Novel gene rearrangement in the mitochondrial genome of Coenobita brevimanus (Anomura: Coenobitidae) and phylogenetic implications for Anomura

Journal Pre-proof Novel gene rearrangement in the mitochondrial genome of Coenobita brevimanus (Anomura: Coenobitidae) and phylogenetic implications for Anomura

Li Gong, Xinting Lu, Zhifu Wang, Kehua Zhu, Liqin Liu, Lihua Jiang, Zhenming Lü, Bingjian Liu PII:

S0888-7543(19)30523-3

DOI:

https://doi.org/10.1016/j.ygeno.2019.10.012

Reference:

YGENO 9381

To appear in:

Genomics

Received date:

6 August 2019

Revised date:

17 October 2019

Accepted date:

18 October 2019

Please cite this article as: L. Gong, X. Lu, Z. Wang, et al., Novel gene rearrangement in the mitochondrial genome of Coenobita brevimanus (Anomura: Coenobitidae) and phylogenetic implications for Anomura, Genomics (2018), https://doi.org/10.1016/ j.ygeno.2019.10.012

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© 2018 Published by Elsevier.

Journal Pre-proof Novel gene rearrangement in the mitochondrial genome of Coenobita brevimanus (Anomura: Coenobitidae) and phylogenetic implications for Anomura Li Gong1,2,3, Xinting Lu1,2, Zhifu Wang4, Kehua Zhu1,2, Liqin Liu1,2, Lihua Jiang1,2, Zhenming Lü1,2, Bingjian Liu1,2 1. National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang Ocean University, 316022, Zhoushan, China; 2. National Engineering Research Center for Facilitated Marine Aquaculture, Marine Science and Technology College, Zhejiang Ocean University, 316022, Zhoushan, China; 3. Guangxi Key Lab for Marine Biotechnology, Guangxi Institute of Oceanography, Guangxi Academy of Sciences, Beihai, Guangxi, 536000, China; 4. Key Laboratory of Engineering Oceanography, Second Institute of Oceanography, Ministry of Natural Resources, 310000, Hangzhou, China.

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Abstract The complete mitochondrial genomes (mitogenomes) can indicate

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phylogenetic relationships among organisms, as well as useful information about the process of molecular evolution and gene rearrangement mechanisms. However,

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knowledge on the complete mitogenome of Coenobitidae (Decapoda: Anomura) is

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quite scarce. Here, we describe in detail the complete mitogenome of Coenobita brevimanus, which is 16,393 bp in length, and contains 13 protein-coding genes, two

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ribosomal RNA, 22 transfer RNA genes, as well as a putative control region. The genome composition shows a moderate A+T bias (65.0%), and exhibited a negative

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AT-skew (-0.148) and a positive GC-skew (0.183). Five gene clusters (or genes) involving eleven tRNAs and two PCGs were found to have rearranged with respect to

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the pancrustacean ground pattern gene order. Duplication-random loss and recombination models were determined as most likely to explain the observed largescale gene rearrangements. Phylogenetic analysis placed all Coenobitidae species into one clade. The polyphyly of Paguroidea was well supported, whereas the nonmonophyly of Galatheoidea was inconsistence with previous findings on Anomura. Taken together, our results help to better understand gene rearrangement process and the evolutionary status of C. brevimanus and lay a foundation for further phylogenetic studies of Anomura. Keywords: Terrestrial hermit crab; Mitogenome; Gene rearrangement; Phylogenetic analysis 

Corresponding author. E-mail address: [email protected] (L. Gong)；[email protected] (B. Liu)

 These authors contributed equally to this paper. 1

Journal Pre-proof 1. Introduction The metazoan mitochondrial genome (mitogenome) is usually 14-20 kb in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (12S and 16S) and one control region (CR, also called D-loop region). The mitogenome can provide useful information on rearrangement trends and phylogenetic analyses mainly because of its conserved gene content, small size, and rapid evolutionary rate [1]. Nowadays, complete mitogenome sequence is becoming

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increasingly common used for population genetic, comparative genomic, molecular

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evolution, and phylogenetic research.

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Generally, gene arrangement in vertebrate mitogenomes is relatively conserved and

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fewer gene arrangements are discovered [2,3,4,5]. In contrast, gene arrangements in invertebrate mitogenomes are relatively common. For example, abundant gene

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rearrangement is evident in sea cucumbers [6], cephalopods [7], bivalves [8], insects

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[9], and crabs [10]. Although a gradually growing number of gene rearrangement in hermit crabs has been discovered [11,12,13,14], it is surprising that gene

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rearrangements in terrestrial hermit crab were overlooked until 2018, when it was found that large-scale gene rearrangements existed in the mitogenome of this group, different from those reported in other crabs [15]. Mitochondrial gene rearrangements have been demonstrated to effectively resolve relationships among various animal groups [15,16,17,18], which provide an additional source for phylogenetic studies. Terrestrial hermit crabs belong to the family Coenobitidae of Anomura, which includes only two genera: the land hermit crab genus Coenobita Latreille, 1829, with 16 species, and the coconut crab genus Birgus Leach, 1816, with only one species, B. latro (Linnaeus, 1767) [19]. It has been indicated that among the 16 Coenobita species, C. carnescens Dana, 1851 could be in fact the juvenile form of C. perlatus 2

Journal Pre-proof Edwards, 1837, and that C. olivieri Owen, 1839 is only a variety of C. spinosus Edwards, 1837 [20,21]. Complete mitogenomes have been commonly used for species identification, especially for the controversial classifications which possess similar morphology. However, there has been only one report on the complete mitogenome of Coenobitidae so far [15]. Coenobita brevimanus Dana, 1852 (Anomura: Coenobitidae) occurs primarily in Indo-West Pacific coastal regions [22,23] but are present in many countries because

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of ornamental tourism [24]. This species and its congeners have been severely

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depleted on most inhabited islands because of over-harvest and habitat degradation. It

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was listed as “vulnerable” under the World Conservation Union (IUCN) Red List and

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was regarded as natural monument in Japan [25,26]. Like other terrestrial hermit crabs, although C. brevimanus is fully terrestrial, the females return to the sea to hatch their

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eggs and their larvae develop through planktonic zoeal stages to a megalopa [27,28],

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which is similar to the life cycle of other marine hermit crab species. So far, most of the studies have focused on the morphology and reproduction [28,29]. However, there

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is less information about its molecular characteristic [20,30]. Although a previous complete mitogenome of C. brevimanus has been published (GenBank accession number KY352233), the authors hardly described the mitogenome features. Herein, the complete mitogenome of a new C. brevimanus individual was sequenced and described in detail. Large-scale gene rearrangements were found in this mitogenome and possible rearrangement mechanisms were discussed. The available complete mitogenomes of Anomura retrieved from the Genbank database were used to provide insight into the phylogenetic relationship of C. brevimanus and related species (Table 1). These results will help to better understand the gene arrangement features of

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Journal Pre-proof coenobitid crab mitogenomes and lay the foundation for further phylogenetic study of Anomura.

2. Materials and methods 2.1. Sampling, DNA extraction, PCR amplification and sequencing An individual specimen of C. brevimanus was collected in Taiwan, China (23°30'8"N, 120°52'53"E). A portion of the epaxial musculature was excised and the

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total genomic DNA was extracted using the SQ Tissue DNA Kit (OMEGA) following

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the manufacturer’s protocol. The mitogenome of C. brevimanus was sequenced by

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next-generation sequencing paired reads of length 150 bp (Illumina HisSeq 4000;

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Shanghai Origingene Bio-pharmTechnology Co. Ltd. China). Raw sequence data were deposited in Short Read Archive (SRA) database

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(https://www.ncbi.nlm.nih.gov/sra/) with the accession no. PRJNA526588. Clean data

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without sequencing adapters were de novo assembled by the NOVOPlasty 2.7.2 software [31]. We compared the assembled genome with three confirmed sequences

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(COI, 12S and 16S rRNA) by PCR and Sanger sequencing methods to evaluate the single-base accuracy. The PCR was carried out in a 25 ul reaction volume containing 2.0 mM MgCl2, 0.4 mM of each dNTP, 0.5 uM of each primer, 1.0 U of Taq polymerase (Takara, China), 2.5 ul of 10 × Taq buffer, and approximately 50 ng of DNA template. PCR cycling conditions included an initial denaturation at 95 °C for 2 min, 45 cycles at 95 °C for 30 seconds, an annealing temperature at 52 °C for 2.5 min, elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min. 2.2 Sequence annotation and analysis The complete mitogenome was annotated using the software of Sequin (version 15.10, http://www.ncbi.nlm.nih.gov/Sequin/). The boundaries of ribosomal RNA 4

Journal Pre-proof genes were performed using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov). Transfer RNA genes and their potential cloverleaf structures were identified using tRNAscanSE [32]. The gene map of the C. brevimanus mitogenome was generated using CGView Server V 1.0 [33]. The base composition and the relative synonymous codon usage (RSCU) was obtained using MEGA 7.0 [34]. Strand asymmetry was calculated using the formulae: AT-skew=(A-T)/(A+T); GC-skew=(G-C)/(G+C) [35].

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2.3 Phylogenetic analyses

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Thirty-eight anomura mitogenomes were downloaded from the GenBank (https://www.ncbi.nlm.nih.gov/genbank/) for phylogenetic analysis, as well as two

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Portunidae species to serve as the outgroup (Table 1). The 13 PCGs were

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concatenated for phylogenetic analysis. Sequences were aligned using Clustal X 2.0 [36] with the default parameters and manually checked with BioEdit [37]. The

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concatenated sequences were used for maximum likelihood (ML) analyses

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implemented in RAxML version 8.0.0 [38], and Bayesian inference (BI) was conducted in MrBayes 3.2.6 [39]. The best-fit evolutionary models were determined using Modeltest 3.7 from 56 models for ML analyses [40] and MrModeltest 2.2 from 24 models for BI analyses [41]. A general time-reversible model with sites following a discrete gamma distribution (GTRGAMMA) was used. A rapid bootstrap (BS) analysis was conducted with 200 replications, and the software produced a bestscoring ML tree with BS probabilities. The Bayesian analysis ran as 4 simultaneous MCMC chains for 2,000,000 generations, sampled every 100 generations, and a burnin of 500,000 generations was used. To guarantee the stationarity had been reached, the average standard deviation of split frequencies was set below 0.01. 5

Journal Pre-proof Table 1. List of 31 Anomura and two Brachyura species used in this paper Family

Superfamily

Length (bp)

Accession No.

Reference

Lithodes nintokuae Paralithodes camtschaticus Paralithodes brevipes Pagurus japonicus Pagurus filholi Pagurus minutus Pagurus gracilipes Pagurus nigrofascia Pagurus lanuginosus Pagurus maculosus Pagurus sp. Pagurus longicarpus Pylocheles mortensenii Lomis hirta Aegla aff. longirostri Kiwa tyleri Gastroptychus roger Gastroptychus investigatoris Stemonopa insignis Clibanarius infraspinatus Birgus latro Coenobita brevimanus Coenobita brevimanus Coenobita perlatus Coenobita variabilis Coenobita rugosus Petrolisthes haswelli Neopetrolisthes maculatus Shinkaia crosnieri Munida gregaria Munida isos Tubuca polita Tubuca capricornis

Lithodidae Lithodidae Lithodidae Paguridae Paguridae Paguridae Paguridae Paguridae Paguridae Paguridae Paguridae Paguridae Pylochelidae Lomidae Aeglidae Kiwaidae Chirostylidae Chirostylidae Albuneidae Diogenidae Coenobitidae Coenobitidae Coenobitidae Coenobitidae Coenobitidae Coenobitidae Porcellanidae Porcellanidae Munidopsidae Munididae Munididae Ocypodidae Ocypodidae

Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Lomoidea Aegloidea Kiwaoidea Galatheoidea Galatheoidea Hippoidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Paguroidea Galatheoidea Galatheoidea Galatheoidea Galatheoidea Galatheoidea Ocypodoidea Ocypodoidea

15731 16720 16303 16401 15674 14939 16051 15423 14632 15420 14648 15630 15093 17239 15387 16865 16504 16423 15596 16504 16411 16390 16393 16447 16421 16427 15348 15324 15182 16326 17910 15672 15629

NC_024202 NC_020029 NC_021458 LC222532 LC222528 LC222533 LC222534 MH756635 LC222527 LC222524 LC222535 NC_003058 KY352242 KY352239 MF457407 NC_034927 KY352238 KY352237 KY352240 NC_025776 KY352241 KY352233 MK310257 KY352234 KY352236 KY352235 NC_025572 NC_020024 NC_011013 NC_030255 NC_039112 MF457400 MF457401

unpublished [12] unpublished [13] [13] [13] [13] [11] [13] [13] [13] [14] [15] [15] [15] [42] [15] [15] [15] [43] [15] [15] This study [15] [15] [15] [44] [45] [46] [47] [15] [15] [15]

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Species

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3. Results and discussion 3.1. Genome structure and composition Although the newly determined complete mitogenome of C. brevimanus is almost identical with (98.6% similarity) the published one (GenBank accession number KY352233), the authors mainly focused on the phylogenetic analyses, while hardly described the mitogenome features [15]. Hence, we described the complete

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mitogenome of a new individual (MK310257) in detail and focused on the process

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and mechanism of gene rearrangements. The sequence is 16,393 bp in length, almost

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the same length with that of the published one (16,390 bp). It comprises 13 PCGs, 22

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tRNAs, two rRNAs and one CR (Fig. 1, Table 2), which is identical with that in most crabs [11,15,48]. The size of C. brevimanus mitogenome presented in this study falls

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within the range of other Anomura mitogenomes from 14,632 bp in Pagurus

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lanuginosus (LC222527) to 17,910 bp in Munida isos (NC_039112). The overall nucleotide composition is 27.7% A, 37.3% T, 20.7% G, and 14.3% C, respectively

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(Table 3). The AT-skew and GC-skew are -0.148 and 0.183, respectively (Table 3), suggesting an obvious bias toward the use of Ts and Gs.

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Fig. 1. Gene map of the Coenobita brevimanus mitogenome.

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Journal Pre-proof Table 2. Features of the mitochondrial genome of Coenobita brevimanus Position Gene

From

Length Amino Start/Stop (bp) acid codon

To

Anticodon

Intergenic region*

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ATG/TAA COI 1 1539 1539 512 -5 TAG Leu(L2) 1535 1600 66 7 ATG/TAG COII 1608 2297 690 229 9 TTT Lys(K) 2307 2371 65 7 CAT Met(M) 2379 2445 67 11 GAT Ile(I) 2457 2522 66 16 ATT/T ND2 2539 3574 1036 345 0 GTC Asp(D) 3575 3639 65 0 ATC/TAG ATP8 3640 3798 159 52 -7 GTG/TAA ATP6 3792 4466 675 224 -1 ATG/TAA COIII 4466 5257 792 263 15 TCG Arg(R) 5273 5333 61 0 GTT Asn(N) 5334 5399 66 5 TTC Glu(E) 5405 5470 66 3 GAA Phe(F) 5474 5538 65 13 ATA/TAA ND5 5552 7249 1698 565 18 GTG His(H) 7268 7334 67 48 ATG/TAA ND4 7383 8723 1341 446 -7 TTG/TAA ND4L 8717 9001 285 94 20 TGT Thr(T) 9022 9089 68 10 GTG/TAA ND6 9100 9633 534 177 -17 ATA/T Cyt b 9617 10748 1132 377 0 TGA Ser(S2) 10749 10814 66 -1 TGG Pro(P) 10814 10879 66 1 ATC/TAA ND1 10881 11807 927 308 0 16S 11808 13217 1410 0 TAC Val(V) 13218 13285 68 0 12S 13286 14082 797 0 CR 14083 15459 1377 0 TCT Ser(S1) 15460 15525 66 4 TGC Ala(A) 15530 15593 64 16 ATG/TAG ND3 15610 15960 351 116 0 TCC Gly(G) 15961 16026 66 3 TAA Leu(L1) 16030 16095 66 -1 GTA Tyr(Y) 16095 16161 67 5 TCA Trp(W) 16167 16235 69 14 TTG Gln(Q) 16250 16319 70 3 GCA Cys(C) 16323 16389 67 0 *Intergenic region: non-coding bases between the feature on the same line and the line below, with a negative number indicating an overlap.

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Strand H H H H H H H H H H H H H H L L L L L H H H H L L L L L H L L L L L H L L L

Journal Pre-proof Table 3. Composition and skewness of Coenobita brevimanus mitogenome

3.2 PCGs, tRNAs, rRNAs, and CR

A+T% 65.0 63.7 61.4 62.9 65.5 69.2 66.6 62.5 63.3 63.1 61.8 68.1 63.9 63.7 62.1 67.4 72.5 69.0 62.0

AT-skew -0.148 -0.221 -0.326 -0.291 -0.438 -0.145 -0.378 -0.366 0.014 -0.005 -0.181 -0.319 -0.383 -0.080 -0.072 -0.009 0.049 0.076 -0.031

GC-skew 0.183 0.041 0.264 0.283 0.256 0.390 0.201 0.221 -0.275 -0.214 -0.211 0.248 0.175 -0.179 -0.261 0.145 0.052 0.036 0.042

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C% 14.3 17.4 14.2 13.3 12.8 9.4 13.3 14.6 23.4 22.4 23.2 12.0 14.9 21.4 23.9 13.9 13.0 14.9 18.2

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G% 20.7 18.9 24.4 23.8 21.6 21.4 20.0 22.9 13.3 14.5 15.1 19.9 21.2 14.9 14.0 18.6 14.5 16.1 19.8

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T% 37.3 38.9 40.7 40.6 47.1 39.6 45.9 42.7 31.2 31.7 36.5 44.9 44.2 34.4 33.3 34 34.5 31.9 31.9

Length(bp) 16393 11159 1539 690 1036 159 675 792 1698 1341 285 534 1132 927 351 1457 1410 797 1377

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A% Mitogenome 27.7 PCGs 24.8 COI 20.7 COII 22.3 ND2 18.4 ATP8 29.6 ATP6 20.7 COIII 19.8 ND5 32.1 ND4 31.4 ND4L 25.3 ND6 23.2 Cyt b 19.7 ND1 29.3 ND3 28.8 tRNAs 33.4 16S 38.0 12S 37.1 CR 30.1

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The mitogenome of C. brevimanus contains 13 PCGs, with a total length of 11,159

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bp. Eight PCGs (COI, COII, ND2, ATP8, ATP6, COIII, ND6 and Cyt b) are encoded on the heavy strand (H-strand), while the rest (ND5, ND4, ND4L, ND1, and ND3) are

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encoded on the light strand (L-strand). Typically, the ND3 gene is encoded on the Hstrand. Interestingly, it is inverted to the L-strand, which to our best knowledge, is a quite rare phenomenon only occurring in Coenobitidae mitogenomes [15]. Totally, it encodes 3,708 amino acids. The most frequently used amino acids are Leu (15.6%), Phe (9.0%), Ile (8.2%) and Val (7.6%), while the least common amino acids are Cys (1.1%), Arg (1.6%), Gln (1.9%), and Asp (1.9%) (Fig. 2A). Relative synonymous codon usage (RSCU) values for the third positions of the 13 PCGs is shown in Fig. 2B. The usage of both two- and four-fold degenerate codons is biased toward the use of codons abundant in T or A, in accord with other crabs. The AT content of the 13

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Journal Pre-proof PCGs is 63.7%. The AT-skew and GC-skew are -0.221 and 0.041, respectively (Table 3). Like most crab mitogenomes, the C. brevimanus mitogenome contains a set of 22 tRNA genes [11,49,50]. In most crab mitogenomes, eight tRNAs are encoded on the L-strand and the other 14 tRNAs are encoded on the H-strand [11,49,50]. However, the number of tRNAs encoding on the two strands is equal for the C. breviamanus mitogenome (Fig. 1, Table 2). The tRNA genes range in size from 61 bp (Arg) to 70

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bp (Gln) and the total length of them is 1,457 bp (Tables 2, 3). It shows a moderate

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AT bias (67.4%), a slight skew of T versus A (AT-skew = -0.009), and strong skew of

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G versus C (GC-skew = 0.145) (Table 3). The 16S rRNA is 1,410 bp between ND1

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and Val while 12S rRNA is 797 bp between Val and CR (Fig. 1, Table 2). The ATskew (0.049 and 0.076, respectively) and GC-skew (0.052 and 0.036, respectively) of

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the two rRNA genes were both positive (Table 3), indicating clearly that more As and

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Gs than Ts and Cs in rRNAs. The CR is located between 12S rRNA and Ser1, with a slight AT bias (62.0%). The AT-skew and GC-skew is -0.031 and 0.042, respectively

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(Table 3), indicating an obvious bias toward the use of Ts and Gs.

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Fig. 2. Amino acid composition in C. brevimanus mitogenome (A); Relative synonymous codon usage in C. brevimanus mitogenome (B).

3.3 Gene rearrangement The gene arrangement in the complete mitogenome of C. brevimanus is shown in Fig. 3. Compared with the gene order in ancestral crustaceans (the pancrutacean ground pattern) mitogenomes [51], the gene order in C. brevimanus mitogenome undergoes a large-scale rearrangement. In total, at least five gene clusters (or genes) dramatically differ from the typical order, involving eleven tRNA genes (G, A, S1, P, L1, I, Q, M, W, C, Y), and two PCGs (ND3 and ND2). If not considering these gene 12

Journal Pre-proof rearrangements, the gene order COI- L2- COII- K- A- ATP8- ATP6- COIII- R- N- EF- ND5- H- ND4- ND4L- T- ND6- Cyt b- S2- ND1-16S -V- 12S- CR remains the same arrangement as that in ancestral crustaceans. Of these five gene rearrangements, GND3- A- S1 cluster is inverted from the downstream of COIII in the H-strand to downstream of the CR in the L-strand (Fig. 3①). A single P moves from the downstream of T to downstream of the S2 (Fig. 3②). A single L1 moves to the position between S1- A- ND3- G cluster and Y- W- Q- C cluster, which is located downstream

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the CR and forms a large-scale rearranged area (Fig. 3③). I- Q- M- ND2 cluster is

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divided into two sections, one (I, M and ND2) is shifted to downstream of K. The

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other (Q) is shifted to the end of linear mitogenome (Fig. 3④).The W- C- Y cluster

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order is changed into Y- W- C order, accompanied with W and Y inversion (Fig. 3⑤). Several common used mechanisms have been proposed to explain the

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mitochondrial gene rearrangements, including duplication-random loss [52],

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duplication-nonrandom loss [53] and recombination [54]. Here, we propose that duplication-random loss and recombination result in the generation of the C.

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brevimanus mitogenome. Firstly, three gene clusters undergo a complete copy, forming three dimeric blocks, (G- ND3- A- R- N- S1- E)- (G- ND3- A- R- N- S1- E) (Fig. 4A①), (I- Q- M- ND2)- (I- Q- M- ND2) (Fig. 4A②), and (W- C- Y)- (W- C- Y) (Fig. 4A③). Consecutive copies are then followed by a random loss of the duplicated genes. G- ND3- A- R- N- S1- E- G- ND3- A- R- N- S1- E, I- Q- M- ND2- I- Q- M- ND2, and W- C- Y- W- C- Y (underline denotes the deleted gene). Then three new gene blocks are formed, (G- ND3- R- N- E- A- S1), (Q- M- I- ND2), and (Y- W- C) (Fig. 4A). Tandem duplication followed by random loss has been widely used to explain this type of translocation of mitochondrial genes [11,55,56], hence, we ascertain that duplication-random loss model is the most likely explanation for these three gene 13

Journal Pre-proof block rearrangements. Subsequently, the two new gene blocks undergo a translocation. (G- ND3- R- N- E- A- S1) block is translocated downstream to the CR, leaving R- N- E in the original position. (Q- M- I- ND2) block is translocated to the K and D junction (Fig. 4A). According to the reported rearrangements [11,49,57], two independent recombination events seem to be the most plausible explanation for these translocations. In the second step, four genes or gene clusters are translocated (Fig. 4B). Q is translocated to the position of W and C junction (Fig. 4B④), P is translocated

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to the downstream of S2 (Fig. 4B⑤), L1 is translocated to the downstream of S1 (Fig.

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4B⑥), G- ND3- A- S1 order is reversed to S1- A- ND3- G in the original position (Fig.

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4B⑦). Also, recombination events appear to account for these translocations. Finally,

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the ultimate gene arrangement of the C. brevimanus mitogenome is shown in Fig. 4C.

Fig. 3. Gene rearrangements in C. brevimanus mitogenome. PCGs and CR are indicated with boxes, and tRNAs are indicated with columns. Genes labeled above the diagram are encoded on the H-strand and those below the diagram on the L-strand. The gene rearrangement steps are labeled with Figs. (A) The ancestral gene arrangement of crustaceans; (B) The gene order in the C. brevimanus mitogenome.

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Fig. 4. Inferred intermediate steps between the ancestral gene arrangement of crustaceans and C. brevimanus mitogenome. (A) Duplication-loss and translocation in the ancestral mitogenome of crustaceans. The duplicated gene block is boxed in dash and the lost genes are labeled with gray. (B) Translocation. (C) The final gene order in the C. brevimanus mitogenome.

3.4 Phylogenetic analysis

To further investigate the phylogenetic relationships of Anomura and the position of C. brevimanus, two phylogenetic trees (ML tree and BI tree) were constructed based on the nucleotide sequences of the 13 concatenated PCGs. In this study, both trees were largely congruent with each other except for few negligible differences in the relationship within Lithodidae; consequently, only the BI topology wass shown, but both the ML bootstrap values and BI posterior probabilities were shown (Fig. 5). It was obvious that two C. brevimanus species clustered together and four Coenobita

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Journal Pre-proof species formed a clade. The largest terrestrial crab, Birgus latro, had the closest relationship with Coenobita, and formed a Coenobitidae clade with high support value. The current phylogenetic analysis of Anomura recovered a polyphyletic Paguroidea, in concordance with previous studies [58,59,60], with the Coenobitidae + Diogenidae clade dissociated from the other paguroids (Lithodidae + Paguridae + Pylochelidae). The Coenobitidae + Diogenidae clade (Coenobita +Birgus + Clibanarius) was similar to what was reported by McLaughlin et al. [58] based on morphological characters

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and by Tan et al. [15] based on the amino acid dataset of 13 PCGs. While the other

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paguroids clade (Lithodidae + Paguridae + Pylochelidae) differed from most

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morphological results [58,61,62] and Tan et al.’s [15] molecular result. In these

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studies, Lithodidae was excluded from Paguroidea and belonged to a new superfamily Lithodoidea. The phylogenetic tree also recovered polyphyletic groups for

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Galatheoidea. In this study, Galatheoidea consisted of two clades: (Porcellanidae +

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Munidopsidae + Munididae) formed a clade and dissociated from the single Chirostylidae clade. This result was consistent with Tan et al.’s [15] phylogenetic

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relationship. However, in Tan et al.’s [15] opinion, they treated Chirostylidae as a new superfamily (Chirostyloidea), while not the previous Galatheoidea [58,63,64]. Hence, Galatheoidea formed a monophyletic group in their findings [15].

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Fig. 5. Phylogenetic tree of Anomura species inferred from the 13 PCGsbased on Bayesian inference (BI) and maximum likelihood (ML) analysis. * at each node indicates 100% supporting

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value and the number indicates the maximum likelihood bootstrap value. The number after the species name is the GenBank accession number. Superfamilies as recognized by McLaughlin et al.

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[58]

4. Conclusion

In this study, we determined and described the complete mitogenome of C. brevimanus, which contained 37 genes and a putative control region, as is typical of metazoan mitogenomes. However, the gene order of this species underwent a largescale gene rearrangements compared with that in the ancestral crustaceans. Five gene clusters (or genes) involving eleven tRNAs and two PCGs were found to have rearranged with respect to the pancrustacean ground pattern gene order. The observed large-scale gene rearrangements could be explained by the duplication-random loss and recombination models. Our phylogenetic trees both showed similarities and 17

Journal Pre-proof controversies with the previous studies. Mitogenome gene rearrangements could provide insights into phylogeny where tree-based relationships are poorly resolved or in conflict. With the growing number of rearranged mitogenomes, it might provide potential phylogenetic markers in future studies. Acknowledgements This work was supported by the Basic Scientific Research Operating Expenses of Zhejiang Provincial Universities (2019J00022) and Scientific Research Foundation

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for the Introduction of Talent of Zhejiang Ocean University.

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