In the wild hybridization of annual Datura species as unveiled by morphological and molecular comparisons
© Tsialtas et al.; licensee BioMed Central Ltd. 2014
Received: 6 March 2014
Accepted: 10 June 2014
Published: 23 June 2014
The present work aimed to verify whether intermediate variants were natural crosses between Datura species (D. stramonium forms and D. ferox). Their existence has been long ago insinuated but has not been studied using morphological features and molecular tools. The variants differed in stem coloring, upper bearing forks, and fruit characters.
Principal Components Analysis of 11 morphological characteristics showed that D. ferox and D. stramonium (forms stramonium and tatula) were quite different and the putative hybrids were intermittent. The D. ferox × D. stramonium f. tatula was closer to the latter of its parents. Sequencing analysis revealed identical amplified trnL intron in all variants and a 100% homology with D. stramonium accession number EU580984.1 suggested that this plastid cannot discern Datura variants. However, genomic analysis with URP markers indicated that the hybrids had >60% genetic makeup similarity with both parents suggesting that the intermediate variants were putative inter-specific hybrids. Moreover, the dendrogram stemmed from cluster analysis of the fingerprint profile of variants placed D. stramonium and D. ferox in different branches indicating their genetic differentiation from each other as well as from their hybrids.
The findings suggest that the natural hybridization of annual Datura species occurs. Extrapolating, this hybridization could be the first step for speciation. More possibly, it can alter population composition, its weediness and adaptability to local conditions.
KeywordsFierce thornapple Hybrids Jimsonweed Solanaceae Self fertilization
Genus Datura, family Solanaceae, consists of nine (annual and tree) species, originating from the New and Old World . In Greece, Datura species are considered to be invasive , known since antiquity for their narcotic and medicinal actions [3, 4]. Nowadays in Greece, D. stramonium L. forms (f. stramonium and f. tatula), D. ferox L., and D. innoxia L. coexist in mixed populations in various combinations and relative ratios. Datura stramonium f. stramonium is the most common variant found as spring weed in fields, roadsides and dumps and usually coexists with the recently identified to occur in Greece D. stramonium f. tatula L. . Fierce thornapple (D. ferox L.) is the dominant Datura species in some sites in northern Greece where it shares habitat with D. stramonium. Finally, D. innoxia, commonly used as ornamental, is found as feral, usually, in dumps .
Datura stramonium is a predominantly self-fertilized species but cross-pollination is feasible to some extent by insects like hawkmoths and honeybees . The predominance of self-fertilization is ascribed to anther-stigma overlapping and results in inbreeding [7, 8], which found to reduce vigor and increase herbivory damages . However, within Datura populations, there are plants showing herkogamy (anther-stigma separation), which permits outcrossing at low rates ranging from 1.3% to 8.5% [6, 10]. The existence of anthocyanin in the purple-stemmed, violet-flowered D. stramonium f. tatula does not affect outcrossing rates .
In tree Datura species, inter-specific crossing is feasible (D. aurea × D. candida) descending hybrids with increased alkaloid content, which could be of economic interest . Regarding annual species, Husaini & Iwo  reported the existence of cytological compatibility between D. stramonium and D. ferox. Weaver & Warwick , reporting the findings of Rietsema , stated that these two Datura species are the only annual species, which gave identified hybrids in the wild, collected in South America. Since then, no relative report exists, making morphological and molecular confirmation of analogous findings necessary. Given that crosses between the annual Datura species yielded viable seeds , possible outcrossing in mixed communities would diversify the existing populations to new ones, with unpredicted features regarding competitiveness, resistance to herbivores, alkaloid content and herbicide tolerance.
During September 2011, in mixed Datura swards in northern Greece, specimens were found showing morphological features intermediate to those of the coexisting D. stramonium forms and D. ferox. Thus, the aim of this study was to verify naturally occurring Datura crosses and unmask the possible hybridization by morphological features and molecular tools.
Means (±standard errors) for the characteristics determined in situ in the five Datura variants
D. stramonium f. stramonium
D. stramonium f. tatula
D. ferox × f. stramonium
D. ferox × f. tatula
PCA results for the selected characteristics of 30 Datura individuals
Long spine length (LSL)
Medium spine length (MSL)
Short spine length (SSL)
Leaf width (LW)
Capsule length (CL)
Capsule width (CW)
Leaf length (LL)
The PC1 was mainly correlated (positively or negatively) with long spine length, medium spine length, spines/capsule, short spine length, capsule length, and leaf length (Table 2). The PC2 was mainly loaded (positively or negatively) by the purple color, leaf width, length/width, and capsule width. The green color had almost the same negative loading on both components.
Characteristics of the bands amplified using 12 URP primers in the examined variants
No of bands
Molecular data proximity matrix constructed from the Jaccard's similarity coefficients for the variants
Jaccard similarity coefficients
The sequencing analysis revealed identical amplified trnL intron in all variants. BLAST similarity search of the GenBank for homologous sequences revealed that a sequence of D. stramonium with accession number EU580984.1 had 100% homology over the 505 nucleotide amplified region (data not shown).
In the wild, inter-specific hybridization is a common phenomenon especially among plant species and may become the first step in the process of speciation [16, 17]. Sympatric coexistence, intermediate characters, inter-fertility, and biochemical additivity are criteria determining the feasibility of inter-specific hybridization , and several of them were met in the present study.
The first clue for this work was the intermediate characters of specimens found to coexist with their putative parents (D. ferox and D. stramonium). Despite the high percentage of self-fertilization, annual Datura species are cytological compatible , show herkogamy [6, 10] and attract insect pollinators . In particular, under Greek conditions, f. tatula is highly attractive for honeybees .
The possible existence of hybrids of annual Datura species in the wild goes back to 1950’s . Nevertheless, exempting its insinuation, no proof has been provided. Nowadays, a combination of morphological and molecular characters is used to provide evidence for the existence of a putative hybrid [19–23]. Selecting an adequate number, in statistical terms (six specimens per variant), of a coherent plant cohort, we found out that the Datura variants were mainly different in stem coloring, upper bearing forks, and fruit characters that were determined. PCA revealed that D. ferox and f. stramonium were different and their putative hybrid was intermediate. The other putative hybrid, D. ferox × f. tatula, was closer to the latter of its putative parents. However, morphological intermediates can also be derived from convergent evolution or environmental selection and this makes ambiguous the confirmation of putative hybrid individuals based solely on morphological evidence. Use of molecular methods provides a number of advantages over morphological analysis, among others being the large number of available markers, their apparent selective neutrality, and the low levels of non-heritable variation . Ideally, additive molecular markers displaying co-dominant inheritance (e.g. SSR), combined with morphological analysis, should be applied to decipher putative hybridization. The lack of such SSR markers for Datura species makes their development cumbersome and time-consuming. Nevertheless, genetic analyses by dominant markers, like RAPDs and AFLPs, solitary or flanked by morphological analysis, have been routinely employed with ultimate success in inter-specific hybrid verification [25–27].
In the present work, genetic distances of the examined variants were estimated using URP primers, which have been proved valuable in genomic fingerprinting of a variety of organisms including plants, animals, and microorganisms . The URP markers were selected for their advantages over RAPDs (high annealing temperature leading to high specificity and reproducibility) and AFLPs (less laborious and equally specific) in genetic analysis. The applied URP primers were employed for first time in Datura species genetic fingerprinting, and proved to be successful in amplification of polymorphic fragments that are sufficient to discriminate their phylogenetic origin.
The fact that the sequencing analysis revealed identical amplified trnL intron in all variants and a 100% homology with D. stramonium accession number EU580984.1  indicates that the typically used plant plastid phylogenetic marker trnL intron is not useful for discrimination, since it is identical between D. stramonium and D. ferox and, consequently, in their putative inter-specific hybrids.
The molecular characterization of the different Datura accessions provided the most compelling evidence on the genetic relationships and genetic makeup of the putative inter-specific hybrids. Using Jaccard’s coefficient for comparison, the similarities determined between the bands of the examined variants bolstered the possibility of putative hybrids (D. ferox × f. stramonium and D. ferox × f. tatula) to be indeed hybrids between annual Datura species. Datura ferox was equally related to both of the putative hybrids with 62% similarity, while D. stramonium was somewhat more similar to D. ferox × f. stramonium (67%) than to D. ferox × f. tatula (60%). The two species were similar at a level of 46% while D. ferox × f. stramonium and D. ferox × f. tatula share 86% similarity. In accordance, Dymshakova et al. , using genetic distances estimated by AFLPs, showed that F1 hybrids were intermediate between the parentals Saxifraga sibirica and S. cernua. It is clear from the results that the putative hybrids were highly similar to each other, meaning that share common ancestry, and a high percentage of their genetic makeup is equally similar to both D. stramonium and D. ferox at a level higher than 60%, and hence those were possibly their progenitors. This evidence supports the hypothesis that the two intermediate accessions may be inter-specific hybrids between D. ferox and D. stramonium.
The natural hybridization of annual Datura species confers putative implications in biological and agronomic terms. In extreme, it could be the first step for speciation but more possibly, it could change local population composition, which in turn could affect Datura weed competitiveness and its susceptibility to chemical or mechanical control. Finally, this hybridization could raise alkaloid content leading to commercial interest of its extraction.
Site description and morphological measurements
During September 2011, mixed swards of Datura species were identified at the locales of Eukarpia (40°32′N, 22°60′E, 14 m a.s.l.) and Agios Demetrios (40°53′N, 23°41′E, 66 m a.s.l.) in Serres region, northern Greece. The predominant D. ferox was coexisting with the typical green-stemmed, white-flowered D. stramonium f. stramonium (f. stramonium) and the purple-stemmed, violet-flowered D. stramonium f. tatula (f. tatula). The swards emerged postharvest, after erratic summer rains, in winter cereal fields. The preceding crop was cotton, the spring crop where D. ferox mainly resides. The soil of the fields in the wider area was derived from alluvial deposits and the climate is Mediterranean, with dry and hot summers.
Plant material sampling and molecular determinations
The plant samples included Datura ferox (F), D. stramonium forms (S), and their putative inter-specific hybrids (D. ferox × f. tatula: A and D. ferox × f. stramonium: B).
Upper forks of each specimen were selected, sealed in a plastic bag, put in a portable refrigerator and transferred to the laboratory for DNA extraction, which was performed from 100 mg ground leaf tissue using the cetyl trimethylammonium bromide (CTAB) method according to the protocol outlined in the NucleoSpin®Plant II kit (Macherey Nagel GmbH & Co. KG, Düren, Germany).
Oligonucleotide primers and PCR conditions for fingerprinting and analysis of the cpDNA trnL intron
Characteristics of 12 URP and the P1 and P2 primers used for amplifying trnL intron
For amplification of the cpDNA trnL intron, the PCR primes P1 and P2 were used (Table 5). PCR setup and instruments were as above. Amplification profile was: one cycle of 2 min at 94°C; 10 cycles of 30 sec at 94°C, 30 sec at 58°C (touchdown: -0.5°C per cycle), 50 sec at 72°C; 30 cycles of 30 sec at 94°C, 30 sec at 53°C, 50 sec at 72°C; one cycle of a final extension for 10 min at 72°C. DNA fragments were detected by staining with ethidium bromide on a 3% MetaPhor™ agarose (Cambrex Bio Science, Copenhagen, Denmark) gel in TBE buffer. The bands of the trnL intron were excised from the gel, DNA was recovered using a modified freeze-squeeze method  and sent for direct sequencing with the primers used in PCR.
Statistical analysis of morphological characteristics
With the exception of corolla and calyx lengths in D. ferox × f. tatula for which measurements were not taken due to the lack of appropriate samples, morphological trait means and standard errors were computed for summarizing the distributions of the corresponding variables. On the basis of morphological data (except stem colour), the five variants were compared with the Analysis of Variance (ANOVA) method. The Duncan’s multiple range test was used for means’ comparisons.
Principal Components Analysis (PCA) with varimax rotation was applied on the correlation matrix between the morphological variables (except corolla and calyx lengths) in order to study the groupings, similarities, and differences between the individuals of the five variants. Significant components were determined by the parallel analysis (PA) method . Since the stem colour was a nominal categorical variable, with three categories (purple, green, and grey-green), two dummy variables with binary coding (0, 1) were entered in the PCA; one variable for the purple colour and one for the green. A third dummy variable for the grey-green colour is redundant since it would be dependent on and negatively correlated with the other two dummy variables. Generally, the number of dummy-coded variables needed is one less than the number of modalities of the corresponding categorical variable. Since PCA is not a modelling but only a descriptive variance summarizing method, the use of binary along with scale variables is legitimate . The significance level for all hypotheses testing procedures was preset at p < 0.05. IBM SPSS package v. 20 (IBM Corp., New York, USA) was used for the analyses. Parallel analysis was conducted with the RanEigen v. 2.0 software .
Analysis of molecular data
Gel photographs were scored using the gel image analysis software GelAnalyzer 2010a (http://www.gelanalyzer.com/). The bands were binary coded with 1 or 0 for their presence or absence in each genotype, respectively, and the coded data were subjected to statistical analysis. Estimates of similarity among all genotypes were calculated from the Jaccard’s similarity coefficient using the IBM SPSS package v. 20 (IBM Corp., New York, USA). Hierarchical cluster analysis based on the Jaccard similarity matrix with the unweighted pair group method based on arithmetic averages (UPGMA)  was conducted using the software MEGA5 .
Amplified fragment length polymorphism
Analysis of variance
Cetyl trimethylammonium bromide
Long spike length
Medium spike length
Principal components analysis
Polymerase chain reaction
Random amplified polymorphic DNA
Short spike length
Simple sequence repeat
Unweighted pair group method based on arithmetic averages.
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