Abstract The Phylogeny of the Genus Mirabilis

This summer I studied the phylogeny of the genus Mirabilis to try to elucidate relationships within the group and learn more about the evolution of gypsum Julia Olivieri. Professor: Michael Moore. Department of Biology endemic species in the clade. I isolated DNA from some individuals using 61 PhytoPure and CTAB procedures, and sequenced the ITS, matK, ndhF, ycf1 63 4100-5500, and ycf1 5500-6300 DNA regions for over fifty individuals using PCR. I matK, ndhF, and 81 Figure 3. Mirabilis nesomii 62 then used Geneious to edit the sequences, and created phylogenetic trees using ycf1 concatenated parsimony with my data. These trees suggest that the species Mirabilis nesomii,

the only known gypsum endemic Mirabilis, may be polyphyletic. The specific 93 95 68 adaptations necessary for living on gypsum might have caused morphological Results/Discussion 95 convergence that led to this mistaken classification. 97 100 • The ITS and chloroplast trees have different topologies. This is common 66 100 in closely related species, due such factors as matrilineal inheritance of 74

100 chloroplasts, incomplete lineage sorting, and gene flow. 57 Figure 2. Natural habitat of Mirabilis nesomii 75 81 Gypsophily: • Sequences of the gypsophilic Mirabilis nesomii are not monophyletic in 91 Section Key 99 92 either tree. This is most likely due to misidentification of M. nesomii

95 Mirabilis collection GBH 25567, which may be M. linearis (its placement in both

63 98 trees supports this) For morphological similarity see fig. 1. 59 Mirabilopsis 98 • The two sequences that are assumed to be correctly identified M. 89 98 nesomii are monophyletic in both trees with bootstrap support >90%, 100 63 Quamoclidion and though they consistently group with M. glabrifolia and significantly 67 100 long branches leading to them, suggesting that M. nesomii has diverged 52 Oxybaphoides into a distinct species. 66

99 Oxybaphus Biogeography:

100 100 • Mirabilis himalaica, the only Mirabilis species native outside of the 100 85 86 Americas, appears monophyletic but nested deeply within Mirabilis. It

100 does possess a significant number of autoapomorphies, placing the divergence of the lineage perhaps around several million years ago.

Paraphyly and polyphyly in sections of Mirabilis:

Parsimony-based phylogenetic tree using concatenated matK, ndhF, and ycf1 regions. Bootstrap values are • Sect. Mirabilis sect. Oxybaphoides, sect. Mirabilopsis and sect. given above each branch; values <50% are not shown. Arrows indicate the gypsophile Mirabilis nesomii. Quamoclidion are almost monophyletic in both trees, though in the Figure 5. Clockwise from top left: Mirabilis jalapa, M. longiflora, M. multiflora and M. bigelovii chloroplast tree sect. Quamoclidion is mixed in with sect. Oxybaphoides • Parts of sect. Oxybaphus seem to be largely monophyletic (the clade containing M. texensis, M. linearis, and M. melanotricha) in both trees, Introduction 52 Nuclear ITS while the inclusion of genera such as M. viscosa and M. glabrifolia make 72 it polyphyletic. The genus Mirabilis (Nyctaginaceae) is part of the incredibly diverse order 95 Caryophyllales, which also includes cacti, beets, and many carnivorous plants. • There is no support for the backbone of the ITS tree, suggesting a rapid 77 radiation near the beginning of the genus. Nyctaginaceae contains many species that are tolerant of or live exclusively on 100 gypsum (hydrous calcium sulfate), a substrate that some have hypothesized is difficult to adapt to due to the hard crust typical of gypseous soils which can 94 prevent germination. Nyctaginaceae is unusual in that it contains at least 25 99 100 gypsophilic species across 7 genera. Mirabilis contains one known gypsum 99 endemic, Mirabilis nesomii (see fig. 2 for habitat). The 54 species of Mirabilis are 57 spread across North and South America with one species in the Himalayas, but its 63 main center of distribution is in the arid western regions of North America. 92

Mirabilis jalapa (common name four o’clock) is in widespread cultivation, and its 86 63 common name comes from the fact that the hawkmoth-pollinated flowers of 92 Mirabilis do not open until the afternoon or evening. Plants of Mirabilis are 78 herbaceous and suffrutescent, with large floral bracts and tuberous underground storage structures. 61 62 GOALS: Determine whether the lineage of Mirabilis nesomii is monophyletic, and Figure 4. Comparison of leaf width and assess whether it is differentiated enough to be acknowledged as a distinct succulence between M. nesomii (right) and species. Analyze whether the sections of Mirabilis are monophyletic. non-gypsophilic Mirabilis (left)

77 100

96 Materials and Methods 83

98 66 61 93 96 • 66 individuals representing 36 species of Mirabilis sampled 59 Figure 1. Comparison of floral bract, fruit, and leaves Top: Mirabilis linearis Bottom: M. nesomii 69 99 • Nuclear ITS and portions of three chloroplast genes (matK, ndhF, and two regions of 100 75 91 Acknowledgements ycf1) were sequenced using Sanger sequencing 57 • Outgroups included representatives of other genera of tribe Nyctagineae Many thanks to Professor Michael Moore for mentoring me and granting me a spot in my • All full-length ITS sequences of Mirabilis from GenBank were included lab, as well as to the NSF for their grant. Thanks also to my labmates Erin Johnson, Noah • DNA isolated from silica-dried, field-collected specimens or from herbarium specimens Last, and Vera Hutchinson, and to Evan Finch for his help at the beginning of the summer. using either the CTAB procedure or the PhytoPure kit • Chromatogram sequences edited and aligned using MAFFT in Geneious, alignments Parsimony-based phylogenetic tree using nuclear ITS. Bootstrap values are given above each branch; Works Cited values <50% are not shown. Arrows indicate the gypsophile Mirabilis nesomii. checked by eye DOUGLAS, N. A. and P. S. Manos. 2007. Molecular Phylogeny of Nyctaginaceae: Taxonomy, Biogeography, and Characters Associated with a • For several taxa, individuals from multiple populations were sequenced Radiation of Xerophytic Genera in North America. American Journal of Botany 94(5): 856-872. LEDESMA, P. H. 2011. Sistemática de Mirabilis L. (Nyctaginaceae). • The three chloroplast regions were concatenated for analysis; ITS was kept separate LEVIN, R. A. 2000. Phylogenetic Relationships Within Nyctaginaceae Tribe Nyctaginaceae: Evidence from Nuclear and Chloroplast Genomes. • Used PAUP to find the most parsimonious trees with a bootstrap search Systematic Botany 25(4): 738-750. LEVIN, R. A. 2003. The Systematic Utility of Floral and Vegetative Fragrance in Two Genera of Nyctaginaceae. Systematic Botany 52(3): 334-351. TURNER, B. L. 1991. A New Gypsophilic Species of Mirabilis (Nyctaginaceae) from Nuevo Loén, México. Phytologia 70(1): 44-46.