Supplementary Information for Vincent Manzanilla1, Irene Teixidor

Home , Anacyclus valentinus, Anthemideae, Otanthus

Supplementary Information for

USING TARGET CAPTURE TO ADDRESS CONSERVATION CHALLENGES: POPULATION-

LEVEL TRACKING OF A GLOBALLY-TRADED HERBAL MEDICINE

Vincent Manzanilla1, Irene Teixidor-Toneu1, Gary J. Martin2, Peter M. Hollingsworth3, Hugo J. de Boer1, Anneleen Kool1

1 Natural History Museum, University of Oslo, Sars gate 1, 0562 Oslo, Norway

2 Global Diversity Foundation, 37 St. Margarets Street, Canterbury, Kent CT1 2TU, England

3 Royal Botanic Garden Edinburgh, Inverleith Row, Edinburgh EH3 5LR, Scotland

Corresponding author: Vincent Manzanilla

Email: [email protected]

SI MATERIAL AND METHODS ...... 3

Morphological estimation of adulteration ...... 3 Marker design, skimming data and denovo assembly ...... 3 Nuclear genes filtering ...... 4 Results ...... 5

SI FIGURE AND TABLE CAPTIONS ...... 6

SI REFERENCES ...... 8

SI FIGURES ...... 11

Figure S1...... 11 Figure S2...... 12 Figure S3...... 13 Figure S4...... 14 Figure S5...... 15 Figure S6...... 16 Figure S7...... 17 Figure S8...... 18 Figure S9...... 19 Figure S10...... 20 Figure S11...... 21 Figure S12...... 22 Figure S13...... 23

SI TABLES ...... 24

Table S1...... 25 Table S2...... 26 Table S3...... 28 Table S4...... 29 Table S5...... 29

Table S6. 29

Table S7...... 30

2 SI Material and Methods

Morphological estimation of adulteration. To estimate adulteration, collected trade samples were screened for plant parts that could be morphologically identified. Identifications were supported by data from previous studies on adulterants for A. pyrethrum (de Boer, Ouarghidi, Martin, Abbad, & Kool, 2014; Kool et al., 2012). Based on the shape, colour, and morphological structure of the root cross-section, roots were grouped into ‘possibly belonging to the genus Anacyclus’ or ‘definitely adulterated’, and the two subsets were subsequently weighed. All roots identified as ‘possibly belonging to the genus Anacyclus’ species were selected for molecular identification.

Marker design, skimming data and denovo assembly. Nuclear markers for molecular identification of the Matricariinae sub-tribe (Asteraceae) were designed using novel skimming data of an accession of A. radiatus subsp. radiatus (voucher MV54) (Table S1). The Hyb-Seq pipeline (Schmickl et al., 2016) was used to find conserved DNA regions of sufficient length and to exclude multiple copy genes and transposable elements. The A. radiatus subsp. radiatus voucher (MV54) yielded 2.86 µg of DNA measured on a Qubit 2.0 fluorometer (Invitrogen/Life Technologies, Carlsbad, CA, USA). DNA integrity and concentration were assessed using a Fragment Analyser (Advanced Analytical, Heidelberg, Germany) and the High Sensitivity genomic DNA Reagent Kit (50–40,000 bp). The sample was sequenced on an Illumina NextSeq 500 paired-end system using a TruSeq DNA PCR- Free library kit. Library adapter sequences and low quality reads were removed with Trimmomatic v. 0.32 (Bolger, Lohse et al. 2014) with a quality threshold set at Q20 with a sliding window of 10 bp.

Prior to the denovo nuclear genome assembly, the plastid genome was assembled with the trimmed reads using MITObim v1.8 (Hahn, Bachmann, & Chevreux, 2013) using the plastid genome of Chrysanthemum indicum L. (NC_020320) as a reference. Protein-coding genes in the plastid genome were annotated with DOGMA (Wyman, Jansen, & Boore, 2004), and after visual inspection, a gene map was drawn using OGDRAW v1.2 (Lohse, Drechsel, & Bock, 2007). Selective filtering of the plastid and mitochondrial genomes as well as nrDNA were done using BWA v0.7.5a (Langmead & Salzberg, 2012). The previously assembled plastid and mitochondrial genomes of Helianthus annuus L. (NC_023337.1) and the nrDNA from Anacyclus valentinus L. (GU818490) were used as references. The nuclear genome of A. radiatus subsp. radiatus was assembled using SOAPdenovo2 vr223 (Xie et al., 2014) with

3 nine kmer values between 20 and 100. The best genome assembly was determined using Quast v2.3 (Gurevich, Saveliev, Vyahhi, & Tesler, 2013).

Low-copy nuclear markers (600-1000 bp in length) were identified using the Hyb-Seq pipeline based on the skimming assembly of A. radiatus, and the transcriptome assembly of a close relative outgroup, Matricaria matricarioides (Less.) Porter (voucher ALTA132745) (Matasci et al., 2014). The original Hyb-Seq pipeline was adapted to identify introns as well as exons (Schmickl et al., 2016). The modified script used for the selection of the low-copy nuclear markers is available on Open Science Framework, Anacyclus project folder (https://osf.io/9bh3p/).

Transcriptome and skimming data were pre-processed to ensure selection of sufficiently long nuclear regions as markers using the Hyb-Seq pipeline, filtering out plastid mitochondrial sequences using Helianthus annuus NC_023337.1 as a reference, and nrDNA with the reference of the A. radiatus assembly. A length threshold was applied on the remaining data and transcripts below 120 bp (RNA probe size) and contigs from the skimming data below 600 bp were discarded. Subsequently, the contigs were mapped against the M. matricarioides transcriptomes using Blat v3.5 (Kent, 2002), and alignments were selected with a minimum length of 80% of the contig size. Alignments with more than 10% divergence and contigs with more than one match against the M. matricarioides transcriptomes were discarded. The obtained preliminary set of markers was mapped with the Burrows-Wheeler Aligner (BWA) v0.7.5a-r405 (Li & Durbin, 2010) against the reads from the A. radiatus nuclear genome assembly. We extracted the coverage from this alignment using BEDtools v2.17 (Quinlan, 2014), and contigs with a higher coverage than average were discarded because they were suspected to be multiple copy genes or contain transposable elements. A total of 872 putative low copy nuclear markers were retained. To ensure that the probes targeted only the nuclear genome, we mapped the probes against the previously assembled organelles and nrDNA with BWA and discarded those that matched. From the selected loci, 19,246 RNA probes with a length of 120 bp and a tilling density of 3.4X were designed and produced by Arbor Bioscience (Ann Arbor, Michigan, USA).

Nuclear genes filtering. Nuclear gene trees were reconstructed for each individual nuclear locus. Samples with >7% missing data across markers were removed from the entire dataset, as well as markers with >5% missing data, as these were considered to have insufficient enrichment success. Retained matrices were re-aligned using MUSCLE v3.8.31 (Edgar, 2004) and filtered with Gblock (Talavera & Castresana, 2007). The final set consisted of 443

4 low-copy nuclear markers with two alleles per individual, with a minimum length of 400bp, no missing samples and less than 5% missing data. For each of these markers, we inferred a gene tree using RaxML v8.0.26 (Stamatakis, 2006) with 1000 bootstrap replicates under the GTRGAMMA model. Out of the 872 nuclear markers, 429 were discarded because we suspected to have transposons within these markers. Plants with huge genomes are known to have many transposon and low-complexity regions. Without a fully annotated genome, like in this case, it is difficult to assess quality of the designed markers before sequencing them.

Results. The best assembly of the A. radiatus subsp. radiatus genome was obtained with kmer values set at 70 bp, resulting in an N50 score of 869 bp with a total contig length of 1.4 Gbp. Assuming that the genome size of Anacyclus is 16 Gb (Bennett & Leitch, 2005; Humphries, 1981), the assembly represents approximately 8% of the A. radiatus subsp. radiatus genome. Raw data are deposited in NCBI Bioproject PRJNA631886. The plastid genome assembly yielded a final annotated genome of 150,925 bp with an average coverage of 350X (Table S1).

5 SI Figure and table captions

Fig. S1. Geographical mapping of the sampled populations. Dots show the locations of the different populations and the colors represent different species.

Fig. S2. Bioinformatics workflow to retrieve the ITS, plastome, standard and nuclear marker datasets.

Fig. S3. Number of reads per sample before and after trimming.

Fig S4. Maximum Likelihood ITS phylogenetic tree reconstructed from the reference dataset only.

Fig. S5. Maximum Likelihood ITS phylogenetic tree with reference and traded samples. GenBank reference sequences are written in plain text, trade samples are in grey and reference samples from our study in bold. The vertical lines on the right of the phylogeny represent supported clades with the associated species names.

Fig. S6. Maximum Likelihood plastome phylogenetic tree reconstructed from the reference dataset only.

Fig. S7. Maximum Likelihood plastome phylogenetic tree with reference and traded samples. Traded samples are indicated in the phylogram by their accession number.

Fig. S8. Maximum Likelihood matK phylogenetic tree with reference and traded samples. Traded samples are indicated in the phylogram by their accession number.

Fig. S9. Maximum Likelihood rbcL phylogenetic tree with reference and traded samples. Traded samples are indicated in the phylogram by their accession number.

Fig. S10. Maximum Likelihood trnH-psbA phylogenetic tree with reference and traded samples. Traded samples are indicated in the phylogram by their accession number.

Fig. S11. Maximum Likelihood trnL phylogenetic tree with reference and traded samples. Traded samples are indicated in the phylogram by their accession number.

Fig. S12. Multispecies Coalescent (MSC) phylogenetic tree based on the nuclear loci dataset with the reference dataset only. The vertical lines on the right of the phylogeny represent supported clades with the associated species names.

6 Fig. S13. Analysis workflow of root samples in trade.

Table S1. Voucher specimens for the reference database. The herbaria where the reference material is deposited is indicated using Index Herbariorum abbreviations: BC for Institut Botànic de Barcelona, Spain; HUJ for Hebrew University, Israel; MA for Real Jardín Botánico, Spain; MARK for Cadi Ayyad University, Morocco; P for Muséum National d'Histoire Naturelle, France; O for Botanical Museum, University of Oslo, Norway; UPNA for Universidad Pública de Navarra; Spain; VIT for Museo de Ciencias Naturales de Alava, Spain.

Table S2. Voucher specimens for traded samples and their taxonomic identification. The first section of the table lists the samples identified from target capture using a phylogenetic framework, and the second section the samples identified using blastn.

Table S3. Reference sequences for the ITS tree with their respective article reference: 1(Guo, Ehrendorfer, & Samuel, 2004), 2(Ata, Abd El-Twab, Helmey, & Dahy, 2017), 3(Priya, Malik, & Babbar, 2018), 4(Oberprieler, 2004), 5(Sonboli, Stroka, Osaloo, & Oberprieler, 2012), 6(Ghorbani, Saeedi, & De Boer, 2017).

Table S4. Basic statistics on the number of reads per sample for the target sequencing and the genome skimming sequencing runs. The table is summarized in Figure S3.

Table S5. Average coverage, length of the matrices and missing data for the standard barcodes, ITS, the plastome and low copy nuclear markers.

Table S6. Percentage of adulteration and molecular identification of the different varieties of A. pyrethrum for each of the value chain stakeholders. This table is summarized in Figure 4.

Table S7. Sequencing recovery and identification success for the traded samples for each dataset. This table is summarized in Figure 1.

7 SI References

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10 SI Figures

Figure S1.

11 Figure S2.

12 Figure S3.

13 Figure S4.

9 3 Tanacetum Matricaria 100 Tripleurospermum 100 Otanthus 9 3 100 Anacylcus atlanticus

Anacyclus radiatus subsp. radiatus 100

Anacyclus radiatus subsp. coronatus

7 3 9 6 Anacyclus monanthos Anacyclus valentinus Anacyclus clavatus

Anacyclus homogamos

Anacyclus valentinus 7 5

Anacyclus clavatus

Anacyclus homogamos

Anacyclus pyrethrum var. depressus

6 6

9 9 Anacyclus maroccanus

Anacyclus pyrethrum var. depressus

Anacyclus pyrethrum var. pyrethrum

0.02

14 Figure S5.

Tanacetum

8 1 Achillea clusiana AY603228.1 Achillea oxyloba AY603238.1 8 3 9 7 Achillea atrata AY603227.1 Achillea virescens AY603211.1 Achillea absinthoides AY603213.1 Achillea clavennae AY603229.1 Achillea holosericea AY603222.1 Achillea ageratum AY603214.1 Achillea euxina AY603202.1 Achillea fraasii AY603232.1 Achillea styriaca AY603190.1 Achillea pannonica AY603184.1 Achillea distans AY603188.1 Achillea alpina AY603206.1 Achillea lanulosa AY603205.1 Achillea millefolium 2011943w 8 9 1 Achillea monticola AY603210.1 Achillea asplenifolia AY603201.1 Achillea roseoalba AY603200.1 Achillea collina AY603198.1 Achillea pratensis AY603199.1 Achillea asiatica AY603208.1 Achillea wilsoniana AY603207.1 Achillea ceretanica AY603203.1 Matricaria chamomilla KC816562.1 100 Matricaria chamomilla MV262 100 Matricaria aurea MV109 8 5 Matricaria discoidea MB2 Matricaria aurea KT954177.1 9 8 Achillea acuminata AY603243.1 Achillea salicifolia AY603244.1 Achillea ptarmica 20025048aS Achillea pyrenaica AY603247.1 Tripleurospermum sp. T1 100 Tripleurospermum sp. KR150169.1 Otanthus maritimus MNHNPP04305585 100 Otanthus maritimus AY603257.1 100 A. atlantica MV25 A. atlantica AJ748782.1 A. atlantica MV187 A. radiatus subsp. radiatus MV409 A. atlanticus 100 A. radiatus subsp. radiatus MV388 A. radiatus subsp. radiatus VIT76273 A. radiatus subsp. radiatus MV413 A. radiatus subsp. radiatus MV403 A. radiatus subsp. coronatus BC860742 A. radiatus subsp. coronatus A. radiatus subsp. coronatus 126066 A. radiatus subsp. radiatus MV392 A. radiatus subsp. radiatus 3061672 A. radiatus subsp. radiatus 7 5 A. radiatus subsp. radiatus 3061671 A. radiatus subsp. radiatus MV391 A. radiatus subsp. radiatus WPT441 A. radiatus subsp. radiatus MV387 7 5 A. clavatus AY603258.1 7 3 A. valentinus UPNA11846 A. clavatus WPT512 A. clavatus WPT49 7 8 A. clavatus VIT56515 A. monanthos MNHNPP03692979 100 A. monanthos KT954174.1 9 7 A. homogamos MV354 A. valentinus BC878628 A. valentinus GU818490.1 A. homogamos MV366 A. clavatus A. valentinus VIT83639 A. homogamos MV358 7 0 A. valentinus VIT88094 A. homogamos A. valentinus VIT91146 A. valentinus VIT78628 A. homogamos MV280 A. valentinus A. clavatus MV433 A. clavatus MV468 8 9 A. homogamos MV322 A. clavatus MV472 A. clavatus MV323 A. clavatus MV448 A. clavatus MV463 A. homogamos MV284 A. clavatus MV324 A. clavatus MV470 A. clavatus MV337 A. clavatus MV289 A. clavatus MV349 A. pyrethrum var. depressus IA2334 106 197 69 101 162 161 64 149 78 186 89 102 138 165 65 8 21 203 66 210 54 77 113 126 174 204 9 0 189 9 154 213 172 150 160 58 136 100 14 125 43 52 124 A. pyrethrum var. pyrethrum 126070 208 137 19 A. maroccanus MV257 A. maroccanus MV490 9 9 A. maroccanus MV489 30 A. homogamos MV101 1 A. homogamos MV103 7 2 A. homogamos MV475 27 A. maroccanus MV194 A. maroccanus MV480 A. maroccanus MV154 A. maroccanus MV180 A. maroccanus MV195 A. maroccanus MV234 A. maroccanus MV246 A. maroccanus MV241 A. maroccanus MV143 A. maroccanus MV168 A. maroccanus 40 A. pyrethrum var. pyrethrum MV344 8 7 A. pyrethrum var. pyrethrum MV224 A. pyrethrum var. depressus 181 A. pyrethrum var. pyrethrum MV203 127 A. pyrethrum var. pyrethrum 141 82 103 198 A. pyrethrum var. depressus MV222 A. pyrethrum var. depressus MV330 20 38 A. pyrethrum var. depressus MV272 Anacyclus pyrethrum KM887397.1 Anacyclus pyrethrum KM887396.1 A. pyrethrum var. depressus MV188 A. pyrethrum var. depressus MV313 112 196 148 7 26 A. pyrethrum var. depressus MV276 202 130 A. pyrethrum var. depressus MV278 36 156 76 108 177 A. pyrethrum var. depressus MV213 60 A. pyrethrum var. depressus MV214 79 83 A. pyrethrum var. depressus MV277 131 155 153 88 173 107 216 7 5 84 24 93 18 37 53 179 Anacyclus pyrethrum KM887358.1 Anacyclus pyrethrum KM887400.1 33 A. pyrethrum var. depressus MV293 15 34 32 Anacyclus depressus KY397478.1 178 68 A. pyrethrum var. depressus AQ3531 29 59 0.03 184 132 35 28 13

15 Figure S6. Tripleurospermum sp. 100 Matricaria discoidea MB2 100 Matricaria chamomilla MV262 Matricaria aurea MV109 Otanthus maritimus MNHNPP04305585 100 Achillea ptarmica 20025048aS Achillea millefolium 2011943w A. clavatus MV323 9 8 100 A. atlanticus MARK7336 A. atlanticus MARK6397 A. maroccanus MV182 100 A. radiatus subsp. radiatus 126066 A. maroccanus MV180 A. maroccanus MV143 100 8 9 A. maroccanus MV195 100 A. maroccanus MB2 7 8 A. maroccanus MV480 A. pyrethrum var. pyrethrum MV468 A. pyrethrum var. pyrethrum MV308 100 A. pyrethrum var. pyrethrum MV224 A. pyrethrum var. pyrethrum MV305 A. pyrethrum var. pyrethrum MV203 7 9A. pyrethrum var. pyrethrum MV222 100 8 2 A. pyrethrum var. pyrethrum MV330 9 3A. pyrethrum var. pyrethrum MV309 A. pyrethrum var. pyrethrum MV344 A. radiatus subsp. radiatus MV51 10A.0 clavatus MV448 100 A. maroccanus MV154 A. homogamos MV101 A. clavatus MV463 A. homogamos MV475 A. homogamos MV103 9 2 A. radiatus subsp. radiatus BC860742 A. radiatus subsp. radiatus 306167 100 A. radiatus subsp. radiatus MV387 A. radiatus subsp. radiatus MV409 A. maroccanus MV234 A. radiatus subsp. radiatus MV433 9 5 A. maroccanus MV257 A. clavatus MV470 A. maroccanus MV490 A. maroccanus MV194 100 A. maroccanus MV241 A. maroccanus MV168 A. clavatus MV472 9 3 A. maroccanus MV143 A. radiatus subsp. radiatus MV391 A. radiatus subsp. radiatus MV392 A. radiatus subsp. radiatus MV388 A. radiatus subsp. radiatus MV403 A. radiatus subsp. radiatus MV413 A. radiatus subsp. radiatus WPT441 A. homogamos MV322 A. maroccanus MV246 A. maroccanus MV489 A. pyrethrum var. depressus MV313 A. pyrethrum var. depressus MV213 7 7 A. pyrethrum var. depressus MV214 A. pyrethrum var. depressus MV188 A. pyrethrum var. depressus AQ3531 A. pyrethrum var. depressus MV272 A. homogamos MV284 A. clavatus MV337 A. homogamos MV366 A. clavatus MV349 A. homogamos MV354 A. homogamos MV358 A. valentinus VIT83639 9 3 A. valentinus VIT88094 A. valentinus VIT78628 A. valentinus VIT91146 A. pyrethrum var. depressus MV278 A. pyrethrum var. depressus MV276 A. pyrethrum var. depressus MV273 A. pyrethrum var. depressus MV277 A. valentinus BC878628 A. pyrethrum var. depressus MV302 A. pyrethrum var. depressus MV303 A. clavatus MV289 A. homogamos MV280 A. pyrethrum var. depressus MV300 7 6 A. pyrethrum var. depressus MV293 100 A. pyrethrum var. depressus IA2334 A. pyrethrum var. depressus 126070 100 A. valentinus WPT512 8 7 A. monanthos MNHNPP03692979 7 8 A. clavatus WPT49 A. valentinus UPNA11846 0.002 9 0 A. clavatus VIT56515

16 Figure S7.

Matricharia chamomilla MV262 Matricaria aurea MV109 100 Achillea millefolium 2011943w 8 1 Achillea ptarmica 20025048aS 8 3 A. clavatus MV323 Otanthus maritimus MNHNPP04305585 100 A. atlanticus MARK6397 A. atlanticus MARK7336 A. maroccanus MV182 100 100 A. maroccanus MV180 A. maroccanus MV195 100 A. maroccanus MV480 A. radiatus subsp. coronatus 126066 A. pyrethrum var. pyrethrum MV224 100 198 100 103 181 9 9 127 A. pyrethrum var. pyrethrum MV203 9 6 2 0 9 0 3 8 8 2 9 9 3 4 100 7 2 9 8 4 A. clavatus MV448 A. clavatus MV463 100 A. maroccanus MV154 9 5 2 7 9 5 A. homogamos MV103 100 A. homogamos MV101 100 9 2 137 6 4 8 7 A. radiatus subsp. radiatus MV409 8A.9 radiatus subsp. radiatus BC860742 100 A. radiatus subsp. coronatus 306167 A. maroccanus MV168 A. maroccanus MV257 8 6 A. maroccanus MV241 A. maroccanus MV234 A. clavatus MV470 A. maroccanus MV490 8 8 8 5 A. maroccanus MV194 A. maroccanus MV194 9 1 8 6 A. maroccanus MV489 A. homogamos MV322 A. radiatus subsp. radiatus MV403 A. radiatus subsp. radiatus WPT441 9 7 A. radiatus subsp. radiatus MV388 8 8 A. radiatus subsp. radiatus MV391 A. radiatus subsp. radiatus MV392 A. radiatus subsp. radiatus MV413 A. clavatus MV472 A. clavatus MV324 180 100 3 3 3 5 1 5 9 9 130 8 0 7 9 A. pyrethrum var. depressus IA2334 A. pyrethrum var. pyrethrum126070 126 9 9 113 9 6 6 5 161 9 9 8 106 196 100 6 9 172 154 7 5 136 8 6 1 9 6 6 165 7 8 3 0 148 2 6 8 4 A. pyrethrum var. depressus MV278 8 9A. pyrethrum var. depressus MV273 9 6 8 6A. pyrethrum var. depressus MV277 100 9 7 A. pyrethrum var. depressus MV276 188 7 5 A. homogamos MV366 A. homogamos MV358 9 4 A. clavatus MV349 A. homogamos MV354 A. monanthos MNHNPP03692979 A. clavatus WPT49 A. clavatus WPT512 9 5 145 7 4 A. clavatus MV337 A. homogamos MV280 8 3 A. clavatus MV289 9 6 160 8 5 A. pyrethrum var. depressus MV313 177 9 4 9 3 100 141 124 216 131 3 2 5 2 2 4 A. pyrethrum var. depressus MV272 1 3 A. pyrethrum var. depressus MV214 153 A. pyrethrum var. depressus AQ3531 8 8 112 108 3 7 1 6 8 2 8 6 0 7 6 0.002 155

17 Figure S8.

Achillea millefolium 2011-94/3w 85 Achillea ptarmica 2002-5048/a-S 61 Anacyclus atlanticus MARK7336 187 99 Anacyclus atlanticus MARK6397 25 Otanthus maritimus MNHN-P-P04305585 41 14 172 165 208 100 178 69 136 154 78 19 79 198 38 127 78 20 Anacyclus radiatus coronatus 126066 22 181 78 103 184 37 180 93 27 64 126 137 Anacyclus radiatus radiatus BC860742 67 Anacyclus radiatus coronatus 306167-1 18 Anacyclus radiatus radiatus WPT44.1 134 34 29 82 153 8 Anacyclus valentinus UPNA11846 164 68 161 80 66 186 33 60 216 88 35 Anacyclus monanthos MNHN-P-P03692979 23 30 15 108 84 7 141 124 160 177 77 106 58 65 Anacyclus valentinus VIT83639 188 131 Anacyclus clavatus VIT56515 183 13 112 130 196 76 113 155 Anacyclus clavatus WPT51.2 182 28 32 Anacyclus pyrethrum var. depressus IA2334 81 26 Tree scale: 0.001 148 52

18 Figure S9.

Achillea millefolium 2011-94/3w 85 Achillea ptarmica 2002-5048/a-S 61 180 35 130 126 216 52 78 8 Anacyclus clavatus VIT56515 183 37 68 196 178 14 13 141 60 112 66 161 148 186 33 28 113 15 32 106 79 100 19 Anacyclus valentinus VIT83639 188 30 26 172 Anacyclus valentinus UPNA11846 164 153 136 155 100 108 177 77 131 Anacyclus clavatus WPT51.2 182 184 Anacyclus radiatus subsp. radiatus WPT44.1 134 127 160 69 181 103 Anacyclus radiatus subsp. coronatus 126066 22 Anacyclus atlanticus MARK6397 25 Anacyclus atlanticus MARK7336 187 Otanthus maritimus MNHN-P-P04305585 41 198 20 38 64 34 208 Anacyclus radiatus subsp. radiatus BC860742 67 Anacyclus radiatus subsp. coronatus 306167-1 18 137 58 154 88 76 Anacyclus monanthos MNHN-P-P03692979 23 93 65 Tree scale: 0.001 165 Anacyclus pyrethrum var. depressus IA2334 81 29 27 7 82 124 84

19 Figure S10.

Achillea ptarmica 2002-5048/a-S 61 155 137 Anacyclus clavatus VIT56515 183 82 208 34 58 Anacyclus valentinus UPNA11846 164 68 161 177 14 106 124 8 65 Anacyclus radiatus subsp. coronatus 126066 22 79 113 160 131 186 196 103 30 154 69 Anacyclus radiatus subsp. radiatus WPT44.1 134 60 Anacyclus clavatus WPT51.2 182 Anacyclus pyrethrum var. depressus IA2334 81 181 127 130 35 33 Anacyclus valentinus VIT83639 188 148 Anacyclus atlanticus MARK7336 187 Anacyclus atlanticus MARK6397 25 93 66 108 180 20 32 78 38 141 77 26 27 37 76 Anacyclus monanthos MNHN-P-P03692979 23 52 88 13 19 Otanthus maritimus MNHN-P-P04305585 41 Achillea millefolium 2011-94/3w 85 112 216 198 184 165 Anacyclus radiatus subsp. radiatus BC860742 67 64 153 172 15 178 136 28 Anacyclus radiatus subsp. coronatus 306167-1 18 100 126 Tree scale: 0.0001 84 29 7

20 Figure S11.

Tree scale: 0.0001

Achillea millefolium 2011-94/3w 85 Achillea ptarmica 2002-5048/a-S 61 Otanthus maritimus MNHN-P-P04305585 41 Anacyclus atlanticus MARK7336 187 93 Anacyclus atlanticus MARK6397 25 Anacyclus valentinus UPNA11846 164 100 52 77 137 Anacyclus radiatus subsp. radiatus WPT44.1 134 82 141 Anacyclus valentinus VIT83639 188 103 181 20 127 38 27 198 Anacyclus radiatus subsp. coronatus 126066 22 33 86 113 Anacyclus radiatus subsp. radiatus BC860742 67 7 34 28 26 106 8 180 79 15 130 35 29 178 186 65 Anacyclus monanthos MNHN-P-P03692979 23 136 14 88 76 177 78 216 93 153 165 60 148 172 32 Anacyclus clavatus WPT51.2 182 68 13 112 37 58 69 160 124 88 184 155 154 131 Anacyclus clavatus VIT56515 183 19 66 208 196 108 30 161 84 Anacyclus pyrethrum var. depressus IA2334 81 Anacyclus radiatus subsp. coronatus 306167-1 18 64 126

21 Figure S12.

Matricaria chamomilla MV262 Matricaria aurea MV109 Matricaria clade Achillea millefolium 2011943w 1 Achillea ptarmica 20025048aS Achillea clade 1 Otanthus maritimus MNHNPP04305585 1 A. atlanticus MARK6397 A. atlanticus 1 A. atlanticus MARK7336 A. monanthos MNHNPP03692979 A. monanthos 1 A. pyrethrum var. pyrethrum MV344 A. pyrethrum var. pyrethrum MV224 1 1 A. pyrethrum var. pyrethrum MV305 A. pyrethrum var. pyrethrum 1 A. pyrethrum var. pyrethrum MV309 1 0.50 A. pyrethrum var. pyrethrum MV308 1 A. pyrethrum var. pyrethrum MV203 A. pyrethrum var. depressus IA2334 A. pyrethrum var. depressus MV214_REF_166 1 A. pyrethrum var. depressus AQ3531 1 A. pyrethrum var. depressus MV276 0.93 1 A. pyrethrum var. depressus MV302 0.55 0.50 A. pyrethrum var. depressus MV313 A. pyrethrum var. depressus 0.63 A. pyrethrum var. depressus MV300 A. pyrethrum var. depressus MV272 A. pyrethrum var. depressus MV273 0.50 0.68 A. pyrethrum var. depressus MV303 A. pyrethrum var. depressus MV277 A. pyrethrum var. depressus MV278 1 A. radiatus subsp. coronatus 126066 A. radiatus subsp. coronatus 3061671 A. radiatus subsp. coronatus A. radiatus subsp. radiatus WPT441 0.69 0.88 1 A. radiatus subsp. radiatus BC860742 A. radiatus subsp. radiatus MV409 1 A. radiatus subsp. radiatus A. radiatus subsp. radiatus MV413 A. radiatus subsp. radiatus MV403 A. radiatus subsp. radiatus MV392 0.57 1 A. radiatus subsp. radiatus MV391 0.63 A. radiatus subsp. radiatus MV387 0.77 A. radiatus subsp. radiatus MV388 A. maroccanus MV168 A. maroccanus MV480 1 A. maroccanus MV257 0.87 A. maroccanus MV489 A. maroccanus MV154 A. maroccanus A. maroccanus MV246 A. maroccanus MV241 A. maroccanus MV490 1 A. maroccanus MV195 0.57 A. maroccanus MV194 A. maroccanus MV234 A. maroccanus MV182 0.93 A. maroccanus MV180 A. clavatus WPT49 1 A. valentinus UPNA11846 1 A. clavatus WPT512 A. clavatus VIT56515 A. homogamos 1 A. homogamos MV366

0.81 A. valentinus VIT83639 1 A. homogamos MV358 0.63 1 A. homogamos MV354 0.54 A. valentinus VIT88094 0.63 A. valentinus VIT91146 0.76 A. valentinus VIT78628 0.91 A. homogamos MV475 A. valentinus 0.87 A. homogamos MV103 1 A. homogamos MV101 0.78 A. clavatus MV463 A. clavatus MV448 0.94 A. clavatus MV472 1 A. clavatus MV470 A. clavatus MV337 A. clavatus MV349 A. clavatus 0.97 A. clavatus MV323 0.52 A. clavatus MV324 0.76 A. clavatus MV289 A. homogamos MV280 A. homogamos MV322 0.9 A. homogamos MV284

22 Figure S13.

ITS 102 identified as Anacyclus sp. (genus level) 0 identified to species level 8 no sequences recovered

With standard barcodes, ITS & Plastome plastomes : 62 identified as Anacyclus sp. (genus level) 1 as Primulaceae, 7 identified to species level 1 as Plumerieae 48 no sequences recovered 9 ID as Plantago Standard barcodes 0-61 identified as Anacyclus sp. (genus level) 0 identified to species level 49-110 no sequences recovered -Anacyclus

11 non shotgun sequencing shotgun sequenced 66 X 25 g 39/66 contain Bulk morphological 110 roots selected 99 Anacyclus sequenced via 10 failed to sequence for Root batches non-Anacyclus sorting for DNA analysis shotgun + capture all methods from trade adulterants 89 worked with target capture

Nuclear Markers 50 identified as A. pyrethrum var depressus 37 identified as A. pyrethrum var. pyrethrum 2 identified as A. homogamos complex

60/89 samples assigned to population of origin

SI Tables

24 Table S1.

subsp. / Index ID during Date GPS Genus Species Collection ID Country var. Herbariorum sequencing collected ° N ° E Achillea millefolium 2011-94/3w O 85 2015 59,92 10,77 Norway Achillea ptarmica 2002-5048/a-S O 61 2015 59,92 10,77 Norway Anacyclus atlanticus MARK6397 MARK 25 16/07/2008 31,03 -7,57 Morocco Anacyclus atlanticus MARK7336 MARK 187 2005 31,28 -7,38 Morocco Anacyclus clavatus MV289 O 121 18/04/2015 33,16 -5,07 Morocco Anacyclus clavatus MV323 O 171 18/04/2015 33,70 -4,84 Morocco Anacyclus clavatus MV324 O 143 18/04/2015 33,70 -4,84 Morocco Anacyclus clavatus MV337 O 145 19/04/2015 34,17 -4,01 Morocco Anacyclus clavatus MV349 O 104 19/04/2015 34,11 -4,05 Morocco Anacyclus clavatus MV448 O 95 22/04/2015 33,28 -5,62 Morocco Anacyclus clavatus MV463 O 122 22/04/2015 33,03 -5,62 Morocco Anacyclus clavatus MV470 O 168 23/04/2015 32,93 -5,56 Morocco Anacyclus clavatus MV472 O 86 23/04/2015 32,93 -5,56 Morocco Anacyclus clavatus VIT 56515 VIT 183 1997 43,30 -2,98 Spain Anacyclus clavatus WPT49.1 O 158 23/06/2015 36,91 -3,48 Spain Anacyclus clavatus WPT51.2 O 182 23/06/2015 37,16 -3,44 Spain Anacyclus homogamos MV101 O 176 04/04/2015 31,16 -7,46 Morocco Anacyclus homogamos MV103 O 200 04/04/2015 31,16 -7,46 Morocco Anacyclus homogamos MV280 O 75 18/04/2015 33,22 -5,06 Morocco Anacyclus homogamos MV284 O 170 18/04/2015 33,16 -5,07 Morocco Anacyclus homogamos MV322 O 48 18/04/2015 33,70 -4,84 Morocco Anacyclus homogamos MV354 O 71 19/04/2015 34,45 -3,91 Morocco Anacyclus homogamos MV358 O 73 19/04/2015 34,78 -3,79 Morocco Anacyclus homogamos MV366 O 2 20/04/2015 34,14 -4,06 Morocco Anacyclus homogamos MV475 O 194 23/04/2015 32,63 -5,97 Morocco Anacyclus maroccanus MV154 O 175 09/04/2015 31,17 -8,08 Morocco Anacyclus maroccanus MV168 O 6 09/04/2015 31,18 -8,09 Morocco Anacyclus maroccanus MV180 O 123 10/04/2015 31,41 -7,95 Morocco Anacyclus maroccanus MV182 O 1 10/04/2015 31,41 -7,95 Morocco Anacyclus maroccanus MV194 O 94 11/04/2015 31,80 -7,15 Morocco Anacyclus maroccanus MV195 O 118 11/04/2015 31,80 -7,15 Morocco Anacyclus maroccanus MV234 O 199 12/04/2015 32,01 -8,45 Morocco Anacyclus maroccanus MV241 O 11 12/04/2015 31,69 -7,98 Morocco Anacyclus maroccanus MV246 O 56 12/04/2015 32,17 -8,17 Morocco Anacyclus maroccanus MV257 O 144 12/04/2015 32,01 -8,45 Morocco Anacyclus maroccanus MV480 O 169 23/04/2015 32,23 -6,67 Morocco Anacyclus maroccanus MV489 O 193 23/04/2015 31,68 -7,76 Morocco Anacyclus maroccanus MV490 O 50 23/04/2015 31,68 -7,76 Morocco Anacyclus monanthos MNHN-P-P03692979 P 23 28/04/1990 35,61 3,94 Algeria Anacyclus pyrethrum depressus AQ3531 MA 109 07/04/2015 38,65 -2,31 Spain Anacyclus pyrethrum depressus IA2334 MA 81 07/04/2015 34,18 -2,54 Morocco Anacyclus pyrethrum depressus MV214 O 166 11/04/2015 31,79 -4,09 Morocco Anacyclus pyrethrum depressus MV272 O 12 18/04/2015 33,39 -5,17 Morocco Anacyclus pyrethrum depressus MV273 O 201 18/04/2015 33,31 -5,11 Morocco Anacyclus pyrethrum depressus MV276 O 10 18/04/2015 33,31 -5,11 Morocco Anacyclus pyrethrum depressus MV277 O 55 18/04/2015 33,31 -5,11 Morocco Anacyclus pyrethrum depressus MV278 O 129 18/04/2015 33,31 -5,11 Morocco Anacyclus pyrethrum depressus MV313 O 167 18/04/2015 33,07 -4,99 Morocco Anacyclus pyrethrum pyrethrum 126070 HUJ 206 20/04/2015 35,41 6,38 Algeria Anacyclus pyrethrum pyrethrum MV203 O 47 11/04/2015 31,82 -6,36 Morocco Anacyclus pyrethrum pyrethrum MV224 O 3 11/04/2015 31,82 -6,39 Morocco Anacyclus pyrethrum pyrethrum MV344 O 146 19/04/2015 34,11 -4,05 Morocco Anacyclus radiatus coronatus 126066 HUJ 22 2001 32,92 35,09 Israel Anacyclus radiatus coronatus 306167-1 HUJ 18 2000 32,35 34,99 Israel Anacyclus radiatus radiatus BC860742 BC 67 2009 43,53 -5,56 Spain Anacyclus radiatus radiatus MV387 O 4 22/04/2015 35,02 -5,47 Morocco Anacyclus radiatus radiatus MV388 O 5 22/04/2015 35,02 -5,47 Morocco Anacyclus radiatus radiatus MV391 O 128 22/04/2015 35,02 -5,47 Morocco Anacyclus radiatus radiatus MV392 O 205 22/04/2015 35,02 -5,47 Morocco Anacyclus radiatus radiatus MV403 O 120 22/04/2015 34,79 -5,55 Morocco Anacyclus radiatus radiatus MV409 O 17 22/04/2015 34,79 -5,55 Morocco Anacyclus radiatus radiatus MV413 O 152 22/04/2015 34,61 -5,53 Morocco Anacyclus radiatus radiatus MV54 O 15/06/2014 31,50 -9,70 Morocco Anacyclus radiatus radiatus WPT44.1 O 134 19.06.2015 36,22 -5,78 Spain Anacyclus valentinus UPNA 11846 UPNA 164 24/05/2008 38,61 -0,05 Spain Anacyclus valentinus VIT 78628 VIT 207 2000 39,75 -0,37 Spain Anacyclus valentinus VIT 83639 VIT 188 2005 37,28 -1,75 Spain Anacyclus valentinus VIT 88094 VIT 159 2011 38,38 -0,51 Spain Anacyclus valentinus VIT 91146 VIT 212 1994 43,33 -2,99 Spain Matricaria aurea MV109 O 151 04/04/2015 31,01 -7,23 Morocco Matricharia chamomilla MV262 O 80 12/04/2015 31,95 -8,24 Morocco Otanthus maritimus MNHN-P-P04305585 P 41 2000 42,00 9,45 Morocco

25 Table S2.

ID during GPS coodinates Molecular identification Collection ID Date collected City Country Type of shop sequencing ° N ° E Genus species subsp. / var. HAS425.2 7 2015 11,66 76,26 Wayanad India Herbalist Anacyclus pyrethrum depressus MV503-1 8 01/06/2015 35,79 -5,81 Tanger Morocco Herbalist Anacyclus pyrethrum pyrethrum MV528.2 9 08/06/2015 33,60 -7,62 Casablanca Morocco Herbalist Anacyclus pyrethrum pyrethrum MV466A.2 13 22/04/2015 32,94 -5,67 Khenifra Morocco Herbalist Anacyclus pyrethrum depressus HAS438.2 14 2015 11,48 79,37 Chidambaram India Herbalist Anacyclus pyrethrum depressus MV536-3.2 15 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum depressus HAS368.3 19 2015 14,47 75,92 Davangere India Herbalist Anacyclus pyrethrum depressus MV501-3 20 01/06/2015 35,78 -5,81 Tanger Morocco Herbalist Anacyclus pyrethrum pyrethrum MV517-3 21 04/06/2015 34,03 -6,84 Rabat Morocco Traditional healer Anacyclus pyrethrum pyrethrum HAS368.1 24 2015 14,47 75,92 Davangere India Herbalist Anacyclus pyrethrum depressus HAS418.1 26 2015 26,21 78,20 Gwalior India Herbalist Anacyclus pyrethrum depressus MV536-6.2 27 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus homogamos MV466A.1 28 22/04/2015 32,94 -5,67 Khenifra Morocco Herbalist Anacyclus pyrethrum depressus HAS418.3 29 2015 26,21 78,20 Gwalior India Herbalist Anacyclus pyrethrum depressus MV536-6.3 30 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus homogamos HAS421.1 32 2015 25,62 85,15 Patna India Herbalist Anacyclus pyrethrum depressus MV539.1 33 01/04/2014 31,73 -7,00 Demnate Morocco Wholesaler Anacyclus pyrethrum depressus MV466B.3 34 22/04/2015 32,94 -5,67 Khenifra Morocco Herbalist Anacyclus pyrethrum depressus HAS421.2 35 2015 25,62 85,15 Patna India Herbalist Anacyclus pyrethrum depressus MV539.4 36 01/04/2014 31,73 -7,00 Demnate Morocco Wholesaler Anacyclus pyrethrum depressus MV506-1.3 37 02/06/2015 33,89 -5,57 Meknes Morocco Traditional healer Anacyclus pyrethrum depressus MV501-1 38 01/06/2015 35,78 -5,81 Tanger Morocco Herbalist Anacyclus pyrethrum pyrethrum HAS408.2 43 2015 11,94 79,49 Villupuram India Herbalist Anacyclus pyrethrum pyrethrum HAS433.1 52 2015 16,95 75,72 peth vadgaon, kolhapur India Herbalist Anacyclus pyrethrum depressus MV510-1 53 03/06/2015 34,07 -4,97 Fès Morocco Traditional healer Anacyclus pyrethrum depressus MV528.3 54 08/06/2015 33,60 -7,62 Casablanca Morocco Herbalist Anacyclus pyrethrum pyrethrum MV466A.3 58 22/04/2015 32,94 -5,67 Khenifra Morocco Herbalist Anacyclus pyrethrum depressus MV536-3.3 60 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum depressus MV536-2.2 64 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV502-2 65 01/06/2015 35,79 -5,81 Tanger Morocco Herbalist Anacyclus pyrethrum pyrethrum MV520-3 66 05/06/2015 34,02 -6,84 Rabat Morocco Wholesaler, Anacyclus pyrethrum pyrethrum HAS398.1.2 68 2015 11,94 79,80 Pondicherry India Herbalist Anacyclus pyrethrum depressus HAS374 69 2015 10,94 76,93 Kovaipudur India Herbalist Anacyclus pyrethrum pyrethrum HAS434.3 76 2015 18,81 73,23 Mumbai India Herbalist Anacyclus pyrethrum depressus MV517-2 77 04/06/2015 34,03 -6,84 Rabat Morocco Traditional healer Anacyclus pyrethrum pyrethrum MV534-2 78 08/06/2015 33,57 -7,60 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV300 79 18/04/2015 33,17 -5,07 Timadith Morocco Collector Anacyclus pyrethrum depressus MV466B.4 82 22/04/2015 32,94 -5,67 Khenifra Morocco Herbalist Anacyclus pyrethrum depressus MV536-4.1 84 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum depressus HAS403.1 88 2015 11,75 79,75 Cuddalore India Herbalist Anacyclus pyrethrum depressus MV502-3 89 01/06/2015 34,79 -5,81 Tanger Morocco Herbalist Anacyclus pyrethrum pyrethrum HAS380.1 93 2015 10,26 78,89 Trichy India Herbalist Anacyclus pyrethrum depressus HAS435.1 100 2015 22,53 88,34 Kolkata India Herbalist Anacyclus pyrethrum depressus MV520-1 101 05/06/2015 34,02 -6,84 Rabat Morocco Wholesaler, Anacyclus pyrethrum pyrethrum MV535-2 102 08/06/2015 33,58 -7,60 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV308 103 18/04/2015 33,17 -5,07 Timadith Morocco Collector Anacyclus pyrethrum pyrethrum HAS398.3 106 2015 11,94 79,80 Pondicherry India Herbalist Anacyclus pyrethrum depressus MV536-4.2 108 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum depressus HAS403.2 112 2015 11,75 79,75 Cuddalore India Herbalist Anacyclus pyrethrum depressus MV503-3 113 01/06/2015 35,79 -5,81 Tanger Morocco Herbalist Anacyclus pyrethrum pyrethrum HAS435.2 124 2015 22,53 88,34 Kolkata India Herbalist Anacyclus pyrethrum depressus MV526-1.1 125 05/06/2015 34,02 -6,83 Rabat Morocco Traditional healer Anacyclus pyrethrum pyrethrum MV536-1.2 126 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV309 127 18/04/2015 33,17 -5,07 Timadith Morocco Collector Anacyclus pyrethrum pyrethrum HAS425.1 130 2015 11,66 76,26 Wayanad India Herbalist Anacyclus pyrethrum depressus MV514-2 131 03/06/2015 34,07 -4,98 Fès Morocco Traditional healer Anacyclus pyrethrum depressus MV540 132 01/04/2014 33,52 -5,12 Ifran Morocco Wholesaler Anacyclus pyrethrum depressus HAS430.2 136 2015 12,91 79,33 Vellore India Herbalist Anacyclus pyrethrum depressus MV505-1 137 01/06/2015 35,78 -5,81 Tanger Morocco Herbalist Anacyclus pyrethrum pyrethrum MV526-1.3 138 05/06/2015 34,02 -6,83 Rabat Morocco Traditional healer Anacyclus pyrethrum pyrethrum HAS387.1 141 2015 8,49 76,95 Thiruvanathapuram India Herbalist Anacyclus pyrethrum pyrethrum HAS438.1 148 2015 11,48 79,37 Chidambaram India Herbalist Anacyclus pyrethrum depressus MV526-1.2 149 05/06/2015 34,02 -6,83 Rabat Morocco Traditional healer Anacyclus pyrethrum pyrethrum MV536-2.1 150 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV303 153 18/04/2015 33,17 -5,07 Timadith Morocco Collector Anacyclus pyrethrum depressus HAS425.4 154 2015 11,66 76,26 Wayanad India Herbalist Anacyclus pyrethrum depressus MV514-3 155 03/06/2015 34,07 -4,98 Fès Morocco Traditional healer Anacyclus pyrethrum depressus MV541.1 156 05/09/2015 31,63 -7,99 Marrakech Morocco Herbalist Anacyclus pyrethrum depressus HAS433.2 160 2015 16,95 75,72 peth vadgaon, kolhapur India Herbalist Anacyclus pyrethrum depressus MV506-2.2 161 02/06/2015 33,89 -5,57 Meknes Morocco Traditional healer Anacyclus pyrethrum pyrethrum MV534-3 162 08/06/2015 33,57 -7,60 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV502-1 165 01/06/2015 35,79 -5,81 Tanger Morocco Herbalist Anacyclus pyrethrum pyrethrum HAS439.1 172 2015 11,66 78,17 Salem India Herbalist Anacyclus pyrethrum depressus MV526-2.2 173 05/06/2015 34,02 -6,83 Rabat Morocco Traditional healer Anacyclus pyrethrum pyrethrum MV536-2.3 174 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV302 177 18/04/2015 33,17 -5,07 Timadith Morocco Collector Anacyclus pyrethrum depressus HAS430.3 178 2015 12,91 79,33 Vellore India Herbalist Anacyclus pyrethrum depressus MV541.3 180 05/09/2015 31,63 -7,99 Marrakech Morocco Herbalist Anacyclus pyrethrum depressus MV305 181 18/04/2015 33,17 -5,07 Timadith Morocco Collector Anacyclus pyrethrum pyrethrum HAS437.1 184 2015 12,95 74,87 Mangalore India Herbalist Anacyclus pyrethrum depressus MV535-3 186 08/06/2015 33,58 -7,60 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV528.1 189 08/06/2015 33,60 -7,62 Casablanca Morocco Herbalist Anacyclus pyrethrum pyrethrum MV536-3.1 196 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum depressus MV542.2 198 05/09/2015 31,63 -7,98 Marrakech Morocco Herbalist Anacyclus pyrethrum pyrethrum HAS434.1 202 2015 18,81 73,23 Mumbai India Herbalist Anacyclus pyrethrum depressus MV520-2 204 05/06/2015 34,02 -6,84 Rabat Morocco Wholesaler Anacyclus pyrethrum pyrethrum HAS437.3 208 2015 12,95 74,87 Mangalore India Herbalist Anacyclus pyrethrum depressus MV536-1.3 210 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum pyrethrum MV536-5.2 216 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anacyclus pyrethrum depressus

ID during GPS coodinates Putative identification (blastn) Collection ID Date collected City Country Type of shop sequencing ° N ° E Family Genus species MV466B.2 31 22/04/2015 32,94 -5,67 Khenifra Morocco Herbalist Plantaginaceae Plantago spp. MV600 39 09/08/2015 9,67 80,02 Jaffna Sri Lanka Traditional healer Plantaginaceae Plantago spp. HAS372.2 40 2015 14,62 74,83 Sirsi India Herbalist Anthemidinae MV601.1 42 15/08/2015 6,95 79,87 colombo Sri Lanka Traditional healer MV507-2 44 02/06/2015 33,89 -5,57 Meknes Morocco Traditional healer Plantaginaceae Plantago spp. MV602 45 09/08/2015 9,67 80,02 Jaffna Sri Lanka Traditional healer Primulaceae Primula spp. MV510-2 59 03/06/2015 34,07 -4,97 Fès Morocco Traditional healer Anthemidinae MV510-3 83 03/06/2015 34,07 -4,97 Fès Morocco Traditional healer Anthemidinae MV521-1 90 05/06/2015 34,03 -6,84 Rabat Morocco Traditional healer Plantaginaceae Plantago spp. MV467.2 92 22/04/2015 32,94 -5,67 Khenifra Morocco Herbalist Plantaginaceae Plantago spp. MV514-1 107 03/06/2015 34,07 -4,98 Fès Morocco Traditional healer Anthemidinae MV521-3 114 05/06/2015 34,03 -6,84 Rabat Morocco Traditional healer Plumerieae Plumeria spp. MV467.3 116 22/04/2015 32,94 -5,67 Khenifra Morocco Herbalist Plantaginaceae Plantago spp. HAS380.2 117 2015 10,26 78,89 Trichy India Herbalist MV526-2.1 179 05/06/2015 34,02 -6,83 Rabat Morocco Traditional healer Anthemidinae MV507-1 185 02/06/2015 33,89 -5,57 Meknes Morocco Traditional healer Plantaginaceae Plantago spp. MV526-2.3 197 05/06/2015 34,02 -6,83 Rabat Morocco Traditional healer MV530 203 08/06/2015 33,60 -7,62 Casablanca Morocco Export company Anthemidinae MV511-1 209 03/06/2015 34,01 -4,97 Fès Morocco Traditional healer Plantaginaceae Plantago spp. MV504-3 213 01/06/2015 34,79 -5,81 Tanger Morocco Herbalist Anthemidinae HAS413.1 215 2015 11,77 79,55 Panruti India Herbalist Plantaginaceae Plantago spp.

Table S3.

Collection Reference Genus Species ID - article Achillea absinthoides AY603213.1 1 Achillea acuminata AY603243.1 1 Achillea ageratum AY603214.1 1 Achillea alpina AY603206.1 1 Achillea distans AY603188.1 1 Achillea asiatica AY603208.1 1 Achillea collina AY603198.1 1 Achillea pratensis AY603199.1 1 Achillea asplenifolia AY603201.1 1 Achillea atrata AY603227.1 1 Achillea ceretanica AY603203.1 1 Achillea clavennae AY603229.1 1 Achillea clusiana AY603228.1 1 Achillea euxina AY603202.1 1 Achillea fraasii AY603232.1 1 Achillea holosericea AY603222.1 1 Achillea lanulosa AY603205.1 1 Achillea monticola AY603210.1 1 Achillea oxyloba AY603238.1 1 Achillea pannonica AY603184.1 1 Achillea pyrenaica AY603247.1 1 Achillea roseoalba AY603200.1 1 Achillea salicifolia AY603244.1 1 Achillea styriaca AY603190.1 1 Achillea virescens AY603211.1 1 Achillea wilsoniana AY603207.1 1 Anacyclus clavatus AY603258.1 1 Anacyclus monanthos KT954174.1 2 Anacyclus pyrethrum KM887358.1 3 Anacyclus pyrethrum KM887396.1 3 Anacyclus pyrethrum KM887397.1 3 Anacyclus pyrethrum KM887400.1 3 Heliocauta atlantica AJ748782.1 4 Matricaria aurea KT954177.1 2 Otanthus maritimus AY603257.1 1 Tanacetum argenteum AB683265.1 5 Tanacetum aucherianum AB683268.1 5 Tanacetum bachtiaricum AB683269.1 5 Tanacetum balsamita AB683270.1 5 Tanacetum bamianicum AB683271.1 5 Tanacetum budjnurdense AB608330.1 5 Tanacetum canescens AB608331.1 5 Tanacetum chiliophyllum AB608332.1 5 Tanacetum demetrii AB683278.1 5 Tanacetum dumosum AB683281.1 5 Tanacetum elbursense AB683282.1 5 Tanacetum germanicopolitanum AB683285.1 5 Tanacetum griffithii AB683286.1 5 Tanacetum haradjanii AB683287.1 5 Tanacetum haussknechtii AB683288.1 5 Tanacetum hololeucum AB683289.1 5 Tanacetum joharchii AB523746.1 5 Tripleurospermum sp. KR150169.1 6

28 Table S4

Table S4 Table S4 Table S4. Target enrichment Genome skimming Before Trimming After trimming Before Trimming After trimming Target enrichment Genome skimming Upper whisker 7.20 6.30 6.80 6.20 Before TrimmingTarget enrichmentAfter trimming Before TrimmingGenome skimmingAfter trimming 3rd quartile 4.20 3.70 4.10 3.60 Upper whisker Before Trimming7.20 After trimming6.30 Before Trimming6.80 After trimming6.20 Median 3.10 2.60 2.90 2.50 3rdUpper quartile whisker 7.204.20 6.303.70 6.804.10 6.203.60 1st quartile 2.20 1.90 2.20 1.80 Median3rd quartile 4.203.10 3.702.60 4.102.90 3.602.50 Lower whisker 0.20 0.20 0.40 0.10 1stMedian quartile 3.102.20 2.601.90 2.902.20 2.501.80 Nr. of data points 182 182 182 182 Lower1st quartile whisker 2.200.20 1.900.20 2.200.40 1.800.10 Total reads 626.5 542.6 587.2 507.2 Nr.Lower of data whisker points 0.20182 0.20182 0.40182 0.10182 TotalNr.Average of reads data points 626.51823.46 542.61822.99 182587.23.24 182507.22.80 AverageTotal reads 626.53.46 542.62.99 587.23.24 507.22.80 Average 3.46 2.99 3.24 2.80

Table S5 Table S5 Table S5 Table S5.

Coverage Alignment ReferenceCoverageCoverage and trade samples ReferenceAlignmentAlignment and trade samples TargetReferenceReference capture andand trade tradeGenome samples samples skimming ReferenceReferencelength (bp) and and trade trade samples missingsamples data (%) Target capture Genome skimming length (bp) missing data (%) Nuclear markers Target capture303 Genome skimming12 length289.236 (bp) missing data1.2% (%) NuclearNuclearPlastome markersmarkers 303303- 121220 289.236289.236110.003 1.2%1.2%2.1% PlastomePlastomenrDNA -- - 2020131 110.003110.003633 2.1%2.1%2.7% nrDNAnrDNAmatk -- - 131131 6336331523 2.7%2.7%0.0% matkrbcl - - 15231438 0.0%0.0% matk - 20 1523 0.0% rbclrbcltrnH-psbA -- - 14381438500 0.0%0.0%0.3% 2020 trnH-psbAtrnH-psbAtrnL -- - 500500947 0.3%0.3%4.6% trnLtrnL -- 947947 4.6%4.6%

Table S6 Table S6. TableTable S6 S6

A. pyrethrum var. A. pyrethrum var. Adulteration A.A. pyrethrum pyrethrum var. var. A.A. pyrethrum pyrethrum var. var. A. homogamos Adulteration pyrethrum depressus A.A. homogamos homogamos pyrethrumpyrethrum depressusdepressus Collectors 0% 63% 38% 0% CollectorsCollectors 0%0% 63%63% 38%38% 0%0% Export company 0% 50% 38% 13% ExportExport companycompany 0%0% 50%50% 38%38% 13%13% Herborist - India 22% 14% 63% 0% HerboristHerborist -- IndiaIndia 22%22% 14%14% 63%63% 0%0% Herborist - Morocco 32% 46% 22% 0% HerboristHerborist -- MoroccoMorocco 32%32% 46%46% 22%22% 0%0% Traditional healer - Morocco 57% 22% 21% 0% TraditionalTraditional healerhealer - Morocco 57%57% 22%22% 21%21% 0%0% Wholesaler 2% 31% 67% 0% WholesalerWholesaler 2%2% 31%31% 67%67% 0%0%

Table S7 TableTable S7S7 29 N.A.N.A. GenusGenus SpeciesSpecies PopulationPopulation percentage N.A.# indiv. percentage Genus# indiv. percentage Species# indiv. percentagePopulation# indiv. percentagepercentage # indiv.# indiv. percentagepercentage # indiv.# indiv. percentagepercentage # indiv.# indiv. percentagepercentage# indiv.# indiv. rbcl 100% 110 0% 0 0% 0 0% 0 rbcl 100% 110 0% 0 0% 0 0% 0 rbcltrnH-psbA 100%100% 110110 0%0% 0 0 0%0% 0 0 0%0% 0 0 trnH-psbA 100% 110 0% 0 0% 0 0% 0 trnH-psbAmatk 100%45% 49110 55%0% 610 0%0% 0 0 0%0% 0 0 matk 45% 49 55% 61 0% 0 0% 0 matktrnL 54%45% 5949 46%55% 5161 0%0% 0 0 0%0% 0 0 trnL 54% 59 46% 51 0% 0 0% 0 trnLITS 54%7% 859 93%46% 10251 0%0% 0 0 0%0% 0 0 ITSPlastomeITS 44%7%7% 488 8 56%93%93% 62102102 6%0%0% 7 0 0 0%0% 0% 0 0 0 PlastomeNuclearPlastome markers 44%9%44% 104848 81%56%56% 8962 62 81%6%6% 89 7 7 55%0% 0% 60 0 0 NuclearNuclear markers markers 9%9% 1010 81%81% 89 89 81%81% 89 89 55%55% 60 60 Table S4

Target enrichment Genome skimming Before Trimming After trimming Before Trimming After trimming Upper whisker 7.20 6.30 6.80 6.20 3rd quartile 4.20 3.70 4.10 3.60 Median 3.10 2.60 2.90 2.50 1st quartile 2.20 1.90 2.20 1.80 Lower whisker 0.20 0.20 0.40 0.10 Nr. of data points 182 182 182 182 Total reads 626.5 542.6 587.2 507.2 Average 3.46 2.99 3.24 2.80

Table S5

Coverage Alignment Reference and trade samples Reference and trade samples Target capture Genome skimming length (bp) missing data (%)

Nuclear markers 303 12 289.236 1.2% Plastome - 20 110.003 2.1% nrDNA - 131 633 2.7% matk - 1523 0.0% rbcl - 1438 0.0% 20 trnH-psbA - 500 0.3% trnL - 947 4.6%

Table S6

A. pyrethrum var. A. pyrethrum var. Adulteration A. homogamos pyrethrum depressus Collectors 0% 63% 38% 0% Export company 0% 50% 38% 13% Herborist - India 22% 14% 63% 0% Herborist - Morocco 32% 46% 22% 0% Traditional healer - Morocco 57% 22% 21% 0% Wholesaler 2% 31% 67% 0%

Table S7. Table S7

N.A. Genus Species Population percentage # indiv. percentage # indiv. percentage # indiv. percentage # indiv. rbcl 100% 110 0% 0 0% 0 0% 0 trnH-psbA 100% 110 0% 0 0% 0 0% 0 matk 45% 49 55% 61 0% 0 0% 0 trnL 54% 59 46% 51 0% 0 0% 0 ITS 7% 8 93% 102 0% 0 0% 0 Plastome 44% 48 56% 62 6% 7 0% 0 Nuclear markers 9% 10 81% 89 81% 89 55% 60