Mitogenomic phylogeny of the common long-tailed macaque (Macaca fascicularis fascicularis)
Liedigk et al. BMC Genomics
Mitogenomic phylogeny of the common long- tailed macaque (Macaca fascicularis fascicularis)
Rasmus Liedigk 0
Jakob Kolleck 0
Kai O Bker 0
Badrul Munir Md-Zain
Muhammad Abu Bakar Abdul-Latiff
Anthony J Tosi
Markus Brameier 0
Christian Roos 0
0 Primate Genetics Laboratory, German Primate Center, Leibniz Institute for Primate Research , Kellnerweg 4, 37077 Gottingen , Germany
Background: Long-tailed macaques (Macaca fascicularis) are an important model species in biomedical research and reliable knowledge about their evolutionary history is essential for biomedical inferences. Ten subspecies have been recognized, of which most are restricted to small islands of Southeast Asia. In contrast, the common long-tailed macaque (M. f. fascicularis) is distributed over large parts of the Southeast Asian mainland and the Sundaland region. To shed more light on the phylogeny of M. f. fascicularis, we sequenced complete mitochondrial (mtDNA) genomes of 40 individuals from all over the taxon's range, either by classical PCR-amplification and Sanger sequencing or by DNA-capture and high-throughput sequencing. Results: Both laboratory approaches yielded complete mtDNA genomes from M. f. fascicularis with high accuracy and/or coverage. According to our phylogenetic reconstructions, M. f. fascicularis initially diverged into two clades 1.70 million years ago (Ma), with one including haplotypes from mainland Southeast Asia, the Malay Peninsula and North Sumatra (Clade A) and the other, haplotypes from the islands of Bangka, Java, Borneo, Timor, and the Philippines (Clade B). The three geographical populations of Clade A appear as paraphyletic groups, while local populations of Clade B form monophyletic clades with the exception of a Philippine individual which is nested within the Borneo clade. Further, in Clade B the branching pattern among main clades/lineages remains largely unresolved, most likely due to their relatively rapid diversification 0.93-0.84 Ma. Conclusions: Both laboratory methods have proven to be powerful to generate complete mtDNA genome data with similarly high accuracy, with the DNA-capture and high-throughput sequencing approach as the most promising and only practical option to obtain such data from highly degraded DNA, in time and with relatively low costs. The application of complete mtDNA genomes yields new insights into the evolutionary history of M. f. fascicularis by providing a more robust phylogeny and more reliable divergence age estimations than earlier studies.
Southeast Asia; Sundaland; Sanger sequencing; High-throughput sequencing; DNA-capture
Macaques (genus Macaca) represent one of the most
successful extant primate radiations. They colonized a
large geographic range, from continents to islands,
making them unique among non-human primates . Fossils
indicate that they arose in northern Africa around 7
million years ago (Ma) . During their expansion into Asia
in the late Miocene, the genus diversified into various
species groups and species that have been defined by
morphological, behavioral and molecular characters, and
by their geographic distribution [2-9].
Taxonomic and phylogenetic affiliations of the various
macaque species have been matter of debate for several
decades [1-7,10]. According to current classifications the
genus comprises 22 species, which are divided into seven
species groups [9,11]; among them three monotypic
species groups, (1) the M. sylvanus group, (2) the M.
arctoides group and (3) the M. fascicularis group, and four
polytypic groups, (4) the Sulawesi macaques group with
six species, (5) the M. mulatta group with three species,
(6) the M. sinica group with five species and (7) the M.
silenus group with five species. As with the other species
groups, the species composition of the M. fascicularis
group has changed over time. Delson  and Fooden [3,4]
included four species (M. mulatta, M. cyclopis, M. fuscata,
M. fascicularis), but Groves  moved M. mulatta, M.
cyclopis and M. fuscata in their own species group, the M.
mulatta group, and integrated M. arctoides in the M.
fascicularis group. Zinner et al.  likewise recognized the
members of the M. mulatta group as a distinct species
group and additionally excluded M. arctoides proposing a
monotypic M. fascicularis group.
The long-tailed macaque (M. fascicularis) has the most
discontinuous, and beside rhesus macaques, the largest
distribution of all macaque species. Its range covers the
southern part of the Southeast Asian mainland (Bangladesh,
Myanmar, Thailand, Laos, Vietnam, Cambodia, peninsular
Malaysia) as well as most of Sundaland (the islands of
Borneo, Sumatra and Java, and adjacent islands) and
beyond (islands east of the Wallace Line, Philippines)
(Figure 1). On the basis of differences in pelage coloration
and tail length ten subspecies are currently recognized
[6,8,9,11-15]. Three of them (M. f. aureus, M. f.
fascicularis, M. f. philippinensis) have relatively large
distributions, while all others (M. f. atriceps, M. f. condorensis, M.
f. fuscus, M. f. karimondjawae, M. f. lasiae, M. f. tua, M. f.
umbrosus) are restricted to small islands (Figure 1).
However, genetic data are not yet available for a comparative
assessment of this classification; so far, genetic studies
have included only samples from M. f. fascicularis and M.
f. philippinensis. Given the large and discontinuous range
of M. f. fascicularis, it is not surprising that (genetic)
variation within this subspecies is high [16-26]. In fact,
there is a deep genetic differentiation between M. f.
Figure 1 Geographical distribution of long-tailed macaques (Macaca fascicularis) and geographical origin of samples. Species and subspecies
distributions are depicted according to  and adapted from . The distribution of M. f. aureus, M. f. fascicularis and M. f. philippinensis is indicated
by black, dark grey and light grey regions, respectively; the hatched region indicates the transition zone between the latter two subspecies. Subspecies
on small islands are named in the map as are countries and islands mentioned in the text. Open and filled circles indicate approximate and
exact geographical origin of M. f. fascicularis samples, respectively. ID numbers correspond to those in Figure 2 and Additional file 2: Table S2.
fascicularis from the Asian mainland and from Sundaland
[16-21,23-26]. Interestingly, on Sumatra, Y-chromosomal
lineages from both, mainland and Sundaland are found
. Besides the genetic variation among local long-tailed
macaque populations, molecular studies have also
revealed significant gene flow from rhesus macaques (M.
mulatta) into Indochinese M. f. fascicularis [17,27-29].
Recent genome data indicate that around 30% of the Asian
mainland M. fascicularis genome is of rhesus macaque
origin . This ancient hybridization (gene flow) most
likely occurred unidirectional (male-mediated), from
rhesus into long-tailed macaques and not vice versa, i.e.
analyses of maternally inherited markers such as
mitochondria will not resolve the question of hybridization
[17,27,28,30]. Even today, hybridization between both
species occurs in a wide hybrid zone running from Vietnam,
through Laos, Thailand, and probably into Myanmar
[13,31]. Since the long-tailed macaque is an important
model organism in biomedical research, reliable
knowledge about its evolutionary history and genetic
composition is essential for biomedical inferences, particularly
since local populations show extreme genetic,
physiological and behavioral variation (for an overview see ).
The geographic origin of M. fascicularis and dispersal
scenarios that led to its current distribution are still a
matter of debate. Delson  suggested that macaques
entered Sundaland, probably in the Pliocene, during
periods of low sea level and ancestral M. fascicularis
became isolated there when rising sea levels and geological
activity fragmented Sundaland. During the Pleistocene,
M. fascicularis extended its range again [2,14]. This
largely corresponds to the observed higher level of
nucleotide diversity found in long-tailed macaque
populations from Sundaland compared to the populations from
the Asian mainland and Malay Peninsula [18,23]. This
scenario is also in agreement with the fact that the
earliest fossils of M. fascicularis, or at least those of a close
relative, were found on Java [2,14,33]. Currently, the
species is also found on islands that were never connected
to the Asian mainland or Sundaland, including islands
beyond the Wallace line (e.g., Lombok, Sumbawa, Flores,
Timor) and the Philippines. Accordingly, it was assumed
that humans introduced M. fascicularis to the islands
east of the Wallace line ca. 4,000 year ago , while the
Philippines were most likely naturally colonized during
two independent immigration events . The species
survival in areas, where it has been introduced by
humans (e.g., Hong Kong, Taiwan, Papua New Guinea,
New Britain, and various Pacific islands), indicates its
significant ecological plasticity. Long-tailed macaques
are naturally adapted to riverine and coastal
environments such as mangrove and gallery forests [34,35].
They primarily feed on fruits and seeds , but as
indicated by one of its common names, crab-eating macaque,
they also include crabs, shrimps, clams and fish in their
diet . They are able to swim and even to dive .
Hence, it is likely that long-tailed macaques were able to
cross short distances between islands actively.
The objective of this study is to shed more light on the
phylogeny of M. f. fascicularis, the most widespread
subspecies of the long-tailed macaques, occurring on the
Southeast Asian mainland and Sundaland islands,
including parts of the Philippines and east as far as Timor.
Therefore, we generated complete mitochondrial (mtDNA)
genomes from 40 long-tailed macaque individuals either by
traditional polymerase chain reaction (PCR) amplification
followed by Sanger sequencing or by DNA-capture and
high-throughput sequencing. We expect that the analysis
of complete mtDNA genomes provides a better resolution
of phylogenetic relationships among lineages than only
short mtDNA fragments.
We generated 42 complete mtDNA genome sequences
from 40 M. fascicularis individuals, either by classical
PCR followed by Sanger sequencing (10 individuals) or
by DNA-capture and high-throughput sequencing (32
individuals). For two museum samples (IDs: 20, 31) both
methods were applied which yielded sequences with
100% identity, thus indicating similarly high accuracy of
both methods. For mtDNA genomes that were captured
and sequenced on the Ion PGM sequencing platform we
obtained an average of 97,583 (12,599-230,683) trimmed
reads with an average read length of 96 bp, resulting in
an average 285-fold coverage. Sequences in the
overlapping parts were identical and all protein-coding genes
were correctly translated without any premature stop
codons, indicating that no nuclear mitochondrial-like
sequences (numts) are present in our dataset. All newly
generated mtDNA genomes had a length of 16,561 to
16,567 bp, and consisted of 22 transfer RNA genes, 2
ribosomal RNA genes, 13 protein coding genes and the
For phylogenetic analysis, we generated an alignment
including the 40 newly generated and 20 additional
mtDNA genomes downloaded from Genbank. The
resulting alignment had a length of 16,874 bp, but was reduced
to 15,868 bp after indels and poorly aligned positions were
removed. Some M. f. fascicularis individuals shared the
same haplotype (IDs: 21 = 23, 27 = 28 = 31, 33 = 34). These
were excluded resulting in a final alignment of 56 unique
primate mtDNA genome haplotypes. Both alignments
are available for download (Supplementary data ).
Phylogenetic trees as obtained from maximum-likelihood
(ML) and Bayesian analyses are nearly identical and most
nodes are strongly supported (ML bootstrap values: > 95%,
Bayesian posterior probabilities: 1.0; Figure 2). According to
estimated divergence ages, Hominidae and Cercopithecidae
Figure 2 Ultrametric tree showing phylogenetic relationships and divergence ages among macaques as calculated from complete mtDNA
genome sequences (relationships among non-macaque taxa not shown). Red dots indicate ML bootstrap support of > 95% and Bayesian posterior
probabilities of 1.0; values below are given at respective nodes. Blue bars indicate 95% CIs of divergence times and the time scale below shows million
years before present. IDs correspond to those in Figure 1 and Additional file 2: Table S2. For detailed information about divergence ages and 95% CIs
see Additional file 1: Table S1.
separated 28.60 (95% credibility interval [CI]: 25.31-31.78)
million years ago (Ma) (for estimates and their 95% CIs see
Figure 2 and Additional file 1: Table S1). Among hominids,
Pongo diverged from the Homo + Pan clade 13.82
(12.6814.86) Ma, while the latter split 6.32 (5.73-6.89) Ma. Among
cercopithecids, Colobus diverged first, 19.89 (16.17-23.87)
Ma, and Chlorocebus separated from papionins 12.81
(10.59-15.22) Ma. In the Papionini clade, Theropithecus and
Papio diverged from macaques 10.90 (8.92-12.90) Ma,
while the former two genera split 4.77 (3.87-5.72) Ma.
Within macaques, the African M. sylvanus branched off
first, 6.10 (5.23-6.92) Ma. The remaining, solely Asian
macaque species, diverged into two clades 5.49 (4.69-6.34) Ma,
one comprising M. silenus and M. tonkeana, and the other
M. thibetana, M. arctoides, M. mulatta and M. fascicularis.
In the former clade, M. silenus and M. tonkeana separated
3.70 (2.80-4.54) Ma, while in the latter clade M. thibetana
split off first, 4.16 (3.47-4.85) Ma, followed by M.
fascicularis 3.42 (2.83-4.01) Ma, before finally M. mulatta and M.
arctoides diverged 3.02 (2.42-3.60) Ma. Within M.
fascicularis, an initial split occurred 1.70 (1.36-2.04) Ma,
separating haplotypes from mainland Southeast Asia, Peninsula
Malaysia and North Sumatra (Clade A), and haplotypes
from Borneo, Java, Bangka, Timor, the Philippines and
Mauritius (Clade B). In Clade A, individuals from mainland
Southeast Asia, Peninsula Malaysia and North Sumatra do
not form reciprocally monophyletic clades. Splitting events
in Clade A started 0.96 (0.78-1.16) Ma. In Clade B,
individuals from different geographic regions form monophyletic
clades or represent distinct lineages. The only exception is
the Borneo clade, which also includes the individual from
the Philippines (ID: 42). In clade B, the branching pattern
among main clades/lineages remains unresolved (ML
bootstrap values: <50-50%, Bayesian posterior probabilities:
0.56-0.74) indicating a diversification within a short time
period. In fact, this radiation was estimated to have
occurred between 0.93 Ma (0.74-1.12) and 0.84 (0.67-1.02)
Ma, thus in less than 100,000 years. Individuals from
Bangka (IDs: 17, 18), an island east of Sumatra (Figure 1),
form a monophyletic clade and separated from the Borneo/
Philippines clade 0.61 (0.47-0.75) Ma. The Philippine
individual is nested within the Borneo clade and specifically
clusters with an individual from Sabah (ID: 41); they
diverged from each other 0.21 (0.15-0.28) Ma.
By applying different methods, classical PCR
amplification followed by Sanger sequencing and DNA-capture
with subsequent high-throughput sequencing, we
successfully obtained complete mtDNA genome data from
40 M. fascicularis individuals. Both methods have proven
to be useful to gain such data with similarly high
accuracy, but the DNA-capture and high-throughput
sequencing approach is less costly and time consuming [40-42].
Moreover, DNA extracted from museum material and
fecal material is normally highly degraded. Fortunately,
some of our museum and fecal samples contained DNA
in sufficient quality so that the complete mtDNA genome
could be amplified via 21 overlapping PCRs. Usually, due
to the high degree of DNA degradation, generating
complete mtDNA genomes would require a much larger
number of PCR amplifications. In contrast, DNA-capture
does not need a certain DNA fragment size, because any
size of DNA fragment can be captured and subsequently
sequenced. However, degraded DNA should be solely
investigated in special laboratories and with various
precautions to prevent contamination.
Our results concerning the phylogenetic relationships
among macaque and non-macaque taxa and estimated
divergence ages are largely in line with previous
molecular studies [5,7,10,19,43-48]. For the phylogenetic
relationships among M. fascicularis haplotypes, we obtained
higher statistical support for most nodes in our tree, as
compared to earlier mtDNA studies which used only
fragments of the mtDNA genome [5,7,17-20,22].
Nevertheless, some nodes in our study are still missing
significant statistical support, thus leaving some phylogenetic
relationships, in particular those between populations from
Timor, Java, Mauritius and Bangka/Borneo/Philippines,
unresolved. Such results are common when clades or
lineages diverged within a short time period [42,48-52].
In contrast to Tosi and Coke  who found
Sumatran individuals to be part of the Sundaland clade
(referring to our Clade B), the Sumatran individuals, which
we analyzed, are nested within the Asian mainland clade
(referring to our Clade A). A possible explanation for
this contradiction is the different geographic origin of
studied individuals, with the South Sumatran samples of
Tosi and Coke  clustering with Sundaland sequences
and our North Sumatran samples clustering with
mainland sequences. Thus, both major M. f. fascicularis
mtDNA clades are likely to be present on Sumatra
similar to the presence of both Y chromosomal haplogroups
on the island .
Since mtDNA is only inherited via the maternal line
and macaques live mainly in female philopatric societies
[53,54], mtDNA data can be utilized to reveal insights
into genetic differences among regional populations and
to trace their phylogeographic history . According to
our phylogenetic reconstruction and estimated
divergence ages, M. f. fascicularis initially split into two clades
1.70 (1.36-2.04) Ma, with representatives of both
lineages being found today on Sumatra (according to our
study and ). Possible explanations therefore are (1)
Sumatra is the place of origin of M. f. fascicularis, (2)
Sumatra is the place of origin of only Sundaland M. f.
fascicularis, while long-tailed macaques from the
mainland invaded the island later, or (3) long-tailed macaques
on Sumatra became extinct and the island was later
recolonized from the mainland and other Sundaland
islands. The hypothesis that Sumatra is the place of M. f.
fascicularis origin is supported by the observed high
mtDNA diversity found on the island compared to other
regions where the subspecies occurs (e.g., ). However,
not in support of this hypothesis is the paraphyly of
haplotypes from the mainland and Malay Peninsula, and the
respective branching pattern among them and the Sumatra
haplotypes, which suggests that the northern Sumatra
population came in from the mainland. To test whether
Sumatra or any other island, e.g., Java [2,18] is the place of
origin of Sundaland or all M. f. fascicularis populations,
needs further investigations and, particularly, should
include data of M. f. fascicularis from southern Sumatra.
As in previous studies [7,19,26], we found long-tailed
macaques from the Philippines clustering within the
Borneo clade. Since the Bornean individual, which is
most closely related to the Philippine specimens, is from
the furthest east of Borneo (Sabah, Tawau Hill Park), this
branching pattern fosters the previously proposed
hypothesis of a colonization of the Philippines via Borneo
[1,26]. Within the last million years, the Philippines have
never been connected to the Southeast Asian mainland
or Borneo via a continuous land bridge . One
possible exception is the island of Palawan, which has been
considered to have had a land connection to Borneo
during sea level low-stands in the late Pleistocene .
The previously proposed Philippine colonization
hypothesis [1,26] via Palawan and appending islets seems
plausible (stepping-stone colonization). A recent study suggests
that there may have been at least two dispersal events
from Borneo into the Philippines, first one via Palawan
resulting in M. f. philippinensis found in the north of the
Philippine Archipelago (Figure 1), and a later one via the
Sulu Archipelago that resulted in M. f. fascicularis in
the south . Our Philippine sample most likely belongs
to this southern taxon.
One noteworthy outcome from our study is the early
divergence of a monophyletic Timor clade within the
Sundaland clade (Figure 2). It appears that this clade
diverged some 0.93 (1.12-0.74) Ma from the other
Sundaland lineages. This finding is supported by an analysis of
blood protein polymorphisms from samples across the
Indonesian and Timor island arc, indicating that
populations east of the Wallace Line (Lombok and Sumbawa)
have greatly differentiated from those to the west .
Our mtDNA-based estimate, however, significantly
predates the earliest finds of macaques in Timors
archaeological record, which appear at the same time, i.e. as the
first evidence of pottery and domesticated pig in one site
a few thousand years ago, indicating human
translocations . Similarly, on Flores, an island further west,
but still east of the Wallace Line, long-tailed macaques
only appear in the archaeological record around 7,000 years
ago . It is unclear what underlies the apparent major
discrepancy between the present phylogenetic analysis and
the zooarchaeological record, but an introduction by
humans as proposed by Fooden  seems unlikely,
although the possibility remains that the detected Timor
haplotypes originated from somewhere else in Sundaland,
a place that was not sampled in our study.
Both applied laboratory methods have proven to be
powerful to generate complete mtDNA genome data with
similarly high accuracy, with the DNA-capture and
highthroughput sequencing approach as the most promising
and only practical option to obtain such data from highly
degraded DNA, fast and relatively cheap. Our study
provides new insights into the evolutionary history of M. f.
fascicularis, most prominent we obtained first evidence
for the presence of haplotypes in North Sumatra that
are related to Asian mainland haplotypes and the clearly
distinct and phylogenetically old Timor clade. However,
to fully resolve the phylogeny of long-tailed macaques,
to identify their origin and the dispersal routes leading
to their current distribution, to assess their full genetic
diversity and to explore to which extent secondary gene
flow occurred between local populations, it is
fundamental to include further M. f. fascicularis populations
from throughout their range into future studies. In these
studies both, mitochondrial and a large number of
nuclear loci, should be analyzed. Moreover, to fully
understand the evolutionary history of the species, the other
subspecies of M. fascicularis should be incorporated in
such studies as well. Since long-tailed macaques are an
important model species in biomedical research and
considering intra-specific variation in genetics,
physiology and behavior, more attention should be paid to
the selection of study specimens.
Blood samples were taken during routine health checks
by experienced veterinarians and not specifically for this
study. All research complied with protocols approved by
the Animal Welfare Body of the DPZ in Germany and the
Department of Wildlife and National Parks in Malaysia,
and adhered to the legal requirements of the countries,
in which research was conducted. The study was carried
out in compliance with the principles of the American
Society of Primatologists for the ethical treatment of
nonhuman primates (https://www.asp.org/society/resolutions/
EthicalTreatmentOfNonHumanPrimates.cfm). No animals
were sacrificed for this study.
We collected and sequenced mtDNA genomes from 40
long-tailed macaque individuals originating from 16 sites
throughout the species range in Southeast Asia and
Sundaland, and from the introduced population on
Mauritius (Figure 1, Additional file 2: Table S2).
Thirtyone of our samples (sample IDs: 4, 5, 1131, 3340)
derived from museum specimens housed in the Bavarian
State Collection of Zoology (ZSM) in Munich, Germany.
Respective specimens were collected between 1904 and
1949. Dried muscle tissue attached to the skeleton was
taken with sterilized scalpels and tweezers, and gloves
and masks were worn during sample collection to avoid
contamination. Museum samples were stored dry in
tubes or plastic envelopes. Additionally, we included
seven fresh fecal samples, stored in 90% ethanol, which
were collected during field surveys (IDs: 610, 32, 41).
We further obtained high-quality DNA extracted from
blood samples from each one individual from Covance
Inc. (Mnster, Germany) and the German Primate
Center (DPZ, Gttingen, Germany), which originated from
the Philippines (ID: 42), and Mauritius (ID: 43),
respectively. For all samples, we tried to obtain information
about the exact geographic provenance, but this was not
always possible. While for all fecal samples, GPS
coordinates were recorded, information about the exact origin
of the samples from the Philippines and Mauritius was
not available. Likewise, we were not able to identify the
exact provenance of five Bornean samples (IDs: 3337,
derived from west coast Borneo), while for all other
museum samples the exact origin could be determined.
Thus, 38 samples can be geographically clearly assigned
to M. f. fascicularis (IDs: 441). The individual from
Mauritius (ID: 43) most likely refers also to M. f.
fascicularis because it is believed that this introduced
population originated from Sumatra or at least from Sundaland
[19,61], while the individual from the Philippines (ID:
42) could be either M. f. philippinensis or M. f.
fascicularis (due to its haplotype it is most likely M. f.
fascicularis). The individuals from Timor refer to the holotype
(ID: 24) and paratypes (IDs: 2531) of Pithecus
fascicularis limitis Schwarz, 1913, which is recognized as
synonym of M. f. fascicularis [6,12]. Beside the 40 samples
mentioned above, we obtained a blood sample from an
additional M. f. fascicularis individual (ID: 3) from
Convance Inc. which originated from Vietnam. The mtDNA
genome of this individual was already published ,
but its DNA was used to prepare baits for DNA-capture.
For detailed sample information see Additional file 2:
For the extraction of total genomic DNA we used two
different methods. First, we applied a kit-based method
using the First-DNA All Tissue kit (Gen-Ial). All fecal
and five of the museum samples (IDs: 11, 14, 20, 31, 38)
were extracted with this method following respective
protocols provided by the company. To avoid and check
for cross-sample contamination, all working steps were
carried out in separate laboratories and under Captair
Bio PCR cabinets (Erlab), gloves and masks were
permanently worn, and negative extraction controls were
routinely performed. Further, samples were treated one
by one, and workbenches were decontaminated with UV
light before and after each extraction. After extraction,
DNA concentration was measured on a NanoDrop
ND1000 spectrophotometer and samples were stored at 20C
until further processing.
Secondly, 28 museum samples (IDs: 4, 5, 12, 13, 15
31, 3337, 39, 40) were extracted in a special ancient
DNA laboratory applying a protocol for nondestructive
DNA extraction [62,63] with slight modifications .
All working steps were carried out in Thermo Scientific
Safe 2020 biological safety cabinets. For each step
(sample preparation, DNA extraction) different cabinets were
used, and before and after each sample, cabinets were
cleaned with DNA decontamination solution and treated
with UV light for at least 30 min. Concentration of
extracted DNAs was measured on a Qubit 2.0 fluorometer
and DNA samples were frozen at 20C until further
processing. For comparative reasons, two museum
samples (IDs: 20, 31) were extracted with both methods.
DNA amplification and Sanger sequencing
We generated complete mtDNA genomes from the
high-quality samples from the Philippines and Mauritius
as well as from three of the fecal samples (IDs: 7, 32, 41)
and five of the museum samples (IDs: 11, 14, 20, 31, 38) by
traditional PCR amplification followed by Sanger
sequencing. All working steps (PCR setup, gel electrophoresis,
PCR product purification, sequencing) were conducted in
separate laboratories and under Captair Bio PCR cabinets
to prevent cross-sample contamination. Further, negative
PCR controls (without template DNA) were routinely
conducted. To minimize the risk of amplifying numts for the
two high-quality DNA samples, we produced two
overlapping long-range PCR products (8 kb and 10 kb) followed
by 21 nested PCRs with product sizes of 1.0-1.2 kb and
an overlap of 100300 bp applying methods described
elsewhere . Since DNA extracted from fecal and
museum samples is usually degraded, the complete
mtDNA genome from these samples was directly
amplified via the 21 PCRs mentioned above and not first via
two long-range PCRs. PCR conditions were the same as
for the nested PCRs above, but sometimes the number
of cycles was increased to 60. As template, we added
1050 ng DNA to the reaction. PCR performance and
product sizes were checked on 1% agarose gels, and
after purification, PCR products were sequenced on an
ABI 3130xL sequencer using the BigDye Terminator
Cycle Sequencing kit (Applied Biosystems) and both
amplification primers. Information on primers and PCR
conditions is available upon request. Sequences were
checked with 4Peaks 1.7.1 (www.mekentosj.com) and
mtDNA genomes were assembled with SeaView 4.4.0
. Annotation was performed with DOGMA  and
DNA-capture and high-throughput sequencing
Complete mtDNA genomes from 28 museum (IDs: 4, 5,
12, 13, 1531, 3337, 39, 40) and four fecal samples
(IDs: 6, 810) were generated using a DNA-capture
approach followed by high-throughput sequencing
according to Maricic et al.  with slight modifications (see
below) to adapt the workflow to the Ion PGM
sequencing system (Ion Torrent). To prevent contamination, all
working steps were carried out in dedicated ancient
DNA and/or special high-throughput sequencing
laboratories, and various negative controls were applied. After
DNA extraction and concentration measurement,
barcoded sequencing libraries were established using the
Ion Plus Fragment Library kit and the Ion Xpress
Barcode Adapters. Adapter ligation and the subsequent
amplification of the samples were performed according
to the protocol for Ion Xpress Plus gDNA Fragment
Library Preparation. Afterwards, we pooled the
adapterligated and amplified libraries in equal concentrations to
a total of 2 g. As bait we used mtDNA genomes of each
one long-tailed macaque individual from Vietnam (ID:
3) and Mauritius (ID: 43). The respective complete
mtDNA genomes were amplified via two overlapping
PCR products (see above). Afterwards, we sheared the
PCR products to an average of ca. 1,000 bp fragments
with a Bioruptor Pico. We diluted 1.5 g of PCR product
to a volume of 150 l, split the sample into three (50 l
each) and sonicated each six times with 10 seconds
ON and 90 seconds OFF. One l of the sheared PCR
product was size-checked on the Agilent 2100
Bioanalyzer with the high sensitivity DNA kit. Fragments were
subsequently end-repaired, biotinylated by ligating the
Bio-T/B adapter , and immobilized on
streptavidincoated beads. Bait and the pooled single-stranded
libraries were combined and four phosphorylated blocking
oligos (BO1.P1.F: CCACTACGCCTCCGCTTTCCTCT
CTATGGGCAGTCGGTGAT-phosphate, BO2.P1.R: AT
TAGTGG-phosphate, BO3.A.F: CCATCTCATCCCTGC
GTGTCTCCGACTCAG-phosphate, BO4.A.R: CTGAGT
added. After 48 h of hybridization at 65C, library
molecules that did not hybridize were washed out and the
enriched library pool was eluted. Subsequently, the
concentration of the enriched library pool was measured by qPCR
(Ion Library Quantitation Kit) and sequenced on the Ion
PGM sequencer using a 316v2 or 318v2 chip and the Ion
PGM Sequencing 400 Kit protocol. The raw sequencing
reads were quality-filtered, and adapters and barcodes were
trimmed with the PGM Torrent Suite Software 4.2. The
extracted reads were initially assembled with the Newbler
program (GS Reference Mapper) of the 454 Sequencing
System Software 2.5 from command line with standard
parameters. The mtDNA genome of the Vietnamese M.
f. fascicularis individual (ID: 3) was used as reference.
Batch processing was done by custom Perl scripts. The
resulting contigs, typically ranging from 1 to 4 sequences
per mtDNA genome, were manually assembled into
genomes with SeaView and annotated with DOGMA. All
gaps between contigs could be closed by combining the
results from multiple sequencing runs.
For phylogenetic reconstructions, we expanded our
dataset with additional mtDNA genome sequences
from macaque and non-macaque taxa derived from
Genbank. The dataset comprised 60 mtDNA genomes
including 43 M. fascicularis individuals (3 from
Genbank including ID: 3), at least one representative of the
other six macaque species groups (2 M. sylvanus, 1 M.
arctoides, 3 M. mulatta, 2 M. thibetana, 1 M. tonkeana,
1 M. silenus) and various outgroup taxa (1 Theropithe
cus gelada, 1 Papio hamadryas, 1 Chlorocebus
pygerythrus, 1 Colobus guereza, 1 Pongo abelii, 1 Pan troglodytes,
1 Homo sapiens). For detailed sample information
and Genbank accession numbers see Additional file 2:
Sequences were aligned with Muscle 3.7  as
implemented in SeaView and manually corrected. Indels and
poorly align positions were removed with Gblocks 0.91b
 using standard settings. Identical sequences were
subsequently excluded (IDs: 21 = 23, 27 = 28 = 31, 33 =
34), resulting in a final dataset of 56 unique mtDNA
genome haplotypes. For ML and Bayesian tree
reconstructions, we applied the programs RAxML 0.93 
and MrBayes 3.1.2 [70,71], respectively. ML calculations
in RAxML were run with the CAT-GTR model and 1,000
bootstrapping replications. For Bayesian tree
reconstructions in MrBayes, we conducted four Markov Chain
Monte Carlo (MCMC) runs with a default temperature of
0.2 and the TrN + I + G model as selected as best-fit model
in jModeltest 2.1  under the Bayesian information
criterion (BIC) and the Decision Theory Performance-based
Selection (DT). All repetitions were run for 1 million
generations with tree and parameter sampling setting in every
100 generations. The first 25% of samples were discarded
as burn-in, resulting in 75,001 trees per run. The adequacy
of the burn-in and convergence of all parameters was
assessed via the uncorrected potential scale reduction
factor (PSRF)  as calculated by MrBayes and by visual
inspection of the trace of the parameters across generations
using TRACER 1.5 . To check whether posterior clade
probabilities were also converging, AWTY  was
applied. Posterior probabilities for each split and a phylogram
with mean branch lengths were calculated from the
posterior density of trees.
Divergence ages from the dataset were estimated with
BEAST 1.6.1  applying a Bayesian MCMC method
with a relaxed molecular clock approach . A relaxed
lognormal model of lineage variation and a Birth-Death
Process prior for branching rates was assumed. The
following five fossil-based calibration points were used with
a normal distribution prior for respective nodes: (1) the
Homo Pan split 6.5 Ma with a 95% CI of 0.5 Ma
[78-80], (2) the split between Pongo and the Homo + Pan
clade at 14 Ma (95% CI: 1.0 Ma) , (3) the divergence
of Theropithecus and Papio 5 Ma (95% CI: 1.5 Ma)
[82,83], (4) the split between African (M. sylvanus) and
Asian macaques at 5.5 Ma (95% CI: 1.0 Ma) [83,84] and
(5) the divergence of hominids and cercopithecids at
27.5 Ma (95% CI: 3.5) [85-87]. Four replicates were run
in BEAST for 25 million generations with tree and
parameter sampling occurring every 100 generations.
TRACER was used to assess the adequacy of a 10%
burn-in and the convergence of all parameters via visual
inspection of the trace of the parameter across
generations. Sampling distributions were combined (25%
burnin) using the software LogCombiner 1.6.1. A consensus
chronogram with node height distribution was generated
and visualized with TreeAnnotator 1.6.1 and FigTree
Availability of supporting data
The alignments supporting the results of this article are
available in the Data Dryad repository, http://dx.doi.org/
Additional file 1: Table S1. Estimated divergence ages in Ma and 95%
CIs (in parentheses) among lineages.
Additional file 2: Table S2. Detailed information about studied mtDNA
genomes (geographical origin, source, Genbank accession number,
RL, JK, KOB, DZ and CR participated in the study design. RL, JK, KOB, MB and
CR conducted the experiments and analysed the data. BMM-Z, MABA-L, AA,
ML, PA-P and AJT provided samples and/or helped to draft the manuscript.
RL, EM, AJT, DZ and CR wrote the manuscript. All authors read and approved
the final manuscript.
We are grateful to the Bavarian State Collection of Zoology, Covance Inc.
and the German Primate Center for providing valuable long-tailed macaque
samples, as well as to the Universiti Kebangsaan Malaysia, the Department of
Wildlife and National Parks (PERHILITAN), the Sarawak Forest Department, the
Sabah Wildlife Department and Sabak Parks for sharing fecal samples. This
research was financially supported by the German Primate Center and by grants
to Badrul Munir Md-Zain (FRGS/1/2012/STWN10/UKM/02/3, DLP-2013-006,
ERGS/1/2013/STWN10/UKM/02/1). We further thank Sabine Hutschenreuther,
Christiane Schwarz, Nico Westphal and Jens Gruber for support during sample
collection, laboratory work or data analysis.
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