The new locally endemic genus Yazdana (Caryophyllaceae) and patterns of endemism highlight the high conservation priority of the poorly studied Shirkuh Mountains (central Iran)

Abstract Although mountain ranges are often recognized as global biodiversity hotspots with a high level of endemism, diversity and biogeographic connections of isolated and weakly explored mountains remain poorly understood. This is also the case for Shirkuh Mts. in central Iran. Here, Yazdana shirkuhensis gen. & spec. nov. (Caryophylleae, Caryophyllaceae) is described and illustrated from the high alpine zone of this mountain. Molecular phylogenetic analyses of nuclear and plastid DNA sequence data show that Y. shirkuhensis is related to Cyathophylla and Heterochroa (tribe Caryophylleae). The newly described genus and species accentuate Shirkuh Mts. as a center of endemism, which harbors a high number of narrowly distributed species, mostly in high elevations reaching alpine habitats. As this area is currently not protected, a conservation priority is highlighted for high elevations of Shirkuh Mts.


Introduction
Mountains are biodiversity hotspots (Spehn et al., 2011), which harbor a considerable number of endemic species (Barthlott et al., 1996;Körner, 2003), mostly in the alpine zone (Nagy & Grabherr, 2009;Hobohm et al., 2014). Iran is a mountainous country (Fig. 1), and a high proportion of the Iranian flora (74%) is concentrated or even restricted to mountain ranges (Noroozi et al., 2019b). With increasing elevation, the rate of endemism increases, and in spite of the small area size of the alpine zone relative to lower elevations, a considerable number of Iranian endemic species are restricted to this habitat (Noroozi et al., 2018(Noroozi et al., , 2019a(Noroozi et al., , 2019b. Although larger areas of alpine zone can be found in Alborz and Zagros, there are numerous smaller and isolated high mountains in different parts of the country. One of these isolated mountain systems is Shirkuh in central Iran (4050 m a.s.l. at the highest peak; Fig. 1), west of the city of Yazd. Together with the Kerman massif, Shirkuh Mts. have recently been identified as an area of endemism (Noroozi et al., 2019b). However, the high elevations of the Yazd-Kerman Massifs have been poorly investigated, and in remote regions, it is still possible to find taxa new to the regional flora or even new to science (e.g., Ajani et al., 2010;Noroozi et al., 2010;Rajaei et al., 2011;Mahmoodi et al., 2013;Moazzeni et al., 2014Moazzeni et al., , 2016Doostmohammadi & Kilian, 2017). Indeed, during a field trip to the highest summit of Shirkuh Mts. in summer 2012, an annual species of Caryophyllaceae was collected close to the summit. It could neither be determined with available floras nor could it be unambiguously assigned to any of the Iranian genera of the family, suggesting that it belongs to a new taxon.
Caryophyllaceae is a large mainly Holarctic family of approximately 3000 species of herbs and subshrubs, with its diversity center in the Mediterranean and the adjacent family have shown that many of the traditionally defined genera are not monophyletic (Dillenberger & Kadereit, 2014;Pirani et al., 2014;Sadeghian et al., 2015;Madhani et al., 2018). This is also the case for the tribe Caryophylleae, where, based on morphology (connate sepals, stipitate ovary, and presence of two styles), the plant from the Shirkuh Mts. was suspected to belong to. Madhani et al. (2018), revising tribe Caryophylleae, described three new genera and resurrected one genus. Thus, currently the tribe contains 14 genera As morphological data did not permit assignment of the plants from Shirkuh Mts. to any of the currently recognized genera, we used molecular phylogenetic data to place this taxon within the phylogenetic framework of the tribe Caryophylleae established by Pirani et al. (2014) and Madhani et al. (2018). Hence, by determining the phylogenetic position of the new taxon based on molecular data, we wanted to clarify its taxonomic position. In light of the obtained taxonomic results (i.e., description of a new genus and species: see Section 3) and a lack of a phytogeographic study of endemics in the Shirkuh Mts., we subsequently addressed the floristic relationships of these mountains to other mountain ranges of the Iranian Plateau on the basis of distribution patterns of Iranian endemics.  (Zohary, 1973;Ebrahimi et al., 2010;Djamali et al., 2012), and annual rainfall of 350-400 mm mainly from October to May (Grunert et al., 1978). However, during the Pleistocene ice ages, these mountains were locally glaciated (Haars et al., 1974). Based on the Global Bioclimatic Classification System developed by Rivas-Martínez et al. (1997, 1999, Shirkuh Mts. belong to the Mediterranean Xeric continental, which is surrounded by Mediterranean Desertic continental (Djamali et al., 2011). Shirkuh Mts. are a part of the Yazd-Kerman area of endemism within the Irano-Anatolian biodiversity hotspot (Noroozi et al., 2019b), and they have recently been identified as a priority conservation gap (i.e., a center of endemism that is not or only marginally covered by protected areas; Noroozi et al., 2019a). Steppe vegetation dominates across all elevation zones. The alpine zone is above ca. 3500 m a.s.l. and, similar to other alpine habitats of the region (Noroozi et al., 2008), is covered by thorn-cushion grasslands ( Figs. 2A-2D), rock habitats (Figs. 2E, 2F), screes (Figs. 2G, 2H), and snowbeds (Fig. 2I).

Plant material
Plant material of the new taxon was collected in early July 2012 and, in the course of a trip dedicated to re-collect this species, in mid July 2019. For the molecular investigation, leaves of six individuals (one collected in 2012, taken as a herbarium voucher, and five collected in 2019, stored in silica-gel) were used. For detailed morphological
Sequences were trimmed and assembled in Geneious 6.1.2 (https://www.geneious.com). The 12 newly obtained sequences (six for each marker) were added to the Caryophylleae data set of Madhani et al. (2018) and aligned using the MUSCLE plug-in in Geneious 6.1.2. Thus, the ITS data set contained 132 accessions representing 113 species and the rps16 data set contained 119 accessions representing 86 species (Doc. S1). Following Madhani et al. (2018), the genus Silene (five species sampled) was selected as outgroup. Nuclear and plastid DNA sequence data were analyzed separately and jointly. Combinability of the markers was assessed with the incongruence length difference (ILD) test (Farris et al., 1995) implemented as the partition homogeneity test in PAUP* 4.a164 (Swofford, 2002) at the CIPRES portal using a full heuristic search, 10 random taxon addition replicates, tree bisection and reconnection (TBR) branch swapping, and with MaxTrees set to 100. Moreover, visual inspection of nodes in the separate analyses did not show any mutually strongly contradicted nodes (i.e, with bootstrap support values of at least 70 and posterior probabilities of at least 0.95).
The three datasets (ITS, rps16, and combined datasets) were analyzed using maximum likelihood (ML) and Bayesian methods. The best-fit substitution models for the ITS and the rps16 data, determined using the Akaike Information Criterion (AIC) as implemented in jModelTest 2.1.4 (Darriba et al., 2012), were the GTR + I + G and the GTR + G model, respectively. Maximum likelihood analysis was carried out on the RAxML web server (RAxML-HPC2 on XSEDE; available at the CIPRES portal: http://www.phylo.org/index.php/portal/) using 1000 bootstrap replicates, obtained by the rapid bootstrap algorithm (Stamatakis et al., 2008). Bayesian inference (BI) analyses were performed using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001) using default prior settings and a random starting tree. The analysis consisted of four parallel runs, each with three heated chains and one cold chain that were run for 10 million generations, with each sampling every 1000 generations. The quality of the analysis was checked by comparing likelihood values and parameter estimates from different runs in Tracer 1.6 (Rambaut et al., 2014) and by average standard deviations of split frequencies (less than 0.01), and the first 25% of the trees were discarded as burn-in. The remaining trees were summarized in a 50% majority-rule consensus tree.

Morphological analysis
Morphological characteristics such as plant habit and the color of the flowers were investigated in the natural habitat; measurements and the study of micromorphological characteristics were conducted on the 18 collected individuals (see Section 2.2). Morphological data for Cyathophylla and Heterochroa, the closest relatives of the new taxon, were extracted from literature (Schischkin, 1936;Barkoudah, 1962;Davis, 1966;Strid, 1986;Rechinger & Schiman-Czeika, 1988;Madhani et al., 2018).

Biogeography of Shirkuh Mts.
All endemic species of the Iranian Plateau present (also) in Shirkuh Mts. at elevations above 1400 m a.s.l. were recorded, and their distribution patterns in different mountain ranges of Iran (which are well associated with the identified areas of endemism of this region according to Noroozi et al., 2018Noroozi et al., , 2019b were analyzed. These data were extracted from the database of endemic vascular plant species of Iran (Noroozi et al., 2019b). The distribution patterns were illustrated by ArcGIS 10 (Esri, Redlands, CA, USA; Jenness, 2013).

Phylogenetic analyses and taxonomic treatment
The newly obtained sequences are available in GenBank under accession numbers MK637517 and MN381230-MN381234 for ITS and MK651077 and MN417289-MN417292 for rps16. The newly generated ITS sequences did not contain any polymorphic sites. All accessions of the new taxon form a clade (bootstrap support [BS]/posterior probability [PP] of 84/1.00 from ITS; BS/PP of 91/1.00 from rps16; BS/PP of 92/1.00 from the combined dataset; Doc. S2). The new taxon is placed in tribe Caryophylleae with strong support as closely related to Cyathophylla and Heterochroa (BS/PP of 84/0.98 from ITS; BS/PP of 91/1.00 from rps16; BS/PP of 92/1.00 from the combined dataset; Fig. 3). Whether the new taxon is sister to Heterochroa, as inferred by ITS, or to Cyathophylla, as inferred by rps16 and the combined data, remained unclear due to low support values (Fig. 3). Both genera belong to subtribe Caryophyllineae, whose internal relationships were barely known until the recent molecular study of Madhani et al. (2018). This is also the case for Heterochroa and Cyathophylla, which were previously classified under Gypsophila and Saponaria, respectively (see Madhani et al., 2018, for details on their taxonomic history). Heterochroa includes six perennial species distributed in Kazakhstan, Mongolia, N China, and Russia, whereas Cyathophylla comprises two annual species distributed from Greece to Turkmenistan (Madhani et al., 2018). However, several of these species are only poorly known. The new species from Shirkuh Mts. has dark purple stems and calyces covered with long-stemmed glandular hairs, only slightly clawed bicolored and emarginate petals. As it differs morphologically from both Heterochroa and Cyathophylla in habit, coloration, leaf shape and petal shape, and color, and as the phylogenetic position of the new taxon relative to these two genera remains unclear (contradicting, yet insufficiently supported relationships inferred from the two markers: Fig. 3 Diagnosis: This monotypic genus is similar to Cyathophylla and Heterochroa, but it differs from Cyathophylla by non-perfoliate leaves, bicolored petals, and capsules ±enclosed in the calyx, and from Heterochroa in being annual, possessing dark purple stems, having basal spathulate leaves and capsules ±enclosed in the calyx. A comparison among the three genera is provided in Table 1. Note: The generic and the specific names are published here simultaneously via a single diagnosis (descriptio generico-specifica; see Art. 38.5 of the Shenzhen Code: Turland et al., 2018).
Etymology: The generic name refers to the city of Yazd in central Iran, whereas the specific name refers to Shirkuh Mts. in the vicinity of Yazd.
Distribution: It is found only in Shirkuh Mts., immediately below the highest summit (4050 m a.s.l.) on the northern slope. Habitat: This species grows on limestone screes in the high alpine zone. The accompanying species of Yazdana, based on six vegetation plots (each of 5 × 5 m), are presented in Table 2. Although the number of annual species is usually very low in the high alpine zone of Iranian mountains (Noroozi et al., 2008), there are several annual and geographically restricted species in these small scree patches. A similar situation can be found near the summit of Hezar Mt. in Kerman, where recently a new annual Senecio has been discovered .
Conservation status: The new species is known only from the type locality. In the year 2012, only a few specimens were collected from a small plot (5 × 5 m) without particular attention to the population size. In 2019, the location was well explored to find more individuals of the species and to make more detailed observations on its ecology and accompanying species. The species grows only in the northern slope and in a few scree patches from 3950 up to 4050 m a.s.l. The size of the population was estimated to have been between 100 to 200 individuals in this year. Its conservation status is, thus, given as Critically Endangered (CE, i.e., facing an extremely high risk of extinction in the wild) according to IUCN criterion B (geographic range; IUCN, 2012). Generally, alpine and subnival species are under high pressure due to ongoing global warming Pauli et al., 2012). In the absence of higher elevations or alternative habitats for this species to shift up, with ongoing global warming, it is possibly even more strongly threatened.
Identification key: To allow Yazdana to be distinguished from other genera of tribe Caryophylleae, we present a generic key modified from the one provided by Madhani et al. (2018;modifications

Plant biogeography of Shirkuh Mts.
A total of 125 plant species endemic to the Iranian Plateau are recorded from Shirkuh Mts. above 1400 m a.s.l. (Fig. 6A), with 13 of those being local endemics (Fig. 6B). The full species list and their distributions in different mountain ranges are presented in Table 3. Of the 125 species, 95 species are also recorded from Zagros, 70 species from the Kerman massif, 31 species from Alborz, 20 species from the Azerbaijan Plateau, and 14 species from Kopet Dagh-Khorassan (Fig. 6A). Of these 125 endemic species, 9 species are restricted to the Yazd-Kerman area (Fig. 6C), 27 species are distributed in the Shirkuh Mts. and in Zagros (Fig. 6D), 30 species are found in the Yazd-Kerman and Zagros (Fig. 6E), and 11 species are widely distributed in the Iranian Plateau (Fig. 6F). These data show that Shirkuh Mts. are not only a biodiversity hotspot with a rich local endemism, but they are also floristically well connected to the mountain ranges of the Iranian Plateau. This connection is stronger with the geographically close ranges of Zagros and the Kerman massif (Fig. 6A).
Of the 13 species endemic to Shirkuh Mts., two species are distributed mainly between 1400 and 2000 m a.s.l. (Acantholimon horridum Bunge, Plumbaginaceae; Echinops cervicornis Bornm., Asteraceae), three species between 2000 and 2500 m a.s.l.  Pirani & Noroozi, Caryophyllaceae). A. issatissensis was described as a new species (Mahmoodi et al., 2013) from material collected during the same field trip in 2012 when Y. shirkuhensis was collected for the first time.  Yazdana is closely related to Cyathophylla and Heterochroa, and thus belongs to a group within Caryophyllinae, whose internal relationships and thus taxonomy have been poorly understood until now (Madhani et al., 2018). Finding two species new for science (Y. shirkuhensis and Astragalus issatissensis) in a single trip demonstrates that the plant diversity of this area is still poorly explored. Therefore, detailed studies of flora and vegetation of the Shirkuh Mts., especially in high elevations, are highly recommended.
Naturally, the area size decreases sharply from lowlands to high elevations. Whereas plant diversity increases until mid elevation (ca. 2000 m a.s.l.), it decreases gradually until the nival zone (Noroozi et al., 2018). In Shirkuh Mts., however, the number of local endemic species does not decrease with increasing elevation, which could be due to the isolation of higher elevations fostering allopatric speciation. Moreover, of the 125 endemic species of the Iranian Plateau present in Shirkuh Mts., nearly half, that is, 59, species are found at elevations above 3500 m a.s.l. (Table 3). Also, of the 22 species recorded to accompany Yazdana (Table 2), 13 species are restricted to Yazd-Kerman area or Zagros plus Yazd-Kerman area. As Shirkuh Mts. constitute a "priority conservation gap", which means, a center of endemism that  Table 3 for species list and indication of each species' distribution). Continued is currently not within any protected area (Noroozi et al., 2019a), we suggest protecting the area as efficiently as possible to conserve its unique and vulnerable biodiversity.