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The effects of the direct interaction between hybridization and speciation-two major contrasting evolutionary processes-are poorly understood. We present here the evolutionary history of the Galápagos marine iguana (Amblyrhynchus cristatus) and reveal a case of incipient within-island speciation, which is paralleled by between-island hybridization. In-depth genome-wide analyses suggest that Amblyrhynchus diverged from its sister group, the Galápagos land iguanas, around 4.5 million years ago (Ma), but divergence among extant populations is exceedingly young (less than 50 000 years). Despite Amblyrhynchus appearing as a single long-branch species phylogenetically, we find strong population structure between islands, and one case of incipient speciation of sister lineages within the same island-ostensibly initiated by volcanic events. Hybridization between both lineages is exceedingly rare, yet frequent hybridization with migrants from nearby islands is evident. The contemporary snapshot provided by highly variable markers indicates that speciation events may have occurred throughout the evolutionary history of marine iguanas, though these events are not visible in the deeper phylogenetic trees. We hypothesize that the observed interplay of speciation and hybridization might be a mechanism by which local adaptations, generated by incipient speciation, can be absorbed into a common gene pool, thereby enhancing the evolutionary potential of the species as a whole. © 2015 The Author(s) Published by the Royal Society. All rights reserved.
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Research
Cite this article: MacLeod A et al. 2015
Hybridization masks speciation in the
evolutionary history of the Gala
´pagos marine
iguana. Proc. R. Soc. B 282: 20150425.
http://dx.doi.org/10.1098/rspb.2015.0425
Received: 23 February 2015
Accepted: 7 May 2015
Subject Areas:
evolution, genetics
Keywords:
restriction site-associated DNA (RAD)
sequencing, single-nucleotide polymorphisms,
El Nin
˜o, volcanism, introgressive hybridization,
morphometrics
Author for correspondence:
Sebastian Steinfartz
e-mail: s.steinfartz@tu-bs.de
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2015.0425 or
via http://rspb.royalsocietypublishing.org.
Hybridization masks speciation in the
evolutionary history of the Gala
´pagos
marine iguana
Amy MacLeod1,2, Ariel Rodrı
´guez1, Miguel Vences1, Pablo Orozco-terWengel3,
Carolina Garcı
´a4, Fritz Trillmich2, Gabriele Gentile5, Adalgisa Caccone6,
Galo Quezada7and Sebastian Steinfartz1
1
Zoological Institute, Technische Universita
¨t Braunschweig, Mendelssohnstrasse 4, Braunschweig 38106,
Germany
2
Department of Animal Behavior, University of Bielefeld, Bielefeld 33501, Germany
3
Biomedical Science Department, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK
4
Charles Darwin Foundation, Puerto Ayora, Santa Cruz Island, Gala
´pagos, Ecuador
5
Laboratory of Experimental Ecology and Aquaculture, Department of Biology, University of Rome,
Tor Vergata, Rome 0033, Italy
6
Department of Ecology and Evolutionary Biology, Yale University, PO Box 208106, New Haven,
CT 065208106, USA
7
Gala
´pagos National Park Authority, Central Office, Puerto Ayora, Santa Cruz Island, Gala
´pagos, Ecuador
The effects of the direct interaction between hybridization and speciation—two
major contrasting evolutionary processes—are poorly understood. We present
here the evolutionary history of the Gala
´pagos marine iguana (Amblyrhynchus
cristatus) and reveal a case of incipient within-island speciation, which is
paralleled by between-island hybridization. In-depth genome-wide analyses
suggest that Amblyrhynchus diverged from its sister group, the Gala
´pagos
land iguanas, around 4.5 million years ago (Ma), but divergence among
extant populations is exceedingly young (less than 50 000 years). Despite
Amblyrhynchus appearing as a single long-branch species phylogenetically,
we find strong population structure between islands, and one case of incipient
speciation of sister lineages within the same island—ostensibly initiated by vol-
canic events. Hybridization between both lineages is exceedingly rare, yet
frequent hybridization with migrants from nearby islands is evident. The con-
temporary snapshot provided by highly variable markers indicates that
speciation events may have occurred throughout the evolutionary history
of marine iguanas, though these events are not visible in the deeper phylo-
genetic trees. We hypothesize that the observed interplay of speciation and
hybridization might be a mechanism by which local adaptations, generated
by incipient speciation, can be absorbed into a common gene pool, thereby
enhancing the evolutionary potential of the species as a whole.
1. Introduction
Processes of population differentiation on island systems provided the cornerstone
for the development of Darwin’s and Wallace’s evolutionary theory [1]. If located
far from the mainland, islands are rarely colonized de novo, and typicallyhost only a
limited number of clades which have often diversified across a system of spatially
proximate but independent islands. Therefore, these systems can be seen as
evolutionary laboratories, and provide a more simplified framework to studyevol-
utionary processes than mainland settings. Prime examples of island-based
evolutionary research include well-known adaptive radiations [2–4], Mayr’s
classical work on allopatric species formation [5] and compelling accounts of
within-island speciation [6– 8]. However, island systems do not only provide
useful settings to study diversification processes leading to speciation—they
also reveal insights into the processes which counteract speciation, such as
&2015 The Author(s) Published by the Royal Society. All rights reserved.
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hybridization. It is now broadly accepted that hybridization
can be as complex a process as speciation and is a major force
influencing the evolution of species [9,10].
The Gala
´pagos, a remote oceanic archipelago of pure vol-
canic origin 1000 km west of the South American mainland,
provides an ideal setting to study processes of evolutionary
diversification and hybridization. It consists of 20 islands ran-
ging from 1.6 to 4600 km
2
in size, plus numerous smaller
islets, with ages of emergence dating back 0.064.0 Myr
[11]. This archipelago has been colonized by many taxa, pro-
gressively from older to younger islands, with subsequent
speciation, but also hybridization and gene flow between
islands [12]. The various effects of hybridization have been
documented in Darwin’s finches, which represent one of
the best-studied adaptive radiations worldwide [13]. While
initial speciation following colonization of the archipelago
occurred on distinct islands, secondary contact between
finch species has resulted in diverse outcomes [14]. On
Daphne Island, it was the introgressive hybridization
between Geospiza fortis and G. scandens—rather than conspe-
cific gene flow with immigrants from other islands—that
increased the genetic and morphological variation of resident
populations and enhanced their evolutionary potential,
enabling the species to more rapidly react to environmental
changes [14]. In other cases, hybridization resulted in despecia-
tion of sister species [15], or the complete disappearance of a
species, as was the case for the large tree finch on Floreana
island [16]. Further examples are available from the well-
studied Gala
´pagos giant tortoises, where lineage fusion
through introgressive hybridization was recently revealed on
the largest island of the archipelago, Isabela. Here, two mor-
phologically and genetically distinct evolutionary lineages
colonized the island at different times, coexisted as distinct
entities for a period, and then merged into one lineage [17].
Thus, it seems that hybridization as an evolutionary process
continues to offer new avenues for evolutionary biologists to
explore. In this work, we investigate the evolutionary history
of Gala
´pagos marine iguanas (Amblyrhynchus cristatus) and
reveal a remarkable situation whereby within-island speciation
is paralleled by simultaneous between-island hybridization.
In this system, hybridization masks incipient speciation, lead-
ing to far-reaching consequences for the interpretation of
speciation events based on phylogenetic patterns.
Marine iguanas rank as one of the most remarkable
organisms of the Gala
´pagos. Unique among lizards world-
wide, they alone have adapted to the marine environment
by feeding exclusively on algae in the tidal and subtidal
zones, though reproduction is purely terrestrial [18,19].
Being strong swimmers, these large and highly mobile ani-
mals have colonized all major and minor islands of the
archipelago [20,21]. Amblyrhynchus is a monospecific ancient
lineage forming an archipelago-endemic clade with the three
species of Gala
´pagos land iguanas (genus Conolophus) [22].
Although only a single species of Amblyrhynchus is recog-
nized, high levels of genetic distinctiveness characterize
most of its current island populations [20]. Previous work
on a limited number of specimens [20] even indicated the
existence of two genetically distinct Amblyrhynchus popu-
lations in the northeast (Punta Pitt—PP) and southwest
(Loberı
´a—LO; figure 3c) areas of San Cristo
´bal island, but
further information on their distribution and evolutionary
history was lacking. San Cristobal is thought to be one of
the oldest of the current Gala
´pagos Islands, having emerged
between 2.4 and 4.0 Ma [11], and measuring only 550 km
2
in
surface area, with 140 km of shoreline.
To investigate the contradictory pattern of shallow phylo-
genetic divergence [21] versus strong genetic population
structure [20] in marine iguanas, we first reconstruct a temporal
framework for their evolution. We estimate the age of the
ConolophusAmblyrhynchus split from an iguanine time-tree
based on protein-coding nuclear genes and multiple temporal
calibrations across squamates [23]. As these genes are not
variable within Amblyrhynchus, we additionally use mitochon-
drial DNA (mtDNA), as well as nuclear DNA (nucDNA) from
genome-wide restriction site-associated DNA sequencing
(RADSeq), to infer the age of marine iguana lineages. On the
population level, we derive genetic clusters from an archipe-
lago-wide analysis of microsatellite loci and reconstruct
phylogenetic relationships between these clusters based on
nucDNA. Finally, we search for significant morphometric
differences between the two units on San Cristo
´bal and inves-
tigate the geological past of this island. We find PPand LO to be
reproductively isolated and morphologically differentiated
sister lineages. As such, the San Cristo
´bal system represents
an exceptional case of within-island divergence of a large
and mobile lizard, probably initiated by recurrent volcanism.
Hybridization of PP and LO with populations from other
islands offers further insights into how marine iguanas—an
apparently monospecific lineage—integrate processes of diver-
sification and local adaptation into a common evolutionary
gene pool. This mechanism may enhance the evolutionary
potential of the species, and enable them to withstand severe
climatic oscillations and successfully occupy diverse habitats
along the entire Gala
´pagos archipelago.
2. Material and methods
(a) Sampling
During 2011–2014, the majority of the coastline of San Cristo
´bal
was surveyed for marine iguana colonies. Blood samples were
obtained from 460 specimens at 17 sites spaced at maximum dis-
tances of roughly 10 km, except in the southeast where no
iguanas were located along a major part of the coast. An additional
53 samples were obtained from previous fieldwork in 1993.
(b) Molecular genetic analyses
Seven different molecular datasets (A– H) were assembled for phy-
logenetic and population genetic analysis. For detailed laboratory
and phylogenetic analysis protocols, see the electronic supplemen-
tary material, in which tables S1 and S2 provide an overview of
samples used in each dataset.
(i) Squamate time-tree based on nuclear gene sequences
To identify the closest relative of the Gala
´pagos iguanas and
date their origin on the archipelago, we sequenced the RAG1,
BDNF, R35 and NKTR genes for six focal species, and combined
them with 72 squamates [23] in a concatenated alignment of
3000 bp. Phylogenetic analysis was conducted by partitioned
Bayesian inference (BI) with MRBAYES v. 3.2 [24]. Divergence times
were estimated using BEAST v. 1.7.2 [25] with 18 time-constraints
across squamates [23].
(ii) Time tree of Gala
´pagos iguanas based on mitochondrial DNA
A representative selection of the three main haplotype lineages
within Amblyrhynchus [20], including PP and LO, and all species
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of Conolophus, were sequenced for 5557 bp from seven mitochon-
drial genes. Analyses were performed as for dataset A, with a
secondary time constraint for the AmblyrhynchusConolophus
split based on the estimate for this split obtained from analysis A.
(iii) Mitochondrial differentiation and phylogeography
of marine iguanas
Complete mitochondrial control region sequences of 1181 bp
in length were sequenced from 310 marine iguanas from San
Cristo
´bal and 34 previously unused samples from various small
islands (electronic supplementary material, table S2). These were
added to existing data [20] to give a total of 1491 sequences to
reconstruct a haplotype network (electronic supplementary
material, figure S2) and perform mitochondrial-based assignment
of San Cristo
´bal samples.
(iv) Archipelago-wide microsatellite loci genotyping
of marine iguanas
Alleles of newly genotyped samples were scored with GENEMARKER
(v. 1.95; Applied Biosystems) and added to an existing dataset of
12 microsatellite loci [20], resulting in an available pool of almost
1500 genotyped samples from all major islands. To avoid overre-
presentation of populations [26], the dataset was standardized
by random pruning to approximately 50 samples per island,
excluding islands with fewer than 20 samples. For San Cristo
´bal,
124 samples were used, with 50 each for LO and PP, plus 24 speci-
mens from the previously unsampled East coast. Altogether 614
individuals from 11 islands (electronic supplementary material,
table S2) were included for model-based Bayesian clustering
analysis [27] to infer archipelago-wide population structure.
(v) Microsatellite loci genotyping of San Cristo
´bal marine iguanas
The 513 available samples from San Cristo
´bal were scored at 18
microsatellite loci [28] and any resampled animals or individuals
with more than 6% missing data were removed (n¼39). In order
to identify occasional migrants from other islands, whose orig-
inal population are not represented in the dataset, we used
assignment tests [29] in GENECLASS [30]. Prior to population struc-
ture analysis, 20 individuals who did not assign with a
probability of more than 80% to either of the San Cristo
´bal popu-
lations, or had a mitochondrial haplotype associated with
another island, were removed. We inferred the fine-scale struc-
ture of populations on San Cristobal from the remaining 454
samples. Demographic history was assessed using BOTTLENECK
[31] and MSVAR v. 1.3 [32] with various priors (electronic sup-
plementary material, tables S5 and S6). Evolution under gene
flow versus drift was tested with 2MOD [33].
(vi) Genome-wide nuclear DNA analysis by restriction site
associated DNA sequencing
Samples of eight outgroups and 33 marine iguana individuals
spanning all major islands, including four LO and five PP
samples (electronic supplementary material, table S2), were sub-
jected to RADSeq by Floragenex [34]. Genomic DNA was
digested with SbfI, and libraries were sequenced in two lanes
of an Illumina HISEQ 2000 platform using single-end 90 bp chem-
istry. A mean of 4 981 040 +1 195 064 reads per sample was
obtained and processed with PYRAD v. 2.15 [35]. Base calls
with a Phred quality score of less than 33 were coded as
N. Sorted reads passing the filter (4 556 898 +1 086 834 reads
per sample) were aligned into within-sample clusters using a
similarity threshold of 0.95 (average 50.7 +199.6 reads per clus-
ter). We retained clusters with more than 20 reads coverage, less
than twice the standard deviation of coverage depth, less than
five undetermined sites, less than 5 heterozygous sites and
with two or fewer alleles. Heterozygous sites were coded using
ambiguity codes and consensus sequences aligned across
samples using the same similarity threshold. Loci with identical
polymorphic sites in more than three samples were excluded as
potential paralogues. A sparse alignment containing loci rep-
resented in more than five samples was retained (electronic
supplementary material, figure S5). We conducted maximum-
likelihood phylogenetic inference with a GTR þGmodel in
RAXML v. 8 [36] with 100 rapid bootstrap replicates and calcu-
lated a time-calibrated phylogeny in BEAST v. 2.0 [37], using
calibrations as in dataset B.
(vii) Timetree based on restriction site-associated DNA
sequencing data
We dated splits within Amblyrhynchus with a four-taxon subset of
dataset F, containing one land iguana outgroup and three marine
iguana specimens, representing one of the deepest splits within
marine iguanas, and the PP/LO split. After exclusion of miss-
ing/ambiguous sites, the final matrix contained 1 793 845 sites,
which we analysed in BEAST v. 2.0 under a coalescent tree prior
(constant growth), time-calibrating the root according to analysis
of dataset A at 4.6 Ma (normal prior, standard deviation 0.3) and
with a GTR substitution model.
(viii) Single-nucleotide polymorphism-based species tree analysis
of marine iguanas
To obtain a more detailed picture of marine iguana diversification,
we repeated the PYRAD procedure for a subset of data including
only the 33 ingroup samples. Settings were identical, except for
the last step in which we retained a strict matrix with 6893 loci,
including 579 304 bp for which data were available for all samples.
The bi-allelic genotypes for each individual were used to identify
single-nucleotide polymorphisms (SNPs) and export them with
ADEGENET for R [38]. An SNP-based species tree of marine iguana
populations using the multispecies coalescent method [39] was
inferred using the Snapp algorithm in BEAST v. 2.0, grouping
samples according to dataset E.
(c) Analysis of morphological characters
Measurements and scale counts were taken from a total of 143
microsatellite-genotyped marine iguanas from San Cristo
´bal to
simply demonstrate morphological differences between diverged
lineages. With the exception of body size, none of these morpho-
logical characters are currently known to be directly affected by
environmental conditions. The following were measured in the
field: snout–vent length (SVL) and total length to the nearest
10 mm; width, length and height of the head (HW, HL and
HH), and length of fourth toe (TOEL) to the nearest 0.1 mm;
and weight to the nearest 0.01 kg. Scale counts were performed
on digital photos taken in the field. For a complete list of
counts, see electronic supplementary material; values are reported
here for infralabials (INFL), series of scales below INFL (infra-infra-
labials, INFINF) and number of dorsal crest spines (DORSC1) in
anteriormost part of dorsal crest, and in addition we counted
lamellae under third and fourth toe (LAM3T and LAM4T),
INFL, supralabials and supra-supralabials. Multivariate analyses
of variance and Mann– Whitney U-tests were performed in
STATIS TIC A v. 7.1 (StatSoft Inc.).
3. Results
In a phylogeny based on four single-copy protein-coding
nuclear genes from 78 squamate species covering all iguanine
genera [23], Amblyrhynchus and Conolophus formed a clade
diverging from Ctenosaura around 8.25 Ma (95% credibility
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intervals, 5.85– 11.06 Ma), and diverging from each other at
4.52 (CI 2.766.67) Ma (figure 1a).
Further phylogenetic analysis of 5557 bp of mtDNA corrobo-
rated reciprocal monophyly of Conolophus and Amblyrhynchus,
but revealed differences in their diversification patterns. In
land iguanas, speciation resulted in the evolutionary branching
of Conolophus marthae around 1.52 (CI 0.89–2.19) Ma, followed
by further diversification around 0.29 (CI 0.15– 0.53) Ma. By
contrast, marine iguanas form a single long branch leading to
a very recent and phylogenetically unresolved divergence of
major lineages around 0.23 (CI 0.13–0.40) Ma ( figure 1b).
Genome-wide analysis of 1800 kb of nucDNA derived from
RADSeq suggests that this diversification is even younger,
occurring at 0.03 (CI 0.02–0.06) Ma (figure 1c).
Analysis of 12 nuclear microsatellite loci revealed at least 10
major genetically distinct population clusters of Amblyrhynchus
across the archipelago, and typically identified one cluster
per island (figure 2a). One striking exception was San
Cristo
´bal, where PP and LO clusters represented genetically
distinct lineages. Phylogenetic relationships between major
40 30 20
Ma
(a)
(b)
(c)
10 0
432
Ma
10
0.88/0.89/74
0.20 (0.11–0.34) Ma
0.23 (0.13–0.40) Ma
0.29 (0.15–0.53) Ma
1.52 (0.89–2.19) Ma
4.48 (3.18–5.85) Ma
0.04
0.50/-/-
1/1/98
*
*
Dipsosaurus
Brachylophus
Cyclura
Iguana
Sauromalus
Ctenosaura
Conolophus
Amblyrhynchus
C. subcristatus (Isabela)
Isabela
Santa Cruz
San Cristóbal - LO (Lobería lineage)
San Cristóbal - PP (Punta Pitt lineage)
A. cristatus
C. marthae
C. pallidus (LSF06)
A. cristatus (San Cristóbal, PP)
A. cristatus (San Cristóbal, LO)
A. cristatus (Santa Fé)
C. pallidus (Santa Fé)
C. subcristatus (Fernandina)
C. subcristatus (Seymour)
C. subcristatus (Isabela)
*
*
*
*
*
*
*
*
*
*
*
Figure 1. Temporal framework of iguana evolution on the Gala
´pagos Islands. (a) Partial timetree based on four nuclear genes (3000 bp) time-calibrated using
multiple time constraints applied to a total dataset of 78 squamates (full tree in electronic supplementary material, figure S1). Numbers at nodes indicate support
from partitioned BI analyses (posterior probability values; PP) and maximum-parsimony (MP) bootstrapping (bootstrapping values; BS); asterisks indicate maximum
support. Bars are 95% credibility intervals of time estimates. (b) Maximum credibility tree from a partitioned BI analysis of 5557 bp of mtDNA. Black asterisks
indicate concordant maximum support from a partitioned BI analysis, timetree analysis and MP bootstrap analysis. Small grey asterisks indicate high support
(PP .0.94; BS .70%) from at least two of these analyses. Time estimates and 95% credibility intervals from a timetree analysis are given at selected
nodes. (c) Timetree based on a complete matrix of 1 793 845 nucDNA sequences obtained by RADSeq of three Gala
´pagos marine iguanas (selected to represent
the deepest splits within the species) and one land iguana (Conolophus pallidus), showing the extremely shallow divergences within Amblyrhynchus.
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genetic clusters, reconstructed from nucDNA-derived SNPs,
provided strong support for a northern clade including
populations from Pinta, Marchena and Genovesa, as well as
a San Cristo
´bal clade containing the PP and LO populations
(figure 2b; electronic supplementary material, figure S6).
Spatially fine-scale genetic analysis of 454 individuals from
17 sites on San Cristo
´bal, using 18 microsatellite loci, combined
with mtDNA D-loop haplotype assignment, suggested gener-
alized genetic differentiation between PP and LO lineages—a
congruence which usually characterizes reproductively iso-
lated species (figure 3). Island-wide, 20 individuals were
identified as either migrants from other islands or hybrids
(figure 3; electronic supplementary material, table S3). Genetic
assignment identified migrants as from either Santa Cruz or
Espan
˜ola islands. On the east coast, an entire colony of iguanas
with genetic signatures of Espan
˜ola island was identified (site
SRECA in figure 3; electronic supplementary material, figure
S2). Individuals of the LO and PP lineages hybridized more fre-
quently with these animals or with migrants from Santa Cruz
(in total eight occurrences) than with each other, as evidenced
by just two PP/LO hybrids in almost 500 analysed animals.
Demographic modelling, based on 18 microsatellite loci,
suggested that both PP and LO have experienced dramatic
reductions in their effective population sizes approximately
18003000 years ago (electronic supplementary material,
tables S4 and S6, and figure S3), largely concordant with
the most recent lava formations on this island (figure 3c).
Despite their young evolutionary age, we found morpho-
logical differences between PP and LO (details in electronic
supplementary material, tables S9S18 and figure S7). PP
specimens were on average smaller (SVL in adult males
and females: MannWhitney U-test; n¼67, p¼0.0006; and
n¼24, p¼0.015), had more INFL, INFINFL and spines in
the first portion of the dorsal crest (n¼122, p¼0.002; n¼
112, p¼0.0002; n¼95, p¼0.0017) as well as relatively
longer heads (residuals of head length: n¼81, p¼0.0053)
when compared with those of the LO cluster.
4. Discussion
(a) Evolutionary age of Gala
´pagos iguanas coincides
with the subaerial age of present islands
Our phylogeny of protein-coding nuclear genes (figure 1)
confirms a sister group relationship between Gala
´pagos igua-
nas and the Central American Ctenosaura [41]. Amblyrhynchus
and Conolophus diverged around 4.5 Ma ( figure 1b), whereas
previous estimates based solely on mtDNA [22] suggested
a much older divergence of around 10 Ma on the now-
sunken islands of the archipelago. Our results reconcile
Gala
´pagos iguana divergence with the geological age of the
oldest extant islands (Espan
˜ola and San Cristo
´bal; figure 1b)
and are consistent with similar estimates for other Gala
´pagos
fauna [12], including giant tortoises (34 Ma [42]), lava
lizards (2.8 Ma [43]) and Darwin’s finches (2.02.3 Ma [44]);
though, conversely, the radiation of leaf-toed geckos may
have occurred far earlier (13.2 Ma [45]).
Marine iguanas existed as a monospecific lineage for
several million years, only diverging as recently as the Late
Pleistocene (less than or equal to 0.23 Ma according to
mtDNA) or even later (0.03 Ma based on nucDNA SNP loci).
Possible male-biased dispersal [21] and the earlier coalescence
time of mtDNA [46] might account for these between-
marker differences. Regardless of the discrepancy, divergences
within Amblyrhynchus are remarkably recent, especially
Pinta
Darwin
Darwin
Darwin
Wolf
Fernandina
+ Isabela
Wolf
Marchena
(a)
(b)
Santiago
Fernandina
Isabela
Floreana Española
Floreana
+ Española
50 km
Santa
Cruz Santa
Genovesa
Genovesa
Pinta
*
*
*
Marchena
Santa Cruz
Santiago
Santa Fé
San Cristóbal
San Cristóbal
San Cristóbal—east coast
LO lineage
PP lineage
San Cristóbal
Figure 2. Marine iguana population clusters and phylogenetic relationships.
(a)MapoftheGala
´pagos archipelago with major islands colour-coded according
to their marine iguana population cluster assignment inferred from structure analysis
of 614 individuals genotyped for 12 microsatellite loci (vertical panel in (b)).
(b) Species tree cloudogram based on an analysis of 6257 RADSeq-derived SNPs
in 33 marine iguanas from across the archipelago, including both San Cristo
´bal
lineages. The graph shows the posterior distribution of consensus trees. Asterisks
mark nodes with posterior probability ¼1.0 (all other nodes less than 0.9). Speci-
mens were grouped according to population assignment based on structure analysis.
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compared with Conolophus. This could imply that marine
iguanas experienced a massive archipelago-wide decline in
the Pleistocene. Despite records of catastrophic crashes of
Amblyrhynchus populations through El Nin
˜o events [47], the
effects vary greatly between islands [48], making an archipe-
lago-wide extinction rather unlikely. Therefore, an alternative
LO
26
30
34
38
42
46
50
SVL males
(cm)***
SVL females
(cm)* HL (residual)**
(d)
(a)
(c)
(b)
DORSC1** INFL** INFINFL***
20 29
11
13
15
17
7
9
11
13
15
17
6
10
14
18
22
–10
10
30
50
70
22
24
26
2828
5
19
57
42
68
61
51
54
53
66
N=39
30
32
LO-SRL
SRECA
San
Cristóbal
Santa
Santa
Cruz
Floreana Española
LO-SRPA
LO-SRCH
LO-SRL
LO-SRPA
LO-SRO
LO-SRIL
LO-SRCB
PP-SRBS
PP-SRG
PP-SRS
PP-SRPB
PP-SRIP
PP-SRPC
LO-SRL
(1993)
PP-SRPC
(1993)
PP-SREC
(E/D/C/B)
San
Cristóbal
LO-SRCH
H69
H60
mostly Española migrants
Punta Pitt lineage (PP)
Lobería lineage (LO) 10 km
H81
n= 185
n= 169
68H
DLO-SRIL
DD
PP-SRECB
DPP-SRECC
PP-SRECD
PP-SRECE
PP-SRBS
5
6
64
PP-SRPC
PP-SRPB
PP-SRS
D
PP-SRG PP-SRIP
DDD
LO-SRO
LO-SRCB
34
PP LO PP LO PP LO PP LO PP LO PP
Figure 3. Genetic and morphological differentiation of LO and PP lineages on San Cristo
´bal Island. LO-SRL and PP-SRPC refer to the original Loberı
´a and Punta Pitt
localities, photos show adult LO and PP males. (a) Assignment of 454 individuals based on 18 microsatellite loci, after exclusion of inter-island hybrids and migrants.
Abbreviations show sampling locations and 1993 marks specimens sampled in that year. (b) Haplotype network of control region sequences (mtDNA) for LO and PP
specimens. (c) Map of sampling localities; arrows indicate migrants/hybrids from Santa Cruz (green), Espan
˜ola (orange) and Loberı
´a (blue); dagger symbols denote
locations of within-island hybrids between PP and LO; triangles denote locations of inter-island hybrids. Population SRECA contains Espan
˜ola migrants/hybrids only.
Shaded areas mark lava groups 46 aged less than 0.1 Ma [40]. (c) Mean, standard deviation and range of morphological variables differing between LO and PP.
***p,0.001, **p,0.01, *p,0.05; sample sizes above each plot, details and abbreviations in Results and electronic supplementary material.
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scenario, whereby regular dispersal facilitates gene flow
between islands, is a more probable explanation for this
weak phylogeographic structure; a similar situation is found
for another semi-aquatic Gala
´pagos organism, the Gala
´pagos
sea lion [49].
(b) Incipient speciation of marine iguanas
on San Cristo
´bal
The sharp genetic differentiation observed between the PP and
LO lineages on San Cristo
´bal is in stark contrast to the overall
pattern of a single genetic cluster per island (figure 2a). From
a mitochondrial perspective, PP and LO do not form a mono-
phyletic group (figure 1b). Conversely, phylogenetic analyses
based on genome-wide SNP loci unambiguously reconstruct
them as sister lineages (figure 2b; electronic supplementary
material, figure S6), indicating that a considerable portion of
their current genomic differentiation must have occurred on
San Cristo
´bal. This mismatch could be due to the mitochon-
drial genome reflecting a complex history of introgressive
gene flow in the species’ past [20,21].
Typically, speciation within the geographical boundaries
of one island occurs when gene flow is prevented by a
large island size, low vagility of a species, or both [50]. The
area effect is paramount, and therefore complete within-
island speciation is exceedingly rare on small islands [1]. In
adaptive island radiations of Anolis, for example, which com-
prise small- to medium-sized lizards, no in situ diversification
was detected in Caribbean islands smaller than 3000 km
2
[51]. In birds, most phylogenetic studies of species occurring
on single islands rejected sister-species relationships, thereby
ruling out within-island speciation [52]. Only small-sized or
less mobile organisms, such as palms, provide unambiguous
examples of sympatric speciation on small volcanic islands
[6]. Given that marine iguanas are large and vagile reptiles,
the observed incipient speciation event, occurring in less
than 30 000 years on a small island, is unexpected. This pro-
cess was probably facilitated by an interaction of geological
and environmental factors which separated populations
spatially, and, in parallel, reduced their sizes. The current
geographical distribution of PP and LO (figure 3c) and their
recent phylogenetic divergence match remarkably well with
the distribution of contemporary volcanism on the island. Lava
flows recurrently occurred in central San Cristo
´bal during the
last 0.1 Myr [40], and the severe bottlenecks evident in both
lineages around approximately 1800– 3000 years ago coincide
with the most recent lava formation (figure 3c).
Accordingly, speciation might have initially followed a
micro-parapatric pattern, where repeated volcanic events
caused geographical disruption of marine iguana habitat
and isolated colonies on the northeastern and southwestern
extremes of the island. Yet these events cannot fully account
for their significant morphological differentiation and current
lack of genetic admixture. Local adaptation and/or prezygo-
tic mating barriers may have also contributed to the
consolidation of the divergence process. Difference in body
size, as observed between PP and LO, could be a sign of
differential habitat adaptation. In marine iguanas, body size
is under strong natural selection, and largely depends on
the occurrence and abundance of preferred algae [53].
Migrants and hybrids found in sampling sites within the
PP lineage (electronic supplementary material, table S3) are
larger than pure PP animals, suggesting that smaller body
size in PP is to some extent genetically determined.
(c) Within-island speciation in parallel with between-
island hybridization
Populations of PP and LO are geographically proximate
(approx. 12 km coastline between LO-SRCB and PP-SRBS;
figure 3b), and PP populations show no significant isolation by
distance over distances of more than 30 km (electronic sup-
plementary material, figure S4), indicating that they could
potentially migrate into the nearest LO populations. It is surpris-
ing that of the 474 individuals sampled around the islands’
coastline, not one full migrant individual of either lineage was
detected in the range of the other (figure 3a). Furthermore,
given reports of occasional hybridization occurring even
between marine and land iguanas [54], the discovery of only
two unambiguous PP/LO hybrids (electronic supplementary
material, table S3) is also remarkable. By contrast, between-
island hybridization, evidenced by eight occurrences, was
more common; a notable result, as only 10 migrants from
neighbouring islands (Santa Cruz and Espan
˜ola) were found
on San Cristo
´bal, making opportunities for this type of hybridiz-
ation rare. Therefore, it seems that two distinct evolutionary
processes are acting in parallel on San Cristo
´bal. Incipient
within-island speciation is evident, but at the same time, intro-
gressive hybridization with individuals from other islands
prevents the completion of this process on an archipelago-wide
scale. We hypothesize that these contrary processes have
influenced the evolutionary history of marine iguanas on the
Gala
´pagos archipelago.
Although introgressive hybridization is now increasingly
viewed as a driving force in speciation [9,10], the overall pattern
observed in marine iguanas resembles more the process of des-
peciation, described for Darwin’s finches [14], where one
species is genetically absorbed into another via hybridization
[15], or lineage fusion, as seen in Gala
´pagos giant tortoises
from Volcano Wolf on Isabela [17]. By contrast, the phylogeny
of Gala
´pagos land iguanas reflects ancient and fully completed
speciation, at least in the case of C. marthae [55], which diverged
around 1.52 Ma (figure 1b). Furthermore, Darwin’s finches
diversified into 14 species and subspecies within 1.6 Myr [14].
This is in clear contrast to the lack of phylogenetic bifurcation
of the marine iguana branch for almost 4.5 Myr. Such a pattern
would commonly be interpreted as an absence of speciation
processes, an assumption contradicted in this case by the
strong differentiation of island populations and the case of inci-
pient speciation on San Cristo
´bal. Thus, although A. cristatus
appears as a single phylogenetic species, incipient speciation
events, made visible here via the contemporary snapshot pro-
vided by highly variable markers, may well have also
occurred in the evolutionary past of this species.
Marine iguana populations regularly experience strong
selective pressure during climatic El Nin
˜o oscillations [47,48],
which vary in strength between locations and disproportio-
nately remove larger individuals; such selection may partly
explain the morphological variation among island popu-
lations, especially in terms of body size [53]. Nevertheless,
marine iguanas are highly successful and occur archipelago-
wide, whereas land iguanas currently occur on only four
major islands [55]. The geography of the Gala
´pagos archipe-
lago is particularly conducive to the emergence of novel local
adaptation in geographical isolation, and in a mobile species
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like the marine iguana, new geneticvariants are rapidly assimi-
lated into a common gene pool via introgressive hybridization.
The establishment of such a gene pool, incorporating events of
local adaptation and speciation, might be an important mech-
anism underlying the evolutionary success of marine iguanas.
Insight from these processes might enhance our understanding
of how a species can persist despite frequent and severe cli-
matic oscillations, such as El Nin
˜o events, which can induce
severe population crashes.
Ethics. Sampling of marine iguanas was done with permissions of the
Gala
´pagos National Park (permit numbers PC-26-12, PC-23-13 and
PC-22-14).
Data accessibility. All sequences generated for this study have been depos-
ited in GenBank under accession numbers KR350691– KR351205. Full
alignments, SNP and microsatellite genotypes have been deposited
in the Dryad data repository (http://dx.doi.org/10.5061/dryad.
pp6bm). Further data, extended methods, results and discussion sup-
porting this manuscript have been submitted as part of the electronic
supplementary material.
Authors’ contributions. S.S., A.M., F.T. and M.V. designed the study. A.M.,
C.G. and G.Q. collected field data. G.G. and A.C. contributed data.
A.M., A.R., P.O. and M.V. analysed data. S.S., M.V. and A.M.
wrote the manuscript.
Competing interests. All authors hereby declare that they have no com-
peting interests.
Funding. This work was supported by grants from the Swiss Friends of
the Gala
´pagos, the Gala
´pagos Conservation Trust and the National
Geographic Society (grant no. GEFNE99-13).
Acknowledgements. We are grateful to the Gala
´pagos National
Park authority for research permission; to S. Rea for help with
paperwork; to S. Pasachnik for providing samples; to L. Unsworth,
Maryuri Ye
´pez, L. Cardas, M. D. Astudillo, L. Cruz, D. Toninho
and T. Reinhardt for field assistance; to G. Jimenez for granting
access to preserved specimens; to D. J. Geist for discussion; to
J. C. Marshall for comments on the manuscript; to C.-K. Baillie,
E. Hippauf, G. Keunecke, M. Kondermann, S. Ku
¨nzel and
S. Weißelberg for help with laboratory work; and to S. Herzog
for morphometric measurements. This publication is contribution
number 2115 of the Charles Darwin Foundation for the Gala
´pagos
Islands.
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Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
1
Electronic Supplementary Material
Hybridization masks speciation in the evolutionary history of the Galápagos marine
iguana
Amy MacLeod, Ariel Rodríguez, Miguel Vences, Pablo Orozco-terWengel, Carolina García,
Fritz Trillmich, Gabriele Gentile, Adalgisa Caccone, Galo Quezda, Sebastian Steinfartz
Extended methods
This section provides additional details on field and laboratory protocols as well as statistical
analysis, complementing information in the main Methods section.
Field sampling
Marine iguanas from San Cristóbal were newly sampled in 2011-2014 at 17 sites (Table S1).
We obtained blood samples from 460 marine iguanas from this island between 2011 and
2013, and used these in addition to samples collected for earlier studies from Punta Pitt
(1993; n = 22) and Loberia (1993; n = 31). Animals were captured using poles fitted with a
lasso loop. From each individual ca. 0.1 ml of blood was collected from the caudal vein, with
the individual in dorsal recumbency. Venipunction point was localized at one third of the
distance between the cloacal opening and the tails’ end, following the middle line. 24G (0.80
x 40 mm) needles were used for large males (>3kg body weight), 23G (0.60 x 25mm) needles
for smaller adults, and 26G (0.45 x 25mm) for larger juveniles, while insulin needles were
used for small juveniles. The needle was inserted with a 45°- 90° angle between the needle
and the animal. Blood was stored in 75% ethanol or in a blood buffer (100 mM Tris, 100 mM
EDTA, 2% SDS) and maintained at 4 7°C prior to extraction of genomic DNA.
Phylogenetic analysis and molecular dating: nuclear protein coding genes (dataset A)
To identify the closest relative of the Galápagos iguanas and date their origin on the
archipelago, we selected four nuclear genes (RAG1, BDNF, R35 and NKTR) from a previous
dataset [1] on the basis of their performance in reconstructing iguanid phylogeny. Sequences
were newly determined for three marine iguanas, one land iguana (Conolophus subcristatus)
and one individual each of Cyclura cornata, Iguana iguana and Ctenosaura similis, and
combined with those of another 72 iguanians and other squamates from the dataset of
Townsend et al. [1] to allow for the use of established time calibrations. DNA sequences
were aligned taking into account their amino acid sequences, using the MAFFT [2] tool
employed in Translator-X [3, 4]. Sequences were cleaned with GBlocks [5], using all three of
the ‘less-stringent’ cleaning parameters available in Translator-X, resulting in a final
sequence alignment of 3000 nucleotide positions which were used in analysis. Partitions and
substitution models for analysis were identified using the Bayesian Information Criterion in
Partition Finder [6], using the ‘greedy search’ scheme. The concatenated sequences were
analysed using three approaches. (i) Partitioned Bayesian inference of phylogeny (BI) with
MrBayes 3.2 [7], running two analyses of four chains for 20 million generations, sampling
every 10,000 generation, and calculating a majority-rule consensus tree discarding the first
25% as burn-in. (ii) Simultaneous inference of phylogeny and divergence times was achieved
using Beast 1.7.2 [8], with a set of 18 time constraints across squamates [1] and a Yule
Speciation tree prior, running 200 million generations and calculating a maximum clade
credibility tree with a burn-in of 25%. For this analysis we used an older Beast version (1.7.2)
in order to strictly follow the previously published analysis [1] and to be able to use the same
settings, with the goal of reaching fully comparable results. All Beast runs were given enough
time to allow effective sample size values of all parameters to reach values well above 200.
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(iii) Maximum parsimony bootstrapping was undertaken in Paup 4b2 [9], with 2000 heuristic
search replicates under tree-bisection-reconnection, and with random addition sequence (10
replicates). Convergence of chains, mixing of parameters and appropriateness of burn-in
settings of all Bayesian analyses was confirmed with AWTY [10].
Phylogenetic analysis and molecular dating: mitochondrial (mt) genes (dataset B)
On the basis of an earlier mitochondrial CR haplotype network [11] a representative selection
of marine iguana samples were chosen from across the archipelago for analysis of
mitochondrial phylogeny. Sampling was adjusted to represent the most divergent mtDNA
phylogroups as well as the two San Cristóbal lineages (PP and LO). In total, up to 5557 bp
were sequenced from seven mitochondrial genes (plus 3 adjacent tRNAs) in 20
Amblyrhynchus, six individuals of three species of Conolophus (including C. marthae), and
one individual of Ctenosaura similis. Sequences of the I. iguana mt genome (GenBank
accession number AJ278511) were used as outgroup. Sequences were edited using
CodonCode Aligner (CodonCode Corporation) and aligned using Clustal W [12], as
employed in MEGA (V6.0; [13]). Only single indels were present in the alignment (mainly in
the outgroup) and all sites were therefore included in the analysis. Identification of partitions
and substitution models, as well as phylogenetic analysis was undertaken as in dataset A,
except for the “timetree” analysis with Beast. Here, we specified a coalescent uniform tree
prior, a secondary time constraint with an uniform prior (2.76-6.67 mya) for the
Amblyrhynchus-Conolophus split based on the estimate for this split obtained from analysis
of dataset A (credibility intervals), and modified substitution rate priors to allow variation
over a wide range. Posterior values did not stabilize with site models suggested by Partition
Finder, probably due to overparametrization, and we therefore specified a simple HKY model
for all partitions. Analyses with alternative priors and models (different coalescent or
speciation tree priors; more complex substitution models; no phylogenetic constraints) were,
however, congruent in divergence time estimates, in all cases recovering an age of basal
Amblyrhynchus splits <0.3 mya.
Phylogenetic analysis and molecular dating: DNA sequences from RADSeq (dataset G)
See main Methods section for details of RADSeq analysis and inference of phylogeny based
on SNP data. For molecular dating of splits within Amblyrhynchus we used a 4-taxon subset
of dataset F (Table S2), containing one land iguana outgroup (Santa Fe, sample LSF06) and
three marine iguana specimens (Santa Fe, FES01; San Cristóbal, SRIL10 and SRS19),
representing one of the deepest split within marine iguanas and the PP/LO split. We excluded
in Paup all sites with missing or ambiguous data in one or several taxa, resulting in a final
matrix of 1,793,845 sites, of which 33,452 were variable, and only 330 were variable among
the three marine iguanas. The dataset was analysed in Beast 2.0 under a coalescent tree prior
(constant growth), time-calibrating the root MRCA according to analysis of dataset A at 4.6
mya (normal prior, standard deviation 0.3). We used a GTR substitution model selected by
the Akaike Information Criterion (AIC) in MrModeltest [14]. We ran 2 billion generations,
sampled every 100,000th generation, discarded the first 50% trees as burn-in after
examination of parameters in Tracer, and ascertained that all ESS values were >200.
Alternative explorative runs with the HKY model (selected by AIC for the dataset of three
marine iguana samples only) did not result in relevant differences in time estimates.
Mitochondrial differentiation of marine iguanas (dataset C)
An alignment of the complete mitochondrial control region (CR) sequences, 1181 bp in
length, was used to analyse archipelago-wide mitochondrial phylogeography of marine
iguanas, and mitochondrial differentiation among PP and LO lineages. This gene segment
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
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3
was newly sequenced from 310 individuals of marine iguanas from San Cristóbal and 34 so
far unused samples from Darwin, Wolf, Rocca Redonda and Seymour Norte islands, using
methods from a previous study [11], and leading to a final dataset of 1491 sequences. One
individual from each of the three species of Galápagos land iguana were also sequenced as
outgroups. Sequences were edited and aligned as described above for dataset B.
We visualized CR variation in haplotype networks reconstructed using information from
phylogenetic trees as implemented in Haploviewer [15]. Maximum likelihood trees were
estimated with PHYML [16] using the best-fitting substitution model (GTR +I +G) as
identified by the Akaike information criterion [17] in J-Modeltest [18].
Microsatellite loci genotyping (datasets D and E)
As a basis for the archipelago-wide comparison of population structure (dataset D) we used
microsatellite loci which had largely been determined in previous studies [11, 19], in this
study, 12 loci were used due to omission of locus E17 which had a high failure rate. This
available dataset was extended with some newly genotyped samples from other islands,
collected during previous fieldwork, and with all newly collected San Cristóbal samples.
DNA from previously genotyped samples were genotyped along with the newly obtained
samples, in order to calibrate alleles between different sequencing machines and to ensure
that alleles were correctly scored. This resulted in an available pool of almost 1500 samples
that were all genotyped at 12 loci. All San Cristóbal samples (dataset E; Table S1, S2) were
genotyped for the same 12 loci as the archipelago-wide dataset, plus an additional 6 loci,
yielding data for 18 microsatellite loci described previously [20] excluding locus E17.
Primers, multiplexes, and PCR parameters are detailed elsewhere [20]. Scoring of alleles was
performed with Genemarker (version 1.95; Applied Biosystems).
Population structure analysis (datasets D and E)
A model-based Bayesian clustering method (Structure, v. 2.3.3; [21]) was used to infer
population structure from microsatellite loci data. As this method requires no a priori
sampling information, it is particularly useful for revealing cryptic population structure. For
within-island analysis on San Cristóbal (dataset E), prior number of inferred populations (K)
ranged from 1-5, and for analysis of samples across the archipelago (dataset D), K ranged
from 1-20; both with 10 iterations for each K. Each run used 100,000 MCMC replicates
following a burn-in period of one million replicates. An admixture model was employed, and
the model parameter alpha was inferred from the data in combination with correlated allele
frequencies. Inferred number of populations was obtained [22] via the Structure Harvester
application [23]. Results were permuted using CLUMPP [24] and visualized using Distruct
[25].
Population structure analysis: sample selection for dataset D
Variation in animal density and sampling effort across the archipelago (dataset D; 12
microsatellite loci) mean that within this dataset, certain populations were strongly
overrepresented with respect to others. Since this can lead to artefacts within population
structure analysis [26, 27], prior to analysis, the sample sizes for each island were
standardized to closely match the smallest sample size available for any island (around 50;
Table S2). Any islands where sample size was well below 20 were not considered in this
analysis. However, this applied only to very small islands (e.g. Rabida); located nearby larger
(included) islands and were not found to be harbouring genetically distinct clusters in earlier
studies [11, 19]. This standardization procedure was done by pooling samples from all
sampling locations and occasions on each island, and randomly selecting 50 individuals,
except in the cases of Santiago and Floreana islands where all available samples (47 and 43
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
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4
respectively) were used. Data from San Cristóbal Island were treated slightly differently,
because of the two strongly divergent populations identified previously [11, 19]. Here, 2 sets
of n=50 were created, representing the PP and LO lineages, in addition to a third set collected
from a previously non-sampled area of the island, where the genetic identity of individuals
was unknown. In total, 614 individuals from all islands were included in this analysis (dataset
E).
Population structure analysis: sample selection for dataset E
For analysis within San Cristóbal (dataset E; 18 microsatellite loci) our goal was to assign
populations and specimens to the two lineages (LO and PP), and to identify possible hybrids
among these two lineages. Therefore, in this analysis, we excluded all individuals that were
identified as migrants or possible hybrids with other islands in the previous Structure analysis
(dataset D) or assignment tests (see text section below). Additionally, any individuals
demonstrating a mitochondrial haplotype private to other islands were removed for the same
reason. Further, to exclude artefacts arising from missing data, individuals with missing data
for more than one allele were excluded; this necessitated only a small reduction in sample
size. Additionally, any resampled individuals were identified using Genalex [28] and
removed. In total, 454 individuals were included in the San Cristóbal within-island dataset.
Detection of hybridization on San Cristóbal: datasets C, D and E
Hybrids or migrants from other islands on San Cristóbal were initially identified from the
Structure analysis of dataset D as individuals who demonstrated a lower probability of
belonging to either LO or PP than a predefined threshold (80%). Furthermore, several
individuals had mitochondrial haplotypes (dataset C) predominantly found on other islands.
All of these individuals were submitted to assignment tests [29] based on their microsatellite
loci genotypes, using GENECLASS [30]. This method was chosen as it produced results,
which were in close agreement to the islands associated with mitochondrial haplotypes. We
considered as hybrids or migrants from other islands all individuals that did not assign with a
probability of >90% [31] to either of the two San Cristóbal populations, and/or if they had a
mitochondrial haplotype private to another island. For the assignment tests, a 12-loci
reference dataset was created to include LO (N=151) and PP (N=286), and any other cluster
where San Cristóbal individuals had been assigned to in the earlier analysis with Structure
(dataset D): Española/Floreana (N=128) and Santa Cruz (N=116).
Isolation by distance (IBD) analysis (dataset E)
To test the extent to which isolation by distance (IBD) shaped the genetic diversification on
each of the two San Cristóbal clusters, we estimated the geographic isolation among localities
measuring in GoogleEarthTM, the pairwise coastline distances (in km) between marine iguana
populations of LO (5 localities) and PP (8 localities). The genetic differentiation between
localities was estimated using pairwise RST values, an analogue of FST specifically developed
for microsatellite loci [32]. For these calculations we considered a sampling locality to be a
place where more than two individuals have been genotyped. Suspected hybrids and migrants
(see previous section) were not included in the dataset. We evaluated the association between
the two distance matrices with a Mantel test, as implemented in the R package vegan [33],
evaluating statistical significance with 10000 permutations.
Demographic history of PP and LO on San Cristóbal (dataset E)
Two approaches were used to examine the demographic history of the LO and PP lineages
based on 18 microsatellite loci data (dataset E). Firstly, the software Bottleneck [34] was used
to determine the presence of heterozygote excess in each of the populations using the
standardized difference test and the Wilcoxon-ranked test. We carried out 1,000 simulations
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
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to assess the tests’ significance under the two phased mutation model, assuming 30% of the
mutations were multistep with a variance in mutation size of 30. Secondly, the software
MsVar 1.3 [35] was used to characterize the recent demographic history of the LO and PP
lineages. The method implemented in MsVar uses coalescent simulations to estimate the
current effective population size (N0), the ancestral population size (Nt) and the time at which
a demographic change (t) may have occurred (i.e. an expansion after a bottleneck) following
an exponential model of effective population size. The simulations carried out in MsVar were
conditioned with various a priori combinations for the model parameters, so that stable
scenarios, bottlenecks and expansion were considered, as well as variation in the mutation
rate across microsatellite loci (Table S5). Each MsVar run consisted of 1,010 iterations of the
MCMC algorithm, discarding the initial 20% of the coalescent simulations as burn-in.
Convergence of the chains was assessed with Gelman & Rubi’s diagnostic [36] calculated on
the basis of the seven runs performed each for the PP and LO population with different
priors. Gelman and Rubin’s diagnostic was carried out using the CODA library [37].
We further tested whether the LO and PP lineages have evolved under a model with or
without gene flow, using simulations performed using the software 2mod [38]. We performed
three independent replicates with a total of 1,000,000 coalescent simulations, each using the
MCMC algorithm implemented in 2mod. For each simulation, the likelihood of the drift or
migration model is estimated on the basis of the allele counts in the data. After discarding the
first 20% of the simulations as burn-in of the MCMC, we estimated the posterior probability
of each model as the proportion of simulations that supported each scenario (i.e. drift or
migration).
Morphological characterization of PP and LO
To analyse whether specimens of the two San Cristóbal lineages, PP and LO, are also
morphologically differentiated, we scored morphometric variables and scale counts from
139 Amblyrhynchus specimens collected from San Cristóbal. These individuals were also
genotyped at 18 microsatellite loci, and all specimens identified as hybrids or migrants from
other islands were disregarded. Specimens were assigned to four different age classes (1:
juveniles below 1 year of age; 2: larger juveniles estimated between 1 and 2-3 years; 3: sub-
adults approaching adult size but lacking well developed adult morphology, and 4: adults of
breeding age based on body size and development of external features such as elevated
tubercular scales positioned dorsally on head, femoral pores, and dorsal crests. We sexed only
specimens in age class 4, and considered specimens with obvious male characteristics such as
enlarged dorsal crests, comparatively larger body size, and well-developed femoral pores as
males, and those lacking these features as females. Measurements taken from living
specimens in the field include snout-vent length (SVL) from snout tip to cloaca and total
length (TL) from snout tip to tail tip to the nearest 10 mm, head width (HW; taken at point of
maximum width of head), head length (HL) taken from the snout tip to the edge of the
furthest elevated tubercular scale dorsally on head, maximum head height (HH), and length
of the 4th toe, taken with a calliper to the nearest 0.1 mm, and weight (to the nearest 0.01 kg).
Scale counts were taken from detailed photographs of each of the specimens.
In an initial search for potentially diagnostic differences, we took the following scale counts
from 20 specimens of each genetic lineage: lamellae under the third and fourth toe on
hindlimb (LAM3T, LAM4T), supralabials (SUPL), series of scales above supralabials
(suprasupralabials, SUPSUPL), infralabials (INFL), series of scales below infralabials
(infrainfralabials, INFINFL), minimum transversal count of large scales dorsally on head
(TRHSC), minimum longitudinal count of large scales dorsally on head (LOHSC), innermost
series of scales on upper and lower eyelid (EYELUI, EYELLI), second series of scales on
upper and lower eyelid (EYELUO, EYLLO), femoral pores (males only; FP), number of
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
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scales between large dorsal head scales and start of dorsal crest spines (HCSP), number of
dorsal crest spines in anteriormost part of dorsal crest (DORSC1). All bilateral counts
(LAM3T, LAM4T, SUPL, SUPSUPL, INFL, INFINFL, EYELUI, EYELLI, EYELUO,
EYELLO) were done on both sides of the body whenever possible, the average between both
values was calculated per specimen and used for further analysis. In cases where reliable
counts could only be obtained from either the left or right side of the body, the respective
value was used for analysis. In numerous cases, reliable counts could not be obtained due to
injuries of the specimens (e.g. partly mutilated toes), scales being difficult to distinguish due
to skin shedding, or poor quality of photographs. In all such cases, counts were disregarded.
Our goal here is not to provide a full morphometric analysis of variation in these lizards, but
to instead highlight morphological differentiation among the lineages. We therefore limited
detailed comparisons to a set of variables in which such differentiation was clearly apparent.
We compared the values of the initially analysed 40 specimens with non-parametrical Mann-
Whitney U-tests for pairwise comparisons. Based on this initial evaluation, we identified the
variables SVL, HL, LAM3T, LAM4T, SUPL, SUPSUPL, INFL, INFINFL, and HCSP as
most clearly differentiated between the two genetically differentiated lineages, and therefore
assessed these variables in the total number of 139 individuals. We used U-tests for final
comparisons with the maximum number of available data for each variable. We further
performed multivariate analyses of variance (ANOVAs) with Tukey's post-hoc tests for
single variables. All statistical tests were done in Statistica 7.1 (StatSoft Inc.). As ANOVAs
cannot deal with missing data, these analyses included a reduced number of specimens (i.e.
those with complete sets of data for the variables compared). Morphometric measurements
were size-corrected by linear regression against SVL, and the residuals used for further
analysis.
Extended Results and Discussion
This section provides additional details on the results, as well as statistical analysis and
discussion, complementing information in the main Results and Discussion parts of the paper.
Data Sharing
Single genes from datasets A, B and C are deposited in Genbank
(www.ncbi.nlm.nih.gov/genbank/) under the following accession numbers: NKTR:
KR350691 - KR350697, R35: KR350698 - KR350704, RAG1: KR350705 - KR350711,
BDNF: KR350712 - KR350718, ND4: KR350719 - KR350742, ND2: KR350743 -
KR350767, Cytochrome-B: KR350768 - KR350787, COI: KR350788 - KR350813, 12S:
KR350814 - KR350838, 16S: KR350839 - KR350861, and control region: KR350862 -
KR351205. Other data are deposited in Dryad (datadryad.org) under
doi:10.5061/dryad.pp6bm. Brief information about the files is given here, with Dryad file
names shown in bold. Aligned and concatenated nuclear sequences (including tRNA
sequences not deposited in Genbank) were deposited as dataset_A, aligned mitochondrial
genes are found in Dataset_B_Mitochondrial, and complete aligned mitochondrial control
region sequences are provided in Dataset_C. Microsatellite genotypes representing marine
iguanas from across the archipelago are provided in Dataset_D_Microsats, genotypes from
San Cristobal only are given in Dataset_E_Microsat and a subset of these samples used in
MsVar and Bottleneck analysis are given in Dataset_E_Microsat. Data from RAD
sequencing are given in Datasets_F_G_H with further notes in the README.doc.
Morphometrics used in comparison between two populations of marine iguanas are given in
the file: Morphometry_Amblyrhynchus.
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
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Divergence time estimates
Our timetree, based on four nuclear genes (dataset A; Fig. S1), predicted a younger age for
the split between Galápagos marine and land iguanas than a previous study had [39]. As such,
our study reconciles the age of this endemic clade with the age of the currently extant islands.
We consider our timetree to be more reliable than the one of the previous study, which was
undertaken at a time when sophisticated molecular dating methods were not yet available,
and is based only on mitochondrial genes, which are prone to overestimating divergence
times [40]. Furthermore, this estimate was based on a possibly inadequate strict clock
approach, with a relatively fast substitution rate (2% per my for the mitochondrial
cytochrome b gene) which was derived from data on mammals. Conversely, our estimates are
based on single-copy protein-coding nuclear genes, using multiple squamate calibrations, and
relaxed clock models, which together are known to provide more realistic divergence time
estimates.
Demographic history of LO and PP
The two approaches (i.e. bottleneck and MsVar) to examine the demographic history of LO
and PP detected bottlenecks in the recent history of these lineages (Fig. S3). Both, the
standardized difference test and the Wilcoxon-ranked test identified a significant excess in
heterozygosity, consistent with the hypothesis of a recent bottleneck in the past history of PP
and LO (Table S4). The MsVar approach identified that the bottleneck experienced by both
lineages took place approximately 1,800-3,000 years ago and consisted of a reduction in the
effective population size of ~100,000 individuals to ~1,000 individuals (see Table S6 for
exact data and corresponding confidence intervals).
The demographic analysis with 2mod aiming at determining whether the LO and PP lineages
evolved under a simple model of drift versus a model that included migration between
populations found consistently across replicates of the analysis that the gene flow model was
supported with a posterior probability of ~1.
Morphological analysis
As our dataset contains a large number of missing data and furthermore differences in body
size imply the need for in-depth analysis of possible allometric effects, a simultaneous
analysis of all data in a single multivariate model would either result in very low sample sizes
(if full variable coverage was needed for each individual) or potential artefacts (if missing
data were replaced by mean values). We therefore opted for a mixed strategy, first analysing
pholidotic characters along with body size (SVL), and second, analysing the morphometric
measurements. Rather than adopting a strict Bonferroni correction for multiple testing, we
considered results as robust if they were consistently suggested by separate analyses, i.e. of
independent datasets (males versus females) or of subsets of data (i.e. after excluding large
and small specimens which might be overrepresented in either cluster).
(i) Body size. Both non-parametric Mann-Whitney U-tests (Tables: S9, S11 & S13; Fig. S7)
and ANOVA coupled with Tukey’s post-hoc tests (Tables: S10, S12 & S14) suggested
statistically significant differences in body size between LO and PP, with PP specimens being
smaller. This result remained significant in separate analyses of adult male and female
specimens, despite wide overlap in sizes between the two clusters. Although p-values in U-
tests would be insufficient under Bonferroni correction, the fact that statistical significance is
maintained in the ANOVA post-hoc tests increases our confidence in the biological meaning
of this pattern.
(ii) Pholidotic characters. Univariate pairwise U-tests flagged a large numbers of variables as
statistically different between the two clusters (Tables: S9-S14; Fig. S7), but in many cases
(LAM3T, LAM4T, SUPSUPL, EYELUO) the significance values were between 0.01-0.05,
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and would not remain significant following Bonferroni correction for multiple testing.
However, three variables remained significantly different after Bonferroni correction,
remained so in Tukey's post-hoc tests, and showed similar trends in males and females. These
were: INFL, INFINFL, and DORSC1. We therefore conclude that there is robust evidence for
PP specimens having on average a larger numbers of infralabials, infra-infralabials, and
spines in first part of dorsal crest than specimens of the LO cluster.
(iii) Morphometric characters. Morphometric measurements are as a rule strongly size-
dependent, and this relationship is often near-linear in adult animals. However, many body
proportions are subject to non-linear allometric growth in the transition from juveniles to
adults. For instance in lizards, young specimens have proportionally larger heads, and may
also have proportionally longer limbs than adults. In the case of marine iguanas, LO
specimens grow to larger sizes than PP specimens. If growth of head and limbs would still be
slightly allometric in the subadult or young adult stage, then between-cluster differences in
related measurements could potentially be caused by their different body sizes. An initial
comparison of all adult specimens (males and females) revealed statistically significant
differences in HW, HL and TOEL residuals between the two clusters. Considering separate
tests of males and females, and tests excluding small specimens (<20 cm SVL) as well as
large specimens (>36 cm SVL), only the between-cluster differences in HL residuals were
consistently retrieved, and as such, can be considered robust (Tables S15-S18). Therefore, we
conclude that PP specimens have proportionally longer heads than LO specimens.
Hybridization
Given the large number of highly polymorphic loci used and the strong level of
differentiation between clusters, it is likely that the analysis would have adequately identified
hybrids [30]. Although results from New Hybrids did not unambiguously assign hybrid
individuals to hybrid classes (e.g. F1, F2 etc.; data not shown), the disparate results between
D-loop haplotypes and microsatellite loci clusters indicate that extensive backcrossing has
occurred. For instance, several individuals demonstrated high assignment probabilities for
being PP individuals based on microsatellite loci, whilst harbouring a D-loop haplotype
private to the Espanola/Floreana cluster (Table S3; Fig. S2). This indicates that the
hybridization event occurred several generations ago, but since then, most of the genetic
signature indicative for Espanola/Floreana cluster in the microsatellite loci has been lost.
Isolation by distance (IBD) analysis
Genetic differentiation scales with distance between subpopulations within the Loberia
population but not within the Punta Pitt population, indicating a more recent expansion within
the PP lineage (Fig. S4). Within both clusters, observed maximum geographically separated
subpopulations display a lower genetic distance than geographically closest subpopulations of
different clusters (Tables: S7 & S8), but in the PP cluster no significant IBD is seen,
suggesting that gene flow is maintained over the maximum coastal distance of 30 km.
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Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
12
Table S1. Locations of sampling and number of marine iguana blood samples collected from San Cristóbal
Island. Asterisk denotes samples collected by K. Rassmann, all others collected by the authors.
Name
Code
Latitude
Longitude
Number of blood samples per year
Data available per location
Number
genotyped
Number
sequence
d for D
loop
1991/3*
2004
2011
2012
2013
2014
12 loci only
18 loci
Punta Carola
SRCA
0°53'22.01"S
89°36'45.04"W
15
13
10
La Loberia
SRL
0°55'19.80"S
89°37'15.04"W
31
39
30
40
27
35
111
133
Playa Ochoa
SRO
0°51'50.04"S
89°34'16.06"W
6
1
7
1
Isla Lobos
SRIL
0°51'20.04"S
89°34'5.04"W
7
15
15
33
11
Cerro Brujo
SRCB
0°45'50.10"S
89°27'31.20"W
6
19
3
14
5
Bahía Sardina
SRBS
0°42'11.90"S
89°21'52.30"W
1
1
2
1
La
Galapaguera
SRG
0°41'34.90"S
89°18'10.90"W
20
35
8
66
42
Las Salinas
SRS
0°41'50.09"S
89°16'15.01"W
20
21
1
42
21
Playa Blanca
SRPB
0°41'42.04"S
89°15'27.08"W
22
13
15
50
10
78
72
Islote Pitt
SRIP
0°42'11.01"S
89°14'50.01"W
6
10
10
10
36
9
Playa Café
SRPC
0°42'51.04"S
89°14'30.09"W
5
15
22
22
87
29
Puerto Chino
(East coast)
SRCH
0°55'32.52"S
89°25'33.10"W
1
1
1
East coast A
SRECA
0°51'20.60"S
89°21'55.20"W
8
8
8
East coast B
SRECB
0°47'9.50"S
89°17'53.00"W
5
5
5
East coast C
SRECC
0°45'43.40"S
89°16'38.70"W
2
2
2
East coast D
SRECD
0°44'55.10"S
89°16'5.30"W
6
6
6
East coast E
SRECE
0°43'44.90"S
89°15'3.60"W
2
2
2
Total counts
53
52
24
130
230
119
35
513
369
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the Galápagos marine iguana
13
Table S2. Locations and number of marine iguana blood samples collected from across the Galapagos archipelago utilised in the various molecular datasets (A-H) used
throughout the study.
Island
Sample size per dataset
A
B
C
D
E
F
G
H
Amblyrhynchus cristatus
Darwin
3
1
1
Rocca Redonda
9
1
1
Wolf
12
2
2
Fernandina
179
50
2
1
2
Isabela
1
5
138
50
2
2
Pinzon
12
Rabida
11
Pinta
93
50
2
2
Marchena
78
50
2
2
Genovesa
81
50
2
2
Santiago
72
47
2
2
Santa Cruz
5
116
50
2
2
Seymour Norte
10
Santa Fé
159
50
2
2
Floreana
60
50
2
2
Española
98
43
2
2
San Cristóbal: Loberia
1
5
166
50
157
4
1
4
San Cristóbal: Punta Pitt
1
5
171
50
306
5
1
5
San Cristóbal: East coast
East Coast
23
24
11
Outgroup
Conolophus subcristatus
1
4
2
Conolophus pallidus
1
2
1
Conolophus marthae
Ctenosaura similis
1
2
Ctenosaura pectinata
1
Iguana iguana
1
1
Other squamate species
73
2
Total: A. cristatus
3
20
1491
614
474
33
3
33
Total: all
79
27
1491
614
474
41
4
33
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the Galápagos marine iguana
14
Table S3. List of putative hybrids and migrants identified on Santa Cruz Island. D-loop haplotype numbers are as in a previous study (S11) and used in Fig. 2. Assignment
tests were carried out with GENECLASS.
Loc.
type
Individual
and
gender/size
Size
Class
D loop hap #
Island(s) associated
with haplotype
Microsat.
assignment
probability
of
assignment
Conclusion
Weight
TL
SVL
East
coast
SRECA_01
M4
4
Española
Santa Cruz
62
Inconclusive, possible hybrid: East coast
population (Española origin) / Santa Cruz
4.6
117
45
SRECA_02
F4
4
Española
Floreana/Española
96
Pure East coast population of Española
origin
2.35
97
38
SRECA_03
M4
4
Española
Floreana/ Española
100
Pure East coast population of Española
origin
4.08
105
41
SRECA_04
U2
4
Española
Floreana/ Española
100
Pure East coast population of Española
origin
0.57
47
18
SRECA_05
U2
6
Española
Floreana/ Española
100
Pure East coast population of Española
origin
0.62
58
26
SRECA_06
U2
4
Española
Punta Pitt
76
Hybrid: Punta Pitt/East coast population
(Española origin)
0.68
55
21
SRECA_07
M4
6
Española
Floreana/ Española
100
Pure Española (EC pop)
5.65
123
52
SRECA_08
M4
4
Española
Loberia
100
Hybrid: Loberia / East coast population
(Española origin)
6.46
123
50
SRECB_03
M4
3
Española
Punta Pitt
100
Hybrid: Punta Pitt / East coast population
(Española origin)
4.2
106
37
SRECB_0
F4
3
Española
Punta Pitt
99
Hybrid: Punta Pitt / East coast population
(Española origin)
2.39
81
32
SRECC_01
F4
3
Española
Punta Pitt
100
Hybrid: Punta Pitt / East coast population
(Española origin)
17.3
71
26
Punta
Pitt
SRG12_06
M4
unavailable
Floreana/ Española
63
Inconclusive. Probable genotype signatures
of Española origin
3.6
95
35
SRG13_08
M4
67
Santa Cruz
Floreana/ Española
76
Inconclusive, possible hybrid: East coast
population (Española origin) / Santa Cruz
5.2
108
40
SRG13_11
F4
81
San Cristóbal: Punta Pitt
Loberia
61
Hybrid: Loberia/Punta Pitt
1.58
69
26
SRG13_34
M4
67
Santa Cruz
Santa Cruz
83
Santa Cruz vagrant
3.04
91
34
SRS13_05
M3
81
San Cristóbal: Punta Pitt
Loberia
90
Hybrid: Loberia/Punta Pitt
0.72
57
21
Loberia
SRL13_22
M3
67
Santa Cruz
Santa Cruz
100
Santa Cruz vagrant
2.2
81
31
SRL13_24
M4
20
Santiago, Santa Fe
Santa Cruz
100
Probable Santa Cruz vagrant
5.12
108
40
SRL14_02
F4
unavailable
Santa Cruz
100
Probable Santa Cruz vagrant
2.62
36
90
SRIL13_06
M4
68
San Cristóbal: Loberia
Santa Cruz
89
Hybrid: Loberia/Santa Cruz
1.76
78
33
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
15
Table S4. Results from the Bottleneck Analysis. P-values for the standardized difference and Wilcoxon tests are
shown. In bold is the non-significant p-value after Bonferroni correction.
Test
Loberia
Punta Pitt
Standardized Difference
test
0.049
0.0009
Wilcoxon test
0.017
0.0002
Table S5. Prior distributions for the MsVar analysis. Each row in the table shows the prior distribution of the
parameters of the MsVar analysis for each of the seven scenarios tested. The parameters are N0= Current
Effective Population Size, Nt= Ancestral Effective Population Size, u= mutation rate, t= time at which the
change in effective population size took place. The value under each parameter is the log mean of the
distribution and under the column parameter Var is the variance of the distribution as used in MsVar. The
variance of the means and of the variances in the hyperpriors of all parameters were in all simulations 0 and 0.5,
respectively. Each of these seven models were tested in each population.
Scenario
N0
N0 Var
Nt
Nt Var
u
u Var
t
t Var
1
4
2
4
2
-4
1
5
2
2
5
2
5
2
-4
2
6
2
3
5
2
3
2
-4
2
5
1
4
4
1
5
1
-4
1
5
2
5
3
1
5
1
-4
1
6
1
6
3
2
5
3
-4
3
5
3
7
4
1
5
1
-4
1
3
2
Table S6. Posterior distribution from the MsVar analysis. Data are given as log10.
No
HPD 95%
LO
1000
962139
PP
1039
2823236
NA
HPD 95% LB
LO
141254
44868568351
PP
100000
32643386843
t
HPD 95% LB
LO
1848
1783561
PP
3043
8579755
Table S7. Genetic (below diagonal, in RST values) and geographic (above diagonal, in km) distances between
localities included in the Isolation by Distance analysis of the LO cluster. Locality codes as presented in Fig. 3.
SRL
SRPA
SRO
SRIL
SRCB
SRL
6.5
13.41
15.02
41.52
SRPA
0.020
6.91
8.52
35.02
SRO
0.020
0.021
1.61
28.11
SRIL
0.036
0.007
0.004
26.5
SRCB
0.062
0.051
0.033
0.043
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
16
Table S8. Genetic (below diagonal, in RST values) and geographic (above diagonal, in km) distances between
localities included in the IBD analysis of the PP cluster. Locality codes as presented in Fig. 3.
SRBS
SRG
SRS
SRPB
SRIP
SRPC
SRECD
SRECC
SRBS
10.5
15.53
17.33
19.41
21.43
26.8
30.88
SRG
0.00
5.03
6.83
8.91
10.93
16.3
20.38
SRS
0.02
0.01
1.8
3.88
5.9
11.27
15.35
SRPB
0.01
0.01
0.00
2.08
4.1
9.47
13.55
SRIP
0.08
0.03
0.02
0.02
3.05
8.42
12.5
SRPC
0.00
0.01
0.01
0.01
0.03
5.37
9.45
SRECD
0.00
0.01
0.02
0.01
0.03
0.00
4.08
SRECC
0.01
0.05
0.05
0.06
0.05
0.02
0.00
Table S9. Results of Mann-Whitney U-tests for SVL and scale counts, based on the pooled dataset of all age
classes and sexes. Note that variables in the second part of the tables were only assessed in a limited number of
specimens (see Valid N columns). Group 1 refers to specimens genetically assigned to LO and Group 2 to PP.
Hybrids were not considered in the analysis. Asterisks mark variables with significant differences between PP
and LO. Note that under Bonferroni correction only p values <0.0032 would remain significant at the 0.05 level,
i.e., only INFL, INFINFL, and DORSCI.
Rank
Sum
Group 1
Rank
Sum
Group 2
U
Z
p-level
Z
adjusted
p-level
Valid N
Group 1
Valid N
Group 2
2*1sided
exact p
*SVL
5550.000
4180.000
1902.000
2.14978
0.031574
2.15168
0.031423
72
67
0.031437
*LAM3T
3418.000
3603.000
1338.000
-2.10666
0.035148
-2.12153
0.033878
64
54
0.035039
*LAM4T
3089.500
3465.500
1259.500
-2.04585
0.040772
-2.05537
0.039844
60
54
0.040449
SUPL
3915.500
3959.500
1569.500
-1.82663
0.067756
-1.88981
0.058784
68
57
0.067641
*SUPSUPL
3905.500
3969.500
1490.500
-2.19188
0.028389
-2.20484
0.027466
69
56
0.028033
*INFL
3591.500
3911.500
1245.500
-3.04373
0.002337
-3.10726
0.001888
68
54
0.002149
*INFINFL
2820.000
3508.000
929.000
-3.66035
0.000252
-3.68227
0.000231
61
51
0.000205
*DORSC1
1602.500
2957.500
699.500
-3.09862
0.001944
-3.14417
0.001666
42
53
0.001726
TRHSC
97.000
113.000
47.000
0.18993
0.849361
0.19506
0.845345
9
11
0.881984
LOHSC
157.000
143.000
52.000
1.12976
0.258577
1.17702
0.239186
11
13
0.276668
EYELUI
36.000
100.000
22.000
0.24254
0.808365
0.24470
0.806685
4
12
0.861538
EYELLI
158.000
248.000
67.000
-1.40499
0.160024
-1.41707
0.156464
13
15
0.169623
*EYELUO
227.000
179.000
43.000
2.46046
0.013876
2.47576
0.013296
12
16
0.013016
EYLLO
230.500
234.500
63.500
1.88384
0.059588
1.89997
0.057439
12
18
0.058779
FP
104.000
247.000
68.000
-0.22222
0.824141
-0.22283
0.823666
8
18
0.849089
HCSP
85.500
145.500
40.500
-0.95940
0.337356
-0.98065
0.326767
9
12
0.345103
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
17
Table S10. ANOVA results of test including 8 variables and all age classes and all sexes. Asterisks mark
variables with significant differences between PP and LO.
Test
Value
F
Effect df
Error df
p
Intercept
Wilks
0.002862
2395.123
8
55
0.000000
SPNR
Wilks
0.663590
3.485
8
55
0.002547
Tukey's Post
hoc test:
*SVL
0.042853
SUPSUPL
0.113464
LAM3T
0.104814
*INFL
0.022578
LAM4T
0.090530
*INFINFL
0.002007
SUPL
0.063207
*DORSC1
0.000460
Table S11. Results of Mann-Whitney U-tests for SVL and scale counts, based on males in age class 4 only.
Group 1 refers to specimens genetically assigned to LO and Group 2 to PP. Hybrids were not considered in the
analysis. Asterisks mark variables with significant differences between PP and LO.
Rank
Sum
Group 1
Rank
Sum
Group 2
U
Z
p-level
Z
adjusted
p-level
Valid N
Group 1
Valid N
Group 2
2*1sided
exact p
*SVL
1589.500
688.5000
282.5000
3.34970
0.000809
3.36051
0.000778
39
28
0.000623
LAM3T
999.000
597.0000
366.0000
0.02539
0.979747
0.02555
0.979619
35
21
0.986616
LAM4T
944.000
709.0000
278.0000
-1.65431
0.098066
-1.65929
0.097059
36
21
0.100053
SUPL
1212.500
678.5000
378.5000
0.96701
0.333541
1.02850
0.303717
37
24
0.336203
SUPSUP
1069.000
822.0000
328.0000
-1.62205
0.104793
-1.63600
0.101841
38
23
0.106775
INFL
1052.000
718.0000
349.0000
-0.90914
0.363277
-0.94115
0.346630
37
22
0.370393
INFINF
950.000
646.0000
284.0000
-1.29957
0.193748
-1.30751
0.191042
36
20
0.198540
*DORSC1
281.500
498.5000
110.5000
-2.21149
0.027003
-2.31631
0.020542
18
21
0.025801
Table S12. ANOVA results of test including 8 variables and males of age class 4 only. Asterisks mark variables
with significant differences between PP and LO.
Test
Value
F
Effect df
Error df
p
Intercept
Wilks
0.001500
1414.137
8
17
0.000000
SPNR
Wilks
0.382688
3.428
8
17
0.015616
Tukey's Post
hoc test:
*SVL
0.005099
SUPSUPL
0.503060
LAM3T
0.506114
INFL
0.284878
LAM4T
0.298264
INFINFL
0.223681
SUPL
0.968222
*DORSC1
0.039254
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
18
Table S13. Results of Mann-Whitney U-tests for SVL and scale counts, based on females in age class 4 only.
Group 1 refers to specimens genetically assigned to LO and Group 2 to PP. Hybrids were not considered in the
analysis. Asterisks mark variables with significant differences between PP and LO. Note that under Bonferroni
correction only p values <0.0063 would remain significant at the 0.05 level, i.e., none of the variables would
show significant differences.
Rank
Sum
Group 1
Rank
Sum
Group 2
U
Z
p-level
Z
adjusted
p-level
Valid N
Group 1
Valid N
Group 2
2*1sided
exact p
*SVL
95.50000
204.5000
14.50000
2.34570
0.018992
2.35752
0.018398
5
19
0.015246
LAM3T
44.50000
231.5000
29.50000
-1.15530
0.247968
-1.18773
0.234942
5
18
0.257066
LAM4T
47.00000
206.0000
32.00000
-0.82263
0.410717
-0.83644
0.402906
5
17
0.445584
SUPL
52.00000
179.0000
37.00000
-0.24772
0.804354
-0.25407
0.799445
5
16
0.841712
SUPSUP
56.50000
174.5000
38.50000
0.12386
0.901427
0.12496
0.900556
5
16
0.904713
INFL
34.50000
175.5000
19.50000
-1.57117
0.116144
-1.61103
0.107174
5
15
0.118550
INFINF
35.00000
155.0000
25.00000
-0.50000
0.617075
-0.50310
0.614895
4
15
0.664603
*DORSC1
25.00000
206.0000
10.00000
-2.47717
0.013243
-2.50083
0.012391
5
16
0.011106
Table S14. ANOVA results of test including 8 variables and females of age class 4 only. Asterisks mark
variables with significant differences between PP and LO.
Test
Value
F
Effect df
Error df
p
Intercept
Wilks
0.000355
2464.067
8
7
0.000000
SPNR
Wilks
0.405860
1.281
8
7
0.378523
Tukey's Post
hoc test:
*SVL
0.016457
SUPSUPL
0.962186
LAM3T
0.194163
INFL
0.380532
LAM4T
0.479982
INFINFL
0.469247
SUPL
0.855120
DORSC1
0.096383
Table S15. Results of Mann-Whitney U-tests for residuals (after regression against SVL) of four morphometric
measurements, based on all age classes and both sexes. Group 1 refers to specimens genetically assigned to LO
and Group 2 to PP. Asterisks mark variables with significant differences between PP and LO. Hybrids were not
considered in the analysis.
Residue
Rank Sum
Group 1
Rank Sum
Group 2
U
Z
p-level
Z
adjusted
p-level
Valid N
Group
1
Valid N
Group
2
2*1sided
exact p
*HW
3997.500
3628.500
1417.500
2.35082
0.018732
2.35085
0.018731
57
66
0.018357
*HL
2853.000
4773.000
1200.000
-3.45396
0.000552
-3.45398
0.000552
57
66
0.000480
HH
3771.000
3855.000
1644.000
1.20204
0.229349
1.20206
0.229342
57
66
0.231237
*TOEL
3069.500
4556.500
1416.500
-2.35590
0.018479
-2.35591
0.018478
57
66
0.018103
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
19
Table S16. ANOVA results for four morphometric measurements, based on all age classes and both sexes.
Asterisks mark variables with significant differences between PP and LO.
Test
Value
F
Effect df
Error df
p
Intercept
Wilks
0.996937
0.090650
4
118
0.985233
SPNR
Wilks
0.808862
6.971001
4
118
0.000045
Tukey's Post
hoc test:
*HW
0.040320
HH
0.332132
*HL
0.002088
*TOEL
0.009745
Table S17. Results of Mann-Whitney U-tests for residuals (after regression against SVL) of four morphometric
measurements, based on all age classes and both sexes, but excluding all specimens <20 mm and >36 mm.
Group 1 refers to specimens genetically assigned to LO and Group 2 to PP. Hybrids were not considered in the
analysis. Asterisks mark variables with significant differences between PP and LO.
Residue
Rank
Sum
Group 1
Rank
Sum
Group 2
U
Z
p-level
Z
adjusted
p-level
Valid N
Group 1
Valid N
Group 2
2*1sided
exact p
HW
1204.500
2116.500
631.5000
0.97681
0.328665
0.97684
0.328648
27
54
0.330836
*HL
831.000
2490.000
453.0000
-2.76512
0.005691
-2.76517
0.005690
27
54
0.005277
HH
1076.500
2244.500
698.5000
-0.30557
0.759935
-0.30558
0.759926
27
54
0.761407
TOEL
920.500
2400.500
542.5000
-1.86846
0.061699
-1.86851
0.061692
27
54
0.061413
Table S18. ANOVA results for four morphometric measurements, based on all age classes and both sexes, but
excluding all specimens <20 mm and >36 mm. Asterisks mark variables with significant differences between PP
and LO.
Test
Value
F
Effect df
Error df
p
Intercept
Wilks
0.996937
0.090650
4
118
0.985233
SPNR
Wilks
0.808862
6.971001
4
118
0.000045
Tukey's Post
hoc test:
HW
0.529953
HH
0.757532
*HL
0.034761
*TOEL
0.034362
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
20
Figure S1. Timetree showing iguanine lizards (yellow box) among squamates. Calibrations
and settings used are as in a previous study [1]
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
21
Figure S2. (A) Details of Structure assignment (based on 12 microsatellite loci) of specimens
from San Cristóbal Island (extracted from Fig. 2 in main paper), showing a low incidence of
gene flow between the two island-endemic lineages LO and PP, but several instances of
migrants and hybrids from Española and Santa Cruz Islands. (B) Haplotype network of DNA
sequences from the mt control region incorporating haplotypes and data from a previous
study [11] with newly sequenced samples from San Cristóbal Island (Table S1). Only
haplotypes found in iguanas from San Cristóbal are shown, thus the presence of private
Española and Santa Cruz haplotypes confirms the presence of migrants from these islands on
San Cristóbal, which also is suggested by the microsatellite data. Light grey shows
individuals from Española and Santa Cruz, whilst individuals from San Cristóbal with
haplotypes typical for Española or Santa Cruz are black and marked with small arrows.
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
22
Figure S3. Changes from ancestral to current effective population size (Ne), and time of
inferred change, in PP and LO clusters, based on simulations with MsVar software and based
on data from 18 microsatellite loci.
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
23
Figure S4. Isolation by distance (IBD) analysis for subpopulations of the LO and PP clusters.
Significant genetic differentiation between subpopulations is found for the Loberia, but not
the Punta Pitt, cluster, indicating a more recent expansion within the PP lineage.
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
24
Figure S5. Proportion of RAD-Seq derived loci shared among individuals. Loci shared
between individuals (black circles, off-diagonal cells) or successfully amplifying within a
single individual (red circles along the diagonal) are expressed as the proportion from 0 to 1
of all 60,396 loci scored in this study.
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
25
Figure S6. Maximum likelihood tree of the sparse matrix of RADSeq-derived DNA
sequences. The tree was obtained in RAxML using a GTR + Γ model and an alignment of
5,082,978 bp, branch support was assessed by 100 bootstrap replicates.
Supplementary Material: MacLeod et al. Hybridization masks speciation in the evolutionary history of the
Galápagos marine iguana
26
Figure S7. Box-Whisker plots comparing specimens of LO and PP iguanas for SVL and 15
scale count variables. All age classes and both sexes included. Asterisks indicate significance
in Univariate Mann-Whitney U tests, * P<0.05, ** P < 0.01, ** P<0.001. Note that under
Bonferroni correction only p values <0.0063 would remain significant at the 0.05 level, i.e.,
none of the variables would show significant differences. See Tables S9-S18 for detailed
statistical results.
... Even though many aspects of marine iguana's morphology, physiology and genetics have received much attention [9][10][11][12][13][14][15][16][17], studies on blood parasites have been largely neglected. Previous morphological assessments revealed the presence of haemogregarines in marine iguanas from the island of Santa Cruz, potentially akin to Hepatozoon Miller, 1908 [18]. ...
... Thus, we expected that such high dispersal ability could affect the transmission of parasites across islands. Migrants from neighbouring islands have been found in some populations, indicating that these are not totally isolated units [12]. In any case, migration of individuals is more likely to happen between closely located islands than between far and Animals 2022, 12, 1142 3 of 16 remote ones. ...
... The amphibious lifestyle and adaptation to the sea render marine iguanas as extremely mobile species able to travel long distances. For instance, in San Cristóbal, hybrids originating of the inter-mix from local and nearby islands' populations (e.g., Santa Cruz and Española) have been found indicating that marine iguanas are able to travel between islands [12]. However, despite the striking variation in parasite prevalence among islands, our mantel test revealed no correlation between geographical distance and differences in parasite abundance among sites or populations. ...
Article
Parasitism is among the most common forms of coexistence of organisms of different species. Hemoparasites live in the bloodstream of the host where they complete different life-cycle stages. Members of the phylum Apicomplexa constitute a large portion of all hemoparasites infecting reptiles and their parasite transmitting vectors, including arthropods. In this study, we carried out a survey and molecular identification of hemoparasites in blood samples of the iconic Galápagos marine iguana (Amblyrhynchus cristatus). Major island populations of marine iguanas were sampled to examine large-scale biogeographic patterns of parasite diversity and prevalence. Nested PCRs were used to amplify segments of the 18S rRNA-gene of hemoparasites. Furthermore, ticks attached to marine iguanas were collected and analyzed in the same way to assess their potential use as a non-invasive method for the detection of hemoparasites in vertebrate host species. PCR products were sequenced and a phylogenetic analysis was carried out showing the presence of two genetically distinct clusters of hemoparasites, one more commonly distributed than the other one, belonging to the genera Hepatozoon and/or Hemolivia (Apicomplexa: Eucoccidiorida). Overall, 25% of marine iguanas were infected by hemoparasites. However, infection rates varied strongly among particular island populations (from 3.45% to 50%). Although marine iguanas are an extremely mobile species that has colonized all islands in the Galápagos archipelago, parasite occurrence was not related to geographical distance, suggesting that dispersal behavior has a minor role in parasite transmission. On most islands, females tended to have higher infection rates than males, but this relationship was only significant on one island. Overall, ticks and marine iguanas had similar prevalence and diversity of parasites. However, the infection profiles of ticks and their corresponding hosts (marine iguanas) did not mirror one another, indicating that this method cannot be used reliably to assess marine iguana infection status. Interestingly, we found that hemoparasite prevalence in marine iguanas and ticks tended to be positively correlated across islands. Our results indicate that certain populations of marine iguanas may have special mechanisms and adaptations to cope with parasite infection. In addition, other factors such as vector density, anthropogenic-related activities or the immunological state of marine iguanas could potentially affect the striking variation in hemoparasite prevalence across island populations.
... Taken together, it is possible to imagine that Gal apagos iguanas evolved on older islands and then migrated to younger islands as they emerged, leaving open the possibility of a continental origin for these lizards with subsequent occupation of the modern Gal apagos Archipelago (at least by the ancestor of Amblyrhynchus and Conolophus) due to vicariance rather than dispersal (cf. Cox, 1983;Geist et al., 2014;Grehan, 2001;MacLeod et al., 2015;Merlen, 2014;Peck, 1996). ...
... As fascinating as they are with their peculiar lifestyle, marine iguanas are still poorly understood from an osteological point of view. Most of the studies on this species have focused on physiology or molecular biology to assess phylogenetic relationships (e.g., MacLeod et al., 2015;Miralles et al., 2017;Rassmann, 1997;. There are virtually no studies on the cranial anatomy of Amblyrhynchus, with the few studies available on skeletal adaptations being limited to the post-cranium (Hugi & S anchez-Villagra, 2012;Paparella et al., 2020). ...
... The skull and mandibles of Amblyrhynchus are visibly shortened even when compared to its sister taxon the land iguana of the Gal apagos, Conolophus spp. (de Queiroz, 1987;Etheridge & de Queiroz, 1988;Frost & Etheridge, 1989;MacLeod et al., 2015;Rassmann, 1997). This trend affects in particular the snout region, as reflected, for instance, by the verticalization of the nasal process of the premaxilla and the septomaxillae, the presence of deep concavities along the anteroventral margin of the frontal, and a relatively low dentary-mandible length ratio. ...
Article
Amblyrhynchus cristatus, the marine iguana, is unique amongst the ~7000 species of living limbed lizards as it has successfully evolved adaptations that allow it to live in both terrestrial and marine environments. This species is endemic to the Galápagos Archipelago and has evolved a specialized feeding behaviour, consuming primarily the algae that grow on the rocky seafloor. The intriguing questions arising around the evolution of the marine iguana concerns the use of exaptations of terrestrial features for aquatic and specifically marine adaptations. However, the lack of fundamental information about its anatomy currently prevents us from understanding how it became adapted to such a peculiar lifestyle in comparison to all other iguanids. The goal of this study is to provide the first ever description of the skull, mandible, and hyoid of Amblyrhynchus. We examined several specimens of marine iguana, including skeletal, wet, and ct‐scanned material, and individuals at different ontogenetic stages. We also analyzed specimens of all other modern iguanid genera (Conolophus, Iguana, Ctenosaura, Cyclura, Dipsosaurus, Brachylophus, Sauromalus) in order to make comparisons between Amblyrhynchus and its closest relatives. We were able to identify several autapomorphic features that distinguish the marine iguana from all other iguanids. These unique morphologies are mostly associated with the modified configuration of the snout (nasal chamber), increased muscle attachments in the temporal‐postorbital region of the skull, and dentition. Since Amblyrhynchus is the only non‐ophidian squamate currently able to exploit the ocean at least for some vital functions (i.e., feeding), we used comparisons to fossil marine lizards (e.g., mosasaurids) to discuss some of these unique traits. The new cranial features described for Amblyrhynchus may represent a source of novel morphological characters for use in future phylogenetic analyses of iguanian (or squamate) relationships, which will then serve as the foundation for the exploration of evolutionary patterns and processes that led to the development of such unique adaptations. This article is protected by copyright. All rights reserved.
... Molecular genetic data were used to establish the phylogenetically closest relatives of 10 of the 11 Galápagos' land-vertebrate clades as well as the time at which the divergences occurred (Figs 6-7); with the tortoises (Chelonoidis niger), fossil data also informs the interpretation. Unfortunately, genetic information is lacking for one of the extinct rodents ( †Megaoryzomys curioi); however, a recent anatomical study has linked it to a species in the Ecuadorian Andes (see below (Poulakakis et al., 2012(Poulakakis et al., , 2015(Poulakakis et al., , 2020Sánchez et al., 2017;Kehlmaier et al., 2017Kehlmaier et al., , 2019Kehlmaier et al., , 2021]; land iguanas and marine iguanas [Conolophus and Amblyrhynchus; one clade (MacLeod et al., 2015;Malone et al., 2017)]. In instances where different colonization models can be inferred for a clade, each are considered and the most parsimonious one is highlighted. ...
... phylogenetically nested within C. subcristatus (MacLeod et al., 2015) and this clade is sister to C. marthae. Both C. marthae and C. subcristatus are present on Isabela; however, their divergence at 0.89-2.19 ...
... Both C. marthae and C. subcristatus are present on Isabela; however, their divergence at 0.89-2.19 Mya (MacLeod et al., 2015) may predate the formation of the island (0.8 Mya). Land iguanas and marine iguanas (Amblyrhynchus cristatus) form a single clade (Figs 6, 9;Gentile et al., 2009;MacLeod et al., 2015). ...
Article
Full-text available
Based on a synthesis of new molecular phylogenetic data, a detailed review is presented for the origins of the Galápagos’ native land-locked vertebrates [42 species; 11 clades: geckos (3), lava lizards (2), giant tortoises (1), iguanas (1), racer snakes (1) and oryzomyine rodents (3)]. Nine groups have roots in coastal Ecuador and Peru and would have been transported to the archipelago on rafts, many on the Humboldt Current. Inferring the sources of the giant tortoises, which probably floated over unaided, and the iguanas is more challenging because their closest living relatives occupy ground remote from the Pacific. Acknowledging uncertainties with the age-dating of both the phylogenetic tree nodes and the landmass emergences, seven, probably eight, of the colonizations likely involved beachings on the modern-day islands within the last 4 Myr. Three, possibly four, of the earlier arrivals may have been on now-submerged landmasses that were created by the Galápagos volcanic hotspot. Alternatively, the true sister taxa of the Galápagos species could be extinct and these colonizations, too, are more recent. This is likely for the giant tortoises. The assembled data set hints at the oldest/youngest clades showing the highest/lowest levels of diversification, although other factors also exert an influence.
... Galapagos Land Iguanas (Conolophus spp.) and Marine Iguanas diverged approximately 4.5 million years ago, likely after their common ancestor arrived in the archipelago by rafting from Central America (MacLeod et al. 2015). Spinytailed Iguanas (genera Ctenosaura and Cachryx) are the closest living relatives of Galapagos Iguanas (land and marine) and last shared a common ancestor ~8.3 million years ago (MacLeod et al. 2015;Malone et al. 2017). ...