Molecular Ecology Resources (2016) 16, 694700 doi: 10.1111/1755-0998.

12496

POLYPATEX:
an R package for paternity exclusion in
autopolyploids
ALEXANDER B. ZWART,* CAROLE ELLIOTT, TARA HOPLEY, DAVID LOVELL and
ANDREW YOUNG
*CSIRO Data61, GPO Box 664, Canberra, ACT 2601, Australia, CSIRO National Facilities and Collections, GPO Box 1600,
Canberra, ACT 2601, Australia, Queensland University of Technology (QUT), GPO Box 2434, Brisbane, QLD 4001, Australia

Abstract
Microsatellite markers have demonstrated their value for performing paternity exclusion and hence exploring mating
patterns in plants and animals. Methodology is well established for diploid species, and several software packages
exist for elucidating paternity in diploids; however, these issues are not so readily addressed in polyploids due to
the increased complexity of the exclusion problem and a lack of available software. We introduce POLYPATEX, an R
package for paternity exclusion analysis using microsatellite data in autopolyploid, monoecious or dioecious/bisex-
ual species with a ploidy of 4n, 6n or 8n. Given marker data for a set of offspring, their mothers and a set of candidate
fathers, POLYPATEX uses allele matching to exclude candidates whose marker alleles are incompatible with the alleles
in each offspringmother pair. POLYPATEX can analyse marker data sets in which allele copy numbers are known (geno-
type data) or unknown (allelic phenotype data) for data sets in which allele copy numbers are unknown, compar-
isons are made taking into account all possible genotypes that could arise from the compared allele sets. POLYPATEX is
a software tool that provides population geneticists with the ability to investigate the mating patterns of autopoly-
ploids using paternity exclusion analysis on data from codominant markers having multiple alleles per locus.
Keywords: allele matching, microsatellite, pollen dispersal, polyploid
Received 22 July 2014; revision received 13 November 2015; accepted 19 November 2015

genotypes are available for a given set of unique alleles.

Introduction
Population genetics has utilized microsatellite molecular
markers to understand the patterns of pollen and seed
dispersal and recruitment in plants as well as mating pat-
terns in animals, to provide information to effectively
manage populations, either from a conservation or eradi-
cation perspective (Ouborg et al. 1999). Microsatellites are
a suitable genetic marker system for studying population
genetics as they are mostly neutral and highly variable
(Selkoe & Toonen 2006). These markers have proven to be
ideal for determining parentage within and among popu-
lations (Ashley 2010) however, assigning parentage
requires knowledge of a candidate parents genotype
including knowledge of the exact number of allelic copies
present across all loci. For diploid species, this is straight-
forward and much of the theory and software has been
developed for this ploidy level (Jones et al. 2010).
Determining polyploid genotypes when allele copy
numbers are unknown is more difficult, as multiple
Correspondence: Alexander Zwart, Fax: +61 2 62167007;
E-mail: Alec.Zwart@csiro.au
For example, a hexaploid (6n) with two alleles (AB) can
have five different genotypes (AAAAAB, AAAABB,
AAABBB, AABBBB, ABBBBB), and this ambiguity makes
the search for parents of offspring extremely complex.
The incidence of polyploidy in plant populations is more
common and widespread than initially estimated (Otto
& Whitton 2000; Wood et al. 2009); however, few popula-
tion genetic studies have been conducted on these organ-
isms due to the complexity of analysing this type of data.
Several software packages can deal with autopolyloid
data in terms of data manipulation, determining genetic
distance and population allele frequencies (e.g. POLYSAT-
Clark & Jasieniuk 2011; SPAGEDI-Hardy & Vekemans 2002;
GENODIVE-Meirmans & Van Tienderen 2004; POPDIST-Guld-
brandtsen et al. 2000 and AUTOTET-Thrall & Young 2000).
While programs are available to assign paternity or
parentage to individuals of a ploidy level greater than
diploid, they have limitations. ORCHARD (Spielmann et al.
2015) is limited to tetraploids. COLONY (Jones & Wang
2002), FAMOZ (Gerber et al. 2003) and the SAS scripts of
Riday et al. (2013, 2015) convert data to pseudodiploid-
dominant data before analysis. This is because of the

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 POLYPATEX: P A T E R N I T Y E X C L U S I O N I N A U T O P O L Y P L O I D S 695
ambiguity in determining allele frequencies when deal-
ing with polyploid allelic phenotype data (Jones et al.
2010), unless the data sets are treated as dominant
instead of codominant markers (e.g. Riday et al. 2013,
2015; Wang & Scribner 2014), as allele frequencies are the
foundation for all likelihood-based methods. Wang &
Scribner (2014) transform polyploid codominant geno-
types from microsatellite data to pseudodiploid-domi-
nant genotypes (presence/absence only) and
demonstrate how useful this conversion can be, when
coupled with the program COLONY (Jones & Wang 2002),
for determining parentage and sibship in polyploids.
However, this transformation results in the loss of valu-
able information, and with increasing polyploidy levels,
there is a decrease in parentage assignment and exclu-
sion accuracies (Wang & Scribner 2014).
POLYPATEX is an R (R Core Team 2015) package for con-
ducting paternity exclusion analysis in autopolyploid
monoecious, dioecious or bisexual species having ploidy
level p of 4n, 6n or 8n. Developed in the context of
microsatellite data, POLYPATEX can also be used for other
codominant markers having multiple alleles per locus,
for example allozymes. POLYPATEX is not optimized for
data sets having very large numbers of loci, such as SNP
data. For plants, self-compatible (i.e. mother included as
a candidate father) and self-incompatible breeding sys-
tems are options in the algorithm. POLYPATEX applies allele
matching at each locus to determine whether a candidate
fathers allele set (the (up to p) alleles observed at the
given locus) are compatible with the corresponding allele
sets in an offspringmother pair. POLYPATEX can analyse
marker data sets in which allele copy numbers are
known (genotype data) or unknown (allelic phenotype
data). For data sets in which allele copy numbers are
unknown, POLYPATEX considers all possible genotypes for
each locus arising from the observed allele sets in candi-
date father, offspring and mother, to determine whether
a match is possible. Per-locus comparisons are then sum-
marized across all loci, resulting in tables giving the
identification (ID) and total counts of nonexcluded can-
didate fathers (which we refer to as potential fathers) for
each offspring.
The advantage of the POLYPATEX algorithm over a sim-
pler presence-/absence-based algorithm is that all avail-
able information is utilized to exclude candidates. For
example, consider a tetraploid where the offspring has
the genotype ABCE and the mother ABCD, with one can-
didate father having the genotype EFGG, and another
CEGG. In this situation, the simpler presence/absence
algorithm would exclude neither candidate because both
candidates contain the allele not found in the mother
(E). However, using the POLYPATEX algorithm, the first
candidate would be excluded, because it could not have
contributed a full gamete (two alleles) to the offspring.
2015 John Wiley & Sons Ltd
This advantage also applies to similar phenotypic
situations (i.e. tetraploid offspring BC and mother AB;
candidate excluded with POLYPATEX and included with
presence/absence method CEFG; candidates not
excluded with either method CE or CFG). Therefore, the
genotype and gamete simulations implemented in POLY-
PATEX provide a robust basis for refining the level of
detail to which candidates can be excluded.
The genetic model
POLYPATEX assumes the following genetic model, which is
based on Mendelian rules of inheritance, to exclude can-
didates as potential fathers using incompatibilities
between parent and offspring pairs. In an autopolyploid
species of ploidy p, at a given locus,
p/2 of the p alleles in the offspring are assumed to have
been selected, without replacement, from the mothers
p alleles (the maternal gamete).
The remaining p/2 of alleles in the offspring are
assumed to have been selected, without replacement,
from the fathers p alleles (the paternal gamete).
The relationship between mother and offspring is
known, and the aim of the analysis was to determine
which of one or more candidate fathers is capable of
producing a paternal gamete compatible with the alle-
les in the offspringmother pair.
Comparisons are made on the basis of allele presence/
absence only POLYPATEX does not use population allele
frequencies to compute likelihoods for paternity.
To allow for the possibility of phenomena such as
double reduction that violate the above model (as well as
the inevitable genotyping errors), POLYPATEX functions
potentialFatherIDs and potential
FatherCounts include an argument
mismatches, that can be used to specify a maximum
number of mismatching (and nonmissing) loci between
candidate father and offspring that are allowed, before
the candidate is excluded as a potential father. The
default is to allow no mismatching loci in a nonexcluded
candidate.
Data input format, loading and preprocessing
POLYPATEX functions require data to be presented as a
table with one row per individual (mother, candidate
father or offspring). Initial columns contain individual
IDs, population identifier, ID of each offsprings mother
and for dioecious species, adult gender. For ploidy p and
k observed loci, k further blocks of p columns each con-
tain the allele labels. Cells in these k blocks should be left
blank as necessary when fewer than p alleles were
observed.
696 A . B . Z W A R T E T A L .
POLYPATEX provides the function inputData to
load the data from a comma-separated value (CSV) for-
matted file into R, storing the data set as an R data frame.
inputData passes this dataframe to function
preprocessData, which performs a number of
checks and preprocessing steps to help ensure the valid-
ity of the data set for analysis by other POLYPATEX func-
tions. In particular, preprocessData checks for
mismatches at each locus between mother and offspring.
These arise when the mothers allele set cannot (in the
absence of, say, a mutation) generate a gamete compati-
ble with the offsprings allele set for that locus. The user
may specify the maximum number of such mismatches
(0 to k 1) that are allowed before the offspring is
removed from the data set. When an offspring is not
removed, loci that mismatch with its mother are set to
contain no alleles (i.e. they are set to be missing) in the
offspring. The default is to remove offspring containing
any mismatches with their mothers. Motheroffspring
mismatch details are reported to the users so that they
can investigate these cases for genotyping errors and
check whether the problem may lie in a mothers allele
sets rather than in those of her offspring.
For genotype data in a species of ploidy p, POLYPATEX
requires every allele set to contain exactly p alleles, or
none. When preprocessData finds nonmissing
allele sets with fewer than p alleles in genotype data sets,
they are reported to the user, then are set to be missing.
In allelic phenotype data sets, there is no requirement to
observe exactly p unique alleles, so this adjustment is
only relevant to genotypic data.
After these and other checks, preprocessData
removes individuals from the data set having fewer than
a user-specified minimum number (lociMin) of non-
missing allele sets. When a mother is so removed, all of
her offspring are also removed from the data frame.
Paternity exclusion
Two functions are provided by POLYPATEX for performing
paternity exclusion, one for analysing genotype data
(genotPPE) and one for allelic phenotype data
(phenotPPE).
For comparison of genotypes, genotPPE partitions
each offspring allele set into alleles that also appear in
the mother, and alleles that do not (and hence must be
provided by the father). For a candidate not to be
excluded as a potential father, it must account for all of
the latter alleles, plus as many of the shared alleles as is
needed to make a complete gamete of p/2 alleles. Func-
tion genotPPE takes proper account of allele multi-
plicities in making these comparisons.
In the more common case of allelic phenotype data, a
nonmissing allele set at a given locus may consist of 1 to
p observed alleles. The locus genotype is known when p
alleles are observed, and when only one allele is
observed, the locus genotype can be inferred as compris-
ing p copies of that allele. But for 2 to p 1 observed alle-
les, the phenotype gives rise to more than one possible
genotype. In the allele-matching process, therefore, the
possible combinations of genotypes arising from off-
spring, mother and candidate phenotypes must be
searched for genotype combinations that allow a match,
before a candidate can be claimed as a possible match for
the offspring. For efficiency reasons, this search is imple-
mented in phenotPPE using lookup tables, for each
ploidy covered by POLYPATEX (4n, 6n and 8n at time of
publication).
In either routine, when one or more of the allele sets
in offspring, mother and candidate father are missing,
comparisons cannot be made at that locus, so the
affected locus is subsequently ignored for that trio of
individuals.
In the situation where more than one candidate father
is the potential donor for an offspring, the user needs to
consider the method best suited for their research ques-
tion and species when handling an exclusion analysis
that is unresolved. Some options include the following:
(i) considering the offspring derived from that group of
candidate fathers without a definitive level of exclusion;
(ii) fractionally assign males an equal proportion of an
offspring (Goto et al. 2004); (iii) choose the closest male
(plants or other sessile organisms only) (Hardesty et al.
2006); or (iv) fractionally assign to a male (sessile organ-
isms only) based on their location/distance from mother.
For a general review of methods of parentage analysis,
see Jones et al. (2010).
Exclusion analysis results summaries and
export
Results from either exclusion routine are returned in an R
list structure, whose contents are explained further in the
POLYPATEX documentation. Two further POLYPATEX func-
tions provide more convenient summarizations of the
per-locus exclusion results across loci. Function
potentialFatherCounts returns an R data
frame with columns containing offspring ID, correspond-
ing mother ID and the number of candidates flagged as
potential fathers for each offspring. Function
potentialFatherIDs provides a similar data
frame listing the IDs of these potential fathers. Both func-
tions include the argument mismatches, that allows
a maximum number of mismatching loci between candi-
date father and offspring before the candidate are
excluded as a potential father. Results from the two sum-
mary functions can be exported to file using R functions
such as write.csv.
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 POLYPATEX: P A T E R N I T Y E X C L U S I O N I N A U T O P O L Y P L O I D S 697

Table 1 The first twelve lines of the output table produced by FR_Genotype.csv contains data from seven loci
potentialFatherIDs. For each progeny, each potential in a tetraploid, dioecious species, Salix cinerea (Hopley
father identified by the algorithm is listed, along with the num- 2011). Appendix 1 shows example R code to load the alle-
ber of loci at which a match was made (FLCount Father Locus
lic phenotype data set into R, perform the exclusion anal-
Count), and the total number of loci at which a valid compar-
ison was possible (VLTotal Valid Loci Total). NA is Rs code ysis, and output results from the summary functions
for a missing datum, and appears in columns FLCount and potentialFatherCounts and potential
VLTotal when no potential father has been identified for a given FatherIDs to CSV files for scrutiny in a spreadsheet
offspring application. The code in Appendix 1 assumes that the
data file has been copied to a suitable working directory,
Progeny Mother PotentialFather FLCount VLTotal
and that the working directory of the R session has been
GF1-2310 GF1 None NA NA set to this directory prior to running the code.
GF1-2311 GF1 None NA NA The first 12 lines of the table produced by
GF1-2315 GF1 GF21 6 7 potentialFatherIDs are shown in Table 1.
GF1-2315 GF1 GF6 7 7
GF1-2316 GF1 GF14 7 7
GF1-2316 GF1 GF23 6 7 Performance testing and simulations
GF1-2317 GF1 GF21 6 6
We compared the performance of POLYPATEX (i.e. exclu-
GF1-2317 GF1 GF6 6 6
GF3-2337 GF3 GF2 7 7 sion based) against COLONY (i.e. likelihood based) using
GF3-2338 GF3 GF14 7 7 the phenotypic, autohexaploid example data set of
GF3-2339 GF3 None NA NA E. glabra. We converted the data to pseudodiploid-domi-
GF3-2341 GF3 GF13 7 7 nant genotypes following Wang & Scribner (2014) and
analysed it in COLONY (version 2.0.5.9; Jones & Wang
2002) using several different parameter settings. The type
of analysis method was full-likelihood with polyga-
Example
mous, inbreeding and monoecious as the core param-
The POLYPATEX R package contains two example eters. The data set contained 95 loci, and we defined the
microsatellite data sets in the required input file format. known maternal sibships. POLYPATEX was run as per
Once POLYPATEX is installed, the following R command Appendix 1, except that the number of mismatches
will print out the location of the files: allowed in the father was set to zero.
> system.file(extdata, package = We found that both programs produced similar
PolyPatEx) results for paternity assignments of the majority of the
File GF_Phenotype.csv contains data offspring tested (Table 2). As expected, POLYPATEX was
from seven loci in a hexaploid, monoecious species, more accurate in certain situations (described above),
Eremophila glabra ssp. glabra (Elliott 2010). File whereas COLONY inferred father candidates that could not

Table 2 The number of offspring from the Eremophila glabra example data set (n = 40) that fall into the following paternity assignment
groups: (i) selfing, maternal plant included in the paternity list; (ii) single father, only one father listed; (iii) multiple fathers, more than
one candidate available to sire the offspring; and (iv) no fathers, no candidate fathers assigned from the list of potential fathers, based
on the outcome of POLYPATEX (PPE) and COLONY analysis. COLONY was run at four probabilities that the father was included in the candida-
ture file (pr = 0.20, pr = 0.50, pr = 0.80 and pr = 0.95). The results of COLONY are split into individuals that had the same outcome as PPE
and those that were different to the PPE outcome within each probability class. The ranges in likelihoods from COLONY for each group
(parent pair or maternal only) are in parenthesis (pr = 1.00 if none presented)

Male Outcome compared multiple

Program probability to PPE Selfing Single father fathers No fathers

POLYPATEX 1 6 7 26
COLONY 0.20 Same 1 3 (0.991.00) 4* 23 (0.991.00)
Different 4 5
0.50 Same 1 3 (0.991.00) 4* 23 (0.911.00)
Different 4 5
0.80 Same 1 3 (0.991.00) 4* 23 (0.641.00)
Different 4 (0.991.00) 5 (0.961.00)
0.95 Same 1 4 (0.571.00) 6* 21 (0.551.00)
Different 1 5 (0.861.00) 2 (0.901.00)

*COLONY listed only most likely father (pr = 1.00), but it was one of the fathers listed in PPE.

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698 A . B . Z W A R T E T A L .

Fig. 1 Simulation results showing the

effect of varying the number of loci
observed on the levels of exclusion of can-
didate fathers for allelic phenotype (solid
line) and genotype (dashed lines) data
sets. Tetraploid (4n) data set presented on
the left and hexaploid (6n) data set pre-
sented on the right. The number of possi-
ble alleles at each locus was fixed at five
for these simulations.

Fig. 2 Simulation results showing the

effect of varying the numbers of alleles
per locus on the levels of exclusion of can-
didate fathers, for allelic phenotype (solid
line) and genotype (dashed lines) data
sets. Tetraploid (4n) data set presented on
the left and hexaploid (6n) data set pre-
sented on the right. The number of loci
observed was fixed at five for these simu-
lations.

be candidates based on the original autohexaploid phe- inclusive when there were multiple candidate fathers
notype or did not infer a potential paternal relationship available for an offspring, despite the user needing to
when POLYPATEX did. In addition, POLYPATEX was more decide how to handle these unresolved exclusions. In

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 POLYPATEX: P A T E R N I T Y E X C L U S I O N I N A U T O P O L Y P L O I D S 699
this situation, COLONY provides a likelihood measure to
infer the most likely father, but the majority of these
were listed as a single parent pair (pr > 0.99) and there
was no indication of the occurrence of alternate fathers
with lower likelihood measures. The differences between
the two programs arise because of (i) the transformation
of codominant polyploid data to pseudodiploid-domi-
nant data required by COLONY, (ii) the probabilistic infer-
ence of relationships (parentage or sibling) by COLONY on
this type of data, and (iii) the full compatibility of pheno-
types or genotypes between offspring and parent by
POLYPATEX.
We conducted a set of simulations to examine the
effect of loci number and number of alleles per locus on
the generation of accurate paternity exclusion. As noted
by Wang & Scribner (2014), many combinations of
parameters can be explored in such simulation studies
and it is impractical to consider all of them. Drawing on
Wang & Scribner (2014), we simulated allele data sets for
a dioecious species, containing 5 mothers, 10 progeny
per mother and 30 candidate fathers. Alleles at each
locus in mothers and candidate fathers were simulated
following a triangular distribution, where the probability
of occurrence at a locus of allele i is pi = i/(k(k + 1)/2), k
being the total number of alleles that can occur at the
locus (and was assumed the same for all loci). Genotypic
allele data for each offspring were generated by mating a
randomly chosen candidate father with the relevant
mother, according to the Mendelian genetic model
described above. Allelic phenotype data were formed by
reducing the data to unique alleles only, using the POLY-
PATEX function convertToPhenot. For each value
of the parameter being investigated (number of loci, or
number of alleles per locus), 100 simulated data sets
were analysed by POLYPATEX, and summary statistics on
the number of candidate fathers detected were obtained.
In the absence of genotyping errors or violations of
the assumed genetic model, POLYPATEX does not exclude
the true father of the offspring. By its nature, allelic phe-
notype data contain less information than genotype data;
hence, a greater number of loci (Fig. 1) or more polymor-
phic loci (Fig. 2) must be used to exclude all candidates
except the true father. In turn, higher ploidy levels (e.g.
6n) required a greater number of loci (Fig. 1), and to a
lesser extent polymorphic loci (Fig. 2), to exclude all but
the true father in comparison with lower ploidy levels
(e.g. 4n). This is also due to the greater amount of genetic
information required for higher ploidy levels compared
to lower ploidy levels to isolate the true father. Interest-
ingly, these simulation curves descend to the true father
(i.e. 1) more rapidly in Fig. 2 than Fig. 1, suggesting a
greater power of exclusion can be obtained from observ-
ing more informative polymorphic loci, rather than sim-
ply a greater number of loci.
2015 John Wiley & Sons Ltd
Obtaining POLYPATEX
POLYPATEX is available from CRAN, the Comprehensive R
Archive Network (https://cran.r-project.org/) or one of
its regional mirrors. Assuming connection to the internet,
POLYPATEX and its dependency, package GTOOLS (Warnes
et al. 2015), can be installed by entering the following in
the R console (> indicates the R console prompt and
should not be typed):
> install.packages(PolyPatEx)
POLYPATEX comes with a detailed user manual, which
can be accessed from the R HTML help system, or via the
following R command:
> vignette(PolyPatEx-vignette,
package=PolyPatEx)
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Appendix 1
guide to using and evaluating microsatellite markers. Ecology Letters, 9,
615629. Example R code for paternity analysis of a hexaploid,
Spielmann A, Harris SA, Boshier DH, Vinson CC (2015) ORCHARD:
paternity program for autotetraploid species. Molecular Ecology
monoecious species. (> denotes the R command prompt,
Resources, 15, 915920. + is Rs prompt indicating continuation of a command).
Thrall PH, Young A (2000) AUTOTET: a program for analysis of autote- Arguments to potentialFatherCounts and
traploid genotypic data. Journal of Heredity, 91, 348349.
potentialFatherIDs specify that a comparison
Wang J, Scribner KT (2014) Parentage and sibship inference from markers
in polyploids. Molecular Ecology Resources, 14, 541553. must involve at least two nonmissing loci in mother, off-
Warnes GR, Bolker B, Lumley B (2015) gtools: Various R Programming spring and candidate father for it to be included in the
Tools. R package version 3.5.0. http://CRAN.R-project.org/packa- summary (VLTMin = 2), and that at most one
ge=gtools.
mismatching locus is allowed in a candidate that is
Wood TE, Takebayashi N, Barker MS, Mayrose I, Greenspoon PB, Riese-
berg LH (2009) The frequency of polyploid speciation in vascular flagged as a potential father (mismatches = 1). The
plants. Proceedings of the National Academy of sciences, 106, 1387513879. latter argument provides some allowance for genotyping
errors or mutations in the data set. In this example,
rather than storing the output from potential
A.Y. suggested the concept. A.Z. developed the R pack- FatherCounts and potentialFatherIDs
age. C.E. and T.H. contributed data and aided in testing as R objects, R function write.csv is used to immedi-
and suggesting improvements to the package and docu- ately export these tables as CSV files in the current work-
mentation. D.L. developed theory used in further test- ing directory.
ing/confirmation of the validity of the package. All
authors contributed to the discussions around the devel- > require(PolyPatEx)
opment of the problem and the code. > adata <- inputData(GF_Phenotype.
csv,
+ numLoci=7,
+ ploidy=6,
Data Accessibility + dataType=phenotype,
+ dioecious=FALSE,
The POLYPATEX R package (which includes documentation
+ selfCompatible=TRUE)
and example data sets) is available from the Comprehen-
> pe1 < - phenotPPE(adata)
sive R Archive Network, (https://cran.r-project.org/web/
> write.csv(potentialFatherCounts
packages/PolyPatEx) or one of its regional mirrors. The
(pe1,mismatches=1,VLTMin=2),
example data sets FR_Genotype.csv and
+ potentialFatherCounts.csv)
GF_Phenotype.csv are included with the pack-
> write.csv(potentialFatherIDs(pe1,
age, and are also archived at the Dryad Digital Repository
mismatches=1,VLTMin=2),
(http://datadryad.org/), via doi:10.5061/dryad.64482.
+ potentialFatherIDs.csv)
Also available via this DOI are an R script to implement

2015 John Wiley & Sons Ltd