Table S1. List of universal primers and PCR ...

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Table S1. List of universal primers and PCR conditions for each candidate barcode tested in the study

Marker

Primer Name

rbcL

a_f

Direction

Sequence 5'-3'

1

Forward

ATGTCACCACAAACAGAGACTAAAGC

26

Reverse

GTAAAATCAAGTCCACCRCG

20

Forward

CGTACAGTACTTTTGTGTTTACGAG

25

Reverse

ACCCAGTCCATCTGGAAATCTTGGTTC

27

Forward

CGATCTATTCATTCAATATTTC

22

Reverse

TCTAGCACACGAAAGTCGAAGT

22

Forward

CTGGATYCAAGATGCTCCTT

20

Reverse

GGTCTTTGAGAAGAACGGAGA

21

a_rev

matK_a

3F_KIM

2

1R_KIM matk_b

390f

3

1326r matK_c

NY552 NY1150

1.

Length (bp)

Source

4

PCR conditions 95°C 10 min (94°C 30s, 55°C 30s,72°C 1min) x 35 cycles 72°C 10min Protocol 1 95°C 10 min (94°C 30s, 55°C 30s,72°C 1min) x 35 cycles 72°C 10min Protocol 2 95°C 10 min (94°C 30sec, 55°C 1min,72°C 1min30s) x 35 cycles 72°C 10min Protocol 3 95°C 10 min (94°C 30s, 54°C 30s,72°C 45s) x 35 cycles 72°C 10min Protocol 4 95°C 10 min (94°C 30s, 52°C 45s,72°C 45s) x 35 cycles 72°C 10min

1. Kress WJ, Erickson DL (2007) A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH psbA Spacer Region. PLoS ONE 2(6): e508. doi:10.1371/journal.pone.0000508 *David Erickson’s modified reverse primer (different from the PLOS One paper) 2. CBOL Plant Working Group (2009) A DNA barcode for land plants. PNAS 106, 12794-12797. 3.Cuénoud, P., Savolainen, V., Chatrou, L. W., Powell, M., Grayer, R. J. and Chase, M. W. 2002. Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA 3. and plastid rbcL, atpB, and matK DNA sequences. Am. J. Bot. 89, 132-144. 4. 4. In gymnosperms D.Little (unpublished)


Table S2 List of plant species and GenBank accession numbers Family

Species Scientific name

Amaranthaceae Apiaceae Apocynaceae Apocynaceae Araliaceae Arecaceae Asteraceae Asteraceae Asteraceae Berberidaceae Boraginaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Buxaceae Cannabaceae Caprifoliaceae Caprifoliaceae Caprifoliaceae Caryophyllaceae Cupressaceae Cupressaceae Fabaceae Fabaceae Fagaceae Fagaceae Fagaceae Ginkgoaceae Lamiaceae Lamiaceae Lauraceae Magnoliaceae Magnoliaceae Oleaceae Oleaceae Oxalidaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pittosporaceae Plantaginaceae Poaceae Poaceae Poaceae

Gomphrena Globosa Daucus Carota Nerium oleander Vinca minor Hedera helix Trachycarpus fortunei Cichorium intybus Senecio vulgaris Taraxacum officinale Mahonia aquifolium Pulmonaria officinalis Brassica oleracea Brassica napus Capsella bursa-pastoris Cardamine hirsuta Erophila verna Buxus sempervirens Cannabis sativa Lonicera xylosteum Viburnum lantana Viburnum tinus Cerastium glomeratum Chamaecyparis lawsoniana Calocedrus decurrens Albizia julibrissin Trifolium pratense Quercus robur Quercus rubra Quercus petraea Ginkgo biloba Rosmarinus officinalis Lamium purpureum Laurus nobilis Liriodendron tulipifera Magnolia grandiflora Ligustrum vulgare Olea europaea Oxalis incarnata Abies alba Cedrus atlantica Cedrus deodara Picea abies Pinus nigra Pinus sylvestris Pittosporum tobira Plantago major Chimonobambusa quadrangularis Bromus sterilis Poa annua

GenBank Accession no. Common name

rbcL

matK

Globe amaranth Carrot Oleander Lesser Periwinkle Common Ivy Windmill Palm Common Chicory Common Groundsel Dandelion Oregon Grapeholly Common Lungwort Wild Mustard Rapeseed Shepherd's-purse Hairy Bittercress Common Whitlow Grass Common Box Marjiuana Fly Honeysuckle Wayfaring Tree Laurustinus Sticky Chickweed Lawson Cypress Incense Cedar Silk Tree Red Clover English Oak Northern Red Oak Sessile Oak Common Ginkgo Rosemary Red Deadnettle Bay Laurel Tulip Tree Southern Magnolia Wild Privet Olive Pale Pink-Sorrel Silver Fir Atlas Cedar Deodar Cedar Norway Spruce Black Pine Norway Pine Japanese Cheesewood Common Plantain Bamboo Barren Brome Annual Blue Grass

HQ619725 HQ619726 HQ619727 HQ619728 HQ619729 HQ619730 HQ619731 HQ619732 HQ619733 HQ619734 HQ619735 HQ619736 HQ619737 HQ619738 HQ619739 HQ619740 HQ619741 HQ619742 HQ619743 HQ619744 HQ619745 HQ619747 HQ619746 HQ619748 HQ619749 HQ619750 HQ619751 HQ619752 HQ619753 HQ619754 HQ619755 HQ619756 HQ619757 HQ619758 HQ619759 HQ619760 HQ619761 HQ619762 HQ619763 HQ619764 HQ619765 HQ619766 HQ619767 HQ619768 HQ619769 HQ619770 HQ619771 HQ619772

HQ619789 HQ619790 HQ619791 HQ619792 HQ619793 HQ619794 HQ619795 HQ619796 HQ619797 HQ619798 HQ619799 HQ619800 HQ619801 HQ619802 HQ619803 HQ619804 HQ619805 HQ619806 HQ619807 HQ619808 HQ619809 HQ619810 HQ619811 HQ619812 HQ619813 HQ619814 HQ619815 HQ619816 HQ619817 HQ619818 HQ619819 HQ619820 HQ619821 HQ619822 HQ619823 HQ619824 HQ619825 HQ619826 HQ619827 HQ619828

Table S2 (continued) Family

Species Scientific name

Primulaceae Ranunculaceae Ranunculaceae Rosaceae Rosaceae Rosaceae Rosaceae Rubiaceae Salicaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Solanaceae Solanaceae Solanaceae Taxaceae

Primula vulgaris Helleborus viridis Ranunculus bulbosus Crataegus monogyna Fragaria vesca Prunus cerasifera Prunus laurocerasus Galium mollugo Populus nigra Veronica arvensis Veronica sublobata Veronica persica Nicotiana tabacum Solanum lycopersicum Solanum tuberosum Taxus baccata

GenBank Accession no. Common name

rbcL

matK

Wild Primrose Green Hellebore Common Buttercup Common Hawthorn Woodland Strawberry Cherry Plum Cherry Laurel White Bedstraw Black Poplar Corn Speedwell Ivy Speedwell Bird-eye Speedwell Tobacco Tomato Potato Common Yew

HQ619773 HQ619774 HQ619775 HQ619776 HQ619777 HQ619778 HQ619779 HQ619780 HQ619781 HQ619782 HQ619783 HQ619784 HQ619785 HQ619786 HQ619787 HQ619788

HQ619829 HQ619830 HQ619831 HQ619832 HQ619833 HQ619834 HQ619835 HQ619836 HQ619837 HQ619838 HQ619839 HQ619840 HQ619841 HQ619842


Table S3 List of plant species and corresponding GenBank accession numbers retrieved from the database for sequence diversity analysis at the genus level for rbcL gene. Overall, 28 genera (265 sequences) for rbcL were available from GenBank database for alignment and subsequent statistical computation. Family

Species

Asteraceae

Senecio polypodioides

Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae

Senecio mikanioides Senecio medley-woodii Senecio scandens Senecio aquaticus Senecio deltoideus Brassica juncea Brassica rapa Viburnum sp. SH-2010 Viburnum acerifolium Viburnum opulus Viburnum lentago Viburnum cotinifolium Cerastium fontanum Cerastium capense Albizia lebbeck Albizia saman Trifolium repens Trifolium burchellianum Trifolium subterraneum Quercus virginiana Quercus macrocarpa Quercus cf. velutina Quercus alba Quercus frainetto Quercus pubescens Quercus crenata Quercus ithaburensis Quercus suber Quercus trojana Quercus cerris Quercus ilex Quercus coccifera Quercus nigra Quercus hemisphaerica Quercus gemelliflora Quercus palustris Quercus oidocarpa Quercus lyrata Quercus argentata Quercus mongolica Quercus hondae Quercus miyagii

Brassicaceae Brassicaceae Caprifoliaceae Caprifoliaceae Caprifoliaceae Caprifoliaceae Caprifoliaceae Caryophyllaceae Caryophyllaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae

Genbank Accession No. rbcL EU385017 L13933 GU817783 GQ436472 AY395561 GU817782 GQ436652 GQ184371 AB586172 L01959 EU677071 EU677069 GQ248707 FJ395574 AM234949 GU135158 Z70149 GQ436346 AM235018 EU849487 AF119175 EU676971 EU676969 EU676968 FN675742 FN675737 FN675732 FN675729 FN675728 FN675725 FN675724 FN675719 FN675715 EU002284 AF119176 AB125019 AB125023 AB125022 AB125021 AB125016 AB060584 AB060583 AB060582

Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Oleaceae Oleaceae Oleaceae Oleaceae Oxalidaceae Oxalidaceae Oxalidaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae

Quercus aliena Quercus acuta Quercus dentata Quercus acutissima Quercus sessilifolia Quercus gilva Quercus variabilis Quercus phillyraeoides Quercus myrsinifolia Quercus glauca Quercus salicina Lamium album Lamium amplexicaule Lamium maculatum Lamium garganicum Lamium galeobdolon Magnolia tripetala Magnolia soulangeana Magnolia salicifolia Magnolia hypoleuca Magnolia kobus Magnolia virginiana Magnolia guatemalensis Magnolia macrophylla Ligustrum sp. SH-2010 Ligustrum lucidum Ligustrum sinense Ligustrum quihoui Oxalis dillenii Oxalis latifolia Oxalis corniculata Abies hidalgensis Abies lasiocarpa Abies bracteata Abies magnifica Abies numidica Abies pinsapo Abies nordmanniana Abies nebrodensis Abies procera Abies amabilis Abies concolor Abies grandis Abies fraseri Abies nephrolepis Abies koreana Abies sibirica Abies holophylla Abies fargesii Abies fabri

AB060581 AB060580 AB060579 AB060578 AB060577 AB060575 AB060574 AB060573 AB060572 AB060571 AB060570 FJ395588 AB266225 Z37402 Z37401 Z37400 AF206791 M58393 L12656 L12655 AY743438 GQ248639 GQ248638 X54345 AB586459 GU135151 GU135150 GQ436544 L01938 EU002282 AB233943 EU269028 AY664855 AF456380 EU331458 AB019827 AB029656 AB029654 AB029653 AB029651 AB029650 AB029648 AB029646 AB029644 AB029643 AB029642 AB029641 AB029640 AB029639 AB029638

Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Pinaceae Plantaginaceae Plantaginaceae Plantaginaceae Plantaginaceae Plantaginaceae Plantaginaceae Poaceae

Abies sachalinensis Abies mariesii Abies veitchii Picea mariana Picea crassifolia Picea glehnii Picea Koyamae Picea Meyeri Picea Likiangensis Picea Obovata Picea Omorika Picea Retroflexa Picea Rubens Picea Torano Picea koraiensis Picea jezoensis Picea asperata Picea wilsonii Picea spinulosa Picea schrenkiana Picea pungens Picea morrisonicola Picea farreri Picea brachytyla Picea sitchensis Picea martinezii Picea alcoquiana Picea chihuahuana Picea breweriana Picea orientalis Picea mexicana Pinus tabuliformis Pinus thunbergii Pinus densiflora Pinus kesiya Pinus yunnanensis Pinus hwangshanensis Pinus halepensis Pinus echinata Pinus praetermissa Pinus taeda Pinus leiophylla Pinus contorta Plantago lanceolata Plantago depressa Plantago asiatica Plantago rugelii Plantago ovata Plantago crassifolia Bromus hordeaceus

AB015651 AB015650 AB015649 EF440585 EF440576 EF440580 EF440583 EF440589 EF440590 EF440592 EF440593 EF440599 EF440600 EF440605 EF440582 EF440581 EF440572 EF440606 EF440604 EF440601 EF440597 EF440591 EF440578 EF440573 EF440602 EF440586 EF440571 EF440575 EF440574 EF440594 EF440588.1| AY555717 FJ899562 DQ353731 AY497253 AY555695 FJ906722 FN689375 AY947435 DQ353727 AF119177.1 AB063380 AY497230 L36454 GQ436318 GQ436317 EU676937 GQ248675 AM235049 AY395531

Poaceae Poaceae Poaceae Poaceae Poaceae Poaceae Poaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rubiaceae

Bromus arvensis Bromus arenarius Bromus interruptus Bromus commutatus Bromus erectus Poa compressa Poa trivialis Primula veitchiana Primula cortusoides Primula sikkimensis Primula gaubaeana Primula edelbergii Primula palinuri Primula miyabeana Primula hidakana Primula floribunda Primula cusickiana Primula dryadifolia Primula malacoides Primula alpicola Primula bulleyana Primula chungensis Primula veris Primula modesta Primula ioessa Primula capitata Primula muscarioides Primula verticillata Primula cuneifolia Helleborus orientalis Helleborus thibetanus Helleborus atrorubens Helleborus croaticus Ranunculus lyallii Ranunculus ficaria Ranunculus acris Ranunculus trichophyllus Ranunculus macranthus Crataegus pinnatifida Crataegus columbiana Prunus armeniaca Prunus sibirica Prunus tomentosa Prunus domestica Prunus persica Prunus davidiana Prunus mume Prunus pseudocerasus Prunus padus Galium aparine

AY836154 AY691632 GQ248558 AJ746287 AJ746286 EU676942 AJ746301 AF213802 AF213800 AF213799 AF213798 AF213797 AF213801 AF395003 AF394998 AF394996 AF394995 AF394994 AF394990 AF394987 AF394985 AF394984 AF394982 AF394978 AF394972 AF394971 AF394970 AF394968 AF394962 EU053915 AY954485 GQ248616 GQ248617 EU053921 EU053919 AY395557 L08766 DQ069502 GU363811 U06799 AF411489 GQ436604 AF411495 AF227901 GQ436598 GQ436591 AF411491 GQ436608 GU363800 X81091

Rubiaceae Rubiaceae Rubiaceae Rubiaceae Rubiaceae Rubiaceae Rubiaceae Salicaceae Salicaceae Salicaceae Salicaceae Salicaceae Salicaceae Salicaceae Salicaceae Salicaceae Salicaceae Salicaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae

Galium parisiense Galium corsicum Galium album Galium scabrum Galium palustre Galium lucidum Galium elongatum Populus tremuloides Populus deltoides Populus cathayana Populus alba Populus grandidentata Populus balsamifera Populus tremula Populus tomentosa Populus szechuanica Populus maximowiczii Populus wilsonii Veronica salicifolia Veronica arguta Veronica officinalis Veronica vandewateri Veronica anagallis-aquatica Veronica melanocaulon Veronica salicornioides Veronica odora Veronica macrantha Veronica formosa Veronica cupressoides Veronica benthamii Veronica elliptica Veronica nivea Veronica raoulii Veronica quadrifaria Veronica densifolia Nicotiana petunioides Nicotiana noctiflora Nicotiana sylvestris Nicotiana debneyi Nicotiana tomentosiformis Solanum nigrum Solanum laciniatum Solanum lepidotum Solanum torvum Solanum tampicense Solanum viarum Solanum jamaicense Solanum diphyllum Solanum dulcamara Solanum panduriforme

X81102 X81096 X81090 X81105 X81101 X81100 X81098 AF206812 AF133677 FJ788565 FN689368 EU676961 EU676955 GQ248681 AF527489 AJ418837 AJ418836 AJ418830 AF307921 AY034025 AY034024 AY034023 AY034021 AY034020 AY034019 AY034017 AY034016 AY034015 AY034014 AY034013 AY034011 AY034010 AY034009 AY034008 AY034007 GQ248656 GQ248655 AB237912 D70815 NC_007602 GQ436617 GQ436613 GQ981879 GU135251 GU135238 GU135237 GU135229 GU135208 EU677012 EU213515

Taxaceae Taxaceae Taxaceae Taxaceae Taxaceae

Taxus wallichiana Taxus fuana Taxus canadensis Taxus cuspidata Taxus brevifolia

GQ436390 GQ436389 GQ436388 GQ436387 AF249666


Table S4 List of plant species and corresponding GenBank accession numbers retrieved from the database for sequence diversity analysis at the genus level for matK gene. Overall, 21 genera (276 sequences) for matK were available from GenBank database for alignment and subsequent statistical computation. Family

Species

Amaranthaceae

Gomphrena ferruginea Gomphrena fuscipellita

Amaranthaceae Amaranthaceae Amaranthaceae Amaranthaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Caprifoliaceae Caprifoliaceae Caprifoliaceae Caprifoliaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae

Gomphrena pulchella Gomphrena mandonii Gomphrena haageana Brassica insularis Brassica rapa Brassica incana Brassica montana Brassica hilarionis Brassica cretica Brassica villosa Brassica rupestris Brassica macrocarpa Brassica barrelieri Brassica carinata Brassica nigra Cardamine rivularis Cardamine penzesii Cardamine amara Lonicera maackii Lonicera japonica Lonicera nervosa Lonicera orientalis Albizia lebbeck Albizia harveyi Albizia bermudiana Albizia versicolor Albizia sinaloensis Albizia kalkora Albizia plurijuga Albizia myriophylla Trifolium wormskioldii Trifolium willdenovii Trifolium thalii Trifolium subterraneum Trifolium striatum Trifolium spumosum Trifolium semipilosum Trifolium repens Trifolium patulum Trifolium nanum Trifolium microcephalum Trifolium lupinaster

Genbank Accession No. matK AM887524 AM887525 AY514802 AY514801 AY514800 AY541614 AB354276 AY541613 AY541617 AY541612 AY541611 AY541622 AY541620 AY541615 AB354270 AB354275 AB354272 AF144365 AF144364 AF144337 GU168651 GQ997392 AF446903 AJ430196 EU812047 EU812003 EU812001 AF274210 AF274121 AF523083 AF523080 AB504374 AF522138 AF522137 AF522136 AF522135 AF522134 AF522133 AF522132 AF522131 AF522130 AF522129 AF522128 AF522127

Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Fagaceae Lamiaceae Lamiaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae

Trifolium incarnatum Trifolium hybridum Trifolium hirtum Trifolium gracilentum Trifolium fragiferum Trifolium cheranganiense Trifolium caucasicum Trifolium burchellianum Trifolium resupinatum Trifolium albopurpureum Trifolium beckwithii Quercus tomentella Quercus suber Quercus myrsinifolia Quercus nigra Quercus mongolica Quercus gemelliflora Quercus hondae Quercus palustris Quercus oidocarpa Quercus lyrata Quercus ilex Quercus coccifera Quercus cerris Quercus argentata Quercus miyagii Quercus aliena Quercus acuta Quercus dentata Quercus acutissima Quercus sessilifolia Quercus gilva Quercus variabilis Quercus phillyraeoides Quercus glauca Quercus salicina Lamium maculatum Lamium album Magnolia dealbata Magnolia dodecapetala Magnolia mexicana Magnolia splendens Magnolia wilsonii Magnolia officinalis Magnolia gigantifolia Magnolia pterocarpa Magnolia liliifera Magnolia henryi Magnolia panamensis Magnolia sinica Magnolia pacifica

AF522126 AF522125 AF522124 AF522123 AF522122 AF522120 AF522119 AF522118 AF522117 AF522116 AY386946 FJ185058 FJ185049 FJ185048 EU002188 AB107616 AB125036 AB060074 AB125040 AB125039 AB125038 AB125037 AB125035 AB125034 AB125033 AB060077 AB060073 AB060071 AB060070 AB060069 AB060068 AB060066 AB060065 AB060064 AB060062 AB060061 AF531780 AJ429332 AY009008 AY009005 AY009004 AY009003 AY009002 AY009001 AY009000 AY008999 AY008998 AY008997 AY008996 AY008990 AB055521

Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Oxalidaceae Pinaceae Pinaceae Pinaceae Pinaceae Plantaginaceae Plantaginaceae Plantaginaceae Poaceae Poaceae Poaceae Poaceae Poaceae Poaceae Poaceae Poaceae Poaceae Poaceae Poaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae

Magnolia yoroconte Magnolia portoricensis Magnolia sieboldii Magnolia carsonii Magnolia caricifragrans Magnolia minor Oxalis eckloniana Oxalis tomentosa Oxalis bifida Oxalis capillacea Oxalis polyphylla Oxalis tenella Oxalis flava Oxalis melanosticta Oxalis caprina Oxalis dentata Oxalis purpurascens Oxalis corniculata Oxalis hedysaroides Abies firma Abies holophylla Abies bracteata Abies hidalgensis Plantago rugelii Plantago media Plantago argentea Bromus suksdorfii Bromus inermis Bromus secalinus Bromus erectus Poa billardierei Poa andina Poa alpina Poa compressa Poa pratensis Poa violacea Poa bulbosa Primula veris Primula edelbergii Primula borealis Primula nutans Primula eximia Primula faberi Primula amethystina Primula dryadifolia Primula forbesii Primula capillaris Primula cusickiana Primula angustifolia Primula rusbyi Primula parryi

AB055522 AB055523 AB055526 AB055529 AB055533 AB055535 EU437359 EU437358 EU437357 EU437356 EU437355 EU437354 EU437348 EU437346 EU437342 EU437341 EU437340 EU437337 FM179935

AF143436 AF143441 AF456365 EU269026 DQ317070 AY667474 AJ429344 DQ786894 AF164398 AM23457 AM234570 DQ786935 DQ786934 DQ786933 EU749333 AF164402 AM234605 AM234594 AY647530 AY647528 AY647527 AY647526 AY647525 AY647524 AY647523 AY647521 AY647520 AY647519 AY647518 AY647514 AY647513 AY647512

Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae

Primula suffrutescens Primula nipponica Primula cuneifolia Primula integrifolia Primula hirsuta Primula deorum Primula glutinosa Primula minima Primula marginata Primula clusiana Primula palinuri Primula septemloba Primula aromatica Primula blattariformis Primula partschiana Primula pycnoloba Primula malvacea Primula saturata Primula wangii Primula barbicalyx Primula calyptrata Primula chapaensis Primula rugosa Primula amethystina Primula sikkimensis Primula capitata Primula caveana Primula smithiana Primula geraniifolia Primula obconica Primula sinolisteri Primula sinomollis Primula helodoxa Primula malacoides Primula celsiiformis Primula blinii Primula polyneura Primula deflexa Primula bellidifolia Primula boreiocalliantha Primula poissonii Primula calliantha Primula serratifolia Primula efarinosa Primula watsonii Primula moupinensis Primula secundiflora Primula interjacens Primula latisecta Primula celsiiformis Primula alpicola

AY647510 AY647508 AY647506 AY647501 AY647499 AY647498 AY647496 AY647494 AY647492 AY647491 AY647489 FJ828656 FJ828655 FJ828654 FJ828653 FJ828652 FJ828651 FJ828650 FJ828649 FJ828647 FJ828646 FJ828645 FJ828644 FJ828643 FJ828642 FJ828641 FJ828640 FJ828639 FJ828638 FJ828636 FJ828635 FJ828634 FJ828633 FJ828631 FJ828629 FJ828628 FJ828627 FJ828626 FJ828621 FJ828620 FJ828619 FJ828618 FJ828617 FJ828616 FJ828615 FJ828614 FJ828613 FJ828610 FJ828608 FJ828607 FJ828606

Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Primulaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Ranunculaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae

Primula ovalifolia Primula sertulum Primula pinnatifida Primula yunnanensis Primula chionantha Primula bella Primula rupestris Primula cicutariifolia Primula algida Primula partschiana Primula caldaria Helleborus orientalis Helleborus multifidus Helleborus thibetanus Ranunculus sardous Ranunculus polyanthemoides Ranunculus brutius Ranunculus submarginatus Ranunculus pensylvanicus Ranunculus weyleri Ranunculus polyanthemos Ranunculus fascicularis Ranunculus macounii Ranunculus repens Ranunculus septentrionalis Ranunculus multifidus Ranunculus muricatus Ranunculus peduncularis Prunus lusitanica Prunus cerasus Prunus domestica Prunus avium Prunus virginiana Prunus persica Prunus dulcis Nicotiana goodspeedii Nicotiana x sanderae Nicotiana attenuata Nicotiana alata Nicotiana langsdorfii Nicotiana bonariensis Nicotiana occidentalis Nicotiana simulans Nicotiana umbratica Nicotiana benthamiana Nicotiana exigua Nicotiana nesophila Nicotiana plumbaginifolia Nicotiana longiflora Nicotiana forgetiana Nicotiana megalosiphon

FJ828605 FJ828604 FJ828603 FJ828602 FJ828601 FJ828600 FJ828599 FJ828598 FJ828595 FJ828593 FJ828592 AY515247 AY515246 FJ626500 AY954186 FM242801 FM242795 FM242777 AY954190 AY954189 AY954185 FM242786 FM242764 AY954182 FM242768 EU288384 AY954191 AY954180 FJ899120 FJ899111 FJ899110 FJ899109 AF288118 AF288117 AF288115 AJ585890 AJ585841 AB040009 AB040000 AB039999 AB039986 AJ585889 AB040020 AB040015 AB040014 AJ585856 AJ585854 AB040003 AB040002 AB040001 AJ585855

Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Taxaceae Taxaceae Taxaceae Taxaceae Taxaceae Taxaceae Taxaceae Taxaceae

Nicotiana amplexicaulis Nicotiana velutina Solanum elaeagnifolium Solanum cinereum Solanum linnaeanum Solanum aculeastrum Solanum stupefactum Solanum hoplopetalum Solanum asymmetriphyllum Solanum sejunctum Solanum heteropodium Solanum oedipus Solanum melanospermum Solanum clarkiae Solanum diversiflorum Solanum chippendalei Solanum leopoldense Solanum petraeum Solanum carduiforme Solanum dioicum Solanum tudununggae Taxus brevifolia Taxus wallichiana Taxus canadensis Taxus fuana Taxus floridana Taxus globosa Taxus sumatrana Taxus mairei

AB040019 AJ585887 EU983576 EU983575 EU983574 EU983573 EU983572 EU983571 EU983570 EU983568 EU983567 EU983566 EU983565 EU983564 EU983563 EU983562 EU983560 EU983559 EU983558 EU983554 EU983552 EU078561 EF660663 EF660661 EF660660 EF660652 EF660647 EF660646 AB024001


Table S5 Size distribution of PCR products and number of successfully sequenced individuals in different families Family

All samples Amaranthaceae Apiaceae Apocynaceae Araliaceae Arecaceae Asteraceae Berberidaceae Boraginaceae Brassicaceae Buxaceae Cannabaceae Caprifoliaceae Caryophyllaceae*** Cupressaceae*** Fabaceae Fagaceae Ginkgoaceae Lamiaceae Lauraceae Magnoliaceae Oleaceae Oxalidaceae Pinaceae Pittosporaceae Plantaginaceae Poaceae

Length (bp)

Nb successfully sequenced individuals

rbcL

matK

rbcL

matK

553-554 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553 553-554 553 553 553

825-898 831 834 831-840 828 837 843 840 837 822 837 840 837 − − 837 837 888* 843 837 837 837 837 831 828 825 837

64 1 1 2 1 1 3 1 1 5 1 1 2 1 2 2 3 1 2 1 2 2 1 6 1 1 3

54 1 1 2 1 1 3 1 1 5 1 1 3 0 0 2 3 1 1 1 2 2 1 1 1 1 3

matK primer pair used°

matK_a, matk_b, matK_c matK_a matK_a matK_a matK_a matK_a matK_a matK_a matK_a matK_a matK_a matK_a matK_a matK_a, matk_b matK_a, matk_b, matK_c matK_a matK_a matK_c matK_a, matk_b matK_a matK_a matK_a matK_a matK_a, matk_b, matK_c matK_a matK_a matK_a

Taxon composition

34 families,55 genera, 65 species 1 genera, 1 species 1 genera, 1 species 2 genera, 2 species 1 genera, 1 species 1 genera, 1 species 3 genera, 3 species 1 genera, 1 species 1 genera, 1 species 4 genera, 5 species 1 genera, 1 species 1 genera, 1 species 2 genera, 3 species 1 genera, 1 species 2 genera, 2 species 2 genera, 2 species 1 genera, 3 species 1 genera, 1 species 2 genera, 2 species 1 genera, 1 species 2 genera, 2 species 2 genera, 2 species 1 genera, 1 species 4 genera, 6 species 1 genera, 1 species 1 genera, 1 species 3 genera, 3 species

Family

Primulaceae Ranunculaceae Rosaceae Rubiaceae Salicaceae*** Scrophulariaceae Solanaceae

Length (bp)

Nb successfully sequenced individuals

rbcL

matK

rbcL

matK

553 553 553-554 553 553 553 553 553

892** 840,895** 825-843 895** − 843,898** 837 834

1 2 4 1 1 3 3 1

1 2 4 1 0 2 3 1

Taxaceae * amplicon size with matK_c primers ** amplicon size with matK_b primers

*** Nucleotide sequence for matK not determined (see text) ° Primer name as mentioned in TableS1

matK primer pair used°

Taxon composition

matK_a, matk_b matK_a, matk_b matK_a matK_a, matk_b matK_a, matk_b matK_a, matk_b matK_a matK_a

1 genera, 1 species 2 genera, 2 species 3 genera, 4 species 1 genera, 1 species 1 genera, 1 species 1 genera, 3 species 2 genera, 3 species 1 genera, 1 species


Table S6 Results of the statistical analysis for the three molecular diversity indices calculated at family level rbcL Family (number of species)

Np/Na

Mean number of pairwise differences

matK Nucleotide diversity (average over loci)

12.190 +/- 8.966 Apocynaceae ( 2 spp.) 12 /553 (2.2%) 12.210 +/- 7.647 18/553 (3.2%) Asteraceae (3 spp.) 12.400 +/- 6.767 25/553 (4.5%) Brassicaceae (5 spp.) 2.000 +/- 1.732 2/553 (0.4%) Caprifoliaceae (3 spp.)c 6.000 +/- 4.582 6/553 (1.1%) Cupressaceae (2 spp.)b 43.000 +/- 30.757 43/553 (7.8%) Fabaceae (2 spp.) 0.667 +/- 0.667 1/553 (0.2%) Fagaceae (3 spp.) b 18.000 +/- 13.077 18/553 (3.2%) Lamiaceae (2 spp.) 17.000 +/- 12.369 17/553 (3.1%) Magnoliaceae (2 spp.) 1.000 +/- 1.000 Oleaceae (2 spp.) 1/553 (0.2%) b 24.933 +/- 12.812 62/554 (11.2%) Pinaceae (6 spp.) 28.333 +/- 17.287 42/553 (7.6%) Poaceae (3 spp.) 30.000 +/- 21.564 30/553 (5.4%) Ranunculaceae (2 spp.) 17.000 +/- 9.641 32/554 (5.8%) Rosaceae (4 spp) 14.000 +/- 8.718 21/553 (3.8%) Scrophulariaceae (3 spp.)d 6.667 +/- 4.328 10/553 (1.8%) Solanaceae (3 spp.) a Np= number of polymorphic sites; N= total number of sites compared b Molecular diversity indices for matK not calculated c Stastical values for the marker rbcL included only two of the three species sampled d Stastical values for the marker matK included only two of the three species sampled

Np/Na


Nucleotide diversity (average over loci)

0.022 +/- 0.223 0.022 +/- 0.017 0.022 +/- 0.014 0.003 +/- 0.004 0.011 +/- 0.012 0.078 +/- 0.079 0.001 +/- 0.001 0.032 +/- 0.033 0.031 +/- 0.032

58/840 (6.9%) 40/843 (4.7%) 93/822 (11.3%) 65/837 (7.8%) 109/837 (13%) 5/837 (0.6%) 17/837 (2%)

51.024 +/- 36.432 27.312 +/- 16.677 43.900 +/- 23.089 43.333 +/- 26.253 109.000 +/- 77.427 3.333 +/- 2.323 17.000 +/- 12.369

0.060 +/- 0.061 0.032 +/- 0.025 0.053 +/- 0.032 0.052 +/- 0.039 0.130 +/- 0.131 0.004 +/- 0.003 0.020 +/- 0.021

0.002 +/- 0.003 0.044 +/- 0.026 0.051 +/- 0.039 0.054 +/- 0.055 0.031 +/- 0.021 0.025 +/- 0.020 0.012 +/- 0.010

12/837 (1.4%) 79/837 (9.4%) 98/811 (12.1%) 136/843 (16%) 65/814 (8%) 19/837 (2.3%)

12.000 +/- 8.832 55.000 +/- 33.225 98.000 +/- 69.649 73.167 +/- 40.343 65.000 +/- 46.314 12.667 +/- 7.920

0.014 +/- 0.015 0.066 +/- 0.049 0.121 +/- 0.121 0.086 +/- 0.057 0.080 +/- 0.080 0.015 +/- 0.012

1

2


Table S7 Results of the statistical analysis for the three molecular diversity indices calculated between congeneric species rbcL Np/Na



Senecio (7 spp.)

17/553 (3.1%)

6.952 +/- 3.722

0.012 +/- 0.008

Brassica (4 spp.)

1/553 (0.2%)

0.500 +/- 0.519

0.001 +/- 0.001

Viburnum (7 spp.)

7/553 (1.3%)

2.381 +/- 1.468

0.004 +/- 0.003

Cerastium (3 spp.)

6/553 (1.1%)

4.000 +/- 2.725

0.007 +/- 0.006

Albizia (3 spp.)

7/553 (1.3%)

4.667 +/- 3.127

0.008 +/- 0.007

Trifolium (4 spp.)

17/553 (3.1%)

10.000 +/- 5.811

0.018 +/- 0.012

Quercus (37 spp.)

7/553 (1.3%)

1.252 +/- 0.809

0.002 +/- 0.002

Lamium (6 spp.)

17/553 (3.1%)

7.133 +/- 3.901

0.014 +/- 0.008

Magnolia (9 spp.)

2/553 (0.4%)

0.722 +/- 0.592

0.001 +/ -0.001

Ligustrum (5 spp.)

4/553 (0.7%)

1.600 +/- 1.128

0.003 +/- 0.002

Oxalis (4 spp.)

25/554 (4.5%)

15.833 +/- 9.003

0.028 +/- 0.019

Abies (23 spp.)

3/553 (0.5%)

0.917 +/- 0.658

0.002 +/- 0.001

Picea (29 spp.)

4/553 (0.7%)

1.258 +/- 0.828

0.002 +/- 0.002

Pinus (23 spp.)

7/553 (1.3%)

1.939 +/- 1.144

0.003 +/- 0.002

Plantago (7 spp.)

27/553 (4.9%)

10.571 +/- 5.492

0.019 +/- 0.011

Bromus (7 spp.)

5/553 (0.9%)

1.905 +/- 1.228

0.003 +/- 0.002

Poa (3 spp.)

6/553 (1.1%)

4.000 +/- 2.725

0.007 +/- 0.006

75/553 (13.6%)

15.925 +/- 7.377

0.029 +/- 0.019

Genus (number of spp.)

Primula (23 spp.)

a

matK

Helleborus (5 spp.)

5/553 (0.9%)

2.000 +/- 1.343

0.004 +/- 0.003

Ranunculus (6 spp.)

30/553 (5.4%)

12.667 +/- 6.674

0.023 +/- 0.014

Crataegus (3 spp.)

1/554 (0.2%)

0.667 +/- 0.667

0.001 +/- 0.001

Prunus (11 spp.)

11/553 (2.0%)

3.127 +/- 1.754

0.006 +/- 0.003

Galium (9 spp.)

20/553 (3.6%)

7.111 +/- 3.685

0.013 +/- 0.007

Populus (12 spp.)

26/553 (4.7%)

6.848 +/- 3.469

0.012 +/- 0.007

Veronica (20 spp.)

46/553 (8.3%)

7.158 +/- 3.503

0.013 +/- 0.007

Nicotiana (6 spp.)

3/553 (0.5%)

1.000 +/- 0.774

0.002 +/- 0.001

Solanum (12 spp.)

23/553 (4.1%)

5.470 +/- 2.831

0.010 +/- 0.006

Taxus (6 spp.)

5/553 (0.9%)

1.867 +/- 1.235

0.003 +/- 0.002

Np/Na



Gomphrena (6 spp.)

56/831 (6.7%)

21.600 +/- 11.144

0.026 +/- 0.015

Brassica (13 spp.) Cardamine (4 spp.) Lonicera (5 spp.) Albizia (9 spp.) Trifolium (24 spp.) Quercus (28 spp.) Lamium (3 spp.) Magnolia (20 spp.) Oxalis (14 spp.) Abies (5 spp.) Plantago (4 spp.) Bromus (5 spp.) Poa (8 spp.) Primula (74 spp.) Helleborus (4 spp.) Ranunculus (15 spp.) Prunus (9 spp.) Nicotiana (21 spp.) Solanum (21 spp.) Taxus (9 spp.)

43/822 (5.2%) 22/822 (2.7%) 24/839 (2.9%) 74/867 (8.5%) 90/837 (10.8%) 43/855 (5%) 28/843 (3.3%) 32/837 (3.8%) 144/837 (17.2%) 8/831 (1%) 38/825 (4.6%) 14/837 (1.7%) 43/837 (5.1%) 320/902 (35.5%) 16/896 (1.8%) 19/840 (2.3%) 245/879 (27.8%) 12/837 (1.4%) 60/839 (7.2%) 26/834 (3.1%)

10.769 +/- 5.244 11.167 +/- 6.449 10.000 +/- 5.521 19.167 +/- 9.388 16.619 +/- 7.668 7.058 +/- 3.416 18.667 +/- 11.508 7.347 +/- 3.588 29.813 +/- 13.876 4.400 +/- 2.608 19.333 +/- 10.917 6.600 +/- 3.755 15.714 +/- 7.863 44.904 +/- 19.684 8.500 +/- 4.989 4.352 +/- 2.279 75.417+/- 35.957 2.257 +/- 1.292 8.576 +/- 4.129 6.500 +/- 3.395

0.013 +/- 0.007 0.014 +/- 0.009 0.012 +/- 0.008 0.022 +/- 0.012 0.019 +/- 0.010 0.008 +/- 0.004 0.022 +/- 0.017 0.009 +/- 0.005 0.036 +/- 0.019 0.005 +/- 0.004 0.023 +/- 0.016 0.008 +/- 0.005 0.019 +/- 0.011 0.050 +/- 0.024 0.009 +/- 0.007 0.005 +/- 0.003 0.086 +/- 0.046 0.003 +/- 0.002 0.010 +/- 0.005 0.008 +/- 0.005

Genus (number of species)

Np= number of polymorphic sites; N= total number of sites compared

1


Table S8. BLAST search results of rbcL and matK for the plant species for which corresponding sequences are published in GenBank Investigated species

Best similar sp. of rbcL

Coverage (%)

Similarity (%)

Daucus carota

Daucus carota

100%

100%

1

Daucus carota

100%

100%

1

Nerium oleander

Nerium oleander

100%

100%

1

Nerium oleander

100%

100%

1

Vinca minor

Vinca minor

100%

100%

3

Vinca minor

100%

100%

3

Hedera helix

Hedera helix

100%

100%

18

Hedera helix

100%

100%

15

Trachycarpus fortunei

Trachycarpus fortunei Chamaerops humilis

100%

100%

1


100%

100%

4

Cichorium intybus

Cichorium intybus

100%

100%

1

Cichorium intybus

97%

100%

1

100%

100%

100%

100%

10

Taraxacum platypecidium

100%

99%

100%

100%

100%

100%

7

Brassica oleracea

100%

100%

15

100%

100%

7

Brassica napus

100%

100%

15

Taraxacum officinale Brassica oleracea

Taraxacum officinale Taraxacum platypecidium Brassica oleracea Brassica napus

Coverage of the exact sp. (%)

100%

Similarity with exact sp. (%)

N° of spp. per genus in GenBank

100%

Best similar sp. of matK

Coverage (%)

Similarity (%)


96%


99%

N° of spp. per genus in GenBank

12

Brassica napus

Brassica spp.(3)

100%

100%

Capsella bursa-pastoris


100%

100%

3


100%

100%

3

Albizia julibrissin

Albizia julibrissin

100%

99%

18

Albizia julibrissin

100%

100%

18

Buxus sempervirens

Buxus microphylla Buxus sempervirens

100%

100%

100%

100%

4

Buxus sempervirens

100%

100%

7

Viburnum tinus

Viburnum opulus

100%

99%

87%

100%

113

Viburnum rhytidophyllum

100%

99%

88%

100%

113

Cerastium glomeratum


100%

99%

11

−

−

−

−

−

12

Calocedrus decurrens


100%

100%

4

−

−

−

−

−

4

Quercus robur

Quercus spp. (4)

100%

100%

100%

100%

59

Quercus spp. (8)

100%

100%

100%

100%

84

Quercus rubra

Quercus spp. (10)

100%

100%

100%

100%

59

Quercus spp. (2)

100%

100%

100%

100%

84

Quercus petraea

Quercus spp. (4)

100%

100%

100%

100%

59

Quercus spp. (3)

100%

100%

100%

100%

84

Ginkgo biloba

Ginkgo biloba

100%

100%

1

Ginkgo biloba

100%

100%

Rosmarinus officinalis

Rosmarinus officinalis Salvia spp.(12)

100%

99%

1

−

−

−

−

−

1

Lamium purpureum

Lamium purpureum

100%

100%

100%

99%

100%

99%

14

Laurus nobilis

Laurus nobilis Persea americana

99%

100%

100%

100%

100%

100%

2

Liriodendron tulipifera


100%

100%

Magnolia grandiflora

Magnolia spp. (3)

100%

100%

100%

Chamaecyparis lawsoniana

Chamaecyparis obtusa

100%

99%

97%

100%

99%

10 99%

100%

2

Lamium maculatum Lamium purpureum Laurus nobilis Laurus azorica

1

2


100%

99%

100%

96

Magnolia spp. (4)

100%

100%

100%

100%

112

100%

6

−

−

−

−

−

9

1

2

Table S8 (continued) Investigated species


Coverage (%)

Similarity (%)

Trifolium pratense

Coverage of the exact species (%)

Trifolium pratense

100%

100%

Olea europaea

Olea europaea

100%

100%

Abies alba

Abies spp. (9)

100%

100%

Picea abies

Picea spp. (12)

100%

100%

Pinus nigra

Pinus spp. (10)

100%

100%

100%

Pinus sylvestris

Pinus spp. (10)

99%

100%

99%

Crataegus monogyna

Crataegus monogyna

100%

Fragaria vesca

Fragaria vesca

Prunus cerasifera

100%

Similarity with exact species (%)

N° of species per genus in GenBank

100%


Coverage (%)

Similarity (%)




30

Trifolium pratense

100%

99%

42

1

Olea europaea

100%

100%

17

49 37

−

−

−

−

−

37

100%

102

−

−

−

−

−

112

100%

102

−

−

−

−

−

112

100%

11

Crataegus monogyna

100%

100%

9

100%

100%

23

Fragaria vesca

100%

100%

18

Prunus spp. (3)

100%

100%

132

Prunus spinosa

99%

99%

Prunus laurocerasus

Prunus laurocerasus

100%

100%

132

Prunus laurocerasus

100%

100%

Nicotiana tabacum

Nicotiana spp. (6)

100%

100%

17

Nicotiana spp. (3)

100%

100%

Solanum lycopersicum


100%

100%

77


100%

99%

107

Solanum tuberosum

Solanum tuberosum

100%

100%

77

Solanum tuberosum

100%

100%

107

Taxus baccata

Taxus spp. (3)

100%

100%

100%

100%

13

Taxus baccata

100%

100%

13

Ligustrum vulgare

Ligustrum spp. (7) Olea europea

100%

100%

100%

100%

19

Ligustrum obtusifolium

100%

99%

97%

99%

19

Galium mollugo

Galium mollugo

100%

100%

27

Galium verum

96%

99%

93%

99%

12

Populus nigra

Populus spp. (7)

100%

100%

100%

100%

15

−

−

−

−

−

11

100%

99%

100%

99%

9

Pittosporum undulatum

100%

99%

91%

99%

6

100%

100%

100%

100%

21

Plantago rugelii

100%

99%

96%

100%

14

100%

100%

100%

100%

30

Bromus secalinus

100%

99%

96%

100%

19

100%

100%

16

Poa annua

100%

100%

Pittosporum tobira Plantago major Bromus sterilis Poa annua

Pittosporum tobira Pittosporum pentadrum Plantago major Plantago depressa Bromus sterilis Bromus arenarius Poa annua

100%

100%

100%

100%

2

95%

100%

113 113

100%

100%

62

22



Coverage (%)

Similarity (%)




Primula vulgaris

Primula veris

99%

99%

98%

99%

45

Helleborus viridis

Helleborus croaticus

100%

100%

98%

100%

Veronica arvensis

Veronica arvensis

100%

Veronica persica

Veronica persica

Cedrus atlantica

Coverage (%)

Similarity (%)




Primula vulgaris Primula veris

100%

100%

100%

100%

220

7

Helleborus multifidus

100%

99%

97%

100%

17

100%

37

Veronica arvensis

100%

100%

100%

99%

37

−

−

−

−

−

18

Cedrus atlantica

100%

100%

2

−

−

−

−

−

2

Cedrus deodara

100%

100%

2

−

−

−

−

−

2

Cannabis sativa

100%

100%

1

Cannabis sativa

100%

100%

Chimonobambusa quadrangularis Phyllostachys spp. (3)

100%

100%

100%

100%

6


100%

99%

97%

100%

133

Lonicera xylosteum

Lonicera orientalis

100%

99%

75.6%

100%

27

Oxalis incarnata

Oxalis tomentosa

100%

97%

59%

98%

31

Cedrus deodara Cannabis sativa Chimonobambusa quadrangularis Senecio vulgaris


100%

99%

98%

100%

12


18

1

Ranunculus bulbosus

Ranunculus macranthus

100%

99%

98%

100%

33

Ranunculus bulbosus

100%

100%

Cardamine hirsuta

Cardamine diphylla

100%

99%

98%

100%

13

Cardamine rivularis

100%

98%

93%

100%

13

100%

98%

100%

113

98%

100%

113

98%

98%

99%

3

Viburnum lantana Erophila verna

Viburnum cotinifolium Draba nemorosa

100% 100%

Viburnum rhytidophyllum

100%

99%

Erophila verna

96%

99%

255

3

Gomphrena globosa

Gomphrena ferruginea

100%

98%

84%

Mahonia aquifolium

Berberis bealei

100%

99%

96%

3

99%

7

100%

16


Table S9. BLAST Search results of rbcL and matK for the plant species for which corresponding sequences are not published in GenBank Investigated species

rbcL sequence in database at the level of:


Best similar sp. of rbcL (similarity%)

Gomphrena globosa

Genus

4

Gomphrena serrata (100%)

Mahonia aquifolium

Genus

20

Mahonia japonica (99%)

Pulmonaria officinalis

Genus

1

Lobostemon fruticosus (98%)

Lonicera xylosteum

Genus

3

Oxalis incarnata

Genus

45

Oxalis articulata (99%)

Chimonobambusa quadrangularis

Genus

5

Chimonobambusa marmorea (99%)

Veronica sublobata

Genus

37

Veronica hederifolia (100%)

MatK sequence in database at the level of:


Best similar sp. of MatK (similarity%)

Genus

1

Pulmonaria obscura (98%)

Genus

17


Genus

31

Abies hidalgensis (100%)

−

Abies alba

1


Table S10 BOLD-IDS search results for rbcL and matK for the plant species for which corresponding sequences are recorded in BOLD Investigated species


Coverage (%)

Similarity (%)

Daucus carota

Daucus carota

98%

100%

Populus nigra

Populus spp. (5)

100%

100%

Vinca minor

Vinca minor

100%

100%

100%

100%

100%

100%

99.8%

100%

Hedera helix Olea europaea

Hedera helix Hedera rhombea Olea europaea Olea woodiana


100%


N° of spp. per genus in BOLD


Coverage (%)

Similarity (%)

93.3%

100%

−

−



N° of spp. per genus in BOLD

1

Daucus carota

7

−

1

Vinca minor

94.5%

100%

1

100%

2

Hedera helix

99%

100%

1

99.8%

2

Fraxinus excelsior

99%

99.3%

1

Cichorium intybus

100%

100%

1

6

Crataegus monogyna

98.7%

100%

6

2

Fragaria vesca Fragaria virginiana

94.5%

100%

96.5%

100%

2

Solanum tuberosum

100%

99.2%

96.5%

99.8%

134

100%

1 −

89%

−

99.6%

7

2

Cichorium intybus

Cichorium intybus

100%

100%

Crataegus monogyna

Crataegus spp. (5)

100%

99.8%

Fragaria vesca

Fragaria vesca Fragaria virginiana

100%

100%



100%

100%

134



100%

100%

1


100%

100%

1

Taxus baccata

Taxus spp. (4)

100%

100%

16

Taxus baccata

98.9%

100%

16

100%

100%

100%

100%

29

96.6%

99.6%

85.9%

99.3%

13

100% 100%

100%

99.8% 100%

100%

Ligustrum vulgare

Ligustrum spp. (8)

100%

100%

100%

100%

29

Ligustrum vulgare Ligustrum sinense

Galium mollugo

Galium verum Galium mollugo

100%

100%

100%

100%

13

Galium verum



100%

100%

17

−

−

−

−

−

17



100%

100%

1

−

−

−

−

−

1

Plantago major

Plantago major

100%

100%

5

Plantago major

100%

100%

Quercus rubra

Quercus spp. (3)

100%

100%

100%

100%

33

Quercus macrocarpa

93.5%

99.2%

Bromus sterilis

Bromus sterilis Bromus tectorum

100%

100%

100%

100%

16

Bromus sterilis

95.3%

100%

22

Poa annua

Poa annua

100%

100%

13

Poa annua

97%

100%

14

Primula vulgaris

Primula vulgaris

98%

99.8%

59

Primula vulgaris

99.5%

100%

59

Lamium purpureum

Lamium purpureum

100%

100%

21

Lamium purpureum

98.6%

99.3%

21

Helleborus viridis

Helleborus spp. (3)

100%

100%

4

Helleborus viridis

82.7%

99.7%

2



100%

100%

1


93.3%

99.6%

1


Magnolia spp. (3)

100%

100%

3


94.3%

100%

3

Veronica arvensis

Veronica arvensis

100%

100%

23

Veronica arvensis

96.9%

99.9%

23

100%

100%

100%

100%

1

4 90%

100%

33



Coverage (%)

Similarity (%)

Cannabis sativa

Cannabis sativa

100%

100%

Ranunculus bulbosus

Ranunculus spp. (6)

100%

100%

Erophila verna

Erophila verna

97%

99%

Viburnum lantana

Viburnum spp. (5)

100%

100%

Nerium oleander

Nerium oleander Nerium indicum

100%

100%



100%

Cedrus atlantica

Cedrus atlantica

Ginkgo biloba

Ginkgo biloba



N° of species per genus in BOLD

1 100%

100%

266


Coverage (%)

Similarity (%)


Cannabis sativa

100%

100%

1

Ranunculus bulbosus

96.4%

99.8%

266

Erophila majuscula

95%

98.6%

42

Viburnum spp. (3)

100%

100%

2

Nerium oleander Nerium indicum

100%

100%

100%

100%

2

100%

1

−

−

−

−

−

1

100%

100%

2

−

−

−

−

−

2

100%

100%

1

Ginkgo biloba

100%

100%

100%

100%

100%

100%

46 150

100%

100% 100%

99.6%


3 100%

89.6%


2 42

1

Abies alba

Abies spp. (9)

100%

100%

100%

100%

46

Abies alba Abies hidalgensis

Pinus sylvestris

Pinus spp. (9)

100%

100%

100%

100%

150

−

−

−

−

−

Cedrus deodara

Cedrus deodara

100%

100%

2

−

−

−

−

−

2

Pinus nigra

Pinus spp. (8)

100%

100%

100%

100%

150

−

−

−

−

−

150



100%

100%

9

−

−

−

−

−

9

Cardamine hirsuta

Cardamine hirsuta

100%

100%

20

Cardamine flexuosa

100%

97.8%

91%

100%

20

Buxus sempervirens

Buxus sempervirens

100%

100%

11

Buxus koreana

100%

99.6%

96.8%

100%

11



100%

100%

100%

100%

1


100%

100%

100%

100%

1

Picea abies

Picea spp. (14)

100%

100%

100%

100%

51

−

−

−

−

−

51

Trifolium pratense

Trifolium pratense

100%

100%

46

Trifolium pratense

100%

100%

Viburnum tinus

Viburnum tinus

100%

100%

42

Viburnum sargentii

100%

99.8%

Albizia julibrissin

Albizia julibrissin

100%

99.8%

30

Albizia julibrissin

100%

99.8%

30

100%

100%

100%

100%

2

Laurus nobilis

100%

100%

2

100%

100%

98%

100%

33

Quercus spp. (6)

100%

99.8%

100%

99.8%

33

100%

100%

98%

100%

33

Quercus spp. (5)

100%

100%

100%

100%

33

Laurus nobilis Quercus petraea Quercus robur

Laurus nobilis Lindera spp. (5) Litsea spp. (4) Quercus spp. (3) Castanopsis spp. (2) Quercus spp. (8) Castanopsis spp. (2)

T 2

46 98.4%

100%

42



Coverage (%)

Similarity (%)

Taraxacum officinale



100%

100%

Pittosporum tobira

Pittosporum spp. (3)

100%

100%

Mahonia aquifolium

Mahonia aquifolium

100%

100%

Brassica oleracea

Brassica spp. (3)

100%

100%

100%

Brassica napus

Brassica spp. (3)

100%

100%

100%

100%




Coverage (%)

Similarity (%)




23


99.7%

99.4%

12

Pittosporum undulatum

100%

99.4%

91%

99.8%

12

79

Berberis thunbergii

100%

99.3%

96.7%

100%

79

100%

28

Brassica juncea

100%

99.6%

96.8%

100%

28

100%

28

Brassica oleracea

100%

99.6%

91.2%

100%

28

100%

100%

100%

100%

86

96.5%

99.8%

134

100%

Prunus cerasifera

Prunus spp. (6)

100%

100%

100%

100%

86

Prunus cerasifera Prunus domestica

Solanum tuberosum


100%

98.9%

37%

98%

134


100%

99.2%

Senecio vulgaris

Senecio spp. (3)

100%

100%

100%

100%

36

Senecio vulgaris

100%

100%

Prunus laurocerasus

Prunus laurocerasus

100%

100%

86

Prunus laurocerasus

100%

100%

23

36 86

Oxalis incarnata

Oxalis tomentosa

97%

98.4%

23%

Chimonobambusa quadrangularis

Chimonobambusa quadrangularis Phyllostachys bambusoides

100%

100%

100%

100%

2

−

−

−

−

23

73%

100%

Veronica persica

Veronica spp. (3)

100%

100%

100%

100%

23

−

Nicotiana tabacum

Nicotiana spp. (4)

100%

100%

100%

100%

64

Nicotiana tabacum

100%

100%

Lonicera xylosteum

−

−

−

−

−

46

Lonicera orientalis

99%

99.5%

Gomphrena globosa

Gomphrena celosioides

100%

99.64%

100%

99.46%

9

3

100%

38

64 46


Table S11. BOLD-IDS search results for rbcL and matK for the plant species for which corresponding sequences are not recorded in BOLD Investigated species

rbcL sequence in database at the level of:


Best similar sp. of rbcL (similarity%)

Pulmonaria officinalis

Genus

4

Pulmonaria obscura (100%)

Oxalis incarnata

Genus

37

Oxalis rubra (99.5%)

Gomphrena globosa Chimonobambusa quadrangularis

Genus

1

Chimonobambusa marmorea (99.8%)

Veronica sublobata

Genus

23


1

matK sequence in database at the level of:


Best similar sp. of MatK (similarity%)

Genus

4

Pulmonaria obscura (98.1%)

Genus

9

Gomphrena ferruginea (98.4%)

Genus

23



TableS12.Sequence recovery rate for the barcode regions considered singly or in two-locus combination DNA Barcode regions Taxonomic Category* Species (n=65)

trnH-psbA

trnL-trnF

rbcL

matK

trnH-psbA+trnL-F

trnHpsbA+matK 63 (100%)

trnLF+rbcL 63 (100%)

trnLF+matK 63 (100%)

rbcL+matK

63 (100%)

trnHpsbA+rbcL 63 (100%)

61(96.8%)

63 (100%)

64 (98.5%)

54 (83.1%)

Genus (n=55)

52(98.1%)

53 (100%)

54 (98.1%)

46 (83.6%)

53 (100%)

53 (100%)

53 (100%)

53 (100%)

53 (100%)

55 (100%)

Family (n=34)

32 (97 %)

33 (100%)

34 (100%)

31 (91.2%)

33 (100%)

33 (100%)

33 (100%)

33 (100%)

33 (100%)

34 (100%)

Order (n=24)

23(95.8%)

24 (100%)

23 (95.8%)

23 (95.8%)

24 (100%)

24 (100%)

24 (100%)

24 (100%)

24 (100%)

24 (100%)

*63 species, 53 genera and 33 families for trnH-psbA and trnL-trnF when considered singly or in combinations For each taxonomic category the absolute number of successes is given along with the percentage relative to the total possible number In the combinations of markers, sequence success is defined as the recovery of any marker

65 (100%)


TableS13.BLAST results for frequency of correct species identification of single- and multi-locus barcodes on the subset of species having records available in GenBank and BOLD databases GenBank DNA Barcode Region

Correct assignment at species level (%)

Correct assignment at genus level (%)

Correct assignment at family level (%) Incorrect assignment (%)

rbcL

27/58 (46.6%)

20/58 (34.5%)

4/58 (6.9%)

7/58 (12.1%)

matk

26/51 (51%)

7/51 (13.7%)

1/51 (2.0%)

17/51 (33.3%)

trnH-psbA

31/53 (58.5%)

13/53 (24.5%)

0

9/53 (17%)

trnL-trnF

25/58 (43.1%)

18/58 (31%)

0

15/58 (25.9%)

trnL-trnF+rbcL

34/62 (54.8%)

20/62 (32.3%)

3/62 (4.8%)

5/62 (8.1%)

trnL-trnF+matK

34/62 (54.8%)

16/62 (25.9%)

1/62 (1.6%)

11/62 (17.7%)

trnH-psbA+trnL-trnF

39/63 (61.9%)

14/63 (22.2%)

0

10/63 (15.9%)

trnH-psbA+rbcL

40/60 (66.7%)

14/60 (23.3%)

1/60 (1.7%)

5/60 (8.3%)

trnH-psbA+matK

37/59 (62.7%)

12/59 (20.3%)

1/59 (1.7%)

9/59 (15.3%)

rbcL+matK

33/61 (54.1%)

18/61 (29.5%)

3/61 (4.9%)

7/61 (11.5%)

BOLD DNA Barcode Region

Correct assignment at species level (%)

Correct assignment at genus level (%)

rbcL

28/60 (46.7%)

26/60 (43.3%)

4/60 (6.7%)

2/60 (3.3%)

matk

26/51 (51%)

8/51 (15.7%)

2/51 (3.9%)

15/51 (29.4%)

39/63 (61.9%)

19/63(30.1%)

2/63 (3.2%)

3/63 (4.8%)

rbcL+matk

Correct assignment at family level (%) Incorrect assignment (%)

Additional Files Click here to download Additional Files: Text ESM 1.doc

Supplementary Text ESM 1PCR

was performed in a 10 µl reaction mixture containing approximately 1-5 ng/µl of genomic

DNA template, 2X Qiagen Multiplex Master Mix (Qiagen) and 0.4 µM of each primer on a Veriti 96-well Thermal Cycler (Applied Biosystems) and all samples were amplified under the same cycling conditions for rbcL region (Table S1). MatK required the implementation of 4 different modified PCR protocols to allow successful amplification in most samples by varying annealing condition (Table S1). PCR products were cleaned using ExoSAP-IT kit (USB Corp.,Cleveland, OH, USA). For sequencing, 4 µl of the purified PCR product was used in a 20 µl reaction with BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) according to the manufacturer protocol, in both forward and reverse direction using original primers. Cycle sequencing protocols were the same for all markers (96 °C 1 min, [96 °C 30 sec, 50°C 5 sec, 60 °C 4 min] x 25 cycles). Sequencing reaction products were finally purified from residual dye terminators using DyeEx 2.0 Spin Kit (Qiagen) and sequence reactions were read with POP-4 performance optimized polymer (Applied Biosystems) on automated capillary sequencer ABI 3130 DNA analyser (Applied Biosystems).

2As

explained in details in the previously work [Ferri et al. 24], a correct identification with the

BLAST tool is achieved when the query sequence pasted in the search window corresponds to the first database entry in the output file, defined as maximum scoring segment pair (MSP), which displays the best concordance (Maximum Score) with the query sequence. A further distinction is made when the sequence search returned a “single best match” (where the query sequence was returned as the unique most likely match) or “multiple best matches” (where the query sequence plus one or more identical sequences were returned as equally likely).