New cytological data in Ophrys sect. Pseudophrys Godfery and comparative karyomorphological studies in Ophrys L. (Orchidaceae)

Eight species of Ophrys sect. Pseudophrys (Orchidaceae) were cytogenetically studied. The analysed species possess the most symmetrical karyotypes of the genus (M_CA ranged from 10.21 to 15.87 and CV_CL from 19.61 to 23.93) with 2n = 2x = 36, being composed of mainly metacentric chromosomes. The karyotype formulae were: 36m for Ophrys archimedea, 32m + 4sm for O. flammeola, 32m + 4sm for O. funerea, 36m for O. laurensis, 36m for O. lojaconoi, 34m + 2sm for O. lucifera, 34m + 2sm for O. obaesa and 36m for O. pallida. Karyotype morphometric characters were evaluated by calculating M_CA and CV_CL for the assessment of karyotype asymmetry, and CV_CI for the evaluation of heterogeneity in the position of the centromeres. The relationships between species were thus finally elucidated. The species characterised by wide distribution show greater karyomorphological distance than those with restricted distribution. The possible evolutionary role of chromosomal rearrangements as well as gene mutations in the speciation of Ophrys is discussed.     

Chromosome diversity and evolution in Allium (Allioideae,Amaryllidaceae)

Despite extensive literature on the diversity of karyotypes in Allium is available, no attempt to analyse these data together, within a robust phylogenetic framework, has been carried out so far. Thus, we examined patterns and trends in chromosome evolution across the genus. Based on literature survey, karyo-morphometric features for 207 species belonging to 12 subgenera of Allium were obtained. Included in the data-set were basic chromosome number (x), somatic chromosome number (2n), total haploid (monoploid) chromosome length (THL) and three different measures defining karyotype structure: CV_CI, measuring how heterogeneous are centromeres positions in a karyotype, CV_CL and M_CA, quantifying interchromosomal and intrachromosomal karyotype asymmetry, respectively. Trends in karyotype evolution were analysed by phylogenetic regressions and independent contrasts. Mean karyotypes highlighted differences and similarities in karyotype structure between the 12 subgenera. Further differences were noted when the two parameters for analysing karyotype asymmetry were assessed. In addition, by examining the effects of increasing karyotype dimensions (a proxy for genome size) on karyotype structure and asymmetry, it was shown that in Allium species, the DNA was added proportionally to their arm lengths. Overall, p = 8 and somehow intermediate karyotype asymmetry levels seem to represent plesiomorphic character-states in Allium.     

Reproductive isolation in the Aegean Ophrys omegaifera complex (Orchidaceae)

The orchid genus Ophrys operates a system of sexual deception by which high specificity of pollination is attained. Reproductive isolation in Ophrys mainly rests upon prezygotic isolation mechanisms. The level of genetic separateness of Ophrys taxa with different pollinators is therefore likely determined by the fidelity of pollinators. The present study employs genetic fingerprinting to investigate this in the east Aegean Ophrys omegaifera s.l. complex, also including O. dryis, a west Mediterranean species of this complex. Ophrys fleischmannii, O. basilissa, and the west Mediterranean O. dyris, are found to be well-separated genetic entities whereas O. omegaifera s.str. and the putative hybrid taxon, O. sitiaca, are found to be genetically inseparable across their entire range of co-occurrence. This suggests that specific pollinators have high enough fidelity to act as effective isolating factors in east Aegean O. omegaifera s.l. as a whole, but that the situation in the species pair of O. sitiaca and O. omegaifera is likely to be more complex.     

Comparison of the internal transcribed spacer region (ITS) of the ribosomal RNA genes in wild and cultivated two and six-rowed barleys (Hordeum vulgare L.)

The ITS region of the ribosomal RNA genes from two and six-rowed cultivated barley (Hordeum vulgare subsp. distichon and H. v. subsp. hexastichon, respectively), and its two and six-rowed wild relatives (H. v. subsp. spontaneum and H. v. subsp. agriocrithon, respectively) was isolated and sequenced. The entire ITS region is 598 bp in the two-rowed taxa (H. v. subsp. distichon and H. v. subsp. spontaneum) and 599 bp in the six-rowed ones (agriochriton and hexastichon). The ITS1 is 217 bp in the six-rowed barleys (H. v. subsp. agriochriton and H. v. subsp. hexastichon) and 218 bp in the two-rowed barleys (H. v. subsp. distichon and H. v. subsp. spontaneum). The 5.8S region is 163 bp in all studied H. vulgare taxa. The ITS2 region is 217 bp in the two-rowed barleys (H. v. subsp. distichon and H. v. subsp. spontaneum) and 219 bp in the six rowed ones (H. v. subsp. hexastichon and H. v. subsp. agriochriton). The ITS sequence data of the studied taxa and that of three other wild Hordeum species (H. murinum, H. marinum and H. chilense) were aligned and a phylogeny tree was reconstructed using the Lasergene Program. H. v. subsp. spontaneum was appeared as the ancestor of all other H. vulgare taxa.     

Neue Beobachtungen zur Bestäubung vonOphrys (Orchidaceae) in Südspanien,mit besonderer Berücksichtigung des FormenkreisesOphrys fusca agg.

An investigation of pseudocopulation behaviour in species ofOphrys from southern Spain confirms the close relationship betweenCampsoscolia ciliata (Scoliidae) andOphrys speculum, and betweenEucera nigrilabris (Apoidea) andOphrys tenthredinifera. It could be demonstrated thatEucera barbiventris is the pollinator ofOphrys scolopax subsp.scolopax, whereas other species ofEucera andTetralonia which are active at the same time show no interest at all for the flowers of this species. Special attention was paid to the forms of the taxonomically confusedOphrys fusca group:O. fusca s. str.,O. iricolor, O. omegaifera andO. atlantica. WhileO. fusca s. str. is widespread, small-flowered and has late anthesis,O. iricolor has very large flowers and early anthesis. Each of the four members ofO. fusca agg. in S. Spain is pollinated by a different bee, and selective experiments show that each of these four species of bees is specifically attracted only to one of theOphrys species:Andrena flavipes is the pollinator ofO. fusca s. str.,Colletes cunicularius infuscatus ofO. iricolor, Anthophora atroalba ofO. omegaifera, andChalicodoma parietina ofO. atlantica. These four pollinators belong to 4 different bee families (Andrenidae, Colletidae, Anthophoridae, Megachilidae). As this type of pollination represents a very effective pregamic isolation mechanism, these four taxa ofOphrys fusca agg., at least in southern Spain, behave and should be regarded as genuine species.

     

Karyomorphological studies of theIxeris dentata complex on Mount Ishizuchi

The karyotypes of four taxa in theIxeris dentata complex collected from Mt. Ishizuchi were investigated.I. dentata subsp.dentata var.dentata f.dentata had 2n=21 in all the examined individuals and its karyotypes were classified into four different types. Var.dentata f.amplifolia showed 2n=21 in all individuals and had three different karyotypes.I. dentata subsp.dentata var.albiflora showed 2n=21 and two different karyotypes.I. dentata subsp.alpicola showed three different karyotypes with 2n=21 and four different ones with 2n=28. Judging from the results of the karyotype analyses, the morphological variation of chromosomes in theI. dentata complex is assumed to be in the process of increasing asymmetry in the shape of chromosomes. Following Levitzky's principle, it can be said that the a-, b-, and c-chromosomes exist in different advanced stages in the process of chromosome variation. The c-group chromosomes are at the most advanced stage in morphological variation, the b-group in the second and the a-group in the least advanced stage. The above-mentioned structural diversity of chromosomes may be related to apomictic reproduction of theI. dentata complex.     

Comparative karyotype analysis of populations in the Alstroemeria presliana Herbert (Alstroemeriaceae) complex in Chile

Alstroemeria L., one of the most diverse genera of the Chilean flora and of high floricultural value, is represented by 35 species, most of them distributed between 28–38° S in the Mediterranean zone of Central Chile. There are 24 complex-forming taxa, of which 18 have conservation problems (8 are considered “endangered” and 10 as “vulnerable”). One of these complexes is Alstroemeria presliana Herb. with two subspecies: subsp. presliana and subsp. australis Bayer. Alstroemeria presliana grows in Chile and Argentina: subsp. presliana is distributed from Reserva Nacional Siete Tazas (35°27′ S, Region of Maule) to Antuco, (37°25′ S, Region of Bío-Bío), and is also found in Neuquén, Argentina; subsp. australis is endemic to the Cordillera of Nahuelbuta. A comparative karyotype study was carried out among six populations of A. presliana subsp. presliana and five populations of A. presliana subsp. australis. The eleven populations presented an asymmetric karyotype, with 2n = 2× = 16 chromosomes but with different karyotype formulae. A. presliana subsp. presliana shows the haploid formula 2m + 2m-sat + 1sm-sat + 1st-sat + 1t + 1 t-sat, and A. preslianasubsp. australis presents a formula 1m + 2m-sat + 1sm + 2t + 2t-sat chromosomes. The architecture of the karyotype between the subspecies is very different. The scatter plot among CV_CL vs. M_CA shows different groupings between populations of the two subspecies. According to the results obtained it is possible to consider raising Alstroemeria presliana subsp. australis at species level.     

Polyploidy and aneuploidy inOphrys,Orchis, andAnacamptis (Orchidaceae)

Studies on chromosome numbers and karyotypes in Orchid taxa from Apulia (Italy) revealed triploid complements inOphrys tenthredinifera andOrchis italica. InO. tenthredinifera there is no significant difference between the diploid and the triploid karyotypes. The tetraploid cytotype ofAnacamptis pyramidalis forms 36 bivalents during metaphase I in embryo sac mother cells. Aneuploidy was noticed inOphrys bertolonii ×O. tarentina with chromosome numbers n = 19 and 2n = 38. There were diploid (2n = 2x = 36), tetraploid (2n = 4x = 72), hexaploid (2n = 6x = 108) and octoploid (2n = 8x = 144) cells in the ovary wall of the diploid hybridOphrys apulica ×O. bombyliflora. Evolutionary trends inOphrys andOrchis chromosomes are discussed.     

Karyomorphological analyses and heterochromatin characteristics disclose phyletic relationships among 2n = 32 and 2n = 36 species ofOrchis (Orchidaceae)

The karyotypes of eight taxa ofOrchis L. with 2n = 32 and 2n = 36 have been investigated using morphometrical measurements following staining with Feulgen, Giemsa (C-banding) and the DNA specific fluorochrome Hoechst 33258. The karyotypes ofO. coriophora subsp.fragrans, andO. papilionacea proved to be the most asymmetrical, whileO. morio andO. longicornu exhibited the most symmetrical karyotypes. Using C-banding and the fluorochrome H33258 only the taxa with high asymmetry indices showed the presence of differentially stained chromatin bands. In most chromosomes heterochromatin bands were present at the telomeric position. The present results seem to indicate that the analysed species do not form a homogeneous group and further subdivisions are possible, which, in turn, do not always correlate with divisions based on morphological characters. Both karyomorphology and heterochromatin distribution coincide in indicating a possible evolutionary pathway.     

A critical review and a new proposal of karyotype asymmetry indices

In literature seven different methods of evaluating karyotype asymmetry – the TF%, the As K%, Stebbins' classification, the Rec and the Syi, the A₁ and the A₂, the DI, and the A – are used for the elucidation of phylogenetic relationships and taxonomic treatments within a particular group or taxon. The investigation of these seven methods reveals that the intervals used by Stebbins to separate the different types of karyotype asymmetry are very broad and only one quantitative parameter, the A₂ index, correctly describes the variation in chromosome length in a complement. A new asymmetry index (AI) is proposed to measure karyotype asymmetry and a new parameter, the CV_CI, is offered, that precisely assesses the relative variation in centromere position in a complement. The AI index, the CV_CI and the CV_CL (=A₂ × 100) have the potential to display even minor karyotypic variations. Thus, these three indices together increase the precision of results in comparison with other existing methods. All this has important consequences as regards the interpretation of the results of karyological studies.     

Comparison of the Giemsa C-banded karyotypes of the three subspecies ofPsathyrostachys fragilis,subspp.villosus (2x),secaliformis (2x, 4x), andfragilis (2x) (Poaceae), with notes on chromosome pairing

The karyotypes of diploidP. fragilis subsp.villosus (2n = 2x = 14) and tetraploid subsp.secaliformis (2n = 4x = 28) were studied by Giemsa C- and N-banding, and AgNO₃ staining and compared with the karyotype of subsp.fragilis (2x). The complements of subsp.villosus and subsp.fragilis were similar, with 8 metacentric and 6 SAT-chromosomes, one metacentric and two submetacentric pairs, with small to minute, polymorphic, heterochromatic satellites. The complement of subsp.secaliformis on the whole agreed with a doubling of the complement of diploidP. fragilis, suggesting autopolyploidy. Only the presence of 12 nucleoli in interphases identified 6 SAT-chromosome pairs. In subsp.villosus one or two extra micronucleoli indicated a chromosome pair with very low nucleolusforming activity, bringing the number of SAT-chromosome pairs to 4. This number may be a characteristc ofPsathyrostachys. Besides very small, inconsistently observed bands, the C-banding pattern consisted of 0–3 small bands per chromosome at intercalary and terminal locations, and at NORs. The level of banding pattern polymorphism was low, but enough to indicate that the taxa are outbreeders. Similarities in chromosome morphology and C-banding patterns identified homology of all chromosomes of subsp.villosus, but for 12 pairs only in subsp.secaliformis. Between plants, reliable identification of homology and homoeology (subsp.secaliformis) was possible only for the SAT-chromosomes and the shortest metacentrics. Chromocentres were very small and the amount of constitutive heterochromatin was low. N-banding stained chromosomes uniformly. The basic karyotypes of theP. fragilis taxa were similar to those ofP. juncea, P. lanuginosa, andP. stoloniformis supporting a close relationship and the presence of a common genome, N. NORs had different nucleolus-forming activities. Meiotic analysis demonstrated a high level of bivalent pairing in the three taxa. A chromosomal rearrangement was suggested in subsp.villosus. The low multivalent frequency in subsp.secaliformis indicates the presence of a pairing regulation mechanism. The majority of chiasmata were interstitial. Pollen grain size discriminated between diploid and tetraploid taxa. The existence of a diploid cytotype of subsp.secaliformis is supported by pollen measurements of herbarium material.     

Characterization and distinction of two subspecies of Eryngium duriaei J. Gay ex Boiss., an Iberian endemic Apiaceae, using flow cytometry and essential oils composition

Eryngium duriaei (Apiaceae) is an endemic taxa from Iberian Peninsula. Some doubts exist in the literature about the taxa relationships, especially among Iberian populations at different altitudes. Also, as other Apiaceae, this species presents a large potential for essential oil production. Considering all this, a multidisciplinary study comprising taxonomic, cytological (using flow cytometry and chromosome counts) and chemical (essential oils) analyses was performed with the objective to morphologically characterize this species and to evaluate the cytotaxonomical and chemical diversity of E. duriaei Portuguese populations. FCM and chromosome counts have shown that every individual presented the same ploidy level, i.e., 2n = 2x = 16 chromosomes. However, flow cytometric analyses revealed that individuals of E. duriaei from higher altitudes (»1,700 m) presented a significantly higher genome size than those belonging to E. duriaei populations below 1,700 m (2C = 6.20 ± 0.04 vs. 2C = 5.52 ± 0.05 pg). Moreover, the essential oils analyses revealed that most chemical constituents were sesquiterpenes, but relevant differences in the main components were observed: α-neocallitropsene (28–53 %), β-betulenal (8.5–15.8 %) and 14-hydroxy-β-caryophyllene (5.8–13.7 %) were the main compounds of Eryngium duriaei oil below 1,700 m, whereas caryophyllene oxide (47 %) and E-caryophyllene (6 %) were the major compounds of E. duriaei oil of higher altitude populations. The results provide important evidences to support the taxonomic separation of E. duriaei in two taxa: E. duriaei J. Gay ex Boiss. subsp. duriaei and E. duriaei subsp. juresianum (M. Laínz) M. Laínz, as previously considered by this author.     

Morphological phylogenetic analysis of Ophrys (Orchidaceae): insights from morpho‐anatomical floral traits into the interspecific relationships in an unresolved clade

Reconstructing the phylogeny of the sexually deceptive orchid genus Ophrys is crucial to our understanding of the evolution of its complex floral morphology. Molecular phylogenetic analyses showed that section Pseudophrys forms a well supported clade with Ophrys bombyliflora, O. tenthredinifera and O. speculum, but were unable to elucidate the relationships between these four groups of taxa. Here we conduct a morphological phylogenetic analysis of this unresolved clade of Ophrys based on a data matrix of 45 macro‐ and micromorphological and anatomical floral characters, using maximum parsimony and Bayesian inference. Our cladistic analysis yielded a single most parsimonious tree and a Bayesian 50% majority‐rule consensus tree which differed in their overall topology but agreed that O. tenthredinifera and O. bombyliflora are not sister groups. The phylogenetic placement of O. tenthredinifera was ambiguous since it shares six valid synapomorphies each with the cluster of O. speculum–O. bombyliflora and with section Pseudophrys. In contrast, O. bombyliflora is most likely the sister group to O. speculum, a finding that rejects an earlier morphological phylogenetic hypothesis and favours the existing molecular trees based on nuclear ITS rather than plastid data. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 454–476.     

KARYOTYPE ANALYSIS IN SOME GENERA OF COMPOSITAE. VIII. FURTHER STUDIES ON THE CHROMOSOMES OF ASTER

Huziwara , Y. (Kobe U., Mikage, Kobe, Japan.) Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromosomes of Aster. Amer. Jour. Bot. 49 (2) : 116–119. Illus. 1962.—The karyotypes of 3 Asiatic and 3 American taxa of Aster are reported here for the first time. These are: A. ageratoides subsp. sugimotoi (Kitamura) Kitamura 2n = 36, A. ageratoides subsp. (Taxon AIII) 2n = 72, A. himalaicus C. B. Clarke 2n = 18, A. ericoides L. 2n = 32, A. meritus A. Nels. 2n = 27, A. umbellatus Mill. 2n = 18. Two American taxa, namely, A. meritus and A. umbellatus, are considered to be ancestral taxa which have retained primitive karyotypes similar to those of Asiatic species of Aster.     

Intraspecific phylogenetic relationships in Ixeridium dentatum (Compositae) complex based on genetic and morphological evidence

Ixeridium dentatum (I. dentatum; family Compositae) forms a complex species through intraspecific differentiation. In this study, intraspecific taxa of I. dentatum were analyzed by counting chromosome numbers, sequencing internal transcribed spacer (ITS), and detecting amplified fragment length polymorphisms (AFLP) to explore phylogenetic relationships within I. dentatum. Analysis of ploidy levels showed that I. dentatum subsp. kitayamense and subsp. nipponicum were diploid (2n = 2x = 14), I. dentatum subsp. kimuranum was tetraploid (2n = 4x = 28), and other intraspecific taxa of I. dentatum were triploid (2n = 3x = 21). The ITS sequences contained only 13 variable sites (2.7 %) among intraspecific taxa, suggesting that the I. dentatum complex originated by autoploidy. Our AFLP-based analysis separated these taxa into five groups: (1) I. dentatum subsp. kimuranum and subsp. kitayamense; (2) subsp. nipponicum and var. albiflorum; (3) two types of subsp. dentatum and f. atropurpureum; (4) f. amplifolium; (5) subsp. ozense and new taxa, which were discovered in this study. The identified clusters significantly differed from morphological classifications. For example, morphologically similar variety and forms of I. dentatum subsp. nipponicum were separated into different groups. Taken together with the ploidy data, this study proposes a possible evolutionary history generating the current taxonomic combinations of the I. dentatum complex. The proposed model suggests that diverse taxa recently emerged during migration to lowlands.     

Palynological investigations on some Hypericum taxa (Hypericaceae) growing naturally in Turkey

This study examined the detailed pollen morphological structures of sixteen Hypericum taxa (four endemic, twelve non endemic) including eight sections showing the natural distribution in Turkey: H. sect. Ascyreia (H. calycinum L.), H. sect. Heterophyllum (H. heterophyllum Vent.), H. Sect. Taeniocarpium (Hypericum confertum Choisy subsp. confertum, H. venustum Fenzl, H. linaroides Bosse), H. sect. Drosocarpium (H. montbretii Spach, H. bithynicum Boiss.), H. sect. Crossophyllum (H. adenotrichum Spach, H. orientale L.), H. sect. Olympia (H. olympicum L. subsp. olympicum), H. sect. Origanifolia (H. origanifolium Willd., H. avicularifolium Jaub and Spach subsp. depilatum (Freyn and Bornm.) Robson var. depilatum, H. avicularifolium Jaub. and Spach subsp. byzantinum) and H. sect. Hypericum (H. tetrapterum Fries, H. perforatum L., H. triquetrifolium Tura). These taxa were studied under light microscope and scanning electron microscope for the first time. Of the taxa studied, H. tetrapterum has the smallest pollen grains (on average 15.85–17.20 × 15.45–16.05 μm); and H. olympicum subsp. olympicum the largest grains (on average 22.90–23.10 × 21.40–22.10 μm). The pollen grains of 15 taxa are subprolate and one taxon is prolate-spheroidal. The regular pollen grains of all 16 taxa are 3-zonocolporate. Ornamentation is microreticulate; lumina regularly spaced in eight taxa, tectum perforatum; tectal perforations regularly spaced in seven taxa and tectum perforatum; and tectal perforations grouped together in one taxon. Endoaperture is cruciform porus, with short lateral and meridional extensions in ten taxa, cruciform porus, transversally elongated, with very small lateral extensions in one taxon and lalongate colpus in five taxa. Basic pollen types are ten taxa in type X, five taxa in type IV and one taxon in type II.     

Karyological studies on eight species of <Emphasis Type="Italic">Onobrychis</Emphasis> genus in Turkey

This study used karyological techniques to determine the chromosome numbers and morphology of eight species of Onobrychis L. (O. caput-galli (L.) Lam, O. aequidentata (Sibth. & Sm.) d’ Urv, O. fallax Freyn & Sint. var. fallax, O. lasiostachya Boiss, O. viciifolia Scop., O. oxyodonta Boiss. subsp. armena (Bois. & Huet) Aktoklu, O. hypargyrea Boiss. and O. cappadocica Boiss.). The results of this study determined the chromosome numbers of O. cappadocica as 2n = 16; O. viciifolia as 2n = 28 and the other species as 2n = 14 The karyotypes of species consisted of median-centromeric (m) or submedian-centromeric (sm) chromosomes. However, O. oxyodonta Boiss. subsp. armena (Bois. & Huet) Aktoklu was found to have only the median-centromeric (m) chromosomes. According to the results of the present study, of the eight Onobrychis taxa, only O. hypargyrea has a pair of satellite chromosomes (sat-chromosome). Furthermore, this study detected karyotype asymmetry.     

Karyomorphology of Turkish species in Centaurea sections Centaurea and Phalolepis (Asteraceae) and implications for taxonomy

In this paper, 31 mostly endemic and locally distributed Turkish Centaurea taxa belonging to Centaurea and Phalolepis sections were examined in terms of their karyomorphology. The basic chromosome number for all of the studied species was concurringly determined as 9(x = 9) for both sections, excluding C. hierapolitana. We also determined tetraploid and hexaploid species in spite of many species having diploid chromosome numbers. Twelve chromosome counts are reported for the first time and most of the karyotyping analyses are described for the first time via the KAMERAM program. The karyotypes had a predominance of metacentric (m) chromosomes. However, in the karyotyping of six taxa, submetacentric (sm) chromosomes were dominant. Five quantitative asymmetric indices were used to evaluate karyological features of the species. A meaningful dendrogram was carried out to assess the karyotype-symmetry conditions and describe the karyotyping relationships between different taxa.     

Karyotype analysis and DNA content in some species of Chrysolaena (Vernonieae,Asteraceae)

Cytogenetic characterization by karyotyping and determination of DNA content by flow cytometry of five species of Chrysolaena (Vernonieae, Asteraceae) was performed. This is the first study of nuclear DNA content realized in the genus. The 2C-values were compared with the ploidy level and the total karyotype length (TKL) of each species. Mitotic analysis revealed a base chromosome number x = 10 for all entities and different ploidy levels, from diploid (2n = 2x = 20) to octoploid (2n = 8x = 80). All species showed bimodal karyotypes composed of metacentric and submetacentric chromosomes. The average chromosome size (ML) varied from 1.86 μm to 2.70 μm, while the TKL ranged from 18.65 μm to 80.55 μm. The intrachromosomal asymmetry index (A₁) varied from 0.27 to 0.38, while the interchromosomal asymmetry index (A₂) ranged from 0.19 to 0.25. A new cytotype is reported for the first time for C. propinqua. Accessory chromosomes found in C. verbascifolia, C. cognata, C. flexuosa, and C. propinqua are also reported as new.     

Speciation in sexually deceptive orchids: pollinator-driven selection maintains discrete odour phenotypes in hybridizing species

Ophrys orchids mimic the female sex pheromones of their pollinator species to attract males for pollination. Reproductive isolation in Ophrys is based on the selective attraction of only a single pollinator species. A change of floral odour can result in the attraction of a new pollinator species that acts as an isolation barrier towards other sympatrically occurring Ophrys species. Ophrys lupercalis, Ophrys bilunulata, and Ophrys fabrella grow sympatrically and bloom consecutively on Majorca and are pollinated by three species of Andrena. We investigated variation of phenotypic and genotypic flower traits, aiming to study the role of the floral odour for reproductive isolation and speciation. Using chemical and electrophysiology (gas chromatography coupled with an electroantennographic detector) methods, we show that the three Ophrys species use the same odour compounds for pollinator attraction, but in different proportions. A comparison of the floral odour bouquets in a multivariate analysis revealed a clear grouping of plants from the same species, although with an overlap between species. A comparison of the same plants using molecular markers gave a contrasting result. Although O. lupercalis and O. fabrella were genetically well separated, plants of O. bilunulata did not form a distinct group but were similar to either O. lupercalis or O. fabrella. Our data indicate gene flow and hybridization to occur between O. bilunulata and O. lupercalis as well as between O. bilunulata and O. fabrella. All plants of O. bilunulata, despite having different genotypes, showed a very similar floral odour. This reflects a strong selective pressure by the pollinating males. The overlap of genotypes of O. bilunulata and O. fabrella supports our hypothesis that O. fabrella diverged from O. bilunulata by scent variation and the attraction of a new pollinator species, Andrena fabrella. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 439–451.