Genetic Diversity of Pedunculate Oak (Quercus robur L.) in Clonal Seed Orchards in Croatia, Assessed by Nuclear and Chloroplast Microsatellites

Background and Purpose: Natural stands of pedunculate oak in Croatia have been delineated in seed areas, zones and regions. The current bylaw recommends that the transfer of reproductive material remains limited within zones, but that it is permitted within areas. Clonal seed orchards (CSOs) of pedunculate oak were established to increase genetic quality of seed and to acquire a more regular seed yield than in natural stands. In total 150 plus trees were selected within three seed regions. The selection included a number of favourable traits of tree size and stem quality. Three CSOs were planted with grafted plus-trees. We aimed to establish whether these orchards encompass enough genetic diversity to potentially produce genetically improved and sufficiently diverse reproductive material. We also wanted to assess neutral genetic differentiation between these orchards and compare it with the genetic diversity obtained from chloroplast DNA markers, depicting conserved lineages from recolonization routes. We wanted to investigate spatial genetic structure in the area of our research and provide additional information on the transfer of forest reproductive material. Materials and Methods: Leaves were collected from all clones in the CSOs. Total genomic DNA was extracted and clones were analysed with eight nuclear and ten chloroplast microsatellite markers. Spatial autocorrelation analysis was performed with nuclear microsatellite data and original plus trees’ coordinates, for each CSO separately, to determine whether shared favourable traits among the selected plus trees in smaller distances are the results of relatedness, which the sampling strategy tried to avoid. Results: We found 28 chloroplast haplotypes belonging to four maternal lineages, and significant differentiation between CSOs, indicating origin from different refuges. Nuclear microsatellites’ diversity in the CSOs is quite high and comparable to diversity found within a recent study of Croatian natural populations. Nuclear microsatellites did not show genetic differentiation between CSOs, i.e. between the seed regions and seed zones they represent. No genetic differentiation was found with nuclear microsatellites among haplotypic lineages. We found no genetic structure within the analysed regions. Conclusions: Lack of differentiation between CSOs found with nuclear microsatellites confirms the permission for transfer of reproductive material between zones within the seed area 1 - Lowland Forests. If original differentiation between chloroplast haplotypic lineages was present after recolonization, it was erased by subsequent gene flow. Lack of genetic structure, with nuclear microsatellites within regions indicates successful sampling strategy

Variability of clones in acorn production and its effect on effective population sizes and genetic diversity of crops in clonal seed orchards of pedunculate oak (Quercus robur L.) in Croatia

U ovom radu željeli smo ispitati različite parametre koji nam daju informaciju o učinkovitosti plantaža u proizvodnji genetski raznolikog potomstva. Koristili smo raspoložive inventurne podatke tvrtke “Hrvatske šume” d.o.o. o brojnom stanju i starosti rameta, te zabilježenim urodima u dvije klonske sjemenske plantaže hrasta lužnjaka; KSP “Plešćice” i  KSP “Petkovac”. U slučaju KSP „Plešćice” korišteni su inventurni podaci, te podaci o urodu za 2003.,2006.,2007.,2008.,2009. i 2010. godinu. Nakon 2010. godine zbog organizacijskih se razloga prestalo pratiti urod po klonovima i prešlo se na evidentiranje ukupnog uroda na plantaži, pa je za period 2010.-2017. godine nemoguće procjeniti genetsku raznolikost uroda. U slučaju KSP „Petkovac” korišteni su inventurni podaci, te podaci o urodu za 2003.,2004.,2005.,2006.,2008.,2009. i 2010. godinu. U slučaju KSP „Petkovac” također se nakon 2010. godine napustilo evidentiranje uroda po klonovima.Količine i varijabilnost uroda žira po godinama, kao i projekcije uroda za 2018. i 2019. godinu na temelju polinomijalnih regresijskih krivulja izračunali smo za sve godine evidentiranog uroda za cijelo razdoblje 2003. – 2017. godina. Rezultati su prikazani u tablici 1 i na slikama 1 i 2. Sve druge analize bile su moguće samo za godine kada se sakupljanje žira u plantažama provodilo odvojeno po klonovima. Korelacijama po metodama Pearsona i Spearmana usporedili smo raspodjelu uroda žira između parova istraživanih godina po klonovima, odnosno poredak udjela klonova u urodima žira između istraživanih godina. Obje korelacije bile su većinom statististički značajne između parova godina, sa ponekim iznimkama (tablica 2).Ravnoteža uroda klonova po godinama, opisana kumulativnim krivuljama proizvodnje žira po godinama, po postocima rodnih klonova, prikazana je na slikama 3 i 4. Krivulje ravnoteže uroda klonova za obje plantaže prikazuju izrazito neravnomjernu raspodjelu uroda po klonova, što znači da je manjina klonova u istraživanim godinama doprinosila većinskom količinom uroda. U uvjetima jednake plodnosti i sinkroniziranosti klonova doprinos u urodu bio bi razmjeran zastupljenosti klonova (clone size) tj. klonovi s većim brojem rameta značajnije bi doprinosili urodu. Iz tablice 3. može se primjetiti da je u slučaju KSP “Plešćice” ova korelacija bila slabija nego kod KSP “Petkovac”. Dapače, u tri od šest promatranih godina ona uopće nije bila značajna. U slučaju KSP “Petkovac” korelacija je bila značajna u svim promatranim godinamaProcjena efektivnih veličina populacije i genetske raznolikosti potomstva po godinama prikazani su u tablici 4. isključivo za žensku plodnost klonova.  Prilikom izračuna doprinosa oba roditelja muška plodnost procijenjena je pomoću tri scenarija koja obuhvaćaju raznolik raspon potencijalnog muškog doprinosa u efektivnoj veličini populacije. Rezultati su prikazani u tablici 5. Usporedbom tri scenarija muškog doprinosa ukupnom efektivnom tj. statusnom broju (Np) vidljivo je da su vrijednosti za prvi ,gdje je muški doprinos razmjeran udjelu rameta datog klona u ukupnom broju rameta, intermedijarne u odnosu na druga dva scenarija. Najniži efektivni brojevi roditelja (Np), a time i najniži relativni efektivni brojevi (Nr), te najviše vrijednosti koeficijenta grupnog srodstva (Θ) dobivene su za drugi scenarij u kojem je muški doprinos jednak ženskome. Te su vrijednosti identične vrijednostima za samo žensku plodnost. Najviši su efektivni brojevi i analogno druge vrijednosti dobivene za treći scenarij u kojem se pretpostavlja jednak muški doprinos svih klonova. Za obje plantaže korelacija poretka ženskih efektivnih brojeva klonova za pojedine godine uroda, sa poretkom za količine uroda tih godina (po metodi Spearmana) je statistički značajna na razini p < 0,05.Za primjenu mjera procjene genetske raznolikosti proizvedena u klonskim sjemenskim plantažama potrebna su sukcesivna višegodišnja praćenja cvjetanja/uroda, kao i saznanja o genetskoj dobiti dobivena testiranjem potomstva i uzgojne vrijednosti klonova u nedavno postavljenim testovima potomstva iz plantaža. Zbog svega toga je apsolutno preporučljivo da se daljnje sakupljanje uroda u ovim klonskim sjemenskim plantaža obavlja odvojeno po klonovima, jer se jedino na taj način, bez skupih molekularnih analiza, može doći do relevantnih informacija o doprinosu pojedinih klonova genetskoj raznolikosti potomstva.In this study we wanted to investigate different parameters providing informations about the efficassy of orchards in producing genetically diverse progeny. We used available inventory data from the company „Croatian Forest” ltd. about the numbers and the age of ramets, as well as evidented crops in two clonal seed orchards (CSOs) of pedunculate oak : CSO „Plešćice” and CSO „Petkovac”. For CSO „Plešćice” we used inventory data on ramets and crops for the years 2003, 2006, 2007, 2008, 2009 and 2010. and for CSO „Petkovac” for the years 2003, 2004, 2005, 2006, 2008, 2009 and 2010. After 2010 the collection of crops per clone was abandoned in both orchards and we only have data for overall crops in the orchards, until the year 2017.We calculated the amounts and variability of acorn crops in different years, as well as projections of crops for years 2018 and 2019 based on polynomial regression curves, for all years of evidented crops between years 2003 and 2017. Results are shown in Table 1 and Figures 1 and 2. All other analysis were possible only for years in which crops were collected separately by clones.  We used Pearson’s product-moment and Spearman’s rank correlations to compare distributions of crops per clones between pairs of investigated years, ie. rank of clones in crops production between investigated years. Both correlations were mostly statistically significant with some exceptions (Table 2) Clone fructification balance described by cummulative curves of acorn production in different years, per percentage of fructifying clones is shown in Figures 3 and 4. Clone balance curves in both orchards show irregular distribution of crops per clones, meaning that in investigated years minority of clones contributed to majority of crops. In conditions of equal clone fertility and maximal sinchronicity of clones, clone’s contribution to crops would be in concordance with the number of its ramets (clone size). Table 3 shows that in CSO Plešćice this correlation was weaker then in CSO Petkovac and, unlike in CSO Petkovac, not significant in all years.Estimations of effective population sizes and genetic diversity of crops in different years is shown for female clone fertility in Table 4. For calculations of biparental contribution of clones, we used three scenarios encompassing various versions of potential male contribution in effective population sizes. Results are shown in Table 5. The values of effective (status) number of parents (Np) for the first scenario, where male contribution is proportionate to the percentage of clone’s ramets in the overall number of ramets of all clones, is intermediate between the other two scenarios. Lowest effective (status) number of parents (Np) and highest values of group ancestry coefficients (Θ) result from the second scenario where male contribution is equal to female. The highest effective numbers are gained for third scenario where equal male contribution of all clones is presumed.  In both CSOs, Spearman rank correlation between female effective numbers (Nf) and overall crops in different investigated years is statistically significant at p < 0,05 level. These two CSOs are still filled with new ramets and are still relatively young. With increasing age and ramets’ tree sizes better results in crops and its genetic diversity are expected. For implementation of measures of crops genetic diversity estimations, it is necessary to monitor successively flowering and fructification in the CSOs through the years. The monitoring can then be combined with knowledge on genetic gain and clones’ breeding values obtained through recently established CSOs progeny trials. Therefore, we recommend collection of CSOs’ crops separately by clones, because without expensive molecular analysis it is the only way to get relevant information of individual clones contribution to genetic diversity of crops produced in the CSOs

On the use of foreign forest reproductive material for the regeration of local stands of pedunculate oak from the stand points of a genetics

Uslijed kronične nestašice žira domaćeg podrijetla, na tržištu je tijekom 2023. godine nabavljena velika količinašumskog reprodukcijskog materijala (ŠRM-a) iz inozemstva, a njegovo korištenje za obnovu sastojina hrastalužnjaka u Republici Hrvatskoj (RH) je u tijeku. U neslužbenim su se komunikacijama kao izvori unesenogŠRM-a spominjale provenijencije iz Mađarske, Rumunjske ili Francuske. U konačnici su, koliko nam je poznato,nabavljene sadnice i žir iz Mađarske. Međutim, stvarno podrijetlo uvezenog ŠRM-a nije niti važno jer jeovo priopćenje uglavnom načelnog karaktera i može poslužiti kao uputa za odlučivanje o njegovoj prikladnosti,neovisno o podrijetlu ili vrsti šumskog drveća.Potrebno je naglasiti da je postupak premještanja ŠRM-a (unutar EU) odnosno njegovog uvoza (iz trećih država),te korištenja u RH, reguliran Zakonom o šumskom reprodukcijskom materijalu (Narodne novine 75/09,61/11, 56/13, 14/14, 32/19, 98/19). Navedeni zakon zadovoljavajuće dobro određuje okvir za postupak odlučivanjao tome kakve provenijencije mogu biti prikladne kao izvor ŠRM-a i gdje ih koristiti, u slučaju potrebe.Nažalost, smatramo da postupak odlučivanja o nabavi i korištenju ŠRM-a, primijenjen ove godine, nije odgovaraonekim temeljnim stručnim postavkama. Primijenjena praksa predstavlja visoki rizik za očuvanje jedinstvenosti,bioraznolikosti i produktivnosti naših sastojina hrasta lužnjaka. Cilj je ovog priopćenja ukazati naneke propuste, pojasniti rizike takve prakse, pružiti savjetodavni doprinos iz perspektive naše specijalnosti –genetike šumskog drveća, i time pomoći da se u budućnosti za obnovu naših sastojina koristi prikladni ŠRM

Use of a Common Garden Experiment in Selecting Adapted Beech Provenances for Artificial Stand Restoration

Increased frequency of extreme weather events has seriously affected forestry operations in south-eastern Europe. A precondition for effective artificial restoration of disturbed forest stands is site-adapted forest reproductive material (FRM). Common garden experiments (provenance trials) may assist in selecting such FRM. The main objective of this study was to establish among-provenance variation pattern using data from a beech provenance trial. Usefulness of the results in selecting seed sources for restoration of European beech stands is discussed. The trial was set up in 2007, at a slope of Medvednica mount facing north-west at 730-750 m above sea level. Plant heights were measured and survival scored in 2008 and 2015. Height increments were calculated and processed to determine variance components due to various effects. Highly significant provenance-by-block interaction was revealed, indicating strong microsite effects on provenance performances. Therefore, corrections were made and provenance mean height increments recalculated. Provenance mean height increment multiplied with survival was used as a measure of a provenance’s adaptedness. Regression tree (RT) analysis was used to determine the pattern of among-provenance variations. A set of provenance clus­ters was grown using climatic variables related to the provenance stands of origin as criteria. All analyzed effects were significant (provenance: F=2.07, p750m), had the highest mean AI (143.9±8.4 cm). The lowest mean AI (106.7±14.8 cm) had cluster 1, containing provenances from lower altitudes with lower mean July temperatures (≤18.4°C). Provenances originating from lower elevations with higher mean July temperatures (>18.4°C) were further divided into two clusters due to the SHM variable. Cluster 2 had the second highest mean AI (141.2±1.5 cm) and contained provenances from relatively wetter habitats (SHM≤48.2). Cluster 3, containing provenances from relatively arid habitats (SHM>48.2), had significantly lower mean AI (116.8±8.6 cm). Established among-provenance variation pattern might be used as a tool in selecting seed sources for artificial restoration of beech stands at mount Medvednica. It is advisable to use FRM from higher altitudes and/or from slightly lower altitudes (up to 150 m lower than a restoring site) but featured with warmer and drier conditions compared to the trial. Generally, provenance trials should be utilized as a valuable decision tool in restoring disturbed forest stands but may also be misleading if not well designed and analyzed

Određivanje teških metala u čestim vrstama povrća na Varaždinskoj gradskoj tržnici

The present study was aimed at the estimation of heavy metal content in vegetables sold at the city market of one of the densely populated Croatian cities, Varaždin, and to establish the relationship between their levels and possible sources of contamination. Twenty-eight samples of the most common diet vegetables (red and white potato, onion, carrot, common bean, lettuce, and cabbage) were randomly bought at the market in September and October 2013. Using the atomic absorption spectrometry method, concentrations of nine heavy metals (As, Cd, Cr, Cu, Hg, Mn, Ni, Pb, and Zn) were measured in the selected samples. The results showed that, in five out of 28 samples analysed, six concentrations exceeded the maximum levels provided for in the regulations: five for Pb and one for Cd. Maximum regulated levels for Pb were exceeded in two samples of red potato, two samples of common bean, and one sample of carrot (17.9 %), and for Cd in a sample of red potato (3.6 %). In conclusion, the cause of the overstepping of the maximum levels for Pb and Cd in the vegetables analysed was most likely the contaminated soil. The possible sources of soil contamination include traffic, nearby industry, floodwaters of rivers and streams, and the use of pesticides and fertilisers in agricultural production.Cilj istraživanja bio je procijeniti sadržaj teških metala u povrću dostupnom na gradskoj tržnici jednog od gušće naseljenih hrvatskih gradova, Varaždina, te ispitati povezanost koncentracija teških metala s mogućim izvorima onečišćenja. Ukupno 28 uzoraka često konzumiranog povrća (crveni i bijeli krumpir, luk, mrkva, grah, salata i kupus) nasumično je kupljeno na Varaždinskoj tržnici u rujnu i listopadu 2013. godine. Koncentracije devet teških metala (As, Cd, Cr, Cu, Hg, Mn, Ni, Pb i Zn) izmjerene su metodom atomske apsorpcijske spektrometrije. U pet od 28 ispitanih uzoraka povrća, šest je koncentracija teških metala bilo iznad najvećih dopuštenih količina: pet za Pb i jedna za Cd. Najveće dopuštene količine Pb prekoračene su u dva uzorka crvenog krumpira, dva uzorka graha i jednom uzorku mrkve (17,9 %), a Cd u jednom uzorku crvenog krumpira (3,6 %). Uzrok prekoračenja najvećih dopuštenih količina Pb i Cd u uzorcima povrća onečišćeno je tlo. Mogući uzroci onečišćenja tla uključuju promet, blizinu industrije, poplavne vode rijeka i potoka te upotrebu pesticida i mineralnih gnojiva u poljoprivrednoj proizvodnji

GENERATIVE AND VEGETATIVE ACTIVITY OF WILD CHERRY (Prunus avium L.) IN A CLONAL SEED ORCHARD

Divlja trešnja (Prunus avium L.) vrlo je vrijedna vrsta šumskog drveća koja raste u prekinutom arealu u mješovitim šumama južne, središnje i zapadne Europe U nekoliko zemalja pokrenuti su dugoročni programi oplemenjivanja ove vrste, namijenjeni poboljšanju kvalitete i proizvodnje njenog drveta. U Hrvatskoj je provedena selekcija 27 stabala divlje trešnje na osnovi osam fenotipskih kriterija i osnovana je klonska sjemenska plantaža na području šumarije Kutina. Za potrebe ovog istraživanja u plantaži je odabran uzorak od 14 klonova, predstavljenih sa po četiri ramete. Na odabranim rametama izvršena su mjerenja tijekom dvije godine ; 2010. i 2011. Za procjenu vegetativne aktivnosti vršene su izmjere promjera na 50 cm visine debla. Za procjenu generativne aktivnosti (cvatnje i plodonošenja) na svakoj je rameti odabrana i obilježena po jedna primjerna grana. Izmjerena je puna dužina odabrane grane i svih njenih izbojaka koji su nosili cvjetove i plodove. Na primjernim su granama, na svakoj rameti, u 2010. i 2011. godini u travnju prebrojani svi cvjetovi. U svibnju su na primjernim granama prebrojani svi plodovi. Broj cvjetova ili plodova je za sve izmjerene ramete sveden na 100 cm dužine grane (Slika 1, Slika 3). Zametanje plodova (Fruit set) izračunato je kao omjer cvjetova i plodova (Slika 4). Za sva svojstva prikazani su prosječni koeficijenti unutarklonske varijabilnosti za obje godine (Slika 2). Na temelju meteoroloških podataka za Kutinu 2009., 2010. i 2011. godine izračunati su parametri zadovoljenja potreba biljaka za zimskim inaktivnim temperaturama (Winter chilling), kao i proljetnim temperaturama, potrebnim za pokretanje sokova i početak vegetacijskog perioda (Forcing), po Luedeling i dr. 2013. (The Chilling Hours Model, The Utah Model za „Winter chilling“ i Growing Degree Hours Model za „Forcing“)(Tablica 2). Cilj istraživanja bio je utvrditi raznolikost nekih reproduktivnih svojstava na uzorku grupe klonova divlje trešnje iz klonske sjemenske plantaže Kutina, utvrditi međusoban odnos tih svojstava i odnos sa svojstvima vegetativnog rasta. Pritom se nastojalo zapažene odnose staviti u kontekst podataka o okolišnim uvjetima u vrijeme cvatnje i plodonošenja. Od promatranih generativnih svojstava klonovi su se statistički značajno razlikovali u broju plodova na 100 cm i po zametanju plodova, gledano zbirno za dvije godine (Tablica 1). Te su razlike bile uzrokovane razlikama između klonova s ekstremnim vrijednostima, dok se većina ostalih klonova međusobno nije statistički značajno razlikovala (Tukey Kramer test). Sveukupne nepovoljne okolišne prilike 2011. godine (nedovoljna količina zimskog hlađenja, previsoke temperature i suša u proljeće) rezultirali su slabijim prosječnim zametanjem plodova, nego u 2010. godini, kada su sve navedene prilike bile povoljnije (Tablica 3). Vrijednosti zametanja plodova za 2010. i 2011. godinu bile su u skladu s drugim istraživanjima ili čak više, što upućuje na zadovoljavajući rodni potencijal ovih klonova u slučaju povoljnih vremenskih uvjeta, prisutnosti polinatora i pravovremenog suzbijanja štetnika. Pronađene su visoko statistički značajne korelacije između broja cvjetova na 100 cm i broja plodova na 100 cm, kao i između zametanja plodova i broja plodova na 100 cm za 2010. i 2011. godinu. U ovom istraživanju nisu pronađene statistički značajne korelacije između vegetativnog rasta promjera cijele biljke i reproduktivnih svojstava procijenjenih pomoću primjernih grana (Tablica 3).Wild cherry (Prunus avium L.) is a valuable forest tree species, discontinuously distributed in mixed forests of southern, central and western Europe. Several countries established long-term breeding programs for Wild cherry, to improve the quality and production of its wood. In Croatia, 27 wild cherry trees were selected, on eight phenotypic criteria. These were cloned by grafting and a clonal seed orchard was established in the area of the Forest Office Kutina. For this research, we monitored 14 clones presented with four ramets. Selected ramets were measured over two years; 2010 and 2011. To estimate the vegetative growth we measured the 50 cm height diameter. To estimate the generative activities, one exemplary branch was marked on each ramet. Each selected branch was measured in full length, including the length of its flower and fruit bearing shoots. In April 2010 and 2011 all the flowers were counted on each exemplary branch. In May, all the fruits. The number of flowers and fruits was reduced to 100 cm length (Figure 1, Figure 3). Fruit set was calculated as the ratio of flowers and fruits (Figure 4). For all the traits average coefficients of intraclonal variability are shown for both years (Figure 2). Based on meteorological data for station Kutina in 2009, 2010 and 2011 we calculated parameters for winter inactive temperatures (Winter chilling), and spring temperatures, necessary for the beginning of the vegetation period (Forcing). We used The Chilling Hours model and the Utah model for "Winter chilling" and Growing Degree Days Model for "Forcing" (Luedeling et al. 2013), (Table 2)). The aim of this study was to determine the diversity of some reproductive traits in a sample of wild cherry clones in clonal seed orchard Kutina, correlation between these traits and correlation with vegetative growth. Another aim was to put these relationships into context of the environmental conditions at the time of flowering and fruiting. Statistically significant differences between clones were found for Number of fruits per 100 cm and Fruit set, viewed cumulatively for two years (Table 1). The statistical significance was caused by differences between clones with extreme values, while most other clones did not differ significantly (Tukey Kramer test). Overall adverse environmental conditions in 2011 (insufficient amount of winter chilling, excessive temperatures and drought in the spring) resulted in lower average fruit set then in 2010, when all of these conditions were more favourable (Table 3). The values ​​of fruit set for 2010 and 2011 were in line with other research, or better, which indicates a satisfactory yield potential of these clones in case of favourable weather conditions, presence of pollinators and pest control. We found statistically significant correlation between Number of flowers per 100 cm and the Number of fruits per 100 cm, and between Fruit set and the Number of fruit per 100 cm for 2010 and 2011. In this study we found no statistically significant correlation between the vegetative growth of the diameter of the whole plant and reproductive characteristics estimated using exemplary branch (Table 3)

VARIABILITY OF HEIGHT GROWTH AND SURVIVAL OF PROGENIES FROM PEDUNCULATE OAK (Quercus robur L.) SEED STANDS AT THE FIELD TRIAL ‘JASTREBARSKI LUGOVI’ – FIRST RESULTS

Dosadašnja istraživanja morfoloških i fizioloških svojstava hrasta lužnjaka u Hrvatskoj ukazala su na genetsku diferencijaciju lokalnih populacija, kao i visok stupanj genetske raznolikosti unutar svake populacije. Kako bi se dodatno istražila genetska raznolikost i diferencijacija populacija hrasta lužnjaka u Hrvatskoj, analizirani su visine i preživljenje u novoosnovanom genetičkom testu potomstva iz 16 sjemenskih sastojina i jedne gospodarske u dobi od četiri odnosno pet godina. Preživljenje u prvoj analiziranoj godini bilo je izuzetno visoko kod svih populacija, no nakon sljedećeg vegetacijskog perioda uočen je značajniji pad preživljenja. Analizom varijance za svojstvo visina za dvije uzastopne godine (2010. i 2011.), utvrđene su statistički značajne razlike između populacija. Ovisno o položaju unutar genetičkog testa za istu populaciju ili familiju utvrđeno je i različito "rasipanje" visina oko prosjeka. Proveden je Tukey-Kramer test najmanjih kvadratnih razlika za srednje visine populacija s ciljem utvrđivanja njihove povezanosti, odnosno utvrđivanja eventualnog postojanja geografskog obrasca genetske diferencijacije. Populacija HR 88 (UŠP Našice, Šumarija Koška, G.j. Lacić-Gložđe) se u obje godine diferencirala kao prosječno najviša, dok su se ostale izdvojene grupe populacija međusobno višestruko preklapale. Uspoređujući signifikantnost razlika u prosječnim visinama između populacija s njihovim geografskim položajem nije se mogao uočiti geografski obrazac diferencijacije. Zbog utvrđenog statistički visoko značajnog efekta blokova provedena je analiza fenotipske plastičnosti istraživanih populacija s obzirom na svojstvo visine u dobi od 5 godina (PIv, RDPI), no nije utvrđen geografski obrazac razdvajanja populacija. Populacije su kategorizirane u fenotipski stabilne, nestabilne te prosječne fenotipske plastičnosti s različitim stupnjevima prilagođenosti na testne stanišne uvjete koji su vladali u analiziranim godinama. Za pomlađivanje i pošumljavanje sličnih staništa kao što je bilo stanište pokusnog nasada preporučuje se korištenje šumskog reprodukcijskog materijala iz sjemenskih sastojina, čije je potomstvo iskazalo prosječnu do visoku fenotipsku stabilnost s visokim stupnjem prilagođenosti (u smislu preživljenja i visinskog rasta). Rezultate ovog istraživanja treba shvatiti kao preliminarne i poticaj za nastavak istraživanja.Previous studies of morphological and physiological trials of pedunculate oak in Croatia revealed genetic differentiation of local populations as well as high degrees of genetic diversity within each population. For further research of genetic diversity and differentiation of oak populations in Croatia, height and survival were analysed in a newly genetic test which contains progeny from 16 seed stands and one regular management forest stand (Table 1) at the age of four and five years, respectively (Figure 1 and 2). Survival for first analysed year was extremely good, but after the next vegetation period visibly decreased. Analysis of variance for mean height of two consecutive years (2010th and 2011th) revealed significant differences between populations (Table 2 and 3). Depending on the position within the genetic test same populations or families had different "dispersal" of mean height. We conducted a Tukey-Kramer test of the least square mean difference of population heights in order to determine their relationship, and to determine a possible geographic pattern of genetic differentiation (Table 5 and 6). Population HR 88 (FA Našice, Forestry Office Koška, Working unit Lacić-Gložđe) in both years differentiated as the highest of all, while other isolated groups of populations multiple overlapped. Comparing the significant difference in average height between populations and their geographic location geographic pattern of differentiation could not be observed (Table 5 and 6). An analysis of phenotypic plasticity of the studied populations for population height at age of 5 years was performed (Table 7) because of statistically significant effect of blocks. No geographic pattern of differentiation between populations was observed for the plasticity indices (PIv, RDPI) (Table 8). Populations were categorized into phenotypically stable, unstable, and with average phenotypic plasticity with varying degrees of adaptedness to the test site conditions that prevailed in the analysed years. For reforestation and afforestation of similar habitats as was at the field trial it is recommended to use forest reproductive material from seed stands whose progeny expressed average to high phenotypic stability with a high degree of adaptedness (in terms of survival and height growth). The results of this study should be considered as preliminary and stimulus for further research

Acorn Morphometric Traits and Seedling Heights Variation of Pedunculate Oak (Quercus robur L.) from the Seed Stands in Croatia

Radi verifikacije važećih sjemenskih zona hrasta lužnjaka u Hrvatskoj na temelju znanstvenih spoznaja, započela su nova istraživanja genetske varijabilnosti u području prirodnog areala ove vrste. U jesen 2006. godine prikupljeno je sjeme iz tzv. priznatih sjemenskih sastojina, čija je osnovna uloga izvorište kvalitetnijeg sjemena za potrebe umjetne obnove gospodarskih sastojina. U radu se prikazuju rezultati morfometrijske analize sjemena i visina jednogodišnjih sadnica podrijetlom iz 17 uzorkovanih sastojina, koje reprezentiraju cjelokupni areal hrasta lužnjaka u Hrvatskoj. Iz svake je sastojine prikupljeno sjeme od 25 stabala, a na uzorku od 30 sjemenki po stablu izmjereni su duljina, maksimalna širina i masa žira, te su na temelju duljine i širine izračunati volumen žira (prema formuli za izračunavanje volumena valjka) i indeks žira (omjer duljine i širine). U rasadniku Hrvatskog šumarskog instituta osnovan je rani, rasadnički test, te su tijekom jeseni i zime 2007. godine, starosti biljaka 1+0, obavljene prve izmjere visina. Analiza varijance provedena je MIXED procedurom u SAS-u, a analizirani su: fiksni učinak važećih sjemenskih zona te slučajni učinci populacija unutar zona i stabala unutar populacija. Cilj analize varijance bio je utvrditi značajnost međuzonskih, međupopulacijskih i unutarpopulacijskih razlika za istraživana svojstva sjemena i visine jednogodišnjih sadnica. Tukey-Kramer testom u SAS-u analiziran je obrazac razlika između uzorkovanih populacija. Rezultati su pokazali da se važeće sjemenske zone nisu statistički značajno razlikovale niti za jedno analizirano svojstvo. Uzorkovane sastojine međusobno su se statistički značajno razlikovale u svim istraživanim svojstvima sjemena. Najveće prosječne dimenzije imali su žirevi iz sjemenske sastojine kasnolistajuće formehrasta lužnjaka Domačaj-Kovačevski lug (Šumarija Karlovac), dok su najmanje dimenzije imali žirevi iz sastojine Česma (Šumarija Bjelovar). Utvrđene međupopulacijske razlike nisu odgovarale važećoj sjemenskoj razdjelbi, tj. nisu potvrdile geografski obrazac diferencijacije populacija. Utvrđena je visoko statistički značajna unutarpopulacijska varijabilnost za istraživana svojstva sjemena (razlike između stabala odnosno familija unutar populacija). Prosječne vrijednosti visine jednogodišnjih sadnica bile su najniže kod populacije Česma (Šumarija Vrbovec) i Repaš Gabajeva greda (ŠumarijaRepaš), dok su najviše bile kod populacije Lacić Gložđe (Šumarija Koška) iRečićki lugovi (Šumarija Karlovac). I za svojstvo visine uočena je statstički značajna diferencijacija između populacija, iako je još veći stupanj varijabilnosti utvrđen između familija unutar svake populacije. Provedena je analiza s ciljem utvrđivanja postojanja geografskog obrasca međupopulacijske diferencijacije. Međutim niti za svojstvo visine sadnica nije se mogao uočiti geografski obrazac diferencijacije analiziranih populacija. Provedena korelacijska analiza između visina jednogodišnjih sadnica i morfometrijskih svojstava sjemena nije pokazala statistički značajnu povezanost.Seed zone delineation of pedunculate oak in Croatia was done according to ecological differences, stand productivity and administrative borders of Forest Range Offices (NN 107/08). Oak forests are divided into three seed zones and it is recommended to transfer reproductive material only within zones. Seed delineation was preformed due to Croatian Forest Reproductive Material Transfer Law (NN 75/09) that was aligned with EU Directive on forests reproductive material (1999/105/EC). Seed delineation should be done according to genetic differences between various zones i.e. provenance regions. Aiming at verification of current seed zones in Croatia, and on the basis of general knowledge on pedunculate oak genetic variation, research on genetic variation of the species in Croatia has been started. Due to that, seeds were collected within so called registrated seed stands which are normally used as a seed sources for regeneration of managed oak stands. Results of the study of seed morphometric analyses are presented in this paper. Seed were collected during autumn 2006 below 25 trees within each of 17 seed stands that represented whole distribution range of the species in Croatia (Table 1). 30 acorn from each tree were sampled for morphometric analysis and measured the height of seedlings. Acorn length, widths and masses and seedlings height were measured and acorn volumes were calculated according to formula for cylinder volume (Figure 1, 2). ANOVA was performed by MIXED procedure in SAS, in order to analyze significance of various effects. Results showed that current seed zones were not statistically significant effect (there were no significant differences between them), but there were significant differences between populations within zones, as well as between trees within populations (Table 2). Table 3 shows Type 3 significance F-test for fixed effect of populations within seed zones for studied acorn traits and for one-year seedling height. Determined populations differences did not match current seed delineation i.e. there were no obvious geographic pattern in between population variability for studied acorn traits as well as analyzed seedlings traits. Tukey-Kramer’s test was performed to determine the form and the differentiation which populations are separated statistically significantly. Test did not give results from which we could observe regularity in the geographical separation of populations for analyzed seedlings traits (Table 5). Population HR 88 (Lacići-Gložđe, Koška) was significantly differed from the geographically closed population HR HR 58 (Darda), HR 577 (Poljadijske šume, Požega) and HR 627 (Repaš), while at the same time did not differ from the distant population (HR 387 – Karlovac, Rečićki lugovi and HR 609 – Buzet). Statistically significant differences between populations for all analyzed traits clearly indicate the genetic differentiation of oak populations. However interpopulation differentiation did not correspond to the geographical pattern. Specific character of differentiation indicates ecotypic pattern of genetic differences between oak populations in Croatia, i.e. differentiation that are caused by adaptation to specific micro-enviromental conditions (e.g. humidity, temperature and edaphic features). To confirm the hypothesis of ecotype pattern of genetic diversity is necessary to conduct additional research in genetic tests (provenance and progeny tests). Differences between currently seed zones were not statistically significant for any of the analyzed traits, which has not confirmed valid justification of current seed delineation. Nevertheless, it should be noted that the analysis of seed morphometric traits are not adaptively valuable properties, while the results of the one-year seedling heights are not sufficiently reliable due to a very early age of plants. Therefore these results should be regarded only as a stimulus for further research of oak genetic diversity in Croatia

Kvantitativna genetska diferencijacija populacija hrasta lužnjaka (Quercus robur L.) u pokusnom nasadu »Jastrebarski lugovi«

Hrast lužnjak, kao jedna od triju najrasprostranjenijih vrsta drveća u Republici Hrvatskoj, ima važnu ulogu u ekonomsko-socijalnom i ekološkom smislu. Poznavanje genetske strukture tako značajne vrste šumskoga drveća temeljni je preduvjet njezina očuvanja za budućnost. Jer to, poznavanje genetske strukture, omogućuje naprednije korištenje šumskoga reprodukcijskoga materijala hrasta lužnjaka radi povećanja prilagodljivosti populacija na stresne okolišne prilike. Analiza kvantitativnih (poligenih, metričkih) fenotipskih svojstava u genetičkim testovima jedna je od metoda utvrđivanja genetske strukture vrsta šumskoga drveća. Iz šesnaest sjemenskih i jedne gospodarske sastojine koje reprezentiraju cjelokupan areal hrasta lužnjaka u Hrvatskoj, u rujnu i listopadu 2006. godine skupljen je sjemenski materijal. S dvogodišnjim potomstvom 2008. godine osnovan je genetički pokusni nasad u gospodarskoj jedinici »Jastrebarski lugovi«. Mjeren je visinski rast i određivani su preživljavanje i visinski prirast u trima uzastopnim godinama (2010, 2011. i 2012). Analiza varijance provedena je procedurom PROC MIXED u statističkom paketu SAS te su izračunate komponente varijance i statistička značajnost slučajnih efekata, tj. izvora varijabilnosti (blokovi, populacije, familije unutar populacija, interakcija blokova i populacija te interakcija blokova s familijama unutar populacija). Izračunate komponente varijance poslužile su za izračunavanje parametra QST kojim se određuje razina međupopulacijske genetske diferencijacije. Multivarijantna regresijska stabalna analiza (engl. MRT) korištena je za određivanje obrasca genetske diferencijacije populacija u nasadu, a s obzirom na okolišne (klimatske) varijable njihovih izvornih matičnih sastojina. Parametar QST bio je visok kod sva tri analizirana svojstva, što jasno upućuje na međupopulacijsku genetsku diferencijaciju hrvatskih populacija hrasta lužnjaka. Utvrđeni obrazac adaptivne genetske diferencijacije potvrđuje ekotipski obrazac, odnosno najvjerojatnije je prirodna selekcija u matičnim sastojinama (ponajprije s obzirom na vlažnost i toplinu staništa) uzrokovala genetsku izdiferenciranost lokalnih populacija