The principal goal of my study was to find if ecologically and chemically different populations of lichens in the Physconia muscigena (Ach.) Poelt group belong to several species or a single one. This study focused on the molecular and chemical investigations of mostly European and Canadian populations. I use sequence data from three genes (ITS rDNA, mtSSU rDNA and TEF1) for the reconstruction of phylogenetic trees. I investigate phylogenetic relationships among the closely related species P. muscigena, P. bayeri, P. rossica, and P. isidiomuscigena. Also, I wanted to detect any possible geographical or ecological trends among chemotypes and haplotypes. As an additional goal I checked the recent localities of P. muscigena in the Czech Republic for valorising its conservation status. Results show that: (1) sequenced data of ITS rDNA and TEF1 show high intraspecific variability in P. muscigena samples. This genetic variability does not correlate neither with geographical distribution nor thallus chemistry; (2) P. bayeri is synonymous with P. muscigena; (3) some samples P. muscigena contain new undetermined secondary metabolite, (4) Physconia muscigena has only three recent localities in Czech Republic. Key words: Physconia muscigena, Physconia bayeri, infraspecific variability, taxonomy, TEF1, ITS...

<p>Pored rodova evropskih orhideja koji se odlikuju velikim stepenom diverzifikacije, kao i značajnim brojem novoopisanih vrsta, kao &scaron;to su <em>Ophrys </em>ili<em> Epipactis,</em> rod<em> Anacamptis </em>je ostao poprilično zapostavljen u orhidolo&scaron;kim istraživanjima. Na predstavnicima ovog roda uglavnom su vr&scaron;ena molekularna istraživanja, u kojima je bio uključen i veći broj drugih rodova.Takođe, urađene su i opsežne studije, ali samo na pojedinim vrstama. Morfolo&scaron;ka istraživanja su bila veoma retka i uz par uzuzetaka, ograničena na geografski mala područja i na jednu ili nekoliko morfolo&scaron;ki sličnih vrsta. Analiza morfolo&scaron;ke varijabilnosti ovolikog broja taksona roda<em> Anacamptis</em> na geografski relativno velikom području kakvo je Balkansko poluostrvo, kao i deo Panonske nizije koji se oslanja na njega, do&nbsp; sada nije bilo sprovedeno. Morfolo&scaron;kim analizama bili su podvrgnuti gotovo svi predstavnici ovog roda u rangu vrste i podvrste, a koji se javljaju na području kopnenog dela Balkanskog poluostrva, kao i južnog oboda Panonske nizije. Sprovedene su jednostrane i uporedne morfolo&scaron;ke analize sa detaljnomobradom podataka metodama bazične, univarijantne i multivarijantne statistike. Do sada najvećim istraživanjem taksona roda<em> Anacamptis</em>, po broju obrađenih jedinki i području na kom je sprovedeno, bila je obuhvaćena 2001 jedinka iz 185&nbsp; populacija. Analizirane su prirodne populacije, ali i materijal iz Herbarijuma BUNS. Ukupno je definisano 69 morfolo&scaron;kih karaktera, kvantitativnih i kvalitativnih, a među njima su traženi oni koji imaju potencijalno diferencijalni karakter između opisanih vrsta i infraspecijskih taksona. Analize su obuhvatile i interpopulacionu morfolo&scaron;ku varijabilnost u okviru svakog taksona, a u cilju uočavanja postojanja određenih geografskih obrazaca njenih promena. Morfolo&scaron;ki karakteri koji su se u sprovedenim analizama pokazali kao taksonomski informativni, iskori&scaron;ćeni su za formiranje dihotomog ključa za determinaciju taksona roda <em>Anacamptis</em> prisutnih na istraživanom području. Analizama je utvrđen veliki broj karaktera koji su statistički značajno različiti između analiziranih populacija, podvrsta i vrsta. Većina karaktera je pokazala umerenu ili nisku varijabilnost, a u retkim slučajevima i povećanu. Karakteri cveta međusobno su statistički značajno korelisani,većina slabo do umereno, dok su oni na listićima kacige, kao i između labeluma i drugih delova cveta jako do izrazito jako povezani. Jedini takson koji nije pokazao nikakvu infraspecijsku diferencijaciju, a koji se odlikuje velikom unutari interpopulacionom varijabilno&scaron;ću je<em> A. pyramidalis</em>. Iz istog razloga je pokazano kao neosnovano izdvajanje jedinki iz različitih delova njegovog areala u do sada opisane infraspecijske oblike. Potvrđena je niska morfolo&scaron;ka varijabilnost uskorasprostranjene vrste <em>A. boryi.</em> Kod svih preostalih istraživanih taksona utvrđeno je postojanje određenih obrazaca geografske interpopulacione varijabilnosti i infraspecijske struktuiranosti.Populacije <em>A. laxiflora</em> subsp. <em>laxiflora </em>mogu se podeliti na jadranske i kontinentalne, pri čemu najzapadnije pokazuju i najmanje vrednosti analiziranih karaktera. <em>A. coriophora </em>subsp.<em> fragrans </em>se pokazala kao manje varijabilnom od tipske podvrste, ali je u okviru nje kao zaseban takson izdvojen var. <em>hermae </em>kao fenolo&scaron;ki, ekolo&scaron;ki i morfolo&scaron;ki odvojen u odnosu na ostaleispitivane populacije. Tipska podvrsta pokazuje obrazac variranja ukupne morfolo&scaron;ke varijabilnosti u pravcu zapad-istok. Kao karakteri koji su najznačajniji za diferencijaciju podvrsta vrste<em> A. palustris</em> izdvojeni su dužina i &scaron;irina brakteje, stepen diferenciranosti labeluma na režnjeve, kao i dubina sinusa koja ih razdvaja. Populacije<em> A. palustris</em> subsp. <em>elegans</em> moguće je podeliti na tri geografski i morfolo&scaron;ki definisane grupe &ndash; istočnobalkansku, zapadnobalkansku i periferne populacije sa severa Srbije i iz Slovenije. Kao diferencijalni karakteri između taksona sekcije <em>Laxiflorae</em> pokazali su se odnos između dužine plodnika i brakteje, kao i dužina srednjeg režnja labeluma. Vrsta<em> A. morio </em>je po prvi put jasno morfolo&scaron;ki i arealno podeljena na dve podvrste na istraživanom području. Podvrste pokazuju velika preklapanja vrednosti morfolo&scaron;kih karaktera, ali se obe odlikuju specifičnim karakteristikama cveta, pre svega labeluma na osnovu kojih ih je moguće razlikovati. Definisani su areali podvrsta<em> A.morio </em>na istraživanom području &ndash; tipska podvrsta je prevashodno severnija i zapadnija, dok je subsp.<em> caucasica </em>južnija i istočnija. Prvu odlikuju krupniji cvetovi, bubrežastih labeluma sa srednjim režnjem koji ne prevazilazi značajno dužinu bočnih, dok je druga sa sitnijim cvetovima i snažno isturenim srednjim režnjem. Obe podvrste mogu se podeliti na grupe populacija sa jasno definisanim geografskim raspostranjenjem. Vrsta <em>A. papilionacea </em>je po prvi put morfolo&scaron;ki istaživana na većem broju primeraka, a ne prostim poređenjem pojedinačnih primeraka iz različitih delova areala. Pokazana je morfolo&scaron;ka diferenciranost na dve podvrste &ndash; tipsku i subsp. aegaea, koje se jasno morfolo&scaron;ki razlikuju i zauzimaju različite delove areala vrste. U okviru subsp. <em>papilionacea</em> registrovan je klinalni raspored morfolo&scaron;ke varijabilnosti u pravcu sever-jug. Registrovana su i tri hibrida, nova za područje pojedinih zemalja &ndash; A. <em>&times; gennarii</em> (Severna Makedonija),A. <em>&times; parvifolia</em> (Crna Gora) i A. &times; <em>timbali</em> (Srbija). Hibridi pokazuju generalnu morfolo&scaron;ku intermedijarnost u odnosu na roditelje, ali se odlikuju i novim stanjima karaktera, nezabeleženim kod roditeljskih taksona. Slični obrasci morfolo&scaron;ke varijabilnosti, kako na interpopulacionom nivou pojedinih vrsta, ali iizmeđu podvrsta svake istraživane vrste, pokazuju jasnu vezu između istraživanih taksona i jo&scaron; jednom opravdavaju njihovo zajedničko grupisanje u rod<em> Anacamptis.</em></p> / <p>Unlike the highly diversified and species-rich genera of European orchids, such as<em> Ophrys </em>or <em>Epipac</em>tis, the genus <em>Anacamptis </em>has remained quite neglected in orchidological research. Representatives of this genus, together with many others genera, were investigated mainly by molecular methods. Also, extensive studies have been done, but only on specific species. Morphological studies were very rare, and with a few exceptions, limited to geographically small areas and one or more morphologically similar species. Analysis of the morphological variability of so many taxa of the genus <em>Anacamptis</em>, in a is, in a geographically relatively large area such as the Balkan Peninsula, as geographically relatively large area such as the Balkan Peninsula, as well as the southern part of the Pannonian Plain, has not been conducted so far. Morphological analyses were<br />performed on almost all representatives of this genus,in the species and subspecies rank , occurring in the Balkan Peninsula mainland, as well as in the southern perimeter of the Pannonian Plain. Comparativemorphological analyses were conducted using the methods of basic, univariate and multivariate statistics.So far, the largest study of the genus&nbsp; nacamptis, by the number of processed individuals and the area in which it was conducted, included 2001 individuals from 185 populations. Natural populations and&nbsp; material from the BUNS Herbarium were analyzed. A total of 69 morphological characters, quantitative and&nbsp; qualitative, were defined, and those that have a potentially differential character among analyzed species and infraspecific taxa were selected. The analyzes also included interpopulation morphological variability within each taxon, to identify the existence of specific geographic patterns of its changes. The morphological characters that proved to be taxonomically informative, were used to form the dichotomous key for the determination of the genus Anacamptis members, presented in the study area. The analyzes identified a large number of characters that were statistically significantly different between the analyzed populations, subspecies, and species. Most of the characters showed moderate or low variability, and in rare cases increased variability. Flower&rsquo;s characters were statistically significantly correlated with each other, most of them weakly to moderately, while those of the sepals and petals, as well as between the&nbsp; labellum and other parts of the flower, were very strongly correlated. The only taxon that has not shown any infraspecific differentiation (except <em>A. boryi</em>), and which is characterized by high intra- and interpopulation variability, is A. pyramidalis. For the same reason, it has been shown as unjustified to separate individuals from different parts of species range into infraspecific forms. The low morphological variability of the narrowly distributed A. boryi was confirmed. In all other taxa studied, the existence of certain patterns of geographical interpopulation variability and infraspecific structuring was detected. <em>A. laxiflora</em> subsp. <em>laxiflora</em> populations can be divided into two groups &ndash; Adriatic and Continental, with the lowest characters values measured among the westernmost populations. <em>A. coriophora s</em>ubsp. <em>fragrans</em> proved to be less variable than the type subspecies, but within it, a new variety (var. hermae) as phenological, ecological and morphologically distinct from other studied populations was described. The typesubspecies has a transition of morphological variability in a west-east direction. The characters that are the most important for differentiation of A. palustris subspecies are the&nbsp; bract length and width, the degree of labellum differentiation into lobes, and the depth of the sinuses that separate them. Populations of A. palustris subsp. elegan<em>s </em>can be divided into three geographically and morphologically defined groups &ndash; Eastern Balkan, Western Balkan and peripheral populations from the North Serbia and Slovenia. Differential characters&nbsp; among the Laxiflorae section taxa are ration between the ovary length and bract length, as well as the labellum middle lobe length. For the first time, species <em>A. morio </em>is morphologically and geographically divided into two subspecies, in the&nbsp; study area. Analyzed subspecies have great overlapping values for many morphological characters, but both are characterized by specific flower&rsquo;s characteristics, above all labellum shape. On this basis, it is possible to distinguish them. Areals of <em>A</em>. <em>morio </em>subspecies were defined &ndash; the type subspecies is predominantly northern and western,while subsp. caucasica is southern and eastern. The first is characterized by larger flowers, renal shape labellum with a median lobe that does not exceed significantly the length of the lateral ones, while the second subspecies has smaller flowers and strongly projecting the median lobe. Both subspecies can be divided into groups of populations that have clearly defined geographical distribution. For the first time, A. papilionacea was morphologically investigated on a larger number of specimens, unlike previous&nbsp;&nbsp; researches in which individual specimens from different parts of the range were simply compared. Morphological differentiation into two subspecies was shown &ndash; type and subsp. aegaea. They are morphologically distinct and occupy different parts of the species range. Within the subsp. papilionacea a clinal distribution of morphological variability in the north-south direction was recorded. Three taxa of hybrid origin, new for the area of North Macedonia (A.<em> &times; gennari</em>i), Montenegro (A. <em>&times; parvifolia</em>) and Serbia (A. &times; t<em>imbali</em>) were registered. Hybrids exhibit a general morphological intermediacy between parents but are also distinguished by new character states that were not observed in<br />parental taxa. Similar patterns of morphological variability, both at the interpopulation level ofindividual species and between the subspecies of each species studied, show a clear relationship between the studied taxa and once again justify their grouping into the genus <em>Anacamptis.</em></p>

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-24T13:34:56Z No. of bitstreams: 2 Tese - Joelma Abadia Marciano de Paula - 2011.pdf: 4630470 bytes, checksum: fc7db5b305643bc19e85c94dab8064ff (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-24T15:02:48Z (GMT) No. of bitstreams: 2 Tese - Joelma Abadia Marciano de Paula - 2011.pdf: 4630470 bytes, checksum: fc7db5b305643bc19e85c94dab8064ff (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-24T15:02:48Z (GMT). No. of bitstreams: 2 Tese - Joelma Abadia Marciano de Paula - 2011.pdf: 4630470 bytes, checksum: fc7db5b305643bc19e85c94dab8064ff (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-03-21 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The purpose of this work is to carry out the phytochemical study and evaluate biological activities of the essential oil and extracts of the Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) leaves. Chapter 1 provides a review of the main botanical aspects of the genus Pimenta, its chemical composition and pharmacological potential. Chapter 2 describes the occurrence of infraspecific variability in P. pseudocaryophyllus. For this the essential oils from 12 specimens of P. pseudocaryophyllus that occur naturally in three different locations of the Brazilian Cerrado underwent a qualitative and quantitative analysis by gas chromatography-mass spectrometry (GC/MS). The multivariate statistical analysis identified three chemotypes of P. pseudocaryophyllus, characterized by the predominance of citral, (E)-caryophyllene and (E)-methyl isoeugenol constituents. Chapter 3 presents the phytochemical assessment of the hexane, dichloromethane, ethyl acetate and aqueous fractions obtained from the crude ethanol extract of P. pseudocaryophyllus leaves. Chromatographic and spectrophotometric (1H and 13C) methods enabled the isolation and identification of lupeol, α-and 􀈕-amyrin, oleanolic, ursolic and betulinic acids, quercetin, quercitrin, isoquercitrin, afzelin, avicularin, guaijaverin, catechin, ellagic acid and gallic acid. Chapter 4 assesses the antimicrobial activity of the ethanol extracts, fractions (hexane, dichloromethane, ethyl acetate and aqueous), essential oils and isolated compounds of citral (CQ) and (E)-methyl isoeugenol (MQ) chemotypes of P. pseudocaryophyllus. The broth microdilution test was applied to determine the minimum inhibitory concentration (MIC) against strains of Gram(+) and Gram(-) bacteria, and of Candida and Cryptococcus spp fungi. The CQ and MQ dichloromethane fraction, CQ ethyl acetate fraction, and CQ essential oil were the most active (MIC = 62.5 to 500 mg / mL) against Gram(+). The oleanolic acid proved to also be responsible for this action. The CQ and MQ extracts of P. pseudocaryophyllus were active (MIC = 7.8 to 500 mg / mL) against most strains of Candida spp and Cryptococcus spp, where gallic acid, ellagic acid and quercitrin contributed to this action. The data obtained indicate the antimicrobial potential of CQ and MQ P. pseudocaryophyllus against some infectious diseases. Chapter 5 describes the antinociceptive activity, in mice, of CQ and MQ ethanol extracts, CQ fractions and CQ essential oil, and the anti-inflammatory activity of CQ crude ethanol extract of P. pseudocaryophyllus. The abdominal writhing test induced by intraperitoneal administration (ip) of acetic acid (1.2% v / v) and the ear edema test induced by croton oil (2.5% V/V) were performed. The CQ crude ethanol extract (2000, 1000 and 500 mg/kg, po) showed a dose-dependent antinociceptive effect. Among the fractions, only the aqueous fraction was not active. The CQ essential oil (60, 200 and 600 mg / kg, po) presented significant antinociceptive activity, probably because of its high citral concentration. Pretreatment with CQ crude ethanol extract (2000, 1000 and 500 mg / kg) reduced edema by 14.3 ± 0.4 mg (control) to 10.9 ± 0.5, 11.3 ± 0, 4, 10.5 ± 0.5 mg, respectively. The analgesic effect may be related to an anti-inflammatory action observed in the ear edema test. / O presente trabalho teve como objetivos realizar o estudo fitoquímico e avaliar atividades biológicas do óleo essencial e de extratos da folha de Pimenta pseudocaryophyllus (Gomes) L. R. Landrum (Myrtaceae). O Capítulo 1 traz uma revisão bibliográfica do gênero Pimenta onde são abordados os principais aspectos botânicos, a composição química e o potencial farmacológico. O Capítulo 2 descreve a ocorrência de variabilidade infraespecífica em P. pseudocaryophyllus. Para tanto, foi realizada a análise qualitativa e quantitativa, por cromatografia gasosa acoplada a espectrometria de massas (CG/EM), do óleo essencial das folhas de 12 espécimes de P. pseudocaryophyllus que ocorrem naturalmente em três diferentes localidades do Cerrado brasileiro. A análise estatística multivariada dos dados identificou três quimiotipos de P. pseudocaryophyllus, caracterizados pela predominância dos constituintes citral, (E)-cariofileno e (E)-metilisoeugenol. O Capítulo 3 apresenta os resultados da avaliação fitoquímica das frações hexano, diclorometano, acetato de etila e aquosa obtidas a partir do extrato etanólico bruto das folhas de P. pseudocaryophyllus. A partir da utilização de técnicas cromatográficas e espectrofotométricas (RMN 1H e 13C) foi possível isolar e identificar os triterpenos pentacíclicos lupeol, α- e 􀈕-amirina, os ácidos oleanólico, ursólico e betulínico, os flavonóides quercetina, quercitrina, isoquercitrina, afzelina, avicularina, guaijaverina e catequina, e os ácidos gálico e elágico. O Capítulo 4 expõe a avaliação da atividade antimicrobiana dos extratos etanólicos brutos, frações (hexano, diclorometano, acetato de etila e aquosa), óleos essenciais e substâncias semipurificadas de P. pseudocaryophyllus quimiotipos citral (QC) e (E)- metilisoeugenol (QM). Foi utilizado o teste da microdiluição em caldo, que permitiu determinar a Concentração Inibitória Mínima (CIM) frente a cepas de bactérias Gram(+), Gram(-) e fungos Candida spp. e Cryptococcus spp. As frações diclorometano (QC e QM), acetato de etila (QC) e o óleo essencial do quimiotipo citral foram os mais ativos (CIM = 62,5-500 μg/mL) frente as bactérias Gram(+), o ácido oleanólico mostrou ser um dos responsáveis por essa ação. O QC e QM foram ativos (CIM = 7,8 – 500 μg/mL) frente à maioria das cepas fúngicas, sendo que ácido gálico, ácido elágico e quercitrina contribuíram para essa ação. Os dados obtidos indicaram que P. pseudocaryophyllus QC e QM apresentam potencial antimicrobiano. O Capítulo 5 descreve a avaliação da atividade antinociceptiva, em camundongos, dos extratos etanólicos brutos (QC e QM), frações e óleo essencial (QC) de P. pseudocaryophyllus e a atividade anti-inflamatória do extrato etanólico bruto (QC) de P. pseudocaryophyllus. Foram utilizados os testes das contorções abdominais induzidas pela administração intra-peritonial (i.p.) de ácido acético (1,2%, v/v) e do edema de orelha induzido pelo óleo de cróton (2,5%, V/V) em camundongos. O extrato etanólico bruto QC (2000, 1000 e 500 mg/kg, v.o.) demonstrou efeito antinociceptivo dose-dependente. Dentre as frações, apenas a aquosa não foi ativa. O óleo essencial QC (60, 200 e 600 mg/kg, v.o.) demonstrou significativa atividade antinociceptiva, provavelmente por causa do seu elevado teor em citral. O pré-tratamento com o extrato etanólico bruto QC nas dose 2000, 1000 e 500 mg/kg reduziu o edema de 14,3±0,4 mg (controle) para 10,9±0,5; 11,3±0,4; 10,5±0,5 mg, respectivamente. A ação analgésica pode estar relacionada a uma ação anti-inflamatória, observada no teste de edema de orelha.