Original Article

Cytogenet Genome Res Accepted: March 13, 2018

by M. Schmid
DOI: 10.1159/000490481
Published online: ■■■

Contrasting Rates of LINE-1

Amplification among New World
Primates of the Atelidae Family
Akash Sookdeo a Manuel Ruiz-García b Horacio Schneider c
Stéphane Boissinot d
a Department of Biology, New York University, New York, NY, USA; b Departamento de Biología, Facultad de
Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia; c Universidade Federal do Pará, Campus de Bragança,
Bragança, Brazil; d New York University Abu Dhabi, Abu Dhabi, UAE

Please note also the following remarks of one of the reviewers:

It would be appropriate to cite Jurka (2011) in the last paragraph of the Discussion when discussing stochastic changes
that may result from population demography.
I found the speculation about life-spans and reproductive rate impacts as they relate to L1 accumulation to be just that,
speculative and would rather see it removed.

Keywords extent of L1 amplification in Atelidae remains overall lower

Alouatta · Ateles · Atelidae · Brachyteles · Lagothrix · LINE-1 ·
New World monkeys · Primates · Retrotransposon
Abstract
LINE-1 (L1) retrotransposons constitute the dominant cate-
gory of transposons in mammalian genomes. L1 elements
are active in the vast majority of mammals, and only a few
cases of L1 extinction have been documented. The only pos-
sible case of extinction in primates was suggested for South
American spider monkeys. However, these previous studies
were based on a single species. We revisited this question
with a larger phylogenetic sample, covering all 4 genera of
Atelidae and 3 species of spider monkeys. We used an en-
richment method to clone recently inserted L1 elements and
performed an evolutionary analysis of the sequences. We
were able to identify young L1 elements in all taxa, suggest-
ing that L1 is probably still active in all Atelidae examined.
However, we also detected considerable variations in the
proportion of recent elements indicating that the rate of L1
amplification varies among Atelidae by a 3-fold factor. The
than in other New World monkeys. Multiple factors can af-
fect the amplification of L1, such as the demography of the
host and the control of transposition. These factors are dis-
cussed in the context of host life history.
© 2018 S. Karger AG, Basel
LINE-1 (L1) retrotransposons constitute the domi-
nant category of transposable elements in mammalian
genomes and have profoundly affected their size and
function [Tollis and Boissinot, 2012; Warren et al., 2015].
They have been a source of evolutionary novelties by pro-
viding regulatory or coding sequences that can be co-opt-
ed by the host for its own benefit [Warren et al., 2015;
Mita and Boeke, 2016]. Despite a strong cis-preference,
the L1 machinery can also act on other transcripts and is
responsible for the retrotransposition of processed pseu-
dogenes [Esnault et al., 2000] and Alu elements [Dewan-
nieux et al., 2003], which can also be domesticated by the
host [Brosius, 1999; Häsler and Strub, 2006; Marques et
al., 2005]. Thus, there is no doubt that L1 activity consti-

© 2018 S. Karger AG, Basel Stéphane Boissinot

New York University Abu Dhabi
Saadiyat Island
E-Mail karger@karger.com
PO 129188, Abu Dhabi (UAE)
www.karger.com/cgr
E-Mail sb5272 @ nyu.edu

CGR490481.indd 1 19.06.2018 07:46:04

 tutes one of the dominant driving forces acting on the
genome. It has even been proposed that the level of L1
activity in a lineage contributes to the adaptability of this
lineage by providing evolutionary novelties on which se-
lection can act [Oliver and Greene, 2009, 2011]. However,
L1 can also have a strong negative effect on the fitness of
its host [Boissinot et al., 2001, 2006] by disrupting gene
function [Ostertag and Kazazian, 2001; Belancio et al.,
2009] and by mediating chromosomal rearrangements
through ectopic recombination [Burwinkel and Kili-
mann, 1998; Song and Boissinot, 2007].
The effect L1 has on its host, negative or positive, is
intrinsically dependent on the level of L1 activity. Com-
parisons among vertebrate genomes have revealed that
the rates of L1 activity differ drastically among species
and through time [Pascale et al., 1990; Furano, 2000; Bois-
sinot et al., 2004; Khan et al., 2006; Ivancevic et al., 2016].
For instance, the mouse genome contains about 3,000 po-
tentially active L1 copies [Goodier et al., 2001; Sookdeo et
al., 2013], whereas only 80–100 copies are potentially ac-
tive in human [Sassaman et al., 1997; Brouha et al., 2003].
This difference results in a 30-fold higher rate of L1 inser-
tion in mouse relative to human [Ostertag and Kazazian,
2001]. In the human lineage, the rate of L1 amplification
was considerably higher at the time of the split between
Old World primates and hominoids (∼35 Mya), and the
reduction in L1 activity occurred only during the last 12
Myr of human evolution [Khan et al., 2006]. The reasons
why the rate of amplification differs among species and
through time are not well understood and might be re-
lated to the rate of transposition, the control of transposi-
tion by the host, selection against new inserts, and the
demographic history of the host [Tollis and Boissinot,
2012].
In primates, L1 activity varies among extant species
[Liu et al., 2003; Boissinot et al., 2004; Lee et al., 2007].
The largest difference, however, is found among New
World primates [Boissinot et al., 2004]. In the squirrel
monkey Saimiri sciureus and in the cotton-top tamarin
Saguinus oedipus, the rate of L1 amplification is very high
and resulted in the evolution of species-specific families.
In contrast, L1 appears to be extinct in the spider monkey
Ateles paniscus as no evidence of recent amplification or
no Ateles-specific families were found. The extinction of
L1 in Ateles would be rather surprising and unique among
primates [Ivancevic et al., 2016]. In fact, L1 extinction has
been documented with certainty in a small number of ro-
dents, bats, perissodactyls, and cetacean species [Casa-
vant et al., 2000; Grahn et al., 2005; Cantrell et al., 2008;
Platt and Ray, 2012; Ivancevic et al., 2016].
2 Cytogenet Genome Res
DOI: 10.1159/000490481
CGR490481.indd 2
The proposed extinction of L1 in New World monkeys
was found in a single species of Atelidae, A. paniscus, and
thus requires confirmation using a larger phylogenetic
sampling. We decided to revisit this possible instance of
L1 extinction in primates by analyzing L1 activity in all
genera of Atelidae and in multiple Ateles species. Deter-
mining with certainty that L1 is extinct in a specific taxon
is difficult because it requires high quality genomic data
[Gallus et al., 2016], which are not available in many spe-
cies. Instead, we used a cloning strategy designed to detect
young L1 elements in a genome. We found evidence of
recent L1 activity in most taxa examined. We conclude
that L1 is not extinct in Atelidae. However, the rate of L1
amplification seems to differ considerably among Ateli-
dae as L1 appears far more active in the Alouattinae sub-
family (the howler monkeys; genus Alouatta) than in the
Atelinae subfamily (spider monkeys, woolly monkeys,
and muriquis; genera Ateles, Lagothrix, and Brachyteles).
Materials and Methods
Samples
The Atelidae family contains 2 subfamilies that diverged 16
Mya: the Alouattinae and the Atelinae (Fig. 1) [Opazo et al., 2006;
Wildman et al., 2009; Morales-Jimenez et al., 2015; Schneider and
Sampaio, 2015]. The Alouattinae subfamily is represented by a sin-
gle extant genus, the genus Alouatta that includes all species of
howler monkeys. We studied 2 species of Alouatta, A. belzebul
from Brazil and A. macconnelli from French Guiana. The Atelinae
subfamily contains 3 genera: Ateles (spider monkeys), Lagothrix
(woolly monkeys), and Brachyteles (muriquis). We obtained and
analyzed DNA samples from 3 Ateles species, A. paniscus (ob-
tained from the Wuppertal Zoological Garden), A. fusciceps (from
the Pacific Chocó area of Colombia) and A. geoffroyi (purchased
from the Coriell Institute for Medical Research), 1 Lagothrix spe-
cies, L. lagothricha (from the Colombian Amazon), and 1 Bra-
chyteles species, B. arachnoides (from Brazil).
Experimental Approach
We amplified 2 regions of the second ORF of L1 by PCR using
the primers and methods described in Boissinot et al. [2004] and
Cantrell et al. [2000]. Region A corresponds to a 355-bp fragment
located near the extremity of ORF2, from position 3,326 to 3,681
of the modern human ORF2 consensus. Region A primers are lo-
cated in a conserved region of ORF2 and are useful for collecting
elements of different ages and consequently for analyzing the long-
term amplification of L1 elements in a species. Region A amplicons
were cloned into the pGEM vector (Promega) and between 23 and
27 clones per species were randomly selected and sequenced. Re-
gion B corresponds to a 575-bp fragment located in the reverse
transcriptase domain of ORF2, from position 2,114 to 2,688 of the
modern human ORF2 consensus. Region B was amplified by PCR
using degenerate primers. PCR products were cloned in-frame
into LacZ. Clones with intact reading frames produce L1/beta-
galactosidase fusion proteins yielding blue colonies. The assump-
Sookdeo/Ruiz-García/Schneider/Boissinot
19.06.2018 07:46:24
 Fig. 1. Evolutionary relationships between the primate species used in this study. The age of the speciation events
is based on the work of Opazo et al. [2006] and Morales-Jimenez et al. [2015].
tion was that clones with interrupted reading frames (blue clones)
are more likely to represent young elements than clones with stop
codons (white clones). For each species, at least 13 blue clones and
13 white clones were sequenced. All region A and B clones were
sequenced in both orientations by Sanger chemistry by Macrogen
(Seoul, Korea). The quality of the chromatograms was assessed vi-
sually. Reverse and forward reads were assembled into contigs that
were used for downstream analyses.
Data Analysis
Sequences were aligned and manipulated using the BioEdit
program [Hall, 1999]. Recently inserted L1 elements are expected
to retain the features of their progenitor more faithfully than older
elements that have accumulated mutations since the time of their
insertion. Thus, we assessed the level of conservation of L1 se-
quences as an indicator of their age using several criteria: the pres-
ence of an intact open reading frame (ORF), the occurrence of
insertions/deletions (indels), the conservation of CpG dinucleo-
tides, and the conservation of critical amino acid residues. CpG
dinucleotides are considered hypermutable as the C in a CpG mu-
tates to T at a rate 10–50 times higher than a C in another context.
Thus the conservation of CpG in an L1 element is an indicator of
its young age. Region A contains 2 CpG sites that are conserved
across all primate L1 families, at positions 3,475–3,476 and 3,583–
3,584 of the human ORF2 sequence. Region B contains 1 con-
served CpG at position 2,563–2,564. The proportion of sequences
with intact CpG or intact ORF was compared among species and
tested using the χ2 statistics. As L1 insertions accumulate muta-
tions at the neutral rate, some of these mutations should affect the
amino acid sequence of ORF2. Thus, recently inserted elements
should have retained a larger fraction of the amino acids critical
for retrotransposition. Critical amino acids in region A and B were
inferred by comparing L1 elements across a wide range of mam-
mals. Thirty-eight conserved amino acids in region B were previ-
ously described in Grahn et al. [2005]. The sequence of the con-
L1 Activity in Atelidae
CGR490481.indd 3
served amino acids in region A is shown here starting at amino-
acid position 1,113 of the human ORF2 sequence, where the
numbers in parentheses indicate the numbers of amino acids be-
tween conserved residues: H(1)TP(1)R(3)I(7)CWRGC(3)GTL(1)
H(1)WW(1)C(1)L(1)QP(1)W(3)W(10)P(1)DPAI(1)LL(14)
TC(3)F(1)AA(4)A(2)W(4)CP(4)W(2)K(1)W. Changes at these
sites were determined by returning each sequence to its original
frame, removing insertions and filling gaps with N. Phylogenetic
analyses were performed using the neighbor-joining and maxi-
mum likelihood methods. The divergence of families was calcu-
lated by computing Kimura’s 2-parameter distance between each
element and the consensus of the family [Kimura, 1980]. Distanc-
es and neighbor-joining trees were calculated using the MEGA 5.0
program [Tamura et al., 2011] and maximum likelihood trees were
built with the PhyML program [Guindon and Gascuel, 2003].
Results
Analysis of Region A
We analyzed 174 PCR-derived clones in 7 Atelidae
species (Table 1). We determined the relative proportion
of young elements by assessing the decay of L1 sequences.
In each taxon, we examined the number of clones with
intact ORFs, the number of clones with indels, and the
conservation of CpG dinucleotides. These data were
compared with sequences from 2 Cebidae species which
are known to host active L1 families, S. sciureus and S.
oedipus, and 2 hominoids, Homo sapiens and Pan troglo-
dytes. For all criteria examined, the proportion of young
sequences in Atelidae is much lower than in Cebidae and
Cytogenet Genome Res 3
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 Table 1. Conservation of region A cloned sequences

Family Clones, Clones with Sequences Intact CpG, n (%) New World monkey-specific
Species n intact ORF, with indels, sequences, n (%)
n (%) n (%)
151 – 152 259 – 260 both sites old clade new clade all

Atelidae
Ateles paniscus 26 4 (15) 16 (62) 12 (46) 4 (16) 16 (31) 4 (16) 2 (8) 10 (38)
Ateles fusciceps 24 2 (8) 12 (50) 5 (21) 9 (38) 14 (29) 4 (17) 2 (8) 11 (45)
Ateles geoffroyi 27 5 (18) 13 (48) 7 (26) 12 (44) 19 (35) 4 (15) 4 (15) 14 (52)
Total Ateles 77 11 (14) 41 (53) 24 (31) 25 (32) 49 (32) 12 (16) 8 (10) 35 (45)
Lagothrix lagothricha 25 3 (12) 13 (52) 14 (56) 4 (16) 18 (36) 1 (4) 7 (28) 13 (52)
Brachyteles arachnoides 25 2 (8) 16 (64) 11 (44) 7 (28) 18 (36) 2 (8) 2 (8) 15 (60)
Total Atelinae 127 16 (13) 70 (55) 49 (39) 36 (28) 85 (33) 15 (12) 17 (13) 63 (50)
Alouatta macconnelli 23 5 (22) 13 (57) 9 (39) 13 (56) 22 (48) 0 (0) 8 (35) 11 (48)
Alouatta belzebul 24 1 (4) 14 (58) 5 (21) 3 (12) 8 (17) 2 (8) 2 (8) 8 (33)
Total Alouatta 47 6 (13) 27 (57) 14 (30) 16 (34) 30 (32) 2 (4) 10 (21) 19 (40)
Cebidae
Saimiri sciureus 26 7 (27) 7 (27) 13 (50) 12 (46) 25 (48) 0 (0) 16 (62) 17 (65)
Saguinus oedipus 26 10 (38) 12 (46) 12 (46) 15 (58) 27 (52) 0 (0) 18 (69) 19 (73)
Hominidae
Pan troglodytes 21 7 (33) 10 (48) 11 (52) 13 (62) 24 (57) NA NA NA
Homo sapiens 25 8 (32) 12 (48) 10 (40) 11 (44) 21 (42) NA NA NA
Total Hominidae 46 15 (33) 22 (48) 21 (46) 24 (52) 45 (49) NA NA NA

Hominoidae. For instance, the fraction of elements with
intact ORF is significantly lower in Atelidae (∼13%) than
in Cebidae (∼33%; p = 0.002) and in hominoids (∼33%;
p = 0.005). Similarly, only 33% of CpG dinucleotides are
intact in Atelidae versus 50% on average in Cebidae (p =
0.001) and Hominidae (p = 0.027). The lower proportion
of recent elements in Atelidae indicates that L1 activity
has been reduced in the Atelidae family relative to other
New World primates. Among Atelidae, the proportion of
young sequences is similar among species with the nota-
ble exception of A. macconnelli. In A. macconnelli, the
proportion of elements with intact CpG (48%) is signifi-
cantly (p = 0.022) higher than in other Atelidae (∼31%).
Similarly, the fraction of clones with intact ORF in A.
macconnelli (22%) is higher than in other Atelidae (11%),
but this difference is not statistically significant (p =
0.159). The higher fraction of young elements in A. mac-
connelli is similar to the one in S. sciureus, a species whose
genome contains an active L1 family [Boissinot et al.,
2004], and suggests that L1 activity is higher in A. mac-
connelli than in other Atelidae.
Sequences were then classified into families based on
the diagnostic characters defined in Boissinot et al. [2004].
New World primate genomes contain 2 L1 lineages, called
L1nwm-old and L1nwm-modern, which diverged after the sepa-
ration between Old World and New World primates but
before the split between Cebidae and Atelidae [Boissinot
et al. 2004]; (Fig. 2A, online suppl. material 3; see www.
karger.com/doi/10.1159/000490481 for all online sup-
pl. material). These 2 lineages coexisted in ancestral
New World monkeys until L1nwm-old became extinct. The
L1nwm-modern lineage persisted and is currently the active
lineage in extant New World monkeys. Thus, the number
of clones belonging to L1nwm-modern can also be used as a
proxy for the recent level of L1 activity. The proportion
of L1nwm-modern clones in Atelidae (16%) is significantly
lower (p < 0.0001) than in the 2 Cebidae species used for
comparison (>60%), confirming that L1 is much less ac-
tive in Atelidae than in other New World primates. Be-
cause the number of recent sequences is small in Atelidae,
the number of old sequences recovered, particularly from
the L1nwm-old family, is proportionally larger than in other
New World monkeys. The proportion of L1nwm-modern se-
quences is consistently low in Atelidae species, except in
A. macconnelli (35%) and in L. lagothricha (28%). The
higher proportion of L1nwm-modern elements in these 2
species is significantly (p = 0.004 and 0.019 for A. mac-
connelli and L. lagothricha, respectively) higher than in
other Atelidae (8–15%). This observation suggests that
L1nwm-modern could have been recently more active in
A. macconnelli and L. lagothricha than in other Atelidae
species. This conclusion is supported in A. macconnelli

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 A B
Fig. 2. Phylogenetic trees of L1 elements based on region A of
ORF2. Trees were built using the neighbor-joining method based
on Kimura’s 2-parameter distances. The number at a particular
node indicates its percentage of appearance in 1000 bootstrap rep-
licates. Only values >75% are indicated. A Tree showing the clus-
where some of the L1nwm-modern elements show a very low
(<1%) level of divergence from their consensus indicating
that they have not resided in the A. macconnelli genome
long enough to accumulate neutral mutations. In con-
trast, L1nwm-modern elements in L. lagothricha are quite di-
vergent from their consensus (2.1–6.6%) and do not rep-
resent recent insertions.
We searched for species-specific L1 families using a
phylogenetic approach. We built a tree using all the Ate-
lidae sequences belonging to the L1nwm-modern lineage
(Fig. 2B). Within Atelidae, we detected a single well-sup-
ported monophyletic group of closely related sequences
belonging to the species A. macconnelli. This suggests that
L1 Activity in Atelidae
CGR490481.indd 5
tering of sequences into taxon-specific clades. For clarity, the tree
presented was built using representative sequences from each tax-
on, but a phylogenetic tree built using all sequences is available in
online supplementary material 3. B Tree built using all Atelidae
L1nwm-modern elements. Note the lack of an Atelinae-specific clade.
L1 amplified and evolved into a species-specific family in
A. macconnelli. In contrast, other Atelidae sequences do
not cluster into species-specific or genus-specific groups
and exhibit long terminal branches, suggestive of the ac-
cumulation of mutations since insertion. We then exam-
ined the average divergence of elements within each Ate-
lidae family. Consensus sequences were built for the
A. macconnelli-specific family, the Atelinae L1nwm-modern
lineage (minus the Alouatta-specific family), and the
L1nwm-old family. The divergences from consensus were
calculated for each sequence and are plotted in Figure 3.
Elements belonging to the A. macconnelli-specific family
are characterized by a very low divergence from consen-
Cytogenet Genome Res 5
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 A 0.6 ଶ Alouatta sp.
ଶ L1nwm-old
0.5 ଶ L1nwm-modern

0.4

Frequency
0.3

0.2

0.1

0
0–1 1–2 2–3 3–4 4–5 5–6 6–7 7–8 8–9 9–10 10–11 11–12
Divergence from consensus, %

B 0.6 ଶ Alouattinea sp.

ଶ Atelinae sp.
0.5

0.4
Frequency

0.3

0.2

0.1

0
0–1 1–2 2–3 3–4 4–5 5–6 6–7 7–8 8–9 9–10 10–11 11–12
Divergence from consensus, %

Fig. 3. Frequency distribution of the divergence between members of L1 families and their respective family-
specific consensus sequences. A Region A. B Region B.

sus (0.3–1.8%). Translated into time using a New World
monkey pseudogene rate of 0.21%/Myr, this level of di-
vergence suggests that the Alouatta-specific family began
amplifying ∼8 Mya and is probably still active. However,
the age of elements needs to be taken with caution as the
rate of neutral evolution in New World monkeys differs
considerably between species and between genomic re-
gions [Peng et al., 2009]. The current activity of the
Alouatta-specific family is supported by the high level of
conservation of these elements. Three out of 4 sequences
have intact ORFs, none of the conserved CpG dinucleo-
tides are mutated, and half of the sequences have all the
residues that are conserved across all mammals intact
(Table 2). The conservation of Alouatta-specific elements
is reminiscent of the conservation of elements belonging
to the currently active L1 family in human (L1PA1) and
strongly suggests that L1 is indeed active in Alouatta (Ta-
ble 2). In contrast, L1nwm-modern elements from Atelinae
species range in divergence from 1.2 to 9.2%, the most
recent element being ∼6 Myr old. The apparent lack of
young, low-divergent elements in Atelinae is consistent
with a very low level of activity or extinction of L1 in this
group. In addition, Atelinae L1nwm-modern elements are
poorly conserved and their level of degradation is compa-
rable to the extinct L1nwm-old family (Fig. 3) and to the hu-
man L1PA4 family that amplified in the human genome
18 Mya and became extinct around 14 Mya (Table 2).
Analysis of Region B
The lack of recent activity in most Atelidae species was
verified using a PCR-based strategy specifically designed
to collect young L1 elements [Cantrell et al., 2000]. This
procedure uses degenerate primers located in a conserved
region of ORF2. PCR products are cloned in-frame into
LacZ, and clones with intact reading frames produce L1/
betagalactosidase fusion proteins yielding blue colonies.
For each species, ∼50% blue and ∼50% white colonies
were collected and sequenced (Table 3). We assessed in

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 Table 2. Conservation of L1 families

Region L1 families Elements, n (%) Intact CpG,

total with intact with indels with intact n (%)
ORF amino acids
A Alouatta-specific 4 3 (75) 1 (25) 2 (50) 8 (100)
L1nwm-old 17 0 (0) 10 (59) 0 (0) 12 (35)
L1nwm-modern Atelidae 17 5 (24) 3 (18) 1 (6) 19 (45)
Saimiri-specific 8 3 (37) 1 (12) 4 (50) 12 (75)
Saguinus-specific 17 10 (59) 5 (29) 6 (35) 25 (74)
L1PA1 (3.1 ± 0.8 Myr) 25 21 (84) 0 (0) 22 (88) 45 (90)
L1PA4 (18.0 ± 4.8 Myr) 25 10 (40) 6 (24) 3 (12) 27 (54)
L1PA7 (31.4 ± 8.4 Myr) 25 0 (0) 19 (76) 0 (0) 7 (14)
B Alouattinae-specific 31 22 (71) 4 (13) 19 (61) 30 (97)
Atelinae-specific 29 25 (86) 2 (7) 15 (52) 23 (79)
L1PA1 (3.1 ± 0.8 Myr) 25 22 (88) 2 (8) 20 (80) 20 (80)
L1PA4 (18.0 ± 4.8 Myr) 25 5 (20) 12 (48) 0 (0) 10 (40)
L1PA7 (31.4 ± 8.4 Myr) 25 0 (0) 24 (96) 0 (0) 4 (16)

Table 3. Conservation of region B cloned sequences

Species Clones Clones, Clones with Sequences Intact CpG, Subfamily Clones with intact
n intact ORF, with indels, n (%) specific, conserved amino
n (%) n (%) n (%) acids, n (%)

Ateles paniscus blue 13 6 (46) 5 (38) 5 (38) 3 (23) 4 (31)

all 26 6 (23) 18 (69) 8 (31) 4 (15) 4 (15)
Ateles fusciceps blue 13 5 (38) 8 (62) 6 (46) 4 (15) 2 (15)
all 26 7 (27) 19 (73) 9 (35) 6 (23) 3 (12)
Ateles geoffroyi blue 39 16 (41) 19 (49) 21 (54) 8 (21) 6 (15)
all 57 18 (32) 38 (67) 30 (53) 9 (16) 6 (11)
Total Ateles blue 65 27 (42) 32 (49) 36 (55) 15 (23) 12 (18)
all 109 31 (28) 75 (69) 39 (36) 19 (17) 13 (12)
Lagothrix lagothricha blue 15 4 (27) 9 (60) 6 (40) 4 (27) 4 (27)
all 28 4 (14) 23 (82) 6 (21) 4 (14) 4 (14)
Brachyteles arachnoides blue 14 2 (14) 10 (71) 7 (50) 1 (7) 2 (14)
all 27 3 (11) 18 (67) 14 (52) 1 (4) 2 (7)
Total Atelinae blue 94 33 (35) 51 (54) 52 (55) 20 (21) 18 (19)
all 164 38 (23) 116 (71) 59 (36) 24 (15) 19 (12)
Alouatta macconnelli blue 16 13 (81) 2 (12) 12 (75) 10 (63) 8 (50)
all 32 15 (47) 11 (34) 27 (84) 16 (50) 14 (44)
Alouatta belzebul blue 15 10 (67) 3 (20) 13 (87) 12 (80) 7 (47)
all 26 12 (46) 11 (42) 20 (77) 15 (58) 9 (35)
Total Alouatta blue 31 23 (74) 5 (16) 25 (81) 22 (71) 15 (48)
all 58 27 (47) 22 (38) 47 (81) 31 (53) 23 (40)

each species the level of conservation of L1 sequences by
determining the number of intact ORFs, the presence of
indels, the conservation of CpG dinucleotides, and the
conservation of amino acids believed to be critical for
transposition [Grahn et al., 2005]. Although the proce-
dure we used was designed to enrich recently inserted el-
ements, we were surprised to find that a large fraction of
elements obtained from Atelinae species were quite de-
graded. For instance, only 23% of Atelinae sequences
have intact ORFs and 36% of the CpG sites are intact (Ta-
ble 3). Furthermore, only 12% of the sequences retain all
38 amino acid critical residues. This suggests that the vast

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CGR490481.indd 7 19.06.2018 07:46:26

 A B

Fig. 4. Phylogenetic trees of L1 elements based on region B of ORF2. Trees were built using the neighbor-joining
method based on Kimura’s 2-parameter distances. The number at a particular node indicates its percentage of
appearance in 1000 bootstrap replicates. Only values >75% are indicated. A Tree built using a selection of se-
quences from each taxon and showing the clustering of sequences into taxon-specific clades. B Tree built using
all Atelidae-specific sequences.

majority of the clones retrieved using the enrichment
strategy are not recent. However, a small number (∼12%)
of elements that have the hallmarks of recent insertions
were retrieved (Tables 2, 3). For instance, in Ateles, 13
clones (out of 109) have an intact ORF and have retained
all 38 critical amino acids. In addition, these elements
have very low divergence from their consensus indicating
that they have not resided in their host genome long
enough to accumulate mutations (Fig. 3B). Thus, we can
conclude that L1 elements are not extinct in Atelinae.
In Alouattinae, we found a large proportion of puta-
tively young elements, confirming that L1 has recently
been very active in this group of primates. More than half
of the sequences have intact ORFs, more than 80% of the
CpG are not mutated, and 40% of the sequences retain all
critical amino acids. Thus, the larger proportion of recent
L1 elements in Alouatta indicates that L1 has been much
more active in Alouattinae than in Atelinae, as suggested
by our analysis of region A. The difference between Ateli-
nae and Alouattinae is not due to a bias in the sampling

8 Cytogenet Genome Res Sookdeo/Ruiz-García/Schneider/Boissinot

DOI: 10.1159/000490481

CGR490481.indd 8 19.06.2018 07:46:26

 Fig. 5. Parsimony tree of region B consen-
sus sequences. The branch lengths are pro-
portional to the number of characters sup-
porting each branch.
of clones as the fraction of blue clones we sequenced is
similar in these 2 groups (57 and 53%, respectively). In
fact, the higher conservation of L1 sequences in Alouat-
tinae holds even if only blue clones are compared (Ta-
ble 3).
Region B sequences were used in a phylogenetic anal-
ysis to identify species- or genus-specific L1 families
(Fig. 4A). The tree built using region B sequences reca-
pitulates the phylogenetic relationship among New
World monkeys. A number of Atelidae sequences form a
monophyletic group distinct from the Cebidae. Within
Atelidae, sequences cluster into Atelinae- or Alouattinae-
specific clades, confirming that L1 amplified and evolved
into subfamily-specific lineages in both Atelidae subfam-
ilies. A tree built using all Atelidae sequences (Fig.  4B)
reveals some additional groupings, including a mono-
phyletic group of Ateles sequences and a small group of
Lagothrix sequences (Fig. 4B), suggesting the presence of
genus-specific L1 families in these 2 taxa. These groups of
elements are not well supported by the bootstrap analysis,
but 2 lines of evidence suggest that they represent genus-
specific L1 families. First, elements within each group are
very closely related. Ateles-specific elements are between
0.4 and 0.7% divergent from consensus, which roughly
corresponds to 2–4 Myr of age, which is consistent with
the origin of the genus Ateles 6.7 Mya [Morales-Jimenez
et al., 2015] and the diversification among the current
Ateles species, which is estimated around 3.2–3.6 Mya
[Ruiz-Garcia et al., 2016a, b]. As the genus Ateles split
from the Lagothrix/Brachyteles clade 12 Mya [Opazo et
al., 2006], it is very likely that these elements inserted in
Ateles genomes after this split. Similarly the 3 Lagothrix
elements diverged between 0.2 and 0.7% from their con-
sensus, which also strongly suggests these elements in-
serted in Lagothrix since its separation from Brachyteles
∼10 Mya. Second, Ateles-specific and Lagothrix-specific
elements share common characters that are not found
among other Atelidae sequences. These diagnostic char-
acters are the hallmark of novel, taxon-specific families
L1 Activity in Atelidae
CGR490481.indd 9
(see consensus sequences in online suppl. material 1, 2).
It should be noted that we failed to detect a genus-specif-
ic L1 family in Brachyteles. Based on our analysis of re-
gions A and B, B. arachnoides seems to be the taxon with
the lower proportion of recent L1 copies. We recovered
in this species a single clone that groups within the Ateli-
nae-specific group. This clone has a divergence from con-
sensus of 1.3% (∼6.5 Myr old). Thus, it appears that with-
in Atelinae, B. arachnoides is the species with the lowest
level of L1 activity.
Finally, we examined the evolution of the active L1 lin-
eage in New World monkeys. We determined the consen-
sus sequences (online suppl. material 1, 2) of the most
recently active L1 family in each species, and we recon-
structed the evolutionary relationships among these se-
quences (Fig. 5). The parsimony tree reveals considerable
differences in the rate of evolution of the active L1 lineage
among New World monkeys. Before the diversification
of Atelidae, the L1 lineage accumulated 7 mutations over
∼7 Myr (1 mutation/Myr) but only 5 mutations over 16
Myr in Alouattinae (0.3 mutation/Myr). Since the split
between Ateles and Lagothrix, the L1 lineage accumulated
only 3 and 2 mutations, respectively, although these 2
genera separated ∼12 Mya (0.17–0.25 mutations/Myr).
Thus the low rate of amplification of L1 in Atelidae oc-
curred simultaneously to a reduction in the rate of evolu-
tion of the active L1 lineage. By comparison with other
species, the lower rate of L1 evolution is even more appar-
ent. For instance, the active L1 lineage in Saguinus has
accumulated 31 mutations since its split from Saimiri
(∼1.5 mutations/Myr). Thus, the rate of L1 evolution
could be 5–10 times lower in Atelidae than in Cebidae.
Discussion
Here, we analyzed the dynamics of amplification of L1
elements in 4 genera and 7 species of Atelidae. Using an
enrichment strategy, we recovered young elements sug-
Cytogenet Genome Res 9
DOI: 10.1159/000490481
19.06.2018 07:46:26
 gestive of recent L1 activity in all taxa. Thus, there is no
reason to believe that L1 might be extinct in this group of
primates, as suggested in a previous study limited to a
single Atelidae species [Boissinot et al., 2004]. However,
we observed large differences in the replicative success of
L1 among Atelidae. The proportion of recent elements is
much larger in Alouattinae than in Atelinae, suggesting
that L1 is amplifying in howler monkeys at a much high-
er rate than in Atelinae. Based on the number of recent
clones recovered for regions A and B, we estimate that the
rate of amplification in the Alouattinae lineage is roughly
3 times higher than in Atelinae. However, the level of re-
cent activity of L1 in Atelidae can be considered low in
comparison with other New World monkey species. The
proportion of recent clones recovered from S. sciureus
and S. oedipus far exceeds the proportion recovered in
Atelinae and Alouattinae, and we estimate that the rate of
L1 amplification in Cebidae is approximately 3 times
higher than in Alouattinae and approximately 5 times
higher than in Atelinae. The large differences in the rate
of L1 amplification might seem considerable, yet they are
within the range observed among extinct human families
[Khan et al., 2006]. Such large differences suggest that
New World monkeys present an excellent model to com-
pare the impact L1 families with different levels of ampli-
fication have on their host genome.
The low level of activity we report in Atelidae in gen-
eral and in Atelinae in particular, is consistent with the
sporadic nature of L1 amplification. The alternation be-
tween periods of high and low L1 activity has frequently
been observed [Pascale et al., 1993; Verneau et al., 1998;
Khan et al., 2006]. As all species are on their own evolu-
tionary trajectory, it is expected that some of them are
experiencing high L1 activity while others are experienc-
ing low activity. However, it remains unknown why L1
amplification is sporadic and 2 non-exclusive categories
of reasoning have been proposed. First, variations in L1
amplification may reflect the antagonistic interactions
between L1 and its host. Because L1 activity presents a
genetic load to its host, mammals have evolved a number
of mechanisms to repress L1 activity [Goodier, 2016],
which in turn could trigger evolutionary changes in L1 to
evade repression. Such evolutionary changes have been
reported in the 5′ UTR region [Adey et al., 1994; Khan et
al., 2006] and in ORF1 [Boissinot and Furano, 2001]. In
particular, the unusual mode of evolution of the 5′ UTR
could explain the rapid emergence of novel, replicatively
dominant L1 families. In rodents and primates, L1 fami-
lies frequently acquire completely novel promoter se-
quences that share no or very little homology with the
10 Cytogenet Genome Res
DOI: 10.1159/000490481
CGR490481.indd 10
promoter sequence of the preceding family. A novel L1
element with a completely different promoter sequence
might not be recognized by the host’s repression machin-
ery and will produce far more copies than the previously
dominant L1 families. This process was experimentally
demonstrated in primates where an arms race between
with the KRAB zinc-finger genes ZNF91/93 and the 5′
UTR drives the evolution of L1 [Jacobs et al., 2014]. Un-
der this scenario, phases of vigorous L1 amplification
should be concomitant with the acquisition of novel pro-
moters, as is observed in human and mouse [Khan et al.,
2006; Sookdeo et al., 2013]. This wave of amplification
will persist until the host evolves a successful mechanism
for the repression of the new family. We can’t confirm if
this scenario applies to New World monkeys until more
data on the evolution of the 5′ UTR in this group become
available.
The second category of explanations focuses on a
larger role for demography and natural history of the
host as driver of L1 evolution. The number of potential-
ly active L1 in a genome is relatively small. For instance,
the human genome contains 80–100 potentially active
elements, a majority of which are polymorphic and at
low frequency in the human population [Brouha et al.,
2003; Boissinot et al., 2006]. In addition, most transposi-
tion results from the activity of a very small (<10) num-
ber of “hot” elements [Brouha et al., 2003]. Thus, it is
possible that drastic reduction in population sizes or
population fragmentation caused the stochastic loss of
active copies and consequently a lower rate of transposi-
tion. If a severe bottleneck occurred at the origin of Ate-
lidae and if the genome of the ancestor to all Atelidae had
a limited repertoire of active copies, it is plausible that
many active elements were lost resulting in the low L1
activity we report here. However, Atelidae originated
about 14 Mya, and the split from Cebidae is estimated
between 18.1 and 27.1 Mya using nuclear data [Perelman
et al., 2011] and between 16.3 and 22.1 Mya using mi-
togenomics [Ruiz-Garcia et al., 2016b]. Then, why has
L1 activity remained so low in Atelidae relative to Cebi-
dae over such an extended period of evolutionary time?
All neotropical primates have evolved in tropical South
America for millions of years, and there is no reason to
believe that Atelinae would be more prone to bottlenecks
than Alouattinae and Atelidae than Cebidae. The answer
might reside, in part, in the life history of these species.
Atelidae tend to have a longer generation time than Ce-
bidae. The age at maturity of males ranges from 5 years
in Ateles and Brachyteles to 8 years in Lagothrix (data
obtained from http://pin.primate.wisc.edu/). It is only
Sookdeo/Ruiz-García/Schneider/Boissinot
19.06.2018 07:46:27
 3.5 years and 1.5 years in Saimiri and Saguinus, respec-
tively. Thus, per unit of time, the number of L1 copies
entering the gene pool is larger in Cebidae than in Ate-
lidae and might account for the long-term difference in
the rate of L1 evolution and amplification between Ate-
lidae and Cebidae, the same way it could account for the
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