In our article entitled “An Aqueous Extract of Fagonia cretica Induces DNA Damage, Cell Cycle Arrest and Apoptosis in Breast Cancer Cells via FOXO3a and p53 Expression” (1), we described the effects of plant material from Pakistan that we referred to as Fagonia cretica. Linnaeus in Species Plantarum (2) described three species, namely, cretica, arabica and hispanica, while we now have over 160 names published in the genus (3, 4). The circumscription of species in Fagonia is reported to be complex due to variability of morphological characters (3, 4).
After the publication of our article it was raised to our attention that in light of the widespread distribution of F. indica and the absence from Pakistan of the more narrowly distributed species F. cretica the plant material may originate from F. indica and not F. cretica as indicated in the article. Some reports suggest that in Pakistan that the name F. cretica is used as name for what actually is F. indica var. schweinfurthii Hadidi (5). Recent studies on Fagonia elucidated relationships within the genus using morphological and molecular data (3,4). In light of this, we completed a series of DNA sequencing analyses on different batches of plant material used in our studies in order to establish if the material originated from F. indica or F. cretica. These additional analyses have confirmed that the material used in the study originated from F. cf. indica.
The methodology for the analyses was as follows: DNA was extracted from batches of plant material using a Qiagen DNeasy Plant minikit, following manufacturer’s instructions. The chloroplast trnLeu intragenic spacer was amplified using primers trnL-c and trnL-d (6), and the the rDNA Internal Transcribed Spacer region was amplified using primers ITS4 and ITS5 (7). Amplification, purification and sequencing were performed as described by Houston and Wolff (8). Sequencing was carried out with the Big Dye Terminator cycle sequencing kit v. 3.1 (Applied Biosystems) according to manufacturer’s instructions. Sequencing products were purified using a Genetix column purification and the sequences of these samples were visualised using an ABI 3100 automated sequencer (Applied Biosystems).
The resulting sequences, both trnL (439 bases) and ITS (489 bases), were blasted to sequences available on GenBank and showed the highest similarity with those of F. indica and of F. paulayana, while being clearly different from those for F. cretica (Table 1). The alignment shows that for trnL the query sample differed from F. indica only for the number of repeats in two A-repeats, and one nucleotide substitution with F. paulayana. The trnL sequence differed from the F. cretica sequences for 12 substitutions and 6 insertions/deletions (Table 2). The ITS query sample differed from F. indica and F. paulayana for 1 and 3 substitutions, respectively, while the difference with F. cretica was 36 substitutions and 1 insertion/deletion (Table 3). The alignment of ITS showed ambiguities at certain positions; this is most likely due to a well-known phenomenon of incomplete sequence conversion between the rDNA paralogs, causing intra-individual sequence diversity. Beier et al. (4) describe that F. indica and F. paulayana occur sympatrically and cannot be distinguished on the basis of sequence data, but that a single morphological character (persistence of sepals) discriminates the two species.
Table 1. Sequence similarity of our query sample and most similar sequences available at Genbank for gene for trn-Leu and rDNA Internal Transcribed Spacer.
| trn Leu | Internal transcribed spacer rDNA | ||||
| Species | Similarity | Genbank nr | Species | Similarity | Genbank nr |
| F. indica | 99% | AY641593.1 | F. indica | 99% | AY641631.1 |
| F. indica | 99% | AY300769.1 | F. paulayana | 99% | AY641652.1 |
| F. indica | 99% | AY641592.1 | F. indica | 99% | AY641630.1 |
| F. paulayana | 99% | AY641607.1 | F. subinermis | 99% | AY641642.1 |
| F. paulayana | 98% | AY641606.1 | F. paulayana | 99% | AY641650.1 |
| F. subinermis | 98% | AY641610.1 | F. indica | 99% | AY641632.1 |
| F. mahrana | 96% | AY641600.1 | F. paulayana | 99% | AY641654.1 |
| F. lahovarii | 96% | AY641596.1 | F. mahrana | 98% | AY641639.1 |
| F. hadramautica | 95% | AY641590.1 | F. gypsophila | 98% | AY641627.1 |
| F. gypsophila | 95% | AY641589.1 | F. lahovarii | 97% | AY641635.1 |
| F. acerosa | 95% | AY641579.1 | F. latistipulata | 97% | AY641636.1 |
| F. indica | 95% | AY300770.1 | F. scabra | 97% | AY641645.1 |
| F. harpago | 95% | AY641591.1 | F. charoides | 97% | AY641621.1 |
| F. longispina | 94% | AY641599.1 | F. minutistipula | 96% | AY641641.1 |
| F. latistipulata | 94% | AY641598.1 | F. longispina | 96% | AY641637.1 |
| F. bruguieri | 94% | AY641582.1 | F. glutinosa | 96% | AY641626.1 |
| F. minutistipula | 93% | AY300771.1 | F. bruguieri | 96% | AY641619.1 |
| F. cretica | 92% | AJ387942.1 | F. olivieri | 96% | AY641646.1 |
| F. densa | 92% | AY641587.1 | F. mollis | 95% | AY641643.1 |
| F. laevis | 92% | AY641594.1 | F. bruguieri | 95% | AY641620.1 |
| F. villosa | 92% | AY641611.1 | F. rangei | 95% | AY641647.1 |
| F. mollis | 92% | AY641601.1 | F. harpago | 95% | AY641629.1 |
| F. laevis | 92% | AY641595.1 | F. acerosa | 95% | AY641617.1 |
| F. scabra | 92% | AY300768.1 | F. luntii | 95% | AY641638.1 |
| F. charoides | 91% | AY641583.1 | F. laevis | 95% | AY641634.1 |
| F. orientalis | 91% | AY641603.1 | F. laevis | 95% | AY641633.1 |
| F. rangei | 91% | AY641609.1 | F. palmeri | 95% | AY641653.1 |
| F. cretica | 91% | AY641585.1 | F. orientalis | 94% | AY641648.1 |
| F. arabica | 91% | AY641580.1 | F. boveana | 94% | KF850598.1 |
| sF. cretica | 91% | AY641586.1 | F. pachyacantha | 94% | AY641651.1 |
| F. pachyacantha | 90% | AY641604.1 | F. pachyacantha | 94% | AY641649.1 |
| F. pachyacantha | 90% | AY300772.1 | F. villosa | 94% | AY641640.1 |
| F. zilloides | 90% | AY641612.1 | F. hadramautica | 94% | AY641628.1 |
| F. arabica | 93% | AY641618.1 | |||
| F. chilensis | 93% | AY641622.1 | |||
| F. densa | 93% | AY641625.1 | |||
| F. cretica | 93% | AY641624.1 | |||
| F. scoparia | 90% | AY641644.1 | |||
| F. cretica | 90% | AY641623.1 | |||
Table 2. Aligned sequences of the trn-Leu gene intron for our query sample and a selection of the most similar sequences and most distant sequences within the genus Fagonia available at Genbank.
| 10 | 20 | 30 | 40 | 50 | 60 | |
| trnL intron query | GTGATCACTT | TCAAATTCAG | AGAAACCCTG | GAATTAGAAA | TGGGCAATCC | TGAGCCAAAT |
| AJ387943 F.indica | GTGATCACTT | TCAAATTCAG | AGAAACCCTG | GAATTATAAA | TGGGCAATCC | TGAGCCAAAT |
| AY641592 F.indica | –––––––CTT | TCNAATTCAG | AGAANCCCTG | GAATTAGAAA | TGGGCAATCC | TGAGCCAAAT |
| AY641608 F.paulayana | GTGATCACTT | TCAAATTCAG | AGAAACCCTG | GAATTAGAAA | TGGGCAATCC | TGAGCCAAAT |
| AY641610 F.subinermi | GTGATCACTT | TCAAATTCAG | AGAA-CCCTG | GAATTASAAA | TGGGCAATCC | TGAGCCAAAT |
| AY641600 F.mahrana | GTGATCACTT | TCAAATTCAG | AGAAACCCTG | GAATTAGAAA | TGGGCAATCC | TGAGCCAAAT |
| AY641596 F.lahovarii | ––GATCACTT | TCAAATTCAG | AGAAACCCTG | GAATTAGAAA | TGGGCAATCC | TGAGCCAAAT |
| AJ387942 F.cretica | GTGATCACTT | TCAAATTCAG | AGAAACCCTG | GAATTAGAAA | TGGGCAATCC | TGAGCCAAAT |
Table 3. Aligned sequences of the rDNA Internal Transcribed Spacer for our query sample and a selection of the most similar sequences and most distant sequences within the genus Fagonia available at Genbank.
| 10 | 20 | 30 | 40 | 50 | 60 | ||
| query | ITS | AGAGCATACC | CCTTCCTCGA | GTGTCGGGAG | GGAGACTTCC | TGACATTATA | ACGAACCCCG |
| AY641631 | F.indica | AGAGCATACC | CCTTCCTCGA | GTGTCGGGAG | GGAGACTTCC | TGACATTATA | ACGAACCCCG |
| AY641630 | F.indica | AGAGCATACC | CCTTCCTNGA | GTGTCGGGAG | GGAGACTTCC | NGACATTATA | ACGAACCCCG |
| AY641632 | F.indica | AGAGCATACC | CCTTCCTCGA | GTGTCGGGAG | GGAGACTTCN | TGACATTATA | ACGAACCCCG |
| AY641652 | F.paulayana | AGAGCATACC | CCTTCCTCGA | GTGTCGGGAG | GGAGACTTCC | TGACATTATA | ACGAACCCCG |
| AY641650 | F.paulayana | AGAGCATACC | CCTTCCTCGA | GTGTCGGGAG | GGAGACTTCC | TGACATTATA | ACGAACCCCG |
| AY641654 | F.paulayana | AGAGCATACC | CCTTCCTCGA | GTGTCNGGAG | GGAGACTTCN | TGACATTATA | ACGAACCCCG |
| AY641642 | F.subinermi | AGAGCATACC | CCTTCCTCGA | GTGTCAGGAG | GGAAACTTCC | TGACATTATA | ACGAACCCCG |
| AY641639 | F.mahrana | AGAGCATGCC | CCTTCCTCGA | GTGTCGGGAG | GGAGACTTCN | TGACATCATA | ACGAACCCCG |
| AY641627 | F.gypsophil | AGAGCATACC | CCTTCCTCGA | GTGTCAGGAG | GGAGACTTCC | TGACATTATA | ACGAACCCCG |
| AY641635 | F.lahovarii | --AGCATGCC | CCTTCCTCGA | GTGTTGGGAG | GGAGACTTCC | CGACATCATA | ACGAACCCCG |
| AY641636 | F.latistipu | AGAGCATGCC | CCTTCCTCGA | GTGTTGGGAG | GGAGACTTCC | CGACATCATA | ACGAACCCCG |
| AY641641 | F.minutisti | AGAGCATGCC | CCTTCCTCGA | GTGTCGGGAG | GGAGACTTCC | CGACATCATA | ACGAACCCCG |
| AY641624 | F.cretica | AGAGCATGCC | CCTTCATCGA | GTGTTGAGAG | GGAGACCTCT | CGACATCATA | ACGAACCCCG |
| AY641623 | F.cretica | AGAGCATGCC | CCTTCATCGA | GTGTTGAGAG | GGAGACCTCT | CGACATCATA | ACGAACCCCG |
We conclude that F. indica or its sister species F. paulayana is the genetic identity of the plant material being commonly referred to as F. cretica in Pakistan and which has cytotoxic activity towards breast cancer cells. We conclude that it is more likely to be F. indica than F. paulayana for three reasons. Firstly, our ITS sequence was more similar to that of F. indica. Secondly, the trnL sequence only had repeat number differences with F. indica, which is a more likely evolutionary step than the substitution that was found in the comparison with F. paulayana. Thirdly, the distribution of F. indica encompasses the region of origin of our samples. The article title, abstract and main text should therefore refer to F. cf. indica and not F. cretica. The active compound has now been isolated and is undergoing molecular characterization.
We are grateful to Dr. Schori, an expert plant systematist, who alerted us to the likely misidentification of the species.
Kirsten Wolff was responsible for the additional DNA analyses described in this Correction and she has been added as a co-author. The author list should therefore be revised to read as follows:
Matt Lam, Kirsten Wolff, Helen Griffiths, Amtul Carmichael.
Dr. Wolff is affiliated at School of Biology, Newcastle University and has no competing interests in relation to this work.
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