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The promotion of responsible and sustainable trade in biological resources is widely proposed as one solution to mitigate currently high levels of global biodiversity loss. Various molecular identification methods have been proposed as appropriate tools for monitoring global supply chains of commercialized animals and plants. We demonstrate the efficacy of target capture genomic barcoding in identifying and establishing the geographic origin of samples traded as Anacyclus pyrethrum, a medicinal plant assessed as globally vulnerable in the IUCN Red List of Threatened Species. Samples collected from national and international supply chains were identified through target capture sequencing of 443 low-copy nuclear makers and compared to results derived from genome skimming of plastome, standard plastid barcoding regions and ITS. Both target capture and genome skimming provided approximately 3.4 million reads per sample, but target capture largely outperformed standard plant DNA barcodes and entire plastid genome sequences. Despite the difficulty of distinguishing among closely related species and infraspecific taxa of Anacyclus using conventional taxonomic methods, we succeeded in identifying 89 of 110 analysed samples to subspecies level without ambiguity through target capture. Of the remaining samples, we determined that eleven contained plant material from other genera and families and ten were unidentifiable regardless of the method used. Furthermore, we were able to discern the geographical origin of Anacyclus samples collected in Moroccan, Indian and Sri Lankan markets, differentiating between plant materials originally harvested from diverse populations in Algeria and Morocco. With a recent drop in the cost of analysing samples, target capture offers the potential to routinely identify commercialized plant species and determine their geographic origin. It promises to play an important role in monitoring and regulation of plant species in trade, supporting biodiversity conservation efforts, and in ensuring that plant products are unadulterated, contributing to consumer protection.

[Background and Aims] Changes in the amount of repetitive DNA (dispersed and tandem repeats) are considered the main contributors to genome size variation across plant species in the absence of polyploidy. However, the study of repeatome dynamism in groups showing contrasting genomic features and complex evolutionary histories is needed to determine whether other processes underlying genome size variation may have been overlooked. The main aim here was to elucidate which mechanism best explains genome size evolution in Anacyclus (Asteraceae). [Methods] Using data from Illumina sequencing, we analysed the repetitive DNA in all species of Anacyclus, a genus with a reticulate evolutionary history, which displays significant genome size and karyotype diversity albeit presenting a stable chromosome number. [Key Results] By reconstructing ancestral genome size values, we inferred independent episodes of genome size expansions and contractions during the evolution of the genus. However, analysis of the repeatome revealed a similar DNA repeat composition across species, both qualitative and quantitative. Using comparative methods to study repeatome dynamics in the genus, we found no evidence for repeat activity causing genome size variation among species. [Conclusions] Our results, combined with previous cytogenetic data, suggest that genome size differences in Anacyclus are probably related to chromosome rearrangements involving losses or gains of chromosome fragments, possibly associated with homoploid hybridization. These could represent balanced rearrangements that do not disrupt gene dosage in merged genomes, for example via chromosome segment exchanges., This work was supported by the Catalan government (grant number 2017SGR1116) and by the Spanish government [grant numbers CGL2010-18039, CGL2013-49097-C2-1-P, CGL2013-49097-C2-2-P, CGL2016-75694-P (AEI/FEDER, UE), RYC-2014–16608 to S.G. and RYC-2016–21176 to O.H.]

Premise of research. Interspecific hybridization was hypothesized to explain the remarkable floral phenotype variation found in overlapping areas of distribution for several Anacyclus species. We aimed to investigate genome size in natural populations of three diploid Anacyclus species with special emphasis in their contact areas to explore patterns of variation as additional evidence supporting current hybridization in these areas. Methodology. Flow cytometry was used to estimate the genome size of 564 individuals of the species complex of A. clavatus, A. homogamos, and A. valentinus from 30 sites. Additionally, genome size variation of 173 first-generation synthetic hybrids between these three species was also studied and compared with the estimates obtained in sympatric sites. Pivotal results. Differences in genome size between A. clavatus and A. valentinus were significant in non-overlapping areas of species distribution, whereas in overlapping areas, the variation increased, preventing a clear differentiation between species. In sympatric sites of A. clavatus and A. valentinus, individuals with intermediate genome sizes between them were also observed and were significantly similar to those obtained from the first-generation experimental hybrids between these species. Genome sizes of A. clavatus and A. homogamos did not differ enough to allow discrimination between these species. Conclusions. The patterns of genome size variation observed in sympatric populations of A. clavatus and A. valentinus support the occurrence of current gene flow between these species and the existence of contact areas in overlapping distribution areas where phenotypic and genomic variation increases.

Premise of the Study: Ray flowers commonly observed in daisies’ flowering heads are a well-known example of advertising structures for enhancing pollinator attraction. Despite this, ray loss has occurred in multiple lineages, which still rely on pollinators, suggesting that rayless phenotypes could also be adaptive for animal-pollination. Here, we investigate the benefits and costs of these specialized floral advertising structures by comparing rayed and rayless phenotypes in two hybridizing closely related species. Methods: We assessed the advantages and costs of ray production in terms of floral visitor's attraction, pollen limitation, and female reproductive success using the broad natural variation on ray size and number at the contact zone of A. clavatus (rayed) and A. valentinus (rayless). In addition, we experimentally explored the effect of rays under controlled neighborhoods and the effect of ray removal on fruit production. Key Results: In sympatry, rayed phenotypes attracted significantly more visitors than rayless plants, in which seed production was pollen limited. However, rayed phenotypes did not show higher fruit set or seed production than rayless phenotypes. Fruit set and seed production benefited from denser neighborhood displays and larger individual floral displays, respectively. The removal of ray florets did not appear to enable resource reallocation to fruit production. Conclusions: Rayless heads compensated their lower visitation rate by means of a higher number of flowers per head achieving similar fecundity levels to rayed plants. The larger size of rayless heads might thus indicate an inflorescence-level trade-off between attraction and fertility., This research was supported by the project “Evolution in Anacyclus L. (Anthemideae, Compositae): Analysis of the A. clavatus (Desf.) Pers. and A. valentinus L. contact zone,” cofunded by the Ministry of Science and Innovation, Spain (Plan Nacional de I+D+I, CGL2010‐18039) and the European Regional Development Fund. SC and AAf were supported by the Portuguese Foundation for Science and Technology (Starting Grant IF/01267/2013 and SFRH/BD/108451/2015, respectively), RT and AAg were supported by the Ministry of Education, Spain (BVA 2010‐0375 for R.T.; BES‐2011‐048197 for AAg).

BACKGROUND AND AIMS: Interstitial telomeric repeat (ITR) sites, consisting of tandem repeats of telomeric motifs localized at intrachromosomal sites, have been reported in a few unrelated organisms including plants. However, the causes for the occurrence of ITRs outside of the chromosomal termini are not fully understood. One possible explanation are the chromosomal rearrangements involving telomeric sites, which could also affect the location of other structural genome elements, such as the 45S rDNA. Taking advantage of the high dynamism in 45S rDNA loci previously found in Anacyclus (Asteraceae, Anthemideae), the occurrence and patterns of variation of ITRs were explored in this genus with the aim of finding common underlying causes. METHODS: In total, 132 individuals from 44 populations of nine species were analysed by fluorescence in situ hybridization using an Arabidopsis-type telomeric sequence as a probe. KEY RESULTS: Variable presence of ITR sites was detected in six out of nine species of Anacyclus, ranging from two to 45 sites and showing contrasting chromosomal locations and a differential presence of the ITR site on homologous chromosome pairs. At the intraspecific level, the ranges were as large as 0–12 ITR sites. Although only 26 % of the total observed ITR sites were localized in chromosomes bearing 45S rDNA loci, all cases of interstitial 45S rDNA reported in a previous work co-occurred with ITRs in close proximity in the same chromosome arms. CONCLUSIONS: High levels of ITR polymorphism within a single species have not been previously reported in plants and suggest that this pattern might have been overlooked due to insufficient sampling. Although ancient Robertsonian translocations or the amplification of terminal 45S rDNA sites cannot, on their own, explain all of the levels of variability in ITRs reported here, there are suggestions that they may have been involved in the evolutionary history of this genus or its ancestors in Anthemideae.

Homologs of the CYC/TB1 gene family have been independently recruited many times across the eudicots to control aspects of floral symmetry The family Asteraceae exhibits the largest known diversification in this gene paralog family accompanied by a parallel morphological floral richness in its specialized head-like inflorescence. In Asteraceae, whether or not CYC/TB1 gene floral symmetry function is preserved along organismic and gene lineages is unknown. In this study, we used phylogenetic, structural and expression analyses focused on the highly derived genus Anacyclus (tribe Anthemidae) to address this question. Phylogenetic reconstruction recovered eight main gene lineages present in Asteraceae: two from CYC1, four from CYC2 and two from CYC3-like genes. The species phylogeny was recovered in most of the gene lineages, allowing the delimitation of orthologous sets of CYC/TB1 genes in Asteraceae. Quantitative real-time PCR analysis indicated that in Anacyclus three of the four isolated CYC2 genes are more highly expressed in ray flowers. The expression of the four AcCYC2 genes overlaps in several organs including the ligule of ray flowers, as well as in anthers and ovules throughout development., This work was supported by grants from the Spanish Ministry of Economy and Competitivness, Plan Estatal de I+D+I and European Social Fund to JF, IA, and PC (CGL2007-66516, CGL2013-49097-C2-2-P and BIO2014-57011-R), “Juan de la Cierva” program co-financed by the European Social Fund to MB (JCI-2010-07374) and Proyecto Intramural CSIC 201430E023 to JF.

Torices, R., Agudo, A. & Alvarez, I. 2013. Not only size matters: achene morphology affects time of seedling emergence in three heterocarpic species of Anacyclus (Anthemideae, Asteraceae). Anales Jard. Bot. Madrid 70(1): 48-55. The production of two or more distinct fruit types by an individual, i.e. hete rocarpy, is considered as a mixed dispersal strategy in which a proportion of the offspring is able to colonize new sites, whilst others remain near the maternal location. Here, we aimed to explore the effects of achene morphology (winged vs. unwinged achenes) and achene size –measured here as achene mass– on post-dispersal life-history traits (probability and time of seedling emergence) in three heterocarpic Anacyclus species (Anthemideae, Asteraceae). Morphology, size and germination performance were studied in achenes from six populations of Anacyclus clavatus (Desf.) Pers., A. homogamos (Maire) Humphries, and A. valentinus L. Our results show that achene morphology and size were related to their position within the capitulum, such that outer, winged achenes were significantly heavier than the inner, unwinged ones. Additionally, winged achenes germinated faster than unwinged ones. This pattern may be related to the sequential achene time of release displayed by these species. Finally, our findings cast doubt on the role of wings as structures that favor dispersal by wind in these three species of Anacyclus.

Background and aims Most of the diversity in the pseudanthia of Asteraceae is based on the differential symmetry and sexuality of its flowers. In Anacyclus, where there are (1) homogamous capitula, with bisexual, mainly actinomorphic and pentamerous flowers; and (2) heterogamous capitula, with peripheral zygomorphic, trimerous and long-/short-rayed female flowers, the floral ontogeny was investigated to infer their origin. Methods Floral morphology and ontogeny were studied using scanning electron microscope and light microscope techniques. Key results Disc flowers, subtended by paleae, initiate acropetally. Perianth and androecium initiation is unidirectional/simultaneous. Late zygomorphy occurs by enlargement of the adaxial perianth lobes. In contrast, ray flowers, subtended by involucral bracts, initiate after the proximal disc buds, breaking the inflorescence acropetal pattern. Early zygomorphy is manifested through the fusion of the lateral and abaxial perianth lobes and the arrest of the adaxials. We report atypical phenotypes with peripheral 'trumpet' flowers from natural populations. The peripheral 'trumpet' buds initiate after disc flowers, but maintain an actinomorphic perianth. All phenotypes are compared and interpreted in the context of alternative scenarios for the origin of the capitulum and the perianth identity. Conclusions Homogamous inflorescences display a uniform floral morphology and development, whereas the peripheral buds in heterogamous capitula display remarkable plasticity. Disc and ray flowers follow different floral developmental pathways. Peripheral zygomorphic flowers initiate after the proximal actinomorphic disc flowers, behaving as lateral independent units of the pseudanthial disc from inception. The perianth and the androecium are the most variable whorls across the different types of flowers, but their changes are not correlated. Lack of homology between hypanthial appendages and a calyx, and the perianth double-sided structure are discussed for Anacyclus together with potential causes of its ray flower plasticity.

An N-alkylamide profiling from an ethanolic Anacyclus pyrethrum DC. root extract was performed using a gradient reversed phase high performance liquid chromatography/UV/electrospray-ionization ion-trap mass spectrometry (HPLC/UV/ESI-MS) method on an embedded polar column. MS1 and MS2 fragmentation data were used for identification purposes while UV was used for quantification. Thirteen N-alkylamides (five N-isobutylamides, three N-methyl isobutylamides, four tyramides and one 2-phenylethylamide) were detected. Six of them, identified as undeca-2E,4E-diene-8,10-diynoic acid isobutylamide, undeca-2E,4E-diene-8,10-diynoic acid N-methyl isobutylamide, tetradeca-2E,4E-diene-8,10-diynoic acid tyramide, deca-2E,4E-dienoic acid N-methyl isobutylamide, tetradeca-2E,4E,XE/Z-trienoic acid tyramide and tetradeca-2E,4E,XE/Z,YE/Z-tetraenoic isobutylamide, are novel compounds which have never been reported before from Anacyclus pyrethrum.

The essential oil from the flowerheads of Anacyclus cyrtolepidioides, a wild Aceraceae herb, gathered in the south of Tunisia, was extracted and separated into four fractions F1-F4. Chemical constituents of those fractions were characterized. Seventy-five compounds, representing 70.2 % of the oil, were identified by GC and GC-MS. Monoterpene hydrocarbons constituted the most abundant terpene family of this oil, by occurrence of α-Pinene in highest percentage in the four fractions. Oxygenated monoterpenes (Compholenal) and Sesquiterpenes (β-Cubebene) were found in F2 at a highest value. Decanal, was found at the highest value in F2, and four carboxylic acids, which values were much important in F2. The crude oil and its four fractions were tested for their antibacterial activity. All the tested samples found to be active towards the gram-positive used strains. The composition-activity relationship is discussed on the basis of the profile of the crude essential oil and its fractions.

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation.

Dissertação de mestrado em Ecologia, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências da Universidade de Coimbra. The evolutionary mechanism behind flowers and its pollinators is generally understood to be a gradual co-adaptive process where the plant specializes to its most efficient pollinator, which exerts selective pressures on specific traits, driving floral evolution. Still, most flowering plants in nature are visited by a wide array of pollinator species, i.e. are generalist plants. However, the role of pollinators as significant drivers of floral evolution in generalist plants has been questioned due to the potential conflicting selection regimes exerted by different pollinators. Taking this into account, using a combination of observation and manipulative experiments, we assessed pollinator preference in a natural contact zone where the generalist rayed species Anacyclus clavatus and the rayless A. valentinus co-exist and hybridize, forming intermediate phenotypes. These contact areas show a remarkably high phenotypic variation, with the intermediate phenotype bridging both phenotypes and forming an exceptional micro-evolutive framework to explore how generalist pollinators could be driving the evolution of floral phenotypes. We found that the production of rays influenced the probability of being visited by specific insect groups, in particular by Dipteran groups; whereas bees showed no preference for rayed phenotypes and their visitation patterns were mainly driven by the number of capitula simultaneously blooming in the plant. In addition, we found support for the importance of the neighbours’ phenotype when assessing pollinator preference on a focal individual. Rayed plants benefited from having other conspicuous neighbours, whereas rayless and intermediate phenotypes significantly competed for pollinators. In conclusion, all these differential behavioural patterns of floral visitors might affect gene flow within the hybrid zone between A. clavatus and A. valentinus influencing the degree of reproductive isolation and floral evolution between both species. Os mecanismos evolutivos que atuam nas flores e respetivos polinizadores são normalmente descritos como processos de co-adaptação gradual onde a planta se especializa no seu polinizador mais eficiente, que por sua vez, exerce pressões evolutivas em características específicas e dessa forma guia a evolução da flor. Ainda assim, a maior parte das plantas com flor são polinizadas por um leque diversificado de espécies de polinizadores, denominando-se assim plantas generalistas. No entanto, em plantas generalistas o papel dos polinizadores na evolução floral tem sido questionado devido a potenciais conflitos na selecção exercida pelas diferentes espécies de polinizadores que visitam a flor. Tendo isto em conta, abordagens observacionais e manipulativas foram utilizadas para avaliar as preferências dos polinizadores numa zona de contacto onde as espécies generalistas Anacyclus clavatus (com lígulas) e a espécie A. valentinus (sem lígulas) coexistem e hibridizam, formando fenótipos intermédios. Estas áreas possuem uma variação fenotípica notável, com o fenótipo intermédio a representar o cruzamento entre as duas espécies, garantindo um cenário microevolutivo excecional para estudar de que forma os polinizadores conduzem a evolução fenotípica em espécies generalistas. Os resultados obtidos revelaram que a produção de lígulas influenciou a probabilidade das plantas serem visitadas por grupos específicos de insetos, em particular por dípteros; por sua vez, as abelhas não revelaram preferências por um fenótipo em particular, preferindo maioritariamente plantas com um elevado número de capítulos em flor. Além disto, os nossos resultados evidenciaram também que a composição fenotípica da vizinhança poderá desempenhar um papel importante na atração de uma planta focal específica; em particular, plantas liguladas beneficiaram em ter outros vizinhos com lígulas, enquanto que os fenótipos sem lígulas e intermédios competiram significativamente por polinizadores. Os diferentes padrões de comportamento diferentes por parte dos visitantes florais observados neste estudo podem afetar o fluxo genético na zona híbrida entre A. clavatus e A. valentinus, influenciando o grau de isolamento reprodutivo e evolução floral entre as duas espécies.

Matricaria pyrethrum Linnaeus, Amoenitates Academicae 4: 491. 1759, nom. nud. Type not relevant. Current name: Anacyclus pyrethrum (L.) Lag. (Asteraceae). Note: As noted by Stearn (in Geck & Pressler, Festschr. Claus Nissen: 631. 1974), this ���name��� resulted from the omission of the generic name Anthemis from a table in a revised version of the 1756 thesis, Flora Monspeliensis. It is not validly published., Published as part of Jarvis, Charlie, 2007, Chapter 7: Linnaean Plant Names and their Types (part M), pp. 651-689 in Order out of Chaos. Linnaean Plant Types and their Types, London :Linnaean Society of London in association with the Natural History Museum on page 658, DOI: 10.5281/zenodo.291971

Matricaria valentina Linnaeus, Amoenitates Academicae 4: 491. 1759, nom. inval. Type not relevant. Current name: Anacyclus radiatus Loisel. (Asteraceae). Note: As noted by Stearn (in Geck & Pressler, Festschr. Claus Nissen: 631. 1974), this ���name��� resulted from the omission of the generic name Anthemis from a table in a revised version of the 1756 thesis, Flora Monspeliensis. It is not validly published., Published as part of Jarvis, Charlie, 2007, Chapter 7: Linnaean Plant Names and their Types (part M), pp. 651-689 in Order out of Chaos. Linnaean Plant Types and their Types, London :Linnaean Society of London in association with the Natural History Museum on page 659, DOI: 10.5281/zenodo.291971

Tanacetum monanthos Linnaeus, Systema Naturae, ed. 12, 2: 541; Mantissa Plantarum: 111. 1767. "Habitat in Oriente. Forsk��hl.��� RCN: 6120. Lectotype (Humphries in Bull. Brit. Mus. (Nat. Hist.), Bot. 7: 116. 1979): Herb. Linn. No. 987.8 (LINN). Current name: Anacyclus monanthos (L.) Thell. (Asteraceae)., Published as part of Jarvis, Charlie, 2007, Chapter 7: Linnaean Plant Names and their Types (part T), pp. 878-905 in Order out of Chaos. Linnaean Plant Types and their Types, London :Linnaean Society of London in association with the Natural History Museum on page 879, DOI: 10.5281/zenodo.291971

Anacyclus valentinus Linnaeus, Species Plantarum 2: 892. 1753. "Habitat ad Reg. Valentini agros & vias." RCN: 6470. Lectotype (Humphries in Bull. Brit. Mus. (Nat. Hist.), Bot. 7: 109. 1979): Herb. Clifford: 417, Anacyclus 1 (BM-000647221). Generitype of Anacyclus Linnaeus (vide Green, Prop. Brit. Bot.: 182. 1929). Current name: Anacyclus valentinus L. (Asteraceae)., Published as part of Jarvis, Charlie, 2007, Chapter 7: Linnaean Plant Names and their Types (part A), pp. 252-342 in Order out of Chaos. Linnaean Plant Types and their Types, London :Linnaean Society of London in association with the Natural History Museum on page 288, DOI: 10.5281/zenodo.291971