# assignR Examples

#### November 10, 2020

We will introduce the basic functionality of assignR using data bundled with the package. We’ll review how to access data for known-origin biological samples and environmental models, use these to fit and apply functions estimating the probability of sample origin across a study region, and summarize these results to answer research and conservation questions. We’ll also demonstrate a quality analysis tool useful in study design, method comparison, and uncertainty analysis.

Let’s load assignR and two other packages we’ll need.

library(assignR)
library(raster)
library(sp)

data("naMap")
plot(naMap)

Let’s do the same for a growing season precipitation H isoscape for North America. Notice this is a RasterBrick with two layers, the mean prediction and a standard deviation of the prediction. The layers are from waterisotopes.org, and their resolution has been reduced to speed up processing in these examples. Full-resolution global growing season isoscapes are included in the package as d2h_world.rda and d18o_world.rda.

data("d2h_lrNA")
plot(d2h_lrNA)

The package includes a database of H and O isotope data for known origin samples (knownOrig.rda), which consists of three features (sites, samples, and sources). Let’s load it and have a look. First we’ll get the names of the data fields available in the tables.

data("knownOrig")
names(knownOrig$sites) ## [1] "Site_ID" "Site_name" "State" "Country" ## [5] "Site_comments" names(knownOrig$samples)
##  [1] "Sample_ID"       "Sample_ID_orig"  "Site_ID"         "Dataset_ID"
##  [5] "Taxon"           "Group"           "Source_quality"  "Age_class"
##  [9] "Material_type"   "Matrix"          "d2H"             "d2H.sd"
## [13] "d18O"            "d18O.sd"         "Sample_comments"
names(knownOrig$sources) ## [1] "Dataset_ID" "Dataset_name" ## [3] "Citation" "Sampling_method" ## [5] "Sample_powdered" "Lipid_extraction" ## [7] "Lipid_extraction_method" "Exchange" ## [9] "Exchange_method" "Exchange_T" ## [11] "H_cal" "O_cal" ## [13] "Std_powdered" "Drying" ## [15] "Analysis_method" "Analysis_type" ## [17] "Source_comments" The sites feature is a spatial object that records the geographic location of all sites from which samples are available. plot(assignR:::wrld_simpl) points(knownOrig$sites, col = "red")

Now lets look at a list of species names available.

unique(knownOrig$samples$Taxon)
##  [1] "Danaus plexippus"            "Setophaga ruticilla"
##  [3] "Turdus migratorius"          "Setophaga coronata auduboni"
##  [5] "Poecile atricapillus"        "Thryomanes bewickii"
##  [7] "Thryothorus ludovicianus"    "Spizella passerina"
##  [9] "Geothlypis trichas"          "Setophaga pensylvanica"
## [11] "Baeolophus bicolor"          "Vermivora chrysoptera"
## [13] "Catharus guttatus"           "Setophaga citrina"
## [15] "Geothlypis formosa"          "Geothlypis tolmiei"
## [17] "Oreothlypis ruficapilla"     "Cardinalis cardinalis"
## [19] "Oreothlypis celata"          "Junco hyemalis oregonus"
## [21] "Seiurus aurocapilla"         "Vireo olivaceus"
## [23] "Melospiza melodia"           "Catharus ustulatus"
## [25] "Catharus fuscescens"         "Vireo griseus"
## [27] "Cardellina pusilla"          "Hylocichla mustelina"
## [29] "Icteria virens"              "Setophaga petechia"
## [31] "Melozone aberti"             "Vermivora cyanoptera"
## [33] "Passer domesticus"           "Aimophila ruficeps"
## [35] "Poecile carolinensis"        "Troglodytes aedon"
## [37] "Dumetella carolinensis"      "Mniotilta varia"
## [39] "Lanius ludovicianus"         "Anthus spragueii"
## [41] "Euphagus carolinus"          "Empidonax minimus"
## [43] "Oreothlypis peregrina"       "Aythya affinis"
## [45] "Cyanistes caeruleus"         "Phasianus colchicus"
## [47] "Lagopus lagopus"             "Tetrao tetrix"
## [49] "Dryocopus maritus"           "Serin serin"
## [51] "Vanellus vanellus"           "Corvus corone"
## [53] "Turdus merula"               "Corvus monedula"
## [55] "Columba palumbus"            "Turtle Dove"
## [57] "Tetrastes bonasia"           "Perdix perdix"
## [59] "Anas platyrhyncos"           "Branta canadensis"
## [61] "Columba livia"               "Numenius arguata"
## [63] "Turdus pilaris"              "Turdus iliacus"
## [65] "Turdus philomelos"           "Fringilla coelebs"
## [67] "Buteo lagopus"               "Accipiter striatus"
## [69] "Falco sparverius"            "Accipiter gentillis"
## [71] "Accipiter cooperii"          "Buteo jamaicensis"
## [73] "Buteo platypterus"           "Buteo swainsoni"
## [75] "Circus cyaneus"              "Falco columbarius"
## [77] "Falco mexicanus"             "Falco perigrinus"
## [79] "Wilsonia citrina"            "Oporornis tolmiei"
## [81] "Wilsonia pusilla"            "Homo sapiens"
## [83] "Charadrius montanus"         "Anas platyrhynchos"
## [85] "Locustella luscinioides"     "Acrocephalus arundinaceus"
## [87] "Acrocephalus scirpaceus"     "Pipilo maculatus"

Load H isotope data for North American Loggerhead Shrike from the package database.

Ll_d = subOrigData(taxon = "Lanius ludovicianus", mask = naMap)
## mask was reprojected
## 524 samples are found from 427 sites
## 524 samples from 427 sites in the transformed dataset

By default, the subOrigData function transforms all data to a common reference scale (defined by the standard materials and assigned, calibrated values for those; by default VSMOW-SLAP) using data from co-analysis of different laboratory standards Magozzi et al., 2021. The calibrations used are documented in the function’s return object.

Ll_d$chains ## [[1]] ## [1] "OldEC.1_H_1" "EC_H_7" "EC_H_9" "VSMOW_H" Information on these calibrations is contained in the stds.rda data file. Transformation is important when blending data from different labs or papers because different reference scales have been used to calibrate published data and these calibrations are not always comparable. In this case all the data come from one paper: Ll_d$sources[,1:3]
##   Dataset_ID            Dataset_name
## 2          2 Hobson et al. 2012 Plos
##                                                                                                                                                                                                         Citation
## 2 Hobson KA, Van Wilgenburg SL, Wassenaar LI, Larson K. 2012. Linking hydrogen (d2H) isotopes in feathers and precipitation: sources of variance and consequences for assignment to isoscapes. Plos One 7:e35137

If we didn’t want to transform the data, and instead wished to use the reference scale from the original publication, we can specify that in our call to subOrigData. Keep in mind that any subsequent analyses using these data will be based on this calibration scale: for example, if you wish to assign samples of unknown origin, the values for those samples should be reported on the same scale.

Ll_d = subOrigData(taxon = "Lanius ludovicianus", mask = naMap, ref_scale = NULL)
## mask was reprojected
## 524 samples are found from 427 sites

Ll_d$sources$H_cal
## [1] "OldEC.1_H_1"

For a real application you would want to explore the database to find measurements that are appropriate to your study system (same or similar taxon, geographic region, measurement approach, etc.) or collect and import known-origin data that are specific to your system.

# Single-isoscape Calibration and Probability of Origin Estimates

We need to start by assessing how the environmental (precipitation) isoscape values correlate with the sample values. calRaster fits a linear model relating the precipitation isoscape values to sample values, and applies it to produce a calibrated, sample-type specific isoscape.

d2h_Ll = calRaster(known = Ll_d, isoscape = d2h_lrNA, mask = naMap)
## known was reprojected
##
##
## ---------------------------------------
##         ------------------------------------------
## rescale function uses linear regression model,
##         the summary of this model is:
## -------------------------------------------
##         --------------------------------------
##
## Call:
## lm(formula = tissue.iso ~ isoscape.iso[, 1], weights = tissue.iso.wt)
##
## Residuals:
##      Min       1Q   Median       3Q      Max
## -148.787  -16.584    0.353   19.239  107.727
##
## Coefficients:
##                   Estimate Std. Error t value Pr(>|t|)
## (Intercept)        0.92452    1.77787    0.52    0.603
## isoscape.iso[, 1]  1.11204    0.03966   28.04   <2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##
## Residual standard error: 32.4 on 522 degrees of freedom
## Multiple R-squared:  0.601,  Adjusted R-squared:  0.6002
## F-statistic: 786.2 on 1 and 522 DF,  p-value: < 2.2e-16

Let’s create some hypothetical samples to use in demonstrating how we can evaluate the probability that the samples originated from different parts of the isoscape. The isotope values are drawn from a random distribution with a standard deviation of 8 per mil, which is a pretty reasonable variance for conspecific residents at a single location. We’ll also add made-up values for the analytical uncertainty for each sample and a column recording the calibration scale used for our measurements. If you had real measured data for your study samples you would load them here, instead.

id = letters[1:5]
set.seed(123)
d2H = rnorm(5, -110, 8)
d2H.sd = runif(5, 1.5, 2.5)
d2H_cal = rep("UT_H_1", 5)
Ll_un = data.frame(id, d2H, d2H.sd, d2H_cal)
print(Ll_un)
##   id        d2H   d2H.sd d2H_cal
## 1  a -114.48381 2.456833  UT_H_1
## 2  b -111.84142 1.953334  UT_H_1
## 3  c  -97.53033 2.177571  UT_H_1
## 4  d -109.43593 2.072633  UT_H_1
## 5  e -108.96570 1.602925  UT_H_1

As discussed above, one issue that must be considered with any organic H or O isotope data is the reference scale used by the laboratory producing the data. The reference scale for your unknown samples should be the same as that for the known origin data used in calRaster. Remember that the scale for our known origin data d is OldEC.1_H_1. Let’s assume that our fake data were normalized to the UT_H_1 scale. The refTrans function allows us to convert between the two.

Ll_un = refTrans(Ll_un, ref_scale = "OldEC.1_H_1")
print(Ll_un)
## $data ## id d2H d2H.sd d2H_cal ## 1 a -127.8800 3.374984 OldEC.1_H_1 ## 2 b -125.0795 2.970657 OldEC.1_H_1 ## 3 c -109.6746 2.808334 OldEC.1_H_1 ## 4 d -122.5874 3.016841 OldEC.1_H_1 ## 5 e -121.7123 2.594943 OldEC.1_H_1 ## ##$chains
## $chains[[1]] ## [1] "UT_H_1" "UT_H_2" "US_H_5" "US_H_6" "VSMOW_H" ## [6] "EC_H_9" "EC_H_7" "OldEC.1_H_1" ## ## ## attr(,"class") ## [1] "refTrans" Notice that both the d2H values and the uncertainties have been updated to reflect the scale transformation. Now we will produce posterior probability density maps for the unknown samples. For reference on the Bayesian inversion method see Wunder, 2010 Ll_prob = pdRaster(d2h_Ll, unknown = Ll_un) Cell values in these maps are small because each cell’s value represents the probability that this one cell, out of all of them on the map, is the actual origin of the sample. Together, all cell values on the map sum to ‘1’, reflecting the assumption that the sample originated somewhere in the study area. Let’s check this for sample ‘a’. cellStats(Ll_prob[[1]], 'sum') ## [1] 1 Check out the help page for pdRaster for additional options, including the use of informative prior probabilities. # Multi-isoscape Analysis We can also use multiple isoscapes to (potentially) add power to our analyses. We will start by calibrating a H isoscape for the monarch butterfly, Danaus plexippus. Dp_d = subOrigData(taxon = "Danaus plexippus") ## 208 samples are found from 32 sites ## 150 samples from 31 sites in the transformed dataset d2h_Dp = calRaster(Dp_d, d2h_lrNA) ## known was reprojected ## ## ## --------------------------------------- ## ------------------------------------------ ## rescale function uses linear regression model, ## the summary of this model is: ## ------------------------------------------- ## -------------------------------------- ## ## Call: ## lm(formula = tissue.iso ~ isoscape.iso[, 1], weights = tissue.iso.wt) ## ## Weighted Residuals: ## Min 1Q Median 3Q Max ## -11.1010 -3.4064 0.3067 3.0780 8.7990 ## ## Coefficients: ## Estimate Std. Error t value Pr(>|t|) ## (Intercept) -51.86981 1.98424 -26.14 <2e-16 *** ## isoscape.iso[, 1] 0.75543 0.04154 18.19 <2e-16 *** ## --- ## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1 ## ## Residual standard error: 4.469 on 148 degrees of freedom ## Multiple R-squared: 0.6908, Adjusted R-squared: 0.6888 ## F-statistic: 330.7 on 1 and 148 DF, p-value: < 2.2e-16 Our second isoscape represents 87Sr/86Sr values across our study region, the state of Michigan. It was published by Bataille and Bowen, 2012, obtained from waterisotopes.org, cropped and aggregated to coarser resolution, and a rough estimate of uncertainty added. In this case, we do not have any known-origin tissue samples to work with. However, our isoscape was developed to approximate the bioavailable Sr pool, and Sr isotopes are not strongly fractionated in food webs. Thus, our analysis will assume that the isoscape provides a good representation of the expected Sr values for our study species without calibration. Let’s look at the Sr isoscape and compare it with our butterfly H isoscape. data("sr_MI") plot(sr_MI$weathered.mean)

proj4string(sr_MI)
## [1] "+proj=aea +lat_0=40 +lon_0=-96 +lat_1=20 +lat_2=60 +x_0=0 +y_0=0 +datum=NAD83 +units=m +no_defs"
proj4string(d2h_Dp$isoscape.rescale) ## [1] "+proj=longlat +ellps=WGS84 +towgs84=0,0,0,0,0,0,0 +no_defs" Notice that the we have two different spatial data objects, one for Sr and one for d2H, and that they have different extents and projections. In order to conduct a multi-isotope analysis, we’ll first combine these into a single object using the isoStack function. In addition to combining the objects, this function resolves differences in their projection, resolution, and extent. It’s always a good idea to check that the properties of the isoStack components are consistent with your expectations. Dp_multi = isoStack(d2h_Dp, sr_MI) lapply(Dp_multi, proj4string) ## [[1]] ## [1] "+proj=longlat +ellps=WGS84 +no_defs" ## ## [[2]] ## [1] "+proj=longlat +ellps=WGS84 +no_defs" lapply(Dp_multi, bbox) ## [[1]] ## min max ## s1 -91.49772 -80.50972 ## s2 40.74959 48.91699 ## ## [[2]] ## min max ## s1 -91.49772 -80.50972 ## s2 40.74959 48.91699 Now we’ll generate a couple of hypothetical unknown samples to use in our analysis. It is important that our isotopic markers appear here in the same order as in the isoStack object we created above. Dp_unk = data.frame("ID" = c("A", "B"), "d2H" = c(-86, -96), "Sr" = c(0.7089, 0.7375)) We are ready to make our probability maps. First let’s see how our posterior probabilities would look if we only used the hydrogen isotope data. Dp_pd_Honly = pdRaster(Dp_multi[[1]], Dp_unk[,-3]) We see pretty clear distinctions between the two samples, driven by a strong SW-NE gradient in the tissue isoscape H values across the state. What if we add the Sr information to the analysis? The syntax for running pdRaster is the same, but now we provide our isoStack object in place of the single isoscape. The function will use the spatial covariance of the isoscape values to approximate the error covariance for the two (or more) markers and return poterior probabilities based on the multivariate normal probability density function evaluated at each grid cell. Dp_pd_multi = pdRaster(Dp_multi, Dp_unk) Note that the addition of Sr data greatly strengthens the geographic constraints on our hypothetical unknown samples: the difference between the highest and lowest posterior probabilities is much larger than with H only, and the pattern of high probabilities reflects the regionalization characteristic of the Sr isoscape. This is especially true for sample B, which has a fairly distinctive, high 87Sr/86Sr value. # Post-hoc Analysis ## Odds Ratio Many of the functions in assignR are designed to help you analyze and draw inference from the posterior probability surfaces we’ve created above. For the following examples we’ll return to our single-isoscape, Loggerhead shrike analysis, but the tools work identically for multi-isoscape results. The oddsRatio tool compares the posterior probabilities for two different locations or regions. This might be useful in answering real-world questions…for example “is this sample more likely from France or Spain?”, or “how likely is this hypothesized location relative to other possibilities?”. Let’s compare probabilities for two spatial areas - the states of Utah and New Mexico. First we’ll load the SpatialPolygons and plot them. data("states") s1 = states[states$STATE_ABBR == "UT",]
s2 = states[states$STATE_ABBR == "NM",] plot(naMap) plot(s1, col = c("red"), add = TRUE) plot(s2, col = c("blue"), add = TRUE) Get the odds ratio for the two regions. The result reports the odds ratio for the regions (first relative to second) for each of the 5 unknown samples plus the ratio of the areas of the regions. If the isotope values (& prior) were completely uninformative the odds ratios would equal the ratio of areas. s12 = rbind(s1, s2) oddsRatio(Ll_prob, s12) ##$P1/P2 odds ratio
##        a        b        c        d        e
## 930.9960 664.1272 108.5820 492.6174 443.7568
##
## $Ratio of numbers of cells in two polygons ## [1] 2 Here you can see that even though Utah is quite a bit smaller the isotopic evidence suggests it’s much more likely to be the origin of each sample. This result is consistent with what you might infer from a first-order comparison of the state map with the posterior probability maps, above. Comparisons can also be made using points. Let’s create two points (one in each of the Plover regions) and compare their odds. This result also shows the odds ratio for each point relative to the most- and least-likely grid cells on the posterior probability map. pp1 = c(-112,40) pp2 = c(-105,33) pp12 = SpatialPoints(coords = rbind(pp1,pp2)) proj4string(pp12) = proj4string(naMap) oddsRatio(Ll_prob, pp12) ## inputP was reprojected ##$P1/P2 odds ratio
##         a         b         c         d         e
## 6379.0797 4049.3574  333.1072 2702.7184 2345.0588
##
## \$Odds relative to the max/min pixel
##    ratioToMax.a ratioToMax.b ratioToMax.c ratioToMax.d ratioToMax.e
## P1 3.000293e-01 3.610181e-01  0.873534828 0.4521961178 0.4764907062
## P2 4.737476e-05 9.279195e-05  0.002662611 0.0001606466 0.0002027243
##    ratioToMin.a ratioToMin.b ratioToMin.c ratioToMin.d ratioToMin.e
## P1 3892540380.1    6889107.5   1114047733 2.000513e+09  834748676.6
## P2     209585.7     162559.2     34544368 3.065530e+03     258540.9

The odds of the first point being the location of origin are pretty high for each sample, and much higher than for the second point.

## Assignment

Researchers often want to classify their study area in to regions that are and are not likely to be the origin of the sample (effectively ‘assigning’ the sample to a part of the area). This requires choosing a subjective threshold to define how much of the study domain is represented in the assignment region. qtlRaster offers two choices.

Let’s extract 10% of the study area, giving maps that show the 10% of grid cells with the highest posterior probability for each sample.

qtlRaster(Ll_prob, threshold = 0.1)

## class      : RasterStack
## dimensions : 17, 38, 646, 5  (nrow, ncol, ncell, nlayers)
## resolution : 2.999999, 2.999999  (x, y)
## extent     : -165, -51.00005, 20.58329, 71.58327  (xmin, xmax, ymin, ymax)
## crs        : +proj=longlat +ellps=WGS84 +towgs84=0,0,0,0,0,0,0 +no_defs
## names      : a, b, c, d, e
## min values : 0, 0, 0, 0, 0
## max values : 1, 1, 1, 1, 1

Now we’ll instead extract 80% of the posterior probability density, giving maps that show the smallest region within which there is an 80% chance each sample originated.

qtlRaster(Ll_prob, threshold = 0.8, thresholdType = "prob")

## class      : RasterStack
## dimensions : 17, 38, 646, 5  (nrow, ncol, ncell, nlayers)
## resolution : 2.999999, 2.999999  (x, y)
## extent     : -165, -51.00005, 20.58329, 71.58327  (xmin, xmax, ymin, ymax)
## crs        : +proj=longlat +ellps=WGS84 +towgs84=0,0,0,0,0,0,0 +no_defs
## names      : a, b, c, d, e
## min values : 0, 0, 0, 0, 0
## max values : 1, 1, 1, 1, 1

Comparing the two results, the probability-based assignment regions are broader. This suggests that we’ll need to assign to more than 10% of the study area if we want to correctly assign 80% or more of our samples. We’ll revisit this below and see how we can chose thresholds that are as specific as possible while achieving a desired level of assignment ‘quality’.

## Summarization

Most studies involve multiple unknown samples, and often it is desirable to summarize the results from these individuals. jointP and unionP offer two options for summarizing posterior probabilities from multiple samples.

jointP calculates the probability that all samples came from each grid cell in the analysis area. Note that this summarization will only be useful if all samples are truly derived from a single population of common geographic origin.

jointP(Ll_prob)

## class      : RasterLayer
## dimensions : 17, 38, 646  (nrow, ncol, ncell)
## resolution : 2.999999, 2.999999  (x, y)
## extent     : -165, -51.00005, 20.58329, 71.58327  (xmin, xmax, ymin, ymax)
## crs        : +proj=longlat +ellps=WGS84 +towgs84=0,0,0,0,0,0,0 +no_defs
## source     : memory
## names      : Joint_Probability
## values     : 1.117089e-47, 0.02106095  (min, max)

unionP calculates the probability that any sample came from each grid cell in the analysis area. In this case we’ll save the output to a variable for later use.

Ll_up = unionP(Ll_prob)

The results from unionP highlight a broader region, as you might expect.

Any of the other post-hoc analysis tools can be applied to the summarized results. Here we’ll use qtlRaster to identify the 10% of the study area that is most likely to be the origin of one or more samples.

qtlRaster(Ll_up, threshold = 0.1)

## class      : RasterLayer
## dimensions : 17, 38, 646  (nrow, ncol, ncell)
## resolution : 2.999999, 2.999999  (x, y)
## extent     : -165, -51.00005, 20.58329, 71.58327  (xmin, xmax, ymin, ymax)
## crs        : +proj=longlat +ellps=WGS84 +towgs84=0,0,0,0,0,0,0 +no_defs
## source     : memory
## names      : layer
## values     : 0, 1  (min, max)

# Quality analysis and method comparison

How good are the geographic assignments? What area or probability threshold should be used? Is it better to use isoscape A or B for my analysis? These questions can be answered through split-sample validation using QA.

We will run quality assessment on the Loggerhead shrike known-origin dataset and precipitation isoscape. These analyses take some time to run, depending on the number of stations and iterations used.

qa1 = QA(Ll_d, d2h_lrNA, valiStation = 8, valiTime = 4, by = 5, mask = naMap, name = "normal")
## known was reprojected

We can plot the result using plot.

plot(qa1)

The first three panels show three metrics, granularity (higher is better), bias (closer to 1:1 is better), and sensitivity (higher is better). The second plot shows the posterior probabilities at the known locations of origin relative to random (=1, higher is better). More information is provided in Ma et al., 2020.

A researcher might refer to the sensitivity plot, for example, to assess what qtlRaster area threshold would be required to obtain 90% correct assignments in their study system. Here it’s somewhere between 0.25 and 0.3.

How would using a different isoscape or different known origin dataset affect the analysis? Multiple QA objects can be compared to make these types of assessments.

Let’s modify our isoscape to add some random noise.

dv = getValues(d2h_lrNA[[1]])
dv = dv + rnorm(length(dv), 0, 15)
d2h_fuzzy = setValues(d2h_lrNA[[1]], dv)
plot(d2h_fuzzy)

We’ll combine the fuzzy isoscape with the uncertainty layer from the original isoscape, then rerun QA using the new version. Obviously this is not something you’d do in real work, but as an example it allows us to ask the question “how would the quality of my assignments change if my isoscape predictions were of reduced quality?”.

d2h_fuzzy = brick(d2h_fuzzy, d2h_lrNA[[2]])
qa2 = QA(Ll_d, d2h_fuzzy, valiStation = 8, valiTime = 4, by = 5, mask = naMap, name = "fuzzy")
## known was reprojected

Now we can plot to compare.

plot(qa1, qa2)

## NULL

Assignments made using the fuzzy isoscape are generally poorer than those made without fuzzing. Hopefully that’s not a surprise, but you might encounter cases where decisions about how to design your project or conduct your data analysis do have previously unknown or unexpected consequences. These types of comparisons can help reveal them!