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The complete mitochondrial genomes of the Galápagos iguanas, Amblyrhynchus cristatus and Conolophus subcristatus

DOI:10.3109/19401736.2015.1079863
Authors:
Amy Macleod at University of Leipzig
Amy Macleod
Iker Irisarri at Leibniz Institute for the Analysis of Biodiversity Change
Iker Irisarri
Miguel Vences at Technische Universität Braunschweig
Miguel Vences
Sebastian Steinfartz at University of Leipzig
Sebastian Steinfartz
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Abstract and Figures

The Galápagos iguanas are among the oldest vertebrate lineages on the Galápagos archipelago, and the evolutionary history of this clade is of great interest to biologists. We describe here the complete mitochondrial genomes of the marine iguana, Amblyrhynchus cristatus (Genbank accession number: KT277937) and the land iguana Conolophus subcristatus (Genbank accession number: KT277936). The genomes contain 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs genes, as well as a control region (CR). Both species have an identical gene order, which matches that of Iguana iguana. The CR of both Galápagos iguanas features similar tandem repeats units, which are absent in I. iguana. We present a phylogeny of the Iguanidae based on complete mitochondrial genomes, which confirms the sister-group relationship of Galápagos iguanas. These new mitochondrial genomes constitute an important data source for future exploration of the phylogenetic relationships and evolutionary history of the Galápagos iguanas.
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ISSN: 1940-1736 (print), 1940-1744 (electronic)
Mitochondrial DNA, Early Online: 1–2
!2015 Taylor & Francis. DOI: 10.3109/19401736.2015.1079863
MITOGENOME ANNOUNCEMENT
The complete mitochondrial genomes of the Gala
´pagos iguanas,
Amblyrhynchus cristatus and Conolophus subcristatus
Amy MacLeod
1
, Iker Irisarri
2
, Miguel Vences
1
, and Sebastian Steinfartz
1
1
Department of Evolutionary Biology, Zoological Institute, Technische Universita
¨t Braunschweig, Braunschweig, Germany and
2
Laboratory for
Zoology and Evolutionary Biology, Department of Biology, University of Konstanz, Konstanz, Germany
Abstract
The Gala
´pagos iguanas are among the oldest vertebrate lineages on the Gala
´pagos
archipelago, and the evolutionary history of this clade is of great interest to biologists. We
describe here the complete mitochondrial genomes of the marine iguana, Amblyrhynchus
cristatus (Genbank accession number: KT277937) and the land iguana Conolophus subcristatus
(Genbank accession number: KT277936). The genomes contain 13 protein-coding genes, 22
transfer RNAs, and two ribosomal RNAs genes, as well as a control region (CR). Both species
have an identical gene order, which matches that of Iguana iguana. The CR of both Gala
´pagos
iguanas features similar tandem repeats units, which are absent in I. iguana. We present a
phylogeny of the Iguanidae based on complete mitochondrial genomes, which confirms the
sister-group relationship of Gala
´pagos iguanas. These new mitochondrial genomes constitute
an important data source for future exploration of the phylogenetic relationships and
evolutionary history of the Gala
´pagos iguanas.
Keywords
Evolutionary relationships, Gala
´pagos
archipelago, Iguanidae, Islands,
phylogenetics
History
Received 15 July 2015
Revised 20 July 2015
Accepted 29 July 2015
Published online 3 September 2015
The Gala
´pagos iguanas are sister taxa that diverged from a
common ancestor on the Gala
´pagos archipelago some 4.5 million
years ago (MacLeod et al., 2015). This clade, including one
species of marine iguana (genus Amblyrynchus) and three species
of land iguana (genus Conolophus), represents one of the most
ancient endemic clades of the Gala
´pagos, and molecular studies
of them have produced important insights into evolutionary
processes (e.g. MacLeod et al., 2015; Rassmann et al., 1997;
Steinfartz et al., 2009). We describe here the first complete
mitochondrial genome sequences of Amblyrhynchus cristatus and
Conolophus subcristatus. In addition, we present a mitogenomic
phylogeny of the Iguanidae, which illustrates the position of the
Gala
´pagos iguanas in relation to other species for which complete
mitochondrial genomes are available.
Genomic DNA was isolated from blood samples collected with
permission of the Gala
´pagos National Park service. A. cristatus
was sampled on San Cristo
´bal Island, and C. subcristatus on
Fernandina Island. Mitochondrial genomes were amplified and
sequenced using a primer-walking approach. PCR involved
primers available from the literature, as well as new primers
developed for the study (details available upon request).
Sequences were assembled in CodonCode (V4.2.5; CodonCode
Corporation, Dedham, MA) and full length mitogenomes
were annotated with the MITOS pipeline (Bernt et al., 2013)
and manually checked by comparison to Iguana iguana
(Genbank accession number: NC002793). Nucleotide gene
alignments were performed with TranslatorX (Abascal et al.,
2010) and MAFFT (Katoh & Standley, 2013) for protein-coding
and ribosomal RNA genes respectively, and concatenated into a
single matrix for phylogenetic analysis. Maximum likelihood
phylogenetic reconstruction was performed with RAxML v.8.1.16
(Stamatakis, 2014) using independent gene partitions under
GTR + G and 100 independent maximum likelihood searches.
Branch support was assessed with 1000 replicates of non-
parametric bootstrapping. A Bayesian analysis was performed
with PhyloBayes MPI v.1.5 (Lartillot et al., 2013) under the CAT-
GTR model and by running two MCMC chains until convergence.
The mitogenomes of A. cristatus and C. subcristatus are,
respectively, 16 897 bp and 16 892 bp long. Gene order was
identical in both species and matches exactly with that of I.
iguana, corresponding to the ancestral vertebrate mitochondrial
gene order. Our findings confirm the repetitive elements found in
the control region in previous studies (Hanley & Caccone, 2005),
with both Gala
´pagos iguanas displaying four repetitive regions of
73 and 74 bp for Conolophus and Amblyrynchus, respectively.
These repetitive sequences have a high sequence identity (uncor-
rected p-distance: 0.35), implying that they have probably evolved
from a common ancestor. However, such repetitive elements are
absent in the CR of both I. iguana (Janke et al., 2001) and Cyclura
pinguis (Genbank accession number: NC027089), and may prove
interesting for future phylogenetic applications. Our phylogenetic
reconstruction confirms the sister-group relationship between the
Gala
´pagos iguanas with strong support, and indicates a relatively
close relationship with Iguana and Cyclura (Figure 1), confirming
earlier work (Pyron et al., 2013). The mitogenomes presented here
will enhance further study into the evolutionary relationships of
the Gala
´pagos iguanas as mitogenomes of additional species and
lineages of these lizards become available.
Correspondence: Amy MacLeod, Department of Evolutionary Biology,
Zoological Institute, Technische Universita
¨t Braunschweig,
Mendelssohnstr. 4, 38106 Braunschweig, Germany. E-mail:
ms.amymacleod@gmail.com
Downloaded by [208.167.254.208] at 08:12 11 September 2015
Acknowledgements
The authors thank K. Rassmann and F. Trillmich for sample collection,
M. Kondermann, J. Juras and L. M. Ko
¨pping for assistance with the
laboratory work, and A. Meyer for logistic assistance. This publication is
contribution number 2119 of the Charles Darwin Foundation for the
Galapagos Islands.
Declaration of interest
This study was supported by grants from the Swiss Friends of the
Gala
´pagos Islands. I. Irisarri was supported by postdoc fellowships of the
Alexander von Humboldt Foundation and EMBO. The authors declare
that they have no conflicting interests.
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Figure 1. Maximum likelihood (RAxML) tree
from full mitogenome data including a
selection of iguanid species and two out-
groups (Chamaeleo and Brookesia), which
are not shown in the figure for the sake of
clarity. Numbers at nodes are support values
from non-parametric bootstrap, and posterior
probabilities from the Bayesian analysis
(PhyloBayes), both transformed into percent.
Both trees agree on the close sister-relation-
ship of the Galapagos iguanas,
Amblyrhynchus cristatus and Conolophus
subcristatus. Genbank accession numbers:
Cyclura pinguis NC027089, Urosaurus
nigricaudus NC026308, Uta stansburiana
NC027261, Anolis carolinensis NC010972,
Polychrus marmoratus NC012839,
Chalarodon madagascariensis NC012836,
Leiocephalus personatus NC012834,
Gambelia wislizenii NC012831, Basiliscus
vittatus NC012829, Oplurus grandidieri
NC012827, Sceloporus occidentalis
NC005960, Iguana iguana NC002793, and
Anolis carolinensis EU747728.2.
2A. MacLeod et al. Mitochondrial DNA, Early Online: 1–2
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Background Every year the human population encounters epidemic outbreaks of influenza, and history reveals recurring pandemics that have had devastating consequences. The current work focuses on the development of a robust algorithm for detecting influenza strains that have a composite genomic architecture. These influenza subtypes can be generated through a reassortment process, whereby a virus can inherit gene segments from two different types of influenza particles during replication. Reassortant strains are often not immediately recognised by the adaptive immune system of the hosts and hence may be the source of pandemic outbreaks. Owing to their importance in public health and their infectious ability, it is essential to identify reassortant influenza strains in order to understand the evolution of this virus and describe reassortment pathways that may be biased towards particular viral segments. Phylogenetic methods have been used traditionally to identify reassortant viruses. In many studies up to now, the assumption has been that if two phylogenetic trees differ, it is because reassortment has caused them to be different. While phylogenetic incongruence may be caused by real differences in evolutionary history, it can also be the result of phylogenetic error. Therefore, we wish to develop a method for distinguishing between topological inconsistency that is due to confounding effects and topological inconsistency that is due to reassortment. Results The current work describes the implementation of two approaches for robustly identifying reassortment events. The algorithms rest on the idea of significance of difference between phylogenetic trees or phylogenetic tree sets, and subtree pruning and regrafting operations, which mimic the effect of reassortment on tree topologies. The first method is based on a maximum likelihood (ML) framework (MLreassort) and the second implements a Bayesian approach (Breassort) for reassortment detection. We focus on reassortment events that are found by both methods. We test both methods on a simulated dataset and on a small collection of real viral data isolated in Hong Kong in 1999. Conclusions The nature of segmented viral genomes present many challenges with respect to disease. The algorithms developed here can effectively identify reassortment events in small viral datasets and can be applied not only to influenza but also to other segmented viruses. Owing to computational demands of comparing tree topologies, further development in this area is necessary to allow their application to larger datasets.
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Abascal F, Zardoya R, Telford MJ.. TranslatorX: Multiple alignment of nucleotide sequences guided by amino acid translations. Nucleic Acids Res 38: W7-W13
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We present TranslatorX, a web server designed to align protein-coding nucleotide sequences based on their corresponding amino acid translations. Many comparisons between biological sequences (nucleic acids and proteins) involve the construction of multiple alignments. Alignments represent a statement regarding the homology between individual nucleotides or amino acids within homologous genes. As protein-coding DNA sequences evolve as triplets of nucleotides (codons) and it is known that sequence similarity degrades more rapidly at the DNA than at the amino acid level, alignments are generally more accurate when based on amino acids than on their corresponding nucleotides. TranslatorX novelties include: (i) use of all documented genetic codes and the possibility of assigning different genetic codes for each sequence; (ii) a battery of different multiple alignment programs; (iii) translation of ambiguous codons when possible; (iv) an innovative criterion to clean nucleotide alignments with GBlocks based on protein information; and (v) a rich output, including Jalview-powered graphical visualization of the alignments, codon-based alignments coloured according to the corresponding amino acids, measures of compositional bias and first, second and third codon position specific alignments. The TranslatorX server is freely available at http://translatorx.co.uk.
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Development of primers to characterize the mitochondrial control region of Gal�pagos land and marine iguanas (Conolophus and Amblyrhynchus)
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Primers were developed for the amplification and sequencing of the mitochondrial control region of Galápagos land (Conolophus) and marine (Amblyrhynchus) iguanas. Sequences were obtained for four land iguana samples from two islands and for 28 marine iguana samples from three islands. A series of 70–80 bp tandem repeats adjacent to the control region are described and preliminary quantification of intra- and interspecific sequence divergence is included.
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The microevolution of the Galapagos marine iguana Amblyrhynchus cristatus assessed by nuclear and mitochondrial analyses
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Marine iguanas may have inhabited the Galápagos archipelago and its former, now sunken islands for more than 10 million years (Myr). It is therefore surprising that morphological and immunological data indicate little evolutionary divergence within the genus. We utilized mitochondrial DNA (mtDNA) sequence analyses and nuclear DNA fingerprinting to re-evaluate the level and pattern of genetic differentiation among 22 marine iguana populations from throughout the archipelago. Both genetic marker systems detect a low level of within-genus divergence, but they show contrasting levels of geographical subdivision among the populations. The mitochondrial gene pools of populations from different regions of the archipelago are isolated, and the mtDNA pattern appears to follow the sequence in which the islands were colonized by marine iguanas. Conversely, the nuclear DNA study indicates substantial interpopulational gene exchange, and the geographical distribution of the nuclear markers seems to be determined by isolation by distance among the populations. The natural history of marine iguanas suggests that the contrasting nuclear and mitochondrial DNA patterns result from an asymmetric migration behaviour of the two sexes, with higher (active and passive) interisland dispersal for males than females. Separate genetic analyses for the sexes appear to support this hypophesis. Based on these findings, a scenario is proposed that explains the marine iguanas' low genetic divergence, notwithstanding their long evolutionary history in the Galápagos archipelago.
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