Morfološka i fitohemijska karakterizacija predstavnika sekcija Pulegium (Mill.) Lam. & DC. 1805 i Mentha (Mentha L., Lamiaceae) sa Balkanskog poluostrva i južnog dela Panonske nizije

Abstract

Među više od 220, do sada poznatih rodova porodice Lamiaceae, rod Mentha se može okarakterisati kao jedan od najbolje istraženih,barem u pogledu njegovog hemijskog sastava. Ono što nanama daje na značaju i omogućava široku tradicionalnu i komercijalnu primenu, jeste visok etarskih ulja i fenolnih jedinjenja.Ova jedinjenja su produkti sekundarnog metabolizma biljaka, koja su prepoznata kao antioksidantni, antiinflamatorni, antifungalni,antibakterijski, antiviralni i insekticidni agensi.Etarska ulja i fenoli su često upotrebljivani u hemotaksonomskim studijama različitih vrsta, rodova, pa i potporodica iz porodice Lamiaceae.Rod Mentha je taksonomski izuzetno kompleksan zbog česte hibridizacije, izražene fenotipske plastičnosti, gajenja, različitog broja hromozoma i nivoa ploidnosti, vegetativnog razmnožavanja i pojave muške sterilnosti u cvetovima. Kao posledica interspecijske hibridizacije, ali i zbog povratnog ukraštanja novostvorenih hibrida sa roditeljskim vrstama,identifikacija i definisanje varijabilnosti taksona su veoma komplikovani, posebno u tipskoj sekciji. Dodatno, smatra se da je taksonomska situacija u okviru grupe Spicata na Balkanskom poluostrvu veoma nejasna i da zahteva detaljniju analizu. Sistem polnog razmnožavanja koji oduvek fascinira naučnike, opisuje i omogućava reproduktivni uspeh polovine vrsta porodice Lamiaceae, ali i uvodi zabunu u nomenklaturu, taksonomiju i sistematiku, jeste ginodioecizam.Ovaj model se navodi kao tipičan za rod Mentha, a podrazumeva redukciju, nepotpuno razvijanje i sterilnost prašnika u cvetovima određenog procenta jedinki u populacijama, odnosno,koegzistenciju funkcionalno ženskih sa hermafroditnim jedinkama, čiji su cvetovi uobičajeno građeni i dvopolni. Iako se prilikom opisa roda uvek navodi postojanje ovog mehanizma, retko je ta pojava detaljno analizirana, a na području Balkanskog poluostrva i u južnom delu Panonske nizije nije nikada. Zbog svega navedenog, istraživanja predstavnika dve sekcije roda Mentha (sect. Pulegium i sect. Mentha), sa Balkanskog poluostrva i južnog dela Panonske nizije,realizovana su u tri pravca: 1) preliminarna analiza strukture populacija četiri vrste (M.pulegium, M. longifolia, M. microphylla i M.spicata), u cilju potvrđivanja postojanja ginodiecizma i utvrđivanja frekvencije funkcionalno ženskih i hermafroditnih jedinki u populacijama, 2) morfološka istraživanja karaktera vegativnih i generativnih organa i komparativna taksonomska analiza tri vrste (M. longifolia, M. microphylla i M. spicata), u cilju definisanja njihove varijabilnosti, kao i intra- i interspecijskih razlika, i 3) primarna, sekundarnai tercijarna hemijska karakterizacija različitih taksona roda Mentha (M. aquatica, M. arvensis,M. longifolia, M. microphylla, M. spicata, M. pulegium, M. × dumetorum, M. × gentillis, M. × piperita, M. × piperita ‘Alba’, M. × piperita ‘Crispa’, M. × piperita ‘Ruska crna’ i M. × verticillata) radi ukazivanja na nedovoljno eksploatisane potencijalne prirodne resurse, kao i zbog utvrđivanja sličnosti i razlika u hemijskom sastavu uzoraka različitih taksona, u cilju eventualne primene fenolnih i isparljivih jedinjenja u hemotaksonomiji. Biljni materijal korišćen u svim analizama roda Mentha,prikupljen je tokom terenskih istraživanja sprovedenih na Balkanskom poluostrvu i u južnom delu Panonske nizije, a većinski potiče sa teritorija Bosne i Hercegovine, Bugarske, Crne Gore, Grčke, Severne Makedonije i Srbije.Pored toga, studija obuhvata i pojedinačne uzorke iz Albanije i Slovenije, kao i nekoliko uzorka gajenih oblika roda sa privatnih parcela, i uzorke porekom sa oglednih parcela Instituta za ratarstvo i povrtarstvo, (Odeljenje za alternativne kulture i organsku proizvodnju, Bački Petrovac). Na osnovu rezultata preliminarne analize ginodiecizma, sprovedene na 1.080 jedinki iz 36 populacija, ustanovljeno je da su sve populacije vrste M. pulegium hermafroditne sa jedinkama koje imaju normalne, dvopolnim cvetove čije su antere krupne, nedeformisane i sadrže veliku količinu polena. Za razliku od njih, 11 populacija vrsta M. longifolia, M. microphylla i M. spicata pored hermafroditnih, uključivale su i funkcionalno ženske jedinke, zbog koji su one okarakterisane kao ginodiecke. U populacijama vrsta M. longifolia i M. spicata zabeležena je po jedna intermedijerna jedinka, koja istovremeno ima i dvopolne i funkcionalno ženske cvetove, a u populacijama vrste M. longifolia bile su prisutne i jedinke nejasnog statusa, čiji cvetovi su imali smanjene prašnike smeštene unutar krunice sa smanjenim anterama koje deluju suvo, a čiji polen zahteva dodatne analize fertilnosti i vijabilnosti. Zabeležena je različita frekvencija funkcionalno ženskih jedinki u populacijama,koja varira u velikom opsegu, a morfometrijskim analizama cvetova, ustanovljeno je da su funkcionalno ženski cvetovi sitniji u odnosu na dvopolne, što se ogleda prevenstevno razlikama u veličini prašnika, a potom i čašice i krunice, dok jasnije diferencijacije u dužinama plodnika, stubića i žiga nema. Morfološka istraživanja kvantitativnih i kvalitativnih karaktera stabla, lista i cvasti ukupno su obuhvatila 95 populacija i 2.023 jedinke, a na nivou cveta i brakteje analizirano je 38 populacija i 989 jedinki tri vrste (M. longifolia, M. microphylla i M. spicata). Stablo, list i cvast analizirani su uz 11 kvantitativnih i 11 kvalitativnih karaktera, dok su za analizu cveta i brakteje isključivo korišteni kvantitativni karakteri (34). Podaci su obrađeni metodama osnovne, univarijantne i multivarijantne statistike Na osnovu analiza morfometrijskih karaktera pokazano je da se stablo, list, cvasti i brakteja karakterišu izraženom varijabilnošću. Nasuprot tome karakteri cveta su bili sličniji i manje varijabilni.Izdvojeni su morfometrijskih karakteri primenljivi za intra- i interspecijsku diferencijaciju i diskriminaciju analiziranih taksona, a utvrđen je i njihov obrazac varijabilnosti. Rezultati upućuju da sporan takson označen kao vrsta „M. microphylla“, koji je prema poslednjoj podeli u vodu sinonima uključen u M. spicata subsp. condensata pokazuje osobine jasno definisane vrste i zaslužuje detaljnu reviziju. Na istraživanom području potvrđena je kompleksnost populacija vrsta M. longifolia i M. spicata, koja se pre svega ogleda u njihovoj velikoj sličnosti i teškoj identifikaciji sa postojećim ključevima za determinaciju. Stoga su kao dodatni karakteri definisani ključevi za razdvajanje ove tri vrste na osnovu morfometrijskih karaktera delova cveta. Na osnovu analize kvalitativnih karaktera ustanovljeno je da uzorak nije uniforman i da se a priori definisane vrste jasno odvajaju, kao i neke populacije unutar njih. Primarna hemijska karakterizacija obuhvatila je određivanje prinosa ekstrakcije i sadržaja ukupnih fenola iflavonoida, gravimetrijskim i spektrofotometrijskim tehnikama, u 163 vodenoalkoholna ekstrakta taksona roda Mentha i rezultati su pokazali veliko bogatstvo. Sekundarana hemijska karakterizacije je podrazumevala analizu prisustva i sadržaja 12 fenolnih jedinjenja, primenom visokoefikasne tečne hromatografije (HPLC) na ukupno 138 ekstrakata. Izdvojena su jedinjenja koja su zabeležena kod svih taksona, njihove učestalosti i rasponi vrednosti. Kod vrsta predstavljenih većim brojem uzoraka poreklom sa velikog geografskog područja, uočljivo je postojanje značajnih razlika u pogledu prinosa, sadržaja ukupnih fenola i flavonoida, kao i 12 analiziranih jedinjenja. Tercijarna hemijska karakterizacija je svedena na analizu isparljivih komponenti u 12 uzoraka suvog biljnog materijala, korišćenjem headspace gasne hromatografije–masene spektrometrije (GC–MS). Identifikovano je prisustvo 54 isparljiva jedinja, grupisanih u pet klasa. Dobijeni su novi podaci o hemijskom diverzitetu roda Mentha, a potvrđeni su i prethodno publikovani. Na osnovu kvalitativnog i kvantitativnog sadržaja ovih isparljivih jedinjenja, a prema rezultatima klaster analize, bio je uočljiv drugačiji hemotipski obrazac hibridnih taksona u odnosu na roditeljske vrste.Na osnovu jednog ili dva najzastupljenija jedinjenja, analizirani taksoni su podeljeni u pet hemotipova, tj. tri zasebne jedinice i dve grupe taksona. Ovo je prva studija koja se bavila pojavom ginodiecizma u populacijama roda Mentha, morfološkom varijabilnošću veoma varijabilnih i sličnih vrsta.

Among more than 220 genera, so far described and accepted in the family Lamiaceae, the genus Mentha can be regarded as one of the best studied, at least in view of its chemical content. The mints owe its importance and wide spectra of traditional and commercial use to the high content of essential oils and phenolics. These compounds are products of plant secondary metabolism, and they acting as antioxidant, antibacterial, antifungal, antiinflammatory, anti-insect and antiviral agents. Essential oils and phenolics have been frequently used as markers in chemotaxonomy of various species, genera, as well as subfamilies of the family Lamiaceae The taxonomy of the genusMentha is extremely complex due to ease of hybridization, strong phenotypic plasticity, cultivation, different chromosome numbers and ploidy levels, vegetative propagation and occurrence of male sterility in flowers. As a consequence of interspecific hybridization and backcrosses of newly formed progenies with their parents, identification of separate taxa and definition of their variability are challenging, particularly in the typical section. Additionally, taxonomy of the group Spicata in the Balkan peninsula is consider to be unclear and demands more detailed analyses. The system of sexual reproduction that fascinates scientist since ever, describes and enables reproductive success of the half of Lamiaceae species, but surely had brought confusion to nomenclature, taxonomy and systematics, is gynodioecism. This mating system is cited as typical trait of the genus Mentha and it consider reduction, incomplete development and sterility of stamens in flowers of some percent of individuals in populations, i.e. coexistence of females (male-sterile) and hermaphrodites, whose flowers are normally developed and bisexual.Although the occurrence of this mechanism in description of the genus Mentha has always been noted, it is quite rarely investigated, and there is no single record of it in the Balkan Peninsula and southern part of Pannonian plain. Taking everything into account, research of the genus Mentha (sect. Pulegium i sect. Mentha) representatives from the Balkan peninsula and southern part of Pannonian plain has been designed as follows: 1) preliminary analysis of populations’ structure of four species (M. pulegium, M. longifolia, M. microphylla and M. spicata) with aim to confirm gynodioecism and determinate frequencies of females and hermaphrodites in populations, 2) morphometric analysis of vegetative and generative organs and comparative taxonomical analysis of three species (M. longifolia, M. microphylla and M. spicata), in order to define their variability, as well as intraand interspecific differences, and 3) primary, secondary and tertiary chemical characterizatioof various taxa of the genus Mentha (M. aquatica, M. arvensis, M. longifolia, M. microphylla, M. spicata, M. pulegium, M. × dumetorum, M. ×gentillis, M. × piperita, M. × piperita ‘Alba’, M. × piperita ‘Crispa’, M. × piperita ‘Ruska crna’ and M. × verticillata) with the goal to indicate insufficiently exploited potential natural resources and to evaluate similarities and differences in chemical content of different taxa samples, for the purpose of possible application of analyzed compounds in chemotaxonomy. Plant material used in all analyses of the genus Mentha was collected during field trips conducted in Balkan peninsula and southern part of Pannonian Plain, and its major part originated from Bosnia and Herzegovina, Bulgaria, Montenegro, Greece, North Macedonia and Serbia. Furthermore, the study included one sample per Albania and Slovenia, respectively, as well as several samples of cultivated forms of the genus obtained from private (Padej, Serbia) and experimental parcels of the Institute of Field and Vegetable Crops (Alternative Crops and Organic Cultures Department, Bački Petrovac, Serbia). According to the results of the preliminary analysis of gynodioecism conducted on 1.080 individuals from 36 populations it was founded that all investigated populations of the species M. pulegium were hermaphroditic, because all recorded individuals had perfect flowers, with normally developed pistils and large anthers containing great amount of pollen grains. In contrast to that, 11 populations of three species (M. longifolia, M. microphylla and M. spicata) contained hermaphrodite and female individuals, hence they were distinguished as gynodioecious. Besides that, in one population per species M.longifolia and M. spicata respectively, there were intermediate individuals, which had both, perfect and female (pistilate) flowers. Moreover, in some populations of the species M. longifolia there were individuals of uncertain status, whose flowers had reduced stamens within corollas, with small,collapsed and often dry anthers, which pollen grains require fertility and viability tests in orderto be properly classified. Different frequencies of hermaphrodites and females were noted, with great variations in and among populations. It was confirmed that female flowers are smaller than perfect flowers, with differences were primarily visible in parameters of stamens, as well as in calyx and corolla dimensions, but unfortunately without clear differentiation in length of ovaries, styles and stigmas. Quantitative and/or qualitative morphological analyses of stem, leaf, inflorescence and bract were performed on 95 populations and 2.023 individuals, while dimensions of flowers and bracts were analyzed on 989 individuals of three species (M. longifolia, M. microphylla and M. spicata). Stem, leaf, inflorescence and bract were analyzed with 13 quantitative and 11 qualitative characters, while flower and bract were solely easured using 34 quantitative characters. Comparative morphological analyses were conducted employing methods of basic, univariate and multivariate statistics. It was shown that stems, leaves, inflorescences and bracts were significantly variable in view of obtained values of morphometric characters. In contrast, morphometric characters of flowers were significantly similar and variable in smaller extent. Morphometric characters with possible application in intra- and interspecies differentiation and discrimination of the analyzed taxa were distinguished, and pattern of their variability was established. The results of morphometric analyses of flower part pointed out that disputable taxon labelled as species “M. microphylla”, in the latest classification included as a synonym within M. spicata subsp. condensata showed features of clearly defined species and therefore deserves detailed revision. Also, large complexity of M. longifolia and M. spicata populations was confirmed in the investigated area, primarily seen in their similarity, and difficult identification with existing identification keys. Therefore, according to dimensions of flower parts, additional identification keys were constructed for easier determination of thosespecies. Based on analyses of qualitative characters, it was found that samples were not homogenous, and that a priori defined species can be clearly separated, as well as some of populations within them. Primary chemical characterization was included determination of yield of extraction and total content of phenols and flavonoids, with gravimetric and spectrophotometric techniques in 163 aqueousalcoholic extracts, and it confirmed richness of the genus Mentha. Secondary chemical characterization was involved analysis of presence and content of 12 phenolic compounds, with highperformance liquid chromatography (HPLC) in 138 aqueous-alcoholic extracts. Compounds that were identified in all analyzed taxa, their ranges and frequencies were listed. Great differences in yield of extraction, total content of phenols and flavonoids, as well as 12 phenolic compounds were the most obvious in species represented by large number of samples, originated from wide geographical territory. Tertiary chemical characterization covered evaluation of qualitative and quantitative volatile content in 12 samples of dried plant material, using headspace gas chromatography-mass spectrometry (GC–MS). There were 54 different volatiles, grouped in five classes. Many previous data were confirmed, but there was also a new data in light of volatiles recorded in investigated area. According to the quantitative and qualitative investigation of identified volatiles (mainly acc. to one or two dominant compounds) performed in cluster analysis there was different chemical pattern of hybrids in respect to parental species. Henceforth, analyzed taxa were divided into five chemotypes, i.e. three separate lines and two groups.