untitled ACTA BOT. CROAT. 75 (1), 2016 53 Acta Bot. Croat. 75 (1), 53–59, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0007 ISSN 0365-0588 eISSN 1847-8476 Alive and kicking, or, living on borrowed time? – Microsatellite diversity in natural populations of the endangered Ulmus minor Mill. sensu latissimo from Croatia Marko Zebec1*, Marilena Idžojtić1, Zlatko Šatović2, Igor Poljak1, Zlatko Liber3 1 University of Zagreb, Faculty of Forestry, Department of Forest Genetics, Dendrology and Botany, Svetošimunska 25, HR-10000 Zagreb, Croatia 2 University of Zagreb, Faculty of Agriculture, Department of Seed Science and Technology, Svetošimunska 25, HR-10000 Zagreb, Croatia 3 University of Zagreb, Faculty of Science, Department of Biology, Division of Botany, Marulićev trg 9, HR-10000 Zagreb, Croatia Abstract – The main objective of this research was to assess the genetic diversity of 5 natural fi eld elm popu- lations in Croatia. The study results suggest that the observed populations are characterized by a satisfactory amount of heterozygosity, and that the impact of the Dutch elm disease on the amount of genetic diversity in the sampled populations is currently negligible. However, one population displayed a signifi cant excess of heterozygosity, implying a genetic bottleneck. The existence of a very clear genetic differentiation between the continental and the Mediterranean populations of Ulmus minor in Croatia was noticed. Keywords: bottleneck event, endangered species, genetic diversity, natural populations, nuclear microsatel- lites, Ulmus minor * Corresponding author, e-mail: mzebec@sumfak.hr Introduction The majority of contemporary studies of elms are based on the need to preserve their genetic diversity and to re- solve the complex situation in their classifi cation through research into genetic variability and morphological analy- ses (Heimler et al. 1990, Jeffers 1999, Nielsen and Kjær 2010). The reduction in genetic diversity of European elms is largely due to the anthropogenic-induced destruction of habitat, introduction of new elm species and spontaneous hybridization with ornamental species (U. pumila L.). Like- wise, the extremely high susceptibility of elms of the Ul- mus Heybr. section to the Ophiostoma novo-ulmi Brasier pathogenic fungus, played an important role in the cata- strophic wilting of adult elm trees through Europe (Collin et al. 2000). The Dutch elm disease (DED) caused by the fungus was fi rst observed in France and the Netherlands in around 1910 and rapidly spread worldwide afterwards (Spierenburg 1921, Buisman 1928). The European fi eld elm (Ulmus minor Mill. sensu latis- simo) is a noble hardwood which, together with the Euro- pean white elm (Ulmus laevis Pall.) and the wych elm (Ul- mus glabra Huds.), belongs to the European segment of the Ulmus L. genus. U. minor s.l. is present in most of Europe except in northern areas, and also appears along the Medi- terranean coast, on most of the islands of the Mediterranean Sea, as well as in Northern Africa, Asia Minor, Caucasus and Transcaucasia (Richens 1976). The natural populations of the fi eld elm are characterized by a very wide ecological tolerance, and therefore it occupies a wide range of habi- tats, from fl oodplain forests to dry Mediterranean forest communities (Richens 1983, Namvar and Spethmann 1985). The taxonomic status of the fi eld elm in Europe could be viewed in two, mutually completely exclusive, ways. The fi rst, proposed by Melville (1975, 1978) implies the existence of a larger number of small species (micro- species treatment). The second, suggested by Richens (1968, 1980) affi rms the existence of only one collective species of the fi eld elm on the European continent, U. mi- nor Mill. sensu latissimo, as adopted in this paper. In contemporary research on fi eld elm genetic diversity, classical sampling methods, due to the small number of adult trees as a result of DED, have not been used by a ma- jority of researchers. Therefore, in recent studies, “a popu- ZEBEC M., IDŽOJTIĆ M., ŠATOVIĆ Z., POLJAK I., LIBER Z. 54 ACTA BOT. CROAT. 75 (1), 2016 lation” consists of set of trees, originating from the same geographic region and the researched samples include a mixture of trees derived from clonal plantations, ex situ col- lections as well as DED tolerant clones and trees from parks and hedges (Machon et al. 1997, Cogolludo-Agustín et al. 2000). Hence, the heterogeneity of sampled material is ex- tremely great and yet the genetic diversity and structure of natural elm populations remains unknown. There is no dis- pute about the indigenous nature of the fi eld elm in Croatia. This taxon occupies a well-defi ned ecological niche, with the possibility of hybridization with the wych elm reduced to a minimum. Indeed, the only issue in Croatia is the prob- lem of assessing the variability of the taxon. The fi eld elm appears in the continental and the Medi- terranean part of Croatia, where the ecological conditions of the distribution range are very different, which is mani- fested by the high variability of morphological traits (Zebec 2010). Pure stands of the fi eld elm are also frequent, partic- ularly in anthropogenic habitats, as well as on abandoned farmland and pastures. A clear separation of the continental and the Mediterranean populations of the fi eld elm accord- ing to leaf morphology was stated by Zebec (2010), who conducted the fi rst extensive research on the entire area of this species’ spread in Croatia. As microsatellite markers for the fi eld elm were not de- veloped until 2004, most of the research on genetic diversi- ty to date has been conducted using isozymes (Machon et al. 1995, Cogolludo-Agustín et al. 2000), allozymes (Ma- chon et al. 1997), RAPD technique and ISSRs (Coleman et al. 2000, Goodall-Copestake et al. 2005) and cpDNA PCR- RFLP markers (Collin et al. 2004). Microsatellites found their application in the resolving of the taxonomic status of U. procera Salisb. (Gil et al. 2004) and recently in assess- ing whether the fi eld elm is or is not indigenous to the Bale- aric Islands (Fuentes-Utrilla et al. 2014). The aim of this research was to assess the level of ge- netic diversity between and within the fi eld elm populations in Croatia, and thus determine the degree of the negative impact of DED on the biodiversity of the species. There are several advantages of performing this research in Croatia. Firstly – since the Balkan region is considered a glacial re- fugium, Croatia by its geographical position remains a place of presumed high biodiversity. Secondly – Croatia combines aspects of both the continental and the Mediter- ranean area of elm distribution. Thirdly – in the southern area of its distribution (including Croatia) the fi eld elm fa- vors sexual reproduction and since there were a suffi cient number of individuals for the research to be performed, we defi ned the term “population” in this study in the standard biological way. Material and methods Plant material The quantifi cation of genetic diversity of the fi eld elm was conducted by an analysis of genotypes of 96 individu- als from 5 populations (Đurđevac, Zagreb, Pula, Nin, Neretva). The populations were carefully selected, in order to enable, in concert with their geographic location and cli- matic adaptation, precise insight into the genetic variability of the fi eld elm in Croatia (Fig. 1). Taxonomical identifi ca- tion of sampled plant material was confi rmed by Dr. Zlatko Liber, a plant taxonomist of the Department of Botany of the University of Zagreb and voucher specimens of plants (IDs 37109 – 37113) have been deposited with the Herbari- um Croaticum (ZA), University of Zagreb, Croatia (On-line Suppl. Tab. 1). DNA extraction, PCR amplifi cation and microsatellite genotyping Total genomic DNA was extracted from silica-dried leaf terminal buds using a DNeasy® Plant Mini Kit (Qiagen®) according to the manufacturer’s instructions. For this pro- cedure 25 mg of dry tissue of single plants was used. The quality and concentration of the DNAs were checked by electrophoresis in 0.8% (w/v) agarose gel and additionally by Qubit 2.0® Fluorometer using Quant-iTTM dsDNA BR Assay Kit (Invitrogen®). Five SSR markers were used in the study (Whiteley et al. 2003, Collada et al. 2004): Ulm2 (AY300797), Ulm8 (AY300800), Ulmi1–21 (AY520827), Ulmi1–98 (AY520829) and Ulmi1–165 (AY520830) as presented in Tab. 1. Ampli- fi cation was performed in a GeneAmp PCR System 9700 (Applied Biosystems®). The PCR conditions described by Collada et al. (2004) were used for the PCR amplifi cations of three microsatellite loci Ulmi1–21 (AY520827), Ulmi1– 98 (AY520829) and Ulmi1–165 (AY520830), whereas the amplifi cation protocol proposed by Whiteley et al. (2003) was used for microsatellite loci Ulm2 (AY300797) and Ulm8 (AY300800), respectively. Amplifi cation products were separated and detected on an automated sequencer (ABI PRISM 3130 DNA sequencer, Applied Biosystems, Foster City, CA, USA) at Macrogen Inc. (Korea). Fragment sizes were determined using the program GENEMAPPER® 4.0 (Applied Biosystems® 2005). Fig. 1. Geographical position of sampled populations. Populations are indicated by following numbers: 1 – Đurđevac; 2 – Zagreb; 3 – Pula; 4 – Nin; 5 – Neretva. GENETIC DIVERSITY OF ULMUS MINOR MILL. SENSU LATISSIMO IN CROATIA ACTA BOT. CROAT. 75 (1), 2016 55 Data analysis By means of descriptive statistical analysis, performed on original microsatellite data matrix using POWER- MARKER 3.23 software (Liu 2002), the total number of alleles per locus (Na), the observed heterozygosity (Ho), the expected heterozygosity or gene diversity (He) and the polymorphism information content (PIC) for each micro- satellite locus as well as the average number of alleles Nal, Ho, He in each population across loci were calculated. In order to assess allelic richness (Nar) and to estimate the sig- nifi cance of genetic differentiation (FST) between popula- tion pairs we used the FSTAT 2.9.3.2 program package (Goudet 1995). GENEPOP 3.4 (Raymond and Rousset 1995) was used to test genotypic frequencies for each locus in each popula- tion for conformance to Hardy-Weinberg (HW) expecta- tions as well as for calculation of the inbreeding coeffi cient (f) for each locus in each population following Weir and Cockerham (1984). The probability test was based on the Markov chain method (Guo and Thompson 1992, Rousset and Raymond 1995) using 10,000 de-memorization steps, 100 batches and 5,000 iterations per batch. Sequential Bon- ferroni adjustments (Holm 1979, Rice 1989) were applied to correct for the effect of multiple tests using SAS 8.02. The program BOTTLENECK 1.2.02 (Cornuet and Lui- kart 1996, Piry et al. 1999) was used to test for evidence of recent bottleneck events on the basis of this theoretical ex- pectation. The gene diversity observed (He) was compared to the gene diversity expected at mutation-drift equilibrium (Heq) and calculated from the observed number of alleles under different mutation models: infi nite allele model – IAM (Kimura and Crow 1964), stepwise mutation model – SMM (Ohta and Kimura 1973) and an intermediate two- phase model – TPM (Di Rienzo et al. 1994). The TPM model was applied assuming 30% – TPM 1 (Pascual et al. 2001, Hoelzel et al. 2002, Kuehn et al. 2003) and 5% multi- step changes – TPM 2 (Piry et al. 1999). Based on the num- ber of loci in our dataset, the Wilcoxon sign-rank test (Lui- kart et al. 1998) was chosen for the statistical analysis of heterozygote excess or defi ciency as recommended by Piry et al. (1999). With the intention of depicting genetic rela- tionships among elm trees visually, a factorial correspon- dence analysis (FCA) was carried out using GENETIX 4.05 (Belkhir et al. 2004). Finally, pairwise Nei’s standard genetic distances (Nei 1972) were calculated and an unroot- ed phylogenetic tree was constructed using a neighbor-join- ing algorithm (Saitou and Nei 1987) with 10,000 bootstraps (Felsenstein 1985) over microsatellite loci as implemented in the PHYLIP 3.6b software package (Felsenstein 2004). Results Microsatellite diversity In all, 54 alleles were detected by surveying 96 Ulmus trees from 5 populations using 5 SSR markers. The number of alleles per SSR marker ranged from seven (Ulmi1–98) to eighteen (Ulmi1–165) with an average of 10.80 alleles per locus. PIC values varied for a single locus from 0.468 to 0.858, with an average value of 0.628, by which it can be concluded that all microsatellite loci scored in this research, displayed a suffi cient level of polymorphism. Overall ob- served heterozygosity values (per marker) ranged from 0.462 to 0.777, with a mean value of 0.594. The results showed some elevated levels of expected heterozygosity compared to those of observed heterozygosity, with values ranging from 0.487 to 0.871 and a mean value of 0.658 (Tab. 1). Intrapopulation diversity and HW equilibrium The mean number of alleles per locus in the populations ranged from 3.2 (Nin) to 7.4 (Đurđevac). The number of alleles per locus independent of sample size (the allelic richness) ranged from 3.06 (Nin) to 6.80 (Đurđevac). The observed heterozygosity per population varied from 0.530 (Zagreb) to 0.709 (Pula) and the expected heterozygosity per population varied from 0.418 (Nin) to 0.642 (Pula). Ex- pected heterozygosity values were higher than observed heterozygosity values in the Đurđevac and Zagreb popula- tions or vice versa in the Pula and Nin populations. The Neretva population had similar values of Ho and He (Tab. 2). Signifi cant deviations from HW equilibrium using the inbreeding coeffi cient (f) were found for the Pula popula- tion at most loci except Ulm8 and also for the Nin popula- tion but only at loci Ulm2 and Ulmi1–165. The inbreeding coeffi cient values were negative in the discussed cases, in- dicating excess of heterozygotes in a population compared to HW equilibrium, while other populations exhibited no signifi cant deviations from HW equilibrium (Tab. 3). Tab. 1. Source, repeat motifs, size ranges, number of alleles (Na), observed (Ho) and expected heterozygosity (He) and polymorphic in- formation content (PIC) for fi ve microsatellite loci used in fi ve Ulmus populations (n = 96). Source Locus Repeat motif Size range (bp) Na Ho He PIC Whiteley et al. (2003) Ulm2 (AY300797) (CAG)8 98–110 8 0.625 0.662 0.610 Ulm8 (AY300800) (GCT)12CTT(GCT)2 cca. 180 10 0.472 0.487 0.468 Collada et al. (2004) Ulmi1–21 (AY520827) (CT)10 204–220 11 0.634 0.736 0.699 Ulmi1–98 (AY520829) (CT)6N14(CT)7 124–156 7 0.462 0.533 0.507 Ulmi1–165 (AY520830) (CT)9 128–166 18 0.777 0.871 0.858 Mean 10.80 0.594 0.658 0.628 Min 7 0.462 0.487 0.468 Max 18 0.777 0.871 0.858 ZEBEC M., IDŽOJTIĆ M., ŠATOVIĆ Z., POLJAK I., LIBER Z. 56 ACTA BOT. CROAT. 75 (1), 2016 Test for mutation-drift equilibrium at all observed poly- morphic loci under four mutation models in fi ve Ulmus populations revealed a recent reduction in effective popula- tion size (genetic bottleneck) only in the Pula population. Accordingly, the Pula population displayed signifi cant (p < 0.05) gene diversity excess (He > Heq) on average across all 5 polymorphic loci under IAM and TPM1, or across 4 poly- morphic loci under TPM2 and SMM, respectively. The Đurđevac population showed signs of population expansion regarding signifi cant (p < 0.05) gene diversity defi ciency (He < Heq) on average across all polymorphic loci under mutation models TPM1, TPM2 and SMM. Other popula- tions did not show statistically signifi cant departures from optimal heterozygosity level, assumed by mutation-drift equilibrium (Tab. 4). Genetic differentiation among populations The results of FCA are graphically presented by Fig. 2, which delineates the projection of the individuals and popu- lation barycenters on the plane defi ned by the fi rst two FCA axes, thus providing a better insight into genetic relation- ship between studied populations. The fi rst two axes ac- counted for 35.32% and 31.68% of the total inertia, respec- tively. Clear differentiation between continental (Đurđevac, Zagreb) and Mediterranean (Nin, Neretva) populations was notable along the fi rst axis, whilst the Pula population lo- cated centrally with a barycenter closer to the continental group. There was some minor overlap in positioning of trees from the Đurđevac and Zagreb populations as well as Nin and Neretva. Poor differentiation between continental (Đurđevac, Zagreb) and Mediterranean (Nin, Neretva) pop- ulations was evident along the second FCA axis, whereas the Pula population occupied a discrete, distinct area of multivariate space. By means of Nei’s standard genetic distance (DNEI72) and index of genetic differentiation (FST) values, a similar pattern of differentiation among observed populations was expressed (Tab. 5). The greatest DNEI72 values were detected between Pula and Neretva (0.409), whilst the lowest values were observed between Zagreb and Đurđevac (0.106). In- triguingly, lower DNEI72 values were more remarkable be- tween the Pula and Zagreb populations (0.278) than be- tween those of Pula and Nin (0.371). Relatively small genetic distance was determined among the Mediterranean populations of Nin and the Neretva; likewise DNEI72 values Tab. 2. Population, sample size and genetic variability estimates based on data from fi ve microsatellite loci in fi ve Ulmus popula- tions. No – number of populations; n – sample size; Na – mean number of alleles; Nar – number of alleles per locus independent of sample size (allelic richness); observed (Ho) and expected hetero- zygosity (He). No. Population n Na Nar Ho He 1 Đurđevac 20 7.4 6.80 0.566 0.619 2 Zagreb 20 6.8 6.21 0.530 0.626 3 Pula 20 3.8 3.74 0.709 0.642 4 Nin 19 3.2 3.06 0.626 0.418 5 Neretva 17 4.2 4.12 0.547 0.561 Tab. 3. Inbreeding coeffi cients (f) across fi ve microsatellite loci in fi ve Ulmus populations. Signifi cant deviations from Hardy-Wein- berg equilibrium after sequential Bonferroni corrections: ** – sig- nifi cance at the 1% nominal level; * – signifi cance at the 5% nom- inal level; ns – non-signifi cant values. No – number of populations; a – monomorphic locus. No. Population Ulm2 Ulm8 Ulmi 1–21 Ulmi 1–98 Ulmi 1–165 1 Đurđevac 0.177 ns –0.166ns –0.072 ns 0.215 ns 0.306 ns 2 Zagreb 0.166 ns –0.048 ns 0.263 ns 0.230 ns 0.125 ns 3 Pula –0.045** 0.111ns 0.209** –0.596** –0.099* 4 Nin –0.615** –0.245 ns –0.397 ns –a –0.581* 5 Neretva 0.491 ns –0.141 ns 0.032 ns –0.067 ns 0.017 ns Tab. 4. Number of loci showing heterozygosity defi ciency / het- erozygosity excess. Test for mutation-drift equilibrium at poly- morphic loci in fi ve Ulmus populations under four mutation mod- els: infi nite allele model (IAM), two phase model assuming 30% multistep changes (TPM1), two phase model assuming 5% multi- step changes (TPM2), stepwise mutation model (SMM). * – sig- nifi cant (p < 0.05) gene diversity defi ciency (He < Heq) on average across all polymorphic loci using Wilcoxon’s test – sign of popu- lation expansion. # – signifi cant (p < 0.05) gene diversity excess (He > Heq) on average across all polymorphic loci using Wilcox- on’s test – sign of population bottleneck. No. Population Mutation model IAM TPM1 TPM2 SMM 1 Đurđevac 3 / 2 5*/ 0 5*/ 0 5*/ 0 2 Zagreb 1 / 4 2 / 3 3 / 2 3 / 2 3 Pula 0 / 5# 0 / 5# 1 / 4# 1 / 4# 4 Nin 2 / 2 2 / 2 3 / 1 3 / 1 5 Neretva 2 / 3 2 / 3 3 / 2 3 / 2 Fig. 2. Factorial correspondence analysis (FCA) of 96 Ulmus trees belonging to fi ve populations. Each individual genotype is indi- cated by a small sign, while the population barycenters are re- presented by larger ones. Populations are depicted by following symbols: Đur đe vac – fi lled squares; Zagreb – fi lled rhombs; Pula – un fi lled squares; Nin – unfi lled rhombs; Neretva – fi lled triangles. GENETIC DIVERSITY OF ULMUS MINOR MILL. SENSU LATISSIMO IN CROATIA ACTA BOT. CROAT. 75 (1), 2016 57 among continental populations of Zagreb and Đurđevac were also low. Correspondingly to the calculated DNEI72 val- ues for the 5 studied populations, it is evident that the dis- tances between all population pairs within the same region were less than the genetic distance between pairs of popula- tions belonging to different regions (continental/Mediterra- nean). Statistically signifi cant FST values were present be- tween most population pairs, excluding Đurđevac and Zagreb. Calculated signifi cant values ranged from 0.098 between Zagreb and Pula to 0.210 concerning Nin and Pula. According to the values of FST, pronounced genetic differentiation among populations belonging to different re- gions was apparent. Interestingly, only Pula stands out from this trend of population division (Tab. 5). The unrooted neighbor-joining tree visually elucidates the pattern of genetic differentiation between the observed populations, suggesting the existence of a clear genetic dif- ferentiation between continental and Mediterranean popu- lations of the fi eld elm in Croatia (Fig. 3). The above men- tioned differentiation is supported by high bootstrap values, resulting in grouping of populations based on an eco-geo- graphical principle. Bootstrap support value of 68% con- fi rmed the separation between continental (Đurđevac, Za- greb) and Mediterranean (Nin, Neretva) populations, whilst the separation among geographically southern (Nin, Neret- va) and northern (Zagreb, Đurđevac, Pula) populations was verifi ed by even higher bootstrap support values of 99% (Fig. 3). Discussion This research revealed abundant allelic variation over fi ve loci and high overall genetic diversity in natural fi eld elm populations in Croatia. The presented results are con- gruent with those obtained by studies of allozymes (Ma- chon et al. 1997), isozymes (Machon et al. 1995, Cogolludo et al. 2000), RAPDs and ISSRs (Coleman et al. 2000, Goodall-Copestake et al. 2005) and SSRs (Fuentes-Utrilla et al. 2014). Although studies of the fi eld elm on the Euro- pean level suggest a high degree of intrapopulation genetic diversity, they also suggest conclusions concerning the small genetic differentiation between populations belong- ing to different geographic regions. As each individual au- thor uses a different system of molecular markers and de- fi nes the term “population” differently, one should exercise a great deal of caution when comparing results. Therefore, comparison of results obtained in this study with results from above mentioned studies in Europe, is possible only for the purpose of orientation. The values of descriptive statistics parameters (Na, Nar, Ho, He) suggest a native status of the fi eld elm in Croatia (Tab. 2). In continental populations (Đurđevac, Zagreb), higher values of Nal are present, as well as of Nar in relation to Mediterranean populations (Pula, Nin, Neretva). The Đurđevac, Zagreb and Neretva populations did not deviate signifi cantly from HW equilibrium, unlike the Pula and Nin populations, where an excess of heterozygotes was found for specifi c loci (Tab. 3). As elm populations in Croatia do not demonstrate a statistically signifi cant deviation from HW equilibrium, it could be claimed that the danger of the appearance of inbreeding within observed populations is relatively small. The Pula population somewhat deviates from the obtained results, which unequivocally suggest the separation of continental from Mediterranean populations. The reasons for the deviations of the Pula population from HW equilibrium, as well as for a signifi cant excess of het- erozygosity should be sought in the recent bottleneck event (Tab. 4). According to Cornuet and Luikart (1996), a recently bottlenecked population is expected to show fewer rare al- leles, as they are lost most quickly. Allelic diversity is re- duced faster than heterozygosity during a bottleneck, be- cause rare alleles are lost rapidly and have little effect on heterozygosity, thus producing a transient excess in hetero- zygosity relative to that expected in a population of con- stant size with the same number of alleles. Namely, the Pula locality is situated in the area of a former pure elm forest which was turned into an agro-biotope by intensive felling and clearing of land, which has a direct and negative impact on the gene diversity of this population. On the other hand, a lack of heterozygosity was found in the Đurđevac popula- tion, which thus shows signs of expansion. The causes of Tab. 5. Nei’s standard genetic distance (above diagonal) and pair- wise FST values (below diagonal) between fi ve Ulmus populations. Pairwise signifi cance after sequential Bonferroni corrections: ** – signifi cance at the 1% nominal level, * – signifi cance at the 5% nominal level, ns – non-signifi cant values. No – number of popu- lations. No. Population 1 2 3 4 5 1 Đurđevac 0.106 0.293 0.208 0.312 2 Zagreb 0.029ns 0.278 0.332 0.406 3 Pula 0.105** 0.098** 0.371 0.409 4 Nin 0.140** 0.195** 0.210** 0.134 5 Neretva 0.130** 0.160** 0.158** 0.102** Fig. 3. Unrooted neighbor-joining tree based on Nei’s standard ge- netic distance between fi ve Ulmus populations. Numbers above branches indicate bootstrap support percentage over 50% in 10,000 pseudoreplicates. ZEBEC M., IDŽOJTIĆ M., ŠATOVIĆ Z., POLJAK I., LIBER Z. 58 ACTA BOT. CROAT. 75 (1), 2016 population expansion can be various. As this is a larger for- est complex, a stand in which economic measures are im- plemented, there is a possibility of a recent introduction of new individuals. The reasons for this could be to stimulate the natural renewal of the fi eld elm in that locality or im- prove the economic quality by forcing the planting of plus trees. If a population has been severely attacked by DED and consequently been under the infl uence of a genetic bot- tleneck, it will strive to establish as soon as possible a new, lower mutation-drift equilibrium by the process of expan- sion. Although the incidence of DED in Croatia is high, the parameters of genetic diversity of observed populations do not deviate signifi cantly from the ideal state of HW equilib- rium. Consequently, one could conclude that the disease has not had a signifi cant impact on the degree of genetic diversity, i.e. on the stability of genetic resources of the fi eld elm in Croatia. However, if we take into account the fact that genetic diversity in natural populations of forest trees is very slowly decreasing, as well as the presence of a very small number of adult trees of the fi eld elm in the re- searched area, concern about the stability of this species’ genofund acquires a new dimension. This study, conducted in Croatia, revealed a high genet- ic diversity of the fi eld elm at the intrapopulation level, but also discovered a signifi cant differentiation at the interpop- ulation level. Although the separation of populations at such a high level could generate certain taxonomic implica- tions, the fi nal decision about the taxonomic validity of hy- pothetical entities i.e. conservation units within the U. mi- nor s. l. complex in Croatia is possible solely if this issue is resolved on the European level. In such research, a system- atic study of fructifi cation, modalities of dispersion and the monitoring of phenological phenomena over a certain time period would be necessary. Acknowledgements This research was carried out with the fi nancial support of the Ministry of Science, Education and Sports of the Re- public of Croatia. Grant No. 068-0242108-2773 and partly Grant No. 119-1191193-1232. References Belkhir, K., Borsa, P., Chikhi, L., Raufaste, N., Bonhomme, F., 2004: GENETIX 4.05, logiciel sous Windows TM pour la gé- nétique des populations. Laboratoire Génome, Populations, Interactions, CNRS UMR 5000, Université de Montpellier II, Montpellier, France. 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