Phylomitogenomics reconfirm the phylogenetic position of the genus Metaplax inferred from the two grapsid crabs (Decapoda: Brachyura: Grapsoidea)

Two new complete mitogenomes of the grapsids, Metaplax longipes Stimpson, 1858 and Nanosesarma minutum (De Man, 1887) were sequenced using next-generation sequencing (NGS). By analyzing a combination of 75 Brachyura taxa, our phylomitogenomic inferences suggested that Metaplax crab seperated earlier from the sesarmid crabs and closely related to the varunids with respect to Nanosesarma crab. It reconfirmed that the Metaplax should be removed from the Sesarmidae and assinged to the Varunidae. Additional mitogenomic comparisons including gene rearrangement and genomic organization were conducted among the 33 taxa of Grapsoidea and Ocypodoidea, and a shared rearrangement pattern between Metaplax longipes and the varunids were recovered, which also strongly supported the inference for the phylogenetic position of the Metaplax.


Introduction
The genus Metaplax H. Milne Edwards, 1852, which currently contains 12 species [1], along with three other genera, Cyclograpsus, Chasmagnathus and Helice, found in the China Seas, were historically assigned to the Sesarminae [2][3], based on the presence of a hairy crest on the third maxilliped and distinct characters in the pterygostomian and ventrolateral regions of the carapace [3][4][5][6]. However, the cladistic analysis proposed by Sternberg and Cumberlidge [7] found that Metaplax and the five genera Chasmagnathus De Haan, 1833, Helice De Haan, 1833; Cyclograpsus H. Milne Edwards, 1837; Paragrapsus H. Milne Edwards, 1853; and Helograpsus Campbell & Griffin, 1966 fail to conform to the other sesarmine genera. The early molecular phylogenetic analysis based on partial sequences of 12S rRNA and/or 16S rRNA revealed that these six genera were more closely related to other varunid crabs and suggested that Metaplax and the five related genera were excluded from the Sesarminae and were included in the Varuninae [8][9][10][11]. The validity of the character of a hairy crest on the 3 rd PLOS ONE | https://doi.org/10.1371/journal.pone.0210763 January 25, 2019 1 / 11 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 maxilliped for intergeneric taxonomy, therefore, needs to be reevaluated, as proposed by Schubart et al [8,9]. The morphological characters, such as the adult morphological characters, i.e., the location of the male genital openings [12], typically a stridulatory suborbital crest that is relatively straight and extends some distance across the lateral branchial region [13] and several larval characters, i.e., the 2, 2 seta l pattern on the endopod of the maxilla and the type of antenna and telson [14], provide strong support that these genera are included to the Varunidae. Therefore, these six genera, including Metaplax, combined with Helicana K. Sakai & Yatsuzuka, 1980, in the updated system of the Brachyurans of the world, were assigned to the Cyclograpsinae and Grapsidae by Davie in 2002 [13] and, more recently, to the Cyclograpsinae, Varunidae, and Grapsoidea by Ng et al. in 2008 [1]. However, the updated position of the genus Metaplax that proposed by Ng et al. was not commonly accepted, as in recent years the genus still has been included within the Grapsidae rather than in the Varunidae in some researchs [15,16]. The phylogenetic position of the Metaplax needs to be further confirmed. Mitogenomic data, including both primary sequences and gene rearrangements, have been shown to contain useful phylogenetic information for inferring the evolutionary relationships among eubrachyurans [17][18][19][20][21][22]. Compared to the data on the partial fragments of genes, phylogenetic reconstruction using mitogenomic data could help avoid the interference of nuclear pseudogenes [23]. Gene order patterns have the potential to act as synapomorphies for specific lineages and taxonomic groups, providing support for the hypotheses for phylogenetic relationships [21,24]. Here, to reconfirm the taxonomic assignment of the genus Metaplax, we first sequenced and annotated the complete mitogenome of Metaplax longipes Stimpson, 1858. We also added the mitogenome of Nanosesarma minutum (De Man, 1887), representing the first species from the Nanosesarma (Grapsoidea: Sesarmidae), not only to increase the taxon, but also to make additional comparisons in the nucleotide contents and the gene rearrangements of mitogenome among related groups by which to provide new evidence for taxonomic position of the Metaplax. As typical semiterrestial species, Grapsoidea live in mangrove swamps or on rocky shores, muddy and sand shores or other swampy grouds, but in this kind of large ecological regions there are various niches [3]. For example, the M. longipes likes intertidal mud flats and sand beach, while the N. minutum prefers to living on the muddy ground or under stones. Obtaining a stable phylogenetic position of M. longipes and N. minutum based on mitogenomic data will help us to understand the their habitatal preference.

Ethics statement
No specific permits were required for crab collection and research in the selected locations. The sampling locations were not privately owned or protected in any way. The crabs used for the experiments were not considered protected or endangered, and their collection is legal in China.

DNA extraction and sequencing
Total genomic DNA was extracted using a Cell and Tissue DNA Extraction Kit according to the manufacturer's instructions (Generay Biotech, Shanghai, China). The quality of the extracted DNA was examined by 1% agarose gel electrophoresis and was then sent to Novogene (Beijing, China) for sequencing using the Illumina MiSeq Desktop Sequencer (2 × 150 bp paired-end reads). The average insert size of the sequencing libraries was approximately 300 bp.

Mitochondrial genome assembly and annotation
Mitochondrial genome assemblies were conducted in Geneious 11.1.2 using parameter settings described previously [22,25]. Protein-coding genes (PCGs) were identified by finding the ORFs (https://www.ncbi.nlm.nih.gov/orffinder) using the invertebrate mitochondrial genetic code. The secondary structure and anticodons of transfer RNA (tRNA) genes were identified using the results of both tRNAscan-SE [26] and MITOS Web Server (http://mitos2. bioinf.uni-leipzig.de/index.py) analyses [27]. Similarly, the large and small rRNA subunits (srRNA and lrRNA) were determined using MITOS Web Server or alignment to closely related references. The graphical map of the mitogenomes was drawn using the online mitochondrial visualization tool Organellar Genome DRAW (https://chlorobox.mpimp-golm.mpg.de/ OGDraw.html) [28]. The nucleotide composition was calculated in MEGA 6.0 [29]. The mitogenomes of M. longipes and N. minutum were deposited under the following respective Gen-Bank accession numbers: MH899176 and MH899177.

Phylogenetic analysis
To infer the phylogenetic position of the studied species, we used a 75-taxon set as ingroups from divergent lineages comprising the 55 genera, 25 families and 15 superfamilies of Brachyura (S1 Table). Eight species from the Gebiidea and Anomura were also included in the analyses as outgroups (S1 Table). After removing all termination codons, the putative amino acid (AA) sequences of the 13 PCGs and sequences for two rRNAs were individually aligned using MAFFT 7.215 [30]. The ambiguously aligned regions from each gene were removed by Gblocks V. 0.91b with default settings [31]. The pruned, aligned AA sequences were then used as a backbone to align the corresponding nucleotide (NT) sequences using DAMBE 5.3.15 [32]. The final NT data was composed of a concatenation of the 13 PCGs and two rRNAs. We then conducted phylogenetic analyses using Maximum likelihood (ML) and Bayesian Inference (BI). The best-fit partitioning schemes and the substitution model for each partition were selected by PartitionFinder 1.1.1 [33] and ModelFinder [34] using a greedy search with Bayesian information criteria (BIC). The ML tree was reconstructed in IQTREE v1.6.3 with 1,000 ultrafast bootstrap (BS) replicates [35,36]. Bayesian inference (BI) was performed using MrBayes 3.2.2 [37] through the Cipres Science Gateway [38]. The Markov chain Monte Carlo (MCMC) was started with one million generations, with every 1000 generations sampled, and the first 25% of the generations were discarded as burn-in. The standard deviation of split frequencies was 0.0089 (below 0.01) after 1,500,000 generations, which reflected that the two runs strongly indicated convergence.

Mitogenomic general characters
The mitogenomes of M. longipes and N. minutum were determined to contain an entire set of 37 genes plus a larger main noncoding region (mNCR), and were 16,305 and 15,637 bp in length, respectively (Table 1; Fig 1). Notably, the length of the M. longipes mitogenome was similar to that of the varunid crabs sampled (ranging from 16,170 to 16,212 bp; Fig 1; S2 Table). We found that the length of varunid crabs mitogenomes was longer than the average length of the other grapsid mitogenomes (15,698 bp) and ranged from 15,406 to 15,920 bp.  Table), while the A+T content of the N. minutum mitogenome was the highest among the sesarmid crabs (avg. 76.03%), which is greater than the average value for the other grapsids, even ocypodid crabs.

Gene rearrangement
Of the two species presented in this study, M. longipes presented a major gene rearrangement pattern identical to that of varunid crabs, including three tRNA gene pairs (C-Y, K-D, and E-F), two tRNA genes (Q and V) and a mNCR translocated into the nad5-nad4 gene junction; furthermore, a four-gene block (nad1-L1-lrRNA-srRNA) and a tRNA gene pair (T-P) were rearranged into the location between nad3 and nad5, while N. minutum exhibited a typical tRNA gene cluster (Q-I-M) rearrangement identical to that of the other five sesarmid crabs (Fig 2).

Phylogenetic analysis
The ML and BI phylogenetic trees produced similar topologies, but slightly different in several species (i.e. Dynomene pilumnoides, Scylla serrate, Cyclograpsus granulosus, and Gaetice depressus) (Fig 3). Within Thoracotremata, the samples from Grapsoidea and Ocypodoidea did not form monophyletic groups. For example, Macrophthalmus species as a distinct lineage from other ocypodids that was sister to grapsid species with high support (BPP = 1.00, BS = 99). As well, the two dotillid crabs were more closely related to Grapsoidea than Ocypodoidea, with strong nodal support in the BI analysis (BPP = 1.00) and ML analysis (BS = 96). As to the grapsid species newly presented in these trees, the M. longipes separated from the sesarmid crabs and formed a clade with other eight publicly available species of Varunidae, while N. minutum is sister to Parasesarma tripectinis (Shen, 1940) and then formed a strongly supported monophyletic clade (BPP = 1.00, BS = 100) with other four species from Sesarmidae.

Discussion
Comparisons of mitogenomes, usually using both gene orders and DNA sequences (PCGs and the two rRNAs), provide strong support for brachyuran phylogenies [17,18,[21][22][23]39]. The length of the mitogenome is a basic character for each animal organism, with the entire set of 37 genes (except for several groups), which are closely packed and probably reflect a strong purifying selection over the evolutionary history, being highly conserved [17,40,41]. However, it was found here that there was an increase in the genome size of the mitogenome of the Metaplax crab. In addition, this length is similar to that found in the varunid crabs known to date. Further comparisons of gene orders and intergenic spacers revealed a consistent gene rearrangement pattern shared by the Metaplax and varunid crabs (Figs 3 and 4) with concordant multiple minor noncoding regions scattered in the mitogenomes. The genome organization with multiple intergenic spacers was first discovered in a varunid crab, Eriocheir, and was recognized as retention resulting from gene duplication and incomplete deletion, which further resulted in the gene rearrangement and increased genome size [17]. Phylomitogenomics reconfirm the phylogenetic position of the genus Metaplax A recent reevaluation of the partial mtDNA sequences and morphological data for the phylogenetic position of Metaplax and other related crabs suggested that contrary to earlier studies positing this genus in the Sesarmidae family of grapsids, this genus presented a closer relationship with varunid genera [8-12, 14, 42]. We report here the first complete mitogenome of Metaplax crabs. Our mitogenome-based phylogenetics indicate that M. longipes was closely related to the other representative varunid crabs and was separated earlier from the "Sesarmidae" and "Grapsidae" clades. This result strongly supported the suggestion that Metaplax should be removed from Sesarmidae [8][9][10][11], and assigned to Varunidae [1]. This relationship was also supported by the shared gene rearrangements among Metaplax and the varunid species sampled and genome organization recovered in the present study. The result has helped to clarify that the morphologies located at the 3 rd maxilliped are not a synapomorphy for supporting the genus Metaplax as a taxon within the Sesarmidae, which appear to have symplesiomorphy.
Previous studies have pointed out there are four distinct patterns of gene rearrangements in the available mitogenomes from Grapsoidea and Ocypodoidea (Fig 4) [39]. Gene order patterns can act as synapomorphies for specific lineages at family level, and show potential in providing additional phylogenetic markers [21,24]. In this study, the 2 nd pattern of gene rearrangement shared by all available mitogenomes from Sesarmidae. Noticeably, species from different superfamilies (Ocypodoidea and Grapsoidea) shared the 4 th gene rearrangement pattern, i.e. Macrophthalmidae and Varunidae, which supports a sister relationship presented in phylogenetic trees. The result also agreed with the previous suggestion that the polyphyly of Grapsoidea and Ocypodoidea [39,43]. Considering that only species from Grapsoidea and Ocypodoidea within Thoracotremata were sampled, the validity of their relationship needs to be reconfirmed by further taxonomic sampling.

Conclusion
In this study, we first determined the complete mitogenomes of the grapsid crabs M. longipes and N. minutum. Further, phylomitogenomic inferences suggested that N. minutum formed a clade with other sesarmids, while the M. longipes seperated earlier from the sesarmid clade. It reconfirmed that the Metaplax should be removed from the Sesarmidae and assinged to the Varunidae that proposed by Ng et al. or even older literature [1,[8][9][10][11]. New evidence that a consistent rearrangement pattern between M. longipes and the varunids was recovered, which also strongly supported the inference for the phylogenetic position of the Metaplax.
Supporting information S1