Ann. For. Res. 58(2): 347-355, 2015 ANNALS OF FOREST RESEARCH DOI: 10.15287/afr.2015.389 www.afrjournal.org Genetic diversity within a newly identified popula- tion of Adenophora liliifolia (L.) A.DC. in Romania: implications for conservation A. Manole, C. Banciu, A. Indreica Manole A., Banciu C., Indreica A., 2015. Genetic diversity within a newly identi- fied population of Adenophora liliifolia (L.) A.DC. in Romania: implications for conservation. Ann. For. Res. 58(2): 347-355. Abstract. Adenophora liliifolia is a relict plant species, endangered at Eu- ropean level. Its occurrence in Romania is limited to a very few sites. The amount of genetic diversity of plant species is a valuable indicator of popu- lation, being the baseline in developing proper strategies for their conserva- tion. Inter-simple sequence repeats (ISSRs) markers were used to analyze polymorphism in A. liliifolia genome and to evaluate the genetic diversity and accordingly, the state of a recently identified population. Five ISSR primers, specially designed for plants, and containing different simple se- quence repeat motifs were tested. A total of 52 ISSR fragments were gen- erated of which 41 were polymorphic (78.84%) and 32 (61.53%) specific to Adenophora genus. The value of Shannon’s index of genotypic diver- sity was 0.812. Jaccard similarity coefficient was calculated for pair wise comparisons among all individuals and ranged from 0.17 to 0.83. The ge- netic variability between individuals was 78.84% which suggests a rela- tive high genetic differentiation. Although the level of genetic variability is moderate to high, the population is declining and exposed to demographic stochasticity. A possible cause is species germination requirements ham- pered by modification in vegetation structure and abundance. The popula- tion survival and reinforcement is conditioned by urgent measures for forest management in order to reduce herbaceous and shrubby vegetation and to limit mowing and grazing. Ex situ conservation measures are also proposed. Keywords Adenophora liliifolia, ISSR markers, genetic diversity, conserva- tion Authors. Anca Manole ([email protected]), Cristian Banciu - Institute of Biology Bucharest, 296 Splaiul Independenţei, 060031, Bucharest, Romania; Adrian Indreica - Transilvania University of Braşov, 1 Şirul Beethoven, 500123 Braşov, Romania. Manuscript received February 02 2015; revised May 12, 2015; accepted May 20, 2015; online first May 21, 2015. 347 Ann. For. Res. 58(2): 347-355, 2015 Research note Introduction liliifolia in a dry-mesic oak forest. To date, worldwide there is a little informa- Assessment of the level and distribution of tion available on the genetic variability of this genetic diversity within endangered species species. Although the critical conservation sta- is critical to their management and conserva- tus of the species was known since nineties, at tion (Ge et al. 1999). Adenophora liliifolia (L.) European level are no studies upon the level A.DC. (Campanulaceae) is a continental-eura- of genetic variation among or within popula- siatic plant species being the only species of tions. The only studies upon population genet- genus Adenophora identified in Romania. As ics in A. liliifolia were made by Boronikova listed in Annex II (b) of the Habitats Directive and refer to four populations from Ural region (Anonymous1992) is considered a plant spe- (Perm) (Boronnikova 2009). The same author cies of European Community interest whose developed also four IRAP (Inter-Retrotrans- conservation requires designation of special posons Amplified Polymorphisms) markers areas of conservation (Natura 2000 sites). as tools for future genetic assessment of A. Moreover, it is considered species indicator liifolia populations (Boronikova & Kalen- of thermophilous forest hotspots signaling dar 2010). Moreover, the lack of information remnant pools of biodiversity (Kiedrzyński & about genetic variability concerns the entire Jakubowska-Gabara 2014). Species conserva- genus Adenophora where except Boroniko- tion status at European level was assessed as va’s studies there are only a few and refers to “Unfavourable bad”, for both reporting peri- A. lobophylla, A. potaninii (Ge et al. 1999), A. ods (2001-2006 and 2007-2012) under article pallustris (Masumoto et al. 2011) and A. gran- 17 of Habitats Directive (European Topic Cen- diflora (Chung & Epperson 1999). tre on Biological Diversity). Taking into account the lack of studies upon The main habitats for the species are open genetic variability within Adenophora genus woodlands and fields with damp light (sandy) in general and A. liliifolia in particular, the to medium (loamy) acid, neutral or basic (alka- present work was focused on genetic charac- line) soils, namely wet meadows of Molinion, terization of a newly identified population in dry grassland of Festuco-Brometea and for- order to assess its state. We have considered ests of Quercetalia pubescenti-petraea (Ghişa for assessment only the new indentified popu- 1964, Ciosek 2006). Even though this species lation because in the last 10 years, at national was not included in any national red list/book level was not identified another population sta- in Romania, it is obvious that natural sites with ble, in a favorable conservation status, or with A. liliifolia dramatically decreased over the last more individuals. The assessment was made at 50 years. More precisely, out of the 34 sites DNA level using the Inter-simple sequence re- with A. liliifolia listed by E.V.Ghişa in 1964, peat (ISSR) method. ISSR amplification uses only a number of 6 were reconfirmed after the PCR amplification of DNA to identify the ge- nineties (Sârbu 2006). Moreover, in the last 5 netic differences between repeated motives of years we have reconfirmed only one site out microsatellite sequences occurring within cod- of the 6 reported, where only one small size ing regions, both centromeric and telomeric, population was identified (unpublished data). that are highly polymorphic (Ziekiewicz et al. Apart from the previously reported sites, two 1994). The primers anneal to simple-sequence new additional sites were identified in Transyl- repeats that are abundant throughout the eu- vania but unfortunately, only one was recently karyotic genome (Tautz & Renz 1984) and this confirmed (Indreica 2011). This new site was method does not require prior knowledge of identified near Herculian village (Covasna DNA sequence for primer design. Compared County) and encloses a small population of A. with RAPD, ISSRs can be highly variable 348 Manole et al. Genetic diversity within a newly identified population ... within species and have the advantage in long- primers - others than those tested by Boronnik- er primers, allowing for more precise anneal- ova (2009) - only 5 were selected as suitable ing temperatures and generating a much higher for Adenophora species. Reactions were car- number of polymorphic fragments (Bornet & ried out in a total volume of 20 µl consisting Branchard 2001). Accordingly, ISSR have of 9.4 µl nuclease free water, 10 µl Phire Plant been widely used to detect polymorphism and PCR Buffer, 0.4 µl Phire Hot Start II Polymer- to evaluate genetic diversity in many plant ase, 0.2 µl primer (0.5 µM), and 0.5mm punch species including A. liliifolia (Boronnikova of plant sample. PCR was performed using an 2009). Eppendorf thermal cycler (Mastercycler Gra- This is the first study upon the genetic struc- dient). Amplifications were performed using ture of a natural population of A. liliifolia at the following programme: pre-de naturation European level. The aim of the present study for 2 min at 94°C, 35 cycles of 30 s at 94°C, was to reveal the level of genetic diversity and 45 s at annealing temperature (44°C), 30 s at to assess the population status, in order to un- 72°C, and 10 min at 72°C for final extension. derstand the causes of population decline and PCR products were electrophoretically sepa- accordingly to suggest some forest manage- rated in 1.5 % agarose gels buffered with 1 X ment measures and conservation strategies. TBE containing ethidium bromide (0.5µg x The data gained may contribute particularly to ml-1) at constant voltage of 120V for one hour. the population survival and reinforcement, and O’GeneRulerTM 50bp (1000 – 50 bp) DNA further for species conservation. Ladder (Fermentas) was used as size marker. PCR products were visualized and analyzed under GENi Gel Documentation System from Materials and methods SynGene. Band scoring and data analyses were The plant material was collected in late performed using Gene Tools software (Syn- July 2014, from a natural population recently Gene, Cambridge, UK, version 4.02). Each identified near Herculian village (Covasna amplification fragment was considered as a county) (Indreica 2011). The population was dominant allele for a given locus. The finger- small consisting of 12 individuals scattered print pattern of the bands was transformed into over an area of about 2500 square meters. a binary character matrix with 1 for presence Fresh young leaves from each individual were and 0 for absence of a band of a particular po- dried in silica gel and stored at -20˚C until sition in a lane. For each polymorphic profile processing. In order to evaluate the level of ge- were generated similarity matrices. Genetic nus specificity of the primers an outlier sample similarities among individuals were quanti- of Campanula rapunculoides L. was added to fied with the Jaccard similarity coefficient and those of