Alexandre Cormier

Rémi Wattier

Maria Teixeira

Thierry Rigaud

Richard Cordaux
email: richard.cordaux@univ-poitiers.fr

The complete mitochondrial genome of Gammarus roeselii (Crustacea, Amphipoda): insights into mitogenome plasticity and evolution

Keywords: Control Region, Duplication, phylogeny, polymorphism

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Introduction

The gammarid Gammarus roeselii (Amphipoda) is a freshwater peracarid crustacean [START_REF] Väinölä | Global diversity of amphipods (Amphipoda; Crustacea) in freshwater[END_REF]. As the related morphospecies Gammarus pulex and Gammarus fossarum, G. roeselii is widespread in European continental freshwaters [START_REF] Karaman | Freshwater gammarus species from Europe, North Africa and adjacent regions of Asia (Crustacea-Amphipoda). Part II. Gammarus roeselii-group and related species[END_REF], playing a key functional role in the ecosystem, through leaf litter recycling [START_REF] Piscart | An acanthocephalan parasite increases the salinity tolerance of the freshwater amphipod Gammarus roeselii (Crustacea: Gammaridae)[END_REF]. In addition, G. roeselii is often used in multi-topic research at the crossroads of ecophysiology, ecotoxicology, biological invasions, behavioral ecology and evolutionary parasitology [START_REF] Haine | Conflict between parasites with different transmission strategies infecting an amphipod host[END_REF][START_REF] Moret | Is the host or the parasite the most locally adapted in an amphipod-acanthocephalan relationship? A case study in a biological invasion context[END_REF][START_REF] Tain | Differential influence of Pomphorhynchus laevis (Acanthocephala) on brain serotonergic activity in two congeneric host species[END_REF][START_REF] Sornom | Effect of gender on physiological and behavioural responses of Gammarus roeselii (Crustacea Amphipoda) to salinity and temperature[END_REF][START_REF] Gerhardt | Gammarus: Important Taxon in Freshwater and Marine Changing Environments[END_REF][START_REF] Böttger | Closer to reality -the influence of toxicity test modifications on the sensitivity of Gammarus roeselii to the insecticide imidacloprid[END_REF]Gismondi et al., 2012bGismondi et al., , 2012cGismondi et al., , 2012a[START_REF] Gismondi | Do male and female gammarids defend themselves differently during chemical stress?[END_REF][START_REF] Gergs | Different ammonia tolerances may facilitate spatial coexistence of Gammarus roeseliii and the strong invader Dikerogammarus villosus[END_REF][START_REF] Andreï | Silver nanoparticles impact the functional role of Gammarus roeselii (Crustacea Amphipoda)[END_REF].

Although G. roeselii is often considered as a native species all over Europe, [START_REF] Jażdżewski | Biogéographie de Gammarus roeselii Gervais en Europe, en particulier répartition en France et en Pologne[END_REF] proposed that the Balkans should be considered as the native area of this species, as expansion outside this area is only recent, being associated with postglacial expansion and/or even more recent human-mediated invasion. Recent invasion is likely as G. roeselii geographic distribution has been recorded to increase over the last 50 years [START_REF] Grabowski | Alien Crustacea in Polish waters -Amphipoda[END_REF][START_REF] Paganelli | The increasing distribution of Gammarus roeseliii Gervais, 1835: first record of the non-indigenous freshwater amphipod in the sub-lacustrine Ticino River basin[END_REF]. Consistently, based on mitochondrial (mt) cytochrome c oxidase subunit I (COI) DNA barcodes, it has been recently established that the morphospecies G. roeselii in the Balkans is characterized by extensive cryptic diversity, with 13 molecular operational taxonomic units (MOTUs) which diversification occurred from Miocene to Pleistocene epochs [START_REF] Grabowski | Neogene paleogeography provides context for understanding the origin and spatial distribution of cryptic diversity in a widespread Balkan freshwater amphipod[END_REF]. By contrast, G. roeselii outside the Balkans is associated with a single COI-MOTU also present in the Northern Balkans (M. Grabowski and R. Wattier, unpublished results, see also [START_REF] Moret | Is the host or the parasite the most locally adapted in an amphipod-acanthocephalan relationship? A case study in a biological invasion context[END_REF]). However, deeper exploration of G. roeselii expansion dynamics would benefit from the availability of additional mitochondrial markers.

Omics resources in amphipods are still relatively scarce or, at least, have been building up only recently [START_REF] Kao | The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion[END_REF], especially for the genus Gammarus [START_REF] Krebes | The mitogenome of Gammarus duebeni (Crustacea Amphipoda): A new gene order and non-neutral sequence evolution of tandem repeats in the control region[END_REF][START_REF] Gismondi | Transcriptome of the freshwater amphipod Gammarus pulex hepatopancreas[END_REF][START_REF] Trapp | High-throughput proteome dynamics for discovery of key proteins in sentinel species: Unsuspected vitellogenins diversity in the crustacean Gammarus fossarum[END_REF]Macher et al., 2017a). Amphipod nuclear genomes tend to be quite large, which constitutes a limiting factor for assembly processes [START_REF] Kao | The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion[END_REF]. By contrast, the small and circular mt genome is more easily accessible. As a consequence, more than 20 amphipod mt genomes have been sequenced, although many are incomplete [START_REF] Bauzà-Ribot | The complete mitochondrial genome of the subterranean crustacean Metacrangonyx longipes (Amphipoda): A unique gene order and extremely short control region[END_REF], 2012;[START_REF] Ito | The complete mitochondrial genome of Caprella scaura (Crustacea, Amphipoda, Caprellidea), with emphasis on the unique gene order pattern and duplicated control region[END_REF][START_REF] Ki | Complete mitochondrial genome sequence of the Arctic gammarid, Onisimus nanseni (Crustacea; Amphipoda): Novel gene structures and unusual control region features[END_REF][START_REF] Kilpert | The mitochondrial genome of the Japanese skeleton shrimp Caprella mutica (Amphipoda: Caprellidea) reveals a unique gene order and shared apomorphic translocations with Gammaridea[END_REF][START_REF] Krebes | The mitogenome of Gammarus duebeni (Crustacea Amphipoda): A new gene order and non-neutral sequence evolution of tandem repeats in the control region[END_REF][START_REF] Shin | Complete mitochondrial genome of the Antarctic amphipod Gondogeneia antarctica (Crustacea, amphipod)[END_REF][START_REF] Pons | Next-generation sequencing, phylogenetic signal and comparative mitogenomic analyses in Metacrangonyctidae (Amphipoda: Crustacea)[END_REF][START_REF] Romanova | The complete mitochondrial genome of Baikalian amphipoda Eulimnogammarus vittatus Dybowsky, 1874[END_REF][START_REF] Romanova | The complete mitochondrial genome of a deep-water Baikalian amphipoda Brachyuropus grewingkii (Dybowsky, 1874)[END_REF][START_REF] Romanova | Evolution of mitochondrial genomes in Baikalian amphipods[END_REF][START_REF] Stokkan | Mitochondrial genome rearrangements at low taxonomic levels: three distinct mitogenome gene orders in the genus Pseudoniphargus (Crustacea: Amphipoda)[END_REF][START_REF] Aunins | Comparative mitogenomic analyses of three North American stygobiont amphipods of the genus Stygobromus (Crustacea: Amphipoda)[END_REF][START_REF] Lan | The deepest mitochondrial genome sequenced from Mariana Trench Hirondellea gigas (Amphipoda)[END_REF]Macher et al., 2017a).

The mt genome of amphipods is a canonical bilaterian mitogenome, defined as a single circular molecule ranging in size from 14 to 18 kb, with a mean size around 16 kb [START_REF] Boore | Animal mitochondrial genomes[END_REF]. The gene content generally is highly conserved, with 37 genes encoding for 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one control region (CR) containing regulatory elements for transcription and replication (Wolstenholme, 1992). In amphipods, mitogenomic features such as alterations in gene order, strand bias reversion, presence of additional tRNA genes and CR duplication have been reported and could be used to explore amphipod evolutionary history at various taxonomic levels [START_REF] Bauzà-Ribot | The complete mitochondrial genome of the subterranean crustacean Metacrangonyx longipes (Amphipoda): A unique gene order and extremely short control region[END_REF][START_REF] Ito | The complete mitochondrial genome of Caprella scaura (Crustacea, Amphipoda, Caprellidea), with emphasis on the unique gene order pattern and duplicated control region[END_REF][START_REF] Ki | Complete mitochondrial genome sequence of the Arctic gammarid, Onisimus nanseni (Crustacea; Amphipoda): Novel gene structures and unusual control region features[END_REF][START_REF] Kilpert | The mitochondrial genome of the Japanese skeleton shrimp Caprella mutica (Amphipoda: Caprellidea) reveals a unique gene order and shared apomorphic translocations with Gammaridea[END_REF][START_REF] Krebes | The mitogenome of Gammarus duebeni (Crustacea Amphipoda): A new gene order and non-neutral sequence evolution of tandem repeats in the control region[END_REF][START_REF] Pons | Next-generation sequencing, phylogenetic signal and comparative mitogenomic analyses in Metacrangonyctidae (Amphipoda: Crustacea)[END_REF][START_REF] Romanova | The complete mitochondrial genome of Baikalian amphipoda Eulimnogammarus vittatus Dybowsky, 1874[END_REF][START_REF] Aunins | Comparative mitogenomic analyses of three North American stygobiont amphipods of the genus Stygobromus (Crustacea: Amphipoda)[END_REF].

In this study, we present the complete mt genome sequence, annotation and analysis of the amphipod G. roeselii, obtained from individuals collected in France, i.e. in the west-most area of the species geographic range. A mitogenome of a G. roeselii individual collected in Germany has recently been reported (Macher et al., 2017b) but it appears to be a partial sequence (see Results section below). Moreover, this mitogenome was produced as part of a methodological study and no annotation or analysis has been performed (Macher et al., 2017b). Our aims were: (i) to gain insights into mt genome evolutionary dynamics in G.

roeselii with a focus on CR organization, and (ii) to perform a phylogenetic reconstruction testing for the relevance of mt coding genes to investigate relationships in the genus Gammarus and the family Gammaridae, also helping understanding the evolutionary dynamics of the CR.

Materials and Methods

Genome sequencing, assembly and annotation

The mt genome of G. roeselii was identified from data generated as part of an ongoing sequencing project in our laboratories. Briefly, total genomic DNA was extracted from two individuals (Ou3 and Ou53) sampled in Trouhans (Burgundy, France) in the Ouche river. For each individual, a paired-end library with ~500 bp inserts was prepared and sequenced on Illumina MiSeq (2x300 bp) and HiSeq2500 instruments (2x125 bp). Data quality was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and raw reads were trimmed and filtered using Trimmomatic v0.33 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF]. De novo assembly of the pooled reads was performed using SOAPdenovo2 with a k-mer size of 67 [START_REF] Luo | SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler[END_REF]. Identification of mt sequences was performed by searching for sequence similarity between scaffolds and the available mt genome of Gammarus duebeni (NC_017760) [START_REF] Krebes | The mitogenome of Gammarus duebeni (Crustacea Amphipoda): A new gene order and non-neutral sequence evolution of tandem repeats in the control region[END_REF] using Blast [START_REF] Altschul | Basic local alignment search tool[END_REF]. This analysis recovered scaffolds which were subsequently used as seeds for independent assembly of Ou3 and Ou53 mt genomes.

Ou3 and Ou53 mt genomes were assembled using NOVOPlasty v2.6.3, with a k-mer size of 55 and previously identified scaffolds [START_REF] Dierckxsens | NOVOPlasty: de novo assembly of organelle genomes from whole genome data[END_REF]. Structural annotation was performed using the MITOS2 WebServer [START_REF] Bernt | MITOS: Improved de novo metazoan mitochondrial genome annotation[END_REF]. Reads were mapped against their respective assemblies using Bowtie2 v2.2.9 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF]) to identify and correct potential errors and to estimate genome sequencing depth. The sequencing depth for Ou3 and Ou53 was ~11,000x and ~18,000x, respectively. Reverse mapping was also used to identify structural variation between the two mt genomes. Because the complete mt genomes from both individuals were highly similar (see Results section below), a single sequence was submitted to public databases. Accordingly, the complete mt genome sequence of Ou3 is available at DDBJ/ENA/GenBank under accession number MG779536.

To compare CR structure among Gammaridae, mt genomes listed in Table 1 were manually reannotated using the same criteria (i.e. CR starting with a poly-T stretch and ending with the start/end of the next annotated gene). Repeat proportion in CRs was calculated using RepeatMasker 4.0.0 (developed by A.F.A. Smit, R. Hubley, and P. Green; see http://www.repeatmasker.org/) in slow search mode.

PCR validation and sequencing of duplicated control region

To experimentally test the presence of the CR duplication, PCR primers were designed as shown in Figure 1 (Gr_mt2R: 5'-GCTTAAGCCGAGAATCATATATGTCA-3' and Gr_mt3F: 5'-TAGGCTAGCGCTGTTTCAGG-3'). This diagnostic PCR assay is expected to yield a single product of 371 bp if there are two copies of the CR, or no product if there is a single copy of the CR (Figure 1). DNA was extracted from 23 G. roeselii individuals sampled in the Ouche river at Trouhans and 15 individuals sampled in the Morthe river at Citey (Burgondy, France), using a EZ-10 96 Well Plate Genomic DNA Isolation kit (Biobasic Inc., Markham, Canada) according to manufacturer's instructions. PCR were performed in a total volume of 20 µL with 2 μL of genomic DNA, 200 nm of each primer, 200 μM of dNTPs, 1 U Taq DNA polymerase (HotMaster TM Taq DNA polymerase, 5PRIME, Gaithersburg, USA) in 1× Taq buffer. Thermal cycling consisted of an initial denaturation at 95°C for 2 min, followed by 35 cycles (94°C for 20 s, 50°C for 20 s and 65°C for 90 s) and a final 5 min extension at 65°C. PCR products were run on a 2 % agarose gel in 1X TAE buffer to be analyzed for size and subsequently sent for direct Sanger sequencing (Genewiz, UK), using forward and reverse primers.

Phylogenetic analysis

Phylogenetic reconstruction was performed using 20 species selected because of the availability of sequence information for the 13 mt protein-coding genes (Table 1).

Specifically, 18 species represent the ingroup, including 7 species from the Gammaridae family and 11 species belonging to five families proposed by [START_REF] Hou | A review of Gammaridae (Crustacea: Amphipoda): the family extent, its evolutionary history, and taxonomic redefinition of genera[END_REF], based on partial COI sequences and 3 nuclear genes, to represent a broader definition of Gammaridae i.e. Gammarridae sensu lato. Two additional species, Parhyale hawaiiensis and Pseudoniphargus sorbasiensis, were used as outgroup to root the resulting tree [START_REF] Romanova | Evolution of mitochondrial genomes in Baikalian amphipods[END_REF]Macher et al., 2017a) (Table 1). Data were obtained from either annotated mt genomes (NCBI GenBank), unannotated genome assemblies (NCBI WGS), unannotated transcriptome assemblies (NCBI TSA) and from raw transcriptome data assembled anew (NCBI SRA) as presented by Macher et al (2017a) (Table 1).

For each protein-coding gene, nucleotide sequences were aligned using MAFFT v7.299b in automatic mode [START_REF] Katoh | MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability[END_REF]. Single-gene alignments were then concatenated into a single alignment using FASconCAT 1.0 [START_REF] Kück | FASconCAT-G: extensive functions for multiple sequence alignment preparations concerning phylogenetic studies[END_REF], resulting in a total of 10,629 aligned nucleotides. The best model of nucleotide substitution was chosen with jModelTest2 [START_REF] Darriba | jModelTest 2: more models, new heuristics and parallel computing[END_REF]. A Bayesian phylogenetic tree was constructed using MrBayes v.3.2.6 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF] with 0.5M iterations (Effective Sample Size > 200).

Results

General features of the G. roeselii mitochondrial genome

The complete mt genomes of Ou3 and Ou53 from G. roeselii are circular, with respective sizes of 16,073 and 16,072 bp. The two genomes only differ by a single nucleotide polymorphism (SNP) at position 1,736 (C/T) in the cox2 gene and a 1-bp indel at position 14,844 (A/-) in a non-coding region. Because both mt genomes are nearly identical, all analyses and results presented hereafter in the manuscript are based on the Ou3 mt genome (unless specified otherwise). The G. roeselii mt genome is enriched in A and T nucleotides (66.8%). The mt gene set corresponds to the canonical bilaterian gene set: 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes (Table 2). Of these 37 genes, 23 are encoded on the forward strand and 14 on the reverse strand. Nineteen pairs of genes are overlapping by 1 to 60 bp: trnL2-cox2, cox2-trnK, trnK-trnD, atp8-atp6, atp6-cox3, cox3-nad3, trnA-trnS1, trnN-trnE, trnE-trnR, trnR-trnF, trnF-nad5, trnH-nad4, trnT-trnP, nad6-cob, cob-trnS2, nad1-trnL1, trnV-rrnS, trnY-trnQ and nad2-trnW. Protein-coding genes show the same order and transcriptional polarity compared to the reference pancrustacean ground pattern [START_REF] Boore | Gene translocation links insects and crustaceans[END_REF]. However, the arrangement is modified for 6 tRNA genes: trnS1, trnR, trnQ, trnQ, trnC and trnW (Figure 2). The mt genome also contains 15 intergenic regions with length ranging from 1 to 1,029 bp.

A comparative analysis of gene order among Gammarus species mitogenomes indicated that protein-coding genes of all species present the same order and transcriptional polarity compared to the Pancrustacean ground pattern. The tRNA genes are more labile and the Pancrustacean ground pattern is not conserved within the Gammarus genus (Figure 2).

The translocation of trnG forming the tRNA gene string trnW-trnG, the translocation of trnS1 and trnR forming the tRNA gene string trnA-trnS1-trnN-trnE-trnR and the translocations of trnY, trnQ and trnC forming the tRNA gene string trnY-trnQ-trnC next to the CR.

Unusual structure of the control region

The CR is the most important non-coding part of the mt genome and it is involved in replication and transcription [START_REF] Saito | Replication Origin of Mitochondrial DNA in Insects[END_REF]. The CR usually consists of a single locus, which is typically associated with several specific features in invertebrates, including presence of a poly-T stretch, tandemly repeated sequences and AT-rich composition [START_REF] Hassanin | Evidence for Multiple Reversals of Asymmetric Mutational Constraints during the Evolution of the Mitochondrial Genome of Metazoa, and Consequences for Phylogenetic Inferences[END_REF][START_REF] Saito | Replication Origin of Mitochondrial DNA in Insects[END_REF][START_REF] Shao | Evolution of Duplicate Control Regions in the Mitochondrial Genomes of Metazoa: A Case Study with Australasian Ixodes Ticks[END_REF][START_REF] Wei | New Views on Strand Asymmetry in Insect Mitochondrial Genomes[END_REF][START_REF] Doublet | Inverted Repeats and Genome Architecture Conversions of Terrestrial Isopods Mitochondrial DNA[END_REF]. The putative CR of G. roeselii Ou3 and Ou53 is unusual in that it is duplicated (see below), spanning the two longest intergenic regions hereafter named CR1 and CR2 (Figure 1), located between rrnS and trnaI (Figure 2). CR1 and CR2 both start with a T-stretch of 13 bp and they are enriched in A and T nucleotides (78.4%). CR1 and CR2 are separated by a short region (169 bp) containing three tRNA genes (trnY, trnQ and trnC) (Figure 1, Figure 2). Interestingly, CR1 and CR2 are nearly identical, differing only by a SNP at position 13,312 (G/A) and a 2-bp indel (AA/--) at positions 13,621 (relative to CR1), strongly suggesting that the observed CR structure of G. roeselii is due to a duplication of the ancestral CR (Figure 2).

CR duplication was independently confirmed using both bioinformatic analyses and molecular biology experiments. Specifically, Inspection of the CR1/CR2 region of the mt genome showed that Illumina sequencing depth in this region is higher than in the rest of the mitogenome (Figure 3). For both Ou3 and Ou53 Illumina datasets, sequencing depth in the CR appeared to be nearly twice as high as mitogenome mean sequencing depth, assuming a single copy of the CR is present. Indeed, Ou3 mitogenome mean sequencing depth was ~11,000x while CR mean sequencing depth in CR was ~18,000x (Figure 3A). Similarly, Ou53 mitogenome mean sequencing depth was ~18,000x while CR mean sequencing depth was ~32,000x (Figure 3B). These convergent observations in two different individuals suggested that the CR duplication is unlikely to be an artifact of the de novo genome assembly process.

To independently test the authenticity of the CR duplication, we designed a diagnostic PCR assay yielding a single band (371 bp) if the duplication is present and no PCR product if the duplication is absent. Testing of 23 G. roeselii samples caught in the Ouche river resulted in all individuals amplifying a single band of expected 371 bp size (Figure 4). Specific PCR amplification was confirmed by Sanger sequencing. This result independently validated the authenticity of the CR duplication and provided direct evidence that the CR is actually duplicated in all tested G. roeselii individuals. CR duplication was also observed in another population (Morthe river), in which all 15 individuals tested were found to amplify a single 371 bp band.

To further investigate the unusual G. roeselii CR structure, we compared the Ou3 mt genome to that produced by Macher et al. (2017b). The two genomes were highly similar at the nucleotide level, only differing by 6 SNPs and a 1-bp indel (Table 3). The only major difference between the two genomes was a 1,181 bp deletion in the Macher et al (2017b) genome relative to the Ou3 genome. This deletion encompassed tRNA genes trnY, trnQ and trnC and one full copy of the CR. We were unable to ascertain if the missing CR corresponds to the full CR1 or CR2, or parts of both CR1 and CR2, because of the high nucleotide similarity of CR1 and CR2. This configuration suggested that the Macher et al (2017b) assembly has been collapsed due to the CR1/CR2 duplication, leading to the concomitant and artefactual deletion of three tRNA genes. This explanation is supported by the fact that lacking three tRNA genes would be deleterious for the mt genome; the deletion thus most likely cannot have a biological origin. Overall, these observations indicate that the unusual CR structure of the G. roeselii mt genome is authentic and that the mt sequence of G. roeselii generated by Macher et al. (2017b) is a partial assembly.

Extensive variation in CR length has previously been reported in Gammaridae sensu lato (Figure 5). Indeed, while average CR size is 1,234 bp, CR varies in size among species from 181 bp in Eulimnogammarus cyaneus to 2709 bp in Brachyuropus grewingkii. This effectively means that there is a 15-fold variation of CR size among species. We found that repeat content varies between CR (from 0 to 15%) but no correlation was observed between CR size and repeat percentage (r = 0.35, p = 0.314) (Figure 5). The AT nucleotide content also varies substantially between species, from 67% in B. grewingkii to 81% in G. duebeni and Pallaseopsis kessleri. No correlation between CR size and AT nucleotide content was observed (r = -0.55, p = 0.078).

Phylogenetic relationships of gammarids

The Bayesian phylogenetic reconstruction (Figure 5) showed that while G. roeselii, G. fossarum and G. pulex form a well-supported clade, the genus Gammarus is not monophyletic, as the three other Gammarus species (i.e. G. lacustris, G. cheuvreuxi and G. duebeni) are more closely related to species from families Eulimogammaridae, Acanthogammaridae, Micruropodidae, Pallaseidae and Crypturopodidae than to other Gammarus species (but see discussion). Second, the family Gammaridae is not monophyletic. Support for this conclusion comes from the aforementioned results on Gammarus species and the fact that Echinogammarus veneris is more related to Pandorites podoceroides (family Pontogammaridae) than to other Gammaridae species.

Discussion

The analysis of the mitogenome of G. roeselii reveals that the order and transcriptional polarity is conserved compared to the Pancrustacean ground pattern for protein-coding genes and modified for six tRNA genes. Despite a common structure, the mt genomes of G. roeselii, G. duebeni and G. fossarum show important size variation. This is mainly due to variation in the size of intergenic regions, and more specifically in the CR. The unusually large size of the G. roeselii mt genome is the result of an event of duplication of the CR. This is the first verified report of a duplicated CR in the superfamily Gammaridea and, overall, one of a relatively few examples described in animal mitogenomes.

Previously, one potential case of CR duplication was reported in the acanthogammarid Garjajewia cabanisii [START_REF] Romanova | Evolution of mitochondrial genomes in Baikalian amphipods[END_REF]. However, the CR was only partially sequenced in that study, which prevented any firm conclusion to be drawn [START_REF] Romanova | Evolution of mitochondrial genomes in Baikalian amphipods[END_REF]. In amphipods, CR duplication has been reported in two related species: Caprella mutica [START_REF] Kilpert | The mitochondrial genome of the Japanese skeleton shrimp Caprella mutica (Amphipoda: Caprellidea) reveals a unique gene order and shared apomorphic translocations with Gammaridea[END_REF] and Caprella scaura [START_REF] Ito | The complete mitochondrial genome of Caprella scaura (Crustacea, Amphipoda, Caprellidea), with emphasis on the unique gene order pattern and duplicated control region[END_REF]. As in G. roeselii, both Caprella species possess highly similar duplicated CR copies. Beyond amphipods, CR duplications have been observed in several animal species, including ticks [START_REF] Black | Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes[END_REF][START_REF] Campbell | The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: fivefold tandem repetition of a coding region[END_REF], ostracods [START_REF] Ogoh | Complete mitochondrial DNA sequence of the sea-firefly, Vargula hilgendorfii (Crustacea, Ostracoda) with duplicate control regions[END_REF], sea cucumbers [START_REF] Arndt | Mitochondrial gene rearrangement in the sea cucumber genus Cucumaria[END_REF], katydids [START_REF] Yang | Mitochondrial genomes of four katydids (Orthoptera: Phaneropteridae): New gene rearrangements and their phylogenetic implications[END_REF], birds [START_REF] Abbott | An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in Thalassarche albatrosses[END_REF][START_REF] Morris-Pocock | Concerted evolution of duplicated mitochondrial control regions in three related seabird species[END_REF][START_REF] Schirtzinger | Multiple independent origins of mitochondrial control region duplications in the order Psittaciformes[END_REF], turtles (Parham et al., 2006a(Parham et al., , 2006b;;[START_REF] Peng | Complete mitochondrial genome of Chinese big-headed turtle, Platysternon megacephalum, with a novel gene organization in vertebrate mtDNA[END_REF], snakes [START_REF] Kumazawa | Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster[END_REF][START_REF] Kumazawa | The complete nucleotide sequence of a snake (Dinodon semicarinatus) mitochondrial genome with two identical control regions[END_REF][START_REF] Dubey | Complete mitochondrial genome sequence from an endangered Indian snake, Python molurus molurus (Serpentes, Pythonidae)[END_REF] and fishes [START_REF] Lee | The complete DNA sequence of the mitochondrial genome of the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the first description of duplication of a control region in fish[END_REF][START_REF] Tatarenkov | Rapid concerted evolution in animal mitochondrial DNA[END_REF][START_REF] Shi | A novel model of double replications and random loss accounts for rearrangements in the Mitogenome of Samariscus latus (Teleostei: Pleuronectiformes)[END_REF]. Sometimes, CR duplications extend beyond the sole CR, as exemplified by terrestrial isopods in which the entire mt genome is duplicated [START_REF] Marcadé | Structure and Evolution of the Atypical Mitochondrial Genome of Armadillidium vulgare (Isopoda, Crustacea)[END_REF][START_REF] Chandler | Multiple Conserved Heteroplasmic Sites in tRNA Genes in the Mitochondrial Genomes of Terrestrial Isopods (Oniscidea)[END_REF][START_REF] Peccoud | Untangling Heteroplasmy, Structure, and Evolution of an Atypical Mitochondrial Genome by PacBio Sequencing[END_REF]. Thus, although reported in a diverse array of species, CR duplication appears to be a relatively rarely documented cause of mt structural variation, especially when considering the high number of animal mitogenomes that have been sequenced to date.

The mechanisms associated with the occurrence of CR duplications are not fully elucidated but several hypotheses have been proposed. Potential mechanisms that could duplicate CR include illegitimate recombination [START_REF] Lunt | Animal mitochondrial DMA recombination[END_REF], dimeric mitogenome [START_REF] Boore | Computational Biology[END_REF] and tandem duplication [START_REF] Boore | Computational Biology[END_REF]. Illegitimate recombination is characterized by recombination between non-homologous or short homologous sequences, but the exact associated biological mechanisms remain poorly understood [START_REF] Rijk | Molecular Mechanisms of Exon Shuffling: Illegitimate Recombination[END_REF]. In the case of dimeric mitogenomes, dimeric molecules are formed by two monomers linked head-to-tail, which produces a fully duplicated mt genome. Another mechanism is tandem duplication, in which case an identical segment of a sequence is inserted next to the original sequence. Interestingly, the duplicated CR of G. roeselii show very high sequence similarity, a pattern that has also been noted in several other species with duplicated CR. It has been proposed that this may be the result of concerted evolution, which leads to sequence homogenization of duplicated CR [START_REF] Liao | Concerted Evolution: Molecular Mechanism and Biological Implications[END_REF][START_REF] Li | Concerted Evolution of Duplicate Control Regions in the Mitochondria of Species of the Flatfish Family Bothidae (Teleostei: Pleuronectiformes)[END_REF].

The potential advantages of the presence and conservation of duplicated CR remains largely unknown and unexplored. Based on studies in mammals and fruit flies, it has been proposed that mt genomes with duplicated CR may have a selective advantage over mt genomes with a single CR, by allowing a higher replication rate [START_REF] Shao | Evolution of Duplicate Control Regions in the Mitochondrial Genomes of Metazoa: A Case Study with Australasian Ixodes Ticks[END_REF].

Moreover, in some avian families, it was demonstrated that duplication of the CR forms a pseudo-CR which is associated with extended lifespan [START_REF] Skujina | Duplication of the mitochondrial control region is associated with increased longevity in birds[END_REF]. It is unclear whether any of these potential advantages may apply to G. roeselii and this issue represents an interesting avenue for future research in G. roeselii mitochondrial biology and, more generally, to further investigate the evolutionary forces at work and implications of CR duplications in animal mt genomes. 3). This is promising with respect to the relevance of next-generation sequencing in assessing polymorphism at very low taxonomic level and disentangling the evolutionary history of G. roeselii. The value of such a strategy for mitogenome sequencing as an approach to study intraspecific expansion/invasion is increasingly recognized [START_REF] Rius | Applications of next-generation sequencing to the study of biological invasions[END_REF] and it has been successfully applied to many organisms (e.g. [START_REF] Wu | Analyses of Mitogenome Sequences Revealed that Asian Citrus Psyllids (Diaphorina citri) from California Were Related to Those from Florida[END_REF]. The optimal strategy for G. roeselii remains to be found, being a compromise between sample size and genome coverage. The mitogenome enrichment strategy recently proposed by Macher et al. (2017b) may be highly relevant in this context. Alternatively, a more affordable strategy may consist in extending sampling in a pilot assay to identify mt genome regions with high information content, which may then be targeted by PCR using primers designed for indexed multiplex amplicon sequencing [START_REF] Maddock | Next-Generation Mitogenomics: A Comparison of Approaches Applied to Caecilian Amphibian Phylogeny[END_REF][START_REF] Meimberg | A new amplicon based approach of whole mitogenome sequencing for phylogenetic and phylogeographic analysis: An example of East African white-eyes (Aves, Zosteropidae)[END_REF].

Until recently (but see

In our phylogenetic analysis, G. roeselii, G. fossarum and G. pulex form a wellsupported clade, a feature also observed in other studies based on either a limited set of partial mt DNA and nuclear DNA sequences (e.g. [START_REF] Hou | Eocene habitat shift from saline to freshwater promoted Tethyan amphipod diversification[END_REF] or a set of 13 mt protein-coding genes (Macher et al., 2017a). However, the genus Gammarus does not appear to be monophyletic in the present study as the three other Gammarus species G. lacustris, G. cheuvreuxi and G. duebeni are more closely related to Baikalian families. However, the phylogenetic status of Gammarus fluctuated over the recent years, sometimes being considered monophyletic [START_REF] Hou | Eocene habitat shift from saline to freshwater promoted Tethyan amphipod diversification[END_REF], monophyletic but with low support (Macher et al., 2017a) or paraphyletic [START_REF] Hou | A review of Gammaridae (Crustacea: Amphipoda): the family extent, its evolutionary history, and taxonomic redefinition of genera[END_REF]. These discrepancies might be caused by incomplete taxon sampling and/or limited informative content of used markers. Testing these hypotheses is beyond the scope of the present study but it is noteworthy that both the present study and Macher et al 2017a, were unable to provide a fully resolved tree for this taxonomic issue, despite use of 13 protein-coding genes and ~11,000 nucleotide-long datasets.

At a higher taxonomical level, the families included in our study are targets of molecular phylogenetic reassessments [START_REF] Hou | A review of Gammaridae (Crustacea: Amphipoda): the family extent, its evolutionary history, and taxonomic redefinition of genera[END_REF][START_REF] Romanova | Evolution of mitochondrial genomes in Baikalian amphipods[END_REF]. Our results, along with Macher et al (2017a), clearly support the conclusion that Gammaridae is not a monophyletic family. Based on partial mt and nuclear sequences, [START_REF] Hou | A review of Gammaridae (Crustacea: Amphipoda): the family extent, its evolutionary history, and taxonomic redefinition of genera[END_REF] proposed that the monophyly of Gammaridae could be assured but to avoid the paraphyly of the family, they proposed the omission of Pontogammaridae, Typhlogammaridae and all Baikalian families. Our results suggest that Gammaridae is paraphyletic with Pontogammaridae and Baikalian families (Eulimogammaridae, Acanthogammaridae, Microropodidae, Pallaseidae, Crypturopodidae). Based on 13 mt protein-coding genes, [START_REF] Romanova | Evolution of mitochondrial genomes in Baikalian amphipods[END_REF] proposed that Baikalian families should be considered as monophyletic, which is consistent with our results. However, as the sister family Gammaridae is paraphyletic, further investigations are warranted to confirm this conclusion.

In conclusion, our study presents the first case of verified duplication of the CR in the Gammarus genus. Comparisons with other G. roeselii MOTUs and gammarid species will provide information on whether this type of structural variation is unique to MOTU of G. roeselii present outside the Balkans or a more general feature of gammarids. The discovery of this CR duplication also offers the opportunity to explore the molecular dynamics of mt genomes, for example with respect to regulation of mt genome replication or the origin of mt gene translocations. Finally, the characterization of multiple mt haplotypes from closely related G. roeselii individuals constitutes a promising avenue to constitute a valuable set of markers for phylogeographic analyses of G. roeselii.

  Ramonova et al 2016, Macher et al 2017a), the only mt markers used in Gammarid phylogeny and phylogeography were the COI barcode (~625 bp) and a small region rnnS (~300 bp) (e.g. Grabowsky et al 2017, Moret et al 2007). In G. roeselii, two complete mt genomes (Ou3 and Ou53 from France, this study) and one partial genome (from Germany, Macher et al 2017b) are now available. Despite the three genomes all sharing the same COI and rrnS haplotype, multiple SNPs were identified in four protein-coding genes and rrnL (Table

Figure legends Figure 1

 legends1 Figure legends

Figure 2

 2 Figure 2 Organization of mitochondrial genomes in three species of the genus Gammarus.

Figure 3

 3 Figure 3 Per nucleotide sequencing depth of G. roeselii mitochondrial genome assuming a

Figure 4

 4 Figure 4 PCR test assessing the presence or absence of the duplicated control region in G.

Figure 5

 5 Figure 5Bayesian phylogenetic tree of 20 gammarid species inferred from 13 mt protein-

Table 1

 1 Summary of amphipod mitogenomes used in this study. (p) indicates partial genome.

	Species name	Family	Accession Number	Size (bp)
	Acanthogammarus victorii	Acanthogammaridae KX341962	17,424 (p)
	Brachyuropus grewingkii	Acanthogammaridae KP161875	17,118
	Crypturopus tuberculatus	Cryturopodidae	KX341963	13,864 (p)
	Echinogammarus veneris	Gammaridae	TSA GARO01000000 n/a
	Eulimnogammarus cyaneus	Eulimnogammaridae KX341964	14,370
	Eulimnogammarus verrucosus Eulimnogammaridae KF690638	15,315
	Eulimnogammarus vittatus	Eulimnogammaridae KM287572	15,534
	Gammarus chevreuxi	Gammaridae	TSA HADC01000000 n/a
	Gammarus duebeni	Gammaridae	JN704067	15,651
	Gammarus fossarum	Gammaridae	KY197961	15,989
	Gammarus lacustris	Gammaridae	SRA SRR3467069	n/a
	Gammarus pulex	Gammaridae	WGS FJVI01000000 n/a
	Gammarus roeselii	Gammaridae	MG779536	16,073
	Garjajewia cabanisii	Acanthogammaridae KX341965	17,576 (p)
	Gmelinoides fasciatus	Microropodidae	KX341966	18,114
	Linevichella vortex	Microropodidae	KX341967	11,444 (p)
	Pallaseopsis kesslerii	Pallaseidae	KX341968	15,759 (p)
	Pandorites podoceroides	Pontogammaridae	SRA SRR3467097	n/a
	Parhyale hawaiiensis	Hyalidae	AY639937	12,224 (p)
	Pseudoniphargus sorbasiensis Speudoiphargidae	LN871175	15,460 (p)

Table 2

 2 Annotation of the complete mitochondrial genome of Gammarus roeselii.

	620
	621
	Gene Position	Strand Size	Start	Stop	Intergenic
				(bp)	codon	codon	nucleotides
	cox1	1-1,540	+	1,540	ATT	T	0
	trnL2	1,544-1,603	+	60			3
	cox2	1,601-2,285	+	685	ATA	T	-3
	trnK	2,283-2,341	+	59			-3
	trnD	2,341-2,405	+	65			-1
	atp8	2,406-2,564	+	159	ATA	TAA	0
	atp6	2,558-3,229	+	672	ATG	TAA	-7
	cox3	3,229-4,014	+	786	ATG	TAG	-1
	nad3	4,011-4,364	+	354	ATA	TAG	-4
	trnA	4,372-4,432	+	61			7
	trnS1	4,432-4,483	+	52			-1
	trnN	4,486-4,548	+	63			2
	trnE	4,546-4,608	+	63			-3
	trnR	4,603-4,662	+	60			-6
	trnF	4,661-4,720	-	60			-2
	nad5	4,691-6,404	-	1,714	ATT	T	-30
	trnH	6,423-6,482	-	60			18
	nad4	6,466-7,695	-	1,230	ATG	TAA	-17
	nad4l	7,788-8,078	-	291	ATG	TAG	92
	trnT	8,082-8,141	+	60			3
	trnP	8,141-8,201	-	61			-1
	nad6	8,213-8,707	+	495	ATT	TAA	11
	cob	8,707-9,843	+	1,137	ATG	TAG	-1
	trnS2	9,842-9,896	+	55			-2
	nad1	9,926-10,864	-	939	ATG	TAG	29
	trnL1	10,859-	-	62			-6
		10,920
	rrnL	10,921-	-	977			0
		11,897
	trnV	11,901-	-	52			12
		11,952
	rrnS	11,952-	-	646			-1
		12,597
	CR1	12,594-		1,029			-4
		13,622
	trnY	13,623-	-	64			0
		13,686
	trnQ	13,683-	-	57			-4
		13,739
	trnC	13,740-	-	54			0
		13,793
	CR2	13,792-		1,027			0
		14,818

Table 3

 3 Sequence variation between G. roeselii mitochondrial haplotypes. Ou3 is used as a

	reference sequence.
	Nucleotide	Type Variation	Genomic locus	Haplotype with variation
	coordinate
	1,736	SNP C/T	cox2	Ou53
	4,836	SNP C/A	nad5	Macher et al 2017b
	5,877	SNP C/A	nad5	Macher et al 2017b
	6,961	SNP C/T	nad4	Macher et al 2017b
	9,442	SNP G/A	cob	Macher et al 2017b
		(Gly/Ser)
	11,143	SNP G/A	rrnL	Macher et al 2017b
	11,207	SNP C/T	rrnL	Macher et al 2017b
	14,843	Indel A/-	Intergenic	Macher et al 2017b;
			region	Ou53

Acknowledgments

We thank Isabelle Giraud for preparation of DNA samples used for sequencing, Amine Chebbi and Bouziane Moumen for assistance with bioinformatic analyses, and Clément Gilbert for discussions. We also thank the Roscoff Bioinformatics platform ABiMS (http://abims.sb-roscoff.fr) for providing additional computational resources. We also thank the editors and reviewers for their comments on an earlier version of the manuscipt. This work was funded by Agence Nationale de la Recherche Grant ANR-15-CE32-0006 (CytoSexDet), Centre National de la Recherche Scientifique (CNRS) PEPS ExoMod Grant (MicroFem) to R.C. and T.R., the 2015-2020 State-Region Planning Contract and European Regional Development Fund, and intramural funds from the CNRS and the University of Poitiers.


Yan Brailowsky

From Genesitic Curiosity to Dangerous Gynocracy in Sixteenth-Century England

The decade between 1550 and 1560 was a particular period in Western European history: many monarchies in the region were led by women, either as queens (Mary Tudor in England, Mary Stuart in Scotland) or regents (Catherine de' Medici in France, Joanna of Austria in Spain, Catherine of Austria in Portugal). To this list, one could add other female figures acting as kings, such as Mary of Hungary, also known as Mary of Austria, who was governor of the Spanish Netherlands from 1531 to 1555. This situation was unheard of; for some, it was even monstrous, unnatural, contumely to God. To all effects and purposes, gynocracy or gynecocracy, 'the rule by women', was as a sixteenth-century 'curiosity', an exceptional, singular, odd, novel situation. 1 This may explain why gynocracy gave rise to a heated discussion reaching back to the origins of mankind, i.e. the Book of Genesis, particularly with Eve's appetite for knowledge -another meaning of 'curious' 2 -which famously led to man's Fall. As this essay contends, in the eyes of many writers in the sixteenth century, the perils of gynocracy were linked with Genesitic curiosity, women's power with the pursuit of forbidden knowledge, original sin with death.

Women and Genesitic Curiosity

To underline the link between Eve and epistemology, on the one hand, and the link between the consequences of her action, i.e. her subjection to her husband, and sixteenth-century political theory, on the other, one must start from the beginning. In the beginning, 'the Lord God said, It is not good that the man should be himself alone; I will make him a help meet for him' (Ge 2:18). 3 A few verses later, man's 'help', Eve, is tempted by the serpent and she tastes of the fruit of the forbidden tree. 1 Sense II.16.a of 'curious' in the OED. 2 Sense I.5.a in the OED.

3 Unless noted otherwise, all Biblical quotes are from the Authorized Version (1611). Similarly, unless noted otherwise, all emphasis in Biblical quotes is mine.

As recalled by Philip Almond, most early modern writers and theologians attributed the Fall to man's curiosity, among a host of other sins. 4 In The Historie of Adam (1606), for instance, Henry Holland lists curiosity as the third sin committed by Adam and Eve: for 'they seeke after strange knowledge, not contented with Gods holy word.' 5 A similar point was made by Elnathan Parr in The Grounds of Divinitie (1615) and Edward Leigh in A Systeme or Bodie of Divinitie (1654). 6 Other writers, however, singled out woman's agency in the Fall. In his Anatomy of Melancholy (1638), Robert Burton compares Adam's transgression with Pandora's box, linking the androcentric Biblical account with the gynocentric Greek myth. 7 In Hexapla in Genesin (1633), Andrew Willet goes one step further, arguing that Eve's curiosity is the fruit of her desire, further linking this desire with her vain hunger for knowledge: 'The woman seeth the tree to be good for meat, there is her voluptuous desire: pleasant to the eyes, there is her curiosity: and to be desired for knowledge, there is the vanity of her minde.'8

This reading would have been influenced by several translations of Ge 3:6, in which what constitutes man's original sin is revealingly described as the linking of desire with knowledge. In the 1561 Geneva Bible, the verse is rendered thus: 'So the woman (seeing that the tree was good for meat, and that it was pleasant to the eyes, and a tree to be desired to get knowledge) took of the fruit thereof, and did eat, and gave also to her husband with her, and he did eat.' In the 1611 Authorized Version: 'And when the woman saw that the tree was good for food, and that it was pleasant to the eyes, and a tree to be desired to make one wise, she took of the fruit thereof, and did eat, and gave also unto her husband with her; and he did eat.' Interestingly, other, earlier versions of this verse do not emphasize the idea of knowledge. The Vulgate, for instance, is descriptive: 'quod bonum esset lignum ad vescendum et pulchrum oculis aspectuque delectabile.' The Wycliffe Bible translates this nearly verbatim, speaking of a 'tree [which] was good, and sweet to eat, and fair to the eyes, and delightable in beholding.' After this transgression, God addresses both Adam and Eve, punishing them in turn. Again, Eve's desire comes into the picture, this time to dictate its subjection to her husband: 'thy desire shall be subject to thy husband, and he shall rule over thee' (Ge 3:16). In the verse immediately following, God berates Adam for listening to his wife: 'Because thou hast hearkened unto the voice of thy wife, and hast eaten of the tree, of which I commanded thee, saying, Thou shalt not eat of it: cursed is the ground for thy sake' ( 17) and so on. In other words, in the Genesitic account of the beginning of mankind, mankind is punished for having gained the knowledge of good and evil; Eve's subordination to her husband is a punishment for her sin; and knowledge and curiosity are intimately linked with desire and subjection. This reading raises a number of issues.

First, theologians were divided on the question of whether Eve is naturally, or only momentarily subordinate. Luther and Calvin, for instance, seemed to hesitate between the two notions. [START_REF] Mattox | Luther on Eve, Women, and the Church[END_REF] Genesis does not say that women are inherently weaker than man, although the idea is present elsewhere in the Bible. Rather, God says Eve is subordinate to her husband as a consequence of her transgression. It does not suggest that her subjection should extend to fathers, brothers and kinsmen, for instance; nor does it suggest that she was subjected to Adam before the Fall. This distinction between natural or absolute, and historical or contextual subjection is of consequence for early modern writers who distinguished the body natural (the perishable flesh which is liable to 'desire') and the body politic (the eternal soul which 'rule[s]' over men).

Secondly, the question remains whether knowledge, and the pursuit thereof, is naturally sinful, or a rememoration of the original sin, or whether once acquired, knowledge should be used and even desired to improve man's fallen state. God punishes Adam and Eve for their transgression, but this does not necessarily mean that he condemns knowledge per se. This point will be important for humanists and reformists who called on the Church to educate its flock and clergy in order to avoid heresies which could imperil one's salvation. 10 In other words, the thirst or desire for knowledge is not necessarily sinful.

Finally, an analysis of the Genesitic account should ask whether God had planned the Fall all along to underline the perils of rebellion and ill-gotten knowledge, stressing the virtues of obedience to authority, however tyrannous, whimsical or arbitrary. The notion that God had foreknowledge that Adam and Eve would be tempted even before He created Eve is suggested by theologians like St Augustine in his Two Books on Genesis Against the Manichees. As noted by Angelamatilde Capodivacca, 'Although Eve had not yet been created when God gave the warning not to eat the forbidden fruit, He formulated this warning in the plural, anticipating Eve's fall':11

[T]he conclusion of the commandment clearly shows it was not addressed just to one person; what he says, you see, is this: but on the day you all take a bite from it, you all shall die the death (Ge 2:17). He is already starting on the explanation of how the woman came to be made, and how she is said to have been made as a help for the man [...]12 Augustine goes on to suggest that the creation of woman was made to show how man must 'subject the soul's appetite or desire', i.e. woman, to 'the interior mind [...] manly reason' (ibid.). The point is important for thinkers who claimed that, although gynocracy was unnatural, the situation may have been willed by God and that man owed obedience to divinely ordained rulers, good or evil. In an English context, the issue is compounded by the adoption of Calvinism in the second half of the sixteenth century, and its attendant belief in predestination.

Knox and the 'Monstrosity' of Gynocracy

This Biblical context and the issues it raises inform many of the early modern debates on the link between Eve's curiosity and the dangers of gynocracy. These discussions were not new. As recalled by Neil Kenny and others, for centuries 'woman' and 'curiosity' were virtually a pleonasm: 'not for nothing is curiositas a feminine noun.'13 A Shakespearean-styled quip could posit that 'curiosity, thy name is woman'.

Discussions over the virtues and dangers of gynocracy had developed since the publication of Christine de Pisan's Cité des Dames and the so-called querelle des femmes in the 15th century.14 Typical views on women included those of Machiavelli who claimed in The Discourses (1531) that women were invariably the harbingers of chaos: we see that women have been the cause of great dissensions and much ruin to states, and have caused great damage to those that govern them. We have seen, in the history of Rome, that the outrage committed upon Lucretia deprived the Tarquins of their throne, and the attempt upon Virginia caused the Decemvirs the loss of their authority. Thus, Aristotle mentions as one of the first causes of the ruin of tyrants the outrages committed by them upon the wives and daughters of others, either by violence or seduction; […] absolute princes and rulers of republics [therefore] […] should well reflect upon the disorders that may arise from such causes. 15 What was new in the sixteenth-century was the historical context mentioned earlier, one which explains why John Knox published his First Blast of the Trumpet Against the Monstruous Regiment of Women while in exile in Geneva in 1558. Knox's famous pamphlet seemed to illustrate common beliefs on the dangers of female rule. Speaking in 'most plain and few words', he began his 'First Blast' with the following declaration:

To promote a woman to bear rule, superiority, dominion or empire above any realm, nation, or city, is repugnant to nature, contumely to God, a thing most contrarious to his revealed will and approved ordinance, and finally it is the subversion of good order, of all equity and justice. 16 The pamphlet goes on to justify this opening salvo by developing each idea in succession. In the course of his pamphlet, Knox used arguments from Aristotle and Augustine. From the Greek philosopher he took the notion of natural order, 'an immutable order of ranks, excluding women from powerful stations'. 17 Women being 'naturally' weak, like the blind, they ought not to rule, 'For who can deny but it repugneth to nature, that the blind shall be appointed to lead and conduct such as do see?' 18 From the bishop of Hippo, Knox took Biblical proof, citing Ge 3, 1 Tim, and 1 Cor. For Augustine, 'the man rules and the woman obeys: the Fall recurs each time this hierarchy is overturned'. 19 Further, Augustine underlines women's paradoxical nature, both curious and skeptical, interpreting Ge 3:4-5 as follows:

Then the serpent told the woman that she would not die. God forbade it only because He knew that on the day you eat it your eyes will open and, then, you will be like gods, having the knowledge of good and evil. How could the woman ever believe these words that told her that God had forbidden something good and useful unless she already had inherent in her mind that love for an independent authority and an arrogant presumption of herself -thus it was she who was to be condemned and punished through this temptation. Not being satisfied by the serpent's words, Eve examined the tree. 20 Albeit Knox's reasoning was mostly rooted in Biblical history, which was supposed to lend it an absolute, timeless authority, the English-language publication was aimed at two particular, pernicious and contemporary examples obtaining in the British Isles: that of Mary of Guise, the Scottish regent, and Mary I, the murderous English Queen who persecuted her subjects and concluded a dangerous alliance with Catholic Spain -a woman who behaved, in short, as a modern-day Jezabel. Similar remarks were made by Christopher Goodman in a pamphlet published in Geneva earlier the same year, sparking a series of misogynist treatises.21 Knox's pamphlet was ill-timed, however, as Mary died just a few months after the publication of The First Blast, to be succeeded by another woman, Elizabeth I. Understandably, the new queen was not amused by the fiery Scottish pamphleteer, and Knox was never to be pardoned. He did not set foot in England, his adoptive land, again.

Knox attempted to distance himself from his own work, repeatedly arguing that he was speaking of Mary and that he welcomed Elizabeth's accession as a sign of the workings of divine Providence. In point of fact, his private letters are 'free of gendered rhetoric', encouraging women to be critical and independent,22 and his pamphlet was in great part aimed at justifying rebellion against tyranny, rather than against female rulers per se. 23 But his insistence in The First Blast on women's gender, railing in his Preface against 'this monstriferous empire of women, (which amongst all enormities, that this day do abound upon the face of the whole earth, is most detestable and damnable)', precluded mitigating interpretations of his analysis in the eyes of the new queen. 24 Half-lucidly, he had foreseen such persecution, using the term 'curious' as one who is 'Unduly minute or inquisitive' (OED, sense I.10.a) when he claimed that he would 'be called foolish, curious, despiteful, and a sower of sedition: and one day perchance (although now I be nameles) I may be attainted of treason.' 25 Knox's insistence on the 'monstrosity' of women's rule was perhaps his own, and what he viewed as monstrous was for others simply something odd, an exception -a curiosity in the other sense. 26 This is what other reformers on the Continent argued when they tried to distinguish themselves from Knox. As recalled by John Lee Thompson, Calvin, like Knox, ruled in favor of women's subordination. However, the Genevan preacher had a different strategy than that of Knox, distinguishing canon and civil interpretations of female rule:

[Calvin] tried to distance himself from Knox, first by dedicating the second edition of his commentary on Isaiah to Elizabeth, then by writing a conciliatory letter to her secretary, William Cecil. [...] In both letters, Calvin makes clear his belief that women's rule is contrary to the legitimate order of nature and is sent by God to punish the indolence of men; [...] but Calvin will not counsel revolt against a woman ruler any more than he would consider rebelling against a tyrant: "Private persons have no right to do anything but to deplore [such rule]. Indeed, gynecocracy -like tyranny -is a bad arrangement which must be tolerated until God sees fit to overthrow it." [...] For Calvin, female rule -whether that of Mary Tudor or Elizabeth -always remained a kind of tyranny and, as such, it was to be neither welcomed nor resisted; it was only to be endured. 27 Similarly, as made clear in his Sermon on the Epistle of St Paul to the Ephesians (on Eph. 5:21-5), Calvin argued that women ought to bear their subjection with equanimity rather than rebellion. Their subordination was the divine punishment for Eve's curiosity, something women were called on never to forget. According to Calvin: there is no other shift but that women must stoop and understand that the ruin and confusion of mankind came in on their side, and through them we be all forlorn and accursed and banished the kingdom of heaven: when women do understand that all this came of Eve and of womankind (as St Paul telleth us in another place [1 Tim. 2:14]), there is none other way but for them to stoop and bear patiently the subjection that God hath laid upon them, which is nothing else but a warning to them to keep themselves lowly and mild.28

Celebrating Eve

As noted by contemporaries who wrote books in defense of gynocracy, such as Laurence Humphrey, John Aylmer or Henry Howard, Knox's arguments were contradictory.29 To take but one example, his argument of the 'immutable rank and order -man above woman -is immediately contradicted by his other belief in God's full control over all things.' 30 Others went further, defending Eve's transgression as a reason to celebrate life, rather than death, as in a 1591 sermon on Ge 2.18 ('The Creation of Eve') by bishop Lancelot Andrewes at St Paul's Cathedral:

But if any shall complain yet further of the woman's hurt and fault; let us know that this woman was made by the counsel of God, the means and occasion by which amends was made, and that with advantage for the evil, for all the evil which she had first done, for as she brought forth sin and death, so she was a means to bring forth a holy seed, which should bring eternal righteousnesse and life unto all, for as the Serpent should deceive the woman: So it was God's purpose, that the seed of woman should destroy the Serpent and his works; wherefore we must not so much with grief marvel that the woman's sin was made the occasion of all our misery, as with and comfort to wonder, that God made the seed of the woman to save us from sin, and to bring us to felicity. [START_REF] Lancelot | Lancelot Andrewes: Selected Sermons and Lectures[END_REF] This benevolent interpretation of Genesis found additional support in readings which sought to contextualize the Biblical model. John Aylmer, in his Harborowe for Faithfull and Trewe Subjects (1559), helped read scripture as a topical, historical document, viewing women's subjection as a social construct, rather than an absolute necessity; the point was also developed by other writers such as Bruni da Pistoia in Difesi delle Donne (Milan, 1559). 32 Among defenders of women, however, most continued to acknowledge the doxa of their relative weakness, due in part to what was still perceived as a constitutive weakness and tendency to be carried away by affection, rather than reason. To counter these failings, Sir Thomas Smith advocated the use of advisors in 1583: 'such personages [such as Queens should] never lack the counsel of such grave and discrete men as be able to supply all other defects'. [START_REF] Sir | De Republica Anglorum: A Discourse on the Commonwealth of England[END_REF] Ironically, however, such recommendations suggest only a partial understanding of the Genesitic model: Eve, being ignorant, could be deceived by the serpent who may well be considered as woman's first advisor. Incidentally, this prompted some writers to wonder whether the serpent was gendered, some suggesting that it was female, i.e. sweet-tongued, spreading false and dangerous rumours. In a painting by Johann Brabender in Münster, Sündenfall vom Paradies des Doms (1550), the serpent has exposed breasts; in the mid-fifteenth-century Bracacci Chapel in Florence, the serpent is distinctly feminine. [START_REF] Charlesworth | The Good And Evil Serpent: How a Universal Symbol Became Christianized[END_REF] Others insisted on the serpent's masculinity, underlining the sexual interpretations of Eve's temptation by the beast, even claiming that Eve gave birth to his brood, Cain and Abel, partly explaining why one of the brothers turned out a fratricide. [START_REF] Almond | [END_REF] More generally, views such as those of Sir Thomas Smith emphasized the need to provide a decent education and a sense of history to women of the elite, and Aylmer found solace in the idea that in England any monarch's potential shortcomings could be compensated by Parliament's sound counsel. [START_REF] John | Harborowe for Faithfull and Trewe Subjects[END_REF] In 1559, Queen Elizabeth seemed to be a case in point. She had Roger Ascham as her tutor, who claimed the princess was very knowledgeable in Latin, developing a beautiful style, notably in her translations, for instance of Plutarch's essay on curiosity -skills which would turn to her advantage when she ascended the throne -and her early years suggested a certain deference to Parliamentary procedures. [START_REF] Levin | Mary Wroth, Lucy Russell (the Countess of Bedford), Mary Herbert (the Countess of Pembroke), Elizabeth Cary, and Dorothy Shirley are but a few of the well-known examples of Renaissance women who were responsible for creating "a significant index of the importance of women for the drama[END_REF] Others criticized her choice of advisors who eventually served as proxies to stamp out any criticism against the regime.

Curiosity, Gynocracy and Dynastic Change

The representation of female kingship was fraught with peril in early modern England. While Elizabethan censorship limited direct criticism of female rule and several writers sought to justify it, most pamphlets continued to link contemporary wars and chaos to the 'unnatural' nature of gynocracy, claiming such troubles descended from the Biblical curse for which Eve's curiosity was the prime culprit. The death of Elizabeth in 1603 radically changed the prevailing atmosphere. The Virgin Queen was succeeded by an experienced, mature male heir, allowing English subjects to look forward to a return to a stable, long-term, male-led monarchy, one which no longer elicited the 'curiosity of nations'. 38 The dynastic change was also felt in the drama of the time, as evidenced by Shakespeare's early Jacobean productions. In the first decade of James I's reign, female kingship was represented onstage in a manner which, far from extolling the virtues of female rule, suggested either their weakness or wickedness with a series of memorably dangerous queens, such as Lady Macbeth in Macbeth (c. 1606), Regan and Goneril in King Lear (c. 1606), or the Queen in Cymbeline (c. 1609). In these plays, the dramatist depicts the evil queens' malice using the topos of women as 'leaking vessels', actually materializing their malevolence with liquids: poison in Cymbeline (1.6 et al.), the unending effort to washing off blood in Macbeth (5.1), and a combination of the two motifs in King Lear, as Goneril stabs herself after having poisoned her sister Regan (5.3). These common modi operandi are in keeping with a Genesitic reading of women's nature which claimed that women had achieved their aims serpent-like, by indirect means, convincing or coaxing men to perform treasonous acts, or furnishing them with equally devious, or 'curious', means to do away with (male or female) rivals. These plays seemingly presented female rule as a monstrous 'curiosity'. In doing so, and in line with the works of Knox or Goodman on the right to rebel against tyranny, they also subtly contributed to questioning the prevailing system of government, which might have potentially paved the ground for the later demise of the monarchy and the Civil War. This critical perspective on history was further questioned by the spectacular increase in the number of women 'prophets' and 'curious' women in the decades preceding the Civil War -two phenomena challenging the hitherto male-dominated fields of religious prophecy and scientific research. Despite a return to patriarchy and gender orthodoxy with the advent of the Stuart monarchy, the gynocratic curiosity of sixteenth-century English history may thus have paved the way to the development of women's epistemological curiosity in the seventeenth century.

4

  Almond P. C., Adam and Eve in Seventeenth-Century Thought (Cambridge: 2008) 194.5 Holland Henry, The Historie of Adam, or the foure-fold state of man, vvell formed in his creation,

deformed in his corruption, reformed in Grace, and perfected in glory, RSTC 13587 (T[homas] E[ast] for Thomas Man, London: 1606) 10. 6 Almond P. C., Adam and Eve 194-195. 7 Burton Richard, The Anatomy of Melancholy: What it Is, with All the Kinds, Causes, Symptomes, Prognostickes, and Seuerall Cures of it (Henry Cripps, London: 1638) 1.1.2.

  16 Knox John, The First Blast of the Trumpet Against the Monstruous Regiment of Women, RSTC 15070 ([J. Poullain and A. Rebul], Geneva: 1558) 9r. Spelling modernized.

	17 Chavura S. A., Tudor Protestant Political Thought 1547-1603 (Leiden -Boston: 2011) 64.
	18 Knox, First Blast 9v.
	19 Augustine, On Genesis II 11,15.
	20 De Genesi ad Literam XI 30, 11, quoted by Capodivacca, Curiosity 48.

Willet Andrew, Hexapla in Genesin & Exodum: that is, a sixfold commentary upon the two first bookes of Moses, being Genesis and Exodus Wherein these translations are compared together, RSTC 25685(John Haviland, London: 1633) 29. Emphasis in the original.

The point is made, inter alia, by Augustine in his On Christian Learning.

Capodivacca A., Curiosity and the Trials of the Imagination in Early Modern Italy (Berkeley: 2007) 48.

Augustine, On Genesis, trans. Edmund Hill, O.P., ed. John E. Rotelle, O.S.A (New York: 2002) II 11,15. Sixteenth-century translations used the pronoun thou.

Happel, quoted by Kenny N., The Uses of Curiosity in Early Modern France and Germany (Oxford: 2004) 384.

Levin C. -Sullivan P. A. (eds.), Political Rhetoric, Power, and Renaissance Women (Albany: 1995). On early modern discussions on gynocracy, see, among others, Jordan C., "Woman's Rule in Sixteenth-Century British Political Thought", Renaissance Quarterly 40, 3 (1987) 421-451; Jansen S. L., The Monstrous Regiment of Women: Female Rulers in Early Modern Europe (New York: 2002); Levin C. -Carney J. E. -Barrett-Graves D. (eds.), 'High and Mighty Queens' of Early Modern England: Realities and Representations, The New Middle Ages (New York: 2003); Wanegffelen T., Le pouvoir contesté: souveraines d'Europe à la Renaissance (Paris: 2008).

15 Book 3, chap. 26, quoted by Jankowski T. A., Women in Power in the Early Modern Drama (Urbana: 1992) 56.

Goodman Christopher, How superior powers oght to be obeyd of their subiects and wherin they may lawfully by Gods Worde be disobeyed and resisted. Wherin also is declared the cause of all this present miserie in England, and the onely way to remedy the same, RSTC 12020(John Crispin, Geneva: 1558).

Felch S. M., "The Rhetoric of Biblical Authority: John Knox and the Question of Women", The Sixteenth Century Journal 26, 4 (1995) 805-822; idem, "'Deir Sister': The Letters of John Knox to Anne Vaughan Lok" Renaissance andReformation 19, 4 (1995) 47-68.

Dubois-Nayt A., "La tyrannie travestie, ou le genre au service du tyrannicide" Cercles 16, 2 (2006) 33-43; and "'La différence des sexes': construction et fonction du 'genre' dans la pensée politiquede John Knox" Cités 34, 2 (2008) 163-164, 168-169.

Knox, First Blast 5r.

Calvin John, Sermons on the Epistle to the Ephesians [1577], trans. A. Golding (Edinburgh: 1973) 569.

Caney A., "Let He Who Objects Produce Sound Evidence: Lord Henry Howard and the Sixteenth Century Gynecocracy Debate", Electronic Theses, Treatises and Dissertations 97 (2004), and Shephard A., Gender and Authority in Sixteenth-Century England: the Knox debate (Keele: 1994).

Shakespeare William, King Lear, ed. Foakes R. A., Arden Shakespeare (London: 1997) 5.2.4. The line is spoken by Edmund, the Duke of Gloucester's bastard son, as he complains about the consequences of his illegitimate birth.

30 Chavura, Tudor Protestant Polical Thought 67. Knox is countered in this sense by Laurence Humphrey, who claimed: 'There is a fixed order, both a state of things and an ordering of kingdoms. Nor are states constituted first of all without laws, without leader, rashly and by chance; neither are kings or those who are in charge for them, thus constituted. But as once the kings of Judah, so now ours, are anointed by the command and will of God, whether they are good or bad or men or women. For there is no power but of God.' Laurence Humphrey, On the Preservation of Religion and its True Reformation (1559), Janet Kemp (trans.), in Kemp J. K., Laurence Humphrey, Elizabethan Puritan: His Life and Political Theories, PhD thesis (West Virginia: 1978), quoted by Chavura 67.

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