GENETIC DIVERSITY of Iris Pontica Zapal., in MOVILA LUI BURCEL Ornamental Hybrids (Niketić Et Al., 2018)

AgroLife Scientific Journal - Volume 9, Number 2, 2020

ISSN 2285-5718; ISSN CD-ROM 2285-5726; ISSN ONLINE 2286-0126; ISSN-L 2285-5718 pumila L. and I. aphylla (Kunth) Kuntze, (M. Bieb.) Fisch., Kengia bulgarica (Bornm.) resulting in a multitude of new created Packer, Crocus reticulatus Steven ex Adams, GENETIC DIVERSITY OF Iris pontica Zapal., IN MOVILA LUI BURCEL ornamental hybrids (Niketić et al., 2018). Dianthus capitatus Balb. ex DC., Galium Currently, the species is cultivated as an volhynicum Pobed., Goniolimon besserianum PROTECTED AREA: IMPLICATION FOR SPECIES CONSERVATION ornamental plant, all over the world, mostly in (Schult. ex Rchb.) Kusn. Hyacinthella private gardens (Cullen et al., 2011) but also in leucophaea (K. Koch) Schur, Iris aphylla L., 1 2 1 1 Ioana Cătălina PAICA , Irina IRIMIA , Cristian BANCIU , Mihnea VLADIMIRESCU , botanical gardens as University Botanic Garden Iris pontica Zapal., Salvia aethiopis L. and 1 1 Gabriel Mihai MARIA , Anca MANOLE Ljubljana, St. Petersburg Botanical Garden, Salvia nutans L. (Irimia et al., 2015).

1 Pruhonice Botanic Garden etc. The latest assessment of the protected area Institute of Biology Bucharest, Plant and Animal Cytobiology Department, 296 Independenței Spl., revealed some endangering anthropic District 6, 060031, Bucharest, Romania, Phone: +4 021 221 9092, Fax: +4 021 221 9071 influences like the plantations invading 2 “Alexandru Ioan Cuza” University, Faculty of Biology, 20 A, Carol I Blvd, 700505 concurrent species black locust (Robinia Iași, Romania, Phone: +40232201072, Fax: +40232201472, pseudoacacia) and Russian olive (Elaeagnus Email: [email protected] angustifolia), grazing, wastes and fire hearths. All these interventions have resulted in habitat Corresponding author email: [email protected] changes that endanger the rare indigenous flora from the protected area (Irimia et al., 2015). Abstract The newly identified population is very small 2 Irises are valued as ornamental and medicinal plant species, the wild populations being valuable sources of material and spread over an area of about 50 m . Taking for horticulture and/or for secondary metabolites extraction. Iris pontica is an endangered plant species with a into account species rarity and population size, restricted distribution range from Eastern Europe to Caucasus region. Until recently, the species was recorded in we have considered that an assessment of Romania from a few counties from Transylvania only, but a new population was reported, thus extending the species genetic diversity will bring new information national occurrence to East. The aim of present study was to evaluate the genetic diversity of the newly reported regarding population conservation status. population of I. pontica using Inter-simple sequence repeats (ISSR). The results showed low level of genetic diversity within population as well as a homogeneous spatial distribution of genotypes. We can conclude that supplementary in Molecular markers have been widely used to situ conservation measure should be taken as well as some complementary ex situ approaches in order to preserve the Figure 1. I. pontica in natural populations from Movila characterize wild plant populations (Rakoczy- valuable genetic resources of this ornamental and medicinal species. lui Burcel protected area Trojanowska and Bolibok, 2001; Laurentin,

pumila L. and I. aphylla (Kunth) Kuntze, (M. Bieb.) Fisch., Kengia bulgarica (Bornm.) resulting in a multitude of new created Packer, Crocus reticulatus Steven ex Adams, GENETIC DIVERSITY OF Iris pontica Zapal., IN MOVILA LUI BURCEL ornamental hybrids (Niketić et al., 2018). Dianthus capitatus Balb. ex DC., Galium Currently, the species is cultivated as an volhynicum Pobed., Goniolimon besserianum PROTECTED AREA: IMPLICATION FOR SPECIES CONSERVATION ornamental plant, all over the world, mostly in (Schult. ex Rchb.) Kusn. Hyacinthella private gardens (Cullen et al., 2011) but also in leucophaea (K. Koch) Schur, Iris aphylla L., 1 2 1 1 Ioana Cătălina PAICA , Irina IRIMIA , Cristian BANCIU , Mihnea VLADIMIRESCU , botanical gardens as University Botanic Garden Iris pontica Zapal., Salvia aethiopis L. and 1 1 Gabriel Mihai MARIA , Anca MANOLE Ljubljana, St. Petersburg Botanical Garden, Salvia nutans L. (Irimia et al., 2015).

2009). Inter-simple sequence repeats (ISSR) Key words: Iris, endangered, ornamental, genetic diversity. Until recently, in Romania, species occurrence are dominant markers amplified through was limited to 5 counties from Transylvania polymerase chain reaction (PCR) from regions INTRODUCTION medicinal use. Theophrast (371-287 BC) was (Alba, Bistrița-Năsăud, Cluj, Mureș and Sibiu), of genomic DNA located between two probably one of the first who left written but since 2013 the occurrence was extended to The genus Iris L. (Iridaceae) was differentiated identical, adjacent and inversely oriented documents regarding medicinal properties of Eastern Romania when the species was from a clade originate to Africa and Lord Howe microsatellites (Assefa et al., 2003). ISSR iris rhizome. Both rhizomes and leaves are identified from Movila lui Burcel, a protected Island about 45 million years ago (Goldblatt et sequences are reproducible, abundant and recognised as rich sources of secondary area from Miclești commune, Vaslui county al., 2008) and first occurred in Eastern and polymorphic in plant genomes (Bornet and metabolites, especially flavonoids. In only one (Irimia and Mânzu, 2013). The Movila lui Southeastern Asia (Rodionenko, 1987). From Branchard, 2001) and their use do not require decade, between 1999-2008 over 90 flavonoid Burcel botanical reserve - Site of Community Asia, the range was expanding in the temperate data of microsatellite regions, thus are constituents have been discovered and Importance in Natura 2000 Network Northern Hemisphere consequently with frequently used to investigate both wild and characterized from Iris species (Wang et al., (ROSCI0117) - is a remnant of Western-Pontic species differentiation. Currently, the genus cultivated plant species. Using ISSR markers is 2010). Currently, Iris genus is estimated as the steppe meadow (Habitat 62C0* - Ponto- comprises more than 300 species being the assessed as a reliable, informative, rapid, richest in isoflavonoids from monocotyledons Sarmatic steppes) being a habitat with high largest from Iridaceae family (Makarevitch et simple, inexpensive and reproductible method (Kaššák, 2013). conservation value (Mănoiu and Brânzan, al., 2003). for studies on population genetic diversity Iris pontica Zapal. (Figure 1), is a critically 2013). The taxa of community interest found in Due to their beauty and wide distribution range, (Rosa et al., 2017; Christopoulos et al. 2010; endangered iris species belonging to subgenus the botanical reserve are two species of irises are valued from ancient times as Bonet and Branchard, 2001; Rakoczy- Limniris and series Spuriae. It grows in dry cormophytes (Echium russicum and Iris ornamental plants. The early history of Trojanowska and Bolibok, 2001). grasslands and meadows of steppes from aphylla ssp. hungarica), and a species of cultivates irises date from Egyptian Pharaohs Eastern Europe (Romania, Ukraine, Republic Currently, are available only a few studies mammals (Spermophilus citellus). Moreover, a and continues in our days when extensively of Moldova, North-Eastern Turkey) and regarding genetic structure of Iris species and range of rare phytotaxa grows on the reserve grown all over the world. There were Central and South-Western Asia - Caucasus among them very few concerns wild area: Adonis vernalis L., Adonis volgensis developed large collections with thousands of region (Webb and Chatter, 1980; Dihoru and populations, but none refers to I. pontica. Steven ex DC., Adonis x hybrida C. F. Wolff iris plants, as those of Presby Memorial Iris Negrean, 2009). (Artyukova et al., 2001; Bublyk et al., 2013; ex Nyman, Allium moschatum L., Astragalus Garden (New Jersey, USA) and Giardino dell Since 20th century I. pontica was included into Brothers et al., 2013; Cornman et al., 2004; dasyanthus Pall., Astragalus glaucus M. Bieb., Iris (Florence, Italy). the breeding together with I. lutescens Lam., I. Kostrakiewicz and Wróblewska, 2008; Lamote Astragalus pubiflorus DC., Centaurea Apart of their ornamental value, many species reichenbachii Heuff., I. imbricata Lindl., I. et al., 2002; Meerow et al., 2007; Taylor et al., of irises have a long history of traditional marschalliana Spreng., Cirsium serrulatum 195 2013; Wang et al., 2009; Wroblewska et al., extension. Negative controls with water estimated to be 0.317 at the population level, The clusters resulted from genetic distances are 2003). replacing the template DNA were used to while the Shannon index (I) was 0.483. These consistent with the spatial distribution showing Based on the lack of information about genetic monitor for contamination. PCR products were results show a relatively low level of genetic that genetically related individuals are nearly structure of I. pontica population from separated by electrophoresis in 1.5% agarose diversity. located into the habitat (Figure 2). Moreover, Romania, the aim of the present study was to gels buffered with 1X TBE at a constant The dendrogram of genetic distances (Figure this correlation shows that the main fulfill this gap assessing genetic diversity voltage of 4V/cm, for three hours. 1Kb Plus 2A) showed that individuals clustered in 5 reproductive strategy within the population is within the newly discovered eastern population. DNA Ladder (Invitrogen) was used as size slightly separate groups as follows: (1, 2, 5), (6, vegetative propagation which also explains the The information gathered will enable to marker. PCR products were visualized under 7, 9), (3, 4, 10), (13), and (8, 11, 12). When relatively low level of genetic diversity. evaluate population adaptative potential to GENi Gel Documentation System from compared, using the Mantel test, the genetic current both anthropogenic and climate SynGene. The bands were identified by image distances and spatial distances show a low pressure. analysis software (Gene Tools software positive correlation (R2 = 0.1235). (SynGene, Cambridge, UK, version 4.02). MATERIALS AND METHODS Table 1. Sequences of selected primers Plant material Primer Sequence Leaf material was collected from a total of 13 UBC855 ACA CAC ACA CAC ACA CYT individuals which represents the entire UBC866 CTC CTC CTC CTC CTC CTC UBC880 GGA GAG GAG AGG AGA population from Movila lui Burcel botanical UBC889 DBD ACA CAC ACA CAC AC reserve (46º50ʹ52ʺN, 27°48ʹ07ʺE – 46°50ʹ54ʺN, UBC890 VHV GTG TGT GTG TGT GT 27°48ʹ07ʺE). Data analysis A B DNA extraction ISSR bands were scored as presence or absence Figure 2. Genetic relations (A) and corresponding spatial distribution (B) of the 13 I. pontica samples The fragments of fresh leaf were frozen and of binary characters. The resulting (Color correspond to clusters) stored at -20°C until DNA extraction. DNA presence/absence data matrix was analysed was extracted using a Wizard® Genomic DNA using GenAlEx 6.5 (Peakall and Smouse 2006, The population is located in the upper hill zone Unfortunately, wild populations of irises are Purification Kit (#A1125, Promega) in 2012) in order to estimate the percentage of on the North-Western slope but the area decreasing all over Europe (Crișan et al., 2017) accordance with the manufacturer’s protocol. polymorphic loci (P) and expected covered by the iris individuals is very small and with every lost population, a valuable The concentration and purity of the total heterozygosity (HE). The presence/absence data (Irimia and Mânzu, 2013), showing that resource for plant breeding is also lost. In this extracted DNA were assessed using a matrix was also used to generate a pairwise population territorial extension is limited by context, an understanding of population NanoDrop 1000 spectrophotometer. genetic distance matrix for binary data (D) some specific habitat requirements. Most diversity is important also, for an effective which was visualized using a cluster analysis probably, these specific requirements consists utilization and preservation of the genotypes PCR amplification and electrophoresis (unweighted pair-group method with arithmetic in soil chemistry and composition, I. pontica still available to breeders (Jabbarzadeh et al., Two individual DNA samples material were averages, UPGMA) and illustrated in a being a species which grows onto limestone 2010). Thus, there is a need for increased initially amplified using a number of 18 ISSR dendrogram (Figure 2A) using MEGA version and chalky slopes. According to Costică and attention and awareness concerning the primers from the UBC primer set no. 9, 7 (Tamura et al., 2013). Stoleriu report (2012) onto Movila lui Burcel protection of natural populations of irises. In (Biotechnology Laboratory, University of In order to test for putative correlations protected area the land is highly fragmented the case of I. pontica from Movila lui Burcel British Columbia). Following the initial between genetic distances (D) and spatial and only a limited surface with western protected area, the conservation measure screening, 5 of these primers noted as UBC no. distances (estimated in meters) amongst the location is characterized by a calcareous should consist in both in situ and ex situ 855, 866, 880, 889 and, 890 (Table 1) individuals, a Mantel test was performed with substrate. approaches. In situ conservation measures generated clear distinguished bands and were GenAlEx 6.5. The spatial distribution of the Under currently multiple pressures, both should be conducted mainly to mitigate selected for further analysis. individuals is illustrated in a plot designed in anthropic (Irimia et al., 2015) and climatic, anthropic pressure by excluding waste Polymerase Chain Reaction (PCR) was carried Excell 2018 using the original geospatial data correlated with specific substrate requirements, disposals, fire activities and grazing, onto the out in a total volume of 20 µL, containing 1X (Figure 2B). the adaptive response of the population to the territory of the protected area. Another urgent AmpliTaq Gold® 360 Master Mix (Applied environment is limited by the low genetic measure should be an effective control of the Biosystems), 3 µL 360 GC Enhancer (Applied RESULTS AND DISCUSSIONS variance and could not ensure population continuous extending of planted species Biosystems), 0.5 µM primer, 1 µL template persistence in time and space. The assumption Robinia pseudoacacia and Elaeagnus DNA, and PCR grade water. PCR was The 5 primers chosen for analysis produced a that population is not in a favorable angustifolia that covers more than 50% of the performed with an Eppendorf thermal cycler total of 59 reproducible ISSR bands at an conservation status was previously rereported protected area surface (Costică and Stoleriu, (Mastercycler Gradient) using the following average of 11.8 bands per primer. Al 59 scored based on field observations (Irimia and Mânzu, 2012) and soon will invade the population program: initial denaturation for 10 min at bands were polymorphic with an average of 2013) and are sustained by our current results territory. These measures should regard mostly 95°C, 35 cycles of 30 s at 95°C, 45 s at 50°C, 2 0.44 per locus. Assuming Hardy-Weinberg concerning genetic variation within the the locations characterized by calcareous soils min at 72°C, and 10 min at 72°C for the final equilibrium, expected heterozygosity (HE) was population. in order to facilitate iris population spatial

196 2013; Wang et al., 2009; Wroblewska et al., extension. Negative controls with water estimated to be 0.317 at the population level, The clusters resulted from genetic distances are 2003). replacing the template DNA were used to while the Shannon index (I) was 0.483. These consistent with the spatial distribution showing Based on the lack of information about genetic monitor for contamination. PCR products were results show a relatively low level of genetic that genetically related individuals are nearly structure of I. pontica population from separated by electrophoresis in 1.5% agarose diversity. located into the habitat (Figure 2). Moreover, Romania, the aim of the present study was to gels buffered with 1X TBE at a constant The dendrogram of genetic distances (Figure this correlation shows that the main fulfill this gap assessing genetic diversity voltage of 4V/cm, for three hours. 1Kb Plus 2A) showed that individuals clustered in 5 reproductive strategy within the population is within the newly discovered eastern population. DNA Ladder (Invitrogen) was used as size slightly separate groups as follows: (1, 2, 5), (6, vegetative propagation which also explains the The information gathered will enable to marker. PCR products were visualized under 7, 9), (3, 4, 10), (13), and (8, 11, 12). When relatively low level of genetic diversity. evaluate population adaptative potential to GENi Gel Documentation System from compared, using the Mantel test, the genetic current both anthropogenic and climate SynGene. The bands were identified by image distances and spatial distances show a low pressure. analysis software (Gene Tools software positive correlation (R2 = 0.1235). (SynGene, Cambridge, UK, version 4.02). MATERIALS AND METHODS Table 1. Sequences of selected primers Plant material Primer Sequence Leaf material was collected from a total of 13 UBC855 ACA CAC ACA CAC ACA CYT individuals which represents the entire UBC866 CTC CTC CTC CTC CTC CTC UBC880 GGA GAG GAG AGG AGA population from Movila lui Burcel botanical UBC889 DBD ACA CAC ACA CAC AC reserve (46º50ʹ52ʺN, 27°48ʹ07ʺE – 46°50ʹ54ʺN, UBC890 VHV GTG TGT GTG TGT GT 27°48ʹ07ʺE). Data analysis A B DNA extraction ISSR bands were scored as presence or absence Figure 2. Genetic relations (A) and corresponding spatial distribution (B) of the 13 I. pontica samples The fragments of fresh leaf were frozen and of binary characters. The resulting (Color correspond to clusters) stored at -20°C until DNA extraction. DNA presence/absence data matrix was analysed was extracted using a Wizard® Genomic DNA using GenAlEx 6.5 (Peakall and Smouse 2006, The population is located in the upper hill zone Unfortunately, wild populations of irises are Purification Kit (#A1125, Promega) in 2012) in order to estimate the percentage of on the North-Western slope but the area decreasing all over Europe (Crișan et al., 2017) accordance with the manufacturer’s protocol. polymorphic loci (P) and expected covered by the iris individuals is very small and with every lost population, a valuable The concentration and purity of the total heterozygosity (HE). The presence/absence data (Irimia and Mânzu, 2013), showing that resource for plant breeding is also lost. In this extracted DNA were assessed using a matrix was also used to generate a pairwise population territorial extension is limited by context, an understanding of population NanoDrop 1000 spectrophotometer. genetic distance matrix for binary data (D) some specific habitat requirements. Most diversity is important also, for an effective which was visualized using a cluster analysis probably, these specific requirements consists utilization and preservation of the genotypes PCR amplification and electrophoresis (unweighted pair-group method with arithmetic in soil chemistry and composition, I. pontica still available to breeders (Jabbarzadeh et al., Two individual DNA samples material were averages, UPGMA) and illustrated in a being a species which grows onto limestone 2010). Thus, there is a need for increased initially amplified using a number of 18 ISSR dendrogram (Figure 2A) using MEGA version and chalky slopes. According to Costică and attention and awareness concerning the primers from the UBC primer set no. 9, 7 (Tamura et al., 2013). Stoleriu report (2012) onto Movila lui Burcel protection of natural populations of irises. In (Biotechnology Laboratory, University of In order to test for putative correlations protected area the land is highly fragmented the case of I. pontica from Movila lui Burcel British Columbia). Following the initial between genetic distances (D) and spatial and only a limited surface with western protected area, the conservation measure screening, 5 of these primers noted as UBC no. distances (estimated in meters) amongst the location is characterized by a calcareous should consist in both in situ and ex situ 855, 866, 880, 889 and, 890 (Table 1) individuals, a Mantel test was performed with substrate. approaches. In situ conservation measures generated clear distinguished bands and were GenAlEx 6.5. The spatial distribution of the Under currently multiple pressures, both should be conducted mainly to mitigate selected for further analysis. individuals is illustrated in a plot designed in anthropic (Irimia et al., 2015) and climatic, anthropic pressure by excluding waste Polymerase Chain Reaction (PCR) was carried Excell 2018 using the original geospatial data correlated with specific substrate requirements, disposals, fire activities and grazing, onto the out in a total volume of 20 µL, containing 1X (Figure 2B). the adaptive response of the population to the territory of the protected area. Another urgent AmpliTaq Gold® 360 Master Mix (Applied environment is limited by the low genetic measure should be an effective control of the Biosystems), 3 µL 360 GC Enhancer (Applied RESULTS AND DISCUSSIONS variance and could not ensure population continuous extending of planted species Biosystems), 0.5 µM primer, 1 µL template persistence in time and space. The assumption Robinia pseudoacacia and Elaeagnus DNA, and PCR grade water. PCR was The 5 primers chosen for analysis produced a that population is not in a favorable angustifolia that covers more than 50% of the performed with an Eppendorf thermal cycler total of 59 reproducible ISSR bands at an conservation status was previously rereported protected area surface (Costică and Stoleriu, (Mastercycler Gradient) using the following average of 11.8 bands per primer. Al 59 scored based on field observations (Irimia and Mânzu, 2012) and soon will invade the population program: initial denaturation for 10 min at bands were polymorphic with an average of 2013) and are sustained by our current results territory. These measures should regard mostly 95°C, 35 cycles of 30 s at 95°C, 45 s at 50°C, 2 0.44 per locus. Assuming Hardy-Weinberg concerning genetic variation within the the locations characterized by calcareous soils min at 72°C, and 10 min at 72°C for the final equilibrium, expected heterozygosity (HE) was population. in order to facilitate iris population spatial

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Botanical Journal complementary ex situ conservation measures, plants: a manual for the identification of plants Peakall, R., Smouse, P.E. (2012). GenAlEx 6.5: genetic of the linnean Society, 142(1), 65-72. like cultivation in other favourable cultivated in Europe, both out-of-doors and under analysis in Excel. Population genetic software for environments should be taken, in order to glass (Vol. 1). Cambridge University Press, pg. 256. preserve its valuable genetical resources. Dihoru, Gh., Negrean, G. (2009). Cartea roşie a plantelor vasculare din România. Editura Academiei Moreover, establishing an in vitro collection of Române, București. I. pontica with samples from all Romanian Goldblatt, P., Rodriguez, A., Powell, M.P., Davies, J.T., population, could be an efficient strategy to Manning, J.C., Van der Bank, M., Savolainen, V. maintain ex situ, in a controlled environment, (2008). Iridaceae'out of Australasia? 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Ex situ measures could be variability of Iris setosa. Molecular Biology, 35(1), genetic diversity analysis of rose species. African teaching and research-an update. Bioinformatics, 28, cultivation in protected habitats, less exposed 134-138. Journal of Biotechnology, 9(37), 6091-6095. 2537-2539. Assefa, K., Merker, A., Tefera, H. (2003). Inter simple Kaššák, P. (2013). Secondary metabolites of the choosen Rakoczy-Trojanowska, M., Bolibok, H. (2004). to endangering factors, ensuring species sequence (ISSR) analysis of genetic diversity in tef genus Iris species. Acta Universitatis agriculturae et Characteristics and a comparison of three classes of specific requirements, like botanical gardens, [Eragrostistef (Zucc.) Trotter]. Hereditas, 139, 174- silviculturae Mendelianae brunensis, 60(8), 269-280. microsatellite-based markers and their application in private collections, parks, recreative spaces, 83. Kostrakiewicz, K., Wróblewska, A. (2008). Low genetic plants. 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(2013). Catalogul habitatelor, species Iris nelsonii and one of its progenitors, Iris simple sequence repeat (ISSR) markers. Scientia speciilor şi siturilor Natura 2000 în România. hexagona. Heredity, 110(1), 63. CONCLUSIONS Horticulturae, 125, 584-92. Editura Fundaţia Centrul Naţional pentru Dezvoltare Wang, H., Cui, Y., Zhao, C. (2010). Flavonoids of the Cornman, R.S., Burke, J.M., Wesselingh, R.A., Arnold, Durabilă, Bucureşti. genus Iris (Iridaceae). Mini reviews in medicinal M.L. (2004). Contrasting genetic structure of adults Meerow, A.W., Gideon, M., Kuhn, D.N., Motamayor, chemistry, 10(7), 643-661. Summing up, using ISSR polymorphism is an and progeny in a Louisiana Iris hybrid population. J.C. Nakamura, K. (2007). Genetic structure and gene Wang, K., Kang, J., Zhou, H., Sun, Y., Yang, Q., Dong, efficient method to assess genetic diversity Evolution, 58(12), 2669-2681. flow among south Florida populations of Iris J., Meng, L. (2009). Genetic diversity of Iris lactea within the irises population and could be Costică, T.M., Stoleriu, C.C. (2012). Raport final: Studiu hexagona Walt. (Iridaceae) assessed with 19 var. chinensis germplasm detected by inter-simple successfully used for further genetic analyses de evaluare a stării de conservare a speciilor şi a microsatellite DNA loci. International Journal of sequence repeat (ISSR). African Journal of of other populations of I. pontica. habitatului din cadrul sitului ROSCI0117 Movila lui Plant Sciences, 168(9), 1291-1309. Biotechnology, 8(19), 4856-4863. Burcel. https://www.fundatiacorona.ro (accessed on Niketić, M., Tomović, G., Siljak-Yakovlev, S. (2018). A Webb, D.A., Chater, A.O. (1980). Iris L.In: T. G. Tutin, Even though the population was identified into 12.10.2019). new spontaneous hybrid between the cultivated and V. H. Heywood, N. A. Burges, D. M. Moore, D. H. a protected area, its conservation status is not Crișan, I., Vidican, R., Stoian, V., Stoie, A. 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Botanical Journal complementary ex situ conservation measures, plants: a manual for the identification of plants Peakall, R., Smouse, P.E. (2012). GenAlEx 6.5: genetic of the linnean Society, 142(1), 65-72. like cultivation in other favourable cultivated in Europe, both out-of-doors and under analysis in Excel. Population genetic software for environments should be taken, in order to glass (Vol. 1). Cambridge University Press, pg. 256. preserve its valuable genetical resources. Dihoru, Gh., Negrean, G. (2009). Cartea roşie a plantelor vasculare din România. Editura Academiei Moreover, establishing an in vitro collection of Române, București. I. pontica with samples from all Romanian Goldblatt, P., Rodriguez, A., Powell, M.P., Davies, J.T., population, could be an efficient strategy to Manning, J.C., Van der Bank, M., Savolainen, V. maintain ex situ, in a controlled environment, (2008). Iridaceae'out of Australasia? Phylogeny, the germplasm of this critically endangered biogeography, and divergence time based on plastid DNA sequences. Systematic Botany, 33(3), 495-508. plant species. Irimia, I., Ambrono, V., Stratu, A. (2015). Aspects Regarding The Anthropic Influence On The “Movila ACKNOWLEDGEMENTS Lui Burcel” Protected Area (Vaslui County). Present Environment and Sustainable Development, 9(2), The study was funded by project no RO1567- 159-172. IBB08/2019 supported by Romanian Academy. Irimia, I., Mânzu, C. (2013). Iris pontica Zapal. in Moldova’s Flora (Romania). Analele Științifice ale Universității 'Al I Cuza' din Iași. (Serie Nouă) s. II a. REFERENCES Biologie Vegetală, 59(1), 45-51. Jabbarzadeh, Z., Khosh-Khui, M., Salehi, H., Artyukova, E.V., Kozyrenko, M.M., Ilyushko, M. V., Saberivand, A. (2010). Inter simple sequence repeat Zhuravlev, Y.N., Reunova G.D. (2001). Genetic (ISSR) markers as reproducible and specific tools for 199