Phylogenetic Relationships of Burmeistera (Campanulaceae): Using High-Throughput Sequencing to Improve Resolution in a Recent Radiation

Phylogenetic relationships of Burmeistera (Campanulaceae): using high-throughput sequencing to improve resolution in a recent radiation

1 Simon Uribe-Convers , 2 1 3 1 Monica Carlsen , Laura Lagomarsino , Eric Knox , Nathan Muchhala 1University of Missouri - St. Louis, 2Smithsonian Institution, 3Indiana University

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@uribe_convers Systematic/Evolutionary Question

@uribe_convers The genomics era

Systematic/Evolutionary Question

@uribe_convers The genomics era

PCR Target Enrichment GBS/RADSeq and HTS Sequence Capture (Microfluidics)

Systematic/Evolutionary Question

Transcriptomes Genome Skimming

Whole Genome

@uribe_convers The genomics era

PCR Target Enrichment GBS/RADSeq and HTS Sequence Capture (Microfluidics)

Systematic/Evolutionary Question

Transcriptomes Genome Skimming

PCR Target Enrichment Whole Genome and Sanger

@uribe_convers The genomics era

Sanger data = HTS data

@uribe_convers Burmeistera (Campanulaceae)

Triana (1854) ~116 spp. Guatemala - Peru Colombia & Ecuador 50% spp. Wimmer (1943): 2 sections Barbatae and Imberbes

L.Lagomarsino L.Lagomarsino Burmeistera (Campanulaceae)

Barbatae L.Lagomarsino Imberbes L.Lagomarsino Burmeistera (Campanulaceae)

Anoura (Phyllostomidae)

N. Muchhala

N. Muchhala Burmeistera (Campanulaceae)

N. Muchhala

N. Muchhala Burmeistera (Campanulaceae)

Anoura caudifer: 3.7cm (n=6) Anoura geoffroyi: 3.9cm (n=5) Anoura fistulata: 8.5cm (n=4)

N. Muchhala Anoura fistulata N. Muchhala Burmeistera (Campanulaceae)

Anoura geoffroyi Camilo Calderón Burmeistera (Campanulaceae)

Burmeistera: ~116 spp.

Centropogon: ~210 spp.

Siphocampylus: ~230 spp. Lagomarsino et al., 2014 Burmeistera (Campanulaceae) 2.6 Myr (2.4–3.5 Myr)

Laura’s Talk *Tuesday 9:15am*

Lagomarsino et al., 2016 Burmeistera (Campanulaceae)

Antonelli, 2008 Knox et al., 2008

Lagomarsino et al., 2014 Lagomarsino et al., 2016 Sanger data = HTS data Antonelli, 2008; Knox et al., 2008 Lagomarsino et al., 2014, 2016

trnG GCC tRNA

psbZ CDS

S trnS GGA tRNA

trnL UAA tRNA C trnF GAA tRNA b

p psbD CDS trnT GGU tRNA

petN CDStrnC GCA tRNA

trnS UGA tRNA rps14 CDS trnfM CAU tRNA psaB CDS psaI CDS psaA CDS

pafII CDS pafI CDS

cemA CDS rps4 CDS trnT UGU tRNA petA CDS ndhJ CDS

ndhK CDS psbM CDS ndhC CDS rpoB CDS

trnE UUC tRNAtrnY GUA tRNA trnD GUC tRNA petL CDS petG CDS rpoC1 CDS psaJ CDS psbJ CDS rpl33 CDS psbL CDS rpoC2 CDS rps18 CDS psbF CDS rps2 CDS psbE CDS atpI CDS trnR UCU tRNA trnW CCA tRNA 48kb trnG UCC tRNA trnP UGG tRNA 32kb atpH CDS atpF CDS psbI CDS psbK CDS rpl20 CDS LSC atpA CDS rps12 CDS psbB CDS clpP CDS psbT CDS 64kb trnS GCU tRNA psbH CDS 16kb trnQ UUGrps16 tRNA CDS petB CDS psbN CDS trnK UUU tRNA trnV UAC tRNA petD CDS matK CDS atpE CDS atpB CDS trnM CAU tRNA rpoA CDS rps11 CDS rpl36 CDS rps8 CDS rbcL CDS rpl14 CDS 80kb rpl16 CDS Burmeistera borjensis Jeppesen psbA CDS rps3 CDS 0kb trnH GUG tRNA rpl22 CDS 163,961 bp rpl2 CDS rps19 CDS rpl2 CDS trnI CAU tRNA trnI CAU tRNA ycf2 CDS

IRb ycf2 CDS 96kb IRa trnL CAA tRNA trnL CAA tRNA trnV GAC tRNA ndhB CDS ndhB CDS 144kb trnI GAUrrn16 tRNA rRNA rps7 CDS rps7 CDS SSC 112kb trnA UGC rrn23 rRNA 128kb trnR ACGrrn4.5 tRNA rRNA rrn5 rRNA

ycf1 CDS trnV GACrrn16 tRNA rRNA t rps15 CDS r ndhH CDS trnN GUU tRNA n ndhA CDS L

ndhG CDS ndhI CDS trnN GUU tRNA trnA UGC U trnI GAU tRNA rpl32 CDS

rrn23 rRNA R

rrn4.5rrn5 rRNA rRNA A

trnR ACG tRNA

ycf1 CDS ndhF CDS

rps15 CDS ndhH CDS

ndhA CDS ndhI CDS

ndhG CDS ndhE CDS psaC CDS

ndhD CDS 200+ accessions 19 plastomes 7 cpDNA loci The everything approach Complete plastomes

50 species

Locus 1 Locus 2 Locus 3 … Locus n The everything approach

Missing Data

7 loci (9,300bp) are only ~7% of the plastome (~133Kb) The everything approach

94 Burmeistera_borjensis_Muchhala_154 88 Burmeistera_oyacachensis_Muchhala_171 97 Burmeistera_vulgaris_Santamaria_S_980 45 Burmeistera_refracta_Muchhala_110 6 Burmeistera_glabrata_Muchhala_165

18 Burmeistera_holmnielsenii_Muchhala_134 98 Burmeistera_auriculata_Muchhala_120 97 30 Burmeistera_truncata_Muchhala_201 Burmeistera_rubrosepala_Muchhala_220 98 Burmeistera_draconis_Muchhala_464 35 Burmeistera_cyclostigmata_Muchhala_145 41 Burmeistera_succulenta_K_Bacon_297 94 28 Burmeistera_toroensis_Lagomarsino_92 100 Burmeistera_crispiloba_Muchhala_123 22 Burmeistera_sodiroana_Muchhala_115

31 Burmeistera_resupinata_Muchhala_223

53 Burmeistera_crassifolia_Muchhala_121 Burmeistera_cylindrocarpa_Muchhala_202 Burmeistera_multiflora_Muchhala_114 95 Burmeistera_almedae_Lagomarsino_86 94 Burmeistera_zurquiensis_Lagomarsino_26 70 91 Burmeistera Burmeistera_obtusifolia_Lagomarsino_61 39 Burmeistera_loejtantii_F_2190436

69 Burmeistera_succulenta_E_Muchhala_447 96 Burmeistera_succulenta_W_Muchhala_214 59 62 5 Burmeistera_smaragdi_Muchhala_205 5 Burmeistera_tenuiflora_Lagomarsino_276 Burmeistera_microphylla_Lagomarsino_34 94 Burmeistera_zamorensis_Muchhala_467 91 Burmeistera_crebra_Lagomarsino_70 8 Burmeistera_panamensis_Muchhala_494 66 Burmeistera_dukei_Cl12662 66Burmeistera_parvifolia_Lagomarsino_53 92 Burmeistera_chiriquiensis_Santamaria_8996 84 52 Burmeistera_dendrophila_Lagomarsino_255 Burmeistera_utleyi_Lagomarsino_253 81 Burmeistera_mcvaughii_Lagomarsino_257 55 91 Burmeistera_morii_Lagomarsino_273 79 Burmeistera_minutiflora_Bacon_262 Burmeistera_pirrensis_Muchhala_491 100 Burmeistera_variabilis_Bacon_298 100 90 Burmeistera_racemiflora_Madison_7145 94 Burmeistera_fuchsioides_Bacon_258 Burmeistera_aff_bullatifolia_Bacon_264A 100 Burmeistera_fuscoapicata_Cl_12955 99 Burmeistera_ceratocarpa_Muchhala_177 7 Burmeistera_lutosa_Muchhala_133 Burmeistera_domingensis_Muchhala_102 Centropogon_nigricans_Muchhala_118 Centropogon Siphocampylus_krauseanus_Smith_and_Leiva_509 Siphocampylus 0.006 Extraction

Locus 1 Locus 2 Locus 3 … Locus n Extraction

Locus 1 Locus 2 Locus 3 … Locus n Extraction Extracted data from complete plastomes

Locus 1 Locus 2 Locus 3 … Locus n

~30% Missing Data Extraction 95 Burmeistera_oyacachensis_Muchhala_171 94 98 Burmeistera_borjensis_Muchhala_154 Burmeistera_vulgaris_Santamaria_S_980 60 Burmeistera_refracta_Muchhala_110 95 Burmeistera_truncata_Muchhala_201 39 95 Burmeistera_auriculata_Muchhala_120 9 Burmeistera_rubrosepala_Muchhala_220 Burmeistera_glabrata_Muchhala_165 23 100 Burmeistera_draconis_Muchhala_464 Burmeistera_cyclostigmata_Muchhala_145 29 Burmeistera_holmnielsenii_Muchhala_134 31 Burmeistera_toroensis_Lagomarsino_92 90 13 100 Burmeistera_crispiloba_Muchhala_123 Burmeistera_succulenta_K_Bacon_297 22 Burmeistera_sodiroana_Muchhala_115 31 Burmeistera_resupinata_Muchhala_223 15 Burmeistera_microphylla_Lagomarsino_34 Burmeistera_crassifolia_Muchhala_121 Burmeistera_cylindrocarpa_Muchhala_202 97 Burmeistera_zurquiensis_Lagomarsino_26 11 89 93 Burmeistera_almedae_Lagomarsino_86 56 Burmeistera_obtusifolia_Lagomarsino_61 77 Burmeistera_loejtantii_F_2190436 77 90 Burmeistera_succulenta_E_Muchhala_447 97 Burmeistera_smaragdi_Muchhala_205 Burmeistera 82 Burmeistera_succulenta_W_Muchhala_214 Burmeistera_tenuiflora_Lagomarsino_276 Burmeistera_multiflora_Muchhala_114 67 Burmeistera_chiriquiensis_Santamaria_8996 88 Burmeistera_parvifolia_Lagomarsino_53 77 77 93 95Burmeistera_dendrophila_Lagomarsino_255 71 Burmeistera_utleyi_Lagomarsino_253 Burmeistera_zamorensis_Muchhala_467 67 48 Burmeistera_mcvaughii_Lagomarsino_257 41 Burmeistera_morii_Lagomarsino_273 41 94 70 Burmeistera_minutiflora_Bacon_262 43 Burmeistera_pirrensis_Muchhala_491 Burmeistera_crebra_Lagomarsino_70 100 Burmeistera_domingensis_Muchhala_102 100 Burmeistera_dukei_Cl12662 Burmeistera_panamensis_Muchhala_494 98 Burmeistera_variabilis_Bacon_298 82 89 Burmeistera_racemiflora_Madison_7145 Burmeistera_fuchsioides_Bacon_258 15 Burmeistera_aff_bullatifolia_Bacon_264A 100 Burmeistera_fuscoapicata_Cl_12955 100 Burmeistera_ceratocarpa_Muchhala_177 Burmeistera_lutosa_Muchhala_133 Centropogon_nigricans_Muchhala_118 Centropogon Siphocampylus_krauseanus_Smith_and_Leiva_509 Siphocampylus

0.006 Better use of HTS data? Better use of HTS data? Complete plastomes

Partitioned by coding and non-coding (79 and 103, respectively)

PartitionFinder (11)

Partitioned analysis Complete plastome tree

1 Burmeistera_borjensis_Muchhala_154 1

1 Burmeistera_oyacachensis_Muchhala_171 1

1 Burmeistera_auriculata_Muchhala_120 1 1 1 Burmeistera_rubrosepala_Muchhala_220

1 1 Burmeistera_cyclostigmata_Muchhala_145

1 Burmeistera_crispiloba_Muchhala_123 1 1

1 Burmeistera_sodiroana_Muchhala_115

1 1 Burmeistera_resupinata_Muchhala_223

1 1 Burmeistera_cylindrocarpa_Muchhala_202 Burmeistera 1 Burmeistera_loejtantii_F_2190436 1 1 1 Burmeistera_smaragdi_Muchhala_205

1 Burmeistera_parvifolia_Lagomarsino_53 1

1 Burmeistera_pirrensis_Muchhala_491 1

1 Burmeistera_ceratocarpa_Muchhala_177 1

1 1 Burmeistera_fuscoapicata_Cl_12955

1 1 0.996 Burmeistera_lutosa_Muchhala_133

1 Burmeistera_domingensis_Muchhala_102

1 Centropogon_nigricans_Muchhala_118 Centropogon 1 Siphocampylus_krauseanus_Smith_and_Leiva_509 Siphocampylus

0.002 1 Burmeistera_borjensis_Muchhala_154 1

1 Burmeistera_oyacachensis_Muchhala_171 1

1 Burmeistera_auriculata_Muchhala_120 Extracted-Constrained 1 1 1 Burmeistera_rubrosepala_Muchhala_220

1 1 Burmeistera_cyclostigmata_Muchhala_145

1 Burmeistera_crispiloba_Muchhala_123 1 1

1 Burmeistera_sodiroana_Muchhala_115

1 1 Burmeistera_resupinata_Muchhala_223

1 1 Burmeistera_cylindrocarpa_Muchhala_202

1 Burmeistera_loejtantii_F_2190436 1 1 1 Burmeistera_smaragdi_Muchhala_205

1 Burmeistera_parvifolia_Lagomarsino_53 1

1 Burmeistera_pirrensis_Muchhala_491 1

1 Burmeistera_ceratocarpa_Muchhala_177 1

1 1 Burmeistera_fuscoapicata_Cl_12955

1 1 0.996 Burmeistera_lutosa_Muchhala_133

1 Burmeistera_domingensis_Muchhala_102

1 Centropogon_nigricans_Muchhala_118

1 Siphocampylus_krauseanus_Smith_and_Leiva_509

0.002

Locus 1 Locus 2 Locus 3 … Locus n Extracted-Constrained

100 Burmeistera borjensis 100 98 Burmeistera oyacachensis Burmeistera vulgaris 43 Burmeistera refracta 96 Burmeistera truncata 31 96 Burmeistera auriculata 9 Burmeistera rubrosepala Burmeistera glabrata 100 Burmeistera draconis 30 Burmeistera cyclostigmata 24 Burmeistera succulentaK 86 100 Burmeistera crispiloba 13 4 Burmeistera toroensis Burmeistera sodiroana 22 Burmeistera holmnielsenii Burmeistera crassifolia 18 34 Burmeistera microphylla Burmeistera resupinata Burmeistera cylindrocarpa 98 Burmeistera almedae 98 78 92 Burmeistera zurquiensis Burmeistera obtusifolia 62 Burmeistera loejtantii 89 Burmeistera succulentaW 84 Burmeistera 88 Burmeistera smaragdi 6 Burmeistera tenuiflora Burmeistera succulentaE Burmeistera multiflora 72 86Burmeistera dendrophila 89 Burmeistera utleyi 93 81 Burmeistera chiriquiensis 44 Burmeistera parvifolia 71 Burmeistera zamorensis Burmeistera mcvaughii 31 76 Burmeistera morii 97 67 Burmeistera minutiflora Burmeistera pirrensis Burmeistera crebra 100 Burmeistera variabilis 66 100 93 Burmeistera racemiflora Burmeistera fuchsioides Burmeistera aff 100 Burmeistera ceratocarpa 100 34 Burmeistera fuscoapicata Burmeistera lutosa 40 Burmeistera panamensis 100 Burmeistera dukei Burmeistera domingensis Centropogon nigricans Centropogon Siphocampylus krauseanus Siphocampylus

0.005 Extracted-Constrained

100 Burmeistera borjensis 100 98 Burmeistera oyacachensis Burmeistera vulgaris 43 Burmeistera refracta 96 Burmeistera truncata 31 96 Burmeistera auriculata No sections by Wimmer 9 Burmeistera rubrosepala Burmeistera glabrata 100 Burmeistera draconis 30 Burmeistera cyclostigmata 24 Burmeistera succulentaK 86 100 Burmeistera crispiloba 13 4 Burmeistera toroensis Burmeistera sodiroana 22 Burmeistera holmnielsenii Burmeistera crassifolia 18 34 Burmeistera microphylla Burmeistera resupinata Burmeistera cylindrocarpa 98 Burmeistera almedae 98 Clade C 78 92 Burmeistera zurquiensis Burmeistera obtusifolia 62 Burmeistera loejtantii 89 Burmeistera succulentaW 88 84 Burmeistera smaragdi 6 Burmeistera tenuiflora Burmeistera succulentaE Burmeistera multiflora 72 86Burmeistera dendrophila 89 Burmeistera utleyi 93 81 Burmeistera chiriquiensis 44 Burmeistera parvifolia 71 Burmeistera zamorensis Burmeistera mcvaughii 31 76 Burmeistera morii 97 67 Burmeistera minutiflora Burmeistera pirrensis Burmeistera crebra 100 Burmeistera variabilis 66 100 93 Burmeistera racemiflora Burmeistera fuchsioides Colombia Burmeistera aff Clade B: 100 Burmeistera ceratocarpa 100 34 Burmeistera fuscoapicata Burmeistera lutosa 40 Burmeistera panamensis 100 Burmeistera dukei Clade A: Darien Burmeistera domingensis Centropogon nigricans Siphocampylus krauseanus

0.005 Extracted-Constrained

100 Burmeistera borjensis 100 98 Burmeistera oyacachensis Burmeistera vulgaris 43 Burmeistera refracta 96 Burmeistera truncata 31 96 Burmeistera auriculata Bibracteate pedicels 9 Burmeistera rubrosepala Burmeistera glabrata 100 Burmeistera draconis 30 Burmeistera cyclostigmata 24 Burmeistera succulentaK Recurved petals 86 100 Burmeistera crispiloba 13 4 Burmeistera toroensis Burmeistera sodiroana Inflated fruit 22 Burmeistera holmnielsenii Burmeistera crassifolia 18 34 Burmeistera microphylla Burmeistera resupinata Burmeistera cylindrocarpa 98 Burmeistera almedae 98 Clade C 78 92 Burmeistera zurquiensis Burmeistera obtusifolia 62 Burmeistera loejtantii 89 Burmeistera succulentaW 88 84 Burmeistera smaragdi 6 Burmeistera tenuiflora Burmeistera succulentaE Burmeistera multiflora 72 86Burmeistera dendrophila 89 Burmeistera utleyi 93 81 Burmeistera chiriquiensis 44 Burmeistera parvifolia 71 Burmeistera zamorensis Burmeistera mcvaughii 31 76 Burmeistera morii 97 67 Burmeistera minutiflora Burmeistera pirrensis Burmeistera crebra 100 Burmeistera variabilis 66 100 93 Burmeistera racemiflora Burmeistera fuchsioides Colombia Burmeistera aff Clade B: 100 Burmeistera ceratocarpa 100 34 Burmeistera fuscoapicata Burmeistera lutosa 40 Burmeistera panamensis 100 Burmeistera dukei Clade A: Darien Burmeistera domingensis Centropogon nigricans Siphocampylus krauseanus

0.005 100 Burmeistera borjensis 100 98 Burmeistera oyacachensis Burmeistera vulgaris 43 Burmeistera refracta 96 Burmeistera truncata Limitations 31 96 Burmeistera auriculata 9 Burmeistera rubrosepala Burmeistera glabrata 100 Burmeistera draconis 30 Burmeistera cyclostigmata 24 Burmeistera succulentaK 86 100 Burmeistera crispiloba 13 4 Burmeistera toroensis Burmeistera sodiroana 22 Burmeistera holmnielsenii Burmeistera crassifolia 18 34 Burmeistera microphylla Burmeistera resupinata Burmeistera cylindrocarpa Only 50% species collected 98 Burmeistera almedae 98 78 92 Burmeistera zurquiensis Burmeistera obtusifolia 62 Burmeistera loejtantii 89 Burmeistera succulentaW 88 84 Burmeistera smaragdi Single locus phylogeny 6 Burmeistera tenuiflora Burmeistera succulentaE Burmeistera multiflora 72 86Burmeistera dendrophila 89 Burmeistera utleyi 93 81 Burmeistera chiriquiensis 44 Burmeistera parvifolia No coalescence processes 71 Burmeistera zamorensis Burmeistera mcvaughii 31 76 Burmeistera morii 97 67 Burmeistera minutiflora Burmeistera pirrensis Burmeistera crebra 100 Burmeistera variabilis 66 100 93 Burmeistera racemiflora Burmeistera fuchsioides Burmeistera aff 100 Burmeistera ceratocarpa 100 34 Burmeistera fuscoapicata Burmeistera lutosa 40 Burmeistera panamensis 100 Burmeistera dukei Burmeistera domingensis Centropogon nigricans Siphocampylus krauseanus

0.005

Maddison, 1997 Degnan & Rosenberg, 2009 Acknowledgements

Muchhala Lab Michael McKain

N Muchhala

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