A new species of Dianthus (Caryophyllaceae) from Antalya, South Anatolia, Turkey

Abstract Dianthus multiflorus from Gazipaşa (Antalya), south Anatolia (Turkey), is described as a new annual species with verrucose calyx. The morphological differences from the species within the same group with Dianthus multiflorus, which are Dianthus aydogdui, Dianthus cyri and Dianthus tripunctatus, are discussed. The International Union for Conservation of Nature (IUCN) threat category and observations on the ecology of the populations are noted. The karyology and seed micromorphology of Dianthus multiflorus and Dianthus tripunctatus were examined by light microscopy and scanning electron microscopy.


Introduction
Amongst all its neighbouring countries, Turkey is the richest in terms of plant taxa, being home to 9996 plant species (11707 taxa) (Güner et al. 2012). Th e fl oristic richness of the country is partially due to the high number of endemic and rare species present. Th e Mediterranean region is one of the important centers of endemism in Turkey, and Antalya is the richest province of Turkey in terms of plant diversity, hosting 773 of the country's endemic species. Amongst these endemic species, about 244 are best described as locally endemic, being found only in Antalya (Deniz and Aykurt 2014).
After Silene L., Dianthus L. is the second largest genus of Caryophyllaceae. Th is genus, containing approximately 300 species, is mainly distributed in the Mediterranean region of Europe and Asia (Reeve 1967;Bittrich 1993). Th e most comprehensive study on Dianthus species in the Flora of Turkey and East Aegean Islands was carried out by Reeve (1967) wherein 67 species were recorded. Since that date, new species and records have been added and the total number of Dianthus species recorded in Turkey has increased to 81 (Shishkin 1985, Davis et al. 1988, Gemici and Leblebici 1995, Güner 2000, Menemen and Hamzaoğlu 2000, Aytaç and Duman 2004, Özhatay and Kültür 2006, Vural 2008, Yılmaz et al. 2011, İlçim et al. 2013, Hamzaoğlu 2012, Hamzaoğlu and Koç 2015, Hamzaoğlu et al. 2014, 2015a, 2015b, 2015c. Among the annual Dianthus species recognized by Reeves (1967), there are only two species (D. cyri Fisch. & C.A.Mey. and D. tripunctatus Sm.) that have a verrucose calyx.
Dianthus aydogdui Menemen & Hamzaoğlu, which resembles D. cyri and D. tripunctatus, has been described from Salt Lake (Central Anatolia) province by Menemen and Hamzaoğlu (2000). Th e new species described here, Dianthus multifl orus Deniz & Aykurt, was collected from Gazipaşa province in Antalya. It is distributed on stony sliding slopes and serpentine soils in clearings of Pinus brutia forest. Dianthus multifl orus is included in the same group along with D. aydogdui, D. cyri and D. tripunctatus. Dianthus multifl orus shows distinct diff erences from these species by its habit, number of fl owers on the stem, calyx and petal features.

Plant samples and morphological studies
Dianthus multifl orus specimens were collected from Gazipaşa province (Antalya) during fi eld studies within the scope of the project "EXPO 2016 Endemic and Rare Flowers of Antalya". New species and its morphologically most similar species, D. tripunctatus were observed during fi eld studies and their morphological characteristics were recorded both in the fi eld and in the laboratory. Specimens collected were comprehensively evaluated by the use of the literature (Velenovsky 1891;Post 1932;Tutin 1964;Reeve 1967;Shishkin 1985;Strid 1986;Rechinger 1988) and the specimens present in GAZI, ISTE, and Akdeniz University herbariums. Th e overall morphology of the new species was examined with stereo-binocular microscope.
Th e seed micromorphology of D. multifl orus and D. tripunctatus was investigated using scanning electron microscopy (SEM) techniques. Seeds were attached to SEM stubs, coated with gold conjugate following the manufacturer specifi cations and examined with a Zeiss LEO-1430 scanning electron microscope.

Karyological studies
Chromosome number and karyological features of the Dianthus multifl orus and D. tripunctatus were determined from plant material collected from Antalya. All karyological observations were carried out on root tips. Root-tip meristems were provided from seeds by germinating them on wet fi lter paper in petri dishes at the temperature of 23°C. Firstly, root tips were pretreated for 24 h in a-monobromonaphthalene at 4 °C, fi xed in 3:1 absolute alcohol-glacial acetic acid. Root tips were then hydrolyzed with 1 N HCL for 13 min. at 60 °C, stained in Feulgen solution, and squashed in acetoorcein. For karyotype analysis, the photographs were taken using OLYMPUS BX53 microscope with camera Kameram 12 CCD attachment. Chromosome counts in mitosis metaphase and karyotype analyses were obtained based on fi ve root tips, fi ve metaphase cells for each individual. Measurements of somatic chromosomes were made with the program KAMERAM, they were calculated with formula of the relative variation in chromosome length CV CL (Paszko 2006), mean centromeric asymmetry (M CA ) according to Peruzzi and Eroğlu (2013) and chromosome total haploid length (THL) (Peruzzi et al. 2009). Chromosomes were classifi ed to the nomenclature following Levan et al. (1964) and asymmetry types following Stebbins (1971).

Results
With the recognition of Dianthus multifl orus as a new species, there are now four annual species of Dianthus, (D. multifl orus, D. tripunctatus, D. cyri and D. aydogdui) that can be separated from other annual Dianthus species on the basis of their verrucose calyxes in Turkey. A detailed comparision of the morphological and ecological features of these species is shown in the Table 1. Annual, many-stemmed, divaricately branched herbs. Stem erect to ascending, fragile, slender, 20-80 cm, branching from the base, glabrous or minutely scabridulous especially toward base, usually purplish at base, many-fl owered (30-)80-250(-300). Basal leaves linear-lanceolate to lanceolate-spatulate, 25-35 × 2.5-4.5 mm, with scabridulous edges especially near base, obtuse at apex. Cauline leaves linear-narrowly triangular to linear-lanceolate, gradually smaller upwards, fl attened, distinctly nervous, glabrous, with scabridulous margin and narrowly membranous toward base, acute to acuminate at apex, their sheaths shorter than the internodes; lower cauline leaves linear-lanceolate, 20-50 × 2-2.2 mm, longer or shorter than internodes, swollen and usually purplish at base; upper cauline leaves linear to linear-narrowly triangular, 4-20 × 0.5-1.5 mm, slightly swollen at base. Infl orescence dichotomously branched; fl owers almost always solitary, occasionally two or three fl owers borne on the same nod; branches usually minutely scabridulous;   Phenology. Dianthus multifl orus was observed fl owering in June and July, and mature fruits are produced in July to middle of August.

Dianthus multifl orus
Etymology. Th e species epithet is derived from its abundant fl owers representing one of the main characters that distinguishes it from other similar species.
Proposed conservation status. Dianthus multifl orus is included in the Critically Endangered category according to IUCN criteria ver. 11 (IUCN 2014). Th e species was determined at only a single location and the extent of occurrence (EOO) value of the species was determined to be 7 km 2 taking into account location of occupancy and the area contained within the shortest continuous imaginary boundary. Additionally, the area of occupancy (AOO) value in this area was calculated as 4 km 2 [CR B1ab(i)+CR B2b(ii)].
Seed testa micro-morphology. A detailed comparison of seed micromorphology based on SEM analysis was made (Table 2). Th e seeds of D. multifl orus and D. tripunctatus are black, minutely cuspidate at apex, granular, and have anticlinal walls represented by shallow and wide grooves, with U-like undulations, while the periclinal walls are distinctly papillose. Th e seeds of D. multifl orus are covered by irregularly polygonal or rectangular cells, and the cells of ventral surface are more elongated and larger, conversely the seeds of D. tripunctatus are covered by irregularly rectangular cells and the cell size of ventral and dorsal surfaces are similar (Fig. 3).
Karyology. Th e chromosome number of Dianthus multifl orus and D. tripunctatus is 2n = 30 (Fig. 4). Th e shortest chromosome length for D. multifl orus is 0.54 μm, the longest is 1.24 μm, and total haploid chromosome length (THL) is 12.21 μm. Th e karyotype formula of D. multifl orus consists of 28 median pairs and 2 submedian pairs. As for karyotype asymmetry, its karyotype was classifi ed according to the symmetry classes of Stebbins (1971) as 3B. Intrachromosomal asymmetry (M CA ) is 6.26 and the interchromosomal asymmetry index (CV CL ) is 19.93. Our study showed that the shortest chromosome length for D. tripunctatus is 0.77 μm, the longest is 1.21 μm, and total haploid chromosome length (THL) is 14.09 μm. Th e karyotype formula of this species consists of 28 median pairs and 2 submedian pairs. As for karyotype asymmetry, the karyotype of this species is classifi ed according to the symmetry classes of Stebbins (1971) as 3A. Intrachromosomal asymmetry (M CA ) is 6.42 and the interchromosomal asymmetry index (CV CL ) is 12.57. Th e karyogram is given in Figure 4 and ideogram was drawn based on the centromeric index (Fig. 4).  According to Stebbin's (1971) classifi cation, the karyotypes of D. multifl orus belong to type 3B, whereas the karyotypes D. tripunctatus belong to type 3A. Th e asymmetry indices also reveal some small diff erences between the two species. Nevertheless, according to interchromosomal asymmetry index (CV CL ), D. multifl orus is more asymmetric than D. tripunctatus. Th e total haploid chromosome length (THL) in D. multifl orus is 12.21 μm and that of D. tripunctatus is 14.09 μm (Table 2).
Diagnostic key to the annual Dianthus species with verrucose calyx in Turkey

Discussion
Of the annual Dianthus species that occur in Turkey, there are only four species that have verrucose calyxes; Dianthus aydogdui, D. cyri, D. multifl orus and D. tripunctatus. Dianthus aydogdui was recently described from Salt Lake province (Menemen and Hamzaoglu 2000), and this species is distinct from D. tripunctatus and D. Cyri due to its short and single stems, and shorter epicalyx scales in relation to its calyx tube. Dianthus multifl orus is also closely related to D. tripunctatus and D. cyri. In particular, the habit and fl oral characteristics of D. multifl orus are quite diff erent from other species. Its divaricately branched stems bear numerous fl owers unlike those of D. tripunctatus and D. cyri. Contrary to the pink and dentate petal-limbs of D. tripunctatus and D. cyri, D. multifl orus has white petals with distinct dark purplish venation and emarginate with shallowly sinuate margins. Dianthus multifl orus shows more resemblance to D. tripunctatus than D. cyri by the length of its epicalyx scales and the features of calyxes. Th e calyx tubes of D. multifl orus and D. tripunctatus are nervose-striate whereas those of D. cyri are enervose. However, the calyx length of D. multifl orus is shorter than that of D. tripunctatus. Further, the epicalyx scales are shorter to almost equaling in the calyx tube in D. multifl orus whereas they are equaling in D. tripunctatus and shorter in D. cyri. Dianthus multifl orus and D. tripunctatus have acuminate calyx teeth compared to their being aristate in C. cyri. Besides morphological characteristics, both seed micromorphological and karyological features of D. multifl orus and D. tripunctatus were also identifi ed within the present study. According to the results of the seed micromorphological studies, the main diff erence between the seeds of D. multifl orus and D. tripunctatus is the shape and size of the coat cells. According to the results of the karyological studies, there are some karyomorphological diff erences between the two species. According to Levin (2002), the correlation between THL and 1C values within and between species in related genera, THL is considered a good proxy for genome size. On this basis, Peruzzi and Altınordu (2014) proposed a standardized method, taking into account six quantitative parameters, in order to establish relationships among taxa. THL is one of these parameters and the total haploid chromosome length (THL) in D. multifl orus was comparatively lower than that of D. tripunctatus.

Conclusion
Th e most important reason for the high endemism values in the Antalya and Mediterranean regions of Turkey is the sudden climatic and topographic diff erences. Th e Taşeli Plateau, which is one of the endemism centers of the eastern part of the Antalya province, is at an altitude of approximately 2200 meters and is just 20 km from the Mediterranean Sea. Th e lower slopes of the region are characterized by a typical Mediterranean climate and vegetation types, but the climatic conditions are continental at higher altitudes. Th e distribution area of Dianthus multifl orus is located in these climatic and topographic transition regions, and discovery of the new species contributes to a better understanding the richness of the Turkish Flora. With this study, the total number of species belonging to the genus Dianthus has risen to 82, the study provides material and data to aid further research on Dianthus, an important member of the Caryophyllaceae.