The More the Merrier? Bioprospecting the bitterness of cytogenetic variants from creosote bush (Larrea tridentata (DC.) Coville, Zygophyllaceae) in the Sonoran and Chihuahuan Desert

Sarah Ramirez1,2, I. Guzman1 and S. Fuentes-Soriano2,3

1Department of Plant and Environmental Sciences, 2NMSU Herbaria & 3Department of Animal and Range Sciences, New Mexico State University , Las Cruces, NM, 88003

Larrea tridentata or creosote in the Zygophyllaceae plant family is a typical and prolific shrub in North American Deserts. This shrub is widely used in traditional Native American medicine and has a unique geographic distribution of populations differing in ploidy levels (n=13, 26, or 39). The role of ploidy variation in the specific medicinal properties of the species has not been explored. Therefore, the primary goal of this project is to test the hypothesis that there is a correlation between ploidy and differences in the bioactive compounds present in the species.

BA

12

10

6

14

Tota

l Phe

nolic

s (m

g/g)

8

4

2

0 Chihuahuan Sonoran

Characters measured:

HYPOTHESIS We hypothesize that there is a correlation between ploidy and differences in the bioactive compounds present in the species.

A B

Figure 2. Distribution map of cytotypes and populations searched in this study. A. Populations collected in AZ (tetraploids in red, diploids in green). B. Creosote population to the north of Pusch Ridge Wilderness Area of the Santa Catalina Mountains, located in the Coronado National Forest north of Tucson, Arizona, in the United States, Pima Co., AZ. Left - Sarah Ramirez, right Sara Fuentes-Soriano (Photo by I. Guzman, March, 2019).

INTRODUCTION

in the Chihuahuan Desert and

RESULTS AND CONCLUSIONS Morphological Analyses Structural morphological features of the plants were key to indirectly confirm ploidy conditions of samples (Fig.

A B 5).

Figure 5. Morphological variation of leaves & fruits in creosote populations from the Chihuahuan & Sonoran Desert. A. Leaf length (mm). B. Fruit length (mm).

Medicinal Phytochemistry

Total Phenolics

Total phenolics were taken using Galic acid as a standard. OBJECTIVES Samples had TP averages of 1. Petal length

• To identify and collect representative creosote populations 2. Petal width 12.64 mg/g Dried Weight (DW) with different chromosome number (cytotypes) or ploidy 3. Filament length

• To profile bioactive chemical compounds of diploid and 4. Fruit length 11.36 mg/g DW in the Sonoran tetraploid cytotypes 5. Leaflet length

Desert (Fig. 6). 6. Leaflet width • To compare chemical profiles of cytotypes in creosote Figure 6. Comparison of Total Phenolics (mg/g DW) 7. Internode length among populations of creosote in the Chihuahuan and

MATERIALS AND METHODS (Fig. 1) Sonoran Desert. Figure 3. Morphological characters measured in this study, A. Petal length (1); petal width (2); filament length (4). B. Leaflet length (6) and leaflet width (3 and 4). Scale bar = 1cm (figure modified from Laport & Ramsey (2015)). Essential Oils

Identification of medicinal Selection of one target Medicinal Phytochemistry Gas Chromatography found 95 total compounds, 18 of the most plants species by ◆ Total Phenolics (TP) were extracted with acidic methanol from dry leaves & stems

◆ Essential Oils extracted with hexane and analyzed with Gas Chromatography/ A B

Mass spectra (GC-MS).

medicinal plant species abundant represented 40.73% of the total identified volatiles (Fig. 7). reviewing ethnobotanical (creosote) and measured using a spectrophotometer. literature

• Leaves and stems collection

• Materials freeze dried

• Materials grinded • 0.5g. DW used

• Spectrometer • Calculate avg.

Extraction(3x) • 80% MeOH • Sonicate 40°C • Centrifuge 4°C

Search for patterns of morphological variation

and cytotypes geographic distribution

(Fig. 2)

Measurement of 8 morphological plant

features to identify ploidy variants in Chihuahuan (2n) and Sonoran (4n)

Deserts (Fig. 3)

• Leaves and stems collection

• Materials freeze dried

• Materials grinded • 0.5g. DW used

Extraction(3x) • 80% MeOH • Sonicate 40°C • Centrifuge 4°C

• Spectrometer • Calculate avg.

samples to have a greater understanding of the physiological and genetic Figure 4. Protocol to extract Total Phenolics of leaves and stems in creosote populations in Chihuahuan (Left, Doña Ana Co., NM) and Sonoran Desert (Right, Pima Co. AZ). Photos from left to right Sarah Ramirez, Sara Fuentes, and Ivette Guzman (Photos by D. Anderson and S. Fuentes-Soriano, March, 2019). mechanisms behind the medicinal properties of creosote.

Figure 7. Top 15 essential oils in leaves of creosote populations from the Chihuahuan desert. A. Main essential oils compounds identified .B. Abundance of essential volatiles according to GC-MS. Upper graph total compounds, lower graph Pinane fractions.

FUTURE DIRECTIONS Preliminary morphological measurements and phenolic totals where moderately Field exploration to Chemical profiling by collect diploid and Total Phenolics and tetraploid populations GC-MS assays (Fig. 4) throughout AZ & NM

Figure 1. General workflow of the study.

different in Chihuahuan and Sonoran Desert. We aim to conduct additional morphological measurements, karyotyping, and essential oil analysis of plant

4. PubMed.2018 Available at https://www.ncbi.nlm.nih.gov/ [accessed April 2019]. References Acknowledgements and Additional Funding 5. Laport R.G. and J. Ramsey. 2015. Morphometric analysis of the North American creosote bush (Larrea tridentata, Zygophyllaceae) and the microspatial 1.Laport R.G, R.L Minckley, and J. Ramsey. 2012. Phylogeny and cytogeography of the North American Creosote Bush (Larrea tridentata, Zygophyllaceae). distribution of its chromosome races. Plant Systematics and Evolution 301: 1581-1599. Systematic Botany 37(1): 153-164. Louis Stokes Alliance for Minority Participation (LSAMP) HRD # 1305011 6. GEOLocate: Platform for georeferencing natural history collections data. http://www.geo-locate.org/ [accessed April 2019]. 2. Kono C., Z.-Z. Lu, H.-Z. Xue, C.A.J. Erdelmeir, D. Meksuriyen, C.-T. Che, G.A. Cordell, D.D. Soejarto, D.P. Waller, and H.S. Fong. 1990. Furanoid Lignans from

7. Martins S., C.N. Aguilar, J.A. Teixeira, and S.I. Mussatto. 2012. Bioactive compounds (phytoestrogens) recovery from Larrea tridentata leaves by solvent Larrea tridentata. Journal of Natural Products 53(2): 396-406. extracts. Separation and Purification Technology 88: 163-167. USDA Natural Resources Career Tracks (NRCT) 3. SEINet. 2017. Southwest Environmental Information Network-Arizona Chapter. Available online at http://swbiodiversity.org/seinet/ [accessed April 2019].