A new subspecies of Saxifraga artvinensis V.A.Matthews (Saxifragaceae) from northeastern Anatolia

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.

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 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. 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.

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 m 2 . Consequently, the threat category of this new subspecies has been assessed as critically endangered [CR B1ab(ii,iii,iv)    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.

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. We would also like to thank Dr. Richard Milne (University of Edinburgh, School of Biological Sciences) for his critical reading of the manuscript and Dr. Fatih Satıl from Balıkesir University who suplied materials belonging to Saxifraga sancta.