Can phylogeny predict chemical diversity and potential medicinal activity of plants? A case study of amaryllidaceae

  • Nina Rønsted1Email author,

    Affiliated with

    • Matthew R E Symonds3,

      Affiliated with

      • Trine Birkholm2,

        Affiliated with

        • Søren Brøgger Christensen2,

          Affiliated with

          • Alan W Meerow4, 5,

            Affiliated with

            • Marianne Molander2,

              Affiliated with

              • Per Mølgaard2,

                Affiliated with

                • Gitte Petersen1,

                  Affiliated with

                  • Nina Rasmussen2,

                    Affiliated with

                    • Johannes van Staden6,

                      Affiliated with

                      • Gary I Stafford1 and

                        Affiliated with

                        • Anna K Jäger2

                          Affiliated with

                          BMC Evolutionary Biology201212:182

                          DOI: 10.1186/1471-2148-12-182

                          Received: 10 May 2012

                          Accepted: 7 September 2012

                          Published: 14 September 2012

                          Abstract

                          Background

                          During evolution, plants and other organisms have developed a diversity of chemical defences, leading to the evolution of various groups of specialized metabolites selected for their endogenous biological function. A correlation between phylogeny and biosynthetic pathways could offer a predictive approach enabling more efficient selection of plants for the development of traditional medicine and lead discovery. However, this relationship has rarely been rigorously tested and the potential predictive power is consequently unknown.

                          Results

                          We produced a phylogenetic hypothesis for the medicinally important plant subfamily Amaryllidoideae (Amaryllidaceae) based on parsimony and Bayesian analysis of nuclear, plastid, and mitochondrial DNA sequences of over 100 species. We tested if alkaloid diversity and activity in bioassays related to the central nervous system are significantly correlated with phylogeny and found evidence for a significant phylogenetic signal in these traits, although the effect is not strong.

                          Conclusions

                          Several genera are non-monophyletic emphasizing the importance of using phylogeny for interpretation of character distribution. Alkaloid diversity and in vitro inhibition of acetylcholinesterase (AChE) and binding to the serotonin reuptake transporter (SERT) are significantly correlated with phylogeny. This has implications for the use of phylogenies to interpret chemical evolution and biosynthetic pathways, to select candidate taxa for lead discovery, and to make recommendations for policies regarding traditional use and conservation priorities.

                          Keywords

                          Amaryllidaceae Phylogeny Chemical diversity Prediction Lead discovery

                          Background

                          During evolution, plants and other organisms have developed a diversity of chemical defence lines, leading to the evolution of various groups of specialized metabolites such as alkaloids, terpenoids, and phenolics, selected for their endogenous biological function [17]. Intuitively, a correlation between phylogeny and biosynthetic pathways is sometimes assumed [1, 810] and could offer a predictive approach enabling deduction of biosynthetic pathways [6, 1115], defence against herbivores [16, 17], more efficient selection of plants for the development of traditional medicine and lead discovery [1822] as well as inform conservation priorities [23].

                          Several studies have confirmed the usefulness of specialized metabolites such as glucosinolates, iridoids, sesquiterpene lactones, flavonoids, and phenolics to support molecular based phylogenies contradicting morphologic patterns [11, 12, 2429]. On the contrary, several studies have found inconsistency of specialized metabolite profiles at various taxonomic levels and indicated that specialized chemistry and anti-herbivore defence syndromes tend to be poorly correlated with plant phylogeny [6, 7, 13, 30].

                          Lack of congruence between specialized chemistry and phylogeny may be caused by several different phenomena. One contributing factor is convergent evolution by which the same or similar traits originate independently in taxa that are not necessarily closely related, often in response to similar environmental challenges [17, 31]. A striking example of convergent evolution is the common use of the sex pheromone (Z)-7-dodecen-l-yl acetate by over 120 species of primarily Lepidopteran insects and female Asian elephants, Elephas maximus[32]. In relation to plants, well known convergent morphological adaptations are the occurrence of prickles, thorns, and spines, which have evolved to avoid or limit herbivory [33], succulence as adaptation to dry environments in both North American Cactaceae and African Euphorbia[34, 35], and insectivorous plants, which have evolved several times in response to a nitrogen-deficient environment [36]. Likewise, chemical defence lines may also have arisen independently in unrelated taxa, and convergent evolution in plant specialized metabolism appears to be surprisingly common [6, 17, 31, 37]. For example, the ability to produce cyanogenic glycosides appears to have evolved independently in many different plant families [17, 31].

                          However, convergent evolution can be difficult to verify because absence of evidence is not evidence of absence and it is possible that some compounds presently considered to be limited to some lineages are indeed more universally found in plants [31]. Specialized compounds are not continuously expressed, but may be produced as a response to herbivory or other damage, the expression may also be dependent on the environment [38] and plants often use a combination of several defensive traits [7, 17]. In addition, chemosystematic data are scattered in the literature and negative results are often not reported. Absence or presence of a compound is also dependent on the amount of plant material investigated as well as the detection limit of the analytical methods [27, 39]. Finally, the existence of several different phytochemical methods can cause inconsistence in the results reported in the literature.

                          Nevertheless, reports of incongruence between phytochemistry and phylogeny have questioned the degree of correlation between phylogeny and specialized metabolites, indicating that such a correlation cannot simply be assumed [6]. However, this relationship has rarely been tested because of lack of accurate estimates of phylogeny and corresponding chemical data; lack of tradition for interdisciplinary studies bridging botany, chemistry, and molecular systematics; and appropriate statistical tools. Consequently, the potential predictive power is unknown [17]. In the present study, we use Amaryllidaceae subfamily Amaryllidoideae as a model system for testing the correlation between phylogenetic and chemical diversity and biological activity.

                          Amaryllidaceae subfamily Amaryllidoideae sensu APG III [40] (formerly recognized as a separate family, Amaryllidaceae J.St.-Hil.) is a widely distributed subfamily of 59 genera and about 850 species. Amaryllidoideae has centres of diversity in South Africa, South America, particularly in the Andean region, and in the Mediterranean, three of the recognized hotspots of biodiversity on Earth [41, 42] (http://​www.​biodiversityhots​pots.​org). Plants of the Amaryllidoideae are used in traditional medicine to treat mental problems, primarily in Southern Africa [43, 44]. The traditional use of plants of Amaryllidoideae has been related to their unique and subfamily specific alkaloid chemistry (Figure 1). Over 500 alkaloids have been described from various species and have been subdivided into 18 major types based on hypothetical biosynthetic pathways [4547]. Extracts or isolated alkaloids of Amaryllidaceae species have shown activity in vitro in a range of assays related to disorders of the central nervous system, primarily Alzheimers disease (inhibition of acetylcholinesterase, AChE) [21, 4851], and anxiety and depression (affinity to the serotonin re-uptake transporter, SERT) [21, 52, 53]. Galanthamine is registered in a number of countries as an AChE inhibitor (Reminyl or Rezadyne; Janssen Pharmaceutica) [54]. Another Amaryllidaceous alkaloid, sanguinine (9-O-demethylgalanthamine), is shown to be a ten times more potent inhibitor of AChE than galanthamine in vitro[55].
                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-182/MediaObjects/12862_2012_2122_Fig1_HTML.jpg
                          Figure 1

                          Alkaloid types recovered in the present study. Alkaloids were classified to type according to the hypothetical biosynthetic pathways proposed by Jin [45, 46]. Marvin was used for drawing and displaying chemical structures, Marvin 5.9.0, 2012, ChemAxon (http://​www.​chemaxon.​com).

                          Amaryllidaceae subfamily Amaryllidoideae is therefore an ideal model system for comparing phylogenetic and chemical diversity with bioactivity. Previous molecular phylogenetic studies based on plastid gene regions (rbcL, trnLF, and ndhF) have confirmed Amaryllidoideae as monophyletic and resolved many taxa into geographically confined monophyletic groups [42, 56]. The African tribe Amaryllideae has been well supported as sister group to the remaining taxa. However, the relationship among several other early diverging lineages, in particular the African tribes Haemantheae and Cyrtantheae, and the Australasian Calostemmateae are not well supported by previous studies and remain problematic [42]. In a study by Meerow and Snijman [42] based on parsimony analysis of plastid ndhF sequences, Amaryllideae also resolved as sister to the remainder of the subfamily. The next major split resolved a clade with American and Eurasian subclades, and an African/Australasian clade with Cyrtantheae as sister to a Haemantheae/Calostemmateae clade. However, this African/Australasian clade was not supported by bootstrap analysis.

                          The objectives of the present study were: (1) to produce a comprehensive and well supported phylogenetic hypothesis of Amaryllidaceae subfamily Amaryllidoideae based on total evidence from DNA regions from all three plant genomes; (2) to test for correlation between phylogenetic and chemical diversity and central nervous system (CNS) related activities.

                          Results

                          Phylogeny

                          The ITS region was the most informative region followed by the matK region (Table 1). The trnLF and the nad1 regions resolved 10% or less of the clades with strong support defined as ≥ 90% bootstrap support. There were no strongly supported conflicts among any regions (Bayesian consensus tree with posterior probabilities and parsimony bootstrap consensus tree for the total evidence analysis and bootstrap consensus trees of individual regions are provided as Additional file 1: Figures S1-S7). The number of resolved clades (87%; Table 1) and the number of resolved clades supported by  90% bootstrap (62%) was highest in the total evidence analysis, which was also the only analysis that resolved all the major lineages. The Bayesian analysis of the total evidence matrix provided the same overall topology as parsimony analysis and all major clades were strongly supported by Bayesian analysis (Figure 2; Additional file 1: Figures S1 and S2).
                          Table 1

                          Details of the matrices included in this study

                          Matrix

                          # taxa

                          # aligned characters

                          # of PPI1(%)

                          # MP trees

                          Length of MP trees

                          CI2

                          RI3

                          Percent clades with > 50% BS4

                          Percent clades with ≥ 90% BS4

                          ITS

                          105

                          953

                          502 (53)

                          6520

                          2537

                          0.42

                          0.82

                          78

                          55

                          trnLF

                          106

                          1163

                          185 (16)

                          470

                          601

                          0.70

                          0.86

                          33

                          10

                          matK

                          105

                          2019

                          295 (15)

                          9940

                          922

                          0.74

                          0.90

                          67

                          39

                          Plastid combined

                          107

                          3182

                          480 (15)

                          3620

                          1544

                          0.71

                          0.88

                          76

                          45

                          nad1

                          104

                          1726

                          53 (3)

                          8330

                          275

                          0.79

                          0.97

                          28

                          7

                          Total evidence

                          109

                          5861

                          1086 (19)

                          554

                          4454

                          0.53

                          0.85

                          87

                          62

                          1PPI: potentially parsimony informative characters. 2CI: Consistency index. 3RI: Retention index. 4Percent of resolved clades in the Bootstrap consensus tree with > 50% BS (bootstrap support) and with  90% BS are proportions of the possible number of clades (# taxa −1).

                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-182/MediaObjects/12862_2012_2122_Fig2_HTML.jpg
                          Figure 2

                          Phylogenetic hypothesis for Amaryllidaceae subfamily Amaryllidoideae. Obtained after 1,000,000 replicates of Bayesian inference. Parsimony bootstrap percentages and Bayesian posterior probabilities (BS/PP) are indicated for major clades only. Examples of members are illustrated on the right hand side.

                          The topology (Figure 2) of the total evidence analysis largely supports previous studies [42, 56]. The African tribe Amaryllideae (100% BS; PP = 1.00) is sister to the remainder of the Amaryllidoideae (100% BS; PP = 1.00), and this is strongly supported by all analyses. The next major split resolves an American clade (66% BS; PP = 0.99) and a Eurasian clade (65% BS; PP = 1.00) as sisters (100% BS; PP = 1.00) and a clade (66% BS; PP = 1.00) with the African monogeneric tribe Cyrtantheae (100% BS; PP = 1.00), and tribe Haemantheae (100% BS; PP = 1.00) as sisters (100% BS; PP = 1.00), and the Australasian tribe Calostemmateae (100% BS; PP = 1.00) as sister to these.

                          In the ITS analysis ( Additional file 1: Figure S3), tribe Amaryllideae (100% BS) is sister to the remainder of Amaryllidoideae (61% BS). Within the remainder of Amaryllidoideae, tribe Calostemmateae (100% BS) is sister to a clade (54% BS) including tribes Cyrtantheae (100% BS), Haemantheae 95% BS) and the American and European Amaryllidoideae. Tribes Cyrtantheae and Haemantheae are sisters (96% BS). In the combined plastid analysis ( Additional file 1: Figure S6), Cyrtantheae is sister to the remainder of Amaryllidoideae except tribe Amaryllideae (75% BS). In both the matK ( Additional file 1: Figure S4 supporting online material) and the combined plastid analysis tribe Calostemmateae is sister to tribe Haemantheae (matK: 70% BS; plastid: 94% BS).

                          The low bootstrap support (65%) for the Eurasian clade in the total evidence analysis (Figure 2, Additional file 1: Figure S2) may be caused by uncertainty in the placement of the genus Lycoris. The remainder of the Eurasian clade is strongly supported in all analyses except trnLF and nad1, which are the two regions providing the least resolution and support in general ( Additional file 1: Figures S3-S7 in the supporting online material).

                          Relationship of phylogeny to chemistry and bioactivity

                          The relationship of individual types of compounds and biological activity could be assessed using the D metric developed to deal with discrete binary characters [57]. D is equal to 1 if the observed chemical component has a random distribution (i.e. no phylogenetic signal). D is equal to 0 if the component is distributed exactly as would be predicted under a Brownian motion model of gradual divergent evolution (i.e. strong phylogenetic signal). See Materials and Methods for details. Of the seven alkaloid types which are found in more than one species we found evidence for some phylogenetic signal of five types (Table 2a) [57]. With the exception of tazettine and galanthindole, D values were significantly different from that expected under a random distribution of the components across the phylogenetic tree (D = 1). Inhibition of acetylcholinesterase (AChE) and binding to the serotonin re-uptake transporter (SERT) were used as proxies for biological activity. Both measures of bioactivity, AChE activity and SERT activity also displayed significant phylogenetic signal (Table 2b). However, in none of the above cases was the phylogenetic signal sufficiently strong to be considered as indistinguishable from a Brownian motion model of evolution, where traits are strongly ‘clumped’ on the phylogeny (and D = 0) [57].
                          Table 2

                          Phylogenetic signal in chemistry and biological activity determined using Fritz and Purvis’s[57]D metric (see Materials and Methods for details)

                          a) Chemical components

                          Alkaloid group

                          D

                          P (D = 1)

                          P (D = 0)

                          Crinine

                          0.6768

                          0.021

                          0

                          Galanthamine

                          0.549

                          0

                          0.009

                          Lycorine

                          0.77

                          0.018

                          0

                          Galanthindole

                          1.091

                          0.621

                          0.011

                          Homolycorine

                          0.769

                          0.021

                          0

                          Montanine

                          0.572

                          0.002

                          0.01

                          Tazettine

                          0.852

                          0.094

                          0

                          b) Biological activity

                          Measure

                          D

                          P (D = 1)

                          P (D = 0)

                          AChE

                          0.679

                          0.004

                          0.001

                          SERT

                          0.634

                          0.037

                          0.044

                          D is equal to 1 if the observed chemical component has a random distribution (i.e. no phylogenetic signal). D is equal to 0 if the component is distributed exactly as would be predicted under a Brownian motion model of gradual divergent evolution (i.e. strong phylogenetic signal). P values represent the probability that the observed D value is equal to 1 or 0, respectively (P >0.05 indicates that the observed value is not significantly different from these values).

                          There was a statistically highly significant correlation between differences in chemical profile and phylogenetic distance, indicating that closely-related species tend to have more similar chemical profiles than more distantly-related species, although the effect was not strong (Mantel test: r = 0.085, p = 0.002). There was also statistically significant correlation between chemical profile and phylogenetic distance in the genus level comparison (Mantel test: r = 0.090, p = 0.024), although the effect was also weak.

                          Discussion

                          Phylogeny of Amaryllidoideae

                          For the purpose of the present study, we consider the total evidence approach to provide the best estimate of phylogeny and all major lineages are supported by both parsimony and Bayesian analyses. The present study has doubled previous sampling of Amaryllidoideae from 51 species [56] to 108 and from combined analysis of two DNA regions [42, 56] to four DNA regions representing all three genomes. The only previous study resolving relationships among basal lineages was based on plastid ndhF sequences [42] and resolved Calostemmateae and Haemantheae as sisters and tribe Cyrtantheae as sister to these. However, in the present study (Figure 2), the African tribes Cyrtantheae and Haemantheae are strongly supported by both bootstrap and Bayesian posterior probabilities as sister clades. Tribe Calostemmateae is sister to these, although this was only weakly supported by the bootstrap, but strongly supported by Bayesian posterior probabilities. A sister group relationship of the two African tribes Cyrtantheae and Haemantheae and the Australasian tribe Calostemmateae as sister to these appears more convincing than the alternative based on biogeography. However, in terms of morphology there may be some room to question this relationship. The indehiscent capsule of Calostemmateae has more in common with the indehiscent baccate fruit of Haemantheae (resembling the unripe fruit of Clivia, Scadoxus, Haemanthus, and Cryptostephanus) than with the dehiscent capsule of Cyrtanthus.

                          Phylogenetic signal of chemical diversity and bioactivity

                          Our approach to quantify overall correlation between chemical and phylogenetic diversity has previously been applied to show positive correlations between pheromone differences and nucleotide divergence in Bactrocera fruit flies [58] and phylogenetic correlation of cuticular hydrocarbon diversity in ants [59]. We have now shown the potential application of this approach to explore correlations between phylogenetic and chemical diversity of medicinal plants.

                          We found significant phylogenetic signal for five out of seven tested individual alkaloid types and for both AChE and SERT bioactivity proxies, although the signal was not strong enough to be indistinguishable from a Brownian model of evolution, where traits are strongly clumped on the phylogeny [57]. There was also a highly significant correlation between differences in chemical profiles and phylogenetic distance in both species and generic level analyses. Despite this, there was often still considerable difference in chemical make-up even between phylogenetically very close species (Figure 3) with the average number of alkaloid types differing between congeners being 2.26. Whereas members of some genera such as Crinum and Galanthus have generally similar chemical profiles, other genera such as Hippeastrum and Narcissus have striking diversity.
                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-182/MediaObjects/12862_2012_2122_Fig3_HTML.jpg
                          Figure 3

                          Relationship between phylogenetic distance and chemical diversity (number of alkaloid group differences) for Amaryllidaceae subfamily Amaryllidoidae. A) Scatter plot showing distribution of points and best-fit regression line. The size of each point is proportional to the log-transformed number of data points contributing to it. B) The same data showing mean (±s.e.) number of differences for categories of phylogenetic distance (number of substitutions per site) for ease of visualization. The overall relationship is significant. There is a statistically highly significant correlation between differences in chemical profile and phylogenetic distance, although the effect is not strong (Mantel test: r = 0.085, p = 0.002), indicating that closely related species tend to have more similar chemical profiles than more distantly related species.

                          An explanation for the moderate correlation found could be either methodological artefacts or underlying ecological or genetic differences [38]. We minimized methodological artefacts by using the same plant accessions for both phylogenetic, chemical, and bioactivity studies, and by analysing our data with consistent methods. Chemical profiles were based on types deducted from hypothetical pathways and could be an oversimplification of the chemical diversity contained by over 500 individual alkaloid structures known from the subfamily [45, 47].

                          The strength of correlation could be dependent on taxonomic scale. Whereas alkaloids derived from norbelladine and its derivatives are almost exclusively restricted to the subfamily Amaryllidoideae [45], and alkaloids with AChE activity appear to be phylogenetically constrained within Narcissus [18], the considerable variation at the species and genus level found in this study corresponds well with within species variation of alkaloid profiles in for example Galanthus [51, 60].

                          Evaluation of extensive historical drug data, marine natural products, medicinal plants and bioactive natural products suggests that drugs are derived mostly from pre-existing drug-productive families that tend to be clustered rather than randomly scattered in the phylogenetic tree of life [61]. Zhu et al. [61] further suggest that efforts to identify new potential drugs can therefore be concentrated on exploring a number of drug-productive clusters. However, based on our results, such a strong presumed correlation between phylogeny and bioactivity appears to be an oversimplification, at least at the taxonomic scale tested in our study. Based on our data for the medicinally important plant subfamily Amaryllidoideae, it appears that phylogeny can predict chemical diversity and bioactivity, but considerable caution must be emphasized. We also suggest that phylogenetic correlation of chemical traits of interest may need to be assessed for a particular phylogenetic framework before it is used for prediction of occurrence in un-investigated taxa.

                          Application of phylogenetic prediction and in silico data mining

                          A predictive approach could enable deduction of biosynthetic pathways, defence against herbivores, more efficient selection of plants for the development of traditional medicine and lead discovery as well as inform conservation priorities as outlined in the introduction. A plethora of data on phylogenetic relationships, chemical constituents and bioactivity are available through public databases (e.g., GenBank) and in the literature. Systematic in silico data mining could enable more efficient use of predictive approaches to speed up all of the above applications [22, 6165].

                          However, a methodological framework still needs to be developed. In the present study, we have suggested an approach for testing correlations between phylogenetic and chemical diversity and biological activity using experimental data generated for this purpose. One method for subsequent identification of specific nodes in phylogenies with high bio-screening potential has been proposed by Saslis-Lagoudakis et al.[22] using tools from community ecology. In this approach, a matrix of ethnomedicinal use was composed and used to identify nodes in a phylogeny of Pterocarpus (Fabaceae), which have more medicinal taxa related to a specific category of use than expected by chance. This approach could be useful for identifying alternative resources or substitute taxa in cases where supply of a medicinal plant or natural product of interest is limited or where species in use are subject to conservation concerns [22]. However, for the purpose of increasing the chance of making truly new discoveries such as new compounds and/or new activity profiles, it may be more relevant to identify clades that possess activity of interest and at the same time do not correspond to well known compounds with well known activity profiles [66].

                          Other methods for predictive in silico data mining may be combined with a phylogenetic selection approach, e.g., exploration of natural product chemical space as developed by Backlund and co-workers [65, 67, 68]. Another computerized geospatial tracking tool linking bioactive and phylogenetic diversity has been developed for microorganisms [63]. The concept of virtual parallel screening developed for natural products by Rollinger [64], which simultaneously enables fast identification of potential targets, insight into a putative molecular mechanism and estimation of a bioactivity profile, could allow for optimal selection of relevant targets.

                          Conclusion

                          In conclusion, we have shown significant correlation between phylogenetic and chemical diversity and biological activity in the medicinally important plant subfamily Amaryllidoideae. However, a correlation cannot be assumed for other study systems without considerable caution or testing. This has implications for the use of phylogenies to interpret chemical evolution and biosynthetic pathways, to select candidate taxa for lead discovery, and to make recommendations for policies regarding traditional use and conservation priorities. Phylogenetic prediction of chemical diversity and biological activity may provide an evolutionary based tool alone or in combination with other recently developed tools for in silico data mining of natural products and their bioactivity.

                          Methods

                          Taxon sampling

                          Specimens were collected in their natural habitat or obtained from botanical gardens or specialist nurseries. Sampling included 108 (over 10%) of circa 850 species in Amaryllidaceae subfamily Amaryllidoideae [69] with Agapanthus campanulatus L. used as outgroup ( Additional file 2). Sampling represents 43 of circa 60 genera and all currently recognized tribes except Griffineae Ravenna [41, 42, 56]. Samples from tribes Galantheae and Haemantheae were partly retrieved from previous studies [21, 51]. The same accessions of plant material were used for both molecular, chemical, and bioactivity analysis to minimize effects of intraspecific and ecological variation.

                          Phylogenetic analyses

                          DNA was extracted using the Qiagen DNeasy kit (Qiagen, Copenhagen, Denmark) from 20 mg of dried leaf fragments. Amplification and sequencing of the nuclear encoded ITS and plastid encoded matK and trnLF regions followed Larsen et al.[51]. Amplification and sequencing of the mitochondrial nad1 region followed Cuenca et al.[70]. Primers used for amplification and sequencing are listed in Additional file 3. Both strands were sequenced for each region for all taxa whenever possible. Sequences were edited and assembled using Sequencer 4.8TM software (Gene Codes, Ann Arbor, MI, USA). All sequences are deposited in GenBank and accession numbers JX464256- JX464610 are listed in Additional file 2. Sequences were aligned using default options in MUSCLE [71] as implemented in the software SeaView [72].

                          Phylogenetic analyses were conducted using both parsimony and Bayesian inference. Most parsimonious trees (MP) were obtained with PAUP v. 4.0b10 [73] using 1,000 replicates of random taxon addition sequence and TBR branch swapping saving multiple trees. All characters were included in the analyses and gaps were treated as missing data. We analysed the four regions separately to identify strongly supported phylogenetic conflicts among the regions, prior to performing a combined total evidence analysis. By using total evidence the explanatory and descriptive power of the data is maximized [74]. Bootstrap analyses [75] of the four individual datasets and the combined dataset were carried out using 1,000 replicates. Bayesian analysis of the combined dataset was performed with MrBayes 3.1.2 [76]. We first selected the best fitting model (GTR + I + G; Parameters: lset NST = 6 RATES = gamma) of molecular evolution using the Akaike criterion (AIC) in Modeltest v. 3.8 [77]. The analysis was performed with 1,000,000 generations on four Monte Carlo Markov chains. The average standard deviation of the split frequencies was 0.01 after 232,000 generations and < 0.005 after 1 million generations corresponding to an effective sample size of 115 using the software Tracer v. 1.5.0 [78]. The first 2,500 (25%) trees of low posterior probability were deleted and all remaining trees were imported into PAUP. A majority rule consensus tree was produced showing the posterior probabilities (PP) of all observed bi-partitions. We also performed a partitioned analysis allowing different models for the three genomes. However, in consideration of the limited information present in our plastid and mitochondrial datasets (Table 1), partition rich strategies are not always the best ones and in some cases less complex strategies have performed better [79, 80]. Although the Bayesian MCMC approach is good at handling complex models, there is a risk of over-parameterization, which can result in problems with convergence and excessive variance in parameter estimates [81].

                          Chemical diversity

                          Alkaloids were extracted from 300 mg dried bulb scales using 0.1% H2SO4 and clean-up on ion-exchange solid phase columns as described by Larsen et al.[51]. All extracts were concentrated under vacuum until dryness and re-dissolved to a standard concentration of 5 mg ml-1 in MeOH. Alkaloid profiles were obtained by gas chromatography–mass spectrometry (GC-MS) as described by Larsen et al.[51] using a method developed by Berkov et al.[82]. Alkaloids were identified to type by comparison with the NIST 08 Mass Spectral Search Program, version 2.0 (NIST, Gaithersburg, Maryland) and with published spectral data. Alkaloid structures were scored to one of eighteen types (Figure 1) proposed by Jin [45, 46] based on hypothetical biosynthetic pathways [51]. Only nine of the eighteen alkaloid types were recorded in the present study (Figure 1). Each type show characteristic fragmentation patterns in the MS-spectra [83]. In most of the cases, the database proposals with highest similarity could therefore be used to score the candidate structure indirectly to one of the types. Candidate structures were excluded from the profile if they could not be scored unambiguously to types.

                          In vitro biological activity

                          AChE inhibition and SERT affinity of the standardized alkaloid extracts were tested using published methods [21]. AChE activity was conducted using isolated acetylcholinesterase (Electrophorus electricus, Sigma, Germany) and SERT activity using homogenates of whole rat brains except cerebellum. Galanthamine and fluoxetine hydrochloride were used as positive standards in the AChE and SERT assays, respectively. Data were analysed with the software package GraFit 5 (Erithacus Software Ltd.). Activity values are means of three individual determinations each performed in triplicate. In an initial screening, AChE inhibition was defined as minimum 50% inhibition at a concentration of 1.0 μg ml-1. Subsequently IC50 values were determined for all extracts deemed active according the initial screening. IC50 values < 50 μg ml-1 was considered active for the analysis. SERT activity was defined as more than 85% binding of extract at 5 mg ml-1. Subsequently IC50 values were determined for all extracts deemed active according the initial screening. IC50 values < 50 μg ml-1 was considered active for the analysis. These activity levels were designed to reflect the observed level of activity in the present study and do not necessarily reflect levels of pharmacological relevance, but within the range of proposed ecological relevance [84]. SERT activity data were not determined for eight species of Narcissus and these samples were pruned from the phylogenetic trees in the correlation tests.

                          Phylogenetic signal

                          We assessed the relationship of phylogeny to chemical diversity and biological activity by calculating the phylogenetic signal present for individual alkaloid types and types of biological activity. Each alkaloid type was coded as being either present (1) or absent (0) for each species. Likewise, for biological activity, species were scored for the presence or absence of AChE inhibition or SERT binding activity. Two of the alkaloid traits (belladine and cherylline) are found only in one species each, rendering calculation of phylogenetic signal for these traits meaningless, so they were not included in this part of the analysis.

                          To quantify phylogenetic signal we used the recently developed D metric [57], specifically developed to deal with discrete binary traits. D is calculated as follows:
                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-182/MediaObjects/12862_2012_2122_Equ1_HTML.gif

                          where s obs is the observed number of changes in the binary trait (here, a chemical component) across the ultrametric phylogeny, http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-182/MediaObjects/12862_2012_2122_IEq1_HTML.gif is the mean number of changes generated from 1000 random permutations of the species values at the tips of the phylogeny, and http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-182/MediaObjects/12862_2012_2122_IEq2_HTML.gif is the mean number of changes generated from 1000 simulations of the evolution for the character by a Brownian motion model of evolution with likelihood of change being specified as that which produces the same number of tip species with each character state as the observed pattern. A D value of 1 ( http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-182/MediaObjects/12862_2012_2122_IEq3_HTML.gif ) indicates that the trait has evolved in a way that cannot be distinguished from a random manner (i.e., no phylogenetic signal), whilst a D value of 0 ( http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-182/MediaObjects/12862_2012_2122_IEq4_HTML.gif ) indicates that the trait has evolved in a phylogenetically highly correlated manner. Estimation of whether D differs significantly from 1 or 0 is achieved by evaluating where the observed number of changes (s obs ) fits within the distribution of the 1000 generated s r and s b values respectively. Thus if 95% or more values of s r are greater than s obs then P (D = 1) ≤ 0.05 and the trait is significantly more phylogenetically structured than random expectation. Calculation of D was carried out using the packages caper [85] and ape [86] in the R v2.14.0 framework [87].

                          We also quantified the relationship between overall chemical profile and phylogeny following an approach used to study the evolution of pheromone chemical diversity [57, 58]. We constructed pairwise matrices; one of ultrametric phylogenetic distances (summed branch lengths) between species, and the other of chemical difference calculated as the binary squared Euclidean distance (i.e., the total number of alkaloid types that are absent in one taxon but present in another and vice versa). In addition to using all included species as terminal taxa, we also pruned the phylogenetic tree to genera and compared the resulting distance matrix to summed chemical profiles for each genus. In the case of polyphyletic genera both clades were retained. The correlation between phylogenetic distance and chemical difference was calculated using Mantel tests, with rows and columns of the distance matrix being randomly perturbed and the correlation coefficient recalculated 999 times to generate a null frequency distribution. These tests were performed using the program GenAlEx [88].

                          Declarations

                          Acknowledgements

                          K. Krydsfeldt and C. Hansen are thanked for technical assistance. This research was supported by a Steno grant ´Phylodrugs´ (N° 09–063972) to NR and a grant (N° 272-06-0436) to Ole Seberg and GP, both grants from the Danish Council for Independent Research - Natural Sciences; a Marie Curie Incoming Fellowship (N°235167) from the European Commission to GS and NR; and grants from the Lundbeck Foundation, Aase and Ejnar Danielsens Foundation and Brødrene Hartmanns Foundation to NR.

                          Authors’ Affiliations

                          (1)
                          Botanic Garden, Natural History Museum of Denmark
                          (2)
                          Natural Products Research, Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen
                          (3)
                          Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University
                          (4)
                          USDA-ARS-SHRS, National Germplasm Repository
                          (5)
                          Fairchild Tropical Garden
                          (6)
                          Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg

                          References

                          1. Abbott HCDS: The chemical basis of plant forms. In Studies in Plant and Organic Chemistry, and Literary Papers. Edited by: Michael HA. Cambridge, MA: The Riverside Press; 1907.
                          2. Stahl E: Pflanzen und Schnecken. Jena Z Med Naturwiss 1888, 22:557–684.
                          3. Ehrlich PR, Raven PH: Butterflies and plants - a study in coevolution. Evolution 1964, 18:586–608.View Article
                          4. Berenbaum MR, Zangeri AR, Nitao JK: Constraints on chemical coevolution: wild parsnips and the parsnip webworm. Evolution 1985, 40:1215–1228.View Article
                          5. Bennett RN, Wallsgrove RM: Secondary metabolites in plant-defense mechanisms. New Phytol 1994, 127:617–633.View Article
                          6. Wink M: Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry 2003, 64:3–19.PubMedView Article
                          7. Agrawal AA, Fishbein M: Plant defense syndromes. Ecology 2006, 87:S132-S149.PubMedView Article
                          8. Hegnauer R: Chemotaxonomie der Pflanzen, Vol. 1–6. Basel: Birkhäuser Verlag; 1962–1973.
                          9. Dahlgren R: Angiospermernes taxonomi, Vol. 1–4. Copenhagen: Akademisk Forlag; 1974–1976.
                          10. Dahlgren R, Clifford HT, Yeo P: The families of the monocotyledons: Structure, evolution, and taxonomy. Berlin and New York: Springer-Verlag; 1985.View Article
                          11. Rodman JE, Soltis PS, Soltis DE, Sytsma KJ, Karol KG: Parallel evolution of glucosinolate biosynthesis inferred from congruent nuclear and plastid gene phylogenies. Am J Bot 1998, 85:997–1006.PubMedView Article
                          12. Rønsted N, Franzyk H, Mølgaard P, Jaroszewski JW, Jensen SR: Chemotaxonomy and evolution of Plantago . Pl Syst Evol 2003, 242:63–82.View Article
                          13. Wink M, Mohamed GIA: Evolution of chemical defense traits in the Leguminosae: mapping of distribution patterns of secondary metabolites on a molecular phylogeny inferred from nucleotide sequences of the rbcL gene. Biochem Syst Ecol 2003, 31:897–917.View Article
                          14. Albach DC, Jenen SR, Ozgokce F, Garyer RH: Veronica: Chemical characters for the support of phylogenetic relationships based on nuclear ribosomal and plastid DNA sequence data. Biochem Syst Ecol 2005, 33:1087–1106.View Article
                          15. Larsson S: The "new" chemosystematics: phylogeny and phytochemistry. Phytochemistry 2007, 68:2904–2908.PubMedView Article
                          16. Becerra JX, Noge K, Venable DL: Macroevolutionary chemical escalation in an ancient plant-herbivore arms race. PNAS 2009, 106:18062–18066.PubMedView Article
                          17. Agrawal AA: Current trends in the evolutionary ecology of plant defence. Funct Ecol 2011, 25:420–432.View Article
                          18. Rønsted N, Savolainen V, Mølgaard P, Jäger AK: Phylogenetic selection of Narcissus species for drug discovery. Biochem Syst Ecol 2008, 36:417–422.View Article
                          19. Schmitt I, Barker K: Phylogenetic methods in natural product research. Nat Prod Rep 2009,2009(26):1585–1602.View Article
                          20. Bohlin L, Goransson U, Alsmark C, Weden C, Backlund A: Natural products in modern life science. Phytochem Rev 2010, 9:279–301.PubMedView Article
                          21. Bay-Smidt MGK, Jäger AK, Krydsfeldt K, Meerow AW, Stafford GI, van Staden J, Rønsted N: Phylogenetic selection of target species in Amaryllidaceae tribe Haemantheae for acetylcholinesterase inhibition and affinity to the serotonin reuptake transport protein. S Afr J Bot 2011, 77:175–183.View Article
                          22. Saslis-Lagoudakis CH, Klitgaard B, Forest F, Francis L, Savolainen V: The use of phylogeny to interpret cross-cultural patterns in plant use and guide medicinal plant discovery: an example from Pterocarpus (Leguminosae). PLoS One 2011, 6:e22275.PubMedView Article
                          23. Forest F, Grenyer R, Rouget M, Davies TJ, Cowling RM, Faith DP, Balmford A, Manning JC, Proche S, van der Bank M, Reeves G, Hedderson TAJ, Savolainen V: Preserving the evolutionary potential of floras in biodiversity hotspots. Nature 2007, 445:757–760.PubMedView Article
                          24. Grayer RJ, Chase MW, Simmonds MSJ: A comparison between chemical and molecular characters for the determination of phylogenetic relationships among plant families: An appreciation of Hegnauer's "Chemotaxonomie der Pflanzen". Biochem Syst Evol 1999, 27:369–393.View Article
                          25. Jensen SR, Franzyk H, Wallander E: Chemotaxonomy of the Oleaceae: iridoids as taxonomic markers. Phytochemistry 2002, 60:213–231.PubMedView Article
                          26. Aguilar-Ortigoza CJ, Sosa V: The evolution of toxic phenolic compounds in a group of Anacardiaceae genera. Taxon 2004, 53:357–364.View Article
                          27. Zidorn C: Sesquiterpene lactones and their precursors as chemosystematic markers in the tribe Chichorieae of the Asteraceae. Phytochemistry 2008, 69:2270–2296.PubMedView Article
                          28. Sareedenchai V, Zidorn C: Flavonoids as chemosystemtic markers in the tribe Cichorieae of the Asteraceae. Biochem Syst Ecol 2010, 38:935–957.View Article
                          29. Stanstrup J, Rusch A, Agnolet S, Rasmussen HB, Mølgaard P, van Staden J, Stafford GI, Staerk D: Itoside A and 4-hydroxytremulacin from Dovyalis caffra and Dovylais zeyheri . Biochem Syst Ecol 2010, 38:346–348.View Article
                          30. Becerra JX: Insects on plants: macroevolutionary chemical trends in host use. Science 1997, 276:253–256.PubMedView Article
                          31. Pichersky E, Lewinsohn E: Convergent evolution in plant specialized metabolism. Ann Rev Plant Biol 2011, 62:549–566.View Article
                          32. Rasmussen LEL, Lee TD, Roelofs WL, Zhang A, Doyle Davies G Jr: Insect pheromone in elephants. Nature 1996, 379:684.PubMedView Article
                          33. Brodie ED: Convergent evolution. Pick your poison carefully. Curr Biol 2010, 20:R152-R154.PubMedView Article
                          34. Landrum JV: Four succulent families and 40 million years of evolution and adaptation to xeric environments: What can stem and leaf anatomical characters tell us about their phylogeny? Taxon 2002,2002(51):463–473.View Article
                          35. Bennici A: The convergent evolution in plants. Riv Biol 2003, 96:485–489.PubMed
                          36. Albert VA, Williams SE, Chase MW: Carnivorous plants: phylogeny and structural evolution. Science 1992, 257:491–1495.View Article
                          37. Jensen NB, Zagrobelny M, Hjerno K, Olsen CE, Houghton-Larsen J, Borch J, Moller BL, Bak S: Convergent evolution in biosynthesis of cyanogenic defence compounds in plants and insects. Nat Commun 2011, 2:273.PubMedView Article
                          38. Gatehouse J: Plant resistance towards insect herbivores: a dynamic interaction. New Phytol 2002, 156:145–168.View Article
                          39. Nyman T, Julkunen-Tiitto R: Chemical variation within and among six northern willow species. Phytochemistry 2005, 66:2836–2843.PubMedView Article
                          40. Chase MW, Reveal JL, Fay MF: A subfamilial classification for the expanded asparagalean families Amaryllidaceae, Asparagaceae and Xanthorrhoeaceae. Bot J Linn Soc 2009, 161:132–136.View Article
                          41. Meerow AW, Snijman DA: Amaryllidaceae. In The families and genera of vascular plants. Edited by: Kubitzki K. Berlin: Springer-Verlag; 1998:pp. 83-pp. 110. vol. 3
                          42. Meerow AW, Snijman DA: The never-ending story: multigene approaches to the phylogeny of Amaryllidaceae, and assessing its familial limits. Aliso 2006, 22:353–364.
                          43. Neuwinger HD: African Traditional Medicine. A dictionary of plant use and applications. Stuttgart: Medpharm Scientific Publishers; 2000.
                          44. Stafford GI, Pedersen ME, van Staden J, Jäger AK: Review on plants with CNS-effects used in traditional South African medicine against mental diseases. J Ethnopharmacol 2008, 119:513–537.PubMedView Article
                          45. Jin Z: Amaryllidaceae and Sceletium alkaloids. Nat Prod Rep 2007, 24:886–905.PubMedView Article
                          46. Jin Z: Amaryllidaceae and Sceletium alkaloids. Nat Prod Rep 2009, 26:363–381.PubMedView Article
                          47. Jin Z: Amaryllidaceae and Sceletium alkaloids. Nat Prod Rep 2011, 28:1126–1142.PubMedView Article
                          48. López S, Bastida J, Viladomat F, Codina C: Acetylcholinesterase inhibitory activity of some Amaryllidaceae alkaloidsand Narcissus extracts. Life Sci 2002, 71:2521–2529.PubMedView Article
                          49. Elgorashi EE, Stafford GI, van Staden J: Acetylcholinesterase enzyme inhibitory effects of Amaryllidaceae alkaloids. Planta Med 2004, 70:260–262.PubMedView Article
                          50. Jäger AK, Adsersen A, Fennell CW: Acetylcholinesterase inhibition of Crinum spp. S Afr J Bot 2004, 70:323–325.
                          51. Larsen MM, Adsersen AA, Davis AP, Lledó MD, Jäger AK, Rønsted N: Using a phylogenetic approach to selection of target plants in drug discovery of acetylcholinesterase inhibiting alkaloids in Amaryllidaceae tribe Galantheae. Biochem Syst Ecol 2010, 38:1026–1034.View Article
                          52. Elgorashi EE, Stafford GI, Jäger AK, van Staden J: Inhibition of [ 3  H] citalopram binding to the rat brain serotonin transporter by Amaryllidaceae alkaloids. Planta Med 2006, 72:470–473.PubMedView Article
                          53. Neergaard JS, Andersen J, Pedersen ME, Stafford GI, van Staden J, Jäger AK: Alkaloids from Boophone disticha with affinity to the serotonin transporter. S Afr J Bot 2009, 75:371–374.View Article
                          54. Heinrich M, Teoh HL: Galanthamine from snowdrop – the development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. J Ethnopharmacol 2004, 92:147–162.PubMedView Article
                          55. Bores GM, Huger FP, Petko W, Mutlib AE, Camacho F, Rush DK, Selk DE, Wolf V, Kosley RW, Davis L, Vargas HM: Pharmacological evaluation of novel Alzheimer’s disease therapeutics: Acetylcholinesterase inhibitors related to galanthamine. J Pharmacol Exp Ther 1996, 227:728–738.
                          56. Meerow AW, Fay MF, Guy CL, Li Q-B, Zaman FQ, Chase MW: Systematics of Amaryllidaceae based on cladistic analysis of plastid rbc L and trn L-F sequence data. Am J Bot 1999, 86:1325–1345.PubMedView Article
                          57. Fritz SA, Purvis A: Selectivity in mammalian extinction risk and threat types: a new measure of phylogenetic signal strength in binary traits. Conserv Biol 2010, 24:1042–1051.PubMedView Article
                          58. Symonds MRE, Moussalli A, Elgar MA: The evolution of sex pheromones in an ecologically diverse genus of flies. Biol J Linn Soc 2009, 97:594–603.View Article
                          59. van Wilgenburg E, Symonds MRE, Elgar MA: Evolution of cuticular hydrocarbon diversity in ants. J Evol Biol 2011, 24:1188–1198.PubMedView Article
                          60. Berkov S, Sidjimova B, Evstatieva L, Popov S: Intra-specific variability in the alkaloid metabolism of Galanthus elwesii . Phytochemistry 2004, 65:579–586.PubMedView Article
                          61. Zhu F, Qin C, Tao L, Liu X, Shi Z, Ma X, Jia J, Tan Y, Cui C, Lin J, Tan C, Jiang Y, Chen Y: Clustered patterns of species origins of nature-derived drugs and clues for future bioprospecting. PNAS 2011, 108:12943–12948.PubMedView Article
                          62. Larsson S, Backlund A, Bohlin L: Reappraising a decade old explanatory model for pharmacognosy. Phytochemistry Lett 2008, 1:31–134.View Article
                          63. Pacharawongsakda E, Yokwai S, Ingsriwang S: Potential natural product discovery from microbes through a diversity-guided computational framework. Appl Microbiol Biotech 2009, 82:579–586.View Article
                          64. Rollinger JM: Accessing target information by virtual parallel screening – the impact on natural product research. Phytochem Lett 2009, 2:53–58.View Article
                          65. Backlund A: Topical chemical space relation to biological space. In Comprehensive Natural Products II Chemistry and Biology. Edited by: Mander L, Lui H-W. Oxford: Elsevier; 2010:pp.47-pp.79. volume 3
                          66. Bruhn JG, Bohlin L: Molecular pharmacognosy: an explanatory model. Drug Discov Today 1997, 2:243–246.View Article
                          67. Larsson J, Gottfries J, Muresan S, Backlund A: ChemGPS-NP: Tuned for navigation in biologically relevant chemical space. J Nat Prod 2007, 70:789–794.PubMedView Article
                          68. Rosén J, Lövgren A, Kogej T, Muresan S, Gottfries J, Backlund A: ChemGPS-NPweb – chemical space navigation online. J Comp Aided Mol Des 2009, 23:253–259.View Article
                          69. Govaerts R, Kington S, Snijman DA, Marcucci R, Silverstone-Sopkin PA: World Checklist of Amaryllidaceae. The Board of Trustees of the Royal Botanic Gardens, Kew. 2007. Published on the Internet; http://​www.​kew.​org/​wcsp/​ accessed 22 September 2011
                          70. Cuenca A, Petersen G, Seberg O, Jahren AH: Genes and processed paralogs coexist in plant mitochondria. J Mol Evol 2012, 74:158–169.PubMedView Article
                          71. Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004, 35:1792–1797.View Article
                          72. Gouy M, Guindon S, Gacuel O: SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Conserv Biol 2010, 27:221–224.
                          73. Swofford DL: PAUP*: Phylogenetic Methods Using Parsimony (*and other methods), version 4.0b.10. Sunderland, Massachusetts: Sinauer; 2002.
                          74. Kluge AG, Wolf AJ: Cladistics: What’s in a word? Cladistics 1993, 9:183–199.View Article
                          75. Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985, 39:783–791.View Article
                          76. Ronquist F, Hulsenbeck JP: MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003, 19:1572–1574.PubMedView Article
                          77. Posada D, Crandall KA: Modeltest: testing the model of DNA substitution. Bioinformatics 1998, 14:817–818.PubMedView Article
                          78. Rambaut A, Drummond AJ: Tracer v1.4. 2007. Available from http://​beast.​bio.​ed.​ac.​uk/​Tracer
                          79. Lemmon AR, Moriarty EC: The importance of proper model assumption in Bayesian phylogenetics. Syst Biol 204, 53:265–277.View Article
                          80. Kergoat GJ, Silvain J-F, Delobel A, Tuda M, Anton K-W: Defining the limits of taxonomic conservatism in host–plant use for phytophagous insects: Molecular systematics and evolution of host–plant associations in the seed-beetle genus Bruchus Linnaeus (Coleoptera: Chrysomelidae: Bruchinae). Mol Phyl Evol 2007, 43:251–269.View Article
                          81. Ronquist F, Huelsenbeck JP, van der Mark P: MrBayes 3.1. 2011. Manual. http://​mrbayes.​sourceforge.​net/​wiki/​index.​php/​Evolutionary_​Models_​Implemented_​in_​MrBayes_​3
                          82. Berkov S, Bastida J, Viladomat F, Codina C: Analysis of galanthamine-type alkaloids by capillary gas chromatography–mass spectrometry in plants. Phytochem Anal 2008, 19:285–293.PubMedView Article
                          83. Bastida J, Lavilla R, Viladomat F: Chemical and biological aspects ofNarcissusalkaloids. In The alkaloids Chemistry and Biology. Edited by: Cordell GA. Amsterdam: Academic Press; 2006:pp. 87-pp. 179. vol. 63
                          84. Wink M: Interference of alkaloids with neuroreceptors and ion channels. In Studies in Natural Product Chemistry. Edited by: Atta-ur R. Amsterdam: Elsevier Science; 2000:pp. 3-pp. 122. vol. 21
                          85. Orme D, Freckleton R, Thomas G, Petzoldt T, Fritz S, Isaac N: Caper. 2011. Comparative Analyses of Phylogenetics and Evolution in R. R package version 0.4. URL: http://​CRAN.​R-project.​org/​package=​caper
                          86. Paradis E, Claude J, Strimmer K: APE: analyses of phylogenetics and evolution in R language. Bioinformatics 2004, 20:289–290.PubMedView Article
                          87. R Development Core Team: R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2011. URL: http://​www.​R-project.​org/​
                          88. Peakall R, Smouse PE: GenAlEx 6: genetic analysis in excel. Population genetic software for teaching and research. Mol Ecol Notes 2006, 6:288–295.View Article

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                          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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