Introduction

Brachyura crab is the largest clade in the Decapod crustacean group, with more than 7250 known species, including 98 families of marine, freshwater, and terrestrial habitats, most of which are economically important1. However, the phylogenetic relationships among members of Brachyura and their evolutionary origin continue to be controversial due to the high morphological similarity and ecological diversity2,3,4. Initially, Brachyura was divided into Podotremata, Heterotremata, and Thoracotremata5. Subsequently, it was segmented into Dromiacea and Eubrachyura (including Thoracotremata, Raninoida, and Heterotremata)6. However, the latest classification scheme divides Brachyura into Cyclodorippoida, Eubrachyura, Dromicea, and Raninoida7,8. Although the phylogenetic relationship within Brachyura is still uncertain, the current classification system has been recognized by most scholars.

According to WoRMS (http://www.marinespecies.org/), the family Grapsidae has 8 genera and 49 species in total. However, only five species sequences of Grapsidae have been published4,9,10,11,12. The herbivorous crab (Grapsus albolineatus) is one of the marine crustaceans that live on rocky shores which belongs to the phylum Arthropod, subphylum Crustacea, order Decapoda, infraorder Brachyura, clade Thoracotremata, family Grapsidae, genus Grapsus. They are mainly distributed in Japan, Hawaii, Australia and China’s Guangdong, Hainan Island, Xisha Islands, Taiwan. So far, most studies of this species have focused on the morphology and growth13,14. Although there are few studies on the molecular level, most of them were based on partial mitochondrial and nuclear ribosomal RNA gene sequences15.

The mitochondrial genome (mitogenome) of metazoans is usually 14–20 kb in size and encoded with a set of 37 genes, including 13 protein coding genes (cox1-3, cob, nad1-6, nad4L, atp6, and atp8), 2 ribosomal RNA genes (rrnl and rrns), 22 transport RNA genes (tRNAs), and an AT-rich region (also called control region, CR) which contains some initiation sites for transcription and replication of the genome16. Mitochondrial DNA forms a separate unit of genetic information that evolved independently from the nuclear genome. Due to its haploid properties, matrilineal inheritance, limited recombination, and rapid rate of evolution17, the mitogenome is increasingly being used in evolutionary and phylogenetic studies. With the rapid development of sequencing technology, next-generation sequencing has become a fast and low-cost method to provide complete mitotic genomes18.

Gene rearrangements in the mitogenomes of crabs are relatively common1,19,20. So far, several hypotheses have been suggested to help explain gene rearrangements in animal mitogenomes. Recombination model and tandem duplication/random loss (TDRL) model are more commonly accepted. Recombination models are involved in the breaking and reconnecting of DNA strands21. The TDRL model assumes that the rearranged gene order occurs via tandem duplications followed by random deletion of certain duplications22. This model has been widely used to explain the translocation of genes encoded on the same strand23. Model tRNA mis-priming model and the tandem duplication/non-random loss model (TDNL) are less commonly used.

In this study, we successfully sequenced the complete mitogenome of G. albolineatus and used existing complete mitogenomes to compare it with other Brachyura species. In addition, a phylogenetic analysis of 70 brachyuran species was conducted based on the nucleotide sequences of 13 PCGs (Protein-coding gene). These results will help us to understand features of the G. albolineatus mitogenome and the evolutionary relationships within Brachyura.

Results and discussion

Genome structure and composition

The complete mitogenome sequence of G. albolineatus is a typical closed-circular molecule of 15,583 bp in size (GenBank accession number MZ262276), which is similar in length to the published Grapsidae mitogenomes4,9,10,11,12, a size range from 15,406 to 15,920 bp (Table 1). The mitogenome contents of G. albolineatus is the same as most other published Brachyura which includes 37 genes, 13 PCGs, 22 tRNAs, and 2 rRNA (rrnl and rrns), as well as a brief non-coding region, all the genes were identified (Fig. 1, Table 2). Most of the 37 genes are located on the heavy (H-) strand, except 4 PCGs (ND5, ND4, ND4L, ND1), 8 tRNAs (tRNA-Cys, Tyr, Gln, Val, Leu, Pro, Phe, and His), and 2 rRNA which are located on the light (L-) strand (Fig. 1, Table 2). There are 13 regions with overlap in the total G. albolineatus mitogenome, with 3 of them more than 10 bp (trnT (41 bp), trnL1 (25 bp), and cox2/trnS2 (20 bp)) and the other 10 shorter than 10 bp (nad4 (7 bp), atp8 (4 bp), cox3/atp6/rnK/nad6/trnW (1 bp), trnG (3 bp), and nad3/nad2 (2 bp)) (Table 2). The G. albolineatus mitogenome also contains 328 bp of intergenic spacers located in 17 regions, ranging from 1 to 122 bp (Table 2) and indicating the occurrence of tandem duplications and the deletions of redundant genes. GC-skew of the complete mitogenomes of 6 Grapsidae species were calculated and compared (Tables 3, 4). The nucleotide composition of the G. albolineatus mitogenome is A (33.4%), T (34.04%), G (12.02%), and C (20.54%), with a high A–T bias. The A + T (%) content of the mitogenomes was 66.74%. The AT-skew and GC-skew value are calculated for the chosen complete mitogenomes (Table 3). Both AT-skew and GC-skew of the G. albolineatus mitogenome are slightly negative, −0.009 and −0.262, informing T’s and C’s are more abundant than A’s and G’s. Similar results were observed for the other selected Grapsidae mitogenomes. In general, the AT-skew and GC-skew of the overall mitogenomes, nucleotide composition, and gene lengths of the G. albolineatus were the same as those of the other Grapsidae species4,9,10,11,12.

Table 1 List of Brachyuran species with their GenBank accession numbers.
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Figure 1
figure 1

Circular mitogenome map of Grapsus albolineatus. Protein coding, ribosomal, and tRNA genes are shown with standard abbreviations. Arrows indicate the orientation of gene transcription. The inner circles show the G–C content and GC-skew, which are plotted as the deviation from the average value of the entire sequence.

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Table 2 Nucleotide composition and skewness of Grapsus albolineatus mitochondrial genome.
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Table 3 Organization of the Grapsus albolineatus mitochondrial genome.
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Table 4 Nucleoride composition in regions of the mitogenomes of six Grapsidae species.
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PCGs and codon usage

The initial and terminal codons of all PCGs of G. albolineatus are listed in Table 2. G. albolineatus has 13 PCGs in the typical order found in Brachyuran species, containing 7 NADH dehydrogenase (nad1-nad6, nad4L), 3 cytochrome c-oxidases (cox1–cox3), two ATPases (atp6, atp8), and cytochrome b (cob). The total length of the 13 PCGs is 11,323 bp. The length of the 13 PCGs range from 303 to 1371 bp (Tables 2, 3).

The average A + T content is 65.26%, ranging from 39.63% (ND5) to 74.21% (ATP8) (Table 3). The AT-skew and GC-skew are −0.159 and −0.034, respectively (Table 3). All of the PCGs are initiated by the start codon ATN (ATT, ATG, and ATC), except ATP8 (GTG). The majority of the PCGs are terminated with TAA, whereas the other three PCGs (cox1, nad1, and nad2) use TAG as the stop condon (Table 2). The most frequently used amion acid in G. albolineatus is Leu, and the least common anion acid is Trp (Fig. 2). The relative synonymous codon usage (RSCU) values for G. albolineatus of the 13 PCGs are shown in Table 5 and Fig. 224. The three most frequently detected codons are GCU (Ala), UCU (Ser2), and GUA (Val), whereas GCU (Ala) is the least common codon. Based on CDspT and RSCU, comparative analyses showed that the codon usage pattern of G. albolineatus is conserved. The codon usage patterns of 13 PCGs are similar to those of other Grapsidae species.

Figure 2
figure 2

Codon usage patterns in the mitogenome of Grapsus albolineatus CDspT, codons per thousand codons. Codon families are provided on the x-axis (A), and the relative synonymous codon usage (RSCU) (B).

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Table 5 The codon number and relative synonmous codon usage in the mitochondrial genome of Grapsus albolineatus.
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Transfer RNAs and ribosomal RNAs

Like most Grapsidae species, G. albolineatus mitogenome contains 22 tRNA genes20,25,26. Fourteen of them are encoded by the heavy strain (H-) and the rest are encoded by the light strain (L-). In the whole mitogenome, the size of tRNAs range from 50 to 73 bp and have a total length of 1402 bp, with an obvious AT bias (71.54%) (Table 2). The AT-skew and GC-skew are −0.009 and 0.158, respectively, showing a slight bias toward the use of Ts and an apparent bias toward Cs (Table 3).

The 12S and 16S rRNA genes are 1331 and 827 bp, respectively, which are typically separated by tRNA-Val (Table 2). These sizes are similar to those of other Grapsidae species15,16,17,18,19. The A-T content of rRNAs is 72.57%. The AT-skew and GC-skew are −0.001 and 0.284, respectively, suggesting a slight bias toward the use of Ts and an apparent bias toward Cs (Table 3). As most typical mitogenomes of other crabs, CR is located between 12S rRNA and tRNA-Ile. The 617 bp CR is obviously AT biased (77.63%). The AT-skew and GC-skew are 0.173 and −0.203, respectively (Table 3), indicating an obvious bias toward the use of A’s and C’s. The index of substitution saturation (Iss) was measured as an implemention in DAMBE 5 and the GTR substitution model25. Iss is for the combined dataset of all PCGs of the 59 Brachyura mitogenomes and was significantly lower (Iss = 0.674) than the critical values (Iss, cSym = 0.859). The genes are not saturated, so the reconstructed phylogeny was reliable.

Gene rearrangement

Mitochondrial gene rearrangement is an important molecular marker and is considered to be an effective tool for studying mitochondrial evolution26. A large number of studies and results have shown that gene rearrangements in metazoan mitochondrial genomes are conserved20 and the occurrence of gene rearrangements is relatively random and rare1,19,20,27. However, it can be used as direct evidence of evolutionary relationships between species28. Mapping the gene layout based on the complete mitochondrial sequences of 70 species. Through comparison and analysis with the ancestor of Decapoda (Fig. 3A), we found that G. albolineatus and another 5 species from Grapsidae have a trnH translocation4,9,10,11,12,13, which the trnH shifted into trnE and trnF instead of the usual location between nad5 and nad4 (Fig. 3C). It is widely believed that the tandem duplication/random loss model (TDRL) can explain the movement of trnH, occur from tandem duplication in the region between trnE and nad4, followed by deletions of redundant genes producing trnH-trnF-nad5. Additionally, 45 species from 14 families (Grapsidae, Mictyridae, Ocypodidae, Bythograeidae, Calappidae, Dotillidae, Matutidae, Menippidae, Oziidae, Xanthidae, Oregoniidae, Geryonidae, Portunidae, and Carpiliidae) had the same gene rearrangement, which are consistent with the ancestral of Brachyura (Fig. 3B). However, the gene order in 4 families (Sesarmidae, Varunidae, Macrophthalmidae, and Xenograpsidae)30,32 displayed 4 patterns of gene rearrangements. The family Sesarmidae observed trnQ and trnI invertred, which has been described in previous studies (Fig. 3D)3,19,20,33. The gene order of the Varunidae (Grapsoidea) and Macrophthalmidae (Ocypodoidea) have the same high level rearrangementa (Fig. 3E). It is worth noting that the two families come from two different superfamilies, but they form a sister clade in phylogenetic trees. The gene order of the Xenograpsidae have a more complex rearrangement and such within-genus rearrangements were infrequent34 (Fig. 3F,G), which seems to be related to their particular habitat. Xenograpsidae have been found thus far only in shallow-water, volcanically active, and sulphur-rich hydrothermal vents35.

Figure 3
figure 3

Linear representation of gene arrangements of an (A) ancestor of Decapoda, (B) ancestor of Brachyura, (C) gene arrangement of Grapsus albolineatus and 13 familes, (D) gene arrangement of Sesarmidae, (E) gene arrangement of Varunidae amd Macrophthalmidae, (F) gene arrangement of Xenograpsus testudinatus, and (G) gene arrangement of Xenograpsus testudinatus. Gene arrangement of all genes are transcribed from left to right. The green box indicates the duplicated gene. 16S rRNA and 12S rRNA are the large and small ribosomal RNA subunits, respectively. The rearranged gene blocks are underlined and compared with ancestral gene arrangement of Brachyura. The genes encoded on the light strand are highlighted in red.

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Phylogenetic relationships

In the present study, the phylogenetic relationships were analyzed based on the sequences of the 13 PCGs to clarify the relationships in Brachyura. G. albolineatus and other 68 known brachyuran specie were analyzed, with P. nigrofascia and P. gracilipes as outgroups. The two phylogenetic trees (Maximum Likelihood (ML) tree and Bayesian Inference (BI) tree) resulted in identical topological structuring with different supporting value. Then, only one topology (ML) with both support values was presented displayed (Fig. 4). Both trees showed that all the species of Grapsidae clustered together as a solid monophyletic group and consist of three sister clades ((Grapsus + Pachygrapsus) + Metapograpsus). It is obvious that G. albolineatus had the closest relationship with G. tenuicrustatus, and that these two species form a sister clade with high support values (BI posterior probabilities PP = 1, ML bootstrap BP = 100), constituting a Grapsus group. However, recent molecular studies, including our dataset, have not reached an agreement about closest relatives in Grapsidae. Our phylogenetic tree showed that Grapsidae and Dotillidae form a sister clade, which was in concordance with Wang et al.10. While Wang et al. and Ng, N. K. et al. found that Grapsidae do not have any close relatives9,35, Li et al.36 found that Grapsidae and Ocypodidae form a sister clade.

Figure 4
figure 4

The phylogenetic tree was inferred from the nucleotide sequences of 13 mitogenome PCGs using BI and ML methods. Numbers on branches indicate posterior probability (BI) and bootstrap support (ML). The node marked with a solid citcle indicates 100 ML bootstrap support (BS) and 100% BI posterior probability (PP).

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Among the 21 families included in our phylogenetic tree, except Menippidae, each family in the tree forms a monophyletic clade with high nodal support values. At a higher level of classification, most Brachyura superfamilies were found to be monophyletic, except Ocypodoidea, Grapsoidea and Eriphioidea, which is in line with previous studies9,10,37. It showed that Grapsidea was divided into three clades (((Seasamidae + Gecarcinidae + Xengrapsidae) + Grapsidae) + Varunidae), Ocypodoidea was divided in three clades ((Ocypodidae + Dotillidae) + Macrophthalmidae + Mictyrisae) and Eriphioidea was divided into two clades (Oziidae + Menippidae). Within Thoracotremata, the superfamilies Ocypodoidea and Grapsoidea supported paraphletic and 9 families showed the following relayionship: ((((Seasamidae + Gecarcinidae) + Xengrapsidae) + Ocypodidae) + (Grapsidae + Dotillidae) + (Varunidae + Macrophthalmidae) + Mictyrisae) (Fig. 4).

The main phylogenetic structure of our tree is consistent with previous results, but some controversial findings were observed. Here, the families Macrophthalmidae and Varunidae were grouped into one clade, and Mictyridae as basal group which supports the previous findings revealed in Wang et al. and Zhang et al.9,33. However, previous researchers revealed that Macrophthalmidae and Varunidae were grouped into one clade, then into another clade with Varunidae ((Macrophthalmidae + Varunidae) + Mictyridae)38,39, which was conflict with our results. The classification of Grapsoidea and Ocypodoidea has long been controversial. Previous studies based on morphological characteristics considered them to be monophyletic branches. However, an increasing number of molecular studies, including ours, challenge the inconsistent views on the traditional classification system that are put forward. Although the polyphyly of Grapsoidea, Ocypodoidea, and Eriphioidea is well supported, the phylogenetic relationships of these superfamilies need to be further analyzed by integrating additional molecular data32,33,34,35,36. Previous studies on mitochondrial phylogeny have confirmed the importance of mitochondrial genomic data in elucidating the Grapsidae phylogeny13,19. On the contrary, many families contained only one representative, which may produce unstable phylogenetic relationships. Therefore, it is necessary to perform further mitogenome sequence studies to obtain a more comprehensive taxon sampling and understand the phylogeny and evolution of Grapsidae.

Materials and methods

Sampling and DNA extraction

A specimen of G. albolineatus was collected from Yangjiang, Guangdong Province, China (21°28′45″ N, 111°16′35″ E). The specimen was immediately preserved in absolute ethanol after collection and then stored at −20 °C. This specimen was identified by morphology and fresh tissues were dissected from the operculum and preserved in absolute ethanol before DNA extraction. The total genomic DNA was extracted using the salt-extraction procedure with a slight modification40 and stored at −20 °C.

Genome sequencing, assembly, and annotation

The mitogenomes of G. albolineatus was sequenced by Origin gene Co. Ltd., Shanghai, China and was sequenced on the Illumina HiSeq X Ten platform. HiSeq X Ten libraries with an insert size of 300–500 bp were generated from the genomic DNA. About 10 Gb of raw data was generated for each library. Low-quality reads, adapters, and sequences with high “N” ratios and length less than 25 bp were removed. The clean reads were assembled using the software NOVOPlasty (https://github.com/ndierckx/NOVOPlasty)42, annotated, and manually corrected on the basis of the complete mitogenome sets assembled de novo by using MITOS tools (http://mitos2.bioinf.uni-leipzig.de/index.py)43. To confirm the correct sequences, we compared the assembled mitochondrial genes with those of other Grapsus species and identified the mitogenomic sequences by checking the cox1 barcode sequence with NCBI BLAST43. The abnormal start and stop codons were determined by comparing them with the start and stop codons of other marine gastropods. Then, the reads were reconstructed using the de novo assembly program. The complete mtDNA was annotated using the software Sequin version 16.0 (https://trace.ncbi.nlm.nih.gov/Traces/sra). The mitogenome map of the G. albolineatus was drawn using the online tool CGView Server (http://cgview.ca/)45. The secondary structures predicted of the tRNA genes were plotted by using MITOS Web Server. The relative synonymous codon usage (RSCU) values and substitution saturation for the 13 PCGs, calculated by DAMBE 545, were analyzed with MEGA 746. The GC-skews and AT-skews were used to determine the base compositional difference and strand asymmetry among the samples. According to the following formulas46, composition skew values were calculated as AT-skew = A − T/A + T and GC skew = G − C/G + C. Substitution saturation for the 13 PCGs was calculated by DAMBE 545.

Phylogenetic analysis

The phylogenetic relationships within Brachyura were reconstructed using the sequences of the 13 PCGs of a total of 57 complete mitogenome sequences downloaded from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) and adding two species of Paguridae to serve as the outgroup (Table 1). The phylogenetic relationships were analyzed with Maximum Likelihood (ML) by using IQ-TREE 1.6.2 and Bayesian Inference (BI) methods in MrBayes 3.2 version program47,48,49. The ML analysis was inferred with 1000 ultrafast likelihood bootstrap replicates by using IQ-TREE 1.6.2. The best-fit model for each partition was GTR + F + R6, selected according to the Bayesian information criterion (BIC). BI was performed in MrBayes 3.2, and the best-fit evolutionary models were determined using MrMTgui50. MrMTgui was used to associate PAUP, ModelTest, and MrModelTest across platforms. MrBayes settings for the best-fit model (GTR + I + G) were selected by Akaike Information Criterion (AIC) in MrModelTest 2.351,52. The Bayesian phylogenetic analyses were performed using the parameter values estimated with the commands in MrModelTest or ModelTest (nst = 6, rates = invgamma)53. With three hot chains and one cold chain, they were run simultaneously twice by Markov Chain Monte Carlo (MCMC) sampling, and the posterior distribution was estimated. The MCMC chains were set for 2,000,000 generations and sampled every 1000 steps, with a relative burn-in of 25%. The convergence of the independent runs was evaluated by mean standard deviation of the split frequencies (< 0.01). The phylogenetic trees were visualized and edited using Figure Tree v1.4.3 software54.

Conclusions

In this study, the mitogenome of G. albolineatus was sequenced by next-generation sequencing, thereby generating new mitochondrial data for Grapsidae and confirming its ancestral gene order. The G. albolineatus mitogenome is a typical closed-circular molecule including 13 PCGs, 22 tRNA genes, two rRNA genes, and a CR. The AT-skew and GC-skew are both negative in the mitogenome of G. albolineatus, showing an obvious bias towards the use of T’s and C’s, consistent with published findings in most Brachyura crabs. G. albolineatus exhibits a novel gene rearrangement, which is similar to G. tenuicrustatus, P. crassipes, P. marmoratu, M. frontalis, and M. quadridentatus. Compared with the pan-crustacean ground pattern, the trnH of G. albolineatus shifted into trnE and trnF instead of the usual location between nad5 and nad4. By adding 62 Brachyura mitochondrial genomes, rearrangement and the phylogeny of Brachyura was reanalyzed. The phylogenetic analyses indicated that G. albolineatus has close relationships with G. tenuicrustatus, P. crassipesand, P. marmoratu, M. frontalis, and M. quadridentatus, belonging to Grapsoidea, part of the Grapsidae family.