Region Functions As a Highly Effective and Efficient

Supporting Information for Original Article The large single-copy (LSC) region functions as a highly effective and efficient molecular marker for accurate authentication of medicinal Dendrobium species Ludan Lia, Yu Jianga, Yuanyuan Liua, Zhitao Niua, Qingyun Xuea, Wei Liua, Xiaoyu Dinga,* aCollege of Life Sciences, Nanjing Normal University, Nanjing 210023, China Received 29 October 2019; received in revised form 19 December 2019; accepted 13 January 2020 *Corresponding author. Tel./fax: +86 25 85891605. E-mail address: [email protected] (Xiaoyu Ding).

Table S1 Sampling localities and accession numbers of the plastomes and ITS used in the authentication analyses of 23 Dendrobium species of “Fengdou” (DSFs).

No. Sample Locality Plastome accession ITS accession number number

1 D. huoshanense_006 Huoshan, Anhui province LC490207 MN108045 2 D. huoshanense_052 Huoshan, Anhui province LC490373 MN108046 3 D. huoshanense_173 Huoshan, Anhui province LC490384 MN108048 4 D. huoshanense_085 Huoshan, Anhui province LC490375 MN108046 5 D. huoshanense_139 Huangshan, Anhui province LC490382 MN108046 6 D. huoshanense_054 Huangshan, Anhui province LC490374 MN108046 7 D. huoshanense_129 Longhushan, Jiangxi province LC490380 MN108046 8 D. huoshanense_114 Longhushan, Jiangxi province LC490379 MN108046 9 D. huoshanense_088 Shangrao, Jiangxi province LC490378b MN108047 10 D. huoshanense_086 Shangrao, Jiangxi province LC490381 MN108046 11 D. huoshanense Anhui province LC269310a KF143476a 12 D. huoshanense NC_028430a KC331011a 13 D. wilsonii_023 Simao, Yunnan province LC490388b MN108049 14 D. wilsonii_097 Dehua, Fujian province LC490389 MN108049 15 D. wilsonii_168 Enshi, Hubei province LC490392 MN108049 16 D. wilsonii_231 Lechang, Guangdong province LC490394 MN108050 17 D. wilsonii_102 Laibin, Guangxi province LC490391 MN108049 18 D. wilsonii NC_035330a KF143526a 19 D. moniliforme_016 Wenshan, Yunnan province LC490386b MN108051

1 20 D. moniliforme_153 Qujing, Yunnan province LC490377 MN108052 21 D. moniliforme_042 Longlin, Guangxi province LC490652 MN108053 22 D. moniliforme_201 Xinyi, Guangdong province LC490387 MN108051 23 D. moniliforme NC_035154a KF143489a 24 D. xichouense_055 Xichou, Yunnan province LC490656 MN108054 25 D. xichouense_192 Xichou, Yunnan province LC490658 MN108055 26 D. xichouense_080 Xichou, Yunnan province LC490657b MN108054 27 D. xichouense_036 Xichou, Yunnan province LC490655 MN108056 28 D. xichouense NC_035341a KF143527a 29 D. fanjingshanense_106 Fanjingshan, Guizhou province LC490372b MN108057 30 D. fanjingshanense_132 Fanjingshan, Guizhou province LC490653 MN108058 31 D. fanjingshanense_189 Fanjingshan, Guizhou province LC490654 MN108058 32 D. fanjingshanense_008 Chibi, Hubei province LC490405 MN108058 33 D. fanjingshanense_067 Chibi, Hubei province LC490660 MN108058 34 D. fanjingshanense_069 Xianning, Hubei province LC490407 MN108058 35 D. fanjingshanense NC_035344a KF143459a 36 D. lituiflorum_205 Debao, Guangxi province LC490679 MN108059 37 D. lituiflorum_215 Tianlin, Guangxi province LC490680 MN108059 38 D. lituiflorum_213 Zhenkang, Yunnan province LC490681b MN108059 39 D. devonianum_064 Longlin, Guangxi province LC490383b MN108060 40 D. devonianum_078 Xingyi, Guizhou province LC490385 MN108061 41 D. devonianum NC_035325a KF143453a 42 D. devonianum LC317045a KT778760a 43 D. hercoglossum_116 Jianghua, Hunan province LC490400 MN108062 44 D. hercoglossum_099 Xinyi, Guangdong province LC490398b MN108063 45 D. hercoglossum_122 Lingyun, Guangxi province LC490402 MN108063 46 D. hercoglossum NC_035328a KF143471a 47 D. gratiosissimum_177 Jinghong, Yunnan province LC490662 MN108064 48 D. gratiosissimum_156 Simao, Yunnan province LC490659 MN108065

49 D. gratiosissimum_020 Mengla, Yunnan province LC490406b MN108065 50 D. gratiosissimum NC_035327a KF143464a 51 D. primulinum_079 Longling, Yunnan province LC490397 MN108066 52 D. primulinum_072 Wenshan, Yunnan province LC490376 MN108067 53 D. primulinum_046 Simao, Yunnan province LC490399b MN108067 54 D. primulinum NC_035321a KF143499a

55 D. crystallinum_134 Mengla, Yunnan province LC490677b MN108068 56 D. crystallinum_145 Menghai, Yunnan province LC490678 MN108068 57 D. crystallinum_225 Jinghong, Yunnan province LC490676 MN108068

2 58 D. loddigesii_061 Napo, Guangxi province LC490673 MN108069 59 D. loddigesii_038 Luodian, Guizhou province LC490674 MN108069 60 D. loddigesii_015 Luofushan, Guangdong province LC490396a MN108069a 61 D. loddigesii_029 Mengla, Yunnan province LC490401b MN108070 62 D. loddigesii NC_036355a KF143481a 63 D. aphyllum_148 Funing, Yunnan province LC490671 MN108071 64 D. aphyllum_182 Xingyi, Guizhou province LC490669b MN108071 65 D. aphyllum_186 Longlin, Guangxi province LC490390 MN108071 66 D. aphyllum NC_035322a AB593539a 67 D. falconeri_221 Zixing, Hunan province LC490408 MN108072 68 D. falconeri_150 Lingui, Guangxi province LC490393 MN108072 69 D. falconeri_208 Shiping, Yunnan province LC490395b MN108073 70 D. falconeri NC_035326a KF143458a 71 D. wardianum_195 Wenshan, Yunnan province LC490661 MN108074 72 D. wardianum_199 Mengzi, Yunnan province LC490664 MN108074 73 D. wardianum_210 Tengchong, Yunnan province LC490666b MN108074 74 D. wardianum NC_035329a AB593686a 75 D. lohohense_237 Lingyun, Guangxi province LC490670b MN108075 76 D. lohohense_243 Shimen, Hunan province LC490668 MN108075 77 D. lohohense NC_035338a KF143482a 78 D. crepidatum_228 Zhenkang, Yunnan province LC490675 MN108076 79 D. crepidatum_119 Menghai, Yunnan province LC490403b MN108077 80 D. crepidatum_130 Xingyi, Guizhou province LC490404 MN108077 81 D. crepidatum NC_035331a KJ944628a 82 D. chrysanthum_222 Anlong, Guizhou province LC490684b MN108078 83 D. chrysanthum_256 Pingbian, Yunnan province LC490682 MN108079 84 D. chrysanthum_260 Baise, Guangxi province LC490683 MN108080 85 D. chrysanthum NC_035336a KJ944627a 86 D. pendulum_232 Simao, Yunnan province LC490663 MN108081

87 D. pendulum_247 Jinghong, Yunnan province LC490665 MN108081 88 D. pendulum_267 Mengla, Yunnan province LC490698b MN108082 89 D. strongylanthum_281 Yingjiang, Yunnan province LC490685 MN108083 90 D. strongylanthum_272 Jinghong, Yunnan province LC490672b MN108084 91 D. officinale Liancheng, Fujian province LC348725a,b MG779627a 92 D. officinale Guilin, Guangxi province LC348525a MG779623a

93 D. officinale Huoshan, Anhui province LC348527a MG779624a 94 D. officinale Guangnan, Yunnan province LC348520a MG779623a 95 D. officinale Yandangshan, Zhejiang province NC_024019a MG779625a

3 96 D. flexicaule Ganluo, Sichuan province LC348856a MG779629a 97 D. flexicaule Ganzi, Sichuan province LC348965a,b MG779630a 98 D. flexicaule Shennongjia, Hubei province LC348854a MG779630a 99 D. aduncum Luofushan, Guangdong province LC348858a,b MG779634a 100 D. aduncum Taoyuan, Hunan province LC348859a MG779634a 101 D. aduncum Xingyi, Guizhou province LC348857a MG779634a 102 Cymbidium sinense* NC_021430a KJ597850a 103 Oncidium sphacelatum* NC_028148a AF350810a 104 Phalaenopsis equestris* NC_017609a AY912225a 105 Masdevallia picturata* NC_026777a AF262775a 106 Cattleya crispata* NC_026568a AY008665a 107 Calanthe triplicata* NC_024544a KM025154a aStands for the sequences downloaded from GenBank. bDenotes the species used in the repeat sequence analysis. *Represents the species serving as outgroups.

Table S2 SNPs, indels, repeats and GC content in 260 nonoverlapping bins of 600 bp each through the alignment of 101 complete plastomes of 23 DSFs.

Position SNPs Indels Repeats GC content

1–600 26 28 3 0.392

601–1200 14 0 0 0.432 1201–1800 44 33 16 0.324 1801–2400 47 0 2 0.341

2401–3000 28 0 1 0.309 3001–3600 32 12 8 0.329 3601–4200 57 67 65 0.291 4201–4800 24 13 26 0.311 4801–5400 51 19 25 0.309

5401–6000 48 15 20 0.328 6001–6600 64 13 13 0.323 6601–7200 76 50 25 0.201 7201–7800 35 28 12 0.314 7801–8400 51 17 11 0.291 8401–9000 48 35 12 0.282 9001–9600 37 29 14 0.367 9601–10200 59 35 21 0.246 10201–10800 44 16 6 0.319

4 10801–11400 27 21 11 0.367

11401–12000 25 6 8 0.357

12001–12600 9 1 0 0.415 12601–13200 23 5 3 0.396 13201–13800 34 1 15 0.348 13801–14400 36 28 14 0.273 14401–15000 18 1 2 0.358 15001–15600 46 18 8 0.329 15601–16200 44 10 1 0.347 16201–16800 25 7 2 0.350 16801–17400 39 4 4 0.354 17401–18000 42 8 24 0.348

18001–18600 13 1 0 0.407 18601–19200 33 0 1 0.337 19201–19800 38 0 0 0.348 19801–20400 41 3 0 0.360 20401–21000 22 0 1 0.354 21001–21600 16 0 0 0.419 21601–22200 18 2 0 0.392 22201–22800 16 6 11 0.361

22801–23400 20 0 1 0.423 23401–24000 17 2 2 0.386 24001–24600 30 20 21 0.367 24601–25200 24 0 0 0.389

25201–25800 11 0 0 0.411 25801–26400 14 0 0 0.385 26401–27000 32 0 2 0.403 27001–27600 21 0 0 0.382 27601–28200 17 1 1 0.353 28201–28800 27 1 0 0.334 28801–29400 39 40 6 0.304 29401–30000 68 20 15 0.365 30001–30600 75 35 20 0.339 30601–31200 35 67 36 0.307 31201–31800 67 48 25 0.292

31801–32400 46 23 17 0.286 32401–33000 26 28 2 0.393 33001–33600 75 35 18 0.339

5 33601–34200 49 48 38 0.308

34201–34800 64 21 22 0.342 34801–35400 42 17 32 0.314 35401–36000 22 0 1 0.409

36001–36600 8 0 0 0.432 36601–37200 12 0 0 0.429 37201–37800 12 0 0 0.451 37801–38400 13 10 23 0.417 38401–39000 22 12 11 0.393 39001–39600 45 20 3 0.361 39601–40200 26 4 1 0.396

40201–40800 21 1 3 0.396 40801–41400 10 0 0 0.410 41401–42000 10 0 5 0.427 42001–42600 13 2 0 0.422

42601–43200 7 0 0 0.418 43201–43800 11 0 0 0.436 43801–44400 13 0 3 0.430 44401–45000 32 2 4 0.411 45001–45600 25 23 24 0.316 45601–46200 21 10 2 0.337 46201–46800 17 3 3 0.372 46801–47400 28 13 7 0.354 47401–48000 41 10 7 0.322 48001–48600 46 16 21 0.359

48601–49200 29 1 0 0.384 49201–49800 59 47 37 0.305 49801–50400 53 91 75 0.331 50401–51000 42 82 52 0.320

51001–51600 50 29 27 0.306 51601–52200 42 15 9 0.318 52201–52800 25 19 10 0.392 52801–53400 26 54 42 0.290 53401–54000 47 25 10 0.327 54001–54600 31 9 0 0.377

54601–55200 35 7 0 0.367 55201–55800 17 9 0 0.403

55801–56400 8 0 0 0.434

6 56401–57000 16 0 0 0.444 57001–57600 37 18 14 0.335 57601–58200 72 42 63 0.200 58201–58800 17 1 2 0.412

58801–59400 12 0 0 0.409 59401–60000 48 15 16 0.422 60001–60600 47 28 33 0.320 60601–61200 47 7 1 0.302

61201–61800 28 0 1 0.351 61801–62400 39 12 0 0.348 62401–63000 54 22 47 0.285 63001–63600 58 10 2 0.332 63601–64200 32 7 4 0.398 64201–64800 36 25 22 0.333

64801–65400 20 1 4 0.336 65401–66000 19 0 3 0.334

66001–66600 16 1 0 0.394 66601–67200 52 15 12 0.304 67201–67800 64 18 26 0.307 67801–68400 18 5 2 0.378 68401–69000 35 10 3 0.366 69001–69600 81 40 34 0.278 69601–70200 44 15 14 0.295 70201–70800 38 12 3 0.367 70801–71400 56 17 37 0.325 71401–72000 66 10 25 0.272 72001–72600 33 13 12 0.331 72601–73200 26 23 7 0.340 73201–73800 31 14 13 0.370

73801–74400 30 5 0 0.383 74401–75000 34 7 8 0.343 75001–75600 19 15 6 0.375 75601–76200 50 29 32 0.270 76201–76800 33 30 33 0.333 76801–77400 56 67 66 0.282

77401–78000 20 4 0 0.430 78001–78600 15 3 0 0.460 78601–79200 28 19 21 0.396

7 79201–79800 47 86 69 0.218

79801–80400 27 5 2 0.371 80401–81000 51 4 5 0.294 81001–81600 19 7 3 0.377 81601–82200 30 3 0 0.378 82201–82800 32 14 17 0.352 82801–83400 29 8 2 0.364 83401–84000 36 12 3 0.338

84001–84600 26 0 4 0.349 84601–85200 37 1 9 0.418 85201–85800 34 8 11 0.319 85801–86400 28 1 1 0.352 86401–87000 33 24 17 0.335 87001–87600 19 7 4 0.371 87601–88200 43 26 10 0.320 88201–88800 56 71 68 0.297 88801–89400 35 23 33 0.328 89401–90000 36 5 2 0.296 90001–90600 28 18 7 0.340 90601–91200 14 2 4 0.399 91201–91800 11 3 4 0.443 91801–92400 8 0 1 0.414 92401–93000 9 0 0 0.393 93001–93600 13 3 0 0.384

93601–94200 8 0 0 0.403 94201–94800 10 1 3 0.401 94801–95400 13 2 2 0.388 95401–96000 9 4 2 0.358 96001–96600 17 1 2 0.351

96601–97200 7 3 0 0.383 97201–97800 7 0 2 0.382 97801–98400 8 2 12 0.378 98401–99000 10 1 0 0.382 99001–99600 13 3 1 0.403 99601–100200 4 2 1 0.403

100201–100800 12 4 0 0.423 100801–101400 14 10 0 0.445 101401–102000 32 4 3 0.371

8 102001–102600 16 2 4 0.391 102601–103200 14 3 3 0.392 103201–103800 7 2 0 0.383 103801–104400 11 3 3 0.355 104401–105000 7 1 0 0.401 105001–105600 10 1 0 0.413 105601–106200 15 20 33 0.381 106201–106800 25 21 42 0.392 106801–107400 21 22 21 0.417 107401–108000 16 9 0 0.438

108001–108600 4 0 0 0.532

108601–109200 2 0 0 0.545 109201–109800 10 3 0 0.564 109801–110400 14 0 5 0.538

110401–111000 11 3 0 0.471 111001–111600 9 1 2 0.517 111601–112200 10 2 0 0.502

112201–112800 6 0 0 0.529 112801–113400 1 0 0 0.552 113401–114000 2 0 0 0.547

114001–114600 1 0 0 0.578 114601–115200 1 2 0 0.535 115201–115800 8 2 0 0.487 115801–116400 25 7 1 0.429 116401–117000 36 8 8 0.363 120601–121200 62 22 22 0.250 121201–121800 93 40 38 0.260 121801–122400 44 58 33 0.321

122401–123000 28 1 0 0.346 123001–123600 37 25 2 0.285 123601–124200 10 7 0 0.371 124201–124800 50 9 2 0.369 124801–125400 37 5 0 0.381 125401–126000 83 29 29 0.229 126001–126600 59 20 2 0.313 130801–131400 51 38 43 0.266

131401–132000 42 17 5 0.288

132001–132600 47 0 9 0.290

9 132601–133200 72 5 15 0.280

133201–133800 58 6 4 0.259 133801–134400 78 8 11 0.255 134401–135000 54 10 22 0.294 135001–135600 74 5 14 0.323 135601–136200 57 3 16 0.281 136201–136800 59 9 12 0.379 136801–137400 35 4 5 0.359 137401–138000 12 5 2 0.398 138001–138600 25 6 0 0.477 138601–139200 2 4 0 0.479

139201–139800 0 0 0 0.558 139801–140400 2 0 0 0.579 140401–141000 2 0 0 0.552 141001–141600 2 0 0 0.533 141601–142200 6 1 0 0.512 142201–142800 15 2 0 0.521 142801–143400 4 2 0 0.487 143401–144000 11 7 0 0.501 144001–144600 18 3 4 0.540

144601–145200 2 0 0 0.563 145201–145800 2 0 1 0.556 145801–146400 11 0 0 0.492 146401–147000 15 9 1 0.407 147001–147600 21 24 20 0.410 147601–148200 37 29 39 0.361 148201–148800 6 3 1 0.423 148801–149400 11 1 1 0.374 149401–150000 8 3 1 0.403

150001–150600 12 2 0 0.355 150601–151200 8 3 1 0.398 151201–151800 11 0 4 0.388 151801–152400 29 3 0 0.388 152401–153000 23 9 4 0.392 153001–153600 9 7 1 0.470

153601–154200 10 2 2 0.382 154201–154800 6 2 1 0.420

154801–155400 12 0 1 0.392

10 155401–156000 10 3 13 0.387

156001–156600 9 0 2 0.372 156601–157200 4 0 1 0.381 157201–157800 11 3 2 0.372 157801–158400 14 3 2 0.363 158401–159000 13 3 2 0.362 159001–159600 13 2 2 0.403 159601–160200 10 0 0 0.387 160201–160800 9 1 0 0.403 160801–161400 11 2 5 0.371 161401–162000 6 0 1 0.410 162001–162600 7 3 3 0.408 162601–163200 10 1 1 0.454 163201–163800 28 19 15 0.352

The bins located in coding regions are printed in bold.

Figure S1 Maximum likelihood (ML) trees of 23 DSFs inferred from three differently filtered alignments of the complete plastome. (A) The strictly filtered; (B) the lightly filtered; (C) the unfiltered. Bootstrap support values > 50% are shown above branches.

Figure S2 ML trees of 23 DSFs inferred from three differently filtered alignments of LSC. (A) The strictly filtered; (B) the lightly filtered; (C) the unfiltered. Bootstrap support values > 50% are shown above branches.

Figure S3 ML trees of 23 DSFs inferred from three differently filtered alignments of IR. (A) The strictly filtered; (B) the lightly filtered; (C) the unfiltered. The colored branches represent unsuccessfully identified individuals; blue ones denote the conspecific individuals not forming a species-specific monophyletic clade, whereas orange ones stand for the conspecific individuals forming a monophyletic clade but with the support values < 70%. Bootstrap support values > 50% are shown above branches.

Figure S4 ML trees of 23 DSFs inferred from three differently filtered alignments of SSC. (A) The strictly filtered; (B) the lightly filtered; (C) the unfiltered. The orange branches represent the unsuccessfully identified individuals that form a species-specific monophyletic clade but with the support values < 70%. Bootstrap support values > 50% are shown above branches.

Figure S5 ML trees of 23 DSFs inferred from 12 datasets of commonly used DNA markers (A–L). The coloured branches represent unsuccessfully identified individuals; blue ones denote the conspecific individuals not forming a species-specific

16 monophyletic clade, whereas orange ones stand for the conspecific individuals forming a monophyletic clade but with the support values < 70%. Bootstrap support values > 50% are shown above branches (I, ITS; I2, ITS2; M, matK; R, rbcL; T, trnH-psbA; A, atpF-atpH; P, psbK-psbI; TT, trnT-trnL; R32, rpl32-trnL; C, clpP-psbB; TI, trnL intron; R16, rps16-trnQ).

Figure S6 Sliding window analysis of the LSC regions of DSFs (window length: 600 bp, step size: 200 bp). X-axis: position of the midpoint of a window, Y-axis: nucleotide diversity (Pi) of each window.

Figure S7 ML trees of 23 DSFs inferred from the combination of 6 highly variable fragments of LSC region (rps16-trnQ+ndhJ-trnV+atpB-rbcL+psbB-psbT+trnT-psbD+trnT-trnL). The colored branches represent unsuccessfully identified individuals; blue ones denote the conspecific individuals not forming a species-specific monophyletic clade, whereas orange ones stand for the conspecific individuals forming a monophyletic clade but with the support values < 70%. Bootstrap support values > 50% are shown above branches.