<p><em>Prospero&nbsp; autumnale&nbsp;</em> kompleks&nbsp; je&nbsp; taksonomski&nbsp; najintrigantniji&nbsp; član&nbsp; roda&nbsp; <em>Prospero,</em>&nbsp; sa&nbsp; centrom rasprostranjenja u Mediteranu. Kompleks se rasprostire i na obalama Atlantskog okeana u Francuskoj, sve do južnih&nbsp; delova Velike&nbsp; Britanije, zatim&nbsp; na Balkanskom poluostrvu, u Panonskoj&nbsp; niziji, sve&nbsp; do Krima, Kavkaza&nbsp; i&nbsp; delova&nbsp; Irana&nbsp; na&nbsp; istoku.&nbsp; Areal&nbsp; kompleksa&nbsp; se&nbsp; preklapa&nbsp; sa&nbsp; arealima&nbsp; ostale&nbsp; dve&nbsp; vrste&nbsp; roda&nbsp; &ndash;&nbsp; u<br />zapadnom Mediteranu sa <em>P. obtusifolium</em>, a u istočnim delovima Mediterana sa&nbsp;<em> P. hanburyi. </em>Za razliku od <em>P. obtusifolium&nbsp; i&nbsp; P. hanburyi,&nbsp; P. autumnale</em>&nbsp; kompleks se odlikuje visokom kariolo&scaron;kom varijabilno&scaron;ću.Razlikuju se četiri osnovna broja hromozoma&nbsp; x&nbsp; = 5, 6, 7 i četiri različita tipa genoma (A, B⁵, B ⁶ i B⁷ )&nbsp; i diploidna&nbsp; citotipa&nbsp; (AA,&nbsp; B ⁵B⁵,&nbsp; B ⁶B⁶i&nbsp; B ⁷B ⁷ )&nbsp; koja&nbsp; se&nbsp; razlikuju&nbsp; u&nbsp; odnosu&nbsp; na&nbsp; osnovni&nbsp; broj&nbsp; hromozoma, veličinu&nbsp; i&nbsp; morfologiju&nbsp; hromozoma.&nbsp; Genom&nbsp; B ⁵ ima&nbsp; x&nbsp; =&nbsp; 5,&nbsp; B⁶ x&nbsp; =&nbsp; 6,&nbsp; a&nbsp; genomi&nbsp; A&nbsp; i&nbsp; B⁷x&nbsp; =&nbsp; 7. Najrasprostranjeniji&nbsp; genom&nbsp; je&nbsp; B ⁷ ,&nbsp; koji&nbsp; je&nbsp; zabeležen&nbsp; u&nbsp; celom&nbsp; arealu&nbsp; kompleksa,&nbsp; dok&nbsp; je&nbsp; A&nbsp; zastupljen&nbsp; u zapadnom Mediteranu, B⁵ u Libiji, a B ⁶je endemičan za Krit. U okviru B ⁷ genoma se, dalje, razlikuju dve linije nastale kao posledica duplikacija 5S rDNK lokusa, pri čemu linija I ima jedan lokus, a kod linije II taj&nbsp; lokus&nbsp; je&nbsp; duplikovan.&nbsp; Osim&nbsp; diploidnih&nbsp; citotipova,&nbsp; poznato&nbsp; je&nbsp; i&nbsp; nekoliko&nbsp; ploidnih&nbsp; nivoa,&nbsp; od&nbsp; kojih&nbsp; su najučestaliji&nbsp; tetraploidi&nbsp; (2n=4x=28)&nbsp; i&nbsp; heksaploidi&nbsp; (2n=6x=42).&nbsp; Za&nbsp; <em>P.&nbsp; autumnale&nbsp;</em> kompleks&nbsp; je karakteristična varijabilnost u veličini genoma između različitih citotipova i unutar pojedinih citotipova. Genomsko restrukturiranje, koje je imalo najveću ulogu u evoluciji i diverzifikaciji kompleksa, nije imalo velikog uticaja na&nbsp; morfolo&scaron;ku varijabilnost, te je&nbsp; iz tog razloga kompleks&nbsp; morfolo&scaron;ki skoro uniforman. Ipak karakteri kao &scaron;to su: boja tunike lukovice, visina biljke, oblik i dimenzije lista, broj cvetova u cvasti i boja cvetova pokazuju izvesnu varijabilnost. Taksonomske nejasnoće nastaju zbog opisanih desetak vrsta između kojih ne postoje jasne morfolo&scaron;ke razlike i kod kojih je prisutno preklapanje vrednosti karaktera.Kako&nbsp; je&nbsp; do&nbsp; sada&nbsp; kompleks&nbsp; najvi&scaron;e&nbsp; bio&nbsp; predmet&nbsp; kariolo&scaron;kih&nbsp; istraživanja,&nbsp; pri&nbsp; čemu&nbsp; su&nbsp; izostale&nbsp; detaljne analize morfolo&scaron;ke i/ili anatomske varijabilnosti, uzorkovan je biljni materijal sa 37 lokaliteta na području Panonske nizije i Balkanskog poluostrva sa ciljem utvrđivanja morfolo&scaron;ke, anatomske, kao i kariolo&scaron;ke varijabilnosti. Određivan&nbsp; je broj hromozoma, nivoi ploidije&nbsp; i veličina genoma. Primenom univarijantne statističke&nbsp; metode,&nbsp; ispitana&nbsp; je&nbsp; varijabilnost&nbsp; morfolo&scaron;kih&nbsp; i&nbsp; anatomskih&nbsp; karaktera,&nbsp; a&nbsp; upotrebom multivarijantne&nbsp; statističke&nbsp; metode&nbsp; su&nbsp; testirane&nbsp; razlike&nbsp; između&nbsp; unapred&nbsp; definisanih&nbsp; grupa,&nbsp; koje&nbsp; su&nbsp; se odnosile na populacije i ploidne nivoe. Korespodentnom analizom su analizirani kvalitativni morfolo&scaron;ki i anatomski&nbsp; karakteri.&nbsp; Ukupno&nbsp; je&nbsp; analizirano&nbsp; 65&nbsp; karaktera&nbsp; (33&nbsp; morfolo&scaron;ka&nbsp; i&nbsp; 32&nbsp; ana tomska).&nbsp; Rezultati kariolo&scaron;ke&nbsp; analize&nbsp; se&nbsp; potvrdili&nbsp; varijabilnost&nbsp; kompleksa&nbsp; na&nbsp; istraživanom&nbsp; području.&nbsp; Detektovana&nbsp; su&nbsp; tri ploidna&nbsp; nivoa&nbsp; (diploidi,&nbsp; tetraploidi&nbsp; i&nbsp; heksaploidi),&nbsp; a&nbsp; potvrđena&nbsp; je&nbsp; i&nbsp; varijabilnost&nbsp; u&nbsp; veličini&nbsp; genoma. Poređenjem monoploidne veličine genoma između tri ploidna nivoa je detektovano smanjivanje veličine genoma&nbsp; sa&nbsp; povećanjem&nbsp; nivoa&nbsp; ploidije.&nbsp; U&nbsp; odnosu&nbsp; na&nbsp; koeficijent&nbsp; varijabilnosti,&nbsp; konstatovano&nbsp; je&nbsp; da&nbsp; se većina&nbsp; morfolo&scaron;kih&nbsp; karaktera&nbsp; nalazi&nbsp; u&nbsp; zoni&nbsp; umerene&nbsp; varijabilnosti&nbsp; (22),&nbsp; a&nbsp; samo&nbsp; pet&nbsp; karaktera&nbsp; je visokovarijabilno.&nbsp; Anatomski&nbsp; karakteri&nbsp; su&nbsp; raspoređeni&nbsp; u&nbsp; četiri&nbsp; kategorije&nbsp; (niskovarijabilni, umerenovarijabilni, visokovarijabilni i veoma visokovarijabilni), pri čemu većina karaktera (20) pripada umerenovarijabilnoj&nbsp; kategoriji. Četiri&nbsp; kvalitativna&nbsp; morfolo&scaron;ka (oblik lukovice, boja tunike, boja cveta i oblik&nbsp; plodnika)&nbsp; i&nbsp; sedam&nbsp; kvalitativnih&nbsp; anatomskih&nbsp; karaktera&nbsp; (oblik&nbsp; poprečnog&nbsp; preseka&nbsp; lista,&nbsp; oblik&nbsp; ćelija epidermisa na licu i&nbsp; naličju,&nbsp; oblik ćelija palisadnog tkiva na licu&nbsp; i&nbsp; naličju, prisustvo papila&nbsp; i&nbsp; kristala u ćelijama&nbsp; parenhima)&nbsp; su&nbsp; pokazala&nbsp; varijabilnost.&nbsp; U&nbsp; korespodentnoj&nbsp; analizi,&nbsp; zasnovanoj&nbsp; na&nbsp; kvalitativnim morfolo&scaron;kim&nbsp; karakterima,&nbsp; izdvojile&nbsp; su&nbsp; se&nbsp; tri&nbsp; grupe.&nbsp; Za&nbsp; prvu&nbsp; grupu&nbsp; su&nbsp; bili&nbsp; zajedničke&nbsp; &scaron;irokojajaste&nbsp; i uzanojajaste&nbsp; lukovice&nbsp; sa&nbsp; ružičastom&nbsp; tunikom,&nbsp; jedinkama&nbsp; u&nbsp; drugoj&nbsp; grupi&nbsp; su&nbsp; bili&nbsp; svojstveni&nbsp; jajasti&nbsp; i uzanojajasti plodnici i lukovice sa braon tunikom, dok su za treću grupu bili karakteristični &scaron;irokojajasti plodnici, loptaste i pljosnate lukovice. U odnosu na kvalitativne anatomske karaktere najvi&scaron; e se izdvojila jedna&nbsp; grupa&nbsp; za&nbsp; koju&nbsp; su&nbsp; bile&nbsp; karakteristične&nbsp; gusto&nbsp; raspoređene&nbsp; papile&nbsp; i&nbsp; prisustvo&nbsp; četvorougaonih&nbsp; i okruglastih epidermalnih ćelija na abaksijalnoj strani lista. Rezultati multivarijantnih analiza, zasnovanih na&nbsp; populacijama&nbsp; kao&nbsp; unapred&nbsp; definisanim&nbsp; grupama&nbsp; i&nbsp; kvantitivnim&nbsp; karakterima,&nbsp; ukazali&nbsp; su&nbsp; na&nbsp; složenu varijabilnost&nbsp; uzorka&nbsp; i&nbsp; izostanak&nbsp; jasne&nbsp; separacije&nbsp; grupa.&nbsp; Naznake&nbsp; razdvajanja&nbsp; grupa&nbsp; su&nbsp; bile&nbsp; uočljive&nbsp; u analizi&nbsp; morfo-anatomske&nbsp; matrice.&nbsp; Izdvojili&nbsp; su&nbsp; se&nbsp; sledeći&nbsp; karakteri:&nbsp; visina&nbsp; stabla,&nbsp; dužina&nbsp; cvasti,&nbsp; broj cvetova,&nbsp; povr&scaron;ina&nbsp; poprečnog&nbsp; preseka&nbsp; lista,&nbsp; ukupna&nbsp; povr&scaron;ina&nbsp; palisadnog&nbsp; tkiva&nbsp; i&nbsp; ukupna&nbsp; povr&scaron;ina sunđerastog tkiva. Najjasnija razdvajanja su uočena u analizama gde su ploidni nivoi predstavljali&nbsp; a priori definisane&nbsp; grupe,&nbsp; naročito&nbsp; u&nbsp; kombinaciji&nbsp; morfolo&scaron;kih&nbsp; i&nbsp; anatomskih&nbsp; karaktera.&nbsp; Karakteri&nbsp; sa diskriminacionim&nbsp; potencijalom&nbsp; između&nbsp; jedinki&nbsp; koje&nbsp; pripadaju&nbsp; različitim&nbsp; ploidnim&nbsp; nivoima&nbsp; jesu:&nbsp; broj<br />cvetova,&nbsp; &scaron;irina&nbsp; listića&nbsp; perigona&nbsp; unutra&scaron;njeg&nbsp; kruga&nbsp; i&nbsp; filamenta&nbsp; spolja&scaron;njeg&nbsp; kruga,&nbsp; povr&scaron;ina&nbsp; poprečnog preseka&nbsp; lista,&nbsp; ukupna&nbsp; povr&scaron;ina&nbsp; palisadnog&nbsp; tkiva,&nbsp; povr&scaron;ina&nbsp; ćelija&nbsp; palisadnog&nbsp; tkiva,&nbsp; visina&nbsp; i&nbsp; &scaron;irina&nbsp; ćelija palisadnog tkiva i udeo epidermisa. Od sva tri ploidna nivoa, najvi&scaron;e izdvojene su bile diploidne jedinke, koje&nbsp; se&nbsp; od&nbsp; tetraploida&nbsp; i&nbsp; heksaploida&nbsp; mogu&nbsp; razlikovati&nbsp; na&nbsp; osnovu:&nbsp; dužina&nbsp; filamenata&nbsp; spolja&scaron;njeg&nbsp; i unutra&scaron;njeg&nbsp; kruga&nbsp; cveta,&nbsp; povr&scaron;ine&nbsp; ćelija&nbsp; palisadnog&nbsp; tkiva,&nbsp; visine&nbsp; i&nbsp; &scaron;irine&nbsp; ćelija&nbsp; palisadnog&nbsp; tkiva,&nbsp; kao&nbsp; i ukupne&nbsp; povr&scaron;ine&nbsp; palisadnog&nbsp; tkiva.&nbsp; Tetraploidi&nbsp; i&nbsp; heksaplodi&nbsp; se&nbsp; najvi&scaron;e&nbsp; međusobno&nbsp; razlikuju&nbsp; na&nbsp; osnovu karaktera u regionu cveta: prečnik otvorenog perigona, dužine listića perigona spolja&scaron;njeg kruga cveta i &scaron;irine filamenta spolja&scaron;njeg&nbsp; kruga cveta.&nbsp; Uočene razlike&nbsp; između tri analizirana ploidna&nbsp; nivoa (diploidi, tetraploidi&nbsp; i&nbsp; heksaploidi),&nbsp; u&nbsp; taksonomskom&nbsp; smislu&nbsp; se&nbsp; mogu&nbsp; tumačiti&nbsp; pre&nbsp; kao&nbsp; jedna&nbsp; od&nbsp; infraspecijskih kategorija, a ne kao kategorija koja bi odgovarala vrsti.</p> / <p>Prospero autumnale&nbsp; complex is the most taxonomically intricate member of the genus&nbsp; Prospero&nbsp; with the centre of distribution on the Mediterranean Basin, westwards to&nbsp; the Atlantic coast in France as far north&nbsp; as&nbsp; Great&nbsp; Britain,&nbsp; northwards&nbsp; to&nbsp; the&nbsp; Pannonian&nbsp; Basin&nbsp; and&nbsp; Crimea,&nbsp; and&nbsp; eastwards&nbsp; to&nbsp; Iran.&nbsp; The distribution area of the complex overlaps with distribution areas of the other two species of the genus<br />Prospero-P.&nbsp; obtusifolium&nbsp; is&nbsp; present&nbsp; in&nbsp; the&nbsp; western&nbsp; parts&nbsp; of&nbsp; the&nbsp; Mediterranean,&nbsp; while&nbsp; P.&nbsp; hanburyi&nbsp; is distributed in the eastern Mediterranean. Unlike&nbsp; P. obtusifolium and P. hanburyi, P. autumnale complexis characterized by extraordinary karyological&nbsp; cariation. It encompasses&nbsp;&nbsp; four distinct genomes (A, B&nbsp; 5 , B 6 , B 7 ) and four distinct diploid cytotypes (AA, B 5 B 5 , B 6 B 6 , B 7 B 7 ), each with a unique combination of<br />basic chromosome&nbsp; number (x&nbsp; = 5, 6, 7).&nbsp; An additional difference is related to&nbsp; chromosome&nbsp; size and morphology. The B 5 genome&nbsp; has&nbsp; x&nbsp; = 5, B 6 x&nbsp; = 6, and A and B 7x&nbsp; = 7 basic chromosome numbers. The B 7 genome&nbsp; is&nbsp; present&nbsp; in&nbsp; the&nbsp; whole&nbsp; distribution&nbsp; area&nbsp; of&nbsp; P.&nbsp; autumnale&nbsp; complex,&nbsp; while&nbsp; genome&nbsp; A&nbsp; is distributed in the western Mediterranean, B 5 is restricted to Libya, and B 6 is endemic to&nbsp; Crete. Within diploids and tetraploids&nbsp; of&nbsp; B 7 genome, two&nbsp; lineages&nbsp; occur as a consequence&nbsp; of&nbsp; duplication&nbsp; of the 5S rDNA locus. Type I (lineage) has a single locus, while in type II (lineage) 5S rDNA locus is duplicated. Besides diploid cytotypes, the polyploids cytotypes are also known. The most common polyploids are tetra- (2n=4x=28) and hexaploids (2n=6x=42). Variability in the genome size within, as well as between cytotypes, is also characterized for the complex. Genomic restructuring, which played the b iggest role in evolution&nbsp; and&nbsp; diversification&nbsp; of&nbsp; the&nbsp; P.&nbsp; autumnale&nbsp; complex,&nbsp; did&nbsp; not&nbsp; have&nbsp; a&nbsp; major&nbsp; impact&nbsp; on morphological&nbsp; variability.&nbsp; Only&nbsp; slight&nbsp; variation&nbsp; has&nbsp; been&nbsp; detected&nbsp; in&nbsp; plant,&nbsp; tepal&nbsp; and&nbsp; filament&nbsp; size, flower&nbsp; and&nbsp; seed&nbsp; color,&nbsp; shape&nbsp; and&nbsp; size&nbsp; of&nbsp; leaves&nbsp; a nd&nbsp; color&nbsp; of&nbsp; bulbs.&nbsp; Taxonomic&nbsp; ambiguities&nbsp; are<br />additionally&nbsp; caused&nbsp; by&nbsp; description&nbsp; of&nbsp; the&nbsp; new&nbsp; ten&nbsp; species&nbsp; of&nbsp; the&nbsp; complex.&nbsp;&nbsp; morphological&nbsp; differences between those species are unclear, with overlapping values of the most morphological traits. Until now, P. autumnale&nbsp; complex was mostly karyologicaly investigated, without detail analyses of morphological and/or&nbsp;&nbsp; anatomical&nbsp; variability.&nbsp; The&nbsp; aim&nbsp; of&nbsp; the&nbsp; present&nbsp; study&nbsp; was&nbsp; to&nbsp; investigate&nbsp; karyological, morphological and anatomical variability on individuals collected on 37 localities across the Pannonian Basin and&nbsp; Balkan Peninsula. Basic chromosome&nbsp; number, ploidy&nbsp; levels, as well as&nbsp; genome size&nbsp; were determined. The variability of morphological and anatomical characters was examined using univariate statistical&nbsp; methods.&nbsp; Differences&nbsp; between&nbsp; predefined&nbsp; groups&nbsp; (populations&nbsp; and&nbsp; ploidy&nbsp;&nbsp; levels)&nbsp; were investigated&nbsp; using&nbsp; multivariate&nbsp; statistics&nbsp; with&nbsp; the&nbsp; attempts&nbsp; to&nbsp; identify&nbsp; morphological&nbsp; and&nbsp; anatomical characters&nbsp; with&nbsp; discriminatory&nbsp; potential.&nbsp; For&nbsp; analysing&nbsp; qualitative&nbsp; morphological&nbsp; and&nbsp; anatomical characters, correspondence analysis was conducted. In total 65 traits were analyzed (33&nbsp; morphological and&nbsp; 32&nbsp; anatomical).&nbsp; The&nbsp; results&nbsp; of&nbsp; karyological&nbsp; analysis&nbsp; confirmed&nbsp; the&nbsp; high&nbsp; variation&nbsp; of&nbsp; the&nbsp; P. autumnale&nbsp; complex&nbsp; in the study area. Three&nbsp; ploidy&nbsp; levels (diploids, tetraploids and&nbsp; hexaploids)&nbsp; were detected,&nbsp; and&nbsp; the&nbsp; genome&nbsp; size&nbsp; variation&nbsp; was&nbsp; confirmed.&nbsp; Genome&nbsp; downsizing&nbsp; was&nbsp; observed&nbsp; by comparing&nbsp; monoploid&nbsp; genome&nbsp; sizes&nbsp; between&nbsp; three&nbsp; ploidy&nbsp; levels.&nbsp; According&nbsp; to&nbsp; the&nbsp; oefficient&nbsp; ofc variation,&nbsp; most&nbsp; morphological characters show&nbsp; moderate&nbsp; variation (22). Only&nbsp; five traits showed&nbsp; high variation.&nbsp; Anatomical&nbsp; characters&nbsp; were&nbsp; classified&nbsp; into&nbsp; four&nbsp; categories&nbsp; according&nbsp; to&nbsp; the&nbsp; coefficient&nbsp; of variation (with low, moderate, high and very high variation), but the&nbsp; most traits (20) showed moderate<br />variation. Variation in qualitative morphological (bulb shape and color, flower color and ovary shape) and seven&nbsp; qualitative anatomical characters (leaf cross-sectional area shape, shape&nbsp; of the adaxial and abaxial&nbsp; epidermal&nbsp; cells,&nbsp; shape&nbsp; of&nbsp; the&nbsp; adaxial&nbsp; and&nbsp; abaxial&nbsp; palisade&nbsp; cells,&nbsp; presence&nbsp; of&nbsp; the&nbsp; papillae&nbsp; and crystals&nbsp; in&nbsp; the&nbsp; parenchyma&nbsp; cells)&nbsp; were&nbsp; recorded.&nbsp; Three&nbsp; groups&nbsp; of&nbsp; populations,&nbsp; as&nbsp; a&nbsp; result&nbsp; of&nbsp; the correspondence&nbsp; analysis,&nbsp; based&nbsp; on&nbsp; qualitative&nbsp; morphological&nbsp; characters,&nbsp; were&nbsp; formed.&nbsp; Populations&nbsp; of the first group showed common characteristics such as broadly and narrowly ovate shaped bulbs with pink&nbsp; outer&nbsp; tunic.&nbsp; The&nbsp; second&nbsp; group&nbsp; was&nbsp; characterized&nbsp; by&nbsp; bulbs&nbsp; with&nbsp; brown&nbsp; outer&nbsp; tunic,&nbsp; ovate&nbsp; and narrowly ovate ovary, while the third group had broadly ovate ovaries, globose and flattened bulbs. In relation&nbsp; to&nbsp; qualitative&nbsp; anatomical&nbsp; characters,&nbsp; only&nbsp; one&nbsp; group&nbsp; was&nbsp; the&nbsp; most&nbsp; distinguished&nbsp; and&nbsp; was characterized by densely distributed papillae, squared and rounded shape of abaxial epidermal cells. The results&nbsp; of&nbsp; multivariate&nbsp; analyses&nbsp; of&nbsp; quantitative&nbsp; traits,&nbsp; based&nbsp; on&nbsp; populations&nbsp; as&nbsp; presumptive&nbsp; groups, revealed&nbsp; similar&nbsp; patterns&nbsp; without&nbsp; structuring&nbsp; and&nbsp; with&nbsp; no&nbsp; specific&nbsp; groupings.&nbsp; The&nbsp; tendency&nbsp; toward&nbsp; a separation&nbsp; of&nbsp; the&nbsp; populations&nbsp; was&nbsp; noticeable&nbsp; in&nbsp; the&nbsp; analysis&nbsp; based&nbsp; on&nbsp; combined&nbsp; morpho-anatomical characters. The highest correlation with the canonical axes showed: stem height, inflorescence length,number of flowers, leaf cross-sectional area, palisade and spongy tissue area. The clearest separationsbetween groups were observed with ploidy levels as presumptive groups. Morpho-anatomical traits with discriminatory potential among ploidy levels were: number of flowers, inner tepal width, outer filament width, leaf cross-sectional area, palisade tissue area, the cross-sectional area of palisade cells, height and width&nbsp; of palisade cells and&nbsp; epidermis percentage. The&nbsp; most&nbsp; distinct&nbsp; group among three ploidy&nbsp; levels were diploids and could be distinguished from tetra-&nbsp; and hexaploids by outer and inner&nbsp; filament width, the&nbsp; cross-sectional area&nbsp; of palisade cells, the&nbsp; width of palisade cells, the&nbsp; height&nbsp; of palisade cells and palisade tissue area. Tetra-&nbsp; and&nbsp; hexaploids&nbsp; differed&nbsp; mostly&nbsp; in floral characters: open flower diameter, outer tepal length and outer filament width. For taxonomic purposes, the level of overlap indicates that the ploidy levels could be regarded as showing intraspecific variation.</p>

Cotoneaster is an ornamental shrub valued for showy flowers, berries and architecture as well as the ability tolerate adverse conditions under which other taxa fail. Cotoneaster is a highly diverse genus of over 400 species, of which few are available in the US nursery trade. Some species commercially available have been identified as potentially invasive in the state of Oregon and also are susceptible to the bacterial disease fire blight. Cotoneaster selections with reduced fertility and disease resistance would be desirable characteristics for low input landscape plants. A goal of my research was to characterize Cotoneaster spp. to provide information for the rationale planning and development of novel clones to meet these horticultural goals. In the first study, genome sizes were estimated using flow cytometery and ploidy levels were inferred using holoploid genome size. Observed differences in monoploid genome sizes translate to a difference in chromosome size. Differences in chromosome size may present a reproductive barrier when they are large. This may pose a challenge in wide crosses, but may be utilized to achieve sterility in the F1 interspecific hybrid population. Differences in genome size are not related to taxonomic ordering, so wide inter sectional and inter subgeneric crosses may be possible. In the second study, susceptibility of Cotoneaster to fire blight was measured on plants inoculated with Erwinia amylovora strain Ea153. In greenhouse assays conducted over two years, plants were inoculated by cutting leaves with scissors infested with the pathogen. Some species were 'highly susceptible' to fire blight where plants were killed to the ground, and others were rated 'highly resistant' and no lesions were observed. Seventeen accessions were rated as resistant to fire blight This research provides the first report of ploidy, genome sizes, and susceptibility of species of Cotoneaster to fire blight. Collectively this research provides a toolbox for a breeder to tackle the challenge of creating disease resistant cultivars with reduced fertility. / Graduation date: 2013

Made available in DSpace on 2015-03-26T13:42:26Z (GMT). No. of bitstreams: 1 texto completo.pdf: 881362 bytes, checksum: 0e498263c87a06e3c091110afa80ddfe (MD5) Previous issue date: 2012-07-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Tissue culture techniques are often associated with the occurrence of somaclonal variation, characterized by a genetic/epigenetic alteration found in cells, embryos, organs or in vitro grown plants, due to stress conditions imposed by the system. The propagation process involving the calogenesis step has a higher susceptibility to variation, because of many cell divisions. Calli are disorganized cell masses, whose cells divide at different rates depending on where they are located in relation to the culture medium, therefore, they are not considered homogeneous materials. Flow cytometry is a tool that has been used to evaluate the competence of somatic embryogenesis in callus, both to identify the different types as possible and to detect early changes in DNA content of their cells. However, there are no reports in the literature of the flow cytometry use to determine the existence of heterogeneity in relation to the DNA content in callus. In this perspective, the present work adapted this methodology for friable Passiflora cincinnata calli in order to verify the existence of vertically regionalized heterogeneity in relation to DNA ploidy level, and relate it to factors inductors of somaclonal variation, such as cultivation time and subculture numbers. Initially, P. cincinnata cotyledonary leaves were inoculated onto semi-solid medium containing 2, 4-D and BA, for friable embryogenic callus induction. The calli were multiplied and subcultured at different times, thus generating three callus types: new, intermediate and old. Twenty calli of each type were subjected to sagittal cuts resulting two layers (I, II) in new calli, and three layers (I, II and III) in intermediate and old calli, totalizing 160 layers, which were analyzed by flow cytometry. Six different DNA ploidy levels and multiploidies were detected in the layers. In new calli, 25% were heterogeneous, i.e. at least one callus layer showed different DNA ploidy level from the others, differently from intermediate and old calli, which 75% and 63% were heterogeneous, respectively. The homogeneous new calli were predominantly diploid. The highest DNA ploidy levels were observed in the layers in contact with the culture medium on intermediate calli, and 4C and 4C/8C cells were more evident in older calli. These observations suggest that the appearance of new DNA ploidy levels between the layers and between the different callus types is related to the exposure time to the medium containing relatively high concentration of 2,4-D. It was also possible to verify that the layer in contact with the culture medium is more proliferative when compared the percentages of the G2 peaks in homogeneous diploid calli. The heterogeneity in DNA ploidy level between the layers was observed, and it implies that parts of a callus cannot infer about a whole callus. This work suggests that time influences the appearance of different DNA ploidy levels in calli induced in vitro. / Técnicas de cultura de tecidos estão frequentemente associadas com a ocorrência de variação somaclonal, caracterizada pela alteração genética e/ou epigenética encontrada em células, embriões, órgãos ou plantas cultivadas in vitro, em decorrência das condições de estresse impostas pelo sistema. O processo de propagação que envolve a etapa de calogênese apresenta maior susceptibilidade à ocorrência de variação, em função das inúmeras divisões celulares. Calos são massas celulares não organizadas, cujas células se dividem a diferentes velocidades dependendo de onde se localizam em relação ao meio de cultura; portanto, não são considerados materiais homogêneos. A citometria de fluxo é uma ferramenta que tem sido utilizada para avaliar a competência da embriogênese somática em calos, tanto para identificar seus diferentes tipos quanto para detectar precocemente possíveis alterações no conteúdo de DNA de suas células. No entanto, não há relatos na literatura da utilização da citometria de fluxo para verificar a existência da heterogeneidade quanto ao conteúdo de DNA em calos. Nesta perspectiva, o presente trabalho adaptou essa metodologia para calos friáveis de Passiflora cincinnata, a fim de verificar a existência de heterogeneidade verticalmente regionalizada, em relação à ploidia de DNA, e relacioná-la com fatores de indução de variação somaclonal, como tempo de cultivo e número de subcultivos. Inicialmente, folhas cotiledonares de P. cincinnata foram inoculadas em meio semissólido contendo 2,4-D e BA, para indução de calos embriogênicos friáveis. Os calos foram multiplicados e subcultivados em diferentes tempos, gerando, assim, três tipos de calos, denominados como novos, intermediários e velhos. Vinte calos de cada tipo foram submetidos a cortes sagitais que resultaram duas camadas (I e II) em calos novos, e três camadas (I, II e III) em calos intermediários e velhos, totalizando 160 amostras que foram analisadas por citometria de fluxo. Seis diferentes ploidias de DNA e multiploidias foram detectadas nas camadas. Em calos novos, 25% deles foram heterogêneos, ou seja, pelo menos uma camada apresentou nível de ploidia de DNA diferente das demais, diferentemente dos calos intermediários e velhos, os quais 75% e 63% foram heterogêneos, respectivamente. Os calos novos homogêneos foram predominantemente diploides. As ploidias de DNA mais altas foram observadas nas camadas em contato com o meio de cultura em calos intermediários, e células 4C e 4C/8C foram mais evidentes em calos velhos. Essas observações sugerem que o surgimento de novos níveis de ploidia entre as camadas e entre os calos de diferentes tipos está relacionado com o tempo de exposição ao meio contendo concentração relativamente alta de 2,4-D. Foi possível, também, verificar que a camada em contato com o meio é mais proliferativa quando se comparou os percentuais dos picos G2 de calos diploides homogêneos. A heterogeneidade quanto ao nível de ploidia de DNA entre as camadas foi constatada e isto implica que partes de um calo não podem inferir sobre um calo inteiro. Esse trabalho sugere que o tempo influenciou o surgimento de diferentes ploidias de DNA em calos induzidos in vitro.

Made available in DSpace on 2015-03-26T13:42:26Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1076965 bytes, checksum: 39f371186cedd4587ea0ad27b341a26b (MD5) Previous issue date: 2012-07-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The anther culture has been applied in tomato as an attempt to induce callogenesis followed by regeneration of haploid and doubled haploid plants. By this technique, homozygous lines can be obtained, which has been considered relevant to breeding programs and molecular techniques. The androgenetic response can be influenced by several factors, especially the development stage of microsporogenesis. In this perspective, this study adapted a protocol by associating of flow cytometry and cytogenetic techniques in order to relate the development stages of microsporogenesis in tomato anthers to their size, aiming to select anthers profiles to be inoculated into culture medium. After a flow cytometry protocol was adapted for calli obtained in vitro, in order to monitor the DNA ploidy level. The nuclear suspensions analyzed by flow cytometry were obtained from digestion of anther cells and further homogenization using a mini mixer. The histograms showed low CV s, being considered suitable for cytometric analysis. Thus, anthers small (0.5 0.7 mm), showing cells in interphase/prophase I, exhibited a histogram profile similar to that observed in diploid leaf, and anthers large (1.6 1.9 mm), containing tetrad and microspores, showed an haploid peak and a G2 peak enlarged due to the presence of tetrads. From these analyzes, anthers of flower buds 1 5 mm, with size corresponding to those analyzed by flow cytometry and cytogenetics (0.5 1.9 mm), were inoculated in the callogenesis induction medium, to identify the phases more responsive to the condition in vitro. The basal MS medium supplemented with 2 mg L-1 AIA and 1 mg L-1 2 ip, induced 21.7% of callogenesis, and the highest percentage of calli was obtained from anthers of flower buds 2.0 3.9 mm, corresponding to the phases prophase I to anaphase II. The calli were analyzed by flow cytometry to identify the DNA ploidy. The chopping performed on calli afforded the nuclei isolation, and histograms with CV s considered suitable were obtained. The cytometric analysis revealed that the all calli were multiploids, each of which presented one of five classes of DNA ploidy levels, namely: 2C-4C-8C-16C; 2C-4C-8C-16C-32C; 4C-8C; 4C-8C-16C; 8C-16C-32C. A total of 44.4% of calli analyzed showed five levels of DNA ploidy. A statistical test of interaction showed that the size of flower buds had no effect on polyploidization occurred in the calli. These data evidence the somaclonal variation occurrence, which, probably, results from the interaction of genotype with growth regulators added to the induction medium. / A cultura de anteras tem sido aplicada em tomate como tentativa à indução de calogênese seguida da regeneração de plantas haplóides e duplo-haplóides. Por meio desta técnica, linhas homozigóticas podem ser obtidas, o que tem sido considerado relevante para programas de melhoramento e técnicas moleculares. A resposta androgenética pode ser influenciada por diversos fatores, destacando-se, dentre eles, o estádio de desenvolvimento da microsporogênese. Nesta perspectiva, este estudo adaptou um protocolo associando técnicas de citometria de fluxo e citogenética para relacionar as fases de desenvolvimento da microsporogênese em anteras de tomate com o tamanho das mesmas. O objetivo desta associação foi selecionar perfis de anteras a serem inoculadas em meio de cultura. Posteriormente, adaptou-se um protocolo de citometria de fluxo para os calos obtidos in vitro, a fim de monitorar o nível de ploidia de DNA. As suspensões nucleares analisadas pela citometria de fluxo foram obtidas com digestão das anteras e posterior homogeneização das células usando um mini mixer. Os histogramas obtidos mostraram baixos CV s, sendo considerados adequados para a análise citométrica. Dessa forma, anteras pequenas (0,5 0,7 mm), na fase de intérfase/prófase I, apresentaram um perfil de histograma semelhante ao observado em folha diplóide, e anteras grandes (1,6 1,9 mm) na fase de tétrades e micrósporos apresentaram um pico haplóide e um aumento em G2 em função da presença de tétrades. A partir destas análises, as anteras de botões florais de 1 5 mm, com tamanho correspondente aos daquelas analisadas por citometria de fluxo e citogenética (0,5 1,9 mm) foram inoculadas em meio de indução de calogênese, visando identificar as fases mais responsivas a esta condição in vitro. O meio MS basal, suplementado com 2 mg L-1 de AIA e 1 mg L-1 de 2 ip, induziu 21,7% de calogênese, e o maior percentual de calos foi obtido a partir de anteras de botões florais com 2,0 3,9 mm, correspondendo às fases prófase I a anáfase II. Os calos foram analisados pela citometria de fluxo a fim de identificar a ploidia de DNA. O chopping realizado nos calos proporcionou o isolamento dos núcleos, e histogramas com CV s considerados adequados foram obtidos. A análise citométrica evidenciou que todos os calos eram multiplóides, sendo que cada um apresentou uma das cinco classes de níveis de ploidia de DNA, a saber: 2C-4C-8C-16C; 2C-4C-8C-16C-32C; 4C-8C; 4C-8C-16C; 8C-16C-32C. Um total de 44,4% dos calos analisados apresentaram cinco níveis de ploidia de DNA. Um teste estatístico de interação mostrou que o tamanho dos botões florais não influenciou na poliploidização ocorrida nos calos. Estes dados evidenciam a ocorrência de variação somaclonal que, provavelmente, é resultado da interação do genótipo com os reguladores de crescimento acrescidos ao meio de indução.

Presence and range of phenotypic plasticity in the Agrostis stolonifera polyploid complex (Poaceae) was studied in the territory of the Czech Republic. Plants were cultivated under different experimental conditions. Stomatal size of different Agrostis stolonifera cytotypes was measured. Flow cytometry was applied for genome size estimation. Ploidy levels were determined for more than 150 samples of Agrostis stolonifera as well as several specimens of Agrostis canina, A. capillaris, A. gigantea, A. rupestris and A. vinealis. Absolute DNA content was estimated in all studied species. Isozyme analysis was used to test the possibility of the hybrid origin of pentaploid individuals of Agrostis stolonifera species.

The mode of reproduction can greatly influence the demography and the evolutionary success of the taxon. In the case of autonomous asexual formation seeds are apomictic taxa fully independent of pollinators and compatible partners. For sexual taxa with strict autoincompatibility it is the opposite, i.e. sexual taxa need pollinators and compatible partners for birth of offspring. Therefore, in marginal population and for more extreme situation with lower pollinating activity will have apomictic taxa a higher level of reproductive assurance than sexual taxa vascular plants. This hypothesis was tested in natural populations of apomictic and sexual taxa. In the diploma thesis we therefore investigate the method mode of reproduction and reproductive assurance of 52 taxa of the genus Hieracium s. str. (family Asteraceae) in Europe. Of these, 12 were diploid sexually diploid taxa and 42 polyploid apomictic reproductive taxa. From these taxa we harvested seeds from fully developed capitulum and we determined the potential (total number of seeds in the capitulum) and the realized (the percentage of well-developed seeds at the capitulum). The ploidy of the offspring (the embryos and the seedling) and method origins of seeds we examined using flow cytometry. The results show that the plants of diploid species...

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No description available.

Introdução O diagnóstico de derrame pleural maligno (DPM) se baseia no achado de células tumorais no líquido ou no tecido pleural. Resultados falsos positivos ou falsos negativos influenciam na escolha da melhor conduta terapêutica a ser tomada, além de alterar substancialmente o prognóstico desses pacientes. A sensibilidade do exame citológico é geralmente inferior a 70%, motivo pelo qual, métodos complementares são frequentemente associados. Fatores como tipo histológico, sítio primário e grau de invasibilidade do tumor são os principais responsáveis por esta variação. Dentre os exames complementares propostos, destacam-se a dosagem de marcadores tumorais no líquido pleural (LP), as técnicas citoquímicas, imunocitoquímicas e de marcadores de proliferação celular em células do LP, a análise da ploidia de DNA por citometria de fluxo (CF) ou estática (CE) e, mais recentemente, as técnicas de citogenética e de biologia molecular, como a técnica de hibridação in situ por fluorescência (FISH) e a técnica de amplificação multiplex por sondas ligação - dependentes (MLPA) estas, capazes de detectar alterações em regiões gênicas consideradas \"alvo\" para o desfecho neoplásico. Objetivos 1) Padronizar as técnicas de DNA ploidia, FISH e MLPA em amostras frescas de líquido pleural; 2) Testar a eficiência diagnóstica dos métodos da DNA ploidia e da FISH no diagnóstico de derrame pleural maligno e 3) Avaliar alterações no número de cópias no gene EGFR em metástases pleurais utilizando a técnica de MLPA. Métodos Foram incluídos 200 pacientes adultos portadores de derrame pleural (DP) com indicação de toracocentese. O diagnóstico histológico foi o padrão ouro para malignidade. Características clínicas, radiológicas, histológicas e de seguimento clínico foram considerados para a exclusão de malignidade, de maneira que 130 casos foram classificados como malignos e 70 como benignos. As 200 amostras de LP foram submetidas ao exame citológico e à FISH utilizando sondas centroméricas para os cromossomos 11 e 17. A análise da ploidia de DNA por CF foi realizada em 45 casos de DP e a MLPA com o kit do gene do receptor do fator de crescimento epidérmico (EGFR) em 50 casos. Resultados A análise da ploidia de DNA por CF apresentou sensibilidade inferior ao exame citológico, com especificidade próxima (57,0%vs 96,2%; 70,0% vs 66,7%, respectivamente). A FISH isoladamente apresentou sensibilidade de 98,5% e especificidade de 98,6% e de 98,0% e 99,% quando associada ao exame citológico, com apenas um caso falso positivo e dois casos falsos negativos. A técnica de MLPA, padronizada para LP, demonstrou alterações na sequência do gene do EGFR em 28,2% dos casos malignos. Nenhuma amostra de líquido pleural dos casos benignos (controle) apresentou alteração no número de cópias e/ou rearranjos estruturais. Conclusão A análise citogenética de amostras frescas de líquido pleural por FISH é um valioso complemento ao exame citológico no diagnóstico de derrame pleural maligno, particularmente nos casos em que o resultado da citologia oncótica é inconclusiva / Introduction The diagnosis of malignant pleural effusion (MPE) is based on the finding of tumor cells in the pleural fluid or tissue. False positive or false negative results influence the choice of the best therapeutic approach to be used with these patients and substantially change their prognosis. The sensitivity of the cytology is generally lesser than 70%, for which complementary methods are often associated. Factors such as tumor histological type, staging, primary site and potential of invasiveness are responsible for this variation. Among the proposed ancillary tests, we highlight the dosage of tumor markers in pleural fluid (PF), the cytochemical and immunocytochemical techniques, including markers of cell proliferation, DNA ploidy analysis by flow cytometry (FC) or static cytometry (EC) and more recently, the cytogenetics and molecular techniques, as the fluorescence in situ hybridization (FISH) and the multiplex ligation - dependent probe amplification (MLPA), capable of detecting changes in gene regions considered \"target\" for the neoplastic outcome. Objectives 1) To standardize the techniques of DNA ploidy, FISH and MLPA in fresh samples of pleural fluid; 2) To test the diagnosis efficiency of DNA ploidy and FISH in the diagnosis of malignant pleural effusion and 3) To evaluate changes in the copy number of the EGFR gene by using the MLPA technique in cases of pleural metastases. Methods We included 200 adult patients with pleural effusion and clinical indication for thoracentesis. The histological diagnosis was considered the gold standard for malignancy. Clinical follow-up, radiological and histological characteristics were considered for exclusion of malignancy, which ranked de cases as 130 malignant effusions and 70 as benign ones. All cases were submitted to cytology and FISH using centromeric probes for the chromosomes 11 and 17. Analysis of DNA ploidy by FC was performed in 45 cases and the MLPA for epidermal growth factor receptor (EGFR) gene in 50 cases. Results DNA ploidy analysis showed less sensitivity than PF cytology, with similar specificity (57.0% vs 96.2% and 70.0% vs 66.7%, respectively). FISH alone had a sensitivity of 98.5% and specificity of 98.6%, and of 98.0% and 99% when associated with cytology. Only one false positive and two false negative cases were observed. The MLPA technique, standardized for PF, showed changes in the EGFR gene in 28.2% of the malignant cases. No samples of pleural fluid from benign cases (control) showed changes in copy number and/or structural rearrangements. Conclusion The cytogenetic analysis of fresh pleural fluid samples by FISH seems to be a valuable method to be associated to cytology in the diagnosis of malignant pleural effusion, particularly in cases of inconclusive cytological results

Introdução O diagnóstico de derrame pleural maligno (DPM) se baseia no achado de células tumorais no líquido ou no tecido pleural. Resultados falsos positivos ou falsos negativos influenciam na escolha da melhor conduta terapêutica a ser tomada, além de alterar substancialmente o prognóstico desses pacientes. A sensibilidade do exame citológico é geralmente inferior a 70%, motivo pelo qual, métodos complementares são frequentemente associados. Fatores como tipo histológico, sítio primário e grau de invasibilidade do tumor são os principais responsáveis por esta variação. Dentre os exames complementares propostos, destacam-se a dosagem de marcadores tumorais no líquido pleural (LP), as técnicas citoquímicas, imunocitoquímicas e de marcadores de proliferação celular em células do LP, a análise da ploidia de DNA por citometria de fluxo (CF) ou estática (CE) e, mais recentemente, as técnicas de citogenética e de biologia molecular, como a técnica de hibridação in situ por fluorescência (FISH) e a técnica de amplificação multiplex por sondas ligação - dependentes (MLPA) estas, capazes de detectar alterações em regiões gênicas consideradas \"alvo\" para o desfecho neoplásico. Objetivos 1) Padronizar as técnicas de DNA ploidia, FISH e MLPA em amostras frescas de líquido pleural; 2) Testar a eficiência diagnóstica dos métodos da DNA ploidia e da FISH no diagnóstico de derrame pleural maligno e 3) Avaliar alterações no número de cópias no gene EGFR em metástases pleurais utilizando a técnica de MLPA. Métodos Foram incluídos 200 pacientes adultos portadores de derrame pleural (DP) com indicação de toracocentese. O diagnóstico histológico foi o padrão ouro para malignidade. Características clínicas, radiológicas, histológicas e de seguimento clínico foram considerados para a exclusão de malignidade, de maneira que 130 casos foram classificados como malignos e 70 como benignos. As 200 amostras de LP foram submetidas ao exame citológico e à FISH utilizando sondas centroméricas para os cromossomos 11 e 17. A análise da ploidia de DNA por CF foi realizada em 45 casos de DP e a MLPA com o kit do gene do receptor do fator de crescimento epidérmico (EGFR) em 50 casos. Resultados A análise da ploidia de DNA por CF apresentou sensibilidade inferior ao exame citológico, com especificidade próxima (57,0%vs 96,2%; 70,0% vs 66,7%, respectivamente). A FISH isoladamente apresentou sensibilidade de 98,5% e especificidade de 98,6% e de 98,0% e 99,% quando associada ao exame citológico, com apenas um caso falso positivo e dois casos falsos negativos. A técnica de MLPA, padronizada para LP, demonstrou alterações na sequência do gene do EGFR em 28,2% dos casos malignos. Nenhuma amostra de líquido pleural dos casos benignos (controle) apresentou alteração no número de cópias e/ou rearranjos estruturais. Conclusão A análise citogenética de amostras frescas de líquido pleural por FISH é um valioso complemento ao exame citológico no diagnóstico de derrame pleural maligno, particularmente nos casos em que o resultado da citologia oncótica é inconclusiva / Introduction The diagnosis of malignant pleural effusion (MPE) is based on the finding of tumor cells in the pleural fluid or tissue. False positive or false negative results influence the choice of the best therapeutic approach to be used with these patients and substantially change their prognosis. The sensitivity of the cytology is generally lesser than 70%, for which complementary methods are often associated. Factors such as tumor histological type, staging, primary site and potential of invasiveness are responsible for this variation. Among the proposed ancillary tests, we highlight the dosage of tumor markers in pleural fluid (PF), the cytochemical and immunocytochemical techniques, including markers of cell proliferation, DNA ploidy analysis by flow cytometry (FC) or static cytometry (EC) and more recently, the cytogenetics and molecular techniques, as the fluorescence in situ hybridization (FISH) and the multiplex ligation - dependent probe amplification (MLPA), capable of detecting changes in gene regions considered \"target\" for the neoplastic outcome. Objectives 1) To standardize the techniques of DNA ploidy, FISH and MLPA in fresh samples of pleural fluid; 2) To test the diagnosis efficiency of DNA ploidy and FISH in the diagnosis of malignant pleural effusion and 3) To evaluate changes in the copy number of the EGFR gene by using the MLPA technique in cases of pleural metastases. Methods We included 200 adult patients with pleural effusion and clinical indication for thoracentesis. The histological diagnosis was considered the gold standard for malignancy. Clinical follow-up, radiological and histological characteristics were considered for exclusion of malignancy, which ranked de cases as 130 malignant effusions and 70 as benign ones. All cases were submitted to cytology and FISH using centromeric probes for the chromosomes 11 and 17. Analysis of DNA ploidy by FC was performed in 45 cases and the MLPA for epidermal growth factor receptor (EGFR) gene in 50 cases. Results DNA ploidy analysis showed less sensitivity than PF cytology, with similar specificity (57.0% vs 96.2% and 70.0% vs 66.7%, respectively). FISH alone had a sensitivity of 98.5% and specificity of 98.6%, and of 98.0% and 99% when associated with cytology. Only one false positive and two false negative cases were observed. The MLPA technique, standardized for PF, showed changes in the EGFR gene in 28.2% of the malignant cases. No samples of pleural fluid from benign cases (control) showed changes in copy number and/or structural rearrangements. Conclusion The cytogenetic analysis of fresh pleural fluid samples by FISH seems to be a valuable method to be associated to cytology in the diagnosis of malignant pleural effusion, particularly in cases of inconclusive cytological results