82 ACTA BOT. CROAT. 80 (1), 2021 Acta Bot. Croat. 80 (1), 82–90, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2021-008 ISSN 0365-0588 eISSN 1847-8476 Crocus adamioides (Iridaceae) in the Bulgarian flora Tsvetanka Raycheva¹, Kiril Stoyanov¹*, Samir Naimov2, Elena Apostolova-Kuzova2 1 Agricultural University – Plovdiv, Department of Botany and Agrometeorology, Plovdiv, Bulgaria 2 University of Plovdiv, Department of Plant Physiology and Molecular Biology, Plovdiv, Bulgaria Abstract – Crocus adamioides Kernd. et Pasche, as it is currently known, was originally treated as C. biflorus Mill. subsp. adamii B. Mathew in the flora of Bulgaria by Mathew (1982) and verified by Rukšāns (2017). The taxon was af- terwards described as a separate species (Kerndorff et al. 2012), the holotype being collected in Kırklareli Province, European Turkey. The species was for the first time mapped in two floristic regions of Bulgaria. The diagnostic charac- ters based on the general morphology and leaf anatomy were defined from the natural populations of the species and compared to the type specimen and relevant data from the literature. The phylogenetic position of the species was clarified by sequencing of the internal transcribed spacer region (ITS: ITS1 + 5.SsrDNA + ITS2) and comparison of the obtained sequence with those annotated in NCBI. A phylogenetic tree was built using Bayesian phylogeny. Results have shown the highest degree of phylogenetic similarity with C. adamioides from Turkey. The closest relative C. ranjeloviciorum Kernd., Pasche, Harpke et Raca remains in the proximity. Our morphological, anatomical and mo- lecular analyses have revealed that the Bulgarian population shows a peculiar combination of characters specific to C. adamioides. Keywords: anatomy, Bulgaria, chorology, Crocus, ITS region, morphology Introduction The genus Crocus currently consists of about 230 taxa according to Rukšāns, (2017). It is distributed from Western Europe and Northwestern Africa to Western China, with the centre of the species diversity being on the Balkan Pen- insula and west Turkey (Mathew 1982, Ranđelović et al. 2012, Harpke et al. 2014, 2016, Rukšāns 2017). As a result of this pattern Turkey represents an especially rich territory in terms of Crocus species, currently comprising approximate- ly 140 taxa (Yüzbaşıoğlu 2019, Çiftçi et al. 2020). The num- ber of endemic species recorded in these areas is increasing continuously, and to date there are approximately 40 Balkan endemic species (Miljković et al. 2016, Spirios et al. 2019). Most of the newly described species do not have a clear in- frageneric position in the existing system of classification. Moreover, the conception about the volume of the polymor- phic groups in the genus is complicated and debatable. One of the rather heterogeneous groups is Crocus biflorus s.l., belonging to C. sect. Nudiscapus B. Mathew, ser. Biflori B. Mathew. Recent phylogenetic analyses have proved several units of this series to be para- or polyphyletic, causing sub- sequent taxonomic problems (Petersen et al. 2008, Harpke et al. 2013). A DNA-based investigation showed that sever- al subspecies of C. biflorus s.l. according to Mathew (1982) have been ranked as species instead of subspecies (Harpke et al. 2016). According to recent Bulgarian floristic literature, the ge- nus Crocus is represented by 9 species, growing wild (As- syov and Petrova 2012). The genus Crocus was studied taxonomically more than 50 years ago (Velchev 1964). Tra- ditionally, all Bulgarian taxa with white or lilac flowers, with three to five striking purple longitudinal stripes on the out- er tepals have been treated under the name C. biflorus (Vele- novský 1898). In the 3rd edition of Flora of Bulgaria (Sto- janov and Stefanov 1948) and Flora of the P.R. of Bulgaria (Velchev 1964), the variability of C. biflorus is represented as a single taxon – var. violaceus Boiss. This taxon is is con- sidered synonymous with C. biflorus subsp. adamii (J.Gay) Mathew, with distributional area covering the territory be- tween Bulgaria and Iran (Mathew 1982). In the 4th edition * Corresponding author e-mail: nomtax@gmail.com CROCUS ADAMIOIDES IN BULGARIAN FLORA ACTA BOT. CROAT. 80 (1), 2021 83 Thracian Lowland 35TLG86, Protected locality “Propadnaloto blato”, near the village of Golyamo Asenovo, N42.12545 E25.64678, 145 m. 2019-01-29, 2019-02-02 (coll. V. Trifonov and K. Stoya- nov) SOA 062524, 062525. Toundja hilly plain (Sakar Mountain, a new record for the country) 35TMG43, Oak forest near Dervishka Mogila, N41.9073056 E26.3345, 404 m, 2019-02-02 (coll. Ts. Ray- cheva and K. Stoyanov) SOA 062626. Oak forest, near the village of Moustrak, N42.0811944 E26.356, 322 m, (coll. Ts. Raycheva and K. Stoyanov) SOA 062627. 35TMG44, Oak forest between the villages of Planinets and Oustrem, N41.9756111 E26.3885, 303 m, 2019-02-02 (coll. Ts. Raycheva and K. Stoyanov) SOA 062625* (se- quence MN955426). 35TMG45, Dry shrubs with Quercus sp. div. and Pali- urus near the town of Topolovgrad, N42.0811944 E26.356, 322 m, 2019-02-02 (coll. Ts. Raycheva and K. Stoyanov) SOA 062624. 35TMG54 . Village of Stoudena, N41.9166667 E26.4013889, 295 m, 2019-02-21 (coll. V. Trifonov) SOA 062628. Morphological investigations The collected fresh plants of C. adamioides were photo- graphed in detail. The morphological measurements (Tab. 1) were made on both fresh and dried samples from three populations (35TMG43, 35TMG44, 35TMG54), 30 individ- uals from each population. The morphological measurements, after statistical anal- ysis (mean, standard deviation, minimum and maximum), were compared with those of C. randjeloviciorum Kernd., Pasche, Harpke et Raca as a rather similar and closely re- lated species (Harpke et al. 2017). Anatomical investigations Developed leaves from flowering plants were used in the anatomical study. In order to examine the leaf anatomy, five individuals per population, from 3 populations in total (35TLG86, 35TLG86, 35TMG44) were collected and con- served in 75% ethanol. Five transverse cross sections and epidermal areas of leaves from each individual were made manually from the middle part. The microscope slides were prepared in glycerine jelly. Photographs of the microscope slides were taken using a Carl Zeiss Amplival microscope with an external attached ocular camera, calibrated using a micrometric slide. The measurements of the characters were done using Micam 2.4 software (Van Westen 2018). Nineteen anatomical features, in the 30 leaf transverse sections from each population were measured: section width and height, arm length, white stripe, vascular bundle count (magnifica- tion 3.2×), palisade tissue height, height and width of pali- of Flora of Bulgaria (Stojanov et al. 1966), C. biflorus com- prises three varieties: var. biflorus, var. adamii (Gay.) Bak, and var. albus Herb. Subsequently, Kerndorff et al. (2012), on the basis of phylogenetic studies in the ITS region, de- scribed C. adamioides as a new species, which is neither closely allied to C. biflorus nor to its subspecies adamii. The conventional morphological approach is not reliable enough for sufficient differentiation of taxa in polymorphic groups and needs to be combined with the molecular one. Тhe internal transcribed spacer region (ITS: ITS1 + 5.8SrDNA + ITS2) and 5’ external transcribed spacer (ETS) of the nuclear ribosomal DNA (rDNA) have proven to be useful phylogenetic markers in Crocus (Harpke et al. 2014). However, as a multigene family consisting of hundreds of tandemly repeated units in single or multiple clusters in the genome (NOR), the rDNA is subjected to concerted evolution. Processes like unequal crossing-over (Eickbush and Eickbush 2007) and gene conversion (Li et al. 2011) can lead to the homogenization of the different units. Moreover, frequent intra-individual polymorphic rDNA regions are reported for a few cases in Crocus (Harpke et al. 2013, 2014) and are often caused by e.g. hybridization or polyploidy (Petersen et al. 2008). In January 2018, we found a population of plants that was identified as Crocus cf. adamii B. Mathew. The morpho- logical analysis revealed that this population has a combi- nation of specific characters of C. adamioides, instead of C. adamii (Kerndorff et al. 2012). Consequently, we decided to study the Bulgarian population of C. adamioides in detail (including its morphology, anatomy, and molecular data). This paper provides full insight into the chorology and taxonomy of C. adamioides for the flora of Bulgaria. Materials and methods Plant material After extensive field studies, the presence of the species was observed in two floristic regions – the Thracian Low- land and Toundja hilly plain (Mt Sakar). The plant mate- rial was collected in January-February 2019. Data about the habitat and populations of the species were derived from personal observations. On the other hand, the information about the distribution was based on relevant literature sources as well as on the authors’ field observations. The sampling localities are represented on a map (BGMountains Project 2019). The voucher specimens are deposited in the herbarium of the Agricultural University – Plovdiv (SOA). A list of them is given below by floristic regions in Bulgaria, then by quadrant code of MGRS coordinates (10 × 10 km). Each specimen contains information about the description of the habitat and toponym, followed by coordinates (WGS84), altitude, dates of collections, collectors’ names, herbarium code, and voucher numbers. The specimen used for molecular analysis is marked with an asterisk (*). RAYCHEVA T., STOYANOV K., NAIMOV S., APOSTOLOVA-KUZOVA E. 84 ACTA BOT. CROAT. 80 (1), 2021 sade and spongy cells (magnification 10×), and the height and width of the adaxial and abaxial epidermal cells (mag- nification 40×). The length and width of adaxial epidermal cells were measured (magnification 3.2×). Abaxial epider- mal cells were measured in the area of the stomatal rows (between the ribs). The size of stomata (both stomatal cells) was also measured (magnification 10×). The values were processed using basic statistical analysis – mean, standard deviation, maximum and minimum, and compared with the data for C. randjeloviciorum (Harpke et al. 2017). Molecular methods The genomic DNA was isolated using DNeasy Plant Mini Kit (QIAGEN). Briefly, 50 µg of plant material was ground in liquid nitrogen and processed according to the manufacturer’s requirements. DNA concentration and quality were determined spectrophotometrically at a wave- length of 260 nm using Epoch microtiter plate reader and T3 plate protocol. The ITS region (ITS1, 5.8S rDNA, ITS2) was amplified using the following primers: ITS-A (5'-GGAAGGAGAAGTCGTAACAAGG-3') and ITS-B (5'-CTTTTCCTCCGCTTATTGATATG-3'), as described in Tirel et al. (1996). The reaction was carried out in a 50 µL reaction mixture containing: 1x reaction buffer, 200 µM dNTPs, 0.2 µM of each primer, 100 ng plant genomic DNA, and 1 unit of Q5 High Fidelity DNA polymerase (New Eng- land Biolabs). The PCR thermal cycler steps were as follows: initial melting of the reaction mixtures at 94 °C for 45 sec, followed by 30 cycles at 94 °C for 10 sec for denaturation, 10 sec at 62 °C for primer annealing, 30 sec. at 72 °C for primer extension, and a final elongation step of 2 min at 72 °C. Amplified PCR products were separated by 0.8% agarose gel electrophoresis, excised from the gel, and purified using a QIAquick Gel Extraction Kit (QIAGEN). The purified DNA fragments were subsequently sequenced with Microsynth Company (Switzerland) technology. Chromatograms were corrected manually with DNAStar software (Lasergene, USA). The sequence was submitted in NCBI Gene database under accession number MN955426 (voucher SOA 062625). Phylogenic analyses The obtained nucleotide sequence was blasted against nucleotide sequences from the NCBI Nucleotide database (BLAST 2019). The best hits, all deposited by Harpke, were downloaded and used for the phylogenetic analysis. The alignment of the sequences was done using the ClustalW Multiple alignment (Thompson et al. 1994). The phyloge- netic analysis was done using Bayesian phylogenetic infer- ence with MrBayes 3.2 (Ronquist et al. 2012). The parame- ters of the analysis were the same as described by Harpke et al. (2017) – 2 times 4 chains for 2 million generations, nu- clear data set ГТР+G+I, sampling tree per 1000 generations, 2 independent runs. The result was visualized as a tree us- ing TreeGraph 2 (Stöver and Müller 2010). The analysis in- cluded 31 nucleotide sequences, cited as numbers of entries in the phylogenetic tree. Results Crocus adamioides Kernd. et Pasche, Stapfia 97: 11 (2012). Holotypus: Turkey, Kırklareli Province, Yildiz Dağları, 900-1100 m, 26.01.2009, HKEP 0904 (Gatersleben, GAT 7136!) Description (based on Bulgarian materials): Perenni- al synanthous geophyte, 6-18 mm in height (Fig. 1). Corm subglobose 9-18.5 mm in width and 7-19 mm in height; tu- nics coriaceous, inner softer; neck bristly, up to 5 mm long, Tab. 1. Comparison of morphological characters of Crocus adamioides Kernd. et Pasche (both Bulgarian – this study, and the Turkish populations – Kerndorff et al. 2012) and C. ranjeloviciorum Kernd., Pasche, Harpke et Raca (Serbia, Harpke et al. 2017). Characters C. adamioides C. ranjeloviciorum Bulgaria Turkey Serbia Corm height/diameter, mm 9-18.5 10-15 9-11 Edge of the basal rings Clearly toothed, with distance between the teeth 0.5-2.5 mm Clearly toothed Smooth, rarely with tiny teeth Teeth length, mm 0.5-2 up to 1 0-0.5 Leaf indumentum glabrous glabrous, sometimes slightly ciliated papillose Leaf width, mm 0.7-1.6 1-1.5 1.2-2.5 Main color of flower segments Whitish, rarely pale liliac White or liliac Violet-blue, rarely whitish Outer perygone segments (length × width, mm) 13.5-23-31.9 × 3.2-7.1-13.7 15-19-25 × 6-8.7-13 21-25-30 × 6-9.5-14 Inner perygone segments (length × width, mm) 13.7-21.7-33.9 × 2.4-7.5-12.5 14-18-24 18-23-28 × 6-10.2-14 Anthers – length, mm 5.6-9-14 6-8.8-12 7-11.5-15.2 Filaments glabrous glabrous hairy CROCUS ADAMIOIDES IN BULGARIAN FLORA ACTA BOT. CROAT. 80 (1), 2021 85 split into broad segments; ring present, basal detachable, with small teeth 0.5-1.5(-2) mm long, evenly spaced (Fig. 2C). The tunic teeth details depend on age. In young corms (3-4 years old), the teeth of the basal rings are sparsely situ- ated, 1-1.3 mm long and the distance between them is about 2.5 mm. In mature corms (9-10 years old), the teeth are 1.5- 1.7(-2) mm long, with the distance between them about 1.5 mm. Cataphylls silvery-whitish to yellowish. Leaves (2-)3-4 during flowering time, dark green. Bract 1.5-2 mm wide, skinny, yellowish. Bracteole 3-4 mm wide, membranous, transparent. Flowers 1-2(-3). Perigone segments white to pale blue or lilac, usually with acute apical regions. Outer perigone segments 13-32 mm long and 3-14 mm wide, with 3 violet-lilac stripes on the outside. Inner perigone segments 13.5-34 mm long and 4.5-12.5 mm wide, without stripes, but most of them with a distinct blotch on the outside. Peri- gone tube yellow, smooth, 20-50 mm long. Anthers 5-14 mm long, yellow, divided by a distinct white connective. Stigma trilobate, stylodia 2-6 mm, equal, or longer com- pared to the stamens (Fig. 2A, B). Habitat and distribution In Bulgaria, the species occurs in the Thracian Lowland and on Sakar Mountain (the floristic region of Toundja hilly plain), on hilly terrain at an altitude between 140 and 410 m a.s.l. (Fig. 3, see also the description in Materials and meth- ods) The flowering period of the collected samples starts at the end of January and lasts until the end of February. The accompanying species are: Quercus cerris L., Q. robur subsp. pedunculiflora (K.Koch) Menitsky, Paliurus spina-christi Mill., Fagus sylvatica L., Viola odorata L., Dipsacus fullonum L., Centaurea salonitana Vis., Teucrium capitatum L., San- guisorba minor Scop., Euphorbia amygdaloides L., Iris sin- Fig. 1. Crocus adamioides Kernd. et Pasche – specimen SOA 062626 near the village of Moustrak, Toundja hilly plain (Sakar Mountain), southeastern Bulgaria. Fig. 2. Crocus adamioides, specimen SOA 062626 near village of Moustrak, Toundja hilly plain (Sakar Mountain), southeastern Bulgaria. A, B – flower; C – corm. Fig. 3. Distribution of Crocus adamioides: numbered – new re- cords in Bulgaria. HKEP 0904 – type locality in Turkey, R1 – data provided by J. Rukšāns (personal communication). RAYCHEVA T., STOYANOV K., NAIMOV S., APOSTOLOVA-KUZOVA E. 86 ACTA BOT. CROAT. 80 (1), 2021 tenisii Janka, Fragaria viridis Weston, Verbascum phoeni- ceum L., Clematis viticella L., Asparagus officinalis L., A. verticillatus L., Teucrium chamaedrys L., etc. Leaf anatomy The cross-section of the leaf is typical of the genus Cro- cus with a central square or rectangular “keel” and two lat- eral “arms” (Fig. 4A). Each arm has 2 well-defined ribs. The cross-section width is 1053-1931 µm and height 216-610 µm. Large, thin-walled parenchymal cells are oriented in the central part of the keel, forming a lacuna area, visible as a longitudinal white strip on the top of the leaf. The total of 9 – 12 – 15 collateral vascular bundles are positioned in one row on the abaxial leaf side along the leaf mesophyll; the 6-10 located on the lateral arms and the 4-5 in the keel. Four of these bundles are larger than the other, with well-developed sclerenchyma tissue like a “cap”. The biggest pair (width 92-155 µm and height 68-127 µm) of the vascular bundles is located at the ends of the arms (Fig 4B). Another pair of big vascular bundles (width 78-158 µm and height 66-135 µm) is positioned in the corners of the keel. The xylem is oriented towards the adaxial side with the phlo- em beneath. The epidermal cells of both surfaces have thick- ened external cell walls, a thick cuticular layer and uniform micropapillary relief (Fig. 4C). The assimilating parenchyma has a distinct palisade layer of 1-2 rows of cells. The spongy parenchyma is located in the zones around the stomata (Fig. 4D) between the ribs of the vascular bundles. The measured anatomical characters are listed in Tab. 2 and compared with those of C. randjeloviciorum (Harpke et al. 2017). The epidermal cells are elongated, rectangular to ellipti- cal, with straight anticlinal walls (Fig. 5). The cells of the adaxial epidermis (Fig. 5A) are 264-785 μm long and 10-39 µm wide, distinctly longer than those of the abaxial epider- mis. The abaxial epidermis (Fig. 5B) is represented by basic cells (length 44-111 μm and width 15-29 μm) and stomata. The stomata are anomocytic, Amaryllis – type (length 23- 34 µm and width 15-26 µm), situated only on the lower sur- face of the leaf, in the zone of the leaf arms and in the lat- eral zone of the keel, between the nerves. ITS sequences The BLAST analysis of the obtained ITS sequence com- pared with the Nucleotide data from NCBI displayed simi- larity (99.34%) with the sequences LT222361 (Crocus adamioides, Harpke et al. 2016, GAT7136, holotypus) and Fig. 4. Crocus adamioides from Toundja hilly plain (voucher SOA 062625): A – leaf cross sections (3.2×), B – arm detail (10×), C – ad- axial epidermis (40×), D – abaxial epidermis (40×). Abbreviations: ad – adaxial side, ab – abaxial side, la – lacuna area, e – epidermis, pp – palisade parenchyma, sp – spongy parenchyma, sc – sclerenchyma cap, ph – phloem, xy – xylem). CROCUS ADAMIOIDES IN BULGARIAN FLORA ACTA BOT. CROAT. 80 (1), 2021 87 HE664018 (C. biflorus subsp. pulchricolor, Harpke et al. 2013), followed byMF766260 (99.18%, C. randjeloviciorum, Harpke et al. 2017). The region 5.8s rRNA was identical in the whole genus and this fact allows use of the neutral evo- lutionary model in the phylogenetic analysis of the whole ITS1-5.8SrRNA-ITS2 sequence. The resulting tree looks similar to that published by Harpke et al. (2017). The Bul- garian specimen was placed in a clade together with the specimens designated as C. adamioides and C. pulchricolor, in proximity with the clade containing C. randjeloviciorum. The specimens of C. biflorus subsp. adamii s.str. remain as an outgroup (Fig. 6). Tab. 2. Leaf anatomy character measurements (range and mean ± standard deviation (SD)) of Bulgarian collections of Crocus adamioides Kernd. et Pasche, compared to the data of C. randjeloviiciorum Kernd., Pasche, Harpke et Raca (Harpke et al. 2017). Crocus adamioides Crocus randjeloviciorum Range Mean ± SD Range Mean ± SD Section width, µm 1053 – 1931 1555 ± 308 2644 – 3668 3154 ± 311 Section height, µm 216 – 610 399 ± 103 446 – 692 563 ± 75 Arm width, µm 358 – 681 530 ± 105 1164 – 1726 1393 ± 170 Vascular bundles, count 9 – 17 12 ± 3 11 – 21 18 ± 3 Palisade tissue – height, μm 25 – 84 51 ± 13 51 – 90 68 ± 11 Spongy tissue – height, μm 22 – 63 42 ± 9 37 – 68 50 ± 8 White stripe, µm 224 – 291 275 ± 24 323 – 599 434 ± 91 Adaxial epidermal cell – length, µm 264 – 785 531 ± 131 . . Adaxial epidermal cell – height, µm 16.5 – 30.5 22 ± 3.2 14 – 19 16 ± 2 Adaxial epidermal cell – width, µm 7.7 – 18.8 12.6 ± 2.3 13 – 18 16 ± 1 Palisade cell – height, µm 24.4 – 42.8 35 ± 4.7 34 – 61 46 ± 6 Palisade cell – width, µm 10.3 – 16.9 12.8 ± 1.6 13 – 19 17 ± 1 Spongy cell – height, µm 8 – 23.7 14.5 ± 3.7 14 – 22 18 ± 2 Spongy cell – width, µm 12 – 25.3 19.2 ± 4.2 21 – 33 26 ± 3 Abaxial epidermal cell – height, µm 7 – 18 12.1 ± 2.9 12 – 20 16 ± 2 Abaxial epidermal cell – width, µm 7 – 21 14.8 ± 3.5 13 – 22 18 ± 2 Abaxial epidermis cell – length, µm 44 – 111 76 ± 19 . . Stomata – length, µm 24 – 34 28 ± 3 . . Stomata – width, µm 15 – 26 21 ± 2 . . Fig. 5. Crocus adamioides from Toundja hilly plain (voucher SOA 062625). Leaf epidermis: A – adaxaial epidermis (10×), B – abaxial epidermis (10×). RAYCHEVA T., STOYANOV K., NAIMOV S., APOSTOLOVA-KUZOVA E. 88 ACTA BOT. CROAT. 80 (1), 2021 Discussion The genus of Crocus consists of critical taxa, and their discussion remains open. After the monograph of Mathew (1982), the status of the infraspecific taxa in the polymor- phic groups was reassigned as species (Mathew et al. 2009). The first report about C. adamioides was from Turkey, the mountain Yıldız Dağları in the province of Kırlkareli, at an altitude between 900 and 1100 m a.s.l. (Kerndorff et al. 2012). Later, Rukšāns confirmed the species with a locality near Kofçaz – a place very close to the border with Bulgaria. He believed that the mountain ridge where it was found stretches further into adjacent Bulgaria, and C. ada- mioides can be found there, too (Rukšāns 2017). Along a straight line, Bulgarian localities of C. adami- oides are found 90 km northwest of the locus classicus. Not only the distance but also the lack of high mountains func- tioning as a dividing system are the reasons for the uninter- rupted distribution range of the species on Bulgarian territory, outlining the presence of C. adamioides in the southern part of the Balkan Peninsula. This plant is not un- common in the investigated Bulgarian localities, where most of the populations have numerous individuals. Nev- ertheless, we did not find specimens of this taxon deposited in the national herbaria in Bulgaria, or as a species men- tioned in the Bulgarian floristic literature. However, in a study of C. adamioides, Rukšāns (2017) commented on his observations on the morphology of this species, based on material from Bulgaria. It is plausible that the species in Bul- garia had been previously unnoticed due to its inconspicu- ous habit. We suppose that it is due to the ephemeral flow- ering period, that the species has remained unnoticed until now. The flowering period of the collected samples starts at the end of January and lasts until the end of February, sim- ilarly to the samples described from Kırklareli province. Moreover, when the weather is cold or cloudy, the flowers remain closed and almost invisible. The described facts sug- gest the high probability that the area of distribution is wid- er than represented here, occurring in similar habitats in other localities in the country. Crocus adamioides has morphological similarities with C. randjeloviciorum. The main morphological differences between the two species are listed in Tab. 1. One of the clearly defined morphological features that distinguish C. adamioides from the closely related species C. randjelovi- ciorum is the distinct separation of the teeth on the basal rings of the corm. The teeth of C. adamioides are much lon- ger (0.5-1.5-2 mm) and more evenly spaced than those of C. randjeloviciorum, which are tiny (less than 0.5 mm) and rarely present. The flower segments of the specimens from Bulgaria have similar dimensions to those from Turkey, while the flower size of C. randjeloviciorum has greater val- ues. Another clearly distinctive morphological character is the indumentum of the filaments – hairy in C. randjelovi- ciorum, and glabrous in C. adamioides. The investigated plants have glabrous filaments. The anatomical cross-section of the leaf is a specific tax- onomical marker in the genus Crocus. It is a useful character for the determination of the species, especially in the lack of discretion between the morphological criteria. The anatom- ical sections of the studied plants are similar to those of C. randjeloviciorum. The quantitative morphological charac- ters of the leaves show higher values in C. randjeloviciorum than those from the Bulgarian populations (Tab. 2). The keel is not squared but slightly curved. The arms are at least twice as short as those of C. randjeloviciorum. The white stripe takes up 15-21% of the leaf cross section, while the white stripe of the leaves of C. randjeloviciorum takes 12-16% of the cross section. There are few vascular bundles than in C. randjeloviciorum. The adaxial epdermis is thicker than that of C. randjeloviciorum. The biggest vascular bundles in the sections of the collected material of C. adamioides from Bul- garia are terminal in the arms (Fig. 4 B). In comparison, the sections of C. randjeloviciorum have a terminal smaller bun- dle. Papillae are not presented but the cuticles all adaxial epidermal cells are slightly as elongated like micropapillae (Fig. 4 C), not longer than the height of the epidermal cells. Because of the limited distribution of the taxon, data on the anatomical quantitative and qualitative characteristics of the Fig. 6. Phylogenetic tree obtained by Bayesian phylogenetic infer- ence of the nuclear rDNA ITS regions using the methodology of Harpke et al. (2017). A – clade of Crocus adamioides, R – clade of C. randjeloviciorum, O – clade of C. biflorus subsp. аdamii s.str. Posterior probabilities designated by numbers. CROCUS ADAMIOIDES IN BULGARIAN FLORA ACTA BOT. CROAT. 80 (1), 2021 89 leaf in C. adamioides are not reported in the literature. We believe that they will be useful in future studies of other closely related species in the Nudiscapus section. Genetic distances in the genus Crocus frequently cor- relate with geographic distances (Kerndorff et al. 2017). This suggests that a combination of polyploidy and geographical speciation may have driven cladogenesis in the group. Despite the large number of species identified in the last decade, supported solely by ITS sequences, intra- and inter- population variability of these species have not been investigated, making it difficult to comment on our results in the general model of the Nudiscapus polymorphic group. The results show a relationship between the polymorphism and the genetic diversity of the C. biflorus group. The sam- ples of C. adamioides remain in one node with C. randjelovi- ciorum but in distinct clade (Fig. 6). The phylogenetic anal- ysis based on ITS sequences confirms that the investigated population belongs to C. adamioides. The represented analyses confirm the presence of C. ad- amioides in Bulgaria. On the other hand, the presence of the taxa belonging to the putative C. biflorus group, which was considered widespread in the Balkans – from Greece in the south to Rhodes, Turkey (Rukšāns 2017) and Bulgaria (Velchev 1964) – remains questionable. Even though taxo- nomic revisions have led to the description of the new species in the neighbouring Balkan regions (Kerndorff et al. 2013, Harpke et al. 2017), the taxonomic structure of the taxa in Bulgaria is still unresolved, since no nomenclatural or taxonomic revisions of the species composition of the genus have been carried out. Future research should be fo- cused on taxa from the C. biflorus s.l. group with unclear phylogenetic relationships and with no clearly distinguish- ing morpho-anatomical features. Revision of all specimens deposited as C. biflorus in many collections in Bulgaria is necessary. In order to disentangle the complex taxonomic status and relationships, the traditional morphological ap- proach should be combined with molecular studies. Such an analysis would confirm the hypothesis that it is an aggregate of species with a severe morphological syndrome, possibly due to ongoing hybridization, species formation processes, and recent divergence. Acknowledgements The authors would like to thank Dr. Pistrick, curator at IPK Gatersleben Herbarium, for the kindly given scanned copies of the holotype herbarium specimen of C. adamioi- des. Also, many thanks to Dr. Rukšāns for his kindly sent mapped data of Crocus adamioides, and to the anonymous reviewers for their useful comments that helped to improve the manuscript. 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