ACTA BOT. CROAT. 77 (2), 2018 181 Acta Bot. Croat. 77 (2), 181–188, 2018 CODEN: ABCRA 25 DOI: 10.2478/botcro-2018-0022 ISSN 0365-0588 eISSN 1847-8476 Genetic structure of populations of several endangered and endemic Dianthus species revealed by microsatellite markers Anca Butiuc-Keul1,2*, Cornelia Crăciunaș1,2, Irina Goia3,2, Anca Farkas1,2, Liliana Jarda4, Victoria Cristea4,2 1 Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeş-Bolyai University, 400084 Cluj-Napoca, Romania 2 Laboratory of Plant Biology, Genetics, Microbiology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Babeş-Bolyai University, 400006 Cluj-Napoca, Romania 3 Department of Taxonomy and Ecology, Faculty of Biology and Geology, Babeş-Bolyai University, 400015 Cluj-Napoca, Romania 4 Alexandru Borza Botanical Garden, Babeş-Bolyai University, 400015 Cluj-Napoca, Romania Abstract – In order to develop a proper conservation programme for several endangered, rare or endemic spe- cies of Dianhtus from Romania, molecular characterization by simple sequence repeat (SSR) markers has been accomplished. Amplification of SSR loci in individuals belonging to different populations of D. callizonus, D. glacialis ssp. gelidus, D. henteri, D. nardiformis and D. tenuifolius revealed 23 polymorphic alleles. D. callizonus and D. tenuifolius showed particular sets of SSR alleles. D. glacialis ssp. gelidus, D. henteri and D. nardiformis proved to share almost the same alleles in most of the loci. The highest genetic diversity was observed in D. gla- cialis ssp. gelidus and D. tenuifolius in locus MS-DINMADSBOX. Allelic patterns across Dianthus species indi- cate that the mean number of different alleles was highest in D. glacialis ssp. gelidus, while the number of effec- tive alleles was highest in D. tenuifolius. There are no particular differences in individuals belonging to the same species. Genetic diversity is generally low, ranging from 0.18 (D. callizonus) to 0.44 (D. henteri). Regarding the genetic diversity within populations of the same species, no differences were revealed by the use of the SSR markers tested in the present study. Key words: Dianthus, endemic species, genetic structure, microsatellites, SSR markers, polymorphism * Corresponding author, e-mail: anca.keul@ubbcluj.ro Introduction In the last decades, habitat modifications induced by cli- mate change and human activity led to the extinction of ma- ny plant species. Species conservation programs targeting large areas should focus on the most valuable populations and so require a better understanding of species biology and ecology as well as knowledge of the genetic diversity distri- bution within and among populations. Genetic diversity is important because it influences the populations’ ability to adapt to a changing environment conditions. A characteris- tic of rare or endemic plants is the maintenance of low lev- els of genetic variation. Limited genetic diversity has been reported for many rare plant species (Hamrick et al. 1991, Frankham 1996, Leimu et al. 2006, Szczecińska et al. 2016). The genetic diversity within an endangered population is lost due to relatively faster genetic drift, which is exacerbated by limited gene flow. This can lead to inbreeding depression and higher homozygosity, which results in reduced adaptive potential (Reed and Frankham 2003, Johansson et al. 2007). Genetic diversity is often correlated with plant fitness (Leimu et al. 2006, Ilves et al. 2013). Genetic structure of plant populations, the level of genet- ic polymorphism within and among populations could offer information of value in the development of proper strategies for their conservation. The aim of conservation programs is to preserve the highest number of populations, as well as their genetic structure and variability. The genetic structure and variability of endemic and rare plant populations were BUTIUC-KEUL A., CRĂCIUNAŞ C., GOIA I., FARKAS A., JARDA L., CRISTEA V. 182 ACTA BOT. CROAT. 77 (2), 2018 intensively studied by izoenzymes (Gitzendanner and Soltis 2000) and nuclear DNA markers (Pritchard et al. 2000, Cru- zan 2001, Kang et al. 2005, °Cuan et al. 2006, Breinholt el al. 2007). Among these markers, simple sequence repeats (SSR) are frequently used to investigate genetic structure and vari- ability in plant populations because of their cost effectiveness (Zietkiewicz et al. 1994, Varshney et al. 2005). There are ma- ny previous data regarding the usefulness of SSR in genetic studies in rare and endangered plants (Katoh et al. 2007, So- sa et al. 2010). Molecular characterization of the carnation was previously reported by sequence-related amplified poly- morphism (SRAP) and inter simple sequence repeat (ISSR) markers (Fu et al. 2008). SSR markers have been also used for genotyping of carnation varieties (Smulders et al. 2003). Dianthus is one of the most diverse Mediterranean-type plant genera; more than 300 species are distributed through- out Eurasia and Africa. Over 100 species of Dianthus species occur in Europe, and more than 70 are endemic (Valente et al. 2010). In Romania, 58 Dianthus taxa have been record- ed (Ciocârlan 2009), of which 8 are endemic. Recent works have analysed the genetic variability of two endemic and en- dangered species of Dianthus from Romania (D. spiculifolius Schur; D. giganteus d'Urv. subsp. banaticus (Heuff ) (Cristea et al. 2014, Jarda et al. 2014, Gabel et al. 2017). In this paper we investigate the genetic diversity of 5 en- demic and threatened Romanian Dianthus species using SSR markers. D. callizonus Schott & Kotschy, a Dacian element, is strictly endemic to the Curvature Carpathian Mountains. Previously classified as Vulnerable (VU) by Oprea (2005), it is currently considered as Low Risk (LR) species in the Red Book of Vascular Plants from Romania (Dihoru and Negre- an 2009). D. callizonus grows on alpine and subalpine calcar- eous rocks. D. glacialis Haenke ssp. gelidus (Schott, Nyman & Kotschy) Tutin is a taxon endemic to the Eastern and South- ern Carpathians (Oprea 2005). It is sporadic in alpine com- munities on calcareous rocks. D. henteri Heuff. ex Griseb. & Schenk is endemic to the Southern Carpathians. This spe- cies grows on rocky soils, in the beech and spruce belt (Sâr- bu et al. 2013). D. nardiformis Janka grows on rocky areas, in the Lower Danube Basin and in the Dobrudja region. This species is considered VU (Oprea 2005, Dihoru and Negre- an 2009). D. tenuifolius Schur is an endemic species in the Romanian Carpathians and grows in meadows, on skeletal soil. It is considered a Least Concern (LC) species by Oprea (2005). All taxa are presumably diploids (2n=30), according to Carolin (1957), a ploidy feature that may allow an easy in- terpretation of their SSR patterns. All the above mentioned taxa are perennial and were identified in different Natura 2000 sites from Romania. Besides D. nardiformis, which is found not only in eastern Romania but also in northern Bul- garia, all the other 4 species are found only in Romania and are strictly endemic to the above mentioned areas. Materials and methods Plant material The plant material was collected from different popula- tions of the studied Dianthus taxa from Romania (Fig. 1). Five individuals were collected from each location. Thus, D. callizonus was collected from 2 locations from Pia- tra Craiului Mountains: P1-Spîrlea refuge and Zăplaz (45º31'42.60" N, 25º12'00.17" E) and Diana refuge and Pa- dina Popii (45°33'29.18" N, 25°14'29.75" E); D. glacialis ssp. gelidus from 3 populations from Bucegi Mountains: P1- Omu Peak (45°22'51.00" N, 25°30'40.00" E), P2-Bâlea Lake, Făgăraş Mountains (45°31'40.00" N, 24°44'26.00" E), P3- Obârşia, Bucegi Mountains (45°22'50.00" N, 25°30'39.00" E); D. henteri from 3 populations from Vâlcea county: P1-Cornet (45°23'19.82" N, 24°18'28.54" E), P2-Călineşti Valley (45°22'19.34" N, 24°17'23.23" E), P3-Jiului Gorge (45°16'46.00" N, 23°23'19.00" E); D. nardiformis from 3 pop- ulations from Tulcea county: P1-Allah Bair (44°29'01.42" N, 28°13'24.88" E), P2-Consul Hill (45°10'55.19" N; 28°16'15.77" E), P3-Măcin (45°14'22.71" N, 28°35'01.01" E); D. tenuifo- lius from 2 populations from Suceava county: P1-Stânişoarei Mountains (47°21'50.87" N, 25°36'10.88" E), P2-Bistriţei Mountains (47°23'30.00" N, 25°30'16.65" E). SSR analysis Genomic DNA was isolated from leaves using the CTAB method described by Doyle and Doyle (1987). For SSR anal- ysis, a total of five primer pairs (On-line Suppl. Tab. 1) were used (Smulders et al. 2000). PCR amplifications were per- formed in a 0.2 mL tube containing 2 mM MgCl2, 1 μM of each primer, 200 μM of each dNTP, 1.5 U of Taq (Fermen- tas) and 25 ng of genomic DNA in a final volume of 25 μL. DNA amplification was performed according to the follow- ing program: 1. T = 94 °C, 5 min; 2. T = 94 °C, 45 s; 3. primer annealing at 55 °C, 45 s; 4. elongation T = 72 °C, 45 s; steps 2–4 were repeated 35 times. Amplicons were separated on 1.5% agarose gel, stained with 0.5 μg mL–1 ethidium bromide. At least 2 independent PCR amplifications were performed for each primer. Data analyses Several genetic diversity parameters were calculated for each locus and per Dianthus species using POWERMARK- ER 3.25 (Liu and Muse 2005). These included gene diversity (expected heterozygosity), observed heterozygosity (referred Fig. 1. Sampling locations for Dianthus populations in Romania. GENETIC STRUCTURE OF POPULATIONS OF SOME DIANTHUS SPECIES ACTA BOT. CROAT. 77 (2), 2018 183 to as heterozygosity), and polymorphism information con- tent (PIC) value. These parameters take into account allele frequencies. Within species, richness and evenness were al- so explored using GENALEX6.5 (Peakall and Smouse 2006, 2012). The number of alleles per species (arithmetic mean across loci), the number of alleles with a frequency greater than 5%, the effective number of alleles, the number of pri- vate alleles and the number of locally common alleles oc- curring in less than 50% of the populations were explored. Genetic diversity was assessed via Shannon’s information in- dex, on a single-locus basis, based on natural logarithmic al- lele frequencies. The evolutionary history was inferred using the neighbour-joining method (Saitou and Nei 1987). The optimal tree with the sum of branch length = 100.13433838 was constructed. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary dis- tances used to infer the phylogenetic tree. The evolutionary distances were provided by the user. Evolutionary analyses were conducted in MEGA 6 (Tamura et al. 2013). Principal coordinate analysis (PCoA) was used to explore multivariate Tab. 1. Genetic characterization of Dianthus species based on SSR alleles. Different types of alleles: a-BSY-150, b-BSY-100, c-BSY-75, d- BSY-50, e-DIA-200, f-DIA-175, g-DIA-100, h-DIA-75, i-DIA-50, j-DINCA-200, k-DINCA-150, l-DINCA-100, m-DINCA-50, n-DINGS- TA-200, o-DINGSTA-175, p-DINGSTA-100, r-DINGSTA-75, s-BOX-300, t-BOX-175, u-BOX-150, v-BOX-100, x-BOX-75, z-BOX-50, - absence of allele. Sp ec ie s/ po pu la tio n Lo ca tio n N o. o f i nd iv id ua ls M S- D C A M C R B SY M S- D C D IA 30 M S- D IN C A R A C C M S- D IN G ST A M S- D IN M A D SB O X D. callizonus P1 Spîrlea refuge and Zăplaz, Diana refuge and Padina Popii, Piatra Craiului Mountains 5 1-2 alleles: 100:75 (-b, bc) 2 alleles: 100:75 (gh) 1 allele: 100 (l) 1 allele: 100 (p) 1 allele: 100 (v) D. glacialis ssp. gelidus P1 Omu Peak, Bucegi Mountains 5 0-1 alleles: 100 (b-) 1-2 alleles: 200:50 (ei, -i) 1-2 alleles: 200:50 (jm, -m) 1 allele: 75 (r) 0-2 alleles: 150:100:75 (vx, ux, --, x-) P2 Bâlea Lake, Făgăraş Mountains 5 1-2 alleles: 100:75 (b-, bc) 1-2 alleles: 200:50 (ei, -i) 1-2 alleles: 200:50 (jm, -m) 1 allele: 75 (r) 1-2 alleles: 150:75:50 (uz, x-) P3 Obârşia, Bucegi Mountains 5 1-2 alleles: 150:50 (ad, b-) 1-2 alleles: 200:50 (-i, ei) 1-2 alleles: 200:50 (jm, -m) 1 allele: 75 (r) 1-2 alleles: 150:75:50 (uz, -z, x-) D. henteri P1 Cornet, Vâlcea county 5 2 alleles: 100:50 (bd) 1-2 alleles: 200:75 (eh, -h) 2 alleles: 200:150:100 (jk, kl) 2 alleles: 200:75 (nr) 1-2 alleles: 150:100 (uv, -v) P2 Călineşti Valey, Vâlcea county 5 2 alleles: 100:50 (bd) 1-2 alleles: 200:75 (eh, -h) 2 alleles: 200:150:100 (jk, kl) 2 alleles: 200:75 (nr) 1-2 alleles: 150:100 (uv, -v) P3 Jiului Gorge 5 2 alleles: 100:50 (bd) 0-1 alleles: 75 (-h) 2 alleles: 150:100 (kl) 1-2 alleles: 200:75 (nr, -r) 0-1 alleles: 100 (-v) D. nardiformis P1 Allah Bair, Tulcea county 5 0-1 alleles: 100 (-b, --) 1-2 alleles: 200:75 (-h, eh) 1-2 alleles: 150:100 (-l, kl) 1-2 alleles: 200:100 (n-, np) 1-2 alleles: 150:75 (-u, ux) P2 Consul Hill, Tulcea county 5 0-1 alleles: 150:100 (a-, -b) 1-2 alleles: 200:75 (-h, eh) 1-2 alleles: 150:100 (kl, k-) 1-2 alleles: 200:100 (n-, np) 1-2 alleles: 150:75 (-u, ux) P3 Măcin, Tulcea county 5 0-1 alleles: 100 (a-) 1-2 alleles: 200:75 (-h, eh) 1-2 alleles: 150:100 (-l, kl) 1-2 alleles: 200:100 (-p, np) 1-2 alleles: 150:75 (-x, ux) D. tenuifolius P1 Stânişoarei Mountains, Suceava county 5 0-1 alleles: 100 (a-) 2 alleles: 175:75 fh 0-1 alleles: 100 (l, --) 1-2 alleles: 200:175:75 (or, r-, no) 1-2 alleles: 300:150:75 (-x, ux, su) P2 Bistriţei Mountains, Suceava county 5 1-2 alleles: 150:75 (a-, ac, -c) 0-1 alleles: 75 (-h) 0-1 alleles: 100 (l, -) 1-2 alleles: 175:75 (or, -r) 2 alleles: 300:150 (su) BUTIUC-KEUL A., CRĂCIUNAŞ C., GOIA I., FARKAS A., JARDA L., CRISTEA V. 184 ACTA BOT. CROAT. 77 (2), 2018 relationships among inter-individual genetic distances with- in each species and among Dianthus populations. Addition- ally, divergence statistics were computed using the analysis of molecular variance (AMOVA) implemented in ARLEQUIN 3.5 (Excoffier and Lischer 2010). Results Genetic polymorhism All loci amplified well in nearly all individuals tested. A total of 23 polymorphic alleles were amplified. The num- ber of alleles per locus and the frequency in each species is shown in On-line Suppl. Tab. 2. The average number of al- leles was 4.6. The genetic variation within populations is gen- erally low. Almost all the individuals belonging to a popula- tion share the same alleles. The allelic composition of each SSR locus based on the results of all five Dianthus species is detailed in Tab. 1. The most frequent allele in the locus BSY was b in all species except D. tenuifolius and the allele d had the lowest frequency, being present only in D. henteri in- dividuals. In the locus DIA the most frequent allele was h, while the alleles f and g had the lowest frequencies. In the lo- cus DINCA the most frequent allele was l and the least fre- quent was m. The most frequent allele in the locus DINGSTA was r and the lowest frequency was observed for the allele o. In the locus BOX the highest frequency was observed for al- lele x and the lowest frequency for allele z. The alleles b, l and v were present in most of the species, while the allele g was present only in D. callizonus. Individuals of D. glacialis ssp. gelidus showed a particular combination of alleles in most of the loci, the alleles i and m. Other alleles, j and r, were pres- ent only in some individuals of D. henteri and D. tenuifolius. The individuals belonging to D. henteri species showed the particular allele d and the allele v which was present in D. cal- lizonus as well. D. tenuifolius showed a particular combina- tion of alleles, the allele a which was also present only in D. nardiformis and the allele c also present in D. callizonus. The alleles f, o and s, were present only in D. tenuifolius. In our study the gene diversity is low, ranging from 0.18 to 0.44, the lowest gene diversity being observed in D. calli- zonus (Tab. 2). The proportion of the heterozygous individu- als (observed heterozygosity) ranged between 0.3 and 0.57, the lowest value being observed again in D. callizonus. The distribution power of the SSR marker, estimated by the poly- morphism information content (PIC) value ranged between 0.14 (D. callizonus) and 0.34 (D. henteri) (Tab. 2). High similarity among Dianthus species collected from Romania was observed in this study. Allelic patterns across Dianthus species, as revealed in Fig. 2, indicate that the mean number of different alleles was highest in D. glacialis ssp. gelidus, while the number of effective alleles was found to be higher in D. tenuifolius. The amount of alleles with a fre- quency equal to or higher than 5% was similar in all five spe- cies, excepting for D. callizonus, where it was slightly lower. Except for D. nardiformis, private alleles were detected in all Dianthus populations. The strongest genetic divergence was observed among D. callizonus and D. tenuifolius. The num- ber of locally common alleles with a frequency higher than 5% revealed that D. henteri and D. nardiformis share almost the same alleles in most of the loci. Shannon’s information index (I) reveals low levels of genetic diversity in all popula- tions, especially in D. callizonus (I = 0.251). D. henteri was characterized by the highest genetic diversity (I = 0.626). Moreover, Shannon’s index was closely related to the expect- ed heterozygosity. A high similarity in allelic patterns was observed in case of D. henteri and D. tenuifolius. Genetic structure and evolutionary analysis A neighbour-joining tree showed the evolutionary re- lationships of taxa (Fig. 3). Thus, some of the individuals belonging to D. henteri, D. glacialis and D. callizonus clus- tered independently while an overlapping distribution was observed in case of D. nardiformis and D. tenuifolius and other individuals of previous species. This distribution was confirmed by principal coordinate analysis (PCoA). This analysis shows a relatively high diversity of patterns in D. nardiformis and D. glacialis ssp. gelidus. Consistent with the low levels of genetic diversity, a low variety of allelic profiles was observed in D. callizonus population (Fig. 4). The first, the second and the third principal coordinates accounted for 32.27%, 18.69% and 11.99%, respectively, explaining for 62.96 of the total genetic variation across individuals belong- ing to different Dianthus species. Tab. 2. Mean gene diversity (He), heterozygosity (Ho) and poly- morphism information content (PIC) values of the 5 studied Dian- thus species. Species He Ho PIC D. callizonus 0.18 0.3 0.14 D. glacialis ssp. gelidus 0.31 0.32 0.26 D. henteri 0.44 0.57 0.34 D. nardiformis 0.35 0.32 0.28 D. tenuifolius 0.4 0.47 0.32 Fig. 2. Distribution of allelic patterns across Dianthus species. Na – number of different alleles; Na Freq ≥ 5% – number of different alleles with a frequency ≥ 5%; Ne – number of effective alleles; I – Shannon's information index; No. Private Alleles – number of alleles unique to a single species; No. LComm Alleles – number of locally common alleles with a frequency ≥ 5% found in 50% or fewer species; He – Expected heterozygosity. Error bars represent standard deviations. GENETIC STRUCTURE OF POPULATIONS OF SOME DIANTHUS SPECIES ACTA BOT. CROAT. 77 (2), 2018 185 firmed the results of the overall AMOVA and revealed that the greatest amount of genetic variance occurred among all five Dianthus species (50.88%). Genetic variance found with- in Dianthus species was 49.12% (Tab. 3). Tab. 3. Results of analysis of molecular variance (AMOVA) based on FST from data of five microsatellite loci and Gini-Simpson index of Dianthus species. Source of variation Degrees of freedom Sum of squares Variance components Percentage variation (p < 0.001) AMOVA analysis for all loci Among species 4 66.876 0.748 51.48 Within species 105 74.042 0.705 48.52 Locus by locus AMOVA Among species 83.317 0.961 50.88 Within species 94.336 0.927 49.12 Fig. 3. Evolutionary relationships of Dianthus taxa in Romania. Fig. 4. A two-dimensional plot of the principal coordinate analysis (PCoA) of SSR data showing the clustering of five Dianthus species in Romania. Analysis of molecular variance (AMOVA) for all loci showed that the highest amount of genetic variance (51.48%) occurred among Dianthus species and a variance of 48.52% was found within the species. Locus-by-locus AMOVA con- Discussion Genetic diversity of Romanian Dianthus taxa Molecular investigation of Dianthus species from Europe has recently aroused interest in several aspects. Analysis of the genetic diversity and structure of populations of endan- gered, rare or endemic species of Dianthus are useful tools in conservation planning. Molecular analysis could aid espe- cially in the case of plant species capable of clonal propaga- tion as Dianthus. In many cases, endangered, rare or endem- ic species with large populations can be genetically similar or identical. In spite of the high number of individuals, the low genetic diversity variation is due to clonal multiplication or bottlenecks. Thus, data about genetic structure and vari- ability should be useful before the development of proper conservation strategies. There are few data about molecular analysis of rare or endemic Dianthus species (Cristea et al. 2014, Jarda et al. 2014) despite the numerous data regarding the conservation of these species. In Romania these plant species were preserved by in vitro cultures (Butiuc-Keul et al. 2001, Miclăuş et al. 2003, Cristea et al. 2006, 2010, Marcu et al. 2006, Jarda et al. 2011, 2014). Our study revealed the genetic diversity structure of 5 endangered or endemic spe- cies of Dianthus found in different sites in Romania. Most of these locations have been designated NATURA 2000 Sites. By the use of SSR markers developed with the 5 primer pairs tested in this study, 10–11 alleles from a total of 23 polymor- phic alleles amplified were identified in most of the species. The population of D. callizonus showed the lowest diversi- ty, probably as a result of limited distribution (this is a local endemic species). According to these results, D. callizonus conservation should be a priority for the administration of the Piatra Craiului Natural Park. A single allele was identi- fied in each of the loci MS-DINCARACC, MS-DINGSTA and MS-DINMADSBOX and 2 alleles were identified in loci MS-DCAMCRBSY and MS-DCDIA30. The highest diversity was revealed in the populations of D. glacialis ssp. gelidus, especially in locus MS-DINMADS- BUTIUC-KEUL A., CRĂCIUNAŞ C., GOIA I., FARKAS A., JARDA L., CRISTEA V. 186 ACTA BOT. CROAT. 77 (2), 2018 BOX (4 from the 6 alleles observed). This highest diversity can be explained by its larger distribution area. Regarding the genetic diversity among populations of the same species, no differences were revealed by SSR markers tested in the present study. The highest genetic diversity was observed in D. glacialis ssp. gelidus and D. tenuifolius in lo- cus MS-DINMADSBOX. The genetic diversity is generally low in the Dianthus species studied here. This may be related to several factors. Mountain and alpine plants have to cope with harsh envi- ronmental conditions, e.g. short vegetation periods, unsta- ble and low-fertility soils, desiccating winds, and high so- lar radiation, thus a population with low genetic diversity has limited possibilities to fight against harsh environmen- tal conditions. In the short term, a loss of genetic diversity can reduce plant performance and population viability and may limit the potential for further adaptive evolution (Ell- strand and Elam 1993). Low genetic diversity could also be explained by the clonal propagation of many plants, as in Dianthus species grown in severe environmental conditions (Stöcklin 1992). Clonal propagation usually produces a rap- id, but spatially limited spread of genotypes. Along an alti- tudinal gradient, the vegetative growth is increased, because reproduction by seeds may be hampered by the harsh alpine conditions (Young et al. 2002, Gabel et al. 2017). The low values of gene diversity, heterozygosity and PIC found in our study are in accord with other data showing low diversity and polymorphism of Dianthus species from the Iberian Peninsula (Balao et al. 2010) and Iran (Farsi et al. 2013). These data seem to suggest that Dianthus species may be in general very young and the Romanian species are not an exception to the pattern. It is well known that small populations isolated in frag- mented habitats contain less genetic diversity than larg- er populations because over time, individuals will become more homozygous due to the low amounts of available ge- netic diversity within the population and increased inbreed- ing (Duminil et al. 2007). There are also complex interac- tions between small population size, genetic diversity and individual fitness (Aguilar et al. 2008) with several conse- quences, such as inbreeding depression (Jolivet et al. 2013). Human activity and land use changes was followed by small- scale fragmentation of grassland habitats in most regions of central Europe, thus small populations are facing extinction due to local maladaptation in remnant habitats (Busch et al. 2016). In Romania, populations of these endangered species of Dianthus seems to be also such small populations with low adaptation to the habitats. On the other hand in the case of D. gratianopolitanus, genetic variation within populations seems to be much more affected by population density and size than by isolation. Thus, genetic variation decreases with increasing population density across all studied populations, which may be due to the effect of gene flow within popula- tions, which should be stronger in dense populations. Ge- netic variation was higher among populations of D. gratiano- politanus in Germany than in Switzerland. In the German region with a higher magnitude of isolation, genetic varia- tion within populations depended on population size. In the Swiss study region, with a lower magnitude of isolation, the higher genetic variations may be attributed to the gene flow among populations even within smaller populations (Putz et al. 2015). Other studies showed that the historical landscape structure may be more important for genetic diversity than present habitat conditions. Thus, populations that were per- sisting in abandoned grassland fragments significantly influ- ence the genetic variability of those species even under de- teriorating habitat conditions. All of these studies may lead to the conclusion that individuals from different populations should be included in approaches to preserve the genetic variation of such plant species (Reisch et al. 2017). Evolutionary framework and genetic variation of the studied carnations Dianthus genus is characterized by its extensive morpho- logical and genetic variability (Erhardt 1990, 1991, Fried- man et al. 2001, Bloch et al. 2006). Many phylogenetic stud- ies revealed that evolutionary radiations have occurred in Dianthus (Balao et al. 2010, Valente et al. 2010). Polyploi- dy, hybridization, and genome duplication, are common evolutionary forces in plants and act as potential drivers of plant radiation (Otto 2007, Paun et al. 2009, Soltis and Soltis 2009). Although interspecific hybridization is feasible in sev- eral clades of Dianthus (Collin and Shykoff 2003), it seems, however, that the incomplete lineage sorting and/or ances- tral polymorphism processes have played a prominent roles in its recent evolutionary history (Balao et al. 2010, Valente et al. 2010). Hybridization is exceptionally high in Dianthus compared to many genera, partly in consequence of the re- cent origin of most species. High rates of hybridization have possibly contributed to fast diversification by providing evo- lutionary potential via lineage fusions and may explain the shared alleles. One of the phylogenetic studies of the genus revealed that rates of diversification in Mediterranean Dianthus have been exceptionally high (Valente et al. 2010) and suggest that a combination of polyploidy and geographical speciation may have driven cladogenesis in the group. The major patterns of variation within and among populations of particular spe- cies on mountains have not been clearly described to date, and thus it has been difficult to discuss our results of specif- ic case studies in relation to general patterns. However a re- cent study based on morphological and molecular data and analyses suggest that Dianthus binaludensis, distributed on Binalud Mountain, northeast Iran, also displays a local mor- phological divergence (Farsi et al. 2013). In conclusion, the gene diversity is low in all of the five endemic or rare species of Dianthus. The genetic structure is similar in their different populations, D. callizonus and D. tenuifolius show particular sets of SSR alleles, the other 3 spe- cies of D. glacialis ssp. gelidus, D. henteri and D. nardiformis share almost the same alleles in most of the loci. Our results of evolutionary neighbour-joining analysis show overlapping distribution in the case of D. nardiformis, D. tenuifolius and several individuals of D. henteri, D. glacialis and D. callizonus. GENETIC STRUCTURE OF POPULATIONS OF SOME DIANTHUS SPECIES ACTA BOT. CROAT. 77 (2), 2018 187 This phenomenon could occur from the evolutionary pro- cesses in recently differentiated species such as Dianthus species (Balao et al. 2010, Valente et al. 2010). From these da- ta we suggest that D. henteri, D. glacialis have not yet fully di- verged. Although these two species clustered independently, several individuals clustered together with other species. Acknowledgments This work was supported by the projects 31-008/2007 and 71/2014, Partnerships in priority areas – PN II, financed by Romanian Ministry of National Education (CNMP and UEFISCDI). Many thanks to Dr. Irina Holobiuc, Dr. Mihai Puşcaş and Dr. Adrian Oprea for collecting plant material. 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