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R E S E A R C H Open Access
Alu insertion polymorphisms shared by
Papio baboons and Theropithecus gelada
reveal an intertwined common ancestry
Jerilyn A. Walker
1†
, Vallmer E. Jordan
1†
, Jessica M. Storer
1
, Cody J. Steely
1
, Paulina Gonzalez-Quiroga
1
,
Thomas O. Beckstrom
1
, Lydia C. Rewerts
1
, Corey P. St. Romain
1
, Catherine E. Rockwell
1
, Jeffrey Rogers
2,3
,
Clifford J. Jolly
4
, Miriam K. Konkel
1,5
, The Baboon Genome Analysis Consortium and Mark A. Batzer
1*
Abstract
Background: Baboons (genus Papio) and geladas (Theropithecus gelada) are now generally recognized as close
phylogenetic relatives, though morphologically quite distinct and generally classified in separate genera. Primate
specific Alu retrotransposons are well-established genomic markers for the study of phylogenetic and population
genetic relationships. We previously reported a computational reconstruction of Papio phylogeny using large-scale
whole genome sequence (WGS) analysis of Alu insertion polymorphisms. Recently, high coverage WGS was
generated for Theropithecus gelada. The objective of this study was to apply the high-throughput “poly-Detect”
method to computationally determine the number of Alu insertion polymorphisms shared by T. gelada and Papio,
and vice versa, by each individual Papio species and T. gelada. Secondly, we performed locus-specific polymerase
chain reaction (PCR) assays on a diverse DNA panel to complement the computational data.
Results: We identified 27,700 Alu insertions from T. gelada WGS that were also present among six Papio species,
with nearly half (12,956) remaining unfixed among 12 Papio individuals. Similarly, each of the six Papio species had
species-indicative Alu insertions that were also present in T. gelada. In general, P. kindae shared more insertion
polymorphisms with T. gelada than did any of the other five Papio species. PCR-based genotype data provided
additional support for the computational findings.
Conclusions: Our discovery that several thousand Alu insertion polymorphisms are shared by T. gelada and Papio
baboons suggests a much more permeable reproductive barrier between the two genera then previously suspected.
Their intertwined evolution likely involves a long history of admixture, gene flow and incomplete lineage sorting.
Keywords: Retrotransposon, Evolutionary biology, Primate phylogeny, Alu element
Background
The phylogenetic position of the gelada (Theropithecus
gelada) has been debated since the species was first scien-
tifically described in 1835 by Rüppell. Originally named
Macacus gelada, it was later placed in a genus of its own
by I. Geoffroy Saint-Hilaire (1843) [1] where it remains
today as the only extant species of Theropithecus [2]. By
contrast, there are currently six recognized species of
Papio baboons distributed across most of sub-Saharan
Africa [3–5]. Evidence from morphological comparisons
and mitochondrial and whole genome sequencing (WGS)
all support a primary phylogenetic division into northern
(P. anubis,P. papio and P. hamadryas) and southern (P.
ursinus,P. cynocephalus and P. kindae)clades[5–7]. The
genetics of the baboon species complex have been studied
much more extensively [4–14] than that of the mountain
dwelling geladas of the Ethiopian highlands [15–20]. Ther-
opithecus is estimated to have diverged from a Papio-like
ancestor about 4–5 million years ago (mya) based on fossil
evidence [2,4,21,22] and analysis of mitochondrial DNA
[23], while extant Papio species began to diversify about 2
mya [5,7,9,24].
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
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(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
* Correspondence: mbatzer@lsu.edu
†
Jerilyn A. Walker and Vallmer E. Jordan contributed equally to this work.
1
Department of Biological Sciences, Louisiana State University, 202 Life
Sciences Building, Baton Rouge, Louisiana 70803, USA
Full list of author information is available at the end of the article
Walker et al. Mobile DNA (2019) 10:46
https://doi.org/10.1186/s13100-019-0187-y
Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-n5ohlkrjdes50
A complex history of evolution has been reported among
extant species within the genus Papio [5–7,25–28], in
which mitochondrial and phenotypically-based phyloge-
nies of the six currently recognized extant species fre-
quently conflict. Recently, the Baboon Genome Analysis
Consortium published a study of the complex population
history of Papio baboons based on whole genome se-
quences, providing evidence for multiple episodes of
introgression and admixture throughout radiation of the
genus and a long history of genetic exchange among
diverging lineages that were presumably phenotypically
distinct [6].
Primate specific Alu retrotransposons are well-
established genomic markers for the study of population
genetic and phylogenetic relationships [27,29–40]. Alu
element insertions are considered unique events, have a
known directionality where the ancestral state is known
to be the absence of the element, and are relatively inex-
pensive to genotype [33,41–45]. Alu insertions shared
by individuals or species are widely accepted as largely
being inherited from a common ancestor. The amplifica-
tion of Alu elements has been ongoing in primate
genomes since the origin of the Order, about 65 mya
[42,46,47]. Alu elements mobilize via a “copy and
paste”mechanism through an RNA intermediate, a
process termed “target-primed reverse transcription”
(TPRT) [48]. We recently reported a computational re-
construction of Papio phylogeny using 187,000 Alu in-
sertions identified through a large-scale whole genome
sequence analysis [26]. This study not only determined
the most likely branching order within Papio with high
statistical support, but also quantified the number of Alu
insertions supporting alternative topologies, demonstrat-
ing the efficacy of whole genome computational analysis
of Alu polymorphisms to identify and investigate com-
plexities in phylogenetic relationships.
During the early stages of the Baboon Genome Analysis
Consortium [6] an analysis of the [Panu_2.0] genome of
Papio anubis revealed an occasional Alu element insertion
that appeared to be present in T. gelada DNA based on
PCR, while also remaining polymorphic among the six
Papio species. Although intriguing given the estimated 4–
5 mya divergence between the two genera, with no other
WGS data available at the time for further computational
screening, these insertions were set aside as being unin-
formative for resolving phylogenetic relationships within
Papio. Recently, we have generated high coverage WGS
data for an individual Theropithecus gelada (Sample name
36168, BioProject PRJNA251424, submitted by Baylor
College of Medicine). Therefore, the objective of this study
was to apply the “polyDetect”method [26]tocomputa-
tionally determine the number of Alu insertion polymor-
phisms shared by the representative T. gelada genome
and 12 individuals representing the genus Papio.Our
approach targeted recently integrated Alu insertions
present in T. gelada yet polymorphic within Papio and
absent from rhesus macaque [Mmul8.0.1]. Alu insertions
recent enough to remain polymorphic among Papio spe-
cies would be expected to have integrated after the split
from Theropithecus and therefore be absent from Thero-
pithecus. Similarly, Theropithecus, with a much smaller
effective population size [20], would be expected to have
its own set of lineage-specific insertions. Observations of a
large number of Alu insertions present in both genera that
remain unfixed in all species would suggest a long history
of ancient admixture, extensive incomplete lineage sort-
ing, or on-going hybridization [44]. Here, we have compu-
tationally ascertained a dataset of Alu insertions present in
the Theropithecus gelada WGS data that also remained
polymorphic among 12 Papio baboons representing all six
species. This analysis prompted a reciprocal computa-
tional comparison of WGS of each Papio individual to
determine the number of Alu insertion polymorphisms
shared exclusively between each Papio species and T.
gelada.
Locus specific PCR analyses were performed on a DNA
panel which included samples from all six Papio species,
T. gelada and rhesus macaque (Macaca mulatta)asan
outgroup to provide experimental support for the compu-
tational findings.
Methods
WGS samples
Whole-genome sequencing was performed by the Baylor
College of Medicine Human Genome Sequencing
Center. All samples were sequenced to an average cover-
age of 32.4x and minimum of 26.3x [6]. The same data-
set described in Jordan et al. (2018) [26] for 12 Papio
individuals was used in this analysis along with WGS
from a representative T. gelada genome. These samples
are listed in Additional file 1. We used two individuals
from each of the six extant Papio species (we randomly
selected two individuals from P. anubis and P. kindae)
to conduct our computational analysis; along with WGS
data from the rhesus macaque sample used to build
the recent M. mulatta assembly [Mmul8.0.1] and
WGS data for one Theropithecus gelada (isolate
891096; sample name 38168; adult female captive
born at the Bronx Zoo; NCBI BioProject PRJNA251424;
Accession: SAMN06167567). WGS data were accessed
from the NCBI-SRA database as described previously [26].
Computational Alu detection
We used the “polyDetect”computational pipeline [26]to
perform our analysis. Our approach targeted recently in-
tegrated Alu insertions present in T. gelada yet poly-
morphic within Papio and absent from rhesus macaque
[Mmul8.0.1]. The approximate chromosomal position of
Walker et al. Mobile DNA (2019) 10:46 Page 2 of 13
each candidate insertion was estimated using a split-read
method as described previously [26]. Briefly, for the
alignment phase, we used BWA-MEM version 0.7.17-
r1188 [49] to map the sequencing reads to a consensus
AluY sequence obtained from Repbase [50]. The Alu
portion of each candidate split-read was cleaved allowing
the remaining unique flanking sequence to be aligned to
the rhesus macaque genome assembly [Mmul8.0.1] using
bowtie2 version2.3.2 [51]. Split-reads were categorized
as sequences that mapped uniquely to the AluY consen-
sus sequence and the [Mmul8.0.1] assembly. The result-
ing genotypes, generated for all individuals in our panel,
isolated thousands of phylogenetically informative
markers. Data for these loci were sorted by the number
of Alu insertions common to T. gelada and any two to
twelve Papio individuals. For purposes of the present
analyses, those present in all 12 Papio individuals were
considered fixed present in the dataset and eliminated
from this portion of the study. For the reciprocal
comparison, the Alu insertions detected in both individ-
uals of a single Papio species, as reported previously in
Jordan et al. (2018) [26], were sorted by their
[Mmul8.0.1] predicted insertion coordinates and cross-
referenced with coordinates from the T. gelada WGS
reads to identify candidate shared insertion polymor-
phisms. These are listed in Additional file 1, Worksheet
“Papio-Theropithecus.”
Statistical analysis of Alu insertion polymorphisms
Alu insertions predicted to be shared by T. gelada and
any two to eleven of the twelve Papio individuals were
considered polymorphic in the genus Papio and retained
for further analysis. To determine if any particular
species or clade had significantly different numbers of
shared insertions with T. gelada, we performed a one-
way analysis of variance (ANOVA) in Excel (alpha set at
0.05). A separate ANOVA was performed for each of the
ten data bins representing two to eleven individuals.
ANOVA “groups”were defined as either six Papio spe-
cies with two individuals each, or two Papio clades
(North / South) with six individuals each. If a significant
‘between group’difference was detected, we followed
with a Bonferroni post-hoc test in Excel, selecting the “t-
Test: Two-sample assuming equal variances”function to
perform a two-tailed t-test for P≤0.05. All Pvalues were
recorded in Additional file 1: Table S1.
Candidate Alu element selection and oligonucleotide
primer design
We randomly selected 150 candidate Alu insertion poly-
morphisms from the first comparison (A: ascertained
from the T. gelada WGS and polymorphic among Papio
baboons) for in-house oligonucleotide primer design as
described previously [52]. From the second comparison
(B: present in WGS of both individuals of a single Papio
species and shared in T. gelada) we randomly selected
about 10% of the candidate loci identified from each of
the six Papio species, but no less than five loci from each
species, for primer design. Oligonucleotide primers for
PCR were designed using the predicted insertion coordi-
nates from the rhesus macaque genome [Mmul8.0.1]
since that was the “reference”genome used to map the
T. gelada and Papio WGS reads. Suitable primer pairs
were then analyzed against the Papio anubis baboon
genome [Panu_2.0] using the “In-Silico PCR”tool in
BLAT [53] through the University of California Santa
Cruz (UCSC) Genome Browser [54]. If no PCR product
was identified due to mismatches in the primer se-
quence, the primer pairs were analyzed by In-Silico PCR
using the [Mmul8.0.1] assembly to obtain the predicted
PCR product. This entire amplicon sequence was then
analyzed using BLAT against the P. anubis genome
[Panu_2.0] and checked for mismatches in order to de-
sign alternative oligonucleotide primers to help ensure
PCR amplification in Papio baboons. Using this method
we obtained estimates for our expected PCR product
sizes in [Mmul8.0.1] and [Panu_2.0] (Additional file 2).
Oligonucleotide primers for PCR were obtained from
Sigma Aldrich (Woodlands, TX).
Polymerase chain reaction assays
The primate DNA panel used for PCR analyses was com-
prised of three P. anubis,oneP. hamadryas, two P. papio,
two P. cynocephalus, two P. ursinus,twoP. kindae,oneT.
gelada,andaMacaca mulatta. A human (HeLa) sample
was used as a positive control and TLE (10 mM Tris / 0.1
mM EDTA) was used as a negative control. Information
about the samples is provided in Additional file 2includ-
ing their common name, origin, and ID.
A total of 172 Alu insertion polymorphisms were
retained in the dataset for PCR analyses. We used a subset
of the computationally-derived Alu insertion polymor-
phisms ascertained from either A) T. gelada WGS and
predicted to be shared in Papio,(N=96); or B) Papio
species WGS and predicted to be shared in T. gelada,
(N= 52). We also included N=24 Alu loci previously
ascertained from the reference genome of Papio anubis
[Panu_2.0] (12 loci each from [6,52]) in which PCR re-
sults indicated the Alu insertion was present in T. gelada
while remaining polymorphic among the six Papio
species.
Oligonucleotide primers for PCR were designed using
Primer3 software, either manually [55] for most of the
Panu_2.0 derived candidate loci or using a modified ver-
sion [56]. PCR amplifications were performed in 25 μl
reactions containing 25 ng of template DNA; 200 nM of
each oligonucleotide primer; 1.5 mM MgCl
2
, 10x PCR
buffer (1x:50 mM KCl; 10 mM TrisHCl, pH 8.4); 0.2 mM
Walker et al. Mobile DNA (2019) 10:46 Page 3 of 13
dNTPs; and 1–2U Taq DNA polymerase. PCR reactions
were performed under the following conditions: initial
denaturation at 94 °C for 60 s, followed by 32 cycles of de-
naturation at 94 °C for 30 s, 30 s at annealing temperature
(57 °C –61 °C), and extension at 72 °C for 30 s. PCRs were
completed with a final extension at 72 °C for 2 min.
Twenty microliter of each PCR product were fractionated
by size in a horizontal gel chamber on a 2% agarose gel
containing 0.2 μg/ml ethidium bromide for 60 min at 185
V. UV-fluorescence was used to visualize the DNA frag-
ments and images were saved using a BioRad ChemiDoc
XRS imaging system (Hercules, CA). Following gel
electrophoresis, genotypes were recorded in an Excel
spreadsheet as (1, 1) for homozygous present, (0, 0) for
homozygous absent, or (1, 0) for heterozygous. “Missing
data”was coded as (−9, −9). Genotypes for these 172 loci
are shown in Additional file 2;Worksheet“Genotypes.”
Validation of computational predictions
Our DNA panel for locus-specific PCR analyses did not
include samples from every WGS individual analyzed.
Because our representative T. gelada individual differed
from that supplying the WGS sample used for Alu
ascertainment, we used genotype data from PCR ana-
lyses for ten Papio individuals on our DNA panel to
estimate the validation rate of the computational predic-
tions (Additional file 3). Based on these results, we im-
plemented an additional filtering step on the data in an
attempt to minimize the number of false predictions,
while continuing to ensure that our interpretation of the
computational results was correct. This filter involved
re-analyzing the read files for the dataset of Alu inser-
tions present in T. gelada WGS and imposed a mini-
mum length requirement of 30 bp of unique 5′flanking
sequence adjacent to the predicted Alu insertion for the
call to be retained. These post-filtered data were sorted as
before for the number of shared Alu insertions between T.
gelada and any two to twelve Papio individuals. The set of
candidate loci determined to be present in both individ-
uals of a single Papio species (as reported previously in
Jordan et al. 2018), that were also computationally pre-
dicted to be shared with T. gelada, were also subjected to
the filtering step and those retained were then screened
against the [Panu_2.0] baboon genome to eliminate those
shared in the P. anubis reference genome.
Alu subfamily analysis
Papio lineage-specific Alu subfamilies evolved from
older AluY subfamilies after the baboon stem lineage di-
verged from its common ancestor with the rhesus ma-
caque [52]. Identification of Alu subfamilies and the
corresponding sequence divergence can provide insight
regarding the approximate age of an Alu insertion event
[52,57]. This study included 24 loci ascertained from
the baboon genome assembly [Panu_2.0] and another 16
ascertained from the T. gelada WGS with complete Alu
sequence available. PCR data indicated that 15 of the 24
[Panu_2.0] set and 8 of the 16 WGS set met the study
criteria of being polymorphic among Papio baboons and
shared by T. gelada. These 23 polymorphic loci were
analyzed for Alu subfamily affiliation. Using the genome
coordinates in BED format we uploaded a custom track to
the UCSC Genome Browser [54] using the Table Browser
function. The complete Alu sequence was obtained in
FASTA format. Subfamily identification for these ele-
ments was determined using an in-house RepeatMasker li-
brary [58](http://www.repeatmasker.org; last accessed
November 2019) developed in Steely et al. (2018) [52].
Results
Computational Alu detection
Our split-read methods predicted 27,700 Alu insertions
in T. gelada WGS data shared among the 12 Papio indi-
viduals but absent from rhesus macaque [Mmul8.0.1]
(Additional file 4). Because our objective was to target
recently integrated Alu insertions present in T. gelada
yet polymorphic within Papio, we eliminated 14,744
(53%) that were present in all twelve Papio individuals.
We retained the remaining 12,956 shared by any of two
to eleven of the twelve Papio individuals for further ana-
lysis. To determine if any particular Papio species or
clade was favored or excluded for shared insertion
events with T. gelada, we sorted the raw output for the
number of shared Alu elements in each bin of 2 to 11
individuals (Table 1). Then we counted the number of
times a shared insertion was predicted in each Papio in-
dividual (Table 1). For example, when an Alu insertion
was predicted to be present in any 5 of the 12 Papio in-
dividuals and absent from the other 7, we found 294
instances where one of the five individuals with the in-
sertion was P. anubis LIV5. All 12 Papio individuals
shared hundreds of Alu insertion polymorphisms with
T. gelada in all categories. The average of the two indi-
viduals of each species + / - the standard deviation is
plotted in Fig. 1. A one-way ANOVA with Bonferroni
correction detected significant between-group differ-
ences for test bins 2 to 10, but not for bin 11. In bin 2,
P. hamadryas has more shared insertions with T. gelada
than do P. anubis, P. papio,orP. cynocephalus, while in
bin 6, P. cynocephalus has more shared insertions than
the three northern species (Fig. 1; Additional file 1:
Table S1). As a group, the northern and southern clades
appear to have similar representation overall except as
detected in bins 5 and 6 (of 12) in which the southern
clade has significantly more shared insertions, on aver-
age, than the northern clade (P≤0.05; Additional file 1:
Table S1). However, the most consistent statistical find-
ing across all bins was for the two P. kindae individuals.
Walker et al. Mobile DNA (2019) 10:46 Page 4 of 13
Table 1 Number of T. gelada Alu insertion polymorphisms shared in Papio individuals
A. B. Northern clade Southern clade
P. anubis P. hamadryas P. papio P. cynocephalus P. ursinus P. kindae
LIV5 L142 97124 97074 28547 30388 16066 16098 28697 28755 34449 34474
2 1139 112 122 192 179 136 146 127 140 155 111 486 372
3 989 174 169 210 205 166 184 227 231 249 185 537 430
4 944 296 248 268 261 206 259 282 297 343 247 567 502
5 839 294 290 248 280 241 294 342 375 413 310 574 534
6 938 396 396 360 381 370 396 491 497 531 421 727 662
7 851 495 466 448 456 395 430 497 480 505 428 702 655
8 991 626 638 631 645 546 617 623 663 677 584 849 929
9 1171 899 865 830 851 811 869 824 862 894 759 1040 1035
10 1881 1659 1635 1531 1522 1442 1516 1501 1563 1590 1405 1732 1714
11 3213 2980 2971 2890 2907 2966 3104 2811 2884 3025 2636 3079 3090
12,956
The number of Alu insertion polymorphisms ascertained from T. gelada and not fixed in all 12 Papio individuals was calculated to be 12,956. The distribution of
these when shared between any of 2 to 11 of the 12 Papio individuals (column A, 2 to 11) is shown in column B. The sum of the values in column B is 12,956. The
ID for each Papio individual is shown at the top of the twelve adjacent columns, for each of the six Papio species, separated by northern and southern clades. The
numbers in each column represent the number of times that the shared insertion with T. gelada was predicted in that individual. For example, when an Alu
insertion was predicted to be shared in 4 of the 12 individuals and absent from the other 8, one of the four (column A, row 4) was P. anubis LIV5 296 times and
one of the four was P. kindae 34474 (BZ11050) 502 times. All 12 Papio individuals share hundreds of Alu insertion polymorphisms with T. gelada in all categories.
No Papio individuals are preferentially excluded from having shared insertions with T. gelada. ANOVA detected between-group differences in bins 2–10, but not
bin 11. P. kindae has significantly more shared insertion events with T. gelada than all other five Papio species in bins 2 to 4 and 7 to 8, while significantly more in
all except P. ursinus in the remaining bins 5, 6, 9 and 10. See Fig. 1
Fig. 1 The number of times a T. gelada-ascertained Alu insertion polymorphism was predicted to be shared in a Papio species when shared in
any of 2 to 11 of the 12 Papio individuals. Vertical bars are the average of the two individuals of a given species +/−the standard deviation (error
bars). No Papio individuals are preferentially excluded from having shared insertions with T. gelada in any category. In bin two, P. hamadryas has
significantly more shared insertions than P. anubis,P. papio, and P. cynocephalus (+: P≤0.05). In bin six, P. cynocephalus has significantly more
shared insertions than the three northern species, P. anubis,P. hamadryas and P. papio (+: P≤0.05). Across bins 2 to 10 shared insertions are
predicted in P. kindae significantly more often than all other five Papio species (*) or all except P. ursinus (#) (P≤0.05)
Walker et al. Mobile DNA (2019) 10:46 Page 5 of 13
P. kindae has significantly more shared Alu insertions
with T. gelada than all other five Papio species in bins 2
to 4 and 7 to 8, while significantly more in all except P.
ursinus in the remaining bins 5, 6, 9 and 10 (Fig. 1;
Additional file 1: Table S1).
These findings prompted us to perform the reciprocal
database comparison (B) between the Papio WGS Alu
analyses reported in Jordan et al. (2018) [26] and the
current WGS Alu database for T. gelada. In that study,
P. kindae was found to have the most ‘species-indicative’
Alu insertions with 12,891 elements identified in both P.
kindae individuals and absent from both the two individ-
uals of all the other five Papio species. We cross-
referenced those 12,891 P. kindae Alu loci with the data-
base of 27,700 T. gelada Alu loci to determine if any
were shared exclusively between P. kindae and T. gelada
and identified 236 (1.83%) cases. We performed the
same cross-reference analyses for the other five Papio
species and found that each of the six Papio species had
Alu insertions shared exclusively with T. gelada.P. kin-
dae had significantly more shared insertions than the
other five Papio species (P< 0.05) (Table 2). The pre-
dicted insertion coordinates and sample IDs are listed in
Additional file 1, Worksheet “Papio-Theropithecus.”
Candidate loci and PCR analyses
A subset of 150 T. gelada computationally-derived
candidate Alu insertion events were selected for PCR
analyses. The oligonucleotide primer design pipeline se-
lected suitable primer pairs using the [Mmul_8.0.1] gen-
ome as the mapped reference. After screening these
primer pairs against the baboon genome assembly
[Panu_2.0], a total of 105 loci were analyzed by PCR for
Alu presence / absence within Papio and T. gelada, with
96 generating interpretable results (Additional file 2).
PCR based genotypes revealed that 60 of these 96 loci
(62%) met the objective criteria of being polymorphic for
insertion presence / absence among Papio baboons and
also being shared in a representative T. gelada individ-
ual, KB10538 from the San Diego Zoo (DNA was not
available for WGS individual 38168). Allele frequency
calculations on these 60 loci showed that P. hamadryas
sample 97124 and P. kindae sample 34474 (BZ11050)
had the highest counts of shared insertions with 25 and
24%, respectively, while the average across the other
Papio samples was 18% (Additional file 2, Worksheet
“allele frequency”). Given that these loci were randomly
selected from thousands of candidates, the fact that PCR
shows P. kindae to have one of the highest frequencies
of alleles shared with T. gelada supports the computa-
tional predictions reported in Table 1.
The second subset of PCR candidates was selected
from the dataset of Papio species-indicative elements
shared with T. gelada (Table 2). Because we did not have
DNA samples from every WGS sample analyzed,
including the T. gelada, we randomly selected approxi-
mately 10% of the candidate loci from each Papio spe-
cies for PCR analysis, with a minimum of five per
species. A total of 52 loci from this dataset were ana-
lyzed by PCR with 49 generating interpretable results
(Additional file 2). PCR results confirmed 26 of these
loci contained the candidate Alu insertion in the pre-
dicted Papio species and the representative T. gelada
individual KB10538 (Additional files 2and 3). Al-
though 26 of 49 is only about a 53% confirmation
rate from within the candidate loci selected, they pro-
vide clear evidence that this particular phenomenon
of shared Alu insertion polymorphisms exists in na-
ture, and that each Papio species has multiple Alu in-
sertions also shared in T. gelada but not yet observed
in the other five Papio species. An example of this
scenario for each of the six Papio species is illustrated
with an agarose gel image in Fig. 2.
In addition to the candidate Alu insertion polymor-
phisms computationally ascertained in this study, subsets
A and B, we also retained 24 loci from previously
published studies (12 loci each from [6,52]) that
were ascertained from the olive baboon genome
[Panu_2.0] in which PCR experiments indicated the
Alu insertion might be shared by Papio and
Table 2 Number of Papio species-indicative Alu insertion polymorphisms shared with Theropithecus gelada
Papio species Number of species-
indicative
Alu insertions
Number shared in
T. gelada
% Z-score one-tailed
P-value
P. anubis 4645 34 0.73% −0.6821 0.2476
P. hamadryas 8060 101 1.25% 0.1802 0.4285
P. papio 10,873 68 0.63% −0.2445 0.4036
P. cynocephalus 2794 26 0.93% −0.7851 0.2162
P. ursinus 9545 57 0.60% −0.3861 0.3498
P. kindae 12,891 236 1.83% 1.9176 0.0276 *
The number of Alu insertion polymorphisms shared in WGS of Theropithecus gelada that were reported [26] to be exclusive to one Papio species and absent from
the other five. P. kindae has significantly more such shared elements with T. gelada than the other five Papio species (*P< 0.05). Z-scores are calculated based on
the mean, 87, and standard deviation equal to 77
Walker et al. Mobile DNA (2019) 10:46 Page 6 of 13
Theropithecus. PCR results using the current DNA
panel confirmed that 15 of these 24 met the objective
criteria of being polymorphic for insertion presence /
absence among Papio baboons while also being
shared in our representative T. gelada sample. All
172 loci in this study (96 + 52 + 24) were confirmed
by PCR to be absent in rhesus macaque.
Validation of computational predictions
We analyzed genotype data for the Papio individuals on
our DNA panel to determine the validation rate of the
computational predictions (Additional file 3). Of the 96
loci in this dataset that were ascertained from WGS of
T. gelada, a total of 206 instances of a filled allele being
shared with a Papio individual on our DNA panel were
predicted computationally. No PCR amplification oc-
curred in 3 cases, leaving 203 predicted shared cases to
analyze. 145 (71%) of the 203 were confirmed by PCR
while 58 (29%) of the 203 were shown by PCR to be false
predictions. Of the 58 false predictions, nearly three-
quarters (N= 43) occurred within 22 loci in which all in-
dividuals genotyped as absent for the insertion. A review
of all the read files, split-reads and paired-end reads used
to make these predictions, provided some clues as to
why some predictions were validated by PCR while
others were not. PCR-validated predictions typically had
multiple supporting reads with at least 40–50 bp of
unique 5′flanking sequence adjacent to the head of the
Alu insertion. Predictions not confirmed by PCR tended
to have very short (≤25 bp) 5′flanking sequence. This
suggests that a lack of flanking sequence to accurately
map the split-reads to unique sequence is the likely
cause for the majority of the false predictions. Seven
(7.3%) of the 96 loci were considered ‘false negative’in
that they were not computationally detected in all 12
Papio individuals (considered polymorphic), but the
PCR results indicated the insertion was present in all the
Papio individuals on the DNA panel (Additional file 3).
This type of error is likely caused by a lack of supporting
reads for those individuals such that the insertion is sim-
ply not detected, rather than being “predicted absent”by
the polyDetect method.
To determine the role of 5′flanking sequence length
on the number of false predictions, we re-analyzed the
dataset of 27,700 Alu insertions present in T. gelada
WGS that were computationally predicted to be present
in any of two to twelve Papio individuals and absent
from rhesus macaque [Mmul8.0.1]. We implemented a
‘read filter’requiring a minimum of 30 bp of 5′flanking
sequence adjacent to the predicted Alu (See Methods).
These post-filtered data were sorted as before for the
number of Alu insertions shared by T. gelada and any
two to twelve Papio individuals. The post-filtered
equivalent of Table 1is available in Additional file 1:
Table S2 and the associated Pvalue for each bin is
shown on the same worksheet as Additional file 1: Table
S3. The post-filtered equivalent of Fig. 1, using data
from Additional file 1: Table S2, is shown in Additional
file 1: Figure S1. The number of acceptable candidate
loci dropped from 27,700 to 22,875, with 10,422 (45.6%)
of those determined to be present in all 12 Papio indi-
viduals and the remaining 12,453 (54.4%) were
Fig. 2 Papio species-indicative Alu insertion polymorphisms shared
in Theropithecus gelada. Lanes: 1- 100 bp ladder, 2- TLE (negative
control), 3- Human (HeLa), 4- P. anubis (27861 Panu_2.0 reference
individual), 5- P. anubis (L142), 6- P. anubis (LIV5), 7- P. hamadryas
(97124), 8- P. papio (28547), 9- P. papio (30388), 10- P. cynocephalus
(16066), 11- P. cynocephalus (16098), 12- P. ursinus (28697), 13- P.
ursinus (28755), 14- P. kindae (34474; BZ11050), 15- P. kindae (34472;
BZ11047), 16- T. gelada (KB10538), 17- Macaca mulatta.aolive
baboon locus AnuGel_12; bhamadryas locus HamGel_76; cGuinea
baboon locus PapioGel_38; dYellow baboon locus YelGel_11; e
chacma baboon locus ChacmaGel_43; fkinda baboon locus
KindaGel_199. Green bars outline the Papio species with the Alu
present (upper band); the blue bar outlines the Alu present band in
T. gelada
Walker et al. Mobile DNA (2019) 10:46 Page 7 of 13
determined to be polymorphic among any two to eleven
Papio individuals. Although the number of elements in
any particular bin shifted somewhat with gains or losses
due to the filter requirement, the overall results and in-
terpretation of those results remained the same. All 12
Papio individuals share dozens of Alu insertion polymor-
phisms with T. gelada. Also, as with the original ana-
lyses, P. kindae still has significantly more shared Alu
insertions with T. gelada than any of the other five
Papio species in most bins while significantly more in all
except P. ursinus in bins 4–6. The observable conse-
quences of the filtering step appear to be a reduction in
the number of acceptable reads for P. anubis sample
L142, as compared to the other Papio individuals. Also,
the mean values of shared insertions with T. gelada now
favor the southern clade over the northern clade more
consistently (bins 3–7) than in the previous analyses
(bins 5–6). Of the 22 loci containing 43 of the 58 false
predictions in the previous analyses, 16 loci and 34 of
the 43 false calls were omitted by the filtering step. The
number of false predictions was reduced from 58 to 22
and the false prediction rate dropped from 29 to 11%
(Additional file 3). Only one previously validated call
was erroneously filtered out. Therefore, the filtered re-
sults improved the overall validation rates within this
study.
However, the effect of the 30 bp flanking requirement
on data reported in Table 2was more informative. The
filter reduced the number of acceptable calls in P. anubis
sample L142, thus reducing the number found in both
P. anubis individuals, LIV5 and L142. The consequence
was that some loci were eliminated that had already
been PCR validated (i.e. Anu-12 and Anu-6; Additional
file 3). Alternatively, the number of predicted P. hama-
dryas indicative elements included 7 new loci that were
not in the original set because they had previous calls in
L142 or other Papio individuals that now had been fil-
tered out. Therefore, not only were some reads elimi-
nated, as expected, but this in turn erroneously added
loci to each “Papio-indicative”category due to previously
called reads in other Papio individuals that were no lon-
ger acceptable under the filter conditions. To obtain a
value for each Papio species with “high confidence”
following the filtering step, we retained only those
post-filtered loci also present in the original analyses
reported in Table 2, that were also not present in the
Panu_2.0 genome. (Additional file 1:TableS4).As
before, P. kindae still has significantly more shared
Alu insertions with T. gelada than do the other five
Papio species (*P< 0.05).
In our attempt to minimize the number of false pre-
dictions and improve the validation rate of the polyDe-
tect output in this study, we also inadvertently increased
the number of ‘false negative’calls dramatically. That is,
the absence of a call (no detection in a WGS individual)
does not necessarily mean the “predicted absence”of the
Alu insertion, only a lack of acceptable mapped reads.
Therefore, the filtered results were far less accurate for
this metric of the study compared to the first analysis.
Also, the errors induced by the filter were more prob-
lematic to the overall results of the study than the rela-
tively minor impact of the initial false prediction rate.
This highlights the importance of validating methods for
data filtering and downstream data processing, and its
potential impact on data interpretation. In this case, hav-
ing a large dataset with overwhelming numbers meant
that the overall interpretation was robust to the identi-
fied issues.
Papio Alu subfamily distribution
Of the 172 elements PCR-analyzed in this study, only 23
were suitable for Alu subfamily analysis. They had the
complete Alu sequence available from the [Panu_2.0]
reference genome and met the study criteria of being
polymorphic for insertion presence / absence among
Papio baboons while also being shared in T. gelada.
These sequences were analyzed for Papio Alu subfamily
assignment using an in-house RepeatMasker [58] library
developed by Steely et al. (2018) [52]. The RepeatMasker
output is available in Additional file 2, Worksheet “RM
output”. Most of these subfamilies are generally older
ancestral subfamilies as shown by their location near the
central nodes of the clusters reported in Steely et al.
(2018) [52]. The percent divergence from the respective
consensus sequences ranged from 0.3 to 3.9% with the
average being 1.8% (≤2% divergence is considered rela-
tively young) [59,60]. Of the 23 loci analyzed, 7 were
assigned directly to subfamily AluMacYa3, the central
node of cluster 1 matching subfamily 0 [52] and the an-
cestral node originally discovered in Macaca mulatta.
Another 11 loci were assigned to Papio Alu subfamilies
that derived from AluMacYa3. One locus derived from
AluY (3.2% divergence) while the remaining four loci
represented different subfamily clusters but were gener-
ally from older rhesus macaque subfamilies such as
AluYRa4 (Additional file 2).
Discussion
The close evolutionary relationship between savanna ba-
boons, genus Papio, and geladas, genus Theropithecus,is
well documented [2,5] although recognized as separate
genera based on numerous differences in morphology,
social behavior and ecology [4,16,19,21]. Our finding
that about half (47–54%) of Alu insertions ascertained
from a representative T. gelada genome have not
reached fixation in the Papio species is unexpected given
aTheropithecus / Papio divergence time dating back to
4–5 mya. We also find that each of the six Papio species
Walker et al. Mobile DNA (2019) 10:46 Page 8 of 13
possesses several species-indicative Alu insertions
(present in both individuals of that species while absent
from all ten individuals from the other five species) that
are shared inter-generically with T. gelada. This implies
a long history of incomplete lineage sorting, admixture
and gene flow.
During most of the Plio-Pleistocene, Theropithecus
was present throughout much of non-rainforest Africa.
Three subgenera are currently recognized: T. (Thero-
pithecus), T. (Simopthecus), and T. (Omopithecus).Of
these, T. (Theropithecus), including only the extant T.
gelada, is unknown as a fossil, and may have always been
restricted to the Ethiopian highlands. T. (Omopithecus)
includes only a single recognized species, T.brumpti,
confined to the Early Pleistocene of East Africa. The
third subgenus, T.(Simopithecus), including T. oswaldi
and closely related species, is extensively distributed in
time and space, from ~ 4 mya to ~ 100 kya, and from
southern Africa to Algeria, extending into southern
Europe and western Asia [2,4,22,61,62]. Late popula-
tions of T.(S.) oswaldi were probably too large in body
mass to breed successfully with Papio baboons, but for
most of its history, T.(Simopithecus) was comparable in
mass to extant baboons.
Some observations of extant baboons and geladas
suggest that even after 4 mya of separate evolution, the
possibility of gene flow between them is not completely
excluded by an intrinsic barrier. A suspected hybrid
individual has been observed in a natural gelada-olive
baboon overlap zone [63]. In a zoo environment, com-
pletely viable first-generation hamadryas baboon x gel-
ada hybrids of both sexes are reliably reported. While
the hybrid males are suspected to be infertile, female
hybrids have produced viable offspring by backcrossing
to Papio hamadryas [64]. Especially during the earlier
phases of their long period of co-existence, Papio x
Theropithecus matings (including with T. oswaldi) may
have allowed ongoing, low-frequency genetic exchange.
Our Alu insertion polymorphism data support this
hypothesis.
In this study, we also report that P. kindae baboons
share more Alu insertions with T. gelada than do the
other Papio baboons. The reason for this is not well
understood and may require further study. Each of the
12 Papio genomes was sequenced to an average read
depth of 32.4x coverage with minimum coverage 26.3x
[6] and therefore it is unlikely that this finding can be
attributed to differences in sequence coverage. An Alu-
based phylogeny of Papio species placed P. cynocephalus,
not P. kindae, as most basal within the southern clade
[26]. The modern ranges of P. kindae and T. gelada are
geographically far apart [5,7]. If they adjoined or over-
lapped, it might suggest recent hybridization between
the two taxa. Moreover, all of the Papio individuals
investigated had dozens of shared insertions with T. gel-
ada, including multiple species-indicative loci. None
were preferentially excluded. This suggests that modern
geography and habitat are not contributing factors to
this finding. Using whole genome comparisons within
Papio, the P. kindae genome was found to harbor more
species-indicative Alu insertions than the other five spe-
cies and also found to share more Alu insertions with
members of the northern clade that were absent from
the other southern clade members [26]. The history of
P. kindae is reportedly quite unique among baboons. As
part of the Baboon Genome Analysis Consortium [6],
the best fitting model using coalescent hidden Markov
methods indicated that the history of P. kindae includes
an ancient admixture event involving a lineage related to
extant P. ursinus from the southern clade (52% contribu-
tion to extant P. kindae), with the remaining 48% contri-
bution to extant P. kindae originating from an ancient
lineage, possibly extinct, belonging to the northern clade
[6]. However, other scenarios may also be possible. If
extant P. kindae is the (now geographically restricted)
descendent of a geographically widespread ancestral
population that exchanged genes with ancestral popula-
tions in the Theropithecus lineage and also gave rise to
small spin-off populations that expanded one to the
north and another to the south, this might also be con-
sistent with the Alu evidence presented in this study.
Our analyses of Alu subfamily distribution are also
consistent with a complex evolutionary history for Papio.
The ancestral lineages of Asiatic and African papionin
monkeys diverged about 8 mya [23]. Alu subfamilies
rooted with rhesus macaque, meaning that these sub-
families were active prior to the divergence of Thero-
pithecus /Papio from Macaca, such as AluMacYa3,
were shown in this study to have recently integrated
progeny elements in Theropithecus /Papio. Many of the
23 Alu insertion polymorphisms analyzed for subfamily
assignment had < 2% divergence from their respective
consensus sequences, providing support for their recent
integration. The observation that generally older Alu
subfamilies have produced the majority of the relatively
recent integration events is consistent with the overall
estimated divergence timeframe of 4–5 mya. Low Alu
sequence variation combined with ongoing persistent
levels of insertion polymorphism suggest that the Alu
retrotransposition rate among these lineages has been
relatively uniform over a long period of time, possibly
driven by a lack of reproductive isolation [65].
This study suggests that Papio baboons and Thero-
pithecus have a long history of intertwined evolutionary
ancestry that likely includes episodes of intergeneric
introgression. A precedent for this among other African
primates is available by examining the complex origins
of the kipunji, Rungwecebus kipunji. The kipunji is a
Walker et al. Mobile DNA (2019) 10:46 Page 9 of 13
papionin primate discovered in Tanzania in 2003. It was
initially assigned to the genus Lophocebus (arboreal
mangabey) based on general morphology and arboreal
behavior [66] but genetic studies based on mtDNA from
a single specimen from Mount Rungwe indicated the
new species was more closely related to baboons, genus
Papio [67,68]. The arboreal mangabey-like phenotype of
the kipunji combined with a mtDNA profile similar to a
yellow baboon, suggested that Rungwecebus kipunji orig-
inated from a hybridization event between a female yel-
low baboon (Papio cynocephalus) and a Lophocebus
male mangabey [69]. It was not until genetic material
became available from a kipunji individual from the
Ndundulu population about 350 km away that new evi-
dence suggested that the two kipunji populations likely
have different evolutionary histories [70]. The Ndundulu
haplotype is considered to be the ancestral or “true”
mitochondrial haplotype while the Mount Rungwe
population has experienced more recent and perhaps
persistent localized introgression from Papio, introdu-
cing the observed Papio mtDNA haplotype [71]. The
example of the kipunji provides a biological precedent
with regard to intergeneric introgression among African
primates, similar to our findings between genus Papio
and genus Theropithecus.
Following Groves (2001) [3], the tribe Papionini in-
cludes macaques (Macaca), mandrills (Mandrillus), ter-
restrial mangabeys (Cercocebus) and the Highland
mangabeys (Rungwecebus kipunji) along with three
closely related genera Papio,Theropithecus and Lophoce-
bus [23]. Phylogenetic studies of Papionini have gener-
ally separated the genera into two clades, one with
Macaca basal to sister taxa Cercocebus and Mandrillus
and a second clade consisting of Theropithecus, Papio
and Lophocebus, subgenus Papionina [23,72].Phylogen-
etic relationships among the three Papionina genera re-
main unresolved [23,73]. Some studies have placed
Theropithecus basal to a clade consisting of Papio and
Lophocebus [73,74], while other analyses have placed
Theropithecus and Papio as sister taxa, with Lophocebus
diverging first [23]. The fact that extensive molecular
evidence has yet to resolve this phylogeny suggest pos-
sible admixture, reticulation and short internode inter-
vals that facilitate incomplete lineage sorting, and
possibly inter-generic hybridization among the lineages.
The increasing availability of vast amounts of WGS
data has led to many recent studies being conducted
based exclusively on computational analyses, without
wet-bench experimental validation to support the gen-
omic comparisons [75,76]. Although these reports are
informative, this study demonstrates the need to inter-
pret such results with caution. It is important to keep in
mind that although “figures don’t lie”, all forms of data
filtering and downstream processing have consequences,
some of which are obvious while others are more obscure.
Computational data alone may produce interpretable re-
sults, but the biological significance of such interpretation
should be anchored with experimental evidence when
possible. This is especially important when investigating
complex phylogenies with an extensive history of admix-
ture and hybridization. Even high quality WGS data from
limited sample sizes may not necessarily be representative
of the species or genus as a whole, thus molecular valid-
ation and adequate sampling are required to support the
findings. It is undeniable, however, that the burgeoning
availability of WGS data allows greater resolution of com-
plex phylogenies while also recognizing and addressing
the impact of confounding factors.
Conclusions
In this study, we computationally identified over twelve
thousand Alu insertions polymorphic in Theropithecus
and Papio. Even after incorporating our initial 71% valid-
ation rate and possible 7.3% false negative error rate, at
least 8500 Alu insertions have not reached fixation
among the two genera. PCR sequencing based on a small
subset of these insertions confirmed over one hundred
such cases in support of the computational findings. We
also computationally identified over 500 Papio species-
indicative Alu insertions polymorphisms (present in
WGS of both individuals of one Papio species while be-
ing absent from two samples from each of the other five
species) that were determined to be shared in T. gelada.
PCR evidence confirmed numerous cases of this unex-
pected phenomenon. All six Papio species have many
Alu insertion polymorphisms shared with T. gelada,
while P. kindae has the largest number. This study sug-
gests that Papio baboons and Theropithecus have a long
history of intertwined evolutionary ancestry that likely
includes episodes of intergeneric introgression.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s13100-019-0187-y.
Additional file 1. An Excel file containing different worksheets for the
WGS sample list, Tables S1 –S4, Figure S1 and “Papio-Theropithecus”.
(XLSX 173kb)
Additional file 2. An Excel file with worksheets for DNA samples,
oligonucleotide PCR primers, genomic coordinates, genotype data for the
PCR experiments, allele frequency and RepeatMasker output. (XLSX 89kb)
Additional file 3. An Excel file summarizing the PCR validation of
computational predictions. (XLSX 45.6kb)
Additional file 4. An Excel file with a list of the 27,700 T. gelada / Papio
shared Alu insertions. (XLSX 1.7MB)
Abbreviations
bp: Base pairs; kya: thousand years ago; mya: million years ago;
PCR: Polymerase chain reaction; TPRT: Target primed reverse transcription;
WGS: Whole genome sequence
Walker et al. Mobile DNA (2019) 10:46 Page 10 of 13
Acknowledgements
The authors would like to thank all the members of the Batzer Lab and the
Baboon Genome Analysis Consortium for all their helpful suggestions. We
also thank Drs. Oliver A. Ryder and Leona G. Chemnick at the San Diego Zoo
Institute for Conservation Research for providing T. gelada sample KB10538.
Author List: The Baboon Genome Analysis Consortium.
Jeffrey Rogers
1,2
, R. Alan Harris
1,2
, Muthuswamy Raveendran
1
, Yue Liu
1
,
Shwetha Murali
1*
,
Tauras P. Vilgalys
3
, Jerilyn A. Walker
4
, Miriam K. Konkel
4
, Vallmer E. Jordan
4
,
Cody J. Steely
4
, Thomas O. Beckstrom
4
, Gregg W.C. Thomas
5
, Kymberleigh A.
Pagel
6
, Vikas Pejaver
6
, Claudia R. Catacchio
7
, Nicoletta Archidiacono
7
, Mario
Ventura
7
, Alessia Marra-Campanale
7
, Antonio Palazzo
7
, Oronzo Capozzi
7
,
Archana Raja
8
, John Huddleston
8
, Veronica Searles Quick
9
, Anis Karimpour-
Fard
9
, Dominik Schrempf
10
, Marc de Manuel Montero
11
, Konstantinos Billis
12
,
Fergal J. Martin
12
, Matthieu Muffato
12
, Georgios Athanasiadis
13
, Christina Ber-
gey
14
, Andrew Burrell
15
, Jade Cheng
13
, Laura Cox
16
, James Else
17
, Yi Han
1
,
Gisela H. Kopp
18,19
, Maximilian Kothe
20
, Kalle Leppälä
13
, Angela Noll
19
, Jera
Pecotte
21
, Lenore Pipes
22
, Karen Rice
21
, Christopher E. Mason
22
, Todd Diso-
tell
15
, Jane Phillips-Conroy
23
, Lutz Walter
20
, Kasper Munch
13
, Thomas Mai-
lund
13
, Mikkel Schierup
13
, Carolin Kosiol
10
, Tomas Vinar
24
, James M. Sikela
9
,
Dietmar Zinner
18
, Christian Roos
19
, Clifford J. Jolly
15
, Predrag Radivojac
6
, Ros-
coe Stanyon
25
, Mariano Rocchi
7
, Evan E. Eichler
8,26
, Bronwen Aken
12
, Matthew
W. Hahn
5
, Mark A. Batzer
4
, Tomas Marques-Bonet
11
, Jenny Tung
3
, Donna M.
Muzny
1
, Richard A. Gibbs
1,2
, Kim C. Worley
1,2
.
Author Affiliations by Institution.
1
Human Genome Sequencing Center, Baylor College of Medicine.
2
Dept. of Molecular and Human Genetics, Baylor College of Medicine.
3
Dept. of Evolutionary Anthropology, Duke University.
4
Dept. of Biological Sciences, Louisiana State University.
5
Dept. of Biology, Indiana University.
6
Dept. of Computer Science and Informatics, Indiana University.
7
Dept. of Biology, University of Bari, Bari, Italy.
8
Dept. of Genome Sciences, Univ. of Washington.
9
Dept. of Biochemistry and Molecular Genetics, Univ of Colorado Anschutz
Medical Campus.
10
Institute of Population Genetics, University of Veterinary Medicine, Vienna.
11
ICREA at Institut de Biologia Evolutiva, Universitat Pompeu Fabra, Barcelona.
12
European Molecular Biology Laboratory, European Bioinformatics Institute,
Hinxton.
13
Bioinformatics Research Center, Aarhus University, Aarhus.
14
Dept. of Biological Sciences, Norte Dame University.
15
Dept. of Anthropology, New York University.
16
Dept. of Genetics, Texas Biomedical Research Institute.
17
Emory University.
18
Cognitive Ethology Laboratory, German Primate Center, Gottingen.
19
Dept. of Biology, Univ. of Konstanz, Konstanz.
20
Primate Genetics Laboratory, German Primate Center, Gottingen.
21
Southwest National Primate Research Center, Texas Biomedical Research
Institute.
22
Dept. of Physiology and Biophysics, Weill Cornell Medical College, New
York and HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Inst. of
Computational Biomedicine, Weill Cornell Medicine.
23
Dept. of Neuroscience, Washington Univ. School of Medicine and Dept. of
Anthropology, Washington University.
24
Faculty of Mathematics, Physics and Informatics, Comenius University,
Bratislava.
25
Dept. of Biology, University of Florence, Florence, Italy.
26
Howard Hughes Medical Institute.
Authors’contributions
VEJ, JAW, PG-Q and MAB designed the research and wrote the paper; JAW,
VEJ, CJS, PG-Q, CPS, LCR, CER and JMS conducted the experiments and ana-
lyzed the results; MKK and TOB performed the Alu repeat analysis of the
[Panu_2.0] genome assembly; JMS designed in-house python scripts for data
filtering and analyzed the results; VEJ, CJS and TOB performed the computa-
tional analysis of the whole genome sequencing data downloaded from the
Baylor College of Medicine Human Genome Sequencing Center File Transfer
Database; JR contributed the research samples, supervised the work of the
Baboon Genome Consortium and edited the manuscript; CJJ revised the
manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by National Institutes of Health Grant R01 GM59290
(M.A.B.).
Availability of data and materials
The algorithms used in this study are available on GitHub (https://github.
com/papioPhlo/polyDetect). The Additional Information files are available on
the online version of this paper and through the Batzer Lab website under
publications, https://biosci-batzerlab.biology.lsu.edu/. Additional file 1is an
Excel file containing a WGS sample list, Additional file 1: Tables S1-S4, Add-
itional file 1: Figure S1 and worksheet “Papio-Theropithecus.”Additional file 2
is an Excel file with worksheets for DNA samples, oligonucleotide PCR
primers, genomic coordinates, genotype data for the PCR experiments, allele
frequency and RepeatMasker output. Additional file 3is an Excel file summar-
izing the PCR validation of computational predictions. Additional file 4is an
Excel file with a list of the 27,700 T. gelada / Papio shared Alu insertions.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Biological Sciences, Louisiana State University, 202 Life
Sciences Building, Baton Rouge, Louisiana 70803, USA.
2
Human Genome
Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA.
3
Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA.
4
Department of Anthropology, New York University,
New York, NY 10003, USA.
5
Department of Genetics & Biochemistry, Clemson
Center for Human Genetics, Clemson, SC 29634, USA.
Received: 10 September 2019 Accepted: 1 November 2019
References
1. Geoffroy S-HI. Description des Mammifères Nouveaux ou Imparfaitement
Connus de la Collection du Muséum d’Histoire Naturelle, et Remarques sur
la Classification et les Caractères des Mammifères. Arch Mus Hist Nat Paris.
1843;2:485–592.
2. Jablonski NG. Theropithecus: the rise and fall of a primate genus.
Cambridge: Cambridge University press, 2005; 2005.
3. Groves C. Primate taxonomy. Washington, DC: Smithsonian Institution Press;
2001.
4. Jolly CJ. Species, subspecies and baboon systematics. In: LBM WHK, editor.
Species, Species concepts and Primate Evolution. New York: Plenum Press;
1993. p. 67–107.
5. Zinner D, Groeneveld LF, Keller C, Roos C. Mitochondrial phylogeography of
baboons (Papio spp.): indication for introgressive hybridization? BMC Evol
Biol. 2009;9:83.
6. Rogers J, Raveendran M, Harris RA, Mailund T, Leppala K, Athanasiadis G,
Schierup MH, Cheng J, Munch K, Walker JA, et al. The comparative
genomics and complex population history of Papio baboons. Sci Adv. 2019;
5(1):eaau6947.
7. Zinner D, Wertheimer J, Liedigk R, Groeneveld LF, Roos C. Baboon
phylogeny as inferred from complete mitochondrial genomes. Am J Phys
Anthropol. 2013;150(1):133–40.
8. Jolly CJ, Burrell AS, Phillips-Conroy JE, Bergey C, Rogers J. Kinda
baboons (Papio kindae) and grayfoot chacma baboons (P. ursinus
griseipes) hybridize in the Kafue river valley, Zambia. Am J Primatol.
2011;73(3):291–303.
9. Newman TK, Jolly CJ, Rogers J. Mitochondrial phylogeny and systematics of
baboons (Papio). Am J Phys Anthropol. 2004;124(1):17–27.
10. Phillips-Conroy JE, Jolly CJ, Brett FL. Characteristics of hamadryas-like male
baboons living in anubis baboon troops in the awash hybrid zone. Ethiopia
Am J Phys Anthropol. 1991;86(3):353–68.
Walker et al. Mobile DNA (2019) 10:46 Page 11 of 13
11. Rogers J, Kidd KK. Nuclear DNA polymorphisms in a wild population of
yellow baboons (Papio hamadryas cynocephalus) from Mikumi National
Park. Tanzania Am J Phys Anthropol. 1993;90(4):477–86.
12. Swedell L. African papionins: Diversity of social organization and ecological
flexibility. In: AF CJC, MacKinnon KC, Bearder SK, Stumpf RM, editors.
Primates in Perspective. 2nd ed. New York: Oxford Univ. Press; 2011.
13. Cheney DL, Seyfarth RM. Baboon Metaphysics: The Evolution of a Social
Mind. Chicago: Univ. of Chicago Press; 2008.
14. Kingdon J, Butynski T. Mammals of Africa: Volume 2 Primates. New York:
A&C Black Publ; 2013.
15. Bergman TJ, Kitchen DM. Comparing responses to novel objects in wild
baboons (Papio ursinus) and geladas (Theropithecus gelada). Anim Cogn.
2009;12(1):63–73.
16. Dunbar R, Dunbar P. Social dynamics of gelada baboons. Contrib Primatol.
1975;6:1–157.
17. Gippoliti S. Theropithecus gelada distribution and variations related to
taxonomy: history, challenges and implications for conservation. Primates; J
Primatol. 2010;51(4):291–7.
18. Snyder-Mackler N, Alberts SC, Bergman TJ. The socio-genetics of a complex
society: female gelada relatedness patterns mirror association patterns in a
multilevel society. Mol Ecol. 2014;23(24):6179–91.
19. Tinsley Johnson E, Snyder-Mackler N, Lu A, Bergman TJ, Beehner JC. Social
and ecological drivers of reproductive seasonality in geladas. Behavior Ecol.
2018;29(3):574–88.
20. Zinner D, Atickem A, Beehner JC, Bekele A, Bergman TJ, Burke R,
Dolotovskaya S, Fashing PJ, Gippoliti S, Knauf S, et al. Phylogeography,
mitochondrial DNA diversity, and demograph ic history of geladas
(Theropithecus gelada). PLoS One. 2018;13(8):e0202303.
21. Delson E. Theropithecus fossils from Africa and India and the taxonomy of
the genus. In: Theropithecus: the rise and fall of a primate genus.
Cambridge: Cambridge University Press; 1993. p. 157–89.
22. Gilbert CC, Frost SR, Pugh KD, Anderson M, Delson E. Evolution of the
modern baboon (Papio hamadryas): a reassessment of the African Plio-
Pleistocene record. J Hum Evol. 2018;122:38–69.
23. Liedigk R, Roos C, Brameier M, Zinner D. Mitogenomics of the Old World
monkey tribe Papionini. BMC Evol Biol. 2014;14:176.
24. Boissinot S, Alvarez L, Giraldo-Ramirez J, Tollis M. Neutral nuclear
variation in baboons (genus Papio) pr ovides insights into their
evolutionary and demographic histories. Am J Phys Anthropol. 2014;
155(4):621–34.
25. Jolly CJ. A proper study for mankind: Analogies from the Papionin monkeys
and their implications for human evolution. Am J Phys Anthropol. 2001;
Suppl 33:177–204.
26. Jordan VE, Walker JA, Beckstrom TO, Steely CJ, McDaniel CL, St Romain CP,
Worley KC, Phillips-Conroy J, Jolly CJ, Rogers J, et al. A computational
reconstruction of Papio phylogeny using Alu insertion polymorphisms. Mob
DNA. 2018;9:13.
27. Steely CJ, Walker JA, Jordan VE, Beckstrom TO, McDaniel CL, St Romain CP,
Bennett EC, Robichaux A, Clement BN, Raveendran M, et al. Alu insertion
polymorphisms as evidence for population structure in baboons. Genome
Biol Evol. 2017;9(9):2418–27.
28. Szmulewicz MN, Andino LM, Reategui EP, Woolley-Barker T, Jolly CJ, Disotell
TR, Herrera RJ. An Alu insertion polymorphism in a baboon hybrid zone. Am
J Phys Anthropol. 1999;109(1):1–8.
29. Baker JN, Walker JA, Denham MW, Loupe CD 3rd, Batzer MA. Recently
integrated Alu insertions in the squirrel monkey (Saimiri) lineage and
application for population analyses. Mob DNA. 2018;9:9.
30. Li J, Han K, Xing J, Kim HS, Rogers J, Ryder OA, Disotell T, Yue B, Batzer MA.
Phylogeny of the macaques (Cercopithecidae: Macaca) based on Alu
elements. Gene. 2009;448(2):242–9.
31. McLain AT, Meyer TJ, Faulk C, Herke SW, Oldenburg JM, Bourgeois MG,
Abshire CF, Roos C, Batzer MA. An alu-based phylogeny of lemurs
(infraorder: Lemuriformes). PLoS One. 2012;7(8):e44035.
32. Meyer TJ, McLain AT, Oldenburg JM, Faulk C, Bourgeois MG, Conlin EM,
Mootnick AR, de Jong PJ, Roos C, Carbone L, et al. An Alu-based phylogeny
of gibbons (hylobatidae). Mol Biol Evol. 2012;29(11):3441–50.
33. Perna NT, Batzer MA, Deininger PL, Stoneking M. Alu insertion
polymorphism: a new type of marker for human population studies. Hum
Biol. 1992;64(5):641–8.
34. Ray DA, Batzer MA. Tracking Alu evolution in New World primates. BMC
Evol Biol. 2005;5:51.
35. Ray DA, Xing J, Hedges DJ, Hall MA, Laborde ME, Anders BA, White BR,
Stoilova N, Fowlkes JD, Landry KE, et al. Alu insertion loci and platyrrhine
primate phylogeny. Mol Phylogenet Evol. 2005;35(1):117–26.
36. Salem AH, Ray DA, Xing J, Callinan PA, Myers JS, Hedges DJ, Garber RK,
Witherspoon DJ, Jorde LB, Batzer MA. Alu elements and hominid
phylogenetics. Proc Natl Acad Sci U S A. 2003;100(22):12787–91.
37. Stewart C, Kural D, Stromberg MP, Walker JA, Konkel MK, Stutz AM, Urban
AE, Grubert F, Lam HY, Lee WP, et al. A comprehensive map of mobile
element insertion polymorphisms in humans. PLoS Genet. 2011;7(8):
e1002236.
38. Xing J, Wang H, Han K, Ray DA, Huang CH, Chemnick LG, Stewart CB,
Disotell TR, Ryder OA, Batzer MA. A mobile element based phylogeny of
Old World monkeys. Mol Phylogenet Evol. 2005;37(3):872–80.
39. Xing J, Wang H, Zhang Y, Ray DA, Tosi AJ, Disotell TR, Batzer MA. A mobile
element-based evolutionary history of guenons (tribe Cercopithecini). BMC
Biol. 2007;5:5.
40. Xing J, Witherspoon DJ, Ray DA, Batzer MA, Jorde LB. Mobile elements and
primate evolution. Yearb Phys Anthropol. 2007;50:2–19.
41. Batzer MA, Deininger PL. A human-specific subfamily of Alu sequences.
Genomics. 1991;9(3):481–7.
42. Batzer MA, Deininger PL. Alu repeats and human genomic diversity. Nat Rev
Genet. 2002;3(5):370–9.
43. Batzer MA, Stoneking M, Alegria-Hartman M, Bazan H, Kass DH, Shaikh TH,
Novick GE, Ioannou PA, Scheer WD, Herrera RJ, et al. African origin of
human-specific polymorphic Alu insertions. Proc Natl Acad Sci U S A. 1994;
91(25):12288–92.
44. Ray DA, Xing J, Salem AH, Batzer MA. SINEs of a nearly perfect character.
Syst Biol. 2006;55(6):928–35.
45. Stoneking M, Fontius JJ, Clifford SL, Soodyall H, Arcot SS, Saha N, Jenkins T,
Tahir MA, Deininger PL, Batzer MA. Alu insertion polymorphisms and
human evolution: evidence for a larger population size in Africa. Genome
Res. 1997;7(11):1061–71.
46. Cordaux R, Batzer MA. The impact of retrotransposons on human genome
evolution. Nat Rev Genet. 2009;10(10):691–703.
47. Roy-Engel AM, Batzer MA, Deininger PL. Evolution of Human
Retrosequences: Alu. In: Encyclopedia of Life Sciences (ELS); 2008. p. 1–4.
48. Luan DD, Korman MH, Jakubczak JL, Eickbush TH. Reverse transcription of
R2Bm RNA is primed by a nick at the chromosomal target site: a
mechanism for non-LTR retrotransposition. Cell. 1993;72(4):595–605.
49. Li H. Aligning sequence reads, clone sequences and assembly contigs with
BWA-MEM. Genomics. 2013;1303:1–3.
50. Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J.
Repbase update, a database of eukaryotic repetitive elements. Cytogenet
Genome Res. 2005;110(1–4):462–7.
51. Langmead B, Salzberg SL. Fast gapped-read alignment with bowtie 2. Nat
Methods. 2012;9(4):357–9.
52. Steely CJ, Baker JN, Walker JA, Loupe CD 3rd, Batzer MA. Analysis of lineage-
specific Alu subfamilies in the genome of the olive baboon, Papio anubis.
Mob DNA. 2018;9:10.
53. Ken t WJ. BLAT-- the BLAST-like alignment tool. Genome Res. 2002;12(4):
656–64.
54. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D.
The human genome browser at UCSC. Genome Res. 2002;12(6):996–1006.
55. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for
biologist programmers. Methods Mol Biol. 2000;132:365–86.
56. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M,
Rozen SG. Primer3--new capabilities and interfaces. Nucleic Acids Res.
2012;40(15):e115.
57. Konkel MK, Walker JA, Batzer MA. LINEs and SINEs of primate evolution. Evol
Anthropol. 2010;19:236–49.
58. RepeatMasker Open-3.0. [http://www.repeatmasker.org]. Accessed Nov 2019.
59. Bennett EA, Keller H, Mills RE, Schmidt S, Moran JV, Weichenrieder O, Devine
SE. Active Alu retrotransposons in the human genome. Genome Res. 2008;
18(12):1875–83.
60. Konkel MK, Walker JA, Hotard AB, Ranck MC, Fontenot CC, Storer J, Stewart
C, Marth GT, Batzer MA. Sequence analysis and characterization of active
human Alu subfamilies based on the 1000 genomes pilot project. Genome
Biol Evol. 2015;7(9):2608–22.
61. Delson E, Thomas H, Spassov N. Fossil Old World monkeys (Primates,
Cercopithecidae) from the Pliocene of Dorkovo. Bulgaria Geodiversitas.
2005;27(1):159–66.
Walker et al. Mobile DNA (2019) 10:46 Page 12 of 13
62. Jolly CJ. The classification and natural history of Theropithecus
(Simopithecus) baboons of the African Plio-Pleistocene. Bull Brit Mus (Nat
Hist) Geology. 1972;22(1):1–123.
63. Dunbar RI, Dunbar P. On hybridization between Theropithecus gelada and
Papio anubis in the wild. J Hum Evol. 1974;3(3):187–92.
64. Jolly CJ, Woolley-Barker T, Beyene S, Disotell TR, Phillips-Conroy JE.
Intergeneric hybrid baboons. Int J Primatol. 1997;18(4):597–627.
65. Hedges DJ, Cordaux R, Xing J, Witherspoon DJ, Rogers AR, Jorde LB, Batzer
MA. Modeling the amplification dynamics of human Alu retrotransposons.
PLoS Comput Biol. 2005;1(4):e44.
66. Jones T, Ehardt CL, Butynski TM, Davenport TR, Mpunga NE, Machaga SJ, De
Luca DW. The highland mangabey Lophocebus kipunji: a new species of
African monkey. Science. 2005;308(5725):1161–4.
67. Davenport TR, Stanley WT, Sargis EJ, De Luca DW, Mpunga NE, Machaga SJ,
Olson LE. A new genus of African monkey, Rungwecebus: morphology,
ecology, and molecular phylogenetics. Science. 2006;312(5778):1378–81.
68. Olson LE, Sargis EJ, Stanley WT, Hildebrandt KB, Davenport TR. Additional
molecular evidence strongly supports the distinction between the recently
described African primate Rungwecebus kipunji (Cercopithecidae, Papionini)
and Lophocebus. Mol Phylogenet Evol. 2008;48(2):789–94.
69. Burrell AS, Jolly CJ, Tosi AJ, Disotell TR. Mitochondrial evidence for the
hybrid origin of the kipunji, Rungwecebus kipunji (Primates: Papionini). Mol
Phylogenet Evol. 2009;51(2):340–8.
70. Roberts TE, Davenport TR, Hildebrandt KB, Jones T, Stanley WT, Sargis EJ,
Olson LE. The biogeography of introgression in the critically endangered
African monkey Rungwecebus kipunji. Biol Lett. 2010;6(2):233–7.
71. Zinner D, Chuma IS, Knauf S, Roos C. Inverted intergeneric introgression
between critically endangered kipunjis and yellow baboons in two disjunct
populations. Biol Lett. 2018;14(1):20170729.
72. Harris EE, Disotell TR. Nuclear gene trees and the phylogenetic relationships
of the mangabeys (Primates: Papionini). Mol Biol Evol. 1998;15(7):892–900.
73. Guevara EE, Steiper ME. Molecular phylogenetic analysis of the Papionina
using concatenation and species tree methods. J Hum Evol. 2014;66:18–28.
74. Harris EE. Molecular systematics of the old world monkey tribe papionini:
analysis of the total available genetic sequences. J Hum Evol. 2000;38(2):
235–56.
75. Gao B, Wang S, Wang Y, Shen D, Xue S, Chen C, Cui H, Song C. Low
diversity, activity, and density of transposable elements in five avian
genomes. Funct Integr Genomics. 2017;17(4):427–39.
76. Lammers F, Blumer M, Ruckle C, Nilsson MA. Retrophylogenomics in
rorquals indicate large ancestral population sizes and a rapid radiation. Mob
DNA. 2019;10:5.
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Walker et al. Mobile DNA (2019) 10:46 Page 13 of 13
