untitled ACTA BOT. CROAT. 75 (1), 2016 81 Acta Bot. Croat. 75 (1), 81–88, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0020 ISSN 0365-0588 eISSN 1847-8476 Morphological variability of the Bulgarian endemic Betonica bulgarica Degen et Neič. (Lamiaceae) from Sinite Kamani Natural Park, Eastern Balkan Range Neli H. Grozeva1*, Mariya A. Gerdzhikova2, Dimitar H. Pavlov2, Galia D. Panayotova2, Mima H. Todorova2 1 Trakia University, Faculty of Agriculture, Department of Biology and Aquaculture, Stara Zagora, Bulgaria 2 Trakia University, Faculty of Agriculture, Department of Plant Growing, Stara Zagora, Bulgaria Abstract – Four populations of Betonica bulgarica Degen et Neič. at Sinite Kamani Natural Park were mor- phologically tested. Intrapopulation and interpopulation variabilities were established. The rеlationship be- tween morphological variability, number, area and ecological appurtenance of the studied populations were explored. The results demonstrated that the main source of phenotype variation is intrapopulation variability, mainly due to the age structure of populations. The most variable traits are height of stem and dimensions of leaves. The registered interpopulation variability was affected by the differences in altitude, soil type and dif- ferences in environmental conditions and soil properties. Indumentum and morphology of generative organs had taxonomic signifi cance for distinguishing B. bulgarica from the other species in the genus, including the species that were morphologically most similar to it – Betonica offi cinalis L. Keywords: Betonica bulgarica, morphology populations, variations * Corresponding author, e-mail: grozeva@uni-sz.bg Introduction Endemic plants are an emblematic symbol of the Bul- garian fl ora and one of the most sensitive and vulnerable units in the country’s nature ecosystems (Anchev 2011). They comprise 12.8% of the members of the fl ora of Bul- garia and demonstrate the specifi city and genetic features of its fl ora (Petrova et al. 2005). The Bulgarian endemic Bet- onica bulgarica Degen et Neič. (syn. Stachys bulgarica Hayek), described by the Hungarian botanist A. V. Degen and the Bulgarian botanist I. Neichev in 1906 from materi- als collected in the Central Balkan Range, grows on open, stony and sandy ground in the oak and beech forest belts in the Balkan Range (Central, Eastern) and the Thracian Low- land (Velchev et al. 1992, Petrova 2006, Genova 2011). The species is morphologically similar to Betonica offi cinalis L. (syn. Stachys offi cinalis (L.) Trevis.). Although B. bulgari- ca is a protected species by the Biological Diversity Act of Bulgaria (Anonymous 2002) and most of its populations are found in protected territories – the Central Balkan Na- tional Park, Sinite Kamani Natural Park and in the protect- ed areas of Natura 2000 – recent expert evaluations defi ne it as endangered (Genova 2011). To this day, the species has not been subjected to detailed morphological studies. The polyphenol content in roots and above-ground parts has been studied (Bankova et al. 1999). Data about the state of B. bulgarica populations on the territory of Balkan Range are contained in the management plans of the Central Bal- kan National Park (Yankov et al. 2001) and Sinite Kamani Natural Park (Grozeva et al. 2004). B. bulgarica was fi rst registered in the Eastern Balkan Range by Neichev (1906) but materials were not deposited in Bulgarian scientifi c herbaria (SOM, SO, SOA). Nowa- days the species locality specifi ed by Neichev is part of the territory of the Sinite Kamani Natural Park. Grozeva et al. (2004) registered its population in the Ablanovo area of the park. Currently, the species populations have been regis- tered in three other areas of the park territory – near the Microyazovir in Karandila area, on Slancheva Polyana and near Upper Lift Station (Grozeva et al. 2014). Their state was assessed and their requirements as to soil fertility were studied (Grozeva et al. 2014). The objective of the present study was to establish the morphological population variability of B. bulgarica on the territory of Sinite Kamani Natural Park, to identify the traits that have the greatest taxonomic value and to seek the rela- GROZEVA N. H., GERDZHIKOVA M. A., PAVLOV D. H., PANAYOTOVA G. D., TODOROVA M. H. 82 ACTA BOT. CROAT. 75 (1), 2016 tionship between the degree of morphological variability, area, number and ecological conditions of the studied popu- lations. Materials and methods The subject of this survey were the four currently known populations of B. bulgarica in the territory of Sinite Kamani Natural Park (Tab. 1). Observations were carried out during the vegetation periods of 2013 and 2014, and morphometric tests were performed during the vegetation period of 2014. In each population the sites for taking mor- phological measurements were selected at the beginning of vegetation according to the presence of suffi cient popula- tion density and the presence of B. bulgarica and at least four sites were identifi ed. They were found along the diago- nals in both directions of the population and areas in the central parts were deliberately included. The number of plants measured at each point was at least ten with a total of 50 plants used from each population. Morphometric mea- surements were conducted eight times during the vegeta- tion period from April 5th to September 5th. The morpho- logical analysis included 35 quantitative traits (On-line Suppl. Tabs. 1 and 2). A total of 21 qualitative traits were also analyzed (Tab. 2). Due to lack of variability in the studied 21 quantitative traits, the data obtained from the four investigated populations are presented in a summa- rized form in Tab. 2. Flower and fruit dimensions, specifi cs of leaf epidermis and pollen morphology were recorded in laboratory condi- tions for fl owers, fruit and leaves collected earlier from the population. Ripe fruits collected in isolation bags were measured. After the measurements, nutlets were sown with- in the borders of each population. All other characteristics were recorded directly on site. To study pollen and nutlet morphology and for a more detailed study of leaf epidermis and type of trichomes the electronic microscope method was used. Studies were conducted on dry plant parts in the laboratory at the Faculty of Chemistry and Pharmacy in St. Kliment Ohridski University of Sofi a. At least twenty pol- len grains and ten nutlets and leaves from each population were studied. Pollen terminology followed Erdtman (1952) and Punt et al. (1994). Data from the microscopic measurements of pollen were presented in generalized form for the species due to lack of signifi cant variability within and between populations. The results obtained for the parameters related to the morphological characteristics were statistically processed with Statistica for Windows 10. The statistical reliability of differences among various parameters, the interpopulation and intrapopulation variations of traits were established through ANOVA. Cluster analysis was applied to group populations by similarity of 35 of the quantitative morpho- logical traits. To allocate populations into groups by strength and direction of infl uence of the basic traits (from the included 35 morphological traits) principal component analysis (PCA) was conducted. Results The data on the mean arithmetic values of the studied quantitative traits and the variance values of each trait are presented in On-line Suppl. Tabs. 1 and 2. Interpopulation variability was traced based on variance values. The values differed both by individual traits of each population and among populations. The following traits were the most variable among all the populations: height of stem, basal leaf petiole length and stem leaf petiole length. In the popu- lations from Ablanovo and Slancheva Polyana a higher am- Tab. 1. Studied population of Betonica bulgarica Degen et Neič. Locality Ecological conditions Population Ablanovo area, N 42°42.638’, E 26°17.262’, 540 m a.s.l. An open meadow on the edge of a mixed deciduous forest comprising Carpinus betulus L., Quercus robur L., Ulmus minor Mill., Fraxinus ornus L. and Crataegus monogyna Jacq. The grassland community is dominated by Betonica bulgarica.The terrain is very slightly sloped (3°–4°), non-eroded, facing south-east. The bedrock is limestone, the soil type – Chromic Luvisols (WRBSR, 2006). The area of the population is 1600 m2 and it numbers 440 specimens, incl. 310 fl owering and 50 juvenile ones. Upper lift station, N 42°43.100´, Е 26°21.619´, 1015 m a.s.l. An open meadow on the edge of a forest consisting of Fagus sylvatica L. ssp. moesiaca (K. Maly) Hjelmquist, Picea abies (L.) Karst. and Pinus sylvestris L. The grassland community is dominated by cereal species.The terrain has a slope of 7°, slightly eroded, dry, facing west. The bedrock is limestone, the soil type is Rendzinas (WRBSR, 2006). The plant community is dominated by cereal species. The area of the population is 150 m2 and it numbers 140 specimens, incl. 92 fl owering and 48 juvenile ones. Microyazovir area N 42°42.852´, E 26°22.654´, 945 m a.s.l. An open meadow near the cliffs east of the dams. The terrain is very sloped 11°–20°, highly eroded, dry, facing north. The grassland community is dominated by Sesleria latifolia Degen.The bedrock is quartz porphyry, the soil type – Eutric Cambisols. Due to the reported high level of soil erosion in Karandila area, east of the dam, the soil in the studied area was determined as Regosols, too (WRBSR, 2006). The population area is 950 m2 and it numbers 95 specimens, incl. 74 fl owering and 21 juvenile ones. Slancheva polyana area N 42°43.252´, E 26°21.668´, 1001 m a.s.l. An open meadow on the edge of a mixed forest consisting of Fagus sylvatica ssp. moesiaca, Pinus silvestris, Fraxinus ornus, Prunus cerasifera Ehrh., Acer campestre L., with some bushes of Juniperus communis L., Crataegus monogyna, Rosa canina L., Rubus canescens DC. In the grass layer Festuca valesiaca Schleich. ex Gaudin is dominant. The terrain has a slope of up to 11°, non-eroded, dry, facing west. The bedrock is limestone, the soil type – Eutric Cambisols. The area of the population is 724 m2 and it numbers 215 specimens, incl. 155 fl owering and 60 juvenile ones. BULGARIAN ENDEMIC BETONICA BULGARICA DEGEN ET NEIČ. MORPHOLOGY ACTA BOT. CROAT. 75 (1), 2016 83 plitude of variability was registered for other traits as well: stem leaf petiole length, raceme and branch length. The least variable in all the populations were the fol- lowing traits: basal leaves length/width ratio, raceme leaves petiole length, fi rst raceme leaves width, third raceme leaves width, corolla width base and those characterizing seed (length, width, length/width ratio). In the Ablanovo population a low level of variability was registered for oth- er traits as well: corolla length/width top and calyx length, in the population at Slancheva Polyana – second raceme leaves petiole length, in the population at Upper Lift Station – raceme branch length and third raceme leaves length, and for that at Microyazovir – raceme branch length, second ra- ceme leaves petiole length and calyx length/width ratio. In the population at Slancheva Polyana no variability was found for third raceme leaves length and third raceme leaves length/width ratio or in the population at the Microy- azovir for third raceme leaves length/width ratio. The ANOVA results (On-line Suppl. Tabs. 1 and 2) showed that intrapopulation variability was dominant in the total variability. Higher interpopulation variability was found only for petiole length of basal leaves. The data from the unweighted pairgroup average (UPGA) cluster analysis based on morphological pairwise similari- ties (Euclidean distances between population centroids) showed the greatest similarity in the entire complex of qua li- tative traits among specimens from the populations at Slan- cheva Polyana and Microyazovir (Fig. 1). The greatest dif- ferences were recorded for the population from the Ablanovo area. Factor scores based on the principal component analysis of the distribution of populations according to the 35 mor- phological traits studied showed that the population at Ablanovo had positive values for both factor 1, and factor 2 (On-line Suppl. Tab. 3). The populations at Microyazovir and Slancheva Polyana had positive values for the second Tab. 2. Studied qualitative features of Betonica bulgarica Degen et Neič. Feature 1. Colour of stem Light green. 2. Indumentum of stem Non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; peltate glandular trichomes. 3. Shape of leaf lamina Rounded to elliptic and lanceolate, with a blunt tip, concave base and scalloped margin for annual plants developed from seeds; Lanceolate with a blunt tip, concave or asymmetric base and scalloped edge at the biennial and perennial plants developed from seeds and rhizomes. 4. Colour of leaf lamina Green to light green. 5. Indumentum of leaf lamina Adaxial and abaxial leaf surface – Non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; peltate glandular trichomes. 6. Shape of epidermal cell of leaf lamina Adaxial and abaxial leaf surface – elongated, polygonal, with varied size. 7. Type of stomata Аbaxial leaf surface – anomocytic, diacytic, paracytic. 8. Indumentum of leaf petiole Non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; peltate glandular trichomes. 9. Type of infl orescence Flowers gathered in fl ower vertebrae, forming tight cyme. The bottom vertebra is often separated at a distance from the cluster. 10. Shape of bracts Lanceolate, with smooth edge, awned peak. Persists in the fruit. 11. Indumentum of bracts Adaxial and abaxial leaf surface – Non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes along the edge and the primary vein; peltate glandular trichomes. 12. Shape of calyx segments Sepals fused in a tube, calyx teeth styliform, almost equal to the tube. Calyx retained in the fruit. 13. Colour of calyx segments Light green to dark purple. 14. Indumentum of calyx segments Calyx tube – Non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; non glandular stellate multicellular trichomes with 2–3 rays; peltate glandular trichomes. Calyx teeth – Non glandular multicellular, simple, uniseriate, hard, with smooth surface and peltate glandular trichomes. 15. Shape of corolla segments Corolla tube narrow, ± curved, upper lip entire, sometimes binary, lower lip ternary with wavy middle part and egg-shaped side parts. 16. Colour of corolla segments Light purple 17. Indumentum of corolla segments Non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; non glandular stellate multicellular trichomes with 2–3 rays; peltate glandular trichomes. 18. Colour of anthers Upon opening of the fl ower – yellow, during fl owering – purple. 19. Shape of nutlet Triangular, elongated, the outside almost fl at, the edges with narrow wings, which at the top edge go into irregularly toothed membranous appendage. 20. Colour of nutlet Dark brown 21. Nutlet surface Smooth formed by oblong cells variable in size GROZEVA N. H., GERDZHIKOVA M. A., PAVLOV D. H., PANAYOTOVA G. D., TODOROVA M. H. 84 ACTA BOT. CROAT. 75 (1), 2016 factor but negative ones for the fi rst one. The population at the Upper Lift Station area was negative for both factors. The population from Ablanovo exceeded the other popula- tions by 11 out of a total of 35 studied traits. Height of plants, length, width of basal leaves, petiole length of basal leaves, length and width of stem leaves were signifi cant. The data related to the qualitative traits studied are pre- sented in Tab. 2; Figs. 2, 3A–B and 4. In the four studied populations no signifi cant variability in the types of tri- chomes and stomatas, pollens and nutlet morphology was established. The epidermal surfaces of all examined plants showed both non-glandular and glandular trichomes (Tab. 2). Stem, leaves, bract, calyx and corolla were more or less covered with peltate glandular trichomes (Figs. 2 and 3A–B). On the fresh parts of B. bulgarica small capilate trichomes were found, practically invisible on the herbarised plant parts due to their small size. Stem, leaf petioles, bracts and the calyx tube were covered with non-glandular multicel- lular, simple, uniseriate trichomes with micropapillae cu- ticular surface (Figs. 2A–E and 3A). The calyx and corolla tube of B. bulgarica were covered with non-glandular stel- late multicellular trichomes with two to three rays (Figs. Fig. 1. Dendrogram of the cluster analysis of four Betonica bul- garica Degen et Neič. populations based on 35 traits of vegetative and generative morphological variability (see On-line Suppl. Tabs. 1 and 2). Fig. 2. Scanning electron micrographs of Betonica bulgarica De- gen et Neič.: A) stem surface; B) abaxial leaf surface; C, D) adax- ial leaf surface; E) petiole surface; F) adaxial bract surface; G) abaxial bract surface; H) calyx tube surface; a – non-glandular multicellular uniseriate trichomes, b – peltate glandular trichomes; c – non-glandular stellate multicellular trichomes; d – paracytic stomata; e – anomocytic stomata; f – diacytic stomata. Fig. 3. Scanning electron micrographs of Betonica bulgarica De- gen et Neič.: A) calyx teeth surface; B) corolla surface; C–D) pol- len grains; E) stigmas; F) whole nutlet dorsal face; G) whole nutlet ventral face; H) details of nutlet surface; a – non-glandular multi- cellular uniseriate trichomes, b – peltate glandular trichomes; c – non-glandular stellate multicellular trichomes; d – muri; e – lumina. BULGARIAN ENDEMIC BETONICA BULGARICA DEGEN ET NEIČ. MORPHOLOGY ACTA BOT. CROAT. 75 (1), 2016 85 2H and 3B). By the calyx teeth non-glandular multicellular hard non-branched trichomes of up to 2 mm and branched ones with three to fi ve rays were found (Fig. 3A). Three types of stomata were established in the survey – anomocytic, diacytic and paracytic, the anomocytic being observed most frequently (Tab. 2 and Figs. 2B–D, F–G). Among the four studied populations no signifi cant vari- ability in pollen morphology was established. The pollens of B. bulgarica were monad, medium sized, and 3-zonocol- pate (Figs. 3C–D). Polar axis (P) was between 30.2 and 38.5 μm, equatorial axis (E) 20.4–24.2 μm and P/E rate 1.3–1.6. The coloration was reticulate. The width of muri was 0.3– 0.6 μm (Fig. 3D). The luminas were slightly elongated with length ranging within 0.8–1.6 μm and width – 0.4–1.2 μm. The nutlets of B. bulgarica were dark brown, triangular, elongated, the outside almost fl at, the edges were with nar- row wings, which at the top edge went into irregularly toothed membranous appendage (Tab. 2 and Figs. 3F–H). The three sides of the nutlets had even surfaces formed by oblong cells variable in size and usually with no trichomes (Fig. 3G); peltate trichomes were only found on the mem- branous appendage of single nutlets (Fig. 3F). Discussion Intrapopulation variability was dominant in the pheno- typic variability of B. bulgarica (On-line Suppl. Tabs. 1 and 2). Our studies showed that a possible reason for this was the age structure of the population. Due to the perennial type of the species and the possibilities for seed and vegeta- tive (through roots) reproduction within each population, well-developed perennials were observed. There were also annuals that propagated through seeds and during the fi rst year formed only a leaf patera, as well as biennials that propagated through seeds, which did not always reach fl owering and fruiting. In addition, there were vegetatively propagated plants in which the basal leaves were often re- duced. Morphometric analyses found that the size of vege- tative organs in each of these groups of plants varied sig- nifi cantly within the range of each of the four populations studied. A certain intrapopulation variability was also regis- tered for the size of generative organs in each of the age groups. This determined the dominant role of intrapopula- tion within total variability along the entire complex of morphological traits. The height of stem in the different groups in each popu- lation varied to a great extent, due to the greatest registered trait variance values (On-line Suppl. Tab. 1). The competi- tive relations which arise among the specimens of B. bul- garica and those of other species in the community, infl u- enced the high variability of that trait. In the populations of Upper Lift Station, Microyazovir and Slancheva Polyana, the uneven distribution of moisture due to the ground slope had an additional effect. It was visible that the specimens located on the steepest parts of the population had shorter stems than those located on even ground. Higher variability was registered for the traits petiole length of basal and stem leaves (On-line Suppl. Tab. 1). A typical property of the species is that basal leaves always have petioles longer than the laminae, stem leaves have petioles shorter than the laminae and the upper pair are ses- sile (Koeva 1989). The data from morphometric measure- ments showed that in some of the specimens the petioles of basal leaves were up to 7 times longer than the lamina and for a period of 20 days their dimensions increased mani- fold. Since the vegetative development of B. bulgarica lasts from the end of March to the fi rst half of April, when the majority of the dominant species are well developed, a rap- id extension of leaf petioles results from the competitive relations and provides the leaf laminae with appropriate ac- cess to light. Dominant for this trait in the common vari- ability was the interpopulation one (On-line Suppl. Tab. 1) due to the varied composition of communities in the four populations and the different competitive relationships. The greatest amplitude of variability was registered for the pop- ulation at the Ablanovo area, where the altitude was the lowest and B. bulgarica dominated in the grass community (Tab. 1). In specimens that had competitive relations with dominant species of that population, the length of leaf peti- oles increased up to 2–3 times during a period of 20 days. At the same time in plants that were neighbours to other specimens of B. bulgarica, also at the beginning of their vegetative development, the length of leaf petioles and the growth rate were far smaller. The registered high amplitude of variability led to the higher average values of the petiole length of basal and stem leaves for this population. The conducted two-year study showed that in all populations that were in competitive relations with cereals or other plant specimens of B. bulgarica, leaf petioles of basal and stem leaves had a greater size. Regardless of the registered vari- ability, the variation in the size of leaf petioles of the stud- ied specimens in the four populations was within the spe- cies variability as stem leaves always have shorter petioles than the basal ones and the upper pair of stem leaves are sessile. Variability was also found for the dimensions of leaf laminae within each population. Plants developing from seeds during the fi rst year had smaller, rounded, elliptic to lanceolate laminae with a blunt tip, scalloped edge and con- cave base (Figs. 4A–B). Biennial plants produced from seeds (Figs. 4C–D), as well as the other perennial speci- mens, which reproduced from seeds or vegetatively (Figs. 4E–F), had lanceolate lamina, obtuse apex, asymmetric or concave bases and their dimensions varied. The leaf lami- nae of biennial plants developed from seeds were always smaller than those of the others (Fig. 4). Regardless of the registered variability in the dimensions of leaf laminae, the ratio between their length and width was one of the traits that had lower variability. With annual specimens that de- veloped from seeds the ratio between length and width of leaf laminae was 1–1.5, and in the other age groups – from 1.5 to 4.5. Regardless of the differences in their size and shape, leaf laminae are always covered with trichomes. The laminae of annual specimens developed from seeds had the thickest trichome cover of all the populations. In specimens from the other age groups no regularities were found in the degree of trichome covering. Variability of basal leaves was established for another species from the genus – B. offi cina- lis (Dušek et al. 2010). GROZEVA N. H., GERDZHIKOVA M. A., PAVLOV D. H., PANAYOTOVA G. D., TODOROVA M. H. 86 ACTA BOT. CROAT. 75 (1), 2016 The dimensions of fl oral parts (Fig. 5 and On-line Sup- pl. Tab. 2) in each of the four populations varied slightly and corresponded to those mentioned for the species by Ko- eva (1989) and Genova (2011). Certain differences were found in the colour of anthers. According to data by Koeva (1989) the stamens of the species have yellow anthers. Our observations showed that the anthers were yellow only im- mediately after opening of the fl ower and after that they were purple. The greatest similarity out of the entire set of quantita- tive traits (Fig. 1) was registered between the populations at Slancheva Polyana and Microyazovir. In these two popula- tions, the values of the greater part of the studied quantita- tive traits were very close (see On-line Suppl. Tabs. 1 and 2). Most probably the similarity found was infl uenced by the similarity in soil type and soil reaction that had contrib- uted to the formation of similar ecological conditions with- in their boundaries. The soil type in both areas is Eutric Cambisol. The soil is characterized by sandy clay loam soil texture, weakly structured with granular structure and acid- ic soil reaction (Grozeva et al. 2014). A certain similarity of the populations from Microyazo- vir and Slancheva Polyana was found with the population from Upper Lift Station, which has an intermediate altitude compared to the other two populations, signifi cantly small- er area and a different soil type (Fig. 1 and Tab. 1). Domi- Fig. 4. Variation of leaf lamina of Betonica bulgarica Degen et Neič.: A–B) annual plants developing from seeds; C–D) biennial plants developing from seeds; E–F) perennial plants. Fig. 5. Betonica bulgarica Degen et Neič. – branch with upper leaves and infl orescence (photo M. Srebreva). BULGARIAN ENDEMIC BETONICA BULGARICA DEGEN ET NEIČ. MORPHOLOGY ACTA BOT. CROAT. 75 (1), 2016 87 nant within the community in all three populations were cereal species, and moisture availability of the soil was lower during the greater part of vegetation. The greatest difference compared to the populations of Microyazovir, Upper Lift Station and Slancheva Polyana was observed for the Ablanovo population (Fig. 1), where B. bulgarica was the dominant species. The area of the pop- ulation was considerably bigger and the altitude signifi cant- ly lower (Tab. 1). The soil type also as differed and through- out the entire vegetation soil moisture was moderate. According to our previous investigation (Grozeva et al. 2014), the soil type in that area is Chromic Luvisol (Anony- mous 2006). The studied soil is characterized as moderately structured, with crumb structure in the upper surface hori- zon and a slightly acidic soil reaction with values of pH up to 6.6. The differences in ecological conditions most prob- ably led to the appearance of the registered morphological differences in the species of that population from those of the other three populations. Dušek et al (2010) also evalu- ated the morphological characteristics between plant popu- lations of different origin from another species of genus Betonica – B. offi cinalis. The most conservative traits in all populations were those characterizing trichomes, stomata, pollen and nutlet morphology (Figs. 2 and 3). The taxonomic value of the indumentum and its impor- tance in systematic and phylogenetic relationships was well known in Lamiaceae (Abu-Assab and Cantino 1994, Na- varro and El Oualidi 2000, Dinç and Őztűrk 2007, Giuliani et al. 2008). Trichomes are among the most useful taxo- nomic characters for distinguishing the species of genus Betonica and genus Stachys. The epidermal surfaces of all examined plants of B. bulgarica had both non-glandular and glandular trichomes. The peltate and capillate glandular tri- chomes observed on stem, leaves, bract, calyx and corolla were registered by Giuliani and Maleci Bini (2012) in other spe- cies of genus Stachys and genus Betonica. The peltate tri- chomes established for B. bulgarica (Figs. 2A, C, H, Figs. 3A–B) had the morphological characteristics of the tri- chomes specifi ed by Giuliani and Maleci Bini (2012) as Type A, whereas the capillate trichomes observed in fresh plants were specifi ed as Type B. According to Giuliani and Maleci Bini (2012) Type A peltate trichomes consist of one basal epidermal cell, one neck cell and a multicellular head with a large subcuticular space where the secretion is accumulat- ed, whereas Type B capillate trichomes consist of one basal epidermal cell, one stalk cell and a head of two to four cells with a thin subcuticular space where secretion is accumu- lated. Type A trichomes are mainly essential oil producers, whereas Type B have an exclusive or prevalent polysaccha- ride content (Giuliani et al. 2008). The non-glandular mul- ticellular, simple, uniseriate trichomes observed on stem, leaf petioles, leaves, bracts and calyx tube of B. bulgarica were also found by Naidu and Shan (1981) on B. offi cinalis. The non-glandular stellate multicellular trichomes with two to three rays found on calyx and corolla tube on B. bul- garica (Fig. 2H, Fig. 3B) were similar to the trichomes on calyx and corolla surface of B. offi cinalis and B. scardica as pointed out by Giuliani and Maleci Bini (2012), where, the rays were two to eight. The non-glandular multicellular hard non-branched trichomes up to 2 mm long and branched with three to fi ve rays typical of the calyx teeth of B. bulgarica (Fig. 3A) were not mentioned by Koeva (1989) for the calyx teeth of the morphologically closest Bulgarian species – B. offi cinalis. Salmaki et al. (2009) ob- served numerous types of trichomes of Iranian Stachys taxa (incl. Betonica) that showed considerable variability among different species, but were constant among different popu- lations of one species, and therefore afford valuable charac- ters in the delimitation of sections and species. Stomata were described on the leaves of some Lamiace- ae by different researchers (El-Gazza and Watson 1970, In- ambad and Bhatt 1972, Naidu and Shan 1981). From their study of this family El-Gazza and Watson (1970) reported that the stomata were predominantly diacytic, predominant- ly anomocytic, predominantly anisocytic and a mixture of anomocytics and anisocitycs. Inambad and Bhatt (1972) found that in 33 Lamiaceae species diacytic was the most frequent type of stomata. Naidu and Shan (1981) pointed out that out of 34 Lamiaceae species the diacytic was the most frequent type, and the anomocytic was the next most abun- dant type. Our studies confi rmed the data known from litera- ture since in all studied plants from the four populations 3 types of stomata have been found – anomocytic, diacytic and paracytic, anomocytic being the most frequent (Figs. 2B–D, F–G). Stomata were evenly distributed throughout the epidermis of abaxial surface without any defi nite pattern of orientation. According to Naidu and Shan (1981) the ano- mocytic is the most frequent type in Stachys offi cinalis (syn. Betonica offi cinalis), S. palustris and S. recta. Investigations of pollen morphology in Lamiaceae were essential from the point of view of improving the classifi ca- tion within this family (Abu-Assab and Cantino 1994). Ac- cording to Dinç and Őztűrk (2007) the pollen morphology of the genus Stachys (incl. Betonica) is not well known. Palynological studies on the Bulgarian Betonica taxa have not been conducted so far. Pollen morphology of B. bul- garica (Figs. 3C–D) exhibited the features specifi ed by Moore et al. (1991) as Stachys sylvatica type. Typical for that type are: trizonocolpate pollens with reticulate orna- mentation and lumina more or less uniform in size. Moore et al. (1991) included in the group with Stachys sylvatica type pollen eight of the species studied by them, B. offi cina- lis among them. The systematic importance of nutlet morphology in ge- nus Stachys and genus Betonica was underlined by Dem- issew and Harley (1992), Martin Mosquero et al. (2000), etc. In a comparison of the nutlet characteristics of B. bul- garica with those published by various researchers (Dem- issew and Harley 1992, Martin Mosquero et al. 2000, Dinç and Doĝan 2006, Salmaki et al. 2008, Satil et al. 2012) for other species in the genus, the greatest similarity according to nutlet shape was established with B. offi cinalis. Regard- less of the similarity, nutlets of B. bulgarica differed from those of B. offi cinalis by their size (On-line Suppl. Tab. 2) as well as by lack of differences in the reticulate pattern among the three sides of the nutlet surface and the lack of non-glandular trichomes on it (Figs. 3F–H). In conclusion, the results from the present study demon- strated that the registered intrapopulation variability of the GROZEVA N. H., GERDZHIKOVA M. A., PAVLOV D. H., PANAYOTOVA G. D., TODOROVA M. H. 88 ACTA BOT. CROAT. 75 (1), 2016 four studied populations of the Bulgarian endemic B. bul- garica was based mainly on their age structure, while the established interpopulation variability derived from differ- ences in altitude, soil type and differences in environmental conditions and soil properties. The most taxonomic signifi - cance for distinguishing B. bulgarica from the other species in the genus, including from the species that is morphologi- cally most similar to it – B. offi cinalis – was found in the indumentum and morphology of generative organs, espe- cially calyx segments and nutlets. Acknowledgement This work was fi nancially supported under contract No OPOS 2-36 / 24.07.2013 to project No 5103020-15-658 for Sinite Kamani Natural Park, funded under contract No 5103020-С-002 OPERATIONAL PROGRAMME “ENVI- RONMENT 2007- 2013”. References Abu-Assab, M. S., Cantino, P. D., 1994: Systematic implications of pollen morphology in subfamilies Lamioideae and Pogoste- monoideae (Labiatae). Annals of the Missouri Botanical Gar- den 81, 653–686. Anchev, M., 2011: General characteristics of Bulgarian fl ora and fl oristic regions in Bulgaria. In: Peev, D. (ed.), Red data book of the Republik of Bulgaria. Retrieved from http://e-ecodb. bas.bg/rdb/bg/vol1/4fl ora.html. Anonymous, 2002: Biological diversity act. Darzhaven Vestnik no. 77 from 9 August 2002, 9–42. Amended in Darzhaven Vestnik no. 94 from 16 November 2007 (Bg). Anonymous, 2006: World reference base for soil resources. A framework for international classifi cation, correlation and communication. 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Food and agriculture organization of the United Na- tions – Rome, Italy. BULGARIAN ENDEMIC BETONICA BULGARICA DEGEN ET NEIČ. MORPHOLOGY ACTA BOT. CROAT. 75 (1), 2016 On–line Suppl. Tab. 1. Mean (upper numbers in each row), variance value (lower numbers in each row, %) and percentage of intrapopu- lation variation (SSv, %) in the overall morphological variation of Betonica bulgarica Degen et Neič. populations for each of the 24 ob- served vegetative traits; a,b- differences among parameters in the points mentioned with equal letters are not statistically signifi cant. Ablanovo area Slancheva polyana Upper lift station Microyazovir SSv, % 1. Height of stem, cm 73.93 50.18 60.84 46.39 58.43 224.24 158.79 88.32 196.51 2. Number of basal leaves 5.66 6.22 6.47 6.02 97.46 2.09 3.34 3.59 5.17 3. Basal leaves length, cm 11.12 8.33 7.40 8.01 77.83 9.70 10.23 6.16 7.16 4. Basal leaves width, cm 5.75 4.40 4.14 4.00 77.58 1.91 2.10 1.87 2.14 5. Basal leaves length/width ratio 1.95ab 1.90a 1.86b 2.09 96.60 0.16 0.15 0.17 0.27 6. Basal leaves petiole length, cm 26.30 13.11 12.29 11.20 41.50 46.83 21.88 21.38 32.53 7. Stem leaves length, cm 6.03 5.10 5.28 4.79 95.79 5.73 4.95 4.14 4.12 8. Stem leaves width, cm 3.18 2.91 2.92 2.59 97.46 2.15 1.64 1.45 1.26 9. Stem leaves length/width 2.04 1.87 1.90 1.957 98.68 0.37 0.39 0.25 0.3 10. Stem leaf petiole length cm 8.14 5.77 6.66 5.29 94.40 31.31 14.07 19.90 14.15 11. Raceme length, cm 12.81 7.82 10.45 6.08 82.07 31.14 13.98 71.91 7.58 12. First racemes leaves length, cm 2.64 2.41 2.88 2.19 95.16 1.22 1.05 1.37 0.85 13. First racemes leaves width, cm 1.01 0.89 1.14 0.83 94.86 0.27 0.18 0.27 0.22 14. First racemes leaves length/width ratio 2.91а 2.99а 2.68 2.98а 99.02 1.09 3.39 0.40 1.34 15. Raceme leaves petiole length, cm 0.64 0.39 0.48 0.31 85.41 0.13 0.02 0.10 0.02 16. Branch length, cm 13.03 5.03 12.86 4.94 69.61 70.88 7.20 20.98 2.78 17. Raceme branch length, cm 4.00 1.80 2.66 1.44 73.91 7.03 2.04 0.20 0.24 18. Second raceme leaves length, cm 1.35 1.34 2.08 1.37 84.86 0.68 0.57 0.55 0.39 19. Second raceme leaves width, cm 0.44 0.53 2.36 0.53 77.85 0.09 0.11 9.29 0.08 20. Second raceme leaves length/width ratio 3.19 2.75a 2.21 2.71a 84.81 0.43 0.61 1.37 0.26 21. Second raceme leaves petiole length, cm 0.00 0.21 0.33 0.99 98.18 0.00 0.02 0.49 0.01 22. Third raceme leaves length, cm 1.36 1.00 1.41 1.98 58.69 0.47 0.00 0.19 7.35 23. Third raceme leaves width, cm 0.46 0.30 0.46 0.98 59.76 0.05 0.00 0.01 0.02 24. Third raceme leaves length/width ratio 3.05a 3.33a 3.14a 3.00a 99.28 0.77 0.00 0.51 0.00 1 GROZEVA N. H., GERDZHIKOVA M. A., PAVLOV D. H., PANAYOTOVA G. D., TODOROVA M. H. ACTA BOT. CROAT. 75 (1), 2016 On–line Suppl. Tab. 2. Mean (upper numbers in each row), variance value (lower numbers in each row, %) and percentage of intrapopu- lation variation (SSv, %) in the overall morphological variation of Betonica bulgarica Degen et Neič. populations for each of the 11 ob- served generative traits; a,b- differences among parameters in the points mentioned with equal letters are not statistically signifi cant; L/W – length/width ratio. Ablanovo area Slancheva polyana Upper lift station Mikroyazovir SSv, % 25.Calyx length, cm 7.78a 7.72a 7.93a 7.81a 99.24 0.25 0.46 1.61 0.75 26. Calyx width, cm 2.80a 3.20b 2.89ab 3.15ab 99.24 0.75 0.56 0.33 0.55 27. Calyx L/W 2.72a 2.47b 2.74ab 2.43b 93.21 0.36 0.18 0.43 0.21 28. Corolla length, cm 10.73 11.64a 11.88a 11.83a 89.42 2.11 1.55 2.51 1.44 29. Corolla width base, cm 1.06a 1.09a 1.09a 1.10a 97.49 0.03 0.04 0.05 0.04 30. Corola L/W base 10.27 10.64a 10.89a 10.74a 88.63 4.08 4.63 13.15 5.16 31. Corolla width top, cm 5.45a 5.02a 4.06 5.06a 89.17 2.52 2.59 1.08 2.77 32. Corolla L/W top 1.95a 2.31a 2.92a 2.33a 96.51 0.13 5.94 0.21 5.94 33. Nutlets length, mm 3.93a 3.71a 3.82a 3.85 76.05 0.10 0.11 0.11 0.18 34. Nutlets width, mm 1.75a 1.61b 1.69ab 1.77a 90.66 0.058 0.030 0.029 0.044 35. Nutlets length/width ratio 2.24a 2.38a 2.27a 2.22a 96.82 0.206 0.154 0.176 0.289 On–line Suppl. Таb. 3. Factor scores based on principal compo- nent analysis and correlations of all Betonica bulgarica Degen et Neič. populations – average of 1600 measurements for 35 traits. Factor 1 Factor 2 Factor 3 Ablanovo 1.434345 0.3975 –0.18615 Slancheva polyana –0.38116 0.16364 1.441506 Upper lift station –0.17819 –1.43334 –0.40469 Microyazovir –0.875 0.87221 –0.85067 2