poir., Encycl. Suppl. 3: 195 and fixed in Carnoy solution (3:1 absolute ethanol and (1813) glacial acetic acid), and then the root tips were hydrolyzed Sect. Hesperis with 1 N HCl at room temperature and stained with 2% Type: Tournefort, no. 16 (B, P! KNYA photo.!, GAZI aceto-orcein. Stained root tips were flattened in a drop photo!). Described from Turkey. of 45% acetic acid, and permanent slides were made by Synonyms ≡ Cheiranthus bicuspidatus Willd., Sp. Pl., 3, mounting in Depex. 1: 519 (1800). = Hesperis kotschyana Fenzl, Pug. PI. Nov. 2.3. Molecular analyses Syr., 13 (1842); H. violacea Boiss., Diagn. PI. Orient., ser. Total DNA was obtained from 50–75 mg of dried leaf 1, 5: 80 (1844); H. reuteriana Boiss. & A.Huet, Diagn. PI. tissue from 11 different individuals. DNAs were extracted Orient., ser. 2, 5: 20 (1856); H. unguicularis Boiss., Diagn. using phenol-chloroform (Sambrook et al., 1989) after PI. Orient., ser. 2, 5: 21 (1856); H. aspera E. Fourn., Bull. concentrations were determined by NanoDrop. Sample Soc. Bot. Fr. 13: 352 (1866) syn. nov.; H. tauricola Kotschy DNAs were diluted to 25 ng/µL. Stock DNAs were kept & Boiss., Fl. Orient. 1:232 (1867); H. sintenisii Hausskn. at –86 °C. ISSR primers were used for PCR amplification; & Bornm., Repert. Spec. Nov. Regni Veg. Beih. 89(1): 38 the characteristics of the primers used are given in Table (1936); H. stellata Dvorak, Oesterr. Bot. Z. 112: 610 (1965). 1. Each reaction contained 25 μL. PCR reactions were Hesperis tosyaensis A.Duran sp. nov. (Figures 1–6)

Table 1. The characteristics of the ISSR primers.

Primer Sequences Tm (°C) Length (bp) GC ratio (%) Total bands M1 5ʹ-AGC AGC AGC AGC AGC AGC G-3ʹ 63.10 19 68.4 5 M2 5ʹ-ACC ACC ACC ACC ACC ACC G-3ʹ 63.10 19 68.4 9 M3 5ʹ-AGC AGC AGC AGC AGC AGC C-3ʹ 63.10 19 68.4 9 M5 5ʹ-GAG AGA GAG AGA GAG AGA C-3ʹ 56.70 19 52.6 16 M7 5ʹ-AGA GAG AGA GAG AGA GAG C-3ʹ 56.70 19 52.6 15 M9 5ʹ-ACA CAC ACA CAC ACA CCG-3ʹ 56.00 18 55.6 8 Diplo1 5ʹ-CAC ACA CAC ACA CAC AG-3ʹ 57.19 17 52.9 7 Cucurbita 5ʹ-GAG CAA CAA CAA CAA CAA -3ʹ 53.07 18 33.3 25 Cucurbita2 5ʹ-GAG AGA GAG AGA GAG AGA T-3ʹ 54.78 17 52.9 10

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Figure 1. Hesperis tosyaensis: A- general view, B- mature fruit. Scale bar = 2 cm.

Subgen. Hesperis Sect. Hesperis Description: Perennial, rarely biennial herb. Roots Type: Turkey. A5 Kastamonu: between Tosya-Sekiler thickened taproot, (2.5–)4–16 mm diameter. Stem village, 7 km, 1000 m, under an open Quercus forest, ±erect, 45–90(–110) cm tall (including flowering part), 40°55.62′N, 34°02.06′E, 12.5.2001, A.Duran 5657 & generally purplish below or rarely completely, solitary or Hamzaoğlu (holotype: KNYA, isotypes: GAZI, ANK, 2–4-branched flowering part, terete, smooth, 1.5–9 mm ISTE, ISTF, E, K). in diameter at base; hispid especially in lower part, simple Diagnosis: Hesperis tosyaensis differs from H. ozcelikii hairs at bottom; bifurcate and simple hairs in middle part; by its erect stem, 45–90 cm (not ascending to erect, 25–60 bifurcate, a few trifid, simple, and rarely glandular hairs cm), simple hairs especially in lower part and hispid (not in upper part, long simple hairs 2–2.5 mm long. Leaves with only densely articulated glandular hairs or densely crowded at bottom and middle part; basal leaves ±narrowly glandular, sparsely bifurcate hairs below), hairs 2–2.5 mm lyrate, 5–10(–14) × 0.6–2(–3.5) cm (including 2.5–5 cm long (not ±1.5 mm), pedicel glabrous or sparsely bifurcate petiole), remotely denticulate; cauline leaves decreasing and rarely trifid hairs (not only densely glandular hairs, or to flowering part, all with main midrib ±conspicuous; bifurcate-stellate and glandular hairs), fruit glabrous (not lower cauline leaves bifurcate, glandular, a few simple and densely glandular, sparsely bifurcate hairs), valve narrower 3 branched hairs, simple hairs especially on main midrib than septum (not valve wider than septum). and branched hairs on margin, ±obtuse; middle cauline

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Figure 2. Stem, fruiting pedicel, and flowering pedicel features. Stem hairy: A- H. tosyaensis (A.Duran 5822), B- H. ozcelikii (A.Duran 4636), C- H. bicuspidata (A.Duran 7443); fruiting pedicel (1- pedicel, 2- fruit): D- H. tosyaensis (A.Duran 5822), E- H. ozcelikii (A.Duran 4636), F- H. bicuspidata (A.Duran 7443); flowering pedicel (1- pedicel, 2- calyx): G- H. tosyaensis (A.Duran 5657), H- H. ozcelikii (A.Duran 5262), I- H. bicuspidata (Hamzaoğlu 2649).

Figure 3. Distribution map of Hesperis tosyaensis (■) and H. ozcelikii (●). A- Mediterranean phytogeographical region, B- Euro-Siberian phytogeographical region, C- Iran-Turanian phytogeographical region.

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Figure 4. Metaphase chromosomes of Hesperis tosyaensis (A.Duran 5822) (2n = 14). Scale bar = 5 μm.

Coefficient Figure 5. Dendrogram showing genetic relationships of some Hesperis species using ISSR markers. leaves ±narrowly oblong, oblanceolate, lanceolate, or purplish to violet, veins mostly conspicuous and purplish; lyrate, petiolate or sessile, attenuated at the base, serrate, limb obovate, tapering gradually into the claw, 9–13 × ±acute, densely bifurcate, and sparse or a few simple and 4.5–6 mm, rounded, ±horizontal; claw 9–13 × 1.3–1.7 3–4-branched hairs, simple hairs especially on the petiole mm, clearly exerted sepal. Outer filaments not dilated at and main midrib, and branched hairs on the margin; upper base, 3.8–4.5 mm long, inner filaments dilated at base, cauline leaves very narrowly linear-lanceolate, sessile 6.4–7.2 mm long, glabrous. Anthers all fertile, ±linear, or rarely semiamplexicaule, slightly contracted at the 3–3.6 mm long, yellowish or greenish, basifixed. Stigma base, serrate, acute to acuminate. Inflorescence raceme, with 2 obtuse, decurrent carpidial lobes. Ovary glabrous. rarely waxy panicle, branches ascending to erect, 10–45 × Fruiting pedicels not thickened towards the fruit, graceful, 5–35 cm. All flowers ebracteate. Pedicels ascending, very 0.4–0.5 mm in diameter. Siliquae (20–)40–70(–100) × graceful, 12–22 mm long at anthesis, elongating to 28 mm 1–1.5 mm, terete, dehiscent, torulose, straight or rarely long at most, glabrous or sparsely bifurcate and rarely slightly curved, ascending to erect, glabrous, greenish trifid hairs. Sepals partly or entirely pinkish, inner sepal or partly purplish; valves slightly narrower than septum; oblong-oblanceolate, outer sepal oblanceolate-oblong, septum membranous or semimembranous in seed place, deciduous, 5–8 veins, 7–8.5 × 1.4–2.2 mm, bifurcate, a remaining spongiose, with median veins inconspicuous. few trifid hairs and a few simple hairs only on tips, with Seeds ±dark brown, 2–2.6 × 1–1.3 mm, 1–24 in number. membranous margins, inner sepals strongly saccate. Chromosome number: 2n = 14. Petals obovate or oblanceolate, 19–26 × 4.5–6 mm long,

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(3867) are the same as H. tosyaensis. For this reason, the specimen is called a paratype. IUCN Red List category: The specimens were collected in Kastamonu Province in Turkey, where the species seems to be very rare and local, from an area of ca. 2.5 km2. The population is not in good condition, with approximately 135 specimens. In the latest studies, this species was not collected in Safranbolu and is only known from Tosya. This species is evaluated as Critically Endangered [CR B2ab (i-v)] (IUCN, 2010). Distribution, habitat, and biogeography: Turkey is the most complex country in the Middle East with regard to geographic structure and landforms. It comprises comparatively narrow and long, variously oriented mountain chains separated by deep valleys and also high- and medium-elevational plateaus. Central Anatolia is predominantly a rolling plateau, the bulk of which ranges from 900–1200 m in elevation. While more or less secluded in the north, east, and south by higher mountain ranges, it is open to wide valleys coming from the west (Zohary, 1973). Central Anatolia creates a barrier between the Mediterranean and Black Sea regions. Hesperis ozcelikii, which is a local endemic in southern Anatolia (Isparta Province), is an eastern Mediterranean element, while Hesperis tosyaensis appears to be endemic to the area of Tosya (Kastamonu Province) and Safranbolu (Karabük Province), northern Anatolia, and is a European-Siberian element. Hesperis tosyaensis grows on roadsides, the edges of stony piles, open places, and under open Quercus forest at 900–1200 m (Figure 3).

4. Discussion Hesperis tosyaensis differs from H. ozcelikii by having its Figure 6. Profiles of studied species on agarose gel, amplified stem erect, 45–90 cm (not ascending to erect, 25–60 cm), with ISSR primers. 1-, 2- Hesperis matronalis, 3- H. tosyaensis, hairs 2–2.5 mm long (not ±1.5 mm), inflorescence 10–45 4- H. ozcelikii, 5- H. bicuspidata, 6- H. buschiana, 7- H. bottae, × 5–35 cm (not 4–35 × 3–15 cm), pedicel very graceful 8- H. pendula, 9- Matthiola anchoniifolia, 10- Hesperis hedgei, 11- (not ±thick), glabrous or rarely sparse hairs (only densely H. breviscapa. glandular hairs), ovary glabrous (not hairy), fruiting pedicel 0.4–0.5 mm in diameter (not 1 mm in diameter), fruit glabrous (not densely glandular, sparsely bifurcate Phenology: Hesperis tosyaensis has been found hairs), siliquae 1–1.5 mm wide (1.6–2.2 mm wide). More flowering in May and June, and fruiting in June and July. comprehensive descriptions are given in Table 2. Paratypes: A5 Kastamonu: between Tosya-Sekiler Key to closely related Hesperis species village, 7 km, 1000 m, 21.07.2001, under an open Quercus 1. Perennial, rarely biennial herb, erect, 45–90 cm, hispid forest, 40°55′62″N, 34°02′06″E, A.Duran 5822 & Menemen hairs 2–2.5 mm long, inflorescence 10–45 × 5–35 cm; (KNYA, ANK, HUB, GAZI); Paplagonia, Wilajet pedicel glabrous or rarely sparse hairs, fruiting pedicel Kastambuli (A4 Kastamonu): Tossia (Tosya) in fruticdis ad 0.4–0.5 mm diameter; fruit glabrous, valve narrower Karkun, 24.05.1892, Sintenis 3867 (WU!, BM!, K!, ANK!); than septum ...... H. tosyaensis Anatolia, Safranbolu, Wiedemann s.n. (K!, G!). 1. Biennial, ascending to erect, 25–60 cm, only densely Specimen collected by Sintenis (3867) was identified as articulated glandular hairs, hairs ±1.5 mm long; H. armena by Prof C Haussknecht. After type specimens inflorescence 4–35 × 3–15 cm; pedicel densely glandular of H. aspera were examined in Geneva Herbarium, it hairs, fruiting pedicel ca. 1 mm in diameter; fruit densely was understood that the specimens collected by Sintenis glandular, valve wider than septum ...... H. ozcelikii

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Table 2. The comparison of diagnostic characters of Hesperis tosyaensis with H. ozcelikii.

Diagnostic characters Hesperis tosyaensis Hesperis ozcelikii Hesperis bicuspidata

Life form Perennial, rarely biennial Biennial Perennial

Stem height Erect, 45–90 cm Ascending to erect, 25–60 cm Ascending to erect or erect, 20–50 cm

Generally purplish at bottom, Generally green, rarely purplish at bottom, 2–6 Stem Rarely purplish at bottom, 3–6 cm diameter at base 1.5–9 mm diameter at base mm diameter at base

Only densely articulated glandular hairs or densely Densely short stellate-furcate and very rarely a Stem hairs Simple hair at bottom glandular and sparsely bifurcate hairs below few glandular hairs in lower part

Hairs 2–2.5 mm long ±1.5 mm long up to 1 mm

Leaves Narrowly lyrate Lyrate or sinuate dentate Generally entire

Petiole 2.5–5 cm long 1–3 cm long 1.5–4 cm long

Raceme, rarely waxy panicle, Generally raceme, sometimes branched at base, Inflorescence Generally simple raceme, branches 4–35 × 3–15 cm branches 10-45 × 5–35 cm 5–20 × 5–20 cm

±thick, short stellate-bifurcate and Pedicel Very graceful, glabrous ±thick, only densely glandular hairs rarely patently stalked glandular hairs

12–22 mm long at anthesis, 5–20 mm long at anthesis, elongating to 25 mm 4–12 mm long at anthesis, elongating to 16 mm Pedicel length elongating to 28 mm long at most long at most long at most

Anther 3–3.6 mm long 3.5–4 mm long 2.5–4 mm long

Ovary Glabrous Hairy Glabrous or hairy

Fruiting pedicel Graceful, 0.4–0.5 mm diameter Slightly thickened, ±1 mm diameter Slightly thickened, 0.6–1 mm diameter

Fruit wide 1–1.5 mm 1.6–2.2 mm 1.4–2.2 mm Glabrous or asperous Fruit Glabrous Densely glandular, sparsely bifurcate hairy

Valve Narrower than septum Wider than septum Wider than septum

Hesperis tosyaensis differs from H. bicuspidata by its conspicuously thickened towards the fruit, 0.7–1.3 mm in stem erect, 45–90 cm (not ascending to erect, 20–50 cm), diameter). hairs 2–2.5 mm long (not up to 1 mm), leaves narrowly Chromosome numbers of Hesperis taxa are reported as lyrate (not entire), pedicel very graceful (not ±thick), 12– n = 6, 7, 8, 12, 14, 16, 20, 24 and 2n = 12, 14, 16, 24, 28 22 mm long at anthesis (not 4–12 mm long at anthesis), (Manton, 1932; Khosravi and Maassoumi, 1998; Warwick glabrous or rarely sparse hairs (not short stellate-bifurcate and Al-Shehbaz, 2006; Duran et al., 2008; Ünal et al., 2008). and rarely patently stalked glandular hairs), fruiting pedicel The somatic chromosome number of Hesperis tosyaensis graceful, 0.4–0.5 mm in diameter (not thickened, 1 mm was identified as 2n = 14 (Duran et al., 2008). Our results in diameter), siliquae 1–1.5 mm wide (1.4–2.2 mm wide). are similar to those given in the literature (Figure 4). More comprehensive descriptions are given in Table 2. The ISSR marker system is a PCR-based technique Hesperis tosyaensis is closely related to Hesperis that uses a single amplification primer composed of a buschiana, which is spread throughout northeastern microsatellite motif to target a subset of simple sequence Anatolia. H. tosyaensis mainly differs from H. buschiana repeats (SSRs) or microsatellites (Zietkiewicz et al., in pedicel ascending, very graceful, 12–22 mm long 1994; Gürkök et al., 2013). ISSR-PCR is a simple, quick, at anthesis (not ascending to erect, ±thick, 7–12 mm), and efficient technique. It has high reproducibility. The pedicel elongating to 28 mm long at most (not to 25 mm), amplified products (ISSR markers) are usually 200–2000 pedicel glabrous or sparsely bifurcate and rarely trifid bp long and amenable to detection by both agarose and hairs (not bifurcate and trifid); petals 19–26 mm long (not polyacrylamide gel electrophoresis (Reddy et al., 2002). 17–20 mm); fruiting pedicel not thickened towards the The technique is useful in areas of genetic diversity, fruit, graceful, 0.4–0.5 mm in diameter (not gradually and phylogenetic studies, gene tagging, genome mapping,

93 DURAN and ÇETİN / Turk J Bot and evolutionary biology in a wide range of crop species subclade includes Hesperis breviscapa Boiss., which is (Reddy et al., 2002). placed in section Cvelevia. All flowers ofHesperis breviscapa Twenty ISSR primers were used for PCR amplification. are bracteate. Fruits are flattened siliquae that significantly Nine of them were chosen for ISSR analyses. The GC narrow towards the tip and open easily. The fourth clade percentages of selected primers were within the range of includes Hesperis pendula, which has pendulous fruit. 33.3%–68.4%. A total of 104 bands were produced, of which Hesperis pendula, which is widely distributed in all regions 77 were polymorphic (74.03%). The total number of bands of Turkey, is found in section Pachycarpos. The sixth clade ranged from 5 to 25. The lowest ISSR bands were produced consists of the taxa of section Hesperis. Section Hesperis by primer M1. The highest ISSR bands were produced by is found spread across a wide geographic area, including primer Cucurbita. Genetic similarity was estimated using southern Europe and the Near East, from the mountainous simple matching coefficients (Table 3). Genetic similarity regions of southern Yakutia, western China, and across between studied species ranged from 0.5185 to 0.9814. The Mongolia. The sections in northwestern Iran within Asia highest similarity (0.9814) was observed between different Minor, the Balkan Peninsula, and the Caucasus are especially specimens of Hesperis matronalis. The genetic similarity of rich (Tzvelev, 1959). The sixth clade contains 3 subclades, H. ozcelikii and H. tosyaensis is 0.8518. Hesperis matronalis the first of which is Hesperis buschiana (Figure 5). Hesperis has a high similarity (0.8518) to H. bicuspidata, H. buschiana is endemic to Turkey and is known from only one ozcelikii, and H. tosyaensis. The lowest similarity (0.4814) locality within the region of Artvin Province. The second was observed between Matthiola and Hesperis hedgei. subclade includes Hesperis bicuspidata and H. matronalis. Six main clusters were observed in the dendrogram. Hesperis bicuspidata is a species endemic to Turkey and The first clade includes Matthiola. This species was used is found in the Irano-Turanian phytogeographic region as outgroup. The other clades include sectionHesperis in Turkey. Hesperis bicuspidata is the most widespread taxa. The second clade includes Hesperis bottae Fourn., species of Hesperis in Anatolia. There are wide variations which is found in section Diaplictos (Dvorak) Dvorak. in plant height, leaf shape and size, calyx color and length, Hesperis bottae is distinguished from other Hesperis taxa corolla color, shape, size, venation and fruit hair, and shape by its sepals, petals, and stamens persistent in fruit. The characters among the H. bicuspidata samples collected from third clade incudes 2 subclades. The first subclade includes different localities. The third subclade includes Hesperis Hesperis hedgei Davis, which is found in section Delicate tosyaensis and Hesperis ozcelikii. Results of molecular A.Duran. This taxon has the shortest calyx, corolla, and analyses have confirmed that Hesperis tosyaensis is a distinct fruit among Hesperis taxa. Section Delicate is related to species (see Table 3 and Figure 5). In conclusion, Hesperis section Diaplictos. Section Delicate can be distinguished species studied in this manuscript show a correlation from section Diaplictos by its fruit being glabrous, thinner, between the morphological diagnostic characters and and shorter, pedicel not thick and ascending. The second molecular taxonomic classification in the dendrogram.

Table 3. Similarity matrix of studied species using ISSR markers.

Species H. tosyaensis H. ozcelikii H. bicuspidata H. buschiana H. matronalis H. matronalis-1 H. pendula H. breviscapa H. hedgei H. bottae Matthiola

H. tosyaensis 1

H. ozcelikii 0.8518 1

H. bicuspidata 0.8148 0.7777 1

H. buschiana 0.7592 0.7592 0.6851 1

H. matronalis 0.8333 0.8333 0.8333 0.7777 1

H. matronalis-1 0.8518 0.8518 0.8518 0.7962 0.9814 1

H. pendula 0.6851 0.6481 0.7222 0.6666 0.8148 0.7962 1

H. breviscapa 0.7777 0.7407 0.7037 0.6481 0.7222 0.7407 0.5740 1

H. hedgei 0.7037 0.6666 0.6666 0.6481 0.7222 0.7407 0.5740 0.7037 1

H. bottae 0.6111 0.6111 0.6481 0.5185 0.7037 0.7222 0.5555 0.5370 0.6111 1

Matthiola 0.5925 0.5185 0.6296 0.5370 0.6481 0.6666 0.6851 0.5555 0.4814 0.5370 1

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As a result, comprehensive morphological studies treated as a synonym of H. bicuspidata. The specimens indicate that Hesperis aspera and H. bicuspidata cannot be of H. tosyaensis were examined and compared with separated from each other as different species. According specimens of the related species H. ozcelikii in Turkey, and to the priority rule (Greuter, 2000), H. aspera should be Hesperis tosyaensis is described as a new species.

References

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Appendix. from Gedikli village to Çiçek Mountain, 1750–1850 m, Specimens examined (*also used for resources as 05.06.1999, A.Duran 4578* (KNYA); C6 Kahramanmaraş: DNA samples): – Hesperis tosyaensis: A5 Kastamonu: Tekne Da. between Göksun & Maraş, 1500 m, 04.05.1957, between Tosya-Sekiler village, 7 km, 1000 m, 21.07.2001, rocky limestone slopes, Davis 27541 & Hedge (BM). under an open Quercus forest, 40°55′62″N, 34°02′06″E, Synonyms of H. bicuspidata: – Hesperis stellata: Turkey. A.Duran 5822* & Menemen (KNYA, ANK, HUB, GAZI). A7 Gümüşhane: Prov. Pontus, distr. Gümüşhane, Kop – Hesperis ozcelikii: Turkey. C3 Isparta: Sütçüler, 17th Dagh Pass, 2440 m a.s.l., 3 Aug 1960, rocky slopes, Furse km from Ayvalıpınar to Kesme, 1025 m, 37°36′53″N, & Synge 865 (holotype: W, left-hand plant; isotype: 31°10′26″E, 11.06.1999, A.Duran 4636, H.Özçelik & E!). – Hesperis tauricola: Iter cilicico-kurdicum, 5000′, Sağıroğlu (KNYA); ibid., 04.08.1999, A.Duran 4973 24.v.1859, Kotschy 180 (BM! E!). – Hesperis sintenisii: (KNYA); ibid., 09.06.2000, A.Duran 5262* & H.Özçelik Erzincan: Egin (Kemaliye), Bagir Başı, 3.5.1890, Sint. (KNYA); Isparta: Sütçüler, between Ayvalıpınar-Kesme, 2514 (holotype: JE; isotype: LD). – Hesperis kotschyana: 1300 m, 11.05.1997, H.Özçelik 7714 (GUL). – Hesperis Turkey, in monte Tauro. Aestate, 1524 m, 24.05.1838 bicuspidata: Herbier de Mr. Benj. Delessert, ...., Aucher Kotschy 24 (holoype: K!). – Hesperis violacea: Turkey: 109 (G) (type of Hesperis aspera); Tournefort, Nr. 16 (BM); Mersina, 1843, Pinard s.n. (type: GOET!). – Hesperis A4 Kastamonu: Ilgaz Mountain, Pinus sylvestris forest, reuteriana: Turkey: prope İspir, in incultis, 6/1853, 1500-2000 m, 30.06.1973, H.Peşmen & S.Erik s.n. (HUB); Pavillon s.n. (type: FI photo! JE photo! BM!). – Hesperis A5 Yozgat: Aydıncık, Kazankaya town, Kazankaya canyon unguicularis: Turkey: environs d’Erzeroum, Calvert s.n. pass, 650 m, 15.05.2000, stony places, shrubbery, A.Duran (isotype: JE). – Cherianthus bicuspidatus: Turkey: Herbar 5071 (KNYA); Kastambuli (Kastamonu): Tossia (Tosya), Gundelsheimer (type: B photo!). – Hesperis matronalis: Giaurdagh (Gavur Dağı), 10.06.1892, Sint. 3631 (WU, A6 Giresun: between Giresun-Şebinkarahisar, 44 km, 650 P); A7 (Gümüsckhane) Gümüşhane: 28.05.1894, Sint. m, 15.07.2000, A.Duran 5539* (KNYA); Ardahan: Göle, 5660 (K, WU, P); Gümüşhane: Köse, Kabaktepe village, Kabaktepe, 2100 m, 16.07.2000, steppe, 40°15′94″N, between Tellioğlu-Dörtkilise village, 2000 m, 27.06.1999, 39°40′65″E, A.Duran 5551 (KNYA); Bayburt: between A.Duran 4733* (KNYA); – Hesperis hedgei: C7 Şanlıurfa: Bayburt-Aşkale, Kop Mountain, 2300-2500 m, 31.06.1999, Siverek, northwest of Karacadağ village, 26.05.2000, 1100 stony steppe, 40°02’28″N, 40°30’22″E, A.Duran 4802 & m, 37°44′32″N, 39°38′91″E, A.Duran 5185* & Hamzaoglu Akgül (KNYA); B4 Kırıkkale: Balışeyh, Mehmetbeyobası, (KNYA). – Hesperis buschiana: A8 Artvin: Ardanuç, 1350 m, 25.05.2001, Hamzaoglu 2649; B5 Kayseri: Felahiye, Karlı village, 860 m, 01.06.2000, 41°05′62″N, 42°07′34″E, Büyüktoraman village, Ak Dağ, Hamza Sultan tepe, 2100 A.Duran 5237* & Kandemir (KNYA). – Hesperis bottae: m, 18.07.1999, steppe, A.Duran 4948 & Akgül (KNYA); B7 Elazığ: Harput, 21.06.1999, steppe, 38°42′80″N, B6 Sivas: between Pınarbaşı-Gürün, 1695 m, 08.06.2007, 39°15′85″E, A.Duran 5182* & Hamzaoglu (KNYA). – A.Duran 7443; B7 Erzinghan (Erzincan): Sipikordagh, Hesperis breviscapa: B7 Erzincan: Üzümlü, Keşiş Dağı, 15 05.07.1889, Sint. 1201 (WU, K); B9 Bitlis/Van: 10 km km, 2400-2500 m, 03.07.2014, 40°00′00″N, 35°09′37″E, SE of Pelli, 8500 (2500 m), 8.06.1954, Davis 22597 & A.Duran 4825* & Akgül (KNYA). – Hesperis pendula: B5 Polunin (K); C2 Burdur: Dirmil-Korkuteli road, left of Kayseri: Yahyalı, Aladağlar, 2600 m, 11.08.2001, A.Duran Dirmil throat, 1650 m a.s.l., 09.06.1999, serpentine open 5834 (KNYA). – Matthiola anchoniifolia: B6 Malatya: place, 36°58′08″N, 29°35′11″E, A.Duran 4628 (KNYA); between Darende-Gürün, 1260 m, 13.06.2007, A.Duran C3 Isparta: between Şarkikaraağaç-Yenişarbademli, 7452* (KNYA).

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