Distribution of exudate flavonoids in the genus Plectranthus

Thirty-four species of the genus Plectranthus (including species of the former genera Coleus and Solenostemon, fam. Lamiaceae) were surveyed for exudate flavonoids to see whether the distribution of these compounds would support a recent classification of the genus based on molecular and morphological characters. In this classification two major groups had been identified, the Coleus and Plectranthus clades. Only about 40% of the species, predominantly from the Plectranthus clade, were found to produce exudate flavonoids, which were mainly flavones. Flavanones were restricted to five species of the Plectranthus clade, whereas flavonols were only found in two species of the Coleus clade, Plectranthus montanus Benth. (synonyms Plectranthus marrubioides Hochst. ex Benth. and Plectranthus cylindraceus Hochst. ex Benth.) and Plectranthus pseudomarrubioides R.H.Willemse. Four of these flavonols were isolated from P. montanus and identified by NMR spectroscopy as the 3,7-dimethyl ether and 3,7,4′-trimethyl ether of quercetin and the 3,6,7-trimethyl ether and 3,6,7,4′-tetramethyl ether of quercetagetin. The remaining flavonols and flavones were identified by HPLC–UV and LC–MS of crude extracts on the basis of their UV and mass spectra, retention times and comparison with standards. Most flavonols were 3-methyl ethers and many of the flavones and flavonols were oxygenated at the 6-position. The most common flavones, occurring in both clades, were cirsimaritin and salvigenin, which are methoxylated at the 6- and 7-positions. 6-Hydroxylated flavones such as scutellarein and ladanein were restricted to species of the Plectranthus clade.     

Variation in Phytochemical,Morphological, and Ploidy Levels of Iranian Thymus Species

Thymus is one of the most important genera of the Lamiaceae family. This work was performed to assess inter and intra species variation, which is an indispensable prerequisite for the selection and the exploitation of the germplasm, using yield, secondary metabolites, and ploidy level criteria. Nineteen Iranian populations belonging to 11 Thymus species which includes T. vulgaris were used in this study. The results of cytological observations on the 19 populations revealed the three root-tip chromosome numbers of 2n=2x=30, 2n=4x=56 or 60 (diploid and tetraploid). This study also presents the results of a two-year field experiment that evaluates the agronomic and morphology of the 19 populations of Thymus spp. Cluster analysis grouped the populations into six groups and explained the relationships among ploidy levels, morphological traits, and essential oils (EOs). In general, diploid species belonged to the thymol chemotype, whilst carvacrol chemotype consistently dependent on the gene-dosage effect. Thymus migricus, T. daenensis-2, T. serpyllum, and T. trautvetteri populations with diverse thymol background were the best selection as the parents to improve thymol in a breeding program. Moreover, dry and fresh weight criteria can be used to improve EO content in thyme. Achieving this goal would be expected by crossing T. migricus and T. daenensis-2. Finally, providing relevant information on the ploidy level of Thymus species, with emphasis on morphology and EO components variations, may be recommended for the selection of populations or species to improve bioactive components as well as morphological traits in future breeding programs.     

Individual relationship between aneuploidy of gill cells and growth rate in the cupped oysters Crassostrea angulata, C. gigas and their reciprocal hybrids

The Portuguese oyster, Crassostrea angulata, is taxonomically close to the Pacific oyster, C. gigas, but there are clear genetic and phenotypic differences between these taxa. Among those differences, the faster growth of C. gigas compared with C. angulata has often been observed in the field. Crosses between C. angulata and C. gigas were performed to investigate the relationship between growth variation and somatic aneuploidy at the individual level in the two taxa and their reciprocal hybrids. The different progenies were reared in Ria Formosa (Portugal) under standard farming conditions. Growth rate and survival were significantly higher in C. gigas than in C. angulata, and the hybrids showed intermediate performances. Significant differences were also observed in the proportion of aneuploid cells (PAC) and of missing chromosomes (PMC) between the two taxa, C. angulata showing the highest values. Intermediate values of PAC and PMC were observed in the hybrids, supporting additive genetic bases of these parameters. Our results also confirm the negative correlation between somatic aneuploidy and growth rate at the individual level, as previously reported in C. gigas.     

DNA barcoding to analyse taxonomically complex groups in plants: the case of Thymus (Lamiaceae)

We evaluated the utility of the core barcode regions (matK and rbcL) and the plastid intergenic spacer trnH‐psbA to distinguish between Thymus spp. This is a taxonomically complex group that has been investigated so far mainly using morphological approaches. Thirty‐six samples representing nine different morphospecies were collected and used for molecular analysis. The three markers showed clear amplification and sequencing. However, the genetic variation and the resulting haplotype networks showed that only Thymus capitatus forms a well‐defined ‘barcoding gap’ compared with the other taxa. The identification problems observed in the other Thymus spp. may be related to reduced gene flow among populations, resulting in high intraspecific and low interspecific genetic variation. This situation does not permit the definition of species‐specific barcodes. A second hypothesis suggests that morphological traits used for the delimitation of Thymus spp. do not reflect real biological and molecular species boundaries. If this is the case, the taxonomy of Thymus should be revised through extensive sampling and analyses with different tools (i.e. molecular variability, morphology, geographical distribution, etc.) to define the natural units at the species level. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013 , 171 , 687–699.     

Essential oil variability in Thymbra capitata (L.) Cav. growing wild in Southern Apulia (Italy)

Essential oils of Thymbra capitata (Thymus capitatus) collected from Southern Apulia (Italy) were analysed using gas chromatography and gas chromatography–mass spectrometry techniques, to check for chemical variability. The study showed that among the 75 components of the oils the most recurrent ones were thymol and carvacrol, which always constituted more than 50% of the oils, as well as γ-terpinene, borneol and p-cymene. Cluster analysis led to the identification of three chemotypes: thymol, carvacrol and thymol/carvacrol; this was presumably a crossbreed between the other two chemotypes. Principal component analysis showed the direct correlation among myrcene, α-terpinene and γ-terpinene; anti-correlation between thymol and carvacrol, and the inverse correlation between linalool and myrcene. Moreover, lower thymol concentrations were accompanied by an increase in myrcene, α-terpinene and γ-terpinene.     

Morphological variation within the Edraianthus graminifolius complex (Campanulaceae) from the central Balkan Peninsula – Evidence from multivariate statistical analysis

The Edraianthus graminifolius complex is one of the most interesting groups within the genus Edraianthus DC (Campanulaceae). The plants inhabit a variety of habitats and there is a micro-geographic and ecological differentiation of populations, accompanied by pronounced morphological variation. Hence, it is not surprising that this complex is taxonomically a very controversial group. On the one hand it is described to comprise only two to four taxa, including subspecies, and on the other, a classification comprising 23 taxa of the rank of species and subspecies has been proposed.     

The maintenance of hybrids by parasitism in a freshwater snail

Hybrids have often been labelled evolutionary dead-ends due to their lower fertility and viability. However, there is growing awareness that hybridisation between different species may play a constructive role in animal evolution as a means to create variability. Thus, hybridisation and introgression may contribute to adaptive evolution, for example with regards to natural antagonists (parasites, predators, competitors) and adaptation to local environmental conditions. Here we investigated whether parasite intensity contributes to the continuous recreation of hybrids in 74 natural populations of Melanopsis, a complex of freshwater snails with three species. We also examined, under laboratory conditions, whether hybrids and their parental taxa differ in their tolerance of low and high temperatures and salinity levels. Infections were consistently less prevalent in males than in females, and lower in snails from deeper habitats. Infection prevalence in hybrids was significantly lower than in the parental taxa. Low hybrid infection rates could not be explained by sediment type, snail density or geographic distribution of the sampling sites. Interestingly, infected hybrid snails did not show signs of parasite-induced gigantism, whereas all parental taxa did. We found that hybrids mostly coped with extreme temperatures and salinity levels as well as their parental taxa did. Taken together, our results suggest that Melanopsis hybrids perform better in the presence of parasites and environmental stress. This may explain the widespread and long-term occurrence of Melanopsis hybrids as evidenced by paleontological and biogeographic data. Hybridisation may be an adaptive host strategy, reducing infection rates and resisting gigantism.     

Screening of the essential oil composition of wild Sicilian thyme

Thirty samples of wild thyme collected from as many Sicilian locations have been analysed for their qualitative and quantitative essential oil profiles. The oils, obtained by hydrodistillation, have been analysed by a combination of GC-FID-MS; in all, 46 components, representing more than 98% of the oils, were fully characterized. Monoterpenes, both hydrocarbons and oxygenated, were the most highly represented components: the former with a range of 8–61% and the latter with a range of 31–86%. Carvacrol was the main compound in 29 samples, ranging between 49 and 83%, suggesting that Thymus capitatus Hoff. et Link. [syn. Coridothymus capitatus (L.) Rchb.f., Satureja capitata L., Thymbra capitata (L.) Cav.] is the most widespread wild species in the Sicilian area. Only one sample, identified as Thymus longicaulis C. Presl., collected from the North-East of Sicily showed a different composition, p-cymene and thymol being the main compounds with 40 and 16%, respectively. Statistical analyses allowed establishing a single broad group, confirming the substantial compositional uniformity of the essential oil profiles of the wild Sicilian thyme.     

Three methylated flavones from Thymus vulgaris

Three highly oxygenated flavones were isolated from leaves of Thymus vulgaris. Their structures were determined by spectroscopic methods as 5, 6, 4′-trihydroxy- 7, 8, 3′-trimethoxyflavone (thymonin), 5, 4′-dihydroxy-6, 7, 3′-trimethoxyflavone (cirsilineol) and 5, 4′-dihydroxy-6, 7, 8, 3′-tetramethoxyflavone. These flavones are reported for the first time in the genus Thymus.     

Evolution of the Portulaca oleracea L. aggregate in Egypt on molecular and phenotypic levels revealed by morphology,inter-simple sequence repeat (ISSR) and 18S rDNA gene sequence markers

Molecular markers including inter simple sequence repeats (ISSR) and 18S rDNA gene sequence markers were combined with a detailed morphological analysis to seek characters that discriminate four taxa of Portulaca oleracea s.l. These taxa were identified as the microspecies Portulaca nitida, Portulaca oleracea, Portulaca rausii and Portulaca granulatostellulata. Morphological characters did not provide a clear distinction among the four taxa of P. oleracea s.l. It was found that mixed populations of the taxa occur in several locations and morphological similarities between the microspecies were detected. ISSR analysis indicates that gene flow among populations of different taxa was high and most of the genetic variation (61.9%) occurs within population. These results are inconsistent with the general characteristics of P. oleracea as a self- or 5% cross-pollinated species. A close relationship between P. oleracea, P. rausii and P. granulatostellulata was supported by ISSR and 18S rDNA gene sequence. The ISSR and 18s data support the specific status of P. nitida as a recently evolved taxon. We conclude that P. oleracea exists as a polymorphic species and is not divisible into microspecies based on seed surface as the primary morphological trait, and inter simple sequence repeats (ISSR) and 18S rDNA gene sequence markers. We suggest that the taxa do not merit specific rank as morphological characters and absence of a breeding barrier fail to separate the different populations when they become sympatric.     

Microsatellite variability and heterozygote excess in Elymus trachycaulus populations from British Columbia in Canada

Microsatellite markers were used to assess the genetic diversity and population structure in four populations of Elymus trachycaulus from British Columbia and one population of Elymus alaskanus from Northwest Territories. Fourteen microsatellite loci were used in this study. Our results indicated that E. trachycaulus is highly polymorphic, with an average percentage of polymorphic loci of 96.5% over the four populations. Average expected heterozygosity values (H_E or gene diversity) varied from 0.418 to 0.585 with a mean of 0.497. Most of the genetic variation was found within populations (85%) and the differentiation among populations was found to be 15% (F_st = 0.15). Interpopulation genetic distances corresponded well with the geographic distance between the population sites of origin, as well as morphological characteristics. Tests for Hardy–Weinberg equilibrium (HWE) for all loci and all populations revealed that all loci significantly differ from HWE. Subsequent analysis indicated that departure from HWE at some loci was due to an excess of heterozygotes. Possible explanations for heterozygote excess are discussed. The most likely reason for observed heterozygote excess could be due to the polyploidy nature of the species.     

Interspecific gene flow in a multispecies oak hybrid zone in the Sierra Tarahumara of Mexico

Background and Aims

Interspecific gene flow can occur in many combinations among species within the genus Quercus, but simultaneous hybridization among more than two species has been rarely analysed. The present study addresses the genetic structure and morphological variation in a triple hybrid zone formed by Q. hypoleucoides, Q. scytophylla and Q. sideroxyla in north-western Mexico.

Methods

A total of 247 trees from ten reference and 13 presumed intermediate populations were characterized using leaf shape variation and geometric morphometrics, and seven nuclear microsatellites as genetic markers. Discriminant function analysis was performed for leaf shape variation, and estimates of genetic diversity and structure, and individual Bayesian genetic assignments were obtained.

Key Results

Reference populations formed three completely distinct groups according to discriminant function analysis based on the morphological data, and showed low, but significant, genetic differentiation. Populations from the zone of contact contained individuals morphologically intermediate between pairs of species in different combinations, or even among the three species. The Bayesian admixture analysis found that three main genetic clusters best fitted the data, with good correspondence of reference populations of each species to one of the genetic clusters, but various degrees of admixture evidenced in populations from the contact area.

Conclusions

The three oak species have formed a complex hybrid zone that is geographically structured as a mosaic, and comprising a wide range of genotypes, including hybrids between different species pairs, backcrosses and probable triple hybrids.     

Interspecific hybridization in oysters: Restriction Enzyme Digestion Chromosome Banding confirms Crassostrea angulata × Crassostrea gigas F1 hybrids

The taxonomic status of the two commercially important cupped oysters, Crassostrea angulata, the Portuguese oyster (Lamarck, 1819) and Crassostrea gigas, the Japanese oyster (Thunberg, 1793) has long been in question. The recent observation of the hybridization between C. gigas and C. angulata and the production of fertile F1s led us to search for cytogenetic evidence of both parental genomes in the interspecific hybrids. The cytogenetic characterization of the hybrids was performed by the use of restriction endonuclease treatments. This technique has recently shown the potential for individual chromosome identification by banding in oysters. Chromosomes of C. gigas, C. angulata and their hybrids were treated with two different restriction enzymes (ApaI and HaeIII), stained with Giemsa, and examined for banding patterns. These chromosome markers allowed the parental haploid sets to be identified in the hybrids. The analysis of the banded karyotypes of the interspecific hybrids showed that for each chromosome pair, one of the homologues presented a banding pattern consistent with that of C. gigas and the other homologue presented a banding pattern consistent with that of C. angulata. These cytogenetic results substantiate the reported interspecific hybridization between C. gigas and C. angulata. In view of these results and taking into account the present expansion of C. gigas aquaculture in southern Europe, the question of the need for preservation of pure C. angulata stocks should be raised as only a few populations remain in the south of Spain and Portugal. Recently, changes in the genetic composition of populations in southern Portugal have indeed been observed, showing that human activities have created contact zones between the two taxa while no natural sympatric zones exist in Europe.     

A combined approach using RAPD,ISSR and volatile analysis for the characterization of Thymus caespititius from Flores,Corvo and Graciosa islands (Azores,Portugal)

RAPDs and ISSRs were used as molecular markers and combined with the chemical analysis of the volatiles, aiming at characterizing Thymus caespititius from the islands of Corvo, Flores and Graciosa (Azores). Thirty-one individuals were analyzed using 17 RAPD and 11 ISSRs primers, which generated respectively 199 and 127 polymorphic scorable bands. Volatiles were isolated by distillation–extraction and analyzed by GC and GC–MS. Molecular data obtained from both markers discriminated assessments from Corvo and Flores which clustered separately from plants collected in Graciosa. Volatile data grouped plants in a similar way, giving rise to two groups, depending on their oil type; plants collected on Corvo and Flores yielded carvacrol-rich volatiles while those from Graciosa yielded both carvacrol and α-terpineol-rich oils.     

Chemotaxonomy of Hypericum genus from Portugal: Geographical distribution and essential oils composition of Hypericum perfoliatum, Hypericum humifusum, Hypericum linarifolium and Hypericum pulchrum

The geographical distribution and analysis of the essential oils of species from three sections of Hypericum L. (Guttiferae/Clusiaceae/Hypericaceae) from Portugal are presented. Hypericum perfoliatum (section Drosocarpium) grows wild in the centre and south of Portugal; Hypericum humifusum and Hypericum linarifolium are both from section Oligostema, the former occurring throughout the country, while the second is distributed mainly in the north and centre; Hypericum pulchrum (section Taeniocarpium) is confined to the littoral north of Portugal. The essential oils were obtained by distillation–extraction, hydrodistillation and distillation in a modified Marcusson apparatus from the dried aerial parts of the different populations and were analysed by GC and GC–MS. Monoterpene hydrocarbons constituted the main fraction in all oils (43–69%, 53–85%, 28–45% and 48–65% for H. perfoliatum, H. humifusum, H. linarifolium and H. pulchrum, respectively). Sesquiterpene hydrocarbons (2–13%, 6–18%, 21–27% and 16–18%, respectively) and a third fraction of non-terpenic compounds (20–29%, 3–16%, 2–14% and 5–11%, respectively) from the four species attained relatively high amounts in all oils. Within each species, no major differences were detected in the essential oil composition, despite the fact that different locations, phenological phases and extraction methodologies were used. Notwithstanding the dominance of α-pinene in all four species' oils, cluster and principal components analysis on the identified components showed that the range of α-pinene, β-pinene and n-nonane supported a separation of the four species. The essential oil composition of the four species showed some qualitative resemblances, which correlate well with the taxonomical classification based on morphological characters.     

Asymmetric hybridization in Rhododendron agastum: a hybrid taxon comprising mainly F1s in Yunnan, China

Background and Aims

Rhododendron (Ericaceae) is a large woody genus in which hybridization is thought to play an important role in evolution and speciation, particularly in the Sino-Himalaya region where many interfertile species often occur sympatrically. Rhododendron agastum, a putative hybrid species, occurs in China, western Yunnan Province, in mixed populations with R. irroratum and R. delavayi.

Methods

Material of these taxa from two sites 400 km apart (ZhuJianYuan, ZJY and HuaDianBa, HDB) was examined using cpDNA and internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) loci, to test the possibility that R. agastum was in fact a hybrid between two of the other species. Chloroplast trnL-F and trnS-trnG sequences together distinguished R. irroratum, R. delavayi and some material of R. decorum, which is also considered a putative parent of R. agastum.

Key Results

All 14 R. agastum plants from the HDB site had the delavayi cpDNA haplotype, whereas at the ZJY site 17 R. agastum plants had this haplotype and four had the R. irroratum haplotype. R. irroratum and R. delavayi are distinguished by five unequivocal point mutations in their ITS sequences; every R. agastum accession had an additive pattern (double peaks) at each of these sites. Data from AFLP loci were acquired for between ten and 21 plants of each taxon from each site, and were analysed using a Bayesian approach implemented by the program NewHybrids. The program confirmed the identity of all accessions of R. delavayi, and all R. irroratum except one, which was probably a backcross. All R. agastum from HDB and 19 of 21 from ZJY were classified as F₁ hybrids; the other two could not be assigned a class.

Conclusions

Rhododendron agastum represents populations of hybrids between R. irroratum and R. delavayi, which comprise mostly or only F₁s, at the two sites examined. The sites differ in that at HDB there was no detected variation in cpDNA type or hybrid class, whereas at ZJY there was variation in both.     

Flavonoid as chemotaxonomic markers in endemic/endangered species of Rauvolfia from Southern Western Ghats of India: A preliminary study

Preliminary analysis of flavonoid chromatographic migration profiles of endemic/endangered species of Rauvolfia L from Southern Western Ghats of India were carried out. Paper chromatogram showed maximum separation in the solvent system of forestral. In the paper chromatogram, number of flavonoid spots varied from 9 to 12 in the five taxa studied. The main aglycones detected in high performance liquid chromatography (HPLC) analysis were flavones apigenin and luteolin, flavonol kaempferol, myricetin, quercetin and anthocyanidins such as delphinidin and cyanidin. Flavonol Quercetin was detected in all the five species of Rauvolfia giving a chemotaxonomic significance to its presence at the generic level. The two species Rauvolfia serpentina and Rauvolfia tetraphylla could be regarded as the most primitive in the evolutionary line with respect to the flavonoid pattern. Rauvolfia densiflora has the most advanced pattern of flavonoids. The dendrogram generated by unweighted pair group method with arithmetic average (UPGMA) cluster analysis of chemo metric data showed a clear grouping of five species in three clusters. Flavonoid profiles were efficiently used for the identification of Rauvolfia beddomei, which due to morphological similarity, was erroneously suspected to be the medicinally significant species Rauvolfia micrantha. Flavonoid profiling using paper chromatography, in the solvent system of forestral could suggest an easy and quick procedure for identifying adulteration by substitution in Rauvolfia species.     

Systematic studies in sedum section ternata (Crassulaceae)

An investigation of flavonoid compounds, pollen grains, and seed coats of the four taxa of the genusSedum sectionTernata, S. nevii, S. glaucophyllum, S. pulchellum, andS. ternatum, was undertaken to resolve the taxonomic status ofS. glaucophyllum. Flavonoid compounds were tentatively identified utilizing UV spectroscopy and shift reagents. Both flavones and fla vonols, as apigenin, luteolin, quercetin, and kaempferol aglycones and glycosides, were detected. Pollen grains and seed coats were viewed and photographed with the aid of a scanning electron microscope. Pollen grains of all four taxa were uniformly tricolporate; interspecific variation was negligible. Seed coats of all four taxa differed significantly with respect to ornamentation. Phytochemical and morphological data both support the recognition ofS. glaucophyllum as a species distinct from S.nevii.     

Multilocus sequence and microsatellite identification of intra-specific hybrids and ancestor-like donors among natural Ethiopian isolates of Leishmania donovani

Protozoan parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis is endemic in Ethiopia where it has also been responsible for fatal epidemics. It is postulated that genetic exchange in Leishmania has implications for heterosis (hybrid vigour), spread of virulent strains, resistance to chemotherapeutics, and exploitation of different hosts and vectors. Here we analyse 11 natural Ethiopian Leishmania donovani isolates consisting of four putative hybrids, seven parent-like isolates and over 90 derived biological clones. We apply a novel combination of high resolution multilocus microsatellite typing (five loci) and multilocus sequence typing (four loci) that together distinguish parent-like and hybrid L. donovani strains. Results indicate that the four isolates (and their associated biological clones) are genetic hybrids, not the results of mixed infections, each possessing heterozygous markers consistent with inheritance of divergent alleles from genetically distinct Ethiopian L. donovani lineages. The allelic profiles of the putative hybrids may have arisen from a single hybridisation event followed by inbreeding or gene conversion, or alternatively from two or more hybridisation events. Mitochondrial sequencing showed uniparental maxicircle inheritance for all of the hybrids, each possessing a single mitochondrial genotype. Fluorescence activated cell sorting analysis of DNA content demonstrated that all hybrids and their associated clones were diploid. Together the data imply that intra-specific genetic exchange is a recurrent feature of natural L. donovani populations, with substantial implications for the phyloepidemiology of Leishmania.