Genetic Relationships among Different Chemotypes of Lupinus

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Genetic Relationships among Different Chemotypes of Lupinus sulphureus Daniel Cook,*,†,‡ Ivan W. Mott,†,§ Steven R. Larson,†,§ Stephen T. Lee,‡ Robert Johnson,∥ and Clinton A. Stonecipher‡ ‡

Poisonous Plant Research, Agricultural Research Service (ARS), United States Department of Agriculture (USDA), 1150 East 1400 North, Logan, Utah 84341, United States § Forage and Range Research, Agricultural Research Service (ARS), United States Department of Agriculture (USDA), 690 North 1100 East, Logan, Utah 84322, United States ∥ Stanley L. Welsh Herbarium, Brigham Young University, Provo, Utah 84602, United States ABSTRACT: Lupines (Lupinus spp.) are a common plant legume species found on western U.S. rangelands. Lupinus spp. may contain quinolizidine and/or piperidine alkaloids that can be toxic and/or teratogenic to grazing livestock. Alkaloid profiles may vary between and within a species. The objectives of this study were to (1) further explore the characteristic alkaloid profiles of Lupinus sulphureus using field collections and (2) explore the phylogenetic relationship of the different populations and chemotypes of L. sulphureus using the amplified fragment length polymorphism method of DNA fingerprinting, thus providing possible explanations to the phenomena of multiple chemotypes within a species. A total of 49 accessions of L. sulphureus were classified into seven chemotypes. The DNA profiles showed that one L. sulphureus chemotype, chemotype A, is genetically divergent from the other chemotypes of L. sulphureus, suggesting that it represents an unresolved lupine taxon, possibly a new lupine species. Additionally, the different chemotypes of L. sulphureus represented different genetic groups, as shown by Bayesian cluster analysis and principle component analysis. KEYWORDS: Lupinus sulphureus, lupine, alkaloid, genetic diversity

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INTRODUCTION Lupinus species are common legumes found on western U.S. rangelands.1 There are more than 100 Lupinus species in the Intermountain West and Great Basin, which are found in many habitats from desert to alpine ecoystems.2 Lupinus species may contain a variety of piperidine and/or quinolizidine alkaloids (Figure 1). These alkaloids have been implicated in plant− herbivore interactions and possibly plant−microbe interactions.3 Furthermore, many of these alkaloids are toxic and/or teratogenic to livestock, which leads to economic losses for livestock producers.1,2 In the later part of the 19th century, lupines caused large losses of sheep as a result of acute intoxication, whereas isolated cases with smaller losses of sheep continue today.1,4 Ingestion of some lupine species by cattle can also cause congenital birth defects in calves termed “crooked calf disease”.5 Crooked calf syndrome is caused by reduced fetal movement when lupine is consumed during days 40−100 of gestation, resulting in the development of limbs and spine in misaligned or contracted positions.6−8 The quinolizidine alkaloid anagyrine9−11 and some piperidine alkaloids, including ammodendrine,12,13 can reduce fetal movement; thus, lupines containing these alkaloids have the potential to cause crooked calf syndrome. Lupine-induced crooked calf syndrome continues to pose a problem in several Western States; for example, Lupinus sulphureus and Lupinus leucophyllus have been reported to cause crooked calf syndrome in Oregon and Washington.14 Alkaloid profiles within a lupine species vary among plant taxa, populations, and plant parts.3,15,16 Wink and Carey15 reported © XXXX American Chemical Society

that Lupinus argenteus had multiple alkaloid profiles in the region near Crested Butte, CO, U.S.A. Several other investigators have reported similar observations; for example, L. leucophyllus exhibited at least two different alkaloid profiles among plants collected in southeast Washington and northeast Oregon.14,17 Moreover, Cook et al.18 identified seven distinct L. sulphureus chemotypes throughout the species distribution of Oregon, Washington, and British Columbia. All seven chemotypes, if grazed in sufficient quantity, could pose a risk to grazing livestock. Two chemotypes contained the teratogen anagyrine, while two contained the suspected teratogen ammodendrine. In addition, three chemotypes contained thermopsine, which induces myopathy in livestock.19 Although these seven chemotypes have different geographic distributions, none appears to follow notable geographical features, such as watersheds. The Lupinus genus is taxonomically complex, and few of its species have well-documented taxonomic delineations, phylogenetic relationships, and/or alkaloid composition. Because some Lupinus species have different chemotypes within a similar geographic region, it is important to understand how geography and chemotype influence the relationships between these different plant populations. Several authors14,15,18 have suggested that the presence of multiple chemotypes within a species may be the result of (1) cryptic species, (2) hybridization between Received: Revised: Accepted: Published: A

December 14, 2017 January 25, 2018 January 26, 2018 January 26, 2018 DOI: 10.1021/acs.jafc.7b05884 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of representative piperidine and quinolizidine alkaloids from L. sulphureus. mL of methanol containing 1.3 μg/mL caffeine (internal standard). A portion (∼1 mL) was transferred to 1.5 mL gas chromatography (GC) autosample vials for gas chromatography/flame ionization detector (GC/FID) or gas chromatography/mass spectrometry (GC/MS) analysis. GC/FID Analysis. All samples were analyzed by GC/FID using a Shimadzu GC-2010 gas chromatograph equipped with a Shimadzu AOC-20i autosampler, a J&W DB-5 column (30 m × 0.32 mm, 0.25 μm film thickness), and a FID. Samples (1.0 uL) were injected splitless at 250 °C, and helium was used as the carrier gas at a constant flow rate of 2.0 mL/min. The column oven was temperature-programmed starting at 100 °C for 1 min, increased to 200 °C at 50 °C/min, increased to 260 °C at 5 °C/min, increased to 320 °C at 50 °C/min, and held at 320 °C for 8.8 min for a total run time of 25 min. Chemical profiles were classified into groups by visual analysis of the gas chromatogram based on the presence and absence of major peaks and corresponding relative retention times from the GC/FID analysis. GC/MS Analysis. GC/MS analysis was performed as previously reported.14 In brief, a representative sample (2 μL) of each population was analyzed by GC/MS using a Finnigan MAT GCQ equipped with a split/splitless injector and a DB-5MS (30 m × 0.25 mm, J&W Scientific) column. The injection port temperature was 250 °C and operated in splitless mode. The split vent flow rate was 50 mL/min and purged after 0.80 min. The oven temperature was 100 °C for 1 min, 100−200 °C at 40 °C/min, 200−275 °C at 5 °C/min, and held at 275 °C for 1.5 min. Electron impact ionization (EI) at 70 eV was used with an ion source temperature of 200 °C. Alkaloid Identification. Alkaloid identification was performed as previously reported.14 In brief, six individual alkaloids were identified from authenticated [mass spectrometry (MS) and nuclear magnetic resonance (NMR)] samples of ammodendrine, anagyrine, and thermopsine from the alkaloid collection of the Poisonous Plants Research Laboratory, ARS, USDA, Logan, UT, U.S.A., and from commercially obtained standards (sparteine, lupanine, and D-αisolupanine). The yet to be identified alkaloids were determined from correlation of measured retention times to retention indices (RI) calculated by linear extrapolation from RI values generated from known standards and assigned RI numbers from the literature and their EI mass spectra.16 In addition, alkaloid identification was further supported by correlation of measured relative retention times (RRt) to lupanine and EI mass spectra to those reported in the literature.20 Data Analysis. Global Positioning System (GPS) coordinates from field collections were used to construct the maps showing the distribution of the populations of L. sulphureus, L. leucophyllus and L. polyphyllus using ArcGIS (ESRI, Inc.). In brief, a spreadsheet was created containing GPS points and chemotypes of plants. The spreadsheet was converted to a feature class using ArcCatalog in the ArcGIS program. The feature class was projected in ArcCatalog to match the projections of the state and county boundary shapefiles. Washington State and county boundary data were downloaded from http://fortress.wa.gov/

species to create novel chemotypes, and/or (3) chemical warfare between the plant and herbivores. The objectives of this study were to (1) further explore the characteristic alkaloid profiles of L. sulphureus using field collections and (2) explore the phylogenetic relationships of the different populations and chemotypes of L. sulphureus using amplified fragment length polymorphism (AFLP), thus providing possible explanations to the phenomena of multiple chemotypes within a species. L. sulphureus was selected as a result of our previous studies characterizing its chemotypic diversity and its agricultural importance to livestock producers in parts of Oregon and Washington. L. leucophyllus and Lupinus polyphyllus var. burkei were also included as part of this study as other representative Lupinus species because they occur in the same geographic region.

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MATERIALS AND METHODS

Plant Materials. Field collections of L. sulphureus Douglas ex Hook. (n = 49 accessions) were made throughout its geographical distribution in Oregon, Washington, and British Columbia (Table 1). In addition, limited field collections of L. leucophyllus Douglas ex Lindl. (n = 4 accessions) and L. polyphyllus var. burkei (S. Watson) C.L. Hitchc. (n = 3 accessions) were included from this same geographic region as other representative Lupinus species for the phylogenetic studies because they occur in the same geographic region (Table 1). A single flowering stem from 4 to 8 plants was collected at each location, immediately frozen on dry ice, and stored at −80 °C until lyophilized. Additionally, a voucher specimen was pressed at most locations, which was subsequently determined by the authors. Specimens are retained at the Poisonous Plant Research Laboratory Herbarium (PPRL) and the Stanley L. Welsh Herbarium at Brigham Young University (BRY). Extraction of Alkaloids. Plant material was freeze-dried and ground to pass through a 2 mm screen. Samples from field collections (100 mg) were weighed into a 16 mL screw-top glass test tube. Plant material was extracted using a previously reported procedure.14 In brief, the plant material was extracted by mechanical rotation using the Rugged Rotator (Glas Col, LLC) with a mixture of 1 N HCl (4.0 mL) and CHCl3 (4.0 mL) for 15 min. The samples were centrifuged (5 min), and the aqueous layer was removed. An additional 2.0 mL of 1 N HCl was added to the test tube containing plant material and CHCl3, extracted again by mechanical rotation (15 min), and centrifuged, and the aqueous layer was removed. The aqueous portions were combined into a clean 16 mL screw-top glass test tube. The pH of the aqueous layer was adjusted to 9.0−9.5 with concentrated NH4OH. The basic solution was extracted twice with CHCl3, first with 4.0 mL and then with 2.0 mL. The CHCl3 solutions were combined and filtered through anhydrous Na2SO4 into a clean 16 mL screw-top glass test tube, and the solvent was evaporated under N2 at 60 °C. The alkaloid fraction extracted was reconstituted in 4 B

DOI: 10.1021/acs.jafc.7b05884 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 1. Lupine Species, Populations, Alkaloid Chemotype, Population Collection State/Province, GPS Coordinates, and Voucher Numbers taxa

accession

chemotype

state/province

latitude (N)

longitude (W)

L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. sulphureus L. leucophyllus L. leucophyllus L. leucophyllus L. leucophyllus L. polyphyllus L. polyphyllus L. polyphyllus

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S34 S35 S36 S37 S38 S39 S40 S41 S42 S43 S44 S45 S46 S47 S48 S49 L3 L7 L8 L11 P1 P2 P3

A A A A A A A A A A A A A A D D E E E E E E E E E E E E F F F F F F F F F G G G C C C C C C B B B 1 1 2 2 nd nd nd

BC BC BC WA WA WA WA WA WA WA WA WA WA WA WA WA WA WA WA WA WA WA WA OR OR OR OR OR WA WA WA WA WA WA WA WA WA OR OR OR OR OR OR OR OR OR OR OR OR WA WA OR OR WA WA OR

49.457 49.373 49.349 48.163 47.979 47.971 47.903 47.633 47.598 47.405 47.389 47.334 47.330 47.250 46.108 46.108 46.126 46.319 46.433 46.413 46.203 46.203 46.335 45.573 45.233 45.981 45.373 45.723 46.359 46.262 46.228 46.185 46.122 46.167 46.133 46.091 46.145 45.984 45.861 45.787 45.380 45.732 45.290 45.325 45.601 45.376 45.148 45.297 45.384 46.915 46.373 45.979 45.621 46.320 46.203 45.325

119.730 119.655 119.708 119.380 119.038 119.158 119.700 119.262 119.394 119.085 119.454 118.479 118.671 118.257 117.265 117.270 117.071 117.421 117.433 117.558 117.575 117.575 117.795 117.903 118.014 118.047 118.097 118.196 117.696 117.871 117.953 117.981 117.985 117.994 118.017 118.075 118.079 118.212 118.268 118.281 118.333 118.426 118.459 118.505 118.606 118.648 118.717 118.978 119.235 118.049 117.696 118.047 118.627 117.408 117.575 118.505

dar/app1/datwed/dmmatrix.html (accessed January 2015). Oregon State and county boundary data were downloaded from http://www.

voucher PPRL3146 PPRL3150 PPRL3135 PPRL3154 BRY199751 PPRL3160 PPRL3153 PPRL3151 PPRL3152 PPRL3155 PPRL3156 BRY199748 BRY199748 PPRL4048 PPRL2876 BRY199752 PPRL2967 PPRL2967 PPRL2969 PPRL3157 PPRL3216 BRY198206 PPRL3194 PPRL3195

PPRL3201

PPRL3205 PPRL2975 PPRL3215 PPRL3206 PPRL3212 PPRL2870 PPRL3208 BRY198897 PPRL2971 PPRL3187 PPRL3203

PPRL2968 PPRL3210

oregon.gov/DAS/EISPD/alphalist.shtml (accessed January 2015). The feature class was opened in ArcMap and overlaid onto the state and C


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Figure 2. GC/MS total ion chromatograms of alkaloid profiles from L. sulphureus: (A) chemotype A collected near Ritzville, WA, U.S.A., (B) chemotype B collected near Ukiah, OR, U.S.A., (C) chemotype C collected near Pendleton, OR, U.S.A., (D) chemotype D collected near Anatone, WA, U.S.A., (E) chemotype E collected near Pomeroy, WA, U.S.A., (F) chemotype F collected near Coppei, WA, U.S.A., and (G) chemotype G collected near Tollgate, OR, U.S.A.. Peaks correspond to the following alkaloids: (1) ammodendrine, (2) N-methylammodendrine, (3) gramine, (4) 5,6-dehydrolupanine, (5) lupanine, (6) anagyrine, (7) sparteine, (8) isomer of 11,12-dehydrosparteine, (9) 11,12-dehydrosparteine, (10) epiaphylline, (11) α-isosparteine, (12) 5,6-dehydro-α-isolupanine, (13) α-isolupanine, (14) 11,12-dehydrolupanine, (15) 7-hydroxylupanine, (16) thermopsine, (17) 10,17-dioxo-β-sparteine, (18) 17-oxolupanine, (19) aphylline, (20) isomer of 17-oxolupanine, and (21) dehydrolupanine. Peaks with an asterisk (∗) were always present in each respective chemotype. This figure was modified from a previous report on chemotypes of L. sulphureus.18 county boundary shapefiles. A map was then created with the GPS points representing the different collections. In addition, principle component analysis with the AFLP data was performed using BioNumerics 4.6 (Applied Maths, Inc.). Genetic Analysis. A total of 320 plants from 56 accessions, including 49 of L. sulphureus, 4 of L. leucophyllus, and 3 of L. polyphyllus,

were tested for DNA polymorphism using the AFLP method of DNA fingerprinting21 modified for fluorescent detection. Samples of DNA were extracted using DNeasy 96 extraction kits (Qiagen, Valencia, CA, U.S.A.). The concentration and quality of each DNA sample was assessed by spectrophotometry and agarose gel electrophoresis. The AFLP procedure followed the protocol of Vos et al.,21 using the six D


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Figure 3. Map showing the distribution of L. sulphureus chemotypes A−G (49 accessions) in the states of Washington and Oregon as well as the province of British Columbia. A small inset shows the distribution of L. leucophyllus (4 accessions) and L. polyphyllus (3 accessions). using the Mantel correlation test, using 999 permutations, using GenAlEx 6.501.22,23 The genetic ancestry of individual plants was determined using the model-based Bayesian clustering analysis, allowing for dominant allele admixture, without an assignment of flags for hierarchical groups using the Structure version 2.3 program.24 A total of 10 replicated runs with 1 100 000 iterations and 110 000 burn-in iterations were produced to compare the log probability, Pr(K), of each model, where K is the number of groups.25 The stability of the second-order change in log probability between successive models, ΔPr(K), for K = 2−9, was also calculated26 using Structure Harvester Web version 0.6.94 July 2014.27

selective primer pairs (E.AAG/M.CAC, E.ACC/M.CAG, E.ACT/ M.CAT, E.ACA/M.CAG, E.AAT/M.CGG, and E.AGC/M.CTC), except that the EcoRI selective amplification primers were labeled using 6-carboxy fluorescein (6-FAM) and separated by capillary electrophoresis using an ABI 3730 instrument with the GS-500 LIZ size standards and GeneScan software (Applied Biosystems, Foster City, CA, U.S.A.). Individual AFLP profiles, from each plant, were subsequently visualized and manually scored for the presence (1) or absence (0) of fragments with Genographer version 1.6 software (James Benham and Tom Blake, Montana State University, Bozeman, MT, U.S.A.). Plants with poor amplification profiles for one or more selective primer pairs were removed from the study. Monomorphic loci defined as having either absence or presence frequencies greater than 0.95 were removed from the data matrix. The apportionment of genetic variation within and among accessions, chemotypes, and taxa was investigated using analysis of molecular variance (AMOVA) based on pairwise comparisons of the number of AFLP marker differences (Euclidean distances), with 999 random permutations, among individual plants using GenAlEx 6.501.22,23 Moreover, the relationships between geographical distances and Euclidean genetic distances among individual plants were also tested

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RESULTS AND DISCUSSION Identification of Chemotypes and Alkaloids. Previously, seven alkaloid profiles (chemotypes) of L. sulphureus were identified throughout its geographical distribution.18 This study was based primarily on herbarium specimens and a limited number of field samples. The initial objectives here were to characterize the alkaloid profiles of the 49 accessions of L. sulphureus and to map their distribution. The alkaloid profile of each accession was determined using GC/MS and GC/FID and E


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Figure 4. Map showing the detailed distribution of L. sulphureus chemotypes B−G (35 accessions) in the states of Washington and Oregon.

As part of this study, limited collections of two additional Lupinus species, L. leucophyllus and L. polyphyllus, from the same region were included. The four L. leucophyllus accessions represented two alkaloid profiles previously reported.14,17 Each profile was represented by two accessions. For more details regarding alkaloid identification and the relative frequency of occurrence of each alkaloid in each respective profile, one is referred to the previous report.17 Lastly, no quinolizidine and piperidine alkaloids were detected in the individual plants representing the three accessions of L. polyphyllus. Genetic Analysis. AFLPs have been used as a tool to explore the genetic diversity of various plants, including Lupinus species.28,29 There are a limited number of studies exploring the diversity and evolutionary relationships using AFLPs, chloroplast genes, and nuclear genes among Old World and New World Lupinus species.30−33 To date, there is a single report exploring genetic relatedness of two chemotypes of a Lupinus species.17 Here, AFLP-based phylogenetic analysis was applied to

placed within one of the previously defined seven chemotypes, A−G (Figure 2). The qualitative alkaloid composition of each respective chemotype contained one to three diagnostic alkaloids that were always present in the individual plants from each accession. The diagnostic alkaloids for each respective chemotype were as follows: chemotype A, ammodendrine; chemotype B, ammodendrine and N-methylammodendrine, chemotype C, lupanine and anagyrine; chemotype D, sparteine, lupanine, and anagyrine, chemotype E, isolupanine and thermopsine; chemotype F, aphylline; and chemotype G, sparteine and lupanine (Figures 1 and 2). For more details regarding the relative frequency of occurrence of the other alkaloids in each respective chemotype, one is referred to the previous report.18 Chemotypes A and E were the most common and were represented by 14 and 12 accessions, respectively, while chemotypes B, D, and G were the least common chemotypes and were represented by 3, 2, and 3 accessions, respectively (Table 1). A distribution map of the different accessions and corresponding chemotypes is shown in Figures 3 and 4. F


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Figure 5. Model probabilities from Bayesian cluster analysis of AFLP genotypes from 320 Lupinus samples from 56 accessions, with model probabilities, Pr(K), and stability of the second-order rate change, ΔPr(K), for models with K = 1−10 groups, in which K is the number of groups tested.

Figure 6. Inferred population structure and ancestry coefficients for K = 2 groups, where K is the number of groups tested in each model based on Bayesian cluster analysis of L. sulphureus chemotype A (14 accessions), L. sulphureus chemotypes B−G (35 accessions), L. leucophyllus (L.l.) (4 accessions), and L. polyphyllus (L.p.) (3 accessions). Accessions are numerically sequential within each chemotype (see Table 1).

the multiple accessions representing the seven chemotypes of L. sulphureus. AFLP analysis using six selective primer pairs resulted in scoring a total of 637 polymorphic fragments from 320 individuals representing 49 accessions of L. sulphureus, 4 accessions of L. leucophyllus, and 3 accessions of L. polyphyllus. The total average number of fragments per plant was 157, ranging from 105 to 225. Hierarchical AMOVA apportioned 38% of the total DNA polymorphism among these three Lupinus taxa (p < 0.001) and an average of 14% of the variation among accessions within species (p < 0.001). Considered within taxa, the apportionment of DNA polymorphism among L. sulphureus accessions, 23% (p < 0.001), was higher than L. polyphyllus or L. leucophyllus, which were both about 13% (p < 0.001). However, a hierarchical AMOVA within L. sulphureus apportioned 15% of the variation among chemotypes (p < 0.001), 10% among accessions (p < 0.001), and 75% among plants within accessions (p < 0.001). Thus, chemotypes present within L. sulphureus contribute a significant amount of genetic structure to this

species. Moreover, L. sulphureus chemotype A, in particular, is geographically and genetically distinct from the six other L. sulphureus chemotypes. A hierarchical AMOVA apportioned 18% of the L. sulphureus DNA polymorphism among chemotype A and the other six chemotypes (p < 0.001), 13% among accessions within these two groups (p < 0.001), and 69% remaining among plants within accessions (p < 0.001). Thus, the apportionment of L. sulphureus DNA variation among accessions within chemotype A and the remaining group of six other chemotypes (13%) is very similar to the apportionment of DNA variation among accessions within L. leucophyllus (13%) or L. polyphyllus (13%). However, by all accounts, the vast majority of DNA variation within these Lupinus taxa was present as variation among plants within accessions. With taxa and chemotypes factored out, we found that about 77% of the Lupinus DNA polymorphisms is present among plants within collection sites. A similar percentage of within accession variation has been reported for other out-crossing species.34 G


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Figure 7. Inferred population structure and ancestry coefficients for K = 3−6 groups, where K is the number of groups tested in each model based on Bayesian cluster analysis of L. sulphureus chemotype A (14 accessions), L. sulphureus chemotypes B−G (35 accessions), L. leucophyllus (L.l.) (4 accessions), and L. polyphyllus (L.p.) (3 accessions). Accessions are numerically sequential within each chemotype (see Table 1).

chemotypes B−G are genetically more similar to accessions of L. leucophyllus than they are to those of L. sulphureus chemotype A. It should also be noted that some admixing was observed. For example, chemotypes B and C of L. sulphureus showed admixing with L. sulphureus chemotype A (Figure 7). Although these two groups are geographically distinct, we suspect that this admixing may have occurred at the times of the Missoula floods,35 resulting in genetic material from L. sulphureus chemotype A becoming geographically proximal to chemotypes B and C of L. sulphureus. To further explore the relationships among the different accessions from the three species, the number of groups (K) was evaluated from K = 4−6 in the Bayesian analysis (Figure 7). The three distinct taxa, L. sulphureus, L. leucophyllus, and L. polyphyllus, as well as L. sulphureus chemotype A were observed as distinct ancestral groups in the K = 4 model. In this model, a high proportion of admixing was observed in chemotypes E and G of L. sulphureus with L. leucophyllus. As the number of groups was increased in the Bayesian analysis to K = 5 and/or 6, the chemotypes B−G of L. sulphureus became split into two or three apparent races, showing a large proportion of admixing. The large proportion of admixing may be the result of hybridization and/or incomplete sorting among the chemotypes of L. sulphureus. Little change in the ancestral coefficient composition was observed in L. sulphureus chemotype A, L. leucophyllus, and L. polyphyllus in these models. Generally, collections with the same

Bayesian clustering provides a means to model and test the number and composition of genetic groups present among a given (sample) set of genotypes.24−26 Estimates of the probability of each model are shown in Figure 5. The model K = 3 was best supported by ΔK, while K = 6 was the model with near optimum probability. The results from models greater than K = 6 were not shown because they produced relatively small gains in the model probabilities and much greater genetic complexities that were non-sensical and difficult to interpret. The number and composition of the genetic groups were consistent across the 10 replicated runs, for each model (K value), with the exception of K = 2, where two different outcomes were observed (Figure 6). In the case of this study, for example, it would be expected a priori to obtain high estimates of probability for a three-group model because it should be easy to classify plant genotypes into three distinct groups corresponding to the L. sulphureus, L. leucophyllus, and L. polyphyllus taxa. Although the K = 3 model did in fact show some optimum properties (Figure 5), the actual clustering of the AFLP genotypes for the K = 3 model was not consistent with the current taxonomic understanding of these species (Figure 7). The three ancestral groups defined in this model were as follows: L. polyphyllus, L. sulphureus chemotype A, and L. sulphureus chemotypes B−G together with L. leucophyllus (Figure 7). These data indicate that accessions of L. sulphureus H


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Figure 8. Principle component analysis of the AFLP data from L. sulphureus chemotype A, L. sulphureus chemotypes B−G, and L. leucophyllus. Each symbol represents a single plant from the different accessions of the different species and/or chemotypes, L. sulphureus chemotype A (14 accessions), L. sulphureus chemotypes B−G (35 accessions), and L. leucophyllus (4 accessions).

Figure 9. Principle component analysis of the AFLP data from L. sulphureus chemotypes B−G (35 accessions). Each symbol represents a single plant from the different accessions of the different chemotypes.

I


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In summary, the alkaloid profiles of L. sulphureus were determined throughout its geographical distribution. A total of 49 accessions of L. sulphureus were classified into seven chemotypes; the biogeography of these chemotypes was consistent with previously reported findings.18 Lastly, we explored the phylogenetic relationship among these different chemotypes of L. sulphureus. In summary, the AFLP profiles showed that L. sulphureus chemotype A is most divergent from the other chemotypes of L. sulphureus, suggesting that it represents an unresolved taxon, possibly an undescribed lupine species. Future work will resolve the taxonomic position of L. sulphureus chemotype A. Additionally, the different chemotypes of L. sulphureus represented different genetic groups/races, as shown by Bayesian cluster analysis and principle component analysis. This molecular diversity may in part explain the diversity of chemotypes observed in L. sulphureus.

chemotype had a similar ancestral coefficient composition. Likewise, a strong association was observed between one or two chemotypes and a specific ancestral group. Principle component analysis revealed similar trends to the Bayesian analysis. Principle component analysis separated L. sulphureus chemotype A, L. sulphureus chemotypes B−G, and L. leucophyllus into three distinct groups (Figure 8). Likewise, when L. polyphyllus was included in the principle component analysis, four distinct clusters were observed (data not shown). These data are consistent with the Bayesian analysis with the K = 4 model. Principle component analysis of chemotypes B−G showed that the respective chemotypes generally separated into three groups, chemotypes B and C, chemotypes D and F, and chemotypes E and G (Figure 9), consistent with the K = 6 model from the Bayesian analysis. In summary, the principle component analysis was consistent and supported the Bayesian models. Lastly, significant (p < 0.05) Mantel correlations were detected between genetic distance and geographic distance matrices comparing the taxa and some of the ancestral groups from the Bayesian analysis. The correlation coefficient (R) among all of the data was 0.131 versus 0.44, 0.31, and 0.37 within the three species, L. leucophyllus, L. polyphyllus, and L. sulphureus, respectively. In general, relatively high correlations between genetic and geographic distances are expected within species versus between species because gene flow between species is interrupted. The relatively low correlation coefficient overall among the three taxa is due the fact that genetically distinct species can be found in close proximity. The correlation coefficient (R) within the two principle ancestral groups of L. sulphureus, chemotype A and chemotypes B−G from the different Bayesian models were 0.33 and 0.19, respectively. The low correlation coefficient among L. sulphureus chemotypes B−G is consistent with different genotypes/races in close geographic proximity, which is supported by both the Bayesian analysis and principle component analysis. The correlation coefficients reported here were similar yet lower to some reported in the literature.36−38 Spatial scales ranged from 0 to 487 km for the reported correlation coefficients. Stochastic dispersal events on this limited spatial scale may result in much lower correlation coefficients. Several investigators have suggested that the presence of multiple chemotypes within a species may be the result of (1) cryptic species, (2) hybridization between species to create novel chemotypes, and/or (3) chemical warfare between the plant and herbivores.14,15,18 The AFLP data reported here shows that L. sulphureus chemotype A is very divergent from the other chemotypes of L. sulphureus, suggesting that it may represent a new lupine taxon. Preliminary data suggests that L. sulphureus chemotype A has morphological characters that distinguish it from L. sulphureus chemotypes B−G. The most notable differences from field observation are flower color and leaf architecture, both of which are not as notable on herbarium sheets. AFLP analysis has been used as a tool to investigate relationships within and among different accessions of several plant species that exhibit different chemotypes.39,40 These studies have shown an association while others have not between molecular diversity and secondary compounds. The data reported here show an association between molecular diversity and the unique chemotypes representing chemical diversity. The association of molecular diversity and chemotypes could be used to explore this association in other species with distinct chemotypes, such as Delphinium occidentale.41

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 435-752-2941. Fax: 435-753-5681. E-mail: daniel. [email protected]. ORCID

Daniel Cook: 0000-0001-8568-113X Stephen T. Lee: 0000-0002-0597-8353 Author Contributions †

Daniel Cook, Ivan W. Mott, and Steven R. Larson contributed equally to this work. Funding

This work was supported by direct congressional appropriations via the USDA, which is part of the ARS. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Kimberly Thorsted, Jessie Roper, and Charles Hailes for technical assistance. REFERENCES

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Genetic Relationships among Different Chemotypes of Lupinus

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