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The gene arrangement and phylogeny using mitochondrial genomes in spiders (Arachnida: Araneae)
Journal Pre-proof The gene arrangement and phylogeny using mitochondrial genomes in spiders (Arachnida: Araneae)
Kaomud Tyagi, Vikas Kumar, Nikita Poddar, Priya Prasad, Inderjeet Tyagi, Shantanu Kundu, Kailash Chandra PII:
S0141-8130(19)40035-4
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.01.014
Reference:
BIOMAC 14318
To appear in:
International Journal of Biological Macromolecules
Received date:
6 December 2019
Revised date:
1 January 2020
Accepted date:
3 January 2020
Please cite this article as: K. Tyagi, V. Kumar, N. Poddar, et al., The gene arrangement and phylogeny using mitochondrial genomes in spiders (Arachnida: Araneae), International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/ j.ijbiomac.2020.01.014
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© 2020 Published by Elsevier.
Journal Pre-proof The gene arrangement and phylogeny using mitochondrial genomes in spiders (Arachnida: Araneae) Kaomud Tyagi, Vikas Kumar*, Nikita Poddar, Priya Prasad, Inderjeet Tyagi, Shantanu Kundu, Kailash Chandra Centre for DNA Taxonomy, Molecular Systematics Division, Zoological Survey of India, Kolkata, India
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Corresponding author email id: [email protected]
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Abstract
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The complete mitochondrial genome (mitogenome) of Cheiracanthium triviale was sequenced
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for the first time. The 14,590 bp C. triviale mitogenome contained 37 genes (13 protein coding
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genes, 2 ribosomal RNAs, 22 transfer RNAs) and one control region. The mitogenome of Dysdera silvatica which was available at NCBI GenBank was annotated. The mitogenome of C.
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triviale was compared with 43 previously sequenced spider species to observe the gene
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arrangements, control region and phylogeny. TreeREx analysis identified 19 mitochondrial gene
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rearrangements (11 transposition, 6 inversion, 2 inverse transposition) in spiders as compared with the putative ancestral gene order and lead to form new gene boundaries: trnQ-trnA, trnAtrnM for Loxosceles similis; nad3-trnS1, trnE-trnL2, trnL2-trnA, trnN-trnF for Agelena silvatica; trnN-trnE, trnE-trnA, trnR-trnF, nad4L-trnW, trnW-trnP for Carrhotus xanthogramma; trnQtrnW, trnW-trnG, trnG-trnM for Tetragnatha nitens. Our study revealed that the gene rearrangement in spiders with putative ancestor is accelerated in Araneomorphae as compared to Mygalomorphae. Phylogenetic analysis of spiders using mitochondrial sequence data supports the monophyly of two infraorders, and sister relationship of Cheiracanthiidae with Selenopidae
Journal Pre-proof and Salticidae. The systematic position of the Cheiracanthium species always a controversial issue as this taxa was placed in different families by different authors.
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Key words. Mitogenome; Phylogeny; Gene arrangement.
Journal Pre-proof 1. Introduction The mitogenome of the majority of metazoans are circular, spanning 14-19 kb, contain 37 genes, (13 protein-coding genes, PCGs; two ribosomal RNA genes, rRNA; 22 transfer RNA genes, tRNA) and a noncoding control region [1]. The mitochondrial genes in metazoans evolve rapidly as compared to nuclear genes [2]. Mitochondrial genome has been widely used in phylogenetic reconstruction, population genetics and evolutionary studies [3, 4]. The length of
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mitochondrial genomes, their structure, and gene rearrangements are consistent in vertebrates [5-
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7], whereas, inconsistent in invertebrates [1, 8-10]. However, it has been assumed that extensive
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changes in the gene order occurred due to the high rate of sequence evolution [1, 11]. The
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mitochondrial gene rearrangements are used for studying the phylogenetic relationship, as it was
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hypothesized to be a theoretical hybrid of molecular and morphological characters explaining the ultrastructural changes in the genome [12]. The derived characters are formed due to the
the
present
study,
the
complete
mitogenome
of
family Cheiracanthiidae
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In
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shuffling of neighboring genes (mostly tRNA genes) or translocation of the genes.
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(Cheiracanthium triviale) was sequenced for the first time. Additionally, we have annotated the mitogenome of Dysdera silvatica (family Dysderidae) which was available at NCBI GenBank. Further, C. triviale mitogenome was compared with putative ancestor along with previously published 43 spider species mitogenomes in order to observe gene rearrangement, phylogeny and secondary structure of the control region. The sequence based phylogenetic utility on gene rearrangements has been attempted to identify the derived characters in spider’s mitogenomes. Thus, an extended comparison using mitochondrial genomes of the C. triviale and other members of spiders, especially Clubionidae and Miturgidae would help in understanding their systematic position.
Journal Pre-proof 2. Materials and Methods 2.1 Ethics statement No prior permission was required for the collection as the species is neither endangered nor protected in IUCN Red List or Indian Wildlife Protection Act, 1972. 2.2 Sample collection, and DNA extraction
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The specimen of C. triviale was collected from Gujarat state of (24.85 N 92.56E) India and
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preserved in absolute ethyl alcohol at −80°C in Centre for DNA Taxonomy, Molecular
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Systematics Division, Zoological Survey of India, Kolkata. The specimen was morphologically
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identified by Priya Prasad using available taxonomic keys [13]. Further, this specimen was also compared with the type material illustrations of male genitalia provided by Dankittipakul &
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Beccaloni [13]. The genomic DNA extraction of C. triviale was done by using the XpressDNA
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Tissue Kit (MagGenome, India). The genomic DNA was quantified by using dsDNA high-
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sensitivity kit (Thermo Fisher Scientific, MA, USA) in Qubit fluorometer.
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2.3 Mitochondrial genome sequencing and assembly The whole genome library of genomic DNA was sequenced using Illumina Hiseq2500 (2 × 150 base paired-end reads) (Illumina, USA) platform which yielded ~54 million reads. TruSeq DNA Library Preparation kit (https://support.illumina.com/downloads/truseq) with standard protocols was used for the construction of the paired end library. The NGS-Toolkit [14] was used to remove adapter contamination and low-quality reads with a cutoff of Phred quality scores of Q20 for trimming and filtering of raw sequencing reads. The screening of high quality reads (5.4 million) was done by the Burrows-Wheeler Alignment (BWA) tool [15] using Seqtk (https
Journal Pre-proof ://github.com/lh3/seqtk). These reads were assembled with SPAdes 3.9.0 [16], using Cheliceroides longipalpis mitochondrial genome (NC_041120) as a reference. 2.4 Gene annotation, visualization and comparative analysis Annotation of the assembled mitogenome was done by using MITOS web-server (http://mitos.bioinf.uni-leipzig.de/index.py) to determine the location of PCGs, tRNAs, rRNAs
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and their secondary structures [17]. The boundaries of PCGs and rRNAs was confirmed
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manually by nucleotide-nucleotide BLAST (BLASTn), protein-protein BLAST (BLASTp) [18], and open reading frame finder (ORF finder) in the National Center for Biotechnology
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Information (NCBI) (https://www.ncbi.nlm.nih.gov/orffinder/). Remaining tRNAs in the
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mitogenome was searched by using tRNAscan-SE 1.21 (http://lowelab.ucsc.edu/tRNAscan-SE/)
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[19], but incapable to detect. So, we have used other spider tRNA sequences from GenBank to confirm the locations and boundaries of each tRNAs. The anticodon arm motifs were examined
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manually, which were conserved among all spider species (Table 1). The ClustalX program [20]
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was used to observe the start and stop codon of PCGs in comparison with other spider species.
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MEGAX [21] was used to align the PCGs of C. triviale with other spider species. The assembled mitogenome with gene features was submitted to Banklt (NCBI) to get the accession number (https://www.ncbi.nlm.nih.gov/WebSub/). The CGView online server [22] was used to draw the circular image of C. triviale (http://stothard.afns.ualberta.ca/cgview_server/). The overlapping and intergenic spacer regions of C. triviale were examined manually. The skewness was calculated by the following formula: AT skew= (A−T) / (A+T) and GC skew= (G−C) / (G+C) [23]. We predicted the secondary structure of tRNAs for C. triviale by VARNA 3.93 [24] and control region (CR) by The Mfold web server [25]. The online Tandem Repeats Finder web tool (https://tandem.bu.edu/trf/trf.html) was used to predict the tandem repeats in the Control Region
Journal Pre-proof [26]. Further, we have annotated the complete mitogenome of D. silvatica as it was submitted in GenBank without gene features (Table 2). 2.5 Phylogenetic and gene arrangement analysis Forty three (43) available spider mitogenomes were retrieved from the GenBank and used in the present study [27-56] along with newly generated C. triviale sequence. The Limulus polyphemus
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(order: Xiphosura) mitogenome was used as an out-group [57] (Table S1) Thus, to prepare the
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datasets of 45 taxa, SequenceMatrix 1.7.8 [58] was used for the concatenation of all the 13 PCGs. To eliminate the poorly aligned position and divergent regions, we used TranslatorX
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server [59] using MAFFT algorithm [60]. For phylogenetic analysis, four datasets were made:
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(1) PCGs without GBlock [61], 11645 bp; (2) PCGs without GBlock (third codon position
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excluded), 7768 bp; (3) PCGs with GBlock, 8064 bp; (4) PCGs with GBlock (third codon position excluded), 5376 bp. To find the best substitution models for Bayesian Inference (BI) and
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Maximum Likelihood (ML), the PartitionFinder version 2.1.1 [62], with the greedy algorithm
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was used. The PartitionFinder analyses included 39 partitions for 13 PCGs (codon positions 1, 2,
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3) and 26 partitions for 13 PCGs (codon positions 1, 2) (Table S2). BI phylogeny using Mr. Bayes 3.2.6 [63] and ML analysis using IQ-tree [64] were performed on the online web portal The CIPRES Science Gateway v.3.3 [65] (https://www.phylo.org/). The trees were visualized and edited in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) [66]. TreeREx analysis [67] (extended version of CREx) was performed for allocating rearrangements to the edges of a given phylogenetic tree, and reconstructing ancestral gene orders at the inner nodes (http://pacosy.informatik.uni-leipzig.de/185-0-TreeREx.html). This software is not able to handle missing gene or duplicated genes. So, to keep the uniformity, we have removed the trnG from all
Journal Pre-proof spider species as it was not detected in Pirata subpiraticus. We have also removed the CR2 from the family Dysderidae as it was not present in other spider species (Table S3). 3. Results and Discussion 3.1 Structure of complete mitochondrial genomes The generated complete mitogenome of C. triviale is circular and spans 14,590 bp (accession
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number MN334527). It is encoded by 37 genes, including 13 PCGs, large and small rRNAs, 22
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tRNAs and one non-coding control region (CR) (Fig. 1). Twenty two genes were present on the
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majority strand and remaining on the minority strand. The AT and GC content of the mitogenome is 77.9% and 22.1% with negative AT skewness (-0.109) and positive GC skewness
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(0.194). Further, the annotated D. silvatica mitogenome (accession number CM016944.1),
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spanning 14,440 bp, encoded by 37 genes, and two CRs. The AT and GC content of the
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mitogenome is 69.4% and 30.6% with negative AT skewness (-0.129) and positive GC skewness (0.410). In C. triviale and D. silvatica, nine PCGs were encoded on the majority strand and the
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remaining four PCGs (nad1, nad4, nad4l and nad5) on the minority strand, as reported in other
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spiders species (Table 1, 2). Most of the PCGs used ATN start codon in C. triviale, D. silvatica. Whereas, TTG start codon was used by cox2 (C. triviale, D. silvatica), cox3 (C. triviale); TTT by nad1 (C. triviale). In C. triviale, eight PCGs used TAA termination codon, whereas nad1 and nad4L with incomplete termination codon. No termination codon was found in cox1, nad4 and atp8. In D. silvatica, TAA and TAG termination codon were used by 10 PCGs except cytb and nad5 with incomplete termination codon, nad4L without termination codon. The large ribosomal RNA (rrnL) of C. triviale and D. silvatica was located between trnL1 and trnV and the small ribosomal RNA (rrnS) between trnV and trnQ. The length of rrnL
Journal Pre-proof and rrnS of C. triviale were 1,001 and 727 bp. The length of rrnL and rrnS of D. silvatica were 1,056 and 674 bp. C. triviale has 22 tRNAs (total length 1,304 bp), constituting around 8.9% of the total mitogenome. The length of the tRNAs ranged between 50 bp to 74bp, with the shortest being that of trnS1 and the longest trnN. Total 13 tRNAs were located on the majority strand and nine tRNAs on minority strand. Twelve tRNAs either lack dihydrouracil (DHU) stem or the TψC arm and are simplified down to a loop, so could not be folded into typical cloverleaf secondary
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structures (Fig. S1). The tRNA genes (trnA, trnS1 and trnS2) lack DHU stem, whereas trnD,
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trnG, trnH, trnI, trnK, trnF, trnS1, trnT, trnW and trnY lack TψC arm. trnS1 neither had DHU
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stem nor TψC arm and four tRNAs (trnL1, trnM, trnQ, and trnN) showed the proper cloverleaf
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secondary structure. The Watson Crick base pairings (A=T and G≡C) were observed in most of the tRNAs; however 31 wobble base pairing and 20 mismatches were identified. Twenty tRNAs
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were overlapped with the adjacent gene on either one end or both the ends. The quantity of
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truncation or gene overlapping in each tRNA were varied from species to species. trnE showed overlap of 33 nucleotides (nt) with its neighboring gene (trnR) on the same strand at 5′ end and
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24 nt overlap with trnF on the opposite strand at the 3′ end. After overlapping on both the ends,
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only two nt was remaining which exclusively denoted for trnE. trnY showed overlap of 21 nucleotides (nt) at the 5′ end with its neighboring gene (trnW) on the opposite strand and 21 nt overlap at the 3′ end with trnC on the same strand. After overlapping on both the ends, 13 nucleotides were remaining for trnY. D. silvatica has 22 tRNAs (total length 1,320 bp), with 71.4% AT content and negative AT skewness (-0.054) and positive GC skewness (0.379). The length of the tRNAs ranged between 47 bp to 69bp, with the shortest being that of trnA and the longest trnI. Thirteen genes were located on the majority strand and nine on minority strand. Twelve tRNAs either lack of
Journal Pre-proof dihydrouracil (DHU) stem or TψC arm and could not be folded into typical cloverleaf secondary structures (Fig. S2). The tRNA genes that lack of DHU stem were trnQ, trnS2 and trnV whereas trnC, trnD, trnE, trnH, trnL2, trnM, trnF, trnT and trnY lack of TψC arm. trnA and trnS1 neither had DHU stem nor TψC arm. Only seven tRNAs showed proper cloverleaf secondary structure (trnR, trnG, trnL1, trnK, trnP, trnW and trnI). Thirty eight wobble base pairing and 27 mismatches were identified. Most of the tRNAs were overlapped with the adjacent gene on
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either one end or both the ends except trnP. trnA showed overlap of 12 nt with its neighboring
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gene (trnN) on the same strand at 5′ end and 37 nt overlap with trnS1 on the same strand at the 3′
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end. Further, trnF showed overlap of 24 nt with its neighboring gene (trnE) on the opposite
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strand at 5′ end and 55 nt overlap with nad5 gene on the same strand at the 3′ end. After overlapping on both the ends, no nucleotide was remaining for trnA and trnF. trnY showed
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overlaps of 36 nt at the 5′ end with its neighboring gene (trnW) on the opposite strand and 16 nt
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overlap at the 3′ end with trnC gene on the same strand. After overlapping on both the ends, 4 nt were remaining exclusively for trnY. Further, we have also annotated trnM of Argiope amoena
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between CR and nad2.
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(54 bp) and Argiope bruennichi (58 bp) which was at conserved position in Araneomorphae
3.2 Control region
The control region (CR) of metazoans plays a vital role in the initiation of replication [68-69]. Spider mitogenomes were usually known by one CR except family Dysderidae with two CRs. The first CR was conserved at their position between trnQ and trnM throughout the spider mitogenomes except members of the suborder Mesothelae (Liphistius erawan and Songthela hangzhouensis), where the CR was present between rrnS and trnI. The second CR was present
Journal Pre-proof between trnL2 and trnN in family Dysderidae. The CRs were ranged from 155 bp (Trichonephila clavata, Araneidae) to 2,047 bp (Argyroneta aquatica, Dictynidae). Conserved elements within the CRs have been identified as initiation sites for replication and transcription [68, 70-71] such as AT-rich region, TATA motif, stem and loop, and G(A)nT motif. C. triviale CR spans 985 bp and contains 77.1% AT content with negative AT and GC skewness (-0.048 and -0.15). All the conserved elements were present in a consistent manner as
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compared to other Opisthothelae spiders. Two tandem repeats of (TAATA) and two of (CTTAT)
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were also present at the downstream of GAT motif. The CR of D. silvatica spans 534 bp with
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77.7% AT content with AT and GC skewness (-0.035 and 0.174). In D. silvatica, all the
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conserved elements were present in a consistent manner along with two tandem repeats of
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(TTTTAA). The conserved elements were also detected in all spiders CRs except Liphistius erawan in which TATA motif was absent. The arrangement of these elements was identical in
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most of the species except three species Loxosceles similis, Argyroneta aquatica, Tetragnatha
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nitens, in which tandem repeats were detected between AT-rich region and TATA motif. In few species, the tandem repeats were detected either upstream of AT-rich region or downstream of
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GAT motif (Fig. 2).
3.3 Phylogenetic analyses
Eight phylogenetic trees with two inference methods (BI, ML) using four datasets with the best partition schemes were constructed (Fig. 3). The topologies of all the phylogenies were almost identical (Fig. S4 to S11). The results clearly indicate the monophyly of suborders Opisthothelae, Mesothelae, and infraorder Mygalomorphae. The infraorder Araneomorphae was recovered monophyletic in all the analyses except BI-2, ML-2 (Fig. S5, S9). The observed paraphyly of Araneomorphae in BI-2, and ML-2 (without Gblock excluding 3rd codon) may be due to the
Journal Pre-proof unstable position of ancestral lineages of family Hypochilidae to the other members of spiders. The families of Mygalomorphae (Nemesiidae, Theraphosidae and Euagridae) showed sister relationship to each other. However, the genus Phyxioschema of Mygalomorphae was recently transferred from the family Dipluridae to family Euagridae [72]. The infraorder Araneomorphae have two clades (1) first clade with family Dysderidae, Pholcidae, and Sicariidae (2) second clade with family Dictynidae, Salticidae, Agelenidae, Selenopidae, Oxyopidae, Lycosidae,
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Thomisidae, Tetragnathidae, Araneidae, and Cheiracanthiidae. The family Hypochilidae either
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associated with the first clade of Araneomorphae in four analysis (BI-1, BI-3, ML-1, and ML-3)
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or second clade in two analysis (BI-4, ML-4). The estimated tree from the mitochondrial PCG sequences is well congruent with previously generated multi-locus phylogeny [73]. In both the
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analysis, the ‘Synspermiata’ families Dysderidae, Hypochilidae, Sicariidae are found in sister
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relationship to the ‘Lost Tracheae clade’ family Pholcidae. Further, in the ‘Entelegynae’ taxa, the
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‘Oval Calamistrum clade’ families Salticidae, Selenopidae, Oxyopidae, Lycosidae, Thomisidae are found as sister group to ‘RTA clade’ families Dictynidae and Agelenidae. The previous
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multi-locus phylogeny revealed a close association of Cheiracanthium species with other
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Eutichuridae species [73], however, this family is now considered as a junior synonym of family Cheiracanthiidae [74]. The present mitogenome based phylogeny showed that, Cheiracanthiidae is sister to families Selenopidae and Salticidae in all the analyses with strong posterior probabilities and bootstrap support. The present study suggested that the mitochondrial genome data of different families, especially Clubionidae and Miturgidae are necessary to confirm the phylogenetic position of Cheiracanthiidae. 3.4 Gene arrangement
Journal Pre-proof The features of the gene arrangements are transpositions, inversions, inverse transpositions. The Tandem Duplication-Random Loss (TDRL) is the mechanism to explain the transpositions. The gene order (GO) of C. triviale mitogenome is identical to the majority of spiders families, but differ from the putative ancestor (Limulus polyphemus) (Fig. 4). CREx analysis of C. triviale revealed four transpositions and two TDRL events in comparison to ancestral GO. The transposition of trnC from gene block trnW-trnC-trnY to downstream of trnY occurred in C.
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trivale as compared to ancestor. The transposition of trnA, trnS1 and trnR occurred from gene
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block trnA-trnE and made the new gene boundaries trnA-trnS1, trnN-trnA, trnS1-trnR, and trnR-
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trnE in C. triviale. The transposition of trnL2 occurred from downstream of nad1 to upstream of
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the same gene and made new gene boundaries nad3-trnL2 and trnL2-trnN. CREx analysis of D. silvatica also revealed the four transposition (trnT, trnL2, trnI, trnC) and one TDRL event as
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compared to putative ancestor.The GO of the suborder Mesothelae is similar to putative ancestral
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GO, though they belong to different arthropod classes. Further, the GO of the different spiders families are mostly similar to each other. A very few transpositions, inversions were detected in
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comparison to the putative ancestral GO.
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TreeREx analysis revealed the transpositions of eight tRNAs (trnL2, trnN, trnA, trnS1, trnR, trnQ, trnT, trnY) from the suborder Mesothelae to Opisthothelae at the connecting node of two infra-orders of Opisthothelae (Araneomorphae and Mygalomorphae) (Fig. 4). The inversion of trnI occurred between the taxa of Mygalomorphae in comparison to ancestral GO except Lyrognathus crotalus where trnI is reverted back to the ancestral condition. Further, the transposition of the trnI occurred between the connecting nodes of all the families of Araneomorphae. The transposition of trnI is occurred from CR-trnQ to rrnS-trnQ in family Pholcidae and CR-trnQ to trnM-nad2 in family Dysderidae. This transposition derived the new
Journal Pre-proof gene boundaries trnM-trnI, trnI-nad2 which indicate the synapomorphy for the family Dysderidae. Further the inversion of trnH is observed in all the taxa of family Pholcidae, except Pholcus phalangioides. The inverse-transposition of trnA occurred in Loxosceles similis (Sicariidae) and made new gene boundaries trnQ-trnA, trnA-trnM which indicated the apomorphic character of this taxa. The inversion of trnN occurred at the connecting node of family Dictynidae and Agelenidae, whereas, the trnN undergoes inverse transposition in family
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Dictynidae. The taxa of family Agelenidae is further detected by transposition of three tRNAs
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(trnS1, trnR, trnE), inversion of trnI and inverse transposition of trnN in comparison to ancestor.
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This makes apomorphic character or new gene boundaries nad3-trnS1, trnE-trnL2, trnL2-trnA,
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and trnN-trnF for Agelena silvatica (Agelenidae). The GO of Salticidae taxa are similar to each other except Carrhotus xanthogramma in which transposition of trnE and trnW occurred. The
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transposition of trnE and trnW make the new gene boundaries trnN-trnE, trnE-trnA, trnR-trnF,
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nad4L-trnW, and trnW-trnP, which indicate the apomorphy for C. xanthogramma. Further, the inversion of trnL2 occurred from majority strand to minority strand in S. bursarius. The taxa of
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family Tetragnathidae shared the GO with family Araneidae except Tetragnatha nitens in which
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two inversions of trnY and trnC and one transposition of trnW occurred which makes the new gene boundaries trnQ-trnW, trnW-trnG, trnG-trnM which are apomorphic character for Tetragnatha nitens. 4. Conclusion The complete mitochondrial genome of C. triviale was characterized and compared with other members of the spiders. Moreover, the available mitogenome sequence data of D. silvatica was annotated and characterized. The phylogenetic relationships based on mitochondrial genome data are congruent with morphology and the earlier multi-locus phylogeny. The phylogenetic analysis
Journal Pre-proof indicated the monophyly of infraorder Araneomorphae and Mygalomorphae. The family Cheiracanthiidae showed sister relationship with Salticidae and Selenopidae. The systematic position of the genus Cheiracanthium is always a controversial issue and matter of concern to arachnologists. The genus Cheiracanthium was placed under family Clubionidae without knowing its original placement under family ‘Cheiracanthidae’ [75-76]. Later on, Ramírez et al. in 1997 transferred this genus from Clubionidae to Miturgidae [77] and further revert back in
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Clubionidae by Deeleman-Reinhold in 2001 [78]. Platnick in 2011, transferred the genus under
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family Miturgidae [79]. Ramirez in 2014 again transferred this genus from Miturgidae to
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Eutichuridae [80]. However, Ono and Ogata in 2018 synonymized the family Eutichuridae under
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family Cheiracanthiidae [74]. The systematic position of the genus Cheiracanthium under family Cheiracanthidae, Miturgidae or Clubionidae is still in debate [81-82, 73]. However, in-depth data
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on taxa of Miturgidae and Clubionidae could build up our knowledge towards the rearrangement
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Acknowledgements
na
We are thankful to the Director, Zoological Survey of India, Kolkata, for providing necessary facilities, constant support and encouragement throughout the study. The study is financially
ur
supported by Zoological Survey of India, Kolkata, Ministry of Environment Forest and Climate
Jo
Change under National Faunal Genome Resources (NFGR) Program. Competing Interests.
The authors declare that they have no competing interests. Additional Information Supplementary information accompanies this paper at
Journal Pre-proof Caption of Figures Fig. 1. The CGView of Cheiracanthium triviale mitochondrial genome. . PCGs represented by Royal blue arrows, rRNA genes by turqoise arrows, tRNA genes by orange arrows and CR regions by gray rectangles. Fig. 2. The control region spider mitogenomes including C. triviale. Different colours are used to show the structural elements. Fig. 3. Bayesian Phylogenetic tree (BI-1) inferred by PCGs spider mitochondrial genomes. BI and ML support values of datasets are shown at the nodes by different shape and colors in the following order (BI-1/BI-2/BI-3/BI-4/ML-1/ML-2/ML-3/ML-4). The posterior probabilities
of
(1.0) and bootstrap support (100%) are represented by pink circles. The posterior probabilities
ro
below (0.9-1.0) and bootstrap support (70-100%) are shown by ‘ns’.
Fig. 4. Representation of the output of the TreeREx analysis on Maximum likelihood tree
-p
(ML-1) with gene arrangement. The rearrangements on the branches are given as Transposition (T), Inversion (I), Inverse transposition (iT). Genes on minority strand indicated
lP
re
with red circle and the rearranged genes highlighted with yellow color.
Strand
Location start
-
trnV
-
rrnS
-
trnQ
-
CR
13
Anticodon
Start Codon
Stop Codon
IGN
stop
1013
Jo
rrnL
Size(bp)
ur
Gene
na
Table 1. The annotated mitochondrial genes of C. triviale and its characteristic features. IGN represents (+) values as intergenic nucleotides and (-) values as overlapping regions. CR represents the control region.
1001
1014
1072
59
1067
1793
727
1756
1820
65
1821
2805
985
0 TAC
-6 -38
TTG
0 0
trnM
+
2806
2873
68
CAT
-18
nad2
+
2856
3839
984
trnW
+
3814
3867
54
TCA
-21
trnY
-
3847
3901
55
GTA
-21
trnC
-
3881
3932
52
GCA
0
cox1
+
3933
5447
1515
ATA
not found
23
cox2
+
5471
6133
663
TTG
TAA
-2
trnK
+
6132
6194
63
CTT
-18
trnD
+
6177
6229
53
GTC
-5
ATT
TAA
-26
Journal Pre-proof atp8
+
6225
6380
156
ATT
not found
atp6
+
6377
7045
669
ATG
TAA
3
cox3
+
7049
7834
786
TTG
TAA
-2
trnG
+
7833
7888
56
nad3
+
7881
8234
354
trnL2
-
8205
8262
58
TAA
-6
trnN
+
8257
8330
74
GTG
40
trnA
+
8371
8429
59
TGC
-3
trnS1
+
8427
8476
50
GCT
1
trnR
+
8478
8550
73
TCG
-33
trnE
+
8518
8576
59
TTC
-24
trnF
-
8553
8609
57
GAA
nad5
-
8605
10242
1638
trnH
-
10249
10301
53
nad4
-
10303
11589
1287
nad4l
-
11574
11845
272
trnP
-
11848
11903
56
nad6
+
11916
12347
432
trnI
+
12343
12403
61
cytb
+
12392
13534
trnS2
+
13527
13581
trnT
+
13582
13636
nad1
-
13629
14546
918
L1
-
14527
14595
69
TCC
-8
of
ATT
-p
GTG
ro
TTA
TAA
T(AA)
re
6 1
ATG
not found
ATT
TT(A)
-16 2 12
ATA
TAA
GAT
lP
-30
-5
TGG
1143
-5 -12
ATT
TAA
-8
55
TGA
0
55
TGT
-8 TTT
T(AA)
-20
TAG
Jo
ur
na
-4
Table 2. The annotated mitochondrial genes of D. silvatica and its characteristic features. IGN represents (+) values as intergenic nucleotides and (-) values as overlapping regions. CR represents the control region. Gene
Strand
Location
Size (bp)
start
stop
Anticodon
Start Codon
Stop Codon
IGN
trnM
+
421
482
62
CAT
-17
trnI
+
466
534
69
GAT
8
nad2
+
543
1451
909
trnW
+
1450
1515
66
TCA
-36
trnY
+
1480
1535
56
TAC
-16
trnC
-
1520
1573
54
GCA
-5
cox1
+
1569
3110
1542
ATA
ATA
TAG
TAA
-2
52
Journal Pre-proof cox2
+
3163
3804
642
trnK
+
3805
3865
61
AAA
-47
trnD
+
3819
3903
85
GTC
-8
atp8
+
3896
4045
150
ATA
TAA
-4
atp6
+
4042
4704
663
ATA
TAG
30
cox3
+
4735
5493
759
ATT
TAA
-2
trnG
+
5492
5556
65
nad3
+
5538
5873
336
trnL2
-
5858
5913
56
TTG
TAG
TCC
0
-19 ATT
TAA
-16
TTA
81 -12
5994
81
trnN
5995
6059
65
AAC
trnA
+
6048
6094
47
GCA
trnS1
+
6058
6117
60
AGC
trnR
+
6104
6165
62
CGA
-15
trnE
+
6151
6203
53
TTC
-24
trnF
-
6180
6244
65
nad5
-
6190
7858
1669
trnH
-
7887
7935
nad4
-
7934
9211
nad4l
-
9201
9461
261
trnP
-
9461
9518
58
nad6
+
9522
9971
450
ATG
TAA
cytb
+
9964
11076
1113
ATA
T(AA)
trnS2
+
11078
11130
53
TCA
4
trnT
+
11135
11183
49
ACA
-8
nad1
-
11176
12078
903
trnL1
-
12076
12143
68
rrnL
-
12095
13150
1056
trnV
-
13118
13174
57
rrnS
-
13174
13847
674
trnQ
-
13847
13906
60
13907
14440
1
420
-p
ro
-14
re
GAA
-55 ATT
T(AA)
GTG ATC
TAA
ATT
not found
na
28 -2
lP
49
-37
1278
ur
Jo
CR
of
5914 +
CR
CCA
-11 -1 3
ATA CTA
TAA
-8 1
-3 -49 -33
GTA
-1 -1
TTG
954
Author statement
Kaomud Tyagi, Vikas Kumar: Conceptualization, Methodology, Software
-1
Journal Pre-proof Kaomud Tyagi, Nikita Poddar, Shantanu Kundu: Data curation, Writing- Original draft preparation. Nikita Poddar, Priya Prasad: Visualization, Investigation. Vikas Kumar, Kailash Chandra: Supervision. Kaomud Tyagi, Inderjeet Tyagi: Software, Validation. Kaomud Tyagi, Vikas Kumar: Writing- Reviewing and Editing
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Highlights The complete mitogenome of spider family Cheiracanthiidae was sequenced and annotated for the first time.
There were nineteen mitochondrial gene rearrangement types among 44 spider species.
New gene boundaries as derived characters were recovered as a result of gene arrangements.
Accelerated gene rearrangement is observed in Araneomorphae as compared to Mygalomorphae.
Phylogenetic position of family Hypochilidae is unstable and needs further studies.
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Figure 1
Figure 2
Figure 3
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Kaomud Tyagi, Vikas Kumar, Nikita Poddar, Priya Prasad, Inderjeet Tyagi, Shantanu Kundu, Kailash Chandra PII:
S0141-8130(19)40035-4
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.01.014
Reference:
BIOMAC 14318
To appear in:
International Journal of Biological Macromolecules
Received date:
6 December 2019
Revised date:
1 January 2020
Accepted date:
3 January 2020
Please cite this article as: K. Tyagi, V. Kumar, N. Poddar, et al., The gene arrangement and phylogeny using mitochondrial genomes in spiders (Arachnida: Araneae), International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/ j.ijbiomac.2020.01.014
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© 2020 Published by Elsevier.
Journal Pre-proof The gene arrangement and phylogeny using mitochondrial genomes in spiders (Arachnida: Araneae) Kaomud Tyagi, Vikas Kumar*, Nikita Poddar, Priya Prasad, Inderjeet Tyagi, Shantanu Kundu, Kailash Chandra Centre for DNA Taxonomy, Molecular Systematics Division, Zoological Survey of India, Kolkata, India
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Corresponding author email id: [email protected]
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Abstract
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The complete mitochondrial genome (mitogenome) of Cheiracanthium triviale was sequenced
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for the first time. The 14,590 bp C. triviale mitogenome contained 37 genes (13 protein coding
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genes, 2 ribosomal RNAs, 22 transfer RNAs) and one control region. The mitogenome of Dysdera silvatica which was available at NCBI GenBank was annotated. The mitogenome of C.
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triviale was compared with 43 previously sequenced spider species to observe the gene
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arrangements, control region and phylogeny. TreeREx analysis identified 19 mitochondrial gene
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rearrangements (11 transposition, 6 inversion, 2 inverse transposition) in spiders as compared with the putative ancestral gene order and lead to form new gene boundaries: trnQ-trnA, trnAtrnM for Loxosceles similis; nad3-trnS1, trnE-trnL2, trnL2-trnA, trnN-trnF for Agelena silvatica; trnN-trnE, trnE-trnA, trnR-trnF, nad4L-trnW, trnW-trnP for Carrhotus xanthogramma; trnQtrnW, trnW-trnG, trnG-trnM for Tetragnatha nitens. Our study revealed that the gene rearrangement in spiders with putative ancestor is accelerated in Araneomorphae as compared to Mygalomorphae. Phylogenetic analysis of spiders using mitochondrial sequence data supports the monophyly of two infraorders, and sister relationship of Cheiracanthiidae with Selenopidae
Journal Pre-proof and Salticidae. The systematic position of the Cheiracanthium species always a controversial issue as this taxa was placed in different families by different authors.
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Key words. Mitogenome; Phylogeny; Gene arrangement.
Journal Pre-proof 1. Introduction The mitogenome of the majority of metazoans are circular, spanning 14-19 kb, contain 37 genes, (13 protein-coding genes, PCGs; two ribosomal RNA genes, rRNA; 22 transfer RNA genes, tRNA) and a noncoding control region [1]. The mitochondrial genes in metazoans evolve rapidly as compared to nuclear genes [2]. Mitochondrial genome has been widely used in phylogenetic reconstruction, population genetics and evolutionary studies [3, 4]. The length of
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mitochondrial genomes, their structure, and gene rearrangements are consistent in vertebrates [5-
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7], whereas, inconsistent in invertebrates [1, 8-10]. However, it has been assumed that extensive
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changes in the gene order occurred due to the high rate of sequence evolution [1, 11]. The
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mitochondrial gene rearrangements are used for studying the phylogenetic relationship, as it was
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hypothesized to be a theoretical hybrid of molecular and morphological characters explaining the ultrastructural changes in the genome [12]. The derived characters are formed due to the
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present
study,
the
complete
mitogenome
of
family Cheiracanthiidae
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shuffling of neighboring genes (mostly tRNA genes) or translocation of the genes.
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(Cheiracanthium triviale) was sequenced for the first time. Additionally, we have annotated the mitogenome of Dysdera silvatica (family Dysderidae) which was available at NCBI GenBank. Further, C. triviale mitogenome was compared with putative ancestor along with previously published 43 spider species mitogenomes in order to observe gene rearrangement, phylogeny and secondary structure of the control region. The sequence based phylogenetic utility on gene rearrangements has been attempted to identify the derived characters in spider’s mitogenomes. Thus, an extended comparison using mitochondrial genomes of the C. triviale and other members of spiders, especially Clubionidae and Miturgidae would help in understanding their systematic position.
Journal Pre-proof 2. Materials and Methods 2.1 Ethics statement No prior permission was required for the collection as the species is neither endangered nor protected in IUCN Red List or Indian Wildlife Protection Act, 1972. 2.2 Sample collection, and DNA extraction
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The specimen of C. triviale was collected from Gujarat state of (24.85 N 92.56E) India and
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preserved in absolute ethyl alcohol at −80°C in Centre for DNA Taxonomy, Molecular
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Systematics Division, Zoological Survey of India, Kolkata. The specimen was morphologically
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identified by Priya Prasad using available taxonomic keys [13]. Further, this specimen was also compared with the type material illustrations of male genitalia provided by Dankittipakul &
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Beccaloni [13]. The genomic DNA extraction of C. triviale was done by using the XpressDNA
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Tissue Kit (MagGenome, India). The genomic DNA was quantified by using dsDNA high-
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sensitivity kit (Thermo Fisher Scientific, MA, USA) in Qubit fluorometer.
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2.3 Mitochondrial genome sequencing and assembly The whole genome library of genomic DNA was sequenced using Illumina Hiseq2500 (2 × 150 base paired-end reads) (Illumina, USA) platform which yielded ~54 million reads. TruSeq DNA Library Preparation kit (https://support.illumina.com/downloads/truseq) with standard protocols was used for the construction of the paired end library. The NGS-Toolkit [14] was used to remove adapter contamination and low-quality reads with a cutoff of Phred quality scores of Q20 for trimming and filtering of raw sequencing reads. The screening of high quality reads (5.4 million) was done by the Burrows-Wheeler Alignment (BWA) tool [15] using Seqtk (https
Journal Pre-proof ://github.com/lh3/seqtk). These reads were assembled with SPAdes 3.9.0 [16], using Cheliceroides longipalpis mitochondrial genome (NC_041120) as a reference. 2.4 Gene annotation, visualization and comparative analysis Annotation of the assembled mitogenome was done by using MITOS web-server (http://mitos.bioinf.uni-leipzig.de/index.py) to determine the location of PCGs, tRNAs, rRNAs
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and their secondary structures [17]. The boundaries of PCGs and rRNAs was confirmed
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manually by nucleotide-nucleotide BLAST (BLASTn), protein-protein BLAST (BLASTp) [18], and open reading frame finder (ORF finder) in the National Center for Biotechnology
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Information (NCBI) (https://www.ncbi.nlm.nih.gov/orffinder/). Remaining tRNAs in the
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mitogenome was searched by using tRNAscan-SE 1.21 (http://lowelab.ucsc.edu/tRNAscan-SE/)
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[19], but incapable to detect. So, we have used other spider tRNA sequences from GenBank to confirm the locations and boundaries of each tRNAs. The anticodon arm motifs were examined
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manually, which were conserved among all spider species (Table 1). The ClustalX program [20]
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was used to observe the start and stop codon of PCGs in comparison with other spider species.
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MEGAX [21] was used to align the PCGs of C. triviale with other spider species. The assembled mitogenome with gene features was submitted to Banklt (NCBI) to get the accession number (https://www.ncbi.nlm.nih.gov/WebSub/). The CGView online server [22] was used to draw the circular image of C. triviale (http://stothard.afns.ualberta.ca/cgview_server/). The overlapping and intergenic spacer regions of C. triviale were examined manually. The skewness was calculated by the following formula: AT skew= (A−T) / (A+T) and GC skew= (G−C) / (G+C) [23]. We predicted the secondary structure of tRNAs for C. triviale by VARNA 3.93 [24] and control region (CR) by The Mfold web server [25]. The online Tandem Repeats Finder web tool (https://tandem.bu.edu/trf/trf.html) was used to predict the tandem repeats in the Control Region
Journal Pre-proof [26]. Further, we have annotated the complete mitogenome of D. silvatica as it was submitted in GenBank without gene features (Table 2). 2.5 Phylogenetic and gene arrangement analysis Forty three (43) available spider mitogenomes were retrieved from the GenBank and used in the present study [27-56] along with newly generated C. triviale sequence. The Limulus polyphemus
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(order: Xiphosura) mitogenome was used as an out-group [57] (Table S1) Thus, to prepare the
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datasets of 45 taxa, SequenceMatrix 1.7.8 [58] was used for the concatenation of all the 13 PCGs. To eliminate the poorly aligned position and divergent regions, we used TranslatorX
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server [59] using MAFFT algorithm [60]. For phylogenetic analysis, four datasets were made:
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(1) PCGs without GBlock [61], 11645 bp; (2) PCGs without GBlock (third codon position
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excluded), 7768 bp; (3) PCGs with GBlock, 8064 bp; (4) PCGs with GBlock (third codon position excluded), 5376 bp. To find the best substitution models for Bayesian Inference (BI) and
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Maximum Likelihood (ML), the PartitionFinder version 2.1.1 [62], with the greedy algorithm
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was used. The PartitionFinder analyses included 39 partitions for 13 PCGs (codon positions 1, 2,
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3) and 26 partitions for 13 PCGs (codon positions 1, 2) (Table S2). BI phylogeny using Mr. Bayes 3.2.6 [63] and ML analysis using IQ-tree [64] were performed on the online web portal The CIPRES Science Gateway v.3.3 [65] (https://www.phylo.org/). The trees were visualized and edited in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) [66]. TreeREx analysis [67] (extended version of CREx) was performed for allocating rearrangements to the edges of a given phylogenetic tree, and reconstructing ancestral gene orders at the inner nodes (http://pacosy.informatik.uni-leipzig.de/185-0-TreeREx.html). This software is not able to handle missing gene or duplicated genes. So, to keep the uniformity, we have removed the trnG from all
Journal Pre-proof spider species as it was not detected in Pirata subpiraticus. We have also removed the CR2 from the family Dysderidae as it was not present in other spider species (Table S3). 3. Results and Discussion 3.1 Structure of complete mitochondrial genomes The generated complete mitogenome of C. triviale is circular and spans 14,590 bp (accession
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number MN334527). It is encoded by 37 genes, including 13 PCGs, large and small rRNAs, 22
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tRNAs and one non-coding control region (CR) (Fig. 1). Twenty two genes were present on the
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majority strand and remaining on the minority strand. The AT and GC content of the mitogenome is 77.9% and 22.1% with negative AT skewness (-0.109) and positive GC skewness
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(0.194). Further, the annotated D. silvatica mitogenome (accession number CM016944.1),
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spanning 14,440 bp, encoded by 37 genes, and two CRs. The AT and GC content of the
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mitogenome is 69.4% and 30.6% with negative AT skewness (-0.129) and positive GC skewness (0.410). In C. triviale and D. silvatica, nine PCGs were encoded on the majority strand and the
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remaining four PCGs (nad1, nad4, nad4l and nad5) on the minority strand, as reported in other
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spiders species (Table 1, 2). Most of the PCGs used ATN start codon in C. triviale, D. silvatica. Whereas, TTG start codon was used by cox2 (C. triviale, D. silvatica), cox3 (C. triviale); TTT by nad1 (C. triviale). In C. triviale, eight PCGs used TAA termination codon, whereas nad1 and nad4L with incomplete termination codon. No termination codon was found in cox1, nad4 and atp8. In D. silvatica, TAA and TAG termination codon were used by 10 PCGs except cytb and nad5 with incomplete termination codon, nad4L without termination codon. The large ribosomal RNA (rrnL) of C. triviale and D. silvatica was located between trnL1 and trnV and the small ribosomal RNA (rrnS) between trnV and trnQ. The length of rrnL
Journal Pre-proof and rrnS of C. triviale were 1,001 and 727 bp. The length of rrnL and rrnS of D. silvatica were 1,056 and 674 bp. C. triviale has 22 tRNAs (total length 1,304 bp), constituting around 8.9% of the total mitogenome. The length of the tRNAs ranged between 50 bp to 74bp, with the shortest being that of trnS1 and the longest trnN. Total 13 tRNAs were located on the majority strand and nine tRNAs on minority strand. Twelve tRNAs either lack dihydrouracil (DHU) stem or the TψC arm and are simplified down to a loop, so could not be folded into typical cloverleaf secondary
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structures (Fig. S1). The tRNA genes (trnA, trnS1 and trnS2) lack DHU stem, whereas trnD,
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trnG, trnH, trnI, trnK, trnF, trnS1, trnT, trnW and trnY lack TψC arm. trnS1 neither had DHU
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stem nor TψC arm and four tRNAs (trnL1, trnM, trnQ, and trnN) showed the proper cloverleaf
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secondary structure. The Watson Crick base pairings (A=T and G≡C) were observed in most of the tRNAs; however 31 wobble base pairing and 20 mismatches were identified. Twenty tRNAs
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were overlapped with the adjacent gene on either one end or both the ends. The quantity of
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truncation or gene overlapping in each tRNA were varied from species to species. trnE showed overlap of 33 nucleotides (nt) with its neighboring gene (trnR) on the same strand at 5′ end and
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24 nt overlap with trnF on the opposite strand at the 3′ end. After overlapping on both the ends,
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only two nt was remaining which exclusively denoted for trnE. trnY showed overlap of 21 nucleotides (nt) at the 5′ end with its neighboring gene (trnW) on the opposite strand and 21 nt overlap at the 3′ end with trnC on the same strand. After overlapping on both the ends, 13 nucleotides were remaining for trnY. D. silvatica has 22 tRNAs (total length 1,320 bp), with 71.4% AT content and negative AT skewness (-0.054) and positive GC skewness (0.379). The length of the tRNAs ranged between 47 bp to 69bp, with the shortest being that of trnA and the longest trnI. Thirteen genes were located on the majority strand and nine on minority strand. Twelve tRNAs either lack of
Journal Pre-proof dihydrouracil (DHU) stem or TψC arm and could not be folded into typical cloverleaf secondary structures (Fig. S2). The tRNA genes that lack of DHU stem were trnQ, trnS2 and trnV whereas trnC, trnD, trnE, trnH, trnL2, trnM, trnF, trnT and trnY lack of TψC arm. trnA and trnS1 neither had DHU stem nor TψC arm. Only seven tRNAs showed proper cloverleaf secondary structure (trnR, trnG, trnL1, trnK, trnP, trnW and trnI). Thirty eight wobble base pairing and 27 mismatches were identified. Most of the tRNAs were overlapped with the adjacent gene on
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either one end or both the ends except trnP. trnA showed overlap of 12 nt with its neighboring
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gene (trnN) on the same strand at 5′ end and 37 nt overlap with trnS1 on the same strand at the 3′
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end. Further, trnF showed overlap of 24 nt with its neighboring gene (trnE) on the opposite
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strand at 5′ end and 55 nt overlap with nad5 gene on the same strand at the 3′ end. After overlapping on both the ends, no nucleotide was remaining for trnA and trnF. trnY showed
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overlaps of 36 nt at the 5′ end with its neighboring gene (trnW) on the opposite strand and 16 nt
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overlap at the 3′ end with trnC gene on the same strand. After overlapping on both the ends, 4 nt were remaining exclusively for trnY. Further, we have also annotated trnM of Argiope amoena
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between CR and nad2.
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(54 bp) and Argiope bruennichi (58 bp) which was at conserved position in Araneomorphae
3.2 Control region
The control region (CR) of metazoans plays a vital role in the initiation of replication [68-69]. Spider mitogenomes were usually known by one CR except family Dysderidae with two CRs. The first CR was conserved at their position between trnQ and trnM throughout the spider mitogenomes except members of the suborder Mesothelae (Liphistius erawan and Songthela hangzhouensis), where the CR was present between rrnS and trnI. The second CR was present
Journal Pre-proof between trnL2 and trnN in family Dysderidae. The CRs were ranged from 155 bp (Trichonephila clavata, Araneidae) to 2,047 bp (Argyroneta aquatica, Dictynidae). Conserved elements within the CRs have been identified as initiation sites for replication and transcription [68, 70-71] such as AT-rich region, TATA motif, stem and loop, and G(A)nT motif. C. triviale CR spans 985 bp and contains 77.1% AT content with negative AT and GC skewness (-0.048 and -0.15). All the conserved elements were present in a consistent manner as
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compared to other Opisthothelae spiders. Two tandem repeats of (TAATA) and two of (CTTAT)
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were also present at the downstream of GAT motif. The CR of D. silvatica spans 534 bp with
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77.7% AT content with AT and GC skewness (-0.035 and 0.174). In D. silvatica, all the
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conserved elements were present in a consistent manner along with two tandem repeats of
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(TTTTAA). The conserved elements were also detected in all spiders CRs except Liphistius erawan in which TATA motif was absent. The arrangement of these elements was identical in
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most of the species except three species Loxosceles similis, Argyroneta aquatica, Tetragnatha
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nitens, in which tandem repeats were detected between AT-rich region and TATA motif. In few species, the tandem repeats were detected either upstream of AT-rich region or downstream of
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GAT motif (Fig. 2).
3.3 Phylogenetic analyses
Eight phylogenetic trees with two inference methods (BI, ML) using four datasets with the best partition schemes were constructed (Fig. 3). The topologies of all the phylogenies were almost identical (Fig. S4 to S11). The results clearly indicate the monophyly of suborders Opisthothelae, Mesothelae, and infraorder Mygalomorphae. The infraorder Araneomorphae was recovered monophyletic in all the analyses except BI-2, ML-2 (Fig. S5, S9). The observed paraphyly of Araneomorphae in BI-2, and ML-2 (without Gblock excluding 3rd codon) may be due to the
Journal Pre-proof unstable position of ancestral lineages of family Hypochilidae to the other members of spiders. The families of Mygalomorphae (Nemesiidae, Theraphosidae and Euagridae) showed sister relationship to each other. However, the genus Phyxioschema of Mygalomorphae was recently transferred from the family Dipluridae to family Euagridae [72]. The infraorder Araneomorphae have two clades (1) first clade with family Dysderidae, Pholcidae, and Sicariidae (2) second clade with family Dictynidae, Salticidae, Agelenidae, Selenopidae, Oxyopidae, Lycosidae,
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Thomisidae, Tetragnathidae, Araneidae, and Cheiracanthiidae. The family Hypochilidae either
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associated with the first clade of Araneomorphae in four analysis (BI-1, BI-3, ML-1, and ML-3)
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or second clade in two analysis (BI-4, ML-4). The estimated tree from the mitochondrial PCG sequences is well congruent with previously generated multi-locus phylogeny [73]. In both the
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analysis, the ‘Synspermiata’ families Dysderidae, Hypochilidae, Sicariidae are found in sister
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relationship to the ‘Lost Tracheae clade’ family Pholcidae. Further, in the ‘Entelegynae’ taxa, the
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‘Oval Calamistrum clade’ families Salticidae, Selenopidae, Oxyopidae, Lycosidae, Thomisidae are found as sister group to ‘RTA clade’ families Dictynidae and Agelenidae. The previous
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multi-locus phylogeny revealed a close association of Cheiracanthium species with other
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Eutichuridae species [73], however, this family is now considered as a junior synonym of family Cheiracanthiidae [74]. The present mitogenome based phylogeny showed that, Cheiracanthiidae is sister to families Selenopidae and Salticidae in all the analyses with strong posterior probabilities and bootstrap support. The present study suggested that the mitochondrial genome data of different families, especially Clubionidae and Miturgidae are necessary to confirm the phylogenetic position of Cheiracanthiidae. 3.4 Gene arrangement
Journal Pre-proof The features of the gene arrangements are transpositions, inversions, inverse transpositions. The Tandem Duplication-Random Loss (TDRL) is the mechanism to explain the transpositions. The gene order (GO) of C. triviale mitogenome is identical to the majority of spiders families, but differ from the putative ancestor (Limulus polyphemus) (Fig. 4). CREx analysis of C. triviale revealed four transpositions and two TDRL events in comparison to ancestral GO. The transposition of trnC from gene block trnW-trnC-trnY to downstream of trnY occurred in C.
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trivale as compared to ancestor. The transposition of trnA, trnS1 and trnR occurred from gene
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block trnA-trnE and made the new gene boundaries trnA-trnS1, trnN-trnA, trnS1-trnR, and trnR-
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trnE in C. triviale. The transposition of trnL2 occurred from downstream of nad1 to upstream of
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the same gene and made new gene boundaries nad3-trnL2 and trnL2-trnN. CREx analysis of D. silvatica also revealed the four transposition (trnT, trnL2, trnI, trnC) and one TDRL event as
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compared to putative ancestor.The GO of the suborder Mesothelae is similar to putative ancestral
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GO, though they belong to different arthropod classes. Further, the GO of the different spiders families are mostly similar to each other. A very few transpositions, inversions were detected in
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comparison to the putative ancestral GO.
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TreeREx analysis revealed the transpositions of eight tRNAs (trnL2, trnN, trnA, trnS1, trnR, trnQ, trnT, trnY) from the suborder Mesothelae to Opisthothelae at the connecting node of two infra-orders of Opisthothelae (Araneomorphae and Mygalomorphae) (Fig. 4). The inversion of trnI occurred between the taxa of Mygalomorphae in comparison to ancestral GO except Lyrognathus crotalus where trnI is reverted back to the ancestral condition. Further, the transposition of the trnI occurred between the connecting nodes of all the families of Araneomorphae. The transposition of trnI is occurred from CR-trnQ to rrnS-trnQ in family Pholcidae and CR-trnQ to trnM-nad2 in family Dysderidae. This transposition derived the new
Journal Pre-proof gene boundaries trnM-trnI, trnI-nad2 which indicate the synapomorphy for the family Dysderidae. Further the inversion of trnH is observed in all the taxa of family Pholcidae, except Pholcus phalangioides. The inverse-transposition of trnA occurred in Loxosceles similis (Sicariidae) and made new gene boundaries trnQ-trnA, trnA-trnM which indicated the apomorphic character of this taxa. The inversion of trnN occurred at the connecting node of family Dictynidae and Agelenidae, whereas, the trnN undergoes inverse transposition in family
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Dictynidae. The taxa of family Agelenidae is further detected by transposition of three tRNAs
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(trnS1, trnR, trnE), inversion of trnI and inverse transposition of trnN in comparison to ancestor.
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This makes apomorphic character or new gene boundaries nad3-trnS1, trnE-trnL2, trnL2-trnA,
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and trnN-trnF for Agelena silvatica (Agelenidae). The GO of Salticidae taxa are similar to each other except Carrhotus xanthogramma in which transposition of trnE and trnW occurred. The
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transposition of trnE and trnW make the new gene boundaries trnN-trnE, trnE-trnA, trnR-trnF,
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nad4L-trnW, and trnW-trnP, which indicate the apomorphy for C. xanthogramma. Further, the inversion of trnL2 occurred from majority strand to minority strand in S. bursarius. The taxa of
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family Tetragnathidae shared the GO with family Araneidae except Tetragnatha nitens in which
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two inversions of trnY and trnC and one transposition of trnW occurred which makes the new gene boundaries trnQ-trnW, trnW-trnG, trnG-trnM which are apomorphic character for Tetragnatha nitens. 4. Conclusion The complete mitochondrial genome of C. triviale was characterized and compared with other members of the spiders. Moreover, the available mitogenome sequence data of D. silvatica was annotated and characterized. The phylogenetic relationships based on mitochondrial genome data are congruent with morphology and the earlier multi-locus phylogeny. The phylogenetic analysis
Journal Pre-proof indicated the monophyly of infraorder Araneomorphae and Mygalomorphae. The family Cheiracanthiidae showed sister relationship with Salticidae and Selenopidae. The systematic position of the genus Cheiracanthium is always a controversial issue and matter of concern to arachnologists. The genus Cheiracanthium was placed under family Clubionidae without knowing its original placement under family ‘Cheiracanthidae’ [75-76]. Later on, Ramírez et al. in 1997 transferred this genus from Clubionidae to Miturgidae [77] and further revert back in
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Clubionidae by Deeleman-Reinhold in 2001 [78]. Platnick in 2011, transferred the genus under
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family Miturgidae [79]. Ramirez in 2014 again transferred this genus from Miturgidae to
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Eutichuridae [80]. However, Ono and Ogata in 2018 synonymized the family Eutichuridae under
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family Cheiracanthiidae [74]. The systematic position of the genus Cheiracanthium under family Cheiracanthidae, Miturgidae or Clubionidae is still in debate [81-82, 73]. However, in-depth data
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on taxa of Miturgidae and Clubionidae could build up our knowledge towards the rearrangement
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Acknowledgements
na
We are thankful to the Director, Zoological Survey of India, Kolkata, for providing necessary facilities, constant support and encouragement throughout the study. The study is financially
ur
supported by Zoological Survey of India, Kolkata, Ministry of Environment Forest and Climate
Jo
Change under National Faunal Genome Resources (NFGR) Program. Competing Interests.
The authors declare that they have no competing interests. Additional Information Supplementary information accompanies this paper at
Journal Pre-proof Caption of Figures Fig. 1. The CGView of Cheiracanthium triviale mitochondrial genome. . PCGs represented by Royal blue arrows, rRNA genes by turqoise arrows, tRNA genes by orange arrows and CR regions by gray rectangles. Fig. 2. The control region spider mitogenomes including C. triviale. Different colours are used to show the structural elements. Fig. 3. Bayesian Phylogenetic tree (BI-1) inferred by PCGs spider mitochondrial genomes. BI and ML support values of datasets are shown at the nodes by different shape and colors in the following order (BI-1/BI-2/BI-3/BI-4/ML-1/ML-2/ML-3/ML-4). The posterior probabilities
of
(1.0) and bootstrap support (100%) are represented by pink circles. The posterior probabilities
ro
below (0.9-1.0) and bootstrap support (70-100%) are shown by ‘ns’.
Fig. 4. Representation of the output of the TreeREx analysis on Maximum likelihood tree
-p
(ML-1) with gene arrangement. The rearrangements on the branches are given as Transposition (T), Inversion (I), Inverse transposition (iT). Genes on minority strand indicated
lP
re
with red circle and the rearranged genes highlighted with yellow color.
Strand
Location start
-
trnV
-
rrnS
-
trnQ
-
CR
13
Anticodon
Start Codon
Stop Codon
IGN
stop
1013
Jo
rrnL
Size(bp)
ur
Gene
na
Table 1. The annotated mitochondrial genes of C. triviale and its characteristic features. IGN represents (+) values as intergenic nucleotides and (-) values as overlapping regions. CR represents the control region.
1001
1014
1072
59
1067
1793
727
1756
1820
65
1821
2805
985
0 TAC
-6 -38
TTG
0 0
trnM
+
2806
2873
68
CAT
-18
nad2
+
2856
3839
984
trnW
+
3814
3867
54
TCA
-21
trnY
-
3847
3901
55
GTA
-21
trnC
-
3881
3932
52
GCA
0
cox1
+
3933
5447
1515
ATA
not found
23
cox2
+
5471
6133
663
TTG
TAA
-2
trnK
+
6132
6194
63
CTT
-18
trnD
+
6177
6229
53
GTC
-5
ATT
TAA
-26
Journal Pre-proof atp8
+
6225
6380
156
ATT
not found
atp6
+
6377
7045
669
ATG
TAA
3
cox3
+
7049
7834
786
TTG
TAA
-2
trnG
+
7833
7888
56
nad3
+
7881
8234
354
trnL2
-
8205
8262
58
TAA
-6
trnN
+
8257
8330
74
GTG
40
trnA
+
8371
8429
59
TGC
-3
trnS1
+
8427
8476
50
GCT
1
trnR
+
8478
8550
73
TCG
-33
trnE
+
8518
8576
59
TTC
-24
trnF
-
8553
8609
57
GAA
nad5
-
8605
10242
1638
trnH
-
10249
10301
53
nad4
-
10303
11589
1287
nad4l
-
11574
11845
272
trnP
-
11848
11903
56
nad6
+
11916
12347
432
trnI
+
12343
12403
61
cytb
+
12392
13534
trnS2
+
13527
13581
trnT
+
13582
13636
nad1
-
13629
14546
918
L1
-
14527
14595
69
TCC
-8
of
ATT
-p
GTG
ro
TTA
TAA
T(AA)
re
6 1
ATG
not found
ATT
TT(A)
-16 2 12
ATA
TAA
GAT
lP
-30
-5
TGG
1143
-5 -12
ATT
TAA
-8
55
TGA
0
55
TGT
-8 TTT
T(AA)
-20
TAG
Jo
ur
na
-4
Table 2. The annotated mitochondrial genes of D. silvatica and its characteristic features. IGN represents (+) values as intergenic nucleotides and (-) values as overlapping regions. CR represents the control region. Gene
Strand
Location
Size (bp)
start
stop
Anticodon
Start Codon
Stop Codon
IGN
trnM
+
421
482
62
CAT
-17
trnI
+
466
534
69
GAT
8
nad2
+
543
1451
909
trnW
+
1450
1515
66
TCA
-36
trnY
+
1480
1535
56
TAC
-16
trnC
-
1520
1573
54
GCA
-5
cox1
+
1569
3110
1542
ATA
ATA
TAG
TAA
-2
52
Journal Pre-proof cox2
+
3163
3804
642
trnK
+
3805
3865
61
AAA
-47
trnD
+
3819
3903
85
GTC
-8
atp8
+
3896
4045
150
ATA
TAA
-4
atp6
+
4042
4704
663
ATA
TAG
30
cox3
+
4735
5493
759
ATT
TAA
-2
trnG
+
5492
5556
65
nad3
+
5538
5873
336
trnL2
-
5858
5913
56
TTG
TAG
TCC
0
-19 ATT
TAA
-16
TTA
81 -12
5994
81
trnN
5995
6059
65
AAC
trnA
+
6048
6094
47
GCA
trnS1
+
6058
6117
60
AGC
trnR
+
6104
6165
62
CGA
-15
trnE
+
6151
6203
53
TTC
-24
trnF
-
6180
6244
65
nad5
-
6190
7858
1669
trnH
-
7887
7935
nad4
-
7934
9211
nad4l
-
9201
9461
261
trnP
-
9461
9518
58
nad6
+
9522
9971
450
ATG
TAA
cytb
+
9964
11076
1113
ATA
T(AA)
trnS2
+
11078
11130
53
TCA
4
trnT
+
11135
11183
49
ACA
-8
nad1
-
11176
12078
903
trnL1
-
12076
12143
68
rrnL
-
12095
13150
1056
trnV
-
13118
13174
57
rrnS
-
13174
13847
674
trnQ
-
13847
13906
60
13907
14440
1
420
-p
ro
-14
re
GAA
-55 ATT
T(AA)
GTG ATC
TAA
ATT
not found
na
28 -2
lP
49
-37
1278
ur
Jo
CR
of
5914 +
CR
CCA
-11 -1 3
ATA CTA
TAA
-8 1
-3 -49 -33
GTA
-1 -1
TTG
954
Author statement
Kaomud Tyagi, Vikas Kumar: Conceptualization, Methodology, Software
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Journal Pre-proof Kaomud Tyagi, Nikita Poddar, Shantanu Kundu: Data curation, Writing- Original draft preparation. Nikita Poddar, Priya Prasad: Visualization, Investigation. Vikas Kumar, Kailash Chandra: Supervision. Kaomud Tyagi, Inderjeet Tyagi: Software, Validation. Kaomud Tyagi, Vikas Kumar: Writing- Reviewing and Editing
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Highlights The complete mitogenome of spider family Cheiracanthiidae was sequenced and annotated for the first time.
There were nineteen mitochondrial gene rearrangement types among 44 spider species.
New gene boundaries as derived characters were recovered as a result of gene arrangements.
Accelerated gene rearrangement is observed in Araneomorphae as compared to Mygalomorphae.
Phylogenetic position of family Hypochilidae is unstable and needs further studies.
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