J Mol Evol (2012) 74:81–95 DOI 10.1007/s00239-012-9490-7
Nucleotide Composition of CO1 Sequences in Chelicerata (Arthropoda): Detecting New Mitogenomic Rearrangements Juliette Arabi • Mark L. I. Judson • Louis Deharveng • Wilson R. Lourenc¸o Corinne Cruaud • Alexandre Hassanin
•
Received: 19 October 2011 / Accepted: 2 February 2012 / Published online: 24 February 2012 Ó Springer Science+Business Media, LLC 2012
Abstract Here we study the evolution of nucleotide composition in third codon-positions of CO1 sequences of Chelicerata, using a phylogenetic framework, based on 180 taxa and three markers (CO1, 18S, and 28S rRNA; 5,218 nt). The analyses of nucleotide composition were also extended to all CO1 sequences of Chelicerata found in GenBank (1,701 taxa). The results show that most species of Chelicerata have a positive strand bias in CO1, i.e., in favor of C nucleotides, including all Amblypygi, Palpigradi, Ricinulei, Solifugae, Uropygi, and Xiphosura. However, several taxa show a negative strand bias, i.e., in favor of G nucleotides: all Scorpiones, Opisthothelae spiders and several taxa within Acari, Opiliones, Pseudoscorpiones, and Pycnogonida. Several reversals of strandspecific bias can be attributed to either a rearrangement of the control region or an inversion of a fragment containing the CO1 gene. Key taxa for which sequencing of complete mitochondrial genomes will be necessary to determine the
Electronic supplementary material The online version of this article (doi:10.1007/s00239-012-9490-7) contains supplementary material, which is available to authorized users. J. Arabi M. L. I. Judson L. Deharveng W. R. Lourenc¸o A. Hassanin (&) De´partement Syste´matique et Evolution, UMR 7205, Origine, Structure et Evolution de la Biodiversite´, Muse´um national d’Histoire naturelle, 57, Rue Cuvier, 75005 Paris, France e-mail:
[email protected] J. Arabi A. Hassanin De´partement Syste´matique et Evolution, Service de Syste´matique Mole´culaire, Muse´um national d’Histoire naturelle, CP 26, 43, Rue Cuvier, 75005 Paris, France C. Cruaud Centre National de Se´quenc¸age, Genoscope, CP 5706, 2, Rue Gaston Cre´mieux, 91057 Evry Cedex, France
origin and nature of mtDNA rearrangements involved in the reversals are identified. Acari, Opiliones, Pseudoscorpiones, and Pycnogonida were found to show a strong variability in nucleotide composition. In addition, both mitochondrial and nuclear genomes have been affected by higher substitution rates in Acari and Pseudoscorpiones. The results therefore indicate that these two orders are more liable to fix mutations of all types, including base substitutions, indels, and genomic rearrangements. Keywords Chelicerata Mitochondrial genome Strand bias Rearrangements Inversion Control region Phylogeny
Introduction The mitochondrial genome (mtDNA) presents a relatively conserved structure among metazoans (Gissi et al. 2008). A typical metazoan mtDNA is a circular, double-stranded molecule of 14–20 kb that contains only 37 genes, including 13 protein-coding genes essential for cellular ATP production by oxidative phosphorylation: subunits 6 and 8 of the ATPase (atp6 and atp8); cytochrome c oxidase subunits 1–3 (CO1–3); apocytochrome b (cytb); NADH dehydrogenase subunits 1–6 and 4L (nad1–6 and nad4L); two RNAs of the mitochondrial ribosome (small 12S and large 16S, subunit rRNAs); and 22 transfer RNAs (tRNAs) required for the translation of proteins encoded by the mtDNA. Most mt genomes have only one major noncoding region, named the control region (CR), which contains the main regulatory elements for the initiation of replication and transcription (Gissi et al. 2008). A remarkable feature of metazoan mtDNA is the strandspecific bias in nucleotide composition: one strand (positive
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strand) is characterized by an excess of adenine (A) relative to thymine (T) and an excess of cytosine (C) relative to guanine (G) (i.e., positive AT and CG skews), whereas the other (negative strand), because of base complementarities, is characterized by an excess of T relative to A and an excess of G relative to C (i.e., negative AT and CG skews) (Hassanin et al. 2005; Perna and Kocher 1995). The origin of the strand-specific bias is related to asymmetric mutational constraints generating inequalities between the frequencies of the complementary bases A/T and C/G (Hassanin et al. 2005; Reyes et al. 1998; Tanaka and Ozawa 1994). Two asymmetric processes are potentially involved in strand bias: replication (Cameron et al. 2007; Fonseca et al. 2008; Hassanin et al. 2005; Reyes et al. 1998; Tanaka and Ozawa 1994) and transcription (Cameron et al. 2007; Hassanin et al. 2005). During replication and transcription processes, the strength of the mutational bias is related to the length of time each gene spends in a single-stranded state. The TG-rich strand is longer in single-stranded form than its complement (replication: Brown and Clayton 2006; Goddard and Wolstenholme 1978, 1980; Saito et al. 2005; transcription: Roberti et al. 2006; Taanman 1999). As a consequence, this TG strand is exposed to higher levels of mutation, especially deaminations of A and C nucleotides (Hassanin et al. 2005; Reyes et al. 1998; Tanaka and Ozawa 1994). Hassanin et al. (2005) have suggested that the orientation of the CR is crucial in the establishment of asymmetric mutational constraints because this region contains both replication and transcription origins. In support of that hypothesis, they showed that two kinds of mitogenomic inversions can lead to a reversal in nucleotide composition: (i) inversion of the CR can result in a global reversal of asymmetric mutational constraints; (ii) inversion of a genomic fragment can result in a local reversal of asymmetric mutational constraints. Within Arthropoda, the subphylum Chelicerata is characterized by the presence of chelicerae on the head segment called the prosoma (Brusca and Brusca 2003). Among chelicerates, the class Pycnogonida (sea spiders) is regarded as the sister-group of Euchelicerata, the latter class being subdivided into Merostomata (horseshoe crabs) and Arachnida (e.g., spiders, scorpions, mites, ticks, and daddy-longlegs) (Weygoldt and Paulus 1979). More specifically, Chelicerata can be divided into 12 orders: Pantopoda (Pycnogonida), Xiphosura (Merostomata), and the Arachnida orders Acari, Amblypygi, Araneae, Opiliones, Palpigradi, Pseudoscorpiones, Ricinulei, Scorpiones, Solifugae, and Uropygi (Shultz 2007; Weygoldt and Paulus 1979). In this report, we focused on the subphylum Chelicerata because previous studies have shown that several unrelated taxa in this clade have a mtDNA characterized by a global reversal of strand compositional bias, including one sea spider (Achelia bituberculata : Arabi et al. 2010), opisthothele spiders (Argiope, Caligosa, Habronattus, Hypochylus, Nephila, and
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Ornithoctonus), scorpions (Buthus, Centruroides, Euscorpius, Mesobuthus, and Uroctonus) (Hassanin 2006; Hassanin et al. 2005; Masta et al. 2009), and two unrelated species of mites (Varroa destructor: Hassanin 2006 and Steganacarus magnus: Domes et al. 2008). These observations suggest that many mitogenomic inversions occurred during the evolutionary history of Chelicerata. Furthermore, chelicerates are especially interesting in mitogenomics because they include taxa showing the ancestral arthropod gene order (e.g. Limulus polyphemus: Staton et al. 1997; Damon diadema: Fahrein et al. 2009), as well as taxa with highly rearranged genomes (e.g., Leptotrombidium: Shao et al. 2006 and Unionicola foili: Ernsting et al. 2009). The gene coding the cytochrome c oxidase subunit 1 (CO1) forms the primary barcode sequence for members of the animal kingdom; about 650-nt long, it is used to assign unidentified specimens to a known species (Hebert et al. 2003). In a recent paper on sea spiders, Arabi et al. (2010) have shown that the AT and CG skews at third synonymous positions of CO1 sequences can be used to detect the two kinds of mitogenomic inversions. That is, by comparing the gene order of two pycnogonid species—Achelia bituberculata (Park et al. 2007) and Nymphon gracile (Podsiadlowski and Braband 2006)—with the deduced organization of the common ancestor of Chelicerata, these authors related reverse strand biases in CO1 to (i) a global reversal of asymmetric mutational constraints, associated with the inversion of the CR in Achelia, versus (ii) a local reversal of asymmetric mutational constraints due to the inversion of a genomic fragment including CO1, in Nymphon. The number of CO1 sequences available in nucleotide databases has increased exponentially in the past few years, due to DNA barcoding projects (Ratnasingham and Hebert 2007). These data provide an excellent opportunity to detect additional taxa affected by mitogenomic inversions and are used here to: (1)
(2)
(3)
Identify new examples of chelicerate species affected by reversals of strand compositional bias. We retrieved from GenBank all CO1 sequences longer than 500 nt and sequenced additional taxa. In total, 1,701 CO1 sequences were analyzed for nucleotide composition. Provide a phylogeny of Chelicerata to infer how the nucleotide composition of CO1 evolved in this group. Phylogenetic relationships were investigated using an alignment of 5,218 nt (combining the mt gene CO1 with two nuclear genes, 18S and 28S rRNA) for 180 taxa, representing all 12 chelicerate orders. Reconstruct the organization (gene arrangement) of the ancestral genome for several higher taxa of Chelicerata, to better understand how and when genomic rearrangements generated changes in
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nucleotide composition. Fifty-three complete mtDNA and three partial genomes were coded and analyzed to infer gene-order rearrangements during the evolution of Chelicerata.
Materials and Methods Phylogenetic Analyses Taxa Sampling The evolution of nucleotide composition in CO1 sequences of Chelicerata was studied using a phylogenetic framework, based on a combined analysis of CO1 with the 18S and 28S nuclear rRNA genes. The data set comprises 180 taxa and was constructed to represent as much of the diversity of Chelicerata as possible. For this purpose, new sequences were obtained for 97 taxa. Fifteen species, comprising three non-arthropod Ecdysozoa, nine Pancrustacea, and three Myriapoda, were chosen as outgroups (Online Resource 1). Extraction, PCR, and Sequencing Total DNA was extracted from specimens preserved in 70–95% ethanol using the QIAamp DNA Micro Kit (Qiagen, Germany). Each of the three markers (18S, 28S, and CO1) was amplified and sequenced using the primers employed in previous studies (Arabi et al. 2010; Hassanin et al. 2005), plus three new specific primers for the 28S (R4S: 50 -GAAGACCCTGTTGAGCTTGACT-30 /R4AS: 50 -GATTCTGACTTAGAGGCGTTCA-30 ) and CO1 (U1sco: 50 -TCWACDAATCATAAGGATATTGGDAC-30 ). PCR reactions were carried out in a 30-ll final volume using the following conditions: 109 reaction buffer with MgCl2, 3 ll; dNTP mix (6.6 mM), 3 ll; primers (10 lM), 1.5 ll; H2O, 19.3 ll; Sigma Red Taq DNA polymerase, 0.70 ll, and DNA template, 1 ll. The cycling protocol included an initial denaturation step of 4 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at the appropriate annealing temperature and 60 s at 72°C. Final extension followed for 10 min at 72°C. Purification and cycle-sequencing reactions were performed at the Genoscope (Evry, France). Sequences were edited and assembled using Sequencher 4.7 (Gene Codes Corporation, Ann Arbor, MI, USA). Tree Reconstruction Alignments were performed manually with Se-Al v2.0a11 (Andrew Rambaut, software available at http://tree.bio.ed. ac.uk/software/seal/). All regions in the alignments
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involving ambiguity for gap positions were excluded from the analyses to avoid erroneous hypotheses of primary homology. The alignments of all data sets analyzed for the nucleotide composition and the phylogeny are available upon request from the authors. The three markers (18S: 1,516 nt; 28S: 2,451 nt; and CO1: 1,251 nt) were analyzed separately to evaluate their individual signals. We also combined the two ribosomal genes to detect potential cases of serious incongruence (Bootstrap proportions, BPs [ 50) between nuclear and mitochondrial data. We then analyzed the matrix that combined all the genes (5,218 nt). Separate and combined analyses were analyzed by maximum likelihood (ML) under RAxML v7.0.4 (Stamatakis 2006), using a GTR ? G model for each gene partition and each codon position in the case of CO1. As recommended by Stamatakis (2006), the parameter corresponding to the percentage of invariants (I) was not included in the model. BPs were computed from 1,000 replicates. Analyses of the Nucleotide Composition The strand-specific bias in nucleotide composition was analyzed at third codon positions of CO1 sequences using the approach detailed in Hassanin et al. (2005). Because similar trends were found for two- and fourfold degenerate sites, we applied a simplified method in which all third positions were analyzed together (Arabi et al. 2010). The frequencies of complementary nucleotides were compared and tested for skewness: AT skew = [A - T]/[A ? T] and CG skew = [C - G]/[C ? G]; AT and CG skews were statistically significant if the null hypothesis of symmetry was rejected at p B 0.05. The analyses were conducted on all the 180 CO1 sequences used to obtain the phylogeny. They were complemented by studying all CO1 sequences available for Chelicerata in the GenBank/EMBL/DDBJ nucleotide databases. Sequences were selected according to three criteria: (1) a length superior to 500 nt; (2) no stop codon or reading-frame shift; (3) one sequence per species, except in the case of data obtained in different laboratories or extracted from different subspecies or geographical zones. In total, 1,701 CO1 sequences were included in the analyses of nucleotide composition (Online Resource 2). In order to characterize the genomic inversion responsible for reverse strand-specific bias, i.e., whether a gene or the CR was inverted, we also studied the nucleotide compositions of the 53 complete mitochondrial genomes of Chelicerata, plus three partial genomes of Pycnogonida (Dietz et al. 2011; Masta et al. 2010), available in the nucleotide databases. CG skews were analyzed separately for 11 protein-coding genes (CO1–3, cytb, nad1–6 and nad4L), and for an alignment combining atp6 and atp8,
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because these short atp genes overlap in all studied genomes (Online Resource 3).
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Results Chelicerate Diversity in the Nucleotide Databases
Reconstruction of Ancestral Genome Organization The evolution of mt genome organization was studied by maximum parsimony (MP) using the approach detailed in Hassanin et al. (2005). In order to reconstruct the ancestral mitochondrial gene arrangement for chelicerate subgroups of interest, each of the 56 mtDNA of Chelicerata was coded in a matrix including 74 characters, corresponding to the 50 and 30 ends of each of the 37 mt genes (Online Resource 4). For each character, the state of relative gene location was coded by determining the 50 or 30 end of the adjacent gene. Because gene rearrangements are often homoplastic due to the limited number of genes in the mt genome, our MP analysis used a constraint tree for inferring ancestral genomes. Heuristic searches were performed under PAUP 4.0b10 (Swofford 2003) by keeping only trees compatible with the following constraint tree: ((Colossendeis, ((Nymphon sp., N. gracile), ((Achelia, Tanystylum), (Ammothea carolinensis, A. hilgendorfi)))), ((Limulus, Tachypleus), ((Steganacarus, (Dermatophagoides farinae, D. pteronyssinus)), (((Tetranychus cinnabarinus, T. urticae), (Panonychus citri, P. ulmi)), ((Ascoschoengastia, Walchia, ((Leptotrombidium akamushi, L. deliense), L. pallidum)), (Unionicola foili, U. parkeri)))), (Nothopuga, Eremobates), (((Carios, (Onithodoros moubata, O. porcinus)), (((Ixodes hexagonus, I. persulcatus), (I. holocyclus, I. uriae)), (Amblyomma, (Haemaphysalis, Rhipicephalus)))), (Varroa, ((Metaseiulus, Phytoseiulus), Stylochyrus))), ((Damon, Phrynus), Mastigoproctus, (Heptathela, ((Caligosa, Ornithoctonus), (Habronattus, Nephila), Hypochilus))), (Uroctonus, (Buthus, Centruroides, (Mesobuthus gibbosus, M. martensii))), (Opilio, Phalangium), Pseudocellus)). This constraint tree was established using the ML tree we calculated in Fig. 2 and published phylogenies of Pycnogonida (Arabi et al. 2010), Acariformes, and Parasitiformes (Dermauw et al. 2009; Domes et al. 2008). In agreement with previous studies, we assumed that the arrangement of the ancestral genome of Chelicerata was identical to that of Limulus (Hassanin et al. 2005; Staton et al. 1997). For each node of interest, the ancestral character states were inferred using either Acctran (accelerated transformation) or Deltran (delayed transformation) optimizations in MP. Then, we calculated a consensus sequence, in which ambiguous character-states were coded as ‘‘?’’ (Online Resource 4). In a final procedure, the ancestral sequences were used to reconstruct circular mtDNA, which resolved some ambiguities in the ancestral sequences.
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As summarized in Fig. 1, most of the extant taxonomic diversity of the subphylum Chelicerata occurs in the class Arachnida, with 11,840 genera assigned to 646 families in ten orders. By comparison, the class Pycnogonida is composed of 70 genera in ten families and one order (Pantopoda), and the class Merostomata contains only three genera in a single family and order (Xiphosura). The two largest orders within Arachnida are Acari and Araneae, with more than 100 families and 3,000 genera, whereas Ricinulei is the smallest order, comprising a single family and three genera. CO1 sequences extracted from the nucleotide databases represent only a small part of the taxonomic diversity: 29% of the chelicerate families and 6% of the genera. The diversity of Pantopoda and Xiphosura is well sampled at both familial (90 and 100%, respectively) and generic levels (39 and 100%, respectively). By comparison, the diversity of Arachnida is poorly represented, with 28% of the families and only 6% of the genera. In addition, most CO1 sequences were obtained from Acari and Araneae, with, respectively, 237 and 930 sequences. However, Acari is the worst represented order, with only 10% of the familial and 2% of the generic diversity covered. No data are yet available for the Opilioacariformes, one of the three suborders of Acari. Ricinulei and Amblypygi are the bestsampled orders, with, respectively, 80 and 100% of familial diversity, and 67 and 47% of generic diversity. With about 50% of families represented, other arachnid orders remain poorly sampled at generic level. For instance, only 18% of Pseudoscorpiones, 10% of Araneae, and 4% of Opiliones are represented. Results worth noting in the present analysis are the production of the first CO1 sequences for Palpigradi, a doubling of the number of families represented for Amblypygi and Ricinulei, and an increase of two-thirds in the familial coverage of Solifugae. Representation of the generic diversity of Scorpiones and Mygalomorphae is also increased by a third (Online Resource 2). Phylogenetic Results Phylogenetic analyses were conducted on a matrix of 5,218 characters combining the two nuclear markers 18S and 28S, and the mitochondrial protein-coding gene CO1. The results are presented in Fig. 2a. All nodes found with maximum BP support were also recovered in the ML analysis of nuclear rRNA genes (Online Resource 5). In contrast, four nodes were not found in the mitochondrial CO1 tree: (1) Uropygi, as Thelyphonida and Amblypygi
J Mol Evol (2012) 74:81–95
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45 58%
Opiliones 2 50%
Palpigradi
25 88%
657 29%
Arachnida 646 28%
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Pseudoscorpiones
1 100% 17 47% 12 42% 3 67% 2 50%
Ricinulei Scorpiones Solifugae Uropygi Schizomida
1 100% 10 90% 1 100%
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Thelyphonida Pantopoda Xiphosura
0
8000
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915
4% 17 47%
3821
10%
Araneomorphae
48%
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Chelicerata
Opilioacariformes Parasitiformes
30%
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Acariformes
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7% 5 20%
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1646
4% 6 17% 18% 3 67%
439
163 13% 141 4% 65 15% 47 13% 18 22% 70 39% 3 100%
0
Chelicerata 11,913 6%
Arachnida 11,840 6%
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Fig. 1 Number of families and genera described in all higher taxa of Chelicerata (subphylum, classes, orders, and suborders) and the percentage of taxa sequenced for CO1. Black histograms represent the number of families (on the left) and genera (on the right) described in higher taxa of Chelicerata: the subphylum Chelicerata and the subclass Arachnida; chelicerate orders (bold) and suborders (italics). Taxonomic data were compiled from various bibliographic sources (Arango and Wheeler 2007; Chiu and Morton 2003; Coddington et al.
2004; Harvey 2007; Kury 2010; Murienne et al. 2008; Naskrecki 2008; Pinto-da-Rocha et al. 2007; Platnick 2011; Villarreal-Manzanilla et al. 2008). White histograms indicate the number of families or genera sequenced for CO1 ([500 nt); the percentage of taxonomic coverage is also indicated. In order to facilitate the reading of each graph, scales were modified for values superior to 100 families and 1,000 genera
are grouped together (BP = 72); (2) the sister-group relationship between Chaerilidae and Buthidae, as the former family occupies a basal position within scorpions (BP = 64); (3) Cyphophthalmi Opiliones, as Cyphophthalmus is allied to the ostracod crustacean Vargula (BP = 51); and (4) Psoroptidia within Sarcoptiformes, as Dermatophagoides is closely related to Sennertia (Hemisarcoptoidea) (BP = 55). The position of Pantopoda with respect to Euchelicerata, Myriapoda, and Pancrustacea is uncertain, but pycnogonids with euchelicerates for the monophyly of Chelicerata is the most robust hypothesis (BP = 45). The monophyly of Euchelicerata is highly supported in the combined analyses (BP = 99) and in the 18S tree (BP = 86), but the relationships between Xiphosura and Arachnida orders are unresolved. Within arachnids, all inter-ordinal relationships remain unresolved. An exception is the Tetrapulmonata (Amblypygi ? Araneae ? Uropygi) (combined: BP = 91; nuclear: BP = 99; CO1: not recovered). However, the position of Amblypygi is unstable: they either grouped
with Araneae (nuclear: BP = 67) or with Thelyphonida (CO1: BP = 72). All arachnid orders are found monophyletic (92 \ BP \ 100), with the exception of the Acari. Indeed, the acarine suborder Acariformes (mites) is robustly associated with Solifugae in the combined analysis (BP = 97), whereas the sister-group of the suborder Parasitiformes cannot be determined with our data. A similar result has been found in two recent molecular studies on Acariformes, both based on 18S, 28S, and CO1 sequences (Dabert et al. 2010; Pepato et al. 2010). Our separate analyses highlight that the sistergroup relationship between Acariformes and Solifugae is supported by only one marker, the nuclear 28S rRNA gene (BP = 88), and by only two synapomorphies (in our 28S alignment, transitions T ? C at position 1,026 and A ? G at position 1,415). In addition, this grouping was not recovered in the recent phylogenomic study of Regier et al. (2010), rendering suspicious the reliability of this node. The monophyly of Acari suborders—of Acariformes and of Parasitiformes—is well supported in the combined
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analyses (BP = 100 and 99, respectively), and there is no conflicting hypothesis in separate analyses of rRNA versus CO1. In addition, the suborder Acariformes can be divided into Sarcoptiformes (BP = 97) and Trombidiformes (BP = 93), whereas the suborder Parasitiformes contains two major taxa, Mesostigmata (BP = 100) and Ixodida (BP = 99). Within spiders, Mesothelae are found as sister-group of Opisthothelae (BP = 100). Within Opisthothelae, Araneomorphae are found to be paraphyletic, due to the early divergence of Hypochilus (BP = 63). However, this result was not supported by separate analyses. The clade Atypoidea, which unites all families of Mygalomorphae except Antrodiaetidae and Atypidae, was found to be robust in all analyses (combined: BP = 94; nuclear: BP = 74; CO1: BP = 69). Our phylogeny of Pseudoscorpiones supports the monophyly of the major groups Chthonioidea (combined: BP = 100; nuclear: BP = 100; CO1: BP = 100), Feaelloidea (combined: BP = 100; nuclear: BP = 100; CO1: BP = 60) and Iocheirata (combined: BP = 100; nuclear: BP = 100; CO1: BP = 94). In a recent molecular phylogeny, Murienne et al. (2008) obtained an early divergence of Feaelloidea with respect to Chthonioidea and Iocheirata. Our combined analyses instead support a sistergroup relationship between Feaelloidea and Iocheirata (BP = 93), as proposed on morphological grounds by Weygoldt (1971). This result was also found with the 28S (BP = 90), whereas the 18S rather favors a sister-group relationship between Chthonioidea and Feaelloidea (BP = 55), which is consistent with Harvey’s (1992) hypothesis of relationships. Within Opiliones, three major taxa are found monophyletic in the combined and nuclear analyses: Cyphophthalmi (combined and nuclear: BP = 100), Laniatores (combined and nuclear: BP = 100), and Palpatores (combined: BP = 98; nuclear: BP = 90). Laniatores and Palpatores are found to be sister-groups (combined: BP = 72; nuclear: BP = 87), and the latter is subdivided into two well-supported clades (combined and nuclear: BP = 100): Dyspnoi and Eupnoi. There is no robust signal in CO1 for deep relationships within Opiliones. Evolution of Nucleotide Composition Nucleotide composition was analyzed at third codon positions of 1,701 CO1 sequences (Online Resource 2). Changes in AT and CG skews were inferred using a phylogenetic framework based on 180 taxa (Fig. 2b), to better understand the evolution of nucleotide compositional bias within Chelicerata. For most taxa, AT and CG skews show similar trends, i.e., both are either positive or negative. However, the absolute value of the CG skew is generally higher than that
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J Mol Evol (2012) 74:81–95 Fig. 2 Evolution of the nucleotide composition at third codon c positions of CO1 sequences of Chelicerata. a Phylogeny of Chelicerata. The tree is a 50% majority-rule consensus obtained after 1,000 bootstrap replicates under RAxML. It is based on the analysis of 5,218 nt, representing three different genes: 18S and 28S rRNAs and CO1. Nodes with BP = 100 are indicated with the symbol (m). Major groups of Chelicerata are indicated to the right of the tree. A circle (s) indicates species for which the complete mtDNA sequence is available in the nucleotide databases. Green branches indicate species with a positive strand bias, as indicated by a positive and significant CG skew (i.e., with an excess of C vs. G nucleotides in third codon position of CO1); orange branches indicate taxa with a negative strand bias, as indicated by significant negative AT and CG skews (i.e., with an excess of T and G nucleotides); black branches indicate taxa with no significant strand bias, i.e., A & T and C & G; dotted branches correspond to ambiguous ancestral conditions. b Nucleotide composition at third codon-positions of CO1. Black histograms correspond to CG skews, and white histograms correspond to AT skews. Significant negative and positive values are highlighted in green and orange, respectively, and non-significant values are indicated with an asterisk (Color figure online)
of the AT skew (in 97% of cases). In addition, 84% of the CG skews are significant compared to just 72% for the AT skews. Taxa with a positive and significant CG skew are not necessarily characterized by a positive and significant AT skew (e.g., Amblypygi and Uropygi). By contrast, all taxa with a significant negative CG skew have a significant negative AT skew (e.g., Opisthothelae spiders and Scorpiones). Overall, all these observations suggest that CG skews are more reliable than AT skews for indicating strand-specific bias. Four categories of taxa can therefore be distinguished: (1) species characterized by positive and significant values for CG skew (positive strand bias), indicating an excess of C relative to G nucleotides (highlighted in green in Fig. 2); (2) species characterized by negative and significant values for CG and AT skews, indicating a negative strand bias (highlighted in orange in Fig. 2); (3) species with no strand bias (no significant values for CG and AT skews); and (4) species with a low positive CG skew associated with a significant negative AT skew (e.g., Lagynochthonius, Neopurcella, Sarcoptes). The results show that most taxa of Chelicerata have a positive strand bias, including all species of Amblypygi, Ricinulei, Palpigradi, Solifugae, Uropygi, and Xiphosura (Fig. 2b and Online Resource 2). In addition, our inferences suggest that a positive strand bias was also present in the common ancestors of Arthropoda, Chelicerata, Pycnogonida, Euchelicerata, Tetrapulmonata, and Parasitiformes. In contrast, all species of Scorpiones (55 species) and Opisthothelae spiders (800 species) are characterized by a negative strand bias, indicating that their ancestors underwent a reversal in strand composition bias. Our inferences suggest that two independent reversals of nucleotide composition occurred during the evolution of Pycnogonida: one in Nymphon gracile, and another one in the ancestor of Achelia and Tanystylum (Fig. 2). However,
J Mol Evol (2012) 74:81–95
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(A)
(B) 75
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Trombidiformes Sarcoptiformes
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99
Hydracarina
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70
Ixodida
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89 99
96 98
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85
Mesostigmata
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53
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93 84
63
Mygalomorphae
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60 65 67
99 94
82 81 70
Schizomida Thelyphonida
55
57
66
69
90 57
58 59
Iurida Chaerilida Buthida
Eupnoi
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56
80 84
93
94 90
Cheliferoidea
58
Neobisioidea
98
84
70
Dyspnoi
72
Palpatores Laniatores
96
Iocheirata Feaelloidea
Cyphophthalmi
57
Chthonioidea
99
92
Opisthothelae
91
Tetrapulmonata
Mesothelae
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87
Euchelicerata
77
Arthropoda
97
63
99 54 99 72
99
Priapulus caudatus Peripatoides novaezealandiae Ramazzottius oberhaeuseri Squilla empusa Vargula hilgendorfii Triops longicaudatus Cryptopygus antarcticus Podura aquatica Locusta migratoria Tribolium castaneum Davidius lunatus Pedetontus silvestrii Thyropygus sp. Lithobius forficatus Scutigera coleoptrata Achelia hispida Tanystylum sp. Ammothea sp. Ascorhynchus sp. Endeis spinosa Callipallene novaezealandiae Pseudopallene ambigua Nymphon gracile Colossendeis macerrima Colossendeis sp. Rhopalorhynchus filipes Pycnogonum stearnsi Limulus polyphemus Tachypleus tridentatus Sarcoptiformes sp. Platynothrus peltifer Steganacarus magnus Sennertia japonica Myialges caulotoon Proctophyllodes cetti Dermatophagoides farinae Dermatophagoides pteronyssinus Sarcoptes scabiei Tetranychus urticae Mideopsis roztoczensis Hydrachna conjecta Hydrachna globosa Hygrobates norvegicus Unionicola foili Teutonia cometes Teutonia sp. Hydrodroma torrenticola Torrenticola amplexa Eremobates palpisetulosus Galeodidae sp. Rhagodidae sp1 Rhagodidae sp2 Daesiidae sp. Ornithodoros moubata Ixodes hexagonus Amblyomma triguttatum Haemaphysalis flava Hyalomma dromedarii Rhipicephalus sanguineus Dermacentor marginatus Megisthanus floridanus Varroa destructor Phorytocarpais fimetorum Prodinychus sp. Sarax sp. Stygophrynus sp3 Stygophrynus sp1 Stygophrynus sp2 Damon gracilis Damon johnstonii Damon medius Phrynichodamon Euphrynichus bacillifer Phrynichus orientalis Phrynichus scaber Phrynus sp. Phrynus goesii Heterophrynus alces Heterophrynus longicornis Heptathela hanzgouensis Antrodiaetus unicolor Atypoides riversi Atypus affinis Sophros abboti Stasimopus robertsi Caligosa longitarsis Mygalomorphae sp1 Mygalomorphae sp2 Chilobrachys huahini Aphonopelma seemani Lasiodora parahibana Cyriopagopus schioedtei Haplopelma schmidti Promyrmekiaphila sp. Atrax sp. Araneoidea sp. Araneus diadementus Argiope bruennichi Nephila clavata Cheiracanthium punctorium Habronattus oregonensis Tegenaria atrica Sparassidae sp. Hypochilus thorelli Schizomida sp1 Schizomida sp3 Schizomida sp2 Mastigoproctus giganteus Mastigoproctus giganteus Thelyphonida sp1 Thelyphonida sp2 Thelyphonida sp3 Thelyphonida sp4 Cyphophthalmus minutus Paragovia sironoides Neopurcella minutissima Rakaia denticula Siro rubens Troglosiro longifossa Fangensis insulanus Stylocellus sp. Epedanoidea sp. Gnomulus armillatus Gonyleptoidea sp. Sandokan malayanus Scotolemon sp. Equitius doriae Amelinus aurentiacus Phalangium opilio Leiobunidae sp1 Leiobunidae sp2 Sclerosomatidae sp. Protolophus singularis Megalopsalis sp. Dendrolasma parvulum Trogulus nepaeformis Ischyropsalis pyrenaea Androctonus hoggarensis Buthacus occidentalis Grosphus flavopiceus Lychas mucronattus Parabuthus laevifrons Tityus serrulatus Chaerilus sp2 Chaerilus sp1 Chaerilus borneensis Chaerilus julietteae Belisarius xambeui Euscorpius flavicaudis Liocheles australasiae Thestylus glasioui Heterometrus cyaneus Heteromerus laoticus Pandinus imperator Palpigradi sp. Pseudocellus pearsei Ricinoides atewa Chthonius dacnodes Chthonius ischnocheles Lagynochthonius sp. Tyrannochthonius sp. Feaella anderseni Neopseudogarypus scutellatus Pseudogarypus bicornis Beierochelifer peloponnesiacus Withiidae sp. Withius hispanus Chernes hahnii Lamprochernes savignyi Apocheiridium reddelli Afrosternophorus sp. Calocheiridius termitophilus Garypinus sp. Geogarypus nigrimanus Bisetocreagris sp1 Bisetocreagris sp2 Neobisium geronenses Neobisium sp. Roncus sp. Ideoroncidae sp.
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the analyses of all CO1 sequences available in the databases (Online Resource 2)—103 versus 12 taxa—suggest a more complex evolutionary history of base composition. Indeed, most species exhibit a positive strand bias (63%), but many have a negative strand bias (20%) or no significant strand bias (12%). The highest levels of heterogeneity were found in the families Ammotheidae and Nymphonidae. In Acariformes, Parasitiformes, and Opiliones, we also found that most species are characterized by a positive strand bias for CO1 (53, 51, and 57%, respectively: Online Resource 2), but, once again, many species show a negative strand bias (19, 9, and 12%, respectively: Online Resource 2) or no strand bias (12, 13, and 15%, respectively; Online Resource 2). In addition, a high percentage of taxa exhibit a positive CG skew associated with a significant negative AT skew (16, 27, and 16%, respectively; Online Resource 2). Pseudoscorpiones is the most atypical order for mtDNA base composition. Contrary to the situation observed in other chelicerate orders, the CO1 gene of most species is characterized by strand symmetry, i.e., A & T and C & G (49%; Online Resource 2). Only 19% of the species show a positive strand bias, 13% show a negative strand bias, and 19% exhibit opposite trends for CG and AT skews. Gene Order Evolution Within Chelicerates The mt genome organization was studied by MP analysis using the matrix of 74 characters shown in Online Resource 4. Of the 74 total characters, 71 are parsimonyinformative. By keeping only trees compatible with the constraint tree (see ‘‘Materials and Methods’’), 81 equally parsimonious trees of 599 steps were found (CI 0.8664; RI 0.8940). One of these trees was used for determining unambiguous character-states transformations (Fig. 3). In parallel, CG skews were analyzed for each protein-coding gene of the 56 genomes. The results are listed in detail in Online Resource 3. For many taxa, we deduced an ancestral genome identical to that of Chelicerata. These taxa are Euchelicerata, Xiphosura, Tetrapulmonata, Amblypygi, Araneae, Scorpiones, Solifugae, and Parasitiformes (including Argasidae, Ixodida, Ixodidae, and Ixodes). In all these taxa except Scorpiones (see below), the analyses of CG skews on complete mt genomes indicate that all protein-coding genes of the positive strand have a positive strand bias, whereas all protein-coding genes of the negative strand have a negative strand bias (Online Resource 3). These results therefore show that gene organization and nucleotide composition of the mtDNA have remained stable in most branches of the chelicerate tree. In the case of Scorpiones, the analyses of CG skews of five complete mt genomes indicate that all protein-coding genes of the positive strand have a negative bias, whereas
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all protein-coding genes of the negative strand have a positive bias. The absence of gene rearrangement in the ancestral genome of scorpions supports the hypothesis that the CR was inverted without implication of adjacent genes, resulting in a global reversal of asymmetric mutational constraints. A small number of genomic rearrangements were inferred in other chelicerate lineages. Within Araneae, the CR and the adjacent tRNA-I and tRNA-Q genes were rearranged in the ancestor of Opisthothelae spiders. Our analyses of CG skews indicate that all protein-coding genes of the positive strand have a negative bias, whereas protein-coding genes of the negative strand show a positive bias (Online Resource 3). These results suggest that an inversion of the CR took place in the ancestor of Opisthothelae, which induced a global reversal of asymmetric mutational constraints. For Pycnogonida, our study indicates that the common ancestor of sea spiders had the same protein-coding gene organization as that of the ancestor of Chelicerata, although the position of tRNA-Q is not identified. The analyses of CG skews in Colossendeis indicate that all protein-coding genes of the positive strand have a positive bias and reciprocally for the negative strand (Online Resource 3). Our analyses highlight that the heterogeneity of strand bias observed within Ammotheidae and Nymphonidae is related to multiple genome rearrangements. Both nymphonid species present a negative CG skew for CO1. In the branch leading to Nymphon gracile, the fragment including the three proteincoding genes nad2, CO1, and CO2 was inverted, explaining why a negative CG skew was obtained for these three genes (Online Resource 3). In the case of Nymphon sp., the analyses of CG skews indicate that all protein-coding genes of the positive strand have a negative bias, and all protein-coding genes of the negative strand show a positive bias (Online Resource 3). These results suggest that an inversion of the CR took place in the branch leading to Nymphon sp., resulting in a global reversal of asymmetric mutational constraints. In the ancestral genome of Ammotheidae, tRNA-Q was transposed between the 12S rRNA and the CR. The analyses of CG skews in two Ammothea genomes indicate that all protein-coding genes of the positive strand have a positive bias, and reciprocally for the negative strand (Online Resource 3). These results suggest that the CR was not inverted in the common ancestor of Ammotheidae. In the case of Achelia, the analysis of CG skews indicates that the values of strand bias are not significant for the most proteincoding genes, except nad3, nad6, and cytb. The genomic segment including the nad2, CO1, CO2, atp8/6, CO3, nad3, and nad5 genes is characterized by apparent reversals of strand bias: negative CG and AT skews from nad2 to nad3, and positive skews for nad5 (Online Resource 3). In the case of Tanystylum, the analysis of CG skews indicates that all
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89 CR duplication?
Colossendeis megalonyx Nymphon gracile Q? Nymphonidae Nymphon sp. hypothetical CR inversion Ammothea hilgendorfi Q? Ammotheidae unidentified CR arrangement Ammothea carolinensis Tanystylum orbiculare hypothetical Q CR inversion Achelia bituberculata Limulus polyphemus Tachypleus tridentatus Steganacarus magnus Sarcoptiformes ? Dermatophagoides farinae Dermatophagoides pteronyssus Panonychus citri ? Tetranychidae Panonychus ulmi ? Tetranychus cinnabarinus Tetranychus urticae ? Unionicola foili Trombidiformes Unionicola parkeri Ascoschoengastia sp. ? Parasitengona Walchia hayashii ? Leptotrombidium akamushi Trombiculidae ? Leptotrombidium deliense Leptotrombidium pallidum Eremobates palpisetulosus Nothopuga sp. Carios capensis Argasidae Ornithodoros moubata Ornithodoros porcinus Ixodida Ixodes hexagonus Ixodinae Ixodes persulcatus CR duplication Ixodes holocyclus Ixodidae Ixodes uriae CR duplication Amblyomma triguttatum Metastriata Haemaphysalis flava (nad1-16S-12S) transposition Rhipicephalus sanguineus Phytoseiidae Metaseiulus occidentalis unidentified CR arrangement ? Phytoseiulus persimilis unidentified CR arrangement (CO1-CO2) inversion Ascoidea Styochyrus rarior ? Mesostigmata Varroa destructor CR inversion Damon diadema Phrynus sp. Tetrapulmonata Mastigoproctus giganteus Heptathela hangzhouensis Hypochilus thorelli Entelegynae Opisthothelae Habronattus oregonensis Nephila clavata CR inversion Caligosa longitarsis Ornithoctonus huwena Uroctonus mordax hypothetical CR inversion Buthus occitanus Buthidae Centruroides limpidus Mesobuthus gibbosus Mesobuthus martensii unidentified CR arrangement Opilio parietinus ? Phalangium opilio Pseudocellus pearsei
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Fig. 3 Evolution of the mitochondrial genome organization. The ancestral genome organization of Chelicerata is shown to the left of the tree. The positive strand (?) contains all protein-coding genes with a positive CG skew in third codon positions (in green), whereas the negative strand (-) contains all protein-coding genes with a negative CG skew (in orange). Green branches indicate taxa with a positive strand bias in third codon positions of CO1; orange branches indicate taxa with a negative strand bias for CO1; bold orange branches indicate taxa with a global reversal of strand bias; black branches indicate taxa with non-significant strand bias; dotted
(nad2-CO1-CO2) inversion
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branches indicate ambiguous ancestral skew conditions. The black star (w) indicates that the ancestral genome organization of Chelicerata is totally conserved, and the white star (q) indicates a conservation of the protein-coding gene order. The symbol (d) indicates a rearrangement of protein-coding or rRNA genes, and the stick ( ) indicates tRNA rearrangements. Inversions and duplications of the CR are shown on the branch concerned. A question mark (?) indicates that the ancestral genome organization cannot be inferred (Color figure online)
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protein-coding genes of the positive strand have a negative bias, whereas all protein-coding genes of the negative strand have a positive bias. These results corroborate the hypothesis that an inversion of the CR occurred in the common ancestor of Achelia and Tanystylum, thus inducing a global reversal of asymmetric mutational constraints. Contrary to what was previously suggested by Arabi et al. (2010), the CR was inverted without the implication of adjacent genes, i.e., independently of the tRNA-Q transposition. Both Opiliones (Opilio parietinus and Phalangium opilio: Dyspnoi) have the same gene organization as that of the ancestor of Chelicerata, but the position of tRNA-Q and tRNA-I, as well as the CR arrangement, were not identified. The analyses of CG skews indicate that all proteincoding genes of the positive strand have a positive bias and reciprocally for the negative strand, suggesting that the CR was not inverted in the common ancestor of Opiliones (Online Resource 3). Within Parasitiformes, three genomic fragments were conserved in the ancestor of Mesostigmata (Varroa ? Metaseiulus ? Phytoseiulus ? Stylochyrus): a long fragment, including CO1, from tRNA-I to tRNA-S1, a second fragment from nad1 to tRNA-L1, and a third fragment from 16S to 12S rRNAs (Online Resource 4). In the ancestor of Ascoidea (Metaseiulus ? Phytoseiulus ? Stylochyrus), the long genomic fragment including CO1, from tRNA-I to tRNA-S2, remained conserved, but the CR and its adjacent tRNAs were rearranged. In the genome of Varroa, CG-skew analyses indicate a global reversal of mutational constraints as all protein-coding genes of the positive strand have a negative strand bias and all protein-coding genes of the negative strand have a positive bias. Our results suggest that the CR was inverted either in the ancestor of Mesostigmata or in the branch leading to Varroa (Fig. 3). Within Phytoseiidae, 80% of the 74 character-states were found to be ambiguous. It was therefore impossible to determine how the mitochondrial genome evolved in this part of the tree. Nevertheless, several rearrangements occurred in the branch leading to Phytoseiulus, including four CR duplications, an inversion of nad6 and an inversion of the fragment covering CO1 and CO2, which explains why these three genes show negative CG skews (Online Resource 3). Within Ixodidae, the CR was duplicated in the ancestor of Australian species of Ixodes (I. holocyclus and I. uriae), and independently in the ancestor of metastriate ticks (Amblyomma, Haemaphysalis, and Rhipicephalus). The duplicated CR is localized between tRNA-L2 and 16S rRNA in the case of the two Ixodes species, whereas it is located between tRNA-L2 and tRNA-C in the case of metastriate ticks. Previous studies found that the two CRs evolved in concert in each species of tick (Black and Roehrdanz 1998; Shao et al. 2005). When CG skews were analyzed for metastriate tick
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genomes, we detected a significant reversal of the strand bias for nad5, but CG skews were not found to be significant for nad1, nad3, nad4, and CO3 (Online Resource 3). Taking into account the fact that these genes surround the duplicated CRs, two non-exclusive hypotheses can be proposed to explain these differences in strand-specific nucleotide compositional bias: (i) the CRs have recently been inverted; (ii) mutational constraints are at equilibrium due to the concerted evolution of duplicated CRs, suggesting that both CRs initiate replication/transcription processes. Within Acariformes, 70% of the 74 character-states were found to be ambiguous. As a consequence, it was not possible to understand how the mtDNA evolved in this group. This explains the multiple question marks on the stems in the Acariformes part of the tree in Fig. 3. However, it is evident that the CR has been involved in several genomic rearrangements.
Discussion Basal Radiation within Euchelicerata The combination of the nuclear 18S and 28S rRNAs with the mitochondrial CO1 gene provides strong support (BP [ 90) for the monophyly of most orders of Arachnida, including Amblypygi, Araneae, Opiliones, Pseudoscorpiones, Ricinulei, Solifugae, and Uropygi. Despite our sequencing effort (97 additional taxa), deep divergences within Euchelicerata remain unresolved, including whether Arachnida is monophyletic, as well as most relationships between arachnid orders. The only exception is the clade uniting the three orders Amblypygi, Araneae, and Uropygi (Tetrapulmonata), a result consistent with previous morphological and molecular phylogenies (Mallatt and Giribet 2006; Regier et al. 2010; Shultz 1990, 2007; Weygoldt and Paulus 1979). The lack of a robust signal for basal relationships was also found in the recent study of Regier et al. (2010), in which 62 nuclear markers were sequenced for 20 chelicerate species. This suggests that Euchelicerata underwent a rapid basal radiation in the early Paleozoic Era. That hypothesis is consistent with the fossil record, which indicates that Xiphosura, Acari, Opiliones, and Scorpiones emerged at about the same time, between 440 and 410 Mya (Dunlop 2010). Evolution of Strand-Specific Bias in Chelicerata Hassanin et al. (2005) showed that asymmetric mutational constraints can be reversed through two different mechanisms: (i) inversion of the CR, which results in a global reversal, and (ii) gene inversion, which results in a local
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Within Pycnogonida, three genera include species with opposite trends for strand bias (Online Resource 2): Achelia (A. assimilis/A. sawayai vs. A. bituberculata/A. hispida/A. hoekii) and Ammothella (A. spinifera vs. A. tuberculata) in the family Ammotheidae, and Nymphon (e.g., N. pagophilum vs. N. gracile) in the family Nymphonidae. These results therefore suggest the occurrence of recent genomic inversions involving either CO1 or the CR in some species of both Ammotheidae and Nymphonidae. Comparing the gene organization of the four pycnogonid species characterized by a negative strand bias—Achelia bituberculata and Tanystylum orbiculare (Ammotheidae), Nymphon sp. and N. gracile (Nymphonidae)—with that deduced for the common ancestor of Chelicerata, we inferred that the reversal of strand bias observed in CO1 can be related to (i) a global reversal of asymmetric mutational constraints, associated with the inversion of CR in the case of two ammotheid species and Nymphon sp., and (ii) a local reversal of asymmetric mutational constraints due to the inversion of CO1 in the case of N. gracile (Arabi et al. 2010; Podsiadlowski and Braband 2006). Our hypothesis that a CR inversion is responsible for a global reversal of asymmetric mutational constraints in the common ancestor of Achelia and Tanystylum can be tested by sequencing a closely related species showing a positive strand bias for CO1, such as Achelia assimilis or A. sawayai.
Online Resource 2). In contrast, a positive strand bias was found for the single CO1 sequence available for Mesothelae spiders (Heptathela hanzgouensis), as well as for all CO1 sequences analyzed for Amblypygi (32) and Uropygi (30). This means that a positive strand bias was the plesiomorphic condition for Tetrapulmonata and Araneae, and that the change occurred during the evolution of Opisthothelae. Taking into account that nearly 50% of the opisthothele families are represented in our CO1 data set (Fig. 1), including the most basal families of Araneomorphae (Hypochilidae) and Mygalomorphae (Antrodiaetidae and Atypidae), it seems reasonable to conclude that the reversal of CO1 strand bias occurred in the common ancestor of Opisthothelae (Fig. 3). According to the fossil record, this event would have taken place around 240 Mya (Dunlop 2010). Our analyses of complete mtDNA revealed that asymmetric mutational constraints were globally reversed following an inversion of the CR. All the 66 CO1 sequences of Scorpiones analyzed in this study show a significant negative strand bias (Online Resource 2), suggesting that a strand bias reversal in CO1 occurred in their common ancestor. However, only three of the four parvorders of Scorpiones are represented in our study, namely Buthida, Chaerilida, and Iurida (Soleglad and Fet 2003). The other parvorder is Pseudochactida, for which three competing hypotheses have been proposed for its phylogenetic position: (i) sister-group of all scorpions, (ii) sister-group of Buthida, and (iii) sister-group of Chaerilida (Lourenc¸o 2007). One 18S sequence of this key suborder is available in GenBank (Pseudochactas ovchinnikovi—AY368258) and was integrated in our 18S alignment. The ML analyses favor a sistergroup relationship between Pseudochactida and Chaerilida (BP = 58, data not shown). On the basis of this result, it can be assumed that the strand bias reversal occurred in the common ancestor of Scorpiones. At first sight, this unique and ancient reversal in nucleotide composition of CO1 cannot be explained by a mitogenomic inversion. Indeed, the organization of protein-coding genes was inferred to be identical between the ancestor of Scorpiones and that of Chelicerata as a whole (Fig. 3). However, our study of CG skews on the five genomes of Scorpiones indicates that all protein-coding genes have a reverse strand bias, which implies that the CR was inverted without the implication of adjacent genes. Based on the fossil record, Buthida appeared in the early Triassic and the most ancient remains of scorpions are reported from the Silurian (Dunlop 2010; Lourenc¸o and Gall 2004), suggesting that the CR inversion took place sometime between 428 and 245 Mya.
Ancient Reversals of Strand-Specific Bias in Opisthothelae and Scorpiones
Evidence for Multiple Reversals of Strand-Specific Bias in Acari, Opiliones, and Pseudoscorpiones
All the 930 CO1 sequences of Opisthothelae spiders analyzed in this study show negative AT and CG skews (Fig. 2 and
Within Arachnida, our analyses show that Acari, Opiliones, and Pseudoscorpiones present a complex evolution of
reversal. In addition, a previous study on sea spiders showed that strand bias analyses of CO1 sequences can detect mitogenomic inversions involving either the CR or the CO1 gene (Arabi et al. 2010). Here, we extend the analysis of strand asymmetry to very many CO1 sequences from the entire range of Chelicerata. Mapping reversals of nucleotide composition onto the phylogeny (Fig 2) shows that many independent reversals occurred. Some of these are ancient, such as those that took place in the common ancestor of Opisthothelae spiders and in the ancestral Scorpiones, whereas others are more recent, since they concern only one or a few related species, such as Nymphon gracile in Pycnogonida, Ischyropsalis pyrenaea in Opiliones, and Lamprochernes savignyi in Pseudoscorpiones. Within Euchelicerata, we found the highest heterogeneity in nucleotide composition in the Acari, Opiliones, and Pseudoscorpiones, suggesting a more complex evolution in these clades. Evidence for Multiple Reversals of Strand-Specific Bias in Pycnogonida
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nucleotide composition. In all these orders, four distinct categories of nucleotide composition can be observed: (1) positive strand bias, (2) negative strand bias, (3) no strand asymmetry, and (4) uncommon strand bias characterized by a positive CG skew associated with a significant negative AT skew (Fig. 2 and Online Resource 2). Within Opiliones, a comparison of the mitochondrial genomes of Opilio parietinus and Phalangium opilio (suborder Dyspnoi) indicates that these taxa have the same protein-coding gene organization as the common ancestor of euchelicerates. None of the 13 protein-coding genes show deviation from the usual strand composition bias (Online Resource 3). The analyses of ten CO1 sequences show that all species of the suborder Dyspnoi have positive AT and CG skews, suggesting that no major genomic rearrangement occurred during the evolution of this taxon. In contrast, the three other harvestman suborders contain species with a negative strand bias (Fig. 2 and Online Resource 2): Cyphophthalmi with Paragovia sironoides (Neogovidae) and Suzukielus sauteri (Sironidae), Laniatores with species of Travunioidea, and Eupnoi with species of Ischyropsalidoidea. We also found both positive and negative strand biases for different specimens identified as Paragovia sironoides (AY639581 vs. DQ518131 and DQ825650). These results suggest ancient and recent strand bias reversals due to independent mtDNA rearrangements involving either CO1 or the CR during the evolution of these harvestmen lineages. Palpatores comprise the suborders Eupnoi and Dyspnoi. In view of the heterogeneity in nucleotide composition found in CO1 sequences, it would be interesting to compare the mt genome organization of Phalangium opilio (Dyspnoi) with complete mt genomes from a wider variety of taxa in the Eupnoi subtree, such as Ischyropsalis pyrenaea (negative strand bias) and Trogulus nepaeformis (positive strand bias). In the case of Acari, the notion of complexity also refers to the multiple genomic rearrangements manifest in the course of their evolution (Fig. 3). Within Parasitiformes, a reversal of strand bias was identified in the CO1 gene of two Dermanyssina (Mesostigmata): Varroa destructor (Varroidae) and Phytoseiulus persimilis (Phytoseiidae). As previously suggested by Hassanin (2006), our analysis of complete mtDNA confirms that a global reversal of strand composition bias has occurred in Varroa (Online Resource 3). The mitochondrial genome of Phytoseiulus presents multiple protein-coding gene rearrangements compared with the ancestral organization of euchelicerates, as well as four duplications of the CR (Dermauw et al. 2010). We infer that the reverse strand bias observed in CO1 is related to a local reversal of asymmetric mutational constraints due to the transposition of CO1 on the negative strand. The study of all CO1 sequences extends the reversal to two
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genera of Dermanyssina: Hypoaspis and Stratiolaelaps (Laelapidae) (Online Resource 2). Additional genomic data for taxa widely separated in the Mesostigmata subtree, such as Stratiolaelaps (Laelapidae; negative bias), Typhlodromus pyri (Phytoseiidae; positive CG skew and negative AT skew), and Prodinychus (Uropodina; positive bias), are needed to better understand the evolution of the CR in this lineage. In the case of metastriate ticks, we established that the reversal of the strand bias (Online Resource 3) is correlated with a CR duplication affecting mutational constraints (concerted evolution and/or CR inversion). These results highlight a complex evolution of the mitochondrial genomes for certain Parasitiformes lineages, which involves CR duplications and/or inversions, and rearrangements of protein-coding genes. Within Acariformes, the study of CO1 sequences reveals a reversal of strand asymmetry in the common ancestor of Sarcoptiformes (Fig. 2). However, mtDNA of Acariformes have undergone multiple major rearrangements, which hamper the reconstruction of ancestral genome organizations (Fig. 3). We are therefore unable to identify mitogenomic rearrangements that would explain shifts of the strand-specific bias within Acariformes. The Acari constitute the most diverse chelicerate order, with more than 50,000 species arranged in 5,539 genera and 426 families (Coddington et al. 2004). With only 2% of the generic diversity available in the nucleotide databases (Fig. 1), a huge sampling effort needs to be carried out to improve our knowledge of acarine mt genome evolution and to better understand the origins of such a diversity of organization. The nucleotide composition within Pseudoscorpiones is quite atypical compared to other groups, because most taxa have a non-significant strand bias, i.e., A & T and C & G. Only a few taxa have a positive or negative strand bias, and they are widely scattered within the phylogeny of Pseudoscorpiones. Such a pattern suggests that their evolutionary history was punctuated by multiple events (ancient and recent) of mitogenomic inversion. It is noteworthy that there is no mitochondrial genome of Pseudoscorpiones in the nucleotide databases, meaning we cannot yet prove this. Our hypothesis could be tested by sequencing additional species of the following groups showing divergent patterns of nucleotide composition bias: Chthonius (no strand bias) for Chthonioidea; Lamprochernes (negative strand bias) and Withius (no strand bias) for Cheliferoidea; Feaella (positive strand bias) and Neopseudogarypus (no strand bias) for Feaelloidea. Higher Rates of Mutations in Acari and Pseudoscorpiones Looking at the ML trees obtained with the CO1 gene, it is evident that Acariformes, Mesostigmata within Parasitiformes, and Pseudoscorpiones show higher evolutionary
J Mol Evol (2012) 74:81–95
rates than do other groups of Euchelicerata (Online Resource 5, part B). Interestingly, Acariformes, Mesostigmata, and Pseudoscorpiones are characterized by large variability in nucleotide composition and several independent events of strand-specific bias reversal. We observed that Acariformes and Mesostigmata present highly rearranged mt genomes (Fig. 3 and Online Resource 4). Although no mtDNA data are available for Pseudoscorpiones, our analyses of nucleotide composition of CO1 sequences suggest that multiple major protein-coding gene rearrangements also occurred during the evolution of this group. These results support previous studies on Arthropoda, which have shown that taxa with more highly rearranged mt genomes tend to have higher rates of sequence evolution (Shao et al. 2003; Hassanin 2006; Xu et al. 2006). Shao et al. (2003) have proposed that increased rates of nucleotide substitution may lead to increased rates of gene rearrangement in the mt genomes. In contrast, Hassanin (2006) suggests that genomic rearrangements, such as gene inversion, gene translocation, and duplication of the CR, generate changes in pattern and rates of substitution. Four biological factors can also raise the rates of nucleotide substitution: body size, generation time, population size, and metabolic rates (Fontanillas et al. 2007; Gillooly et al. 2007; Glazier 2008; Welch et al. 2008). Comparative analyses with nuclear genes are useful for determining whether one or several of these factors are involved in accelerated rates of molecular evolution observed in the mtDNA sequences of Acari and Pseudoscorpiones. For this purpose, we re-examined our nuclear data from 18S and 28S rRNAs. ML trees obtained from nuclear data show that Acariformes, Mesostigmata and Pseudoscorpiones consistently have long branches with respect to other Euchelicerata (Online Resource 5, part A). Such rapid evolution was also found in the phylogenomic study of Regier et al. (2010), based on 62 nuclear genes and including one representative for two of these groups: Idiogaryops (Pseudoscorpiones) and Dinothrombium (Acariformes). The comparison between mitochondrial and nuclear data suggests a global accelerating rate of molecular evolution, i.e., higher substitution rates in both mitochondrial and nuclear genomes for these three separate lineages. In the case of nuclear rRNAs, the acceleration of substitution rates is accompanied by an increase of indel events. Pairwise comparisons of 18S sequences reveal 18.9, 7.8, and 7% of indels within Mesostigmata, Acariformes, and Pseudoscorpiones, respectively, whereas slowly evolving groups like Amblypygi and Opiliones show only 0.7 and 2.8% of indels, respectively. Our analyses therefore indicate that Acari and Pseudoscorpiones are more liable to fix mutations of all types, including base substitutions, indels, and genomic rearrangements.
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Acari and Pseudoscorpiones are small arachnids: one of the smallest Acariformes (mites) measures only 0.1 mm; the largest Parasitiformes (ticks) swell to 20–30 mm in length during a blood meal and Pseudoscorpiones rarely exceed 7 mm (Brusca and Brusca 2003). However, other orders of small-sized Euchelicerates, such as Palpigradi or Ricinulei, which are \3- and \10-mm long, respectively, are not affected by accelerated rates of mitochondrial-gene evolution. It seems therefore obvious that a reduced body size alone is not responsible for the higher rates of molecular evolution observed in Acari and Pseudoscorpiones. One possible explanation is that Acari and Pseudoscorpiones could be more adaptable to change than other arachnid orders, which may have resulted in higher rates of speciation during their evolutionary history. In support of that hypothesis, both groups occur worldwide, at almost all altitudes and latitudes—including Antarctica in the case of acarines—and both have colonized a great variety of habitats—some mites even invaded aquatic environment (Brusca and Brusca 2003). Acknowledgments We are grateful to the following colleagues who kindly provided samples and/or contributed to chelicerate identification: Michel Baylac, Michel Bertrand, Renaud Boistel, Magalie Castelin, Re´gis Cleva, Cyrille d’Haese, Arnaud Faille, Reinhard Gerecke, Cle´ment Gilbert, Pedroso Giupponi, Ton van Haaren, Ce´line Houssin, Michae¨l Manuel, Patrick Mare´chal, Bertrand Margat, Aure´lien Miralles, Piotr Naskrecki, Eric Ollivier, Eric Que´innec, Christine Rollard, Anne Ropiquet, Harry Smit, Christian Vanderbergh and Peter Weygoldt. We also thank members of the ‘‘Groupe d’Etude des Arachnides’’, directed by Olivier Dupont, for their contribution to the sampling. This work was supported by the MNHN programs ‘‘Etat et Structure de la Biodiversite´ Actuelle et Fossile’’ and ‘‘Cordille`re Annamitique’’, and the ‘‘Consortium National de Recherche en Ge´nomique’’. It forms part of agreement no. 2005/67 between the Genoscope and the MNHN on the project ‘‘Macrophylogeny of life’’, directed by Guillaume Lecointre. Conflict of interest of interest.
The authors declare that they have no conflict
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