Maren Möller-Krull

Frédéric Delsuc
email: delsuc@isem.univ-montp2.fr

Gennady Churakov

Claudia Marker

Mariella Superina

Jürgen Brosius

Emmanuel J P Douzery

Jürgen Schmitz

Maren Krull

Retroposed elements and their flanking

Keywords: DAS-SINE, retroposon, phylogeny, rare genomic changes, maximum likelihood

Introduction

Sloths, anteaters, and armadillos are among the most morphologically specialized mammals. These three lineages belong to Xenarthra, a mammalian order whose monophyly is undisputed [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF][START_REF] Van Dijk | The virtues of gaps: Xenarthran (Edentate) monophyly supported by a unique deletion in alpha A-crystallin[END_REF]). On molecular grounds, xenarthrans constitute one of the four major placental clades [START_REF] Madsen | Parallel adaptive radiations in two major clades of placental mammals[END_REF]Murphy et al. 2001a;[START_REF] Delsuc | Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting[END_REF]. Their evolutionary radiation began about 65 million years ago while South America was becoming isolated from other continental land masses [START_REF] Patterson | The fossil mammal fauna of South America[END_REF]. Thirty-one living species of xenarthrans (21 armadillos, four anteaters and six sloths) have been described [START_REF] Wetzel | The identification and distribution of recent Xenarthra (=Edentata)[END_REF][START_REF] Vizcaíno | Identificación específica de las "mulitas", género Dasypus L. (Mammalia, Dasypodidae), del noroeste argentino. Descripción de una nueva espécie[END_REF][START_REF] Anderson | A new species of three-toed sloth (Mammalia: Xenarthra) from Panamá, with a review of the genus Bradypus[END_REF]).

Today's limited xenarthran species number is in sharp contrast with their past diversity, as more than 200 fossil genera are recognized [START_REF] Mckenna | Classification of Mammals above the species level[END_REF]. The most impressive xenarthrans, such as giant ground sloths and glyptodonts, disappeared at the end of the last Pleistocene glaciations (about 10,000 years ago; [START_REF] Patterson | The fossil mammal fauna of South America[END_REF]. Remnants of this ancient evolutionary radiation, most extant xenarthran species are, nevertheless, poorly studied and uncertainties remain concerning their conservation status (da Fonseca and Aguiar

2004).

As curious as xenarthrans are, so disputed is their place in the placental evolutionary tree. Whereas nuclear gene concatenations have been frustratingly inconclusive (Murphy et al. 2001b;[START_REF] Delsuc | Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting[END_REF][START_REF] Amrine-Madsen | A new phylogenetic marker, apolipoprotein B, provides compelling evidence for eutherian relationships[END_REF][START_REF] Nikolaev | Early history of mammals is elucidated with the ENCODE multiple species sequencing data[END_REF]), the classical morphology-based Epitheria hypothesis, placing Xenarthra as the sister group of all placental orders [START_REF] Mckenna | Toward a phylogenetic classification of the Mammalia[END_REF], has found some support from retroposed elements [START_REF] Kriegs | Retroposed elements as archives for the evolutionary history of placental mammals[END_REF]). However, [START_REF] Murphy | Using genomic data to unravel the root of the placental mammal phylogeny[END_REF] have recently found some rare insertion/deletion events that represent contradictory evidence in support of a sister-group relationship between Xenarthra and Afrotheria instead.

Living xenarthrans are divided into three phylogenetically distinct groups, the arboreal sloths (Folivora: Megalonychidae and Bradypodidae), the toothless anteaters (Vermilingua: Myrmecophagidae), and the shelled armadillos (Cingulata: Dasypodidae). Folivora and Vermilingua form the suborder Pilosa for which there is unequivocal support from both morphological [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF][START_REF] Patterson | The ear region in xenarthrans (=Edentata, Mammalia). Part II. Sloths, anteaters, palaeanodonts, and a miscellany[END_REF]) and molecular [START_REF] Delsuc | The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua[END_REF][START_REF] Delsuc | Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting[END_REF] data.

The phylogenetic relationships and taxonomy of the living pilosans are well resolved [START_REF] Gaudin | The phylogeny of Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF][START_REF] Delsuc | The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua[END_REF][START_REF] Delsuc | Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting[END_REF]. Folivora contains the two morphologically distinct tree-sloth families Megalonychidae (Choloepus, two-toed sloths) and Bradypodidae (Bradypus, three-toed sloths) [START_REF] Webb | The interrelationships of tree sloths and ground sloths[END_REF][START_REF] Höss | Molecular phylogeny of the extinct ground sloth Mylodon darwinii[END_REF][START_REF] Greenwood | A molecular phylogeny of two extinct sloths[END_REF]). Among Vermilingua, myological [START_REF] Reiss | Myology of the feeding apparatus of myrmecophagid anteaters (Xenarthra: Myrmecophagidae)[END_REF]) and morphological [START_REF] Gaudin | The phylogeny of Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua[END_REF] studies supporting an early emergence of the pygmy anteater (Cyclopes didactylus) have been confirmed by molecular data [START_REF] Delsuc | The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua[END_REF][START_REF] Delsuc | Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting[END_REF].

These studies therefore group together the giant anteater (Myrmecophaga) and tamanduas (Tamandua).

The armadillos are the oldest and most speciose lineage of xenarthrans. Their phylogeny has been difficult to reconstruct from morpho-anatomical data with incongruent results among the rare studies [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF][START_REF] Patterson | The ear region in xenarthrans (=Edentata, Mammalia). Part I. Cingulates[END_REF][START_REF] Abrantes | Proposta filogenética para os Dasyposidae (Mammalia: Cingulata)[END_REF][START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF]. Molecular evidence [START_REF] Delsuc | Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting[END_REF][START_REF] Delsuc | Molecular systematics of armadillos (Xenarthra, Dasypodidae): contribution of maximum likelihood and Bayesian analyses of mitochondrial and nuclear genes[END_REF] has, nevertheless, clearly defined three major lineages compatible with the subfamilies of [START_REF] Mckenna | Classification of Mammals above the species level[END_REF]

classification:

Dasypodinae (the single genus Dasypus), Tolypeutinae (genera Tolypeutes, Cabassous, and Priodontes), and Euphractinae (genera Euphractus, Chaetophractus, and Zaedyus). A close relationship between Tolypeutinae and Euphractinae was also strongly supported, with Dasypodinae emerging first within Cingulata [START_REF] Delsuc | The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua[END_REF][START_REF] Delsuc | Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting[END_REF][START_REF] Delsuc | Molecular systematics of armadillos (Xenarthra, Dasypodidae): contribution of maximum likelihood and Bayesian analyses of mitochondrial and nuclear genes[END_REF]. These relationships are congruent with the study of spermatozoa [START_REF] Cetica | Evolutionary sperm morphology and morphometry in armadillos[END_REF]) but contradict morphological studies [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF][START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF]. However, even molecular data have failed to resolve relationships within the subfamilies Tolypeutinae and Euphractinae, with contradictory results obtained from mitochondrial versus nuclear genes suggesting that speciation events occurred very rapidly within each of these two armadillo lineages [START_REF] Delsuc | Molecular systematics of armadillos (Xenarthra, Dasypodidae): contribution of maximum likelihood and Bayesian analyses of mitochondrial and nuclear genes[END_REF]. Within tolypeutines, a close relationship between Cabassous and Priodontes would be consistent with their classification in the tribe Priodontini based on their similar morphologies [START_REF] Mckenna | Classification of Mammals above the species level[END_REF][START_REF] Abrantes | Proposta filogenética para os Dasyposidae (Mammalia: Cingulata)[END_REF][START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF] and a study of their spermatozoa [START_REF] Cetica | Evolutionary sperm morphology and morphometry in armadillos[END_REF]. Within euphractines, the grouping of Euphractus and Chaetophractus to the exclusion of Zaedyus would be congruent with a recent cladistic study of craniodental characters [START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF] whereas another cladistic study including also postcranial characters rather supports a close relationship between Chaetophractus and Zaedyus [START_REF] Abrantes | Proposta filogenética para os Dasyposidae (Mammalia: Cingulata)[END_REF]. Uncertainty remains in morphological evidence because of the highly derived morphology of Tolypeutes within Tolypeutinae, and the very similar morphologies exhibited by extant members of Euphractinae.

At present, the only armadillo subfamily for which no molecular data is available is

Chlamyphorinae (fairy armadillos). It includes only two living species classified in the genus

Chlamyphorus: Chlamyphorus truncatus (pink fairy armadillo) and Chlamyphorus retusus (chacoan or greater fairy armadillo). These two species are morphologically specialized to a subterranean lifestyle with notably reduced eyes and reinforced forearms supporting enlarged digging claws. Some cladistic analyses of morphological characters including fossils have supported a close relationship of fairy armadillos with Zaedyus [START_REF] Engelmann | The phylogeny of the Xenarthra[END_REF][START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF]. However, when only extant taxa were considered in an analysis of craniodental characters, Chlamyphorus grouped with Euphractinae [START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF], a result also obtained by [START_REF] Abrantes | Proposta filogenética para os Dasyposidae (Mammalia: Cingulata)[END_REF] in their morphological study. Yet, the phylogenetic affinities of fairy armadillos have never been tested with molecular data, as both species are fairly elusive and rare in the field [START_REF] Cuéllar | The tatujeikurajoyava (Chlamyphorus retusus) in the Izozog communities of the Bolivian Gran Chaco[END_REF][START_REF] Superina | New information on population declines in pink fairy armadillos[END_REF].

The genomic insertion patterns of various retroposed mobile elements provide an extraordinary archive from which to elucidate the evolutionary history of organisms [START_REF] Shedlock | SINE insertions: powerful tools for molecular systematics[END_REF]. SINEs (Short INterspersed Elements) and other retroposed elements, shared by some taxa but missing from the genomes of others, are highly valuable, essentially homoplasy-free indicators of relatedness [START_REF] Okada | SINEs: Short interspersed repeated elements of the eukaryotic genome[END_REF][START_REF] Nikaido | Phylogenetic relationships among cetartiodactyls based on insertions of short and long interpersed elements: hippopotamuses are the closest extant relatives of whales[END_REF][START_REF] Salem | Alu elements and hominid phylogenetics[END_REF][START_REF] Roos | Primate jumping genes elucidate strepsirrhine phylogeny[END_REF][START_REF] Kriegs | Retroposed elements as archives for the evolutionary history of placental mammals[END_REF][START_REF] Nishihara | Pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions[END_REF], with an extremely large possible number of basically unique character states [START_REF] Steel | Maximum parsimony and the phylogenetic information in multistate characters[END_REF]. A specific tRNA-derived retroposed SINE family, called DAS-SINEs, has been discovered in the Dasypus novemcinctus genome [START_REF] Borodulina | PCR-based approach to SINE isolation: simple and complex SINEs[END_REF][START_REF] Churakov | A novel abundant family of retroposed elements (DAS-SINEs) in the nine-banded armadillo (Dasypus novemcinctus)[END_REF], with average genomic copy numbers of about 467,000 calculated from the ~71% of available genomic information (EnsEMBL GeneBase version 45.1c). Using a specific strategy to compare nested DAS-SINEs, distinct historical waves of activity of six DAS-SINE subfamilies were defined [START_REF] Churakov | A novel abundant family of retroposed elements (DAS-SINEs) in the nine-banded armadillo (Dasypus novemcinctus)[END_REF]. This information was used for the first time in the present study to screen for a maximum variety of young -and respectively old -phylogenetically informative elements to provide the first retroposon presence/absence support for terminal -and respectively deep -splits in xenarthrans.

Furthermore, low complexity, rare genomic changes (RGCs), such as random insertions or deletions (indels) provide additional capability in molecular systematics [START_REF] Rokas | Rare genomic changes as a tool for phylogenetics[END_REF]. Although simple random indels are of lower reliability than complex retroposon insertions, they have often been shown to be evolutionarily conserved and phylogenetically informative [START_REF] Murphy | Using genomic data to unravel the root of the placental mammal phylogeny[END_REF]). Retroposed elements with their characteristic flanking target site duplications (direct repeats -DR), diagnostic mutations, and often specific truncations are exceptionally informative RGCs [START_REF] Ray | SINEs of a nearly perfect character[END_REF]) and, compared to simple random indels, are virtually ambiguity-free. In the present study, we combined different strategies to find phylogenetically informative retroposed elements and indels to serve as cladistic molecular markers for reconstructing the phylogeny of all xenarthran genera including the previously unsampled genus Chlamyphorus. Moreover, phylogenetic analyses of the flanking regions of these loci enabled us to reconstruct the evolutionary history of xenarthrans using a large data set of non-protein coding DNA. The resulting phylogenetic tree was used as a framework for mapping the presence/absence of retroposed elements and indels providing independent evidence for further resolving the phylogeny of this major placental clade.

Materials and Methods

Computational strategies

To identify phylogenetically informative presence/absence patterns of retroposed elements, we applied three different in silico search strategies.

Strategy 1:

We downloaded all available Dasypus novemcinctus sequences from the NIH Intramural Sequencing Center at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide (20.1 million bases) and used the local version of RepeatMasker (Smit and Green at http://www.repeatmasker.org) containing a specific library comprising all DAS-SINE subfamily consensus sequences to scan for DAS-SINEs. All detected DAS-SINEs were extracted along with 250 nucleotides of their flanking sequences (2023 loci). For outgroup comparison and to construct conserved PCR primers the selected loci were aligned against human genomic sequences using the UCSC Server (http://genome.ucsc.edu/cgi-bin/hgBlat). Only sequence alignments with more than 80% identity were used to generate conserved PCR primers (80 loci).

For estimating the genomic number of DAS-SINEs in Dasypus novemcinctus we downloaded available sequence information from http://www.ensembl.org/info/data/download.html (Dasypus_novemcinctus.0.ARMA.45.dna.nonchromosomal.fa.gz) and used our specific DAS-SINE library for a local RepeatMasker screen. We extrapolated the number of all detected elements to the expected full size of the genome. Strategy 3: To identify randomly inserted xenarthran-specific retroposed elements, we PCR amplified 171 highly conserved mammalian loci that were previously analyzed by [START_REF] Kriegs | Retroposed elements as archives for the evolutionary history of placental mammals[END_REF].

Statistical analyses

To test the validity of clade hypotheses at various nodes of the phylogenetic tree we used the method of [START_REF] Waddell | A phylogenetic foundation for comparative mammalian genomics[END_REF]. Assuming the sequence-based tree reconstruction as a prior hypothesis at any given node, a minimum of three integration sites against zero for alternative branching hypotheses are required for a significance level of p < 0.04.

Tissue sampling

We isolated genomic DNA from tissue samples conserved in 95% ethanol from the following species using standard protocols [START_REF] Sambrook | Molecular cloning: A laboratory manual[END_REF]: Folivora:

PCR amplification

We initiated our screening with high-throughput PCR for all selected 283 loci, using The raw concatenation of the 39 alignments yielded a combined dataset of 28,869 sites for the 16 species. For phylogenetic reconstructions from these flanking regions of retroposed elements, we adopted a conservative approach. First, regions containing the retroposed elements were entirely removed from the concatenation to make subsequent phylogenetic inferences fully independent of the retroposed insertions. Then, ambiguously aligned sites and highly gapped/repeated regions were excluded, reducing the dataset to 12,488 sites of which The homogeneity of base composition across taxa was evaluated by the χ 2 test implemented in PAUP* 4.0b10 [START_REF] Swofford | PAUP*. Phylogenetic analysis using parsimony (and other methods)[END_REF]. This test did not reject the null hypothesis of overall base composition homogeneity (P = 0.64) in this A+T rich (62%) non-protein coding dataset. ML phylogenetic analyses were conducted with PAUP* through heuristic searches with Tree Bisection Reconnection (TBR) branch swapping starting from a Neighbor-Joining (NJ) tree. ML model parameters were estimated with a successive approximation approach [START_REF] Sullivan | Evaluating the performance of a successive-approximations approach to parameter optimization in maximumlikelihood phylogeny estimation[END_REF]) using three rounds of optimization, starting from a NJ topology derived from ML distances obtained from a GTR+Γ 8 model (α = 0.5) and followed by ML heuristic searches by TBR branch swapping under the best fitting TVM+Γ 8 model.

Reliability of nodes was estimated by bootstrap resampling with 500 pseudo-replicate datasets generated by the program SeqBoot of the PHYLIP package [START_REF] Felsenstein | PHYLIP (PHYLogeny Inference Package)[END_REF]. The 500 corresponding ML heuristic searches were performed using PAUP* with the previously estimated ML parameters, NJ starting trees, and TBR branch swapping. They were parallelized on a Beowulf-class cluster supercomputer comprising 16 processor Weon 5160

Dual Core AMD Athlon computing nodes enabling simultaneous running of up to 64 processes. ML bootstrap percentages were obtained from the majority-rule consensus tree of the 500 bootstrap-generated trees using the latest version of TreeFinder [START_REF] Jobb | TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics[END_REF].

Bayesian phylogenetic analyses of the concatenated flanking sequence dataset were implemented using MrBayes 3.1.2 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF]. Two independent runs of four incrementally heated Metropolis-coupled Markov chains Monte Carlo (MCMCMC)

were launched from random starting points. We used Dirichlet priors for base frequencies

(1,1,1,1) and for GTR parameters (1,1,1,1,1) scaled to the G-T substitution rate, a Uniform (0.05,50.00) prior for the Γ distribution, and an Exponential (10.0) prior for branch lengths.

The four MCMCMC were simultaneously run for 1,000,000 generations using the program default priors as starting values for the GTR+Γ 8 model parameters. Parameters and trees were sampled every 100 generations and the convergence of MCMCMC was checked by monitoring values of the marginal likelihood and model parameters through generations.

Bayesian Posterior Probabilities (PP) were obtained from the 50% majority rule consensus of the 2 x 9,000 trees sampled at stationarity after removing the first 1,000 trees as the "burn-in" stage.

Results

From ∼7400 investigated loci, 283 were selected for high-throughput PCRs in representatives of all major xenarthran branches and an afrotherian outgroup (Materials and Methods). 244 loci did not contain xenarthran-specific RGCs or were not amplifiable in all representative lineages. For the remaining 39 informative loci, we extended PCR screening and sequencing of retroposed elements and their flanking regions including all available species (Materials and Methods). These 39 informative loci contained 35 phylogenetically informative retroposed element markers, 14 autapomorphic retroposon insertions, and 39 phylogenetically informative indels other than retroposons (fig. 1; Supplementary table 2, Supplementary Material online). For most of the diagnostic retroposed elements, including spliceosomal U snRNAs (retropseudogenes generated by a variant L1 retroposition mechanism [START_REF] Schmitz | A novel class of mammalian-specific tailless retropseudogenes[END_REF])), we also detected the short direct repeats (DR) and the singular target sites at orthologous positions in species devoid of the retroposons. Moreover, examination of the phylogenetically conserved random indels occurring in flanking regions was restricted to those with a minimum length of three nucleotides.

The ML tree reconstructed from the concatenated 12,488 sites of retroposon flanking sequences resulted in a well-resolved phylogeny (fig. 2). All but four nodes were supported by Bootstrap Probabilities (BPs) of 100 and PPs of 1.0. This ML topology strongly supported xenarthran monophyly and their division into the three major groups Folivora (sloths), Vermilingua (anteaters), and Cingulata (armadillos). Anteaters and sloths were firmly

Discussion

SINE activity in xenarthran genomes

Of the retroposed elements, 36 were Cingulata-specific DAS-SINEs. As our search strategies were based predominantly on available sequence information from D.

novemcinctus, most diagnostic insertions were found in the lineage leading to this species.

Diagnostic insertions on other xenarthran branches were found as additional insertions in the investigated loci. Unfortunately sufficient sequence data to enable similar searches are not yet available for other xenarthran species. This is expressed in the limited significant phylogenetic support from discrete retroposon data (fig. 2b). The generation of large-scale genomic sequence information (e.g., by BAC or Whole Genome Shotgun (WGS) libraries)

will provide a multidirectional source for additional lineage-specific informative loci in the future. The DAS-SINEs preferentially investigated in this study spread in characteristic waves of activity that correspond to the evolutionary scenario proposed by [START_REF] Churakov | A novel abundant family of retroposed elements (DAS-SINEs) in the nine-banded armadillo (Dasypus novemcinctus)[END_REF].

Simple monomeric DAS-SINEs like DAS-Ia were active in ancient armadillo lineages and complex elements like DAS-III3b were active in species of some terminal branches.

The armadillo-specific distribution of DAS-SINEs was confirmed by screening all available genomic sequences of C. hoffmanni using a DAS-SINE specific library (RepeatMasker). Not a single copy of DAS-like sequences was detected. Interestingly, we found no other lineage-specific SINE elements for other xenarthrans than those for Cingulata.

A more elaborated search (e.g., for SINE specific promoter boxes) will be necessary to fill this gap. However, we did find lineage-specific L1-like and L1MB2 elements in Cingulata and L1MC2 and L1MB8 elements in Pilosa.

Improved resolution of xenarthran phylogeny

Reconstructing phylogenetic relationships among living xenarthran genera has been the subject of previous molecular studies using a combination of three nuclear and two mitochondrial genes [START_REF] Delsuc | Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting[END_REF][START_REF] Delsuc | Molecular systematics of armadillos (Xenarthra, Dasypodidae): contribution of maximum likelihood and Bayesian analyses of mitochondrial and nuclear genes[END_REF]. These Chaetophractus and Euphractus supported by 5 synapomorphies [START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF], but it agrees with the relationships proposed by [START_REF] Abrantes | Proposta filogenética para os Dasyposidae (Mammalia: Cingulata)[END_REF] based on additional postcranial evidence.

Our results appeared less clear-cut for the relationships within Tolypeutinae, as no diagnostic retroposed element insertions or indel events were found (fig. 3). Sequence-based phylogenetic analyses, nevertheless, favor the monophyly of Cabassous and Tolypeutes to the exclusion of Priodontes (fig. 2a). This is also in contrast to the morphological evidence for grouping Cabassous with Priodontes [START_REF] Abrantes | Proposta filogenética para os Dasyposidae (Mammalia: Cingulata)[END_REF][START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF].

However, in this case it is possible that morphologically based cladistic reconstructions might have been confounded by the highly derived morphology of Tolypeutes relative to other members of Tolypeutinae. Obviously, more work is needed to understand the reasons of these discrepancies between molecular and morphological characters. The occurrence of two embedded deletions in the same region casts doubt on the potential phylogenetic significance of these deletion events that might be homoplasic. We also cannot fully exclude neither lineage sorting nor paralogy effects, but the probability of comparing paralogs is very low with respect to the highly conserved regions compared (this conservation encompasses xenarthrans and afrotherians up to human) and the definitively singular fragments in PCRs of all compared species. The contradiction presented by this random indel emphasizes the necessity to search for additional support by more reliable retroposon insertions using experimental lineage-specific search strategies as outlined in [START_REF] Endoh | Total DNA transcription in vitro: a procedure to detect highly repetitive and transcribable sequences with tRNA-like structures[END_REF] or by random sequencing and subsequent Zoo-PCR.

The inclusion of the second, morphologically divergent, species of fairy armadillo (C.

retusus) in future molecular phylogenetic analyses might help to confirm the position of Chlamyphorinae within the xenarthran tree by breaking up the branch leading to C. truncatus.

The results of the present study make Xenarthra the first major placental group to have its phylogenetic history fully resolved at the genus level by using multiple independent molecular and genomic evidences. These results provide a robust phylogenetic framework for future evolutionary studies of comparative genomics, taxonomy, cytology, and development in this mammalian order. It might also be useful for defining conservation priorities for its most endangered species (da Fonseca and Aguiar 2004).

Figure legends

Strategy 2 :

 2 For finding markers for other xenarthran branches in which DAS-SINEs were possibly not active (deep xenarthran splits), we also searched for other retroposed elements (i.e., Long INterspersed Elements (LINEs) or retroposed small nuclear (sn) RNAs such as spliceosomal U snRNAs) in conserved regions. We downloaded all available ftp.ncbi.nih.gov/pub/TraceDB/ (4.6 gigabases for D. novemcinctus and 4.2 gigabases for C. hoffmanni) and detected 5206 potentially phylogenetic informative loci with embedded L1MB4 (2609), L1MB5 (625), L1MB7 (1891) elements that were potentially active in xenarthrans[START_REF] Kriegs | Retroposed elements as archives for the evolutionary history of placental mammals[END_REF], as well as 81 snRNA retrogenes. These loci were screened using trace blast (http://www.ncbi.nlm.nih.gov/BLAST/mmtrace) in three different ways: (1) for the presence of retroposed elements in C. hoffmanni that were absent in D. novemcinctus in order to find potential integrations on the branch leading to sloths, (2) for the presence of elements in D. novemcinctus that were absent in C. hoffmanni in order to find potential integrations specific to Cingulata, and (3) for the presence of elements in both species in order to find potential markers indicating xenarthran monophyly. From the 5206 candidates, only 32 contained insertions meeting at least one of the above criteria. They were used to generate conserved PCR primers (12 L1MB4, four L1MB5, four L1MB7, and 12 spliceosomal RNAs).

  Bradypus tridactylus (pale-throated three-toed sloth) and Choloepus hoffmanni (Hoffmann's two-toed sloth); Vermilingua: Tamandua tetradactyla (southern tamandua), Myrmecophaga tridactyla (giant anteater), Cyclopes didactylus (silky anteater); Dasypodinae: Dasypus novemcinctus (nine-banded armadillo), Dasypus kappleri (greater long-nosed armadillo); Euphractinae: Euphractus sexcinctus (six-banded armadillo), Zaedyus pichiy (pichi), Chaetophractus vellerosus (screaming hairy armadillo); Chlamyphorinae: Chlamyphorus truncatus (pink fairy armadillo); Tolypeutinae: Tolypeutes tricinctus (three-banded armadillo), Cabassous unicinctus (southern naked-tailed armadillo), Priodontes maximus (giant armadillo); and Afrotheria (outgroup): Loxodonta africana (African savanna elephant) and Trichechus manatus (Caribbean manatee).

  grouped together into Pilosa (BP = 99; PP = 1.0). Within anteaters, Myrmecophaga and Tamandua were sister-groups (BP = 100; PP = 1.0) to the exclusion of the early divergingCyclopes.Within armadillos, phylogenetic analyses unambiguously supported (BP = 100; PP = 1.0) the respective monophyly of the three previously identified subfamilies: Dasypodinae, Euphractinae and Tolypeutinae. Among Euphractinae, the sister group of Zaedyus and Chaetophractus was strongly supported (BP = 99; PP = 1.0), whereas relationships were less clear within Tolypeutinae, where Tolypeutes and Cabassous are sister-groups (BP = 81; PP = 0.99). Finally, Chlamyphorus, representing the previously unsampled subfamily Chlamyphorinae, showed clear phylogenetic affinities with the clade Tolypeutinae (BP = 93; PP = 1.0).In the 39 informative presence/absence loci examined, we found independent support for ten internal branches of the ML tree (fig.3): [1] Xenarthra: Five independent insertions of retroposed elements (U6, U5-L1MB2-L1MB4-chimer, L1MB4, L1MB5, L1ME3A) confirm the monophyly of xenarthrans. The corresponding elements were clearly absent in Afrotherian outgroup species. [2] Pilosa: One LINE element (L1MC2) and two indels were present in all five representative species of Bradypodidae, Megalonychidae and Myrmecophagidae and were absent in all species of Dasypodidae, thus providing evidence for the monophyly of Pilosans. [3] Folivora: A diagnostic 27 nt deletion was detected in the Bradypodidae and Megalonychidae species that was not present in other Pilosa. [4] Myrmecophagidae (Vermilingua): We found one LINE element (L1MB8) and a 24 nt deletion in all three representatives of Myrmecophagidae. [5] Myrmecophaginae: Four indels present in Myrmecophaga and Tamandua indicate a common ancestry of these species to the exclusion of Cyclopes. [6] Dasypodidae (Cingulata): Seventeen diagnostic retroposons (10× DAS-Ia, 3× DAS-IIa, 2× DAS-IIb, U2, L1-like element) and three indels were found in the Dasypodidae that were absent in all pilosans. [7] Dasypodinae: Eight retroposons (3× DAS-Ia, 2× DAS-IIa, DAS-III1b, U2, L1MB2) and 16 indels were present in only the two investigated Dasypus species. [8] Euphractinae + Chlamyphorinae + Tolypeutinae: We detected one DAS-IIb element and three indels in the Euphractinae, Chlamyphorinae and Tolypeutinae species that were not present in Dasypodinae. [9] Euphractinae: One DAS-IIb insertion and seven indels were present only in the Euphractinae species. [10] Chaetophractus vellerosus + Zaedyus pichiy: A DAS-III3b element insertion and a 19 nt diagnostic deletion support the grouping of Chaetophractus and Zaedyus.

  studies, including all extant genera except Chlamyphorus, resulted in a comprehensive phylogenetic framework with only two remaining uncertainties within armadillos. The present study represents the first ever evidence from retroposed elements for resolving xenarthran phylogeny. Also, the concatenation of flanking sequences offered the first large-scale, nonprotein coding dataset from which the evolutionary history of all living xenarthran genera could be inferred. The results from the present work independently corroborated previous studies and provided strong additional support for most internal branches within Xenarthra including the areas of uncertainty. Indeed, previous molecular data failed to resolve relationships within the subfamilies Tolypeutinae and Euphractinae. Contradictory results were obtained from mitochondrial versus nuclear genes resulting in low statistical support values when analyzing their concatenation[START_REF] Delsuc | Molecular systematics of armadillos (Xenarthra, Dasypodidae): contribution of maximum likelihood and Bayesian analyses of mitochondrial and nuclear genes[END_REF]. These difficult-to-resolve trifurcations were interpreted as evidence for the occurrence of two radiation events possibly triggered by environmental changes[START_REF] Delsuc | Influence of Tertiary paleoenvironmental changes on the diversification of South American mammals: a relaxed molecular clock study within xenarthrans[END_REF]. Consensus network analyses of the phylogenetically conflicting signals present in the combination of two mitochondrial and three nuclear genes (Delsuc and Douzery in press) revealed that two of the three competing hypotheses coexisted with almost equal support within each subfamily: Chaetophractus + Euphractus versus Chaetophractus + Zaedyus within Euphractinae and Cabassous + Priodontes versus Cabassous + Tolypeutes within Tolypeutinae. Phylogenetic analyses of the more than 12 kb dataset of non-protein coding DNA provided unambiguous support for the grouping of the genera Chaetophractus and Zaedyus within Euphractinae (fig. 2a). Furthermore, C. vellerosus and Z. pichiy shared a diagnostic DAS-III3b element insertion and a 19 nucleotide-long deletion providing independent evidence for their direct common ancestry to the exclusion of E. sexcinctus (fig. 3) (Supplementary figure 1, Supplementary Material online). This result is in contradiction to a cladistic study of craniodental characters favoring a sister-group relationship between

  The present work provides the first molecular data to resolve the phylogenetic position of the armadillo subfamily Chlamyphorinae (fairy armadillos) within Cingulata. In contrast with cladistic analyses of morphological characters[START_REF] Abrantes | Proposta filogenética para os Dasyposidae (Mammalia: Cingulata)[END_REF][START_REF] Gaudin | The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis[END_REF], the probabilistic phylogenetic analyses of the flanking region dataset strongly supported a close relationship of C. truncatus with Tolypeutinae instead of Euphractinae (fig.2a). However, no diagnostic RGC was found to confirm this unexpected result (fig.3). Future extensive wet-bench approaches focusing on retroposons in these and other lineages will help to strengthen the sequence-based relationships and balance biased in silico methods that depend on available sequence sources. However, for the present work only very little tissue was available from the rare species. This constrained our analyses to preferentially in silico methods instead of the tissue intensive wet-bench approaches. The inclusion of the second, morphologically divergent, species of fairy armadillo (C. retusus) in future sequence-based phylogenetic analyses might also help to confirm the position of Chlamyphorinae within the xenarthran tree by breaking up the branch leading to C. truncatus. Of all the retroposed elements and indels identified, only one indel (#25') contradicted our ML tree. This 19 nt deletion was found in C. truncatus and C. unicinctus and not in other species. It is nevertheless worth noting that in this region two species of Dasypus also contained a 57 nt deletion encompassing the one shared by Chlamyphorus and Cabassous.

Fig. 1 Presence

 1 Fig. 1

Fig. 2

 2 Fig.2

Fig. 3

 3 Fig.3

Figure 3

 3 Figure 3

  5,530 were variable. Alignments are available upon request.Phylogenetic analyses were conducted using Maximum Likelihood (ML) and Bayesian approaches. The best fitting model of sequence evolution was selected using Modeltest 3.7[START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF]. Among 56 competing models, the Akaike

Information Criterion (AIC; Akaike 1974) identified the transversional model (TVM) with among-site rate heterogeneity modeled by a gamma distribution with eight discrete categories (+Γ 8 ) as the best fitting model for our concatenated dataset. The TVM model is a restriction of the most general, General Time Reversible (GTR) model. It allows different base compositions and different substitution rate probabilities for the four types of transversions, whereas the two types of transitions have the same rate.

Acknowledgments

We thank Pablo Beldomenico, Benoit de Thoisy, Martin S. Fischer, Jorgue Omar García, Eric Hansen, Rodney L. Honeycutt, Jan Ole Kriegs, Jean-François Mauffrey, Bernhard Neurohr, Rodolpho Rearte, Sylvia Rolle, Erica Taube, Jean-Christophe Vié, and Heike Weber for providing us with tissue samples. We are grateful to François Catzeflis for providing access to the Mammalian Tissue Collection of the Institut des Sciences de l'Evolution de Montpellier.

We also thank Marsha Bundman for editorial assistance. Two anonymous reviewers and the associated editor provided helpful suggestions on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SCHM 1469 to J.S. and J.B.). This is contribution ISEM 2007-XXX of the Institut des Sciences de l'Evolution de Montpellier (UMR5554-CNRS).

Supplementary Material

Supplementary tables 1 -2 and supplementary figure 1 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org).


domain: phys spi

text:
S Bourdarie

P Calvel

C Barillot

L Rey

T Parrinello

B Hoyos

Member, IEEE R Ecoffet

How Much Do Solar Cycle Variations Impact Long-Term Effect Predictions at LEO?

Keywords:

An eight-year long flight database from an error detection and correction (EDAC) counter implemented onboard an altimeter flying on CryoSat-2 spacecraft at 715-km altitude is analyzed to investigate the impact of solar cycle variations on long-term effect predictions. In situ observations are then compared to various specification models including legacy models as well as models under developments.

This article addresses this issue for trapped protons at Low Earth Orbit (LEO). To do so, flight data from error detection and correction (EDAC) counter collected over several years at LEO will be used. Then predictions obtained with AP8 min and max, AP9 Mean, and GREEN-p will be compared to flight data to deduce the most appropriate model to be used at LEO.

In Section II, flight data are presented as well as support data necessary to perform effect calculations, in Section III, model predictions will be given, and in Section IV, results will be discussed.

II. FLIGHT DATA

CryoSat-2 is a European Space Agency (ESA) satellite flying on an LEO (near circular orbit: 715-735 km, inclination: 92 • , 14.52 revolutions/day). It was launched on April 8, 2010. The evolution of perigee, apogee, and inclination from launch date to April 4, 2019, is given in Fig. 1. One can see that the orbit is almost the same over this time range, that is, the spacecraft will encounter the same environment along the mission life time.

In [START_REF] Bourdarie | Benchmarking ionizing space environment models[END_REF], single-event phenomena (bit flips) recorded by an EDAC onboard the SAR Interferometer Radar Altimeter (SIRAL) being built by Thales Alenia Space (TAS) were analyzed. These errors are automatically corrected, listed by the EDAC counter, and downloaded on ground station every month. The memory map of this payload is made of 10 static random access memory (SRAM) (1-MB SRAM M65608, 0.5-μm CMOS process, developed by ATMEL in cooperation with the ESA).

The total number of single-event upset (SEU) recorded by the EDAC from November 1, 2010, to March 31, 2019, is 6927. The longitude-latitude event distribution is given in Fig. 2. The number of events is sufficient to nicely image the South Atlantic Anomaly (SAA). Following [START_REF] Bourdarie | Benchmarking ionizing space environment models[END_REF], SEU events can be sorted out to identify those induced by trapped protons in the radiation belts (McIlwain L < 1.9) to those induced by galactic cosmic rays (GCRs) or solar energetic particles (SEPs) (McIlwain L > 1.9). As already reported in [START_REF] Bourdarie | Benchmarking ionizing space environment models[END_REF], most SEUs are recorded in the SAA (87.3%), while the SEUs at high and low latitudes can be attributed to GCRs and SEPs (12.7%). Note that since the time coverage is longer here than in [START_REF] Bourdarie | Benchmarking ionizing space environment models[END_REF], the percentages obtained here are slightly different (∼1%). The mean SEU rates then obtained are 1.88 and 0.28 SEU/day attributed to trapped protons and GCRs plus SEPs, respectively, over the full time period.

The time evolution of cumulated SEUs as well as the year average daily SEU rates attributed to trapped protons and GCRs plus SEP are plotted versus time in Fig. 3. The cumulated SEU values (top two panels) confirm that events are dominated by the trapped proton contribution. From the cumulative SEU values, it is then possible to compute year average daily SEU rates attributed to trapped protons (third panel from top) and GCR plus SEP (fourth panel from top). In both cases, the solar cycle modulation in SEU rates can be clearly seen. Note that such variations cannot be attributed to an orbit change as apogee and perigee only evolve by 5 km from 2011 to 2019 and the inclination remains unchanged. As expected, the SEU rate attributed to GCR plus SEP is the lowest at solar cycle maximum phase (April 2014) and is increasing by a factor of 2 from solar maximum (0.22 SEU/day in 2014) to almost solar minimum (0.48 SEU/day in 2019). The SEU rate attributed to trapped protons is decreasing slowly in time from year 2011 (2.1 SEU/day) to year 2015 (1.65 SEU/day), that is, all along solar maximum phase a 30% decrease is found. Then from year 2015 (1.65 SEU/day) to year 2019 (2.22 SEU/day), a slow increase in SEU daily rates is observed, that is, all along declining phase a 30% increase is found.

During SEP events, January 27-29, 2012, and March 7-11, 2012, a net increase (two events can be attributed to each SEP event) is found in cumulated SEU values attributed to GCR plus SEP, while no significant changes are visible in cumulated SEU values attributed to trapped protons (Fig. 4).

To find out how accurate trapped proton specification models are cumulated as well as SEU daily rates will be evaluated is explained in Section III. To calculate an accurate SEU rate of mass memory of the SIRAL payload, the 3-D shielding around the SRAM device and the device cross section versus proton energy must be well known.

The distribution of shielding thicknesses seen by the SRAM device onboard CryoSat-2/SIRAL was calculated by a sector analysis carried out by TAS Company using the FASTRAD software [6]. The solid angle viewed from SRAM M65608 is decomposed into 1800 sectors of equal value, that is, 30 steps in polar angles and 60 steps in azimuthal angles (see [START_REF] Bourdarie | Benchmarking ionizing space environment models[END_REF] for more details). The 3-D CAD model of the spacecraft is then simplified to the distribution of shielding in the 4π steradian obtained with FASTRAD.

The M65608 SRAM has been tested under heavy ions and proton beams by ESA. In particular, three test campaigns under proton beam were performed and are denoted by "ESA 1997" [START_REF] Poivey | Radiation characterisation of commercially available 1 Mbit/4 Mbit SRAMs for space applications[END_REF], "RADECS 2005" [START_REF] Novak | In flight SEU tests on the European SMART-1 spacecraft[END_REF], and "TRAD 2013" [START_REF] Vandevelde | Proton test report: Single event effects M65608E[END_REF]. The SEU cross section versus proton energy is given in Fig. 5 and can be fitted by a Weibull fit function. Two fits can be defined, one "high" which is pessimistic for prediction (the solid line in Fig. 5) and one "low" which is optimistic for prediction (the dashed line in Fig. 5), with the following parameters: W = 18, s = 1.5, an energy threshold of 15 MeV and a saturated cross section of 5.28•10 -8 cm 2 /device and W = 30, s = 1, an energy threshold of 15 MeV and a saturated cross section of 4.8•10 -8 cm 2 /device can be found, respectively.

To compute SEU cumulated values out of environment specification models, the same strategy is applied here as in [START_REF] Bourdarie | Benchmarking ionizing space environment models[END_REF].

III. SPECIFICATION MODEL PREDICTIONS

Although AP8 has been widely used to design spacecraft for several decades, alternatives like AP9 and GREEN-p are now I.

To compute the trapped proton environment encountered by CryoSat-2 spacecraft, its orbit parameters (altitude, latitude, and longitude) are computed with a 20-s time resolution from two line elements [START_REF] Celestrak | [END_REF] using SGP4 orbit propagator [START_REF] Celestrak | [END_REF] from launch date to April 1, 2019. Then the proton fluxes are estimated at all orbital locations from AP8 min with ESA interpolations, AP8 max with ESA interpolations, AP9 V1.30.001 Mean, AP9 V1.50.001 Mean, and GREEN-p. All calculations are performed using the Benchmark of Ionizing Space Environment tool [START_REF] Bourdarie | Benchmarking ionizing space environment models[END_REF].

A comparison of the mission average differential trapped proton spectrum (assumed to be isotropic at the spacecraft) and transmitted (at the chip level) deduced from AP8 min with ESA interpolation, AP8 max with ESA interpolation, AP9 Mean V1.30.001, AP9 Mean V1.50.001, and GREEN-p models are given in Figs. 6 and7, respectively. Note that the transmitted proton fluxes at the chip level are calculated from a MCNPx V2. Comparison of the cumulated SEUs measured by the EDAC onboard CryoSat-2/SIRAL and predicted from AP8 min with ESA interpolation, AP8 max with ESA interpolation, AP9 Mean V1.30.001, AP9 Mean V1.50.001, and GREEN-p models. Solid lines are obtained when the Weibull fit "high" is used, while the dashed lines are obtained when the Weibull fit "low" is considered.

highest ones, while AP8 max fluxes are the lowest ones, the differences being close to a factor 2.

A comparison of cumulated SEUs predicted from AP8 min with ESA interpolation, AP8 max with ESA interpolation, AP9 Mean V1.30.001, AP9 Mean V1.50.001, and GREEN-p is shown in Fig. 8. At the end of the time period under study, it is found that -AP8 min with ESA interpolation underestimates the cumulated SEUs by 12.2%-28.6% according to the Weibull fit "high" or "low" being considered, respectively; -AP8 max with ESA interpolation underestimates the cumulated SEUs by 29.5%-42.5% according to the Weibull fit "high" or "low" being considered, respectively; -AP9 Mean V1.50.001 underestimates the cumulated SEUs by 15.6%-30.7% according to the Weibull fit "high" or "low" being considered, respectively; -GREEN-p underestimates the cumulated SEUs by 4.7%-22.4% according to the Weibull fit "high" or "low" being considered, respectively; -AP9 Mean V1.30.001 overestimates the cumulated SEUs by 83.9%-52.7% according to the Weibull fit "high" or "low" being considered, respectively.

AP9 models offer other options to investigate the long-term space environment. In particular, the "perturbed mean" and "Monte-Carlo" modes allow defining the trapped particle flux for a given confidence level (CL). In this prospect, the AP9 V1.50.001 "perturbed mean" model has been used following the same strategy as for the other engineering models. The median, 75th percentile, and 95th percentile proton fluxes were computed out of 40 scenarios. Then the cumulated SEU events have been deduced using the Weibull fit "high" (Fig. 9). The following has been found. To analyze which energy range of the input transported spectrum at the cell level is the most relevant to predict the total SEU number in the SRAM, the cumulative percentage of total SEU is plotted versus energy in Fig. 10. Fifty percent of events are attributed to protons with energy greater than 112-150 MeV depending on the specification model and the Weibull fit being considered. Full details are provided Fig. 10. Cumulative percentage of total SEU versus energy predicted from specification models. Solid lines are obtained when the Weibull fit "high" is used, while the dashed lines are obtained when the Weibull fit "low" is considered.

TABLE II ENERGY [MeV] AT WHICH 50% OF CUMULATED EVENTS IS PREDICTED VERSUS THE SPECIFICATION AND THE WEIBULL FIT CONSIDERED

in Table II. Note that in all cases, 50% of events are attributed to protons with energies corresponding to the saturated device cross section. While AP8 solar min and solar max provide proton spectrum up to 300 MeV (i.e., 276 MeV at the sensitive cell), AP9 Mean V1.30.001, AP9 Mean V1.50.001, and GREEN-p indicate that 14.2%, 9.57%, and 12.3% of events are attributed to protons with incident energy greater than 300 MeV. It is also found that 99% of events are predicted if the spectrum is truncated at 500 MeV from AP9 Mean models and GREEN-p and for both Weibull fits. This is clearly indicating that a proton specification model with a spectrum defined up to 500 MeV is sufficient in the present case study.

To investigate which model reflects best the solar cycle modulation, daily SEU rates (averaged over each calendar year) can be deduced from those predictions. Results are given in Fig. 11 for each specification model. The solid lines are deduced when the Weibull fit "high" is considered, that is, it provides upper predicted values because the device cross section is pessimistic in this case and the dashed lines are computed using the Weibull fit "low," that is, it leads to lower predicted values because the device cross section is optimistic in this case. By definition, AP8 min and max do not vary along the solar cycle. Although there is an offset down in AP8 predictions, the amplitude between AP8 min and AP8 max is quite representative of what to expect between solar max and solar minimum phase. While AP9 V1.30.001 Mean and AP9 V1.50.001 do not include solar cycle variations, results exhibit a general trend: predicted SEU rates are declining along years. This feature cannot be attributed to an orbital effect as CryoSat-2 orbit remains unchanged throughout the time period under study. Predictions from the GREEN-p model track quite nicely in-flight data from 2011 to 2019. Nevertheless, a more or less similar trend as in AP9 predictions is suspected. In 2011, GREEN-p overestimates the SEU daily rates a little bit, while it underestimates them in 2019. This feature is suspected to be related to International Geophysical Reference Field (IGRF)-12 which is being used in AP9 and in GREEN-p. IGRF-12 is extrapolated after 2015-2020 and may not represent accurately the real Earth's internal magnetic field. Definitive IGRF coefficients to be used between 2015 and 2020 will be made available only during year 2020.

As shown before, SEU rate predictions obtained from AP8 solar min, AP8 solar max, AP9 Mean V1.50.001, and GREEN-p are closer to observations when the Weibull fit "high" is being used.

SEU measured onboard CryoSat-2/SIRAL payload and predicted by GREEN-p model is given in a longitude-latitude map for two years of the solar cycle in Fig. 12. The bin size in each plot is 4 • in longitude and latitude. In the flight data as well as in GREEN-p model prediction, it is found that the heart of the SAA (where events are more numerous) is more extended in 2011 than in 2015, that is, the SAA shrink in solar maximum phase.

To validate specification models in deep details, predicted SEU along all the period under study is plotted in a longitude-latitude map and compared to flight data (Fig. 13). The bin size in each plot is 2 • in longitude and latitude. The color plot provides the total number of events recorded (or predicted) in a bin from November 1, 2010, to March 31, 2019. AP9 Mean V1.50.001 and GREEN-p locate the SAA at the right place, while AP8 min locates the SAA too far eastward and southward. This is not a surprise and these results are attributed to the magnetic field model being implemented in the specification models. AP9 and GREEN-p are defined with a magnetic field model depending on the year of interest, while AP8 min is forced to use a magnetic field model from the 1970s. The shape of the mapping in the SAA obtained also differs somehow from model to model. Note that the overall shape predicted by AP9 Mean and GREEN-p looks very similar to each other and to the flight data.

Nevertheless, they exhibit differences in the shape of the heart of the SAA: GREEN-p is predicting isocontour more oval than AP9 Mean. This feature is also found in the flight data. Finally, the peak SEU predicted by GREEN-p is the closest to flight data (underestimating by 14.7%), while AP9 Mean V1.50.001 and AP8 min models underestimate the peak by 40.6% and 46.5%, respectively. To better illustrate this result, a vertical (-54 • < longitude < -52 • ) and horizontal cut (-24 • < latitude < -22 • ) in the longitude-latitude maps from Fig. 13 is given in Fig. 14 where the three specification model outputs are compared to flight data. Note that AP8 min prediction is well below flight data but this discrepancy is mainly due to the wrong SAA location found with AP8 min and does not reflect a peak-to-peak discrepancy.

A detailed look at each energy contribution to the number of SEUs over the full time period is given at two locations, one in the heart of the SAA and one at the outer edge of the SAA in Fig. 15. It turns out that GREEN-p predicts a higher contribution at all energies in the heart of the SAA and a lower one at the outer edge of the SAA than AP9 Mean V1.50.001. This is consistent with results shown in Figs. 6 and7.

IV. DISCUSSION

Solar cycle variations of trapped protons flux at low altitudes have been investigated by several authors [START_REF] Qin | Solar cycle variations of trapped proton flux in the inner radiation belt[END_REF]- [START_REF] Miyake | An empirical modeling of spatial distribution of trapped protons from solar cell degradation of the Akebono satellite[END_REF]. A common feature was found: the long-term trapped proton flux exhibits a modulation along solar cycle larger than predicted by the models and out of phase with the solar radio flux F10.7. A lag time on the order of several hundred days (depending on the proton energy) is also pointed out. Moreover, years after years, the SAA is drifting westward.

This study concentrates on trapped proton environment with energies greater than 40 MeV at 715-km altitude along the synchronous orbit of Sun for eight years. The long-term prediction of SEU events recorded by an EDAC implemented on a TAS's payload flying onboard CryoSat-2 spacecraft are within 30% to flight data (except for AP8 max) when AP8 min, AP9 Mean V1.50.001, or GREEN-p are considered. Note that all three specification models have been developed independently and by different groups. So, having all three long-term predictions close one to the other suggest that they should be very much representative to the long-term (multiyears) true state. On shorter time scales (one year), a 30% modulation along solar cycle 24 is found from CryoSat-2/SIRAL/EDAC data. Such a result reinforces previous results [START_REF] Qin | Solar cycle variations of trapped proton flux in the inner radiation belt[END_REF], [START_REF] Underwood | Long term trend in the LEO radiation environment as measured by the radiation monitors on-board three UoSAT-class micro-satellites[END_REF], [START_REF] Miyake | An empirical modeling of spatial distribution of trapped protons from solar cell degradation of the Akebono satellite[END_REF]. So far, among AP9 Mean V1.50.001, AP8 min, AP8 max, and GREEN-p, only GREEN-p model appears to be the most suitable proton specification model to reproduce the solar cycle modulation with a one-year time resolution seen in flight observations. Such a detailed knowledge of the space environment allows revisiting the margin policy that must be applied to design space missions. In particular, it allows considering margins attributed to specification model uncertainties well below the de facto factor 2 being often suggested at least at LEO. Accessing the solar cycle modulation of the space environment also allows to better access margins attributed to the environment for mission duration shorter than a solar cycle.

Fig. 1 .

 1 Fig. 1. CryoSat-2 orbital parameters out of two line elements versus time.

Fig. 2 .

 2 Fig. 2. SEU recorded by the EDAC implemented on the CryoSat-2/SIRAL payload from November 1, 2010, to March 31, 2019.

Fig. 3 .

 3 Fig. 3. (Top two panels) Cumulated SEUs as well as the (second and third panels from bottom) SEU rates attributed to trapped protons and GCRs plus SEP versus time. The F10.7 solar radio flux is given to illustrate the solar cycle evolution.

Fig. 4 .Fig. 5 .

 45 Fig. 4. (Bottom two panels) Cumulated SEUs attributed to trapped protons and GCRs plus SEP from January 15, 2012, to March 15, 2012, and SEPs measured by Geostationary Operational Environmental Satellite (GOES) spacecraft.

Fig. 6 .

 6 Fig. 6. Differential proton flux at CryoSat-2 deduced from AP8 min, AP9 Mean V1.30.001, AP9 Mean V1.50.001, and GREEN-p models.

  7.0 Monte-Carlo run accounting for the 3-D distribution of shielding surrounding the chip. For protons with energy greater than 40 MeV at the spacecraft or greater than 10 MeV at the chip, AP9 Mean V1.30.001 fluxes are the

Fig. 7 .

 7 Fig. 7. Transmitted differential proton flux at the CryoSat-2/SIRAL/ chip level deduced from AP8 min, AP9 Mean V1.30.001, AP9 Mean V1.50.001, and GREEN-p models.

Fig. 8 .

 8 Fig. 8.Comparison of the cumulated SEUs measured by the EDAC onboard CryoSat-2/SIRAL and predicted from AP8 min with ESA interpolation, AP8 max with ESA interpolation, AP9 Mean V1.30.001, AP9 Mean V1.50.001, and GREEN-p models. Solid lines are obtained when the Weibull fit "high" is used, while the dashed lines are obtained when the Weibull fit "low" is considered.

Fig. 9 .

 9 Fig. 9. Comparison of the cumulated SEUs measured by the EDAC onboard CryoSat-2/SIRAL and predicted from AP9 Perturbed Mean V1.50.001 median (CL = 50), 75th percentile (CL = 75), and 95th percentile (CL = 95). The Weibull fit "high" is used.

-

  AP9 V1.50.001 "Perturbed Mean" median provides exactly the same result as the one deduced from AP9 V1.50.001 Mean model. It underestimates the cumulated SEUs by 15.6%. -AP9 V1.50.001 "Perturbed Mean" 75th percentile overestimates the cumulated SEUs by 28.9%. -AP9 V1.50.001 "Perturbed Mean" 95th percentile overestimates the cumulated SEUs by 119.5%. -Flight data correspond to an CL comprises between 50 and 75 according to AP9 "Perturbed Mean" V1.50.001 model.

Fig. 11 .

 11 Fig. 11. (Bottom) Daily SEU rates from in-flight data and predicted with AP8 min with ESA interpolation, AP8 max with ESA interpolation, AP9 Mean V1.30.001, AP9 Mean V1.50.001, and GREEN-p. Solid lines are obtained when the Weibull fit "high" is used, while the dashed lines are obtained when the Weibull fit "low" is considered. (Top) Solar radio flux at 10.7 cm, F10.7.

Fig. 12 .

 12 Fig. 12. SEU measured (left) onboard CryoSat-2/SIRAL payload and (right) predicted by GREEN-p model in a longitude-latitude map for two years (2011 early rising phase of the solar cycle and 2015 one year after solar maximum phase).

Fig. 13 .

 13 Fig. 13. SEU onboard the CryoSat-2/SIRAL payload from November 1, 2010, to March 31, 2019, in a longitude-latitude map, measured in situ (flight data on top panel), predicted from specification models using the Weibull fit "high" (second from top: GREEN-p, third from top: AP9 Mean V1.50.001, and bottom: AP8 min).

Fig. 14 .

 14 Fig. 14. (Top) SEU latitudinal profile at -53 • longitude and (bottom) SEU longitudinal profile at -23 • from flight data and specification model predictions.

Fig. 15 .

 15 Fig. 15. Number of events per MeV predicted by AP9 Mean V1.50.001 and GREEN-p at two locations (one in the heart of the SAA: top two curves; and one at the outer edge of the SAA: bottom two curves).

TABLE I MAIN

 I CHARACTERISTICS OF TRAPPED PROTON SPECIFICATION MODELS

ACKNOWLEDGMENT

The authors would like to thank the CNES R&T program for their full support to this activity.

This work was supported in part by the Centre National d'Etudes Spatiales Research and Technology (CNES R&T) Program (R-S17/MT-0003-180) under Grant 4500055131/DIA094. S. Bourdarie is with the ONERA-The French Aerospace Lab/