Chloroplast primers for clade-wide phylogenetic studies of Thalictrum.

PREMISE: Chloroplast primers were developed for phylogenetic and comparative studies in Thalictrum (Ranunculaceae). METHODS AND
RESULTS: We assembled and annotated the complete plastome sequence of T. thalictroides by combining multiple whole genome sequencing libraries. Using transcriptome-sequencing libraries, we also assembled a partial plastome of the related species T. hernandezii. From the newly assembled plastomes and one previously sequenced plastome, we designed and validated 28 primer pairs to target variable portions of the chloroplast genome in Thalictrum. Furthermore, we tested the validated primers in 62 species of Thalictrum. The total alignment length of the 28 regions was 15,268 bp with 2443 variable sites and 92% character occupancy.
CONCLUSIONS: The newly developed chloroplast primer pairs improve the phylogenetic resolution (bootstrap support and tree certainty) in Thalictum and will be a useful resource for future phylogenetic and evolutionary studies for species in the genus and in close relatives in Thalictroideae.

The chloroplast genome (cpDNA) has been particularly useful for resolving evolutionary relationships in plants for the past 30 years (reviewed in Gitzendanner et al., 2018). High‐throughput sequencing has facilitated the development of various approaches for collecting multiple regions or complete sequences of this genome (reviewed in Twyford and Ness, 2016). Furthermore, approaches based on PCR target enrichment in combination with high‐throughput sequencing (e.g., Uribe‐Convers et al., 2016) have proven to be a cost‐effective approach for sequencing multiple chloroplast regions simultaneously, and have been successfully applied in phylogenetic studies (e.g., Jacobs et al., 2018; Morales‐Briones and Tank, 2019).

Thalictrum L. (Thalictroideae, Ranunculaceae) is a clade of ca. 190 species of herbaceous perennials distributed primarily in northern temperate regions (Tamura, 1995) with a diversity of sexual systems (hermaphroditic, dioecious, andromonoecious, or gynomonoecious [Boivin, 1944]), pollination mode (insect or wind [Soza et al., 2012; Wang et al., 2018]), and ploidy (from 2x =14 to 30x = 210 [Soza et al., 2013]). To date, molecular phylogenetic studies of Thalictrum have relied on sequences of the nuclear ribosomal DNA (nrDNA) cistron, especially the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions, and up to five cpDNA regions (Soza et al., 2012, 2013; Wang et al., 2018). Although Soza et al. (2013) surveyed several cpDNA regions from Shaw et al. (2007), only one was identified as sufficiently variable for phylogenetic analyses in Thalictrum. Here, we assembled and annotated the complete plastome of T. thalictroides (L.) A. J. Eames & B. Boivin, assembled a partial plastome of T. hernandezii Tausch ex J. Presl, and designed and validated PCR primers that target highly variable chloroplast regions in Thalictrum to aid in future phylogenetic studies of this group and close relatives.

METHODS AND RESULTS

Plastome assembly

Genomic libraries of T. thalictroides and transcriptome libraries of T. hernandezii were downloaded from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (Appendix 1). Due to low cpDNA coverage in the transcriptome libraries, a reference‐guided assembly of T. hernandezii was carried out using Alignreads version 2.5.2 (Straub et al., 2011) with T. coreanum H. Lév. as a reference (Park et al., 2015), with one inverted repeat (IR) removed. We obtained a plastid consensus sequence made of 151 contigs representing 116,700 bp (after removal of regions not covered in the reference) for T. hernandezii. Given the availability of multiple genome sequencing libraries for two samples of T. thalictroides (Appendix 1), we performed de novo assemblies for this species. Assemblies were carried out for the two individuals of T. thalictroides (WT478 and WTBG) separately using the Fast‐Plast version 1.2.6 pipeline (McKain and Wilson, 2017). Single contigs representing the complete chloroplast genome of T. thalictroides were obtained. The resulting complete plastomes of T. thalictroides were annotated using CpGAVAS (Liu et al., 2012). Genes encoding for tRNAs were verified using tRNAscan‐SE version 2.0 (Lowe and Chan, 2016). Annotations were verified and edited in Geneious version 7.1.9 (Kearse et al., 2012) using other available Ranunculaceae plastomes as references (Appendix 1). The genome map was drawn with OGDraw version 1.2 (Lohse et al., 2013). Characterization of the T. thalictroides plastome and its comparison with the plastome of T. coreanum (Park et al., 2015) can be found in Appendix S1.

Primer design

Plastome sequences of T. thalictroides, T. hernandezii, and T. coreanum (all with one IR removed) were aligned using MAFFT version 7.017b (Katoh and Standley, 2013). The most variable regions of the alignment, spanning 400–600 bp, were identified using a custom R script (Uribe‐Convers et al., 2016). Primer design was carried out using Primer3 (Untergasser et al., 2012) following the specifications of the microfluidic PCR Access Array system protocol (Fluidigm, San Francisco, California, USA), with an annealing temperature of 60°C (±1°C) and no more than three continuous nucleotides of the same base. Primer validation followed Uribe‐Convers et al. (2016) by simulating the four‐primer reaction of the microfluidic PCR. We used our target‐specific primers and 5′ conserved sequence (CS) tags to provide annealing sites for Illumina sequencing adapters and sample‐specific barcodes. PCR validations were done using genomic DNA from three Thalictrum species (Appendix 2) and a negative control that did not contain DNA. Amplicons were visualized in a QIAxcel Advanced System (QIAGEN, Valencia, California, USA) and scored following Uribe‐Convers et al. (2016).

A total of 81 primer pairs were designed, of which 28 passed the validation step (Table 1), 32 failed (i.e., failed to amplify in one or more species, produced significant primer dimers, and/or produced multiple amplicons), and 21 were not validated (Appendix S2). In order to test cross‐amplification of the 28 validated PCR primer pairs, we amplified and sequenced these regions on 75 individuals of Thalictrum representing 62 species from 13 of 14 described sections of the genus (Tamura, 1995). Our sampling represents 33% of Thalictrum species and 70% of the sampling used in the most recent molecular phylogeny of the genus (Wang et al., 2018). Additionally, we amplified the newly designed regions from one individual each of three related genera in Thalictroideae (Aquilegia L., Leptopyrum Rchb., and Paraquilegia J. R. Drumm. & Hutch.; Appendix 2) using previously extracted DNA samples by Soza et al. (2012). Microfluidic PCR was carried out on the Access Array system (Fluidigm) following the manufacturer's protocol. PCR amplicons were multiplexed, and sequenced in an Illumina MiSeq (San Diego, California, USA) with 300‐bp paired‐end reads. Raw reads were cleaned, demultiplexed, and merged using the dbcAmplicons pipeline (Uribe‐Convers et al., 2016). Consensus sequences for each sample in all amplicons were generated using the ‘reduce_amplicons’ R script (part of dbcAmplicons). Each chloroplast region was aligned with MAFFT and alignment summary statistics were calculated with AMAS (Borowiec, 2016).

Table 1

Sequence of validated primers for the Thalictrum chloroplast genome.a

Locus	Primer sequences (5′–3′)b	Amplification region	Chloroplast region	Amplicon length, bpc
thal‐13	F: GCAATAAGTCCGGTTTGCAT	atpA, (atpA‐atpF) IGS, atpF	LSC	550
	R: GGGCGATGAAAGAAATAAACG
thal‐15	F: ACATGGCTTTCTTCCATAACG	(atpH‐aptI) IGS, atpI	LSC	574
	R: GAATCCATGGAGGGTCATCA
thal‐44	F: TGAAGTGATAGCCCGATTCC	(rbcL‐accD) IGS, accD	LSC	518
	R: TTTCCAGTTCATTCCGATCA
thal‐45	F: TGATGGGTCTAAGAGTGACAATCA	accD	LSC	419
	R: CGATTCTTTCTGAACTGCTCATT
thal‐46	F: TTTGCAGCATTGAGTAAGGAAC	(ycf4‐cemA) IGS	LSC	577
	R: CCCGAACGAGTCATTTCAA
thal‐47	F: GAGAAGGTTCAATTGTCCGAAA	petA, (petA‐psbJ) IGS, psbJ	LSC	572
	R: GGTATTCTTGTGATCGGTTTACTAGG
thal‐50	F: TGAGGTGATTGGATTTGCAC	(rpl20‐clpP) IGS, clpP	LSC	558
	R: CGAAGACATGGAAAGGGATG
thal‐51	F: AACCCTTGTGAGGGTTTCG	clpP	LSC	541
	R: GAGGCCTCTTTCCAATATTTATGTTA
thal‐52	F: TTACATATTGCGAAGGCATAGTCT	clpP	LSC	414
	R: TGAACCGTATGCATCCAAAG
thal‐53	F: AAGAATCAATGTGCTGATTCCA	clpP	LSC	534
	R: GTATCCAGGCTCCGTTCAGA
thal‐54	F: TCTGAACGGAGCCTGGATAC	clpP, (clpP‐psbB) IGS	LSC	560
	R: TTCGTAGGAACAAAGATAAGCAGA
thal‐55	F: TGCTCTTGTATCTTTCGCCTCT	(psbB‐psbT) IGS, psbT, (psbT‐psbN) IGS, psbN, (psbN‐psbH) IGS	LSC	525
	R: CATTGCGGTCTTGCAATTT
thal‐57	F: CTGGCTCCGTAAGATCCAGT	petD	LSC	513
	R: CGAAGGAACCGGACATGATA
thal‐58	F: GGAGCAACATTGCCTATTGATAA	petD, (petD‐rpoA) IGS, rpoA	LSC	546
	R: CAATCAAGGCAGGGTTACTTTAC
thal‐59	F: TAACCCTGCCTTGATTGTCC	rpoA	LSC	565
	R: GGAACATGTATCACACGAGCA
thal‐61	F: TCGAATTGTTATTCAACCCTATAGAA	(rpl16‐rps3) IGS, rps3	LSC	597
	R: AATCGATCTGATCCAGGTCATAA
thal‐62	F: CCCTCGGTCTATTAGTGAACCA	ycf2	IR	562
	R: CCAAGCTCGAAGTACCATTTG
thal‐64	F: ATATGCGCCCTCCACCTAC	ndhF	SSC	379
	R: TTTGATTGGTATGAATTTGTGAGAA
thal‐65	F: ATGGATCCGACGAACAAAGT	ndhF	SSC	541
	R: GGCTCTTATGGGCGGTTTA
thal‐68	F: TGTGTGGATCATTATTATCAGTAGCTC	ccsA	SSC	506
	R: TGAACCATAACTATGCAGCCCTA
thal‐69	F: AAAGGTCTTACAAATCCAATACGC	(ccsA‐ndhD) IGS, ndhD	SSC	581
	R: CTCGATGGCTTCTCTTGCAT
thal‐70	F: CCCAGAACTCCCATTAAGAGAA	ndhD	SSC	483
	R: TTTCCCTCATAGAGGAAATAAGGTT
thal‐72	F: CCGATGGATAATAAATAGGCACTC	ndhE, (ndhE‐ndhG) IGS, ndhG	SSC	591
	R: TGTGATGTTCATCAATGGTTCA
thal‐74	F: TCCGCTTAGCTTAACCCTTG	ndhA	SSC	525
	R: TCGTTTATTCAGTATCGGACCA
thal‐75	F: AACACTCCGATCTCCTATCAGAA	ndhH	SSC	530
	R: GGATAGATAAATGTTTGGATTTCTGTG
thal‐78	F: TGCGGCACTAATCTAGACCATC	ycf1	SSC	542
	R: TCCCGACTAATACGTAAATGTCAC
thal‐80	F: TCTGAATACCGTCGATTAACCA	ycf1	SSC	503
	R: ATGCGTGCTCAAAGACGTAA
thal‐81	F: CGTATCAAAGCCACTTCGTCT	ycf1	SSC	578
	R: CATCGCGGAACAATCAAA

IR = inverted repeat region; LSC = large single copy region; SSC = small single copy region.

Primer pairs were designed for an annealing temperature of 60°C (±1°C). Validation consisted of successful (single amplicon) amplification on three test species and absence of (or minimal) primer dimer detection.

Conserved sequence tags CS1 (5′‐ACACTGACGACATGGTTCTACA) and CS2 (5′‐TACGGTAGCAGAGACTTGGTCT) were added to each primer to make target‐specific primer for microfluidic PCR.

Estimated from three Thalictrum species, including primer length.

The cross‐amplification and sequencing resulted in regions with 15–75 (mean 69) consensus sequences of Thalictrum. Two regions, thal‐53 and thal‐55, had lower amplification success, with 15 and 40 sequences, respectively (Table 2, Fig. 1, Appendix S3). The amplification success per sample ranged from 19 to 28 (mean 26) regions. The amplification success in Aquilegia, Leptopyrum, and Paraquilegia was 12, 26, and 23 regions, respectively (Fig. 1), showing the potential utility of the newly developed primers on related genera in Thalictroideae. Thalictrum‐only alignment lengths ranged from 335 to 658 bp (mean 525 bp; Appendix S3), with the number of variable sites ranging from nine to 195 (mean 73). Alignments including the other Thalictroideae genera ranged from 335 to 725 bp (mean 544; Appendix S3) in length and contained 29–218 (mean 86) variable sites (Table 2). The total alignment length of the 28 regions (including all genera) was 15,268 bp, with 2443 variable sites and 92% character occupancy.

Table 2

Alignment summary statistics for 28 amplified chloroplast regions in Thalictrum and relatives.

Locus	Thalictrum					Thalictrum + Aquilegia + Leptopyrum + Paraquilegia
	Alignment length, bp	No. of sequences	Sequence length range, bp (mean)	Pairwise identity, %	Variable sites, bp (PI)	Alignment length, bp	No. of sequences	Sequence length range, bp (mean)	Pairwise identity, %	Variable sites, bp (PI)
thal‐13	515	75	509–515 (509)	99.50	28 (12)	531	78	509–522 (509)	99.40	42 (14)
thal‐15	605	75	434–544 (525)	93.70	85 (36)	632	78	434–544 (524)	93.00	128 (45)
thal‐44	496	53	466–479 (473)	98.50	33 (20)	500	55	457–479 (472)	98.10	45 (22)
thal‐45	372	75	372–372 (372)	99.00	24 (15)	372	77	345–372 (371)	98.50	30 (15)
thal‐46	615	74	500–561 (521)	96.10	142 (34)	630	76	500–561 (520)	95.80	152 (43)
thal‐47	658	75	150–554 (518)	83.50	195 (140)	725	78	150–554 (518)	83.40	218 (150)
thal‐50	545	75	499–517 (506)	97.50	30 (14)	556	77	495–517 (506)	97.20	43 (14)
thal‐51	594	73	461–557 (512)	85.20	190 (140)	599	75	461–557 (511)	85.20	197 (142)
thal‐52	381	75	366–377 (370)	99.60	16 (11)	571	77	366–558 (372)	98.10	45 (11)
thal‐53	585	15	469–560 (520)	85.60	126 (78)	NA	NA	NA	NA	NA
thal‐54	567	72	505–558 (514)	98.30	76 (17)	575	73	505–558 (514)	98.20	78 (18)
thal‐55	504	40	474–495 (486)	97.30	28 (19)	524	41	474–501 (486)	97.10	32 (19)
thal‐57	495	73	464–480 (473)	99.00	36 (23)	496	75	464–480 (473)	98.90	45 (25)
thal‐58	568	74	482–556 (485)	98.10	39 (19)	573	75	458–556 (485)	97.90	48 (21)
thal‐59	524	74	518–524 (524)	99.40	33 (18)	524	77	518–524 (524)	99.20	53 (18)
thal‐61	590	70	532–553 (552)	96.10	153 (107)	591	72	532–553 (552)	96.00	165 (110)
thal‐62	519	75	519–519 (519)	99.80	9 (7)	525	78	519–525 (519)	99.70	29 (7)
thal‐64	335	72	335–335 (335)	98.50	50 (30)	335	74	335–335 (335)	98.40	58 (36)
thal‐65	579	75	496–563 (504)	96.80	77 (34)	586	78	496–563 (504)	96.70	93 (39)
thal‐68	456	73	447–456 (456)	98.90	47 (29)	474	76	447–468 (456)	98.60	71 (38)
thal‐69	616	72	521–558 (545)	96.00	96 (64)	620	75	462–558 (544)	95.30	111 (62)
thal‐70	436	75	436–436 (436)	99.20	38 (20)	436	78	436–436 (436)	99.00	52 (24)
thal‐72	658	68	530–556 (542)	97.10	66 (30)	662	70	530–556 (542)	96.70	103 (42)
thal‐74	495	74	454–488 (482)	98.40	38 (21)	593	76	454–560 (483)	97.60	50 (21)
thal‐75	488	75	249–480 (477)	97.70	104 (15)	486	78	249–480 (477)	97.80	82 (23)
thal‐78	508	73	469–502 (484)	98.10	54 (33)	556	75	469–502 (485)	97.50	83 (35)
thal‐80	460	72	454–460 (460)	98.10	105 (40)	460	73	454–460 (460)	97.90	118 (44)
thal‐81	545	71	502–545 (529)	97.40	121 (62)	551	74	502–545 (529)	97.00	146 (67)

PI = parsimony informative; NA= not applicable.

Figure 1

Cross‐amplification performance of chloroplast primers for phylogenetic studies in Thalictrum. Amplification results in 62 species of Thalictrum and one species of Aquilegia, Leptopyrum, and Paraquilegia (outgroups) with the 28 validated primer pairs. Darker shades of blue represent longer amplification products; white represents failed amplification. Thalictrum clades sensu Soza et al. (2012, 2013).

To test the usefulness of the newly generated chloroplast primers for improving phylogenetic resolution within Thalictrum, we inferred a phylogeny of 62 Thalictrum species (one individual per species) and three outgroups using all 28 regions and compared it to an inferred phylogeny of the same species using six chloroplast regions (ndhA, rbcL, rpl16, rpl32‐trnL, trnL‐trnF, and trnV‐ndhC) (Soza et al., 2012, 2013; Wang et al., 2018). For each concatenated matrix, we searched for the best partition scheme followed by maximum likelihood tree inference and 1000 ultrafast bootstrap replicates for node support using IQ‐Tree version 1.6.10 (Nguyen et al., 2014). As an additional measure of tree resolution, we estimated internode certainty scores (Salichos et al., 2014) using the majority rule consensus tree across 1000 bootstrap replicates in RaxML version 8.2.11 (Stamatakis, 2014). The six‐region matrix had an aligned length of 6650 bp and 363 parsimony‐informative sites, whereas the 28‐region matrix had an aligned length of 15,268 bp and 1045 parsimony‐informative sites. Mean bootstrap values of the 28‐region trees were higher than those of the six‐region trees (89% and 79%, respectively; Fig. 2A). Moreover, mean internode certainty scores were also higher in the 28‐region tree (0.68 and 0.51, respectively; Fig. 2B). In summary, these results show that the 28‐region chloroplast matrix produces a tree with overall higher node support than the six‐region matrix, and is therefore suitable for improved phylogenetic studies in Thalictrum and close relatives.

Figure 2

Overall performance of chloroplast primers for phylogenetic studies in Thalictrum. (A) Bootstrap value distribution of the 28‐region (blue) and six‐region (yellow) phylogenies. Dashed lines represent mean values. (B) Internode certainty scores distribution of the 28‐region (blue) and six‐region (yellow) phylogenies. Dashed lines represent median scores.

CONCLUSIONS

Here, we contribute chloroplast primers for phylogenetic and comparative studies of Thalictrum and its close relatives in Thalictroideae. Furthermore, we demonstrate the utility of whole genome and transcriptome libraries as a source of chloroplast sequence data for PCR primer design. Out of the 81 chloroplast primer pairs reported here, 28 were successfully validated for use with the high‐throughput, Fluidigm‐based microfluidic PCR system. Finally, although this was not directly tested here, these primers could also be used for traditional PCR.

APPENDIX S1. Characterization of the Thalictrum thalictroides plastome and comparison to the plastome of T. coreanum.

Click here for additional data file.

APPENDIX S2. Primer sequences of the Thalictrum chloroplast genome that failed to pass our validation criteria or were not validated.

Click here for additional data file.

APPENDIX S3. Length (in base pairs) of 28 amplified chloroplast regions in Thalictrum and relatives.

Click here for additional data file.

Species	Sample code	Usage	Accession no.	Notes
Thalictrum hernandezii Tausch ex J. Presl	WT441	Partial plastome assembly and primer design	SRA: SRR6869419, SRR6869420	Transcriptome libraries. Libraries represent staminate and hermaphrodite flowers from one andromonoecious individual.
T. thalictroides (L.) A. J. Eames & B. Boivin	WT478	Whole plastome assembly and primer design	SRA: SRR6869426, SRR6869425, SRR6869418	Genomic libraries. Each library has different insert size.
T. thalictroides	WTBR	Whole plastome assembly	SRA: SRR6869424, SRR6869423, SRR6869421	Genomic libraries. Each library has different insert size.
T. coreanum H. Lév.	—	Primer design and plastome annotation	GenBank: NC026103
Aconitum chiisanense Nakai	—	Plastome annotation	GenBank: NC029829
Megaleranthis saniculifolia Ohwi	—	Plastome annotation	GenBank: NC012615
Ranunculus macranthus Scheele	—	Plastome annotation	GenBank: DQ359689

Species	Sample code	Voucher (Herbarium)	Locality
Aquilegia formosa Fisch. ex DC.	Aqfor1	V. Di Stilio 128 (WTU)	Di Stilio Garden, Seattle, WA, USA
Leptopyrum fumarioides (L.) Rchb.	Lefum1	A. Liston 819‐13 (RSA)	Xinjiang, China
Paraquilegia microphylla (Royle) J. R. Drumm. & Hutch.	Pamic1	I. Smirnov 277 (RSA)	Irkutsk, Arshan, Russia
Thalictrum alpinum L.	Thalp2	D. E. Boufford et al. 32249 (F)	Xizang (Tibet), China
Thalictrum alpinum L.	Thalp3	V. Di Stilio 115 (WTU)	Cultivated from Ion Exchange Nursery, Iowa, USA
Thalictrum amurense Maxim.	Thamu1	Unvouchered	Cultivated at UC Botanical Garden at Berkeley, CA, USA
Thalictrum aquilegiifolium L.	Thaqu1	V. Di Stilio 108 (WTU)	Cultivated material from Cricklewood Nursery, CA, USA
Thalictrum arsenii B. Boivin	Thars1	A. Liston 1128 (OSC)	Mpio. Morelia, Jaripeo, Michoacan, Mexico
Thalictrum atriplex Finet & Gagnep.	Thatr1	D. E. Boufford et al. 32557 (GH)	Xizang (Tibet), China
Thalictrum baicalense Turcz.	Thbai1	Unvouchered	Cultivated at University of Washington Botany Greenhouse, Seattle, USA, from seeds from B & T World Seeds
Thalictrum baicalense Turcz.	Thbai2	R. Zhang 20120614‐01 (PE)	Ecological station, Dong Lin Mtn., China
Thalictrum baicalense Turcz.	Thmeg1	D. E. Boufford et al. 37958 (GH)	Sichuan, Danba Xian, China
Thalictrum cooleyi H. E. Ahles	Thcoo1	Unvouchered	The State Botanical Garden of Georgia, Athens, GA, USA
Thalictrum coriaceum (Britton) Small	Thcor3	A. Floden s.n. (TENN)	Greene Co., TN, USA
Thalictrum cultratum Wall.	Thcul1	D. E. Boufford et al. 31166 (F)	Xizang (Tibet), China
Thalictrum cultratum Wall.	Thcul4	D. E. Boufford et al. 31233 (GH)	Xizang (Tibet), China
Thalictrum dasycarpum Fisch. & Avé‐Lall.	Thdas1	V. Di Stilio 110 (WTU)	Cultivated from Ion Exchange Nursery, Iowa, USA
Thalictrum decipiens B. Boivin	Thdec3	L. Galetto 2089 (CORD)	Pampa de Achala, Córdoba, Argentina
Thalictrum delavayi Franch.	Thdel1	D. E. Boufford et al. 30452 (F)	Sichuan, Xiangcheng Xian, China
Thalictrum delavayi Franch.	Thdel2	V. Di Stilio 121 (WTU)	Cultivated from seed from B & T World Seeds, Aigues‐Vives, France
Thalictrum diffusiflorum C. Marquand & Airy Shaw	Thdif1	A. Liston 1161 (OSC)	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum dioicum L.	Thdio3	M. Sain 60 (WIS)	University of Wisconsin campus, Muir Woods, Madison, WI, USA
Thalictrum dioicum L.	Thdio2	V. Di Stilio 101 (A)	Lithia Springs, South Hadley, MA, USA
Thalictrum fendleri Engelm. ex A. Gray	Thfen4	Unvouchered	Cultivated at UW Botany Greenhouse, Seattle, USA
Thalictrum fendleri Engelm. ex A. Gray	Thfen3	V. Soza 1920 (WTU)	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum filamentosum Maxim.	Thfil2	V. Di Stilio 104 (WTU)	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum finetii B. Boivin	Thfin1	D. E. Boufford et al. 33172 (GH)	Sichuan, Jiulong Xian, China
Thalictrum flavum L.	Thfla2	V. Di Stilio 109 (WTU)	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum foetidum L.	Thfoe5	Unvouchered	Cultivated from Arrowhead Alpines nursery, Michigan, USA
Thalictrum foetidum L.	Thfoe2a	V. Soza 1923 (WTU)	Cultivated from Arrowhead Alpines nursery, Michigan, USA
Thalictrum grandiflorum Maxim.	Thgra1	Unvouchered	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum grandifolium S. Watson	Thgra2	G. B. Hinton 20254 (TEX)	Coahuila, Mexico
Thalictrum heliophilum Wilken & DeMott	Thhel1	Mary Waters s.n. (CS)	Cathedral Bluffs, Rio Blanco County, CO, USA
Thalictrum henricksonii M. C. Johnst.	Thhen1	J. Henrickson 13417 (RSA)	Zacatecas, Mexico
Thalictrum hernandezii Tausch ex J. Presl	Thher2a	A. Liston 1125 (OSC)	Temascaltepec, State of Mexico, Mexico
Thalictrum ichangense Lecoy. ex Oliv.	Thich2	A. Floden 13116 (TENN)	Sapa, Lao Cai Province, Vietnam
Thalictrum isopyroides C. A. Mey.	Thiso1	V. Di Stilio 111 (WTU)	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum kiusianum Nakai	Thkiu1	J. Brunet s.n. (OSC)	Cultivated at Corvallis, OR, USA
Thalictrum lecoyeri Franch.	Thlec1	D. E. Boufford et al. 37972 (GH)	Sichuan, Danba Xian, China
Thalictrum leuconotum Franch.	Thleu1	D. E. Boufford et al. 42102 (GH)	Yunnan, Zhongdian Xian, China
Thalictrum lucidum L.	Thluc1	V. Di Stilio 122 (WTU)	Cultivated from Arrowhead Alpines nursery, Michigan, USA
Thalictrum macrostylum Shuttlew. ex Small & A. Heller	Thmac2	Unvouchered	“Serpentine Barrens,” Chunky Gal Mountain, NC, USA
Thalictrum minus L.	Thmin1	H. W. Rickett & F. A. Stafleu 742 (OSC)	Gelderland, Netherlands
Thalictrum minus L.	Thmin2	V. Soza 1910 (WTU)	Cultivated at University of Washington Botany Greenhouse, Seattle, WA, USA
Thalictrum occidentale A. Gray	Thocc2	K. A. Beck 200712 (WTU)	Lower Boundary Dam Reservoir, Pend Oreille River, WA, USA
Thalictrum omeiense W. T. Wang & S. H. Wang	Thome2	A. Liston 1166 (OSC)	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum petaloideum L.	Thpet3	Unvouchered	Dong Ling Mt., Betula Forest, Beijing, China
Thalictrum petaloideum L.	Thpet2	Unvouchered	Tong Ling Mt., Yin Ranje, Beijing, China
Thalictrum pinnatum S. Watson	Thpin1	R. M. Straw & M. Forman 1857 (RSA)	Chihuahua, Mexico
Thalictrum podocarpum Kunth ex DC.	Thpod1	M. Weigend 2000/623 (OSC)	Ancash, Huaylas, Pampanomas, Peru
Thalictrum polycarpum (Torr.) S. Watson	Thpol3	B. Keller s.n. (UC)	Cultivated at University of California Botanical Garden at Berkeley, CA, USA
Thalictrum polycarpum (Torr.) S. Watson	Thpol2	J. F. Smith 4572 (WTU)	Cassia County, Idaho, USA
Thalictrum przewalskii Maxim.	Thprz1	D. E. Boufford et al. 36521 (GH)	Sichuan, Dege Xian, China
Thalictrum pubescens Pursh	Thpub1	D. Baum & D. Howarth 375 (A)	Arnold Arboretum, Jamaica Plain, MA, USA
Thalictrum punctatum H. Lév.	Thpun1	V. Di Stilio 117 (WTU)	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum ramosum B. Boivin	Thram1	A. Larsen s.n. (UC)	Cultivated at UC Botanical Garden at Berkeley, CA, USA
Thalictrum reniforme Wall.	Thren1	B. Keller s.n. (UC)	Cultivated at University of California Botanical Garden at Berkeley, CA, USA
Thalictrum reticulatum Franch.	Thret1	D. E. Boufford et al. 42802 (GH)	Sichuan, Muli Xian, China
Thalictrum revolutum DC.	Threv2	A. Floden 1347 (TENN)	Campbell County, TN, USA
Thalictrum revolutum DC.	Threv1	V. Soza 1917 (WTU)	Cultivated from USDA AMES #28275
Thalictrum rhynchocarpum Quart.‐Dill. & A. Rich.	Thrhy3	W. Kindeketa 820 (MO)	Arusha, Tanzania
Thalictrum rutifolium Hook. f. & Thomson	Thrut2	D. E. Boufford et al. 32127 (GH)	Xizang (Tibet), Riwoqe Xian, China
Thalictrum simplex L.	Thsim2	V. Soza 1914 (WTU)	Cultivated from USDA GRIN AMES #23805
Thalictrum smithii B. Boivin	Thsmi4	D. E. Boufford et al. 28205 (A)	Sichuan, Daocheng Xian, Gongling, China
Thalictrum sparsiflorum Turcz. ex Fisch. & C. A. Mey.	Thspa1	M. Williams 1630 (OSC)	7 miles from Seward, AK, USA
Thalictrum squamiferum Lecoy.	Thsqu2	D. E. Boufford et al. 32003 (GH)	Xizang (Tibet), Riwoqe Xian, China
Thalictrum squarrosum Stephan ex Willd.	Thsqu3	X. Duan 20120617 (PE)	Cultivated at CAS Botanical Garden, Beijing, China
Thalictrum steyermarkii Standl.	Thste1	T. B. Croat 40494 (MO)	Chiapas, Mexico
Thalictrum tenue Franch.	Thten1	W. Zhai 20120615 (PE)	Wanging, Henan, China
Thalictrum thalictroides (L.) A. J. Eames & B. Boivin	Ththa10a	V. Di Stilio 124 (WTU)	Cultivated from Arrowhead Alpines nursery, Michigan, USA
Thalictrum thalictroides (L.) A. J. Eames & B. Boivin	Ththa9	V. Di Stilio 123 (WTU)	Cultivated from natural population from Massachusetts, USA
Thalictrum tripeltiferum B. Boivin	Thtri1	L. E. Detling 8788 (ORE)	Jalisco, Mexico
Thalictrum uchiyamae Nakai	Thuch4	V. Di Stilio 113 (WTU)	Cultivated from seed from B & T World Seeds, Aigues‐Vives, France
Thalictrum uncatum Maxim.	Thunc2	D. E. Boufford et al. 30691 (GH)	Sichuan, Xiangcheng Xian, China
Thalictrum urbainii Hayata = T. fauriei Hayata	Thfau1	A. Liston 1162 (OSC)	Cultivated from Heronswood Nursery, Kingston, WA, USA
Thalictrum venulosum Trel.	Thven2	Voucher lost at OSC	NA
Thalictrum virgatum Hook. f. & Thomson	Thvir1	D. E. Boufford et al. 30496 (GH)	Sichuan, Xiangcheng Xian, China
Thalictrum zernyi Ulbr.	Thzer2	R. E. Gereau & C. J. Kayombo 3976 (MO)	Iringa, Ludewa, Tanzania

NA = not available; s.n. = unnumbered.

Sample used for primer validation and cross‐amplification.