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2019. Journal of Arachnology 47:190–201
Cost effective microsatellite isolation and genotyping by high throughput sequencing
Henrik Krehenwinkel
1,2,3
,Susanne Meese
4,*
,Christoph Mayer
5
,Jasmin Ruch
6
,Jutta Schneider
6
,Trine Bilde
7
,Sven K ¨
unzel
8
,
James B. Henderson
3
,Joseph Russack
3
,Warren Brian Simison
3
,Rosemary Gillespie
2
and Gabriele Uhl
4
:
1
Department
of Biogeography, University of Trier, Trier, Germany; E-mail: krehenwinkel@uni-trier.de;
2
Department of
Environmental Science, Policy and Management, University of California, Berkeley, USA;
3
Center for Comparative
Genomics, California Academy of Sciences, San Francisco, California, USA;
4
General and Systematic Zoology,
Zoological Institute and Museum, University of Greifswald, Germany;
5
Zoologisches Forschungsmuseum Alexander
Koenig, Bonn, Germany;
6
Institute of Zoology, Behavioural Biology, Universit¨
at Hamburg, Germany;
7
Department of
Bioscience, Aarhus University, Denmark;
8
Max Planck Institute for Evolutionary Biology, Pl ¨
on, Germany
Abstract. High throughput sequencing (HTS) has emerged as a valuable tool for the rapid isolation of genetic markers
for population genetics and pedigree analysis. HTS-based SNP (single nucleotide polymorphism) genotyping protocols like
RAD (Restriction-site associated DNA) sequencing or hybrid capture allow for the isolation of thousands of markers from
any non-model organism. However, these protocols are relatively laborious and expensive and the resulting high marker
density is not always necessary. Since HTS technology has also greatly simplified the process of microsatellite marker
isolation and genotyping, we develop microsatellite markers as a cost-efficient and simple alternative to SNP genotyping.
We present low coverage genome sequencing data from seven distantly related spider species (Argiope bruennichi (Scopoli,
1772), Larinia jeskovi Marusik, 1987, Oedothorax retusus (Westring, 1851), Pisaura mirabilis (Clerck, 1757),
Australomisidia ergandros (Evans, 1995), Cheiracanthium punctorium (Villers, 1789), Theridion grallator Simon,1900)
and show the utility of HTS for microsatellite isolation. We also present a simple Illumina amplicon sequencing protocol to
genotype microsatellites from multiplex PCR amplicons in the Hawaiian happy face spider T. grallator. We discuss
advantages and drawbacks of the use of microsatellites for a range of research questions, and highlight an unexpectedly
fast decay and gain of repeat loci for T. grallator.
Keywords: Genome, paternity assessment, population genetics, amplicon sequencing
High throughput sequencing is currently revolutionizing
molecular ecology and systematics. Thousands of markers can
be isolated from any non-model organism with protocols like
RAD (Restriction-site associated DNA) sequencing (Peterson
et al. 2012) or sequence capture (Smith et al. 2013; Mayer et al.
2016) and whole genome and transcriptome sequencing are
now feasible (Ekblom & Galindo 2011; Ellegren 2014). The
resulting marker density has greatly contributed to an in-depth
understanding of evolutionary and ecological processes,
including in the field of arachnology (Brewer et al. 2014).
Several spider genomes have recently been sequenced (Sang-
gaard et al. 2014; Babb et al. 2017; Schwager et al. 2017), the
spider tree of life has been tackled using high-density markers
(Bond et al. 2014; Ferna
´ndez et al. 2014, 2018) and genomic
and transcriptomic analyses have provided insights into
evolutionary divergence in spiders (Croucher et al. 2013;
Bechsgaard et al. 2015; Krehenwinkel et al. 2015; Settepani et
al. 2017). The recent isolation of Ultra Conserved Elements
(Starrett et al. 2017) and application of ddRAD (double
digest) sequencing protocols (Burns et al. 2017; Settepani et al.
2017) have additionally contributed a wealth of genetic
markers for spider research. However, RAD sequencing
(Burns et al. 2017) or sequence capture (Smith et al. 2013;
Cotoras et al. 2018) protocols are relatively laborious and
expensive and the analysis of such high throughput genotyping
data requires considerable computational resources. Depend-
ing on the question of research, a density of thousands of
SNPs may not always be necessary, and thus a simpler and
more cost-efficient approach is desirable for some types of
studies. In this regard, microsatellites are noteworthy: They
are repeated short sequences of DNA that evolve rapidly and
hence are powerful markers for analyzing mating rates and
paternity success, for population and conservation genetics, as
well as pedigree analyses and recent evolutionary divergence
(e.g., Sch¨
afer et al. 2008; Tuni et al. 2012; Krehenwinkel &
Tautz 2013; Zimmer et al. 2014; Krehenwinkel et al. 2016b).
While their isolation used to be laborious, microsatellites are
now routinely isolated from any species by shotgun sequenc-
ing of genomic DNA (Castoe et al. 2010; Malausa et al. 2011).
Thus, while little used for arachnids in the past (Brewer et al.
2014), microsatellites are becoming available for an increasing
number of spider species (R ¨
utten et al. 2001; Bilde et al. 2009;
da Silveira & Bonatto 2009; Hataway et al. 2011; Esquivel-
Bobadilla et al. 2013; Parmakelis et al. 2013; Planas et al.
2014). High throughput sequencing has not only simplified the
isolation of microsatellite markers but is also well-suited for
genotyping of microsatellites (Cao et al. 2014; Darby et al.
2016). In particular, Illumina amplicon sequencing provides a
rapid, accurate and very cost-efficient alternative to the
laborious and expensive capillary electrophoresis protocols,
which were commonly used for microsatellite genotyping (Cao
et al. 2014; Darby et al. 2016). Large-scale amplicon
sequencing of multiplex PCRs can be routinely performed
for hundreds of samples in parallel (Fadrosh et al. 2014) and
microsatellite alleles can be directly called from amplicon
sequencing data by analyzing the read length distribution per
specimen. Several software solutions for microsatellite geno-
typing from Illumina amplicon sequencing data have been
published or are under development (Suez et al. 2016; Zhan et
* current address: Department of Environmental Systems Science,
ETH Zurich, Switzerland
190
al. 2017; Henderson, Russack, Krehenwinkel & Simison
unpublished data). Against this background, it is worthwhile
to consider the utility of microsatellite markers as a simple and
cost-efficient alternative to high throughput SNP genotyping.
Here, we provide an assessment of the isolation and
genotyping processes of microsatellite markers. Our aims are
twofold. We first analyze the feasibility of high throughput
sequencing for microsatellite marker isolation. For this, we
present low coverage genome sequencing data for seven
distantly related spider species from six families. These species
are important models in different fields of arachnology, from
behavioral ecology, to phylogeography and population
genetics. We identify tandem repeat contents in the analyzed
spider genomes, isolate markers from the genomic data and
present structural similarities of the repeat content between
genomes. Based on these results, we discuss the necessary
sequencing depth for microsatellite marker discovery in
spiders. We then provide a set of established markers for the
studied species. Secondly, we present an overview of the
workflow for microsatellite genotyping using Illumina ampli-
con sequencing, from PCR set up, to library preparation and
sequencing, and genotyping from raw read data, using the
Hawaiian happy face spider Theridion grallator Simon, 1900.
The phylogeography of the happy face spider has been studied
using mitochondrial sequence information and allozyme data
(Croucher et al. 2012); thus, by applying the approach to T.
grallator, we can compare directly the effectiveness of
microsatellite markers for recovering genetic differentiation
among populations. The well-understood genetic history of
the species also offers the potential to investigate the evolution
of repeat loci over time, e.g., decay or gain of microsatellites
between different populations. Based on our results, we
discuss promises and drawbacks of Illumina based microsat-
ellite analyses and highlight unique features of tandem repeat
evolution in spiders. Our results demonstrate the feasibility of
high throughput sequencing based microsatellite isolation and
genotyping, and may help in establishing microsatellites as a
more commonly used marker type for genetic studies on
spiders.
METHODS
Target species.—We targeted seven distantly related spider
species from six families to assess the utility of high
throughput sequencing for microsatellite marker isolation.
Argiope bruennichi (Scopoli, 1772) (Araneidae) serves as a
model species in research on the evolution of sexual
cannibalism and its consequences for mating systems (Fromh-
age et al. 2003; Schneider & Andrade 2011; Schneider 2014;
Schneider et al. 2015; Uhl et al. 2015) and has recently gained
importance as a model for evolutionary divergence during
contemporary range expansions (Krehenwinkel & Tautz 2013;
Krehenwinkel et al. 2015, 2016a). Larinia jeskovi Marusik,
1987 (Araneidae) is remarkable due to its mating behavior,
since males mutilate the female genitalia to prevent further
insemination by rival males (Mouginot et al. 2015, 2017).
Oedothorax retusus (Westring, 1851) (Linyphiidae) has re-
ceived considerable attention due to its mating strategy, in
which males perform gustatory courtship and plug the
female’s genital opening with secretion (Kunz et al. 2012,
2014). Pisaura mirabilis (Clerck, 1757) (Pisauridae) is known
as the nuptial gift spider, in which males provide females with
a prey item prior to mating that results in sexual conflict (Bilde
et al. 2007; Albo et al. 2013; Ghislandi et al. 2018).
Cheiracanthium punctorium (Villers, 1789) (Cheiracanthiidae)
is known for being the only medically important spider in
Central Europe and is currently rapidly expanding its range
(Muster et al. 2008; Krehenwinkel et al. 2016b). The thomisid
species Australomisidia ergandros (Evans, 1995) shows com-
munal hunting behavior (Ruch et al. 2014, 2015; Dumke et al.
2016) and brood care by which the female provides herself to
her offspring as food (Evans 1998). The happy face spider,
Theridion grallator Simon, 1900 (Theridiidae), is widely-
distributed across the rainforests of the Hawaiian Islands of
Oahu, Molokai, Maui, and Big Island, and is well known for
its conspicuous and eponymous color morphs that occur as a
balanced polymorphism in every population (Gillespie &
Oxford 1998). The diverse research questions ranging from
paternity and relatedness to population structure require fast
evolving genetic markers.
Microsatellite isolation by high throughput sequencing.—
Genomic DNA from six spider species was extracted from leg
muscle tissue using the Qiagen DNeasy blood and tissue kit
according to the manufacturer’s protocol (Qiagen, Hilden,
Germany). The taxonomy and origin of samples are shown in
Table 1. The untreated genomic DNA of five species was
sequenced, each on1/8th flow cell of a 454 GS FLXþflow cell
in 2010 and 2011. Library preparation and sequencing were
performed according to the manufacturer’s protocols (Roche,
Basel, Switzerland; see Krehenwinkel & Tautz (2013) for more
details on the 454 sequencing and analyses). An additional
data set of the yellow sac spider Cheiracanthium punctorium
was generated by sequencing pooled genomic DNA on two
full flow cells of an Illumina Miseq in 2014 (Illumina, San
Diego, CA, USA). Library preparation and sequencing were
performed using the V3 chemistry according to the manufac-
turer’s protocols and sequencing 300 bp paired end reads. The
454 and Illumina sequencing runs were both performed at the
Max Planck Institute for Evolutionary Biology in Pl¨
on,
Germany. The paired end Illumina reads were quality trimmed
using PoPoolation (Kofler et al. 2011) with a minimum quality
threshold of 20 and adapters removed using Trimmomatic
(Bolger et al. 2014). A de novo assembly was generated
including both Illumina libraries and using CLC genomic
workbench at a minimum contig length of 1000 and including
a scaffolding step (CLC Bio, Boston, USA). For the happy
face spider Theridion grallator, we used a subset of ~20 Mb of
contigs from a preliminary genome assembly, based on
Illumina paired end sequencing data (Croucher, unpublished
data). We estimated the GC content of reads and assemblies
using UNIX.
We identified tandem repeats in the Illumina assemblies of
C. punctorium and T. grallator and the raw 454 reads of the
remaining spiders using MSat Commander (Faircloth 2008)
with a minimum length of 10 repeats for mononucleotide
repeats, 10 repeats for di-, 6 repeats for tri-, 5 repeats for
tetra-, 4 repeats for penta- and hexanucleotide repeats. We
counted the number of repeat loci for each repeat class and
calculated the genome wide content of different repeat motifs
per megabase of sequence data (Table 1, 2). In order to
identify the tandem repeat content, we designed primers for
KREHENWINKEL ET AL.—MICROSATELLITES AND GENOTYPING FOR SPIDERS 191
all possible di, tri and tetranucleotide repeat loci using the
Primer3 plugin (Rozen & Skaletsky 1999) of MSat Com-
mander. A subset of the microsatellite loci was tested for
variability using fluorescently labeled primers and following
genotyping as described in Krehenwinkel & Tautz (2013).
Amplicon sequencing for microsatellite genotyping.—We
used several Hawaiian populations of the happy face spider
Theridion grallator to explore the utility of Illumina amplicon
sequencing for microsatellite genotyping. Specimens of T.
grallator were collected in March 2015 on the Hawaiian
Islands of Oahu, Maui, Molokai and Hawai’i by beating
vegetation and hand collecting from the underside of leaves.
Two separate subpopulations per island were sampled (see
Table 3). All specimens were stored in 99%ethanol and
brought back to the University of California Berkeley for
further analyses. DNA extractions were performed on the
whole prosoma of each specimen using the Qiagen Puregene
Tissue kit according to the manufacturer’s protocol (Qiagen,
Valencia, USA). The DNA from 96 specimens was quantified
using a Qubit Fluorometer (Thermo Scientific, Waltham,
USA), diluted to approximately 20 ng/ll and distributed
among wells of a 96-well plate. Fifty primer pairs were tested
in a subset of three specimens from each island for
determining PCR amplification efficiency. The targeted loci
contained di-, tri- or tetranucleotide repeats and the targeted
amplicons were less than 400 bp long, to achieve a good
overlap during read merging. Each primer pair was tested in
an annealing temperature gradient from 50–60 8C in incre-
ments of 2.5 8C. Gradient PCR were run for each primer pair
using the Qiagen Multiplex PCR kit according to the
manufacturer’s protocol and with 30 cycles. No Q-solution
was added. PCR products were screened on a 1.5 %agarose
gel. The 25 primer pairs which most consistently amplified
specimens from all four islands were chosen for further
analyses (see Supplementary Table 1, online at http://dx.doi.
org/10.1636/JoA-S-16-017.s1 for details on primer pairs and
repeat loci). This selection was done to avoid priming bias and
drop out of loci by sequence divergence between populations.
PCR reactions with primers of similar optimal annealing
temperature were then combined to multiplex reactions. We
added a 50tail to each primer and amplified the 25 primer
pairs in six multiplex PCRs of 30 cycles for all of the 96
specimens and otherwise as described above. PCRs were run in
10 ll volumes, with 0.5 ll of each 10 lM primer and 1 ll of the
20 ng/ll template. The added 50tails served as a priming site
for a second PCR round of 5 cycles, in which dual indices
(short, unique sequences for individual identification) and
Illumina Truseq adapters were introduced. The concept of
PCR-based library preparation followed that described in
Lange et al. (2014). Before the second PCR, all multiplexes
were pooled into a single 96-well plate according to specimen
and placed for an approximate quantification on a 1.5 %
agarose gel. A second PCR round was run with 8 forward
times 12 reverse combinations of indexed primers. This
indexing PCR was run as described above, but with only 5
cycles at 55 8C annealing temperature, 0.25 ll of each 10 lM
Table 1.—The upper table shows the taxonomy and sampling origin of the sequenced spider species, sequencing platform used, number of
sequenced bases, number of sequenced reads and estimates for the genome-wide GC-content for the six species. The lower table presents the
assembly statistics for the two assemblies used for marker isolation.
Family Genus Species Origin Platform Sequenced bases No. reads GC-content (%)
Araneidae Argiope bruennichi Germany 454 30.29*10
6
80,031 29
Araneidae Larinia jeskovi Poland 454 30.39*10
6
101,616 31
Linyphiidae Oedothorax retusus Germany 454 24.79*10
6
86,424 34
Pisauridae Pisaura mirabilis Germany 454 23.12*10
6
81,970 34
Theridiidae Theridion grallator Hawaii -- - 27
Thomisidae Australomisidia ergandros Australia 454 24.18*10
6
84,722 33
Cheiracanthiidae Cheiracanthium punctorium Baltic States MiSeq 18.78*10
9
19,673,693 34
Cheiracanthiidae Cheiracanthium punctorium Mediterranean MiSeq 12.37*10
9
14,150,027 35
Family Genus Species Origin Platform Assembly size
Median
contig size (bp) GC-content (%)
Cheiracanthiidae Cheiracanthium punctorium Mediterranean MiSeq 148.60*10
5
1024 32
Theridiidae Theridion grallator Hawaii HiSeq 22.00*10
6
6290 27
Table 2.—Coverage of different tandem repeat motifs (TR) per megabase (Mb) of sequenced DNA for all spider species studied. The last two
columns show the number of primer pairs which could be designed on the recovered tandem repeats per Mb of DNA and the %of recovered
repeat motifs on which primers could be designed.
Species TR motifs/Mb Mono/Mb Di/Mb Tri/Mb Tetra/Mb Penta/Mb Hexa/Mb Primers/Mb %motifs with primers
A. bruennichi 340.84 293.89 35.55 7.13 2.48 1.39 0.45 3.93 8.36
L. jeskovi 103.30 78.79 17.67 3.16 1.48 2.01 0.07 1.88 7.71
O. retusus 118.20 102.3 3.15 4.36 5.28 2.22 0.89 2.74 17.23
P. mirabilis 225.90 113.74 94.27 5.02 9.09 3.37 0.43 9.13 8.14
A. ergandros 248.30 200.18 25.93 6.86 13.44 1.61 0.33 8.15 16.92
C. punctorium 464.78 434.22 10.91 9.65 8.08 1.83 0.11 19.18 62.72
T. grallator 218.19 203.22 13.02 0.6 0.75 0.55 0.05 2.5 16.70
192 JOURNAL OF ARACHNOLOGY
primer and 0.5 ll of the PCR template from the first PCR
round. After each PCR, the products were purified from
remaining primer using AMpure XP beads (Beckman Coulter,
Brea, USA). The purified and dual indexed PCR products
were quantified using a Qubit, and then 10 ng of each sample
were pooled. The sample was sequenced on approximately 1/4
of a MiSeq flow cell using the Illumina V3 chemistry, with 300
bp paired reads and according to the manufacturer’s protocol
(Illumina, San Diego, USA). The remainder of the flow cell
contained microbial 16S and arthropod mitochondrial COI
samples. A ‘‘spike-in’’ of 15 %PhiX was added to the run.
Adapter sequences were trimmed from the raw reads using
Trimmomatic (Bolger et al. 2014). Paired reads were then
merged using PEAR (Zhang et al. 2014) with a minimum
overlap of 75 bp and a minimum quality of 30. Only those
assembled reads with at least 90 %of bases with Q30 or higher
were transformed into fasta files using the fastx toolkit
(Gordon & Hannon 2010). All sequences, starting with the
forward primer and ending with the reverse primer of each
specimen and each of the 25 microsatellite loci were filtered
and saved to new files. This step was performed using UNIX
and served to demultiplex all separate loci. For allele-calling,
we measured the length of each amplicon and counted the
abundance of each length per specimen and per locus using the
programming language awk. By the previous filtering of
sequences, which start and end exactly at the PCR primers, all
fragments can be measured in exact relation to each other. We
plotted the distribution of different fragment lengths for each
specimen and locus using a custom-made software (Hender-
son, Russack, Krehenwinkel & Simison unpublished data).
The approach is very comparable to that implemented in
classic fragment length analysis software for dye-labelled
allele-calls, e.g., Genemapper (Thermo Scientific, Waltham,
USA). However, here we plotted the abundance of reads for
different fragment lengths instead of the intensity of dye
fluorescence (Fig. 1 A, B). The final version of our simple and
user-friendly software solution for allele-calling will perform
all tasks from sequence adapter trimming and assembly, to
demultiplexing, allele-calling and exporting the called alleles in
a user-friendly graphical interface (Henderson, Russack,
Krehenwinkel & Simison unpublished data). Alleles were
called for each specimen and locus and the fragment size
distributions for each locus were manually inspected to edit
allele calls. Due to locus and population specific stutter
patterns and the possibility of flanking indels contributing to
repeat length, this manual curation step proved essential. We
used a minimum coverage of 40 reads per locus and specimen
to call alleles. We additionally inspected a random subset of
unassembled reads for their microsatellite pattern and
compared the result with that identified based on assembled
reads pairs. This step served to exclude assembly-based
artifacts. MEGA (Tamura et al. 2013) was used to visualize
repeat containing sequences and to align subsamples of
sequences. We visually inspected the sequences of every
specimen for the presence of repeat motifs, indels outside of
repeat motifs and flanking SNPs.
The genetic structure of T. grallator across the Hawaiian
Islands was evaluated by a STRUCTURE (Pritchard et al.
2000; Falush et al. 2003) analysis. STRUCTURE was run with
an admixture model, k-values from 1–10, 10 replicates per k-
value, 150,000 MCMC generations of which 50,000 were
removed as burnin. The optimal number of clusters was
identified using STRUCTURE HARVESTER (Earl et al.
2012) with the method described in Evanno et al. (2005).
Pairwise F
ST
values between populations were calculated in
Genepop (Raymond & Rousset 1995).
RESULTS
Microsatellite isolation by high throughput sequencing.—The
results of sequencing and assembly can be found in Table 1.
The 454 runs yielded about 100,000 reads and 30,000,000 bp
per species, the MiSeq runs between 15–20 million reads. The
assembly resulted in 148,602 contigs over 1 kb long, with a
median size of 1,024 and a total size of ~232 million bases
(Table 1). All spider genomes were characterized by a
relatively low GC content between 27–34 %. Depending on
the species, we recovered between 103–465 microsatellites per
megabase (Mb) of sequenced DNA (Table 2). The repeat
content in spider genomes was highly biased towards
mononucleotides and was variable between species. As
mononucleotide repeats are hard to genotype, we did not
design primers for this repeat type. Excluding mononucleo-
tides, only between 15–112 microsatellites could be recovered
per Mb. Due to the high AT content of spider genomes
(Sanggaard et al. 2014; Krehenwinkel et al. 2015), AT rich
repeats dominate in the isolated loci (Supplementary Table 1).
A relatively small subset of the identified repeat loci
contained sufficient flanking sequences to design primers.
Between 2–19 microsatellite markers could be recovered per
sequenced Mb of DNA. This corresponds to between 8–62 %
of the total number of recovered repeat loci. Moreover, a
considerable proportion of designed primer pairs had to be
dropped after initial analyses. On average, we could establish
Table 3.—Collection sites and sample numbers per population for the eight sampled populations of Theridion grallator on the Hawaiian
Islands.
Island Population Locality N H
obs
H
exp
Big Island HiBiK15 Saddle Road, Kipuka15 12 0.277 0.351
Big Island HiBiM21 Saddle Road, Milemarker 21 8 0.265 0.388
Maui HiMaWA Waikamaoi, outside preserve 13 0.485 0.676
Maui HiMaWB Waikamoi, upper preserve 7 0.424 0.678
Molokai HiMoKA Kamakou, TNC cabin 17 0.324 0.508
Molokai HiMoKB Kamakou, boardwalk 14 0.318 0.463
Oahu HiOhPA Pahole 10 0.369 0.485
Oahu HiOhPB Pahole 10 0.332 0.474
KREHENWINKEL ET AL.—MICROSATELLITES AND GENOTYPING FOR SPIDERS 193
11 highly variable microsatellite markers out of 50 primer
pairs, which we designed per species and tested in PCR assays.
This low yield was due to two problems. First, some markers
showed highly population specific amplification biases, leading
to dropouts if genetically distant populations were analyzed.
This problem was most evident in the analyses of divergent
Eastern and Western Palearctic populations of A. bruennichi
(Krehenwinkel et al. 2016a). Second, many repeat loci lacked
size variation in some spider species, particularly O. retusus
and A. ergandros. We provide a list of established primer
sequences, examples for their application and amplification
bias or repeat variation issues in Supplementary Table 1.
Amplicon sequencing for microsatellite genotyping.—After
trimming and assembly, we recovered 26,425 high-quality
sequences per T. grallator specimen on average (68,787
standard deviation). Four specimens had to be removed from
the analysis due to low sequence coverage (12–16 sequences
only). We found 1,001 reads on average (61,488 standard
deviation) per microsatellite locus and specimen. One locus
was removed from the analysis because coverage was too low
(16 sequences per specimen on average). Our final dataset
consisted of 92 specimens and 24 loci. Many recovered
microsatellite loci showed variable repeat motifs within and
among populations (Fig. 1A, B).
A STRUCTURE analysis of the microsatellite dataset
supported k ¼4 populations (Fig. 2), each of them
corresponding to one of the Hawaiian Islands and in line
with recent research by Croucher et al. (2012). An F
ST
analysis
corroborated these results (Table 4). While populations within
islands showed little to no differentiation, we found consid-
erable divergence between islands.
Alignments of the microsatellite amplicons allowed more
detailed insights into the distribution of repeat patterns,
flanking indels and SNPs in different populations of T.
grallator, and in comparison to the population from Maui, for
which the loci were designed (Fig. 3). Repeat sequences were
often imperfect, e.g., containing additional bases which break
the repeat pattern (70 %of loci; Fig. 3). One of the amplified
loci did not contain any tandem repeat, despite being
identified as a dinucleotide repeat in Msatcommander. Many
microsatellites appeared to be population-specific and were
absent in other populations. Specimens from Maui, which
were also used as template to design microsatellites, carried
repeat motifs for 23 out of 24 analyzed loci. In most other
populations, we found an absence of many repeat loci, with
about 1/3 of loci not showing the repeat in comparison to
Maui (Fig. 4, Table 5). Overall, 13 out of 24 loci showed a
missing repeat in at least one population. The decay of repeats
was often part of the standing variation, with some specimens
in a population carrying repeats and others not. Most of the
loci, for which repeats were absent in other populations,
carried the loss of the repeat pattern in the standing variation
Figure 1.—(A) Subsection of genotyping plots for the allelic distribution of a CT-dinucleotide repeat locus (TG_MS41) for two happy face
spider specimens from Maui. The plots show the abundance (Y-axis) of reads of different fragment length (X-axis). Each bar indicates the
abundance of one fragment length in a mixture of sequences, with the red dot indicating the called allele length. The upper specimen is
heterozygous and the lower is homozygous.
194 JOURNAL OF ARACHNOLOGY
on Maui. Across the phylogeny of the happy face spider
(Croucher et al. 2012), our data suggest considerable gains and
losses of repeat loci within about 1 million years of inter-island
colonization of the species (Table 5, Fig. 4).
Apart from losses of repeats, additional factors contributed
to size differences in PCR amplicons (Fig. 3, Table 5). About
25 %of the analyzed loci carried a second tandem repeat
motif, often right next to the targeted one. Moreover, indels
outside of the targeted tandem repeat pattern had a
substantial contribution to amplicon size differences. Depend-
ing on the population, up to 90%of the analyzed loci carried
flanking indels in the amplicon. Even after a complete loss of
the repeat motif, these indels contributed to variable fragment
sizes. This was particularly important in populations outside
of Maui, where a significant proportion of the repeat motifs
decayed.
DISCUSSION
Microsatellite isolation by high throughput sequencing.—Our
results show that microsatellite markers can be routinely
isolated by low coverage sequencing from any spider genome.
Simple high throughput sequencing of untreated genomic
DNA will yield sufficient markers for certain types of
population studies in spiders. It is important to aim for long
reads to provide sufficient flanking regions for primer design.
The best combination of read length and high output is
currently offered by the Illumina MiSeq system. With its V3
Figure 1.—(B) Subsection of genotyping plots for the allelic distribution of an AT-dinucleotide repeat (TG_MS1) for two happy face
specimens from Hawaii Island. The plots show the abundance (Y-axis) of reads of different fragment length (X-axis). Each bar indicates the
abundance of one fragment length in a mixture of sequences, with the red dot indicating the called allele length. The upper specimen is
heterozygous and the lower is homozygous.
Table 4.—Pairwise F
ST
between Hawaiian populations of the happy face spider Theridion grallator for the microsatellite fragment length
dataset generated by Illumina amplicon sequencing. The population names correspond to those in Table 3.
HiBiK15 HiBiMM21 HiMaWaA HiMaWaB HiMoKaA HiMoKaC HiOhPeA
HiBiMM21 0.002
HiMaWaA 0.302 0.277
HiMaWaB 0.348 0.315 0.000
HiMoKaA 0.372 0.341 0.250 0.255
HiMoKaC 0.403 0.375 0.273 0.275 0.000
HiOhPeA 0.497 0.463 0.337 0.354 0.437 0.460
HiOhPeB 0.517 0.483 0.346 0.365 0.446 0.467 0.012
KREHENWINKEL ET AL.—MICROSATELLITES AND GENOTYPING FOR SPIDERS 195
Figure 2.—Results (right) of a STRUCTURE analysis of the microsatellite dataset assuming k ¼4 populations. Colors correspond to that of
islands in the sampling map (left).
Figure 3.—Alignment of sequences of happy face spiders (Theridion grallator) from different Hawaiian Islands for a CT-dinucleotide repeat
(MS41). Each island is represented with 8 sequences. The repeat motif is only found in populations on Maui and absent on all other islands. The
repeat motif is not perfect, with a Thymine inserted instead of Cytosine in some specimens. Flanking SNPs support the signal of the repeat
pattern. A flanking deletion is present in all specimens from Big Island and a subset of those from Oahu.
196 JOURNAL OF ARACHNOLOGY
chemistry, it is possible to obtain up to 20,000,000 paired reads
of 2 3300 bp per run. About 50 microsatellite loci should
provide sufficient resolution for some population genetic
applications depending on the question in focus, as well as
for paternity analysis. Considering the recovery of 2–19 primer
pairs per sequenced Mb of DNA, this translates to a minimum
of 25 Mb of DNA that needs to be sequenced for
microsatellite recovery. Our analyses in T. grallator suggest a
very fast turnover of repeat loci in spider genomes. Due to the
fast evolution of the repeat content, it is probably not possible
to predict the expected repeat content for a target species from
other related taxa. The repeat content has to be determined de
novo for each species. We found a high dropout rate due to the
lack of variation in repeat length for some taxa, e.g., many loci
could not be used for further analysis in these taxa.
Consequently, we recommend a higher sequencing coverage
of about 250 Mb per specimen for the development of
microsatellite markers. Considering the throughput of an
Illumina MiSeq, this still translates to 80 separate spider
species, for which microsatellites could be isolated in a single
sequencing run. At a cost of about $1,600 USD per run,
microsatellites for any spider species can be isolated for about
$50 USD, including the cost for library preparation. Using
enrichment protocols, the output of isolated loci could be
increased substantially (Malausa et al. 2011).
Microsatellite genotyping using Illumina amplicon sequenc-
ing.—Microsatellite amplicons can be rapidly sequenced and
genotyped using paired-end Illumina sequencing of multiplex
PCRs. High throughput sequencing approaches come with
many advantages over traditional capillary electrophoresis of
dye-labelled amplicons. At the same time, the results of high
throughput sequencing approaches are highly congruent with
those of capillary electrophoresis (Vartia et al. 2016; Zhan et
al. 2017). The high coverage of Illumina sequencing avoids the
need to balance amplification of every single marker in a
multiplex reaction. While the fluorescence of a single,
overrepresented marker can outshine all other loci in a
multiplex, the read count information for each locus is
independent from that of the others. Even without previous
optimization of multiplexes, we recover a very high number of
loci (24 out of 25) and specimens (92 out of 96) in our analysis.
There is also no need to select non-overlapping groups of
marker sizes for multiplex reactions, as is typical for dye-
labelled reactions. The only limit of an Illumina based
approach is the current maximum size of paired read lengths
of 2 3300 bp. Assuming that each sequence is supposed to
read through the tandem repeat, a large overlap is desired for
read merging. Thus, amplicons should not exceed 400bp. Our
approach of plotting read length abundance profiles per locus
(Henderson, Russack, Krehenwinkel & Simison unpublished
data) is comparable to classic allele-calling software. Recent
work suggests automated calling options (Suez et al. 2016) or a
Figure 4.—Losses and gains of tandem repeat loci over the microsatellite-based phylogeny of the four happy face spider populations analyzed.
The root of the tree and the divergence ages for the major clades are in accordance with Croucher et al. (2012). The bar plot shows the number of
loci with a repeat motif present in all specimens (black), present in only a subset of the population, e.g., part of the standing variation of the
population (grey) and absent (white).
Table 5.—Proportion of loci, out of 24 sequenced loci, for
Theridion grallator populations from four islands, which (1) contain
the tandem repeat motif targeted by primer design, (2) contain an
additional tandem repeat motif, (3) contain indels outside of the
tandem repeat motif, (4) carry variable SNPs.
Big Island Maui Molokai Oahu
Repeat motif present 0.71 0.96 0.71 0.63
Second repeat motif present 0.25 0.29 0.25 0.25
InDels present 0.67 0.92 0.83 0.75
SNPs present 0.96 0.96 1.00 1.00
KREHENWINKEL ET AL.—MICROSATELLITES AND GENOTYPING FOR SPIDERS 197
mixture of automated and manual curation of such datasets
(Zhan et al. 2016).
For the genotyping of small to moderate numbers of
markers, sequencing of microsatellite amplicons will be
cheaper and require less laboratory experience than current
SNP genotyping protocols, e.g., RAD sequencing. To generate
the 24-locus dataset for each specimen, we required six
multiplex PCRs, two clean up reactions and one indexing
PCR, adding to $4 USD. Sequencing required another $4
USD per specimen. However, after further optimization of our
multiplex PCRs, the number of PCRs and necessary
sequencing coverage and processing cost could be consider-
ably reduced. Recovering a higher genetic diversity and having
a better population assignment power at low marker densities
(Yang et al. 2011), microsatellites might even be preferable
over SNPs for small-scale population studies. A combination
of microsatellite isolation and genotyping by high throughput
sequencing could allow the rapid and cost-efficient analysis of
paternity or population structure.
While microsatellites can be valuable markers, microsatellite
repeats can show highly complex and unpredictable patterns
of evolution (Ellegren 2000) making modelling difficult. This
can hamper the interpretation of microsatellite data and
requires careful evaluation, e.g., by manual curation of the
data before downstream analysis. Furthermore, it should be
investigated whether more traditional enrichment marker
design protocols as described in Nolte et al. (2005) and Leese
et al. (2008) could be combined with HTS techniques, in order
to further increase the effectiveness of the procedure.
Implications of a rapid turnover of microsatellite repeat loci in
spiders.—The decay and possible gain of tandem repeat loci is
a prominent pattern in our data, with almost half of the
investigated loci not showing the repeat in at least one
population of T. grallator. Only populations from Maui, on
which the microsatellite loci used in this study were designed,
consistently show all repeat loci. Recent molecular analyses
suggest a stepping stone mode of colonization of the spiders
from Oahu to Maui Nui (Maui and Molokai) and on to the
Big Island within the past million years (Croucher et al. 2012).
With only a few thousand years of coalescence time, the happy
face spider is a young taxon. As an annual species, this
corresponds to only a few thousand generations. A turnover of
almost half of all repeat loci in this timeframe suggests a rapid
decay and gain of repeat loci. The fast turnover may also be
the reason for the lack of variation we found in many
microsatellite sequences for some spider species, particularly
O. retusus and A. ergandros. The lack of variation may also be
the result of ascertainment bias (Ellegren et al. 1995). By
choosing relatively long repeat motifs in our primer design, the
maximum repeat motif length for some loci may be reached
already. It is then likely by chance that evolution will decrease
the size of repeat motifs in distant groups.
A study on interspecific decay rates of repeat motifs in
Diptera and Hymenoptera, suggest losses of motifs in 30–40 %
of loci after 2 million generations (Stolle et al. 2013). Spiders
might thus show a faster rate of repeat motif evolution than
other arthropod taxa. In contrast to Diptera and Hymenop-
tera, all spiders have large genome sizes (Gregory 2001). This
might result in more genomic regions with little functional
constraint, allowing for a faster random evolution and decay
of repetitive sequences. Moreover, spider genomes generally
show a low GC content (Sanggaard et al. 2014; Krehenwinkel
et al. 2015). A low GC content in tandem repeats and their
flanking sequence has been suggested to possibly contribute to
an increased mutation rate (Schl¨
otterer 2000). Microsatellites
are often associated with transposable elements (Ellegren
2004; Megl´
ecz et al. 2007). Their emergence by repeat
mobilization could explain the rapid appearance and losses
of repeats in T. grallator. However, little is currently known
about microsatellite evolution in invertebrates (Chapuis et al.
2015), and future studies will have to explore this topic in more
detail.
Practically, the rapid decay and gain of repeat loci means
that called allele sizes for microsatellites in distant populations
are often only based on flanking indels and not the evolving
repeat. This urges care in cross-species amplification, still a
popular approach (Moodley et al. 2015). This problem is
probably not specific for spiders. In particular, distances based
on models of evolution for the actual repeat motif might be
biased by repeat loss. Another practical issue with the lack of
size variation in repeat motifs is the need to explore large
numbers of microsatellite loci to identify sets of variable
markers for some spider species. However, due to the
simplicity of multiplex PCRs and the high throughput of
current amplicon sequencing protocols, the scoring of large
numbers of microsatellite markers is still a worthwhile
approach for studying population genetics in spiders.
Current high throughput sequencing technology allows the
rapid and cost-efficient isolation of large numbers of
microsatellite markers from spiders as we have shown in
seven distantly related spider species. Moreover, Illumina
amplicon sequencing is well suited for genotyping of
microsatellite markers. As we highlight in an exemplary
species, amplicon sequencing based microsatellite genotyping
offers a greatly simplified workflow over currently used
capillary electrophoresis-based protocols. Provided that mi-
crosatellite data are carefully analyzed, our results demon-
strate that microsatellite markers can be a useful alternative to
SNP genotyping for population genetics and pedigree
analyses.
ACKNOWLEDGMENTS
We cordially thank Thoomke Br¨
uning, Sarah Frehse, Nadja
Gogrefe and Nicole Thomsen for assistance during lab work.
We thank Andrew Rominger, Ellie Armstrong and Nate Yuen
for help collecting happy face spider specimens. Anna Sellas
and the California Academy of Sciences’ Center for Compar-
ative Genomics, are acknowledged for support during
sequencing. Jun Ying Lim kindly provided a map of the
Hawaiian Archipelago. We acknowledge the Nature Conser-
vancy and the Hawaiian authorities for providing the
necessary permits to collect happy face spiders. Natalie
Graham provided helpful comments on the manuscript.
Diethard Tautz provided helpful discussion and access to
high throughput sequencing machines. HK was funded by a
PhD fellowship of the Studienstiftung des Deutschen Volkes
and by a postdoctoral fellowship of the German Research
Foundation (DFG). The research was supported by NSF
grant DEB 1241253 to RG and by the DFG to GU (Uh/87-7,
198 JOURNAL OF ARACHNOLOGY
RTG 2010). TB was supported by The Danish Council for
Independent Research grant number 4002-00328B.
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SUPPLEMENTAL MATERIAL
The following data sets are available in the Dryad Digital repository
(Online at doi:10.5061/dryad.c4d7cc0)
1. 454 reads and Assemblies for all studied species
2. Isolated primer sequences for all studied spider species
3. Primer sequences, which have already been tested for
variability and PCR amplification success.
4. Illumina reads for microsatellite genotyping of Hawaiian
happy face spider populations.
KREHENWINKEL ET AL.—MICROSATELLITES AND GENOTYPING FOR SPIDERS 201
