View metadata,citationandsimilarpapersatcore.ac.uk This article isThis article by protected copyright. All rightsreserved. 10.1002/cbdv.201700093doi: differences to lead between the of thisversion c and Version Record.Please copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review buthasnot [email protected] * Corresponding phone: author: +381 “ Terpenes are useful markers indifferentiationTerpenes areuseful ofnatural Acceptede Article Univers d c f Zorica S.MitićZorica Institute forInstitute Medici a University University of , University University of Faculty of Belgrade, ofBotanyBiology, Institute andBotanical Garden University University of SciencesandMathematics, Niš,Faculty Despota Stefana 142,11060Belgrade, (e ; Ecology, b Institute of Forestry, KnezaInstitute ofForestry, Višeslava 3,11000Belgrade, Serbia(e ity of Institutefor Belgrade, “Siniša Biological Stanković”,Research Boulevard ” , Studentski 16,11000 trg Belgrade, Serbia

Višegradska 33, 18000Niš,Serbia (e , a , * Belgrade, Serbia (e Belgrade,

Biljana Belgrade, Serbia Belgrade, nal Plantnal Pančić”, ResearchTadeuša Košćuška “Dr.Josif 1,11000

Pinus heldreichii Faculty of Faculty Chemistry, Studentski trg16,P.O. 158,11000 Box

M. Bojović Nikolić Article Type:Article FullPaper smikit - 18 - ,

e - (e 533 ,

mail: b and [email protected]) -

Mihailo Mihailo mail: , - P. nigra 015, fax: +381

Petar D. Petar Marin

[email protected] [email protected] S.

and Ristić - mail: [email protected]) - mail:

P. peuce -

(e , c 18 f

- Vele

Department ofBiology and [email protected]) mail: - populations 533 V.

- [email protected] 014, e )

Tešević )

- mail: ite thisarticle as

of relict provided byFacultyofChemistryRepository-Cherry - , mail: d

Srdjan Srdjan

R. brought toyouby )

CORE This article isThis article by protected copyright. All rightsreserved. classifications later all in subgenera distinct two considered bundles), fibrovascular two (with Diploxylon fibrovascu one only (with Haploxylon lineages, two into Hemisphere. Northern the in of Introduction Keywords: isefforts, it tonecessary performmorphological, additional biochemical andgenetic studies. phylog of positions precise high by characterized were monoterpene six Pinus area. same results our hybridize, and similarities morphological nigra P. divergence analyses statistical Multivariate nigra P. C A omparative analysis of terpene diversity and differentiation of differentiation and diversity terpene of analysis omparative Accepted Article bstract ,

ntc re a wl a dvlp utbe osrain taeis n ftr breeding future and strategies conservation suitable develop as well as tree, enetic we assumed that assumed we Pinus and ,

In accordance with recently proposed concept of concept proposed recently with accordance In and among Pinus heldreichii

L., with over 100 extant species, is the largest and the most widespread genus widespread most the and largest the is species, extant 100 over with L., . [3 . peuce P.

s . heldreichii P. - , is more basal than those of hard pines pines hard of those than basal more is ,

6] the species from different from species pine the . h frt subgenus first The . heldreichii P. indicated

the terpene profile of soft pine pine soft of profile terpene the from the the from , otn levels content

P

the the .

differen

nigra showed (subgenus eta Balkan central at that fact , , . nigra P.

P [1] tiation of their of tiation

. ht composi that

peuce A century ago, Shaw ago, century A -

f any sesquiterpene mainly of P. nigra P. Strobus Pinus

,

and Terpenoids subgenera: subgenera: s

). was

sf pns i caatrzd y non by characterized is pines) (soft . peuce P. and n diin d addition, In reflects terpenes needle of tion P. peuce P. populations naturally populations

performed P. nigra P. P. heldreichii P. “ , a bnl i te ede and needle) the in bundle lar flavonic evolution flavonic

P. peuce P. Molecular dive Molecular

[2]

reli , defined by high by defined , within the taxonomic and and taxonomic the within

proposed to split the genus the split to proposed

ct pines ct and

t h pplto level population the at s

. (subgenus

sie h described the espite In order to establish establish to order In P. heldreichii P.

may spontaneously spontaneously may

Pinus heldreichii Pinus rsity growing in the the in growing ” in the genus the in ” Strobus . amounts

, which which ,

clear ) vs. vs. ) of

- . ,

This article isThis article by protected copyright. All rightsreserved. Accepted (subsection that indicated had studies molecular former to contributed undoubtedly have Bosn spontaneous of Discoveries the between position statu taxonomic distinct that revealed nigra contentious been have subgenus the of member a is which , and , Serbia, Macedonia, , Articleand subgenus the the of representatives through both Sea; Black Africa the to eastwards North and Mediterranean from extends that range fragmented highly a with and , southern the in mountains high at areas fragmented occupying naturally subendemic aut fasciclepersistent sheaths subgenus an bases cataphyll the at pulvini decurrent ochthonous pines of Tertiary origin: Tertiary of pines ochthonous iii ia and Herzegovina and ia .

After Shaw, After Mcdna pn ( pine Macedonian ) o a og ie t time, long a For The Strobus -

territory of central Balkans is inhabited by many species, including three three including species, conifer many by inhabited is Balkans central of territory Pinaster Pinus . heldreichii P. ii ) Black pine ( pine Black ) . [7][8]

[2] (hard pines) has decurrent pulvini at the cataphyll bases and usually usually and bases cataphyll the at pulvini decurrent has pines) (hard ) than to the the to than )

who considered Bosnian pine only as a variety of variety a as only pine Bosnian considered who Eurasian and the “ the and Eurasian

particularly , s of Bosnian pine has been confirmed been has pine Bosnian of s ,

. e pnos n hlgntc positions phylogenetic on opinions he as well as several varieties and transitional forms of forms transitional and varieties several as well as [6] . peuce P.

Bosnian pine hybrids with hybrids pine Bosnian a a opeey ifrn tree opsto. lhuh the Although composition. terpene different completely a has P. nigra P. Eurasian hard pine lineage (subsection lineage pine hard Eurasian relations

P. heldreichii P. Griseb),

Arnold), one of the the of one Arnold), true” Mediterranean true” i ) Bosnian pine ( pine Bosnian ) perplexities d deciduous fascicle sheaths, while the second second the while sheaths, fascicle deciduous d is between hips Bla endemic Balkan a

is more closely related t related closely more is . [13] P. nigra P.

Pinus heldreichii Pinus

However, the majority of recent recent of majority the However, most widespread pine widespread most ad pines hard hard pines pines hard

i [11] n all subsequent studies, its studies, subsequent all n of

f h hi the of

and these P. nigra P. . heldreichii P. remains P. mugo P. Pinus relict Christ), a Balkan Balkan a Christ), h onan of mountains gh o Mediterranean Mediterranean ) that includes includes that ) , Mirov , P. heldreichii P.

debatable pine s in Europe, Europe, in s

Turra N

orthern species and [9] Pinus [12]

first .

[10] P. in ,

This article isThis article by protected copyright. All rightsreserved. the pinetowards black Montenegro, that considering inhabiting the define to order in pattern terpene the use to intends study te heldreichii conditi necessary the are isolation terpene and material storing and harvesting in procedure to procedure. isolation terpene particular ecologic tissue, or organ of type development, ontogenetic plant’s of phases factors: endogenous and exogenous various . syste and evolution variability, geographic for importance their out pointing markers, genetic as terpenes significantly qualitative terpene the that fact the on according tochloroplast the heldreichii nigra P.

AcceptedMontenegro. and Serbia of rritory Article ons thatmust met be toachievevalid inorder interpretation of studyresults hmdvriy of Chemodiversity as The useofterpenes

. n h ohr ad tee is there hand, other the On [6][10][14] , the [13][18] eog o ifrn sbetos ( subsections different to belong

influenced by environmental factors environmental by influenced which are are which

central part of the Balkan Peninsula. Balkan the of part central

Hence, P. nigra P. . heldreichii P. . [8] regarded as var. var. as regarded

in modern infrageneric infrageneric modern in [19] [6][10]

needle and l atr, rcdr o poesn te ln mtra, and material, plant the processing of procedure factors, al chemotaxonomic

and nuclear DNAand nuclear sequences P. peuce P.

ouain fo suhetr Sri ad northern and Serbia southwestern from populations Therefore, with respect to the already published data, this data, published already the to respect with Therefore,

terpenes [17] eti vraiiy n epn composition terpene in variability certain

pančići Therefore the proper sampling and strict adherence strict and sampling proper the Therefore [20] profile ais epcal i suis f oie order conifer of studies in especially matics,

, has been studied at the population level on the the on level population the at studied been has Pinu markers has a very long tradition and is based longandisbased markers avery tradition has obtained classifications of the genus, the of classifications Fukarek .

[15][16] s under is s

This opens m opens This and

Hanover

, [21][22] Pinaster by strong .

relationships among relationships [14] n

-

represent a transitional form form transitional a represent [17] pentane researches

rs. o section of resp.) , any interesting questions, interesting any

discussed the efficacy of efficacy the discussed eei cnrl n not and control genetic extraction

of biodiversity, biodiversity, of nigra P. , caused by by caused , . relict

,

and of

Pinus pines P. P. P.

This article isThis article by protected copyright. All rightsreserved. and (60.5%) hydrocarbons pine soft were ratios from pines with diversity Species respect to abundanceofterpene classescompounds and majorterpene and Results sp pine of evolution and phylogeny biogeography, chemotaxonomy, concerning in stands occurring from P. nigra a analysis). discriminate heldreichii peuce peuce pines investigati on given terpene selected statistically Balkan

Accepted Articlenew : ca. . set of terpene compounds which could could which compounds terpene of set P. heldreichii P. s ecies. ae led be investigated, been already have Although the differences in the terpene composition between between composition terpene the in differences the Although Mono Bearing D

, (with ifferentiation P. heldreichii 15 . peuce P.

the central Balkans central the about about

population

D populations -

iscussion n ssutree wr te an opud lse i nels of needles in classes compound main the were sesquiterpenes and ie omorika Picea all 1. . hs n id te epn poie wr suid on studied were profiles terpene the mind, in this 5

on of terpene of on and h cnrl Balkans central the Namely, :1 for both hard pines: pines: hard both for :1

The aim of this study was to was study this of aim The between and

n ue one used and P. nigra P. of P. peuce s . heldreichii P. [23] particula

. peuce P. , ačć (Purk.) Pančić

but and P. , as well as to their to as well as , differentiation

. heldreichii in different ratios different in

n - rly alkanes

[23] a shown has more Fg 1 (Fig. , oxygen [13][18]

P. nigra P. in this paper we included included we paper this in help to elucidate to help

.

as outgroup), has already been approved approved been already has outgroup), as [24] and utvrae ttsia aayi (canonical analysis statistical multivariate

,

. nigra P. between two morphologically similar morphologically two between

- al 1 Table otiig (O containing However, i However,

find individual relationships individual sihl higher slightly a . peuce P. and

(Fig. 2). The total mono/sesquiterpene mono/sesquiterpene total The 2). (Fig. , through ,

P. heldreichii P. [19] ) .

h rsls c results The and

n diversity populations from the the from populations the present the - . peuce P. several otiig monoterpenes containing)

, and , ee o monoterpene of level . heldreichii P. t and differentiation and wo

statis ud e important be ould one [20]

- these three relict relict three these work accent was accent work about 3 about year with

from naturally naturally from tical additional - old soft pine pine soft :1 for the the for :1 analyses analyzed analyzed

needle central central and hard hard

by P. P. P. P. of of s

,

This article isThis article by protected copyright. All rightsreserved. terpene with volatiles were D germacrene (α high in found noticeable a terpenes is there analyzed, are the (>10%) amounts only if Nevertheless, limonene. without but profile similar ( D, germacrene pinene, mono were them of all and concentration respectively, in detected compounds four, and five by characterized side, other the On pine. soft this in monoterpenes containing oxygenated β camphene, D, germacrene pinene, 5%. over concentration compoun of number highest the by characterized was (Table metabolites terpene major the of profiles diterpenes w pines in while (2.4%), diterpenes total of content higher slightly a by characterized to compared when resp.) cl but resp.), 52.8%, and in class compound main the represented also hydrocarbons (9.5%)

Accepted- Article iee n grarn D in D) germacrene and pinene C in addition to addition in comp omparison of dominant terpenes among the studied the among terpenes dominant of omparison

ooepn (onl ctt) rslig in resulting acetate), (bornyl monoterpene o unds ere detectedere mostly intraces.

for each species for eachspecies were already reported.

the lowest level of sesquiterpene hydrocarbons (24.6%). hydrocarbons sesquiterpene of level lowest the

E n diint temi mono main the to addition In ) sr o h lvl f eqiepn hdoabn (16 n 37.6%, and (41.6 hydrocarbons sesquiterpene of level the to oser - caryophyllene and β and caryophyllene . peuce P. . nigra P. high - . iee n ( and pinene

oee, ede of needles However, contents -

and and

- pinene in pinene

2 eqiepn hdoabn: ioee α limonene, hydrocarbons: sesquiterpene . peuce P.

; Fig. 3 Fig. ; eodd in recorded

predominance of the same two volatiles two same the of predominance E ds (six) detected in an average average an in detected (six) ds ) -

-

cnieal hge cnet f O of content higher considerably a caryophyllene),

n sesquiterpene and P [13][18 ). The terpene profile of profile terpene The ). . heldreichii . wie ioee α limonene, while , P. nigra P. P. h P. pines - 20] . heldreichii P. eldreichii P. heldreichii heldreichii P.

has shown highly distinct highly shown has

and , while , it P. heldreichii heldreichii P. s

and hydrocarbons (α hydrocarbons also ihr hn 5 than higher P. nigra P. Aons of Amounts . P. nigra P. the M - include iee and pinene onoterpene ee also were

other two other P. peuce P. relative

had a had (56.5 were were s

%, an an - - -

This article isThis article by protected copyright. All rightsreserved. of populations peuce P. reveale percen explain axes component principal two first in the among relationships 2 (PCA,Multivariate analysis AHC) CDA and heldreichii of chemotypes wherein chemotypes, terpene different to attributed be may pines three peuce (i.e. volatiles of number of separation indicated five six Further, of each for profiles terpene distinct of existence the to pointing pines, γ and D germacrene humulene, ( limonene, pinene, D germacrene and terpinolene for except volatiles these all N unequal for (Table 0.5% than higher concentrations ofAnalysis variance anddifferentiation ofstudiedVariability pinesbased onte 6

Accepted Article characters), was performed in order to determine the overall chemical variation and and variation chemical overall the determine to order in performed was characters), . tages of 25. of tages Based on the number of compounds which indicate divergence indicate which numbercompounds of the Basedon d good grouping of individuals within the species, and clear separation of the soft pine pine soft the of separation clear and species, the within individuals of grouping good d Principal component analysis (PCA), based on a selected data set (412 individuals x x individuals (412 set data selected a on based (PCA), analysis component Principal of profiles terpene The

from the hard pines hard the from and other post P. peuce P. P. peuce

2 volatiles indicated volatiles -

hoc E

and 2 and ) - β

test) indicated statistical indicated test) form a group at the at group a form - are theare m cmn, onl ctt, α acetate, bornyl ocimene, three 4 dividuals from analyzed populations of all three pine three all of populations analyzed from dividuals . 3 %, resp.) %, P. nigra P. . peuce P. ) indicated separat indicated ) - all three pine species pine three all cadinene) cadinene) ost distinctive. divergence

. The scatter plot in the projection of the first two axes axes two first the of projection the in plot scatter The . and

vs. 2 ). have P. heldreichii P. Analysis of variance of Analysis . nigra P.

negative

shown significant differences among all three all among differences significant shown 4

ly significant differences between means for means between differences significant ly 9 of o of ion . 4 P. heldreichii P. o te oa variation total the of % -

and epnl ctt, ( acetate, terpinyl part included

. nigra P.

(Fig. 4A). (Fig. s rpene markers . heldreichii P.

of both - 2 4 - 6

-

ol. from

ANOVA (with (with ANOVA vs. compounds ax Namely, samples from all all from samples Namely,

, it was assumed all was that it , The e P. nigra nigra P. s . heldreichii P. , E

wie h smallest the while , w

) - the species tested. tested. species the most hile individuals of individuals hile aypyln, α caryophyllene,

recorded and

wt similar (with

terpene species Tukey P. peuce P.

and

. s HSD HSD with with The (α P. P. P. P. - - , This article isThis article by protected copyright. All rightsreserved. 1 scatter The function. δ and β first, the while only affected functions, considerably both on impact significant had ol), of terpenes 6 by represented was function first the participated functions two first the that shown o based CDA The other. each from differentiated chemically are that groups discrete of composed was sample analyzed the isopimarol ( ( caryophyllene abundance ( mainly ( acetate terpinyl ( phellandrene 4B) (Fig. variability described. already PCA the in trends main the than weaker was 1 axis along trend this but evident, was Serbia from the Within 1. axis for values negative of those axis: 2. nigra P.

1 Numerals initalics referto the entriesinTable 1.

Accepted Article1 However, individuals of two hard pines were additionally differentiated along the first first the along differentiated additionally were pines hard two of individuals However, ) and germacrene D ( D germacrene and ) Canonical discriminant analysis (CDA) was done in order to check the hypothesis that hypothesis that the check to order in done discriminantwas (CDA)analysis Canonical asd by caused

and

of eight eight of 2 these pines these P. heldreichii P. 6 P. heldreichii P. 7 )) 1 ), β ), . 15 6

), α ), high )) - phellandrene ( phellandrene

. - compounds influenced separation of separation influenced

humulene ( humulene plot obtained by CDA suggested suggested CDA by obtained plot H (α amounts igh

2 - 1 Sever

pinene, β pinene, axis of part positive the at groups adjacent two as separated were ) have shown mainly po mainly shown have ) contents .

n 15 populations of populations 15 n Finally, l epn caatr wr rsosbe o te e the for responsible were characters terpene al

mostly , 19 of 10

), - n mntree n oe sequit one and monoterpene one ), bornyl acetate ( acetate bornyl ),

pinene, limonene, germacrene D and germacrene D germacrene and D germacrene limonene, pinene,

γ

P. heldreichii P. of - P. heldreichii P. muurolene ( muurolene 2

.

8 six sesquiterpene (Table % -

3 P. peuce P. monoterpene - to carene and aromadendrene only the second second the only aromadendrene and carene

9 sitive, while those of those while sitive, 1 P. heldreichii P. 2 .

6

group, some separation of population V population of separation some group, 0 individuals were individuals 3 % of the total discrimination, of which which of discrimination, total the of % ), γ ),

). Five compounds, mostly dominant dominant mostly compounds, Five ). 13 individuals s

- ), terpinen ), the c ( ycn ( myrcene adinene ( adinene metabolites -

hladee n β and phellandrene xsec o tre chemically three of existence , . P. nigra P.

Diversity of Diversity - 2 4 6 3 - erpene

ol , ioee ( limonene ), ), characterized by high high by characterized

P. nigra P. - cmhn ( (camphene δ acetate ( acetate -

cadinene ( cadinene and

( ( E P. peuce P. P. nigra P.

) have shown have - - 14 β gurjunene gurjunene - xplained ocimene ) and α and ) 9 2 3 , ( ), 4 ) ) and ) 1

α , was was has E - 4 ) - - - - This article isThis article by protected copyright. All rightsreserved. demarcation their suggested that markers first the were metabolites Pinus heldreichii f responsible mainly were compounds monoterpene several of levels content high However, of products the therefore and molecules, volatile of skeleton carbon larger that assume characterized pines hard of those than basal more is monoterpenes, terpinen of levels high pine soft a of profile terpene the concept, this accept we if Therefore, Strobus other to According clear Therefore, analyses. nigra for while axis, first the for values positive shown have to similar entities differentiated or separation of separation or

Accepted Articlemed atthegroups part negative two ofaxis1,mostly separated thesecondaxis. along . heldreichii P.

and iegne ewe soft between divergence with On the other hand, other the On separated clearly (AHC) analysis cluster hierarchical Agglomerative

- may be considered be may 4 - , in same ofour the results wayasin analysis. P. heldreichii P. ol

highly highly . “ - terpene evolution terpene Nik acetate by high content high by six , olić et al. et olić

P. peuce P. monoterpene . peuce P. op

all three analyses three all htceia markers phytochemical oe and posed n α and

populations

[23] P. nigra P.

and high amounts of sesquiterpene volatiles for separation of separation for volatiles sesquiterpene of amounts high and - “ n one and epnl acetate terpinyl s ancestral

reported somewhat different terpenes that influen that terpenes different somewhat reported of mainly sesquiterpene compounds (Fig. 4B). Hence, we may we Hence, 4B). (Fig. compounds sesquiterpene mainly of ” s

those obtained by PCA (Fig. 5). (Fig. PCA by obtained those

in these species went in the direction toward synthesis of a of synthesis toward direction the in went species these in (camphene, α (camphene, uncertain and

and oie seis rm ifrn genus different from species conifer

(Fig. 6) (Fig. ” suggested ad ie ( pines hard

in reference to the Laurasian origin of the genus the of origin Laurasian the to reference in P. heldreichii P. hlgntc eainhp. neetnl, terpene Interestingly, relationships. phylogenetic and ) , in agreement with the other two multi two other the with agreement in , peoi dt) te ie fr pines the data), (phenolic - phellandrene, β phellandrene,

that composi that ,

generally subgenera P. nigra P. P. nigra P.

represent two species of species two represent

,

a

,

tion of needle terpenes reflects terpenes needle of tion

and and - greater structural diver structural greater h highest the p

Strobus hellandrene, bornyl acetate, acetate, bornyl hellandrene, All populations of populations All P. heldreichii P. P. heldreichii P. , [9] P. peuce P.

and

s rvosy the previously as om the subgenus subgenus the om ( P. peuce P. amount ie omorika Picea Pinus the ced diversity ced , which were which ,

, defined by by defined , populations

subgenus

P. peuce P. , resp.). resp.). ,

f total of from variate variate sity of sity

. [25] P. P. P. ) .

This article isThis article by protected copyright. All rightsreserved. ty different of accumulation preferential the identifying allowed oils essential the in skeletons subsect different section of taxa 27 of al. et Mitić terpene results our hybridize, spontaneously can pines black and Bosnian nee ( form nigradermis var to assigned and nuclear and genus of classifications sylvestris subgenus in place markers RAPD using species conifer ten between relationships the with to related closely more remained (subsect. the Eurasian between position its however confirmed; been has pine Bosnian of status taxonomic of variety a only considered was pine Bosnian

AcceptedC sesquiterpene of pes Article ls n/r cones and/or dles Pinaster P. Nevertheless, ifrnito o their of differentiation

nigra unresolved “ [29] (species divider genomes

, resp.) of section of resp.) , uae ad iaoi and Vidaković and Fukarek

compared composition of essential oils obtained by obtained oils essential of composition compared .

f. pančići s .

leucodermoides ”

( Pinus

Pinus

Pinus . . f subsect. of oiin between position [14] . [10] [8] . heldreichii P. Pinus , Pinus

P. nigra P.

, but , is - ept te ecie mrhlgcl iiaiis and similarities morphological described the Despite In the older literature sources, sources, literature older the In

skeleton ad h “ the and ) and

similar to similar

and

, it was it Pinaster P. nigra P. Pinus

populations ,

ofre pstos of positions confirmed

P. sylvestris P. s Fukarek and Nikolić) based on morphology of branches, of morphology on based Nikolić) and Fukarek

Pinus between the examined subsects. examined the between

, at the same the at ,

P. nigra P. rm otwsen eba and Serbia southwestern from according to the DNA sequences from sequences DNA the to according , resp , true

and hs to groups two these )

. ” P. P. heldr P.

.). Furthermore, within the recent infrageneric infrageneric recent the within Furthermore,

growing Mediterranean

x and to some extent to two natural hybrids ( hybrids natural two to extent some to and

Interes and other Eurasian hard pines hard Eurasian other and P. nigra P. mugodermis time eichii tingly, naturally naturally equally distant equally . P. heldreichii P.

[2]

. nigra P. belong to different subsect different to belong

. I [27] n all subsequent studies, distinct studies, subsequent all n

percentages of occurrence of C of occurrence of percentages ad pines hard uae) n oe intermediate one and Fukarek) ,

[28] n h sm are same the in In hydrodistillation

P. heldreichii P. and peetd here, presented , the

i.e., subsect. subsect. i.e.,

northern

was either either was from . heldreichii P.

nlss f genetic of analysis (subsect. P. nigra P. chloroplast [26]

Montenegro, Montenegro, the a. Recently, Recently, a. was found a a found was or a species a or

considered considered

Pinus of Pinaster s

at that fact . showed

needles needles

and ( wi Pinus

[6][10] P. was was thin thin P.

x - )

This article isThis article by protected copyright. All rightsreserved. Accepted group.any plant still is terpenoids on based taxonomi Resolving unknown. completely is most constituents of roles the regarding knowledge overall known, are terpenoids of functions many point changes evolve also metabolites secondary other and terpenes of function as species America North and more are flavonols methylated of an hot under growing of concept proposed recently the with consistent largely are results our manner this In diterpene). one even and sesquiterpenes to high comparison the in by basal more Article (characterized is sequiterpene) one and monoterpene peuce inconcentrationwere detected lower 0.5%orevendetected than not guaiol and by subsect. in However, while unique. was guaiane → germacrane cadinane, → germacrane route the favoring as occur unique a by characterized n - ed pentane extraction were a were extraction pentane , it may be assumed that the terpene profile of profile terpene the that assumed be may it , constit specialized plant of class largest the constitute terpenoids As B . out that they play a wide range of roles in plant metabolism. In spite In metabolism. plant in roles of range wide a play they that out [ sd on ased 30 ]

el s hi ancestors their as well ta soe sgiiat ifrne bten subsects. between differences significant showed (that

individual individual ). [25] d dry climates (Mediterranean region) and containing very high levels levels high very containing and region) (Mediterranean climates dry d

However, any attempt to determine connections connections determine to attempt any However, a

relatio very difficult issue not only within the genus the within only not issue difficult very

nalyzed and compounds such as such compounds and nalyzed amounts renc evolved

nships “ ut osdr ht uig h eouin f pce the species of evolution the during that consider must e of the route germacrane → bicyclogermacrane as well well as bicyclogermacrane → germacrane route the of e lvnc evolution flavonic

f ih compounds: eight of

of than those from cold and/or wet regions ( regions wet and/or cold from those than studied studied i n the present study present the n P. nigra P. hard pines pines hard i genus in ”

c and phylogenetic relationships relationships phylogenetic and c

(defined by high levels of one of levels high by (defined bicyclogermacrene, d as such pine soft a with

in line with environmental with line in w mntree, five monoterpenes, two

Pinus . terpene profiles obtained profiles terpene [13][18 Pinus Pinaster - , stating that pines pines that stating , 20] uents, it should be should it uents,

Pinus

and ewe extant between of . heldreichii P.

, but also in also but , Pinaster the fact that fact the te route the , α - Eurasia cadinol [29] P. P. )

This article isThis article by protected copyright. All rightsreserved. Acceptedinfluenc anthropogenic negative from spared was trees), 200 tha fact the to primarily thanks decades, few last the over increasing individuals of number (over Serbia in species this of population natural largest the represents var. as described enclaves formtwo of in southwest, the in only wild the in growing Serbia, of territory anddifferentiation diversity Terpene pines ofstudied at the taxonomic their consequently, and, pines of evolution the highlight better could which terpenoids, of i further in found be might side, between sesquiterpenes Article andtheirplants taxonomic relevance of research further during account o role the and synthesis of aspects molecular and physiological phytochemical, into taken be should route “alternative” This of triterpenes. finding and sesquiterpenes as such terpenoids cytosolic for substrate of source cytosol/ the to restricted be to seems pathway mevalonate the while etc.), carotenoids diterpenes, monoterpenes, as i found is route non discovered was new, route) phosphate/pyruvate a syntesized), are terpenoids which (from units noting worth is It process. iu heldreichii Pinus and monoterpenes in contents in difference sharp for explanation possible Thus, obviously is terpenoids of diversification and Evolution

and phylogenetic and phylogenetic relationships. n the plastids of all vascular (for the plastid the (for plants vascular all of plastids the n pančići P. heldreichii P.

nolsi reticulum endoplasmic (Fig. 1 (Fig. s a is that in addition to mevalonate route for the synthesis of the C5 C5 the of synthesis the for route mevalonate to addition in that akn subendemite Balkan nvestigation of enzymes and genes involved in biosynthesis in involved genes and enzymes of nvestigation , Table .

and

1 ).

.

P. nigra P. [ The population from Mt. Revuša (population V) V) (population Revuša Mt. from population The 31 ]

codn to According Te eaoae ot my be may route mevalonate The . es (discussed in Bojović et al. et Bojović in (discussed es

with an extremely narrow range in the the in range narrow extremely an with rmoe and one, from

population level a - mevalonate (glyceraldehyde (glyceraldehyde mevalonate eet report, recent - associated terpenoids such terpenoids associated a

P. peuce P. ey ope ad long and complex very

[ from the other the from f terpenoids in terpenoids f 32 ]

[ 18 the second second the ] ). On the the On ). the

that are that

main with t it it t

This article isThis article by protected copyright. All rightsreserved. closest the connection between the two most spatially distant populations IV. I and level. species their influence not do but level population on detected be may which factors, climatic of influence the under composition terpene in differences small of presence show an of populations combined the between comparison of results However, year. humid more and cooler a generally was which 2009, in conditions climatic Europe. Western in mostly August, and July in recorded were levels heat highest the and 1540, least at since Europe in record on summer hottest the to led 2003 of wave heat European Europe. populations studied other (Table conditions collected fr material plant that is explanation possible The III). as var. alsodescribed IV), (population ofstudiedrest populations that largely over the from separation of degree certain a shown has V) (population population Serbian largest time. first Montene from populations wild three with together populations, bowls). of and manufacturing resin, kindling, for used was wood resinous (the impact anthropogenic by destroyed between trees individual and groups little scattered some only includes IV) (population population Serbian second the hand, other

Accepted2009) d Article This result was somewhat unexpected, considering that the second Serbian population Serbian second the that considering unexpected, somewhat was result This [33] rm ae umr o al fall early to summer late from

and Therefor codn t te obnd eut o al utvrae analyses multivariate all of results combined the to According

In the previous study that only included populations I populations included only that study previous the In

populations of populations 1 ): for the largest Serbian population (population V) in 2009, while for all for while 2009, in V) (population population Serbian largest the for ): e, certain separation of Serbian population V could be caused by different different by caused be could V population Serbian of separation certain e, (I – n h peet td, epn poie o tee w Serbian two these of profiles terpene study, present the In IV P. ) in )

nigra

2003, which was the year of the immense heat wave in wave heat immense the of year the was which 2003, pančići t Zaio ad etr plate Pešter and Zlatibor Mt.

(collected in 2009) and 2009) in (collected lapped other. witheach n w yas ih opeey different completely with years two in , was grouped with Montenegrowasgrouped , with om these two Serbian populations was populations Serbian two these om

P. heldrechii P. P. peuce P. gro, were compared for the the for compared were gro,

– au, IV

,

which were almost almost were which [ (collected in 2003) in (collected

13 (collected in 2003 in (collected ]

AHC Fg. 4 (Figs. populations (I populations

placement at at placement

suggested climatic - 6) , the the , –

This article isThis article by protected copyright. All rightsreserved. Accepted closer connection between populationsand when II III population comparedto I. t detached AHC while 5), and 4A (Figs. populations of overlapping total shown have CDA and dissi var. as suggested had AHC territor the within taxa of circumscription support to failed that mitochondrial) and (plastid data molecular characters chemotaxonomic as compounds wax epicuticular using by obtained Articleinfraspecific different of populations close geographically the between hybridization natural by explained be may overlap population observed The group. single a formed populations Serbian studied for studied zone contact the represents Serbia of territory the As 6). (Fig. populations pine black Serbian within similarity of degree highest the confirmed also AHC while 5), and 4A (Figs. of populations the among overlap significant a was there plots scatter CDA and Ionescu Đorđević subsp. taxa: infraspecific four as (determined Serbia from populations milar to results the of thepresent asshownstudy, inthedendrogram. In the case of case the In wit differentiation and variability terpene of levels detected The ; banatica P. nigra P.

Fig. 1 Fig. subsp. , he population II as the most distant. On t On distant. most the as II population he . nigra P. , Table Table

ws h ms dsat Ti arneet of arrangement This distant. most the was ) taxa (including borders of their ranges) perhap ranges) their of borders (including taxa pallasiana the P. peuce P. 1 y of Serbia of y

) aa wih r no are which taxa, existence of three population groups where population VII (assigned VII population where groups population three of existence

were lower than in populations of other two pines. Namely, in PCA PCA in Namely, pines. two other of populations in than lower were

, only three natural populations were analyzed. Results of PCA PCA of Results analyzed. were populations natural three only , Lm. Holmboe) (Lamb.) . [ 35 ]

However, in our previous study of terpene markers terpene of study previous our in However, rpoutvl ioae. iia rsls were results Similar isolated. reproductively t , and var. var. and , he other hand, Nikolić et al. et Nikolić hand, other he

banatica . nigra P. s it is not surprising that all all that surprising not is it s

(Endl.) Georgescu et et Georgescu (Endl.)

populations is quite quite is populations nigra , var. var. , , hin [ [ 20

3 4 ] ]

reported a reported as well as as well as . nigra P. . nigra P. gocensis P. nigra P. (Fig. 6) (Fig. [ 19 ]

This article isThis article by protected copyright. All rightsreserved. collection 1 Fig. in presented indicating area, study the of map of populations three of populations five Serbia, MaterialPlant Experimental strategies and futurebreedingefforts. conservation suitable develop subsequently and tree, phylogenetic and taxonomic the within of positions precise establish to order in necessary are studies genetic and phytochemical morphological, Additional compounds. volatile the of s compounds. sesquiterpene of those than basal monoterpe genus of concept proposed recently the with accordance In markers. their of differentiation pines (subgenus subgenera: different from species Conclusions Accepted of carbonynthesis larger the skeleton therefore, ofmoleculesgreaterstructural and, diversity Article Pinus . nigra P. Plant material Plant reflected terpenes needle of composition The e eaoie ad eeal te highest the generally and metabolites ne for every population are listed in the Table Table the in listed are population every for Pinus ,

we assumed that the that assumed we

Section

) n diin d addition, In . and , P. nigra P. P. peuce P. and t and

(fresh needles) (fresh

. heldreichii P.

p opulations naturally naturally opulations he P. heldreichii P. Hence

corresponding geographic and geologic data as well as date of date as well as data geologic and geographic corresponding

and (two from Montenegro and one from Serbia) were analyzed. were Serbia) from one and Montenegro from (two espite the morphological similarities and the fact that hard that fact the and similarities morphological the espite

P. heldreichii P. P. pe P.

terpene profile of profile terpene , h locations the “

epn evolution terpene

from seven populations of populations seven from uce a sotnosy yrdz, u rsls show results our hybridize, spontaneously may

(three from Montenegro and two from Serbia), and Serbia), from two and Montenegro from (three

(subgenus rwn i te ae ra bsd n terpene on based area, same the in growing , characterized by high by characterized , f sele of P. peuce P. amount Strobus . heldreichii P. ” td populations cted 1

clear

. in these species might go toward toward go might species these in

Two

, defined by high content high by defined , ) f oa mntree, s more is monoterpenes, total of

iegne ewe te pine the between divergence vs. - year P. nigra P. “ lvnc evolution flavonic P. nigra P. , -

old needles old . nigra P. abundance f tde pines studied of

naturally grown in in grown naturally

and

and P. heldreichii P.

from

of mainly of . peuce P. ” in the the in ” s

of lower lower , six

ed A is

This article isThis article by protected copyright. All rightsreserved. AcceptedSeries Technologies analysis. H gas, carrier 280°; temp., detector 250°; temp., injector 4°/min; at 280° to 40 from rising linearly programmed was temp. oven The µm). a and (MSD), (ALS), sampler liquid automatic respectively apparatuses, with out carried were GC chromatography withsolid caps inarefrigerator vials until solvent) /ml needles 2 of pieces into cut were extraction, Article until Isolation was identifiedbyDr.Srdjan analyzed at stored and freezer a to b polyethylene from and 15 with VII population ofcrownthird - FID and GC FID and

late summer to early fall in 2003 and 2009. and 2003 in fall early to summer late ), a flame ionization detector (FI detector ionization flame a ), The GC The Two [13][18][20]

of terpenes 195 - year , Santa Clara, CA, USA) CA, Clara, Santa , P. nigra P.

HP of ags ( ags - FID -

- 30 randomly select 30 randomly MS old n h suy of study the In - 5 MS 5 label

Te xrcs ee et t 4 at kept were extracts The . and analyses needles , 127 , Hewlett

e

fused

GC - d 20°C prior to further analysis. further to prior 20°C (

Hewlett with data on sample plot, date of collection, locality), transferred locality), collection, of date plot, sample on data with P. heldreichii P. R. - P. heldreichii P.

MS of every individual tree, stored separately stored tree, individual every of

-

silica cap. column (30 m x 0.25 mm i.d., film thickness 0.25 0.25 thickness film i.d., mm 0.25 x m (30 column cap. silica Bojović and Dr. Bojović and Biljana - akr G1800C Packard a analyses - . nigra P. flame ionization detector (FID), a ma a (FID), detector ionization flame Packard ed individuals in each of ed individualseach in population the

equipped with equipped D), a mass selective detector (MSD), and a and (MSD), detector selective mass a D),

and 90 and

of terpenes of terpenes of - population IV with 7 individuals) were individuals) 7 with IV population

m lnt ad xrce with extracted and length mm 3 Pl At, A USA), CA, Alto, Palo , terpenes 2

P. peuce P. in GC in - The entire plant material was material plant entire The - GC 6°C for 24h, then filtered, and stored in in stored and filtered, then 24h, for 6°C , an auto an [19]

M. and -

FI

In the present the In P. heldreichii P.

gln 7890A Agilent D (1.0 ml/min) and He in GC in He and ml/min) (1.0 D further analysis. individuals individuals Nikolić. - Hewlett injection system( injection

-

akr G1800C Packard oh qipd ih an with equipped both in total in paper, we merged and merged we paper, [13][18] in a freezer at freezer a in ss selective detector selective ss

paau ( apparatus [13][18 s

(except and . n Agilent 7683B Agilent Plant material Plant - -

etn ( g (1 pentane 20] deposited in deposited P. peuce P.

HP collected P. nigra Agilent - - - 5 MS 5 GCD 20°C - MS MS [20]

This article isThis article by protected copyright. All rightsreserved. analysis discriminant canonical (PCA), analysis component Multivariate principal characteristics. included chemical analysis their of values mean on based species, analyzed post A (ANOVA). variance of analysis univariate the by determined was species studied the between differences of significance The al. all while 0.5%, than more in present were the account into taken had we matrix, a form to fact, In variables. original as t needle of percentage) (in content the included matrices Statistical USA). OK, Tulsa, analysisStatistical Thelibraries. obtained result reported those Compound Identification determined byGC/FID. m/z 150°; tem injector min; 10 for 300° at isothermal then and 3°/min at 300° to 60 from rising linearly programmed was temp. fused

Accepted, Article [ 3 range 40 range - 7 hoc ] -

silica cap. column (30 m x 0.25 mm i.d., film thickness 0.25 µm) 0.25 thickness film i.d., mm 0.25 x m (30 column cap. silica carrier gas, He. Electron He. gas, carrier contents <0.5%) were not considered. All selected co selected All considered. not were <0.5%) contents Statistical data processing was carried out by STATISTICA 8 software ( software 8 STATISTICA by out carried was processing data Statistical spect mass their of comparison the on based identified were components The

et a ue i odr o evaluate to order in used was test - 450 y Adams by [13][18][20]

[13][18 p., 250°; detector temp., 300°; temp., detector 250°; p.,

or 40 or [3 6 s were withthecorrelated retention ( indices ] -

20] r ih hs cmie i the in compiled those with or -

- impact mass spectra (EI spectra mass impact 550. Tukey [19]

For quantification purposes, area purposes, quantification For

honest significant difference (HSD) for unequal N unequal for (HSD) difference significant honest terpenes

statistical importance of difference between between difference of importance statistical

found in traces (according to Lieutier et Lieutier to (according traces in found source temp., 230°; quadrupole temp., quadrupole 230°; temp., source -

MS; 70eV) were acquired over the over acquired were 70eV) MS; mponents are listed in Table Table in listed are mponents Wiley - 275 RI -

was used. The oven oven The used. was percent values were values percent n the and ). [13][18 Statsoft terpenes - 20 NIST/NBS ]

erpenes erpenes ra with ra ,

Inc that that 2 ., .

This article isThis article by protected copyright. All rightsreserved. Accepted had already inbeen done previous investigations material authors listed o approval final gave and manuscript Marin D. Petar manuscript. statistical revised analyses,in andrevised helped their interpretation Srdjan results. obtained of interpretation and classes terpene of determination an Tešević Mihailo group. research the of supervision general made and version design and concept to contribution first the drafted Articleand interpretation their and analyses statistical performed data, final all merged Mitić S. Zorica AuthorStatement Contribution DevelopmentTechnological ofSerbia. Republic ofthe This Acknowledgement and used was (UPGMA) mean arithmetic with method hierarchical agglomerative and (CDA) Euclidean work ,

determination of species, of determination

alyzed chromatographic data up to the level of statistical processing and helped in in helped and processing statistical of level the to up data chromatographic alyzed a spotd by supported was ,

in accordanc in distances asdiversity assessment inAHC criteria analysis.

eso of version e to their closer spec closer their to e participated G ra

nt isolation

the No of f the version to be published. published. be to version the f

the to the to 173029 by the Ministry of Education, Science and and Science Education, of Ministry the by 173029 aucit Blaa . ioi gv substantial gave Nikolić M. Biljana manuscript. lse aayi (H) Uwihe pi group pair Unweighted (AHC). analysis cluster

aucit ciial rdce i utl h final the until it redacted critically manuscript, of

general research design, design, research general terpenes , too. ialties,

as a criterion for the cluster the for criterion a as

participated , and/or biochemical analyses biochemical and/or ,

the in n diin a addition, In the S.

critically redacted critically draf

Ristić and Vele and Ristić collecting collecting ted version ted

development, R. lmost Bojović of

which

of plant the the all all V. V.

This article isThis article by protected copyright. All rightsreserved. (Sarajevo) Hercegovini u Prenju planini na Vid.) et Fuk. [ rps18 ( pines Eurasian of relationships [ [ 1982 [ M. O. A. Burges, [ of [ CambridgeRichardson, Press, Cambridge University [ Garden,NewYork,Botanical 1 [ ServiceForest Miscellaneous Publication 1144, Washington, D.C. [ Riverside [ Richmond,UK, 2001. [1] References 7 6 5 4 3 2 11 10 9 8 ] ] ] ] ] ] ] ] Pinus

Accepted Article ] ] Walters, D. A. N. T. ʹ Mirov, R. D. A. Farjon, Farjon, A. H. A. M. Vidaković M. E.

G. P. . X.

spacer, and A.

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Vidaković W. Styles ,

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– , B genus

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Cambridge Uni , 29 87 (in BHS).87 (in . S.

, ʹ ,

Critchfield Pinus Edmondson, V. H. V. Edmondson,

. H.

Pinus – M , ʹ ,

997. Pinus 42. Strauss orphology and variation and orphology Nal Pinus ʹ ,

. Ron ()

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Moore, D. H. D. Moore, , 1969.

– 1993, ʹ . Szmidt E. Z , Eds. , Pinus

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matK

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This article isThis article by protected copyright. All rightsreserved. 1377 peuce Pinus [ from Serbia of populations natural in composition terpene the of significance [ heldreichii ʹ [ 159 [ 147 biodivers for tool of classification a the on as study case a analysis informatics: data multivariate and resonance magnetic nuclear [ sylvestris [ – Martinez [ 2007 heldreichii Pinus [ Croatian)(in [ aiblt i ceia cmoiin n audne f h rr Trir relict Tertiary rare the of abundance and composition chemical in Variability 17 16 15 14 13 12 20 19 18

Accepted Article566. ] ] ] ] ] ] ] ] ] , 947

– Z. D. G D. B. B. P. S. J , G. P – . 178. 4

.

W 1388. , 905 Fukarek Šarac, S. Šarac,

Bojović, B. Bojović, Nikolić, M. Nikolić, Nikolić, net, A. Incerti, aaa, R Baradat, , ʹ ,

– rivet, J. Climent, M. Zabal M. Climent, J. rivet, . grown sites ondifferent

Adaptive evolution of Mediterranean pines Mediterranean of evolution Adaptive 954. Hanover in Serbia ʹ

, from different populations in Montenegro and Serbia and Montenegro in populations different from . –

Chem. Biodivers

916.

, ʹ ,

M. Ristić, S. Bojović, P. Bojović, S. Ristić, M. Bojović, B. Bojović,

Nova svojta borova u Bosni u borova svojta Nova rm ifrn ppltos n otngo n Serbia and Montenegro in populations different from

, oao P. Romano, Ristić, S. Ristić, Nikolić, M. Nikolić, . ʹ

, ʹ Applications of terpene analysis in forest genetics forest in analysis terpene of Applications Chem. Biodivers. Yazdani

Nikolić,

Bojović, P. Bojović, . ʹ ,

2013

Ristić, eei epeso fr ooepns n clones in monoterpenes for expression Genetic emln, V. Termolino, ʹ, Scand. J.For.Res. - , Aguirre, Aguirre, 10 V.

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Lanzotti 1520. B. Rosa

M. Neale, Neale, ʹ Variability of the needle essential oils of oils essential needle the of Variability Variability of the n the of Variability ʹ, –

1988 rđević, P. D. Marin,ʹ D. rđević, P. Veselinović, Veselinović,

Mol. Phylogenet. Evol. Phylogenet. Mol. 1765.

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ʹ Metabolomic fingerprinting using using fingerprinting Metabolomic G. damascena , 3

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AcceptedPinus Article

] ] ] ] ] ] ] ] ] Marin

B. B. D. B. , S. S. Kaundun S. S. Z. S. Mitić, S. Č. Jovanović, Jovanović, Č. S. Mitić, S. Z. P. . lu, F. Klaus, W. M. 162

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Plant Syst. Plant Evol.

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Godišnjak Biološkog Instituta (Sarajevo) Instituta Biološkog Godišnjak 10

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286. 5 4 Accepted7 Article ] ] ] ] ] ]

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mevalonate isoprenoid biosynthesis: enzymes, genes and and genes enzymes, biosynthesis: isoprenoid mevalonate 1991 , , , A. Wiley 28 48

, 785 , 377 Millstein,

Č.

in ʹ in ooi, P. Bojović, – -

Blackwell, Oxford, UK, Oxford, Blackwell,

oaoi, G. Jovanović, Annual Plant Reviews. Plant Annual –

aooi considerations taxonomic –

9.

, 388.

Allured Publishing Co. Publishing Allured (Ascomycetes: Ophiostomatales) and two and Ophiostomatales) (Ascomycetes:

ʹ rlmnr suy f h monoterpene the of study Preliminary

D.

ai, J. Marin,

S. Pinus

tjnvć P. Stojanović,

2010, Vol. 40, 40, Vol. 2010, Biochemistry of Plant Plant of Biochemistry M. ʹ , , Carol Stream, IL, IL, Stream, Carol ,

Aleksić, hm Biodivers. Chem. populations from from populations

C. R. Biol R. C. D.

ʹ

ʹ Genetic Genetic pp. 258 pp. , Marin, Int. J. Int. .

P. peuce P. peuce P. peuce P. heldreichii P. heldreichii P. heldreichii P. heldreichii P. heldreichii P. nigra P. nigra P. nigra P. nigra P. nigra P. nigra P. nigra Taxon Accepted isThis article by protected copyright. All rightsreserved. Article Table Tables

var. var. var. subsp. subsp. subsp. subsp. 1

. Location habitat and descriptionof selected the populations of banatica gocensis gocensis

var. var. pallasiana nigra nigra nigra pančići pančići

Serbia: Mt. Mokra Gora Monten Montenegro: Mt. Zeletin Serbia: Mt. Revuša population Pešter plateau consideredas single trees between Mt. Zlatibor and Serbia: little andindividual groups Montenegro: Bjelasica Montenegro: Zeletin Mon Kovej Serbia: LazarevaReka Canyon, Serbia: Mt. Dukat, Jarešnik Serbia: Mt. Goč, Gvozdac Serbia: Priboj, Crni vrh Serbia: Mt. Tara, Zmajvečki potok Serbia: Mt. Tara, Omar Serbia: Mt. Tara, Banjskastena Locality tenegro: Mt. Lovćen

egro: Mt. Sjekirica

III II I V IV III II I VII VI V IV III II I code Population

42°50' 42°40' 42°37' 43°14'52" 43°30' 43°15' 42°53' 42°37' 42°25' 44°01'02" 42°22'32" 43°33'32" 43°34'56" 43°51'45" 43°54'02" 43°57'04" (N) Latitude

P. nigra -

20°23' 19'50' 19°50' 19°51'39" 19°55' 19°30' 19°45' 19°50' 18°50' 21°55'39" 22°24'10" 20°40'56" 19°35'06" 19°25'05" 19°23'18" 19°24'04" (E) Longitude , P. heldreichii

-

1750 1700 1700 1525 1100 1700 1700 1700 750 1300 790 1100 830 950 1040 (m a.s.l.) Altitude

- - - - - and 1950 1900 1430 1900 1800

P. peuce

Limestone Schist, Serpentinite Serpentinite, Schist Limestone, Neogenesediments Neogenesed Hornestone, Tuff, Limestone, Dolomite, Limestone, Dol Serpentinite, Schist Limestone, Limestone Limestone Crystallineslate Serpentinite Serpentinite Serpentinite Limestone Limestone S ubstratum .

iments omite

30 30 30 30 7 30 30 30 15 30 30 30 30 30 30 analyzed individuals Number of

August 2003 August 2003 August 2003 August 2009 2003 September August 2003 August 2003 August 2003 August August August August August Aug August collection Date of ust ust

2009 2009 2009 2009 2009 2009 2009

taxon. ( significantly differ total the of Bojovi deviation) (mean±standard percentages as given are 1 This article isThis article by protected copyright. All rightsreserved. concentration than greater 0.5%in Table Entry ) F: ANOVA F ANOVA F: ) 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1.

Accepted Article

ć et al., et ć 2 Germacrene D ( α . Results of analysis of variance (ANOVA) variance of analysis of Results . E A - α β ( Terpinen Germacrene D Bornyl acetate Terpinyl acetate ) E α γ β - - α romadendrene - Terpinolene δ γ δ Phellandre Phellandrene Isopimarol - Camphene Caryophyllene - ) α Limonene - - - Terpenes Sabinene Muurolene - α Myrcene β Muurolene - - Gurjunene Humulene - Cadinene Cadinene [18] 3 β acet - - Thujene Pinene - Pinene - - Carene Ocimene test.

and Nikolić et al.; et Nikolić and

ate - Tukey 4

2 -

) P: Level of significance of Level P: ) ol -

ne

4

-

-

ol

HSD for unequal N unequal for HSD

Sesquiterpene monoterpenes Terpeneclass sesquiterpene hydrocarbons hydrocarbons Monoterpene Oxygenated Oxygenated Oxygenated diterpene [13][20]

P. nigra

means with different superscript letters within the same row (a,b,c) row same the within letters superscript different with means

post (*:0.05

)

test);

*** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** P ns ns for * 4 2

) N: number of individuals studied for a given a for studied individuals of number N: ) , ***: P<0.001, ns: not significant). not ns: P<0.001, ***: ,

)

2

ek surface peak 6

and 43.6± 9.1

N = N 195 29.8±9.1 0.0± 0.0 0.4±0.6 2.5±2.8 0.6±0.4 1.2±0.4 7.9±2.6 0.6±0.5 0.0±0.0 0.1±1.1 0.0±0.0 0.8±0.4 6.2 0.3±0.2 1.0±1.2 0.9±0.6 0.4±0.3 0.2±0. 0.5±0.4 0.0±0.0 0.0±0.0 0.3±0.6 0.0±0.0 P. nigra needle terpenes detected in mean in detected terpenes needle 0.6±0.5 0.7±2.3 ±6.4 P. peuce 2 4 b b b b b c a a a a a a a a a a a a a a a a

c

)

c

codn t Šrc t al., et Šarac to according Content (%) P. heldreichii .

15.3±7.5 25.8±9.3 16.0±8.2 10.2±2.6 0.2±0.3 2.1±6.3 2.2±0.6 1.3±0.9 1.4±0.9 1.3±0.9 0.9±0.6 0.7±0.4 1.1±0.8 0.5±0.3 0.1±0.2 0.0±0.0 0.0±0.1 5.2±2.6 0.1±0.2 0.9±0.4 0.2±0.2 0.8±0.5 2.1±1.9 0.0±0.1 N = N 127 0.5±0.5 0.3±0.2 b b b b b b b b b b a a a a a a a c c a

b c a c

3 )

3 36.5±3.6 ) Contents ) 11.4±3.0 P. peuce 4.7±1.0 0.8±0.4 0.7±2.0 8.5±1.9 0.6±0.1 0.5±0.2 1.2±0.4 0.6±0.1 1.1±0.3 1.6±0.8 6.8±1.9 0.0±0.0 0.0±0.0 0.0±0.1 1.0±0.2 6.8±3.7 0.0±0.0 0.8±0.3 0.6±0.1 0.8±0.5 5.2±0.8 0.9±0.4 0.5±0.1 0.5±0.7 N = N 90 [19] b b b b b b b b b b a a a a a c c a a a a a

b a

This article isThis article by protected copyright. All rightsreserved. Accepted Article Significant coefficients boldface. arein terpene Table 3

. Standardized coe Standardized . opud fo te iciiat ucinl nlss f 1 of analysis functional discriminant the from compounds Entry 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1.

% explained variation % explained Terpinen Germacrene D Germacrene ( α E Aromadendrene - α β ( fficients for the first three canonical axes (CA) of variation in variation of (CA) axes canonical three first the for fficients Germacrene D Germacrene Bornyl acetate Bornyl Terpinyl acetate Terpinyl ) E α γ β - α - - Terpinolene δ Eigenvalue γ δ Ph Phellandrene Isopimarol - Caryophyllene Camphene - ) α Limonene - - - Muurolene Sabinene - α Myrcene β Muurolene - - Variable Gurjunene Humulene - Cadinene Cad 3 β - - Thujene ellandrene Pinene - Pinene - - Carene Ocimene 4 inene - ol - acetate

-

4

-

ol

43.98 ------CA1 0.63 0.21 0.14 0.63 0.07 0.08 1.01 0.01 0.30 0.17 0.33 0.24 0.79 0.34 0.34 0.04 0.62 0.01 1.09 0.60 0.12 0.09 1.27 0.30 0.37 0.41 0.37

20.20 ------CA2 0.23 0.25 0.92 0.29 0.04 0.57 0.11 0.16 0.45 0.77 0.54 0.90 0.16 0.22 1.66 0.09 0.55 0.02 0.05 1.75 0.28 0.40 0.08 0.29 0.21 0.30 0.30

------CA3 0.11 0.17 0.95 2.68 0.24 0.27 0.02 0.11 0.20 0.19 1.00 0.07 0.33 0.79 0.11 0.13 1.32 0.15 0.30 0.37 0.12 0.71 0.20 0.16 0.23 0.55 0 0.11 .30 5

priori a

groups. groups. 26

This article isThis article by protected copyright. All rightsreserved. 1. Table represe numbers italic The axes. two first the of plane the populations three from 127 populations, 4. Fig. Camphene; compounds. 3. Fig. populations. (OD). diterpenes and oxygenated (DH), hydrocarbons diterpene (OS), sesquiterpenes monot oxygenated (SH), oxygenated hydrocarbons (MH), hydrocarbons monoterpene 2. Fig. II Zeletin, plateau Pešter and – heldreichii Jarešnik Dukat, Mt. gocensis II stena, Banjska 1. Fig. Figure legends.

small

Acceptedα Article Bornyl acetate; Others.Bornyl acetate; - Pinene;

Location of analyzed populations. populations. analyzed of Location Differences between three pine species (in %) on the basis of main terpene classes: classes: terpene main of basis the on %) (in species pine three between Differences

) rnia cmoet nlss PA o 195 of (PCA) analysis component Principal A) triangles represent triangles : ifrne bten he pn species pine three between Differences IV –

(

Mt. Sjekirica, Mt. III I

– –

Priboj, Crni vrh, Crni Priboj,

Germacrene D;( Mt. ovćen, Mt.

P. heldreichii P. – considered

; t Tr, Omar, Tara, Mt. . nigra P. . B) Representation of Representation B)

II scattered

–

– as single population single as

Mt. Zeletin, Mt. individuals from individuals Mt. Mt. Gora Mokra var. var. and Error bars represent standard deviations of analyzed analyzed of deviations standard represent bars Error E ) banatica V

- Caryophyllene; Caryophyllene; β little groups little

and –

Mt. Goč, Gvozdac; Gvozdac; Goč, Mt.

III Pinus nigra Pinus III : 2 – – VII ) 6

. five Mt. Bjelasica; Mt.

Mt. Tara, Zmajevečki potok; potok; Zmajevečki Tara, Mt.

selected variables (terpene compounds) on on compounds) (terpene variables selected ,

and individual trees between Mt. Zlatibor Mt. between trees individual and – and (in

populations and 90 and populations La

V %) on the basis of major terpene terpene major of basis the on %) aea ea ayn Kovej) Canyon, Reka zareva (

- nt the entries of the components, cf. cf. components, the of entries the nt P. nigra P. Pinene; Limonene; – . nigra P.

Mt. Revuša Mt. erpenes (OM), sesquiterpene sesquiterpene (OM), erpenes P. nigra P.

P. heldreichii P.

subsp

individual subsp. ) . ; ; P. peuce P. nigra P. peuce P. pallasiana

var. : s from seven seven from s . nigra P. I

– pančići individuals individuals

Mt. Tara, Mt.

( ; I : VI : -

: var. Mt. Mt. IV P. –

This article isThis article by protected copyright. All rightsreserved. Accepted Article to populations indicatedinFig.4A. as the for criterion and development clusters a as UPGMA pattern, as terpenes selected of percentages populations: 6. Fig. populations as indicated in Fig. heldreichii 5. Fig.

Canonical discriminant analysis (CDA) based on based (CDA) analysis discriminant Canonical edorm band y H cutrn of clustering AHC by obtained Dendrogram

and . peuce P. Euclidean

ih hi ppltos as populations their with

4A.

distances as diversity assessment criteria assessment diversity as distances

. nigra P. priori a

2 6

selected terpenes of terpenes selected

, . heldreichii P.

groups.

Symbols refer to to refer Symbols .

Symbols refer Symbols and P. nigra P. P. peuce peuce P. , P. P. This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article