Targeted gene sequencing and selection detection in ex situ collections of Brighamia insignis Christina Shehata1, Lauren Audi1,2, Hilary Noble2, Colby Witherup1,2, Jeremie Fant2, Norm Wickett2 1Northwestern University, Evanston, IL 60201; 2Chicago Botanic Garden, Glencoe, IL 60022 Introduction Results B_insignis_107_BI−001 Discussion B_insignis_UNK_BI−005

B_insignis_1_BI−006

B_insignis_132_BI−007 Brighamia insignis (family: , subfamily: Lobelioideae) is a B_insignis_255_BI−014 B_insignis_255_BI−015 B_insignis_255_BI−020 While 206 genes were recovered during assembly, some genes were B_insignis_255_BI−031

B_insignis_255_BI−032

B_insignis_UNK_BI−034

B_insignis_565_BI−052

B_insignis_92_BI−073 species of caudiciform succulent endemic to . Brighamia insignis is B_insignis_256_BI−077 B_insignis_wild_BI−083 B_insignis_wild_BI−084 not captured by the Campanulaceae probes in any of 83 samples (Figure B_insignis_wild_BI−087

B_insignis_wild_BI−095

B_insignis_wild_BI−097

B_insignis_UNK_BI−099

B_insignis_wild_BI−104 extinct in the wild, due to habitat loss, small population sizes, and the loss B_insignis_257_BI−107 B_insignis_1002_BI−109 B_insignis_106_BI−110 2). The probes were designed from erinus, a relatively distant B_insignis_UNK_BI−112

B_insignis_wild_BI−113

B_insignis_UNK_BI−124

1 B_insignis_UNK_BI−129

B_insignis_UNK_BI−131 of its native , Tinostoma smaragditis . Fortunately, due to their B_insignis_UNK_BI−1367 B_insignis_UNK_BI−139 B_insignis_MULT21_BI−153 species from Brighamia insignis, possibly explaining the lower success of B_insignis_UNK_BI−160

B_insignis_UNK_BI−201

B_insignis_UNK_BI−208

B_insignis_UNK_BI−211

B_insignis_UNK_BI−212 novel morphology, Brighamia insignis have been cultivated as ornamental B_insignis_UNK_BI−215 B_insignis_UNK_BI−218 B_insignis_UNK_BI−231 target capture for some particular probes. The overall diversity among B_insignis_UNK_BI−237

B_insignis_UNK_BI−251

B_insignis_wild_BI−252

B_insignis_wild_BI−253

B_rockii_wild_BR−008 and still exist in ex situ collections around the world. B_rockii_wild_BR−016 B_rockii_wild_BR−020

B_rockii_97_BR−077

B_rockii_wild_BR−098 the samples of Brighamia insignis was greater than expected, with

B_rockii_wild_BR−099

B_rockii_UNK_BR−100

B_rockii_UNK_BR−101

B_rockii_UNK_HKM002 Genomic data obtained via next-generation B_insignis_UNK_HKM003 D_rhytidosperma_UNK_HKM004

B_rockii_wild_HW012

Cl_fauriei_wild_HW016 multiple points of divergence observed on the population tree along the

Cl_fauriei_wild_HW025

T_kauaiensiswild_HW027

T_kauaiensiswild_HW031

D_rhytidosperma_UNK_HW040 sequencing methods provide valuable information D_rhytidosperma_UNK_HW041 D_kauensis_wild_HW042

D_kauensis_wild_HW043 Cy_pseudofauriei_wild_HW044 branch leading to the populations of Brighamia insignis (Figure 2). Cy_pseudofauriei_wild_HW046

Cy_leptostegia_wild_HW048

Cy_leptostegia_wild_HW049

Cy_leptostegia_wild_HW050 about putatively expressed regions of the genome. Cy_hardyi_wild_HW051 Cy_fissa_wild_HW052

Cy_fissa_wild_HW053 Cy_fissa_wild_HW054 93 genes were found to have an LSDnorm value greater than 0 (Figure Cy_fissa_wild_HW055

Cl_fauriei_wild_HW059

Cy_hardyi_wild_HW061

B_rockii_wild_HW063

B_rockii_wild_HW064

Identifying those genes under natural selection can D_waianaeensis_UNK_LY002

B_rockii_UNK_ML002 B_rockii_UNK_ML005 3), suggesting that these genes are adaptive candidates within the D_kauensis_UNK_NT005

B_insignis_UNK_QKP005 help conservation scientists understand the sampled populations of Brighamia insignis. While traditional population scaffold_XAEC_2031402 scaffold_XAEC_2009701 scaffold_XAEC_2085436 scaffold_XAEC_2084760 scaffold_XAEC_2014344 scaffold_XAEC_2082868 scaffold_XAEC_2083385 scaffold_XAEC_2085304 scaffold_XAEC_2014228 scaffold_XAEC_2012381 scaffold_XAEC_2013546 scaffold_XAEC_2010914 scaffold_XAEC_2085985 scaffold_XAEC_2072819 scaffold_XAEC_2005335 scaffold_XAEC_2084276 scaffold_XAEC_2085700 scaffold_XAEC_2082107 scaffold_XAEC_2081424 scaffold_XAEC_2085904 scaffold_XAEC_2006755 scaffold_XAEC_2017460 scaffold_XAEC_2000424 scaffold_XAEC_2086239 scaffold_XAEC_2082050 scaffold_XAEC_2085725 scaffold_XAEC_2009935 scaffold_XAEC_2011064 scaffold_XAEC_2001544 scaffold_XAEC_2085482 scaffold_XAEC_2079785 scaffold_XAEC_2016261 scaffold_XAEC_2081872 scaffold_XAEC_2080877 scaffold_XAEC_2011700 scaffold_XAEC_2014153 scaffold_XAEC_2083592 scaffold_XAEC_2014329 scaffold_XAEC_2006092 scaffold_XAEC_2080494 scaffold_XAEC_2085496 scaffold_XAEC_2078669 scaffold_XAEC_2001114 scaffold_XAEC_2071303 scaffold_XAEC_2086058 scaffold_XAEC_2070149 scaffold_XAEC_2003396 scaffold_XAEC_2079367 scaffold_XAEC_2005232 scaffold_XAEC_2082937 scaffold_XAEC_2015246 scaffold_XAEC_2082812 scaffold_XAEC_2081103 scaffold_XAEC_2017079 scaffold_XAEC_2005196 scaffold_XAEC_2017875 scaffold_XAEC_2013884 scaffold_XAEC_2017561 scaffold_XAEC_2001477 scaffold_XAEC_2080606 scaffold_XAEC_2082141 scaffold_XAEC_2009747 scaffold_XAEC_2014175 scaffold_XAEC_2009451 scaffold_XAEC_2081270 scaffold_XAEC_2084298 scaffold_XAEC_2082941 scaffold_XAEC_2064325 scaffold_XAEC_2080433 scaffold_XAEC_2013530 scaffold_XAEC_2004450 scaffold_XAEC_2083526 scaffold_XAEC_2015208 scaffold_XAEC_2006174 scaffold_XAEC_2057767 scaffold_XAEC_2017553 scaffold_XAEC_2001888 scaffold_XAEC_2001550 scaffold_XAEC_2000846 scaffold_XAEC_2011036 scaffold_XAEC_2037036 scaffold_XAEC_2083253 scaffold_XAEC_2017893 scaffold_XAEC_2077649 scaffold_XAEC_2084810 scaffold_XAEC_2006412 scaffold_XAEC_2084741 scaffold_XAEC_2009073 scaffold_XAEC_2017521 scaffold_XAEC_2084670 scaffold_XAEC_2006695 scaffold_XAEC_2085611 scaffold_XAEC_2086014 scaffold_XAEC_2079951 scaffold_XAEC_2083474 scaffold_XAEC_2081492 scaffold_XAEC_2079547 scaffold_XAEC_2017491 scaffold_XAEC_2083079 scaffold_XAEC_2017499 scaffold_XAEC_2017095 scaffold_XAEC_2084829 scaffold_XAEC_2085928 scaffold_XAEC_2083788 scaffold_XAEC_2012087 scaffold_XAEC_2030622 scaffold_XAEC_2011401 scaffold_XAEC_2082878 scaffold_XAEC_2083607 scaffold_XAEC_2012769 scaffold_XAEC_2083953 scaffold_XAEC_2085250 scaffold_XAEC_2016681 scaffold_XAEC_2079242 scaffold_XAEC_2084852 scaffold_XAEC_2085290 scaffold_XAEC_2084227 scaffold_XAEC_2014903 scaffold_XAEC_2009007 scaffold_XAEC_2083270 scaffold_XAEC_2074174 variation that influences the fitness of individuals. XAEC_scaffold_XAEC_2017819 XAEC_scaffold_XAEC_2017706 XAEC_scaffold_XAEC_2083730 XAEC_scaffold_XAEC_2082801 XAEC_scaffold_XAEC_2083964 XAEC_scaffold_XAEC_2017474 XAEC_scaffold_XAEC_2082687 XAEC_scaffold_XAEC_2017899 XAEC_scaffold_XAEC_2003001 XAEC_scaffold_XAEC_2085914 XAEC_scaffold_XAEC_2016820 XAEC_scaffold_XAEC_2084049 XAEC_scaffold_XAEC_2084620 XAEC_scaffold_XAEC_2083751 XAEC_scaffold_XAEC_2007962 XAEC_scaffold_XAEC_2082878 XAEC_scaffold_XAEC_2080889 XAEC_scaffold_XAEC_2084899 XAEC_scaffold_XAEC_2006461 XAEC_scaffold_XAEC_2081183 XAEC_scaffold_XAEC_2004648 XAEC_scaffold_XAEC_2014207 XAEC_scaffold_XAEC_2082730 XAEC_scaffold_XAEC_2085672 XAEC_scaffold_XAEC_2011632 XAEC_scaffold_XAEC_2017888 XAEC_scaffold_XAEC_2085349 XAEC_scaffold_XAEC_2084964 XAEC_scaffold_XAEC_2016838 XAEC_scaffold_XAEC_2081064 XAEC_scaffold_XAEC_2084968 XAEC_scaffold_XAEC_2081539 XAEC_scaffold_XAEC_2083571 XAEC_scaffold_XAEC_2007280 XAEC_scaffold_XAEC_2080356 XAEC_scaffold_XAEC_2001087 XAEC_scaffold_XAEC_2083714 XAEC_scaffold_XAEC_2083540 XAEC_scaffold_XAEC_2082861 XAEC_scaffold_XAEC_2017484 XAEC_scaffold_XAEC_2013983 XAEC_scaffold_XAEC_2084272 XAEC_scaffold_XAEC_2081317 XAEC_scaffold_XAEC_2084904 XAEC_scaffold_XAEC_2085000 XAEC_scaffold_XAEC_2084767 XAEC_scaffold_XAEC_2014103 XAEC_scaffold_XAEC_2015748 XAEC_scaffold_XAEC_2084975 XAEC_scaffold_XAEC_2083564 XAEC_scaffold_XAEC_2084044 XAEC_scaffold_XAEC_2082272 XAEC_scaffold_XAEC_2084608 XAEC_scaffold_XAEC_2016994 XAEC_scaffold_XAEC_2014535 XAEC_scaffold_XAEC_2082633 XAEC_scaffold_XAEC_2083385 XAEC_scaffold_XAEC_2086004 XAEC_scaffold_XAEC_2086273 XAEC_scaffold_XAEC_2083002 XAEC_scaffold_XAEC_2084675 XAEC_scaffold_XAEC_2017242 XAEC_scaffold_XAEC_2081946 XAEC_scaffold_XAEC_2085817 XAEC_scaffold_XAEC_2014436 XAEC_scaffold_XAEC_2084541 XAEC_scaffold_XAEC_2084544 XAEC_scaffold_XAEC_2081519 XAEC_scaffold_XAEC_2085400 XAEC_scaffold_XAEC_2083780 XAEC_scaffold_XAEC_2085975 XAEC_scaffold_XAEC_2085788 XAEC_scaffold_XAEC_2080465 XAEC_scaffold_XAEC_2081799 XAEC_scaffold_XAEC_2082353 XAEC_scaffold_XAEC_2084955 XAEC_scaffold_XAEC_2083584 XAEC_scaffold_XAEC_2009457 XAEC_scaffold_XAEC_2015036 XAEC_scaffold_XAEC_2010142 XAEC_scaffold_XAEC_2084387 XAEC_scaffold_XAEC_2082990 XAEC_scaffold_XAEC_2000468 XAEC_scaffold_XAEC_2084829 XAEC_scaffold_XAEC_2017859 XAEC_scaffold_XAEC_2085828 XAEC_scaffold_XAEC_2084550 XAEC_scaffold_XAEC_2080609 XAEC_scaffold_XAEC_2006542 XAEC_scaffold_XAEC_2007551 XAEC_scaffold_XAEC_2084324 XAEC_scaffold_XAEC_2017292 XAEC_scaffold_XAEC_2017698 XAEC_scaffold_XAEC_2085134 XAEC_scaffold_XAEC_2085135 XAEC_scaffold_XAEC_2084528 XAEC_scaffold_XAEC_2085304 XAEC_scaffold_XAEC_2017586 XAEC_scaffold_XAEC_2086103 XAEC_scaffold_XAEC_2082201 XAEC_scaffold_XAEC_2081679 XAEC_scaffold_XAEC_2014240 XAEC_scaffold_XAEC_2014818 XAEC_scaffold_XAEC_2081723 XAEC_scaffold_XAEC_2016802 XAEC_scaffold_XAEC_2007405 XAEC_scaffold_XAEC_2084246 XAEC_scaffold_XAEC_2082742 XAEC_scaffold_XAEC_2085436 XAEC_scaffold_XAEC_2081748 XAEC_scaffold_XAEC_2085432 XAEC_scaffold_XAEC_2083404 XAEC_scaffold_XAEC_2084005 XAEC_scaffold_XAEC_2015015 XAEC_scaffold_XAEC_2011594 XAEC_scaffold_XAEC_2083748 XAEC_scaffold_XAEC_2082380 XAEC_scaffold_XAEC_2083626 XAEC_scaffold_XAEC_2086049 XAEC_scaffold_XAEC_2016380 XAEC_scaffold_XAEC_2007273 XAEC_scaffold_XAEC_2085656 XAEC_scaffold_XAEC_2009564 XAEC_scaffold_XAEC_2080879 XAEC_scaffold_XAEC_2084988 XAEC_scaffold_XAEC_2083349 XAEC_scaffold_XAEC_2084583 XAEC_scaffold_XAEC_2082599 XAEC_scaffold_XAEC_2084634 XAEC_scaffold_XAEC_2084739 genetic work focuses on putatively neutral loci, genomic data collected by This information may help guide breeding efforts to next-generation sequencing methods provide important information make ensure that extant individuals are genetically 0 0.2 0.4 0.6 0.8 1 regarding functional genetic variation, which could be incorporated into Percent Reference Protein Length Recovered Figure 1. Brighamia diverse, which is essential for future reintroduction future breeding programs for Brighamia. If the species has very low levels insignis collection at the efforts of critically . Figure 2. Heat map representing HybPiper's success at recovering genes at each Chicago Botanic Garden. locus, across 83 samples. of diversity, one of the suggested ways to increase diversity is to cross

Brighamia insignis with its sister species, Brighamia rockii. However, if B_insignis_UNK_BI-201

B_rockii_UNK_ML002

B_rockii_wild_BR-008 B_rockii_UNK_BR-101

B_rockii_97_BR-077

B_rockii_wild_HW063

B_rockii_wild_BR-099

B_rockii_wild_BR-098 B_rockii_wild_BR-020 there are genes that are locally adapted to the sampled populations of B_rockii_wild_BR-016

B_rockii_UNK_ML005 Objectives B_rockii_wild_HW064 B_rockii_UNK_BR-100 Cy_leptostegia_wild_HW050 Brighamia insignis, the information gained from identifying adaptive B_rockii_wild_HW012 Cy_leptostegia_wild_HW048

B_insignis_255_BI-015 B_insignis_wild_BI-083 • Cy_leptostegia_wild_HW049 B_insignis_UNK_HKM003 Evaluate variation and relatedness among Hawaiian lobelioids kept in B_insignis_255_BI-014 B_insignis_UNK_BI-211 B_insignis_wild_BI-084 B_insignis_UNK_BI-099 Cy_pseudofauriei_wild_HW044 B_insignis_565_BI-052 candidate genes can help ensure that these genes are preserved in Cy_hardyi_wild_HW051 B_insignis_wild_BI-253 B_insignis_UNK_BI-005 botanic collections around the world. B_insignis_wild_BI-113 B_insignis_107_BI-001 Cy_hardyi_wild_HW061 B_insignis_255_BI-020 future breeding efforts. B_insignis_132_BI-007 Cy_fissa_wild_HW053 • Sequence protein-coding genes described from the Campanulaceae B_insignis_UNK_BI-237 Cy_fissa_wild_HW052 B_insignis_1002_BI-109 B_insignis_UNK_BI-208 Cy_fissa_wild_HW055 2 B_insignis_wild_BI-087 family using Hyb-Seq , a sequencing method that can capture targeted Cy_fissa_wild_HW054 B_insignis_wild_BI-252 Cl_fauriei_wild_HW025 B_insignis_UNK_BI-112 B_insignis_106_BI-110 Cl_fauriei_wild_HW016 genes in non-model organisms. B_insignis_UNK_BI-231 Cl_fauriei_wild_HW059 Future B_insignis_UNK_BI-215 Directions Cy_pseudofauriei_wild_HW046 B_insignis_UNK_BI-218 • Identify genes that are adaptive candidates in populations of Brighamia B_insignis_256_BI-077 T_kauaiensiswild_HW031 • Sequence the remaining Brighamia samples to complete a genomic B_insignis_257_BI-107 T_kauaiensiswild_HW027 B_insignis_wild_BI-104 insignis and Brighamia rockii. D_kauensis_wild_HW043 Undetermined dataset with representation of all Brighamia insignis individuals. D_kauensis_wild_HW042 B_insignis_UNK_BI-251 B_insignis_92_BI-073 D_kauensis_UNK_NT005 B_rockii_UNK_HKM002 • Use the tested probes on other Hawaiian lobelioids to answer D_rhytidosperma_UNK_HKM004 B_insignis_255_BI-031 Methods D_rhytidosperma_UNK_HW041 B_insignis_1_BI-006 phylogenetic questions pertaining to the Campanulaceae family. D_rhytidosperma_UNK_HW040 B_insignis_wild_BI-095 B_insignis_255_BI-032 B_insignis_UNK_BI-212 Data Collection D_waianaeensis_UNK_LY002 • Test other bioinformatic methods to detect signatures of positive DNA from collected lobeliad samples was extracted following the Figure 3. Phylogenetic tree constructed from individual gene trees. LSD was selection (i.e. dN/dS). standard CTAB protocol. Samples were sheared using the Covaris used to detect signatures of selection along the branches leading to Brighamia Focused-Ultrasonicator. DNA libraries were prepared and amplified insignis (pink) and Brighamia rockii (blue). Literature Cited following the KAPA protocol. Libraries were pooled into groups of 12 Levels of Exclusively Shared Difference0.4 (LSD) in Brighamia insignis 1. Gemmill CEC, Ranker TA, Ragone D, Perlman SP, Wood KR. 1998. Conservation genetics of samples. Campanulaceae baits were hybridized to the pools, which were the endangered Hawaiian genus Brighamia (Campanulaceae). American Journal of Botany then amplified using PCR. The Bioanalyzer was used to evaluate pool 85(4): 528-539. concentrations and calculate average fragment size. Samples were 2. Weitemier K, Straub SCK, Cronn RC, Fishbein M, Schmickl R, McDonnell A, Liston A. 2014. Hyb-seq: Combining target enrichment and genome skimming for phylogenomics. sequenced using Illumina’s MiSeq System. Applications in Plant Sciences 2(9): 1400042. 3. Johnson MG, Gardner EM, Liu Y, Medina R, Goffinet B, Shaw AJ, Zerega NJC, Wickett NJ. 2016. HybPiper: Extracting coding sequence and introns for phylogenetics from high- Post-Sequencing Analysis Count throughput sequencing reads using target enrichment. Applications in Plant Sciences 4(7): Sequenced reads were trimmed with Trimmomatic then processed and 1600016. assembled with HybPiper3. Orthologous gene sequences were aligned 4. Librado P, Orland L. 2018. Detecting signatures of positive selection along defined branches of a population tree using LSD. Molecular Biology and Evolution 35(6): 1520–1535. using MACSE and trimmed with Trimal. FastTree was used to construct a phylogenetic tree for each gene of interest. Astral was used to construct a population tree from the individual gene trees. LSD4 was used to detect Contact Information LSDnorm [email protected] signatures of positive selection among the population tree branch Figure 4. Histogram representing the distribution of normalized LSD values leading to Brighamia insignis. along the branch leading to Brighamia insignis. https://github.com/ChristinaShehata/

REU Site: Plant Biology & Conservation Research Acknowledgements: We would like to thank NSF-REU grant DBI-1757800, Northwestern University’s Experiences for Undergraduates - From Genes to Undergraduate Research Grant Program, and the Chicago Botanic Garden for support and for making this summer experience Ecosystems (Supported by NSF award DBI-1757800). possible. I would also like to thank my mentors Colby Witherup, Lindsey Bechen, Lauren Audi, Jeremie Fant, and Norm Wickett for their continuous guidance and support. Also, thank you to Hilary Noble and Jordan Wood for their support in the laboratory.