An expanded nuclear phylogenomic PCR toolkit for Sapindales.

PREMISE OF THE STUDY: We tested PCR amplification of 91 low-copy nuclear gene loci in taxa from Sapindales using primers developed for Bursera simaruba (Burseraceae). METHODS AND
RESULTS: Cross-amplification of these markers among 10 taxa tested was related to their phylogenetic distance from B. simaruba. On average, each Sapindalean taxon yielded product for 53 gene regions (range: 16-90). Arabidopsis thaliana (Brassicales), by contrast, yielded product for two. Single representatives of Anacardiaceae and Rutacaeae yielded 34 and 26 products, respectively. Twenty-six primer pairs worked for all Burseraceae species tested if highly divergent Aucoumea klaineana is excluded, and eight of these amplified product in every Sapindalean taxon.
CONCLUSIONS: Our study demonstrates that customized primers for Bursera can amplify product in a range of Sapindalean taxa. This collection of primer pairs, therefore, is a valuable addition to the toolkit for nuclear phylogenomic analyses of Sapindales and warrants further investigation.

Low-copy nuclear gene regions offer increased phylogenetic utility for species- and population-level studies of plants as compared to chloroplast and nuclear ribosomal markers (Zimmer and Wen, 2012), yet sampling these regions remains challenging due to the dearth of universal primers and barriers to sequencing whole or partial nuclear genomes from multiple individuals. Consequently, assessing the phylogenetic limits of custom-designed target sequences or primers for low-copy nuclear gene regions is critical to fully realizing their broader impacts for advancing plant systematics. We report the results of a cross-amplification study incorporating primers for 91 low-copy nuclear gene loci created by Gostel et al. (2015) for species-level phylogenetics of Malagasy Commiphora Jacq. (Burseraceae). Primers for these markers were developed using genomic resources from two rosid orders by mapping sequence data from a transcriptome of Bursera simaruba (L.) Sarg. (Burseraceae; Sapindales) (Matasci et al., 2014) to 950 putative low- or single-copy nuclear gene loci of Arabidopsis thaliana (L.) Heynh. (Brassicaceae; Brassicales) (Duarte et al., 2010). Gostel et al. (2015) further optimized the primers for microfluidic PCR-based target enrichment, a method that allows simultaneous and cost-effective amplification of multiple loci (Blow, 2009; Uribe-Convers et al., 2016).

We tested cross-amplification of these primers using 10 taxa that have varying phylogenetic distances from B. simaruba within Sapindales and included A. thaliana as the outermost limit of the survey. Sapindales is a widespread group that includes ca. 6700 species within nine families (Angiosperm Phylogeny Group, 2016) (Fig. 1). Molecular phylogenies of this order often lack sufficient phylogenetic support along their backbone as well as at the species level (e.g., Fine et al., 2014; Grudinski et al., 2014), thus our understanding of Sapindalean systematics could benefit from an expanded phylogenetic toolkit such as that provided by the Gostel et al. (2015) primers.

Fig. 1.

Phylogeny of Sapindalean lineages condensed from Wang et al. (2009), Weeks et al. (2014), and Muellner-Riehl et al. (2016); nodes having low or conflicting support are indicated by dashed branches. Lineages sampled by the current study are noted by open diamonds. Generalized generic phylogeny of Burseraceae does not depict Rosselia or Pseudodacryodes, which have not been included in any molecular phylogenetic analysis; paraphyletic genera are indicated by asterisks.

METHODS AND RESULTS

Taxonomic sampling and molecular methods

Appendix 1 contains accession information for the 11 taxa sampled; Fig. 1 displays their phylogenetic relationships. Bursera simaruba (Bursera Jacq. ex L. subgenus Bursera) and C. grandifolia Engl. were included as positive controls; prior work has shown that all or most of the custom-designed primers amplify PCR product in these two species (Gostel et al., 2015). For experimental taxa, we included B. tonkinensis Guillaumin, which is sister to Commiphora (Weeks and Simpson, 2007), as well as Aucoumea Pierre, the monotypic genus sister to Bursera and Commiphora (Weeks et al., 2014). One species from each of Boswellia Roxb. ex Colebr., Canarium L., and Protium Burm. f. were included, as well as Beiselia Forman, the monotypic genus sister to all other Burseraceae (Weeks et al., 2014). We included one species of Anacardiaceae, the family that is sister to Burseraceae (Weeks et al., 2014), and one species of Rutaceae, which represents the Sapindalean clade sister to Burseraceae–Anacardiaceae–Kirkiaceae (Muellner-Riehl et al., 2016). Arabidopsis thaliana (Brassicales) was included because its genomic resources were used in primer design and can test the applicability of these primers to other closely related rosid lineages (Wang et al., 2009).

Whole genomic DNA was extracted from taxa using the FastPrep FastDNA Spin Kit (Bio101 Systems, La Jolla, California, USA) or the cetyltrimethylammonium bromide (CTAB) method (Weeks et al., 2005). Primer development for the 91 markers is detailed by Gostel et al. (2015); primer sequences are listed in Table 1. Markers were amplified via PCR in 15-μL reactions including: 0.15 μL of forward and reverse primers (50 μM), 0.75 μL spermidine (4 mM), 7.5 μL GoTaq Green Master Mix (Promega Corporation, Madison, Wisconsin, USA), 5.6 μL nuclease-free water, and 1 μL genomic DNA (0.1–25.8 ng/μL). Markers that failed to amplify for B. simaruba and C. grandifolia were then trialed using reaction chemistry based on that recommended for microfluidic PCR-based target enrichment including: 0.15 μL of forward and reverse primers (50 μM); FastStart High Fidelity PCR System reagents (Roche Diagnostics, Mannheim, Germany), composed of 1.5 μL FastStart High Fidelity Reaction Buffer without MgCl2 (10× concentration), 2.7 μL MgCl2 (25 mM), 0.75 μL DMSO, 1.2 μL Nucleotide Mix (10 mM), 0.15 μL FastStart High Fidelity Enzyme Blend (5 U/μL); 0.75 μL Loading Reagent (Fluidigm Corporation, San Francisco, California, USA); 6.8 μL nuclease-free water; and 1 μL genomic DNA.

Table 1.

Primer pair sequences and validation results by taxon.

		GenBank accession no.b
Locus IDa	Primer sequences (5′−3′)a	B. simaruba	C. grandifolia	Amplicon length range among all taxa	Arabidopsis thaliana	Aucoumea klaineana	Beiselia mexicana	Boswellia neglecta	Bursera simaruba	Bursera tonkinensis	Canarium pilosum	Commiphora grandifolia	Phellodendron amurense	Protium guianense	Schinus fasciculatus
AT3G54460c	F: GGACACACCCTTGGCTCTAG	KX767982	KX767983	270–290		X	X	X	X	X	X	X		X
	R: CTCCCATGACTTTTGGTTCTGTC
AT2G04620	F: TCCACCATATTTTGAGTGAGAGGAA	KX76792	KX767929	420–520		X		X	X	X	X	X	X	X	X
	R: AATGGGAGTGGGAATGAGAATGTG
AT4G37510c	F: TTCATTTTGAGACCTCCATTAGATGAC		KX768000	280		X	X	X	X	X	X	X		X	X
	R: GCTTAGCCGGATTATCGTCTCC
AT3G22660c	F: AGATGAAGATGTGAAATTGGTTGAACC	KX767974	KX767975	450			X	X	X	X	X	X		X
	R: TTTCTGCTTAGCTCTCTCTTTCATCT
AT1G21840c	F: TGTTGGAGAAGTTGAAGAGAGAGG			630–640		X	X	X	X	X	X	X		X
	R: CACCATTTATCCCAACCTCCTGAA
AT2G04740d	F: CAAACTCCAAAAACCCTAAACCGG	KX767930	KX767931	460–590		X		X	X	X	X	X		X
	R: TCAAAAGCCTTCAAAAGCTTCCTC
AT4G14605d	F: CTTCTCACTCATAGCAGGCAGAAG	KX767986	KX767987	510–580			X	X	X	X	X	X			X
	R: CTTCTTCACAGCCTTATCAAAGTCA
AT4G19900c	F: GTTCTCTGAGACGATTGAGCTTGA	KX767990	KX767991	350–420			X	X	X	X	X	X	X	X
	R: CTTGTAGAGAGCAGCAAGTCGG
AT4G29590	F: GAGCAATTCCCCTTCAAAGAGGAR: GTGCTTGTATCCTTTTGGGTAATGG	KX767994, KX767996	KX767995, KX767997	490				X	X	X	X	X		X
AT5G04910	F: TAAGAGTCCAACAGAGCATGAGTG	KX768005	KX768006	260			X		X	X	X
	R: TAAAAGAATGATGTCACTCAGCTTCG
AT3G15110e	F: CTCACTGGTGCCATATCTGTCTTC			1560					X
	R: ATTCTCTGTACCTTTGCTTCTGGA
AT1G18060d	F: AACAAGAAAGGTTGCAGTAGAGGA	KX767902	KX767903	740–930			X	X	X		X	X		X
	R: GCTTGGCTCTCTGTCATCTTTTTG
AT2G03667c	F: CTAGTTGGCTCTGGTGCTGATG	KX767926	KX767927	590			X	X	X	X	X	X		X
	R: CACAAAGGAATATCAAGCAAAGTCCT
AT2G40760	F: GGTGTATCATCTGGAAGGGGG	KX768007	KX768008	400		X		X	X	X	X	X		X
	R: CGCTCTCGCCCTCTCTTTC
AT2G20790[c,g]	F: CCAATTGTCAATGGTCTCTGAAGATG	KX767940	KX767941	320–350		X	X	X	X	X	X	X	X	X	X
	R: CCATGGTGCAAATTTAACTGTTCC
AT2G36740	F: AGTCCACAAGAACTGCAGTGAT			640–810			X	X	X	X	X			X
	R: CATCCTTTGAGAAATACCGTATCTGT
AT3G01380d	F: AATCATCATAATAGGGGCAGCCGR: CCAAGAAATATAGAAGTTAGTCGGGAC	KX767958, KX767960	KX767959, KX767961	530–930			X	X	X	X	X	X	X
AT3G10400	F: AGAAGAAAAAGACTAACAGTGACAGC	KX767966	KX767967	340				X	X	X	X	X
	R: CGGTCTTTGAGCACGCTGA
AT1G59990d	F: GCTACTTGGTTCCTTTAATTGATAAGCR: TGACACCACGAATAAAATCCAAGC	KX767908, KX767910	KX767909, KX767911	450–510		X	X	X	X	X	X	X			X
AT2G22370Bc	F: AACCCACATGGACTGTTAAACATG	KX767944	KX767945	610–780			X	X	X	X	X	X		X	X
	R: CATCAGACATAAGAGATGCAGCAG
AT1G31780[c,g]	F: CTTGTCCTTGGGTTACTTGATCCAR: TTGTGGGTCTCAATGATTTCAAGC	KX767904, KX767906	KX767905, KX767907	520–570		X	X	X	X	X	X	X	X	X	X
AT1G31780 (INT)[c,g]	F: CTTGTCCTTGGGTTACTTGATCCA			380–832			X	Xh	X	X	X	X	X	X	X
	R: GGACCCAAAGTGTACTACAGAGAG
AT2G27760	F: GAACCTTAAACCCTAACAATGGAGAA			930					X
	R: GGCGGTTCCGTGACCATAT
AT2G27760 (INT)	F: GAACCTTAAACCCTAACAATGGAGAA			160–470		X		X	X	X	X	X	X	X	X
	R: CGAAATTCCTTAGCAGTGAACTCC
AT1G63160 (INT)	F: GACGCTGTATCTAGGCTCCAAG			220–640					X	X	X		X	X
	R: AAAATGTTGCATGTGAAGTTTGGC
AT1G63160	F: GACGCTGTATCTAGGCTCCAAG			1070–1490			X	X	X	X	X				X
	R: CACCATGAGACGGCCAAGTAT
AT1G65030	F: CGGTTTTCTGTAACTCGGTACAG	KX767912	KX767913	340					X	X	X	X
	R: CGGGGGAAAGAGAGGTTTTGG
AT5G52180	F: CTCCGAGAATTTGGTTGGAAATGT	KX768003	KX768004	460				X	X		X	X
	R: CATACAGAAAGCCGCGTCGATA
AT2G44760d	F: CAGCATGGAATACGTTTGCTAGTA	KX767954	KX767955	530–900		X	X		X	X	X	X	X	X	X
	R: TATCAACTGGACCCCTGGAATAAG
AT2G05320Ac	F: TGCCAAGTGAAACAGATATTTGCT	KX767934	KX767935	440			X	X	X	X	X	X		X	X
	R: TCTCCAAACAGTCTGGTTAAAGGA
AT4G31770c	F: GCGGTGAGAAATGAGAATGACATG	KX767998	KX767999	580–780			X	X	X	X	X	X		X	X
	R: AACAAGTTCCTTCCAATTCCCAAA
AT2G20330[c,g]	F: TCATTGAAGGTTTGGGATTTACGC	KX767938	KX767939	610–750	X	X	X	X	X	X	X	X	X	X	X
	R: ACGACTTGGCTGATCTCTGAATAA
AT1G66080	F: CCTCTTCTCTTCCATAGTGTTGCT			900					X
	R: CCCACAAAACGACTGCATAAAGTT
AT2G05170Bc	F: GCACAGTACATTAACACCATTGGT			430–480		X	X	X	X	X	X	X		X	X
	R: TGGCTTGTGGTCTATGAGAATCTT
AT1G65070	F: CCTAATACTGGAGGGAAAACTGCT	KX767914	KX767915	510–600				X	X	X		X
	R: CAGTACTTCCCCAGAGAATTCGAA
AT5G67220	F: CGGTTAAAAATGCTCTCAGGATCC			690					X
	R: CATCTGCCGAATGAGTAACCTTCT
AT2G17265[c,d,g]	F: TTATGGGAGGTTTCGTTTTGATTCG	KX767936	KX767937	470–1690			X	X	X	X	Xh	Xh	X	X	X
	R: CTAGCACCAACTCTATCCAACCTC
AT2G46890Bd	F: TTCTTTGCTGTCTACCTCTCTCAG			570–780				X	X	X	X	X		X
	R: CGATGTCGTCTTCTGATATAGCCT
AT2G31890B[c,g]	F: CTCTCCAGTGCTCAGTTTTAACAG	KX767946	KX767947	410			X	X	X	X	X	X	X	X	X
	R: CTTGAGAAATGTGTTGGTCCATCA
AT2G46100c	F: TTTAAAGGACTTCGCCGTTTCAAA	KX767956	KX767957	310–370		X	X	X	X	X	X	X	X	X
	R: GGCAGAAAGAATAGGCCTCCAG
AT3G26580[c,g]	F: AGGTGAACGGTGTGGATTATGATGR: GTGACGGTTATTTGCCTCGTAAG	KX767976, KX767978	KX767977, KX767979	660–920		X	X	X	X	X	X	X	X	X	X
AT2G44660B	F: GTTTTTGCAGGAAGGGATGGATTT	KX767952	KX767953	590–1130				X	X	X	X	X			X
	R: TGAAGGTTGTTGCTGGAGTTATCT
AT2G44660B (INT)c	F: GTTTTTGCAGGAAGGGATGGATTT	KX767952	KX767953	520–900			X	X	X	X	X	X		X
	R: TGCCTGAATCTTGAACCCTAGTTT
AT3G49730	F: CCGAAACTGGAGATGGCTTTG			140					X
	R: AATCAACTCAGGCCTTTCTTTTCTC
AT2G44660Af	F: ATCGTATCACAGCACAGACTTTGA	KX767950	KX767951	790					X			X
	R: GCAAAAACAAAACCACCCATCAAA
AT2G21710c	F: TTTCCTCCTTTACTAACATACAGCCTR: CTTGTCTGCAACCTTCTGATTGAA	KX767942 (5′ only)	KX767943 (5′ only)	1040–1360			X	X	X	X	X	X	X	X
AT2G21710 (INT)d	F: TTTCCTCCTTTACTAACATACAGCCT			750–860			X	X	X		X	X		X	X
	R: GCTGCATCCCAAGAGCTCTGG
AT2G22370Ac	F: ATGTTGAGGCCCTTGAGATTCTTC			980–1320			X	X	X	X	X	X		X	X
	R: TAGGTGCTGTTACTTCAACCAGTT
AT1G77930Ad	F: ACCCTAATTCTGTTCTGCGATTTG	KX767924	KX767925	580–740				X	X	X	X	X
	R: GAGCAGTTCATAAGCAGCTTGAAT
AT1G77930A (INT)	F: ACCCTAATTCTGTTCTGCGATTTG			410–460		X		X	X	X	X	X		X
	R: GCATCCCTCTCTAACTCTGCAATT
AT5G02250d	F: CACTTATCCCATGTTTCCAGAGAAC			1240–1680				X	X	X	X	X	X
	R: GGATCTGCCTGGTTTTCAACATAT
AT2G31440	F: GTATGGAGGGCTTTTCTTCCTTTG			1000–1350					X	X
	R: ATTCCTGCAGCAAGATGAACTACA
AT1G77550Ad	F: TGTGAGCTTTTCTATATTGTGGCC	KX767920	KX767921	740–860			X	X	X	X		X
	R: TGATGCTTCATGACCAGACAAGA
AT3G15290e	F: GATGTTGTAGTCGAGGCTATTGTG			1090					X
	R: ATCTGCAAGTTCTAAAGGACCCAT
AT5G11980	F: TTCAACCATGCATCCCAAATTACC			N/A
	R: GACAGAGATCCGCCTTCAGTTATC
AT5G14580e	F: TATACGTATTGGCAGAATTTCCGG			1030–1750				X	X
	R: TCTTGTGCAATCTTATCTAAGGCCT
AT2G31840d	F: AGTGATTGATTGGTGTCCTGATGT			480–1220				X	X		X	X		X	X
	R: CATCTTGGTGAAGGTAGCCTACAG
AT5G57655	F: TTGGTTATGCTCAGTGTAATCCGA			340–1340			Xh	Xh	X		Xh			Xh	X
	R: CTACAGTGCAGATTGGAAAAGCAT
AT2G47760	F: CAGCATGGAATACGTTTGCTAGTA			620–1480					X						X
	R: TATCAACTGGACCCCTGGAATAAG
AT3G29130d	F: TTTGCCGAGGTTTCTGGTGATTR: AAGTACTTCTCTTGTTGATTCATCCG		KX767980, KX767981	980–1720					X		Xh	X
AT3G13200e	F: AACTCATCGGCTTTTTCCTCTCT			1970					X
	R: GAATCATCAGCATCTACATTGCGT
AT4G33030d	F: GATGGTGTCTTTGGTACTGCTTTG			770–1340				X	X		X	X
	R: CCAAGAAACAGTGGGCATTATCTG
AT1G73180d	F: AACTCCTGCCAGTGTCCAAATATA			810–1000				X	X		X	X	X	X	X
	R: AGAATGCCATATCACCAGGTAAGT
AT2G31890A	F: AGATTGGAGGGGAGCTACTTTATT	KX767948	KX767949	450–620			X		X	X	X	X	X	X	X
	R: CCTCCCTATACTGCTCTGAAATCC
AT3G46220d	F: CAATTGAGGAATGAAATGGTGGCT			330–570				X	X		X	X
	R: TCCATTTCTTGCAAAAGCTTCTGT
AT2G05120[c,g]	F: TGTCAAAGCTCTGGTCTCATGAAA	KX767932	KX767933	370–570			X	X	X	X	X	X	X	X	X
	R: CGAAGGAAGAACTGAAGCATCTAG
AT1G73740d	F: TTGATATTGGGAGGCTCTTTGGG			870–1230				Xh	X		Xh	X		Xh
	R: CACCAGCTCTTGAAACAACGAG
AT4G31790d	F: ATTTGGTTGTTCGAGCCAAGAAAA			1620–2180					X		X	X		X
	R: GTCCAAAATCAACCATCTGCAGTT
AT5G10460	F: TGGTCATCATTAGCAATTCTTCACG			1320–1800			X	X	X		X			X
	R: GCTTCTTCAACATTCTCCACAACT
AT4G26980d	F: CTGCTAGTGGTGTTTCTGAATTGG			940–1170				X	X		X	X		X
	R: ACTTCTCAGCCATTGACAACTCAT
AT5G48790d	F: GAGGATTTTGGTTTCACTGAAAAGG			680–1250			X	X	X		X	X	X		X
	R: TCGCGACCTTTAAAATTGTGAATGT
AT5G15680A	F: TTCTCATTCAAACATCATTGGGCC	KX768001	KX768002	560			X	X	X	X	X	X	X		X
	R: GAGGAATTGCATCAGATTCTCGTC
AT3G04650	F: CAAATCGCTTGCTTGGTTCATCAR: CTGTGGCAGTTGGGATGTTTTC	KX767962, KX767964	KX767963, KX767965	490–660			X	X	X		X	X		X	X
AT2G25570d	F: GACAACTCAAATACACATGCCAGG			660–1160				X	X		X	X		X	X
	R: GTCCCTTCTCTGATGCCCTATG
AT2G31040d	F: AAGTACTGGGGTGGAGAAAAAGAG			1230–1690				X	X		X	X	X	X
	R: CCAAGTGTGAGGATTTGCAACTTC
AT4G04955	F: GAACAGATACGGTACAGAGCCAG			440–1170					X	X
	R: TGCAGCTTTAGTCCCTGAAGG
AT3G21540c	F: GTTGCTATTAGTCCTGATGCCAAAR: AATGGTTCTTCAGTACGATCCCAA	KX767970, KX767972	KX767971, KX767973	730–1020			X	X	X	X	X	X		X
AT2G05170Ac	F: GAAAGGAAATGTACCAGTGGAGGA			650–880			X	X	X	X	X	X		X	X
	R: TGAGAAGAATTGGTGGAGCTTCTT
AT2G28450f	F: TTTCTGAGATCATGCTTATAGTTCAGG			1330					X			X
	R: CGCCCAATTGTTCCAGTACCA
AT3G07750d	F: GCTATATTTGTTGATTGCAGCCCT			940–1330			X	X	X		X	X		X
	R: TGGTTGCTCATCGTTTAATGCATC
AT1G76450d	F: TGGTCCGGATTTTACAAGAATGGAR: CGTCCGCACAAAAATCAAAATAGG	KX767916, KX767918	KX767917, KX767919	1330–1570					X			X		X
AT3G10530[c,d]	F: GGTCTTCTTGCATAATGAGCTGTT			700–1960			X	X	X	X	X	X	X	X
	R: TCAAATTTCCGCAAGTCCCAAATC
AT3G61620e	F: ATTTCCCCATTCAAATTCCACTCG			1330					X
	R: ACCTCATGCGATCTCCAGTACTAA
AT4G21770d	F: TGGAGCTGTTTATTATGCCCTTGT	KX767992	KX767993	730–1130			X	X	X		X	X	X
	R: TAGTCCTAGCTAACACAACACAGC
AT3G22990d	F: TTCTAGGTCCATCTCTTCAAGTGC			900–1130			X	X	X		X	X	X		X
	R: TTATGATATTTGAGGCAGCAACGG
AT4G18810B	F: CCTAAAAGGTGATGGTCGAAGGTA	KX767988	KX767989	540–640			X	X	X		X	X	X	X
	R: TCTCCCTTCAACTTGAATGTGAGA
AT1G77550Bd	F: ACAGTTCAGAAGGCACATGGATC	KX767922	KX767923	760–820				X	X			X		X
	R: CTGGTGTGTTCATGTGATTGAGTC
AT5G16690d	F: TGCTTCCTCTAGACAATTGTTCACT			760–820				X	X			X
	R: TCCAAAGAAGCCGCTATGAATTG
AT4G00560	F: CTGCTATGTCTATAAACGTTCCTTCCR: GTCCACCAACATTCAACAGTAACT	KX767984 (5′ only)	KX767985 (5′ only)	900–1180			X		X		X	X		X
AT3G17170d	F: GATGATGAACTATTTTTCCCTGAGGC			630–900				X	X	X	X	X
	R: TCTTGAACTTTCTCATTCACACTGC
AT3G14910c	F: GGAGCTATTTTATCAAAAGTTGTGCC	KX767968	KX767969	360–840	X		X	X	X	X	X	X		X	X
	R: AAAGCAATATACGACCAAGAGAATCTG
Total no. of primers amplified/taxon					2	16	47	68	90	53	71	72	26	54	34

Note: INT = reverse primer is an internal primer for the locus.

Primer originally developed by Gostel et al. (2015).

GenBank accession numbers from loci used in phylogenetic analysis in Gostel et al. (2015). GenBank numbers were only created for loci of Bursera simaruba and Commiphora grandifolia that were used in the phylogenetic analysis in Gostel et al. (2015). Some loci have two GenBank numbers for a species because sequence reads did not cover the full length of the locus. The first GenBank number corresponds to the read from the 5′ end of the locus; the second GenBank number corresponds to the read from the 3′ end of the locus.

Universal Burseraceae primer (excluding Aucoumea).

Primer for which high-fidelity TAQ increased amplification success for Commiphora grandifolia.

Primer for which high-fidelity TAQ increased amplification success for Bursera simaruba.

Primer for which high-fidelity TAQ increased amplification success for Bursera simaruba and Commiphora grandifolia.

Universal Sapindales primer (excluding Aucoumea).

Faint double band observed.

The PCR thermocycler protocol followed that of Gostel et al. (2015) and included three alternating standard and Cot cycles (Mathieu-Daude et al., 1996), beginning with 2 min at 50°C, 20 min at 70°C, and 10 min at 95°C. The first set of 10 standard cycles included a denaturation step at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. Two Cot cycles followed, including four steps consisting of 95°C for 15 s, 80°C for 30 s, 60°C for 30 s, and 72°C for 1 min. Standard and Cot cycles alternated two more times with eight, two, eight, and five cycles, respectively. After 35 cycles, samples were held at 4°C prior to being visually verified via agarose gel electrophoresis (1% agarose; 94 V for 40 min). Low DNA mass ladder (Invitrogen, Carlsbad, California, USA) was included in the first and last wells of each gel to guide length estimation of PCR products.

Marker amplification results

Table 1 contains amplification results for the low-copy nuclear loci, including the range of amplicon lengths for all taxa and GenBank numbers for markers sequenced by Gostel et al. (2015) for B. simaruba and C. grandifolia that had ≥15 sequence reads mapped. Table 2 summarizes marker amplification success for each taxon. Ninety primer pairs amplified product in B. simaruba and, on average, 54 primer pairs worked for other Burseraceae taxa. The low number of markers amplified in Aucoumea (16) was unexpected given its close relationship to Bursera. This result may have been caused by primer mismatch due to increased genetic change within this monotypic genus, as evidenced by its long branch within Burseraceae phylogeny (Weeks et al., 2014). In total, nine primer pairs worked for every Burseraceae taxon tested, and if Aucoumea is excluded as an outlier, the panel of family-universal primer pairs increases to 26. Thirty-four and 26 primer pairs generated product in Anacardiaceae and Rutaceae, respectively, while only two primer pairs worked in Arabidopsis. Comparing the Burseraceae panel to that of Anacardiaceae and Rutaceae reveals 16 and 12 successfully amplified regions in common, respectively, with eight shared among the three families. PCR chemistry may have suppressed amplification of markers, as high-fidelity PCR reagents were not used due to their high cost. Among the positive controls, high fidelity as compared to standard PCR reagents increased amplification success by 8% (Bursera, 83 to 90 primer pairs) and 85% (Commiphora, 39 to 72 primer pairs). Thus, our experimental results report a conservative baseline for the cross-amplification success of these primer pairs.

Table 2.

Number of primer pairs amplified of the 91 primer pairs tested for each of the 11 taxa.

Species tested (Order; Family)	Primer pairs amplified/tested (%)
Arabidopsis thaliana (Brassicales; Brassicaceae)	2/91 (0.02)
Aucoumea klaineana (Sapindales; Burseraceae)	16/91 (17)
Beiselia mexicana (Sapindales; Burseraceae)	47/91 (52)
Boswellia neglecta (Sapindales; Burseraceae)	68/91 (75)
Bursera simaruba (Sapindales; Burseraceae)	90/91 (99)
Bursera tonkinensis (Sapindales; Burseraceae)	53/91 (58)
Canarium pilosum (Sapindales; Burseraceae)	71/91 (78)
Commiphora grandifolia (Sapindales; Burseraceae)	72/91 (79)
Phellodendron amurense (Sapindales; Rutaceae)	26/91 (28)
Protium guianense (Sapindales; Burseraceae)	54/91 (59)
Schinus fasciculatus (Sapindales; Anacardiaceae)	34/91 (37)

CONCLUSIONS

Our study demonstrates that 90 of 91 primer pairs for novel low-copy nuclear loci developed by Gostel et al. (2015) for B. simaruba successfully amplify product in a broad range of Sapindalean taxa and effectively expand the phylogenomic toolkit for this order. Twenty-six markers amplify all Burseraceae taxa (excluding Aucoumea) and eight amplify all Sapindalean groups tested. Our results present a new source for universal targets or primers for phylogenetic reconstruction of taxa within Sapindales. Future efforts will include sequencing amplicons to determine the number of phylogenetically informative characters for each locus.

Appendix 1.

Accession information for taxa used in this study, including voucher information, country of origin, and latitude and longitude coordinate data, if available, and DNA extraction method.

Species	Voucher (Herbarium)	Country of origin	Geographic coordinates	DNA extraction methoda
Sapindales
Burseraceae
Aucoumea klaineana Pierre	Walters et al. 466 (MO)	Gabon	00°07′12″S, 11°42′57″E	1
	McPherson 16293 (MO)	Gabon	00°27′S, 11°45′E	1
Beiselia mexicana Forman	Pell s.n. (TEX)	Mexico	NA	1, 2
Boswellia neglecta S. Moore	Weeks 00-VII-29-1 (TEX)	Ethiopia	NA	2
Bursera simaruba (L.) Sarg.	Weeks 16-VI-16-01 (GMUF)	USA	NA	1
	Goldman s.n. (BH)	USA	NA	2
Bursera tonkinensis Guillamin	Daly et al. 13929 (NY)	Vietnam	20°15′12.6″N, 105°43′2.5″E	1
Canarium pilosum A. W. Benn.	Bogler s.n. (TEX)	Malaysia	NA	2
Commiphora grandifolia Engl.	Gostel 121 (GMUF)	Madagascar	23°39′19.64″S, 44°37′44.36″E	1
	Weeks 10-I-09-10 (GMUF)	Madagascar	12°14′16.14″S, 49°22′12.906″E	1
Protium guianense (Aubl.) Marchand	Miller and Hauk 9391 (MO)	Suriname	04°45′22″N, 056°52′30″W	1
Anacardiaceae
Schinus fasciculatus (Griseb.)   I. M. Johnst.	Silva-Luz 287 (NY)	Argentina	24°52′05.4″S, 65°32′41.4″W	1
Rutaceae
Phellodendron amurense Rupr.	Weeks 15-VII-13-01 (GMUF)	USA	38°49′53.76″N, 77°18′32.04″W	1
Brassicales
Brassicaceae
Arabidopsis thaliana (L.) Heynh.	Gostel s.n. (GMUF)	USA	NA	1

Note: NA = not available.

1 = FastDNA, 2 = CTAB.