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Comparative mitochondrial genome analysis of Varunidae and its phylogenetic implications
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Ying Zhang1, 2, Li Gong1, 2, 3, Xinting Lu1, 2, Zengliang Miao2, Lihua Jiang1, 2, Bingjian Liu1, 2, Liqin Liu1, 2, Pengfei Li3, Xu Zhang4, Zhenming Lü1, 2, *
Acta Oceanologica Sinica | 2022, 41(6) : 119 - 131
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Acta Oceanologica Sinica | 2022, 41(6): 119-131
• Marine biology •
Comparative mitochondrial genome analysis of Varunidae and its phylogenetic implications
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Ying Zhang1, 2, Li Gong1, 2, 3, Xinting Lu1, 2, Zengliang Miao2, Lihua Jiang1, 2, Bingjian Liu1, 2, Liqin Liu1, 2, Pengfei Li3, Xu Zhang4, Zhenming Lü1, 2, *
Affiliations
  • 1 National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang Ocean University, Zhoushan 316022, China
  • 2 Marine Science and Technology College, Zhejiang Ocean University, Zhoushan 316022, China
  • 3 Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530007, China
  • 4 Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
Published: 2022-06-25 doi: 10.1007/s13131-021-1927-7
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Complete mitochondrial genomes (mitogenomes) can indicate phylogenetic relationships, as well as useful information for gene rearrangement mechanisms and molecular evolution. Currently, the phylogenetic location of the genus Varuna (Brachyura: Varunidae) has not been well resolved mainly because of limited representatives (only two extant species). Here, we determined a new mitogenome of this genus (Varuna litterata) and added the published mitogenomes to reconstruct the phylogeny of Varunidae. The 16 368-bp mitogenome contains the entire set of 37 genes and a putative control region. The characteristics of this newly sequenced mitogenome were described and compared with the other 15 Varunidae mitogenomes. All 16 analyzed mitogenomes have identical gene order and similar molecular features. The sliding window and genetic distance analyses demonstrate highly variable nucleotide diversity, with comparatively low variability of COI and COII, and high variability of ND6. The nonsynonymous/synonymous substitution rates (dN/dS ratio) analysis shows that all 13 PCGs are under purifying selection and ATP8 gene evolves under the least selective pressure. Twelve tRNA genes, two rRNAs, one PCG, and the putative control region are found to be rearranged with respect to the pancrustacean ground pattern gene order. Tandem duplication/random loss model is adopted to explain the large-scale gene rearrangement events occurring in Varunidae mitogenomes. Phylogenetic analyses show that all Varunidae species are placed into one group, and form a sister clade with Macrophthalmidae. Nevertheless, the phylogenetic relationships within Varunidae are not completely consistent based on the two different datasets used in this study. These findings will contribute to a better understanding of gene rearrangement and molecular evolution in Varunidae mitogenomes, as well as provide insights into the phylogenetic studies of Brachyura.

varunid crab  /  Varuna litterata  /  mitogenome  /  gene rearrangement  /  tandem duplication/random loss  /  phylogeny
Ying Zhang, Li Gong, Xinting Lu, Zengliang Miao, Lihua Jiang, Bingjian Liu, Liqin Liu, Pengfei Li, Xu Zhang, Zhenming Lü. Comparative mitochondrial genome analysis of Varunidae and its phylogenetic implications[J]. Acta Oceanologica Sinica, 2022 , 41 (6) : 119 -131 . DOI: 10.1007/s13131-021-1927-7
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1 Introduction
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The typical metazoan mitochondrial genome (mitogenome) is a closed-circular molecule of 14–20 kb. It generally contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (12S and 16S), and an AT-rich region (also called control region, CR) (Boore, 1999). The mitogenome has been widely used as an ideal tool for population genetics, comparative genomics, and phylogenetic studies. The reasons for its popularity are as follows: (1) the small genome size and simple gene structure are convenient for PCR amplification and sequencing; (2) the strict maternal inheritance can avoid the complexity of parental inheritance; (3) the relatively high evolutionary rate and low level of recombination are suitable for conducting evolutionary and phylogenetic analysis (Gyllensten et al., 1991; Sato and Sato, 2013; Ma et al., 2015; Sanchez et al., 2016; Tan et al., 2018). Besides, comparative analyses of the complete mitogenomes of closely related taxa can provide key information for gene rearrangements and phylogenetic studies (Liu and Cui, 2010; Zhuang and Cheng, 2010; Xin et al., 2017). With the advent and maturation of next-generation sequencing (NGS) technologies, comparative mitogenomics has become an important method for adaptive evolution and phylogenetic analysis (Li et al., 2020; Wang et al., 2020; Zhang et al., 2020b). For instance, Wang et al. (2020) demonstrated the common origin of Macrophthalmidae and Varunidae, and supported the previous hypotheses of paraphyly of Ocypodoidea and Grapsoidea. In Zhang et al. (2020b), comparative analysis of mitogenome of a deep-sea crab Chaceon granulates revealed positive selection and novel genetic features, which should be useful for studies on crab evolution and adaptive mechanisms.
The true crabs (infraorder Brachyura), with over 7250 known species inhabiting marine, freshwater, and terrestrial habitats, are one of the most successful aquatic invertebrate taxa worldwide (Basso et al., 2017; Chen et al., 2018; Ma et al., 2019). However, the extreme morphological and ecological diversity have made them difficult to be identified, and thus darken the real phylogenetic relationships (Tan et al., 2018; Camargo et al., 2020). The Grapsoidea is one of the largest superfamilies within Brachyura, and its classification and phylogeny have long been disputed. It is traditionally recognized as a monophyletic group based on morphological characteristics (Martin and Davis, 2001; Ng, 2008; Davie et al., 2015); however, an increasing number of molecular studies have challenged the monophyly of this taxon (Chen et al., 2018, 2019; Tan et al., 2018; Lu et al., 2020). Moreover, members of the current family Varunidae (Brachyura; Grapsoidea) were previously grouped as a subfamily (Varuninae) of Grapsoidae (Alcock, 1900). Afterward, it was elevated to family level based on larval morphology and molecular data (Schubart et al., 2000, 2002). Among them, Varuna is a commercially important genus that is widely distributed in the Indo-West Pacific (Jamieson et al., 1996; Ng, 2006). According to World Register of Marine Species (http://www.marinespecies.org/), there are only two extant species in this genus (V. yui and V. litterata). Currently, most studies of this genus have focused on the morphology and growth (Tu, 1992; Ng, 2006), and there have been few researches on molecular phylogeny (Lin et al., 2018). To date, the phylogenetic location of the genus Varuna has not been well resolved mainly because of limited representatives.
Accordingly, in the present study, we determined a new mitogenome of this genus (V. litterata) and added the published mitogenomes to reconstruct the phylogeny of Varunidae. A detailed comparison of 15 published Varunidae mitogenomes was also performed to reveal the gene rearrangements and genomic evolution. Additionally, the most comprehensive molecular phylogenetic analysis of 109 Brachyuran species was conducted based on the nucleotide and amino acid sequences of 13 PCGs. These results will help to better understand the rearrangement processes and possible mechanisms of Varunidae mitogenomes and lay a foundation for further evolutionary relationships within Brachyura.
2 Materials and methods
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2.1 Ethics statement
The species was not involved in the endangered list of International Union for Conservation of Natural Resource (https://www.iucnredlist.org/). Specimen collection and maintenance were performed in strict accordance with the recommendations of Animal Care Quality Assurance in China. All experimental protocols were approved by the Institutional Ethics Committee of Zhejiang Ocean University.
2.2 Sample collection and identification
An individual specimen of V. litterata was collected from Hainan Province, China (18°20'18''N, 109°30'50''E). The specimen was immediately preserved in absolute ethanol after collection and then stored at –20°C. Identification of this specimen was performed with a stereo dissecting microscope based on the key morphological features of crabs (Aiyun and Siliang, 1991; Ng, 2006). After identification, we measured the width and length of its carapace, which were about 3.2 cm and 2.9 cm, respectively. Also, the total body weight of this specimen was about 7.2 g.
2.3 DNA extraction, mitogenome sequencing and assembly
The SQ Tissue DNA Kit (OMEGA) was used to extract the total genomic DNA following the manufacturer's instructions. The mitogenome was obtained using a next-generation sequencing method with Illumina Hiseq 4000 at Shanghai Origingene Biopharm Technology Co. Ltd. Raw sequence data were deposited in Short Read Archive database (https://www.ncbi.nlm.nih.gov/sra/) with the accession No. SRX7809615. Clean data without sequencing adapters were de novo assembled by the NOVOPlasty 2.7.2 software (Dierckxsens et al., 2017). To assess the single-base accuracy of the assembled genome, we compared it with three confirmed sequences by PCR and Sanger sequencing methods.
2.4 Mitogenome annotation and sequence analyses
The complete mitogenome was manually annotated using the software of Sequin (version 15.10, http://www.ncbi.nlm.nih.gov/Sequin/). The PCGs were determined by the Open Reading Frame following the invertebrate mtDNA translation table. The boundaries of rRNA and tRNA genes were performed using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov) and tRNAscan-SE 1.21 (Lowe and Chan, 2016), respectively, comparing with the related species. Transfer RNA genes were manually plotted, according to the secondary structure predicted by tRNAscan-SE 1.21 (Lowe and Chan, 2016) and MITOS Web Server (Bernt et al., 2013) with invertebrate mitochondrial genetic codes. The control region was determined by the locations of adjacent genes. The mitogenome map was drawn by CGView Server version 1.0 (Stothard and Wishart, 2005). The tandem repeats in the control region were detected using Tandem Repeats Finder 4.09 (Benson, 1999). The base composition was obtained using MEGA X (Kumar et al., 2018). The following formulae were used to calculate strand asymmetries: AT-skew = (A−T) / (A+T), GC-skew = (G−C) / (G+C) (Perna and Kocher, 1995).
Sixteen complete mitogenomes including the newly sequenced one were selected to better understand the mitogenomic evolution of Varunidae species. The concatenated sequences of the aligned 13 PCGs and two rRNAs from these Varunidae mitogenomes were used to perform the DNA polymorphism sliding window analysis. The nucleotide diversity (Pi) of each PCG and rRNA gene and a sliding window of 200 bp at a step size of 20 bp were conducted using DnaSP version 6.12.03 (Rozas et al., 2017). The nonsynonymous (dN) and synonymous (dS) substitution rates were deduced from the data of PCGs sequences alignment of 16 varunid species using Mega X (Kumar et al., 2018). The genetic distances of 13 PCGs were also calculated using Mega X with Kimura-2-parameter. Besides, pairwise comparisons of gene order among Decapoda, Brachyura, and Varunidae were executed by CREx program (Bernt et al., 2007). The rearrangement mechanism for Varunidae was deduced using the common intervals parameter, which considers events of transpositions, inverse transpositions, inversions, and tandem duplication random loss.
2.5 Phylogenetic analysis
One hundred and ten complete mitogenome sequences downloaded from Genbank database (https://www.ncbi.nlm.nih.gov/genbank), and one newly determined sequence (V. litterata) was used to reconstruct the phylogenetic relationships among Brachyura (Table S1). The nucleotide and amino acid sequences of 13 PCGs for each species were extracted from the GenBank files using PhyloSuite (Zhang et al., 2020a). All genes were aligned in batches with MAFFT (Katoh et al., 2002) integrated into PhyloSuite, using normal-alignment mode. Gblocks (Talavera and Castresana, 2007) was used to identify and remove the ambiguously aligned sequences using default settings. The sequences were then concatenated and used to generate input files (phylip and nexus format) for phylogenetic analyses. The best-fit model was selected by ModelFinder (Kalyaanamoorthy et al., 2017) based on the Bayesian Information Criterion (BIC). GTR+F+I+G4 and mtMet+F+R6 were selected as the best-fit models for nucleotide and amino acid sequences, respectively. Both maximum likelihood (ML) and Bayesian inference (BI) were employed for phylogenetic analyses. The ML analysis was conducted using IQ-TREE (Nguyen et al., 2015), under an ML+rapid bootstrap (BS) algorithm with 1000 replicates. The BI analysis was carried out in MrBayes 3.2.6 (Ronquist et al., 2012) with default parameters. Two independent runs of four Markov chain Monte Carlo (MCMC) chains (one cold chain and three hot chains) were simultaneously run for 3×106 generations, with sampling conducted every 1000 generations. The final average standard deviation of split frequencies fell to 0.0018, which was considered to reach convergence (<0.01). The first 25% samples were discarded as burn-in, and the remaining trees were used to calculate Bayesian posterior probabilities (BPP) in a 50% majority-rule consensus tree.
3 Results
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3.1 Sequence features of V. litterata mitogenome
The complete mitogenome of V. litterata is a circular double-stranded DNA molecule of 16 368 bp in length (Fig. S1). The sequence has been deposited in GenBank under accession number MT193719. It contains 13 PCGs, 2 rRNAs, 22 tRNAs, as well as a putative CR (Fig. S1, Table 1). The majority of 37 genes are encoded by the heavy (H-) strand, except 4 PCGs and 2 rRNAs (Table 1). The overall nucleotide composition is 35.2% A, 36.2% T, 10.8% G, and 17.8% C, which reveals a strong AT bias (71.4%) (Table 2). The nucleotide skew statistics show negative AT-skew (−0.014) and negative GC-skew (−0.243) (Table 3). Overall, the genes in V. litterata mitogenome are closely arranged with overlapping (eight overlappings totaling 22 bp) and interval (19 intergenic spacers totaling 382 bp) phenomena (Table 1).
All the 13 PCGs are initiated by the canonical start codon ATN. Most PCGs terminate with TAA or TAG, whereas COI and Cyt b use an incomplete stop codon T (Table 1). Incomplete stop codons are common in metazoan mitogenomes and may be recovered via post-transcriptional polyadenylation (Ojala et al., 1981). Both the AT-skew and GC-skew of the 13 PCGs are negative, −0.177 and −0.011; respectively (Table 2), showing an obvious bias toward the use of Ts and a slight bias toward Cs in the entire protein-coding gene sequence. Besides, the GC-skew values of four PCGs (ND5, ND4, ND4L, and ND1) are positive, indicating they are encoded by the L-strand, whereas the remaining nine exhibit negative values, indicating they are encoded by the H-strand (Table 2).
The 22 tRNA genes are interspersed throughout the entire genome. The total length is 1454 bp, with each tRNA gene ranging from 63 bp to 73 bp. Most tRNAs can fold into the typical cloverleaf structure except of tRNA-Ser (TCT) that lacking the dihydrouridine arm (Fig. S2), which seems to be a common feature in metazoan mitogenomes (Wang et al., 2015; Gong et al., 2019, 2020). Except for the Watson-Crick base pairs (A-T and G-C) and G-U matches, a total of three mismatched base pairs are found, including two U-U base pairs in tRNA-Asn and tRNA-His, and one C-A base pair in tRNA-Met. Such mismatches are probably corrected through posttranscriptional RNA editing (Lavrov et al., 2000; Masta and Boore, 2004). The 16S and 12S rRNA genes are located between tRNA-Leu (L1) and tRNA-His (H), with a total length of 2262 bp (Fig. S1, Table 1). Both of them exhibit positive GC-skew (Table 2), indicating they are encoded by the L-strand.
3.2 Comparative analysis of Varunidae mitogenomes
The sequence features of the new mitogenome in this study (V. litterata) and the other 15 Varunidae mitogenomes are compared. The mitogenomes possess variations in size that range from 15 915 bp (V. yui) to 16 898 bp (Pseudohelice subquadrata). The concatenated PCGs, tRNAs, and rRNAs are quite conserved in length except the unusually larger size of rRNAs in P. subquadrata (Fig. S3, Table S1). The maximum length diversification is detected in the rapidly evolving CR. It ranges from 643 bp to 1096 bp, which is the primary contributor to variations in Varunidae mitogenome sizes. Besides, the size of mitogenome is also related to the overlapping regions and intergenic spacers dispersing in the mitogenomes.
The nucleotide composition of 16 Varunidae mitogenomes shows that all of them are rich in As and Ts, with the A+T content ranging from 67.7% (P. subquadrata) to 74.6% (Neoeriocheir leptognathus) (Table 3). Skew metric shows that all mitogenomes are highly consistent with negative base skewness, except Metaplax longipes and P. subquadrata exhibit positive AT-skew values (Table 3). Further analysis reveals that GC-skew values of PCGs in the L-strand are much higher than those in the H-strand (Fig. 1a). In contrast, there is no significant difference in AT-skew values between the two strands. Most AT-skew values are negative, except that of COII and ATP8 genes in M. longipes mitogenome (Fig. 1b).
In the ancestral crustacean mitogenomes without gene rearrangements, CR is typically located between 12S rRNA and tRNA-Ile genes. However, it moves to the location between tRNA-Val and tRNA-Gln genes in all 16 Varunidae mitogenomes. Tandem repeat elements are one of the most common structures in CR (Li and Liang, 2018; Li et al., 2019). Here, each mitogenome of Varunidae has tandem repeat units except Cyclograpsus intermedius. The motif (consensus nucleotide) length varies from 2 bp to 113 bp and the copy number ranges from 2 to 139.5 (Fig. 2). The tandem repeat units differ in nucleotide composition, length, and copy number across these varunid species, thus leading to the length heteroplasmy of CR (Fig. 2).
Nucleotide diversity of 16 Varunidae mitogenomes is conducted using the DNA polymorphism sliding window analysis. Due to the instability of tRNA and non-coding CR, we only calculate the nucleotide diversity of the 13 PCGs and 2 rRNAs. The result indicates a highly variable nucleotide polymorphism of these genes. Among them, ND6 gene presents the highest polymorphism (Pi=0.267), followed by ATP8 (Pi=0.240) and ND2 (Pi=0.237). Inversely, COII (Pi=0.161) and COI (Pi=0.163) show the lowest values, suggesting they are the conserved genes. Two rRNA genes are moderately polymorphic, with nucleotide diversity values of 0.204 in 16S rRNA and 0.178 in 12S rRNA, respectively (Fig. 3a). Congruent results are observed based on the K2P genetic distance analysis. Both COI and COII genes possess the least genetic distance (average 0.187), and ND6 gene captures the largest value (average 0.348), representing the most conserved and variable genes, respectively (Fig. 3b).
To investigate the selective pressure imposed on the PCGs in these varunid species, we perform dN/dS analyses for each PCG. All of the dN/dS ratios are lower than one (<1), indicating that all 13 PCGs are evolving under purifying selection. The dN/dS ratios of individual genes are different, reflecting the different levels of functional constraints (Muse, 2000). COI gene exhibits the strongest purifying selection with the lowest dN/dS value (0.024), whereas ATP8 gene exhibits a highly relaxed purifying selection with the highest dN/dS value (0.469) (Fig. 3b). The dN/dS ratio analysis indicates that the evolution of Varunidae mitogenomes has been dominated by purifying selection.
3.3 Gene rearrangement
Compared with the gene arrangements in the mitogenomes of ancestral crustaceans (the pancrustacean ground pattern) (Boore et al., 1998), the gene order in V. litterata and 15 other Varunidae mitogenomes underwent a large-scale rearrangement. At least nine gene rearrangements dramatically altered the gene order, involving 12 tRNA genes (K, D, E, F, H, T, P, L1, V, Q, C, and Y), 2 rRNAs (16S and 12S rRNA), 1 PCG (ND1), and a putative CR (Fig. S4). Of these gene rearrangements, 3 tRNA gene pairs (K-D, E-F, and C-Y) and 2 single tRNA genes (V and Q) are moved into the ND5 and ND4 junction, forming an 8-tRNA cluster (V-Q-C-Y-K-D-E-F) if CR is not considered. The CR is shifted from the typical area between 12S rRNA and I to the V and Q junction. In addition, the ND1-L1-16S-12S gene cluster, 1 tRNA gene pair (T-P), and a single H gene are moved to the position between ND3 and ND5. Correspondingly, the A-R-N-S1-E-F cluster order is changed into A-R-N-S1-T-P order.
3.4 Phylogenetic analysis
To investigate the phylogenetic status of the genus Varuna within Varunidae and the phylogenetic relationships of Brachyura, the phylogenetic analysis of 109 Brachyuran species was conducted based on the concatenated nucleotide and amino acid sequences of 13 PCGs using ML and BI methods (Figs 4 and 5, Figs S5 and S6). All trees show that V. litterata is most closely related to the species of the same genus, V. yui. All 16 Varunidae species cluster into a clade and form a sister group with Macrophthalmidae, supporting the recent molecular researches (Wang et al., 2019, 2020; Lu et al., 2020). Nevertheless, the phylogenetic relationships within Varunidae are not completely consistent with each other and the support values are relatively low (Fig. 4). The nucleotide sequences place Gaetice depressus at the most basal position in both ML and BI trees, whereas it is replaced by Metaplax longipes and nests within the Varunidae clade in the amino acid trees. Furthermore, Cyclograpsus granulosus and C. intermedius belonging to the same genus cluster together as the closest relatives in the amino acid trees as a matter of course, however, P. subquadrata replaces C. intermedius’s position and C. intermedius scatters in the clade of the nucleotide trees.
In the family-level relationship within Brachyura, the phylogenetic trees (ML tree and BI tree) based on nucleotide sequences show an identical topology; thus, only one topology (BI) with both support values is displayed (Fig. 5). However, the phylogenetic trees based on amino acid sequences are not consistent, and both trees are slightly different from the nucleotide trees (Figs S5 and S6). Of the 29 families included in this phylogeny, the monophyly of each family is well supported except Xanthidae and Homolidae (Fig. 5, Figs S5 and S6). However, it is worth noting that the monophyly of Gecarcinidae is presented in the amino acid trees, whereas it consists of two clades in the nucleotide trees, one of which forms a sister clade with Sesarmidae (Fig. 5, Figs S5 and S6). Viewed from a higher taxonomic level, most superfamilies of Brachyura are found to be monophyletic, with the exception of Eriphioidea, Ocypodoidea, and Grapsoidea. Although the polyphyly of the above three superfamilies is well supported in both the amino acid and nucleotide trees, the interrelationships of the three groups are inconsistent (Fig. 5, Figs S5 and S6). An in-depth analysis of a larger number of Brachyuran samples is required to further elucidate the phylogenetic relationships of Eriphioidea, Ocypodoidea, and Grapsoidea.
4 Discussion
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4.1 Possible gene rearrangement mechanism
How did the mitogenome structure of Varunidae species emerge? Four dominating mechanisms have been proposed to account for mitogenomic rearrangements, including tandem duplication/random loss (TDRL) model (Moritz and Brown, 1987), tandem duplication/non-random loss (TDNL) model (Lavrov et al., 2002), recombination model (Lunt and Hyman, 1997), and tRNA mis-priming model (Cantatore et al., 1987; Jacobs et al., 1989). With the help of CREx, three TDRL events are found to have occurred in the mitogenomes of 16 varunid crabs. The hypothesized intermediate steps are as follows. Firstly, one single tRNA gene H underwent a transposition from the F-ND5-H to H-F-ND5 order, which is a common phenomenon in the ancestral mitogenomes of Brachyura and is widely explained by the TDRL model (Xin et al., 2017; Chen et al., 2019; Wang et al., 2020). The gene block (F-ND5-H) tandemly duplicated and generated two sets of the same gene cluster (F-ND5-H)-(F-ND5-H). Subsequently, a novel H-F-ND5 gene order was formed after a random loss of the duplicated genes (Fig. 6b). In the second rearrangement event, the gene block from K to Y underwent a complete copy, forming a dimeric block. Consecutive copies were then followed by a random loss of supernumerary genes, forming a new gene block (K-, D-, ATP8-, ATP6-, COIII-, G-, ND3-, A-, R-, N-, S1-, E-, F-, ND4-, ND4L-, T-, P-, ND6-, Cyt b-, S2-, ND1-, L1-, 16S-, 12S-, I-, M-, ND2-, W-, H-, ND5-, V-, CR-, Q-, C-, and Y) (Fig. 6b). In the following step, the newly formed gene block from K to Y underwent a second copy and likewise experienced a random loss of redundant genes (Fig. 6b). Finally, the ultimate gene arrangements in Varunidae mitogenomes was generated, namely COI-, L2-, COII-, ATP8-, ATP6-, COIII-, G-, ND3-, A-, R-, N-, S1-, T-, P-, ND1-, L1-, 16S-, 12S-, H-, ND5-, V-, CR-, Q-, C-, Y-, K-, D-, E-, F-, ND4-, ND4L-, ND6-, Cyt b-, S2-, I-, M-, ND2- and W (Fig. 4c). Summarily, all the rearrangement events mentioned above can be explained by TDRL model, which supposes that the rearranged gene order occurs via tandem duplications followed by random deletion of certain duplications (Moritz et al., 1987; Arndt and Smith, 1998). The hypothesis of TDRL model leading to rearrangements in Varunidae mitogenomes accords with Chen et al. (2018) and Wang et al. (2020).
4.2 Phylogenetic relationship within Varunidae and Brachyura
At present, it seems to be a common phenomenon that the intergeneric relationship within Varunidae is unstable. In Tang et al. (2018), the genus Eriochei form a sister clade with Cyclograpsus. In contrast, Eriochei are closely relative to Hemigrapsus, while Cyclograpsus are placed at the most basal branch in Chen et al. (2018). Besides, the genus Varuna have a relatively distant relationship with Eriocheir in our phylogenetic trees, while in Wang et al. ( 2020), Varuna yui and the genus Eriocheir cluster together as a sister group. Limited representative genus and scarce samples of each genus might be responsible for the inconsistent relationships of Varunidae species. Accordingly, large-scale taxonomic samplings are needed to resolve the interrelationships within Varunidae in future studies.
The main topological structures in our phylogenetic trees follow the previous results, but some differences are also observed. Here, all trees show that Varunidae and Macrophthalmidae form a sister group and are quite distantly related to Mictyridae, which is congruent with most previous studies (Chen et al., 2019; Tan et al., 2019; Wang et al., 2020). While the phylogenetic relationships of these three families were exhibited as ((Macrophthalmidae + Mictyridae) + Varunidae) in recent study (Tan et al., 2018). Furthermore, the amino acid trees support the viewpoint of Chen et al. (2019) that Sesarmidae and Gecarcinidae are more closely related, and Dotillidae is the sister clade to Grapsidae. While the sister-group relationship between Sesarmidae and Dotillidae is revealed in the nucleotide trees, supporting the findings of Basso et al. (2017) and Tan et al. (2018). The unstable topology structures in phylogenetic trees might be caused by the limited representatives of the main taxa. In future studies, extensive samples, more accurate molecular markers, and integrated molecular and morphological data are needed to deduce the authentic phylogenetic relationships within Brachyura.
5 Conclusions
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In this article, we determined and described the complete mitogenome of V. litterata. The 16 368-bp mitogenome contains 37 genes and 1 AT-rich region, as is typical of metazoan mitogenomes. This newly sequenced mitogenome shares similar genomic features with 15 other previously reported Varunidae mitogenomes. Evolutionary analysis reveals that all 13 PCGs evolve under purifying selection, while the ATP8 gene evolves under a highly relaxed selection. All these 16 Varunidae mitogenomes capture the same gene rearrangements, which can be well explained by the TDRL model. Phylogenetic analyses indicate that all Varunidae species cluster into a clade, and form a sister clade with Macrophthalmidae. The polyphyly of three superfamilies (Eriphioidea, Ocypodoidea, and Grapsoidea) is reconfirmed. In future researches, large-scale taxonomic samplings and more classification markers are still required to further investigate the mitogenomic evolution in Varunidae and to better resolve the taxonomy and phylogenetic relationships within Brachyura.
Funding
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  • The Natural Science Foundation of Zhejiang Province under contract No. LY21C190007.
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Year 2022 volume 41 Issue 6
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doi: 10.1007/s13131-021-1927-7
  • Receive Date：2021-02-07
  • Online Date：2025-11-21
  • Published：2022-06-25
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  • Received：2021-02-07
  • Accepted：2021-03-29
Funding
The Natural Science Foundation of Zhejiang Province under contract No. LY21C190007.
Affiliations
    1 National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang Ocean University, Zhoushan 316022, China
    2 Marine Science and Technology College, Zhejiang Ocean University, Zhoushan 316022, China
    3 Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530007, China
    4 Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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