Mitogenomic architecture of the multivalent endemic black clam (Villorita cyprinoides) and its phylogenetic implications

The Indian black clam Villorita cyprinoides (Family: Cyrenidae), an extractive commercially exploited species with aquaculture importance contributing more than 70% of clam fishery in India, is endemic to the Indian peninsula. Currently, there is very sparse information, especially on the molecular data of Villorita. The present study aims to provide a comprehensive knowledge of mitogenome architecture and assess the phylogenetic status of Cyrenidae. This has resulted in reporting the first complete mitogenome of V. cyprinoides using next-generation sequencing technology. The A+T circular mitogenome was 15,880 bp long, exhibiting 13 protein-coding genes (PCGs) including ATP8 (absent in several bivalves), 22 transfer RNA, and two ribosomal RNA genes residing in the heavy strand in a clockwise orientation and a gene order akin to Corbicula fluminea. The molecular phylogeny inferred from a concatenated multi-gene sequence [14 mitochondrial (12 PCGs, rrnS and rrnL) and two nuclear genes (Histone H3, 18S rRNA)] from 47 representative species of superorder Imparidentia, clustered V. cyprinoides and Cyrenid clams to a single clade supporting the monophyly of Cyrenidae. The subsequent mitochondrial gene order analysis substantiates the close relationship of V. cyprinoides and C. fluminea, analogous to phylogenetic output. The multilocus tree topology calibrated with verified fossil data deciphered the origin and diversification of Cyrenid clams during late Triassic-early Jurassic. The data derived from this study shall contribute remarkably for further insights on cryptic species identification, molecular characterization of bivalve mitogenomes and mitochondrial evolutionary history of genus Villorita. Moreover, complete mitogenome can aid in potential marker development for assessing the genetic health of black clam populations.

In 2018, global molluscan aquaculture production was 17.7 million tons 1 , elucidating its astounding potential as a food resource of international importance. The practice of inclusion of bivalves in the diet has had an exquisite role in human ethos for ages 2,3 . The dependence on bivalves for food, industries including jewellery, medical, and ecosystem services challenged the scientific community to enrich the information on various aspects including phylogeny, evolutionary history, genetic improvement and breeding of superior strains for enhancing aquaculture production [4][5][6] .
Mitochondrial DNA (mt-DNA), the lone double-stranded circular extranuclear genome in eukaryotes prevailed by endosymbiosis, is of crucial importance because of its ATP synthesis in the cell through oxidative phosphorylation 7,8 . Besides energy production, small size, high copy number, conserved genes, high rate of evolution, low recombination and maternal inheritance enables mitochondria as a potential marker in molecular studies 8,9 . Bivalve mitogenome as metazoans typically consists of 13 protein-coding genes for OXPHOS, 2 rRNA, 22 tRNAs and an A+T rich region controlling replication and transcription 10 . However, peculiar mitochondrial features like extensive genome reorganisation and occurrence of Doubly Uniparental Inheritance (DUI) in certain bivalves extended supplementary information on evolutionary patterns across species and among conspecifics [11][12][13] . The popularity of mitogenome as a marker of choice for phylogenetic studies is on arise due www.nature.com/scientificreports/ barrages results in restricted gene flow within conspecifics 29 . The detrimental effect of the decline in black clam population in the southern side of the Vembanad Lake affecting the livelihood of people depending on clam fishery urged the Govt. of Kerala to take immediate actions for rehabilitation through clam relaying (G.O(Rt) No. 460/16/F&PD of Fisheries and Ports (C) Department, Thiruvananthapuram, dated 01.08. 16) to replenish the stock so as to ensure a sustainable supply of food, as implemented in many developed countries 30,31 . Also, the dredging for the sub-fossil deposit of clamshell for industrial use 32 , pollution due to effluents from shrimp processing plants, eco-tourism, urban and agricultural runoff and infestation of the invasive Eichhornia crassipes engender threats to the clam 33 in its natural habitat. The economic and ecological eminence of Villorita cyprinoides results in continuous demand leading to overexploitation of the species including juveniles. Extensive harvesting of clam from the wild, niche alterations and exploitation of juveniles could be a potential threat to the existence of the species 34 . The selective pressure applied in stock by overfishing cannot be reversed through genetic selection on phenotypic traits 35 . In-depth studies on the exploitation of juvenile black clams suggested implementation of Minimum Legal Size (MLS) in clam fishery to reduce economic loss and preserve genetic variance 36 . The ecosystem degradation as well as overexploitation, if continued at the present level, may result in the reduction of effective population size leading to increased homozygosity, reduction in fitness due to faster erosion in genetic variation of related traits and ultimate genetic drift in far or near future. Along with genetic information, the need for breeding standardisation and proper farming technique is inevitable for a sustainable black clam fishery.
Villorita cyprinoides, a resource of paramount importance in meeting the country's need for a cheap and balanced diet, has received less attention at the genetic level. The present status of black clam fishery demands genetic investigation and analysis for the proper implication of management and conservation. The present work is a genuine attempt to determine the mitogenome architecture of V. cyprinoides thereby delivering ample molecular data for genus Villorita. The multi-gene phylogeny from mitochondrial and nuclear genes was attempted to allocate V. cyprinoides in the phylogenetic network and resolving the phylogenetic status of Cyrenidae. Additionally, mitochondrial gene order (GO) analysis and estimation of divergence time were also inferred using multiple genes and fossil data.

Results and discussion
Mitogenome characteristics. The female mt-genome of Villorita cyprinoides is 15,880 bp in length, comparable to previously reported bivalve mitogenomes. Gene content of V. cyprinoides displayed 37 typical sets of genes found in metazoan mitogenome 10 constituting 13 protein-coding genes (PCGs); encoding three electron transport chain (ETC) complexes (nd1-nd6, nd4l, cytb and cox1-cox3) and F 0 F 1 ATP synthase complex (ATP6 and ATP8) in OXPHOS. Besides PCGs two ribosomal RNA (rRNA) and 22 transfer RNA (tRNA) genes were observed with the putative control region (CR). All the 37 genes are located on major (H-strand) strand with forward orientation like in other Heterodont bivalves 37 . Figure 2 plots the circular map of black clam mitogenome.
The base composition of the mt-genome holds A = 25.1%, T = 42.9%, G = 22.8%, C = 9.2% with A+T content representing 68% of the total genome. A positive GC skew (0.424) and negative AT skew (− 0.262) were noticed indicating a bias towards Ts and Gs. Comprehensive analysis of mitogenomes, PCGs and rRNAs amongst 47 species from orders Veneroida, Myida, and Cardiida disclosed rich A+T content and low C content along with positive GC and negative AT skew (Supplementary Table S1).
Most of the PCGs employ ATN (ATG, ATA and ATT) as an initiation codon. In addition to the classical metazoan start codon, GTG (Valine) initiated translation [38][39][40] of ATP6 and nad2 gene in Cyrenid clams 41 including V. cyprinoides. Ten PCGs were terminated by TAG, and TAA codons whilst remaining were terminated by truncated codons T-and TA-which presumably functions as complete termination codon after post-transcriptional polyadenylation 42,43 (Table 2).
ATP8 gene, which lacks in most of the heterodont bivalves was identified in V. cyprinoides, encoding a protein of 37 amino acids. No putative conserved domains were detected by pBLAST, whereas conserved amino acid block (WW**KR*Y*F) were identified as in certain species in family Veneridae 44 and Cardiidae 45 (Supplementary Figure S2).
Following the Relative Synonymous Codon Usage (RSCU) value of V. cyprinoides, all the 62 codons (excluding stop codon, TAA and TAG) were utilised in unusual frequencies (Table 3). In V. cyprinoides third codon position (75.1) contains high A+T content compared to first (62.6) and second (64.6) position. Bias towards A+T in the third codon position might be interconnected with degeneracy 46 or natural selection on Wobble nucleotide 47 . Codon usage bias indicates TTT (Phe) is the most likely used codon after GTT (Val) whereas, CTC (Leu), CAC (His), TGC (Cys) were least used. It is evident that frequently used codons are A+T rich; while the lowest used were abundant in G+C content. The commonly used amino acid is Leucine than Phenylalanine, while Glutamine is less used.  AGN) and Leucine (UUR, CUN) genes. All tRNAs can be folded to typical clover-leaf secondary structure except for trnS genes. The secondary structure of 22 predicted tRNA genes were summarised in Fig. 3. The cloverleaf structure of tRNAs consists of 6-8 nucleotide pairs in aminoacyl acceptor stem, 5-6 pairs in anticodon and 4-6 pairs in DHU (dihydrouridine) and TψC stem with mismatched Watson-Crick base pairing. Anticodon of all tRNA genes was succeeded by Uracil and preceded by Guanine or Adenine in the anticodon loop of V. cyprinoides. The DHU stem of least conserved trnS1 and trnS2 genes 48 were replaced by a large DHU loop, an accepted feature in molluscs 44,49 . DHU stem of trnY gene is reduced to one nucleotide pair.

Ribosomal and transfer
BLAST searches confirmed MITOS annotated ribosomal RNA genes, rrnL and rrnS in V. cyprionoides with length 1183 bp and 861 bp respectively (12.8% of mitogenome). Large ribosomal RNA, considered as the largest rRNA locus (rrnL) in bivalves 50 flanked between the genes cytb and ATP8 as in genus Corbicula, Calyptogena, Cyclina, Dosinia and Meretrix (except Meretrix meretrix). Also, the position of small ribosomal RNA (rrnS), between trnT and trnM gene was consistent with genus Corbicula, Calyptogena, Cyclina, and Dosinia (Fig. 2). The exact boundaries of rRNA genes can only be determined by transcript mapping. Therefore, the indefinite length of rrnS and rrnL were resolved by comparison of their sequence similarity with species in family Cyrenidae and Veneridae.

Figure 2.
Mitochondrial genome organisation of Villorita cyprinoides visualised with DNA Plotter. The outer black circle represents the complete mitogenome of V. cyprinoides (scale denoted in base pair). The second ring represents mitochondrial proteins encoding genes, and the third circle denotes both rRNAs and tRNAs wherein all genes predicted in a positive frame. Colour in the box codes for specific categories of mitochondrial genes that are abbreviated according to standard usage. Fourth circle represents the distribution of GC % following yellow colour as below average and green colour, above-average values. The inner-circle represents the distribution of GC skew %, displaying blue colour as below average and red colour, above-average values.
Gene rearrangement and synteny. Gene rearrangement, accepted as a marker of common ancestry 52 , varies unpredictably owing to single strand gene distribution in bivalves 50 . The gene rearrangement explained through TDRL (Tandem Duplication Random Loss) model is believed to transpire in bivalves as in vertebrates 53 . Though overall gene order is not well conserved within bivalve superfamilies, gene arrangement is substantially similar after excluding tRNA genes. Conversely, gene arrangements in Imparidentian bivalve families are nearly identical except for tRNA duplication, and translocation of genes, but variable between families. The conserved gene order or synteny blocks displayed in superfamilies are shown in Fig. 4.
The mitogenome order of bivalves in genus level alters dramatically 54 albeit, comparative mt-genome analysis of Imparidentian clams revealed conserved gene order within the genus level. V. cyprinoides presented highly conserved synteny block with Corbicula fluminea for all 37 genes, but variable in gene length. Alternatively, species belonging to family Vesicomyidae (Archivesica sp. and Calyptogena magnifica) displays exact gene order as V. cyprinoides after removing tRNA genes though they represent a different family. These results reflect Vesicomid clams are related to family Cyrenidae than other families. Also, V. cyprinoides and Corbicula fluminea may be evolved recently and are closely related since gene rearrangement in closely related species is less than in distant 55 . The gene orders conserved in Cyrenid clams were identified as R-cytb-rrnL-ATP8-nad4, ATP6-nad3, and L-nad1-L-V. Species within genus Donax, Solen, Dosinia, shared identical gene order with less rearrangement like Tridacna, Paphia, and Lutraria (purging ATP8). The gene order within Cardioidea is highly rearranged and least conserved compared to other superfamilies.
Phylogenetic analysis. Amplification of Histone H3 and small ribosomal rRNA (18S rRNA) nuclear genes resulted in a generation of 350 bp and 1725 bp amplicons respectively. The phylogenetic analysis was assessed after the exclusion of ambiguous loci using Gblock upon alignment. DNA sequences of mitochondrial (12 PCGs except for ATP8, rrnS and rrnL) and nuclear protein-coding (Histone H3), and non-coding genes (18S rDNA) after Gblock analysis were concatenated bestowing 11,767 base sequences for further phylogenetic reconstruction. The best partitioning scheme and best-fit substitution models were generated for concatenated multi-gene data providing 17 best subset partitions (Supplementary Table S4). Phylogenetic estimation incorporating the subset partitions and evolutionary models for both Maximum likelihood (ML) and Bayesian Inference (BI) analysis generated a well-resolved tree with similar topology. All major clades were well supported with poste- Table 1. Base composition, AT skew, and GC skew of the whole mt-genome, PCGs, rRNA, and tRNA genes of Villorita cyprinoides.   Fig. 5. The phylogeny of Venerida, the most debated order in monophyletic subclass Heterodonta, is still undergoing extensive revision [56][57][58][59] . The present study considered 47 species, including 30 Venerida, 16 Cardiida, and one Myida confirmed the monophyly of the Venerid family, Cyrenidae 60 . Family Cyrenidae (Superfamily: Cyrenoidea) form a well-supported clade corroborating monophyly, placing G. coaxans towards the stem, and V. cyprinoides and C. fluminea to its crown. Cyrenidae co-clusters with Arcticoidae establishing Arcticoidae as immediate sister taxon, whereas Vesicomyidae (Glossoidea) lineage form a separate clade.
D. polymorpha (family: Dreissenidae), freshwater bivalve belonging to order Myida, found nested within Venerid taxa, questioning the phylogenetic status of the orders, and subdivision of Henterodonta to Venerida and Myida 61 . In contrast, this result substantiates the separate radiation of Cyrenidae and Dreissenidae to the freshwater lineage from marine ancestors 18 . The monophyletic status of Mactridae (Superfamily: Mactroidea) was confirmed, which appeared as a sister group to Dreissenidae 62 . Mactridae clustered with major clade [(Veneridae + Vesicomyidae) + (Cyrenidae + Arcticoidae)].
Phylogenetic analysis of Veneridae, the most diverse and economically important taxon in Venerida recovered the monophyletic status 63 forming Vesicomyidae as a sister group. Although phylogenetic analysis suggests Vesicomyidae as a sister group, Veneridae shares close kin with Cyrenidae and Arcticoidae. The close relation of Veneridae with Vesicomyidae, Arcticoidae and Cyrenidae was supported by the presence of hinge characters, a morphological synapomorphy 64 . The major clade of Veneridae split separately, forming two distinct subclades  65 . Furthermore, Pharidae and Solenidae supported as the sister group to each other, converged to form a wellresolved clade pointing to the monophyly of superfamily Solenoidea as expected 66 .
Being sister taxon to each other, Tellinoidea and Cardioidea validities the monophyly of order Cardiida. Five Tellinoidean families aggregated to a single clade with high PP and bootstrap value indicating monophyly of Tellinoidea but failed to resolve familial relationship 62 . However, species from Donacidae clustered to a branch, forming a sister group with other Tellinoid families 9 . Conversely, the monophyletic status of Carridae and Tridacnidae were evident in our phylogenetic analysis 67 , wherein Cardioidea itself is monophyletic.
The monophyletic status of Cardiida and Solenoidea were well supported, but we could not ratify the exact phylogenetic status of Tellinoidean families. Like Cardioidea and Solenoidea, the monophyly of Mactridae was validated, but recent studies support paraphyletic evolution 66 . This disparity might be due to limited taxon sampling because of less available mitogenome and nuclear gene data, exemplifying the importance of using multiple genes in phylogenetics.

Divergence time estimation.
The multi-gene tree topology after phylogenetic analysis were employed for molecular dating. Evolutionary ages and divergence were estimated through calibration of the molecular clock using multiple fossil data ( Table 4). The time-calibrated phylogenetic tree is shown in Fig. 6.
The splitting of vesicomyid clams is dated near to the early Cenozoic era i.e., 47 mya (HPD: 14-102 mya) corroborating previous investigation 69 . Also, the evolutionary radiation of Imparidentian bivalves is found to be in the Mesozoic Era in tune with preceding researches 57,58 . The evolutionary radiations of major superfamilies

conclusion
Even though enormous mitogenomes of clams have been characterised, there is dearth in molecular data for economically and ecologically significant clam, Villorita cyprinoides. The present study determined the first complete mitochondrial genome of multivalent endemic black clam, Villorita cyprinoides, which can be considered as keystone species that can perform top-down and bottom-up control 70 . Further, characterisation of mitogenome of V. cyprinoides resembles typical metazoan mitogenome architecture including the ATP8 gene, which is absent in several heterodont bivalves. Besides, conserved gene order (GO) analysis states the similarity of V. cyprinoides to C. fluminea, which is acceptable with detected phylogeny, revealing that both are closely associated and only recently diverged from an immediate common ancestor. The comparative GO analysis also revealed the conserved gene order of protein-coding and rRNA genes of V. cyprinoides and Vesicomid clams like Archivesica sp. and C. magnifica. The present phylogenetic analysis using both mitochondrial and nuclear genes resolved the monophyletic status of family Cyrenidae confirming a sister relationship with the Arcticoid family. This study also estimated the diversification of Cyrenid clams during the early Jurassic-Cretaceous period. Resolving phylogenetic uncertainty and estimation of divergence time in family Cyrenidae is important, as it is a potential taxon to trace bivalve radiation to the inland water system because it retains both brackish water and freshwater genera 18 . Though we verified monophyly of Cyrenidae and enriched the transparency of phylogenetic status, integrated investigation of other Cyrenid clams with dense species sampling is required to conserve our conclusion and to provide evolutionary divergence among and between taxon. www.nature.com/scientificreports/ Mitogenome sequences can be utilised for comparative phylogenomic studies within family Cyrenidae and development of genetic markers for identifying cryptic species if any. Apart from this, the genetic markers developed from this genome sequence can be used to study the population structure of V. cyprinoides since structure analysis is mandatory before the effective implementation of clam relaying programmes 71 . Even though no report on family Cyrenidae for the presence of an alternative mitochondrial inheritance pattern, Doubly Uniparental Inheritance (DUI) 72 , needs to be determined in the genus Villorita. At present, a reliable fishery-dependant data is available for stock assessment of black clam 18 and the mitogenome characterised here has scope to provide genetic markers to have fishery-independent estimates of stock biomass. The findings shall provide a better base to evaluate if stock biomasses are at sustainable levels.    www.nature.com/scientificreports/ Mt-DNA isolation, library preparation, and sequencing. Mt-DNA was isolated and validated with mt-DNA specific genes for qualitative analysis. The concentration of the mt-DNA was measured using Qubit Fluorometer (Life Technologies, USA).

Methods
The paired-end sequencing library was prepared using QlAseq FX DNA Library Kit. 100 ng DNA was enzymatically sheared into smaller fragments. DNA fragments with an average length of 700 bp were end-repaired, 3′ adenylated and ligated to Illumina adapters. Adapter-ligated libraries were PCR amplified, quantification and size evaluation of the library was performed in Bioanalyzer 2100 using High Sensitivity DNA chip (Agilent Technologies) and sequenced on Illumina sequencing platform (Illumina HiSeq 2500, USA).
Mitogenome assembly. Raw sequences generated after sequencing (2 × 150 PE) were retrieved in the FASTQ format. The quality of the reads was evaluated using FastQC software 79 . Illumina adapters, sequences less than 60 bp and low-quality sequences (Phred scores < 30) were removed using Trimmomatic 80 leaving 17 million (PE) high quality reads. High-quality reads were subjected to De novo assembly using CLC workbench 6 (Qiagen, Germany) with a read map back algorithm. Similarity search of high coverage scaffolds (> 15kbps) was carried out against NCBI's Nucleotide (NT) database using the BLASTN 81 algorithm.
Sequence annotation and analysis. The scaffold identified as mitogenome of Villorita cyprinoides was annotated using MITOS2 82 web server substituting translation table 5. Besides MITOS2, Protein-Coding Genes (PCGs) were identified using the online ORF Finder Tool (https ://www.ncbi.nlm.nih.gov/orffi nder/). Ribosomal subunit (rrnS and rrnL) genes were confirmed using NCBI BLAST (https ://www.nchi.nlm.nih.gov/BLAST / ) searches. Validation of tRNA gene, structure prediction, and visualisation was accomplished with tRNAScan-SE v2.0 83 and ARWEN v1.2 84 under invertebrate mitochondrial settings. The cloverleaf structures of predicted tRNA genes were drawn using PseudoViewer v.3.0 (https ://pseud oview er.inha.ac.kr/) 85 by sequence and structure input format and off numbering draw option. Control Region (CR) was annotated using DNA folding form from the Mfold web server (https ://unafo ld.rna.alban y.edu/?q=mfold /DNA-Foldi ng-Form) 86 retaining all the options as default except folding temperature (20 °C) and exterior loop-type (flat). Boundaries of MITOS2 annotated genes were refined based on the above software results using MEGA 7 87 . DNAPlotter 88 was used to generate a circular visual overview of annotated mitogenome. The curated mitogenome sequence (Accession No: MK481950) was submitted to the NCBI database through the BankIt sequence submission tool (https ://www. ncbi.nlm.nih.gov/BankI t/). Conserved gene order analyses of mt-genomes in superfamily level were conducted in superorder Imparidentia.
Nucleotide composition statistics of PCGs excluding stop codon and mitochondrial ribosomal RNAs were calculated separately by Geneious v11.1.5 89  cyprinoides concerning superorder Imparidentia was appraised. For better-resolved molecular phylogeny, 47 bivalve mitogenomes belonging to 15 families from superorder Imparidentia and their available nuclear (18S rRNA and Histone H3) genes from GenBank database were used (Supplementary Table S5). The nucleotide sequence of all the 12 protein-coding genes excluding stop codons was used whereas the ATP8 gene was omitted from analysis owing to its lack in most of the bivalves. Besides coding genes, sequences of the small and large ribosomal subunit of mitogenome coupled with nuclear small ribosomal subunit and Histone H3 were incorporated. Seven species belonging to class Polyplacophora, Gastropoda and from order Pectenoida served as outgroups.
The amino acid sequence of 12 protein-coding genes and nucleotide sequences of rrnL, rrnS and nuclear 18S rRNA, Histone H3 genes were separately aligned using Clustal W 92 implemented in MEGA7 with a default setting for PCGs. The aligned amino acid sequences of protein-coding genes were manually corrected and backtranslated to nucleotide sequences. On the contrary, gap opening and extension costs for ribosomal gene alignments were set to 20/4 due to the high sequence variability in analysed taxa. After alignment, Gblocks v0.91b 93 under default condition, changing the type of sequence, t = dna, identified and eliminated the ambiguous loci from mitochondrial and nuclear (18S) ribosomal RNA gene.
Appropriate subset partitions and evolutionary models for genes (Supplementary Table S4) were selected by PartitionFinder v.1.1.1 94 under the greedy algorithm, unlinked branch length, and Bayesian information criterion (BIC) parameters. Protein coding genes and non-coding genes are considered as separate partitions. The best-fit model for partitions was chosen to perform Maximum likelihood (ML) and Bayesian Inference (BI). Phylogenetic analysis using the Maximum likelihood method was inferred using IQ-tree with 1,000 ultrafast bootstrapping replicates 95 . Bayesian analysis was implemented using MrBayes v.3.2 96 . The programme was run for 2 × 10 7 generations comprising two simultaneous analysis with four chains, sampling every 100 generations. The average standard deviation of split frequency beneath 0.01 was considered as the stationarity phase, which is evaluated in Tracer v.1.6 (https ://tree.bio.ed.ac.uk/softw are/trace r/), and the 50% majority rule summarised trees after discarding the first 25% as burn-in. Remaining trees were used to construct a consensus tree, edited and visualised in FigTree v.1.4.3 (https ://tree.bio.ed.ac.uk/softw are/figtr ee/). Estimation of divergence time between species and clade were determined using Bayesian relaxed clock models and MCMC algorithms 97,98 implemented in the mcmctree programme of PAML software v4.9 99 . While the rooted tree from the previous analysis was used to calibrate evolutionary rates by incorporating multiple fossil ages spanning from the earliest bivalve fossil to fossil from family Cyrenidae ( www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.