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Spectrophotometry of Artemisia tridentata to Quantitatively Determine Subspecies

Authors:
  • USDA Forest Service

Abstract

Ecological restoration is predicated on our abilities to discern plant taxa. Taxonomic identification is a first step in ensuring that plants are appropriately adapted to the site. An example of the need to identify taxonomic differences comes from big sagebrush (Artemisia tridentata). This species is composed of three predominant subspecies occupying distinct environmental niches, but overlap and hybridization are common in ecotones. Restoration of A. tridentata largely occurs using wildland collected seed, but there is uncertainty in the identification of subspecies or mix of subspecies from seed collections. Laboratory techniques that can determine subspecies composition would be desirable to ensure that subspecies match the restoration site environment. In this study, we use spectrophotometry to quantify chemical differences in the water-soluble compound, coumarin. Ultraviolet (UV) absorbance of A. tridentata subsp. vaseyana showed distinct differences among A.t. tridentata and wyomingensis. No UV absorbance differences were detected between A.t. tridentata and wyomingensis. Analyses of samples from > 600 plants growing in two common gardens showed that UV absorbance was unaffected by environment. Moreover, plant tissues (leaves and seed chaff) explained only a small amount of the variance. UV fluorescence of water-eluted plant tissue has been used for many years to indicate A.t. vaseyana; however, interpretation has been subjective. Use of spectrophotometry to acquire UV absorbance provides empirical results that can be used in seed testing laboratories using the seed chaff present with the seed to certify A. tridentata subspecies composition. On the basis of our methods, UV absorbance values < 2.7 would indicate A.t. vaseyana and values > 3.1 would indicate either A.t. tridentata or wyomingensis. UV absorbance values between 2.7 and 3.1 would indicate a mixture of A.t. vaseyana and the other two subspecies.
Spectrophotometry of Artemisia tridentata to Quantitatively
Determine Subspecies
Bryce A. Richardson
a,
, Alicia A. Boyd
a
, Tanner Tobiasson
a
, Matthew J. Germino
b
a
US Department of Agriculture (USDA) Forest Service, Rocky Mountain Research Station, Provo, UT 84606, USA
b
US Geological Survey, Forest and Rangeland Ecosystem Science Center, Boise, ID 83706, USA
abstractarticle info
Article history:
Received 27 March 2017
Received in revised form 27 June 2017
Accepted 10 July 2017
Available online xxxx
Key Words:
mixed-effect model
sagebrush
seed certication
ultraviolet uorescence
Ecological restorationis predicated on our abilities to discern planttaxa. Taxonomic identication is a rst step in
ensuring that plants are appropriately adapted to the site. An example of the need to identify taxonomic
differences comes from big sagebrush (Artemisia tridentata). This species is composed of three predominant
subspecies occupying distinct environmental niches, but overlap and hybridization are common in ecotones.
Restoration of A. tridentata largely occurs using wildland collected seed, but there is uncertainty in the
identication of subspecies or mix of subspecies from seed collections. Laboratory techniques that can determine
subspecies composition would be desirable to ensure that subspecies match the restoration site environment. In
this study, we use spectrophotometry to quantify chemical differences in the water-soluble compound,
coumarin. Ultraviolet (UV) absorbance of A. tridentata subsp. vaseyana showed distinct differences among
A.t. tridentata and wyomingensis. No UV absorbance differences were detected between A.t. tridentata and
wyomingensis. Analyses of samples from N600 plants growing in two common gardens showed that UV
absorbance was unaffected by environment. Moreover, plant tissues (leaves and seed chaff) explained only a
small amount of the variance. UV uorescence of water-eluted plant tissue has been used for many years to
indicate A.t. vaseyana; however, interpretation has been subjective. Use of spectrophotometry to acquire UV
absorbance provides empirical results that can be used in seed testing laboratories using the seed chaff present
with the seed to certify A. tridentata subspecies composition. On the basis of our methods, UV absorbance values
b2.7 would indicate A.t. vaseyana and values N3.1 would indicate either A.t. tridentata or wyomingensis.UV
absorbance values between 2.7 and 3.1 would indicate a mixture of A.t. vaseyana and the other two subspecies.
Published by Elsevier Inc. on behalf of The Society for Range Management.
Introduction
Distinguishing native plant taxa is fundamental to restoring and
conserving natural ecosystems and has been mandated in the policy of
land management agencies (Olwell and Riibe 2016). However,
the task of determining taxonomy becomes more challenging in taxa
with overlapping distributions and intermediate forms. Taxonomic
complexity across varying spatial scales describes the circumstances
for the subspecies of Artemisia tridentata (big sagebrush). As a species,
A. tridentata occupies a broad environmental niche of the semiarid
cold desert and nonforested uplands of western North America.
This species is of great conservation concern because of loss primarily
from wildre and invasive species. All three predominant subspecies
(A.t. vaseyana [mountain big sagebrush], A.t. wyomingensis [Wyoming
big sagebrush] and A.t. tridentata [basin big sagebrush]) ll distinct envi-
ronmental niches (Mahalovichand McArthur 2004;Still and Richardson
2015). However, overlap along elevation and edaphic gradients is com-
mon and composition of subspecies varies considerably depending on
environmental heterogeneity (McArthur et al. 1988; Goodrich et al.
1999; McArthur and Sanderson 1999). In addition, hybridization is
known to occur between subspecies (Wang et al. 1997; McArthur and
Sanderson 1999). Distinguishing subspecies (or hybrids) in these
transitional areas can be problematic. Subsequently, commercial seed
collection activities may cross these ecotones with little indication of
the loss of subspecies homogeneity in the seed collection. Thus,
A. tridentata seed collections encompassing many hectares can be a mix-
ture of subspecies (Richardson et al. 2015). Seed collections consisting
of subspecies mixtures do not necessarily diminish their value in
restoration, but knowing the composition of subspecies in a collection
is essential information for their appropriate placement on the
landscape, increasing success of establishmentand long-term resiliency.
Ideally, diagnostic characters for sagebrush subspecies would be
compatible with seed testing procedures. Fortunately, techniques that
Rangeland Ecology & Management xxx (2017) xxxxxx
Funding was provided by the Bureau of Land Management, Great Basin Native Plant
Program. Any use of trade, rm, or product names is for descriptive purposes only and
does not imply endorsement by the US government.
Correspondence: BryceA. Richardson, USDA Forest Service,Rocky Mountain Research
Station, 735 N 500 E, Provo, UT 84606, USA.
E-mail address: brichardson02@fs.fed.us (B.A. Richardson).
http://dx.doi.org/10.1016/j.rama.2017.07.004
1550-7424/Published by Elsevier Inc. on behalf of The Society for Range Management.
Contents lists available at ScienceDirect
Rangeland Ecology & Management
journal homepage: http://www.elsevier.com/locate/rama
Please cite this article as: Richardson, B.A., et al., Spectrophotometry of Artemisia tridentata to Quantitatively Determine Subspecies,
Rangeland Ecology & Management (2017), http://dx.doi.org/10.1016/j.rama.2017.07.004
distinguish A. tridentata subspecies can be determined from small
amounts of seed and seed chaff that accompany seed harvest. For exam-
ple, seed weights distinguish A.t. wyomingensisfrom basin big sagebrush
(A.t. tridentata,Richardson et al. 2015), and the elution of plant tissues in
water or ethanol has been shown to be diagnostic for A.t. vaseyana
under an ultraviolet (UV) black light. Specically, subspecies vaseyana
elutions emits a brighter iridescent glow compared with those of the
other subspecies (Stevens and McArthur 1974). The difference in UV
uorescence is caused by differing concentrations of coumarin
compounds, producing a strong iridescent blue color when dissolved
in water and illuminated by black light. Coumarin is found in all
subspecies, but at higher concentrations in A.t. vaseyana (McArthur
et al. 1988). The UV test has been shown to be effective and easy to
use; however, results are subjective and based on the observer
interpretation of the degree of iridescence, usually a scale from 15.
An empirical value for UV testing is needed to standardize seed testing.
Shumar et al. (1982) rst reported on the use of a spectrophotometer
to provide empirical data distinguishing big sagebrush subspecies.
However, spectrophotometry results from a later study showed that
large variance in UV absorbance within subspecies made distinguishing
subspecies using this technique difcult (Spomer and Henderson 1988).
In our current study, a similar approach is employed by using a plate
spectrophotometer to assess UV absorbance spectra among subspecies
of A. tridentata.WeuseA. tridentata plants growing in two common
gardens to assess the effectiveness of UV absorbance spectra in
determining subspecies and evaluate the inuence from environmental
effects and differences between tissue types. Techniques evaluated in
this study have the potential to be used as a rapid and effective seed
certication method for distinguishing A.t. vaseyana from A.t. tridentata
and A.t. wyomingensis.
Methods and Materials
More than 600 big sagebrush samples representing 55 populations
were collected from two common gardens growing at Majors Flat,
Utah (lat 39.339, long 111.520, elev 2 105 m) and Orchard, Idaho,
United States (lat 43.322, long: 115.998, elev 974 m). Common
gardens served to isolate environmental and genetic effects. Orchard is
a warm, dry site and Majors Flat is a cool, relatively mesic site.
Seeds for these common gardens were collected in 2009 from
populations distributed across 11 states in the western United States.
Populations were dened as randomly collected seed from separate
plants within b1 ha. Germinants were reared in 6-inch cone-tainers
for 3 mo before outplanting in the spring of 2010. A combination of
genetic markers, morphology, ploidy, visual-based UV uorescence
(Richardson et al. 2012), and volatile organic compounds (Jaeger et al.
2016) were used to verify subspecies before UV spectra analysis.
For each sample, fresh leaf material was removed from the
stem, weighed to 0.9 g and diced (1 mm
2
)intoa1.5mLtube.
Molecular-grade water (850 μL) was added to each sample and
incubated at room temperature for 3 min. Eluted samples were ltered
(30-μm mesh) into a 1.5-mL tube to remove leaf debris.A 200-μLaliquot
of each samplewas pipetted into the wellof a clear, at-bottom, 96-well
plate. One well was reserved for a negative control (water). Samples
underwent an absorbance spectrum analysis in a Biotek Epoch
microplate spectrophotometer (Winooski, VT), and scanning was
conducted in 10-nm increments from 240 370 nm. The sample
preparation process was repeated for sagebrush seed chaff collected
from the Majors Flat garden to test for similarities or differences in the
absorbance spectrum for different tissues (leaf tissue vs. seed chaff).
Seed chaff included oral parts (e.g., pappus and stem) and leaf
fragments that are obtained during the seed collection process and is
used as inert material during seeding.
Because some seed collections are a mixture of subspecies, we
evaluated the effect of varying ratios of A.t. vaseyana on UV absorbance
values. Three replications of seven different weight ratios of
vaseyana:tridentata leaf tissue totaling 0.9 g were analyzed. These ratios
equated to 10%, 20%, 30%, 50%, 70%, 80%, and 90% A.t. vaseyana compo-
sition. UV absorbance was acquired using the same methods as de-
scribed earlier. In addition to subspecies mixture, two hybrid plants
obtained from controlled crosses were analyzed for UV absorbance
(McArthur et al. 1988).
A linear mixed-effect model (LMM) was used to assess differences in
absorbance spectrum at 340 nm. We chose 340 nm because this data
point showed the lowest absorbance values for A.t. vaseyana (Fig. 1a)
and low variance for all subspecies (data not shown). Absorbance spec-
trum data were compiled according to garden, subspecies, population,
and sample name (Table S1, available online at http://dx.doi.org/10.
1016/j.rama.2017.07.004). An LMM segregated explanatory variables
into xed and random effects. Fixed effects were assigned to subspecies
using the methods described earlier, and random effects were assigned
to garden and the interaction between garden and population. Gardens
were used to assess environmental variance, and population-garden in-
teraction determined genetic × environment interaction (G × E).
In a subsequent experiment, absorbance spectrum data at 340 nm were
analyzed for differences in tissue types: leaf and seed chaff. These data
were collected at one garden, Majors Flat, since environment was found
to be nonsignicant. Data were compiled according to tissue, subspecies,
population, and sample name (Fig. S1, available online at http://dx.doi.
org/10.1016/j.rama.2017.07.004). The statistical analysis was similar as
described earlier except the random effect tissuereplaced garden.
Analyses were conducted in R v3.1.2 (R Core Team 2016) with the
packages LME4 v1.1 (Bates et al. 2015), and signicance for xed effects
Pvalues used Satterthwaites approximation for degrees of freedom cal-
culated with lmerTEST v2.0 (Kuznetsova et al. 2016). Conditional and
marginal R
2
values (Johnson 2014) were calculated in both models
with r.squaredGLMM function in the MuMin package (Barton 2015).
Results and Discussion
UV absorbance of leaf tissue from spectrophotometry distinguished
A.t. vaseyana from A.t. tridentata and wyomingensis. This technique did
not distinguish between the subspecies tridentata and wyomingensis
(Table 1,Fig. 1a). The distinction of A.t. vaseyana in UV absorbance
was apparent from a wavelength between 290 nm and 370 nm with
the greatest separation between 340 and 360 nm, regardless of garden
collection. In contrast, A.t. wyomingensis and tridentata had similar spec-
tra curves and overlapping 95% condence intervals across all spectra
tested (see Fig. 1a). These observations were supported by an LMM.
Subspecies showed a highly signicant difference between A.t. vaseyana
and the other two subspecies at 340 nm, but no signicant difference
was found between A.t. wyomingensis and tridentata (see Table 1,Pb
0.0001). The mixed-effects model explained 55% of the variation
(conditional R
2
= 0.55) in UV absorbance at 340 nm. The majority of
the variation accounted by the model, 78%, was explained by subspecies
(marginal R
2
= 0.43). A smaller proportion of the absorbance variation
(12%) was explained by environment and population × environment
interaction. Garden (i.e., environment) alone showed no signicant
variance; however, population × garden (i.e., genetic × environment
interaction) was signicant (Pb0.0001).
Conditioned sagebrush seed collections are a matrix of seed, seed
chaff, and leaves. Because the matrix of leaves and chaff would likely
vary in composition across seed collections, we evaluated the effect of
tissue types (leaves vs. chaff, Fig. 1b) and conducted LMM on the UV
absorbance at 340 nm. Tissue type accounted for a signicant amount
of the random effect variance in absorbance, but compared with the
differences in xed-effect estimates found between A.t. vaseyana and
other subspecies (0.7), the variance for tissue type is very small
(0.0074, Table 2). These results suggest that variable composition
of leaves and chaff would not compromise the determination of
subspecies composition.
2B.A. Richardson et al. / Rangeland Ecology & Management xxx (2017) xxxxxx
Please cite this article as: Richardson, B.A., et al., Spectrophotometry of Artemisia tridentata to Quantitatively Determine Subspecies,
Rangeland Ecology & Management (2017), http://dx.doi.org/10.1016/j.rama.2017.07.004
Along with varying tissue composition, subspecies composition will also
vary among seed collections. Spatial overlap among subspecies is
common in ecotones between montane and basin habitats (McArthur
et al. 1988; McArthur and Sanderson 1999). To assess how varying
subspecies mixtures and hybrids affected UV absorbance values,
spectrophotometry was conducted on seven different weight ratios
between vaseyana and tridentata and two hybrid plants from control
crosses between A.t. tridentata and vaseyana (McArthur et al. 1988). The
results illustrate that coumarin from A.t. vaseyana can have a strong inu-
ence on the UV absorbance values. Even at low concentrations of A.t.
vaseyana (1020%), the UV absorbance was 2.6 at 340 nm. This value
is within the 95% condence intervals of A.t. vaseyana (see Fig. S1). There-
fore, seed collectors targeting A.t. wyomingensis or A.t. tridentata will have
to take care of the proximity to ecotones and A.t. vaseyana because only a
small amount of this subspecies will greatly affect the absorbance results.
Making several UV uorescence assays with a handheld UV lamp at the
seed collection site would help targetthecorrectsubspecies.
UV uorescence has long been a valuable diagnostic test to deter-
mine A.t. vaseyana. However, the method to date is subjective, relying
on a visual rating system. Shumar et al. (1982) rst demonstrated the
utility of spectrophotometry to assess big sagebrush subspecies. In this
study, we show that determining UV absorbance through spectropho-
tometry provides a fast, empirical-based method to assess the composi-
tion of A.t. vaseyana in seed collections. We also show that this trait is
insensitive to environmental effects and tissue type. In addition to de-
termining seed weights (Richardson et al. 2015), this research
Figure 1. The absorbance spectrum for Artemisia tridentata from 240 nm to 370 nm. (a) Shows absorbance values for three subspecies (tridentata,vaseyana, and wyomingensis)from
populations collected throughout the range of the species and planted at two common gardens (Majors Flat and Orchard). (b) Shows the spectrum for different plant tissues: chaff
and leaf. Vertical error bars indicate the 95% condence intervals.
3B.A. Richardson et al. / Rangeland Ecology & Management xxx (2017) xxxxxx
Please cite this article as: Richardson, B.A., et al., Spectrophotometry of Artemisia tridentata to Quantitatively Determine Subspecies,
Rangeland Ecology & Management (2017), http://dx.doi.org/10.1016/j.rama.2017.07.004
can be used as a seed certication step to inform managers of the
subspecies composition, providing the necessary information to ensure
A. tridentata plants are appropriately adapted to the site.
Supplementary data to this article can be found online at http://dx.doi.
org/10.1016/j.rama.2017.07.004.
Acknowledgments
We thank Dr. Joshua Udall and the Brigham Young University
Genetics laboratory for the use of spectrophotometry equipment,
sample collection from Dr. Brynne Lazarus, and Drs. Matthew Madsen
and Stanley Kitchen and anonymous reviewers for insightful comments.
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Table 1
Random effect v ariances and xed effect estimates from linear mixed-effects model
analysis of ult raviolet absorbance at 340 nm in Artemisia tridentata.Fixedeffectsare
partitioned amo ng subspecies, a nd random effects reect different common gardens
and the interaction between gardens and populations
Random effects obs Variance SD Pvalue
Garden 2 0.0012 0.034 0.3
Population × garden 103 0.0142 0.120 2
e-6
Residual 0.0591 0.2431
Fixed effects Estimate SE Pvalue
Intercept (tridentata) 3.1873 0.0323 6
e-4
vaseyana 0.6822 0.0423 b2
e-16
wyomingensis 0.0246 0.037 0.490
SD, standard deviation; SE, standard error.
Table 2
Random effect variances and xed effect estimatesfrom linear mixed-effects model anal-
ysis of UV absorbance at 340 nm from Artemisia tridentata. Fixed effects are partitioned
among subspecies, and random effects reect two tissue types: leaves and seed chaff
Random effects obs Variance SD Pvalue
Tissue 2 0.0074 0.086 0.02
Population × tissue 95 0.0205 0.143 1
e-13
Residual 0.0594 0.244
Fixed effects Estimate SE Pvalue
Intercept (tridentata) 3.2295 0.0680 0.006
vaseyana 0.7124 0.0443 b2
e-16
wyomingensis 0.0201 0.0456 0.660
SD, standard deviation; SE, standard error.
4B.A. Richardson et al. / Rangeland Ecology & Management xxx (2017) xxxxxx
Please cite this article as: Richardson, B.A., et al., Spectrophotometry of Artemisia tridentata to Quantitatively Determine Subspecies,
Rangeland Ecology & Management (2017), http://dx.doi.org/10.1016/j.rama.2017.07.004
... Sagebrush varies in concentrations of coumarins, a subclass of phenolic compounds, depending on species and growing conditions (e.g., mountain big sagebrush [A. t. vaseyana], black sagebrush, and low sagebrush) (McArthur et al. 1988;Rosentreter 2005;Richardson et al. 2018), and act as an indicator of palatability for some sagebrush-obligate species, such as greater sagegrouse (Rosentreter 2005). ...
... Our results suggest that the more distinct the phytochemical concentration and composition is among morphotypes, the higher the accuracy. For example, the genetic basis for variation in coumarins (Richardson et al. 2018) may explain the higher prediction accuracy for coumarins at Camas where coumarin concentrations were dramatically higher in dwarf patches than in on-or off-mound patches (Fig. 1). Coumarin concentrations are linked to species or subspecies (Rosentreter 2005), and indicate that off-mound dwarf plants are genetically distinct from on-and off-mound Wyoming big sagebrush at Camas, but not at Cedar Gulch. ...
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Tools for performing model selection and model averaging. Automated model selection through subsetting the maximum model, with optional constraints for model inclusion. Model parameter and prediction averaging based on model weights derived from information criteria (AICc and alike) or custom model weighting schemes. [Please do not request the full text - it is an R package. The up-to-date manual is available from CRAN].
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