Pilosella bauhinii (Schult.) Arv.-Touv. and P. cymosa subsp. vaillantii (Tausch) S. Bräut. & Greuter (Asteraceae) from new localities in north-central Poland

We provide information about two species, Pilosella bauhinii and P. cymosa subsp. vaillantii (Asteraceae), from new localities in north-central Poland (ATPOL DC18). All individuals of both species grew on a railway embankment in Górzno-Lidzbark Landscape Park. The ecological preferences for P. cymosa and P. bauhinii estimated according to average Ellenberg values nearly coincided with the original description of Ellenberg. We also sequenced the barcode marker, plastid trnH-psbA intergenic spacer, for two individuals of P. bauhinii, three specimens of P. cymosa subsp. vaillantii, and additionally two individuals of P. officinarum L. and one H. murorum L. growing in close proximity. A pairwise comparison of trnH-psbA sequences showed that each species has a unique haplotype. Taking into account their morphological coherence, it is possible that both P. bauhinii and P. cymosa have not hybridized yet, at least locally, with the more abundant species (e.g., P. officinarum). A search for reference sequences did not provide additional information because of the low quality of the reference database for this group in GenBank. Only 14 sequences of trnH-psbA were available with some apparently being misidentified or of low quality. None were identical to sequences of P. cymosa subsp. vaillantii and P. bauhinii found in this study.


Introduction
The genus Pilosella Vaill.[formerly Hieracium L. subgenus Pilosella (Hill) S.F.Gray] is considered one of the most complicated genera in European flora [1].This is due to hybridization, polyploidization and a complicated reproductive system including apomixis [1,2].Plants from this genus prefer open habitats such as grassland, rocky outcrops and heathlands.Many species, moreover, successfully colonize man-made habitats, for example, railways and roadsides [3].Especially interesting is their occurrence on railways, because these linear landscape elements are considered as migration avenues for many alien as well as rare and endangered species [4][5][6].In Poland, species of Pilosella are often locally rare, and some are considered endangered [7][8][9][10].It is therefore important to document their occurrence to estimate local richness and distribution.
The correct identification of plant material is crucial as mistakes confound interpretation of the biogeographical pattern and preclude correct estimation of local species richness.One useful technique complementary to traditional morphological determination is plant barcoding [11].However, in difficult taxa such as Pilosella, hybridization, polyploidy and speciation via breeding system transition can impede species discrimination.Nonetheless, documenting genetic variation in isolated populations using rapidly evolving markers such as plastid trnH-psbA intergenic spacer [11] can provide information about their origin and putative hybridization when compared to a reference database.A robust database with an extensive taxonomic coverage may also facilitate the identification process providing information on which haplotype should be expected in collected specimens if morphological determination is correct.
In this paper, we provide information about two rare species from the genus Pilosella, P. cymosa subsp.vaillantii (L.) F.W. Schultz & Sch.Bip. and P. bauhinii (Schult.)Arv.-Touv.from new localities in north-central Poland.The population of the former species was discovered in 2003 and of the latter in 2010 during floristic work at Górzno-Lidzbark Landscape Park (ATPOL DC18).We briefly characterize habitat properties and provide trnH-psbA sequences for the discovered species and two other species from the genus Pilosella and Hieracium growing in close proximity to reveal putative hybridization and complement morphological identification.

Material and methods
The Pilosella specimens were initially determined by the first and third author as well as by Dr. Tomasz Załuski (Nicolaus Copernicus University, Poland) as P. cymosa and P. bauhinii.The final determination was performed by Prof. Zbigniew Szeląg (Jagiellonian University) in the case of P. bauhinii and Dr. Franz Schuhwerk (Botanische Staatssammlung München) in the case of P. cymosa ssp.vaillantii.All plant material was deposited in the TRN herbarium (Fig. S1) and high-resolution scans of specimens are available from the first author on request.
We estimated average Ellenberg values for each stand to characterize the ecological requirements of studied species.
Fresh leaves for molecular analyses were sampled from two specimens of Pilosella bauhinii, three specimens of P. cymosa, two individuals of P. officinarum F. Schultz & Schultz-Bip.and one H. murorum L. growing nearby.Total genomic DNA was isolated from ca. 50-100 mg of fresh leaf tissue using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA).The entire intergenic spacer trnH-psbA was amplified using primers psbAF and trnH2 [12].PCR reactions and thermal cycling was set up according to Fazekas et al. [12].The PCR products were sequenced using forward and reverse primers with the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and resolved using a DNA sequencer.All newly obtained sequences were deposited in GenBank (accession numbers: KP890072-KP890079).
BLAST searches were performed against the GenBank database at NCBI for each new DNA sequence to determine its similarity with deposited sequences.

Results and discussion
All individuals of both species grew on the railway embankment approximately 150 m from one another.The railway was surrounded by deciduous forest, wet meadows and willow thickets.Railway embankments are often considered as good migration corridors for thermophilic plants [4] such as members of the genus Pilosella.We observed dozens of individuals of P. cymosa, but only several specimens of P. bauhinii.According to Ellenberg values, individuals of both species occurred in sunlit, dry, warm and more or less infertile and weakly acidic sites in the study area (Tab.1).This characteristic nearly coincided with the original description of ecological preferences for P. cymosa and P. bauhinii [13] (Tab.1).
Both species of Pilosella considered in this study are rare in Poland.Pilosella bauhinii was recorded mainly in the north, south and west of Poland.The newly described stand occur close to three previously reported localities -two historical ones recorded by Abromeit et al. [14] in the vicinity of Olsztyn and Brodnica and a third found near Kwidzyn by Rutkowski [15].According to the literature, this species prefers dry and open places including grasslands, ruderal and pioneering communities [13,16].Similar preferences were encountered in our study (Tab.1).
Pilosella cymosa is rarer than P. bauhinii and has been found only in a few localities in Poland, mainly in Dolny Śląsk [17].It is therefore listed as an endangered species in Poland [10], and included in local red lists for several regions such as Kujawsko-Pomorskie [9], Wielkopolska [8] and Pomorze Gdańskie [7].Our new locality in Górzno-Lidzbark Landscape Park is isolated, although several occurrences of this species in the region were recorded in the 19th century [14,18].Kępczyński and Rutkowski [19], moreover, observed this species near Kwidzyn (ATPOL DB52).However, there is no known record of subsp.vaillantii in Poland [18].
The length of the trnH-psbA sequences varied from 393 bp (H.murorum) to 426 bp (P.bauhinii).All sampled individuals within P. bauhinii, P. cymosa subsp.vaillantii and H. murorum had the same trnH-psbA haplotype.In contrast, no identical haplotypes were identified among species (Tab.2).Hence, our sample included separated entities at both molecular and morphological levels.It is possible that both species have not hybridized yet or served only as a pollen donor for more abundant species (e.g., P. officinarum).Search for reference sequences did not provide additional information because of the low quality of the reference The average Ellenberg values for phytosociological relevés for light (L), temperature (T), moisture (M), soil reaction (R) and nitrogen content (N).The original Ellenberg values for Pilosella bauhinii and P. cymosa s. l. are also provided.databasefor genera Pilosella and Hieracium in GenBank.There were only 14 sequences of trnH-psbA and no identical matches to haplotypes of P. bauhinii and P. cymosa subsp.vaillantii.Moreover, a detailed examination of GenBank resources revealed that the sequences were in some cases misidentified (e.g., H. murorum FR865084 likely confused with Picris L.) or were of low quality (e.g., P. officinarum Tab. 2 A pairwise comparison of trnH-psbA sequences for Pilosella and Hieracium species sampled in the study.Number of nucleotide differences, number and length of indels and poly(A) motifs are provided.P.b. -Pilosella bauhinii; P.c.-P.cymosa subsp.vaillantii; P.o.-P.officinarum; H.m. -Hieracium murorum.