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1 Thapa et al., 1 2 3 4 Thapa et al. - microsatellites 5 6 7 8 9 Development and characterization of microsatellite markers for Antennaria corymbosa 10 11 () and close relatives1 12 13 2 2 2 14 Ramhari Thapa , Randall J. Bayer , Jennifer R. Mandel 15 16 17 18 19 20 21 2 Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA 22 23 24 Email addresses: RT: [email protected] 25 26 27 RJB: [email protected] 28 29 JRM: [email protected] 30 31 32 Number of words: 1194 33 34 1 35 Manuscript received ______; revision accepted ______. 36 37 3Author for correspondence: [email protected] 38 39 40 Acknowledgements 41 42 43 The authors thank members of W. Harry Feinstone Center for Genomic Research, University of 44 45 Memphis, Tennessee for sequencing and technical support, as well as, the High Performance 46 Computing - University of Memphis. This project was partially supported by NSF DEB-1745197 47 48 to JRM and a Graduate Student Research Award from the American Society of 49 50 Taxonomists to RT. 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Thapa et al., 2 2 3 4 ABSTRACT 5 6 Premise of the study: The genus Antennaria has a complex evolutionary history due to dioecism, 7 8 9 excessive polyploidy, and the evolution of polyploid agamic complexes. We developed 10 11 microsatellite markers from A. corymbosa to investigate genetic diversity and population genetic 12 13 14 structure in Antennaria species. 15 16 Methods and Results: Twenty-four novel microsatellite markers (16 nuclear and eight 17 18 19 chloroplast) were developed from A. corymbosa using an enriched genomic library. Ten 20 21 polymorphic nuclear markers were used to characterize genetic variation in five populations of 22 23 24 A. corymbosa. One to four alleles were found per locus, and the expected heterozygosity and 25 26 fixation index ranged from 0.00 to 0.675 and -0.033 to 0.610, respectively. We were also able to 27 28 successfully amplify these markers in five additional Antennaria species. 29 30 31 Conclusions: These markers are promising tools for studying the population genetics of sexual 32 33 Antennaria species and to investigate interspecific gene flow, clonal diversity, and parentage of 34 35 36 Antennaria polyploid agamic complexes. 37 38 39 40 41 Key words: Antennaria corymbosa; Asteraceae; genetic diversity; microsatellites; polyploid 42 43 agamic complexes; population genetics. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Thapa et al., 3 2 3 4 INTRODUCTION 5 6 Antennaria Gaertn. are dioecious perennial herbs distributed mainly in the Holarctic 7 8 9 region with the major center of diversity in the of Western 10 11 (Bayer and Chandler, 2007). The genus comprises 32 known sexual diploid/tetraploid species 12 13 14 and several polyploid agamic complexes that mostly reproduce by forming asexual seeds (Bayer, 15 16 1990). While the sexual species are morphologically distinct, polyploid agamic complexes show 17 18 19 morphological similarities among themselves due to intense hybridization and introgression from 20 21 shared sexual progenitors (Bayer, 1990; Bayer and Chandler, 2007). Because of their 22 23 24 polymorphism, codominant nature, high cross transferability rate, and cost effectiveness, 25 26 microsatellite loci have been used extensively in the study of gene flow, genetic diversity, and 27 28 parentage analysis (Kalia et al., 2011); however, microsatellite markers have not yet been 29 30 31 developed for Antennaria. 32 33 We developed and characterized 16 nuclear and eight chloroplast microsatellite markers 34 35 36 for Antennaria corymbosa E.E. Nelson and demonstrated transferability to other species within 37 38 the genus. These markers will be helpful for population genetic studies of the sexual Antennaria 39 40 41 species, as well as, studies of clonal diversity and parentage analysis of polyploid agamic 42 43 complexes. 44 45 METHODS AND RESULTS 46 47 48 Total genomic DNA from Antennaria corymbosa (Appendix 1) was extracted using the 49 50 CTAB method (Doyle and Doyle, 1987) and quantified using Qubit BR-assay (Life 51 52 53 Technologies, Carlsbad, , USA). A genomic library was prepared with the NEBNext 54 55 Ultra II DNA Library Prep Kit (New England Biolabs, Ipswich, Massachusetts, USA) following 56 57 58 the manufacturer’s protocol. Target enrichment was performed using the myBaits COS 59 60 61 62 63 64 65 1 Thapa et al., 4 2 3 4 Compostiae/Asteraceae 1Kv1 kit (Arbor Biosciences, Ann Arbor, Michigan, USA) that targets a 5 6 Conserved Ortholog Set (COS) of loci in Compositae (Mandel et al. 2014). Sequencing was 7 8 9 performed on an Illumina MiSeq sequencer (paired end, sequencing chemistry V2, 150 bp) at the 10 11 Feinstone Center for Genomic Research, University of Memphis, Tennessee. Raw sequence files 12 13 14 have been deposited to NCBI's Sequence Read Archive (BioProject PRJNA514045). We 15 16 followed the bioinformatics workflow of Mandel et al. (2014) except we used the program 17 18 19 SPAdes v. 3.5 (Bankevich et al., 2012) with k-mer values 99, 111, 115, and 127 for the de novo 20 21 assembly of 2,060,492 quality reads. The 33,457 retained contigs were matched to the COS loci 22 23 24 using the PHYLUCE pipeline v.0.1.0 (Faircloth, 2012) following the workflow of Mandel et al. 25 26 (2014). A perfect motif search for microsatellite loci (di- to hexanucleotide repeats, minimum 27 28 length of 12 base pairs) was carried out using Phobos v. 3.3.11 (Mayer, 2006–2010) as 29 30 31 implemented in Geneious v. R7.1.9 (Kearse et al., 2012). Primers were developed for product 32 33 sizes ranging 150-450 base pairs using Primer3 (Untergasser et al., 2012) with default 34 35 36 parameters. 37 38 Altogether, 19 primer pairs from the contigs matching the COS loci and 22 primers from 39 40 41 the unmatched contigs were developed. Similarly, nine chloroplast microsatellite markers were 42 43 developed from the partial chloroplast genome of A. corymbosa assembled by mapping off-target 44 45 reads to the Artemisia frigida Willd. chloroplast genome (NC_020607). Primers were 46 47 48 synthesized at Integrated DNA Technologies (Coralville, Illinois, USA). Amplification of the 50 49 50 SSR loci was first tested in 12 individuals of A. corymbosa (Appendix 1). Genomic DNA was 51 52 53 extracted from silica gel-dried leaves or herbarium specimens using the E.Z.N.A. SQ Plant DNA 54 55 Kit (Omega Bio-Tek, Norcross, Georgia, USA) and purified with the E.Z.N.A. Cycle Pure Kit 56 57 58 (Omega Bio-Tek, Norcross, Georgia, USA). PCR was carried out in 15 µL single-plex reactions 59 60 61 62 63 64 65 1 Thapa et al., 5 2 3 4 that contained 1.5 µL 10X buffer, 0.5 µL MgCl2 (25 nM), 0.2 µL DNTPs (20 nM), 0.35 µL 5 6 forward primer (5 nM), 0.35 µL reverse primer (20 nM), 0.35 µL unlabeled M13 7 8 9 (CACGACGTTGTAAAACGAC, 10 nM), 0.7 µL Taq, and 1.5 µL DNA. A touch-down PCR 10 11 protocol was used with the following thermal cycler conditions: 95°C initial denaturation for 3 12 13 ° 14 min, 10 cycles of PCR to bolster primer annealing (30 sec at 94 C, 30 sec annealing temp 15 16 decreasing by 1°C from 65°C to 55°C with each cycle, and 60 sec at 72°C), followed by 30 cycles 17 18 ° ° ° 19 of amplification (30 sec at 94 C, 30 sec at 55 C, and 60 sec at 72 C), and a final extension at 20 21 72°C for 10 mins. PCR products were visualized on 1% agarose gels. 22 23 24 A total of 36/50 primer pairs, including 27 nuclear and nine chloroplast primer pairs, 25 26 were selected for genotyping. These 36 loci were re-amplified separately by replacing unlabeled 27 28 M13 with M13 containing VIC, NED, FAM or PET fluorophores in the PCR master mix (Table 29 30 31 1). Amplified products were pooled into dilution plates (four loci, 5 µL each in 30 µL 32 33 volume/sample/well) and run plates were prepared with 1 µL of the pooled sample in Formamide 34 35 36 diluted 6.5 µL of GeneScan 500 LIZ Size Standard (Thermo Fisher Scientific, Carlsbad, 37 38 California, USA) per well. Genotyping was carried out via capillary electrophoresis of the PCR 39 40 41 products on an ABI 3130XL DNA Analyzer (Life Technologies, Carlsbad, California, USA) at 42 43 the Molecular Resource Center, University of Tennessee, Memphis, Tennessee. The 44 45 electropherograms were analyzed with GeneMarker version 2.6.3 (SoftGenetics LLC, State 46 47 48 College, Pennsylvania, USA). Of the 27 nuclear markers, 10 were polymorphic, six were 49 50 monomorphic, and 11 showed ambiguous genotypes. Among the nine chloroplast loci, two 51 52 53 showed very low variability, six were monomorphic, and one was ambiguous. All polymorphic 54 55 and monomorphic loci are listed in Table 1. 56 57 58 59 60 61 62 63 64 65 1 Thapa et al., 6 2 3 4 Genetic diversity within the 10 polymorphic nuclear markers was further tested in 10 5 6 individuals for each of five populations of A. corymbosa (Appendix 1). These populations were 7 8 9 collected from regions representing the major center of diversity for the species in the western 10 11 part of the United States (Bayer, 2006). We could not collect more than 10 individuals per 12 13 14 population because the species produces dense mat-like structures due to stoloniferous growth, 15 16 and hence it was difficult to obtain more than 10 distinct patches in a collection area. Also, we 17 18 19 did not collect individuals from very close patches as they could be clones. Evidence for null 20 21 alleles was not detected by MICRO-CHECKER version 2.2.3 (Van Oosterhout et al., 2004). 22 23 24 Linkage disequilibrium among alleles of the loci in populations was not calculated due to small 25 26 population sizes. Genetic diversity parameters including the number of alleles, observed 27 28 heterozygosity, expected heterozygosity, and Fixation Index were calculated using GenAlEx v. 29 30 31 6.502 (Peakall and Smouse, 2006). Hardy-Weinberg Equilibrium (HWE) for each locus was 32 33 calculated using GENEPOP v. 4.2 (Rousset, 2008) (Table 2). Among the five populations of A. 34 35 36 corymbosa (50 genotyped individuals), the number of alleles per locus ranged from one to four. 37 38 Similarly, the overall observed heterozygosity, expected heterozygosity, and the Fixation Index 39 40 41 ranged from 0.00 to 1.00, 0.00 to 0.675, and -0.033 to 0.610, respectively. After a Bonferroni 42 43 correction for multiple testing, with the adjusted p-value of 0.001, no significant deviations from 44 45 HWE were detected (Table 2). 46 47 48 Cross-species amplification of the markers was also tested in five species of Antennaria 49 50 including A. microphylla Rydb., A. pulchella Greene, A. racemosa Hook, A. rosulata Rydb., and 51 52 53 A. umbrinella Rydb. (Appendix 1). These taxa were chosen as they all occur in the Catipes clade 54 55 and are potential hybridizers (Bayer, 1990; Bayer and Chandler, 2007). Among 10 polymorphic 56 57 58 nuclear markers in A. corymbosa, locus AcSSR506 was monomorphic in all species except A. 59 60 61 62 63 64 65 1 Thapa et al., 7 2 3 4 racemosa, and locus AcSSR655 was monomorphic in A. pulchella and A. rosulata. As in A. 5 6 corymbosa, six nuclear loci were monomorphic in all the five species. Among the eight 7 8 9 chloroplast markers, loci AcCpSSR8110 and AcCpSSR87080 were polymorphic in A. rosulata, 10 11 and locus AcCpSSR85580 was polymorphic in A. umbrinella (Table 3). To assess the ability of 12 13 14 the markers for resolving relationships at the species level, we produced a NeighborNet split 15 16 network using SplitsTree4 v. 4.14.5 (Huson and Bryant, 2006) and we undertook Principal 17 18 19 Coordinate analysis (PCoA) using GenAlEx for 12 samples from each of the six Antennaria 20 21 species (Appendix S1). 22 23 24 CONCLUSIONS 25 26 The novel microsatellite markers described in this study are the first developed in the 27 28 genus Antennaria. These markers will be helpful for the future population genetic studies of 29 30 31 widely distributed A. corymbosa populations. With high cross-species amplification rate, these 32 33 markers will be useful in the study of hybridization, interspecific gene flow, and parentage 34 35 36 analysis of other Antennaria diploid species and polyploid agamic complexes. 37 38 39 40 41 LITERATURE CITED 42 43 Bankevich, A., S. Nurk, D. Antipov, A. A. Gurevich, M.Dvorkin, A. S. Kulikov, V.M. Lesin et 44 45 al. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell 46 47 48 sequencing. Journal of Computational Biology 19(5): 455–477. 49 50 Bayer, R. J. 1990. A phylogenetic reconstruction of Antennaria (Asteraceae: Inuleae). Canadian 51 52 53 Journal of Botany 68(6): 1389–1397. 54 55 Bayer, R. J. 2006. Antennaria. In Flora North America Editorial Committee [ed.], Flora of North 56 57 58 America North of Mexico 19: 388–415. Oxford University Press, New York. 59 60 61 62 63 64 65 1 Thapa et al., 8 2 3 4 Bayer, R.J., and G.T. Chandler. 2007. Evolution of polyploid agamic complexes: A case study 5 6 using the Catipes group of Antennaria, including the A. rosea complex (Asteraceae: 7 8 9 ). In U. Grossniklaus, E. Hörandl, T. Sharbel, and P. van Dijk [ed.]. 10 11 Apomixis: Evolution, Mechanisms and Perspectives (Regnum Vegetabile) 147: 317–336 12 13 14 Lubrecht & Cramer Limited, New York. 15 16 Doyle, J.J., and J.L. Doyle. 1987. A rapid procedure for DNA purification from small quantities 17 18 19 of fresh leaf tissue. Phytochemical Bulletin 19:11–15. 20 21 Faircloth, B. C., J. E. McCormack, N. G. Crawford, M. G. Harvey, R. T. Brumfield, and T. C. 22 23 24 Glenn. 2012. Ultraconserved elements anchor thousands of genetic markers spanning 25 26 multiple evolutionary timescales. Systematic Biology 61(5): 717–726. 27 28 Huson, D.H., and D. Bryant. 2005. Application of phylogenetic networks in evolutionary 29 30 31 studies. Molecular Biology and Evolution 23(2): 254–267. 32 33 Kalia, R. K., M. K. Rai, S. Kalia, R. Singh, and A. K. Dhawan. 2011. Microsatellite markers: an 34 35 36 overview of the recent progress in . Euphytica 177(3): 309–334. 37 38 Kearse, M., A. MoirWilson, S. Stones-Havas, M. Cheung, S. Sturrock, S.Buxton et al. 2012. 39 40 41 Geneious Basic: an integrated and extendable desktop software platform for the 42 43 organization and analysis of sequence data. Bioinformatics 28(12): 1647–1649. 44 45 Mandel, J. R., R. B. Dikow, V. A. Funk, R. R. Masalia, S. E. Staton, A. Kozik, W.M. 46 47 48 Michelmore et al. 2014. A target enrichment method for gathering phylogenetic 49 50 information from hundreds of loci: an example from the Compositae. Applications in 51 52 53 Plant Sciences 2(2): 1300085. 54 55 Mayer, C. 2006-2010. Phobos 3.3.11. Website http://www.rub.de/ecoevo/cm/cm_phobos.htm 56 57 58 59 60 61 62 63 64 65 1 Thapa et al., 9 2 3 4 Peakall, R., and P. E. Smouse. 2006. GenAlEx 6: Genetic analysis in Excel. Population genetic 5 6 software for teaching and research. Molecular Ecology Notes 6: 288–295. 7 8 9 Rousset, F. 2008. Genepop’007: a complete re‐ implementation of the genepop software for 10 11 Windows and Linux. Molecular Ecology Resources 8(1): 103–106. 12 13 14 Untergasser, A., I. Cutcutache, T. Koressaar, J. Ye, B. C. Faircloth, M. Remm, and S. G. Rozen. 15 16 2012. Primer3- new capabilities and interfaces. Nucleic Acids Research 40(15): e115– 17 18 19 e115. 20 21 Van Oosterhout, C., W. F. Hutchinson, D. P. Wills, and P. Shipley. 2004. MICRO‐ CHECKER: 22 23 24 software for identifying and correcting genotyping errors in microsatellite 25 26 data. Molecular Ecology Notes 4(3): 535-538. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Thapa et al., 10 18 19 20 Table 1. Characteristics of developed microsatellite markers for Antennaria corymbosa. 21 22 Repeat Allele size Fluorescent GenBank 23 Locus Primer Sequences Motif range (bp) dye Accession 24 25 Nuclear markers 26 AcSSR655 F: ACGCACCATTAACTCTCCCG (ATT)3 197-212 VIC MK243474 27 R: CGGTGTACGTGATCCTGGAG 28 29 AcSSR652 F: TGGTTGAGTATTGTGGAAGTAACAC (CGGGTT)4 242-260 VIC MK243473 30 R: TGCTACCTTCCTAAAACCGCT

31 AcSSR898 F: AGGAGTCTAAGCTTAAGTTTGTGT (TGG)3 211-223 FAM MK243476 32 33 R: AGAAGCAAGTTTCTCCAACCCT

34 AccSSR208 F: ACTATCAAGATGATGACTTTAGCGA (TTTG)4 281-285 NED MK243463 35 R: GACAGCAAGCCCTCCAAGAA 36 37 AccSSR2209 F: TTCGGGTCAAATACGGGTCG (TTTA)4 197-201 FAM MK243464 38 R: TGGTGTAATCCTGTTGGCCC

39 AcSSR93 F: TGCAAGAAACACACAAAACA (ATC)4 172-181 VIC MK243465 40 41 R: ACGAAGAATCAACCGAAGAT

42 AcSSR731 F: AACCCACCTCCTAAAACATC (ATC)5 199-202 NED MK243475 43 R: TAATCCTTTGCACAATCCCA 44 45 AcSSR546 F: TCATGCTTAACCAGGTCAAA (AACCC)4 377-394 NED MK243472 46 R: AAGTAGGTAAAGCAAGTGCA

47 AcSSR297 F: TTAACAACCCGCTCATGTT (AAAT)4 187-199 FAM MK243470 48 49 R: TTAAATCCGTCAGCACAAGA

50 AcSSR506 F: CGTGGAGTGATTACGGTATT (TTGT)4 254-271 VIC MK243471 51 R: AAGCATGAAAAACAGCAACA 52 53 AcSSR149 F: GTACAAACGGAGCAAAAGAC (AAAG)3 439 PET MK243466 54 R: GGCAACTTTCATTCTCATGG

55 AcSSR170 F: CTTTCTCAAGGAGATGGACC (AGC)4 397 PET MK243467 56 57 R: TTGTAGTCGCTTACTGACAG

58 AcSSR238 F: CGCAATGAGATCACTTCAAG (AAG)4 237 NED MK243468 59 R: TTCCGTTTAGGTATCGGAAC 60 61 62 63 64 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Thapa et al., 11 18 19 20 AcSSR278 F: TTGCTTGTCCCAAATGGCAC (TTTGAC)2 210 FAM MK243469 21 R: AGGGCTCACATTCCAATCTAAA 22 23 AcSSR969 F: TCCTCATCAACGTTATTGCT (TA)6 281 VIC MK243477 24 R: TTACGACGTATGACAGCATT

25 AcSSR1038 F: ATTGATGGGTTCATGCTCTT (TATT)3 207 FAM MK243477 26 27 R: GCCTGTAAAGTTGTAAGGGA

28 Chloroplast markers 29 AcCpSSR66820 F: TGCTAGAGACATAAACAGTCATGGA (ATGAG)3 337 FAM MK243458 30 31 R: GGCCTAGCTGTACCTACCGT

32 AcCpSSR78370 F: CCGATGGTTCTTACTCAGGGA (AAT)5 210-224 VIC MK243459 33 R: CGGGCCTCTTGAATCCTCTC 34 35 AcCpSSR85580 F: TTTGTTCGATGAGCAGATCCA (AAT)12 305 VIC MK243460 36 R: TCTGGATCCAAAGAACCAGTCA

37 AcCpSSR159730 F: ACCCTGTGAATTGTGTGAAAGT (TGAAT)3 239 FAM MK243462 38 39 R: TCAGGAGGAATTTAATGACAGGACA

40 AcCpSSR87080 F: TTTGTATCGCAAAACTACGC (TTA)3 447-451 PET MK243461 41 R: TAAATTTCCGAGGACATGCA 42 43 AcCpSSR36790 F: CATACAACCTTGGCAAGAAC (AAG)4 245 NED MK243457 44 R: TTTTTCAAATCCTGCTGCAG

45 AcCpSSR12160 F: CGCCTGTCAATTCAATGAAT (ATTT)3 329 VIC MK243456 46 47 R: TCGCTTGTATTGTTTGTTGG

48 AcCpSSR8110 F: TGCAACTATGATCTATGCCC (TAA)4 350 VIC MK243455 49 R: TTTGGACTGGAATTGACGAA 50 Note: M-13 (CACGACGTTGTAAAACGAC) is added to the 5' end of forward primers; Optimal annealing temperature is 550C. 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Thapa et al., 12 18 19 20 Table 2. Genetic characterization of 10 polymorphic microsatellite loci for populations of Antennaria corymbosa. 21 22 Cory_3 (N=10) cory_30 (N=10) cory_43 (N=10) cory_45 (N=10) cory_50 (N=10) 23 24 Locus Na Ho He F HWE Na Ho He F HWE Na Ho He F HWE Na Ho He F HWE Na Ho He F HWE 25 AccSSR208 2 0.500 0.375 -0.333 NA 1 0.000 0.000 NA NA 2 0.250 0.219 -0.143 NA 1 0.000 0.000 NA NA 1 0.000 0.000 NA NA 26 27 AccSSR2209 2 1.000 0.500 -1.000 0.010 2 0.889 0.494 -0.800 0.053 2 0.800 0.480 -0.667 0.173 2 0.900 0.495 -0.818 0.045 2 0.700 0.455 -0.538 0.220 28 AcSSR93 2 0.100 0.095 -0.053 NA 1 0.000 0.000 NA NA 2 0.556 0.401 -0.385 1.000 2 0.300 0.255 -0.176 1.000 2 0.200 0.180 -0.111 1.000 29 AcSSR297 2 0.100 0.095 -0.053 NA 2 0.100 0.095 -0.053 NA 3 0.500 0.505 0.010 0.077 3 0.300 0.265 -0.132 1.000 2 0.100 0.095 -0.053 NA 30 31 AcSSR506 1 0.000 0.000 NA NA 3 0.400 0.335 -0.194 1.000 2 0.700 0.455 -0.538 0.220 2 0.600 0.420 -0.429 0.484 2 0.500 0.375 -0.333 1.000 32 AcSSR546 3 0.125 0.320 0.610 0.068 2 0.714 0.459 -0.556 0.438 2 0.200 0.180 -0.111 1.000 2 0.300 0.255 -0.176 1.000 1 0.000 0.000 NA NA 33 AcSSR652 3 0.778 0.648 -0.200 0.750 3 0.600 0.635 0.055 0.469 4 0.600 0.675 0.111 0.366 2 0.200 0.420 0.524 0.133 2 0.800 0.480 -0.667 0.175 34 35 AcSSR655 2 0.300 0.455 0.341 0.483 2 0.200 0.480 0.583 0.079 2 0.200 0.320 0.375 0.304 4 0.900 0.625 -0.440 0.151 2 0.600 0.480 -0.250 1.000 36 AcSSR731 2 0.889 0.494 -0.800 0.054 2 0.900 0.495 -0.818 0.055 2 1.000 0.500 -1.000 0.007 2 1.000 0.500 -1.000 0.007 2 1.000 0.500 -1.000 0.007 37 AcSSR898 2 0.100 0.095 -0.053 NA 1 0.000 0.000 NA NA 2 0.200 0.180 -0.111 1.000 1 0.000 0.000 NA NA 1 0.000 0.000 NA NA 38 39 Note: Na = number of alleles; Ho = observed heterozygosity; He = expected heterozygosity; F= fixation index; HWE = Hardy 40 Weinberg Equilibrium; N = number of individuals sampled; Cory = A. corymbosa. Loci were pooled for genotyping in the following 41 manner: (AccSSR208, AcSSR93), (AcSSR546, AccSSR2209, AcSSR652, AcSSR731), and (AcSSR297, AcSSR506, AcSSR655, 42 43 AcSSR898). 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Thapa et al., 13 18 19 20 Table 3. Cross-species amplification of the 23 microsatellite markers in five additional Antennaria species. 21 22 A. microphylla (N=12) A. pulchella (N=12) A. racemosa (N=12) A. rosulata (N=12) A. umbrinella (N=12) 23 24 allele size allele size allele size allele size allele size Loci success A range (bp) success A range (bp) success A range (bp) success A range (bp) success A range (bp) 25 Nuclear markers (polymorphic) 26 AccSSR208 *** 3 271-285 ** 3 271-289 ** 7 279-301 ** 3 279-285 ** 3 271-285 27 AcSSR93 *** 2 172-181 ** 4 172-189 ** 3 172-189 ** 3 172-181 ** 4 171-285 28 AccSSR2209 *** 2 197-200 * 3 197-202 ** 4 197-207 *** 2 197-200 ** 2 197-200 29 AcSSR546 ** 3 377-388 * 2 387-392 ** 5 382-392 *** 6 388-398 * 4 382-397 30 AcSSR731 ** 2 199-202 ** 2 199-202 *** 2 199-202 *** 2 199-202 ** 3 199-211 31 AcSSR652 *** 3 242-254 *** 4 242-260 *** 2 242-254 *** 4 242-260 *** 3 242-254 32 AcSSR655 *** 3 202-212 ** 1 199 *** 2 199-202 *** 1 199 *** 5 197-210 33 AcSSR506 *** 1 267 * 1 267 *** 4 259-275 *** 1 267 *** 1 267 34 AcSSR297 *** 2 195-199 ** 3 187-204 *** 2 199-204 *** 2 195-199 ** 3 195-204 35 AcSSR898 ** 2 211-223 ** 3 208-213 ** 3 211-226 ** 2 211-223 ** 2 211-223 Nuclear markers (monomorphic) 36 AcSSR149 *** 1 439 *** 1 439 *** 1 439 *** 1 439 *** 1 439 37 AcSSR170 *** 1 397 *** 1 397 *** 1 397 *** 1 397 *** 1 397 38 AcSSR238 *** 1 237 *** 1 237 *** 1 237 *** 1 237 *** 1 237 39 AcSSR278 ** 1 210 ** 1 210 ** 1 210 *** 1 210 *** 1 210 40 AcSSR1038 *** 1 207 *** 1 207 *** 1 207 *** 1 207 *** 1 207 41 AcSSR969 *** 1 301 *** 1 301 *** 1 301 *** 1 301 *** 1 301 42 Chloroplast markers (polymorphic, monomorphic) 43 AcCpSSR8110 *** 1 350 *** 1 350 *** 2 350-356 *** 1 350 *** 1 350 44 AcCpSSR12160 *** 1 329 *** 1 329 *** 1 329 *** 1 329 *** 1 329 45 AcCpSSR36790 *** 1 245 *** 1 245 *** 1 245 *** 1 245 *** 1 245 46 AcCpSSR66820 *** 1 337 *** 1 337 *** 1 337 *** 1 337 *** 1 337 AcCpSSR78370 *** 1 224 *** 1 224 *** 1 224 *** 1 224 *** 1 224 47 AcCpSSR85580 *** 1 305 *** 1 305 *** 1 305 *** 1 305 *** 2 294-305 48 AcCpSSR87080 *** 1 451 *** 1 451 *** 2 451-455 *** 1 451 *** 1 451 49 AcCpSSR159730 *** 1 239 *** 1 239 *** 1 239 *** 1 239 *** 1 239 50 Note: A = number of alleles; N = number of samples; * = amplification and scoring <50%; ** = amplification and scoring >50 %< 51 52 90%; *** = amplification and scoring > 90% 53 54 55 56 57 58 59 60 61 62 63 64 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Thapa et al., 14 18 19 20 Appendix 1. Voucher information for Antennaria corymbosa and five other Antennaria species used in this study. 21 Voucher Species N Collection number/year/collectors Collection locality Geographic coordinates 22 locationd 23 A. corymbosaa 1 M-508/1985/R.J.Bayer & Lebedyk ALTA Beaverhead Co., MT, USA 45°13'48.0"N, 111°27'00.0"W 24 A. corymbosab 10 WY-04001/2004/R.J.Bayer, G.Chandler& B. Robertson MEM Sheridan Co., WY, USA 44°25'47.4"N, 107°45'22.9"W 25 A. corymbosab 10 MT-04028/2004/R.J.Bayer, G.Chandler& B. Robertson MEM Granite Co., MT, USA 46°13'26.8"N, 113°14'54.9"W 26 A. corymbosab 10 CO-07001/2007/R.J.Bayer, G.Chandler, B. Robertson, C. Blanchfield & P. Kemmis MEM Gunnison Co., CO, USA 39°01'25.6"N, 107°03'09.3"W 27 A. corymbosab 10 CO-07003/2007/R.J.Bayer, G.Chandler, B. Robertson, C. Blanchfield & P. Kemmis MEM Gunnison Co., CO, USA 38°50'03.6"N, 106°25'05.8"W A. corymbosab 10 CO-07008/2007/R.J.Bayer, G.Chandler, B. Robertson, C. Blanchfield & P. Kemmis MEM Gunnison Co., CO, USA 38°49'60.0"N, 107°06'00.0"W 28 b 29 A. microphylla 4 CO-07004/2007/R.J.Bayer, G.Chandler, B. Robertson, C. Blanchfield & P. Kemmis MEM Gunnison Co., CO, USA 38°50'03.6"N, 106°25'05.8"W A. microphyllab 4 CO-070011/2007/R.J.Bayer, G.Chandler, B. Robertson, C. Blanchfield & P. Kemmis MEM Saguache Co., CO, USA 37°59'60.0"N, 106°00'00.0"W 30 A. microphyllab 4 CO-17002/2017/R.J.Bayer, R. Thapa, N.P. Prather & S. M. Ballou MEM Conejos Co., CO, USA 37°09'01.0"N, 106°24'58.2"W 31 A. pulchellac 4 CA-720/1987/R.J.Bayer, R. Deluca & D. Lebedyk ALTA Mono Co., CA, USA 37°57'00.0"N, 119°18'00.0"W 32 A. pulchellac 4 CA-724/1987/R.J.Bayer, R. Deluca & D. Lebedyk ALTA Inyo Co., CA, USA 37°10'60.0"N, 118°38'00.0"W 33 A. pulchellac 4 CA-732/1987/R.J.Bayer, R. Deluca & D. Lebedyk ALTA Inyo Co., CA, USA 37°07'60.0"N, 118°34'00.0"W 34 A. racemosab 4 MT-04006/2004/R.J.Bayer, G.Chandler & B. Robertson MEM Gallatin Co., MT, USA 45°53'30.1"N, 110°53'39.6"W 35 A. racemosab 4 MT-04015/2004/R.J.Bayer, G.Chandler& B. Robertson MEM Flathead Co., MT, USA 48°13'08.9"N, 113°19'43.5"W 36 A. racemosab 4 MT-04022/2004/R.J.Bayer, G.Chandler& B. Robertson MEM Deer Lodge Co., MT, USA 46°14'21.9"N, 112°35'08.4"W 37 A. rosulatab 4 NM-17002/2017/R.J.Bayer, R. Thapa, N.P. Prather & S. M. Ballou MEM Rio Arriba Co., NM, USA 36°39'17.8"N, 106°02'30.3"W b 38 A. rosulata 4 CO-17005/2017/R.J.Bayer, R. Thapa, N.P. Prather & S. M. Ballou MEM Saguache Co., CO, USA 38°13'13.9"N, 106°36'25.4"W A. rosulatab 4 AZ-17010/2017/R.J.Bayer, R. Thapa, N.P. Prather & S. M. Ballou MEM Coconino Co., AZ, USA 34°59'10.4"N, 111°27'35.5"W 39 b 40 A. umbrinella 4 WY-04004/2004/R.J.Bayer, G.Chandler& B. Robertson MEM Big Horn Co., WY, USA 44°48'18.9"N, 107°53'34.9"W A. umbrinellab 4 MT-04003/2004/ R.J.Bayer, G.Chandler& B. Robertson MEM Gallatin Co., MT, USA 45°05'35.4"N, 111°12'29.1"W 41 A. umbrinellab 4 MT-04027/2004/R.J.Bayer, G.Chandler& B. Robertson MEM Madison Co., MT, USA 45°04'29.4"N, 111°52'04.9"W 42 43 Note: a = used for Next Generation Sequencing; b = DNA from silica gel dried leaves; c = DNA from herbarium specimens, N = 44 number of individuals sampled; d = Standard herbarium acronyms following Thiers (http://sweetgum.nybg.org/science/ih/). 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Thapa et al., 15 2 3 4 Appendix S1 A. NeighborNet split network for six species of Antennaria. Numbers indicate 5 population number followed by letters for the individual identification number in a population. 6 7 B. Principal Coordinate Analysis for the six Antennaria species based on 10 polymorphic 8 microsatellite markers. 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 $SSHQGL[6                       

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