Turk J Bot (2019) 43: 687-693 © TÜBİTAK doi:10.3906/bot-1901-19 Turkish Journal of Botany http://journals.tubitak.gov.tr/botany/ Research Article A new subspecies of Saxifraga artvinensis V.A.Matthews (Saxifragaceae) from northeastern Anatolia 1, 2 2 Salih TERZİOĞLU *, Kamil COŞKUNÇELEBİ , Murat Erdem GÜZEL Department of Forest Botany, Faculty of Forestry, Karadeniz Technical University, Trabzon, Turkey 2 Department of Biology, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey 1 Received: 18.01.2019 Accepted/Published Online: 18.06.2019 Final Version: 06.09.2019 Abstract: Saxifraga artvinensis V.A.Matthews, which is endemic to Turkey, was discovered on Tiryal Mountain (A8 Artvin, northeastern Turkey). Approximately 175 km from its type locality, a new population of the species was discovered, in the Picea orientalis forest in the Altındere Valley National Park, in A7 Trabzon (northeastern Turkey). A comparison with the known population of S. artvinensis confirmed that this new population represented a new taxon and is described as S. artvinensis subsp. meryemii Terzioğlu & Coşkunç. An identification key and comparison with closely related taxa, based on both morphological and molecular properties, are also provided. The studied samples, belonging to subsp. artvinensis and subsp. meryemii, had identical sequences in the entire internal transcribed spacer region. On the other hand, subsp. meryemii was easily distinguishable from subsp. artvinensis by its hairy flowering stem, 3–8 flowers, subsessile pedicels, and sepals hairy at base. Key words: Biodiversity, endemic, ITS, Saxifraga, taxonomy, Turkey 1. Introduction Saxifraga L., the most species-rich genus of Saxifragaceae, is dominantly distributed in montane to alpine vegetation belts in mountain ranges of the Northern Hemisphere (Soltis, 2007; Tkach et al., 2015). The genus comprises more than 440 species worldwide (Tkach et al., 2015) and 21 species in Turkey (Matthews, 1972; Aslan, 2012; Fırat, 2016). The Caucasus region is recognized as one of the world’s 35 biodiversity hotspots (Marchese, 2015), and the plant biodiversity of this region is facing significant threats (Solomon et al., 2013). The Turkish part of this hotspot holds approximately 3210 vascular plant taxa, of which 465 are endemic to Turkey (Terzioğlu et al., 2015). The mountainous part of the Caucasus, including the northeastern part of Turkey, is among the centers of biodiversity for Saxifraga (Tkach et al., 2015). The Altındere Valley National Park, in northeastern Turkey, is home to many endemic species. This national park was studied floristically by Anşin (1979) for the first time, after which 2 further surveys focused on the subalpine-alpine (Palabaş-Uzun and Anşin, 2006) and forest ecosystems of the valley (Uzun and Terzioğlu, 2008). In the latter study, concentrating on the type location of the present new taxon, 383 vascular taxa were reported, including 16 endemics, and 2 Saxifraga species: S. paniculata Mill. and S. rotundifolia L. During an excursion in the Altındere Valley in April 2013, the authors found dwarf Saxifraga on a damp and shady rock below the Sumela Monastery in the valley (A7 Trabzon). Literature and herbarium surveys revealed that the plants did not belong to any of the taxa recorded from the national park; instead, they represented Saxifraga artvinensis V.A.Matthews, which is endemic to Turkey, and thus only known from the type location (A8 Artvin) (Mathews, 1972). Further comparisons showed that the specimens represented a new subspecies of S. artvinensis. 2. Materials and methods Morphological characters were noted and measured from the authors’ collection from the type locality. To determine the morphological limits of the new and related taxa, additional plant material for morphological and molecular studies was also collected during the field trips (Table 1). All of the morphological measurements were done using at least 3 specimens. An identification key of the new subspecies and related species is therefore given, together with images of both the subspecies and line drawings of useful distinguishing characteristics. Specimens of 2 sub- * Correspondence: sterzi@ktu.edu.tr 687 This work is licensed under a Creative Commons Attribution 4.0 International License. TERZİOĞLU et al. / Turk J Bot species of S. artvinensis were deposited in the Herbarium of the Karadeniz Technical University, Faculty of Forestry (KATO) and Department of Biology (KTUB). The total genomic DNA was extracted from healthy leaves dried with silica gel or the herbarium materials following the modified cetyl trimethylammonium bromide extraction procedure of Doyle and Doyle (1987). Primers internal transcribed spacer ITS4 and ITS5 (White et al., 1990) were used to amplify the nrDNA ITS region. The polymerase chain reaction (PCR) protocol for the amplification of the region followed that of Gültepe et al. (2010). The PCR products were sequenced by Macrogen Inc. (Seoul, Korea) using the ITS4 and ITS5 primers. For the phylogenetic analysis, 9 sequences belonging to Saxifraga were newly generated (Table 1) and 15 more were downloaded from GenBank, comprising 13 of Saxifraga and 2 selected as outgroups (Table 1). Chrysosplenium alternifolium L. and Micranthes nivalis (L.) Small were the chosen outgroup for the phylogenetic reconstruction. All of the sequences (24 accessions) were aligned using Muscle v.3.8.31 (Edgar, 2004) and edited in PhyDE v.0.9971 (Müller et al., 2010). Indels were coded as informative characters according to the Simple Indel Coding method (Simmons and Ochoterena, 2000), as implemented in the program SeqState v.1.40 (Müller, 2005), and added at the end of the sequence data set before being subjected to the maximum parsimony (MP) analysis. The MP analysis was conducted using the parsimony ratchet (Nixon, 1999) with PRAP v.2.0 (Müller, 2004) in combination with PAUP v.4.0b10 (Swofford, 2002). Standard ratchet settings were used (200 ratchet iterations, with 25% of the positions randomly upweighted (weight = 2) during each replicate, and 10 random addition cycles). The generated command files, also including the nexus data matrix, were run in PAUP using the heuristic search option with the following parameters: all of the characters had equal Table 1. Voucher data used in the phylogenetic analysis based on the nrDNA ITS data. Taxa S. adscendens L. ¹Code/²GenBank accession number in the MP tree M. A. Chapman and John M. Burke, Evolution, 2007, 61-7: 1773–1780 ²EF028688 Voucher/reference S. artvinensis subsp. artvinensis Turkey, Coşkunçelebi 1278 (KTUB) ¹C1278/²MN059938 S. artvinensis subsp. artvinensis Turkey, Coşkunçelebi 1279 (KTUB) ¹C1279/²MN059939 S. artvinensis subsp. artvinensis Turkey, Coşkunçelebi 1280 (KTUB) ¹C1280/²MN059940 S. artvinensis subsp. meryemii Turkey, Terzioğlu & Coşkunçelebi 1246 (KTUB) ¹T&C1246/²MN059941 S. bulbifera L. Italy, Fiuminata, Conti (MA) ²AF261166 S. exarata Vill. Austria, Glockturm, Plats D. Gomez 1994 (JACA) ²AJ233861 S. cymbalaria L. S. flagellaris Willd. subsp. stenophylla (Royle) Hulten S. graeca Boiss. & Heldr. Ferguson 1994-04 ²AF087599 Tajikistan, B. Dickore, 18385 (MSB) ²KU524187 Greece, Olimbos, Stauros, Richards (RNG) ²AF261179 S. hederacea L. Italy, Sicily,Trapani ²KU524139 S. hirculus L. Canada, Yukon Territory ²MG237161 S. juniperifolia Adams Turkey, Coşkunçelebi 1284 (KTUB) ¹C1284/²MN059943 S. juniperifolia Adams Turkey, Coşkunçelebi 1244 (KTUB) ¹C1244/²MN059944 S. moschata Wulfen Turkey, Coşkunçelebi 1282 (KTUB) ¹C1282/²MN059942 S. paniculata Mill. Turkey, Coşkunçelebi 1281 (KTUB) ¹C1281/²MN059937 S. rotundifolia L. Turkey, Coşkunçelebi 1283 (KTUB) ¹C1283/²MN059936 S. rotundifolia L. BBG 267-94-80-10 ²AF087598 S. sancta Gris. Greece: M. Roeser 2700 (HAL) ²LN812523 S. sempervivum K.Koch Kew 1984-3743 ²AF087590 S. tridactylites L. M. A. Chapman and John M. Burke, Evolution, 2007, 61-7: 1773–1780 ²EF028687 S. yezhiensis C.Y.Wu China: C.H. Wang, 68389 (HUH) ²KU524203 Micranthes nivalis (L.) Small Denmark, Groenland ²LM654384 Chrysosplenium alternifolium L. Poland, PE: 01505547 688 ²N375572 TERZİOĞLU et al. / Turk J Bot weight, simple addition of sequences, tree bisection and reconnection branching swapping, maxtrees setting to 100 and autoincreased by 100, one nonbinary starting tree arbitrarily dichotomized before branch swapping, and only 1 tree saved. A majority-rule consensus tree was calculated from the most parsimonious trees. Jackknife (JK) support values for the nodes found by the MP analysis were calculated in PAUP by applying the optimal JK parameters according to the method of Farris et al. (1996) with 10,000 JK replicates. 3. Results and discussion Saxifraga artvinensis V.A.Matthews subsp. meryemii Terzioğlu & Coşkunç. subsp. nov. (Figure 1) S. artvinensis subsp. meryemii is easily distinguished from subsp. artvinensis by its pilose flowering stems (not glabrous), flowers 3–8 (not 3–5), pedicels subsessile (not longer than flowers) and sepals hairy at base (not glabrous), and its distribution at altitudes of 1100–1150 m in forest (not in alpine zone higher than 2135 m) (Figures 2 and 3). Type: —Turkey. A7 Trabzon: Altındere Vadisi National Park, around the Sumela Monastery, 1126 m, 31.03.2013, Terzioğlu & Coşkunçelebi 1246 (KATO 9843), holo: KATO; iso: KTUB. Description: —Perennial, caespitose forming cushions up to 10 cm in diameter. Cauline shoots erect, imbricate-leaved, previous years’ dead leaves light brown and remaining in dense rosettes. Leaves lanceolate to linear-lanceolate, glabrous, not lime-encrusted, margin ciliate in lower half only. Flowering stem 1–4 cm long, pilose. Inflorescence racemose with 3–8 flowers, pedicel pubescent, up to 2.5 mm long, shorter than flower. Sepals 2–3.5 mm long, oblong-oval, glabrous, ciliate-margined. Petals 3–5 mm long, linear-spathulate or oblongspathulate, white, shorter than stamens. Fl. 3–4. Damp rocks under oriental spruce forest, 1100–1150 m. Distribution, habitat, and conservation status: —S. artvinensis subsp. meryemii is endemic to Turkey. It belongs to the Euxinian element, and grows on a damp and shady rock in the Picea orientalis (L.) Peterm. forest between 1100 and 1150 m, together with Pachyphragma macrophyllum (Hoffm.) Busch, Geranium robertianum L., Impatiens noli-tangere L., Fragaria vesca L., Saxifraga paniculata Mill., Sanicula europaea L., Telekia speciosa (Schreb.) Baumg., Petasites albus (L.) Gaertn., Cyclamen parviflorum Pobed., Brachypodium sylvaticum (Hudson) P. Beauv. and Festuca drymeja Mert. & Koch. This is very different from the habitat of subsp. artvinensis, which occurs at alpine elevations between 2135 and 2410 m (Figure 4). This new subspecies is a narrow local endemic and is only known from the type locality. There are approximately 175 km between the 2 subspecies of S. artvinensis. Currently, the habitat quality is high, but it is under the negative effects of the religious tourism related to the Sumela Monastery. The entire known population comprises less than 20 individuals, covers a single large rock, and has an area of occupancy smaller than 100 m2. Consequently, the threat category of this new subspecies has been assessed as critically endangered [CR B1ab(ii,iii,iv)+2ab(i,ii)] according to the International Union for Conservation of Nature (2014). Figure 1. Saxifraga artvinensis subsp. meryemii. a- General view (scale bar: 0.5 cm), b- flower (scale bar: 1 mm), c- leaf (scale bar: 1 mm), d- gynoecium (scale bar: 0.5 mm), e- stamen (scale bar: 0.5 mm), f- holotype (Terzioğlu & Coşkunçelebi 1246, KATO 9843). 689 TERZİOĞLU et al. / Turk J Bot Figure 2. Inflorescence and pedicels: a- S. artvinensis subsp. meryemii, b- S. artvinensis subsp. artvinensis, c- S. juniperifolia. Leaves: d- S. artvinensis subsp. artvinensis, e- S.juniperifolia, fS. artvinensis subsp. meryemii. Figure 3. Habitat and habits. a- Saxifraga artvinensis subsp. meryemii, b- S. artvinensis subsp. artvinensis. 690 TERZİOĞLU et al. / Turk J Bot Figure 4. The distribution in northeastern Turkey and habitat of S. artvinensis subsp. meryemii; a- Conifer forest at 1125 m in Trabzon. S. artvinensis subsp. artvinensis; bAlpine rocky slopes at 2410 m in Artvin. Additional specimens examined: —S. artvinensis: A8 Artvin (Çoruh): Tiryal Dağı, above Murgul, 2300 m, shady vertical igneous rocks, 23.06.1957, Davis & Hedge, D. 29957 (Holotype: E photo); Murgul, Tiryal Dağı, 2420 m, damp rocks, 19.06.2018, Terzioğlu & Coşkunçelebi 1278, 1279, 1280 (KTUB). S. juniperifolia Adams A8 Artvin (Çoruh): Şavval Tepe above Murgul, 2800 m, D.32290 (E photo). Trabzon: Çaykara, Uzungöl, Demirkapı Dağı, rocky places, nival zone, 3102 m, 19.09.2013, Coşkunçelebi 1244 (KTUB). A8 Trabzon: Demirkapı Dağı, above Aygır Gölü, 3208 m, cliffs, 20.06.2018, Coşkunçelebi 1284 (KTUB). S. moschata 1781: 128): A8 Trabzon: Demirkapı Dağı, above Aygır Gölü, 3000 m, cliffs, 20.06.2018, Coşkunçelebi 1282 (KTUB). S. paniculata: A8 Artvin: Murgul, Tiryal Dağı, 2306 m, 19.06.2018, Coşkunçelebi 1281 (KTUB). S. rotundifolia: A8 Artvin: Murgul, Tiryal Dağı, 2306 m, 19.06.2018, Coşkunçelebi 1283 (KTUB). S. sancta: B1 Balıkesir: Edremit, Kazdağ, Sarıkız site, limestone, 1600 m, 08.05.2004, F. Satıl 1316 (TTUB). Etymology: —This new subspecies is named in honor of the first author’s mother, who died in 2008. Key to the subspecies of S. artvinensis: —Flowering stem and base of sepal glabrous, pedicels longer than flowers, flowers produced from June–July; alpine plant, at altitudes of 2135–2410 m. subsp. artvinensis —Flowering stem and base of sepal pilose, pedicels as long as or shorter than flowers, flowers produced from March–April; forest plant, at altitudes of 1100–1150 m. subsp. meryemii Relationship and molecular phylogeny: The Saxifraga species distributed in Turkey were not divided into sections by Matthews (1972). However, according to Gornall (1987), S. artvinensis should be classified under the sect. Porphyrion Tausch subsect. Kabschia (Engler) Rouy & Camus. Depending on both the morphological (Table 2) and molecular studies (Figure 5), this new subspecies clearly belongs to S. artvinensis. A comparison of the diagnostic morphological characters of the 2 subspecies of S. artvinensis is given in Table 2. Alignment of the entire nrDNA ITS sequence, including the outgroups, resulted in 731 characters. Identical sequences were observed in the entire ITS regions of samples belonging to subsp. artvinensis (3) and subsp. meryemii (1). In the MP tree, these 4 accessions of S. artvinensis were grouped with a high JK support value (93%) (Figure 5). The MP tree also revealed that S. juniperifolia is sister to S. artvinensis, with maximum JK support (99%) among the studied taxa. Because S. juniperifolia was treated under the sect. Porphyrion by Gornall (1987), our data supported placing S. artvinensis under this section. 691 TERZİOĞLU et al. / Turk J Bot Table 2. Morphological comparison of the subspecies of S. artvinensis and S. juniperifolia. Characters Saxifraga artvinensis subsp. artvinensis S. artvinensis subsp. meryemii S. juniperifolia Flower color White White Yellow Flower number 3–5 3–8 3–10 Sepal Not ciliate Ciliate Ciliate Pedicel Longer than flower (5–15 mm) Shorter than flower (1–2.5 mm) Subsessile Flowering stems Glabrous Pilose Pilose Flowering period June–July Damp cliffs and rock in alpine places, 2135–2410 m March–April Damp shady rock in conifer forest, 1100–1150 m June–August Cliffs and rocks in nival zone, 2700–3300 m Habitat Figure 5. MP tree obtained using PAUP* v.4.0b10. Numbers above the branch are JK support values, in percentages, based on 1000 replicates. Acknowledgments We want to express our special thanks to the Turkish Ministry of Forest and Water Affairs and The Scientific and Technological Research Council of Turkey (TÜBİTAK, project No: 113B201) for their financial support. 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